WO2004018416A1

WO2004018416A1 - 4-[(alkylsulfanyl)methyl]-1, 2-benzenediol, preparation method thereof and antioxidant containing the same

Info

Publication number: WO2004018416A1
Application number: PCT/KR2002/001595
Authority: WO
Inventors: Won Chul Choi; Hongsuk Suh; Jae Sue Choi; Dae-Duk Kim; Yeong-Ok Song; Jae-Kon Kim
Original assignee: Rex Biosciences Inc.
Priority date: 2002-08-22
Filing date: 2002-08-24
Publication date: 2004-03-04
Also published as: AU2002324360A1; KR20040017867A; KR100568191B1

Abstract

The present invention relates to 4-[(alkylsulfanyl)methyl]-1, 2-benzenediol derivative, preparation method thereof and antioxidant containing the same. More particularly, 4-[(alkylsulfanyl)methyl]-1, 2-benzenediol derivative following Formula (1) can inhibit generation of melanin due to its excellent antioxidant activity and can regulate blood cholesterol level by repressing oxidation of low density lipoprotein. Accordingly, the present invention can be useful for treatments of arteriosclerosis and skin deposit by reactive oxygen species.

Description

4-[(ALKYLSULFANYL)METHYL]-l,2-BENZENEDIOL, PREPARATION METHOD THEREOF AND ANTIOXIDANT CONTAINING THE SAME

TECHNICAL FIELD The present invention generally relates to 4-[(alkylsulfanyl)methyl]-l,2- benzenediol derivative, and more specifically, to 4-[(alkylsulfanyl)methyι]-l,2- benzenediol derivative of Formula 1 and pharmaceutically acceptable salt thereof, a preparation method thereof, and composition for antioxidation, whitening and cholesterol regulation containing the same as an effective ingredient: [Formula 1]

wherein is a Cι-C₁₀ alkyl group.

BACKGROUND OF THE INVENTION All the aerobes including human beings are living utilizing the energies obtained from nutrients through aerobic metabolisms using oxygen (0₂). However, various physical, chemical and biological stresses change oxygen into harmful reactive oxygen species such as superoxide anion radical (-0 ^"), hydrogen peroxide (H₂O₂) and hydroxy radical (OH). Phagocytic cell undergoing a respiratory burst produces superoxide anion O^2" to destroy infectious bacteria or viruses. However, if these reactive oxygen species are over-produced by various factors, they may cause fatal physiological disorders or may induce various diseases in the human body, resulting in death.

Human body is believed to have been evolved developing antioxidative responses as a self-defense mechanism to remove reactive oxygen species. However, if reactive oxygen species are overproduced above the defense capacity of the body, they can damage proteins, DNA, enzymes and the factors responsible for the immune system, such as T cells, causing various disorders. In addition, such reactive oxygen species can attack unsaturated fatty acids, which are constituents of cell membrane, causing a peroxidation reaction, and the resulting lipid peroxides accumulated in the body are known to cause aging and disorders such as cancer, multiple atherosclerosis, arthritis and Parkinson's disease.

As the theory that aging and adult diseases are attributed to reactive oxygen species has been widely acknowledged, researches have been directed to antioxidants able to regulate reactive oxygen species. The examples of antioxidants are antioxidation enzymes such as superoxide dismutase, peroxidase, catalase and glutathione peroxidase, and low molecular antioxidation materials derived from natural substances such as tocopherol, ascorbate, carotenoid and glutathione. In particular, synthetic antioxidants such as 2,6-dibutyl-4- hydroxytoluene (BHT), 2,6-dibutyl-4-hydroxyanisole (BHA) are developed and used in food and drug industries. Meanwhile, human skin produces melanin in order to remove naturally- produced reactive oxygen species and free radicals or to prevent ultraviolet transmission. Melanin is made from tyrosine, a kind of amino acid which normally exists in the human body. Tyrosine present in the melanocytes is oxidized by tyrosinase, and then converted into 3,4-dihydroxyρhenylalanine (hereinafter, referred to as "DOPA"). DOPA is further oxidized and converted into DOPA-quinone. Thereafter, DOPA-quinone is naturally oxidized and finally converted via 5,6-dihydroxyindole and indole-5,6-quinone to dark brown melanin [Goldsmith, L.A., Physiology, Biochemistry, and Molecular Biology of the Skin, Oxford University Press (1991)]. Each ofthe steps involved in the melanin biosynthesis can be the target for the inhibition of hyper-pigmentation in the skin. For example, hyper- pigmentation can be inhibited by: 1) protecting skin exposure from ultraviolet; 2) using tyrosinase inhibitors; 3) injecting substances showing toxicity to melanocytes specifically; and 4) stimulating the secretion of produced melanin outside ofthe skin.

The substances preventing hyper-pigmentation of the skin could be classified by their mechanisms: UV absorbents to block ultraviolet irradiated to the skin and UV scattering agents such as inorganic pigments; tyrosinase inhibitors such as vitamin C or kojic acid; substances showing toxicity to melanocytes such as hydroquinone; tocopherol which removes reactive oxygen species or free radicals stimulating melanin production; and alpha-hydroxyl acid (AHA) which removes produced melanin by stimulating the exfoliation of keratin layers. Arbutin, a substance showing toxicity to melanocytes, made by combining saccharides to hydroquinone, is known as a tyrosinase inhibitor like kojic acid, and used in functional cosmetics for skin whitening and anti-aging agent. In addition, retinol and ascorbic acid known to inhibit skin aging have lately attracted considerable attention internationally and domestically, and various products using these substances are already on the market. Although clear mechanisms are not fully known yet, it has been reported that the inhibitory activity ofthe tyrosinase is responsible for the whitening and anti-aging effect of the cell [Curto, E.V. et al., Biochemical Pharmacology, 57, 663-672 (1999); Cabanes, J. et al., J. Pharm. Pharmacol, 46, 982-985 (1994)].

Although tyrosinase inhibitors have been currently developed and used for various whitening agents, they also have many problems at the same time. 4- hydroxyanisol and hydroquinone, topically used for the treatment of excessive pigmentation such as chloasma, freckles, spots, gestational hyper-pigmentation, have powerful inhibitory activity in melanin production. However, they also cause bad effects such as degeneration or death of pigment cells and damage original functions of them. Specifically, compounds of the hydroquinone series used for whitening cream to inhibit melanin biosynthesis are known to induce skin irritation or dermatosis by their cell toxicity. As a result, the usage of the compounds is currently allowed in only certain countries.

Meanwhile, it is reported that arteriosclerosis of the human body caused by abnormal differentiation of the endothelial cells in the artery is highly correlated to a blood cholesterol level [Casteli, W.P. et al., JAMA., 256, 2835- 2845 (1986)]. The amount of cholesterol carried into arterial wall by low density lipoprotein (hereinafter, referred to as 'LDL'), the main substance to carry blood cholesterol, depends on the blood cholesterol level. If LDL moved to the arterial wall is oxidized, that is regarded as inflammatory responses and monocytes moved to this site are differentiated into macrophages by a monocyte-colony stimulating factor (M-CSF) secreted by vascular endothelial cells. The macrophage further stimulates the oxidation of LDL, and the oxidized LDL is accumulated within the macrophages by scavenger receptors. Then, macrophage loses feedback mechanisms required to accumulate cholesterol. As a result, high concentration of cholesterol is accumulated in the macrophages. Here, smooth muscle cells in the middle layers of arterial wall are moved to vascular endothelial cell layer of blood vessel and transformed into foam cells containing accumulated fat like macrophages. The foam cells form fatty streaks, thereby causing an initial stage of arteriosclerosis. The produced foam cells secrete extracellular matrix substances to form fibrous caps. The process of producing and rupturing them is repeated and then plaques are formed in the arterial wall, thereby causing myocardial infarction, heart failure and angina pectoris. The oxidative transformation of LDL is reported to have an important role in this cholesterol- induced endothelial dysfunction, that is, the mechanism of arterial wall overgrowth [Aviram, M. et al., J. Cardiovascular Pharmacology, 31, 39-45 (1998)]. Therefore, the possibility of arteriosclerosis can be increased in the vascular system where LDL is actively oxidized [Miesenock, G. et al., Leaf, A. and Webber, P.C.(ed), Raven Press, New York, vol: 21 , p. 119- 123 ( 1990)] . The oxidation of LDL results in the increase of lipid peroxides and oxygen free radicals, that is, reactive oxygen species. They can cause toxicity to endothelial cells. The oxidized LDL can be attached easily to vascular wall, and damages vascular cells and transforms vascular tissues. Simultaneously, the division of transformed cell is further stimulated, thereby causing oxidized LDLs, blood platelets and macrophages to be more easily attached to vascular wall [Noguchi, N. et al.: Archiv. Biochem. Biphys., 3347, 141-147 (1997)].

Arteriosclerosis is a major cause of death in the Western countries. Korea has a high incidence of cardiovascular diseases, and thus has a great interest in substances preventing arteriosclerosis. The following substances are used to treat arteriosclerosis: Simvastin [Aki Nakai et al.: Biol. Pharm. Bull. 19(9). 1231- 1233 (1996)] and Lovastatin which lower plasma cholesterol levels to treat hyperlipemia; probucol to inhibit oxidation of LDL; and antioxidant such as Vitamin C, Vitamin E and selenium to inhibit LDL oxidation in the body.

The present inventors have developed antioxidizing substances which can effectively inhibit harmful reactive oxygen species, and have founded that the antioxidizing substances can inhibit melanin production and induction of arteriosclerosis partially caused by harmful reactive oxygen species.

DETAILED DESCRIPTION OF THE DISCLOSURE

Accordingly, it is an object of the present invention to provide 4- [(alkylsulfanyl)methyl]-l,2-benzenediol derivative, which shows remarkable antioxidizing activity, pharmaceutically acceptable salt thereof, and a preparation method thereof.

It is another object of the present invention to provide composition for antioxidation, whitening and cholesterol regulation containing the above compound as an effective ingredient.

In order to achieve the objects of the present invention, there is provided 4-[(alkylsulfanyl)methyl]-l,2-benzenediol derivative of Formula 1 and pharmaceutically acceptable salt thereof:

[Formula 1]

wherein R is a Cι-C₁₀ alkyl group.

The compound of Formula 1 can be used as a pharmaceutically acceptable salt, wherein an acid adduct salt formed by pharmaceutically acceptable free acid is preferred for the salt. In other words, those of ordinary skills in the art can prepare pharmaceutically acceptable acid adduct salt comprising the compound of Formula 1. Organic and inorganic acids can be used for the free acid, wherein inorganic acids can be selected from a group consisting of hydrochloric acid, bromic acid sulfuric acid and phosphoric acid, and organic acid can be selected from a group consisting of citric acid, acetic acid, lactic acid, tartaric acid, maleate acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacete acid, benzoic acid, gluconic acid, methanesulfon acid, glycollic acid, succinic acid, 4- toluenesulfon acid, calutron acid, embon acid, glutamic acid and aspartic acid.

Preferred embodiments ofthe compound of Formula 1 are as follows:

4-[(2-propylsulfanyl)methyl]-l,2-benzenediol of Formula la;

[Formula la]

4-[(l-butylsulfanyl)methyl]-l,2-benzenediol of Formula lb; [Formula lb]

4-[(2-butylsulfanyl)methyl]-l,2-benzenediol of Formula lc; [Formula lc]

4-[(l-pentylsulfanyl)methyl]-l,2-benzenediol of Formula Id; [Formula Id]

4-[(isopentylsulfanyl)methyl]-l,2-benzenediol of Formula le; [Formula le]

4-[l-propylsulfanyl]methyl]-l,2-benzenediol of Formula lf; [Formula lf]

There is provided a method for preparing 4-[(alkylsulfanyl)methyl]-l,2- benzenediol derivative of Formula 1 comprising reacting l,3-benzodioxol-5-yl- methanol of Formula 2 and akyltiol of Formula 3 under acidic conditions:

[Formula 2]

[Formula 3]

R-SH; wherein R is a C1-C1₀ alkyl group.

Here, the acid can be selected from the group consisting of A1C1₃, FeBr₃, FeCl₃ and BBr₃.

In addition, the above reaction can be performed in an organic solvent selected from the group consisting of dichloromethane, chloroform, acetonitrile, tetrahydrofuran, dimethylformamide and mixtures thereof. l,3-benzodioxol-5-yl-methanol of Formula 2, a starting material of the above reaction, can preferably be prepared by oxidizing piperonal of Formula 4 under the presence of diisobutylaluminum hydride (DIBAL):

[Formula 4]

There is also provided a composition for antioxidation containing 4- [(alkylsulfanyl)methyl]-l,2-benzenediol derivative of Formula 1 and pharmaceutically acceptable salt thereof as effective ingredient.

There is also provided a composition for whitening containing 4- [(alkylsulfanyl)methyl]-l,2-benzenediol derivative of Formula 1 and pharmaceutically acceptable salt thereof as effective ingredient.

There is also provided a composition for cholesterol regulation containing 4-[(alkylsulfanyl)methyl]-l,2-benzenediol derivative of Formula 1 and pharmaceutically acceptable salt thereof as effective ingredient.

The 4-[(alkylsulfanyl)methyl]-l,2-benzenediol derivative of the present invention can effectively be used for whitening agent by inhibiting melanin production due to its excellent antioxidizing activity, and moreover, for therapeutic agent for arteriosclerosis by inhibiting LDL oxidation and thereby regulating blood cholesterol level. The composition of the present invention can be the forms of solution, suspension or emulsion in oil or aqueous medium, or can be transformed into dry powder which can be dissolved in aseptic water having no bacteria and pyrogens before use. The composition of the present invention can be formulated as dosage units for oral administration or for parenteral administration such as hypodermic, intravenous or intramuscular injections.

In case of oral administration, dosage units such as tablets, troches, saccharated tablets, aqueous or oily suspension, dispersible powder or particles, emulsion, soft or hard capsule, syrup and elixir can be formulated using pharmaceutically acceptable carriers or forming agents. The dosage units for oral administration can suitably be prepared according to the amount and the form of the administration.

Dosage units for parenteral administration can be formulated and injected as a suspension where effective ingredients are suspended in solutions such as sterilized injectable solvent, nonvenomous usable diluent or 1,3-butandiol. Usable excipients, solvents such as water, Ringer's solution and isotonic saline solution, and co-solvents such as ethanol, polyethylene glycol and polypropylene glycol can be used. Sterilized nonvolatile oil can conventionally be used for solvent or suspension. Suppository medicines are injected into rectum after the medicines are mixed with non-stimulant excipients such as cocoa butter or polyethylene glycol. The medicines are solid at room temperature but become liquid at temperature in rectum to be soluble and released into rectum.

When diseases are treated with a composition of the present invention, the amount of the compound of Formula 1, an effective ingredient, can be adjusted by age, weight, health condition, gender, meal, dosage time, excretory speed, medicinal combination and degree of disease during treatment. Preferably, the compound can be administered in total amounts of from 0.01 to 140mg/kg of body weight everyday according to the kind of diseases, and from 0.5mg to 7g per a patient in a day. The amount of the present compound mixed with carrier materials can be adjusted according to the injection methods and patient conditions. For example, dosage units for oral administration may contain carrier materials ranging from 5 to 95wt% of the whole gross and active ingredients ranging from 0.5mg to 5g, while dosage units for parenteral administration may contain carrier materials ranging from 5 to 99wt% of the whole gross and active ingredients ranging from O.lmg to 2.5g.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a graph illustrating experimental results on cell toxicity of compounds according to the present invention.

Fig. 2 is a graph illustrating experimental results on inhibition of melanin secretion of compounds according to the present invention.

THE PREFERRED EMBODIMENTS The present invention will be described in detail with reference to the accompanying preferred embodiments. However, it should be noted that the scope ofthe present invention is not limited to the preferred embodiments.

Example 1. Synthesis of 4-[(2-propylsulfanyl)methyl]-l,2-benzenediol (Step 1) Preparation of l,3-benzodioxol-5-yl-methanol After piperonal (5g) was dissolved in dichloromethane (340mL), IM of

DIBAL solution (200mL) was added in the resulting mixture at -78°C and then stirred at -78°C for 90 minutes. Then, methyl alcohol (lOmL) was added in the resulting mixture, and the temperature of the resulting mixture was raised to room temperature. The resulting solution was diluted with diethylether (250mL) and washed with salt water (50mL). The organic layer was dried with anhydrous magnesium sulfate to remove solvent. A column chromatography was performed using ethyl acetate : n-hexane (1 : 3) as an eluent to obtain the compound of Formula 2 (10.92g) (R_f : 0.24, yield : 89%). The compound was dissolved in CDC1₃ and TMS and then IH NMR was measured. The result was as follows:

1H NMR δ : 4.578 (s, 2H), 5.95 (s, 2H), 6.79 (m, 3H). (Step 2) Preparation of 4-[(2-propylsulfanyl)methyl]-l,2-benzenediol After aluminum chloride (21.9g) was dissolved in anhydrous dichloromethane (70mL), 2-propanethiol (150mL) was added in the resulting mixture at 0°C and then stirred for 20 minutes. The compound obtained from the step 1 (5g) was slowly added in the resulting solution, stirred at 0°C for 30 minutes and then reacted at room temperature for 5 hours. After completion of the reaction, the resulting solution was diluted with dichloromethane (150mL). Ammonium chloride solution was added in the resulting solution until pH of the solution was reached to 2. The resulting mixture was extracted with dichloromethane and water, and dried with anhydrous magnesium sulfate to remove the solvent. A column chromatography was performed using methyl alcohol : dichloromethane (2 : 98) as an eluent to obtain the compound of Formula la (4.78g) (R_f : 0.38, yield : 73%).

The compound was dissolved in CDC1₃ and TMS and then IH NMR was measured. The result was as follows:

1H NMR δ : 1.30 (d, 6H, J = 6.6Hz), 2.77-2.83 (m, IH), 3.64 (s, 2H), 6.7- 6.79 (m, 2H), 6.85 (s, IH). Example 2. Preparation of 4-r(l-butylsulfanyl)methyll-l,2-benzenediol

Aluminum chloride (21.9g) was dissolved in anhydrous dichloromethane (70mL) and 1-butanthiol (176mL) was added in the resulting mixture at 0°C. The resulting solution was stirred for 20 minutes. The compound (5g) obtained from the step 1 of Example 1 was slowly added in the resulting solution, stirred at 0°C for 30 minutes, and then reacted at room temperature for 5 hours. After completion ofthe reaction, the resulting solution was diluted with dichloromethane (150mL). Ammonium chloride solution was added in the resulting solution until pH of the solution was reached to 2. The resulting mixture was extracted with dichloromethane and water, and dried with anhydrous magnesium sulfate to remove the solvent. A column chromatography was performed using methyl alcohol : dichloromethane (2 : 98) as an eluent to obtain the compound of Formula lb (4.85g) (R_f : 0.32, yield : 70%).

1H NMR δ : 0.96 (t, 3H, J = 5.8Hz), 1.29-1.60 (m, 4H), 2.47 (t, 2H, J = 5.8Hz), 3.63 (s, 2H), 6.7-6.79 (m, 2H), 6.85 (s, IH).

Example 3. Preparation of 4-[(2-butylsulfanyl)methyl1-l,2-benzendediol

Aluminum chloride (21.9g) was dissolved in anhydrous dichloromethane (70mL) and 2-butanthiol (170mL) was added in the resulting mixture at 0°C. The resulting solution was stirred for 20 minutes. The compound (5g) obtained from the step 1 of Example 1 was slowly added in the resulting solution, stirred at a temperature of 0°C for 30 minutes, and then reacted at room temperature for 5 hours. After completion of the reaction, the resulting solution was diluted with dichloromethane (150mL). Ammonium chloride solution was added in the resulting solution until pH of the solution was reached to 2. The resulting mixture was extracted with dichloromethane and water, and dried with anhydrous magnesium sulfate to remove the solvent. A column chromatography was performed using methyl alcohol : dichloromethane (2 : 98) as an eluent to obtain the compound of Formula lc (5.6g) (R_f : 0.34, yield : 80%).

1H NMR δ : 0.96 (t, 3H, J = 5.8Hz), 1.22 (d, 3H, J = 6.6Hz), 1.47-1.61 (m, 2H), 2.63(q, IH, J = 6.6Hz), 3.62 (s, 2H), 6.7-6.79 (m, 2H), 6.85 (s, IH). Example 4. Preparation of 4-[(l-pentylsulfanyl)methyl]-l,2-benzenediol

Aluminum chloride (5.5g) was dissolved in anhydrous dichloromethane

(30mL) and 1-pentanthiol (50mL) was added in the resulting mixture at 0°C. The resulting solution was stirred for 20 minutes. The compound (1.28g) obtained from the step 1 of Example 1 was slowly added in the resulting solution, stirred at

0°C for 30 minutes, and then reacted at room temperature for 5 hours. After completion ofthe reaction, the resulting solution was diluted with dichloromethane

(50mL). Ammonium chloride solution was added in the resulting solution until pH of the solution was reached to 2. The resulting mixture was extracted with dichloromethane and water, and dried with anhydrous magnesium sulfate to remove the solvent. A column chromatography was performed using methyl alcohol : dichloromethane (2 : 98) as an eluent to obtain the compound of Formula

Id (1.58g) (R_f : 0.36, yield : 85%).

1H NMR δ : 0.96 (t, 3H, J = 5.8Hz), 1.23-1.35 (m, 4H), 1.58 (m, 2H), 2.33(1, 2H, J = 5.8Hz), 3.60 (s, 2H), 6.7-6.79 (m, 2H), 6.85 (s, IH).

Example 5. Preparation of 4-[(isopentylsulfanyl)methyl]-l,2-bezendediol

Aluminum chloride (21.9g) was dissolved in anhydrous dichloromethane (70mL) and isopentanethiol (50mL) was added in the resulting mixture at 0°C.

The resulting solution was stirred for 20 minutes. The compound (1.123g) obtained from the step 1 of Example 1 was slowly added in the resulting solution, stirred at 0°C for 30 minutes, and then reacted at room temperature for 5 hours.

After completion of the reaction, the resulting solution was diluted with dichloromethane (150mL). Ammonium chloride solution was added in the resulting solution until pH of the solution was reached to 2. The resulting mixture was extracted with dichloromethane and water, and dried with anhydrous magnesium sulfate to remove the solvent. A column chromatography was performed using methyl alcohol : dichloromethane (2 : 98) as an eluent to obtain the compound of Formula Id (2.19g) (R_f : 0.35, yield : 90%).

1H NMR 5 : 0.8 (d, 6H, J = 6.6Hz), 1.45 (t, 2H, J = 5.8Hz), 1.46-1.62 (m, IH), 2.45(t, 2H, J = 5.8Hz), 3.60 (s, 2H), 6.7-6.79 (m, 2H), 6.85 (s, IH).

Example 6. Preparation of 4-[(l-propylsulfanyl(methyl)]-l,2-benzenediol

Aluminum chloride (21.9g) was dissolved in anhydrous dichloromethane (70mL) and 1-propanethiol (148mL) was added in the resulting mixture at 0°C. The resulting solution was stirred for 20 minutes. The compound (5g) obtained from the step 1 of Example 1 was slowly added in the resulting solution, stirred at 0°C for 30 minutes, and then reacted at room temperature for 5 hours. After completion of the reaction, the resulting solution was diluted with dichloromethane (150mL). Ammonium chloride solution was added in the resulting solution until pH of the solution was reached to 2. The resulting mixture was extracted with dichloromethane and water, and dried with anhydrous magnesium sulfate to remove the solvent. A column chromatography was performed using methyl alcohol : dichloromethane (2 : 98) as an eluent to obtain the compound of Formula lf (5.34g) (R_f : 0.37, yield : 82%).

1H NMR δ : 0.98 (t, 3H, J = 5.8Hz), 1.48-1.66 (m, 2H), 2.43 (t, 2H, J = 5.8Hz), 3.60 (s, 2H), 6.7-6.79 (m, 2H), 6.85 (s, IH).

Experimental example 1> DPPH elimination activity l,l-diphenyl-2-picrylhydrazyl (hereinafter, referred to as 'DPPH'), a kind of purple dyes, has been used to measure antioxidizing activity of phenol and aromatic amines [Blois, M.S. Antioxidant determinations by the use of a stable free radical. Nature, 181, 1990-1200 (1958)]. DPPH shows strong absorbing band at 520 nm because of its odd-numbered electrons. When DPPH reacts on hydrogen or electron donor such as phenol, the DPPH receives electrons or hydrogen radicals from the donor and phenol converts into phenoxy radicals. Then, absorption disappears, the DPPH is stabilizing and the color is transformed from dark purple to yellow, resulting a decrease of absorptivity. In other words, radical elimination activity can be assessed by measuring the decrease of absorptivity in the resulting solution [Yokozawa, T. et al., Biochemical Pharmacology, 56, 213-222 (1998); Hatano, T. et al., Chem. Pharm. Bull., 37(8), 2016-2021 (1989)].

The elimination effect on DPPH radical of each samples was measured as follows [Blois, M.S. Nature, 181, 1990-1200 (1958)]. Each sample according to different concentrations (1.25~120μg/mL) was dissolved in methanol. The dissolved samples (each of 4mL) were mixed with 1.5x 10^"4 M of DPPH solution (lmL) dissolved in methanol. The resulting mixture was kept at room temperature for 30 minutes, and then its absorptivity was measured at 520nm. Free radical elimination activity was determined based on percentage by comparing the absorptivity of the sample group with that of the control group containing no samples, and 50% elimination activity (IC₅₀) was calculated. The result was expressed as mean values of three repeating experiments [Yoshida el al., Chem. Pharm. Bull., 37(7), 1919-1921 (1989)].

As a result of measuring DPPH radical elimination effect of the compounds of the present invention, the compound of Example 6 showed the highest activity (see Table 1). DPPH radical elimination effect of other compounds was lower than IC₅₀ value of vitamin C. Especially, for the compound of Example 4, the activity was higher than that of vitamin C by about 55 times. [Table 1]

001595

Experimental example 2> Experiment on cell toxicity

In order to test toxicity ofthe compound of Examples 1~6, 3,4,5-dimethyl thiazole-3,5-diρhenyl tetrazolium bromide (hereinafter, referred to as 'MTT') test was performed by the Mosmann's method using melanocyte [Mosmann, T.: J. Immunol. Methods 65, 55-63, (1983)]. Because living cells can degrade tetrazolium salt, preventing the accumulation of MTT inside of the cells, cell toxicity effect of drugs can be examined by MTT test. MTT stock solution (5mg/ml) was dissolved in phosphate buffer saline (hereinafter, referred to as 'PBS'), filtered with a filter having 0.22μm pore size to remove formazan crystals, and then kept in a darkroom at room temperature. B16 melanoma cells were dispensed into a 96- well plate by a concentration of 3,000 cells/well, and then treated with the compounds of Examples 1~6. After predetermined culturing time, 10 times diluted MTT solution was added to each well. After 4 hours of incubation, the plate was centrifuged in 275 x g for 5 minutes and supernatant (lOOμl) was obtained. In order to remove formazan crystals, DMSO was added to the plate, and the plate was shaken in a microplate shaker until the DMSO was completely dissolved. Then, absorptivity was measured at 560mn using a microplate ELISA reader (El 312e, Bio-Tek). Cell survival rates of each compound- treated group were compared with that ofthe control group. Here, the cell survival rate was represented by % ofthe control (see Fig. 1).

Referring to Fig. 1, arbutin and vitamin C known for having whitening effect showed almost 100%) ofthe cell survival rate to a concentration of lOμg/ml. Hydroquinone showed remarkably high toxicity as was previously known. The compounds of Examples 1~6 showed lower cell toxicity, but some of them had cell toxicity in the concentration of lOμg/ml. However, the compounds of Examples 1~6 showed almost 100% of the cell survival rate under the concentration of lμg/ml.

Experimental Example 3> Experiment on inhibitory effect of melanin secretion using B16 melanoma cell line

The whitening effect of the compounds was examined using B16 mouse melanoma cell line. B16 cells were cultured with Dulbeco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum, streptomycin (100 mg/ml) and penicillin (100 U/ml). Cultured B16 cells were trypsinized and detached from the plate. Then, the cells were dispensed in a 24 well tissue culture plate by a concentration of 1 x 10⁵ cells/ml. After a day, cell culture medium was changed to phenol red-free Dulbeco's modified Eagle's medium containing 2μM α-melanocyte stimulating hormone and 2mM theophylline. Compounds for test were added by various concentrations and the plates were cultured for 7 days. Whitening effect of the compounds was determined by the amount of melanin pigments secreted by the B16 cells. 7 days after compound treatment, each of the cell medium was collected, and cells were removed by centrifugation. The absorptivity was measured by an ELISA reader at 490nm to estimate the amount of secreted melanin. The inhibitory effect of each compound was measured by the following formula:

OD after medicine treatment

%inhibitory activity = ( 1 - ) x 100

OD ofthe control group

As a result, arbutin, vitamin C and hydroquinone showed a tendency to inhibit the secretion of melanin from B16 melanoma cells with concentration- dependent manner. The compounds of Examples 1~6 showed a stronger secretion inhibitory effect than that of the arbutin. Especially, the compounds of Examples 1 and 6 showed 100% inhibitory activity at a concentration of over 0. lμg/ml. When the result was summarized with the previous result of cell toxicity by the MTT test, it was shown that these compounds could completely inhibit the secretion of melanin in a concentration ranging from 0.1 to l.Oμg/ml without cell toxicity (see Fig. 2).

Experimental example 4> Measurement of LDL oxidation inhibition rate After LDL (Sigma No. L-5402) was dialyzed in PBS for 24 hours, the

LDL was sterilized by filtering it using syringe-mountable filter of 0.45μm pore size. The protein density of LDL was determined by Lowry's method using bovine serum albumin (hereinafter, referred to as 'BSA') as a standard.

Final concentration of 5M Cu was added into the reaction solution containing O.lmg protein/ml of LDL and the compounds of the Examples with various concentrations, and then oxidized at 40°C for 4 hours. 0.4% thiobarbituric acid (hereinafter, referred to as 'TBA'), 15% trichloroacetic acid (hereinafter, referred to as 'TCA') and 1ml TBARS (thiobarbituric acid reactive substances) containing 2.5% HC1 were mixed with the oxidized LDL reaction solution (1ml) and standard malondialdehyde reaction solution (hereinafter, referred to as 'MDA') at various concentrations. The resulting mixture was reacted in a water bath at 95-100°C for 20 minutes. Hot samples were vortexed to remove air bubble, cooled and then centrifuged at 2000rpm for 10 minutes to obtain supernatant. The abosorptivity ofthe supernatant was measured at 532nm. The concentration of TBARS in the samples was expressed as nmole of MDA estimated from a standard MDA curve.

In order to investigate the inhibitory effect of the compounds prepared in the Examples on LDL oxidation, the compounds having a concentration of 1, 5, 25 and 50μg/mL, respectively, were added into the LDL reaction solution. The inhibitory effect was compared with that of the vitamin C, a well-known antioxidant. The TBARS concentration of the oxidized LDL (40°C, 4hrs) was 30.35+5.60 nmole MDA/mg protein LDL. The results were expressed as meani standard deviation values of three repeating experiments. The samples were triplicated per each test. As shown in Table 2, the compounds of the present invention prepared in Examples 1~6 showed strong antioxidizing activity against LDL oxidation. When the compound of lμg/mL was added, vitamin C showed about 21% of LDL oxidation inhibition rate while the compounds of Examples 1~6 showed about 72-80% of inhibition rate. As a result, it was found that the compounds of the present invention had remarkably higher antioxidizing effect than vitamin C. [Table 2]

As shown above, preferable examples of the present invention are described. Nonetheless, a number of variations and modification could be possible by a person skilled in the art without a deviation from the spirit and the scope ofthe present invention.

INDUSTRIAL APPLICABILITY

As discussed hereinbefore, the compounds of the present invention, which showed excellent antioxidizing activity, may effectively inhibit production of melanin and oxidation of LDL. As a result, the compounds of the present invention can be used for whitening agents and for cholesterol regulators.

Claims

What is claimed is:

1. 4-[(alkylsulfanyl)methyι]-l,2-benzenediol derivative of Formula 1 and pharmaceutically acceptable salt thereof: [Formula 1]

wherein R is a C1-C1₀ alkyl group.

2. 4-[(alkylsulfanyl)methyl]-l,2-benzenediol derivative and pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of Formula 1 is selected from the group consisting of: 4-[(2-proρylsulfanyl)methyl]- 1 ,2-benzenediol; 4-[(l-butylsulfanyl)methyl]-l,2-benzenediol; 4- [(2-butylsulfanyl)methyl] - 1 ,2-benzenediol; 4-[(l -pentylsulfanyl)methyl]- 1 ,2-benzenediol;

4-[(isopentylsulfanyl)methyl]-l,2-benzenediol; and 4- [( 1 -propy lsulfany l)methyl] - 1 ,2-benzenediol .

3. A method for preparing 4-[(alkylsulfanyl) methyl]- 1,2- benzenediol derivative comprising reacting l,3-benzodioxol-5-yl-methanol of

Formula 2 and alkylthiol of Formula 3 under acidic conditions: [Formula 2]

[Formula 3] R-SH wherein R is a C1-C1₀ alkyl group.

4. The method according to claim 3, wherein the acid is selected from the group consisting of A1C1₃, FeBr₃, FeCl₃ and BBr₃.

5. The method according to claim 3, wherein the reaction is performed using a solvent selected from the group consisting of dichloromethane, chloroform, acetonitrile, tefrahydrofuran, dimethylformamide and mixtures thereof.

6. The method according to claim 3, wherein l,3-benzodioxol-5-yl- methanol of Formula 2 is obtained by oxidizing piperonal of Formula 4 under the presence of diisobutylaluminum hydride (DIBAL): [Formula 4]

7. Antioxidant containing 4-[(alkylsulfanyl) methyl] -1,2-benzenediol derivative of claim 1 and pharmaceutically acceptable salt thereof as an effective ingredient.

8. A composition for whitening containing 4-[(alkylsulfanyl)methyl]-

1,2-benzenediol derivative of claim 1 and pharmaceutically acceptable salt thereof as an effective ingredient.

9. A composition for cholesterol regulation containing 4- [(alkylsulfanyl)methyl]-l,2-benzenediol derivative of claim 1 and pharmaceutically acceptable salt thereof as an effective ingredient.