EFFECT OF CYCLOHEXANE AND BENZENE DIESTER ON MELANOGENESIS 183 it was concluded that 1i, 1m, and 2j would have whitening effects, and these effects were evaluated at the protein and gene levels through Western blotting and RT-PCR. WESTERN BLOTTING Recent reports have demonstrated that many proteins are involved in the regulation of melanin production, including TRP-1, TRP-2, and tyrosinase. To test whether com- pounds 1i, 1m, and 2j regulated the expression of these proteins, we performed Western blot analysis on B16F10 cells. As shown in Figure 1, treatment with 125 μM compounds 1m and 2j led to signifi cant reduction in the expression of TRP-1 and TRP-2, with a slight decrease in tyrosinase. Also, treatment with 125 μM compound 1i resulted in a slight decrease in TRP-1, TRP-2, and tyrosinase. EFFECTS OF CYCLOHEXANE DIESTER DERIVATIVES (1I, 1M) AND BENZENE DIESTER DERIVATIVE (2J) ON EXPRESSION OF MRNA IN B16F10 CELLS To examine whether compounds 1i, 1m, and 2j regulated the expression of those genes, we carried out RT-PCR analysis in B16F10 cells. As portrayed in Figure 2, 1i, 1m, and 2j reduced the expression of mRNA involved in melanin production in a dose-dependent manner. Also, as mentioned in the aforementioned result of Western blotting, treatment with 125 μM compounds, 1i, 1m, and 2j, led to a signifi cant decrease in mRNA such as TRP-1, TRP-2, and tyrosinase. A TYROSINASE INHIBITOR MODEL FOR STRUCTURE-BASED DRUG DESIGN As shown in Figure 3, docking with arbutin and benzene diester derivative 2j was identifi ed and was located at the pocket site of human tyrosinase. Figure 4 shows the docking of the benzene diester derivative 2j with fi ve residues, LYS196, PHE209, SER222, ASN226, and VAL239, of human tyrosinase, and docking of arbutin (used for comparison) with three residues, PHE209, ASN226, and HIS22. The results showed the inhibitory effect of benzene diester derivative 2j on human tyrosinase. Benzene diester derivative 2j acted more effectively as a tyrosinase inhibitor than did arbutin. Using this structure-based drug design, further studies on the inhibitory action of cyclohexane diester derivatives 1i and 1m on tyrosinase will be conducted. Figure 1. Effects of cyclohexane diester derivatives (1i, 1m) and benzene diester derivative (2j) on expression of protein involved in pigmentation in B16F10 cells. (A) 1i, (B) 1m, and (C) 2j.

JOURNAL OF COSMETIC SCIENCE 184 DISCUSSION In summary, a novel series of cyclohexane diester derivatives and benzene diester derivatives were prepared and evaluated for the ability to inhibit mushroom tyrosi- nase and melanin production in B16F10 mouse melanoma cells and were assessed for their structure–cytotoxicity relationships. From the structure–cytotoxicity point of view, it was demonstrated that the binding location of diesters (i.e., 1,2-, 1,3-, 1,4-), change in the carbon number of the side chain, and the difference between the linear and branch types were essential variables for determining cytotoxicity. The melanin inhibitory activities of cyclohexane-1,3-diyl bis(decanoate) (1i), cyclohexane-1,4- diyl dioctanoate (1m), and 1,3-phenylene bis(2-ethylhexanoate) (2j)—having low cytotoxicity while showing whitening effects—were measured at 479.3 ± 1.3 μM, 497.9 ± 3.8 μM, and 474.7 ± 9.4 μM, respectively their potential as whitening agents were further verifi ed using Western blotting and RT-PCR. Figure 2. Effects of cyclohexane diester derivatives (1i, 1m) and benzene diester derivative (2j) on expression of mRNA involved in pigmentation in B16F10 cells. (A) 1i, (B) 1m, and (C) 2j. Figure 3. Docking with arbutin and benzene diester derivative (2j) on human tyrosinase. (A) Arbutin dock- ing, (B) 2j docking, (C) cartoon ligand, and (D) surface ligand.