WO2018038465A1 - Skin-whitening composition containing 3-chloro-n-[trans-4-(methylamino)cyclohexyl]-n-[[3-(4-pyridinyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide - Google Patents

Abstract

One aspect of the present invention relates to a skin-whitening composition containing, as an active ingredient, 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridinyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide, optical or stereoscopic isomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof. Through the one aspect of the present invention, a novel whitening material can be provided to consumers and development of skin whitening-related industries can be promoted.

Description

[Correction 18.09.2017 by Rule 26] 3-Chloro-N- [trans-4- (methylamino) cyclohexyl] -N-[[3- (4-pyridinyl) phenyl] methyl] benzo [b] Skin whitening composition comprising thiophene-2-carboxamide

This specification describes 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide. The present invention relates to a composition for skin whitening comprising a composition for regulating the production and deposition of melanin, and a composition comprising a substance for exerting skin whitening efficacy.

Melanin is a biopolymer of phenols, a complex of black pigments and proteins, that is produced by browning when the cut surface of apples, potatoes, and bananas is exposed to the air, or in the skin feathers, skin, hair, and eyes of animals. Is observed. When melanin is excessively produced, it is deposited on the skin and thus forms blemishes, freckles, and the like, which are directly linked to skin whitening, and the like, and melanin promotes skin aging and skin cancer.

Melanocyte stimulating hormone (MSH) is secreted by UV rays, inflammation, and hormones, and MSH reacts with receptors to enhance cAMP in melanocytes, thereby synthesizing melanin. It is secreted to the outside to protect the skin from ultraviolet rays. Synthesis of melanin is mainly regulated by α-MSH, and MITF, TYR, TRP1, TRP2 and the like are known as proteins involved in the synthesis of melanin.

In one aspect, the invention provides 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2 -To provide a skin whitening composition comprising a carboxamide, an optical or stereoisomer thereof, an acceptable salt thereof, a hydrate thereof, or a solvate thereof as an active ingredient to promote the development of the field of skin whitening, Its purpose is to meet consumer demand.

In one aspect, the invention provides 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2 It provides a composition for skin whitening comprising a carboxamide, an optical or stereoisomer thereof, an acceptable salt thereof, a hydrate thereof, or a solvate thereof as an active ingredient.

In another aspect of the invention, the 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene- 2-carboxamide may be one that inhibits melanogenesis and deposition.

In another aspect of the invention, the composition may be a pharmaceutical composition or a cosmetic composition.

In one aspect, the invention provides 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2 -Carboxamide (3-Chloro- N- [ trans -4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide), its optical Or a method for skin whitening comprising administering a stereoisomer, an acceptable salt thereof, a hydrate thereof, or a solvate thereof to the subject.

The present invention also provides, in another aspect, 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) as an active ingredient for use in skin whitening applications. Phenyl] methyl] benzo [ b ] thiophene-2-carboxamide, optical or stereoisomer thereof, acceptable salt thereof, hydrate thereof, or solvate thereof. In addition, 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene as an active ingredient for skin whitening Provided are non-therapeutic cosmetic uses of 2-carboxamide, optical or stereoisomers thereof, acceptable salts thereof, hydrates thereof, or solvates thereof.

The present invention also provides, in another aspect, 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) for use in the preparation of a skin whitening composition. Phenyl] methyl] benzo [ b ] thiophene-2-carboxamide, optical or stereoisomers thereof, acceptable salts thereof, hydrates thereof, or solvates thereof.

In one aspect, the invention provides 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2 Pioneering and marketing new fields of skin whitening by discovering and providing new skin whitening compositions comprising carboxamides, optical or stereoisomers thereof, acceptable salts thereof, hydrates thereof, or solvates thereof as active ingredients. It can contribute to expansion.

1 shows α-MSH, 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] ti on B16F1 cells. Offen-2-carboxamide, arbutin treatment showed a change in melanin content.

Figure 2 shows α-MSH, 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] ti on B16F1 cells. When treated with offen-2-carboxamide, arbutin was confirmed the effect on the expression of tyrosinase, TRP1.

FIG. 3 shows 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophen-2- in the skin model. After carboxamide treatment, the brightness change was observed.

Figure 4 shows 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophen-2- in the skin model. After carboxamide treatment, the change in brightness was measured with a colorimeter.

As used herein, the term "skin" refers to a tissue covering the body surface of an animal, and is a broad concept including not only tissues covering the body surface such as the face or body, but also the scalp and hair.

As used herein, “3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophen-2-car Radiationamide ”may be expressed as Smo agonist, Smoothened agonist, Smo agonist, or SAG. The 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide is hedge It may include a substance that induces the expression of the smoothened protein that plays a major role in the hedgehog signaling pathway.

As used herein, "isomers" specifically refer to optical isomers (e.g., essentially pure enantiomers, original pure stereoisomers or mixtures thereof, as well as essentially pure diastereomers). , Conformation isomers (ie, isomers that differ only in their angles of one or more chemical bonds), structural isomers, position isomers (in particular, tautomers) or geometric isomers (eg, Cis-trans isomers).

As used herein, “essentially pure”, when used in the context of, for example, an enantiomer, stereoisomer, or diastereomer, includes about 90 specific compounds that may include enantiomers, stereoisomers, or diastereomers. At least%, preferably at least about 95%, more preferably at least about 97% or at least about 98%, even more preferably at least about 99%, even more preferably at least about 99.5% (w / w) It means to exist.

As used herein, “acceptable” means that it can be approved or approved by the government or equivalent regulatory body for use in animals, more specifically in humans, by avoiding significant toxic effects when used in conventional or pharmaceutical dosages. Recognized or enumerated as listed in the Food Code, Health Functional Food Code, or General Pharmacopoeia or as described in other general literature.

As used herein, “acceptable salt” means a salt according to one aspect of the invention, either conventional or pharmaceutically acceptable and having the desired activity of the parent compound. The salt is formed from (1) an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or the like; Or acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) Benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2,2,2] -oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert Acid addition salts formed with organic acids such as butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid; Or (2) salts formed when the acidic protons present in the parent compound are substituted.

As used herein, “hydrate” refers to a compound to which water is bound, and is a broad concept including an inclusion compound having no chemical bonding force between water and the compound.

As used herein, "solvate" means a higher order compound produced between molecules or ions of a solute and molecules or ions of a solvent.

In one aspect, the invention provides 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2 It provides a composition for skin whitening comprising a carboxamide, an optical or stereoisomer thereof, an acceptable salt thereof, a hydrate thereof, or a solvate thereof as an active ingredient.

In one embodiment, the 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophen-2- Carboxamide (3-Chloro- N- [ trans -4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide) is chlorobenzothione As the opene-based compound, may include C ₂₈ H ₂₈ ClN ₃ OS, it may be represented by the following formula.

In another embodiment, the compound may be obtained through synthesis, may be obtained by processing other materials, or may be derived from an organism or a microorganism.

In another embodiment, the 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophen-2- Carboxamide may be one that inhibits melanogenesis or deposition.

According to another aspect of the invention, the 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene The 2-carboxamide may be one that enhances the expression of tyrosinase or Tyrosinase-Related Protein 1 (TRP1). Since the expression of tyrosinase or TRP1 after the compound treatment is less than before the compound treatment, it can be seen that the compound can be used as an active ingredient of the composition for skin whitening. In addition, the treatment of the compound may reduce the number of cells expressing tyrosinase or TRP1, it can also be seen that the compound can be used as an active ingredient of the skin whitening composition.

Melanin (melanin) is observed in the animal's outer skin feathers, skin, hair, eyes, etc., when the excessive production of melanin is deposited on the skin to form blemishes, freckles and the like, promote skin aging, skin cancer may also be induced. The diseases or symptoms caused by the excessive production of melanin include blemishes, freckles, blotch, blemishes, epidermal melanocytic lesions, milky coffee spots (Cafe's au lait macules), birthmarks, Becker's Nevus, and stingles Nevus Spilus, Lentigines, Melanoma, Dermal melanocytic lesions, Mongolian spot, Nevus of Ota, Acquired bilateral nevus of Ota-like macules, Nevus of Ito, Blue nevus, Melanocytic nevus, Junction nevus, Compound nevus, Intradermal nevus Intradermal nevus, Halo nevus, Congenital nevocytic nevus, Spitz nevus, Dysplastic nevus, Melanoma, Malignant black melanoma (Lentigo maligna melanoma) ), Superficially expandable melanoma (Superficial spreading melanoma, Acral lentiginous melanoma, Nodular melanoma, Pigment basal cell carcinoma, Pigmentary dermofibromas, Pigmentary dermal cyst At least one day selected from the group consisting of pigmented keloids, pigmentation by ultraviolet light, pigmentation by drugs, pigmentation after inflammation, and pigmentation resulting from dermatitis, and keratoacanthomas. Can be.

In one embodiment of the present invention, the 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene 2-carboxamide inhibits melanin production and accumulation, so pigments from blemishes, freckles, melanoma, nevus, melanoma, pigmentation by ultraviolet light, pigmentation by drugs, pigmentation after inflammation, and dermatitis It may be to prevent, ameliorate or treat one or more selected from the group consisting of deposition.

In one aspect of the present invention, the process of forming melanin by treating a melanin-forming substance with melanin forming cells is 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3 It was confirmed that it was inhibited by-(4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide.

From these results, we found 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2 Carboxamide can be used as a skin whitening agent. Efficacy of such 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide It is also possible to prevent, ameliorate, and treat the above diseases and symptoms related to melanin.

According to another aspect of the invention, 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b in the composition ] The content of thiophene-2-carboxamide, isomer thereof, acceptable salt thereof, hydrate thereof, or solvate thereof may be from 0.0001% to 20% by weight relative to the total weight of the composition. In one embodiment, the content is at least 0.0001 wt%, at least 0.0005 wt%, at least 0.001 wt%, at least 0.005 wt%, at least 0.01 wt%, at least 0.05 wt%, at least 0.1 wt%, 0.3 wt%, based on the total weight of the composition. At least 0.5 wt%, at least 0.8 wt%, at least 1 wt%, at least 3 wt%, at least 5 wt%, at least 8 wt%, at least 10 wt%, at least 12 wt%, at least 15 wt%, or 18 wt% It may be more than%. In addition, the content is 20 wt% or less, 18 wt% or less, 15 wt% or less, 12 wt% or less, 10 wt% or less, 8 wt% or less, 5 wt% or less, 3 wt% or less, based on the total weight of the composition, 1 wt% or less, 0.8 wt% or less, 0.5 wt% or less, 0.3 wt% or less, 0.1 wt% or less, 0.05 wt% or less, 0.01 wt% or less, 0.005 wt% or less, 0.001 wt% or less, or 0.0005 wt% or less Can be.

According to another aspect of the invention, the 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] ti Dosages of offen-2-carboxamide, isomers thereof, acceptable salts thereof, hydrates thereof, or solvates thereof may be from 0.0001 mg / kg / day to 20 mg / kg / day. In one embodiment, the dosage is at least 0.0001 mg / kg / day, at least 0.0005 mg / kg / day, at least 0.001 mg / kg / day, at least 0.005 mg / kg / day, at least 0.01 mg / kg / day, 0.05 mg / kg / day or more, 0.1 mg / kg / day or more, 0.5 mg / kg / day or more, 0.8 mg / kg / day or more, 1 mg / kg / day or more, 2 mg / kg / day or more, 3 mg / kg / day or more, 5 mg / kg / day or more, 8 mg / kg / day or more, 10 mg / kg / day or more, 12 mg / kg / day or more, 15 mg / kg / day or more, or 18 mg / kg Can be more than / day. In addition, the dosage is 20 mg / kg / day or less, 18 mg / kg / day or less, 15 mg / kg / day or less, 12 mg / kg / day or less, 10 mg / kg / day or less, 8 mg / kg / Day or less, 5 mg / kg / day or less, 3 mg / kg / day or less, 2 mg / kg / day or less, 1 mg / kg / day or less, 0.8 mg / kg / day or less, 0.5 mg / kg / day Or less, 0.1 mg / kg / day or less, 0.05 mg / kg / day or less, 0.01 mg / kg / day or less, 0.005 mg / kg / day or less, 0.001 mg / kg / day or less, or 0.0005 mg / kg / day or less Can be.

According to one aspect of the invention, the composition may be a composition for skin whitening which is a pharmaceutical composition.

The pharmaceutical composition may further contain pharmaceutical supplements such as preservatives, preservatives, stabilizers, hydrating or emulsifying accelerators, salts for regulating osmotic pressure and / or buffers, and other therapeutically useful substances, And can be formulated in various oral or parenteral dosage forms.

The oral dosage forms include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, powders, powders, fine granules, granules, pellets, and the like, and these formulations include surfactants in addition to active ingredients. , Diluents (eg lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), glidants (eg silica, talc, stearic acid and its magnesium or calcium salts and polyethylene glycols). . Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally starch, agar, alginic acid or its sodium salt Pharmaceutical additives such as disintegrants, absorbents, colorants, flavors, and sweeteners. The tablets can be prepared by conventional mixing, granulating or coating methods.

In addition, the parenteral dosage form may be a transdermal dosage form, for example, an injection, drop, ointment, lotion, gel, cream, spray, suspension, emulsion, suppository, patch, or the like. It may be, but is not limited thereto.

The pharmaceutical composition may be administered parenterally, rectally, topically, transdermally, subcutaneously, and the like.

Determination of the dosage of the active ingredient is within the level of those skilled in the art, the daily dosage of the drug may vary depending on various factors such as the progress of the subject to be administered, the onset, age, health condition, complications.

The pharmaceutical composition may be an external preparation for skin, and the external preparation for skin may be included herein as a generic term that may include anything applied outside the skin.

According to another aspect of the invention, the composition may be a composition for skin whitening which is a cosmetic composition.

The cosmetic composition may further include a functional additive and components included in the general cosmetic composition. The functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids and seaweed extract. In addition to the other components included, oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, flavorings, blood circulation And accelerators, cooling agents, limiting agents, purified water, and the like.

The cosmetic composition is not particularly limited in formulation, and may be appropriately selected as desired. For example, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisturizing cream, hand cream, foundation, essence, nutrition essence, pack, soap, cleansing It may be prepared in any one or more formulations selected from the group consisting of foam, cleansing lotion, cleansing cream, body lotion and body cleanser, but is not limited thereto.

When the formulation according to one aspect of the present invention is a paste, cream or gel, the carrier component is animal fiber, vegetable fiber, wax, paraffin, starch, trakant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. And the like can be used.

When the formulation according to one aspect of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component, in particular, in the case of a spray, additionally chlorofluoro Propellants such as hydrocarbon, propane / butane or dimethyl ether.

When the formulation according to one aspect of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Fatty acid esters of propylene glycol, 1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycols or sorbitan.

When the formulation according to one aspect of the invention is a suspension, the carrier component is 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. , Microcrystalline cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the dosage form according to one aspect of the present invention is a surfactant-containing cleansing agent, as a carrier component, an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, methyltaurate, and sarcosy Nates, fatty acid amide ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

Hereinafter, the configuration and effects of one aspect of the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the examples below are provided for the purpose of illustration only to help the understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

Example 1

Cell and skin model preparation

Melan-A, a melanogenesis cell derived from C57BL / 6 J (black, a / a) mice, obtained from normal human epidermal melanocytes (NHEMs, Cascade Biologics, Portland, OR, USA) -A) Cell lines were obtained from Dr. Dorothy C. Bennett (St. George's Hospital Medical School, London, UK). B16F1 mouse melanoma cell line was obtained from ATCC (Manassas, VA, USA).

Human epidermal melanocyte forming cells were maintained under Human Melanocyte Growth Supplements (HMGS) (Cascade Biologics, Inc., Mansfield, UK) in M-254 medium. Melan-A cells were maintained under 10% (v / v) fetal bovine serum, 1% (v / v) penicillin-streptomycin, and 0.2 μM phorbol 12-myristate 13-acetate in RPMI 1640 medium. B16F1 mouse melanoma cell lines were cultured in DMEM with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin.

Three-dimensional human skin substitutes (MelanoDerm ™, MEL-312-B, MatTek, Seoul, Korea) were obtained, followed by keratinocyte growth factor (KGF), fibroblast growth factor (b-FGF), and It was maintained according to the manufacturer's instructions in EPI-100-NMM-113 medium optimized with alpha-melanocyte stimulating hormones (α-MSH).

The brightness change (ΔL) index of the skin model was calculated using an L index (L value, lightness index) measured by a colorimeter (CRimeter, CR-300, Konica Minolta, Tokyo, Japan), and ΔL = day 13 or 20 It was calculated as primary L (treatment skin)-L (control skin). Increasing the ΔL index indicates underdeposition of the pigment by compound induction in the skin (Lee et al., 2013).

reagent

“SAG, which is 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide ”Was obtained from Calbiochem (San Diego, CA, USA). α-MSH, arbutin, and kojic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Melanin Essay

Cells were centrifuged at 1000 x g for 5 minutes after trypsin / EDTA and washed twice with PBS. The final cell pellet in the tube was photographed and the cell pellet was dissolved in 1 N NaOH. Homogenized cell extracts were transferred to 96-well plates and the relative melanin amount was measured at absorbance 405 nm using an ELISA plate reader.

Weston Blot  analysis

All lysates were 2 × Laemmli sample buffer, 62.5 mM Tris-HCl, pH 6.8, 25% [v / v] glycerol, 2% [w / v] SDS, 5% [v / v β-mercaptoethanol, and 0.01% [w / v] bromophenol blue) (Bio-Rad, Hercules, CA, USA). All cellular proteins were measured using Bradford solution (Bio-Rad). The sample was then separated by SDS-PAGE and transferred to PVDF membrane (Bio-Rad). TBST 4% (w / v) skim milk (skim milk) for one rear, the membrane blocking in conjunction with the specific primary antibody in (25 mM Tris, 140 mM NaCl, and 0.05% [v / v] Tween ® 20) Overnight incubation.

Anti-actin antibody (MAB1501, 1: 10,000) was obtained from Millipore (Temecula, Calif., USA), and anti-αPEP7 (tyrosinase) antibody, 1 : 1000) and anti-αPEP1 (TRP1) antibody (anti-αPEP1 (TRP1) antibody, 1: 1000) were obtained from VJ Hearing (NIH, Bethesda, MD). For protein detection, the membrane was incubated with an HRP- conjugated secondary antibody gated signal was measured using a ^{SuperSignal ® West Dura HRP Detection Kit (} Pierce Inc., Rockford, IL, USA).

Statistical analysis

Each data was obtained through at least three independent experiments and expressed as mean ± SE (standard error). Statistical evaluation of the results was performed using one-way ANOVA (* p <0.05, ** p <0.01).

 Experimental Example 1 Melanin Formation Regulation

3-Chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide has the formula It is a substance represented by 1.

[Formula 1]

B16F1 cells were pretreated with α-MSH (1 μM) and after 24 hours 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl ] Benzo [ b ] thiophene-2-carboxamide (5 μΜ) or arbutin (500 μΜ) were treated. After 24 hours, the cellular melanin content, tyrosinase and TRP1 protein expression levels were analyzed by Western blot. As a result, the compound inhibited melanin production in B16F1 cells cultured by treatment with α-MSH (Fig. 1). The compound also reduced the expression levels of tyrosinase, TRP1 protein (FIG. 2).

Experimental Example 2 Artificial Skin Experiment

The regulation of melanin production was tested using a three-dimensional human skin substitute (reconstituted epidermal model comprising normal human epidermal keratinocytes and NHEMs).

Specifically, 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide (5 μM) and kojic acid (KA, 5 mM) were administered to 3D human skin substitutes every 3 days for 13 days and 20 days and the effects on pigmentation were evaluated. Pigmentation was evaluated using a colorimeter, and ΔL in FIG. 4 was calculated by calculating the difference in brightness between the control group and the substance treatment group.

As a result, skin pigmentation was reduced by the compound (FIGS. 3 and 4). Through this, it was confirmed that the compound regulates melanin formation and pigmentation even in the three-dimensional human skin substitute.

Examples of the formulation of the composition according to one aspect of the present invention will be described below, but it is also applicable to various other formulations, which are intended to explain in detail only and not intended to limit the present invention.

Formulation Example 1 Soft Capsule

3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide 20 mg, L -Carnitine 80 ~ 140mg, soybean oil 180mg, palm oil 2mg, vegetable hardened oil 8mg, lead 4mg and lecithin 6mg were mixed and filled into one capsule in a conventional manner to prepare a soft capsule.

Formulation Example 2 Tablet

3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide 20 mg, brown 200 mg of lactooligosaccharide, 60 mg of lactose and 140 mg of maltose were mixed and granulated using a fluidized bed dryer, and then, 6 mg of a sugar ester was added and tableted with a tablet press to prepare a tablet.

Formulation Example 3 Granules

3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide 20 mg, anhydrous 250 mg of crystalline glucose and 550 mg of starch were mixed, molded into granules using a fluidized bed granulator, and then filled into fabrics to prepare granules.

[Formulation Example 4] Drinks

3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide 20 mg, glucose 10 g, citric acid 0.6 g, and liquid oligosaccharide 25 g are mixed, and 300 ml of purified water is added to each bottle and 200 ml is filled. After filling the bottle sterilized for 4-5 seconds at 130 ℃ to prepare a drink.

Formulation Example 5 Injection

3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide 50 mg, main Injectables were prepared in a conventional manner using sterile distilled water titrant and pH adjuster titrant.

Formulation Example 6 Softener (Skin Lotion)

3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide 3.0 wt% , L-ascorbic acid-2-magnesium phosphate salt 1.00% by weight, water-soluble collagen (1% aqueous solution) 1.00% by weight, sodium citrate 0.10% by weight, citric acid 0.05% by weight, licorice extract 0.20% by weight, 1,3-butylene Softener longevity (skin lotion) was prepared using 3.00% by weight of glycol and the remaining amount of purified water.

Formulation Example 7 Cream

3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide 3.00 wt% , Polyethylene glycol monostearate 2.00 wt%, self-emulsifying glycerin monostearate 5.00 wt%, propylene glycol 4.00 wt%, squalene 6.00 wt%, tri2-ethylhexaneglyceryl 6.00 wt%, sphingolipids 1.00 wt%, A creamy formulation was prepared using 7.00 wt% of 1,3-butylene glycol, 5.00 wt% of beeswax, and the balance of purified water.

[Formulation Example 8] Pack

3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide 3.00 wt% , 13.00% by weight polyvinyl alcohol, 1.00% by weight L-ascorbic acid-2-phosphate magnesium salt, 1.00% by weight lauroylhydroxyproline, 2.00% by weight water-soluble collagen (1% aqueous solution), 1,3-butylene The pack was prepared using 3.00 wt% glycol, 5.00 wt% ethanol and the balance of purified water.

Formulation Example 9 Health Food

Health food was prepared by conventional methods according to the compositions described in the table below.

ingredient	content
3-chloro-N- [trans-4- (methylamino) cyclohexyl] -N-[[3- (4-pyridinyl) phenyl] methyl] benzo [b] thiophene-2-carboxamide	10mg
Vitamin mixtures
Vitamin A Acetate	70 μg
Vitamin E	1.0 mg
Vitamin B1	0.13 mg
Vitamin B2	0.15 mg
Vitamin B6	0.5 mg
Vitamin B12	0.2 μg
Vitamin c	10 mg
Biotin	10 μg
Nicotinic acid amide	1.7 mg
Folic acid	50 μg
Calcium Pantothenate	0.5 mg
Mineral mixture
Ferrous sulfate	1.75 mg
Zinc oxide	0.82 mg
Magnesium carbonate	25.3 mg
Potassium phosphate monobasic	15 mg
Dicalcium Phosphate	55 mg
Potassium citrate	90 mg
Calcium carbonate	100 mg
Magnesium chloride	24.8 mg

Although the composition ratio of the vitamin and inorganic mixture is a composition that is relatively suitable for health foods, for example, the composition ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method, and then the conventional method. According to the health food composition can be used.

Formulation Example 10 Health Drink

Health food was prepared by conventional methods according to the compositions described in the table below.

ingredient	content
3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide	15mg
Citric acid	1000 mg
oligosaccharide	100 g
Plum concentrate	2 g
Taurine	1 g
Purified water	Remaining amount
Total volume	900 ml

As shown in Table 2, the remaining amount of purified water was added to a total volume of 900 ml, and the above ingredients were mixed according to a conventional healthy beverage manufacturing method, and then stirred and heated at 85 ° C. for about 1 hour. Obtained in a sterilized 2 liter container, sterilized sealed, and then refrigerated to prepare a healthy beverage.

As described above, specific portions of the present disclosure have been described in detail, and for those skilled in the art, these specific techniques are merely preferred embodiments, and the scope of the present disclosure is not limited thereto. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

1. 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide (3- Chloro- N- [ trans -4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2-carboxamide), its optical or stereoisomer, its acceptable A skin whitening composition comprising a salt, a hydrate thereof, or a solvate thereof as an active ingredient.
2. The compound of claim 1, wherein the 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2 Carboxamide is a composition for skin whitening that inhibits melanin production or deposition.
3. The compound of claim 1, wherein the 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2 -Carboxamide is to enhance the expression of tyrosinase (tyrosinase) or TRP1 (Tyrosinase-Related Protein 1), the composition for skin whitening.
4. The compound of claim 1, wherein the 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2 Carboxamide is one or more selected from the group consisting of blemishes, freckles, melanoma, nevus, melanoma, pigmentation by ultraviolet light, pigmentation by drugs, pigmentation after inflammation, and pigmentation that occurs in dermatitis. A composition for skin whitening which prevents, improves or treats.
5. The compound of claim 1, wherein the composition is 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene. The content of -2-carboxamide, isomer thereof, acceptable salt thereof, hydrate thereof, or solvate thereof is 0.0001% to 20% by weight based on the total weight of the composition.
6. The compound of claim 1, wherein the 3-chloro- N- [trans-4- (methylamino) cyclohexyl] -N -[[3- (4-pyridinyl) phenyl] methyl] benzo [ b ] thiophene-2 The dosage of carboxamide, isomer thereof, acceptable salt thereof, hydrate thereof, or solvate thereof is from 0.0001 mg / kg / day to 20 mg / kg / day.
7. The composition for skin whitening according to any one of claims 1 to 6, wherein the composition is a pharmaceutical composition.
8. The composition for skin whitening according to any one of claims 1 to 6, wherein the composition is a cosmetic composition.

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