Uses of mercapto functional compound having tyrosinase inhibitory activity

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

[0001] The present invention relates to the use of a compound having a mercapto functional group, and more particularly, to 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.

[0002] Tyrosinase, known as a rate-regulating enzyme in melanin biosynthesis, has been recognized 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.

[0003] Recent changes in consumer eating habits, increased health awareness, and a growing preference for fresh and natural foods have led to a surge in consumption of fresh-cut foods, primarily vegetables and fruits. These fresh-cut foods undergo processing processes such as peeling, cutting, cored, and slicing to prepare them for immediate consumption. This process reduces their shelf life, generates ethylene gas, facilitates microbial invasion, and promotes oxidative browning.

[0004] Browning, which occurs due to tissue damage in agricultural products, is caused by tyrosinase oxidation, which converts dihydroxyphenylalanine (DOPA) to O-quinone phenylalanine (DOPA-quinone), which then undergoes further oxidation, continuous condensation, and polymerization to produce a dark brown melanin pigment, which significantly affects quality deterioration. Browning serves as a criterion for consumers to judge freshness and also acts as a purchasing criterion, affecting the marketability of agricultural products. In reality, fresh-cut foods show high distribution losses of approximately 25%. Therefore, minimizing distribution losses by suppressing browning is emerging as an important issue for the food industry, including producers and distributors.

[0005] In addition, melanin absorbs a certain amount of ultraviolet rays and blocks harmful ultraviolet rays from penetrating into the body, thereby protecting the body. However, this increases melanin production, which causes skin pigmentation problems. In order to prevent abnormal pigmentation, a method of reducing melanin production 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 frequently used in the past.

[0006] However, among these, kojic acid has the effect of inhibiting enzyme activity by chelating the copper ions present at the active site of tyrosinase, and although it has good performance, its use is limited due to safety issues when mixed into cosmetics. Hydroquinone is highly irritating to the skin, and ascorbic acid also has safety issues.

[0007] Meanwhile, zebrafish have recently been gaining attention in the domestic pharmaceutical industry as an alternative to animal testing for new drug development. They are already widely used in scientific research in Europe. Zebrafish are tropical fish from India, belonging to the cyprinidae family. They are named zebrafish because of the white stripes on their blue bodies, which resemble spots. They are very small, reaching only about two inches in adulthood, and possess a unique fluorescent pigment. They are also widely raised as ornamental fish.

[0008] Zebrafish are used in animal testing because their genetic structure is over 80% similar to that of humans, making them ideal for genetic modification to study human genetic conditions. This means that genetic mutations can be used to study the impact of specific diseases, including rare ones. Furthermore, zebrafish embryos initially lack melanin production and are transparent. However, within a week, melanin production rapidly develops throughout the body, including in the pupils. Therefore, they are used as an in vivo model for the development of melanin-suppressing compounds. Experiments using zebrafish embryo models can avoid animal testing on higher organisms, potentially alleviating some of the ethical concerns surrounding animals. Consequently, interest in methods for suppressing and depigmenting fish pigmentation is growing, enabling various drug testing using fish as an alternative to animal testing.

[0009] 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.

[0010] 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.

[0011] Another object of the present invention is to provide a composition for inhibiting pigmentation in fish using a compound having excellent tyrosinase inhibitory activity and melanin production inhibitory activity.

[0012] 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-related acceptable salt thereof:

[0013] <Chemical Formula 1>

[0014]

[0015] In the above formula, R 1 , R 2 and R 3 are each 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 five-ring ring, and X can be selected from oxygen, sulfur, or amino.

[0016] The present invention provides a food additive for preventing browning comprising the above-described food browning prevention composition.

[0017] In addition, the present invention provides a method for preventing food browning, comprising a step of treating food with the above-described food browning prevention composition.

[0018]

[0019] 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.

[0020] The present invention provides a health food composition for skin whitening, comprising a compound represented by the above chemical formula 1 or a food-wise acceptable salt thereof.

[0021] The present invention provides a pharmaceutical composition for preventing or treating skin diseases caused by excessive melanin production, comprising a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0022] The present invention provides a cosmetic composition for preventing or improving skin diseases caused by excessive melanin production, comprising a compound represented by the above chemical formula 1 or a pharmaceutically or cosmetically acceptable salt thereof.

[0023] In addition, the present invention provides a health food composition for preventing or improving skin diseases caused by excessive melanin production, comprising a compound represented by the above chemical formula 1 or a food-wise acceptable salt thereof.

[0024]

[0025] In order to achieve another object described above, the present invention provides a feed additive composition for suppressing pigmentation in fish, comprising a compound represented by the chemical formula 1 or a pharmaceutically or food-wise acceptable salt thereof.

[0026] In addition, the present invention provides an animal pharmaceutical composition for suppressing pigmentation in fish, comprising a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0027] 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 used as a composition for preventing food browning of fruits and vegetables or fresh-cut foods using the fruits and vegetables.

[0028] By using the above food browning prevention composition, 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.

[0029] 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 used as a skin whitening composition.

[0030] 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 freckles, liver spots, and melasma caused by excessive melanin production.

[0031] 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.

[0032] 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.

[0033] In addition, the composition according to the present invention can also be used to prevent or treat fish diseases related to pigmentation.

[0034] Figure 1 shows 2-mercaptobenzimidazole, 2-mercaptobenzoxazole, and 2-mercaptobenzothiazole compounds synthesized according to embodiments of the present invention.

[0035] 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.

[0036] Figure 3 shows the results of a docking simulation performed using Schrodinger Suite.

[0037] Figure 4 is a Lineweaver-Burk plot obtained through inhibition of mushroom tyrosinase activity by compounds 4, 6, and 10.

[0038] Figure 5 is a Dixon plot obtained by modifying the Lineweaver-Burk plot of Figure 4.

[0039] Figure 6 is a Lineweaver-Burk plot obtained through inhibition of mushroom tyrosinase activity by compounds 11, 12, and 13.

[0040] Figure 7 is a Dixon plot obtained by modifying the Lineweaver-Burk plot of Figure 6.

[0041] Figure 8 is a Lineweaver-Burk plot obtained through inhibition of mushroom tyrosinase activity by compounds 20, 21, 22, 26, and 28.

[0042] Figure 9 is a Dixon plot obtained by modifying the Lineweaver-Burk plot of Figure 8.

[0043] Figure 10 shows the cytotoxicity results of 2-mercaptobenzimidazole compounds in HEK-293 cells.

[0044] Figure 11 shows the cytotoxicity results of 2-mercaptobenzimidazole compounds in B16F10 cells.

[0045] Figure 12 shows the cytotoxicity results of 2-mercaptobenzimidazole compounds in HaCaT cells.

[0046] Figure 13 shows the cytotoxicity results of 2-mercaptobenzoxazole compounds in B16F10 cells.

[0047] Figure 14 shows the cytotoxicity results of 2-mercaptobenzoicazole compounds in B16F10 cells.

[0048] Figure 15 shows the tyrosinase inhibitory activity of a 2-mercaptobenzimidazole compound in B16F10 cells.

[0049] Figure 16 shows the tyrosinase inhibitory activity of a 2-mercaptobenzoxazole compound in B16F10 cells.

[0050] Figure 17 shows the tyrosinase inhibitory activity of 2-mercaptobenzoic azole compounds in B16F10 cells.

[0051] Figure 18 shows the inhibitory effect of 2-mercaptobenzimidazole compound on melanin production in B16F10 cells.

[0052] Figure 19 shows the inhibitory effect of 2-mercaptobenzoxazole compound on melanin production in B16F10 cells.

[0053] Figure 20 shows the inhibitory effect of 2-mercaptobenzoicazole compound on melanin production in B16F10 cells.

[0054] Figure 21 shows the inhibitory effect of 2-mercaptobenzimidazole compound on zebrafish embryo pigmentation.

[0055] Figure 22 is the result of quantifying the effect of Figure 21 using CS analyzer 3.2 image software.

[0056] Figure 23 shows the inhibitory effect of 2-mercaptobenzoxazole compound on zebrafish embryo pigmentation.

[0057] Figure 24 is the result of quantifying the effect of Figure 23 using CS analyzer 3.2 image software.

[0058] Figure 25 shows the inhibitory effect of 2-mercaptobenzoicazole compound on zebrafish embryo pigmentation.

[0059] Figure 26 shows the inhibitory effect of representative 2-mercaptobenzoic azole compounds (compounds 19, 20, 27, and 28), kojic acid, and PTU on zebrafish embryo pigmentation.

[0060] Figure 27 is the result of quantifying the effect of Figure 25 using CS analyzer 3.2 image software.

[0061] Figure 28 shows the effect of 2-mercaptobenzoxazole compound on tyrosinase expression in B16F10 cells.

[0062] Figure 29 shows the effect of 2-mercaptobenzoicazole compound on tyrosinase expression in B16F10 cells.

[0063] Figure 30 shows the inhibitory effect of 2-mercaptobenzoicazole compound on tyrosinase activity in B16F10 cells using L-dopa staining method.

[0064] Figure 31 confirms the copper ion chelating activity of a 2-mercaptobenzoxazole compound.

[0065] Figure 32 confirms the copper ion chelating activity of a 2-mercaptobenzoic azole compound.

[0066] Figure 33 shows the change in tyrosinase inhibitory activity in the presence or absence of CuSO4 to confirm whether the 2-mercaptobenzoxazole compound chelates with the tyrosinase copper ion.

[0067] Figure 34 shows the change in tyrosinase inhibitory activity in the presence or absence of CuSO4 to confirm whether the 2-mercaptobenzoicazole compound chelates with the tyrosinase copper ion.

[0068] Figure 35 shows the browning inhibition effect of a 2-mercaptobenzimidazole compound in apple slices.

[0069] Figure 36 shows the analysis of the degree of browning of apple slices treated with kojic acid, phenylthiourea (PTU), and 2-mercaptobenzimidazole compounds.

[0070] Hereinafter, the present invention will be described in detail.

[0071]

[0072] 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 these compounds have excellent tyrosinase activity inhibitory ability and melanin production inhibitory ability, thereby completing the present invention.

[0073]

[0074] Composition for preventing food browning

[0075] The present invention provides a composition for preventing food browning, comprising a compound represented by the following chemical formula 1 or a food-related acceptable salt thereof:

[0076] <Chemical Formula 1>

[0077]

[0078] In the above formula, R 1 , R 2 and R 3 are each 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 five-ring ring, and X can be selected from oxygen, sulfur, or amino.

[0079] Preferably, the compound or its food-based acceptable salt is R in the formula 1 , R 2 and R 3is 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 Wow R 3 can be linked to each other to form a five-ring ring or a 1,3-dioxole ring.

[0080] More preferably, the compound or a food-based acceptable salt thereof is 1H-Benzo[d]imidazole-2-thiol, 5-Methyl-1H-benzo[d]imidazole-2-thiol, 5,6-Dimethyl-1H-benzo[d]imidazole-2-thiol, 5-Chloro-1H-benzo[d]imidazole-2-thiol, 5-Methoxy-1H-benzo[d]imidazole-2-thiol, 2-Mercapto-1H-benzo[d]imidazol-5-yl)(phenyl)methanone, 5-Nitro-1H-benzo[d]imidazole-2-thiol, 5-Fluoro-1H-benzo[d]imidazole-2-thiol, 4-Methyl-1H-benzo[d]imidazole-2-thiol, 2-Mercapto-1H-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, 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-Dihydro-5H-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 It may be at least one selected from the group consisting of 6-Methoxybenzo[d]thiazole-2-thiol, and 6-Methylbenzo[d]thiazole-2-thiol, but is not limited thereto.

[0081]

[0082] The above compound can be used in the form of a food-acceptable salt within the range having the same efficacy.

[0083] 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 has a safety and efficacy profile suitable for administration to humans.

[0084] The above salt may be used in the form of either a basic salt or an acid salt acceptable for pharmaceutical, cosmetic, or food applications. For example, the basic salt may include any one of an organic base salt and an inorganic base salt, and may be selected from the group consisting of, but not limited to, sodium salt, potassium salt, calcium salt, lithium salt, magnesium salt, cesium salt, aminium salt, ammonium salt, triethylaminium salt, and pyridinium salt. 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. 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.

[0085] The compound may include all salts, hydrates, and solvates that can be prepared by conventional methods, as well as pharmaceutically, cosmetically, or food-wise acceptable salts, and addition salts may be prepared by conventional methods. For example, the compound may be dissolved in a water-miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile, and an excess of an organic base or an aqueous base solution of an inorganic base may be added, followed by precipitation or crystallization. Alternatively, the compound may be prepared by evaporating the solvent or the excess base from the mixture and then drying to obtain an addition salt, or by suction filtration of the precipitated salt.

[0086]

[0087] The compound according to the present invention or a food-related acceptable salt thereof has excellent tyrosinase inhibitory activity and can be used as a composition for preventing food browning.

[0088] In this specification, “browning” refers to a change in color 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.

[0089] 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.

[0090]

[0091] The composition for preventing food browning according to the present invention may 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, may have safe toxicity and excellent tyrosinase inhibitory activity. The above concentration range may vary depending on the type of food and the degree of browning characteristics.

[0092]

[0093] In the composition for preventing food browning according to the present invention, the food may be fruits and vegetables, fresh-cut foods using the fruits and vegetables, fresh-cut agricultural products, etc., but is not limited thereto and may include all types of foods that may exhibit browning.

[0094] 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 a minimum and the quality to be maximized in order to deliver good quality to consumers. It may include simply processed foods that can be eaten as is, such as agricultural or forest products that have gone through processing processes such as washing, peeling, cutting or mincing, or simple foods or food additives added.

[0095]

[0096] The present invention provides a food additive for preventing browning comprising the above-described food browning prevention composition.

[0097] Corresponding features can be substituted for those described above.

[0098]

[0099] In addition, the present invention provides a method for preventing food browning, comprising a step of treating food with the above-described food browning prevention composition.

[0100] 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 they complement each other to more effectively inhibit browning, but is not limited thereto.

[0101] The step of treating the food may be performed by spraying the composition on the surface of the food or immersing the food in the composition, but is not limited thereto.

[0102]

[0103] Whitening composition

[0104] 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:

[0105] <Chemical Formula 1>

[0106]

[0107] In the above formula, R 1 , R 2 and R 3 are each 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 five-ring ring, and X can be selected from oxygen, sulfur, or amino.

[0108] Preferably, the compound or a pharmaceutically or cosmetically acceptable salt thereof is R in the formula 1 , R 2 and R 3 is 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 Wow R 3 can be linked to each other to form a five-ring ring or a 1,3-dioxole ring.

[0109] More preferably, the compound or a pharmaceutically or cosmetically acceptable salt thereof is 1H-Benzo[d]imidazole-2-thiol, 5-Methyl-1H-benzo[d]imidazole-2-thiol, 5,6-Dimethyl-1H-benzo[d]imidazole-2-thiol, 5-Chloro-1H-benzo[d]imidazole-2-thiol, 5-Methoxy-1H-benzo[d]imidazole-2-thiol, 2-Mercapto-1H-benzo[d]imidazol-5-yl)(phenyl) methanone, 5-Nitro-1H-benzo[d]imidazole-2-thiol, 5-Fluoro-1H-benzo[d]imidazole-2-thiol, 4-Methyl-1H-benzo[d]imidazole-2-thiol imidazole-2-thiol), 2-Mercapto-1H-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, 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-Dihydro-5H-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 It may be at least one selected from the group consisting of 6-Methoxybenzo[d]thiazole-2-thiol, and 6-Methylbenzo[d]thiazole-2-thiol, but is not limited thereto.

[0110]

[0111] The above compound can be used in the form of a pharmaceutically, cosmetically, or food-wise acceptable salt within the same efficacy range.

[0112] Corresponding features can be substituted for those described above.

[0113]

[0114] 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.

[0115] In this specification, “whitening” means the action of improving skin troubles by suppressing, inhibiting, or alleviating excessive deposition of melanin pigments, etc., thereby preventing skin darkening caused by such pigmentation.

[0116] 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.

[0117]

[0118] 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.

[0119] The cosmetic composition for skin whitening according to the present invention may 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, may have safe toxicity and excellent tyrosinase inhibitory activity and melanin production inhibitory activity.

[0120]

[0121] The cosmetic composition according to the present invention may further include one or more ingredients from 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 one or more adjuvants commonly used in the cosmetic field, such as organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, fragrances, surfactants, emulsifiers, fillers, metal ion sequestrants, preservatives, vitamins, blocking agents, humectants, dyes and pigments, hydrophilic or lipophilic active agents, or any other ingredients commonly used in cosmetics.

[0122] 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, etc., 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 may be obtained by a conventional method such as a microbial transformation method, a purification method from a microbial culture, an enzymatic method, or a chemical synthesis method.

[0123] The cosmetic composition according to the present invention may be prepared in any formulation commonly manufactured in the technical field to which the present invention pertains. For example, the cosmetic composition may be formulated as a toner, emulsion, lotion, cream, paste, gel, solution, suspension, oil, wax, pack, powder, foundation, spray, surfactant-containing cleanser, etc., but is not limited thereto.

[0124] 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.

[0125] If the above cosmetic composition is in the form of a cream or gel, it may further include animal oil, vegetable oil, wax, paraffin, starch, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide as a carrier component.

[0126] When the above cosmetic composition is in the form of a solution or emulsion, it may further include 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.

[0127] 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.

[0128] If the above cosmetic composition is in a powder or spray form, it may further include lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder as a carrier component, and particularly if it is in a spray form, it may further include a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether.

[0129] 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, a methyl taurate, a sarcosinate, a fatty acid amide ether sulfate, an alkyl amidobetaine, an aliphatic alcohol, a fatty acid glyceride, a fatty acid diethanolamide, a vegetable oil, a linolenic derivative, or an ethoxylated glycerol fatty acid ester.

[0130]

[0131] The cosmetic composition according to the present invention may be applied alone or in combination, or may be applied in combination with other cosmetic compositions other than those of the present invention. Furthermore, the cosmetic composition according to the present invention may be used according to conventional methods, and the frequency of application may vary depending on the user's skin condition or preference.

[0132]

[0133] The present invention provides a health food composition for skin whitening, comprising a compound represented by the following chemical formula 1 or a food-based acceptable salt thereof:

[0134] <Chemical Formula 1>

[0135]

[0136] In the above formula, R 1 , R 2 and R 3 are each 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 five-ring ring, and X can be selected from oxygen, sulfur, or amino.

[0137] Corresponding features can be substituted for those described above.

[0138]

[0139] In the health food composition according to the present invention, the health food can be manufactured in the form of powder, granules, tablets, capsules, syrup, or beverage, and there is no limitation on the form that the health food can take, and all foods in the conventional sense can be included. 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 all functional foods in the conventional sense can be included. In addition, foods used as feed for animals can also be included.

[0140] The health food composition according to the present invention may further comprise food additives and other suitable auxiliary ingredients commonly used in the art. For example, the health food composition may further comprise flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, colorants, pectic acid, alginic acid, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents, and the like.

[0141] In particular, the natural carbohydrates may 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 may include natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame.

[0142] The effective dosage of the compound contained in the health food according to the present invention can be appropriately adjusted depending on the skin whitening purpose. The composition, made from food ingredients, has the advantage of being free of the side effects that can occur with long-term use of conventional medications. Furthermore, it is highly portable and can be taken as a skin whitening supplement.

[0143]

[0144] 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:

[0145] <Chemical Formula 1>

[0146]

[0147] In the above formula, R 1 , R 2 and R 3 are each 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 five-ring ring, and X can be selected from oxygen, sulfur, or amino.

[0148] Corresponding features can be substituted for those described above.

[0149]

[0150] 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 can be used to prevent or treat skin diseases caused by excessive melanin production.

[0151] The skin disease caused by the above melanin overproduction may be one or more pigment diseases selected from the group consisting of, but not limited to, freckles, liver spots, melasma, and Ota's nevus.

[0152] As used herein, "melasma" refers to a pigmentary disorder characterized by the appearance of irregularly shaped, brown spots of various sizes on exposed areas, particularly the face, which may be aggravated by exposure to sunlight, etc. Melanin pigmentation is mainly deposited symmetrically on the cheeks, forehead, and under the eyes.

[0153] 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.

[0154] In this specification, "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.

[0155] 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.

[0156]

[0157] The pharmaceutical composition according to the present invention can be prepared according to conventional methods in the pharmaceutical field. The pharmaceutical composition can be combined with an appropriate pharmaceutically acceptable carrier depending on the formulation, and, if necessary, can be prepared by further including excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. The appropriate carrier, etc., can be selected differently depending on the dosage form and formulation as long as it does not inhibit the activity or properties of the compound or salt thereof according to the present invention.

[0158] The above pharmaceutical composition can be applied in any dosage form, and more specifically, it can be formulated and used as an oral dosage form, an external preparation, a suppository, and a parenteral dosage form of a sterile injection solution according to a conventional method.

[0159] Among the oral dosage forms, the solid dosage form may be in the form of tablets, pills, powders, granules, capsules, etc., and may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may 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 may be further included.

[0160] Among the above oral dosage forms, 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.

[0161] 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, and glycerogelatin. Without limitation, any suitable formulation known in the art may be used.

[0162] In addition, the pharmaceutical composition according to the present invention may further contain calcium or vitamin D3 to enhance therapeutic efficacy.

[0163]

[0164] The pharmaceutical composition according to the present invention can be administered in a pharmaceutically effective amount.

[0165] As used herein, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing adverse effects.

[0166] 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 and sensitivity of the drug, the method of administration, the time of administration, the route of administration, and the excretion rate, the duration of treatment, the drugs used in combination or concurrently, and other factors well known in the medical field. For example, although not fixed, it may generally be administered once or several times daily at a dosage of 0.001 to 100 mg / kg, preferably 0.01 to 10 mg / kg. The above dosage does not limit the scope of the present invention in any way.

[0167] The above pharmaceutical composition may be administered via an appropriate route of administration depending on the formulation, and may be administered via various routes, either oral or parenteral, as long as it can reach the target tissue. The method of administration is not particularly limited, and may be administered by conventional methods such as oral, rectal, intravenous, intramuscular, dermal application, subcutaneous, respiratory inhalation, intrauterine epidural, or intracerebroventricular injection.

[0168] 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.

[0169]

[0170] 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:

[0171] <Chemical Formula 1>

[0172]

[0173] In the above formula, R 1 , R 2 and R 3 are each 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 five-ring ring, and X can be selected from oxygen, sulfur, or amino.

[0174] Corresponding features can be substituted for those described above.

[0175]

[0176] 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.

[0177]

[0178] In addition, the present invention provides a health food composition for preventing or improving skin diseases caused by excessive melanin production, comprising a compound represented by the following chemical formula 1 or a food-based acceptable salt thereof:

[0179] <Chemical Formula 1>

[0180]

[0181] In the above formula, R 1 , R 2 and R 3 are each 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 five-ring ring, and X can be selected from oxygen, sulfur, or amino.

[0182] Corresponding features can be substituted for those described above.

[0183]

[0184] Composition for inhibiting melanin pigmentation in fish

[0185] The present invention provides 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:

[0186] <Chemical Formula 1>

[0187]

[0188] In the above formula, R 1 , R 2 and R 3 are each 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 five-ring ring, and X can be selected from oxygen, sulfur, or amino.

[0189] Preferably, the compound or a pharmaceutically or food-wise acceptable salt thereof is R in the formula 1 , R 2 and R 3 is 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 Wow R 3 can be linked to each other to form a five-ring ring or a 1,3-dioxole ring.

[0190] More preferably, the compound or a pharmaceutically or food-wise acceptable salt thereof is 1H-Benzo[d]imidazole-2-thiol, 5-Methyl-1H-benzo[d]imidazole-2-thiol, 5,6-Dimethyl-1H-benzo[d]imidazole-2-thiol, 5-Chloro-1H-benzo[d]imidazole-2-thiol, 5-Methoxy-1H-benzo[d]imidazole-2-thiol, 2-Mercapto-1H-benzo[d]imidazol-5-yl)(phenyl)methanone, 5-Nitro-1H-benzo[d]imidazole-2-thiol, 5-Fluoro-1H-benzo[d]imidazole-2-thiol, 4-Methyl-1H-benzo[d]imidazole-2-thiol, 2-Mercapto-1H-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, 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-Dihydro-5H-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 It may be at least one selected from the group consisting of 6-Methoxybenzo[d]thiazole-2-thiol, and 6-Methylbenzo[d]thiazole-2-thiol, but is not limited thereto.

[0191]

[0192] The above compound can be used in the form of a pharmaceutically or food-wise acceptable salt within the range having the same efficacy.

[0193] Corresponding features can be substituted for those described above.

[0194]

[0195] The compound according to the present invention or a pharmaceutically or food-wise acceptable salt thereof has excellent tyrosinase inhibitory activity and can be used as a composition for inhibiting pigmentation in fish.

[0196] 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.

[0197] In addition, according to another experimental example of the present invention, the compounds showed excellent inhibitory ability in a pigmentation inhibition ability test using zebrafish embryos, and thus transparent fish can be produced using these, and the produced transparent fish can be used in drug experiments to easily confirm drug toxicity, side effects, and pharmacological effects of drugs.

[0198] The composition according to the present invention may 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, may have excellent tyrosinase inhibitory activity and melanin production inhibitory activity while being safe from toxicity.

[0199]

[0200] In this specification, “feed additive” is a general term for a substance added in small amounts to feed for nutritional or specific purposes, and in the present invention, it refers to a substance added for the purpose of suppressing pigmentation of fish.

[0201] 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 the individual consuming the feed.

[0202] 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.

[0203] 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.

[0204] The above feed additive composition can be added to feed for fish, preferably farmed fish, but is not limited thereto.

[0205] In the feed additive composition according to the present invention, the effective dose of the compound may be used in accordance with the effective dose of the animal pharmaceutical composition, but in the case of long-term intake, it may be below the above range, and since the effective ingredient does not have any problems in terms of safety, it may also be used in an amount above the above range.

[0206] The dosage of the above feed additive composition can be appropriately adjusted by an expert in the field according to the type, age, weight, degree of pigmentation, desired effect, etc. of the fish to be administered.

[0207]

[0208] The present invention provides an animal pharmaceutical composition for suppressing pigmentation in fish, comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

[0209] <Chemical Formula 1>

[0210]

[0211] In the above formula, R 1 , R 2 and R 3 are each 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 five-ring ring, and X can be selected from oxygen, sulfur, or amino.

[0212] Corresponding features can be substituted for those described above.

[0213]

[0214] The animal 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.

[0215] 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, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

[0216] The formulation of the above-mentioned veterinary pharmaceutical composition may be in a preferred 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. For example, the composition 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.

[0217] When the above-mentioned animal pharmaceutical composition is formulated in the above form, diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, 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, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral 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, fragrances, and preservatives may be included. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. 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, cocoa butter, laurin, and glycerogelatin.

[0218]

[0219] The animal pharmaceutical composition according to the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" refers to an amount sufficient to treat a pigmentation-related disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects. 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 concurrently, and other factors well known in the medical field.

[0220] 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 daily. In addition, the dosage of the compound may be increased or decreased depending on the route of administration, degree of pigmentation, etc. Therefore, the dosage does not limit the scope of the present invention in any way.

[0221]

[0222] In addition, the present invention provides a reagent composition for inhibiting tyrosinase activity, comprising the compound or a pharmaceutically or food-wise acceptable salt thereof.

[0223] The above composition may be for competitive inhibition or mixed inhibition of tyrosinase.

[0224]

[0225] In addition, the present invention provides a reagent composition for inhibiting melanin production, comprising the compound or a pharmaceutically or food-wise acceptable salt thereof.

[0226] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0227]

[0228] <Example 1> Synthesis of 2-mercaptobenzimidazole compound (Compound 1-10)

[0229] To synthesize compounds 1-10, two synthetic methods were used, as shown in Scheme 1 below.

[0230] <Reaction Scheme 1>

[0231]

[0232]

[0233] 1-1. Synthesis of compounds 1, 2, 6, 7, and 9

[0234] o-Phenylenediamine compound [1,2-phenylenediamine in the synthesis of compound 1, 3,4-diaminotoluene in the synthesis of compound 2, 3,4-diaminobenzophenone in the synthesis of compound 6, 4-nitro-1,2-phenylenediamine in the synthesis of compound 7, and 2,3-diaminotoluene in 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 CS2 (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%).

[0235]

[0236] 1-2. Synthesis of compounds 3-5 and 8-10

[0237] o-Phenylenediamine compounds [4,5-dimethyl-1,2-phenylenediamine in the synthesis of compound 3, 4-chloro-1,2-phenylenediamine in the synthesis of compound 4, 4-methoxy-1,2-phenylenediamine in the synthesis of compound 5, 4-fluoro-1,2-phenylenediamine in the synthesis of compound 8, 2,3-diaminotoluene in the synthesis of compound 9, and 3,4-diaminobenzonitrile in the synthesis of compound 10] and sodium N,N-diethyldithiocarbamate trihydrate (2.5 equivalents) 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 AlCl3 (0.1 equivalent).

[0238] To obtain the target product after the reaction, the following operations were performed:

[0239] To obtain compound 3, water was added to the reaction mixture, and then 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.

[0240]

[0241] Compound 1:1H-Benzo[d]imidazole-2-thiol

[0242] 1 H NMR (400 MHz, DMSO-d6)δ12.47 (s, 1H, NH), 7.12-7.04 (m, 4H, 4-H, 5-H, 6-H, 7-H); 13 C NMR (100 MHz, DMSO-d6)δ168.7, 132.8, 122.8, 110.0.

[0243] Compound 2: 5-Methyl-1H-benzo[d]imidazole-2-thiol

[0244] 1 H NMR (400 MHz, DMSO-d6) δ12.35 (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, CH3); 13 C NMR (100 MHz, DMSO-d6) δ168.3, 133.0, 132.2, 130.7, 123.7, 110.1, 109.6, 21.5.

[0245] Compound 3: 5,6-Dimethyl-1H-benzo[d]imidazole-2-thiol

[0246] 1 H NMR (400 MHz, DMSO-d6) δ12.25 (brs, 1H, NH), 6.88 (s, 2H, 4-H, 7-H), 2.18 (s, 6H, 2×CH3); 13 C NMR (100 MHz, DMSO-d6)δ167.7, 131.1, 131.0, 110.6, 20.1.

[0247] Compound 4:5-Chloro-1H-benzo[d]imidazole-2-thiol

[0248] 1 H NMR (400 MHz, DMSO-d6)δ7.18-7.10 (m, 3H, 4-H, 6-H, 7-H); 13 C NMR (100 MHz, DMSO-d6) δ174.6, 138.5, 136.5, 132.0, 127.5, 115.8, 114.5.

[0249] Compound 5:5-Methoxy-1H-benzo[d]imidazole-2-thiol

[0250] 1 H NMR (400 MHz, DMSO-d6)δ12.38 (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, OCH3); 13 C NMR (100 MHz, DMSO-d6) δ168.3, 156.3, 133.6, 126.9, 110.5, 110.3, 95.0, 56.1.

[0251] Compound 6:2-Mercapto-1H-benzo[d]imidazol-5-yl)(phenyl)methanone

[0252] 1 H NMR (400 MHz, DMSO-d6)δ7.65 (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); 13C NMR (100 MHz, DMSO-d6) δ195.6, 171.2, 138.4, 137.5, 133.4, 132.6, 131.1, 129.9, 129.0, 125.6, 111.4, 109.8.

[0253] Compound 7: 5-Nitro-1H-benzo[d]imidazole-2-thiol

[0254] 1 H NMR (400 MHz, DMSO-d6) δ8.03 (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-d6) δ172.3, 143.1, 137.9, 132.8, 119.5, 109.8, 105.2.

[0255] Compound 8:5-Fluoro-1H-benzo[d]imidazole-2-thiol

[0256] 1 H NMR (400 MHz, DMSO-d6)δ7.07 (ddd, 1H,J=8.4, 4.8, 0.8 Hz), 6.95-6.88 (m, 2H); 13 C NMR (100 MHz, DMSO-d6)δ169.2, 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).

[0257] Compound 9:4-Methyl-1H-benzo[d]imidazole-2-thiol

[0258] 1H NMR (400 MHz, DMSO-d6) δ6.99 (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, CH3); 13 C NMR (100 MHz, DMSO-d6) δ167.9, 132.3, 131.9, 124.0, 123.1, 120.4, 107.7, 16.7.

[0259] Compound 10:2-Mercapto-1H-benzo[d]imidazole-5-carbonitrile

[0260] 1 H NMR (400 MHz, DMSO-d6)δ7.52-7.48 (m, 2H, 4-H, 6-H), 7.22 (d, 1H,J=8.8 Hz, 7-H); 13 C NMR (100 MHz, DMSO-d6) δ171.2, 136.2, 132.9, 127.5, 119.8, 113.4, 110.7, 104.8.

[0261]

[0262] <Example 2> Synthesis of 2-mercaptobenzoxazole compound (Compound 11-18)

[0263] Compounds 11-18 were synthesized using the same process as the following reaction scheme 2.

[0264] <Reaction Formula 2>

[0265]

[0266]

[0267] 2-1. Synthesis of compounds 11 and 12

[0268] Sodium N,N-diethyldithiocarbamate trihydrate (2.5 equivalents) was added to a round bottom flask containing 2-aminophenol or 2-amino-5-methylphenol, dissolved in DMF (2 mL per 100 mg of starting material), and stirred at 120°C for 30 minutes in the presence of AlCl3 (0.1 equivalent). 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 obtain compounds 11 (79%) and 12 (55%) as solids, respectively.

[0269]

[0270] 2-2. Synthesis of compounds 13-18

[0271] 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 CS2 (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 (solvents used 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%).

[0272]

[0273] Compound 11: Benzo[d]oxazole-2-thiol

[0274] 1 H NMR (400 MHz, DMSO-d6)δ7.45 (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-d6) δ180.7, 148.7, 131.8, 125.7, 124.3, 111.0, 110.5.

[0275] Compound 12:6-Methylbenzo[d]oxazole-2-thiol

[0276] 1 H NMR (400 MHz, DMSO-d6) δ7.29 (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, CH3); 13 C NMR (100 MHz, DMSO-d6) δ180.4, 148.9, 134.2, 129.6, 126.2, 110.7, 110.6, 21.4.

[0277] Compound 13:5-Methylbenzo[d]oxazole-2-thiol

[0278] 1 H NMR (400 MHz, DMSO-d6) δ7.33 (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, CH3); 13 C NMR (100 MHz, DMSO-d6) δ180.7, 146.9, 135.4, 131.8, 124.9, 111.1, 110.1, 21.4.

[0279] Compound 14:4-Methylbenzo[d]oxazole-2-thiol

[0280] 1 H NMR (400 MHz, DMSO-d6) δ7.26 (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, CH3); 13 C NMR (100 MHz, DMSO-d6) δ180.6, 148.4, 130.9, 126.6, 124.2, 121.7, 107.8, 16.6.

[0281] Compound 15:6-Nitrobenzo[d]oxazole-2-thiol

[0282] 1 H NMR (400 MHz, DMSO-d6) δ7.94 (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-d6) δ189.0, 153.8, 150.8, 140.2, 120.3, 112.8, 102.3.

[0283] Compound 16:5-Nitrobenzo[d]oxazole-2-thiol

[0284] 1 H NMR (400 MHz, DMSO-d6) δ7.86 (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-d6) δ185.9, 156.2, 146.1, 143.7, 117.0, 108.4, 107.2.

[0285] Compound 17:4-Nitrobenzo[d]oxazole-2-thiol

[0286] 1 H NMR (400 MHz, DMSO-d6) δ8.03 (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-d6) δ182.1, 150.3, 131.9, 128.3, 123.9, 120.3, 116.3.

[0287] Compound 18:6-Chlorobenzo[d]oxazole-2-thiol

[0288] 1 H NMR (400 MHz, DMSO-d6) δ7.68 (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-d6) δ180.9, 149.0, 131.0, 128.5, 125.7, 111.9, 111.1.

[0289]

[0290] <Example 3> Synthesis of 2-mercaptobenzothiazole compound (Compound 19-28)

[0291] Compounds 19-28 were synthesized using 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.

[0292] <Reaction Formula 3>

[0293]

[0294]

[0295] 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-1H-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, 2-amino-5-methylthiophenol in the synthesis of compound 28] and NaOH (1.16 equivalents) were added to ethanol (2-aminothiophenol The compound (1.8 mL per 1 mmol) and water (0.6 mL per 1 mmol of 2-aminothiophenol compound) were dissolved and CS2 (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 silica gel column chromatography using hexane:ethyl acetate (18:1), dichloromethane:methanol (20:1), and hexane:ethyl acetate (6:1) as solvents, respectively, to obtain pure compounds, respectively. 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%).

[0296]

[0297] Compound 19:4-Methylbenzo[d]thiazole-2-thiol

[0298] 1H NMR (400 MHz, DMSO-d6)δ7.18 (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, CH3); 13 C NMR (100 MHz, DMSO-d6) δ184.3, 155.1, 137.0, 126.4, 125.4, 120.5, 117.0, 19.1.

[0299] Compound 20: Benzo[d]thiazole-2-thiol was purchased from Sigma-Aldrich and used.

[0300] Compound 21:5-(Trifluoromethyl)benzo[d]thiazole-2-thiol

[0301] 1 H NMR (400 MHz, DMSO-d6) δ7.93 (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-d6) δ191.6, 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.

[0302] Compound 22: 6,7-Dihydro-5H-indeno[5,6-d]thiazole-2-thiol

[0303] 1H NMR (400 MHz, DMSO-d6) δ7.19 (s, 1H, 4-H), 7.09 (s, 1H, 8-H), 2.83 (t, 2H,J= 7.2 Hz, 5-H2), 2.80 (t, 2H,J= 7.2 Hz, 7-H2), 1.99 (quint, 2H,J= 7.2 Hz, 6-H2); 13 C NMR (100 MHz, DMSO-d6) δ184.1, 155.4, 140.4, 136.4, 135.5, 114.7, 113.0, 32.8, 32.4, 26.2.

[0304] Compound 23:6-Phenoxybenzo[d]thiazole-2-thiol

[0305] 1 H NMR (400 MHz, DMSO-d6)δ7.32 (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-d6) δ185.0, 158.9, 153.4, 150.5, 138.8, 130.3, 122.9, 117.8, 117.7, 116.9, 110.9.

[0306] Compound 24: [1,3]Dioxolo[4′,5′:4,5]benzo[1,2-d]thiazole-6-thiol

[0307] 1 H NMR (400 MHz, DMSO-d6) δ6.97 (s, 1H, 8-H), 6.79 (s, 1H, 4-H), 5.89 (s, 2H, 2-H2); 13C NMR (100 MHz, DMSO-d6) δ184.0, 150.7, 145.8, 142.9, 128.8, 100.8, 99.7, 98.6.

[0308] Compound 25: 5,6-Dimethylbenzo[d]thiazole-2-thiol

[0309] 1 H NMR (400 MHz, DMSO-d6) δ7.11 (s, 1H, 4-H), 7.02 (s, 1H, 7-H), 2.18 (s, 3H, CH3), 2.15 (s, 3H, CH3); 13 C NMR (100 MHz, DMSO-d6) δ184.1, 155.1, 134.9, 132.3, 128.6, 119.8, 118.3, 20.2, 19.8.

[0310] Compound 26: 5-Chlorobenzo[d]thiazole-2-thiol

[0311] 1 H NMR (400 MHz, DMSO-d6) δ7.69 (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-d6)δ191.4, 142.8, 132.3, 128.8, 124.5, 123.7, 112.5

[0312] Compound 27:6-Methoxybenzo[d]thiazole-2-thiol

[0313] 1 H NMR (400 MHz, DMSO-d6)δ7.33 (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, OCH3);13 C NMR (100 MHz, DMSO-d6) δ188.9, 157.2, 135.8, 131.3, 115.3, 113.6, 106.4, 56.2.

[0314] Compound 28:6-Methylbenzo[d]thiazole-2-thiol

[0315] 1 H NMR (400 MHz, CDCl3)δ11.5 (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, CH3); 13 C NMR (100 MHz, DMSO-d6) δ190.4, 138.2, 135.0, 130.2, 128.4, 121.5, 111.9, 21.3.

[0316]

[0317] <Experimental Example 1> Confirmation of mushroom tyrosinase activity inhibition effect

[0318] To 10 μL of a sample containing 28 synthesized compounds (compounds 1-28) dissolved in DMSO, 170 μL of 100 mM sodium phosphate buffer (pH 6.5) containing 1 mM substrate L-tyrosine (or L-dopa) was added. 20 μL of a 1000 unit / mL mushroom tyrosinase (Sigma) solution was added and reacted at 37°C for 20 minutes. After the reaction, 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) served as positive controls. The tyrosinase inhibition rate (%) was calculated according to the following equation 1:

[0319] <Formula 1>

[0320] Tyrosinase inhibition rate (%) = [(Abs control -Abs sample ) / Abs control ]×100

[0321] The inhibitory activity of each compound is expressed as the sample concentration at which 50% of the maximum inhibitory effect is achieved (IC 50 ) values ​​were calculated and displayed.

[0322]

[0323] Tables 1 to 3 below show the results of analysis of mushroom tyrosinase inhibitory activity of the compounds synthesized in Examples 1 to 3.

[0324]

[0325]

[0326]

[0327] As a result of confirming the tyrosinase inhibitory activity, all compounds showed more potent mushroom tyrosinase inhibitory activity than kojic acid (KA), and in particular, many compounds (1, 2, 4-8, 10-13, 20-28) showed inhibitory activity similar to or superior to phenylthiourea (PTU).

[0328]

[0329] <Experimental Example 2> Docking simulation analysis between the synthesized compound and mushroom tyrosinase.

[0330] 2-1. Docking simulation using AutoDock Vina

[0331] Docking simulations of compounds 1-28 were performed using AutoDock Vina 1.1.2. The 2D structures of compounds 1-28 were generated using ChemDraw Ultra 12.0, and the generated 2D structures were converted to 3D structures using Chem3D Pro 12.0 software. The MM2 energy minimization process was used to find the energetically most stable 3D structure. Kojic acid was used as a positive control, and the 3D X-ray crystal structure of mushroom tyrosinase (A. Bisporus) was obtained from the Protein Data Bank (PDB, 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).

[0332] Table 4 below shows the binding energies between compounds and mushroom tyrosinase measured by AutoDock Vina.

[0333]

[0334] Referring to Table 4 and Figure 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 hydrogen bonding with His244, the CF3 group was predicted to participate in hydrophobic bonding with Ala286 and Val283, and the benzoic azole ring was predicted to participate in hydrophobic bonding with Phe264 and Val283.

[0335]

[0336] 2-2. Docking Simulation Using Schrodinger Suite

[0337] In silico docking simulations were performed to investigate the chemical interactions between the compounds and mushroom tyrosinase. The simulations were performed using Schr Docking simulations were performed using the Dinger Suite (2021-2) docking simulation program. The X-ray crystal structure of mushroom tyrosinase (A. bisporus) was imported from the PDB (ID: 2Y9X) into the Protein Preparation Wizard of Maestro 12.4. The crystal structure was optimized by deleting unnecessary protein chains, adding hydrogen atoms, and removing water molecules located more than 3 Å 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 structures of the in silico docking compounds were imported into Maestro's entry list in CDXML format, and the structures of the docking compounds were optimized using LigPrep before ligand docking. The chemical structures of the compounds were docked onto the tyrosinase Glide grid using the Glide worklist. The binding affinity and protein-ligand interactions were determined using the Glide standard precision method.

[0338] 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, compounds 1, and compounds 2 were predicted to have binding energies of -4.187, -5.394, and -5.615 kcal / mol, respectively. This indicates that compounds 1 and 2 bind more strongly to the enzyme than kojic acid.

[0339]

[0340] <Experimental Example 3> Study on the Mushroom Tyrosinase Enzyme-Substrate Kinetics of Compounds

[0341] Lineweaver-Burk plots of 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. Compound 13 was used at concentrations of 0, 0.2, 0.6, and 1.8 μM, compound 22 at concentrations of 0, 0.5, 1, and 2 μM, and compound 28 at concentrations of 0, 100, 200, and 400 nM, respectively. In a 96-well plate, 20 μL of mushroom tyrosinase enzyme (200 unit / mL), 10 μL of each compound sample at various concentrations, and 170 μL of potassium buffer (pH 6.5) containing various concentrations of L-dopa substrate (0.125–16 mM) were mixed, and the rate of dopachrome synthesis was measured using a microplate reader (VersaMax™, Molecular Devices, Sunnyvale, CA, USA) at 475 nm (△OD475 / min). The maximum rate (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.

[0342]

[0343] The mechanism of action of 2-mercaptobenzimidazole compounds 4, 6, and 10 was determined by measuring the rate of change in mushroom tyrosinase activity in the presence of these compounds. As shown in Fig. 4, the Lineweaver-Burk plot obtained 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.

[0344] 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 exhibit very low inhibition constants of 9.4, 8.6, and 14.8 nM, respectively, confirming that these compounds bind very strongly to tyrosinase.

[0345]

[0346] The mechanism of action of these compounds was confirmed by measuring the change in the activity rate of mushroom tyrosinase in the presence of 2-mercaptobenzoxazole compounds 11, 12, and 13. As shown in Fig. 6, the Lineweaver-Burk plot obtained confirmed that compound 11 was a competitive inhibitor and compounds 12 and 13 were mixed inhibitors.

[0347] In addition, the Lineweaver-Burk plot was modified to obtain the inhibition constant to obtain the Dixon plot, which is shown in Fig. 7. Referring to Fig. 7, compounds 11, 12, and 13 exhibit very low inhibition constants of 0.057, 0.098, and 0.142 μM, respectively, confirming that these compounds bind very strongly to tyrosinase.

[0348]

[0349] The mechanism of action of these compounds was confirmed by measuring the rate change of mushroom tyrosinase activity in the presence of 2-mercaptobenzoicazole compounds 20, 21, 22, 26, and 28. As shown in Fig. 8, the Lineweaver-Burk plot obtained confirmed that all five compounds were mixed inhibitors.

[0350] In addition, the Lineweaver-Burk plot was modified to obtain the inhibition constant to obtain the Dixon plot, which is shown in Fig. 9. Referring to Fig. 9, compounds 20, 21, 22, 23, 26, and 28 exhibit 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.

[0351]

[0352] <Experimental Example 4> Cytotoxicity analysis of compounds

[0353] 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 supplemented with 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.

[0354] For cytotoxicity measurement, each of the three cell types was dispensed into a 96-well culture vessel, and the compounds were treated at each concentration for 24 hours for HEK-293 cells (final concentration: 0–40 μM) and HaCaT cells (final concentration: 0–20 μM), and for 72 hours for B16F10 cells (final concentration: 0–20 μM). Afterwards, 10 μL of 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.

[0355]

[0356] 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 was observed 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, as shown in Figs. 11 and 12, all measured compounds were not toxic.

[0357] 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.

[0358] Referring to Figure 14, in the case of 2-mercaptobenzoicazole compounds, toxicity was examined in B16F10 cells for up to 72 hours, and all compounds showed no toxicity for 72 hours up to a concentration of 20 μM.

[0359]

[0360] <Experimental Example 5> Measurement of intracellular tyrosinase activity

[0361] 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, 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).

[0362] As a result, as shown in Figures 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.

[0363]

[0364] <Experimental Example 6> Confirmation of intracellular melanin content

[0365] 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, and then treated with 200 μM IBMX and 1 μM α-MSH, followed by incubation 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 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.

[0366] 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 even superior melanin production inhibitory activity than kojic acid at the same concentration.

[0367]

[0368] <Experimental Example 7> Confirmation of the pigmentation-inhibiting effect of compounds in zebrafish embryos.

[0369] 7-1. Zebrafish breeding and zebrafish embryo collection

[0370] Wild-type zebrafish (Danio rerio) were provided by the Zebrafish Disease Modeling Center (ZCDM) at Chungnam National University. Adult zebrafish were maintained at 28°C with a 14:10 h light-dark cycle in a closed-circuit filtration tank. Six adult zebrafish were housed in a 20 L tank and fed three times daily. The fish were maintained in a tank maintained at 28.0°C, the optimal growth temperature. Zebrafish embryos were obtained through natural mating. Male and female adult zebrafish were placed in an egg collection tank the day before egg collection, and natural mating was performed the following morning to obtain zebrafish embryos.

[0371]

[0372] 7-2. Pigmentation inhibition experiment using zebrafish embryos

[0373] ① In the case of 2-mercaptobenzimidazole (1-10) compound, 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.

[0374] ② In the case of the 2-mercaptobenzoxazole (11-18) compound, the chorion of the zebrafish embryos collected through natural mating was removed 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, the animals were treated with compound 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). After storage at 28°C for 48 hours, the effects of the test compounds on the pigmentation of the zebrafish embryos were observed at 75 hpf.

[0375] 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 effects of the test compounds on the pigmentation of the zebrafish embryos were observed at 75 hpf.

[0376] 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 under 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.

[0377]

[0378] As a result, referring to Figures 21 and 22, in the case of 2-mercaptobenzimidazole compounds, most compounds exhibited a pigmentation inhibition effect superior to kojic acid. Specifically, compounds 2 and 4 exhibited a pigmentation inhibition effect similar to that 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 a pigmentation inhibition effect superior to that of 0.2 mM PTU at 0.1 mM.

[0379] Referring to Figures 23 and 24, most of the 2-mercaptobenzoxazole compounds exhibited superior pigmentation inhibition effects compared to kojic acid. In particular, compounds 11, 12, 15, and 18 exhibited pigmentation inhibition activities similar to or slightly superior to PTU at the same concentration.

[0380] Referring to Figures 25 to 27, in the case of 2-mercaptobenzoicazole compounds, all compounds showed a superior pigmentation inhibition effect than kojic acid, and also, when compared considering the concentration treated, compounds 19, 20, 27 and 28 showed a superior pigmentation effect than PTU.

[0381]

[0382] <Experimental Example 8> Confirmation of the inhibitory effect of 2-mercaptobenzoxazole and 2-mercaptobenzoicazole compounds on tyrosinase protein expression in B16F10 cells.

[0383] The inhibitory effects of 2-mercaptobenzoxazole (compounds 11-18) and 2-mercaptobenzoicazole (compounds 19-28) on tyrosinase protein expression were analyzed by 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 protein-transferred membrane was blocked with 5% skim milk (in 10% TBST) for 1 hour at room temperature to prevent nonspecific reactions. Primary antibodies were then incubated overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 hour. Color was then developed using the ECL reagent SuperSignal West Pico Chemiluminescent Substrate kit (Advansta, San Jose, CA, USA), and each labeled protein was visualized using a Davinch-Chemi imager (Davinch-K, Seoul, Korea).

[0384] As a result, referring to FIGS. 28 and 29, compounds 12-16 reduced the tyrosinase expression level by 53, 24, 44, 43, and 48%, respectively, and compounds 20, 23, 27, and 28 reduced the tyrosinase expression level by 50, 26, 25, and 42%, respectively.

[0385]

[0386] <Experimental Example 9> Confirmation of the inhibitory effect of 2-mercaptobenzoicazole compound on tyrosinase activity in B16F10 cells using L-dopa staining method.

[0387] The inhibitory effects of compounds 26 and 28 on intracellular tyrosinase activity in B16F10 cells were confirmed by staining with L-dopa. B16F10 cells were seeded in 24-well plates, each of which was pretreated with each test compound (compounds 26 and 28: 0, 5, 10, and 20 μM; kojic acid: 20 μM) for 1 hour, then treated with 200 μM IBMX and 1 μM α-MSH, and incubated for 72 hours. After 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. Staining was performed with 500 μL of L-dopa (2 mM) per well at 37°C for 2 hours. Photographs were imaged and analyzed using a camera attached to a microscope (Motic, Hong Kong).

[0388] 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 exhibited a more potent inhibition than kojic acid at the same concentration (20 μM). Compound 28 exhibited a potent inhibitory effect on intracellular tyrosinase activity even at a concentration of 10 μM.

[0389]

[0390] <Experimental Example 10> Confirmation of the copper ion chelating activity of 2-mercaptobenzoxazole compounds and 2-mercaptobenzozoazole compounds.

[0391] 10-1. Copper chelating activity using pyrocatechol violet reagent

[0392] Copper chelating activity was measured using the pyrocatechol violet reagent. Ten μL of 2-mercaptobenzoxazole compounds (compounds 11-18; final concentration: 100 μM) and 2-mercaptobenzoxazole compounds (compounds 19-28; final concentration: 100 μM) were added to a 96-well plate, along with 280 μL of sodium acetate buffer (50 mM, pH 6.0), 6 μL of pyrocatechol violet (4 mM), and 10 μL of CuSO4 (1 mg / mL), respectively. Each compound and reaction solution were mixed well, left to stand at room temperature for 20 minutes, and the absorbance was measured at 632 nm using a microplate reader. The copper chelating activity (%) was calculated according to the following equation:

[0393] <Formula 2>

[0394] Copper chelating activity (%) = [(Abs control -Abs sample ) / Abs control ]×100

[0395] Abs here control is the absorbance of the control group and Abs sample is the absorbance of the compound.

[0396]

[0397] Referring to Figure 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 showed the best chelation ability 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 experience steric hindrance with the amino acid residue of tyrosinase, resulting in low tyrosinase inhibitory activity.

[0398] 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 were shown to be most capable of chelating free copper ions.

[0399]

[0400] 10-2. Changes in tyrosinase inhibitory activity in the presence of CuSO4

[0401] To determine whether 2-mercaptobenzoxazole compounds and 2-mercaptobenzoicazole compounds chelate with copper ions of tyrosinase, we investigated whether the tyrosinase inhibitory activity changed in the presence or absence of CuSO4. Among the eight 2-mercaptobenzoxazole compounds, compounds 11-13, which showed the most potent mushroom tyrosinase inhibitory activity, and among the ten 2-mercaptobenzoicazole compounds, compounds 20, 21, 26, and 28, which showed potent tyrosinase inhibitory activity, were evaluated for their ability to chelate with copper ions. 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 or not containing CuSO4 solution (final concentration: 1.25 μM) were added to 20 μL of mushroom tyrosinase enzyme aqueous solution (200 unit / mL) in a 96-well plate. After incubation for 30 min at 37°C, the absorbance was measured at a wavelength of 475 nm using a microplate reader and determined. Tyrosinase activity (%) was calculated according to the following Equation 3:

[0402] <Formula 3>

[0403] Tyrosinase activity (%) = (Abs sample / Abs control )×100

[0404] Abs here control is the absorbance of the control group and Abs sample is the absorbance of the compound.

[0405]

[0406] Referring to Figure 33, compounds 11-13 inhibited tyrosinase activity by more than 92% compared to the control group in the treatment group without CuSO4 solution, but in the presence of CuSO4 solution, compounds 11-13 inhibited tyrosinase activity by only 49-74%. When CuSO4 solution was added, compound 13 showed the greatest decrease in tyrosinase inhibitory activity.

[0407] Referring to Figure 34, compounds 20, 21, 26, and 28 all exhibited tyrosinase inhibitory activity of more than 87% in the treatment group without CuSO4 solution. However, in the presence of CuSO4 solution, tyrosinase inhibitory activity was only up to 20-75%. Compound 20, which exhibited the highest tyrosinase inhibitory activity in the absence of CuSO4 solution, showed the smallest decrease in tyrosinase inhibitory activity in the presence of CuSO4 solution. On the other hand, compound 26, which exhibited the lowest tyrosinase inhibitory activity in the absence of CuSO4 solution, showed the largest decrease in tyrosinase inhibitory activity in the presence of CuSO4 solution. These results support that benzoxazole and benzoicazole compounds chelate or interact with the copper ion of tyrosinase.

[0408]

[0409] <Experimental Example 11> Confirmation of the browning inhibition effect of 2-mercaptobenzimidazole compounds.

[0410] An experiment was conducted to prevent browning of freshly cut apples. Similar-ripe Fuji apples were purchased from E-Mart and cut into uniform 1.5-cm grids. Randomly selected pieces were used in the experiment. The apples were treated with 5 mM sodium salts of compounds 1, 2, and 4-10, and the positive controls, kojic acid or PTU, 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. Because PTU has low solubility, it was treated with DMSO. The browning color scale was measured using a CR-10 (Konica Minolta Sensing, Inc., Osaka, Japan) spectrophotometer, and chromaticity was measured using the average of the a* (red and green), b* (yellow and blue), and L* (brightness) values. The total chromaticity difference (ΔE) was calculated as follows:

[0411] <Formula 4>

[0412] (ΔE) 2 = [(aa initial ) 2 + (bb initial ) 2 + (LL initial ) 2 ]

[0413] The relative degree of browning was measured using CS analyzer 3.2 image analysis software.

[0414]

[0415] Referring to Figure 35, the browning inhibition effect was confirmed at each time point. At 48 hours, the apples exhibited a mushy appearance (indicated by the red arrow). Since mushy appearance also occurred in the untreated group, it appears that the mushy appearance was caused by the apples themselves.

[0416] Fig. 36(A) shows the browning intensity results of the 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 wilt 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.

[0417]

[0418] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for preventing food browning, comprising a compound represented by the following chemical formula 1 or a food-related acceptable salt thereof: <Chemical Formula 1> In the above formula, R 1 , R 2 and R 3 are each 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 interconnected to form a five-ring ring, X is selected from oxygen, sulfur, or amino.

2. In paragraph 1, The above compound or its food-grade acceptable salt is, In the above formula, R 1 , R 2 and R 3 is 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 Wow R 3 A composition for preventing food browning, characterized in that the compounds are linked to each other to form a 5-ring ring or a 1,3-dioxole ring.

3. In paragraph 1, The above compound or its food-grade acceptable salt is, 1H-Benzo[d]imidazole-2-thiol, 5-Methyl-1H-benzo[d]imidazole-2-thiol, 5,6-Dimethyl-1H-benzo[d]imidazole-2-thiol, 5-Chloro-1H-benzo[d]imidazole-2-thiol, 5-Methoxy-1H-benzo[d]imidazole-2-thiol, 2-Mercapto-1H-benzo[d]imidazol-5-yl)(phenyl)methanone, 5-Nitro-1H-benzo[d]imidazole-2-thiol, 5-Fluoro-1H-benzo[d]imidazole-2-thiol, 4-Methyl-1H-benzo[d]imidazole-2-thiol, 2-Mercapto-1H-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,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-Dihydro-5H-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, 6-Methoxybenzo[d]thiazole-2-thiol, and A composition for preventing food browning, characterized in that it comprises at least one selected from the group consisting of 6-methylbenzo[d]thiazole-2-thiol.

4. In paragraph 1, The above compound or its food-grade acceptable salt is, A composition for preventing food browning, characterized by inhibiting tyrosinase activity.

5. In paragraph 1, The above compound or its food-grade acceptable salt is, A composition for preventing food browning, characterized in that it acts as a competitive inhibitor or mixed inhibitor of tyrosinase.

6. In paragraph 1, The above composition, A composition for preventing food browning, characterized in that it comprises the compound or a food-related acceptable salt thereof at a concentration of 0.0001 to 5000 μM.

7. In paragraph 1, The above food is, A composition for preventing food browning, characterized in that it is a fresh-cut food product using vegetables or the above vegetables.

8. A food additive for preventing browning comprising a composition according to any one of claims 1 to 7.

9. A method for preventing browning of food, comprising the step of treating food with a composition according to any one of claims 1 to 7.

10. 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 are each 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 interconnected to form a five-ring ring, X is selected from oxygen, sulfur, or amino.

11. In paragraph 10, The above compound or a pharmaceutically or cosmetically acceptable salt thereof, In the above formula, R 1 , R 2 and R 3 is 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 Wow R 3 A cosmetic composition for skin whitening, characterized in that the compounds are interconnected to form a 5-ring ring or a 1,3-dioxole ring.

12. In paragraph 10, The above compound or a pharmaceutically or cosmetically acceptable salt thereof, 1H-Benzo[d]imidazole-2-thiol, 5-Methyl-1H-benzo[d]imidazole-2-thiol, 5,6-Dimethyl-1H-benzo[d]imidazole-2-thiol, 5-Chloro-1H-benzo[d]imidazole-2-thiol, 5-Methoxy-1H-benzo[d]imidazole-2-thiol, 2-Mercapto-1H-benzo[d]imidazol-5-yl)(phenyl) methanone, 5-Nitro-1H-benzo[d]imidazole-2-thiol, 5-Fluoro-1H-benzo[d]imidazole-2-thiol, 4-Methyl-1H-benzo[d] imidazole-2-thiol, 2-Mercapto-1H-benzo[d]imidazole-5-carbonitrile 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,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-Dihydro-5H-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, 6-Methoxybenzo[d]thiazole-2-thiol, and A cosmetic composition for skin whitening, characterized by comprising at least one selected from the group consisting of 6-methylbenzo[d]thiazole-2-thiol.

13. In paragraph 10, The above compound or a pharmaceutically or cosmetically acceptable salt thereof, A cosmetic composition for skin whitening, characterized by inhibiting tyrosinase activity.

14. In paragraph 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.

15. In paragraph 10, The above compound or a pharmaceutically or cosmetically acceptable salt thereof, A cosmetic composition for skin whitening, characterized by inhibiting melanin production.

16. In paragraph 10, The above composition, A cosmetic composition for skin whitening, characterized in that it comprises the compound or a pharmaceutically or cosmetically acceptable salt thereof at a concentration of 0.0001 to 5000 μM.

17. A health food composition for skin whitening, 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 are each 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 interconnected to form a five-ring ring, X is selected from oxygen, sulfur, or amino.

18. 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 are each 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 interconnected to form a five-ring ring, X is selected from oxygen, sulfur, or amino.

19. In paragraph 18, The above skin disease is, A pharmaceutical composition characterized in that it is at least one pigment disease selected from the group consisting of freckles, blemishes, liver spots, and nevus of Ota.

20. 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 are each 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 interconnected to form a five-ring ring, X is selected from oxygen, sulfur, or amino.

21. A health food composition for preventing or improving skin diseases 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 are each 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 interconnected to form a five-ring ring, X is selected from oxygen, sulfur, or amino.

22. 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 are each 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 interconnected to form a five-ring ring, X is selected from oxygen, sulfur, or amino.

23. In paragraph 22, The above compound or its pharmaceutically or food-wise acceptable salt, In the above formula, R 1 , R 2 and R 3 is 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 Wow R 3 A feed additive composition characterized in that the compounds are linked to each other to form a 5-cyclic ring or a 1,3-dioxole ring.

24. In paragraph 22, The above compound or its pharmaceutically or food-wise acceptable salt, 1H-Benzo[d]imidazole-2-thiol, 5-Methyl-1H-benzo[d]imidazole-2-thiol, 5,6-Dimethyl-1H-benzo[d]imidazole-2-thiol, 5-Chloro-1H-benzo[d]imidazole-2-thiol, 5-Methoxy-1H-benzo[d]imidazole-2-thiol, 2-Mercapto-1H-benzo[d]imidazol-5-yl)(phenyl)methanone, 5-Nitro-1H-benzo[d]imidazole-2-thiol, 5-Fluoro-1H-benzo[d]imidazole-2-thiol, 4-Methyl-1H-benzo[d]imidazole-2-thiol, 2-Mercapto-1H-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,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-Dihydro-5H-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, 6-Methoxybenzo[d]thiazole-2-thiol, and A feed additive composition characterized by comprising at least one selected from the group consisting of 6-methylbenzo[d]thiazole-2-thiol.

25. In paragraph 22, The above compound or its pharmaceutically or food-wise acceptable salt, A feed additive composition characterized by inhibiting tyrosinase activity.

26. In paragraph 22, The above compound or its pharmaceutically or food-wise acceptable salt, A feed additive composition characterized in that it acts as a competitive inhibitor or mixed inhibitor of tyrosinase.

27. In paragraph 22, The above compound or its pharmaceutically or food-wise acceptable salt, A feed additive composition characterized by inhibiting melanin production.

28. 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.

29. 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 are each 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 interconnected to form a five-ring ring, X is selected from oxygen, sulfur, or amino.

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