KR102203722B1 - Novel Compounds, cosmetic composition and pharmaceutical composition comprising the same - Google Patents

Abstract

The present invention relates to a novel compound, a cosmetic composition and a pharmaceutical composition containing the same, according to the present invention, by increasing the expression of autophagy-related proteins to activate autophagy to induce melanosomal degradation, and at the same time A cosmetic composition for whitening and a pharmaceutical composition for the prevention and treatment of melanin 　 pigment 　 hyperdeposited disease that effectively inhibits the inclusion effect and exhibits very excellent effects in the improvement, prevention and treatment of not only whitening effect but also melanin-induced pigmentation Can provide.

Description

Novel Compounds, cosmetic composition and pharmaceutical composition comprising the same

The present invention relates to a novel compound, a cosmetic composition and a pharmaceutical composition comprising the same, and more particularly, a novel compound that induces melanosome decomposition through autophagy activation, and inhibits endocytosis into keratinocytes, It relates to a cosmetic composition for skin whitening comprising the same, and a pharmaceutical composition for the prevention and treatment of melanin hyperpigmentation disease.

Skin color is largely determined by the amount of melanin, hemoglobin, and carotene, among which, melanin plays the most important role. Melanin not only determines the color of a person's skin, but also plays a role of protecting the skin.However, when excessive exposure to ultraviolet rays, air pollution, stress, and inflammation in the skin are excessively generated in the skin, pigmentation occurs. 　The skin color becomes black or blemishes. , Freckles, etc. can also be the cause. Among the external stimulating factors, ultraviolet rays are the largest 　 melanin 　 biosynthetic stimulator, and can affect various processes related to 　 melanin 　 production. In other words, ultraviolet rays act as a major factor in inducing 　melanin 　 hyperproduction through 　 promoting the activation of melanocytes, which are melanocytes, 　 promoting the secretion of melanin biosynthetic stimulating hormones, and promoting the oxidation of melanin or tyrosinase activity.

The biosynthesis of melanin proceeds by the action of various genes such as tyrosinase, TRP-1 (tyrosinase related protein 1), TRP-2 (tyrosinase related protein 2), and Pmel17, and corresponding enzymes. In addition, cytokines such as alpha-MSH hormone ( α - Melanocyte-stimulating hormone ), IL-1, TNF-alpha (tumor necrosis factor- α ), and GM-CSF ( Granulocyte -macrophage colony-stimulating factor ) Interaction is being affected.

Most of the whitening materials currently developed are materials that inhibit tyrosinase activity. However, some whitening materials that inhibit tyrosinase activity cause safety problems such as vitiligo, irritation and allergic reactions. For example, hydroxyquinone, which is known to most effectively inhibit tyrosinase, is known to have a side effect of killing melanocytes. Accordingly, there is an urgent need to develop a whitening material that controls the inclusion of melanocytes in keratinocytes without affecting the expression of tyrosinase and the melanin content of melanocytes.

Autophagy is a mechanism to regenerate energy and remove damaging substances by decomposing old or damaged cellular substances and organs when the intracellular energy source is depleted or excessive intracellular stress factors occur. Means to make. Recently, through various studies, it has been reported that as aging progresses or accelerates aging, intracellular autophagy activity decreases rapidly. In other words, when autophagy is suppressed, excessive accumulation of aging mitochondria or misfolded proteins in the cell increases free radicals and oxidative stress in the cell, resulting in a gene fragment resulting from apoptosis, which stimulates melanocytes. As a result, it is known that the maturation of melanosomes and transport to keratinocytes are induced.

In other words, by activating autophagy, decomposing and recycling old substances and organs in cells, preventing cell death through activation of antioxidant proteins, and quickly removing modified proteins, lipids, and mitochondria, ultimately causing cells to By providing an environment for survival in a more healthy state, it eliminates the maturation factor of melanosomes. In addition, according to a recent report, the skin color is regulated through the activation of autophagy, and there is also a study result that brightens the skin color of artificial skin.

In addition, protease-activated receptor 2 (PAR-2) expressed in keratinocytes is involved in the endocytosis of the influx of melanosomes into keratinocytes, and research results that can control pigmentation through this have been reported. That is, activation of PAR-2 by trypsin causes pigmentation, which can lead to hyperpigmentation or uneven skin tone.

Accordingly, the present inventors effectively decompose melanosomes in keratinocytes through autophagy activation to effectively inhibit skin pigmentation and at the same time promote PAR-2 inhibition to effectively inhibit the influx of melanosomes into keratinocytes. The present invention was completed to provide a novel compound capable of and a whitening use including the same.

Hung et al. Autophagy, 2009, 5, 4, 502-510 Hearing & Jimenez, Int J Biochem, 1987, 19(12), 1141-7 Funasaka et al. J Lipid Res. 1999, Jul;40(7), 1312-6 Qi et al. PLOS one, 2012, 7, 10, e46834 Xilouri et al. Brain, 2013, 136, 2130-2146 Kim et al. The Journal of Clinical Investigation, 2014, 124, 8, 3311-3324 Seiberg et al. Experimental Cell Research, 2000, 254, 25-32

An object of the present invention is to provide a novel compound or a pharmaceutically acceptable salt thereof capable of inducing melanosomal degradation through autophagy activation.

An object of the present invention is to provide a novel compound or a pharmaceutically acceptable salt thereof capable of reducing the influx of melanosomes through inhibition of PAR-2 activity.

An object of the present invention is to provide a cosmetic composition for skin whitening comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

An object of the present invention is to provide a 　 pharmaceutical composition for improving or treating melanin 　 pigment 　 hyperdeposited diseases comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

For the above-described purposes, the present invention provides a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof capable of inducing melanosomal degradation through autophagy activation.

[Formula 1]

[In Formula 1,

AA is an amino acid residue containing substituted or unsubstituted proline.]

In the formula 1, wherein AA; can be hydroxy, C _{1- 10} alkoxy, C _{1- 10} alkyl-carbonyl and amino acid glass containing proline is optionally substituted with one or more substituents selected from carboxy.

Formula 1 may be a compound represented by the following Formula 2 or a pharmaceutically acceptable salt thereof.

[Formula 2]

[In Chemical Formula 2,

R ₁ is C _{1- 10} alkyl carbonyl;

R ₂ is hydrogen, hydroxy, C _{1- 10} alkoxy, C _{1- 10} alkyl, carbonyl or carboxy.]

Formula 1 may be a compound represented by the following Formula 3 or a pharmaceutically acceptable salt thereof.

[Chemical Formula 3]

[In Chemical Formula 3,

R ₃ is hydroxy or C _{1- 10} alkyl;

R ₄ is hydrogen, hydroxy, C _{1- 10} alkoxy, C _{1- 10} alkyl, carbonyl or carboxy.]

For the above-described purposes, the present invention provides a cosmetic composition for skin whitening comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In the cosmetic composition for skin whitening, the active ingredient may be selected from the compound represented by Formula 2 or a pharmaceutically acceptable salt (A) thereof.

In the cosmetic composition for skin whitening, it may be selected from the compound represented by Chemical Formula 3 or a pharmaceutically acceptable salt (B) thereof.

In the cosmetic composition for skin whitening, the active ingredient is a compound represented by Formula 2 or a pharmaceutically acceptable salt thereof (A) and a compound represented by Formula 3 or a pharmaceutically acceptable salt thereof (B). It can be a mixture.

In the cosmetic composition for skin whitening, the active ingredient may be mixed in a weight ratio (A:B) of 1:0.1 to 1:1.

In the cosmetic composition for skin whitening, the active ingredient may be included in an amount of 0.0001 to 10% by weight based on the total weight.

For the above-described purposes, the present invention provides a 　 pharmaceutical composition for improving or treating melanin 　 pigment 　 hyperdeposited diseases comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In the 　 pharmaceutical composition for improving or treating melanin 　 pigment 　 hyperdeposited disease, the active ingredient may be selected from the compound represented by Formula 2 or a pharmaceutically acceptable salt (A) thereof.

In the 　 pharmaceutical composition for improving or treating melanin 　 pigment 　 hyperdeposited disease, the active ingredient may be selected from the compound represented by Chemical Formula 3 or a pharmaceutically acceptable salt (B) thereof.

In a pharmaceutical composition for improving or treating melanin 　 pigment 　 hyperdeposited disease, the active ingredient is a compound represented by Formula 2 or a pharmaceutically acceptable salt thereof (A') and a compound represented by Formula 3 or a pharmaceutical thereof It may be a mixture of acceptable salts (B').

In the 　 pharmaceutical composition for improving or treating melanin 　 pigment 　 hyperdeposited disease, the active ingredient may be mixed in a weight ratio (A':B') of 1:0.1 to 1:1.

In the 　 pharmaceutical composition for the improvement or treatment of melanin 　 pigment 　 hyperdeposited disease, the melanin 　 pigment 　 hyperdeposited disease is freckles, senile spots, liver spots, melasma, brown or black spots, sun pigment spots, blue melanoma, hyperpigmentation after drug use , Gestational brown spots and hyperpigmentation after inflammation may be selected from.

In the 　 pharmaceutical composition for improving or treating melanin 　 pigment 　 hyperdeposited disease, the active ingredient may be included in an amount of 0.0001 to 10% by weight based on the total weight.

The compound according to the present invention or a pharmaceutically acceptable salt thereof can protect cells from oxidative stress by activating autophagy by increasing the expression of autophagy-related proteins. In addition, various diseases and phenomena caused by oxidative stress can be improved, prevented, and treated. In particular, it has the advantage that it is possible to realize a surprising whitening effect through autophagy activation that induces melanosome decomposition and reduces melanin production.

The compound according to the present invention, or a pharmaceutically acceptable salt thereof, induces melanosome decomposition to reduce melanin deposition and at the same time effectively inhibits endocytosis into keratinocytes, thereby preventing the influx of melanosomes. In other words, it is very effective in improving, preventing, and treating hyperpigmentation disorders induced by melanin.

1 is a diagram schematically illustrating a mechanism for inhibiting the pigmentation process of keratinocytes according to the present invention.
2 is a result of confirming whether the example compound according to the present invention inhibits tyrosinase activity.
3 is a view showing the result of performing the MTT assay in melanocytes in order to confirm the treatment concentration without cytotoxicity of the example compound according to the present invention.
4 is a view showing the result of performing the MTT assay in keratinocytes in order to confirm the treatment concentration without cytotoxicity of the example compound according to the present invention.
5 is a result of analyzing whether or not the treatment of the example compound according to the present invention affects the expression change of a gene (Tyrosinase) related to melanosomal maturation.
6 is a result of analyzing whether or not the treatment of the example compound according to the present invention affects the expression change of a gene related to melanosomal maturation (MITF).
7 is a result of analyzing the changed melanin content by treatment of the example compound according to the present invention.
8 is a result of analysis of changes in expression of proteins related to autophagy activation and changes in mRNA of genes related to endocytosis by treatment of the example compounds according to the present invention.
9 is a result of comparative analysis of the effect on melanin deposition by treatment of the Example compound according to the present invention.
FIG. 10 is a confirmation of whether the ability to inhibit melanin deposition is dependent on autophagy by treatment of the Example compound according to the present invention, and changes in melanin content and protein related to autophagy activation in keratinocytes knock-down of ATG5 are shown. This is the result of comparative analysis.
11 is a result of analyzing through a confocal microscope whether or not melanosomes are decomposed through activated autophagy by treatment of an example compound according to the present invention.
12 is a result of quantitatively analyzing whether melanosomes are decomposed through activated autophagy by treatment of the example compound according to the present invention through an electron microscope (scale bar: 2 μm/500 nm/2 μm/500 nm) .
13 is a result of quantitative analysis of the inhibition and decomposition of melanosomes by the treatment of the example compound according to the present invention.
14 is a result of confirming whether melanin deposition is reduced in an artificial skin model by treatment of an example compound according to the present invention.
15 is a result of quantitative analysis through F&M staining that melanin deposition decreases in an artificial skin model by treatment of an example compound according to the present invention.

A novel compound according to the present invention, a cosmetic composition and a pharmaceutical composition containing the same are described below, but unless there are other definitions in the technical and scientific terms used at this time, those having ordinary knowledge in the technical field to which this invention belongs Descriptions of known functions and configurations that have meanings that are commonly understood by the self and may unnecessarily obscure the subject matter of the present invention will be omitted in the following description.

In the present specification, “alkyl”, “alkoxy” and other substituents including “alkyl” moieties include all straight chain or branched forms. In addition, alkyl, alkoxy and the like according to the present invention are preferred to have a straight-chain form of 1 to 10 carbon atoms or a straight-chain form to the carbon number, but having 11 to 30 carbon atoms is also an aspect of the present invention.

In the present specification, “alkylcarbonyl” refers to *-C(=O)alkyl, wherein the alkyl follows the above definition.

In the present specification, "pharmaceutically acceptable salts" are prepared using conventional techniques known in the art, and include salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of the inorganic or organic acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, trifluroacetic acid, Citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, or benzenesulfonic acid. In addition, examples of the base include alkali metals such as potassium and sodium; Alkaline earth metals such as magnesium; Or ammonium; And the like.

In the present specification, "improvement" refers to any action in which the condition is relieved or improved or beneficially changed by application of the composition according to the present invention.

In addition, the singular form used in the present specification may be intended to include the plural form unless otherwise indicated in the context.

In addition, the units used in the present specification are based on weight, and for example, the unit of% or ratio means weight% or weight ratio.

In addition, the numerical range used in the present specification is the lower limit and the upper limit and all values within the range, increments that are logically derived from the shape and width of the defined range, all of the values limited thereto, and the upper limit of the numerical range limited to different forms. Include all possible combinations of lower bounds.

In addition, in the present specification, the expression "comprising" is an open-type description having a meaning equivalent to an expression such as "include", "include", "have", or "feature", and is not listed further. It does not exclude elements, materials or processes.

The inventors of the present invention have devised a novel compound that can effectively induce melanosomal degradation through autophagy activation while researching on whitening materials. The compound according to the present invention has excellent autophagy activation ability, and is expected to be actively utilized in the improvement, prevention and treatment of diseases caused by melanin 　 pigment 　 hyperdeposition.

Specifically, the compound according to the present invention increases the expression of a protein related to autophagy activation, thereby inducing the degradation of melanosomes. Thus, it is possible to effectively inhibit the production of melanosomes. Therefore, a composition comprising this as an active ingredient can exhibit excellent effects in improving, preventing and treating melanin 　 pigment 　 hyperdeposited diseases as well as skin whitening effect.

At the same time, the compound according to the present invention inhibits the activation of protease-activated receptor 2 (PAR-2) expressed in keratinocytes, effectively inhibiting endocytosis of keratinocytes, effectively inhibiting the influx of melanosomes. Can be prevented.

Hereinafter, the present invention will be described in detail.

The compound according to an embodiment of the present invention may be represented by the following Formula 1, and may be a pharmaceutically acceptable salt thereof.

[Formula 1]

[In Formula 1,

AA is an amino acid residue containing substituted or unsubstituted proline.]

The compound represented by Formula 1 or a pharmaceutically acceptable salt thereof activates intracellular autophagy, induces melanosome degradation, and effectively inhibits the expression of MITF (Melanogenesis Associated Transcription Factor). In addition, it inhibits melanosomal maturation of melanocytes, inhibits the inclusion of keratinocytes, and realizes a surprising whitening effect. That is, the compound represented by Formula 1 according to the present invention can be usefully used as a whitening material having an autophagy activation function.

Intracellular autophagy activation occurs in young human tissues and cells, but as aging progresses, the expression level of intracellular autophagy-related proteins rapidly decreases, resulting in a rapid decrease in autophagy activity. Accordingly, aging proteins, lipids, and mitochondria in the cells are not removed in a timely manner, resulting in rapid cell aging. According to the present invention, through the activation of autophagy, aging of each cell, each tissue, and each individual is fundamentally inhibited and various diseases caused by aging can be prevented, improved, and treated.

In addition, the activation of autophagy improves the survival rate of aged cells through the removal of harmful proteins and organs in the cells, and furthermore, has a very close relationship with the increase in lifespan of each individual.

Accordingly, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof inhibits the maturation of melanosomes in melanocytes due to various causes, and acts more usefully in the prevention and treatment of melanin hyperpigmentation diseases.

In the above formula (1), the AA; can be hydroxy, C _{1- 10} alkoxy, C _{1- 10} alkyl-carbonyl and amino acid glass, including optionally substituted with one or more substituents selected from carboxy-proline.

For example, in Formula 1, the AA may be an amino acid residue including proline bound by an amide bond. In this case, the amino acid residues other than proline and proline may be substituted with or not substituted with the above-described substituent. In addition, the amino acid residue may be a combination of two or more amino acids including proline by an amide bond.

For example, in Formula 1, wherein AA is an amino acid residue containing proline bonded by an amide bond, and the amino acid residue is an amino acid containing at least one selected from Asn, Gln, Lys, etc. having two N-terminals. It may be a moiety.

For example, in Formula 1, AA may be an amino acid residue to which proline substituted or unsubstituted with the above-described substituent is directly bonded.

The compound according to an embodiment of the present invention may be a compound represented by Formula 2 below.

[Formula 2]

[In Chemical Formula 2,

R ₁ is C _{1- 10} alkyl carbonyl;

R ₂ is hydrogen, hydroxy, C _{1- 10} alkoxy, C _{1- 10} alkyl, carbonyl or carboxy.]

The compound represented by Formula 2 is excellent in the effect of decomposing melanosomes through activation of autophagy, and in the present specification, it may mean a compound that induces decomposition of melanosomes through activation of autophagy.

For example, in the above Formula 2, wherein R ₁ is C _{1- 4} alkylcarbonyl; Wherein R ₂ may be hydrogen, hydroxy or C _{1- 4} alkoxy.

For example, in the above Formula 2, wherein R ₁ is straight-chain C _{1 - 4} alkylcarbonyl; R ₂ may be hydrogen or hydroxy.

For example, in the above Formula 2, wherein R ₁ is C _{1- 2} alkyl carbonyl; R ₂ may be hydrogen or hydroxy.

In addition, the compound represented by Formula 2 contains one or more chiral asymmetric carbon atoms. Accordingly, the compound represented by Formula 2 may exist in a racemate and optically active form. That is, both diastereomers and enantiomers of the compound represented by Formula 2 are included within the scope of the present invention.

The compound according to an embodiment of the present invention may be a compound represented by the following formula (3).

[Chemical Formula 3]

[In Chemical Formula 3,

R ₃ is hydroxy or C _{1- 10} alkyl;

R ₄ is hydrogen, hydroxy, C _{1- 10} alkoxy, C _{1- 10} alkyl, carbonyl or carboxy.]

The compound represented by Formula 3 can effectively inhibit the influx of melanosomes into keratinocytes through inhibition of endocytosis, and thus, in the present specification, may mean a compound that induces melanosome influx through inhibition.

For example, in the above formula (3), wherein R ₃ is hydroxy or C _{1- 4} alkyl; Wherein R ₄ may be hydrogen, hydroxy or C _{1- 4} alkoxy.

For example, in the above formula (3), wherein R ₃ is hydroxy or straight-chain C _{1 - 4} alkyl; Wherein R ₄ is hydrogen, hydroxy or straight-chain C _{1 - 4} may be an alkoxy group.

For example, in the above formula (3), wherein R ₃ is hydroxy or C _{1- 2} alkyl; R ₄ may be hydrogen or hydroxy.

In addition, the compound represented by Formula 3 contains at least one chiral asymmetric carbon atom. Accordingly, the compound represented by Formula 3 may exist in a racemic form and an optically active form. That is, both diastereomers and enantiomers of the compound represented by Formula 3 are included within the scope of the present invention.

Hereinafter, the use of the present invention will be described in detail.

The use of one aspect according to an embodiment of the present invention may be a cosmetic composition for whitening.

The cosmetic composition for whitening according to an embodiment of the present invention may include the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The compound represented by Formula 1 or a pharmaceutically acceptable salt thereof does not induce cytotoxicity within a concentration range showing activity, and exhibits a whitening effect even in a very low concentration range (eg, 1 νM). In addition, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof does not decrease the stability and storage properties of the formulation even when formulated into a conventional cosmetic composition.

The cosmetic composition for whitening according to an embodiment of the present invention may contain a compound represented by Chemical Formula 2 or a pharmaceutically acceptable salt (A) thereof as an active ingredient. In this case, (A) may be one or a mixture of two or more.

The cosmetic composition for whitening according to an embodiment of the present invention may include a compound represented by Chemical Formula 3 or a pharmaceutically acceptable salt (B) thereof as an active ingredient. In this case, (B) may be one or a mixture of two or more.

The cosmetic composition for whitening according to an embodiment of the present invention is an active ingredient, a compound represented by Formula 2 or a pharmaceutically acceptable salt thereof (A) and a compound represented by Formula 3 or a pharmaceutically acceptable salt thereof. It may be a mixture comprising salt (B).

The cosmetic composition for whitening according to an embodiment of the present invention may contain 0.0001 to 10% by weight of the active ingredient, based on the total weight, in terms of implementing a substantial autophagy activation effect. In contrast to the effect of the content, the active ingredient may be specifically included in 0.0005 to 5% by weight, more specifically 0.001 to 1% by weight, and most specifically 0.001 to 0.1% by weight.

Moreover, the cosmetic composition for whitening according to an embodiment of the present invention is a mixture of a compound that induces melanosome decomposition through autophagy activation and a compound that induces melanosome influx through inhibition of inclusion. It can be included as an active ingredient.

For example, the compound that induces melanosome decomposition through autophagy activation may be at least one compound (A) selected from the compound represented by Chemical Formula 2, and the compound that induces melanosome influx through inhibition of the endocytosis is the It may be at least one compound (B) selected from the compounds represented by Formula 3.

The cosmetic composition for whitening according to an embodiment of the present invention is a compound that induces melanosome decomposition through the activation of autophagy and a compound that induces melanosome influx through inhibition of inclusion in an appropriate weight ratio depending on the application form, purpose of use, and desired effect. Of course, mixtures of can be used.

For example, the mixture may be mixed in a weight ratio (A:B) of 1:0.1 to 1:1.

For example, the mixture may be mixed in a weight ratio (A:B) of 1:0.1 to 1:0.8.

For example, the mixture may be mixed in a weight ratio (A:B) of 1:0.1 to 1:0.5.

The cosmetic composition for whitening may be formulated into a general emulsified formulation and a solubilized formulation, using a conventionally known manufacturing method.

For example, the whitening cosmetic composition may be formulated in a formulation selected from the group consisting of softening lotion, astringent lotion, nutritional lotion, eye cream, nutritional cream, massage cream, powder, essence, pack, and the like.

For example, the cosmetic composition for whitening may be formulated in a formulation selected from the group consisting of soap, cleansing cream, cleansing foam, cleansing water, and the like.

In addition, the cosmetic composition for whitening may further include additional additives as appropriate as desired. For example, the additives are stabilizers, emulsifiers, thickeners, moisturizers, liquid crystal film strengtheners, pH adjusters, antibacterial agents, water-soluble polymers, coating agents, sequestering agents, amino acids, organic amines, polymer emulsions, pH adjusters, skin nutrients, oxidation One or more aqueous additives selected from antioxidants, antioxidants, preservatives, fragrances, and the like; And at least one oily additive selected from fats and oils, waxes, hydrocarbon oils, higher fatty acid oils, higher alcohols, synthetic ester oils and silicone oils; And the like.

In this case, each of the additives may be included in an amount of 0.001 to 20% by weight based on the total weight of the composition, and specifically, may be included in an amount of 0.01 to 10% by weight and 0.05 to 10% by weight, but is not limited thereto.

The use of another aspect according to an embodiment of the present invention may be a pharmaceutical composition for improving or treating melanin 　 pigment 　 hyperdeposited disease.

In addition, the use of one aspect according to an embodiment of the present invention may be a method of alleviating or treating melanin hyperpigmentation disease. Specifically, the method may be to apply an effective amount of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof to the skin of a subject in need thereof, and the skin of a subject in need thereof is excessively deposited with melanin pigment. May be diseased skin.

In this case, the effective amount is not limited as long as the amount of the active ingredient is used.

The 　 pharmaceutical composition for improving or treating melanin 　 pigment 　 hyperdeposited disease according to an embodiment of the present invention may include a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The compound represented by Chemical Formula 1, or a pharmaceutically acceptable salt thereof, exhibits excellent whitening effect and exhibits excellent effects in improving, preventing and treating melanin 　 pigment 　 hyperdeposited diseases. Specifically, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof can protect cells from oxidative stress by activating autophagy by increasing the expression of autophagy related proteins, and various diseases caused by oxidative stress and Symptoms can be improved, prevented and treated. In particular, by inducing melanosome decomposition, reducing melanin production, inhibiting the maturation of melanosomes, effectively inhibiting the inclusion of keratinocytes, interfering with the influx of melanosomes, and improving the pigmentation hyperdeposited disease induced by melanin, It is very effective in prevention and treatment.

The pigment 　 hyperdeposited disease, freckles; Senile spots; Kanban; freckles; Brown or dark spots; Daylight pigment spot; Blue melanoma; Hyperpigmentation after drug use; Gestational brown spot; Or hyperpigmentation after inflammation; And the like, but are not limited thereto.

A pharmaceutical composition for improving or treating melanin 　 pigment 　 hyperdeposited disease according to an embodiment of the present invention may contain a compound represented by Formula 2 or a pharmaceutically acceptable salt thereof (A') as an active ingredient. have. In this case, (A') may be one or a mixture of two or more.

A pharmaceutical composition for improving or treating melanin 　 pigment 　 hyperdeposited disease according to an embodiment of the present invention may contain a compound represented by Formula 3 or a pharmaceutically acceptable salt thereof (B') as an active ingredient. have. In this case, (B') may be one or a mixture of two or more.

The pharmaceutical composition for improving or treating melanin 　 pigment 　 hyperdeposited disease according to an embodiment of the present invention is an active ingredient, a compound represented by Formula 2 or a pharmaceutically acceptable salt thereof (A') and Formula 3 It may be a mixture containing the indicated compound or a pharmaceutically acceptable salt (B') thereof.

In terms of improving or treating melanin 　 pigment 　 hyperdeposition disease according to an embodiment of the present invention, in terms of realizing a substantial autophagy activating effect, based on the total weight, the active ingredient is 0.0001 to 10% by weight. Can include. In contrast to the effect of the content, the active ingredient may be specifically included in 0.001 to 5% by weight, more specifically 0.001 to 1% by weight, most specifically 0.001 to 0.1% by weight.

Moreover, for the improvement or treatment of melanin 　 pigment 　 hyperdeposited disease 　 pharmaceutical composition according to an embodiment of the present invention, for the realization of a more improved effect, the melanosome decomposition-inducing compound through autophagy activation and melanosome through inhibition of inclusion action A mixture of inducing compounds for inducing inflow inhibition may be included as an active ingredient.

For example, the compound that induces melanosome decomposition through autophagy activation may be at least one compound (A′) selected from the compound represented by Formula 2, and the compound that induces melanosome influx through inhibition of inclusion It may be at least one compound (B') selected from the compounds represented by Chemical Formula 3.

The melanin 　 pigment 　 hyperdeposited disease improvement or treatment 　 pharmaceutical composition according to an embodiment of the present invention is a compound that induces melanosome decomposition through the activation of the autophagy in an appropriate weight ratio according to the application form, purpose of use, and desired effect, and inclusion action Of course, a mixture of compounds that induce melanosome influx through inhibition may be used.

For example, the mixture may be mixed in a weight ratio of 1:0.1 to 1:1 (A':B').

For example, the mixture may be mixed in a weight ratio of 1:0.1 to 1:0.8 (A':B').

For example, the mixture may be mixed in a weight ratio of 1:0.1 to 1:0.5 (A':B').

The melanin 　 pigment 　 hyperdeposited disease improvement or treatment 　 pharmaceutical composition may be formulated into a formulation including a pharmaceutically acceptable carrier using a conventionally known manufacturing method.

For example, the 　 pharmaceutical composition for improving or treating melanin 　 pigment 　 hyperdeposited disease may be formulated as a skin external formulation selected from the group consisting of lotions, ointments, gels, creams, patches, sprays, and the like.

For example, the 　 pharmaceutical composition for improving or treating melanin 　 pigment 　 hyperdeposited disease is oral (tablets, capsules, powders), oral, sublingual, rectal, vaginal, nasal, topical or parenteral (intravenous It may be formulated in a dosage form including intramuscular, cavernous, intramuscular, subcutaneous and intraluminal).

In addition, the melanin 　 pigment 　 hyperdeposited disease improvement or treatment 　 pharmaceutical composition may include an additional pharmaceutically acceptable carrier as appropriate as desired. For example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose , Methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil, but is not limited thereto. In addition to the above carriers, carriers such as lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents or preserving agents may be further included.

In this case, each of the carriers may be included in an amount of 0.001 to 20% by weight based on the total weight of the composition, and specifically, may be included in an amount of 0.01 to 10% by weight and 0.05 to 10% by weight, but is not limited thereto.

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

(Example 1) Synthesis of Compound 1

Step 1. Synthesis of compound 1a (Fmoc-Lys(Dde)-O-2-chloro trityl resin)

In a 10 ml reaction vessel, 2-chloro trityl chloride resin (100-200mesh, Novabiochem 100mg, 1 equivalent) and Fmoc-Lys(Dde)-OH(Nα-Fmoc-Nε-Dde-L- lysine,Nα-Fmoc-Nε-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine) (106.5mg, 2 equivalents) and diisopropylamine (DIPEA, 69.7µl, 4 Equivalent) was dissolved in 5 ml of dichloromethane (DCM), added, and reacted at room temperature (23° C.) for 12 hours. The reaction solution was removed by filtering, and the synthesized resin was sequentially washed with 10 ml of dichloromethane, methanol (MeOH), dichloromethane, and dimethylformamide (DMF), respectively, to quantitatively obtain compound 1a .

Step 2. Synthesis of compound 1b (Fmoc-Lys(Fmoc)-Lys(Dde)-O-2-chloro trityl resin)

5 ml of 20% piperidine (in DMF) was added to the reaction vessel of step 1 (compound 1a obtained in step 1) and reacted at room temperature for 5 minutes, followed by filtering to remove the reaction solution. 5 ml of 20% piperidine (in DMF) was added once more and reacted at room temperature for 5 minutes. The reaction solution was removed by filtering, and the synthesized resin was sequentially washed with 10 ml of DCM, MeOH, DCM and DMF, respectively, and then Fmoc-Lys(Fmoc)-OH (236.3mg, 4 equivalents) and HOBt ( Hydroxybenzotriazole , 54.1mg, 4 equivalents) and N,N-diisopropylcarbodiimide (DIC) (61.9µl, 4 equivalents) were dissolved in 5ml of DMF, added, and reacted at room temperature for 2 hours. The reaction solution was removed by filtering, and the synthesized resin was sequentially washed with 10 ml of DCM, MeOH, DCM, and DMF, respectively, to quantitatively obtain compound 1b .

Step 3. Synthesis of compound 1c (tert-butoxycarbonylmethyl)2-Lys(tert-butoxycarbonylmethyl)2-Lys(Dde)-O-2-chloro trityl resin)

In the reaction vessel of step 2 (compound 1b obtained in step 2), 5 ml of 20% peperidine (in DMF) was added and reacted at room temperature for 5 minutes, followed by filtering to remove the reaction solution. 5 ml of 20% piperidine (in DMF) was added once more and reacted at room temperature for 5 minutes. The reaction solution was removed by filtering, and the synthesized resin was washed sequentially with 500 ml of DCM, MeOH, DCM, and DMF, respectively, and then t-butyl bromoacetate (147.7 μl, 10 equivalents) and DIPEA (261.3 µl, 15 eq) was dissolved in 5 ml of DMF, added, and reacted at room temperature for 48 hours. The reaction solution was removed by filtering, and the synthesized resin was sequentially washed with 10 ml of DCM, MeOH, DCM and DMF, respectively, to obtain compound 1c quantitatively.

Step 4. Synthesis of compound 1d (tert-butoxycarbonylmethyl)2-Lys(tert-butoxycarbonylmethyl)2-Lys(NH2)-O-2-chloro trityl resin)

In the reaction vessel of step 3 (compound 1c obtained in step 3), 5 ml of 2% hydrazine (in DMF) was added and reacted at room temperature for 5 minutes. The reaction solution was removed by filtering, and the synthesized resin was washed with 10 ml DMF, and then 10 ml of 10% DIPEA (in DMF) was added and reacted at room temperature for 5 minutes. The reaction solution was removed by filtering, and the synthesized resin was sequentially washed with 10 ml of DCM, MeOH, DCM, and DMF, respectively, to obtain compound 1d quantitatively.

Step 5. Synthesis of compound 1

In the reaction vessel of step 4 (compound 1d obtained in step 4), N-acetyl-L-hydroxyproline (N-Acetyl-L-hydroxyproline, 138.5mg, 8 equivalents), HOBt (180.1mg, 8 equivalents) and DIC (123.9 µl, 8 equivalents) was dissolved in 5 ml of DMF, added, and reacted at room temperature for 3 hours. The reaction solution was removed by filtering, and the synthesized resin was sequentially washed using 10 ml of DCM, DMF, and DCM, respectively.

After vacuum drying, 5 ml of cleavage cocktail (trifluoro acetic acid: triisopropylsilane: DW = 95: 2.5: 2.5) was added and reacted at room temperature for 3 hours. The reaction solution was collected by filtering, and 45 ml of diethyl ether was added thereto to precipitate the product. The solid product was collected using a centrifuge, and washed twice with 45 ml of diethyl ether. The obtained solid product was purified using Prep-HPLC (column C18, 10um, 250mm X 22mm) and then lyophilized to obtain 36.4mg of compound 1 (molecular weight measured by LC mass: 661.66) in a total yield of 55%.

(Example 2) Synthesis of Compound 2

Step 1. Synthesis of compound 2a (Fmoc-Hyp(tBu)-O-2-chloro trityl resin)

In a 10 ml reaction vessel, 2-chlorotrityl chloride resin (100-200mesh, Novabiochem 200mg, 1 equivalent) and Fmoc-Hyp(tBu)-OH (Fmoc-O-tert-butyl-L-hydroxyproline) (163.8mg, 2 Equivalent) and DIPEA (139.3ul, 4 equivalents) were dissolved in 5ml of DCM, added, and reacted at room temperature for 12 hours. The reaction solution was removed by filtering, and the synthesized resin was sequentially washed with 10 ml of DCM, MeOH, DCM, and DMF, respectively, to quantitatively obtain compound 2a .

Step 2. Synthesis of compound 2b (Fmoc-Lys(Fmoc)-Hyp(tBu)-O-2-chloro trityl resin)

In the reaction vessel of step 1 (compound 2a obtained in step 1), 5 ml of 20% piperidine (in DMF) was added and reacted at room temperature for 5 minutes, followed by filtering to remove the reaction solution. 5 ml of 20% piperidine (in DMF) was added once more and reacted at room temperature for 5 minutes. The reaction solution was removed by filtering, and the synthesized resin was sequentially washed with 10 ml of DCM, MeOH, DCM and DMF, respectively, and then Fmoc-Lys(Fmoc)-OH (472.6mg, 4 equivalents) and HOBt (108.1 mg, 4 equivalents) and DIC (123.9ul, 4 equivalents) were dissolved in 5 ml of DMF, added, and reacted at room temperature for 2 hours. The reaction solution was removed by filtering, and the synthesized resin was sequentially washed with 10 ml of DCM, MeOH, DCM, and DMF, respectively, to quantitatively obtain compound 2b .

Step 3. Synthesis of compound 2c (tert-butoxycarbonylmethyl)2-Lys(tert-butoxycarbonylmethyl)2-Hyp(tBu)-O-2-chloro trityl resin)

In the reaction vessel of step 2 (compound 2b obtained in step 2), 5 ml of 20% piperidine (in DMF) was added and reacted at room temperature for 5 minutes, followed by filtering to remove the reaction solution. 5 ml of 20% piperidine (in DMF) was added once more and reacted at room temperature for 5 minutes. The reaction solution was removed by filtering, and the synthesized resin was washed sequentially with 500 ml of DCM, MeOH, DCM and DMF, respectively, and then t-butyl bromoacetate (295.3 µl, 10 equivalents) and DIPEA (522.5 ul, 15 equivalents) was dissolved in 5 ml of DMF, added, and reacted at room temperature for 48 hours. The reaction solution was removed by filtering, and the synthesized resin was sequentially washed using 10 ml of each 10 ml of DCM, DMF and DCM, respectively, to obtain compound 2c quantitatively.

Step 4. Synthesis of compound 2

5 ml of cleavage cocktail (trifluoro acetic acid: triisopropylsilane: DW = 95: 2.5: 2.5) was added to the reaction vessel of step 3 (compound 2c obtained in step 3) and reacted at room temperature for 3 hours. The reaction solution was collected by filtering, and 45 ml of diethyl ether was added thereto to precipitate the product. The solid product was collected using a centrifuge, and washed twice with 45 ml of diethyl ether. The obtained solid product was purified using Prep-HPLC (column C18, 10um, 250mm X 22mm), and then lyophilized to give compound 2 (molecular weight measured by LC mass: 491.45) 59mg in a total yield of 60%.

(Example 3) Confirmation of tyrosinase inhibitory ability

A tyrosinase inhibition assay was performed to analyze whether compounds 1 and 2, obtained in the above examples, inhibit tyrosinase activity.

As a specific experimental method, Mushroom tyrosinase was diluted to 2 unit/ul in 0.1 M sodium phosphate buffer and dissolved in water with 0.03% by weight of tyrosine. Each experimental group described in Table 1 was also diluted to 10, 100, 1000, and 10000 ppm in 0.1 M sodium phosphate buffer and mixed as shown in Table 1. The reaction was performed at 37° C. for 1 hour, and absorbance was measured, respectively (wavelength: 490 nm, Epoch, BioTek). Based on the results, it was calculated as the tyrosinase inhibition rate compared to the control.

The results are shown in FIG. 2.

As can be seen in Figure 2, it was confirmed that Compound 1 and Compound 2 did not inhibit tyrosinase activity at all concentrations.

(Example 4) Confirmation of appropriate treatment concentration

MTT assay was performed to confirm the treatment concentration without cytotoxicity by Compound 1 and Compound 2 obtained in the above example.

As a specific experimental method, melanocytes (human primary melanocyte, HEMn-DP, cat# C2025C, Gibco) and keratinocytes (human primary keratinocyte, HEKn, cat) were used to confirm the cytotoxicity according to the treatment concentration of Compound 1 and Compound 2. # C0015C, Gibco) apoptosis assay was performed. The number of cells was divided into a 96-well plate at 1×10 ⁴ cells/well, and cultured in an incubator at 37° C. and 5% CO ₂ for 24 hours.

Each of the compounds 1 and 2 obtained in the above example was dissolved in distilled water (DW) at a concentration of 100 mM to form a concentrate, and this was diluted with a medium and treated at a concentration of 5, 10, and 50 uM. After 4 days, the medium of the plate was removed and 100 μl of MTT reagent (sigma, 0.5 mg/ml) was treated. After 4 hours, the reagent was removed, dimethyl sulfoxide (DMSO) was added, and the absorbance was measured (wavelength: 570 nm, Epoch, BioTek). At this time, the cell viability was calculated as a percentage of the absorbance of the control group (Blank) not treated with the compound.

The results are shown in FIGS. 3 and 4.

As can be seen in Figures 3 and 4, it was confirmed that Compound 1 and Compound 2 had almost no cytotoxicity even at concentrations of 5, 10, and 50uM. That is, it was confirmed that the compound according to the present invention had no cytotoxicity in both melanocytes and keratinocytes at the above concentration. Therefore, in the subsequent experiment, the experiment was conducted at a concentration of 10 uM.

(Example 5) Confirmation of changes in melanosome maturation-related genes and changes in melanin content

To analyze how compound 1 and compound 2, obtained in the above example, affect melanosome maturation, after treatment of compound 1 and compound 2, the amount of melanosome maturation-related mRNA was analyzed by qPCR, and the melanin content was measured by absorbance. It was measured as.

As a specific experimental method, human-derived melanocytes (HEMn-DP, cat# C2025C, Gibco) were regularly distributed in a ⁶ well plate for culture at a number of 1 X 10 ⁵ cells to M254 (cat# M-254-500, Gibco). Incubated in an incubator under conditions of 37° C. and 5% CO ₂ for 24 hours at. Dissolve each of Compound 1 and Compound 2 in DW at a concentration of 100 mM to form a concentrate, dilute it with a medium and dilute to a concentration of 20 μM, and then add 1 ml of each dilution to each well with 1 ml of medium first. After incubation for 48 hours, RNA was extracted from melanocytes using an RNeasy mini kit (cat# 74106, QIAGEN). The extracted RNA was synthesized by cDNA using a cDNA synthesis kit (cat# FSQ-201, TOYOBO), and MITF (F: TAA CCT GTA CAA CAA CTC TCG ATC TCA; R: GTT GGC CTC AGT CCC AGT TC), Tyrosinase (F : AGC ACC CCA CAA ATC CTA ACT TAC; R: ATG GCT GTT GTA CTC CTC CAA TC) Primer and POWER SYBR MASTER MIX (cat# 4367659, Thermo) were mixed in the recommended ratio with the synthesized cDNA and QuantStudio 3 equipment (cat # A28131, Thermo) qPCR was performed. In the same way, after the experiment was completed, the medium was removed, and the melanocytes were separated from the plate using Trypsin-EDTA (cat# LS-015-09, WELGENE), transferred to a 1.5ml tube, and then spin down. ) To take a picture of the pellet, and measure the absorbance of the solution in which the pellet was dissolved in 1N NaOH (wavelength: 400 nm, Epoch, BioTek).

The results are shown in FIGS. 5 to 7.

As can be seen in FIGS. 5 to 7, it can be seen that the compounds 1 and 2 obtained in the above examples have little effect on the melanosomal maturation activity in melanocytes.

(Example 6) Confirmation of increase in autophagy activity of keratinocytes and decrease in expression of genes related to endocytosis

Western blot for LC3 (light chain 3; autophagy marker) protein was performed for analysis of intracellular autophagy by treatment with Compound 1 obtained in the above example. In addition, qPCR was performed on PAR2 and AAK1 (AP2 Associated Kinase 1) in order to analyze changes in genes related to endocytosis by treatment with Compound 2 obtained in the above example.

As a specific experimental method, compound 1 was treated with keratinocytes (HEKn, cat# C0015C, Gibco) and lysed using a cell lysis buffer containing a protein degradation inhibitor. After fractionating the total solution from the dissolved solution using a centrifuge, only the protein-containing solution was extracted. The extracted protein was quantified by the BCA (Bicinchoninic acid) method. 20 ug of protein was separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The protein-transferred membrane was blocked for 1 hour at room temperature with a TBS-T (Tris-buffered saline/0.05% Tween-20) solution containing 5% non-fat milk to reduce non-specific binding. Thereafter, the primary antibody was reacted overnight at 4° C., and then the secondary antibody was reacted at room temperature for 1 hour. For visualization, an Enhance chemiluminescence system was used.

As a specific experimental method, keratinocytes (HEKn, cat# C0015C, Gibco) treated with Compound 2 extracted RNA using an RNeasy mini kit (cat# 74106, QIAGEN), and cDNA synthesis kit (cat# FSQ-201, TOYOBO), then cDNA synthesized using PAR2 (F: CTC TCC TGC AGT GGC ACC AT; R: GAT GTG CCA TCA ACC TTA CCA A) primer and POWER SYBR MASTER MIX (cat# 4367659, Thermo) The mixture was mixed with the recommended ratio and qPCR was performed with QuantStudio 3 equipment (cat# A28131, Thermo).

The results are shown in FIG. 8.

As can be seen from FIG. 8, it was confirmed that the production of LC3-II (mature autophagosome) was increased by compound 1, and that the expression of PAR-2 gene was decreased by compound 2.

(Example 7) Confirmation of reduction of melanin deposition

In order to analyze how the compounds 1 and 2 obtained in the above example affect melanin deposition, the degree of inhibition of deposition through melanin treatment was analyzed.

As a specific experimental method, human-derived keratinocytes (HEKn, cat# C0015C, Gibco) were uniformly dispensed into a 6well plate for culture at a number of 1 X 10 ⁵ cells, and 37 for 24 hours in EpiLife (cat# MEPI500CA, Gibco). Incubated in an incubator under conditions of °C and 5% CO ₂ . Dissolve each of Compound 1 or Compound 2 obtained in the above example at a concentration of 100 mM to form a concentrate, dilute it with a medium and dilute to a concentration of 20 μM, and then add 1 ml of each dilution solution to each well with 1 ml of medium first. After the treatment was added each, it was incubated for 48 hours, and 10 mg/ml of melanin was added in 2 ul each, followed by additional culture for 2 hours. After the culture is completed, the medium is removed, cells are separated from the plate using Trypsin-EDTA (cat# LS-015-09, WELGENE), transferred to a 1.5ml tube, and then spin down to take a picture of the pellet, and the pellet is 1N The absorbance of the solution dissolved in NaOH was measured (wavelength: 400 nm, Epoch, BioTek).

The results are shown in FIG. 9.

As can be seen in FIG. 9, it was confirmed that melanin deposition on keratinocytes was effectively inhibited by Compound 1 or Compound 2.

In addition, it was found that more melanin deposition was suppressed when compound 1 or compound 2 was mixed than when compound 1 or compound 2 was treated with a single compound.

(Example 8) Analysis of whether melanin deposition inhibitory activity is dependent on autophagy activity

In order to analyze whether the inhibitory activity of melanin deposition on keratinocytes obtained in the above example by treatment with Compound 1 and Compound 2 is dependent on autophagy activity, ATG5, a protein required for autophagy activity, was knock-down followed by melanin. The change in the activity to inhibit deposition through treatment was analyzed.

As a specific experimental method, ATG5 siRNA was mixed according to the method of a transfection reagent (RNAiMAX, cat# 13778-075, Thermo) and then dispensed into keratinocytes (HEKn, cat# C0015C, Gibco). After 8 hours, Compound 1 and Compound 2 were diluted in Epilife (cat# MEPI500CA, Gibco) without supplements and antibiotics to a final concentration of 10 uM, and the cells cultured for an additional 64 hours were treated with a cell lysis buffer containing a proteolytic inhibitor. It was dissolved using (cell lysis buffer). After fractionating the total solution from the dissolved solution using a centrifuge, only the protein-containing solution was extracted. The extracted protein was quantified by the BCA (Bicinchoninic acid) method. 20 ug of protein was separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. To reduce non-specific binding, the protein-transferred membrane was prepared with a TBS-T (Tris-buffered saline/0.05% Tween-20) solution containing 5% BSA (Bovine serum albumin, Gendepot, Cat# A0100-010) at room temperature. After blocking for an hour, the primary antibody was reacted overnight at 4° C., and the secondary antibody was reacted at room temperature for 1 hour. For visualization, an Enhance chemiluminescence system was used. In order to analyze the melanin deposition inhibitory activity, transfection and treatment of the compounds were carried out in the same manner, followed by adding 2ul of Melanin 10mg/ml and incubating for 2 hours. After the culture is complete, remove the culture medium, separate the cells from the plate using Trypsin-EDTA (cat# LS-015-09, WELGENE), transfer to a 1.5ml tube, spin down, and take a picture of the pellet, and the pellet is 1N The absorbance of the solution dissolved in NaOH was measured (wavelength: 400 nm, Epoch, BioTek).

The results are shown in FIG. 10.

As can be seen in FIG. 10, it was confirmed that the amount of melanin in keratinocytes by Compound 1 and Compound 2 was not decreased when ATG5, a protein required for autophagy activity, was knocked down. In addition, it was confirmed that the expression activities of the autophagy proteins LC3-II and ATG5 by Compound 1 and Compound 2 were inhibited under ATG5 knock-down conditions. Accordingly, it was found that the inhibitory activity of compounds 1 and 2 to inhibit melanin deposition was dependent on autophagy activity.

(Example 9) Melanosome decomposition effect

In order to confirm whether melanosomes and autophagosomes were co-localized by the treatment of compounds 1 and 2 obtained in the above example, analysis was performed with an electron microscope and a confocal microscope.

As a specific experimental method, Lysis Buffer (Tris 20mM, NaCl 150mM, EDTA 1mM, Sucrose 250mM in DW) was added to melanocytes (HEMn-DP, cat# C2025C, Gibco) containing high concentrations of melanin derived from humans, and then frozen and dissolved. Break the cell membrane by repeating twice and centrifuge at 1000 xg for 5 minutes to obtain a supernatant. Cushion Buffer (Tris 20mM, NaCl 150mM, EDTA 1mM, Sucrose 1.5M in DW) was placed in an ultracentrifuge tube, and the same amount of supernatant was added, followed by centrifugation at 25,000 rpm, 30 min, 4°C to obtain melanosomes. The concentration of melanosomes was measured using synthetic melanin (cat# M8631, sigma) as a standard. Keratinocytes (HEKn, cat# C0015C, Gibco) were dispensed into 6 wells at 1×10 ⁵ cells/well and cultured for one day. Compound 1 and Compound 2 were treated with 10 μM each and cultured for one day, and then the separated melanosomes (5 ug/well) were added and cultured for 2 more days. After the culture was completed, the culture medium was removed, the cells were fixed using an electron microscope fixative, and fluorescently labeled with antibodies (Pmel17, LC3), and then it was confirmed that melanosomes and autophagosomes co-localized with a confocal microscope. In addition, the cells tested in the same manner were observed with a transmission electron microscope for the appearance of organelles within the cells.

The results are shown in Figs. 11 and 12.

As can be seen in FIG. 11, it was confirmed by confocal microscopy that the autophagosome increased by Compound 1 and Compound 2 co-localized with melanosomes. In addition, it was confirmed that, unlike the control group in which a lot of melanosomes exist in keratinocytes, when the mixture of Compound 1 and Compound 2 was treated, melanosomes were rapidly degraded and the amount thereof was significantly reduced.

In addition, as can be seen in FIG. 12, when the mixture of Compound 1 and Compound 2 was treated, 86% of the total melanosomes contained in the autophagosome could be confirmed with a transmission electron microscope.

(Example 10) Effect of inhibiting deposition of melanosomes

In order to analyze whether the deposition of melanosomes in keratinocytes was inhibited by treatment with Compound 1 and Compound 2 obtained in the above example, melanosomes isolated from keratinocytes were treated, and melanin content and melanosomal index were analyzed.

As a specific experimental method, keratinocytes (HEKn, cat# C0015C, Gibco) were aliquoted into 6 wells at 1x10 ⁵ cells/well and cultured for one day, and then compound 1 and compound 2 were treated with 10 μM each for a day, and Example 9 After adding the separated melanosomes (5 ug/well) as described above, they were further cultured for 2 days. After the culture was completed, a culture solution was obtained, and the cells were separated from the plate using Trypsin-EDTA (cat# LS-015-09, WELGENE), transferred to a 1.5ml tube, and then spin down to take a picture of the pellet, and the pellet was 1N. The absorbance of the solution dissolved in NaOH was measured (wavelength: 400 nm, Epoch, BioTek).

In addition, in order to quantitatively analyze this, synthetic melanin (cat# M8631, sigma) was used as a standard to analyze the amount of melanosomes in the culture medium and cell pellet. The obtained culture solution was freeze-dried, dissolved in 1N NaOH, like a pellet, and then measured for absorbance (wavelength: 400 nm, Epoch, BioTek).

The results are shown in FIG. 13.

As can be seen from FIG. 13, it was confirmed that when the mixture of Compound 1 and Compound 2 was treated, the amount of melanosomes in the keratinocytes decreased and the amount of melanosomes in the culture medium increased. These results indicate that the influx of melanosomes into keratinocytes was effectively suppressed by treatment with a mixture of Compound 1 and Compound 2. Thus, treatment of a mixture of Compound 1 and Compound 2 can significantly inhibit pigmentation by melanosomes.

In addition, the mechanism for inhibiting the pigmentation process of keratinocytes according to the present invention is shown in Figure 1 below.

(Example 11) Melanin deposition inhibitory effect

In order to analyze how the compounds 1 and 2 obtained in the above example affect melanin deposition inhibition histologically, the degree of melanin pigmentation inhibition was analyzed using an artificial skin model.

As a specific experimental method, artificial skin models derived from black people (MelanoDerm™, MEL-300-B, MatTek) were used in a dedicated medium (EPI-100-NMM-PRF) for 15 days in an incubator at 37°C and 5% CO ₂ . Cultured. Compound 1 and compound 2 obtained in the above example were treated at a concentration of 100 ppm on the surface of an artificial skin model at a rate of 25 ul every 24 hours. When compound 1 and compound 2 were treated, the existing compound 1 and compound 2 were removed and then treated, and the medium was also replaced every 24 hours. Photos of the tissue surface were taken on the 1st, 9th, and 15th days of the experiment, and the degree of color change (L*value) of the tissue surface was analyzed using the results.

The results are shown in FIG. 14.

As can be seen from FIG. 14, it was confirmed that melanin pigmentation was statistically significantly reduced in the group treated with the mixture of Compound 1 and Compound 2 compared to the control group.

(Example 12) Melanin decomposition effect

To analyze the melanin decomposition effect of Compound 1 and Compound 2 obtained in the above example, the melanin content was analyzed using an artificial skin model.

As a specific experimental method, the artificial skin model was cultured as described in Example 11. The tissue was obtained on the 15th day and fixed in 4% phosphate buffered formalin for 24 hours. The fixed tissue was washed in running water for 12 hours, dehydrated in the order of increasing alcohol concentration, and then replaced with xylene and embedded in paraffin. The paraffin-embedded tissue was cut to a thickness of 5 μm, and melanin was stained using Fontana-Masson staining and then used for analysis. The pictures of the entire tissue and the pictures of the stratum corneum were analyzed and compared with image J, respectively.

The results are shown in FIG. 15.

As can be seen in FIG. 15, it was confirmed that the melanin was statistically significantly reduced in the group treated with the mixture of Compound 1 and Compound 2 compared to the control group. In addition, it was confirmed that the amount of melanin ascending to the stratum corneum was 43.25%, which was statistically significantly reduced compared to the control group.

From the above-described results, it was confirmed that the compound according to the present invention can effectively induce melanosome decomposition through autophagy activation, and at the same time promote PAR-2 inhibition to effectively inhibit the influx of melanosomes.

Specifically, Compound 1 is a compound that induces melanosome decomposition through autophagy activation, effectively decomposing melanosomes by inducing autophagy activity of melanocytes, and compound 2 is a melanosome as a compound that induces melanosome influx through inhibition of endocytosis. It was confirmed that it exerts an excellent effect on the role of interfering with the entry of keratinocytes. Accordingly, it is expected that it can be used very effectively in relieving, treating and preventing skin pigmentation by ultimately reducing melanin in keratinocytes. In particular, when a compound represented by Chemical Formulas 2 and 3 according to the present invention is mixed and used, it is possible to achieve excellent effects in improving, preventing and treating diseases caused by melanin 　 pigment 　 excessive deposition as well as whitening effect.

(Examples 13 to 14 and Comparative Example 1) Preparation of cosmetic composition for whitening

With the components and contents shown in Table 2 below, it was put into an emulsification reactor and heated to 75° C. while stirring and emulsifying. After the emulsification was completed, the mixture was stirred with a stirrer and gradually cooled to room temperature to prepare a whitening cosmetic composition (o/w cream).

(Example 15) Whitening effect

A clinical test was conducted to find out the whitening effect of the whitening cosmetic composition of Example prepared by the above method.

As a specific experimental method, after inducing darkening of the skin of 20 adult men and women (15 women, 5 men) with an average age of 37 years (36.57±5.90) with hyperpigmentation, as shown in Table 2 above. Creams containing high and low concentrations (test samples) containing compounds 1 and 2 and creams without compounds 1 and 2 (comparative example 1, control sample) were applied twice a day. Each cream was applied at 4 and 8 weeks using a Mexameter and Chromameter to measure according to the device manual.The measured value was tested for normality, and ANOVA was performed for the change according to the evaluation time, and the sample was sampled through a paired t-test. The hypothesis mean difference of 5% (P<0.05) was confirmed before and after application and whether there was a similarity between the test sample/control sample by week. The statistical analysis program used IBM SPSS version 18.0.

The results are shown in Tables 3 and 4 below.

As can be seen in Tables 3 and 4, melanin pigmentation and skin in the group using a cosmetic composition comprising a compound that induces melanosome decomposition through the activation of autophagy of the present invention and a compound that induces melanosome influx through inhibition of inclusion It was confirmed that the brightness change was improved. In addition, it was confirmed that the improvement rate of melanin pigmentation and skin brightness change exhibited more improved effects as the use time elapsed.

As described above, the present invention has been described by specific matters and limited embodiments, but these are provided only to help a more general understanding of the present invention, and the present invention is not limited to the above embodiments, and the field to which the present invention pertains. Those of ordinary skill in the above can make various modifications and variations from these descriptions.

Therefore, the spirit of the present invention is limited to the described embodiments and should not be defined, and all things that are equivalent or equivalent to the claims as well as the claims to be described later belong to the scope of the inventive concept. .

Claims (17)

A compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof:
[Formula 1]

In Formula 1,
AA is an amino acid residue comprising proline, which is unsubstituted or substituted with one or more substituents selected from hydroxy, C _1-10 alkoxy, C _1-10 alkylcarbonyl and carboxy. delete The method of claim 1,
Formula 1,
A compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof:
[Formula 2]

In Chemical Formula 2,
R ₁ is C _{1- 10} alkyl carbonyl;
R ₂ is hydrogen, hydroxy, C _{1- 10} alkoxy, C _{1- 10} alkyl, carbonyl or carboxy. The method of claim 1,
Formula 1,
A compound represented by the following Formula 3 or a pharmaceutically acceptable salt thereof:
[Chemical Formula 3]

In Chemical Formula 3,
R ₃ is hydroxy or C _{1- 10} alkyl;
R ₄ is hydrogen, hydroxy, C _{1- 10} alkoxy, C _{1- 10} alkyl, carbonyl or carboxy. A cosmetic composition for skin whitening comprising a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

In Formula 1,
AA is an amino acid residue comprising proline, which is unsubstituted or substituted with one or more substituents selected from hydroxy, C _1-10 alkoxy, C _1-10 alkylcarbonyl and carboxy. The method of claim 5,
The active ingredient,
A cosmetic composition for skin whitening, which is a compound represented by the following Formula 2 or a pharmaceutically acceptable salt (A) thereof:
[Formula 2]

In Chemical Formula 2,
R ₁ is C _{1- 10} alkyl carbonyl;
R ₂ is hydrogen, hydroxy, C _{1- 10} alkoxy, C _{1- 10} alkyl, carbonyl or carboxy. The method of claim 5,
The active ingredient,
A cosmetic composition for skin whitening, which is a compound represented by the following Formula 3 or a pharmaceutically acceptable salt (B) thereof:
[Chemical Formula 3]

In Chemical Formula 3,
R ₃ is hydroxy or C _{1- 10} alkyl;
R ₄ is hydrogen, hydroxy, C _{1- 10} alkoxy, C _{1- 10} alkyl, carbonyl or carboxy. The method of claim 5,
The active ingredient,
A compound represented by the following formula (2) or a pharmaceutically acceptable salt (A) thereof, and
A cosmetic composition for skin whitening, which is a mixture of a compound represented by the following formula (3) or a pharmaceutically acceptable salt (B) thereof:
[Formula 2]

[Chemical Formula 3]

In Formulas 2 and 3,
R ₁ is C _{1- 10} alkyl carbonyl;
R ₃ is hydroxy or C _{1- 10} alkyl;
R ₂ and R ₄ are each independently hydrogen, hydroxy, C _{1- 10} alkoxy, C _{1- 10} alkyl, carbonyl or carboxy. The method of claim 8,
The active ingredient,
It will be mixed in a weight ratio of 1:0.1 to 1:1 (A:B), a cosmetic composition for skin whitening. The method according to any one selected from claims 5 to 9,
The cosmetic composition,
A cosmetic composition for skin whitening comprising 0.0001 to 10% by weight of the active ingredient, based on the total weight. A pharmaceutical composition for improving or treating melanin hyperpigmentation disease, comprising a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

In Formula 1,
AA is an amino acid residue comprising proline, which is unsubstituted or substituted with one or more substituents selected from hydroxy, C _1-10 alkoxy, C _1-10 alkylcarbonyl and carboxy. The method of claim 11,
The active ingredient,
A pharmaceutical composition for improving or treating melanin hyperpigmentation disease, which is a compound represented by the following Formula 2 or a pharmaceutically acceptable salt (A) thereof:
[Formula 2]

In Chemical Formula 2,
R ₁ is C _{1- 10} alkyl carbonyl;
R ₂ is hydrogen, hydroxy, C _{1- 10} alkoxy, C _{1- 10} alkyl, carbonyl or carboxy. The method of claim 11,
The active ingredient,
A pharmaceutical composition for the improvement or treatment of melanin hyperpigmentation disease, which is a compound represented by the following Formula 3 or a pharmaceutically acceptable salt (B) thereof:
[Chemical Formula 3]

In Chemical Formula 3,
R ₃ is hydroxy or C _{1- 10} alkyl;
R ₄ is hydrogen, hydroxy, C _{1- 10} alkoxy, C _{1- 10} alkyl, carbonyl or carboxy. The method of claim 11,
The active ingredient,
A compound represented by the following formula (2) or a pharmaceutically acceptable salt (A') thereof, and
A pharmaceutical composition for the improvement or treatment of melanin hyperpigmentation disease, which is a mixture of a compound represented by the following Formula 3 or a pharmaceutically acceptable salt (B') thereof:
[Formula 2]

[Chemical Formula 3]

In Formulas 2 and 3,
R ₁ is C _{1- 10} alkyl carbonyl;
R ₃ is hydroxy or C _{1- 10} alkyl;
R ₂ and R ₄ are each independently hydrogen, hydroxy, C _{1- 10} alkoxy, C _{1- 10} alkyl, carbonyl or carboxy. The method of claim 14,
The active ingredient,
A pharmaceutical composition for improving or treating melanin hyperpigmentation disease, which is mixed in a weight ratio of 1:0.1 to 1:1 (A':B'): The method of claim 11,
The melanin hyperpigmentation disease,
Pharmacy for the improvement or treatment of melanin hyperpigmentation disease, which is freckles, senile spots, liver spots, melasma, brown or black spots, sun pigment spots, blue melanoma, hyperpigmentation after drug use, gestational brown spots or hyperpigmentation after inflammation Ever composition. The method according to any one of claims 11 to 16,
The pharmaceutical composition,
A pharmaceutical composition for improving or treating melanin hyperpigmentation disorders comprising 0.0001 to 10% by weight of the active ingredient based on the total weight.

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