TWI508950B - 3-methyl-2-sulfanyl-2,3-dihydro-1H-imidazole-1-carboxylic acid Tertiary butyl ester and its preparation method and use - Google Patents

Description

3-methyl-2-sulfinyl-2,3-dihydro-1H-imidazole-1-carboxylic acid Tertiary butyl ester and preparation method and use thereof

The present invention relates to a novel compound 3-methyl-2-sulfinyl-2,3-dihydro-1H-imidazol-1-carboxylic acid tert -butyl butyl ester ( tert -butyl 3-methyl-2- Sulfanylidene-2,3-dihydro-1H-imidazole-1-carb oxylate), which has been shown to be effective in inhibiting melanogenesis. The invention also relates to a process for the preparation of this compound and to its use in the preparation of a medicament or cosmetic.

Melanogenesis means that tyrosine is catalyzed by tyrosinase in melanocytes [it is a rate-limiting step of melanin production] and a series of The process of conversion to melanin by redox reaction, which is mainly caused by ultraviolet radiation. When the skin is exposed to ultraviolet light, the reactive oxygen species and free radicals generated in keratinocytes induce the expression of tumor protein 53 (p53), which promotes the pro-brain melanocyte The expression of proopiomelanocortin gene (POMC gene) and release of POMC-derived peptides [ Such as α-melanocyte stimulating hormone (α-MSH), which in turn causes the tyrosinase gene in melanocytes to be expressed, eventually causing melanin to accumulate on the skin.

Melanin protects the skin's hypodermis from photodamage caused by ultraviolet light, but may cause skin disorders such as freckles when melanin is accumulated on the skin or is abnormally distributed. (lentigines), freckle, melasma, age spots, and hyperpigmentation.

For the purpose of skin whitening, most people use the melanin synthesis inhibitors to dilute or remove melanin or dark spots that accumulate on the skin. Most of the currently known melanin synthesis inhibitors regulate melanin production via the following mechanisms of action: (1) before melanin production: for example, inhibition of tyrosinase mRNA transcription (such as C2-neuramin ( C2-ceramide), tretinoin and vanillic acid] and glycosylation [such as calcium D-pantetheine-S-sulfonate (PaSSO3Ca)]; (2) During melanin production: for example, inhibition of tyrosinase activity [such as hydroquinone, arbutin, and kokuic acid], acceleration of tyrosinase degradation [such as Linoleic acid and phenylthiourea, as well as promoting the reduction of dopaquinone [such as Ascorbic acid; and (3) after melanin production: for example, promoting melanin decomposition [such as linoleic acid], inhibiting melanosome transfer [such as niacinamide and silk] A serine protease inhibitor, as well as accelerating skin turnover [such as liquiritin and glycolic acid].

In recent years, the demand for skin whitening agents has increased day by day, but the skin whitening agents used today are still not ideal for diluting or removing melanin. Therefore, researchers in the pharmaceutical and cosmetic industries are working to develop skin whitening agents with better whitening efficacy to meet the needs of the market.

Methimazole [chemically named 2-mercapto-1-methylimidazole] is a thiourea derivative having the following chemical formula (I): Methylthiazolone has the utility of inhibiting the synthesis of thyroid hormone and has been widely used as an antithyroid agent. However, in this clinical use, methotrazol has been found to have hepatotoxicity, as well as thyroid hyperplasia, agranulocytosis, thrombocytopenia, and low blood clotting. Hypoprothrombinemia and bleeding, and will cause damage to the fetus across the placental membranes.

In addition, it has been reported that methylthioxazole is also capable of inhibiting the activity of tyrosinase and peroxidase, and in the skin of brown guinea pigs which are topically administered with methylthioxazole. The phenomenon of depigmentation can be observed. Therefore, methylthioxazole is considered to be useful as a depigmentation agent (Kasraee B. et al. (2002), J. Invest. Dermatol . , 118 :205-207).

In Kasraee B. et al. (2004), J. Invest. Dermatol ., 122: 1338-1341, Kasraee B. et al. used B16 melanocytes to analyze the utility of methotrexate in inhibiting melanin production and found Methylthiazolidine is effective in inhibiting melanin production activity of B16 melanocytes. In addition, Kasraee B. et al. have cytotoxicity comparing methiozole to melanin production inhibitors (including hydroquinone, arbutin, and citric acid) which are known to inhibit the activity of tyrosinase. The results showed that hydroquinone had the highest cytotoxicity, while arbutin, citric acid and methotrexate exhibited cytotoxicity at concentrations above 100 μM, above 100 μM and above 880 μM.

WO 2012/020070 A2 discloses a skin depigmentation composition comprising: one of a de-pigmenting effective amount of a methimazole derivative having the following chemical formula (II); at least one selected From the compounds in the group consisting of: primary antioxidants and secondary antioxidants; and one or more dermatologically acceptable carriers: The definition of each group of the methylthioxazole derivative of the formula (II) is as defined in the specification and patent application of WO 2012/020070 A2. In the examples of this patent, the combination of methylthioxazole and butylated hydroxytoluene (BHT) and the combination of methotrexate and dilauryl thiodipropionate (DLTDP) for brown The depigmentation effect of guinea pig skin has proven to be significantly better than that of methimazole, BHT and DLTDP used alone.

Although the above literature has been reported, there is still a need for pharmaceutical chemists and manufacturers in the pharmaceutical industry to develop novel compounds that can be readily prepared, safely and suitable for the supply of melanin.

Summary of invention

Thus, in a first aspect, the invention provides a compound of the following formula (I):

Or a pharmaceutically acceptable salt thereof.

In a second aspect, the present invention provides a process for the preparation of a compound of formula (I) as described above, which comprises a compound having the following formula (A): Reacts with a compound of the following formula (B):

Where R is selected from the group consisting of: , Cl and Br.

In a third aspect, the present invention provides a composition for inhibiting melanin production comprising a compound of formula (I) as described above or a pharmaceutically acceptable salt thereof.

In a fourth aspect, the present invention provides a use of a compound of formula (I) as described above or a pharmaceutically acceptable salt thereof for the preparation of a medicament or cosmetic for inhibiting melanin production.

In a fifth aspect, the present invention provides a method for suppressing black A method of producing a protein comprising administering to a subject in need of inhibition of melanin production of the skin a compound of formula (I) or a pharmaceutically acceptable salt thereof as described above.

The above and other objects, features and advantages of the present invention will become apparent from

Detailed description of the invention

For the purposes of this specification, it will be clearly understood that the words "comprising" means "including but not limited to" and the words "comprises" have a corresponding meaning.

It is to be understood that if any of the previous publications is quoted here, the prior publication does not constitute an acknowledgement that in Taiwan or any other country, the former publication forms a common general in the art. Part of the knowledge.

All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains, unless otherwise defined. A person skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used to practice the invention. Of course, the invention is in no way limited by the methods and materials described.

In order to develop novel compounds which can be easily prepared and suitable for the supply of melanin production, the Applicant has found through various studies that 3-methyl-2-sulfinyl-2,3-dihydro-1H-imidazole- Tricarboxylic acid tert-butyl ester is effective in inhibiting melanin production.

Thus, the present invention provides a compound of the following formula (I):

Or a pharmaceutically acceptable salt thereof.

According to the invention, the compound of formula (I) may be in the form of its free form or a pharmaceutically acceptable salt thereof. Furthermore, the compound of formula (I) according to the invention may also be present as a stereoisomer or as a solvate represented by a hydrate. Therefore, it is expected that such stereoisomers and solvates will fall within the technical concept of the present invention.

Exemplary pharmaceutically acceptable salts include, but are not limited to, salts with inorganic acids such as hydrogen chloride, hydrogen bromide, sulfuric acid, and phosphoric acid; with organic acids such as acetic acid, maleate Salts of tartrate and methanesulfonate; and salts with amino acids such as arginine, aspartic acid and glutamic acid.

The present invention also provides a process for the preparation of a compound of formula (I) as described above, which comprises a compound having the following chemical formula (A): Reacts with a compound of the following formula (B):

Where R is selected from the group consisting of: , Cl and Br.

The compound of the formula (I) according to the present invention has been confirmed by an in vitro test to effectively inhibit melanin production and tyrosinase activity of skin melanoma cells.

Based on the above, the compound of the formula (I) or a pharmaceutically acceptable salt thereof according to the present invention is expected to have an effect of inhibiting melanin production, and thus is applicable to the preparation of a medicament for inhibiting melanin production. use.

Accordingly, the present invention provides a composition for inhibiting melanin production comprising a compound of the formula (I) as described above or a pharmaceutically acceptable salt thereof.

As used herein, the terms "inhibition of melanogenesis" and "inhibition of melanin synthesis", "depigmenting", "lightening the melanin", "whitening" (whitening), "skin color lightening", "bleaching", "whitening", "brightening", "blackening", and "blackening" can be used interchangeably.

The compositions according to the present invention can be made into a dosage form suitable for parenterally, orally or topically, using techniques well known to those skilled in the art, including, But not limited to: injection (for example, sterile aqueous solution or dispersion), sterile powder, tablet, troche, pills (pill), capsule, external preparation, and the like.

The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier which is widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier can comprise one or more agents selected from the group consisting of solvents, buffers, emulsifiers, suspending agents, decomposers. ), a disintegrating agent, a dispersing agent, a binding agent, an excipient, a stabilizing agent, a chelating agent, a diluent (diluent) A gelling agent, a preservative, a wetting agent, a lubricant, an absorption delaying agent, a liposome, and the like. The selection and quantity of these reagents falls within the professional literacy and routine skills of those skilled in the art.

The composition according to the present invention may be selected from a parenteral route in a group consisting of: intravenous injection, subcutaneous injection (subcutaneous injection). Injection), intraepidermal injection, intradermal injection, and intralesional injection. Preferably, the pharmaceutical product is manufactured in a dosage form suitable for administration by intradermal injection.

The compositions according to the present invention can be made into a dosage form suitable for oral administration using techniques well known to those skilled in the art, including, but not limited to, sterile powders, lozenges ( Tablets, troche, lozenge, pellets, capsules, dispersible powders or granules, solutions, suspensions, emulsions Emulsion), syrup, elixir, slurry, and the like.

Compositions in accordance with the present invention may also be fabricated into an external preparation suitable for topical application to the skin using techniques well known to those skilled in the art, including, but not limited to, emulsions, Gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, Serum, paste, foam, drop, suspension, salve, and bandage.

In accordance with the present invention, the external formulation is prepared by mixing the compositions of the present invention with a base well known to those skilled in the art.

According to the invention, the substrate may comprise one or more additives selected from the group consisting of water, alcohols, glycols, hydrocarbons [such as petroleum jelly and white). White petrolatum, wax (such as paraffin and yellow wax), preserving agents, antioxidants, surfactants, absorption enhancers (absorption) enhancers), stabilizers (stabilizing agents), gelling agent (gelling agents) [such as Carbopol ^® 941 (carbopol ^® 941), microcrystalline cellulose (microcrystalline cellulose) and carboxymethyl cellulose (carboxymethylcellulose)], the activity of Active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusive agents Occlusive agents), emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants Colorant (Colorants), and propellant (PROPELLANTS) and the like. The selection and quantity of these additives falls within the professionalism and routine technology of those skilled in the art.

According to the present invention, the composition may also be used in combination with one or more external use agents selected from the following activities: whitening agents [such as tretinoin, catechins) Catechin, citric acid, arbutin and vitamin C], moisturizers, anti-inflammatory agents, bactericides, ultraviolet absorbers, plant extracts Extracts) [such as aloe extract], skin nutrients, anesthetics, anti-acne agents, antipruritic, analgesic, antibiotics Antidermatitis agents, antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents, antiaging agents, anti wrinkles (antiwrinkle) Agents, anti-seborborheic agents, wound-healing agents, corticosteroids, and hormones. The selection and quantity of these external preparations falls within the professional literacy and routine technology of those who are familiar with the technology.

The present invention also provides a method for inhibiting melanin production comprising administering to a subject in need of inhibition of skin melanin production a compound of formula (I) or a pharmaceutically acceptable salt thereof as described above. Preferably, the method comprises administering to the skin of the individual an effective amount of one of the compounds of formula (I) or a pharmaceutically acceptable salt thereof as described above, and for a sufficient period of time until the melanin is Significantly faded. More preferably, the method further comprises the use of sonophoresis to enhance transdermal delivery of a compound of formula (I) or a pharmaceutically acceptable salt thereof as described above.

As used herein, the terms "administering" and "administering" and "application" are used interchangeably.

As used herein, the term "effective amount" It is meant that when the composition of the present invention is administered to an individual in need of inhibition of melanin production on the skin, it is a safe amount sufficient to provide the desired melanin production-inhibiting effect without causing undesired adverse side effects on the skin.

As used herein, the terms "effective amount", "effective dose", "melanogenesis-inhibiting effective amount", and "melanogenesis-inhibiting effective dose (melanogenesis)" -inhibiting effective dose)" can be used interchangeably.

According to the present invention, the effective amount and frequency of application of the composition vary depending on factors such as the initial condition of the skin region to be inhibited by melanin production and the final melanin production desired to be achieved. Inhibitory effect. In general, when the composition according to the present invention is topically applied to the skin, the effective amount per administration is usually from 0.5 to 1 mg per square centimeter of skin area, about two to three times per day. Whereas when the composition according to the invention is administered orally or parenterally, the effective amount per administration is usually from 0.2 to 0.4 mg/kg body weight, about one to two times per day.

Based on the advantageous effect of a compound of formula (I) as described above in inhibiting melanin production, the present invention also contemplates the compound of formula (I) or a pharmaceutically acceptable salt thereof for use in preparation. The use of cosmetics to inhibit melanin production.

Accordingly, the present invention provides a cosmetic composition comprising a compound of the formula (I) as described above or a pharmaceutically acceptable salt thereof.

According to the present invention, the cosmetic composition may further comprise a cosmetically acceptable adjuvant which is widely used in cosmetic manufacturing techniques. For example, the cosmetically acceptable adjuvant may comprise one or more agents selected from the group consisting of solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, chelating agents, Interface agents, coloring agents, thickening agents, fillers, perfumes, and odor absorbers. The selection and quantity of these reagents falls within the professional literacy and routine skills of those skilled in the art.

In accordance with the present invention, the cosmetic composition can be made into a form suitable for skincare or makeup using techniques well known to those skilled in the art, including, but not limited to, aqueous solutions. , an aqueous-alcohol solution or an oily solution, an oil-in-water type, a water-in-oil type or a composite emulsion , gel, ointment, cream, mask, patch, pack, liniment, powder, aerosol, spray, lotion, serum, paste, foam, dispersion, drops, ad Mousse, sunblock, tonic water, foundation, eyeshadow, makeup remover products, soap, and other body cleansing products Wait.

According to the present invention, the cosmetic composition may also be used in combination with one or more external preparations selected from the following activities: whitening agents (such as vitamin A acid, catechin, citric acid, arbutin, and vitamin C) ), insurance Aerosol, anti-inflammatory, bactericide, UV absorber, plant extract (such as aloe extract), skin nutrient, anesthetic, anti-acne, anti-itching agent, analgesic, anti-dermatitis agent, anti-keratosis Agents, anti-dry skin agents, antiperspirants, anti-aging agents, anti-wrinkle agents, anti-serum spills, wound therapeutics, corticosteroids and hormones. The selection and quantity of these external preparations falls within the professional literacy and routine technology of those who are familiar with the technology.

Other features and effects of the present invention will be apparent from the following description of the drawings, wherein: Figure 1 shows the percentage of cell viability measured by B16F10 cells after treatment with different concentrations of the compounds of the invention. Wherein "*" means: when compared with the control group, p <0.05; and Figure 2 shows melanin production induced by α-melanocyte stimulating hormone (α-MSH) [α-melanocyte stimulating hormone (α-MSH) -induced melanogenesis] The melanin content of B16F10 cells measured after treatment with the compound of the present invention, wherein "*" indicates: p < 0.05 when compared with the pathological control group.

Detailed description of the preferred embodiment

The invention is further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting.

Example General experimental materials: 1. Source and culture of mouse skin melanoma cell line B16F10:

The mouse skin melanoma cell line B16F10 (BCRC 60031) used in the following examples is a biological resource conservation and research center (Biosource) purchased from the Food Industry Research and Development Institute (FIRDI) in Taiwan. Collection and Research Center, BCRC).

B16F10 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) [added with 10% fetal bovine serum (Fetal Bovine Serum, FBS) (Gibco), 100 U/mL penicillin) G (penicillin G) (Gibco), 100 μg/mL streptomycin (Gibco), and 0.25 μg/mL amphotericin (Gibco) in a petri dish, followed by culture conditions The culture was carried out in an incubator set at 37 ° C and 5% CO ₂ . After that, fresh medium was replaced approximately every 2 days. When the cell density reached approximately 80% confluence, the medium was removed and the cells were washed twice with phosphate buffered saline (PBS) (Gibco) followed by trypsin-EDTA (trypsin-EDTA) ) to detach the cells from the bottom of the dish. Thereafter, fresh medium is added to neutralize the activity of trypsin and the medium is repeatedly aspirated by a pipette to fully break up the cells, and then the formed cell suspension is dispensed into a new dish and cultured. The conditions were set to be cultured in an incubator of 37 ° C and 5% CO ₂ .

General experimental method: 1. Statistical analysis:

In the following examples, the experiments of each group were repeated 3 times, and the experimental data was expressed by the mean ± standard deviation (SD). All data were analyzed by Duncan's multiple range test followed by analysis of variance (ANOVA) to assess differences between groups. If the statistical analysis obtained is p < 0.05, this indicates statistical significance.

Example 1. Preparation of 3-methyl-2-sulfinyl-2,3-dihydro-1H-imidazole-1-carboxylic acid tert -butyl ester ( tert -butyl 3-methyl-2-sulfanylidene-2, 3-dihydro-1H-imid azole-1-carboxylate) General operating procedures:

Silica gel column chromatography (silica gel column chromatography) using Geduran ^® Si 60 silica gel (silica gel) [230-400 mesh (mesh), produced by the Merck] as a solid phase (solid phase), and with hexane ( Hexane)/ethyl acetate (EtOAc) (1:1, v/v) was carried out as an eluent.

The melting point of the synthesized compound was detected by a Fargo MP-1D melting point apparatus.

¹ H-NMR and ¹³ C-NMR spectra were detected using a Varian Mercury 400 MHz nuclear magnetic resonance spectrometer, and the chemical shifts expressed in δ (in ppm) were CDCl ₃ (7.24 ppm) is an internal standard, and the coupling constant is expressed in J (in Hz).

High-resolution electron impact mass spectra (HREI-MS) were detected using a Finnigan/Thermo Quest MAT 95XL GC-MS mass spectrometer.

Synthesis procedures:

2-mercapto-1-methylimidazole (400 mg, 3.5 mmol) and K ₂ CO ₃ (968 mg, 7 mmol) were combined with N,N-dimethylformamide (N, In N-dimethylformamide (7 mL), di- tert- butyl dicarbonate (1.1 mL, 5.2 mmol) was added. Then, the resulting mixture was stirred at 60 ° C for 30 minutes under a nitrogen atmosphere, followed by partitioning with ethyl acetate (40 mL) and water (20 mL). Next, the ethyl acetate layer was taken and washed with brine, dried over anhydrous MgSO ₄ and concentrated under vacuum. After that, the residue thus obtained was purified by silica gel column chromatography to give a compound (665 mg, yield: 89%).

The obtained purified compound was subjected to recrystallization after EtOH to carry out the analysis of the melting point, ¹ H-NMR and ¹³ C-NMR spectra and high-resolution electron impact free mass spectrometry described in the above "General Procedure". .

result:

The experimental data measured for the recrystallized compound are summarized as follows: Melting point (Mp): 86 to 87 °C. ¹ H NMR (CDCl ₃ , 400 MHz) δ: 1.59 (s, 9H), 3.54 (s, 3H), 6.58 (d, J = 2.8 Hz, 1H), 7.11 (d, J = 2.8 Hz, 1H). ¹³ C NMR (CDCl ₃ , 100 MHz) δ: 27.7, 34.8, 85.5, 114.4, 118.3, 147.0, 164.8. C ₉ H HRMS calcd about ₁₄ N ₂ O ₂ S is: 214.0776; Found: 214.0784.

Based on the measured spectra and mass spectral data, the compound was confirmed to be the title compound having the following chemical structural formula:

The purified title compound was dissolved in PBS to give a 10 mM stock solution for the next experiment.

Example 2. Cell viability analysis of the compounds of the invention

A stock solution of the compound of the present invention obtained in accordance with Example 1 above was subjected to the following cell viability assay to evaluate the cytotoxicity of the compound of the present invention. Further, for comparison, kojic acid [a known melanogenesis inhibitor] was taken for the same experiment.

experimental method:

First, the B16F10 cells subcultured according to the first item "General experimental materials", "Source and culture of mouse skin melanoma cell line B16F10", were divided into 5 groups, including one control group (control), 2 One tannic acid group (ie, tannic acid group 1 and 2) and two experimental groups (ie, experimental groups 1 and 2). Each group of cells was cultured in a volume of 3×10 ⁴ cells/well in 200 μL of DMEM (added with 10% FBS, 100 U/mL penicillin G, 100 μg/mL streptomycin, and 0.25 μg/mL amphiric The cells were cultured in a 96-well plate and cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours.

Next, the cell cultures of each group were replaced with fresh medium, and then an appropriate amount of the stock solution obtained according to the above Example 1 was separately added to the cell cultures of the experimental groups 1 and 2, so that the experimental group 1 and 2 each having a final concentration of 1 mM and 2 mM of the compound of the invention. In addition, cell cultures of citrate groups 1 and 2 were added with citric acid (10 mM) (Sigma-Aldrich) to a final concentration of 1 mM and 2 mM, respectively, while the cell culture of the control group was left untreated. Each group of cell cultures was cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours, after removing the liquid in each well, followed by the addition of 100 μL of 3-[4,5-dimethylthiazole-2- {3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, MTT} (0.5 mg/mL in PBS) and The cultivation lasted for 40 minutes. Thereafter, the liquid in each well was removed, and then 100 μL of DMSO was added and mixed uniformly, and then the absorbance (OD ₅₇₀ ) of each well was measured by an ELISA reader (Tecan) at a wavelength of 570 nm. .

The cell activity percentage (%) is calculated by substituting the measured absorbance value (OD ₅₇₀ ) into the following formula (1): Formula (1): A = (B/C) × 100

Where: A = percentage of cell viability (%)

B = OD ₅₇₀ absorbance measured by each group

C = OD ₅₇₀ absorbance measured in the control group

Thereafter, the obtained experimental data was analyzed in accordance with the method described in the first item "Statistical Analysis" of the "General Experimental Method" above.

result:

Figure 1 shows the percentage of cell viability measured by B16F10 cells after treatment with different concentrations of the compounds of the invention. As can be seen from Fig. 1, the percentage of cell viability of the experimental groups 1 and 2 did not show significant difference compared with the control group, and the percentage of cell viable activity of the citrate group 2 showed a significant decrease. The results of this experiment show that the compounds of the present invention do not cause damage to cells, are therefore not cytotoxic, and are more safe than skin whitening agents (e.g., citric acid) which are currently used.

Example 3. Evaluation of the utility of the compounds of the invention in inhibiting melanogenesis

In this example, Applicants evaluated the utility of the compounds of the invention in inhibiting melanin production by determining melanin content and tyrosinase activity.

experimental method: A, determination of melanin content:

First, the B16F10 cells subcultured according to the first item "General experimental materials", "Source and culture of mouse skin melanoma cell line B16F10", were divided into 4 groups, including one normal control group (normal control). , 1 pathological control, 1 citrate group and 1 experimental group. Each group of cells was cultured in a volume of 1×10 ⁵ cells/well in 200 μL of DMEM (added with 10% FBS, 100 U/mL penicillin G, 100 μg/mL streptomycin, and 0.25 μg/mL amphiric mold). The culture was carried out in a 24-well plate and cultured in an incubator (37 ° C, 5% CO ₂ ) for 48 hours.

Next, the cell cultures of the pathological control group, the citrate group, and the experimental group were replaced with fresh DMEM [added 10% FBS, 100 U/mL penicillin G, 100 μg/mL streptomycin, and 0.25 μg/mL bismuth mold). And 100nM α-melanocyte stimulating hormone (α-MSH)], and cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours, so that B16F10 cells were induced Melanogenesis. In addition, the cell culture of the normal control group was replaced with fresh DMEM (with 10% FBS, 100 U/mL penicillin G, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin) without α-MSH. The culture was carried out in an incubator (37 ° C, 5% CO ₂ ) for 24 hours.

Next, the cell cultures of each group were replaced with fresh DMEM (with 10% FBS, 100 U/mL penicillin G, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin) without α-MSH. Then, an appropriate amount of the stock solution obtained in accordance with Example 1 above was added to the cell culture of the experimental group, so that the experimental group had a compound of the present invention having a final concentration of 1 mM. In addition, the cell culture of the citric acid group was added with citric acid (10 mM) to a final concentration of 1 mM, while the cell cultures of the normal control group and the pathological control group were left untreated. After the cells of each group were cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours, the cell culture was collected and washed with PBS a total of 2 times, followed by the addition of 200 μL of 1 N NaOH (in 10% DMSO) It was thoroughly mixed and then heat-treated at 80 ° C for 1 hour. Thereafter, the resulting mixture was centrifuged at 3,000 rpm for 3 minutes, followed by 100 μL of the supernatant, and the absorbance (OD ₄₀₅ ) of each well was measured by an ELISA reader at a wavelength of 405 nm.

The melanin content (%) is calculated by substituting the measured absorbance value (OD ₄₀₅ ) into the following formula (2): Formula (2): D = (E/F) × 100

Where: D = melanin content (%)

E = measured OD ₄₀₅ absorbance of each group

F = OD ₄₀₅ absorbance measured in the normal control group

Thereafter, the obtained experimental data was analyzed in accordance with the method described in the first item "Statistical Analysis" of the "General Experimental Method" above.

B. Determination of tyrosinase activity:

An appropriate amount of the stock solution obtained in accordance with Example 1 above was separately added to PBS to obtain a sample to be tested containing different concentrations (including 0.1, 1, 2.5, and 10 mM) of the compound of the present invention. Then, 20 μL of each sample to be tested was taken and added to each well of a 96-well plate. In addition, an equal volume of 5 mM dipotassium hydrogen phosphate was used as a control group. Next, 20 μL of tyrosinase (Sigma-Aldrich, T3824) (212.64 μg/mL in 5 mM dipotassium hydrogen phosphate) and 160 μL of L-tyrosine (Sigma-Aldrich) , T8566) (1 mM in 5 mM dipotassium hydrogen phosphate) was added to each well of the 96-well plate. After thorough mixing, the resulting mixture was placed at 25 ° C for 30 minutes for protection from light. Thereafter, the absorbance (OD ₄₇₅ ) of each well was measured by an ELISA reader at a wavelength of 475 nm.

The compound of the present invention inhibits the concentration of 50% tyrosinase activity (50% inhibition concentration, IC ₅₀ ) by calculating the compound of the present invention to reduce the absorbance of tyrosinase by 50% (with the control group) The concentration of the lower phase is determined from the linear portion of the curve and is expressed as the mean ± SD (n = 3).

result: A, determination of melanin content:

Figure 2 shows the melanin content of B16F10 cells induced by melanin production after treatment with the compounds of the invention. As can be seen from Fig. 2, the melanin content of the normal control group was significantly lower than that of the pathological control group, which indicates that α-MSH successfully induced melanin production of B16F10 cells. Compared with the pathological control group, the melanin content of the experimental group was significantly reduced. The results of this experiment show that the compound of the present invention has an excellent inhibitory effect on melanin production by α-MSH-treated B16F10 cells.

B. Determination of tyrosinase activity:

Compounds of the invention of tyrosinase was determined for IC ₅₀ to 1.45 ± 0.14mM. The results of this experiment show that the compound of the present invention is effective in inhibiting tyrosinase activity.

From the above experimental results, it is understood that the compound of the present invention can effectively inhibit melanin production and tyrosinase activity of skin melanoma cells, thereby achieving skin whitening effect. Accordingly, Applicants believe that the compounds of the present invention have a high potential to develop into a melanogenesis inhibitor (i.e., a skin lightening agent).

All of the patents and documents cited in this specification are hereby incorporated by reference in their entirety. In the event of a conflict, the detailed description of the case (including definitions) will prevail.

While the invention has been described with respect to the specific embodiments of the invention, it will be understood that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the invention be limited only by the scope of the appended claims.

Claims (16)

A compound of the following formula (I): Or a pharmaceutically acceptable salt thereof. A composition for inhibiting melanin production comprising a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof. The composition of claim 2, which is in the form of a dosage form for topical administration. The composition of claim 2, which is in a form for parenteral administration. The composition of claim 2, which is in the form of a dosage form for oral administration. A use of a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for inhibiting melanin production. The use of claim 6, wherein the pharmaceutical product further comprises a pharmaceutically acceptable carrier. The use of claim 6, wherein the pharmaceutical product is in a dosage form for topical administration. The use of claim 6, wherein the pharmaceutical product is in a dosage form for parenteral administration. The use of claim 6, wherein the pharmaceutical product is in a dosage form for oral administration. The use of claim 6, wherein the pharmaceutical product is used in combination with one or more external agents selected from the group consisting of whitening agents, moisturizers, anti-inflammatory agents, bactericides, ultraviolet absorbers, Skin nutrient, anesthetic, anti-acne agent, antipruritic, analgesic, anti-dermatitis agent, anti-keratizer, anti-dry skin agent, anti-perspirant, anti-aging agent, anti-wrinkle agent, anti-sebum spill agent, wound Therapeutic agents, plant extracts, corticosteroids, and hormones. A use of a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a cosmetic for inhibiting melanin production. The use of claim 12, wherein the cosmetic further comprises a cosmetically acceptable adjuvant. The use of claim 12, wherein the cosmetic is in a form for topical administration. The use of claim 12, wherein the cosmetic is used in combination with one or more external agents selected from the group consisting of whitening agents, moisturizers, anti-inflammatory agents, bactericides, ultraviolet absorbers, skins. Nutrients, anesthetics, anti-acne agents, antipruritic agents, analgesics, anti-dermatitis agents, anti-keratizers, anti-dry skin agents, anti-perspirants, anti-aging agents, anti-wrinkle agents, anti-serum spills, wound treatment Agents, plant extracts, corticosteroids and hormones. A process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt thereof as defined in claim 1 which comprises a compound of the formula (A): Reacts with a compound of the following formula (B): Where R is selected from the group consisting of: , Cl and Br.