JOURNAL OF COSMETIC SCIENCE 176 Benzenediols in hydroxyl groups are substituted onto a benzene ring. Benzenediols have three isomers: Catechol is commonly known as 1,2-benzenediol (ortho-isomer) resorcinol is 1,3-benzenediol(meta-isomer) and hydroquinone is 1,4-benzenediol (para-isomer). Tyrosinase (EC 1.14.18.1) contains two copper atoms in its active site and binds dioxygen to give oxy- tyrosinase. This form of the enzyme catalyzes ortho-quinone formation by catechol oxidation. Catechols irreversibly inactivate tyrosinase, where the bound catechol deprotonates, leading to the reductive elimination of Cu(0) from the tyrosinase active site (4,5). The resorcinol sub- strate is oxidized via the monooxygenase route, generating a hydroxyl intermediate that un- dergoes deprotonation and results in the irreversible elimination of Cu(0) from the tyrosinase active site (6). Hydroquinone is not a primary substrate for tyrosinase. The presence of the para-hydroxy group prevents binding to the tyrosinase active site (7). Therefore, the tyrosinase inhibitory activity to treat pigmentation disorders has recently been a major subject in numer- ous studies (8–10). Kojic acid and arbutin are well-known depigmenting agents. A4-substi- tuted resorcinols have been reported to be potent tyrosinase inhibitors, and their structure–activity relationships and inhibition mechanisms have been examined in detail (11,12) Also, 4,4’-(ethane-1,2-diyl)bis(resorcinol) is known to show a potent tyrosinase in- hibitory activity that is almost 20-fold stronger than that of kojic acid (13). In this study, we discuss the structure–cytotoxicity relationships, the melanin content, and mushroom tyrosinase inhibitory activities of 30 benzene diester derivatives with wide-ranging acyl group (butanoyl, hexanoyl, octanoyl, decanoyl, and 2-ethylhexanoyl) and hydroxyl group disposition in the benzyl part as whitening agents (see Table I for structures). These benzene diester derivatives have not been studied before in this context. The melanogenesis inhibitory activity of a series of benzene diesters is set here against their structure and cytotoxicity. The results will support the design of novel depigmentation agents. MATERIALS AND METHODS CHEMICALS AND REAGENTS The reagents of 1,2-cyclohexanediol, 1,3-cyclohexanediol, 1,4-cyclohexanediol, 1,2-benzene- diol, 1,3-benzenediol, 1,4-benzenediol, butyl chloride, hexanoyl chloride, octanoyl chloride, decanoyl chloride, 2-ethylhexanoyl chloride, tetrahydrofuran (THF), triethylamine (TEA), anhydrous magnesium sulfate, L-tyrosine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- lium-bromide (MTT), dimethyl sulfoxide (DMSO), and all other chemicals were purchased from Aldrich Chemical Co. (St. Louis, Mo). Dulbecco’s modifi ed Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and trypsin-EDTA were bought from Thermo Scientifi c Co. (San Francisco, CA). Tyrosinase, TRP-1, and TRP-2 antibodies were obtained from Santa Cruz Biotechnology. (Dallas, TX). PD 98059, H-89, KT 5720, RO- 32-0432, and SB 203580 were sourced from Calbiochem (Damstadt, Germany). The reac- tions were monitored by TLC on silica gel F254 (Merck, Damstadt, Germany). Column chromatography was carried out using 230–400 mesh silica gel (Merck). Proton nuclear mag- netic resonance spectra were recorded on a JNM-AL300 (300 MHz JEOL, Tokyo, Japan) spectrometer with tetramethylsilane as an internal standard. A FT-IR spectrum was re- corded on a Scimitar 1000 FTS (Varian, Randolph, MA). SpectraMax 190 (Molecular Devices, Sunnyvale, CA) was used as the absorbance.

EFFECT OF CYCLOHEXANE AND BENZENE DIESTER ON MELANOGENESIS 177 PREPARATION OF CYCLOHEXANE DIESTER DERIVATIVES (1A–1O) AND BENZENE DIESTER DERIVATIVES (2A–2O) The analogs of cyclohexane diester or benzene diester were synthesized by esterifying fi ve types of acyl chlorides with six types of diol (i.e., 1,2-cyclohexanediol, 1,3-cyclohexane- diol, 1,4-cyclohexanediol, 1,2-benzenediol, 1,3-benzenediol, and 1,4-benzenediol). Ten millimoles of diol was dissolved in 50.0 ml THF, with 12 mmol TEA added slowly. The mixture was stirred for 20 min at room temperature. Then, 12 mmol acryl chloride (butyl chloride, hexanoyl chloride, octanoyl chloride, decanoyl chloride, and 2-ethylhexanoyl chloride) was added and stirred for 6 h at room temperature. Upon completion, the reac- tion mixture was added to water and extracted with ethyl acetate. The extract was washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purifi ed by fl ash column chromatography on a silica gel. Cyclohexane diester derivatives were prepared using the aforementioned general procedure. MUSHROOM TYROSINASE INHIBITION ASSAY The inhibitory effect of the test compounds against mushroom tyrosinase was examined using the modifi ed method of Masamoto et al. Mushroom tyrosinase (EC 1.14.18.1) used Table I Chemical Structures of Cyclohexene Diester Derivatives (1a–1o) and Benzene Diester Derivatives (2a–2o) Compounds R n Compounds R n 1a–1o 2a–2o 1a –H 1 2a –H 1 1b –H 3 2b –H 3 1c –H 5 2c –H 5 1d –H 7 2d –H 7 1e –CH2CH3 3 2e –CH2CH3 3 1f –H 1 2f –H 1 1g –H 3 2g –H 3 1h –H 5 2h –H 5 1i –H 7 2i –H 7 1j –CH2CH3 3 2j –CH2CH3 3 1k –H 1 2k –H 1 1l –H 3 2l –H 3 1m –H 5 2m –H 5 1n –H 7 2n –H 7 1o –CH2CH3 3 2o –CH2CH3 3