WO2007052882A1

WO2007052882A1 - Pyridine thiazole carboxamide derivatives, the preparation method thereof, and the composition for skin whitening containing the same

Info

Publication number: WO2007052882A1
Application number: PCT/KR2006/002867
Authority: WO
Inventors: Yong-Joo Na; Soo-Mi Ahn; Hyun-Jung Shin; Ho-Sik Rho; Jae-Sung Hwang; Ih-Seop Chang
Original assignee: Amorepacific Corporation
Priority date: 2005-11-01
Filing date: 2006-07-21
Publication date: 2007-05-10
Also published as: KR100716891B1

Abstract

Disclosed are a pyridine thiazole carboxamide derivative expressed by a following chemistry figure 1 or pharmaceutically acceptable salt thereof, a method for preparing the same and a composition for suppressing c-kit activation or composition for skin whitening containing the same. The compound of the chemistry figure 1 according to the invention suppresses the melanogenesis through the action of inhibiting the c-kit activation, thereby improving the pigmentation to exhibit the whitening effect. Accordingly, the composition for suppressing the c-kit activation or composition for skin whitening containing the same can be usefully used for the cosmetic composition or pharmaceutical composition. Chemistry Figure 1 where R1: phenyl, and n: 0, 1, 2 or 3.

Description

[DESCRIPTION] [Invention Title]

PYRIDINE THIAZOLE CARBOXAMIDE DERIVATIVES, THE PREPARATION METHOD THEREOF, AND THE COMPOSITION FOR SKIN WHITENING CONTAINING THE SAME [Technical Field]

The invention relates to a novel pyridine thiazole carboxamide derivative or pharmaceutically acceptable salt thereof, a method for preparing the same and a composition for suppressing c-kit activation or composition for skin whitening containing the same. [Background Art]

Although a human skin color is complexly determined by red blood cells in blood, carotin and melanin, a difference of the skin colors between human races and hyperchromatism such as chloasma, freckle and the like result from the melanin. The melanin existing in an epidermal layer which is a shell of the skin blocks the ultraviolet, protects skin organs under the dermis, traps free radicals having occurred in the skin and protects proteins and genes in the skin.

However, the melanin having occurred due to stress stimulation from the interior and exterior of the human body is a very stable material that does not disappear until it is discharged through skin keratinization, although the stress is removed. In addition, when the free radicals are much produced in the skin through a polymerization oxidization in vivo wherein tyrosine or DOPA is a substrate and enzyme such as tyrosinase serves as catalyst, when there is an inflammatory response or when the ultraviolet is applied, the melanogenesis is increased. In particular, the ultraviolet increases the melanogenesis, so that the partially increased melanin is developed into chloasma to cause externally undesired results. More severely, it may cause skin cancer and the like which is dangerous to the life.

Due to such reasons, it has been developed and used many melanogenesis inhibitors such as ointment, cream, lotion and the like containing kojic acid, arbutin, glutathione, vitamin A, vitamin C and the like. However, there are no whitening products having a whitening effect satisfying the customers. In addition, in case of the whitening agent containing hydroquinone, it has a whitening effect to some extent, but severely stimulates the skin, so that it is restrictively used.

The c-kit is a cell surface protein belonging to a receptor tyrosine kinase type III family and is known as a receptor of a stem cell factor (SCF). According to research results for white spotting and steel mouse, the SCF/c-kit play an important role in cell maintenance, hematopoiesis, pigmentation, reproduction and the like.

As a result of interaction between the SCF/c-kit, the c-kit protein undergoes the dimerization and is autophosphorylated. It then undergoes the Ras-Raf-MAP kinase cascade in the intracellular signal transfer, and activates the Microphthalmia-Associated Transcription Factor (Mitf), thereby accelerating the tyrosinase synthesis and deriving the melanogenesis in the melanocyte.

The SCF/c-kit signal transfer is also associated with the pigmentation which is derived by the ultraviolet B. It is thought that the mechanism thereof may be related to an increase of the SCF expression in the keratinocyte and an increase of the c-kit expression in the melanocyte. In fact, it can be seen that gene and protein expressions of the SCF and the c- kit are increased when the ultraviolet B stimulation is applied during the cell culture of the keratinocyte and melanocyte.

Such phenomena can be also seen when the ultraviolet B treatment is performed for the epidermis of the human. In other words, the gene and protein expressions of the SCF are significantly increased in the portion exposed to the ultraviolet B, as compared to the portions which are not exposed to it .

An animal experiment of brown guinea pigs, which is a good model of the melanocyte function research, also provides the same result. Specifically, when the specific antibody to the c-kit protein is injected in the subepidermis of the experimental animal, the pigmentation due to the ultraviolet B is perfectly inhibited. Accordingly, it can be seen that the signal transfer of the SCF/c-kit interaction is closely related to the pigmentation ultimately. [Disclosure] [Technical Problem]

In order to solve the problems of the whitening agents, the inventors have researched a new material exhibiting excellent whitening effects through an action of inhibiting the c-kit activation. As a result, the inventors confirmed that a pyridine thiazole carboxamide derivative inhibits the c-kit and thus also suppresses the melanogenesis.

Accordingly, an object of the invention is to provide a novel pyridine thiazole carboxamide derivative or pharmaceutically acceptable salt thereof, as a material exhibiting a new whitening effect.

Another object of the invention is to provide a method of preparing the above compound.

Still another object of the invention is to provide a composition for suppressing c-kit activation or composition for skin whitening containing the above compound. [Technical Solution]

In order to achieve the above objects, according to the invention, there is provided a pyridine thiazole carboxamide derivative expressed by a following chemistry figure 1 or pharmaceutically acceptable salt thereof. [Chemistry Figure 1]

(where R₁: phenyl, and n: O, 1, 2 or 3)

In addition, the invention provides a method for preparing the pyridine thiazole carboxamide derivative expressed by the chemistry figure 1 or pharmaceutically acceptable salt thereof, the method comprising steps of: (1) reflux-reacting ethyl-3-bromo-2-oxo-propionate and pyridine-3-carbothioamide under presence of organic solvent, thereby preparing pyridine thiazole carboxylate; (2) hydrolyzing the pyridine thiazole carboxylate produced -in the step (1) with a base in a reaction solvent, thereby preparing pyridine thiazole carboxylic acid; and (3) reacting the pyridine thiazole carboxylic acid produced in the step (2) with ethylchloroformate, under presence of base, to prepare anhydride and reacting the produced anhydride with amine, thereby preparing pyridine thiazole carboxamide.

In the preparing method, the reaction solvent in the step (2) is a mixed solvent of methanol and tetrahydrofuran mixed in a ratio of 1:1 to 1:5.

In the preparing method, when preparing the anhydride in the step (3), an equivalent ratio of the pyridine thiazole carboxylic acid and the ethylchloroformate is 1:1.1 to 1:1.3, and an amount of the amine reacting with the produced anhydride is such that an equvalent ratio of the pyridine thiazole carboxylic acid and the amine is 1:1 to 1:3.

In addition, the invention provides a composition for suppressing c-kit activation containing the pyridine thiazole carboxamide derivative expressed by the chemistry figure 1 or pharmaceutically acceptable salt thereof.

In addition, the invention provides a composition for skin whitening containing the pyridine thiazole carboxamide derivative expressed by the chemistry figure 1 or pharmaceutically acceptable salt thereof.

In the composition, the pyridine thiazole carboxamide derivative or pharmaceutically acceptable salt thereof is contained in an amount of 0.01-20 wt% for a total composition weight.

The composition is a cosmetic composition or pharmaceutical composition. [Advantageous Effects]

The pyridine thiazole carboxamide derivative or pharmaceutically acceptable salt thereof according to the invention suppresses the melanogenesis through the action of inhibiting the c-kit activation, thereby improving the pigmentation to exhibit the whitening effect. Accordingly, the composition for suppressing the c-kit activation or composition for skin whitening containing the same can be usefully used for the cosmetic composition or pharmaceutical composition. [Description of Drawings]

FIG. 1 is a graph showing an inhibitory effect of SU11248 on the c-kit activation;

FIG. 2 is a graph showing an inhibitory effect of pyridine thiazole carboxamide derivatives of the invention on the c-kit activation;

FIG. 3 shows an inhibitory effect of SU11248 on the c-kit activation in a cell level ; and

FIG. 4 shows an inhibiting effect of pyridine thiazole carboxamide derivatives on the c-kit activation in a cell level, which are prepared in embodiments 1 to 4 of the invention. [Best Mode]

The pyridine thiazole carboxamide derivative compound of the chemistry figure 1 according to the invention is prepared by a method comprising steps of: as shown in a following reaction figure 1, (1) reflux-reacting ethyl-3- bromo-2-oxo-propionate and pyridine-3-carbothioamide under presence of organic solvent, thereby preparing pyridine thiazole carboxylate which is the compound(II) for the reation 1; (2) hydrolyzing the pyridine thiazole carboxylate produced in the step (1) with a base in a reaction solvent, thereby preparing pyridine thiazole carboxylic acid which is the compound(III) for the reation 1; and (3) reacting the pyridine thiazole carboxylic acid produced in the step (2) with ethylchloroformate, under presence of base, to prepare anhydride and reacting the produced anhydride with amine, thereby preparing pyridine thiazole carboxamide (compound (I) of the reaction figure 1) expressed by the above chemistry figure 1. [Reaction Figure 1]

(where Ri: phenyl, and n: 0, 1, 2 or 3)

In the step (1), toluene or benzene may be used as the organic solvent.

In the step (2), alkali metallic hydroxide such as sodium hydroxide, potassium hydroxide and the like may be used as the base.

In addition, water, methanol, ethanol, propanol, tetrahydrofuran or dichloromethane may be used as the reaction solvent, and methanol and tetrahydrofuran are preferably mixed. In this case, the reaction can be completed in the shortest time. At this time, a mixing ratio of the methanol and tetrahydrofuran is 1:1 to 1:5, preferably 1:1. When the mixing ratio of the methanol and tetrahydrofuran is below 1:1 or above 1:5, the hydrolysis is not perfectly carried out. The reason is as follows: when the mixing ratio is below 1:1, the ester compound, which is the compound (II) of the reaction figure 1, is not sufficiently dissolved in the reaction solution, and when the mixing ratio is above 1:5, the alkali metallic hydroxide, which is used as the base, is not sufficiently dissolved, so that there remains the material that is not reacted yet.

In the step (3), acid-halogenation method, active ester method, acid anhydride method can be used as a method for preparing an amide compound, which is the compound (I) of the reaction figure 1, from the pyridine thiazole carboxylic acid which is the compound (III). However, it is most preferred a method of substituting the carboxylic acid into anhydride state using ethylchloroformate. At this time, an equivalent ratio of the acid compound (pyridine thiazole carboxylic acid) and the ethylchloroformate for preparing the anhydride is preferably 1:1.1 to 1:1.3. When the equivalent ratio is below 1:1.1, there remains the pyridine thiazole carboxylic acid that is not reacted yet, so that it should be separated from the object. In addition, when the equivalent ratio is above 1:1.3, there is a difficulty in removing the ethylchloroformate which has been excessively used.

In the invention, the anhydride obtained by reacting the ethylchloroformate and the pyridine thiazole carboxylic acid is again reacted with the amine to prepare the compound (I) expressed by the chemcial formula 1. At this time, an amount of the amine used is preferably such that an equivalent ratio of the pyridine thiazole carboxylic acid, which is a starting material for producing the anhydride, and the amine is 1:1 to 1:3. When the equivalent ratio is below 1:1, an amount of the objective product is too little, and when it is above 1:3, there remains the amine that has been excessively used, so that it is difficult to separate the final product.

In addition, aniline, benzyl amine, phenyl ethyl amine and phenyl propyl amine may be preferably used as the amine reacting with the anhydride.

As the reaction solvent, dichloromethane, acetone, N,N- dimethylformamide, acetonitrile or tetrahydrofuran may be used. It is most preferred to use the N,N-dimethylformamide.

At this time, the reaction temperature may be 10°C to 60°C , preferably about 30^°C.

The pyridine thiazole carboxamide derivative expressed by the chemistry figure 1 or pharmaceutically acceptable salt thereof, which is obtained as described above, may be used as a pharmaceutically acceptable salt form. The salt is not particularly limited as long as it is phamaceutically acceptable, and includes, for example hydrochloric acid, sulfuric acid, nitric acid, O

phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid and the like.

The composition for suppressing the c~kit activation or composition for skin whitening containing the pyridine thiazole carboxamide derivative or pharmaceutically acceptable salt thereof preferably contains the compound in a concentration of 0.01-20.0 wt% for a total composition weight. When the concentration is below 0.01 wt%, it is difficult to obtain the effect, and when it is above 20.0 wt%, it may cause a difficulty in the stability and formulations.

The novel pyridine thiazole carboxamide derivative of the invention can exhibit the whitening effect by suppressing the melanogenesis through the action of inhibiting the c-kit activation.

The inventors confirmed the efficacy of the compound by measuring the inhibitory effect of the pyridine thiazole carboxamide derivative of the invention on the c-kit activation and the whitening effect for the human skin.

The invention provides a cosmetic composition for suppressing the c~kit activation or cosmetic composition for skin whitening containing the pyridine thiazole carboxamide derivative expressed by the chemistry figure 1 or pharmaceutically acceptable salt thereof in an amount of 0.01-20.0 wt% for a total composition weight. When the content is below 0.01 wt%, it is difficult to obtain the effect, and when it is above 20.0 wt%, it may cause problems in stability and formulations.

In addition, the cosmetic composition of the invention may further contain other whitening ingredients capable of preferably providing a synergy effect to the main effect, within a range of not deteriorating the main effect of the inveniton, in addition to the above compound. For example, it may contain kojic acid, arbutin, vitamin C derivative and the like.

The cosmetic composition of the invention can be used in the cosmetics in various manners. The product to which the cosmetic composition of the invention can be added may include cosmetics such as various creams, lotion and the like, cleansing agent, washing agent, soap, cosmetic solution, etc.

The cosmetic composition for suppressing the c-kit activation or cosmetic composition for skin whitening, to which the composition containing the compound is added, may take a form of solution, emulsion, viscous mixture and the like.

In other words, the cosmetics of the invention is not particularly limited with regard to formulations thereof, and may include emulsion, cream, toilet water, essence, pack, gel, powder, makeup base, foundation, lotion, ointment, patch, cosmetic solution, cleansing foam, cleansing cream, cleansing water, body lotion, body cream, body oil, body essence, body washing agent, soap, aerosol and the like.

In the cosmetic compositions of the respective formulations, the other ingredients may be appropriately selected and mixed by one skilled in the art in accordance with formulations or use objects of the cosmetics.

The formulations may contain materials for promoting the skin absorption so as to improve the effects.

In addition, the cosmetics of the invention may contain an ingredient selected from a group consisting of water-soluble vitamin, oil-soluble vitamin, high molecular peptide, high molecular polysaccharide, sphingolipid and seeweed extracts.

The cosmetics of the invention may be mixed with another ingredients, which are typically mixed in the cosmetics, as required, in addition to the essential ingredients.

The other mixed ingredients which can be added may include oil and fat ingredient, moisturizing agent, emollient agent, surfactant, organic and inorganic pigments, organic powder, ultraviolet absorbent, antiseptic, fungicide, antioxidant, plant extract, pH adjuster, alcohol, colorant, flavour, blood circulation accelerant, frigidity agent, anhydrotics, purified water and the like. In addition, the mixing ingredients which can be added are not limited to the above ingredients and any ingredient may be mixed preferably in an amount of 0.01-3 wt% for a total composition weight, within a range of not deteriorating the object and effect of the invention.

In addition, the invention provides a phamaceutical composition for suppressing the c-kit activation or pharmaceutical composition for skin whitening containing the pyridine thiazole carboxamide derivative expressed by the chemistry figure 1 or pharmaceutically acceptable salt thereof in an amount of 0.01-20.0 wt% for a total composition weight.

The phamaceutical composition containing the pyridine thiazole carboxamide derivative expressed by the chemistry figure 1 or pharmaceutically acceptable salt thereof may further comprise appropriate carriers, excipients and diluents which are typically used to prepare the pharmaceutical composition.

The compound of the invention may be used as a pharmaceutically acceptable salt form thereof, and may be used as an individual form or combination with other pharmaceutical active compounds.

The pharmaceutical composition containing the compound of the invention may be formulated into oral-type formulation such as powders, granule, tablet, capsule, suspension, emulsion, syrup, aerosol and the like, external preparations such as ointment, gel, cream, patch, aerosol, etc. and any forms suitable for pharmaceutical formulations, in accordance with typical methods.

The dosage of the pharmaceutical preparation is different depending on ages, sexes, weights and symptoms of the patients, and administration route and the like. In case of the external preparation, it is preferably applied one to five times per a day for one month or more in an amount of 1.0 to 3.0 mi per a day. In addition, in case of the oral-type formulation, although the dosage is different depending on ages, sexes and weights of the patients, it may be administrated one to several times per a day in an amount of 0.1 to 100 mg/kg. In addition, the dosage may be increased or decreased depending on the administration routes, seriousness of the disease, sexes, weights and ages. Accordingly, the dosage does not limit the scope of the invention in any way.

The compound of the invention may be administrated to a mammal including rat, mouse, domestic animal, human and the like in various routes such as non-oral, oral and the like. The compound may be administrated by, for example oral, rectal or vein, muscular, subcutaneous, intrauterine dural or intracerebroventricular injections.

In the mean time, since the compound and pharmaceutically acceptable salt thereof of the invention do not have severe toxicity and side effects, they can be used safely for a long time, for the prevention purpose. [Mode for Invention]

Hereinafter, the invention will be more specifically described with reference to embodiments and experimental examples. However, it should be noted that the invention is not limited thereto.

[embodiment 1] N-phenyl-2-(pyridine-3-il)thiazole-4-carboxamide Ethyl-3-bromo-2-oxo-propionate (10Og, 0.51 mol) and pyridine-3- carbothioamide (7Og, 0.51 mol) were dissolved in toluene 500 rai and then refluxed for 5 hours. After completion of the reaction, the toluene was disti Hated under reduced pressure, and dichloromethanedOOO m-O was drop- added to the remaining material. The mixture was mixed and washed with water 1000 mi, dried with anhydrous manganese (10Og), then filtered and concentrated to be crystallized into hexane and then again filtered, thereby providing ethyl-2-(pyridine-3-il)thiazole-4-carboxylate 99.7g (yield=83%).

Then, the ethyl-2-(pyridine-3-il)thiazole-4-carboxylate (5Og) was dissolved in the mixture solution of tetrahydrofuran and methanol (=1:1), potassium hydroxide (KOH) 10% solution 50 mi was added thereto and then the mixture was stirred for 3 hours. After the stirring, the mixture was neutralized with hydrochloric acid, so that solid was obtained. The solid was filtered, so that 2-(pyridine-3-il)thiazole-4-carboxylic acid (35g, yield=80%) was obtained, which was acid compound. The obtained 2-(pyridine-3-il)thiazole-4-carboxylic acid (20.6g, 0.20 mol) was dissolved in tetrahydrofuran (500ml), triethylamine (13. Ig, 0.20 mol) was then drop-added thereto. The mixture was slowly drop-added with 1.2 equivalent of ethylchloroformate (14.Ig, 0.13 mol) and then stirred for 30 minutes. Then, aniline (9.3g, 0.10 mol) and triethylamine (13.1 ml, 0.13 mol) were dissolved in N,N-dimethylformamide 1500 ml, which was then slowly drop- added to the mixture which was then further stirred for 3 hours. After completion of the reaction, the mixture was filtered and the reaction solution was concentrated, again dissolved in dichloromethane 1500 ml, washed with distilled water 500 mi, again washed with sodium hydrogen carbonate 500 ml, dried with anhydrous manganese (10Og), then filtered and concentrated to be crystallized into hexane and then again filtered, thereby providing N- pheny1-2-(pyridine-3-i 1 )thiazo1e~4-carboxamide 14. Ig (yie1d=70%) .

TLC (acetic acid ethyl:hexane = 1:1); R_f = 0.59

¹HNMR (DMS(HI₆): δ7.52(m, 3H), 7.21(m, 2H), 6.89(m, IH), 6.79(m, 2H),

6.50(s, IH), 6.32(s, IH).

[embodiment 2] N-benzyl-2-(pyridine-3-il)thiazole-4-carboxamide Except that benzyl amine was used instead of the aniline, it was obtained N-benzyl-2-(pyridine-3-il)thiazole-4-carboxamide (18.3g, 62%) in a pale yellow solid form, which was the objective, using the same method as the embodiment 1.

TLC (acetic acid ethyl:hexane = 1:1); R_f = 0.51

¹HNMR (DMSCHI₆): δ7.52(m, 3H), 7.21(m, 5H), 6.51(m, IH), 6.30(s, IH),

3.80(s, 2H).

[embodiment 3] N-phenethyl-2-(pyridine~3-i l)thiazole-4-carboxamide Except that phenyl ethyl amine was used instead of the aniline, it was obtained N-phenethyl-2-(pyridine-3-il)thiazole-4-carboxamide (21.9g, 71%) in a pale yellow solid form, which was the objective, using the same method as the embodiment 1.

TLC (acetic acid ethyl :hexane = 1:1); Rf = 0.54

¹H NMR (DMSO-dg): δ7.52(m, 3H), 7.21(m, 5H), 6.51(m, IH), 6.30(s, IH),

3.OGn, 2H), 2.81(m, 2H).

[embodiment 4] N-(3-phenylpropyl )-2-(pyr idine-3-i l )thiazole-4- carboxamide

Except that phenyl propyl amine was used instead of the ani l ine , i t was obtained N-CS-phenylpropyD^-Cpyr idine-S-i Othiazole^-carboxamide ( 17. Ig, 53%) in a pale yel low sol id form, which was the obj ect ive , using the same method as the embodiment 1.

TLC (acet ic acid ethyl :hexane = 1 : 1) ; R_f = 0.53

¹H NMR (DMSO-de) : δ 7.52(m, 3H) , 7.21(m, 5H) , 6.51(m, IH) , 6.30(s , IH) ,

2.81Gn, 4H) , 1.91(ιn, 2H) .

[experimental example 1] inhibitory effect of pyridine thiazole carboxamide derivative on melanogenesis in the melanocyte

In order to examine inhibitory effects of the compounds obtained in the embodiments 1 to 4 on the melanogenesis in the melanocyte which is a cell for producing melanin pigment, the following experiment was conducted.

A degree of melanogenesis in the cell was measured through a method suggested by Dr. Thomas P. Dooley, hydroquinone and kojic acid were used as a positive control group and the inhibiting extent of the melanogenesis was measured through the comparison with the respective compounds of the embodiments 1 to 4. As a cell line, MeI-Ab cell line (obtained from Dr. Thomas P. Dooley) originating from the skin pigment cell of C57BL/6 was used. The culture was conducted in DMEM culture medium, which contained 10% bovine placenta serum 100 nM 12-0-tetradecanoyl phobol-13-acetate and 1 nM cholera toxin, under conditions of 37°C and 5% CO2. The test material was treated in such a manner that the cultured MeI-Ab cell was detached with 0.25% trypsin- EDTA, disposed in 24-well plates in the same number of 1X10 cells/well and replaced with culture media containing 10 ppm of samples (respective compounds in the embodiments 1 to 4, kojic acid and hydroquinone which were positive control groups) for 3 days from after two days. After 5 days, IN NaOH was applied to dissolve the melanin contained in the cell, so that the amount of the melanogenesis was measured through an absorbency measurement at 400 nm. The result is shown in a table 1. [Table 1]

As shown in Table 1, it can be seen that the respective compounds of the embodiments 1 to 4 suppressed the melanogenesis, particularly the compounds of the embodiments 3 and 4 exhibited good effects similar to that of the hydroquinone.

[experimental example 2] inhibitory effect of pyridine thiazole carboxamide derivatives on the c-kit activation in test tube experiments

2-1. Evaluation of c-kit activity and inhibitory effect of SU11248 on c-kit activation in the test tube experiment

In the experimental example 2-1, in order to verify a system used for an activity evaluation, it was observed the single activity of the c-kit protein and an inhibition degree of the c-kit activation using SU11248, which sticks specifically to the c-kit protein and thus inhibits the activation thereof.

TM

This test was same as the method suggested in the Panvera Z-lyte Kinase assay kit Tyr 4 peptide, but was performed in the optimized conditions with regard to the actual reaction temperature and time, the concentration of the used c-kit, the concentration of ATP and the like.

First, 2X ATP was divided to the 384 well dishes for the fluorescent measurement by 5 μi. Then, SU11248 prepared with 150 nM, 50 nM, 15 nM, 5 nM, 1.5 nM and 0.5 nM were treated with 96-pin dish. To eusure the correctness of the reaction, each sample was prepared with pairs and the control tests of a group having no SU11248/a group to which the c-kit protein was not added/a group of phospho-peptide 0%/a group of phospho-peptide 100% were performed at the same time. After that, the solution in which the c-kit protein and the subtrate peptide therefore were mixed was divided by 5 μi and then the reaction was carried out at 37°C for 45 minutes. After completion of the reaction, the development solution was divided by 5 μi, the reaction was conducted at room temperature for 40 minutes, and the stop solution was divided by 5 μi, thereby completing the reaction. At this time, the final concentrations of the respective materials used for the reaction were ATP 500 μM, the c-kit (kinase) protein 1.5 ng and the peptide 2 μM, and the final concentrations of SU11248 were 30 nM, 10 nM, 3 nM, 1 nM, 0.3 nM and 0.1 nM.

In the mean time, it was measured the fluorescent values for the sample having completed the reaction so as to derive the inhibiting rate of SU11248 for the c-kit protein activation. The activation degree of the enzyme was measured by reading two fluorescent values using the lights activated with 405 nm and 535 nm filters and calculating the ratio between them.

As a result, as shown in Fig. 1, the activation of the c-kit was inhibited dependentIy on the concentrations of SU11248.

2-2. Inhibitory effect of the pyridine thiazole carboxamide derivative on the c-kit activity in the test tube experiment

Based on the verification result of the system for evaluating the c-kit activation in the experimental example 2-1, it was compared the inhibition degrees of the respective compounds of the embodiments 1 to 4 on the c-kit activation. At this time, although the reaction conditions were same as those of the experimental example 2-1, the final concentrations of the respective compounds were 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM and 0.1 μM. The method of calculating the inhibiting rate was as described above, and the result is shown in Fig. 2.

As a result, as can be seen in Fig. 2, the activation of the c-kit was inhibited dependent Iy on the concentrations of the respective compounds of the embodiments 1 to 4.

[experimental example 3] Inhibition of the pyridine thiazole carboxamide derivative for the c-kit activation in the cell level

3.1. Evaluation of c-kit activity and inhibitory effect of SU11248 on c-kit activation in the cell level

In the experimental example 3-1, in order to verify a system used for an activity evaluation, it was observed the single activity of the c-kit protein and an inhibition degree of the c-kit activation using SU11248, which sticks specifically to the c-kit protein and thus inhibits the activation thereof.

At this time, the used cell lines were HM3K0 (which was supplied from Dr. Y Funasaka-Kobe University School of Medicine) which was melanocyte derived from the human, and the culture was carried out in the MEM culture media to which 10% bovine placenta serum was added, under conditions of 37^°C and 5% CO₂. The cultured HM3K0 cells were detached with 0.25% trypsin-EDTA,

again provided to the 6-well plates in the same amount (2.4X10 cells/well) and then left as they were for 24 hours. After that, all of the culture media were removed, replaced with the MEM culture media to which 1% bovine placenta serum was added and then again culured for 24 hours. On the third day, the media were again replaced with MEM culture media to which 1% bovine placenta serum was added, and then SU11248 was treated so that the concentration thereof was made to be 50 nM. Then, after 24 hours, the stem cell factor (SCF), which sticks specifically to the c-kit and thus induces the activation thereof, was applied to the HM3K0 cells with the final concentration of 50 ng/mi for 15 minutes, thereby inducing the activation of the c-kit . At this time, it was compared the increase of the single activity of the c-kit protein and the inhibitory effect of SU11248 on the c-kit activation by using a group which was not treated (notated as UN)/a group which was treated with SCF only (notated as SCF)/a group which was pre-treated with SU11248 and treated with SCF (notated as SU11248). The result is shown in Fig.3.

As can be seen from Fig. 3, it was observed that the activation of the c-kit was inhibited due to SU11248.

3-2. Inhibitory effect of the pyridine thiazole carboxamide derivative on the c-kit activation in the cell level

Based on the verification result of the system for evaluating the c-kit activity in the cell level of the experimental example 3-1, it was compared the inhibition degrees of the respective compounds (notated as 1, 2, 3 and 4, respectively) of the embodiments 1 to 4 on the c-kit activation in the cell level. At this time, although the reaction conditions were same as those of the experimental example 3-1, the final concentrations of the respective compounds were 5 μM, and the result is shown in Fig. 4. In the mean time, in Fig. 4, UN indicates a group which was not treated at all (control group), SCF indicates a group which was treated with SCF only and SU indicates a group which was pre-treated with SU11248 and treated with SCF.

As a result, as can be seen from Fig. 4, the activation of the c-kit was inhibited by the respective compounds of the embodiments 1 to 4. In particular, the compounds of the embodiments 3 and 4 exhibited very excellent effects of inhibiting the c-kit activation.

[experimental example 4] whitening effect of pyridine thiazole carboxamide derivative for human skin

In order to examine the whitening effect of the pyridine thiazole o

carboxamide derivative of the invention for human skin, the following test was conducted.

First, opaque tapes formed with an opening having a diameter of 1.5 cm were attached to the upper arms of 12 health males, and ultraviolet (UVB) was illuminated to about 1.5 to 2 times larger amount than the minimum erythema amount of the examinees so as to induce melanogenesis of the skin. After the illumination of the ultraviolet, 2% respective compounds (solvent: 1, 3- butyleneglycol : ethanol = 7:3) of the embodiments 1 to 4, the hydroquinone and kojic acid, which were positive control groups, were rubbed for 10 weeks. Every week, the color of the skin was measured with a colorimeter (CR-300, Minolta, Japan) to evaluate the increase of a "L" value ("ΔL"). The result is shown in a table 2. At this time, in the evaluation method, it was determined that the larger the values of the brightness degree of the skin color (improvement degree of pigmentation) after the use, the higher the relative whitening effect. [Table 2]

As a result, as shown in the table 2, the compounds of the embodiments 1 to 4 improved the pigmentation, thereby exhibiting the whitening effect of making the skin color bright. In particular, the compounds of the embodiments 3 and 4 exhibited more excellent effects than the hydroquinone and kojic acid which were used as the positive control groups.

Hereinafter, although it will be described formulation examples of the above composition, it should be noted that they are provided to specifically illustrate the invention, not to limit it. Formulation example 1: soap preparation compound of embodiment 1 1.00 (wt%) oil and fat proper quantity sodium hydroxide proper quantity sodium chloride proper quantity perfume small quantity Soap which has the total weight 100 including purified water was prepared in accordance with the mixing ratio

Formulation example 2: lotion preparation compound of embodiment 2 3.00 (wt%)

L-ascorbic acid-2-magnesium phosphate salt 1.00 water-soluble collagen (1% aqueous solution) 1.00 sodium citric acid 0.10 citric acid 0.05 licorice extract 0.20

1,3-butyleneglycol 3.00 Lotion which has the total weight 100 including purified water was prepared in accordance with the mixing ratio

Formulation example 3: cream preparation compound of embodiment 3 1.00 (wt%) polyethyleneglycolmonostearate 2.00 self-emulsified type monostearic acid glycerine 5.00 cetyl alcohol 4.00 squalane 6.00 tri2-ethyl hexanoic acid glyceryl 6.00 sphingoglycolipid 1.00

1,3-butyleneglycol 7.00

Creams which has the total weight 100 including purified water was prepared in accordance with the mixing ratio

Formulation example 4: pack preparation compound of embodiment 4 5.00 (wt%) polyvinylalcohol 13.00

L-ascorbic acid-2-magnesium phosphate salt 1.00 lauroylhydroxyprol ine 1.00 water-soluble collagen (1% aqueous solution) 2.00

*l,3-butyleneglycol 3.00 ethanol 5.00 Pack which has the total weight 100 including purified water was prepared in accordance with the mixing ratio

Formulation example 5: cosmetic solution preparation compound of embodiment 1 2.00 (wt%) hydroxyethylenecellulose (2% aqueous solution) 12.00 xanthan gum (2% aqueous solution) 2.00

1,3-butyleneglycol 6.00 concentrated glycerine 4.00 sodium hyaluronic acid (1% aqueous solution) 5.00

Pack which has the total weight 100 including purified water was prepared in accordance with the mixing ratio

Formulation example 6: ointment preparation compound of embodiment 1 2.00 (wt%) lanolin alcohol 1.00 stearyl alcohol 2.00 ceteareth-20 2.00 perlatum 84.5 lecithin 1.50 carprylic/capric triglyceride 2.00 PEG20 cone glyceride 5.0

[Industrial Applicability]

The pyridine thiazole carboxamide derivative or pharmaceutically acceptable salt thereof according to the invention suppresses the melanogenesis through the action of inhibiting the c-kit activation, thereby improving the pigmentation to exhibit the whitening effect. Accordingly, the composition for suppressing the c-kit activation or composition for skin whitening containing the same can be usefully used for the cosmetic composition or pharmaceutical composition.

Claims

[CLAIMS] [Claim 1]

A pyridine thiazole carboxamide derivative expressed by a following chemistry figure 1 or pharmaceutically acceptable salt thereof. [Chemistry Figure 1]

(where R₁: phenyl, and n: 0, 1, 2 or 3)

[Claim 2]

A method for preparing the pyridine thiazole carboxamide derivative expressed by the chemistry figure 1 or pharmaceutically acceptable salt thereof according to claim 1 comprising:

(1) reflux-reacting ethyl-3-bromo-2-oxo-ρroρionate and pyridine-3- carbothioamide under presence of organic solvent, thereby preparing pyridine thiazole carboxylate;

(2) hydrolyzing the pyridine thiazole carboxylate produced in the (1) with a base in a reaction solvent, thereby preparing pyridine thiazole carboxylic acid; and

(3) reacting the pyridine thiazole carboxylic acid produced in the (2) with ethylchloroformate, under presence of base, to prepare anhydride and reacting the produced anhydride with amine, thereby preparing pyridine thiazole carboxamide.

[Claim 3]

The method according to claim 2, wherein the reaction solvent in the (2) is a mixed solvent of methanol and tetrahydrofuran mixed in a ratio of 1:1 to 1:5. [Claim 4]

The method according to claim 2, wherein when preparing the anhydride in the (3), an equivalent ratio of the pyridine thiazole carboxylic acid and the ethylchloroformate is 1:1.1 to 1:1.3, and an amount of the amine reacting with the produced anhydride is such that an equivalent ratio of the pyridine thiazole carboxylic acid and the amine is 1:1 to 1:3. [Claim 5]

A composition for suppressing c-kit activation containing the pyridine thiazole carboxamide derivative expressed by the chemistry figure 1 or pharmaceutically acceptable salt thereof according to claim 1. [Claim 6]

A composition for skin whitening containing the pyridine thiazole carboxamide derivative expressed by the chemistry figure 1 or pharmaceutically acceptable salt thereof according to claim 1. [Claim 7]

The composition according to claim 5 or 6, wherein the pyridine thiazole carboxamide derivative or pharmaceutically acceptable salt thereof is contained in an amount of 0.01-20 wt% for a total composition weight. [Claim 8]

The composition according to claim 5 or 6, wherein the composition is a cosmetic composition. [Claim 9]

The composition according to claim 5 or 6, wherein the composition is a pharmaceutical composition.