JP4341501B2 - Tyrosinase activity inhibitor - Google Patents

Description

  The present invention relates to N-dihydroferuloyl amino acids and their uses, and the compounds can be suitably used as antioxidants or tyrosinase activity inhibitors in the fields of pharmaceuticals, quasi drugs, cosmetics, foods and the like.

  In recent years, it has become clear that active oxygen and free radicals cause various diseases. Originally, active oxygen is generated in the immune system of the living body, and has a function of attacking pathogenic bacteria that have invaded from the outside. However, if the active oxygen is excessively generated and remains, it reacts with components in the living body, that is, lipids, proteins, DNA, etc., and has an adverse effect. Among them, unsaturated fatty acids that are constituents of biological membranes and the like are easily oxidized and generate lipid peroxides via free radical intermediates. This lipid peroxide is known to induce arteriosclerosis, hypertension, liver dysfunction and the like, and is also said to be closely related to the aging phenomenon. Recently, an oxidative degradation product of unsaturated fatty acid has been cited as a cause of odor (for example, see Non-Patent Document 1). In addition, foods, pharmaceuticals, cosmetics, and the like containing unsaturated fatty acids that are susceptible to oxidation also have a problem that quality deterioration during storage is likely to occur.

  In order to prevent oxidation of such unsaturated fatty acids, it is necessary to use an antioxidant. Many compounds are known as antioxidants, specifically butylhydroxytoluene (BHT), butylhydroxyanisole (BHA), tocopherols, ascorbic acid and its derivatives, ubiquinone and its derivatives, flavone derivatives, gallic acid. Examples include acid derivatives and polyphenols (see, for example, Non-Patent Document 2 and Non-Patent Document 3). However, the synthetic chemicals such as BHT and BHA are not safe for the human body and have application restrictions, and other natural antioxidants are just one step away from performance or are difficult to obtain. There was a problem.

  On the other hand, stains and freckles are generated when melanin pigments are formed in epidermal pigment cells due to exposure to ultraviolet rays and the like, and pigmented on the epidermis. Melanin pigments are synthesized by L-tyrosine, which is a kind of amino acid, being metabolized to L-dopa and L-dopaquinone by the action of tyrosinase, which is an oxidase, and through various processes (for example, Non-Patent Document 4). And Non-Patent Document 5). Therefore, in order to prevent pigmentation such as spots and freckles, it is important to inhibit the activity of tyrosinase, which plays an important role in the synthesis of melanin pigment.

  Conventionally, drugs such as placenta extract, vitamin C and its derivatives, kojic acid, and arbutin have been used for prevention and improvement of stains, freckles, etc., but sufficient effects are not always obtained. In Europe and the United States, hydroquinone is used for the purpose of depigmenting pigment spots, but its use is restricted due to safety problems. More recently, kojic acid has also been pointed out as a possible carcinogen.

  N-feruloylglycine is known as a compound present in barley, and N-feruloylalanine has been synthesized in the study of N-feruloylglycine amide hydrolase, which is a related enzyme of this compound (non-patent literature). 6). Further, it is known that caffeic acid amide derivatives, which are compounds similar in structure to N-dihydroferuloyl amino acids, have antioxidant activity and tyrosinase inhibitory activity (see Patent Document 1). However, both N-feruloylalanine and caffeic acid amide derivatives have a carbon-carbon double bond between the amide group and the phenyl group, and a saturated hydrocarbon is connected between the amide group and the phenyl group. The structural characteristics are different from those of N-dihydroferuloyl amino acids.

JP 2003-192522 A (Claims) Satoru Iida, 7 others, “Analysis and coping with body odor generation mechanism”, Abstracts of the 51st SCCJ Research Discussion Meeting, 2002, p. 64-67 Kenji Fukuzawa, “Pharmacology of Free Radical Defense and Prospects for Drug Development”, Nippon Clinic, October 1988, 46, 10, p. 2269-2276 Sies, H., two others, “Antioxidant Functions of Vitamins”, Annals New York Academy of Sciences, 1992, 669, p. 7-20 Special issue on “Fine Chemical Magazine” Whitening agent development and product development, edited and published by CMMC Publishing, March 15, 1999 Special issue on "Fragrance Journal" Recent whitening agent research and development, edited and published by Fragrance Journal, September 1997 Martens, M. et al., Phytochemistry, 27, 8, p. 2465-2475

An object of the present invention is to provide an excellent tyrosinase activity inhibitor in view of the above situation.

As a result of repeated studies on antioxidants and tyrosinase activity inhibitors, the present inventors have found that N-dihydroferuloyl amino acids have excellent antioxidant activity and tyrosinase inhibitory activity, and have completed the present invention. .
That is, the present invention is a compound represented by the following general formula (A) obtained by condensing L-α-amino acid or a derivative thereof and dihydroferulic acid.

〔式中のＲは、アラニン，セリン，フェニルアラニン，メチオニン，ヒスチジン，チロシンから選択されるＬ−α−アミノ酸およびそのメチルまたはエチルエステル、におけるアミノ基と結合した残基を示す。〕

[R in the formula represents a residue bonded to an amino group in L-α-amino acid selected from alanine, serine, phenylalanine, methionine, histidine, and tyrosine and its methyl or ethyl ester . ]

Furthermore, it is a tyrosinase activity inhibitor characterized by containing the compound represented by the said general formula (A).

  Since the N-dihydroferuloyl amino acid of the present invention has excellent antioxidant activity and tyrosinase inhibitory activity, it is useful as an antioxidant or tyrosinase activity inhibitor in the fields of pharmaceuticals, quasi-drugs, cosmetics and foods. It is.

A compound represented by the general formula (A) R in the general formula (A) is a residue bonded to an amino group in the L-α-amino acid. Examples of the derivative of L-α-amino acid condensed with dihydroferulic acid include L-α-amino acid salt or L-α-amino acid ester in which an amino acid residue is substituted with a salt.
The L-α-amino acid referred to in the present specification is an amino acid (α-amino acid) in which a carboxyl group and an amino group are bonded to the same carbon atom, and the configuration thereof has an L-type. Specifically, L-α-alanine, L-valine, L-α-leucine, L-α-isoleucine, L-α-serine, L-α-threonine, L-α-phenylalanine, L-α-tyrosine L-α-tryptophan, L-α-lysine, L-α-arginine, L-α-histidine, L-α-cysteine, L-α-cystine, L-α-methionine, L-proline, L-3 -Hydroxyproline, L-4-hydroxyproline, L-α-aspartic acid, L-α-glutamic acid, L-α-asparagine, L-α-glutamine and the like. Commercially available L-α-amino acids can be used.

  As the L-α-amino acid salt, the carboxyl group in the amino acid residue is an alkali metal salt such as sodium or potassium, an alkaline earth metal salt such as calcium or magnesium, an ammonium salt, or the like, or tryptophan which is a basic amino acid, A basic residue such as lysine, arginine or histidine forms a salt with an acid component such as inorganic acid or organic acid such as hydrochloric acid, phosphoric acid, sulfuric acid, citric acid or acetic acid.

  L-α-amino acid ester is a compound in which a carboxyl group in an amino acid residue forms an ester bond with alcohols and has an ester group that may have a molecule having 1 to 18 carbon atoms. Examples of the ester group include linear ester groups such as methyl ester group, ethyl ester group, n-butyl ester group and stearyl ester group, ester groups having a branched structure such as isopropyl ester group and 2-ethylhexyl ester group, Examples include ester groups having an unsaturated bond such as a ruester group, a benzyl ester group, a geranyl ester group, and an oleyl ester group, and a methyl ester group, an ethyl ester group, an n-propyl ester group, and an n-butyl ester that are readily available as raw materials. The group, benzyl ester group is preferred.

  In the present specification, the above-mentioned L-α-amino acid, L-α-amino acid salt, and L-α-amino acid ester may be displayed by omitting “α-”.

  The compound represented by the general formula (A) is, for example, a compound represented by the following formula (1) (hereinafter also referred to as compound 1. The following compounds represented by other formulas may also be abbreviated in the same manner). And L-α-amino acid or L-α-amino acid ester can be synthesized. The condensation reaction can be carried out according to conventional methods such as a method using a dehydrating condensing agent, a method in which compound 1 is converted to an active ester compound and then reacted with L-α-amino acid or L-α-amino acid ester.

  As an example of a method for synthesizing the compound represented by the general formula (A), a compound 1 is reacted with N-hydroxysuccinimide to be converted into an active ester compound (compound 2) and then reacted with an L-amino acid ester. explain.

Compound 2 can be prepared by reacting Compound 1 and N-hydroxysuccinimide with a condensing agent in an organic solvent.
The amount of N-hydroxysuccinimide used in this reaction is basically 1 chemical equivalent to compound 1, preferably 0.7 to 1.3 chemical equivalents, more preferably 0.9. -1.1 chemical equivalents.

  Examples of the condensing agent include N, N-dicyclohexylcarbodiimide, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylamipropyl) carbodiimide, 4- (4,6-dimethoxy-1,3,5). -Triazin-2-yl) -4-methylmorpholinium chloride, di-2-pyridyl carbonate, di-2-pyridylthionocarbonate, etc. are exemplified, and N, N-dicyclohexylcarbodiimide is preferably used. Can do. The amount of the condensing agent used is basically 1 chemical equivalent to compound 1, preferably 0.7 to 1.3 chemical equivalents, and more preferably 0.9 to 1.1 chemical equivalents. It is.

This reaction is preferably carried out in an organic solvent, particularly an aprotic solvent, such as tetrahydrofuran, 1,4-dioxane, diethyl ether, 1,2-dimethoxyethane, N, N-dimethylpropyleneurea and these. A mixed solvent or the like can be preferably used.
The temperature of the reaction is preferably 0 to 50 ° C, more preferably 10 to 30 ° C. If this reaction temperature is too low, it is costly to maintain the temperature, and if the reaction temperature is too high, side reactions may proceed.
Although this reaction time varies depending on conditions, it is usually several hours.
After completion of the reaction, compound 2 can be used as it is without purification, and compound 2 can also be isolated and purified by a known method such as recrystallization.

The compound represented by the general formula (A) can be synthesized by reacting the L-amino acid ester with the compound 2 prepared as described above.
The amount of Compound 2 used in this reaction is basically 1 chemical equivalent to L-amino acid ester, preferably 0.7 to 1.3 chemical equivalent, more preferably 0.9. -1.1 chemical equivalents.
This reaction is preferably performed under basic conditions, preferably 0.7 to 5.0 chemical equivalents, more preferably 1.0 to sodium bicarbonate and potassium bicarbonate with respect to the L-amino acid ester. Use ~ 3.0 chemical equivalents.

This reaction is preferably carried out in a solvent that dissolves the basic substance such as Compound 2, L-amino acid ester, and sodium hydrogen carbonate. Tetrahydrofuran, 1,4-dioxane, diethyl ether, 1,2-dimethoxyethane N, N-dimethylpropylene urea, methanol, ethanol, isopropyl alcohol, toluene, methylene chloride, water, and a mixed solvent thereof are preferable.
The temperature of the reaction is preferably 0 to 50 ° C, more preferably 10 to 30 ° C. If this reaction temperature is too low, it is costly to maintain the temperature, and if the reaction temperature is too high, side reactions may proceed.
This reaction time varies depending on the conditions, but is usually from several hours to several tens of hours.
After completion of the reaction, the compound represented by the general formula (A) can be isolated and purified by a known method such as solvent extraction, column chromatography, recrystallization and the like.

  Since the compound represented by the general formula (A) has an antioxidant activity and a tyrosinase inhibitory activity, as an antioxidant or a tyrosinase activity inhibitor in the field of pharmaceuticals, quasi-drugs, cosmetics, foods, for example, foods It can be used for additives and whitening cosmetic ingredients.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

<Synthesis Example 1>
A condensation reaction between dihydroferulic acid (Compound 1) and N-hydroxysuccinimide was carried out to obtain Compound 2.
That is, N, N-dicyclohexylcarbodiimide (25.0 g, 121 mmol) was added to a tetrahydrofuran (250 ml) solution of dihydroferulic acid (23.6 g, 120 mmol) and N-hydroxysuccinimide (13.9 g, 121 mmol). . After stirring at room temperature for 3 hours, 5 ml of distilled water was added and left for 16 hours. Next, the produced insoluble matter was filtered off, and the filtrate was concentrated. The obtained residue was recrystallized from a mixed solvent of ethyl acetate and n-hexane to obtain a colorless crystalline compound (25.0 g, yield 71%). ¹ H-NMR spectrum and infrared absorption (IR) spectrum analysis (hereinafter, unless otherwise specified, IR spectrum is determined by the KBr pellet method), the compound obtained was confirmed to be compound 2.

Compound 2 had a melting point of 140-142 ° C. The chemical shift value of the ¹ H-NMR spectrum of Compound 2 measured in deuterated chloroform is 2.80-2.92 (6H, m), 2.96-3.02 (2H, m), 3.89 (3H, s), 5.57 (1H, s ), 6.70-6.75 (2H, m), 6.85 (1H, d, J = 7.6Hz).
The wave numbers (cm ⁻¹ ) absorbed in the IR spectrum were 3430, 2940, 1820, 1780, 1730, 1520, 1370, 1220, 1070, 820, 650.

<Synthesis Example 2>
Compound 3 was obtained using N-hydroxybenzotriazole instead of N-hydroxysuccinimide used in Synthesis Example 1.

○ Structural formula of Compound 3

The melting point of Compound 3 was 147-149 ° C. Chemical shift values of ¹ H-NMR spectrum measured in a mixed solvent of deuterated chloroform and deuterated methanol of compound 3 are 3.11 (2H, t, J = 7.6 Hz), 3.48 (2H, t, J = 7.6 Hz), 3.88 (3H, s), 6.726.85 (3H, m), 7.66 (1H, t, J = 8.0Hz), 7.86 (1H, t, J = 8.0Hz), 8.03 (1H, d, J = 8.4Hz ), 8.43 (1H, d, J = 8.4Hz).

<Example 1>
The compound 2 obtained in Synthesis Example 1 was reacted with L-α-alanine ethyl ester hydrochloride to synthesize Compound 4 of the present invention.
That is, L-α-alanine ethyl ester hydrochloride (1.54 g, 10.0 mmol) and sodium hydrogen carbonate (850 mg, 10.1 mmol) were dissolved in distilled water (10 ml). Next, a solution of compound 2 (2.94 g, 10.0 mmol) in tetrahydrofuran (30 ml) was added, and the mixture was stirred at room temperature. After 18 hours, the reaction solution was partitioned by adding 20% aqueous sodium carbonate solution (10 ml), saturated brine (20 ml) and ethyl acetate (10 ml). The organic layer was collected and dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography to obtain a colorless liquid compound (2.74 g, yield 93%). ¹ H-NMR spectrum and IR spectrum analysis confirmed that the obtained compound was compound 4.

 ○ Structural formula of Compound 4

The chemical shift value of the ¹ H-NMR spectrum measured in deuterated chloroform of Compound 4 is 1.27 (3H, t, J = 7.2 Hz), 1.35 (3H, d, J = 7.2 Hz), 2.43-2.54 (2H, m), 2.87-2.94 (2H, m), 3.86 (3H, s), 4.19 (2H, q, J = 7.2Hz), 4.56 (1H, t, J = 7.2Hz), 5.58 (1H, br), 5.98 (1H, br), 6.67-6.75 (2H, m), 6.82 (1H, d, J = 8.0Hz).
The wave numbers (cm ⁻¹ ) absorbed in the IR spectrum were 3350, 2980, 1740, 1650, 1520, 1450, 1280, 1210, 1160, 1120, 1060, 1030.

<Example 2>
Compound 5 of the present invention was synthesized using L-serine ethyl ester hydrochloride instead of L-α-alanine ethyl ester hydrochloride used in Example 1.

○ Structural formula of Compound 5

The chemical shift values of ¹ H-NMR spectrum measured in deuterated chloroform of compound 5 are 1.28 (3H, t, J = 7.2Hz), 2.53 (2H, t, J = 7.6Hz), 2.77 (1H, br) , 2.91 (2H, t, J = 7.6Hz), 3.82-3.94 (5H, m), 4.22 (2H, q, J = 7.2Hz), 4.60-4.66 (1H, m), 5.72 (1H, br), 6.46 (1H, br), 6.69 (1H, d, J = 8.4Hz), 6.72 (1H, s), 6.83 (1H, d, J = 8.4Hz).
The wave numbers (cm ⁻¹ ) absorbed in the IR spectrum were 3370, 2940, 1740, 1660, 1520, 1450, 1380, 1280, 1210, 1160, 1030, 820.

<Example 3>
Compound 6 of the present invention was synthesized using L-phenylalanine methyl ester hydrochloride instead of L-α-alanine ethyl ester hydrochloride used in Example 1.

○ Structural formula of compound 6

Compound 6 had a melting point of 113 to 115 ° C. The chemical shift values of ¹ H-NMR spectrum of Compound 6 measured in deuterated chloroform are 2.38-2.53 (2H, m), 2.83-2.92 (2H, m), 3.06 (2H, d, J = 5.6Hz), 3.71 (3H, s), 3.85 (3H, s), 4.85-4.92 (1H, m), 5.53 (1H, br), 5.82 (1H, br), 6.65-6.73 (2H, m), 6.83 (1H, d, J = 8.0 Hz), 6.90-6.96 (2H, m), 7.20-7.25 (3H, m).
The wave numbers (cm ⁻¹ ) absorbed in the IR spectrum were 3420, 3310, 3000, 2940, 1730, 1650, 1550, 1520, 1430, 1280, 1230, 1030, 700.

<Example 4>
Compound 7 of the present invention was synthesized using L-methionine methyl ester hydrochloride instead of L-α-alanine ethyl ester hydrochloride used in Example 1.

○ Structural formula of Compound 7

The chemical shift value of the ¹ H-NMR spectrum of Compound 7 measured in deuterated chloroform is 1.86-1.97 (1H, m), 2.03-2.18 (4H, m), 2.33-2.42 (2H, m), 2.48-2.59. (2H, m), 2.88-2.95 (2H, m), 3.74 (3H, s), 3.87 (3H, s), 4.69-4.75 (1H, m), 5.55 (1H, br), 6.06 (1H, br ), 6.68-6.75 (2H, m), 6.83 (1H, d, J = 8.0Hz).
The wave numbers (cm ⁻¹ ) absorbed in the IR spectrum were 3350, 2950, 1740, 1660, 1520, 1440, 1280, 1120, 1030, 820.

<Example 5>
The compound 2 obtained in Synthesis Example 1 was reacted with L-histidine methyl ester dihydrochloride to synthesize Compound 8 of the present invention.
That is, L-histidine methyl ester dihydrochloride (2.43 g, 10 mmol) and sodium hydrogen carbonate (2.52 g, 30.0 mmol) were dissolved in distilled water (10 ml). Next, a solution of compound 2 (2.94 g, 10.0 mmol) in tetrahydrofuran (30 ml) was added, and the mixture was stirred at room temperature. After 18 hours, the reaction solution was partitioned by adding 20% aqueous sodium carbonate solution (10 ml), saturated brine (20 ml) and ethyl acetate (10 ml). The organic layer was collected and dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography to obtain a colorless powdery compound (3.05 g, yield 97%). ¹ H-NMR spectrum and IR spectrum analysis confirmed that the obtained compound was compound 8.

○ Structural formula of Compound 8

The chemical shift values of the ¹ H-NMR spectrum measured in a mixed solvent of deuterated chloroform and deuterated methanol of compound 8 are 2.46-2.56 (2H, m), 2.80-2.87 (2H, m), 2.97-3.06 (2H, m), 3.70 (3H, s), 3.83 (3H, s), 4.68 (1H, t, J = 7.2Hz), 6.60-6.67 (2H, m), 6.71 (1H, s), 6.75 (1H, d , J = 8.0Hz), 7.47 (1H, s).
The wave numbers (cm ⁻¹ ) absorbed in the IR spectrum were 3290, 1740, 1660, 1520, 1440, 1370, 1280, 1130, 1030, 750.

<Example 6>
The compound 3 of the present invention was synthesized by reacting the compound 3 obtained in Synthesis Example 2 with L-tyrosine methyl ester.
That is, L-tyrosine methyl ester (980 mg, 5.02 mmol) and compound 3 (1.58 g, 5.04 mmol) were dissolved in N, N-dimethylformamide (10 ml) and stirred at room temperature. After stirring for 18 hours, 5% aqueous sodium carbonate solution (80 ml) and ethyl acetate (30 ml) were added and partitioned. The organic layer was collected and dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography to obtain a colorless powdery compound (935 mg, 53% yield).
¹ H-NMR spectrum and IR spectrum analysis confirmed that the obtained compound was compound 9.

○ Structural formula of Compound 9

化合物９の重クロロホルム中で測定した¹Ｈ−ＮＭＲスペクトルのケミカルシフト値は、2.40-2.55(2H,m), 2.79-3.03(4H,m), 3.71(3H,s), 3.81(3H,s), 4.81-4.89(1H,m), 5.72(1H,br), 5.98(1H,br), 6.62-6.85(8H,m)であった。
また、ＩＲスペクトルで吸収のあった波数（ｃｍ^-1）は、3350, 2950, 1740, 1650, 1610, 1520, 1450, 1370, 1230, 1120, 1030, 820であった。

The chemical shift values of ¹ H-NMR spectrum of Compound 9 measured in deuterated chloroform are 2.40-2.55 (2H, m), 2.79-3.03 (4H, m), 3.71 (3H, s), 3.81 (3H, s ), 4.81-4.89 (1H, m), 5.72 (1H, br), 5.98 (1H, br), 6.62-6.85 (8H, m).
The wave numbers (cm ⁻¹ ) absorbed in the IR spectrum were 3350, 2950, 1740, 1650, 1610, 1520, 1450, 1370, 1230, 1120, 1030, 820.

<Example 7>
The compound 10 of the present invention was synthesized by hydrolyzing the compound 5 obtained in Example 2.
That is, compound 5 (1.56 g, 5.00 mmol) was dissolved in isopropyl alcohol (15 ml), 1N aqueous sodium hydroxide solution (5 ml) was added, and the mixture was stirred at room temperature. After 24 hours, 1N hydrochloric acid (5 ml) was added for neutralization. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give a colorless crystalline compound (1.01 g, yield 71%). ¹ H-NMR spectrum and IR spectrum analysis confirmed that the obtained compound was compound 10.

○ Structural formula of compound 10

Compound 10 had a melting point of 137-139 ° C. Chemical shift values of ¹ H-NMR spectrum measured in deuterated dimethyl sulfide of compound 10 are 2.42 (2H, t, J = 7.6 Hz), 2.68 (2H, t, J = 7.6 Hz), 3.55-3.80 (6H , m), 4.23-4.36 (1H, m), 6.58 (1H, d, J = 7.6Hz), 6.65 (1H, d, J = 7.6Hz), 6.77 (1H, s), 8.00 (1H, br) , 8.65 (1H, br).
The wave numbers (cm ⁻¹ ) absorbed in the IR spectrum were 3520, 3310, 2940, 1750, 1710, 1650, 1540, 1520, 1280, 1270, 1220, 1030, 860, 820, 800.

< Reference Example 1 >
Compound 11 of Reference Example was synthesized by hydrolyzing Compound 7 obtained in Example 4. That is, compound 6 (1.71 g, 5.00 mmol) was dissolved in isopropyl alcohol (15 ml), 1N aqueous sodium hydroxide solution (5 ml) was added, and the mixture was stirred at room temperature. After 24 hours, the reaction mixture was concentrated under reduced pressure, and the residue was recrystallized from methanol to obtain a pale yellow crystalline compound (450 mg, yield 43%). It was confirmed that the obtained compound was the compound 11 by 1H-NMR spectrum and IR spectrum analysis.

○ Structural formula of Compound 11

The melting point of Compound 11 was 221 to 222 ° C. Chemical shift values of ¹ H-NMR spectrum measured in a mixed solvent of deuterated dimethyl sulfide and deuterated methanol of compound 11 are 1.73-1.85 (1H, m), 1.91-2.07 (4H, m), 2.29-2.50 (4H , m), 2.73-2.81 (2H, m), 3.79 (3H, s), 4.07-4.14 (1H, m), 6.62 (1H, d, J = 8.0Hz), 6.68 (1H, d, J = 8.0 Hz), 6.81 (1H, s).
The wave numbers (cm ⁻¹ ) absorbed in the IR spectrum were 3370, 2930, 1650, 1650, 1600, 1520, 1400, 1280, 1260, 1230, 1130, 1040, 820.

<Comparative Example 1>
An active ester compound was prepared using trans-ferulic acid instead of dihydroferulic acid (Compound 1) used in Synthesis Example 1, and the resulting active ester compound was reacted with L-histidine methyl ester dihydrochloride. In the same manner as in Example 5, compound 12 was obtained.

○ Structural formula of Compound 12

Chemical shift values of ¹ H-NMR spectrum measured in a mixed solvent of deuterated chloroform and deuterated methanol of compound 12 are 3.08 (2H, m), 3.74 (3H, s), 3.90 (3H, s), 4.80-4.90. (1H, m), 6.44 (1H, d, J = 16.0Hz), 6.81-6.90 (2H, m), 7.01-7.12 (2H, m), 7.50 (1H, s), 7.56 (1H, s) there were.
The wave numbers (cm ⁻¹ ) absorbed in the IR spectrum were 3300, 1740, 1660, 1590, 1510, 1280, 1210, 1130, 980, 820.

<Comparative example 2>
Instead of the compound 2 used in Example 5, 3- (p-hydroxyphenyl) propionic acid N-hydroxysuccinimide ester (manufactured by Wako Pure Chemical Industries, Ltd.) was used to obtain the compound 13.

○ Structural formula of compound 13

The chemical shift value of the ¹ H-NMR spectrum measured in deuterated dimethyl sulfide of Compound 13 is 2.32 (2H, t, J = 8.0 Hz), 2.64 (2H, t, J = 8.0 Hz), 2.79-2.95 (2H , m), 3.58 (3H, s), 4.42-4.53 (1H, m), 6.64 (2H, d, J = 8.8Hz), 6.77 (1H, br), 6.95 (2H, d, J = 8.8Hz) 7.52 (1H, br), 8.21 (1H, br), 9.12 (1H, br), 11.80 (1H, br).
The wave numbers (cm ⁻¹ ) absorbed in the IR spectrum were 3290, 1740, 1660, 1520, 1440, 1370, 1250, 990, 830, 620.

< Reference Example 2 >
As a test for confirming the antioxidant activity, a 1,1-Diphenyl-2-picrylhydrazyl (abbreviated as DPPH) radical scavenging test, which is a conventional method, was performed. That is, using Compound 4 to Compound 10 obtained in the above Example and Compound 11 of Reference Example 1 , and dl-α-tocopherol (manufactured by Tokyo Chemical Industry Co., Ltd.), Compound 12, Compound 13 and L-histidine were used as comparative controls. Then, a DPPH radical elimination test was conducted.
The specific test procedure is as follows.

A 100 mM Tris-HCl buffer (pH 7.4) (80 μl) and an ethanol solution (20 μl) of a test sample (final concentration 50 μg / ml) were dispensed and mixed in each well of a 96-well plate. An 800 μM DPPH ethanol solution (100 μl, final 400 μM) was added thereto and stirred well. This was left to stand at room temperature in a dark place for 20 minutes, and then the absorbance at 540 nm was measured (absorbance of the sample solution).
Absorbance was measured in the same manner as above using 100 mM Tris-HCl buffer (pH 7.4) (80 μl), ethanol (20 μl) and 800 μM DPPH ethanol solution (100 μl) as a control (absorbance of control solution). .
The same measurement was performed four times for each compound, and the average value was substituted into the following formula to calculate the DPPH radical elimination rate.
DPPH radical scavenging rate (%) = [1− (a / b)] × 100
a = absorbance of sample solution
b = absorbance of control solution

  When comparing the structurally similar compound 8 with the compound 12 and the compound 13, the amide group and the phenyl group are saturated with respect to the compound 12 in which the amide group and the phenyl group are a carbon-carbon double bond. It was found that the compound 8 of the present invention connected with a hydrocarbon has a higher radical scavenging rate. Moreover, it turned out that the radical scavenging rate is higher in the compound 8 of the present invention in which the methoxy group is present on the phenyl group than in the compound 13 in which the methoxy group is not present on the phenyl group. Moreover, it turned out that the compound of this invention has the radical scavenging activity more than equivalent to dl- (alpha) -tocopherol. Therefore, it is considered that the compound of the present invention can be used as a scavenger for active oxygen, particularly hydroxy radicals.

< Reference Example 3 >
Using compound 11 and compound 4 to compound 10 obtained in Example described above Reference Example 1, and dl-alpha-tocopherol as a comparative control (manufactured by Tokyo Kasei Kogyo) and L- histidine (manufactured by Wako Pure Chemical Industries) Then, a lipid peroxide production inhibition test in linoleic acid was conducted.

The specific test procedure is as follows.
1) Linoleic acid (0.2 g, manufactured by Tokyo Chemical Industry Co., Ltd.), Compound 4 (0.01 g), and surfactant Nissan OT-221 (0.4 g, taken in a screw vial (capacity 50 ml))
After adding ethanol (2.0 g) to Nippon Oil & Fats Co., Ltd. and dissolving it, distilled water (17.39 g) was added and stirred, sealed, and left in a constant temperature bath at 40 ° C. Two of these were prepared.
The same procedure was performed for compounds 5 to 11, dl-α-tocopherol, L-histidine, and no sample added (blank).
2) The amount of linoleic acid remaining after 10 days, 20 days and 30 days from the start of the test was quantified by HPLC.
HPLC analysis conditions were as follows: detection wavelength: 210 nm, mobile phase: M adjusted to pH 2.6.
mixed solution of cIlvaine buffer and methanol (10:90), flow rate: 1 ml / m
in, column: ODS-80Ts (4.6 φmm × 150 mm), column temperature 40 ° C.
3) Quantification by HPLC was carried out once for each sample, and the linoleic acid residual rate was calculated from the average of the measured values of two times in total. The results are shown in Table 2.

  As a result of the test, it was found that the compound of the present invention has an effect higher than that of dl-α-tocopherol, which is L-histidine and an existing lipid peroxide production inhibitor.

<Example 11>
Using the compounds 4 to 10 obtained above and arbutin (manufactured by Tokyo Chemical Industry Co., Ltd.) and L-histidine (manufactured by Wako Pure Chemical Industries, Ltd.) as comparative controls, a tyrosinase activity inhibition test using L-dopa as a substrate was performed. It was.

The specific test procedure is as follows.
First, the following [1] to [4] solutions were respectively prepared.
[1] Sodium dihydrogen phosphate (1.000 g) and disodium hydrogen phosphate (1.186 g) were dissolved in distilled water (500 ml) to prepare a phosphate buffer.
[2] L-DOPA (32.7 mg) was dissolved in distilled water (200 ml) to prepare a substrate solution.
[3] A test solution was prepared by diluting the dimethyl sulfoxide solution of the specimen 10 times with a phosphate buffer.
[4] Mushroom tyrosinase (3.0 mg, 2400 units) was dissolved in distilled water (7.0 ml) and further diluted 5-fold with a phosphate buffer to prepare a tyrosinase solution.

Next, the test was performed according to the following procedures 1) and 2).
1) L-DOPA solution (80 μl) and test sample solution (80 μl) were dispensed and mixed in each well of a 96-well plate. To this was added tyrosinase solution (80 μl), and 15
Stir for 2 seconds. The resulting solution was kept at 25 ° C., and the absorbance at 490 nm after 1 minute 45 seconds and 2 minutes 45 seconds was measured with a microplate reader.
2) The test procedure of 1) above was performed 4 times for each specimen and blank test (dimethylsulfoxide only), and the obtained value was substituted into the following formula, and the average value was used as the tyrosinase activity inhibition rate ( %).
Tyrosinase activity inhibition rate (%) = [(T1-T2) / T1] × 100
T1 = difference in absorbance of the blank solution after 2 minutes and 45 seconds and after 1 minute and 45 seconds
T2 = difference in absorbance of the test solution after 2 minutes and 45 seconds and after 1 minute and 45 seconds

  Table 3 shows the results when 2.00 mg / ml of Compound 4 to Compound 10, and arbutin and L-histidine of the present invention were used in the enzyme reaction solution.

  As a result of the test, it was found that the compound of the present invention has tyrosinase inhibitory activity, unlike L-histidine, similarly to arbutin which is an existing tyrosinase activity inhibitor.

The compound represented by the general formula (A) of the present invention has excellent antioxidant activity and tyrosinase inhibitory activity. In the fields of pharmaceuticals, quasi drugs, cosmetics, foods, etc., antioxidants or tyrosinase activity It can be used as an inhibitor.

Claims (1)

A tyrosinase activity inhibitor comprising a compound represented by the following general formula (A) obtained by condensing L-α-amino acid or a derivative thereof and dihydroferulic acid .

[R in the formula represents a residue bonded to an amino group in L-α-amino acid selected from alanine, serine, phenylalanine, methionine, histidine, and tyrosine and its methyl or ethyl ester . ]