NOVEL COMPOUND OF 6-METHYL-3-PHENETHYL-3, 4-DIHYDRO-1H- QUINAZOLINE-2-THIONE, ITS PREPARATION AND A DEPIGMENTATION COMPOSITION CONTAINING THE SAME AS AN EFFECTIVE COMPONENT Technical Field The present invention relates to a novel compound of 6-methyl-3-phenethyl- 3, 4-dihydro-lH-quinazoline-2-thione represented by the following formula 1, a preparation method thereof, a medicinal composition for prevention and treatment of hyperpigmentation comprising the same as an effective component, and a skin- whitening cosmetic composition comprising the same as an effective component: Background Art 'The reason why the skin becomes black and darkened is the excessive biosynthesis of melanin, a natural pigment, in melanocyte due to ultraviolet rays, ill- balanced hormones, mental stress, etc. (Nordlund, J. J. et al., The Pigmentation System : Physiology and Pathophysiology. New York, Oxford Univ. Press, pp. 1-1025,1998).

Therefore, a technology to control the melanin biosynthesis comes under the core technology in development of skin-whitening products for preventing excessive melanin pigmentation in the skin or lightening the color of the melanin pigment which has been

already accumulated in the skin to inhibit hyperpigmentation such as discoloration or freckles (Hori, W., Drug Discovery Approaches for Developing Cosmeceuticals.

Advanced Skin Care and Cosmetic Products. Southborough, IBC Library Series, pp.

3.1. 1-3.1. 21,1997).

According to the melanin biosynthesis procedure, tyrosine as a starting material is converted into DOPA and DOPAquinone by tyrosinase. The DOPA undergoes intermediates of 5,6-dihydroxyindole 2-carboxylic acid and 5,6- dihydroxyindole to produce two types of eumelanins. The DOPAquinone reacts with cysteine to produce pheomelanin. The eumelanin derived from 5,6-dihydroindole 2- carboxylic acid is a brown pigment, the eumelanin derived from 5,6-dihydroindole is a black pigment and the pheomelanin is a yellow-red color pigment (Hearing, V. J., FASEB J., 5: 2902-9, 1991 ; Hearing, V. J. , J. Invest. Dermatol., 4: 24-8,1999).

According to studies of gene knockout studies, the lack of tyrosinase causes the albino (white mouse) and the lack of tyrosinase-related protein 2 (TRP-2) causes the slaty. Also, it has been known that the tyrosinase-related protein 1 (TRP-1) plays an important role in development of brown color (Rieder, S., et al., Mamm. Genome, 12: 450-5,2001 ; Tanita, M., et al., J. Hum. Genet., 47: 1-6,2002 ; Kameyama, K., et al., Pigment Cell Res., 8: 97-104,1995). The tyrosinase-related protein 1 does not show any enzymatic activity in the human, though it shows an enzymatic activity of 5,6- dihydroindole 2-carboxylic acid oxidase in the mouse, and is known to participate in the stabilization of tyrosinase (Kobayashi, T., et al., J. Biol. Chem., 272: 31801-5,1998).

The UV rays paly an important role in the synthesis of melanin in the skin.

When the melanocyte is exposed to the UV rays, it activates protein kinase C by diacylglycerol and then, promotes the synthesis of melanin (Gilchrest, B. A., et al.,

Photochem. Photobiol., 93: 1-10,1996 ; Park, H. Y., et al., J. Biol. Chem., 274: 16470-8, 1999). Keratinocyte, when being exposed to solar rays, releases nitrogen monoxide, alpha-melanocyte stimulating hormone, prostaglandin E2 and endothelin-1. These released factors are transferred to the melanocyte, in which nitrogen monoxide increases the synthesis of melanin via signal transduction of cGMP and protein kinase G and the rest of the factors increase the synthesis of melanin via signal transduction of cAMP and <BR> protein kinase A (Romero-Graillet, C. , et al., J. Biol. Chem., 271: 28052-6,1996 ; Rasmussen, N., et al., Neuroendocrinol. Lett., 20: 265-282,1999 ; Scott, M. C., et al., J.

Cell. Sci. 115: 2349-55,2002).

The understanding of the melanin synthesis route is very important in the development of skin-whitening products to inhibit hyperpigmentation (discloration, <BR> freckles, etc. ). Representative examples of melanin biosynthesis inhibitors which are developed so far include kojic acid, arbutin, hydroquinone, vitamin C, mulberry root extract, licorice extract and the like. The kojic acid chelates a copper ion needed for activation of tyrosinase to inhibit the melanin biosynthesis. It has excellent skin- whitening effect. However, recently, it is recognized as a calcinogenic agent and thus, its use is limited. Vitamin C and derivatives thereof show relatively low whitening effect. Hydroquinone is limited in its application in terms of safety due to high skin irritation. In addition, it is known that most of plant extracts show the depigmentation effect only at high concentration but little effect at low concentration.

Therefore, in order to solve the problems involved in the conventional melanin synthesis inhibitors and to seek a more excellent whitening substance, the present inventors have conducted screenings by introducing a multi-polarized activity analysis

to find a novel compound of 6-methyl-3-phenethyl-3, 4-dihydro-lH-quinazoline-2- thione. Also, we have confirmed that medicinal compositions and cosmetic compositions comprising the compound as an effective component have excellent skin- whitening effect and completed the present invention based on these discoveries.

According to the present invention, there are provided a novel compound of 6- methyl-3-phenethyl-3, 4-dihydro-lH-quinazoline-2-thione, a preparation method thereof, and a medicinal composition for prevention and treatment of hyperpigmentation and a skin-whitening cosmetic composition comprising the same as an effective component.

Disclosure of the Invention The present invention is directed to a novel compound of 6-methyl-3- phenethyl-3, 4-dihydro-1H-quinazoline-2-thione represented by the following formula 1, a preparation method thereof, a medicinal composition for prevention and treatment of hyperpigmentation comprising the same as an effective component, and a skin- whitening cosmetic composition comprising the same as an effective component According to the present invention, 6-methyl-3-phenethyl-3, 4-dihydro-1H-

quinazoline-2-thione of the formula 1 may be prepared by reacting 6-methyl-3- phenethyl-3, 4-dihydro-lH-quinazoline-2-one of the formula 4 with Lawesson's reagent as shown in the reaction scheme 1.

Reaction Scheme 1.

In the above reaction scheme 1, 6-methyl-3-phenethyl-3, 4-dihydro-lH- quinazoline-2-one of the formula 4 may be prepared by reducing (5-methyl-2- nitrobenzyl) phenethylamine of the formula 2 to obtain 4-methyl-2- (phenethylaminomethyl) phenylamine of the formula 3, as shown in the following reaction scheme 2, and reacting the product with 1, 1'-carbonyldiimidazole.

Reaction Scheme 2.

In the above reaction scheme 2, (5-methyl-2-nitrobenzyl) phenethylamine of the formula 2 may be prepared by reacting 5-methyl-2-nitrobenzyl chloride with phenethylamine, as shown in the following reaction scheme 3.

Reaction Scheme 3.

The preparation of 6-methyl-3-phenethyl-3, 4-dihydro-lH-quinazoline-2-thione of the formula 1 according to the present invention is summarized as the following reaction scheme 4.

Reaction Scheme 4.

In accordance with another aspect, the present invention is directed to a medicinal composition for prevention and treatment of hyperpigmentation comprising 6-methyl-3-phenethyl-3, 4-dihydro-lH-quinazoline-2-thione. Preferably, the medicinal composition comprises 6-methyl-3-phenethyl-3, 4-dihydro-lH-quinazoline-2-thione in an amount of 0.0001 to 1% by weight based on the total weight of the composition.

The medicinal composition is used for prevention or treatment of melanin hyperpigmentation such as discoloration, freckles, senile pigmentary macule and the like and can be formulated into various formulations with a pharmaceutically acceptable carrier, excipient and/or additive. Representative examples include transdermal formulations such as creams, ointments, gels, lotions, solutions or patches.

In another aspect, the present invention is directed to a skin-whitening cosmetic composition comprising 6-methyl-3-phenethyl-3, 4-dihydro-1H quinazoline-2-thione.

Preferably, the skin-whitening cosmetic composition comprises 6-methyl-3-phenethyl- 3, 4-dihydro-lH-quinazoline-2-thione in an amount of 0.0001 to 1 % by weight based on the total weight of the composition.

The skin-whitening cosmetic composition is used for treatment or prevention of skin pigment anomaly such as discoloration, freckles, senile pigmentary macule and the like and its formulation is not particularly limited. Representative examples of formulation include toners, astringents, nourishing creams, massage creams, essence, packs, gels, lotions, patches, sprays or liquid powders.

The 6-methyl-3-phenethyl-3, 4-dihydro-1H-quinazoline-2-thione according to the present invention synthetically inhibits the melanin biosynthesis via protein kinase C, the melanin biosynthesis via cAMP/protein kinase A, the melanin biosynthesis via cGMP/protein kinase G without cytotoxicity and has skin-whitening effect several hundreds times stronger than kojic acid and arbutin which have been already known to the art.

Brief Explanation of Drawings Fig. la shows the melanin biosynthesis inhibiting effect of 6-methyl-3- phenethyl-3, 4-dihydro-lH-quinazoline-2-thione via cAMP/protein kinase A according to the result of Experimental Example 1 ; Fig. 1 b shows the melanin biosynthesis inhibiting effect of 6-methyl-3- phenethyl-3, 4-dihydro-lH-quinazoline-2-thione via cAMP/protein kinase A, as compared to that of arbutin and kojic acid, according to the result of Experimental Example 1 (IM : the material of the present invention O : Arbutin A : Kojic Acid); Fig. 2a shows the melanin biosynthesis inhibiting effect of 6-methyl-3- phenethyl-3, 4-dihydro-lH-quinazoline-2-thione via cGMP/protein kinase G according to the result of Experimental Example 2; Fig. 2b shows the melanin biosynthesis inhibiting effect of 6-methyl-3-

phenethyl-3, 4-dihydro-lH-quinazoline-2-thione via cGMP/protein kinase G, as compared to that of arbutin and kojic acid, according to the result of Experimental Example 2 (1 : the material of the present invention O : Arbutin A : Kojic Acid); Fig. 3a shows the melanin biosynthesis inhibiting effect of 6-methyl-3- phenethyl-3, 4-dihydro-lH-quinazoline-2-thione via 1-oleoyl-2- acetylglycerol (OAG) /protein kinase C according to the result of Experimental Example 3; Fig. 3b shows the melanin biosynthesis inhibiting effect of 6-methyl-3- phenethyl-3, 4-dihydro-1 H-quinazoline-2-thione via 1-oleoyl-2- acetylglycerol (OAG) /protein kinase C, as compared to that of arbutin, according to the result of Experimental Example 3 (@ : the material of the present invention O : Arbutin); Fig. 4 shows the content of tyrosinase produced by a melanoma B16 cell line treated with alpha-melanocyte stimulating hormone and 6-methyl-3-phenethyl-3,4- dihydro-lH-quinazoline-2-thiones at the same time in DMEM supplemented with 10% fetal bovine serum, relative to 100 of the content of tyrosinase produced by a melanom B16 cell line treated only with alpha-melanocyte stimulating hormone, according to the result of Experimental Example 4; Fig. 5a shows the cell growth of a melanoma B16 cell line group (None) propagated in DMEM supplemented with 10% fetal bovine serum and the cell growth of a melanoma B16 cell line group (a-MSH) proliferated in the same medium with alpha-melanocyte stimulating hormone added, according to the result of Experimental Example 5 (@ : None O : a-MSH only); and Fig. 5b shows the number of surviving cells of melanoma B16 cell line group

proliferated in DMEM supplemented with 10% fetal bovine serum with alpha- melanocyte stimulating hormone added and the number of surviving cells of melanoma B16 cell line group proliferated in the same medium with alpha-melanocyte stimulating hormone and 6-methyl-3-phenethyl-3, 4-dihydro-1H-quinazoline-2-thione (concentration 0.5 to 4 li M) added, at the fourth day of cultivation, according to the result of Experimental Example 5.

Best mode for carrying out the invention Now, the present invention will be illustrated in detail through the following Examples, Formulation Examples and Experimental Examples. However, the present invention is not limited thereto.

Example 1: Preparation of 6-methyl-3-phenethyl-3, 4-dihydro-lH-quinazoline-2- thione Example 1-1: Synthesis of (5-methyl-2-nitrobenzyl) phenethylamine Phenethylamine (2 eq. wt. , 3.38 ml) and triethylamine (3 eq. wt. , 5.6 ml) were dissolved in tetrahydrofuran and 5-methyl-2-nitrobenzyl chloride (2.5 g, 0.0135 mole) dissolved in tetrahydrofuran was added thereto. The mixture was stirred at room temperature for 10 minutes and refluxed at 60 to 70 °C for 12 hours. The reaction was cooled to ambient temperature, followed by vacuum distillation. The organic layer was dehydrated with anhydrous sodium sulfate, vacuum distilled and separated on a column.

The characteristics of (5-methyl-2-nitrobenzyl) phenethylamine are as follows.

Orange oil; Rf=0. 26 (hexane: ethyl acetate=2 : 1) ; Yield 84.8% ; IR (KBr) 3350,3050, 3020,2930, 2850,1610, 1585,1520, 1455,1340, 1265, 1120cm'' ; 'H NMR (CDCl3, 89.45MHz) S 2.40 (s, 3H), 2.88 (m, 4H), 4.03 (s, 2H), 7.13- 7.93 (m, 8H) Example 1-2: Synthesis of 4-methyl-2- (phenethylaminomethvl) phenylamine (5-methyl-2-nitrobenzyl) phenethylamine (3 g) was dissolved in a mixture solution of dichloromethane and methanol (1: 1). Pd carbon (240 mg) was added thereto and the mixture was stirred under a hydrogen pressure of 30 psi for 4 hours.

The reaction was filtered by suction through a celite cake and washed with dichloromethane. The filtrate was vacuum distilled to obtain 4-methyl-2- (phenethylaminomethyl) phenylamine. The characteristics of the product are as follows.

Brown oil; Rf=0. 045 (hexane: ethyl acetate=2: 1) ; Yield 89.7% ; IR (KBr) 3550-3200,3025, 2930,2895, 2750,1620, 1500,1455, 1265cm-1 'H NMR (CDC13, 89.45MHz) 8 2.20 (s, 3H), 2.83 (m, 2H), 2.91 (s, 2H), 3.91 (m, 2H), 4.27 (br, NH2), 6.41-7. 21 (m, 8H) Example 1-3: Synthesis of 6-methyl-3-phenethyl-3, 4-dihydro-1H-quinazoline-2-one 4-methyl-2- (phenethylaminomethyl) phenylamine (2 g, 0.00832 mole) was dissolved in anhydrous tetrahydrofuran. 1, 1'-carbonylimidazole (1.1 eq. wt. , 1.48 g)

was added thereto. The mixture was refluxed at 50 to 60C for 12 hours.

Tetrahydrofuran was removed, followed by extraction with hydrated dichloromethane.

The organic layer was dehydrated with anhydrous sodium sulfate, vacuum distilled and separated on a column. The characteristics of 6-methyl-3-phenethyl-3, 4-dihydro-lH- quinazoline-2-one are as follows.

Yellow solid; Rf=0. 34 (hexane: ethyl acetate=5: 1) ; Yield 81. 9% ; mp 174.8-175. 8 C ; IR (KBr) 3200,3100-3000, 2920,1650, 1610,1500, 1455,1320, 1270,1240, 1180cell ; 'H NMR (CDC13, 89.45MHz) S 2.25 (s, 3H), 2.95 (t, J=7. 5 Hz, 2H), 3.67 (t, J=7.5 Hz, 2H), 4. 31 (s, 2H), 6.57-7. 01 (m, 3H), 7.26 (m, 5H), 7.88 (br, NH) Example 1-4: Synthesis of 6-methvl-3-phenethyl-3, 4-dihvdro-1H-quinazoline-2-thione 6-methyl-3-phenethyl-3, 4-dihydro-lH-quinazoline-2-one (880 mg, 0.0033 mole) was dissolved in toluene and heated. The Lawesson's reagent (0.6 eq. wt. , 802 mg) was added thereto. The mixture was refluxed at 100°C for 8 hours. Toluene was removed by vacuum distillation, followed by extraction with hydrated dichloromethane. The organic layer was dehydrated with anhydrous sodium sulfate, vacuum distilled and separated on a column. The characteristics of 6-methyl-3- phenethyl-3, 4-dihydro-lH-quinazoline-2-thione are as follows.

Light yellow solid; Rt=0. 63 (hexane: ethyl acetate=2: 1) ; Yield 47. 2 % ; mp 177.2-177. 7°C ; IR (KBr) 3200,3020, 2920,1620, 1550-1480,1450, 1325,1265, 1240,1175, 1150cm'1 ;

'H NMR (CDCI3, 89.45MHz) 5 2.27 (s, 3H), 3.10 (t, J=7.7 Hz, 2H), 4.12 (t, J=7. 7 Hz, 2H), 4.36 (s, 2H), 6.60-7. 02 (m, 3H), 7.28 (m, 5H), 8.48 (br, NH); Anal. Calcd. for C17H18N2S C, 72.3 ; H, 6.42 ; N; 9.92 ; S, 11.3 Found: C, 71.9 ; H, 6.89 ; N, 8.64 ; S, 13.1 Formulation of Composition Formulation 1. Medicinal composition for prevention and treatment of hyperpigmentation comprising 6-methyl-3-phenethyl-3, 4-dihydro-lH-quinazoline- 2-thione-Ointment Components Content 6-methyl-3-phenethyl-3, 4-dihydro-1 H 0. 5g quinazoline-2-thione White wax 4.0 g Cholesterol 1. 6 g White Vaseline Balance Cetanol 1. 6 g Total 100 g

Formulation 2. Medicinal composition for prevention and treatment of hyperpigmentation comprising 6-methyl-3-phenethyl-3, 4-dihydro-lH-quinazoline- 2-thione-Gel Components Content 6-methyl-3-phenethyl-3, 4-dihydro-1 H-0. 5 g quinazoline-2-thione Ethanol 18 g Kaolin 5 g 1,3-butandiol 5 g Glycerine 8 g CMC-Na Gelatine 3 g Methylparabene 0.1 g Propylparabene 0.2 g Purified water Balance Total 100 g Formulation 3. Skin-whitening cosmetic composition comprising 6-methyl-3- phenethyl-3, 4-dihydro-lH-quinazoline-2-thione-Nourishing cream Components Content Triethanolamine 0.1 g Glycerine 3. 0 g Butylene glycol 3.0 g Liquid Paraffin 7.0 g Beta-Glucan 7.0 g Carbomer 0. 1 g 6-methyl-3-phenethyl-3, 4-dihydro-1 H-0. 5g quinazoline-2-thione Caprylic capric Triglyceride 3. 0 g Squalane 5.0 g Cetearyl glucoside 1.5 g Sorbitan stearate 0. 4 g Polysorbate 60 1. 2 g Methylparabene 0.1 g Propylparabene 0.1 g Flavoring 0.1 g Purified water Balance Total 100 g Formulation 4. Skin-whitening cosmetic composition comprising 6-methyl-3- phenethyl-3, 4-dihydro-lH-quinazoline-2-thione-Pack Components Content Ethanol 6.0 g Glycerine 4.0 g Polyvinyl alcohol 15.0 g Hyaluronic acid extract 5.0 g Beta-glucane 7.0 g Allantoin 0.1 g 6-methyl-3-phenethyl-3, 4-dihydro-1 H 0. 5g quinazoline-2-thione Nonyl phenyl ether 0.4 g Polysorbate 60 1. 2 g Methylparabene 0.2 g Propylparabene 0.1 g Flavoring 0.1 g Purified water Balance Total 100 g

Experimental Example 1: Inhibiting effect on melanin production via cAMP/protein kinase A A melanoma B16 cell line treated with alpha-melanocyte stimulating hormone produces melanin by signal transduction of cAMP/protein kinase A (Rasmussen, N., et al., Neuroendocrinol. Lett., 20: 265-282,1999 ; Scott, M. C., et al., J. Cell. Sci.

115: 2349-55,2002). The melanoma B16 cells were seeded in DMEM supplemented with 10% fetal bovine serum at a density of 2,500/well of a 96-well plate, under conditions of 37C and 5% CO2. After Incubation of 24 hours, the cells were divided into a non-treatment group, a group treated with alpha-melanocyte stimulating hormone

and a group treated with alpha-melanocyte stimulating hormone (final concentration 1 nM) and 6-methyl-3-phenethyl-3, 4-dihydro-1H-quinazoline-2-thione as an effective agent at the same time. Each group was cultured for 3 days and the amount of melanin released into the medium was determined by measurement of the absorption at wavelength 405 nm.

As a result, the melanoma B16 cells cultured for 3 days in the non-treatment group released 33. 2 1. 4 ug/ml of melanin and the cells cultured in the group treated with alpha-melanocyte stimulating hormone released 143. 9 1.6 llg/ml of melanin.

The cells of the group treated with both alpha-melanocyte stimulating hormone and 6- methyl-3-phenethyl-3, 4-dihydro-lH-quinazoline-2-thione as an effective agent released 37. 9 0. 8//gel of melanin at an agent concentration of 4 u M, 44. 4 9. 5 ug/ml of melanin at 2 u M, 101. 1 1. 0 ug/ml of melanin at 1 u M, 126. 5 5. 5 ug/ml of melanin at 0.5 li M, that is, the cells showed dose-dependently the whitening effect (Fig. la). The concentration at which 50% whitening effect was observed (ECso) was 1.5 u M. In the same experimental model, arbutin and kojic acid showed EC50 values of the whitening effect of 151 11 M and 261 li M, respectively. Therefore, it was noted that the depigmentation effect of 6-methyl-3-phenethyl-3, 4-dihydro-lH- quinazoline-2-thione on the melanin production of melanoma B16 cell line by signal transduction of cAMP/protein kinase A was 100 times higher than arbutin and 170 times higher than kojic acid in terms of the ECso value (Fig. I b).

Experimental Example 2: Inhibiting effect on melanin production via cGMP/protein kinase G A melanoma B 16 cell line stimulated with protoporphyrin IX produces melanin

via cGMP/protein kinase G using nitrogen monoxide (Romero-Graillet, C. , et al., J.

Biol. Chem., 271: 28052-6,1996). The melanoma B16 cells were seeded in DMEM supplemented with 10% fetal bovine serum at a density of 2, 500/well of a 96-well plate, under conditions of 37 C and 5% CO2. After Incubation of 24 hours, the cells were divided into a non-treatment group, a group treated with protoporphyrin IX and a group treated with protoporphyrin IX (final concentration 30 u M) and 6-methyl-3- phenethyl-3, 4-dihydro-1H-quinazoline-2-thione as an effective agent at the same time.

Each group was cultured for 3 days and the amount of melanin released into the medium was determined by measurement of the absorption at wavelength 405 nm.

As a result, the melanoma B16 cells cultured for 3 days in the non-treatment group released 43. 4 0. 8 ug/ml of melanin and the cells of the group treated with protoporphyrin IX released 135. 2 1. 9//gel of melanin. The cells of the group treated with both protoporphyrin IX and 6-methyl-3-phenethyl-3, 4-dihydro-1H quinazoline-2-thione as an effective agent released 52. 5 5. 8 ug/ml of melanin at an agent concentration of 1 li M, 94. 4 6. 0 ug/ml of melanin at 0.5 M, 105. 2 0.9 . ug/ml of melanin at 0.25 p M, 118. 7 3. 2//gel of melanin at 0. 13 11 M, that is, the cells showed dose-dependently the whitening effect (Fig. 2a). The concentration at which 50% whitening effect was observed (ECso) was 0.5 u M. In the same experimental model, arbutin and kojic acid showed EC50 values of the whitening effect of 159 li M and 122 11 M, respectively. Therefore, it was shown that the depigmentation effect of 6-methyl-3-phenethyl-3, 4-dihydro-lH-quinazoline-2-thione on melanin production of melanoma B 16 cell line by signal transduction of cGMP/protein kinase G was 310 times higher than arbutin and 240 times higher than kojic acid in terms of the EC50 value (Fig. 2b).

Experimental Example 3: Inhibiting effect on melanin production via 1-oleoyl-2- acetylglycerol/protein kinase C A melanoma B16 cell line stimulated with 1-oleoyl-2-acetylglycerol produces melanin via protein kinase C (Gilchrest, B. A., et al., Photochem. Photobiol., 93: 1-10, 1996; Park, H. Y., et al., J. Biol. Chem., 274: 16470-8,1999). The melanoma B16 cells were seeded in DMEM supplemented with 10% fetal bovine serum at a density of 2,500/well of a 96-well plate, under conditions of 37 °C and 5% C02. After Incubation of 24 hours, the cells were divided into a non-treatment group, a group treated with 1-oleoyl-2-acetylglycerol and a group treated with 1-oleoyl-2- acetylglycerol (final concentration 200 li M) and 6-methyl-3-phenethyl-3,4-dihydro- 1H-quinazoline-2-thione as an effective agent at the same time. Each group was cultured for 3 days and the amount of melanin released into the medium was determined by measurement of the absorption at wavelength 405 nm.

As a result, the melanoma B16 cells cultured for 3 days in the non-treatment group released 49. 8 1.0 llg/ml of melanin and the group treated with 1-oleoyl-2- acetylglycerol released 109. 4 0. 2//gel of melanin. The group treated with both 1- oleoyl-2-acetylglycerol and 6-methyl-3-phenethyl-3, 4-dihydro-lH-quinazoline-2-thione as an effective agent released 57. 7 2. 9 ug/ml of melanin at an agent concentration of 0.5 Li M, 82. 2+ 6.1 ug/ml of melanin at 0.25 u M, 106. 3 0. 1 ug/ml of melanin at 0.13 M, 109. 7 0. 4 ug/ml of melanin at 0.06 p M, that is, the cells showed dose- dependently the whitening effect (Fig. 3a). The concentration at which 50% whitening effect was observed (EC5o) was 0.3 u M. In the same experimental model, arbutin showed EC5o values of the whitening effect of 473 11 M. Therefore, it was shown that

the depigmentation effect of 6-methyl-3-phenethyl-3, 4-dihydro-1H-quinazoline-2- thione on melanin production of melanoma B16 cell line by signal transduction of protein kinase C was 1,570 times higher than arbutin in terms of the EC$o value (Fig.

3b).

Experimental Example 4: Whitening mechanism reducing tyrosinase content Melanoma B16 cells were seeded in DMEM supplemented with 10% fetal bovine serum at a density of 20,000/well of a 6-well plate, under conditions of 37C and 5% C02 and cultured for 24 hours. The cells were treated with alpha-melanocyte stimulating hormone (final concentration 1 nM) and 6-methyl-3-phenethyl-3,4-dihydro- lH-quinazoline-2-thione as an effective agent at the same time. After incubation for 3 days, the cells were harvested, disrupted and centrifuged. The supernatant was used as an enzyme source. 20 gag of the enzyme source was electrophoresed on polyacrylamide gel containing SDS. Then, the gel was washed with 100 mM phosphate buffer solution (pH 6.8) to remove SDS, transferred to 100 mM phosphate buffer solution (pH 6.8) containing 5 mM DOPA and stored at 37°C under light shielded condition. The content of black-dyed tyrosinase was measured using a densitometer. As compared to the tyrosinase content produced by the melanoma B16 cell line cultured in DMEM supplemented with 10% fetal bovine serum for 3 days, the tyrosinase content produced in the same medium with alpha-melanocyte stimulating hormone added was increased 6 to 7 times. If the tyrosinase content produced by the melanoma B16 cell line treated only by alpha-melanocyte stimulating hormone was assigned to 100%, the tyrosinase content of the group treated with alpha-melanocyte stimulating hormone and 6-methyl-3-phenethyl-3, 4-dihydro-1H-quinazoline-2-thione as

an effective agent at the same time was 17% at an agent concentration of 4 11 M, 35% at 2 u M, 83% at 1 u M, 92% at 0.5 n M, which showed dose-denpendent inhibiting effect (Fig. 4). Therefore, it was noted that 6-methyl-3-phenethyl-3, 4-dihydro-lH- quinazoline-2-thione shows the depigmentation effect by a mechanism reducing tyrosinase content.

Experimental Example 5: cytotoxicity on Melanoma B16 cell line Melanoma B16 cell line suspended in DMEM supplemented with 10% fetal bovine serum were seeded at 2,500/well of a 96-well plate and cultured for 24 hours under conditions of 37C and 5% C02. The cells were treated with alpha-melanocyte stimulating hormone (final concentration 1 nM) and 6-methyl-3-phenethyl-3,4-dihydro- 1H quinazoline-2-thione as an effective agent at the same time. Every 24 hours, the numbers of living cells and dead cells were counted using the trypan blue dye method.

As compared to a melanoma B 16 cell line cultured in DMEM supplemented with 10% fetal bovine serum, the cells cultured in the same medium treated with alpha- melanocyte stimulating hormone showed growth delay but did not show cell killing toxicity (Fig. 5a). At the fourth day, the group treated with both alpha-melanocyte stimulating hormone and 6-methyl-3-phenethyl-3, 4-dihydro-1H-quinazoline-2-thione (concentration 0.5 to 4 u M) showed a substantially equal number of living cells, as compared to the group treated only with alpha-melanocyte stimulating hormone and did not show cell killing toxicity (Fig. 5b).

Industrial applicability

The present invention provides a novel compound of 6-methyl-3-phenethyl- 3, 4-dihydro-lH-quinazoline-2-thione with excellent skin-whitening effect which prevent melanin hyperpigmentation of the skin, or light the color of melanin pigment which has been already accumulated to improve hyperpigmentation such as discoloration and freckles, preparation method thereof, and a medicinal composition and a cosmetic composition comprising the same.