J. Cosmet. Sci., 65, 175–186 (May/June 2014) 175 Effect of novel cyclohexane diester and benzene diester derivatives on melanogenesis JONG WOO CHEON, JI MIN JEON, MOON JEONG CHOI, SI JUN PARK, and SANG YO BYUN, R&D Center, ACT Co., Ltd., 486 Sin-dong (J.W.C., J.M.J., M.J.C., S.J.P.), Suwon, Republic of Korea and Cosmetic Science Major, Department of Applied Biotechnology, Ajou University, Woncheon-dong (J.W.C., S.Y.B.), Suwon 443-749, Republic of Korea. Accepted for publication March 20, 2014. Synopsis In order to investigate potent whitening agents, we synthesized 15 cyclohexane diester derivatives and 15 ben- zene diester derivatives. To evaluate their structure–cytotoxicity relationships, we performed cell cytotoxicity tests on B16F10 mouse melanoma cells. To understand their whitening effects, melanin synthesis inhibitory activities in B16F10 cells and mushroom tyrosinase inhibitory activities were performed. In most cases, cell cytotoxicity was observed to be lower in 1,3-diester than in 1,2- and 1,4-diesters when it came to the structural isomer of the side chain, all derivatives except the 1,2-cyclohexane diester derivatives showed lower cell cyto toxicity in the branch type of the side chain than in the linear type. Among the compounds evaluated, the compounds cyclohexane-1,3-diyl bis(decanoate), cyclohexane-1,4-diyl dioctanoate, and 1,3-phenylene bis (2-ethylhexanoate) emerged as potent melanin synthesis inhibitors. Our goal was to determine the expression levels of proteins involved in melanogenesis, Western blotting and RT-PCR showing that these compounds decreased tyrosinase, TRP-1, and TRP-2 while demonstrating signifi cantly low cytotoxicity. INTRODUCTION Melanin is a major pigment produced by melanocyte cells in the basal layer of human skin and overproduced by chronic sun exposure or other hyperpigmentation diseases (1). Melanin synthesis is regulated by the rate-limiting enzyme of tyrosinase, which is a membrane-bound copper containing glycoprotein that initiates the biosynthetic pathway of melanin by catalyzing the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA). Subsequent reactions in the melanin synthetic pathway—for example, the con- version of DOPA to DOPAquinone as well as other non-enzymatic reactions—are oxida- tive reactions (2). Tyrosinase is known to be a key enzyme for melanin biosynthesis in plants, microorganisms, and human cells. Against this backdrop, many tyrosinase in- hibitors have been tested in cosmetics and pharmaceuticals as a way of preventing any overproduction of melanin in epidermal layers (3). Address all correspondence to Jong Woo Cheon at actcjw@actcos.com.
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 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.
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