WO2025071094A1 - Uses of mercapto functional compound having tyrosinase inhibitory activity - Google Patents

Abstract

The present invention relates to uses of a compound having a mercapto functional group and, more specifically, provides a composition for preventing food browning, a composition for whitening, and a composition for inhibiting melanin pigmentation in fish, comprising the compound having tyrosinase inhibitory activity. The compound is safe without cytotoxicity and has excellent tyrosinase inhibitory activity and melanin production inhibitory activity, and thus can be effectively used for the various uses.

Description

Uses of mercapto functional compounds having tyrosinase inhibitory activity

The present invention relates to the use of a compound having a mercapto functional group, and more specifically, to a composition for preventing food browning, a whitening composition, and a composition for inhibiting melanin pigmentation in fish, comprising the compound having tyrosinase inhibitory activity.

Tyrosinase, known as a rate-controlling enzyme in melanin biosynthesis, has been known as the most important target for the development of agents to inhibit browning of agricultural products such as fruits and vegetables, to suppress pigmentation, and to develop skin whitening agents.

Recently, due to changes in consumers’ eating habits, interest in health, and increasing preference for fresh and natural foods, the consumption of fresh convenience foods with vegetables and fruits as the main ingredients is increasing. These fresh convenience foods are processed by peeling, cutting, cored, and dividing so that they can be consumed immediately by consumers, which reduces their storage life, causes ethylene production, makes it easier for microorganisms to invade, and promotes oxidative browning.

Browning, which is caused by tissue damage in agricultural products, occurs when tyrosinase oxidizes dihydroxyphenylalanine (DOPA) to become O-quinone phenylalanine (DOPA-quinone), which then undergoes further oxidation, continuous condensation, and polymerization to produce black-brown melanin pigments, which has a significant impact on quality loss. Browning serves as a criterion for consumers to judge freshness and also as a criterion for purchase selection, affecting the marketability of agricultural products. In reality, fresh convenience foods show a high distribution loss of about 25%. Therefore, minimizing distribution loss by suppressing browning is emerging as an important issue for the food industry, including producers and distributors.

In addition, melanin absorbs a certain amount of ultraviolet rays and blocks harmful ultraviolet rays from penetrating into the body, thereby protecting the human body. However, this increases melanin production, which causes skin pigmentation problems. In order to prevent abnormal pigmentation, a method of reducing the production of melanin by inhibiting some reactions during the melanin synthesis process is generally and easily performed, and for this purpose, substances such as ascorbic acid, kojic acid, arbutin, and hydroquinone have been often used in the past.

However, among these, kojic acid has the effect of inhibiting enzyme activity by chelating the copper ion present at the active site of tyrosinase, and although it has good performance, its use is restricted due to safety issues when mixed into cosmetics, hydroquinone is highly irritating to the skin, and ascorbic acid also has safety issues.

Meanwhile, in the domestic pharmaceutical industry, zebrafish is gaining attention as an alternative to animal testing for new drug development. It is already widely used in scientific research in Europe. Zebrafish is a tropical fish from India belonging to the cyprinidae family, and is called zebrafish because its blue body has white stripes that look like spots. It is very small, about 2 inches in size when fully grown, and has a unique fluorescent substance. It is also widely raised as an ornamental fish.

The reason why zebrafish are used in animal testing is because their genetic structure is more than 80% similar to that of humans, so they can be genetically modified to suit the study of human genetic conditions. In other words, it is possible to study the effects of specific diseases, including rare diseases, by causing mutations in genes. In addition, zebrafish embryos are initially transparent because they do not produce melanin, but within a week, melanin pigment is rapidly produced throughout the body, including the pupils. Therefore, they are used as an in vivo model for the development of melanin pigment inhibitor compounds. Experiments using zebrafish embryo models can avoid animal experiments using higher organisms, so they can reduce animal ethics issues to some extent. In order to conduct various drug experiments using fish instead of animal experiments, interest in methods that can suppress and depigment fish pigmentation is increasing.

The purpose of the present invention is to provide a composition for preventing food browning and a method for preventing food browning using a compound having excellent tyrosinase inhibitory activity.

Another object of the present invention is to provide a composition for skin whitening using a compound having excellent tyrosinase inhibitory activity and melanin production inhibitory activity, and a composition for preventing, improving, or treating skin diseases caused by excessive melanin production.

Another object of the present invention is to provide a composition for inhibiting pigmentation of fish using a compound having excellent tyrosinase inhibitory activity and melanin production inhibitory activity.

In order to achieve the above purpose, the present invention provides a composition for preventing food browning, comprising a compound represented by the following chemical formula 1 or a food-chemically acceptable salt thereof:

<Chemical Formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, and X may be selected from oxygen, sulfur, or amino.

The present invention provides an anti-browning food additive comprising the above-mentioned anti-browning food composition.

In addition, the present invention provides a method for preventing food browning, comprising a step of treating food with the composition for preventing food browning.

In order to achieve the other objects described above, the present invention provides a cosmetic composition for skin whitening, comprising a compound represented by the chemical formula 1 or a pharmaceutically or cosmetically acceptable salt thereof.

The present invention provides a health food composition for skin whitening, comprising a compound represented by the chemical formula 1 above or a food-wise acceptable salt thereof.

The present invention provides a pharmaceutical composition for preventing or treating skin diseases caused by excessive melanin production, comprising a compound represented by the chemical formula 1 above or a pharmaceutically acceptable salt thereof.

The present invention provides a cosmetic composition for preventing or improving skin diseases caused by excessive melanin production, comprising a compound represented by the chemical formula 1 above or a pharmaceutically or cosmetically acceptable salt thereof.

In addition, the present invention provides a health food composition for preventing or improving skin disease caused by excessive melanin production, comprising a compound represented by the chemical formula 1 above or a food-wise acceptable salt thereof.

In order to achieve another object described above, the present invention provides a feed additive composition for suppressing pigmentation of fish, comprising a compound represented by the chemical formula 1 or a pharmaceutically or food-wise acceptable salt thereof.

In addition, the present invention provides a veterinary pharmaceutical composition for suppressing pigmentation in fish, comprising a compound represented by the chemical formula 1 above or a pharmaceutically acceptable salt thereof.

The compound having a mercapto functional group according to the present invention is safe without cytotoxicity and has excellent inhibitory activity against tyrosinase, which is one cause of food browning, and thus can be utilized as a composition for preventing food browning of vegetables or fresh-cut foods using the vegetables.

By using the composition for preventing food browning above, the quality of the product can be maintained fresh for a long time, making it easy to store for a long time and reducing distribution loss of fresh convenience foods, etc.

The compound having a mercapto functional group according to the present invention or a pharmaceutically/cosmetically/foodstuffally acceptable salt thereof is safe without cytotoxicity and has excellent tyrosinase inhibitory activity and melanin production inhibitory activity, and thus can be utilized as a skin whitening composition.

In addition, the compound or its salt can be used as a composition for preventing or treating skin diseases caused by excessive melanin production, thereby effectively preventing, improving or treating abnormal skin pigmentation diseases such as blemishes, freckles and liver spots caused by excessive melanin production.

In addition, the compound having a mercapto functional group according to the present invention or a pharmaceutically/foodstuffally acceptable salt thereof is safe without cytotoxicity and has excellent tyrosinase inhibitory activity and melanin production inhibitory activity, and thus can be utilized as a composition for inhibiting pigmentation in fish.

A transparent fish or fish embryo is produced using a composition for suppressing pigmentation of fish according to the present invention, and the produced fish or fish embryo can be used for toxicity tests of drugs under development, side effects of drugs, and pharmacological effects of drugs.

In addition, the composition according to the present invention can also be used for the prevention or treatment of fish diseases related to pigmentation.

Figure 1 shows 2-mercaptobenzimidazole, 2-mercaptobenzoxazole, and 2-mercaptobenzothiazole compounds synthesized according to embodiments of the present invention.

Figure 2 shows the interactions between mushroom tyrosinase and compounds (3, 5, 6, 14, 15, 17, and 21) and kojic acid predicted by AutoDock Vina.

Figure 3 shows the results of a docking simulation performed using Schrodinger Suite.

Figure 4 is a Lineweaver-Burk plot obtained through inhibition of mushroom tyrosinase activity by compounds 4, 6, and 10.

Figure 5 is a Dixon plot obtained by modifying the Lineweaver-Burk plot of Figure 4.

Figure 6 is a Lineweaver-Burk plot obtained through inhibition of mushroom tyrosinase activity by compounds 11, 12, and 13.

Figure 7 is a Dixon plot obtained by modifying the Lineweaver-Burk plot of Figure 6.

Figure 8 is a Lineweaver-Burk plot obtained through inhibition of mushroom tyrosinase activity by compounds 20, 21, 22, 26, and 28.

Figure 9 is a Dixon plot obtained by modifying the Lineweaver-Burk plot of Figure 8.

Figure 10 shows the cytotoxicity results of 2-mercaptobenzimidazole compounds in HEK-293 cells.

Figure 11 shows the cytotoxicity results of 2-mercaptobenzimidazole compounds in B16F10 cells.

Figure 12 shows the cytotoxicity results of 2-mercaptobenzimidazole compounds in HaCaT cells.

Figure 13 shows the cytotoxicity results of 2-mercaptobenzoxazole compounds in B16F10 cells.

Figure 14 shows the cytotoxicity results of 2-mercaptobenzoicazole compounds in B16F10 cells.

Figure 15 shows the tyrosinase inhibitory activity of 2-mercaptobenzimidazole compounds in B16F10 cells.

Figure 16 shows the tyrosinase inhibitory activity of 2-mercaptobenzoxazole compounds in B16F10 cells.

Figure 17 shows the tyrosinase inhibitory activity of 2-mercaptobenzoicazole compounds in B16F10 cells.

Figure 18 shows the inhibitory effect of 2-mercaptobenzimidazole compound on melanin production in B16F10 cells.

Figure 19 shows the inhibitory effect of 2-mercaptobenzoxazole compound on melanin production in B16F10 cells.

Figure 20 shows the inhibitory effect of 2-mercaptobenzoicazole compound on melanin production in B16F10 cells.

Figure 21 shows the inhibitory effect of 2-mercaptobenzimidazole compound on zebrafish embryo pigmentation.

Figure 22 is the result of quantifying the effect of Figure 21 using CS analyzer 3.2 image software.

Figure 23 shows the inhibitory effect of 2-mercaptobenzoxazole compound on zebrafish embryo pigmentation.

Figure 24 is the result of quantifying the effect of Figure 23 using CS analyzer 3.2 image software.

Figure 25 shows the inhibitory effect of 2-mercaptobenzoicazole compound on zebrafish embryo pigmentation.

Figure 26 shows the inhibitory effect of representative 2-mercaptobenzoicazole compounds (compounds 19, 20, 27, and 28), kojic acid, and PTU on zebrafish embryo pigmentation.

Figure 27 is the result of quantifying the effect of Figure 25 using CS analyzer 3.2 image software.

Figure 28 shows the effect of 2-mercaptobenzoxazole compound on tyrosinase expression in B16F10 cells.

Figure 29 shows the effect of 2-mercaptobenzoicazole compounds on tyrosinase expression in B16F10 cells.

Figure 30 shows the inhibitory effect of 2-mercaptobenzoicazole compound on tyrosinase activity in B16F10 cells using L-dopa staining method.

Figure 31 shows the copper ion chelating activity of a 2-mercaptobenzoxazole compound.

Figure 32 shows the copper ion chelating activity of a 2-mercaptobenzoicazole compound.

Figure 33 shows the change in tyrosinase inhibitory activity in the presence or absence of CuSO ₄ to confirm whether the 2-mercaptobenzoxazole compound chelates with the tyrosinase copper ion.

Figure 34 shows the change in tyrosinase inhibitory activity in the presence or absence of CuSO ₄ to confirm whether the 2-mercaptobenzoicazole compound chelates with the tyrosinase copper ion.

Figure 35 shows the browning inhibition effect of 2-mercaptobenzimidazole compound in apple slices.

Figure 36 shows the analysis of the degree of browning of apple slices treated with kojic acid, phenylthiourea (PTU), and 2-mercaptobenzimidazole compounds.

Hereinafter, the present invention will be described in detail.

The present inventors synthesized 2-mercaptobenzimidazole, 2-mercaptobenzoxazole and 2-mercaptobenzothiazole compounds as low-molecular-weight compounds having a -SH group based on the fact that tyrosinase is a metalloenzyme having two copper ions and that the mercapto (-SH) group binds well to the copper ions, and confirmed that they have excellent tyrosinase activity inhibitory ability and melanin production inhibitory ability, thereby completing the present invention.

Composition for preventing food browning

The present invention provides a composition for preventing food browning, comprising a compound represented by the following chemical formula 1 or a food-chemically acceptable salt thereof:

<Chemical Formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, and X may be selected from oxygen, sulfur, or amino.

Preferably, the compound or a food-chemically acceptable salt thereof is a compound wherein R ¹ , R ² and R ³ in the formula are selected from hydrogen, halogen, (C1-C2)alkyl, (C1-C2)alkoxy, trifluoromethyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen or phenyl), or R ² and R ³ may be connected to each other to form a five-ring ring or a 1,3-dioxole ring.

More preferably, the compound or a food-chemically acceptable salt thereof is 1 H -Benzo[ d ]imidazole-2-thiol, 5-Methyl-1 H -benzo[ d ]imidazole-2-thiol, 5,6-Dimethyl-1 H -benzo [ d ]imidazole - 2-thiol, 5-Chloro - 1 H -benzo [ d ]imidazole - 2-thiol , 5-methoxy- 1 H -benzo [ d ]imidazole-2-thiol (5-Methoxy-1 H -benzo[ d ]imidazole-2-thiol), 2-Mercapto-1 H -benzo [ d ] imidazol-5-yl)(phenyl)methanone), 5-Nitro - 1 H -benzo[ d ] imidazole -2-thiol, 5-Fluoro - 1 H -benzo [ d ]imidazole-2-thiol, 4-Methyl- 1 H -benzo [ d ]imidazole-2-thiol), 2-Mercapto-1 H -benzo[ d ]imidazole-5-carbonitrile, Benzo[ d ]oxazole-2-thiol, 6-Methylbenzo[ d ] oxazole -2-thiol, 5-Methylbenzo[ d ]oxazole-2-thiol , 4-Methylbenzo[ d ]oxazole-2-thiol, 6-Nitrobenzo[ d ] oxazole - 2-thiol d ]oxazole-2-thiol, 5-Nitrobenzo[ d ]oxazole-2-thiol, 4-Nitrobenzo[ d ]oxazole-2-thiol, 6-Chlorobenzo[ d ]oxazole-2-thiol, 4-Methylbenzo[ d ] thiazole-2-thiol, Benzo[ d ] thiazole-2-thiol, 5-(Trifluoromethyl ) benzo [ d ] thiazole -2- thiol ]thiazole - 2-thiol), 6,7-Dihydro-5 H -indeno[5,6- d ]thiazole-2-thiol, 6-Phenoxybenzo[ d ]thiazole-2-thiol, [1,3 ] Dioxolo[4′,5′:4,5]benzo[1,2- d ]thiazole-6-thiol, 5,6 - Dimethylbenzo [ d ] thiazole -2-thiol, 5-chlorobenzo[ d ]thiazole-2-thiol (5-Chlorobenzo[ d ]thiazole-2-thiol), 6-Methoxybenzo[ d ]thiazole-2-thiol, and 6-Methylbenzo [ d ] thiazole-2-thiol, but is not limited thereto.

The above compound can be used in the form of a food-acceptable salt within the range having the same efficacy.

As used herein, “pharmaceutically, cosmetically, or food-wise acceptable” means a salt that is non-toxic to cells or humans exposed to the composition and thus has a safety and efficacy profile suitable for administration to humans.

The above salt can be used in the form of either a basic salt or an acid salt acceptable from a pharmaceutical, cosmetic or food perspective. For example, the basic salt can include either an organic base salt or an inorganic base salt, and can be selected from the group consisting of a sodium salt, a potassium salt, a calcium salt, a lithium salt, a magnesium salt, a cesium salt, an aluminum salt, an ammonium salt, a triethylaminium salt and a pyridinium salt, but is not limited thereto. The acid salt is useful as an acid addition salt formed by a free acid. Inorganic acids and organic acids can be used as free acids, and inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, sulfurous acid, phosphoric acid, etc., and organic acids include citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid, etc.

The above compound may include all salts, hydrates and solvates that can be prepared by conventional methods, as well as pharmaceutically, cosmetically, or foodologically acceptable salts, and addition salts can be prepared by conventional methods. For example, the compound may be prepared by dissolving it in a water-miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile, adding an excess amount of an organic base or adding an aqueous base solution of an inorganic base, and then precipitating or crystallizing the resulting solution. Alternatively, the compound may be prepared by evaporating the solvent or the excess amount of base from the mixture, followed by drying to obtain an addition salt, or by suction filtration to obtain the precipitated salt.

The compound according to the present invention or a food-related acceptable salt thereof has excellent tyrosinase inhibitory activity and can be utilized as a composition for preventing food browning.

In this specification, “browning” refers to a color change that occurs during food processing, preservation, etc., particularly a change to brown, which is a phenomenon that occurs by generating black-brown melanin pigment through an oxidation reaction by polyphenol oxidase (PPO) or tyrosinase.

According to one experimental example of the present invention, it was confirmed through docking simulation that the compounds bind well to the active site of tyrosinase, and it was confirmed that the compounds act as competitive inhibitors or mixed inhibitors of tyrosinase, and can prevent or suppress browning by inhibiting tyrosinase activity.

The composition for preventing food browning according to the present invention can contain the compound or a food-related acceptable salt thereof at a concentration of 0.0001 to 5000 μM, and by being contained within the above range, can have safe toxicity and excellent tyrosinase inhibitory activity. The above concentration range can vary depending on the type of food and the degree of browning characteristics.

In the composition for preventing food browning according to the present invention, the food may be vegetables, fresh-cut foods using the vegetables, fresh-cut agricultural products, etc., but is not limited thereto and may include all types of foods in which browning may occur.

In this specification, 'fresh convenience food' refers to food that has been stored and distributed in a state that maintains the freshness as much as possible at the time of production, and has been processed to minimize processing and maximize quality in order to deliver good quality to consumers. It may include simply processed foods that can be eaten as is, such as agricultural or forestry products that have gone through processing processes such as washing, peeling, cutting or mincing, or simple foods or food additives added.

The present invention provides an anti-browning food additive comprising the above-mentioned anti-browning food composition.

Corresponding features can be substituted for those described above.

In addition, the present invention provides a method for preventing food browning, comprising a step of treating food with the composition for preventing food browning.

In the above method for preventing food browning, the step of treating the food may be performed by treating the food with only the food browning prevention composition, or by treating the food with a conventional browning prevention agent in combination so that browning can be inhibited more effectively by complementing each other, but is not limited thereto.

The step of treating the food may be performed by, but is not limited to, spraying the composition on the surface of the food or immersing the food in the composition.

Whitening composition

The present invention provides a cosmetic composition for skin whitening, comprising a compound represented by the following chemical formula 1 or a pharmaceutically or cosmetically acceptable salt thereof:

<Chemical Formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, and X may be selected from oxygen, sulfur, or amino.

Preferably, the compound or a pharmaceutically or cosmetically acceptable salt thereof is wherein R ¹ , R ² and R ³ in the formula are selected from hydrogen, halogen, (C1-C2)alkyl, (C1-C2)alkoxy, trifluoromethyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen or phenyl), or R ² and R ³ may be connected to each other to form a five-cyclic ring or a 1,3-dioxole ring.

More preferably, the compound or a pharmaceutically or cosmetically acceptable salt thereof is 1 H -Benzo[ d ]imidazole-2-thiol, 5-Methyl-1 H -benzo[ d ]imidazole-2-thiol, 5,6-Dimethyl-1 H -benzo[ d ]imidazole-2-thiol, 5-Chloro - 1 H -benzo [ d ] imidazole-2-thiol , 5-methoxy - 1 H -benzo [ d ]imidazole-2-thiol (5-Methoxy-1 H -benzo[ d ]imidazole-2-thiol), 2-Mercapto-1 H -benzo[ d ] imidazol-5-yl)(phenyl)methanone), 5-Nitro - 1 H -benzo[ d ]imidazole-2- thiol, 5-Fluoro-1 H -benzo [ d ]imidazole-2-thiol, 4-Methyl- 1 H -benzo [ d ] imidazole- 2 - thiol 2-Mercapto-1 H -benzo[ d ]imidazole- 5 -carbonitrile, Benzo[ d ]oxazole-2-thiol, 6-Methylbenzo[ d ]oxazole-2-thiol, 5-Methylbenzo[ d ]oxazole-2-thiol, 4-Methylbenzo[ d ] oxazole-2-thiol, 6- Nitrobenzo[ d ] oxazole - 2 - thiol ] oxazole-2-thiol, 5-Nitrobenzo[ d ]oxazole-2-thiol, 4-Nitrobenzo[ d ]oxazole-2-thiol, 6-Chlorobenzo[ d ]oxazole-2-thiol, 4-Methylbenzo[ d ] thiazole-2-thiol, Benzo[ d ] thiazole-2-thiol, 5-(Trifluoromethyl ) benzo [ d ] thiazole -2- thiol ]thiazole - 2-thiol), 6,7-Dihydro-5 H -indeno[5,6- d ]thiazole-2-thiol, 6-Phenoxybenzo[ d ]thiazole-2-thiol, [1,3 ] Dioxolo[4′,5′:4,5]benzo[1,2- d ]thiazole-6-thiol, 5,6 - Dimethylbenzo [ d ] thiazole -2-thiol, 5-chlorobenzo[ d ]thiazole-2-thiol (5-Chlorobenzo[ d ]thiazole-2-thiol), 6-Methoxybenzo[ d ]thiazole-2-thiol, and 6-Methylbenzo [ d ] thiazole-2-thiol, but is not limited thereto.

The above compound can be used in the form of a pharmaceutically, cosmetically, or food-wise acceptable salt within the same efficacy range.

Corresponding features can be substituted for those described above.

The compound according to the present invention or a pharmaceutically or cosmetically acceptable salt thereof has excellent tyrosinase inhibitory activity and can be used as a cosmetic composition for skin whitening.

In this specification, “whitening” means the action of improving skin troubles by suppressing, inhibiting, or alleviating excessive deposition of melanin pigments and the like, thereby preventing darkening of the skin caused by said pigmentation.

According to one experimental example of the present invention, it was confirmed through docking simulation that the compounds bind well to the active site of tyrosinase, and it was confirmed that the compounds act as competitive inhibitors or mixed inhibitors of tyrosinase, and can inhibit tyrosinase activity, thereby suppressing melanin production and imparting a whitening effect.

In this specification, "cosmetics" may include functional cosmetics that have the effect of preventing or improving skin pigmentation, etc., and the functional cosmetics, unlike general cosmetics, have specialized functionality with emphasized physiologically active efficacy and effects, and mean cosmetics with emphasized specific efficacy and effects, such as skin whitening, as defined in the Cosmetics Act.

The cosmetic composition for skin whitening according to the present invention can contain the compound or a pharmaceutically or cosmetically acceptable salt thereof at a concentration of 0.0001 to 5000 μM, and by being contained within the above range, can have safe toxicity and excellent tyrosinase inhibitory activity and melanin production inhibitory activity.

The cosmetic composition according to the present invention may further include at least one of the ingredients listed in the list of cosmetic raw materials registered with the Ministry of Food and Drug Safety and the ICID (International Cosmetic Ingredient Dictionary). For example, the cosmetic composition may further include at least one auxiliary agent commonly used in the cosmetic field, such as an organic solvent, a solubilizer, a thickener, a gelling agent, a softener, an antioxidant, a suspending agent, a stabilizer, a fragrance, a surfactant, an emulsifier, a filler, a metal ion sequestering agent, a preservative, a vitamin, a blocking agent, a humectant, a dye and a pigment, a hydrophilic or lipophilic active agent, or any other ingredient commonly used in cosmetics.

The above vitamins may include water-soluble or oil-soluble vitamins, and the water-soluble vitamins are water-soluble vitamins that can be incorporated into cosmetics, and preferably include vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinamide, folic acid, vitamin C, vitamin H, and the like, and more preferably include vitamin C that helps collagen synthesis. In addition, their salts (thiamine hydrochloride, sodium ascorbate, etc.) or derivatives (sodium ascorbate-2-phosphate, magnesium ascorbate-2-phosphate, etc.) may also be included, and the water-soluble vitamins can be obtained by a conventional method such as a microbial transformation method, a purification method from a culture of a microbial substance, an enzymatic method, or a chemical synthesis method.

The cosmetic composition according to the present invention can be prepared in any formulation commonly manufactured in the technical field to which the present invention belongs. For example, it can be formulated as a toner, emulsion, lotion, cream, paste, gel, solution, suspension, oil, wax, pack, powder, foundation, spray, surfactant-containing cleansing, etc., but is not limited thereto.

More specifically, it can be manufactured in the form of a flexible toner, a nourishing toner, an astringent toner, a nourishing cream, a massage cream, a milk lotion, a powder, an essence, an eye cream, a sun lotion, a sunscreen, a makeup primer, a makeup base, a BB cream, a powder foundation, an emulsion foundation, a cleansing cream, a cleansing foam, a cleansing water, a soap, a pack, a stick product, a balm type product, a spray, or a powder.

If the above cosmetic composition is in the form of a cream or gel, it may further contain animal oil, vegetable oil, wax, paraffin, starch, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide as a carrier component.

When the above cosmetic composition is in the form of a solution or emulsion, it may further contain a solvent, solvating agent or emulsifying agent, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, propylene glycol, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

If the above cosmetic composition is in the form of a suspension formulation, it may further include a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth as a carrier component.

If the above cosmetic composition is in a powder or spray formulation, it may further contain lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder as a carrier component, and particularly if it is in a spray formulation, it may further contain a propellant such as chlorofluorohydrocarbon, propane/butane or dimethyl ether.

When the above cosmetic composition is a surfactant-containing cleansing formulation, it may further include, as a carrier component, an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, methyl taurate, a sarcosinate, a fatty acid amide ether sulfate, an alkyl amidobetaine, a fatty alcohol, a fatty acid glyceride, a fatty acid diethanolamide, a vegetable oil, a linolenic derivative, or an ethoxylated glycerol fatty acid ester.

The cosmetic composition according to the present invention can be used by applying it alone or in duplicate, or by applying it in duplicate with another cosmetic composition other than the present invention. In addition, the cosmetic composition according to the present invention can be used according to a conventional method of use, and the number of times it is used can vary depending on the skin condition or preference of the user.

The present invention provides a health food composition for skin whitening, comprising a compound represented by the following chemical formula 1 or a food-chemically acceptable salt thereof:

<Chemical formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, and X may be selected from oxygen, sulfur, or amino.

Corresponding features can be substituted for those described above.

In the health food composition according to the present invention, the health food can be manufactured into powder, granules, tablets, capsules, syrup or beverages, and there is no limitation on the form that the health food can take, and can include all foods in the conventional sense. For example, beverages and various drinks, fruits and processed foods thereof (canned fruits, jams, etc.), fish, meats and processed foods thereof (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.), etc. are possible, and can include all functional foods in the conventional sense. In addition, foods used as feed for animals can also be included.

The health food composition according to the present invention can be manufactured by further including food additives and other appropriate auxiliary ingredients that are commonly used in the art. For example, it can further contain flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, coloring agents, pectic acid, alginic acid, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonating agents, etc.

In particular, the natural carbohydrates that can be used include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol, and the sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame.

The effective dosage of the compound contained in the health food according to the present invention can be appropriately adjusted according to the purpose of skin whitening. The composition has the advantage of being made of food as a raw material and having no side effects that may occur when taking general medicine for a long period of time, and is highly portable, so it can be taken as a supplement for skin whitening.

The present invention provides a pharmaceutical composition for preventing or treating skin diseases caused by excessive melanin production, comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

<Chemical formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, and X may be selected from oxygen, sulfur, or amino.

Corresponding features can be substituted for those described above.

The compound according to the present invention or a pharmaceutically acceptable salt thereof has excellent tyrosinase inhibitory activity and melanin production inhibitory activity, and thus skin diseases caused by excessive melanin production can be prevented or treated using the compound.

The skin disease caused by the above melanin overproduction may be one or more pigment diseases selected from the group consisting of blemishes, freckles, lentigo, liver spots, and Ota's nevus, but is not limited thereto.

In this specification, "melasma" is a pigment disease in which irregularly shaped, brown spots of various sizes appear on exposed areas, especially on the face, and may be worsened by exposure to sunlight, etc. Melanin pigmentation is mainly deposited symmetrically on the cheeks, forehead, and under the eyes.

In this specification, "freckles" are small, yellow-brown pigmented spots that mainly appear on the skin in areas exposed to sunlight. The cause is not exactly known, but it is known to be related to genetic mutations in the melanocortin-1 receptor, and may occur when skin melanocytes are stimulated by ultraviolet rays, increasing the synthesis of melanin pigment.

As used herein, "liver spot, age spot" is a spot on the skin formed by aging and exposure to ultraviolet rays of sunlight, also called solar lentigo or chloasma.

In this specification, "nevus of Ota" refers to a brown or blue-colored spot that often occurs on the skin around the eyes, eyes, mucous membranes, etc.

The pharmaceutical composition according to the present invention can be prepared according to a conventional method in the pharmaceutical field. The pharmaceutical composition can be combined with an appropriate pharmaceutically acceptable carrier according to the formulation, and, if necessary, can be prepared by further including an excipient, a diluent, a dispersant, an emulsifier, a buffer, a stabilizer, a binder, a disintegrant, a solvent, etc. The appropriate carrier, etc., can be selected differently according to the dosage form and formulation as long as it does not inhibit the activity or properties of the compound according to the present invention or its salt.

The above pharmaceutical composition can be applied in any dosage form, and more specifically, it can be formulated and used as a parenteral dosage form of an oral dosage form, an external preparation, a suppository, and a sterile injection solution according to a conventional method.

Among the oral dosage forms, the solid dosage form is in the form of tablets, pills, powders, granules, capsules, etc., and can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc., and in addition to simple excipients, lubricants such as magnesium stearate and talc can also be included. In addition, in the case of a capsule dosage form, in addition to the above-mentioned substances, a liquid carrier such as fatty oil can be further included.

Among the oral dosage forms mentioned above, liquid dosage forms include suspensions, solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.

The above parenteral formulations may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin, and the like. Without being limited thereto, any suitable formulation known in the art may be used.

In addition, the pharmaceutical composition according to the present invention may further contain calcium or vitamin D3 to enhance therapeutic efficacy.

The pharmaceutical composition according to the present invention can be administered in a pharmaceutically effective amount.

As used herein, a “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and without causing any adverse effects.

The effective dosage level of the pharmaceutical composition may vary depending on the intended use, the patient's age, sex, weight and health condition, the type and severity of the disease, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration and the excretion rate, the treatment period, the drugs used in combination or simultaneously, and other factors well known in the medical field. For example, although not constant, it may generally be administered at 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, once or several times a day. The above dosage does not limit the scope of the present invention in any way.

The above pharmaceutical composition can be administered by an appropriate route of administration according to the form of the preparation, and can be administered through various routes, either oral or parenteral, as long as it can reach the target tissue. The method of administration is not particularly limited, and can be administered by conventional methods such as oral, rectal, intravenous, intramuscular, skin application, subcutaneous, respiratory inhalation, intrauterine epidural or intracerebroventricular injection.

The pharmaceutical composition according to the present invention can be used alone for the prevention or treatment of skin diseases caused by excessive melanin production, or can be used in combination with surgery or other drug treatments.

The present invention provides a cosmetic composition for preventing or improving skin diseases caused by excessive melanin production, comprising a compound represented by the following chemical formula 1 or a pharmaceutically or cosmetically acceptable salt thereof:

<Chemical Formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, and X may be selected from oxygen, sulfur, or amino.

Corresponding features can be substituted for those described above.

The compound according to the present invention or a pharmaceutically or cosmetically acceptable salt thereof has excellent tyrosinase inhibitory activity and melanin production inhibitory activity, and thus skin diseases caused by excessive melanin production can be prevented or improved by using the compound.

In addition, the present invention provides a health food composition for preventing or improving skin disease caused by excessive melanin production, comprising a compound represented by the following chemical formula 1 or a food-wise acceptable salt thereof:

<Chemical Formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, and X may be selected from oxygen, sulfur, or amino.

Corresponding features can be substituted for those described above.

Composition for inhibiting melanin pigmentation in fish

The present invention provides a feed additive composition for suppressing pigmentation of fish, comprising a compound represented by the following chemical formula 1 or a pharmaceutically or food-wise acceptable salt thereof:

<Chemical Formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, and X may be selected from oxygen, sulfur, or amino.

Preferably, the compound or a pharmaceutically or food additively acceptable salt thereof is a compound wherein R ¹ , R ² and R ³ in the formula are selected from hydrogen, halogen, (C1-C2)alkyl, (C1-C2)alkoxy, trifluoromethyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen or phenyl), or R ² and R ³ may be connected to each other to form a five-cyclic ring or a 1,3-dioxole ring.

More preferably, the compound or a pharmaceutically or food-wise acceptable salt thereof is 1 H -Benzo[ d ]imidazole-2-thiol, 5-Methyl-1 H -benzo[ d ]imidazole-2-thiol, 5,6-Dimethyl-1 H -benzo[ d ]imidazole-2-thiol, 5-Chloro - 1 H -benzo [ d ] imidazole - 2-thiol, 5-methoxy - 1 H -benzo [ d ]imidazole-2-thiol (5-Methoxy-1 H -benzo[ d ]imidazole-2-thiol), 2-Mercapto-1 H -benzo [ d ] imidazol-5-yl)(phenyl)methanone), 5-Nitro - 1 H -benzo[ d ] imidazole -2-thiol, 5-Fluoro - 1 H -benzo [ d ]imidazole-2-thiol, 4-Methyl- 1 H -benzo [ d ]imidazole-2-thiol), 2-Mercapto-1 H -benzo[ d ]imidazole-5-carbonitrile, Benzo[ d ]oxazole-2-thiol, 6-Methylbenzo[ d ] oxazole -2-thiol, 5-Methylbenzo[ d ]oxazole-2-thiol , 4-Methylbenzo[ d ] oxazole-2-thiol, 6-Nitrobenzo [ d ] oxazole -2-thiol d ]oxazole-2-thiol, 5-Nitrobenzo[ d ]oxazole-2-thiol, 4-Nitrobenzo[ d ]oxazole-2-thiol, 6-Chlorobenzo[ d ]oxazole-2-thiol, 4-Methylbenzo[ d ] thiazole-2-thiol, Benzo[ d ] thiazole-2-thiol, 5-(Trifluoromethyl ) benzo [ d ] thiazole -2- thiol ]thiazole - 2-thiol), 6,7-Dihydro-5 H -indeno[5,6- d ]thiazole-2-thiol, 6-Phenoxybenzo[ d ]thiazole-2-thiol, [1,3 ] Dioxolo[4′,5′:4,5]benzo[1,2- d ]thiazole-6-thiol, 5,6 - Dimethylbenzo [ d ] thiazole -2-thiol, 5-chlorobenzo[ d ]thiazole-2-thiol (5-Chlorobenzo[ d ]thiazole-2-thiol), 6-Methoxybenzo[ d ]thiazole-2-thiol, and 6-Methylbenzo [ d ] thiazole-2-thiol, but is not limited thereto.

The above compound can be used in the form of a pharmaceutically or food-wise acceptable salt within the range having the same efficacy.

Corresponding features can be substituted for those described above.

The compound according to the present invention or a pharmaceutically or food-wise acceptable salt thereof has excellent tyrosinase inhibitory activity and can be utilized as a composition for inhibiting pigmentation in fish.

According to one experimental example of the present invention, it was confirmed through docking simulation that the compounds bind well to the active site of tyrosinase, and it was confirmed that the compounds act as competitive inhibitors or mixed inhibitors of tyrosinase, and can have an activity of inhibiting melanin production by inhibiting tyrosinase activity.

In addition, according to another experimental example of the present invention, the compounds showed excellent inhibitory ability in a pigmentation inhibition ability experiment using zebrafish embryos, and thus transparent fish can be produced using these, and the produced transparent fish can be used in drug experiments to facilitate the confirmation of drug toxicity, side effects, and pharmacological effects of drugs.

The composition according to the present invention can contain the compound or a pharmaceutically or food-wise acceptable salt thereof at a concentration of 0.0001 to 5000 μM, and by being contained within the above range, can have safe toxicity and excellent tyrosinase inhibitory activity and melanin production inhibitory activity.

In this specification, “feed additive” is a general term for substances added in small amounts to feed for nutritional or specific purposes, and in the present invention means a substance added for the purpose of suppressing pigmentation of fish.

In the feed additive composition according to the present invention, the feed is a substance that supplies organic or inorganic nutrients necessary for maintaining the life of an individual and raising the individual, and may include nutrients such as energy, protein, lipid, vitamins, and minerals required by an individual consuming the feed.

The above feed is a feed of a known composition generally used for fish farming, and may include all commercially available general feeds, but is not particularly limited thereto.

The above feed additives may include general feed additives, for example, feed additives for special purposes such as salt, bone meal, calcium phosphate, mineral mixtures, vitamins, amino acids, antibiotics, and hormones.

The above feed additive composition can be added to feed for fish, preferably farmed fish, but is not limited thereto.

In the feed additive composition according to the present invention, the effective dose of the compound can be used in accordance with the effective dose of the animal pharmaceutical composition, but in the case of long-term intake, it can be below the above range, and since the effective ingredient does not have any problems in terms of safety, it can also be used in an amount above the above range.

The dosage of the above feed additive composition can be appropriately adjusted by an expert in the field depending on the type, age, weight, degree of pigmentation, desired effect, etc. of the fish to be administered.

The present invention provides a veterinary pharmaceutical composition for suppressing pigmentation in fish, comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

<Chemical Formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, and X may be selected from oxygen, sulfur, or amino.

Corresponding features can be substituted for those described above.

The veterinary pharmaceutical composition according to the present invention can be manufactured by further including a pharmaceutically acceptable appropriate carrier, excipient or diluent depending on the formulation.

Examples of carriers, excipients or diluents usable in the above animal pharmaceutical composition include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil.

The formulation of the above veterinary pharmaceutical composition may be in a desirable form depending on the method of use, and may be formulated by adopting a method known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to fish in particular. For example, it may be formulated and used in the form of oral formulations such as powders, powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups, aerosols, external preparations, and sterile injectable solutions.

When the above-mentioned animal pharmaceutical composition is formulated in the above form, diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used may be used. Solid preparations for oral administration may be prepared by mixing the compound with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. Non-aqueous solvents or suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

The veterinary pharmaceutical composition according to the present invention can be administered in a pharmaceutically effective amount. The "pharmaceutically effective amount" above means an amount sufficient to treat a pigmentation-related disease at a reasonable benefit/risk ratio applicable to medical treatment and not causing side effects, and the effective dosage level can be determined based on factors including the health condition of the fish, the degree of pigmentation, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration, and the excretion rate, the treatment period, drugs used in combination or simultaneously, and other factors well known in the medical field.

The amount of the compound as an active ingredient in the above animal pharmaceutical composition may vary depending on the age, sex, weight, size, pigmentation or related disease of the fish, but is generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, administered once or several times a day. In addition, the dosage of the compound may be increased or decreased depending on the administration route, degree of pigmentation, etc. Therefore, the dosage does not limit the scope of the present invention in any way.

In addition, the present invention provides a reagent composition for inhibiting tyrosinase activity, comprising the compound described above or a pharmaceutically or food-wise acceptable salt thereof.

The composition may be a competitive inhibitor or a mixed inhibitor of tyrosinase.

In addition, the present invention provides a reagent composition for inhibiting melanin production, comprising the compound described above or a pharmaceutically or food-wise acceptable salt thereof.

Hereinafter, in order to help understand the present invention, examples will be given and described in detail. However, the following examples are only intended to illustrate the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to a person having average knowledge in the art.

<Example 1> Synthesis of 2-mercaptobenzimidazole compound (Compound 1-10)

To synthesize compounds 1-10, two synthetic methods were used, as shown in Scheme 1 below.

<Reaction Formula 1>

1-1. Synthesis of compounds 1, 2, 6, 7 and 9

o-Phenylenediamine compound [1,2-phenylenediamine for the synthesis of compound 1, 3,4-diaminotoluene for the synthesis of compound 2, 3,4-diaminobenzophenone for the synthesis of compound 6, 4-nitro-1,2-phenylenediamine for the synthesis of compound 7, and 2,3-diaminotoluene for the synthesis of compound 9] and NaOH (1.16 equivalents) were dissolved in ethanol (1.8 mL per 1 mmol of o-phenylenediamine compound) and water (0.6 mL per 1 mmol of o-phenylenediamine compound), and CS ₂ (1.16 equivalents) was added. After stirring the mixture at 80°C for 1 to 9 hours, activated carbon (1.5 g per 1 mmol of o-phenylenediamine) was added and stirred at 80°C for 10 minutes. The mixture was filtered while hot, and the filtered solid was washed with hot water (60 to 70°C), and the pH of the filtrate was adjusted to 2 with 50% acetic acid. The filtrate was left in the refrigerator overnight, and the resulting solid was filtered and washed with water to obtain pure 2-mercaptobenzimidazole compounds as solids (Compound 1, 86%; Compound 2, 83%; Compound 6, 77%; Compound 7, 76%; Compound 9, 68%).

1-2. Synthesis of compounds 3-5 and 8-10

o-Phenylenediamine compounds [4,5-dimethyl-1,2-phenylenediamine for the synthesis of compound 3, 4-chloro-1,2-phenylenediamine for the synthesis of compound 4, 4-methoxy-1,2-phenylenediamine for the synthesis of compound 5, 4-fluoro-1,2-phenylenediamine for the synthesis of compound 8, 2,3-diaminotoluene for the synthesis of compound 9, and 3,4-diaminobenzonitrile for the synthesis of compound 10] and sodium N,N-diethyldithiocarbamate trihydrate (2.5 eq.) were dissolved in DMF (4 mL per 1 mmol of o-phenylenediamine) and stirred at 120°C for 0.5 to 9 hours in the presence of AlCl ₃ (0.1 eq.).

To obtain the target product after the reaction, the following operations were performed:

To obtain compound 3, water was added to the reaction mixture, and the resulting solid was filtered to obtain compound 3 as a solid (75%). To obtain compound 4, after evaporating the volatile substances, water was added, the resulting solid was filtered, and the filtered solid was washed with hexane:dichloromethane (2:1) to obtain compound 4 (88%) as a solid. To obtain compounds 5, 8, and 9, extraction was performed between ethyl acetate and water, and the organic layer was dried and evaporated to obtain a solid residue. Water was added to the solid residue, the resulting solid was filtered, and the filtered solid was washed with hexane:dichloromethane (2:1) and ethyl acetate to obtain compound 5 (92%) as a solid, or the filtered solid was washed with dichloromethane to obtain compounds 8 (52%) and compound 9 (32%) as solids, respectively. To obtain compound 10, the volatile substances were evaporated, the resulting solid was extracted with ethyl acetate and water, the organic layer was evaporated, the resulting residue was filtered, and washed with hexane:dichloromethane (1:1) to obtain compound 10 (85%) as a solid.

Compound 1: 1 H -Benzo[ d ] imidazole-2-thiol

¹ H NMR (400 MHz, DMSO -d ₆ ) δ 12.47 (s, 1H, NH), 7.12-7.04 (m, 4H, 4-H, 5-H, 6-H, 7-H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 168.7, 132.8, 122.8, 110.0.

Compound 2: 5-Methyl-1 H -benzo [ d ] imidazole-2-thiol

^1H NMR (400 MHz, DMSO _-d6 ) δ 12.35 (s, 1H, NH), 6.97 (d, 1H, J = 8.0 Hz, 7-H), 6.90 (s, 1H, 4-H), 6.88 (d, 1H, J = 8.0 Hz, 6-H), 2.29 (s, 3H, CH ₃ ); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 168.3, 133.0, 132.2, 130.7, 123.7, 110.1, 109.6, 21.5.

Compound 3: 5,6-Dimethyl- 1 H -benzo[ d ] imidazole-2-thiol

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.25 (brs, 1H, NH), 6.88 (s, 2H, 4-H, 7-H), 2.18 (s, 6H, 2×CH ₃ ); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 167.7, 131.1, 131.0, 110.6, 20.1.

Compound 4: 5-Chloro- 1 H -benzo[ d ] imidazole-2-thiol

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 7.18-7.10 (m, 3H, 4-H, 6-H, 7-H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 174.6, 138.5, 136.5, 132.0, 127.5, 115.8, 114.5.

Compound 5: 5-Methoxy-1 H -benzo [ d ] imidazole-2-thiol

^1H NMR (400 MHz, DMSO- d ₆ ) δ 12.38 (brs, 1H, NH), 7.03 (d, 1H, J = 8.4 Hz, 7-H), 6.72 (dd, 1H, J = 8.4, 2.4 Hz, 6-H), 6.67 (d, 1H, J = 2.4) Hz, 4-H) 3.74 (s, 3H, OCH ₃ ); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 168.3, 156.3, 133.6, 126.9, 110.5, 110.3, 95.0, 56.1.

Compound 6: 2-Mercapto-1 H -benzo[ d ] imidazol-5-yl) ( phenyl)methanone

^1H NMR (400 MHz, DMSO- d ₆ ) δ 7.65 (d, 2H, J = 7.6 Hz, 2′-H, 6′-H), 7.61 (t, 1H, J = 7.6 Hz, 4′-H), 7.54-7.47 (m, 3H, 7-H, 3′-H, 5′-H), 7.44 (s, 1H, 4-H), 7.23 (d, 1H, J = 8.4 Hz, 6-H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 195.6, 171.2, 138.4, 137.5, 133.4, 132.6, 131.1, 129.9, 129.0, 125.6, 111.4, 109.8.

Compound 7: 5-Nitro-1 H -benzo [ d ] imidazole-2-thiol

^1H NMR (400 MHz, DMSO -d ₆ ) δ 8.03 (d, 1H, J = 8.8 Hz, 6-H), 7.85 (s, 1H, 4-H), 7.25 (d, 1H, J = 8.8 Hz, 7-H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 172.3, 143.1, 137.9, 132.8, 119.5, 109.8, 105.2.

Compound 8: 5-Fluoro-1 H -benzo[ d ] imidazole -2-thiol

^1H NMR (400 MHz, DMSO- d ₆ ) δ 7.07 (ddd, 1H, J = 8.4, 4.8, 0.8 Hz), 6.95-6.88 (m, 2H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 169.2, 159.1 (d, J = 235.8 Hz), 133.1 (d, J = 13.2 Hz), 129.2, 110.9 (d, J = 9.8 Hz), 110.1 (d, J = 24.7 Hz), 97.6 (d, J = 28.0 Hz).

Compound 9: 4-Methyl-1 H -benzo [ d ] imidazole-2-thiol

^1H NMR (400 MHz, DMSO- d ₆ ) δ 6.99 (t, 1H, J = 7.6 Hz, 6-H), 6.94 (d, 1H, J = 7.6 Hz, 7-H), 6.89 (d, 1H, J = 7.6 Hz, 5-H), 2.36 (s, 3H, CH ₃ ); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 167.9, 132.3, 131.9, 124.0, 123.1, 120.4, 107.7, 16.7.

Compound 10: 2-Mercapto-1 H -benzo [ d ] imidazole-5-carbonitrile

^1H NMR (400 MHz, DMSO- d ₆ ) δ 7.52-7.48 (m, 2H, 4-H, 6-H), 7.22 (d, 1H, J = 8.8 Hz, 7-H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 171.2, 136.2, 132.9, 127.5, 119.8, 113.4, 110.7, 104.8.

<Example 2> Synthesis of 2-mercaptobenzoxazole compound (compound 11-18)

Compounds 11-18 were synthesized using the same process as the following reaction scheme 2.

<Reaction Formula 2>

2-1. Synthesis of compounds 11 and 12

Sodium N,N-diethyldithiocarbamate trihydrate (2.5 eq) was added to a round bottom flask containing 2-aminophenol or 2-amino-5-methylphenol, which was dissolved in DMF (2 mL per 100 mg of starting material), and the mixture was stirred at 120°C for 30 minutes in the presence of AlCl ₃ (0.1 eq). The solvent was evaporated, and the remaining residue was purified by silica gel column chromatography using dichloromethane:methanol (100:1) as a developing solvent to give compound 11 (79%) and compound 12 (55%) as solids, respectively.

2-2. Synthesis of compounds 13-18

2-Aminophenol compound [2-amino-4-methylphenol in the synthesis of compound 13, 2-amino-3-methylphenol in the synthesis of compound 14, 2-amino-5-nitrophenol in the synthesis of compound 15, 2-amino-4-nitrophenol in the synthesis of compound 16, 2-amino-3-nitrophenol in the synthesis of compound 17, and 2-amino-5-chlorophenol in the synthesis of compound 18] and NaOH (1.16 equivalents) were dissolved in ethanol (1.8 mL per 1 mmol of 2-aminophenol compound) and water (0.6 mL per 1 mmol of 2-aminophenol compound), and CS ₂ (1.16 equivalents) was added. The mixture was stirred under reflux for 1.5 h to 2 days, and then the solvent was evaporated. After adding dichloromethane and filtering, the filtrate was purified by silica gel column chromatography (solvent for each compound: compound 14, hexane:ethyl acetate (1:1); compounds 13, 16, and 17, hexane:ethyl acetate (5:1); compound 15, hexane:dichloromethane (1:1); compound 18, dichloromethane:methanol (30:1)) to obtain compounds 13 to 18 as solids, respectively. The yields of the obtained compounds are as follows: compounds 13 (60%), 14 (81%), 15 (88%), 16 (99%), 17 (45%), 18 (52%).

Compound 11: Benzo[ d ] oxazole-2-thiol

^1H NMR (400 MHz, DMSO- d ₆ ) δ 7.45 (d, 1H, J = 7.6 Hz, 4-H), 7.30-7.12 (m, 3H, 5-H, 6-H, 7-H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 180.7, 148.7, 131.8, 125.7, 124.3, 111.0, 110.5.

Compound 12: 6-Methylbenzo[ d ] oxazole-2-thiol

^1H NMR (400 MHz, DMSO- d ₆ ) δ 7.29 (s, 1H, 7-H), 7.08 (d, 1H, J = 8.0 Hz, 4-H), 7.05 (d, 1H, J = 8.0 Hz, 5-H), 2.32 (s, 3H, CH ₃ ); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 180.4, 148.9, 134.2, 129.6, 126.2, 110.7, 110.6, 21.4.

Compound 13: 5-Methylbenzo[ d ] oxazole-2-thiol

^1H NMR (400 MHz, DMSO -d ₆ ) δ 7.33 (d, 1H, J = 8.8 Hz, 7-H), 7.01 (d, 1H, J = 8.8 Hz, 6-H), 7.01 (s, 1H, 4-H), 2.32 (s, 3H, CH ₃ ); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 180.7, 146.9, 135.4, 131.8, 124.9, 111.1, 110.1, 21.4.

Compound 14: 4-Methylbenzo[ d ] oxazole-2-thiol

^1H NMR (400 MHz, DMSO- d ₆ ) δ 7.26 (d, 1H, J = 8.0 Hz, 7-H), 7.11 (t, 1H, J = 8.0 Hz, 6-H), 7.05 (d, 1H, J = 8.0 Hz, 5-H), 2.33 (s, 3H, CH ₃ ); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 180.6, 148.4, 130.9, 126.6, 124.2, 121.7, 107.8, 16.6.

Compound 15: 6-Nitrobenzo[ d ] oxazole-2-thiol

^1H NMR (400 MHz, DMSO- d ₆ ) δ 7.94 (dd, 1H, J = 8.4, 2.0 Hz, 5-H), 7.90 (d, 1H, J = 2.0 Hz, 7-H), 7.12 (d, 1H, J = 8.4 Hz, 4-H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 189.0, 153.8, 150.8, 140.2, 120.3, 112.8, 102.3.

Compound 16: 5-Nitrobenzo[ d ] oxazole-2-thiol

^1H NMR (400 MHz, DMSO- d ₆ ) δ 7.86 (dd, 1H, J = 8.8, 2.4 Hz, 6-H), 7.83 (d, 1H, J = 2.4 Hz, 4-H), 7.30 (d, 1H, J = 8.8 Hz, 7-H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 185.9, 156.2, 146.1, 143.7, 117.0, 108.4, 107.2.

Compound 17: 4-Nitrobenzo[ d ] oxazole-2-thiol

^1H NMR (400 MHz, DMSO- d ₆ ) δ 8.03 (dd, 1H, J = 8.8, 0.8 Hz, 5-H), 7.89 (dd, 1H, J = 8.8, 0.8 Hz, 7-H), 7.41 (t, 1H, J = 8.8 Hz, 6-H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 182.1, 150.3, 131.9, 128.3, 123.9, 120.3, 116.3.

Compound 18: 6-Chlorobenzo[ d ] oxazole-2-thiol

^1H NMR (400 MHz, DMSO- d ₆ ) δ 7.68 (d, 1H, J = 2.0 Hz, 7-H), 7.30 (dd, 1H, J = 8.4, 2.0 Hz, 5-H), 7.19 (d, 1H, J = 8.4 Hz, 4-H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 180.9, 149.0, 131.0, 128.5, 125.7, 111.9, 111.1.

<Example 3> Synthesis of 2-mercaptobenzothiazole compound (Compound 19-28)

Compounds 19-28 were synthesized by the same process as the following reaction scheme 3. However, benzo[ d ] thiazole -2-thiol (compound 20) was not synthesized but purchased from Sigma-Aldrich and used.

<Reaction Formula 3>

2-Aminothiophenol compound [2-amino-3-methylthiophenol in the synthesis of compound 19, 2-amino-4-trifluoromethylthiophenol in the synthesis of compound 21, 6-amino-2,3-dihydro-1 H -indene-5-thiol in the synthesis of compound 22, 2-amino-5-phenoxythiophenol in the synthesis of compound 23, 6-aminobenzo[ d ][1,3]dioxole-5-thiol in the synthesis of compound 24, 2-amino-4,5-dimethylthiophenyl in the synthesis of compound 25, 2-amino-4-chlorothiophenol in the synthesis of compound 26, 2-amino-5-methoxythiophenol in the synthesis of compound 27, and 2-amino-5-methylthiophenol in the synthesis of compound 28] and NaOH (1.16 equivalents). Ethanol (1.8 mL per 1 mmol of 2-aminothiophenol compound) and water (0.6 mL per 1 mmol of 2-aminothiophenol compound) were dissolved, and CS ₂ (1.16 equivalents) was added. The mixture was refluxed for 3-15 hours, and the solvent was evaporated. A small amount of water was added, filtered, and the filtered solid was washed with hexane:dichloromethane (1-2:1) or dichloromethane to obtain pure compounds 19, 22-25, and 27, respectively, as solids. Through this process, crude compounds 21, 26, and 28 were purified using hexane:ethyl acetate (18:1), dichloromethane:methanol (20:1), and hexane:ethyl acetate (6:1) as solvents, respectively, and were thus obtained purely through silica gel column chromatography. The obtained yields were as follows: compounds 19 (85%), 21 (68%), 22 (87%), 23 (93%), 24 (96%), 25 (96%), 26 (93%), 27 (84%), 28 (70%).

Compound 19: 4-Methylbenzo[ d ] thiazole-2-thiol

^1H NMR (400 MHz, DMSO- d ₆ ) δ 7.18 (dd, 1H, J = 7.6, 1.2 Hz, 7-H), 6.86 (dd, 1H, J = 7.6, 1.2 Hz, 5-H), 6.78 (t, 1H, J = 7.6 Hz, 6-H), 2.38 (s, 3H, CH ₃ ); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 184.3, 155.1, 137.0, 126.4, 125.4, 120.5, 117.0, 19.1.

Compound 20: Benzo[ d ]thiazole-2-thiol was purchased from Sigma - Aldrich and used.

Compound 21: 5-(Trifluoromethyl)benzo[ d ] thiazole-2-thiol

^1H NMR (400 MHz, DMSO- d ₆ ) δ 7.93 (d, 1H, J = 8.4 Hz, 7-H), 7.61 (dd, 1H, J = 8.4, 1.6 Hz, 6-H), 7.48 (d, 1H, J = 1.6 Hz, 4-H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 191.6, 142.0, 134.6, 128.2 (q, J = 32.1 Hz), 124.4 (q, J = 271.0 Hz), 123.6, 121.1 (q, J = 3.6 Hz), 109.2.

Compound 22: 6,7-Dihydro-5 H -indeno[ 5,6- d ]thiazole-2-thiol

^1H NMR (400 MHz, DMSO- d ₆ ) δ 7.19 (s, 1H, 4-H), 7.09 (s, 1H, 8-H), 2.83 (t, 2H, J = 7.2 Hz, 5-H ₂ ), 2.80 (t, 2H, J = 7.2 Hz, 7-H ₂ ), 1.99 (quint, 2H, J = 7.2 Hz, 6-H ₂ ); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 184.1, 155.4, 140.4, 136.4, 135.5, 114.7, 113.0, 32.8, 32.4, 26.2.

Compound 23: 6-Phenoxybenzo[ d ] thiazole-2-thiol

^1H NMR (400 MHz, DMSO- d ₆ ) δ 7.32 (t, 2H, J = 7.6 Hz, 3′-H, 5′-H), 7.22 (d, 1H, J = 8.4 Hz, 4-H), 7.10 (d, 1H, J = 2.4 Hz, 7-H), 7.04 (t, 1H, J = 7.6 Hz, 4′-H), 6.92 (d, 2H, J = 7.6 Hz, 2′-H _, 6′-H), 6.78 (dd, 1H, J = 8.4, 2.4 Hz, 5-H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 185.0, 158.9, 153.4, 150.5, 138.8, 130.3, 122.9, 117.8, 117.7, 116.9, 110.9.

Compound 24: [1,3]Dioxolo[4′,5′:4,5]benzo[1,2- d ]thiazole-6-thiol

^1H NMR (400 MHz, DMSO -d ₆ ) δ 6.97 (s, 1H, 8-H), 6.79 (s, 1H, 4-H), 5.89 (s, 2H, 2-H ₂ ); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 184.0, 150.7, 145.8, 142.9, 128.8, 100.8, 99.7, 98.6.

Compound 25: 5,6-Dimethylbenzo[ d ] thiazole-2-thiol

^1H NMR (400 MHz, DMSO- d ₆ ) δ 7.11 (s, 1H, 4-H), 7.02 (s, 1H, 7-H), 2.18 (s, 3H, CH ₃ ), 2.15 (s, 3H, CH ₃ ); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 184.1, 155.1, 134.9, 132.3, 128.6, 119.8, 118.3, 20.2, 19.8.

Compound 26: 5-Chlorobenzo[ d ] thiazole-2-thiol

^1H NMR (400 MHz, DMSO- d ₆ ) δ 7.69 (d, 1H, J = 8.4 Hz, 7-H), 7.32 (dd, 1H, J = 8.4, 2.0 Hz, 6-H), 7.26 (d, 1H, J = 2.0 Hz, 4-H); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 191.4, 142.8, 132.3, 128.8, 124.5, 123.7, 112.5

Compound 27: 6-Methoxybenzo[ d ] thiazole-2-thiol

^1H NMR (400 MHz, DMSO _-d6 ) δ 7.33 (d, 1H, J = 2.4 Hz, 7-H), 7.21 (d, 1H, J = 8.8 Hz, 4-H), 6.98 (dd, 1H, J = 8.8, 2.4 Hz, 5-H), 3.75 (s, 3H, OCH ₃ ); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 188.9, 157.2, 135.8, 131.3, 115.3, 113.6, 106.4, 56.2.

Compound 28: 6-Methylbenzo[ d ] thiazole-2-thiol

^1H NMR (400 MHz, CDCl ₃ ) δ 11.5 (brs, 1H, SH), 7.26 (d, 1H, J = 2.0 Hz, 7-H), 7.21 (d, 1H, J = 8.4 Hz, 4-H), 7.16 (dd, 1H, J = 8.4, 2.0 Hz, 5-H), 2.40 (s, 3H, CH ₃ ); ¹³ C NMR (100 MHz, DMSO- d ₆ ) δ 190.4, 138.2, 135.0, 130.2, 128.4, 121.5, 111.9, 21.3.

<Experimental Example 1> Confirmation of mushroom tyrosinase activity inhibition effect

170 μL of 100 mM sodium phosphate buffer (pH 6.5) containing 1 mM substrate L-tyrosine (or L-dopa) was added to 10 μL of the sample containing the 28 synthesized compounds (compounds 1-28) dissolved in DMSO. 20 μL of 1000 unit/mL mushroom tyrosinase (Sigma) solution was added and reacted at 37°C for 20 minutes. After the reaction was completed, the absorbance was measured at a wavelength of 475 nm using a microplate reader (VersaMax™, Molecular Devices, Sunnyvale, CA, USA). Kojic acid (KA) and phenylthiourea (PTU) were used as positive controls. The tyrosinase inhibition rate (%) was calculated according to the following Equation 1:

<Formula 1>

Tyrosinase inhibition rate (%) = [(Abs _control -Abs _sample )/Abs _control ]×100

The inhibitory activity of each compound was expressed by calculating the sample concentration at which 50% of the maximum inhibitory effect was achieved (IC ₅₀ ).

Tables 1 to 3 below show the results of analysis of mushroom tyrosinase inhibitory activity of the compounds synthesized in Examples 1 to 3.

As a result of examining the tyrosinase inhibitory activity, all compounds exhibited mushroom tyrosinase inhibitory activity more potent than kojic acid (KA), and in particular, many compounds (1, 2, 4-8, 10-13, 20-28) exhibited inhibitory activity similar to or superior to phenylthiourea (PTU).

<Experimental Example 2> Docking simulation analysis between the synthesized compound and mushroom tyrosinase

2-1. Docking simulation using AutoDock Vina

Docking simulations of compounds 1-28 were performed using AutoDock Vina 1.1.2. The 2D structures of compounds 1-28 were constructed using ChemDraw Ultra 12.0, and the constructed 2D structures were converted to 3D structures using Chem3D Pro 12.0 software, and the energetically most stable 3D structure was converted through MM2 energy minimization process. Kojic acid was used as a positive control, and the 3D X-ray crystal structure of mushroom tyrosinase (A. Bisporus) was obtained from the PDB (Protein Data Bank, PDB ID: 2Y9X). The binding scores between the docking compounds (compounds 1-28 and kojic acid) and tyrosinase were calculated using AutoDock Vina and Chimera 35.91.958. LigandScout 4.4 was used to generate pharmacophore diagrams showing possible interactions between amino acids of tyrosinase and ligands (compounds 1-28 and kojic acid).

Table 4 below shows the binding energies between compounds and mushroom tyrosinase measured by AutoDock Vina.

Referring to Table 4 and Fig. 2 above, the docking simulation results showed that most compounds bind more strongly to the active site of the enzyme than kojic acid. In the case of compound 21, the 2-mercapto group was predicted to participate in a hydrogen bond with His244, the CF ₃ group was predicted to participate in a hydrophobic bond with Ala286 and Val283, and the benzoicazole ring was predicted to participate in a hydrophobic bond with Phe264 and Val283.

2-2. Docking simulation using Schrodinger Suite

dinger Suite (2021-2) 도킹 시뮬레이션 프로그램을 사용하여 수행되었다. 버섯 타이로시네이즈(A. bisporus)의 X-ray 결정 구조를 PDB (ID: 2Y9X)에서 Maestro 12.4의 단백질 준비 마법사로 가져왔다. 버섯 타이로시네이즈 결정 구조에서 불필요한 단백질 사슬이 삭제되었고, 수소 원자를 추가하고 효소에서 3Å 이상 떨어져 있는 물 분자는 제거하여 결정 구조를 최적화하였다. 가져온 X-선 결정 구조에서의 리간드인 트로폴론(tropolone)은 제거를 하고 tropolone이 결합된 위치는 버섯 타이로시네이즈 활성 부위의 활주 격자를 결정하는 데 사용되었다. 인실리코 도킹 화합물의 구조를 Maestro의 항목 목록에 CDXML 형식으로 가져오고 리간드 도킹 전에 LigPrep을 사용하여 도킹 화합물의 구조를 최적화하였다. 글라이드 작업 목록을 사용하여 화합물의 화학 구조를 타이로시네이즈 글라이드 그리드에 도킹하였다. 글라이드 표준 정밀 방법을 사용하여 결합 친화도 및 단백질-리간드 상호작용을 측정하였다.In silico docking simulations were performed to investigate the chemical interactions between the compound and mushroom tyrosinase. The simulations were performed using Schr

The docking simulation program dinger Suite (2021-2) was used. The X-ray crystal structure of mushroom tyrosinase (A. bisporus) was imported from PDB (ID: 2Y9X) into the protein preparation wizard of Maestro 12.4. The crystal structure was optimized by deleting unnecessary protein chains from the mushroom tyrosinase crystal structure, adding hydrogen atoms, and removing water molecules that were more than 3 Å away from the enzyme. The ligand tropolone in the imported X-ray crystal structure was removed, and the tropolone-bound position was used to determine the sliding lattice of the mushroom tyrosinase active site. The structure of the in silico docked compound was imported into the entry list of Maestro in CDXML format, and the structure of the docked compound was optimized using LigPrep before ligand docking. The chemical structure of the compound was docked onto the tyrosinase Glide grid using the Glide task list. The binding affinity and protein-ligand interactions were determined using the Glide standard precision method.

Docking simulations performed using Schrodinger Suite, as shown in Fig. 3, showed that compounds 1 and 2 bind well to the active site of mushroom tyrosinase (PDB ID: 2Y9X). Based on these results, kojic acid, compound 1, and compound 2 were predicted to have binding energies of -4.187, -5.394, and -5.615 kcal/mol, respectively. This means that compounds 1 and 2 bind more strongly to the enzyme than kojic acid.

<Experimental Example 3> Study on mushroom tyrosinase enzyme-substrate kinetics of compounds

Lineweaver-Burk plots of the compounds were performed in the presence of L-dopa substrate. Compounds 4, 6, and 10 were used at concentrations of 0, 5, 10, and 20 nM, and compounds 11, 12, 20, 21, and 26 were used at concentrations of 0, 50, 100, and 200 nM. In addition, compound 13 was used at concentrations of 0, 0.2, 0.6, and 1.8 μM, compound 22 was used at concentrations of 0, 0.5, 1, and 2 μM, and compound 28 was used at concentrations of 0, 100, 200, and 400 nM, respectively. A 96-well plate was filled with 20 μL of mushroom tyrosinase enzyme (200 unit/mL), 10 μL of each compound concentration sample, and 170 μL of potassium buffer (pH 6.5) containing various concentrations of L-dopa substrate (0.125–16 mM). The rate of dopachrome synthesis was measured at 475 nm (△OD475/min) using a microplate reader (VersaMax™, Molecular Devices, Sunnyvale, CA, USA). The maximum velocity (Vmax) was calculated using the Lineweaver–Burk plot (the inverse of the reaction rate (1/V) and the inverse of the substrate concentration (1/[S])). The Lineweaver–Burk plot was modified to obtain the Dixon plot to obtain the inhibition constant.

The mechanism of action of 2-mercaptobenzimidazole compounds 4, 6, and 10 was investigated by measuring the change in the rate of mushroom tyrosinase activity in the presence of these compounds. As shown in Fig. 4, the obtained Lineweaver-Burk plot confirmed that compounds 6 and 10 are competitive inhibitors and compound 4 is a mixed inhibitor. The results of the docking simulation performed above supported the results of these kinetic studies.

In addition, the Lineweaver-Burk plot was modified to obtain the inhibition constant to obtain the Dixon plot, which is shown in Fig. 5. Referring to Fig. 5, compounds 4, 6, and 10 exhibited very low inhibition constants of 9.4, 8.6, and 14.8 nM, respectively, confirming that these compounds bind very strongly to tyrosinase.

The mechanism of action of these compounds was identified by measuring the change in the rate of mushroom tyrosinase activity in the presence of 2-mercaptobenzoxazole compounds 11, 12, and 13. As shown in Fig. 6, the obtained Lineweaver-Burk plot confirmed that compound 11 was a competitive inhibitor and compounds 12 and 13 were mixed inhibitors.

In addition, the Lineweaver-Burk plot was modified to obtain the inhibition constants to obtain the Dixon plot, which is shown in Fig. 7. Referring to Fig. 7, compounds 11, 12, and 13 exhibited very low inhibition constants of 0.057, 0.098, and 0.142 μM, respectively, confirming that these compounds bind very strongly to tyrosinase.

The mechanism of action of these compounds was investigated by measuring the rate of change in mushroom tyrosinase activity in the presence of 2-mercaptobenzoicazole compounds 20, 21, 22, 26, and 28. As shown in Fig. 8, the Lineweaver-Burk plots obtained confirmed that all five compounds were mixed inhibitors.

In addition, the Lineweaver-Burk plot was modified to obtain the inhibition constants to obtain the Dixon plot, which is shown in Fig. 9. Referring to Fig. 9, compounds 20, 21, 22, 23, 26, and 28 showed very low inhibition constants of 0.031, 0.027, 0.306, 0.021, and 0.014 μM, respectively, confirming that these compounds bind very strongly to tyrosinase.

<Experimental Example 4> Analysis of cytotoxicity of compounds

Human embryonic kidney cells (HEK-293), mouse melanoma cells (B16F10), and human keratinocytes (HaCaT) were obtained from ATCC and used. Cells were cultured in DMEM medium containing 10% or 5% fetal bovine serum (FBS) and 1% antibiotics (100 units/mL penicillin, 100 μg/mL streptomycin) at 37°C in a 5% _CO2 incubator.

For cytotoxicity measurement, each of the three types of cells was seeded into a 96-well culture vessel, and the compounds were treated at each concentration for HEK-293 cells (final concentration: 0–40 μM) and HaCaT cells (final concentration: 0–20 μM) for 24 hours, and B16F10 cells (final concentration: 0–20 μM) for 72 hours. After that, 10 μL EZ-cytox solution (Daeillab, Seoul) was added and reacted for 1–2 hours. The cytotoxicity of the compounds was determined by measuring the absorbance at a wavelength of 450 nm using a microplate reader.

Referring to FIG. 10, in the case of 2-mercaptobenzimidazole compounds, compounds 3 and 6 showed slight toxicity at the highest concentration of 40 μM in human embryonic kidney cells (HEK-293), but no toxicity below 10 μM, and the other measured compounds did not show toxicity at any concentration. As a result of measuring the toxicity of 2-mercaptobenzimidazole compounds in B16F10 (mouse melanoma cells) and HaCaT (human keratinocytes) at 72 hours and 24 hours, all measured compounds showed no toxicity, as shown in FIGS. 11 and 12.

Referring to Figure 13, for 2-mercaptobenzoxazole compounds, all compounds showed no toxicity at a concentration of 20 μM for up to 72 hours in B16F10 cells.

Referring to Figure 14, in the case of 2-mercaptobenzoicazole compounds, when toxicity was examined in B16F10 cells for up to 72 hours, all compounds showed no toxicity up to a concentration of 20 μM for 72 hours.

<Experimental Example 5> Measurement of intracellular tyrosinase activity

The intracellular tyrosinase activity of compounds 2, 3, 4, 6, 12, 14, 15, 21, 22, 26, and 28 was evaluated in B16F10 cells. B16F10 cells were pretreated with kojic acid (20 μM) or PTU (20 μM) for 1 h in 6-well plates, followed by treatment with 200 μM IBMX (3-isobutyl-1-methylxanthine) and 1 μM α-MSH (α-melanocyte stimulating hormone), and then cultured for 72 h. After incubation, the cells were washed twice with PBS and lysed with a mixture of 100 μL of potassium buffer (pH 6.5), Triton X-100, and PMSF (phenylmethylsulfonyl fluoride), and then frozen at -80°C for 1 hour. After freezing, the cells were centrifuged at 12,000 g for 10 minutes at 4°C, and 80 μL of the supernatant was mixed with 20 μL of L-dopa (2 mg/mL), and the absorbance at 475 nm was measured using a microplate reader (VersaMax™, Molecular Devices, Sunnyvale, CA, USA).

As a result, as shown in FIGS. 15 to 17, treatment with α-MSH and IBMX significantly increased intracellular tyrosinase activity, but 2-mercaptobenzimidazole compounds 2, 3, 4, and 6 decreased the tyrosinase activity increased by treatment with α-MSH and IBMX in a concentration-dependent manner. The tyrosinase inhibitory activity of these compounds was superior to that of kojic acid and PTU at the same concentration. The tyrosinase inhibitory activity of 2-mercaptobenzoxazole compounds 12 and 15 was superior to that of kojic acid at the same concentration, but slightly weaker than that of PTU, and compound 14 showed weaker inhibitory activity than that of kojic acid and PTU. 2-mercaptobenzoxazole compounds 21, 22, 26, and 28 showed more superior tyrosinase inhibitory activity than kojic acid at the same concentration.

<Experimental Example 6> Confirmation of intracellular melanin content

The intracellular melanin content of compounds 2, 3, 4, 6, 12, 14, 15, 21, 22, 26, and 28 was evaluated in B16F10 cells. B16F10 cells were pretreated with each compound at various concentrations (0, 5, 10, 20 μM; compound 21 at 0, 5, 10 μM) in 6-well plates, kojic acid (20 μM), or PTU (20 μM) for 1 h, then treated with 200 μM IBMX and 1 μM α-MSH, and incubated for 72 h. After incubation, the cells were washed twice with PBS, lysed with 100 μL of 1 N NaOH at 60 °C for 1 h, and then centrifuged at 12,000 g for 10 min at 4 °C. Intracellular melanin content was measured using a microplate reader (VersaMax™, Molecular Devices, Sunnyvale, CA, USA) at 405 nm.

As a result, as shown in Figs. 18 to 20, treatment with α-MSH and IBMX significantly increased intracellular melanin production, but 2-mercaptobenzimidazole compounds 2, 3, 4, and 6 decreased melanin production in a concentration-dependent manner, and the melanin production inhibitory activity of these compounds was superior to kojic acid at the same concentration and similar to or slightly superior to PTU at the same concentration. The melanin production inhibitory activity of 2-mercaptobenzoxazole compounds 12, 14, and 15 was superior to kojic acid at the same concentration but slightly inferior to PTU, and 2-mercaptobenzoxazole compounds 21, 22, 26, and 28 showed more superior melanin production inhibitory activity than kojic acid at the same concentration.

<Experimental Example 7> Confirmation of the pigmentation inhibition effect of compounds in zebrafish embryos

7-1. Zebrafish breeding and zebrafish embryo acquisition (collection)

Wild-type zebrafish (Danio rerio) were provided by the Zebrafish Disease Modeling Center (ZCDM) at Chungnam National University. Adult zebrafish were raised in a closed-circuit filtration tank with a light/dark cycle of 14:10 h and a water temperature of 28℃. Adult zebrafish were placed in a 20 L tank, six at a time, and were fed three times a day. The fish were raised in a tank maintained at the optimal growth temperature of 28.0℃. Zebrafish embryos were obtained through natural mating. Adult zebrafish male and female were placed in an egg collection tank the day before egg collection, and natural mating was performed the next morning to obtain zebrafish embryos.

7-2. Pigmentation inhibition experiment using zebrafish embryos

① In the case of 2-mercaptobenzimidazole (1-10) compounds, zebrafish embryos collected through natural mating were fertilized 9 hours (9 hpf) to remove the chorion of the embryos using pronase, and three embryos were transferred to each well of a 96-well plate containing 200 μL of embryo medium using a pipette. The 96-well plate was placed in an incubator (28°C) until compound treatment. At 21 hpf, each well was treated with compounds 1-10 (0, 0.01, 0.05, 0.1, 0.2, 0.4 mM, 1% DMSO solution), kojic acid (5, 10, 20 mM), and PTU (0.2 mM), and the pigmentation inhibition effect was observed at 69 hpf.

② In the case of 2-mercaptobenzoxazole (11-18) compounds, zebrafish embryos collected through natural mating were removed from the chorion using pronase at 8 hpf, and three embryos were transferred to each well of a 96-well plate containing 200 μL of embryo medium using a pipette and placed in an incubator (28°C) until treatment with the compound. At 27 hpf, compounds 11-18 (0, 2, 10, 50 μM, 1% DMSO solution) or the positive control group, kojic acid (20 mM) and PTU (2, 10, 50 μM) were treated. After storage at 28°C for 48 hours, the effects of the test compounds on the pigmentation of zebrafish embryos were observed at 75 hpf.

③ In the case of the 2-mercaptobenzoicazole (19-28) compound, the chorion of the zebrafish embryos collected through natural mating was removed using pronase at 25 hpf, and three embryos were transferred to each well of a 96-well plate containing 200 μL of embryo medium using a pipette and placed in an incubator (28°C) until treatment with the compound. At 28 hpf, the embryos were treated with compound 19-28 (0, 2, 10, 50 μM, 1% DMSO solution) or the positive control group, kojic acid (20 mM) and PTU (200 μM). After storage at 28°C for 47 hours, the effect of the test compound on the pigmentation of the zebrafish embryos was observed at 75 hpf.

Regardless of the compound series treated, zebrafish embryos were anesthetized with a tricaine methanesulfonate solution purchased from Sigma-Aldrich, mounted on methylcellulose (1%), and photographed using a stereomicroscope SMZ745T (Nikon, Tokyo, Japan) to observe the pigmentation inhibition effect. The relative degree of pigmentation was measured using CS analyzer 3.2 image analysis software.

As a result, referring to FIGS. 21 and 22, in the case of 2-mercaptobenzimidazole compounds, most compounds exhibited superior pigmentation inhibition effects than kojic acid. Specifically, compounds 2 and 4 exhibited pigmentation inhibition effects similar to those of 0.2 mM PTU at 0.1 mM, compound 8 at 0.2 mM exhibited an effect similar to that of PTU at the same concentration, and compound 3 exhibited superior pigmentation inhibition effects than 0.2 mM PTU at 0.1 mM.

Referring to FIGS. 23 and 24, in the case of 2-mercaptobenzoxazole compounds, most of the compounds exhibited better pigmentation inhibition effects than kojic acid. In particular, compounds 11, 12, 15, and 18 exhibited pigmentation inhibition activities similar to or slightly better than PTU at the same concentration.

Referring to FIGS. 25 to 27, in the case of 2-mercaptobenzoicazole compounds, all compounds showed superior pigmentation inhibition effects than kojic acid, and furthermore, when compared considering the treatment concentration, compounds 19, 20, 27 and 28 showed superior pigmentation effects than PTU.

<Experimental Example 8> Confirmation of the inhibitory effect of 2-mercaptobenzoxazole compounds and 2-mercaptobenzozoazole compounds on tyrosinase protein expression in B16F10 cells

The inhibitory effects of 2-mercaptobenzoxazole (compounds 11-18) and 2-mercaptobenzoicazole (compounds 19-28) on tyrosinase protein expression were analyzed through Western blotting. Cells treated with each compound were lysed with 30 μL NP40 buffer (50 mM Tris-HCl pH 8.0, 1% NP40, Triton X-100, 150 mM NaCl, Protease inhibitor cocktail) for 10 minutes, and the concentration of the extracted protein was measured (BCA protein assay, Pierce, Rockford, IL, USA). Cell proteins treated with each compound were subjected to 9% SDS polyacrylamide gel electrophoresis (PAGE), and the proteins present in the gel were transferred to a PVDF membrane using semi-dry transfer (Bio-Rad Laboratories, Hercules, CA, USA) at 25 V for 10 minutes. The membrane onto which the proteins were transferred was blocked with 5% skim milk (in 10% TBST) solution for 1 hour at room temperature to prevent nonspecific reactions. Afterwards, the primary antibody was reacted overnight at 4°C, and the secondary antibody conjugated to HRP was reacted for 1 hour. Then, the color was developed with ECL reagent SuperSignal West Pico Chemiluminescent Substrate kit (Advansta, San Jose, CA, USA), and each labeled protein was confirmed with a Davinch-Chemi imager (Davinch-K, Seoul, Korea).

As a result, referring to FIGS. 28 and 29, compounds 12-16 decreased the tyrosinase expression level by 53, 24, 44, 43, and 48%, respectively, and compounds 20, 23, 27, and 28 decreased the tyrosinase expression level by 50, 26, 25, and 42%, respectively.

<Experimental Example 9> Confirmation of the inhibitory effect of 2-mercaptobenzoicazole compound on tyrosinase activity in B16F10 cells using L-dopa staining method

The inhibitory effect of compounds 26 and 28 on intracellular tyrosinase activity in B16F10 cells was confirmed by the degree of staining using L-dopa. Each test compound (compounds 26 and 28: 0, 5, 10, 20 μM; kojic acid: 20 μM) was pretreated for 1 hour in a 24-well plate seeded with B16F10 cells, followed by treatment with 200 μM IBMX and 1 μM α-MSH, and then incubated for 72 hours. After the incubation, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 40 minutes, washed twice with PBS, and treated with 0.1% Triton X-100 for 2 minutes. L-dopa (2 mM) (500 μL) per well was stained at 37°C for 2 hours. Photographs were imaged and analyzed using a camera attached to a microscope (Motic, Hong Kong).

Referring to Figure 30, treatment with IBMX and α-MSH significantly increased intracellular tyrosinase activity. Compound 26 inhibited intracellular tyrosinase activity in a concentration-dependent manner, and showed more potent inhibition than kojic acid at the same concentration (20 μM). Compound 28 showed a potent inhibitory effect on intracellular tyrosinase activity even at a concentration of 10 μM.

<Experimental Example 10> Confirmation of copper ion chelating activity of 2-mercaptobenzoxazole compound and 2-mercaptobenzozoazole compound

10-1. Copper chelating activity using pyrocatechol violet reagent

Copper chelating activity was measured using pyrocatechol violet reagent. 2-Mercaptobenzoxazole compounds (compounds 11-18; final concentration: 100 μM) and 2-mercaptobenzoxazole compounds (compounds 19-28; final concentration: 100 μM) were taken, and 280 μL of sodium acetate buffer (50 mM, pH 6.0), 6 μL of pyrocatechol violet (4 mM), and 10 μL of CuSO ₄ (1 mg/mL) were added to each 96-well plate. Each compound and the reaction solution were mixed well, left at room temperature for 20 minutes, and the absorbance was measured at a wavelength of 632 nm using a microplate reader to determine the activity. The copper chelating activity (%) was calculated according to the following Equation 2:

<Formula 2>

Copper chelating activity (%) = [(Abs _control -Abs _sample )/Abs _control ] × 100

Here, Abs _control is the absorbance of the control group and Abs _sample is the absorbance of the compound.

Referring to FIG. 31, eight 2-mercaptobenzoxazole compounds (compounds 11-18) exhibited more potent copper chelating activity than kojic acid and PTU, which were used as positive controls. In particular, compound 14 was shown to be the best chelator with free copper ions. However, in the case of compound 14, when forming a chelate with the copper ion of tyrosinase, the methyl group at position 4 of compound 14 appears to exhibit low tyrosinase inhibitory activity due to steric hindrance with the amino acid residue of tyrosinase.

Referring to Figure 32, among the ten 2-mercaptobenzoicazole compounds (compounds 19-28), five compounds (20, 21, 24, 27, and 28) exhibited more potent copper chelating activity than kojic acid and PTU, which were used as positive controls. In particular, compounds 21 and 27 showed the best chelating ability with free copper ions.

10-2. CuSO ₄ Changes in tyrosinase inhibitory activity in the presence of

To determine whether 2-mercaptobenzoxazole compounds and 2-mercaptobenzoxiazole compounds chelate with copper ion of tyrosinase, we investigated whether the tyrosinase inhibitory activity changed in the presence or absence of CuSO ₄ . Among the eight 2-mercaptobenzoxazole compounds, compounds 11-13, which showed the most potent mushroom tyrosinase inhibitory activity, and among the ten 2-mercaptobenzoxiazole compounds, compounds 20, 21, 26, and 28, which showed potent tyrosinase inhibitory activity, were evaluated for their ability to chelate with copper ion. 10 μL of each target compound (final concentration: 500 nM), 170 μL of phosphate buffer (pH 6.5) containing the substrate, and 50 μL of phosphate buffer containing CuSO ₄ solution (final concentration: 1.25 μM) or 50 μL of phosphate buffer without CuSO ₄ solution were added to each 96-well plate. After incubation for 30 minutes at 37°C, the absorbance was measured at a wavelength of 475 nm using a microplate reader to determine the activity. Tyrosinase activity (%) was calculated according to the following Equation 3:

<Formula 3>

Tyrosinase activity (%) = (Abs _sample /Abs _control ) × 100

Here, Abs _control is the absorbance of the control group and Abs _sample is the absorbance of the compound.

Referring to Figure 33, compounds 11-13 inhibited tyrosinase activity by more than 92% compared to the control group in the treatment group without CuSO ₄ solution, but in the presence of CuSO ₄ solution, compounds 11-13 inhibited tyrosinase activity by only 49-74%. When CuSO ₄ solution was added, compound 13 showed the greatest decrease in tyrosinase inhibition activity.

Referring to FIG. 34, compounds 20, 21, 26, and 28 all exhibited tyrosinase inhibitory activity of more than 87% in the treatment group without CuSO ₄ solution. However, in the presence of CuSO ₄ solution, they exhibited tyrosinase inhibitory activity of only 20-75%. Compound 20, which exhibited the highest tyrosinase inhibitory activity in the absence of CuSO ₄ solution, showed the smallest decrease in tyrosinase inhibitory activity in the presence of CuSO ₄ solution. On the other hand, compound 26, which exhibited the lowest tyrosinase inhibitory activity in the absence of CuSO ₄ solution, showed the largest decrease in tyrosinase inhibitory activity in the presence of CuSO ₄ solution. These results support that benzoxazole and benzoicazole compounds chelate or interact with the copper ion of tyrosinase.

<Experimental Example 11> Confirmation of the browning inhibition effect of 2-mercaptobenzimidazole compounds

An experiment on the prevention of browning of freshly cut apples was conducted. Similar ripened apples (Fuji) were purchased from E-Mart and cut into uniform 1.5 cm grid shapes, and randomly selected pieces were used for the experiment. The sodium salts of compounds 1, 2, 4-10 and the positive control kojic acid or PTU were treated at 5 mM concentrations, respectively, and observed for 0, 12, 24, 36, and 48 h. The apple pieces were stored in an incubator maintained at 20°C for up to 48 h. All samples except PTU were prepared as aqueous solutions. PTU was treated with DMSO solution because of its low solubility. The browning color scale was measured using a CR-10 (Konica Minolta Sensing, Inc., Osaka, Japan) spectrophotometer, and the chromaticity was measured using the average of a* (red and green), b* (yellow and blue), and L* (brightness) values. The total color difference (ΔE) was calculated using the following equation 4:

<Formula 4>

(ΔE) ² = [(aa _initial ) ² + (bb _initial ) ² + (LL _initial ) ² ]

The relative degree of browning was measured using CS Analyzer 3.2 image analysis software.

Referring to Figure 35, the browning inhibition effect was confirmed for each time period. At the 48-hour point, the apples showed a mushy appearance (indicated by a red arrow), and since the mushy appearance was also observed in the group that was not treated with the substance, it appears that the mushy appearance was caused by the apples themselves.

Fig. 36(A) is the result of browning intensity of compounds measured using CS analyzer 3.2 image software based on the photograph of Fig. 35. All 2-mercaptobenzimidazole compounds (compounds 1, 2, and 4-10) showed better browning inhibition effects than kojic acid at 36 hours after material treatment, and compounds 1, 8, and 9 inhibited browning of apples better than PTU, and the remaining compounds showed browning inhibition abilities similar to PTU. In addition, as a result of directly measuring the color index related to browning of apples using a CR-10 spectrophotometer, as shown in Fig. 36(B), almost all compounds showed better ΔE values than kojic acid in the time period except for 48 hours when the wilting phenomenon occurred, and many compounds (compounds 1, 2, 4, 6-10) showed ΔE values similar to or better than PTU within 36 hours. Figure 36(C) shows the change in ΔE value of representative compounds that showed excellent browning inhibition effect. These compounds showed a change in ΔE value that was better than kojic acid and similar to or better than PTU.

While the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific description is merely a preferred embodiment and that the scope of the present invention is not limited thereby. In other words, the actual scope of the present invention is defined by the appended claims and their equivalents.

Claims (29)

A composition for preventing food browning, comprising a compound represented by the following chemical formula 1 or a food-chemically acceptable salt thereof: <Chemical formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, X is selected from oxygen, sulfur, or amino. In paragraph 1, The above compound or a food-based acceptable salt thereof, A composition for preventing browning of food, characterized in that in the above formula, R ¹ , R ² and R ³ are selected from hydrogen, halogen, (C1-C2) alkyl, (C1-C2) alkoxy, trifluoromethyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen or phenyl), or R ² and R ³ are connected to each other to form a 5-cyclic ring or a 1,3-dioxole ring. In paragraph 1, The above compound or a food-based acceptable salt thereof, 1 H -Benzo[ d ]imidazole-2-thiol, 5-Methyl-1 H -benzo[ d ]imidazole-2-thiol, 5,6-Dimethyl- 1 H -benzo [ d ]imidazole-2-thiol, 5-Chloro - 1 H -benzo [ d ]imidazole - 2 - thiol, 5-Methoxy- 1 H -benzo [ d ]imidazole-2-thiol), 2-Mercapto-1 H -benzo[ d ]imidazol-5-yl)(phenyl)methanone, 5-Nitro-1 H -benzo[ d ]imidazole-2-thiol, 5-Fluoro- 1 H -benzo [ d ] imidazole-2-thiol, 4-Methyl-1 H -benzo [ d ] imidazole-2-thiol, 2-Mercapto - 1 H -benzo [ d ]imidazole-5-carbonitrile (2-Mercapto-1 H -benzo[ d ]imidazole-5-carbonitrile), Benzo[ d ]oxazole-2-thiol, 6-Methylbenzo[ d ]oxazole-2-thiol, 5-Methylbenzo [ d ]oxazole-2-thiol, 4-Methylbenzo[ d ]oxazole-2-thiol, 6-Nitrobenzo [ d ] oxazole-2-thiol, 5- Nitrobenzo [ d ]oxazole-2-thiol (5-Nitrobenzo[ d ]oxazole-2-thiol), 4-Nitrobenzo[ d ]oxazole-2-thiol, 6-Chlorobenzo[ d ]oxazole-2-thiol, 4-Methylbenzo[ d ] thiazole-2-thiol, Benzo[ d ] thiazole-2-thiol, 5-(Trifluoromethyl ) benzo[ d ] thiazole-2-thiol, 6,7 - dehydro- 5H -Indeno[5,6- d ]thiazole-2-thiol (6,7-Dihydro-5 H -indeno[5,6- d ]thiazole-2-thiol), 6-Phenoxybenzo[ d ]thiazole-2-thiol, [1,3]Dioxolo[ 4 ′,5′:4,5]benzo[1,2- d ]thiazole-6-thiol, 5,6 - Dimethylbenzo [ d ] thiazole -2-thiol, 5-Chlorobenzo[ d A composition for preventing food browning, characterized in that it comprises at least one selected from the group consisting of 6-methoxybenzo[ d ] thiazole-2-thiol, 6-methoxybenzo[ d ]thiazole-2-thiol, and 6-methylbenzo [ d ]thiazole-2-thiol. In paragraph 1, The above compound or a food-based acceptable salt thereof, A composition for preventing food browning, characterized by inhibiting tyrosinase activity. In paragraph 1, The above compound or a food-based acceptable salt thereof, A composition for preventing food browning, characterized in that it acts as a competitive inhibitor or mixed inhibitor of tyrosinase. In paragraph 1, The above composition, A composition for preventing food browning, characterized in that it contains the compound or a food-chemically acceptable salt thereof at a concentration of 0.0001 to 5000 μM. In paragraph 1, The above foods are, A composition for preventing food browning, characterized in that it is a vegetable or a fresh convenience food using said vegetable. An anti-browning food additive comprising a composition according to any one of claims 1 to 7. A method for preventing browning of food, comprising the step of treating a food with a composition according to any one of claims 1 to 7. A cosmetic composition for skin whitening, comprising a compound represented by the following chemical formula 1 or a pharmaceutically or cosmetically acceptable salt thereof: <Chemical formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, X is selected from oxygen, sulfur, or amino. In Article 10, The above compound or a pharmaceutically or cosmetically acceptable salt thereof, A cosmetic composition for skin whitening, characterized in that in the above formula, R ¹ , R ² and R ³ are selected from hydrogen, halogen, (C1-C2) alkyl, (C1-C2) alkoxy, trifluoromethyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen or phenyl), or R ² and R ³ are connected to each other to form a 5-cyclic ring or a 1,3-dioxole ring. In Article 10, The above compound or a pharmaceutically or cosmetically acceptable salt thereof, 1 H -Benzo[ d ]imidazole-2-thiol, 5-Methyl-1 H -benzo[ d ]imidazole-2-thiol, 5,6-Dimethyl- 1 H -benzo [ d ]imidazole-2-thiol, 5-Chloro - 1 H -benzo [ d ]imidazole - 2 - thiol, 5-Methoxy- 1 H -benzo [ d ]imidazole-2-thiol), 2-Mercapto-1 H -benzo[ d ]imidazol-5-yl)(phenyl)methanone, 5-Nitro-1 H -benzo[ d ]imidazole-2-thiol, 5-Fluoro- 1 H -benzo [ d ] imidazole-2-thiol, 4-Methyl-1 H -benzo [ d ] imidazole-2-thiol, 2-Mercapto - 1 H -benzo [ d ]imidazole-5-carbonitrile (2-Mercapto-1 H -benzo[ d ] imidazole-5-carbonitrile), Benzo[ d ]oxazole-2-thiol, 6-Methylbenzo[ d ]oxazole-2-thiol, 5-Methylbenzo [ d ]oxazole-2-thiol, 4-Methylbenzo[ d ]oxazole-2-thiol, 6-Nitrobenzo [ d ] oxazole-2-thiol, 5- Nitrobenzo [ d ]oxazole-2-thiol (5-Nitrobenzo[ d ]oxazole-2-thiol), 4-Nitrobenzo[ d ]oxazole-2-thiol, 6-Chlorobenzo[ d ]oxazole-2-thiol, 4-Methylbenzo[ d ] thiazole-2-thiol, Benzo[ d ] thiazole-2-thiol, 5-(Trifluoromethyl ) benzo[ d ] thiazole-2-thiol, 6,7 - dehydro- 5H -Indeno[5,6- d ]thiazole-2-thiol (6,7-Dihydro-5 H -indeno[5,6- d ]thiazole-2-thiol), 6-Phenoxybenzo[ d ]thiazole-2-thiol, [1,3]Dioxolo[ 4 ′,5′:4,5]benzo[1,2- d ]thiazole-6-thiol, 5,6 - Dimethylbenzo [ d ] thiazole -2-thiol, 5-Chlorobenzo[ d A cosmetic composition for skin whitening, characterized in that it comprises at least one selected from the group consisting of 6-methoxybenzo[ d ] thiazole-2-thiol, 6-methoxybenzo[ d ]thiazole-2-thiol, and 6-methylbenzo [ d ]thiazole-2-thiol. In Article 10, The above compound or a pharmaceutically or cosmetically acceptable salt thereof, A cosmetic composition for skin whitening, characterized by inhibiting tyrosinase activity. In Article 10, The above compound or a pharmaceutically or cosmetically acceptable salt thereof, A cosmetic composition for skin whitening, characterized in that it acts as a competitive inhibitor or mixed inhibitor of tyrosinase. In Article 10, The above compound or a pharmaceutically or cosmetically acceptable salt thereof, A cosmetic composition for skin whitening, characterized by inhibiting melanin production. In Article 10, The above composition, A cosmetic composition for skin whitening, characterized in that it contains the compound or a pharmaceutically or cosmetically acceptable salt thereof at a concentration of 0.0001 to 5000 μM. A health food composition for skin whitening, comprising a compound represented by the following chemical formula 1 or a food-chemically acceptable salt thereof: <Chemical formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, X is selected from oxygen, sulfur, or amino. A pharmaceutical composition for preventing or treating skin diseases caused by excessive melanin production, comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: <Chemical formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, X is selected from oxygen, sulfur, or amino. In Article 18, The above skin diseases are, A pharmaceutical composition characterized in that it is at least one pigment disorder selected from the group consisting of freckles, lentigos, liver spots, and nevus of Ota. A cosmetic composition for preventing or improving skin diseases caused by excessive melanin production, comprising a compound represented by the following chemical formula 1 or a pharmaceutically or cosmetically acceptable salt thereof: <Chemical formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, X is selected from oxygen, sulfur, or amino. A health food composition for preventing or improving skin disease caused by excessive melanin production, comprising a compound represented by the following chemical formula 1 or a food-based acceptable salt thereof: <Chemical formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, X is selected from oxygen, sulfur, or amino. A feed additive composition for suppressing pigmentation in fish, comprising a compound represented by the following chemical formula 1 or a pharmaceutically or food-wise acceptable salt thereof: <Chemical formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, X is selected from oxygen, sulfur, or amino. In paragraph 22, The above compound or a pharmaceutically or food-wise acceptable salt thereof, A feed additive composition, characterized in that in the above formula, R ¹ , R ² and R ³ are selected from hydrogen, halogen, (C1-C2) alkyl, (C1-C2) alkoxy, trifluoromethyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen or phenyl), or R ² and R ³ are connected to each other to form a 5-cyclic ring or a 1,3-dioxole ring. In paragraph 22, The above compound or a pharmaceutically or food-wise acceptable salt thereof, 1 H -Benzo[ d ]imidazole-2-thiol, 5-Methyl-1 H -benzo[ d ]imidazole-2-thiol, 5,6-Dimethyl- 1 H -benzo [ d ]imidazole-2-thiol, 5-Chloro - 1 H -benzo [ d ]imidazole - 2 - thiol, 5-Methoxy- 1 H -benzo [ d ]imidazole-2-thiol), 2-Mercapto-1 H -benzo[ d ]imidazol-5-yl)(phenyl)methanone, 5-Nitro-1 H -benzo[ d ]imidazole-2-thiol, 5-Fluoro- 1 H -benzo [ d ]imidazole-2-thiol, 4-Methyl - 1 H -benzo [ d ] imidazole-2-thiol, 2-Mercapto - 1 H -benzo [ d ]imidazole-5-carbonitrile (2-Mercapto-1 H -benzo[ d ]imidazole-5-carbonitrile), Benzo[ d ]oxazole-2-thiol, 6-Methylbenzo[ d ]oxazole-2-thiol, 5-Methylbenzo [ d ]oxazole-2-thiol, 4-Methylbenzo[ d ]oxazole-2-thiol, 6-Nitrobenzo [ d ] oxazole-2-thiol, 5- Nitrobenzo [ d ]oxazole-2-thiol (5-Nitrobenzo[ d ]oxazole-2-thiol), 4-Nitrobenzo[ d ]oxazole-2-thiol, 6-Chlorobenzo[ d ]oxazole-2-thiol, 4-Methylbenzo[ d ] thiazole-2-thiol, Benzo[ d ] thiazole-2-thiol, 5-(Trifluoromethyl ) benzo[ d ] thiazole-2-thiol, 6,7 - dehydro- 5H -Indeno[5,6- d ]thiazole-2-thiol (6,7-Dihydro-5 H -indeno[5,6- d ]thiazole-2-thiol), 6-Phenoxybenzo[ d ]thiazole-2-thiol, [1,3]Dioxolo[ 4 ′,5′:4,5]benzo[1,2- d ]thiazole-6-thiol, 5,6 - Dimethylbenzo [ d ] thiazole -2-thiol, 5-Chlorobenzo[ d A feed additive composition characterized in that it comprises at least one selected from the group consisting of 6-methoxybenzo[ d ]thiazole-2-thiol, 6-methoxybenzo[ d ]thiazole-2-thiol, and 6-methylbenzo[ d ]thiazole-2-thiol. In paragraph 22, The above compound or a pharmaceutically or food-wise acceptable salt thereof, A feed additive composition characterized by inhibiting tyrosinase activity. In paragraph 22, The above compound or a pharmaceutically or food-wise acceptable salt thereof, A feed additive composition characterized in that it acts as a competitive inhibitor or mixed inhibitor of tyrosinase. In paragraph 22, The above compound or a pharmaceutically or food-wise acceptable salt thereof, A feed additive composition characterized by inhibiting melanin production. In paragraph 22, The above composition, A feed additive composition characterized in that it comprises the compound or a pharmaceutically or food-wise acceptable salt thereof at a concentration of 0.0001 to 5000 μM. A veterinary pharmaceutical composition for suppressing pigmentation in fish, comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: <Chemical formula 1>

In the above formula, R ¹ , R ² and R ³ may be the same or different and are selected from hydrogen, halogen, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) haloalkyl, nitro, nitrile, phenoxy, or C=OR' (wherein R' is selected from hydrogen, (C1-C4) alkyl, (C3-C6) cycloalkyl, or phenyl), or R ¹ , R ² or R ³ are connected to each other to form a pentacyclic ring, X is selected from oxygen, sulfur, or amino.

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