JP2011068632A - Tyrosinase inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new agent which is highly safe and very high tyrosinase-inhibiting action. <P>SOLUTION: The tyrosinase inhibitor includes a compound having a 6- or 7-hydroxyindole skeleton as an effective component. The tyrosinase inhibitor is added to external preparations for the skin or cosmetic bases and used for preventing or treating melanine pigmentation due to aging, preventing suntan caused by ultraviolet rays, and skin-whitening by applying the inhibitor to the skin in the form of the external preparations for the skin or the cosmetics. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a drug that inhibits tyrosinase and prevents melanin deposition in the skin.

  Inhibition of tyrosinase, the key enzyme for melanogenesis, is effective as one of the methods to prevent excessive exposure to sunlight and blackening / browning (melanin deposition) of the skin with aging. Agents have been developed (Patent Documents 1-7, Non-Patent Document 1). Currently, there are arbutin and kojic acid as tyrosinase inhibitors in practical use, but there are problems such as insufficient activity and the need for high concentration administration, and side effects such as mutagenicity have been pointed out. A strong and safe tyrosinase inhibitor is awaited. Recently, the present inventors have found that various serotonin derivatives inhibit tyrosinase, and have revealed that the activity is based on a 5-hydroxyindole nucleus (Non-patent Document 2). Although 5-hydroxyindole itself has already been developed as a tyrosinase inhibitor (Patent Document 8), the test enzyme in that case is a mushroom-derived enzyme. The inhibitory activity against tyrosinase was not necessarily high.

JP 2009-40736 JP 2009-51768 JP2009-40688 JP 2008-179546 JP 2007-131622 A JP 2006-248913 JP JP 2006-16343 A JP-A-9-59123

T.-S. Chang, Int. J. Mol. Sci., 10, 2440 -2475 (2009). Y. Yamazaki et al., Bioorg. Med. Chem. Lett., 19, 4178-4182 (2009)

  An object of the present invention is to develop and put to practical use a novel drug that has a higher inhibitory activity against human tyrosinase than conventional compounds and is safe.

Therefore, as a result of searching for a more potent tyrosinase inhibitor among indole compounds based on the above-mentioned knowledge and prior art, 6-hydroxyindole or 7-hydroxyindole or derivatives thereof are the above-mentioned 5-hydroxyindole and serotonin derivatives. As a result, the present invention was found to have an inhibitory activity one order of magnitude higher than that of the present invention.
That is, the present invention is as shown in the following (1) to (7).

(1) A tyrosinase inhibitor comprising a compound having a 6- or 7-hydroxyindole skeleton represented by the following general formula (I) or (II) as an active ingredient.
[In General Formula (I), R ¹ and R ² each represent a hydroxyl group or a hydrogen atom, at least one of which is a hydroxyl group, and R ³ represents a hydrogen atom, an alkyl group, an alkenyl group, or an R ⁴ —X ¹ — group. (X represents an alkylene group or an alkenylene group, R ⁴ represents an amino group), or R ⁵ —X ² —CONH—X ¹ — group (R ⁵ represents an aromatic group substituted by a hydroxyl group, X ² represents a single bond, an alkylene group or an alkenylene group. ]
[In general formula (II), R ¹ and R ² are the same as in general formula (I). X ³ represents a group forming a condensed ring formed by condensing the benzene ring, the pyridine ring or the dihydropyridine ring by sharing the carbon atoms at positions 2 and ³ of the indole skeleton, and R ⁶ represents a hydrogen atom, an alkyl group or a hydroxyl group. To express. ]

(2) The drug according to (1) above, wherein the compound represented by the general formula (I) is a 6-hydroxyindole represented by the formula (2).
(3) The drug according to (1) above, wherein the compound represented by the general formula (I) is a 7-hydroxyindole represented by the formula (3).
(4) The drug according to (1) above, wherein the compound represented by the general formula (I) is 6-hydroxytryptamine represented by the following formula (4a).
(5) The drug according to (1) above, wherein the compound represented by the general formula (I) is a 6-hydroxytryptamine derivative represented by the following formula (5a) or (10).
(6) The drug according to (1) above, wherein the compound represented by the general formula (II) is 2-hydroxycarbazole represented by the following formula (7a).
(7) The drug according to any one of (1) to (6) above, wherein the use form of the drug is an external preparation for skin or cosmetics.

According to the present invention, it has been found that a compound having a 6- or 7-hydroxyindole skeleton represented by the general formulas (I) and (II) strongly inhibits tyrosinase derived from human melanoma cells. The inhibitory activity in terms of the IC ₅₀ value is one order of magnitude smaller than that of kojic acid that has been used in the past, and is 1/30 to 1/5 the size of 5-hydroxyindole. It has been strengthened. When the drug of the present invention was administered to a human three-dimensional skin model, melanin deposition was clearly reduced as compared with the control. As can be seen from this, it is represented by the above general formulas (I) and (II). The compound prevents the formation of melanin based on inhibition of tyrosinase. Therefore, the chemical | medical agent of this invention can be utilized as a skin external preparation or cosmetics for preventing the blackening and browning of the skin by summer sunburn or aging.

It is a microscope picture when the aqueous solution of 6-hydroxyindole or arbutin is repeatedly added to the human three-dimensional skin model and cultured for 6 days. A is a control, B is a 1 mM aqueous solution containing 6-hydroxyindole, C is a 3 mM aqueous solution containing 6-hydroxyindole, and D is a 37 mM aqueous solution containing arbutin. Tyrosinase was pretreated with N- (3,5-dihydroxybenzoyl) -6-hydroxytryptamine (compound 10), 6-hydroxyindole and kojic acid in the presence of DOPA, and the respective inhibitory activities of the tyrosinase were measured. FIG. N- (3,5-dihydroxybenzoyl) -6-hydroxytryptamine (compound 10), 6-hydroxyindole and kojic acid were added to mouse B16 melanoma cell culture medium, and the amount of melanin produced in the medium was measured. It is a figure which shows a result.

The active ingredient of the tyrosinase inhibitor of the present invention is a compound represented by the following general formula (I) or (II).
[In General Formula (I), R ¹ and R ² each represent a hydroxyl group or a hydrogen atom, at least one of which is a hydroxyl group, and R ³ represents a hydrogen atom, an alkyl group, an alkenyl group, or an R ⁴ —X ¹ — group. (X represents an alkylene group or an alkenylene group, R ⁴ represents an amino group), or R ⁵ —X ² —CONH—X ¹ — group (R ⁵ represents an aromatic group substituted with a hydroxyl group, X ² represents a single bond, an alkylene group or an alkenylene group). ]

[In general formula (II), R ¹ and R ² are the same as in general formula (I). X ³ represents a group forming a condensed ring formed by condensing the benzene ring, the pyridine ring or the dihydropyridine ring by sharing the carbon atoms at positions 2 and ³ of the indole skeleton, and R ⁶ represents a hydrogen atom, an alkyl group or a hydroxyl group. To express. ]

The alkylene group or alkenylene group of X ¹ and X ² in the compounds of the above formulas (I) and (II) is preferably an alkylene group or alkenylene group having 1 to 10 carbon atoms, more preferably an alkylene group or alkenylene group having 1 to 5 carbon atoms. preferable. The alkyl group for R ⁶ is preferably a lower alkyl group having 1 to 5 carbon atoms.

Specific examples of the compound represented by the general formula (I) include compounds represented by the following formulas.
(6-hydroxyindole)

(7-hydroxyindole)

(6-hydroxytryptamine [(R ¹ : hydroxyl group, R ² : hydrogen); compound of the above formula (4a)], 7-hydroxytryptamine (R ¹ : hydrogen, R ² : hydroxyl group)

Derivatives having further increased tyrosinase activity can be synthesized via the amino group of the compound of the above formula (4). Such derivatives are represented by, for example, the following formulas (4-1) and (4-2).
(In the formula (4-1), ^one of R ¹ and R ² represents a hydroxyl group and the other represents hydrogen, and X ₂ represents a single bond, an alkylene group or an alkenylene group.)

(In the formula (4-2), ^one of R ¹ and R ² represents a hydroxyl group and the other represents hydrogen, and X ₂ represents a single bond, an alkylene group or an alkenylene group.)

If these derivatives are shown more specifically, for example, compounds of the following formulas (5), (5 ′) or (6), (6 ′) may be mentioned.
(N- (3,4-dihydroxybenzoyl) -6-hydroxytryptamine (R ¹ : hydroxyl group, R ² : hydrogen) (compound 5a)
(N- (3,4-dihydroxybenzoyl) -7-hydroxytryptamine (R ¹ : hydrogen, R ² : hydroxyl group)

(N- (3,5-dihydroxybenzoyl) -6-hydroxytryptamine (R ¹ : hydroxyl group, R ² : hydrogen) (compound 10)
(N- (3,5-dihydroxybenzoyl) -7-hydroxytryptamine (R ¹ : hydrogen, R ² : hydroxyl group)

(N- [3- (3,4-dihydroxyphenyl) propanoyl] -6-hydroxytryptamine (R ¹ : hydroxyl group, R ² : hydrogen) (compound 6a)
(N- [3- (3,4-dihydroxyphenyl) propanoyl] -7-hydroxytryptamine (R ¹ : hydrogen, R ² : hydroxyl group))

(N- [3- (3,5-dihydroxyphenyl) propanoyl] -6-hydroxytryptamine (R ¹ : hydroxyl group, R ² : hydrogen)
(N- [3- (3,5-dihydroxyphenyl) propanoyl] -7-hydroxytryptamine (R ¹ : hydrogen, R ² : hydroxyl group))

On the other hand, specific examples of the compound of the general formula (II) include compounds represented by the following formulas (7), (8) and (9).
1-hydroxycarbazole ((R ¹ : hydrogen, R ² : hydroxyl)), 2-hydroxycarbazole (R ¹ : hydroxyl, R ² : hydrogen; compound of the above formula (7a))

(Halmarol)

(Hull Mall)

  In the compound of the above formula (7), a hydroxyl group may be further provided at the 7- or 8-position of carbazole, and the compounds of the above formulas (8) and (9) are inferior in tyrosinase inhibitory activity, It is a derived component and has high safety.

The tyrosinase inhibitor of the present invention contains the above-mentioned compound as an active ingredient. Examples of the use form thereof include the above-mentioned compounds, skin external preparations and cosmetic bases such as squalane, glycerin, 1,3-butylene glycol. Can be applied to the skin in the form of an ointment, lotion, cream, milky lotion, etc., and used for prevention, treatment of melanin deposition accompanying aging, sun protection by ultraviolet rays, whitening, and the like. The amount of the compound of the present invention to be applied to the skin or the like, or the amount added to the external preparation for skin and cosmetics may be adjusted within a physiologically safe range depending on the type of compound and the required effect.
6-Hydroxyindole and 7-hydroxyindole are actively used as hair dyeing dye components (JP 2006-1935, JP 6-92828, PCT Int. Appl. WO 2009068830, PCT Int. Appl WO 9320794, PCT Int. Appl. WO 9302655, PCT Int. Appl. WO 2007137676, Eur. Pat. Appl. EP 462883, Fr. Demande FR 2923711). It has been found for the first time in the present invention that it has an inhibitory action.
Examples of the present invention are shown below, but the present invention is not limited thereto.

[Example 1]
Human HMV-II melanoma cell line (purchased from Dainippon-Sumitomo Pharmaceutical Co., Ltd.) was cultured in RPMI-1640 medium containing 15% fetal calf serum for 3 days and then replaced with fresh medium. After 2 days, theophylline was changed to 533 μM. And further cultured for 3 days. Cells were collected with a cell scraper, washed with cold PBS solution, collected by centrifugation, and stored at -85 ° C until use. These cells were sonicated by the method described in the literature (Efdi M. et al., Biol. Pharm. Bull., 30, 1972-1974 (2007)), centrifuged at 12000 rpm for 10 minutes to remove insoluble matter, and centrifuged. Kiyoshi was obtained and used as an enzyme solution. The tyrosinase reaction rate was determined from the increase in absorbance at 475 nm before and after incubation for 5 minutes at 37 ° C. after adding the enzyme using L-DOPA as a substrate based on the method of Non-Patent Document 2. The substrate solution is a mixture of 50 mM phosphate buffer (pH 6.8) containing 2.5 mM L-DOPA and 10 μl ethanol solution (0.3-100 mM) of the test compound or 10 μl ethanol as a control. Solution (50 μl) was added to initiate the reaction. The amount of the enzyme was usually adjusted so that the increase in absorbance at 475 nm was 0.1 in 5 minutes in the control reaction. The% of the enzyme reaction rate in the presence of the test compound relative to the control was determined, and the IC ₅₀ value was determined from the curve obtained by plotting this against the test compound concentration. The difference between the relative reaction rate (%) at 100 μM and 100 was taken as the inhibition rate (%) at this concentration. Furthermore, a similar experiment was performed using a mouse B16 melanoma cell line (purchased from RIKEN Cell Bank).

The above results are summarized in Table 1. The values in this table are the average and standard deviation of 3 or 4 measurements. The test compounds 5-hydroxyindole are from Tokyo Chemical Industry Co., Ltd., 6-hydroxyindole is from Wako Pure Chemical Industries, Ltd., 7-hydroxyindole is from Kanto Chemical Co., Ltd., 2-hydroxycarbazole is Aldrich. From Chemical Co., 6-hydroxytryptamine (creatinine sulfate) was purchased from Toronto Research Chemicals Inc. 6-Hydroxytryptamine (creatinine sulfate) was added to the substrate solution as an aqueous solution, and in this case, the control was also prepared by adding water (the number of measured values of this compound relative to HMV-II tyrosinase is 2). Moreover, it was confirmed that creatinine alone has no tyrosinase inhibitory activity. Looking at the results in Table 1, 6-hydroxyindole and 6-hydroxytryptamine are small compared to kojic acid and 5-hydroxyindole, one-third to one-tenth of tyrosinase in human HMV-II cells. The IC ₅₀ value is shown, indicating that it has a strong inhibitory action. It can also be seen that 7-hydroxyindole and 2-hydroxycarbazole also have stronger inhibitory activity than kojic acid.

[Example 2]
Human HMV-II melanoma cells were seeded on a 96-well plate with 0.2 ml of medium at 1.5 × 10 ⁴ per well. The medium is the same as described above. After culturing for 1 day, the medium in each well was replaced with 0.2 ml of fresh medium containing 500 μM theophylline, and then 2 μl of 6-hydroxyindole (0.1 to 10 mM) or kojic acid (1 to 30 mM) in ethanol or pure ethanol. Added to each well and incubated for 3 days. After removing the medium by suction, each well was washed with 0.2 ml of cold PBS, and the cell viability (% with respect to the control) was measured using a tetrazolium reagent (CellTiter 96, Promega). On the other hand, HMV-II cells were inoculated on a 12-well plate with 2 ml of 2 × 10 ⁵ cells per well. After 1 day, add 40 μM of theophylline aqueous solution to each well in 25 μl, then add ethanol solution of 6-hydroxyindole (3-100 mM) or kojic acid (10-300 mM) or pure ethanol in 2 μl to each well for 3 days. Cultured. The medium was removed by suction, 1 ml of cold PBS was added to each well, the cells were peeled off with a cell scraper, transferred to a 0.5 ml Eppendorf tube for each well, and the cells were collected by centrifugation. 0.2 ml of 1N NaOH was added to each tube, and the cells were ultrasonically disrupted with a UR-20P sonicator manufactured by Tommy Seiko. The absorbance at 415 nm of this treatment solution was measured with a plate reader, and the ratio with the value of the solution obtained by treating the cells in the control well in the same manner was defined as the melanin production rate (%).

  The above results are summarized in Table 2. The values in this table are the mean and standard deviation for 5 wells for cell viability and the mean and standard deviation for 4 wells for melanin production. * And ** in the table indicate that there is a significant difference from the control at p <0.05 and p <0.01 by Student's t-test, respectively. From the results in this table, 6-hydroxyindole inhibits melanin production by 14% without cytotoxicity at 10 μM, which is a low concentration of 1/10 or less of the concentration exhibiting the same inhibitory action of kojic acid, It can be seen that there is a stronger melanin inhibitory effect than kojic acid.

Example 3
Using a human three-dimensional skin model (MEL-312-B kit manufactured by Kurabo Industries Co., Ltd.) consisting of a mixed culture of human keratinocytes and melanocytes, the melanin inhibitory activity of 6-hydroxyindole was tested according to the manual of the kit. After culturing for 4 days, an aqueous solution containing 1 or 3 mM 6-hydroxyindole was cultured for 6 days as a test solution. However, although it does not reach the standard 37 mM arbutin aqueous solution, It was confirmed that cell blackening was clearly suppressed compared to the control. A photomicrograph is shown in FIG.

Example 4
6-Benzyloxytryptamine (0.5 sulfate, manufactured by Toronto Research Chemicals Inc.) was coupled with 3,4-diacetoxybenzoyl chloride or O, O-diacetylcaffeic acid chloride in DMF containing pyridine, followed by benzyl group N- (3,4-dihydroxybenzoyl) -6-hydroxytryptamine (5a) and N- [3- (3,4-dihydroxyphenyl) propanoyl] -6 by removing acetyl and acetyl groups by catalytic reduction and hydrazine treatment, respectively -Hydroxytryptamine (6a) was synthesized. Each ¹ H-NMR spectrum was consistent with the expected structure. The tyrosinase inhibitory activity of these compounds was measured as described above. However, the sample solution of 6-hydroxytryptamine was prepared with an ethanol solution containing a trace amount of water, all other compounds were ethanol solutions, and pure ethanol was used as a control. The results are shown in Table 3. From this result, it can be seen that compounds (5a) and (6a) have a stronger inhibitory activity than human 6-hydroxytryptamine against tyrosinase of human HMV-II cells.

(Example 5)
100 mg of 6-benzyloxytryptamine hemisulfate (Toronto Research Chemicals) was suspended in a mixed solution of 5 ml of dimethylformamide (DMF) and 2 ml of pyridine. To this was added 0.5 ml of a DMF solution in which 100 mg of 3,5-dihydroxybenzoic acid (3,5-DHBA) and 100 mg of dicyclohexylcarbodiimide (DCC) were dissolved. The mixture was sonicated (irradiated with TOMY Seiko UR-20P type Handy Sonic for 5 minutes) and then stirred for about 1 hour. The supernatant and precipitate were separated by centrifugation, 200 mg of 3,5-DHBA and 400 mg of DCC were added to the supernatant, and stirring was continued at room temperature. When the above precipitate was examined by TLC, it contained unreacted 6-benzyloxytryptamine, so it was suspended in a mixture of 1 ml of dimethyl sulfoxide and 0.2 ml of pyridine, and 100 mg of 3,5-DHBA and 200 mg of DCC were added. And stirring was continued at room temperature. After stirring overnight, each of the supernatant reaction product and the precipitation reaction product is filtered, the solvent is removed with an evaporator, the residue is dissolved in 30 ml of ethyl acetate, 10% aqueous citric acid solution, 10% aqueous sodium bicarbonate solution, And washed with saturated brine. After drying over sodium sulfate, the ethyl acetate layers were combined, concentrated, and purified by column chromatography (Wakogel C-300, 1 × 10 cm, hexane / ethyl acetate). The target product eluted with 100% ethyl acetate was concentrated with a rotary evaporator, dissolved in 4 ml of ethanol, 0.2 ml of acetic acid and 30 mg of Pd / C were added, and catalytic hydrogenation was performed at normal pressure. After reacting for 4 hours, the catalyst was filtered off, the filtrate was concentrated, and the residue was applied to a silica gel column (Wakogel C-300, 1.5 × 14 cm, hexane / ethyl acetate), and fractions eluted with 100% ethyl acetate were collected. It was. 62 mg of an oily substance obtained by concentrating this was crystallized from a mixed solution of ethyl acetate and benzene, and 34 mg of N- (3,5-dihydroxybenzoyl) -6-hydroxytryptamine (hereinafter referred to as Compound 10). Was obtained as a crystalline white powder. Yield 34%, decomposition point 213-217 ° C., elemental analysis values: C, 65.61%; H, 5.22%; N, 8.00% [calculated value (C ₁₇ H ₁₆ N ₂ O ₄ ): C, 65.38%; H , 5.16%; N, 8.97%], FAB-MS m / z 312 (M ⁺ ), ¹ H-NMR (270 MHz, acetone-d ₆ ) δ: 2.986 (2H, t, J = 7 Hz, CH ₂ ) , 3.640 (2H, m, CH ₂ N), 6.467 (1H, t, J = 2 Hz, H-4), 6.635 (1H, q, J = 8, 2 Hz, H-5 '), 6.818 (1H , d, J = 2 Hz, H-7 '), 6.841 (2H, d, J = 2 Hz, H-2, 5), 6.992 (1H, m, H-2'), 7.434 (1H, d, J = 8 Hz, H-4 '), 7.563 (1H, br. S, NHCO), 7.823 (1H, br. S, OH), 8.407 (2H, br. S, OH x 2), and 9.636 (1H , br. s, H-1 '). From these analysis results, the structure of Compound 10 was confirmed. The structure of the compound is shown in Formula 10 below.

  The tyrosinase inhibitory activity of compound 10 synthesized in this manner was measured in the same manner as in Example 1. The results are shown in Table 4. This table also shows the results of testing for oxyresveratrol and 5-hydroxyindole derivatives N-caffeoyl serotonin and N-protocatechuoyl serotonin. From the results of Table 4 and Table 1 described above, Compound 10 in which an acyl group having a phenolic hydroxyl group is introduced into 6-hydroxyindole has the strongest tyrosinase inhibitory activity among the 6-hydroxyindole derivatives related to the present invention, and is more similar. Oxyresveratrol (Kim et al., J.), which is considered to be one of the strongest tyrosinase inhibitors as well as N-caffeoyl serotonin and N-protocatechuoyl serotonin, which are 5-hydroxyindole derivatives of the structure Biol. Chem., 277, 16340 (2002)).

Example 6
N- (3,5-dihydroxybenzoyl) -6-hydroxytryptamine (compound 10) is characterized by inhibition of tyrosinase, especially in the presence of the substrate DOPA (dihydroxyphenylalanine), which strongly reduces or deactivates the enzyme. It can be pointed out. In order to investigate the inhibition mechanism, the present inventors conducted an experiment in which tyrosinase was pretreated with the compound of the present invention for a certain period of time prior to the start of the assay. When a low concentration of DOPA was added to the pretreatment solution, the enzyme was It was found that the activity of was significantly reduced. That is, in each well of the 96-well plate, 50 mM phosphate buffer (120 μl) containing or not containing 25 μM DOPA in ethanol solution of compound 10, 6-hydroxyindole or kojic acid (2 μl each) After mixing with enzyme solution (12 μl) and incubating at 37 ° C. for 1 hour, add 120 μl of phosphate buffer containing 5.3 mM DOPA and let stand for 20 hours at room temperature. (Representing the amount of melanin produced). The average of the values measured using two wells for each reaction condition (corrected for the blank value including the enzyme but not containing DOPA at all) was summarized in the graph of FIG. 2 as% of the control.

  In FIG. 2, the numerical value under the horizontal axis is the test compound concentration (μM) in the final mixed solution. The white bars show the results of pretreatment without DOPA, and the black bars show the results of pretreatment in the presence of 25 μM DOPA. Although the coloration decreases as the concentration of the added compound increases, it has been shown that the suppression of coloration is stronger than when there is no DOPA with a low concentration of 25 μM in the pretreatment. This effect is significantly stronger in compound 10 than 6-hydroxy-indole and very weak in kojic acid. Although it was previously known that mouse tyrosinase is inactivated by its substrate, DOPA (Tomita et al., J. Invest. Dermatol., 75, 379 (1980)), it actively promotes this inactivation. There is almost no drug development from this viewpoint. When tyrosinase is induced by ultraviolet irradiation or the like, the enzyme first oxidizes tyrosine to produce DOPA, and further oxidizes DOPA to produce dopachrome as a melanin raw material. Therefore, it can be said that the function of strongly inhibiting or deactivating tyrosinase in the presence of a small amount of DOPA is suitable for achieving complete inhibition. The results in FIG. 2 show that compound 10 is an inhibitor with this purpose.

(Example 7)
Mouse B16 melanoma cells were seeded in 24-well plates at 1 × 10 ⁵ per well with 0.7 ml of medium (DMEM medium containing 10% FBS). After culturing for 1 day, 8.9 μl of 40 mM theophylline aqueous solution was added to each well, and then 2 μl of compound 10 or kojic acid ethanol solution or pure ethanol was added to each well. The concentration of compound 10 in the medium was 3 and 10 μM, and the concentration of kojic acid was 30 and 100 μM. After culturing for 2 days, 100 μl of the medium was placed in a 96-well plate, the absorbance at 490 nm was measured with a plate reader, and the production rate of melanin in the medium (% relative to the control) was determined from the value. Next, after removing the medium remaining on the 24-well plate by suction, each well was washed with 0.5 ml of cold PBS, and the cell viability (% of control) was measured using tetrazolium reagent (CellTiter 96, Promega). did. The above results are summarized in FIG. The values in this figure are the mean and standard deviation for each of the three wells. ** indicates a significant difference from the control at p <0.01 by Student's t-test. The results in this figure show that compound 10 inhibits melanin production in the medium by 26% at 10 μM without cytotoxicity, while kojic acid has little effect under these experimental conditions even at 10-fold higher concentrations. Show. The amount of melanin accumulated in the cells was measured by the same method as in Example 2. However, in this case, a stable result was not obtained for the inhibitory action by Compound 10. However, the function of inhibiting secretion or release of melanin to the outside of cells is one of the important elements of whitening cosmetics. Compound 10 has a function of suppressing the release of melanin to the outside of the cell in addition to the above tyrosinase inhibitory effect.

Claims (7)

A tyrosinase inhibitor comprising a compound having a 6- or 7-hydroxyindole skeleton represented by the following general formula (I) or (II) as an active ingredient.
[In General Formula (I), R ¹ and R ² each represent a hydroxyl group or a hydrogen atom, at least one of which is a hydroxyl group, and R ³ represents a hydrogen atom, an alkyl group, an alkenyl group, or an R ⁴ —X ¹ — group. (X represents an alkylene group or an alkenylene group, R ⁴ represents an amino group), or R ⁵ —X ² —CONH—X ¹ — group (R ⁵ represents an aromatic group substituted by a hydroxyl group, X ² represents a single bond, an alkylene group or an alkenylene group. ]
[In general formula (II), R ¹ and R ² are the same as in general formula (I). X ³ represents a group forming a condensed ring formed by condensing the benzene ring, the pyridine ring or the dihydropyridine ring by sharing the carbon atoms at positions 2 and ³ of the indole skeleton, and R ⁶ represents a hydrogen atom, an alkyl group or a hydroxyl group. To express. ] The drug according to claim 1, wherein the compound represented by the general formula (I) is a 6-hydroxyindole represented by the formula (2).
The drug according to (1) above, wherein the compound represented by the general formula (I) is a 7-hydroxyindole represented by the formula (3).
The compound according to claim 1, wherein the compound represented by the general formula (I) is 6-hydroxytryptamine represented by the following formula (4a).
The drug according to claim 1, wherein the compound represented by the general formula (I) is a 6-hydroxytryptamine derivative represented by the following formula (5a) or (10).
The drug according to claim 1, wherein the compound represented by the general formula (II) is 2-hydroxycarbazole represented by the following formula (7a).
The drug according to any one of claims 1 to 6, wherein the use form of the drug is a skin external preparation or a cosmetic.