A Potent Tyrosinase Inhibitor, (E)-3-(2,4-Dihydroxyphenyl)-1-(thiophen-2-yl)prop-2-en-1-one, with Anti-Melanogenesis Properties in α-MSH and IBMX-Induced B16F10 Melanoma Cells

In this study, we designed and synthesized eight thiophene chalcone derivatives (1a–h) as tyrosinase inhibitors and evaluated their mushroom tyrosinase inhibitory activities. Of these eight compounds, (E)-3-(2,4-dihydroxyphenyl)-1-(thiophen-2-yl)prop-2-en-1-one (1c) showed strong competitive inhibition activity against mushroom tyrosinase with IC50 values of 0.013 μM for tyrosine hydroxylase and 0.93 μM for dopa oxidase. In addition, we used enzyme kinetics study and docking program to further evaluate the inhibitory mechanism of 1c toward tyrosinase. As an underlying mechanism of 1c mediated anti-melanogenic effect, we investigated the inhibitory activity against melanin contents and cellular tyrosinase in B16F10 melanoma cells. As the results, the enzyme kinetics and docking results supports that 1c highly interacts with tyrosinase residues in the tyrosinase active site and it can directly inhibit tyrosinase as competitive inhibitor. In addition, 1c exhibited dose-dependent inhibitory effects in melanin contents and intracellular tyrosinase on α-MSH and IBMX-induced B16F10 cells. Overall, our results suggested that 1c might be considered potent tyrosinase inhibitor for use in the development of therapeutic agents for diseases associated with hyperpigment disorders.


Introduction
Melanogenesis is a process that involves melanin synthesis, transport of melanin, and release of melanosome. Melanin, composed of pheomelanin and eumelanin, is synthesized in melanosomes of melanocytes [1]. The abnormal synthesis of melanin leads to skin conditions such as vitiligo, melasma, chloasma freckles, and inflammatory pigmentation. During melanins production, melanogenic enzymes such as tyrosinase was synthesized [2][3][4][5]. Tyrosinase (EC 1.14.18.1), a multifunctional copper-containing polyphenol oxidative enzyme, is key for melanin biosynthesis and is responsible for melanization in animals and browning in plants [6]. The reactions in the melanin biosynthetic pathway involves the hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) and the oxidation of L-DOPA to DOPA-quinone, which are important in the early stages of melanogenesis [7,8]. Thus, inhibition of tyrosinase could be a prospective target for skin pigmentation. Therefore, development of tyrosinase inhibitors for use as preservatives in a fresh food or a skin-whitening agents is under development.
Chalcones (1,3-diary-2-propen-1-ones), members of the polyphenolic and flavonoid families, exert several biological activities, including anti-bacterial [9], anti-cancer [10,11], anti-inflammatory [12], anti-tumor [13], and anti-oxidant [14] activities. Many researchers reported the inhibitory activity of chalcone-like derivatives from natural origins against mushroom tyrosinase [15][16][17]. Moreover, biological activities of chalcone-like derivatives are significantly determined based on the position and numbers of functional groups attached to benzene ring. In spite of diverse structural moiety of natural tyrosinase inhibitors and available methodology to reasonably design effective synthetic derivatives, many synthetic tyrosinase inhibitors with novel skeletons have been studied [15][16][17][18][19][20][21][22][23][24][25][26]. Especially, Hwang et al. [27] reported the effects of heterocyclic chalcone derivatives containing heterocycles, such as thiophene or furan ring as an isostere of benzene ring on free radical scavenging activity. Recently, Beak et al. [28] revealed that certain synthetic dihydroxychalcones analogues including thiophene ring inhibited cathepsin. Regardless of the evidence suggests that chalcone-like derivatives may be useful as therapeutic agents, inhibitory effect of chalcones bearing thiophene moiety on tyrosinase and melanogenesis associated with hyperpigmentation has not been reported yet. In this regard, the objective of the present work was to identify the anti-tyrosinase activity of chalcone derivatives bearing thiophene using in vitro enzyme assays with kinetics methods, including Lineweaver-Burk and Dixon plots.
Recently, our laboratory discovered and reported several tyrosinase inhibitors [18][19][20][21][22][23][24][25][26]29]. In particular, Kim et al. [22,23] and Lee et al. [24] demonstrated the importance of a thiazolidine derivative bearing 2,4-dihydroxy phenyl moiety (MHY498) which attributed to the inhibitory activity against tyrosinase. Therefore, we designed and synthesized eight compounds (1a-h) of the chalcone skeleton bearing a thiophene ring, which supported our investigation of this particular moiety. Among these synthetic compounds, a thiophene ring with 2,4-dihydroxy group on the B ring chalcone derivative (1c) exhibited the greatest tyrosinase inhibition and was selected for further examination. In this study, we characterize and evaluate of 1c of its tyrosinase inhibitory activity and the modulatory role in melanin synthesis using B16F10 murine melanoma cells. The assays was performed by using a mushroom tyrosinase enzyme and melanin contents on α-MSH and IBMX-induced B16F10 melanoma cells. In addition, an inhibition enzyme kinetics study and an in silico docking program were performed in order to study inhibitory mechanism of 1c to tyrosinase. Since there is no detailed information on the mode of inhibition and molecular interactions between tyrosinase and thiophene ring with 2,4-dihydroxy group of chalcone derivative, this study investigates an approach to develop thiophene ring chalcone derivatives as a potent anti-melanogenic drug candidate by scrutinizing molecular docking analysis and in vitro cell experiments.

Inhibitory Effect of Compound 1c on Mushroom Tyrosinase
3-(Substituted phenyl)-1-(thiophen-2-yl)prop-2-en-1-one derivatives (1a-h) were synthesized as candidate agents for skin whitening and tested for their inhibitory effects on mushroom tyrosinase. As shown in Table 1, 1c demonstrated the most potent tyrosinase inhibitory activity, with 50% inhibition concentration (IC50) values of 0.013  0.64 μM for monophenolase (L-tyrosine) and 0.93 ± 0.22 μM for diphenolase (L-DOPA) as substrates, respectively. For comparison, the IC50 values of positive control kojic acid were 22.84  0.09 μM and 24.57  0.23 μM. In the present study, we found that the 2,4-dihydroxy functional group on the benzylidene (1c) notably inhibited tyrosinase activity. This inhibitory activity was also previously shown for benzylidene-pyrrolidinedione, benzylidene-thiohydantoin, and benzylidenethiazolidinedione derivatives as reported by our laboratory [25][26][27][28][29][30] inconsistent with in this present study. In addition, our structure-activity relationship data also demonstrated that additional hydroxyl group in the presence of a 4-hydroxy group affect tyrosinase inhibitory activity in a structurally dependent manner (1a vs. 1b; 1a vs. 1c). Moreover, 1e significantly inhibited tyrosinase activity, with IC50 values of 17.44  1.81 μM and 28.72  1.98 μM for both substrate L-tyrosine and L-DOPA, respectively. Recently, we reported the importance of a 3-hydroxy-4-methoxybenzylidene moiety, which contributed to improved activity toward tyrosinase [20]. Furthermore, compounds 1a, 1b, 1d, and 1f moderately inhibited tyrosinase activity toward L-tyrosine with IC50 values of 46. 16 However, 1d, 1f, and 1h did not inhibit tyrosinase activity toward L-DOPA at any of the tested concentrations. Our results suggest that the capacity of these compounds to inhibit tyrosinase is affected by number and location of functional groups on the phenyl ring. Out of the tested compounds 1a-h, 1c was most effective in inhibiting tyrosinase activity response to L-tyrosine and L-DOPA as substrates (Table 1). Additionally, 1c dose-dependently inhibited tyrosinase activity

Inhibitory Effect of Compound 1c on Mushroom Tyrosinase
3-(Substituted phenyl)-1-(thiophen-2-yl)prop-2-en-1-one derivatives (1a-h) were synthesized as candidate agents for skin whitening and tested for their inhibitory effects on mushroom tyrosinase. As shown in Table 1, 1c demonstrated the most potent tyrosinase inhibitory activity, with 50% inhibition concentration (IC 50 ) values of 0.013 ± 0.64 µM for monophenolase (L-tyrosine) and 0.93 ± 0.22 µM for diphenolase (L-DOPA) as substrates, respectively. For comparison, the IC 50 values of positive control kojic acid were 22.84 ± 0.09 µM and 24.57 ± 0.23 µM. In the present study, we found that the 2,4-dihydroxy functional group on the benzylidene (1c) notably inhibited tyrosinase activity. This inhibitory activity was also previously shown for benzylidene-pyrrolidinedione, benzylidene-thiohydantoin, and benzylidene-thiazolidinedione derivatives as reported by our laboratory [25][26][27][28][29][30] inconsistent with in this present study. In addition, our structure-activity relationship data also demonstrated that additional hydroxyl group in the presence of a 4-hydroxy group affect tyrosinase inhibitory activity in a structurally dependent manner (1a vs. 1b; 1a vs. 1c). Moreover, 1e significantly inhibited tyrosinase activity, with IC 50 values of 17.44 ± 1.81 µM and 28.72 ± 1.98 µM for both substrate L-tyrosine and L-DOPA, respectively. Recently, we reported the importance of a 3-hydroxy-4-methoxybenzylidene moiety, which contributed to improved activity toward tyrosinase [20]. Furthermore, compounds 1a, 1b, 1d, and 1f moderately inhibited tyrosinase activity toward L-tyrosine with IC 50 values of 46.16 ± 0.55 µM, 75.72 ± 2.46 µM, 98.78 ± 2.11 µM, and 77.91 ± 8.74 µM, respectively, while 1g and 1h were inactive. Likewise, 1a, 1b, and 1g showed moderate activity toward L-DOPA with IC 50 values of 60.05 ± 7.85 µM, 103.44 ± 8.47 µM, and 112.09 ± 14.27 µM, respectively. However, 1d, 1f, and 1h did not inhibit tyrosinase activity toward L-DOPA at any of the tested concentrations. Our results suggest that the capacity of these compounds to inhibit tyrosinase is affected by number and location of functional groups on the phenyl ring. Out of the tested compounds 1a-h, 1c was most effective in inhibiting tyrosinase activity response to L-tyrosine and L-DOPA as substrates (Table 1). Additionally, 1c dose-dependently inhibited tyrosinase activity ( Figure 1). In accordance to IC 50 values, 1c exhibited the highest tyrosinase inhibitory activity, and was used in consequent studies. The inhibitory effect on tyrosinase is conferred the 2,4-dihydroxyl group of benzene ring. Thus, we found that the catechol moiety (2,4-dihydroxyl groups) of 1c plays a crucial role in tyrosinase inhibition. was used in consequent studies. The inhibitory effect on tyrosinase is conferred the 2,4-dihydroxyl group of benzene ring. Thus, we found that the catechol moiety (2,4-dihydroxyl groups) of 1c plays a crucial role in tyrosinase inhibition. The IC50 values (μM) were calculated from a log dose inhibition curve using L-tyrosine a and L-DOPA b as a substrate, respectively, and are as means  standard error of the mean (SEM) of triplicate experiments. c Used as positive control.

1c Inhibition of Tyrosinase and Kinetic Analysis
To determine the inhibitory mechanism of 1c, we used two kinetic models: Lineweaver-Burk plots and Dixon plots [31,32]. Enzyme inhibition was evaluated using various concentrations of 1c different concentrations of L-tyrosine and L-DOPA as substrates, respectively. Thus, we performed kinetic analyses using Lineweaver-Burk and Dixon plots as summarized in Table 2. According to Table 2 and Figures 2A,C, a graphical analysis of the Lineweaver-Burk plot for 1c against tyrosinase showed competitive inhibition, as treatment with 1c resulted in double reciprocal straight-line plots with different slopes that increased the y-axis at the same point. These data suggested that 1c can competitively interact with substrate-binding site in order to inhibit enzyme activity when co-treated with various The IC 50 values (µM) were calculated from a log dose inhibition curve using L-tyrosine a and L-DOPA b as a substrate, respectively, and are as means ± standard error of the mean (SEM) of triplicate experiments. c Used as positive control.
Molecules 2018, 23, x 4 of 14 was used in consequent studies. The inhibitory effect on tyrosinase is conferred the 2,4-dihydroxyl group of benzene ring. Thus, we found that the catechol moiety (2,4-dihydroxyl groups) of 1c plays a crucial role in tyrosinase inhibition. The IC50 values (μM) were calculated from a log dose inhibition curve using L-tyrosine a and L-DOPA b as a substrate, respectively, and are as means  standard error of the mean (SEM) of triplicate experiments. c Used as positive control.

1c Inhibition of Tyrosinase and Kinetic Analysis
To determine the inhibitory mechanism of 1c, we used two kinetic models: Lineweaver-Burk plots and Dixon plots [31,32]. Enzyme inhibition was evaluated using various concentrations of 1c different concentrations of L-tyrosine and L-DOPA as substrates, respectively. Thus, we performed kinetic analyses using Lineweaver-Burk and Dixon plots as summarized in Table 2. According to Table 2 and Figures 2A,C, a graphical analysis of the Lineweaver-Burk plot for 1c against tyrosinase showed competitive inhibition, as treatment with 1c resulted in double reciprocal straight-line plots with different slopes that increased the y-axis at the same point. These data suggested that 1c can competitively interact with substrate-binding site in order to inhibit enzyme activity when co-treated with various

1c Inhibition of Tyrosinase and Kinetic Analysis
To determine the inhibitory mechanism of 1c, we used two kinetic models: Lineweaver-Burk plots and Dixon plots [31,32]. Enzyme inhibition was evaluated using various concentrations of 1c different concentrations of L-tyrosine and L-DOPA as substrates, respectively. Thus, we performed kinetic analyses using Lineweaver-Burk and Dixon plots as summarized in Table 2. According to Table 2 and Figure 2A,C, a graphical analysis of the Lineweaver-Burk plot for 1c against tyrosinase showed competitive inhibition, as treatment with 1c resulted in double reciprocal straight-line plots with different slopes that increased the y-axis at the same point. These data suggested that 1c can competitively interact with substrate-binding site in order to inhibit enzyme activity when co-treated with various concentrations of L-tyrosine or/and L-DOPA as substrates. A Dixon plot is a well-accepted method for analyzing the enzyme inhibition type and determining the K i value for an enzyme-inhibitor complex, wherein the K i is represented by the value of the x-axis. As shown in Table 2, the K i values were 5.01 nM and 1.76 µM, for L-tyrosine and L-DOPA substrates, respectively. The K i values represents concentrations required to form an enzyme inhibitor complex; thus, inhibitors with lower K i values means greater tyrosinase inhibitory activity, suggesting it could be useful in the development of preventive and therapeutic agents. wherein the Ki is represented by the value of the x-axis. As shown in Table 2, the Ki values were 5.01 nM and 1.76 μM, for L-tyrosine and L-DOPA substrates, respectively. The Ki values represents concentrations required to form an enzyme inhibitor complex; thus, inhibitors with lower Ki values means greater tyrosinase inhibitory activity, suggesting it could be useful in the development of preventive and therapeutic agents.

Molecular Docking Simulation of Tyrosinase and Binding Residues Interacting with 1c
To understand the mechanism by which 1c inhibits enzymatic activity of tyrosinase, we conducted in silico molecular docking simulation using AutoDock 4.2 software (SantaFe, NM, USA). Kojic acid has been widely used as a selective competitive inhibitor in numerous studies [33,34]. The docking simulation was effective with significant scores. The binding energies of 1c and kojic acid were −5.9 and −4.21 kcal/mol, respectively. 1c was found to exhibit greater binding affinity than kojic acid. The

Molecular Docking Simulation of Tyrosinase and Binding Residues Interacting with 1c
To understand the mechanism by which 1c inhibits enzymatic activity of tyrosinase, we conducted in silico molecular docking simulation using AutoDock 4.2 software (SantaFe, NM, USA). Kojic acid has been widely used as a selective competitive inhibitor in numerous studies [33,34]. The docking simulation was effective with significant scores. The binding energies of 1c and kojic acid were −5.9 and −4.21 kcal/mol, respectively. 1c was found to exhibit greater binding affinity than kojic acid. The docking score between the ligand and the receptor could be represented by different energy source terms including electrostatic energy, Van der Waals energy, and salvation energy. Furthermore, 1c interacted by hydrogen bonding with MET280 and ASN260 (Figure 3; Table 3). MET257, VAL248, PHE264, VAL283, and ALA286 residues were involved in hydrophobic interactions with 1c ( Figure 3B). Kojic acid was the reported compound, interacted with two active-site amino acid residues, HIS263 and MET280, with two hydrogen bonds. MET 280 participated in one hydrogen bonding interaction with the oxygen atom of kojic acid. These results proposed that 1c is a high affinity enzyme inhibitors capable of binding to the active site of tyrosinase.
Molecules 2018, 23, x 6 of 14 PHE264, VAL283, and ALA286 residues were involved in hydrophobic interactions with 1c ( Figure 3B). Kojic acid was the reported compound, interacted with two active-site amino acid residues, HIS263 and MET280, with two hydrogen bonds. MET 280 participated in one hydrogen bonding interaction with the oxygen atom of kojic acid. These results proposed that 1c is a high affinity enzyme inhibitors capable of binding to the active site of tyrosinase.

1c Inhibited the Melanin Content and Intracellular Tyrosinase Activity in B16F10 Melanoma Cells
We examined whether 1c is cytotoxic to B16F10 melanoma cells. Treatment with 1c did not show any cytotoxicity at concentrations up to 100 μM, as revealed by 48 h cell viability assay (Figure 4). Cytotoxic effect of 1c was determined in B16F10 melanoma cells. The obtained result indicated no significant cytotoxicity up to 100 μM tested concentration in cells. To further test the effect of 1c on anti-melanogenesis, cells were treated with 0.04, 0.2, and 1 μM 1c in the presence of α-MSH and IBMX for 48 h, and analyzed for melanin content and cellular tyrosinase activity. In this experiments, α-MSH and IBMX treatment notably increased melanin content, while 1c treatment noticeably attenuated

1c Inhibited the Melanin Content and Intracellular Tyrosinase Activity in B16F10 Melanoma Cells
We examined whether 1c is cytotoxic to B16F10 melanoma cells. Treatment with 1c did not show any cytotoxicity at concentrations up to 100 µM, as revealed by 48 h cell viability assay (Figure 4). Cytotoxic effect of 1c was determined in B16F10 melanoma cells. The obtained result indicated no significant cytotoxicity up to 100 µM tested concentration in cells. To further test the effect of 1c on anti-melanogenesis, cells were treated with 0.04, 0.2, and 1 µM 1c in the presence of α-MSH and IBMX for 48 h, and analyzed for melanin content and cellular tyrosinase activity. In this experiments, α-MSH and IBMX treatment notably increased melanin content, while 1c treatment noticeably attenuated melanin content in B16F10 melanoma cells in a dose-dependent ( Figure 5A). The melanin contents were 171.30%, after α-MSH and IBMX treatment, and reduced to 118.19%, 107.29%, 98.15%, and 88.09% after treatment with 10 µM kojic acid or 0.04 µM, 0.2 µM, and 1 µM 1c, respectively. According to these results, 1c significantly inhibited melanin biosynthesis. Melanin overproduction was found to stimulate the cellular tyrosinase [35]. For this reason, reduction of tyrosinase activity is an efficient strategy in development of anti-melanogenic agents [36,37]. L-Tyrosine and L-DOPA are sequentially generated substrates that positively regulate melanogenesis and modulates melanocyte function through overlapping substrates [38]. To study this induction, we designed L-DOPA oxidation protocols to examine the inhibitory activity of 1c against tyrosinase in α-MSH and IBMX-induced B16F10 melanoma cells. After 48 h of 1c treatment, we measured the intracellular tyrosinase activity. As demonstrated in Figure 5B, after 48 h of treatment with 1c (0.04, 0.2, and 1 µM) and α-MSH and IBMX, the intracellular tyrosinase activity decreased dose-dependently comparison to control. The levels of intracellular tyrosinase activity were 310.39%, after α-MSH and IBMX treatment, and were reduced to 225.54%, 225.78%, 157.79%, and 132.71% after treatment with 10 µM kojic acid or 0.04 µM, 0.2 µM, and 1 µM 1c, respectively. These results indicated that cellular tyrosinase inhibition by 1c was responsible for its anti-melanogenesis potential. In this study, we used B16F10 cells instead of human normal skin cells (melanocyte), which produce the melanin, because these cells have several common aspects of normal melanocytes [30,[39][40][41]. To the best of our knowledge, this study is the first report on anti-melanogenesis activity of thiophene chalcone derivatives by inhibiting tyrosinase in the cell base experiment system. generated substrates that positively regulate melanogenesis and modulates melanocyte function through overlapping substrates [38]. To study this induction, we designed L-DOPA oxidation protocols to examine the inhibitory activity of 1c against tyrosinase in α-MSH and IBMX-induced B16F10 melanoma cells. After 48 h of 1c treatment, we measured the intracellular tyrosinase activity. As demonstrated in Figure 5B, after 48 h of treatment with 1c (0.04, 0.2, and 1 μM) and α-MSH and IBMX, the intracellular tyrosinase activity decreased dose-dependently comparison to control. The levels of intracellular tyrosinase activity were 310.39%, after α-MSH and IBMX treatment, and were reduced to 225.54%, 225.78%, 157.79%, and 132.71% after treatment with 10 μM kojic acid or 0.04 μM, 0.2 μM, and 1 μM 1c, respectively. These results indicated that cellular tyrosinase inhibition by 1c was responsible for its anti-melanogenesis potential. In this study, we used B16F10 cells instead of human normal skin cells (melanocyte), which produce the melanin, because these cells have several common aspects of normal melanocytes [30,[39][40][41]. To the best of our knowledge, this study is the first report on antimelanogenesis activity of thiophene chalcone derivatives by inhibiting tyrosinase in the cell base experiment system.

Chemistry
All the reagents were achieved commercially and used without further purification. High-resolution MS (HRMS) data were obtained on a 6530 Accurate Mass quadrupole time-of-flight liquid-chromatograph mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). 1 H and 13 C nuclear magnetic resonance (NMR) spectral were recorded in CD 3 OD (δ H 3.30 and δ C 49.0), CDCl 3 (δ H 7.24 and δ C 77.0), and DMSO-d 6 (δ H 2.50 and δ C 39.7) on a Varian Unity INOVA 400 spectrometer or a Varian Unity AS500 spectrometer (Agilent Technologies). 1a, 1b, and 1d Method A: A suspension of substituted benzaldehyde, a, b, or d (0.66~0.82 mmol) and 2-acetylthiophene (1.0 equiv.) in 1.0 M HCl acetic acid solution (1.0 mL/1.0 mmol of benzaldehyde) was stirred at room temperature (a) or 60 • C (b and d) for 27-96 h. Water was added to the reaction mixture, neutralized with a NaHCO 3 aqueous solution and extracted with methylene chloride. The organic layer was dehydrated over MgSO 4 , filtered, and dried in vacuo. The residue was recrystallized to obtain compounds 1a, 1b and 1d in yields of 5~88%.

Synthetic Procedure for Compounds 1e-h
Method B: A 1.0 M NaOH aqueous solution (1.6 mL) was added to a suspension of substituted benzaldehyde, e, f, g or h (0.80 mmol) and 2-acetylthiophene (0.80 mmol) in methanol (2.0 mL). After the reaction mixture was stirred at room temperature for 8 h (e), or 60 • C for 1-6 h (f-h), water was added to the reaction mixture and neutralized with 2.0 M HCl. The precipitates were filtered and eluted with hexane and methylene chloride or refined by silica gel column chromatography using hexane and ethyl acetate to provide compounds 1e-h with yields of 14~32%.

Mushroom Tyrosinase Inhibitory Assay
We investigated the effects of (E)-3-phenylthiophenylprop-2-en-1-one derivatives (1a-h) on oxidization activity of tyrosinase using L-tyrosine or L-DOPA as substrates as described in our aforementioned study with a modified method [20,21]. Concisely, 10 µL of a detailed concentration of each compound (0.0005-50 µM) and 20 µL of mushroom tyrosinase (1000 Units/mL) in 50 mM phosphate buffer (pH 6.5) were added to 170 µL of an assay mixture buffer in a 96-well plate. The ratio of 1mM L-tyrosine or L-DOPA solution, 50 mM potassium phosphate buffer (pH 6.5), and purified water was 10:10:9. The reaction mixtures were keep warm at 37 • C for 20 min. The absorbance of the mixture at 492 nm was measured using a microplate reader. The tyrosinase inhibitory activity (%) was calculated as (1 − Abs sample /Abs control ) × 100%. The degree of inhibition of the sample was stated as the concentration required for 50% inhibition (IC 50 ).

Enzyme Kinetic Analysis in Mushroom Tyrosinase Inhibition
In order to estimate the kinetic mechanism, two kinetic methods using Lineweaver-Burk and Dixon plots were used [21,32]. Using Lineweaver-Burk double reciprocal plots [a plot of 1/enzyme velocity (1/V) vs. 1/substrate concentration (1/[S])], the type of inhibition was determined using various concentrations of L-tyrosine (0.5, 1 and 2 mM) and L-DOPA (0.5, 1, and 2 mM), as substrates, in the absence and presence of various concentrations of 1c (3.2-80 nM; for L-tyrosine, 0.4-10 µM; for L-DOPA). The Dixon plot for inhibition of tyrosinase was obtained using various concentrations of L-tyrosine (0.5, 1 and 2 mM) and L-DOPA (0.5, 1 and 2 mM). The concentrations of 1c were as follows: 3.2-80 nM; for L-tyrosine, 0.4-10 µM; for L-DOPA. The enzymatic procedures comprised of the formerly described tyrosinase test methods. The inhibition constant (K i ) was resolute using Dixon plots.

In Silico Molecular Docking Simulation of Tyrosinase Inhibition
To determine the possible molecular mechanism and the active groups of 1c responsible for inhibition of tyrosinase, molecular docking simulations were accomplished using AutoDock 4.2 program (AutoDock4 and AutoDockTools4, OpenEye Scientific Software, SantaFe, NM, USA). X-ray crystallographic structures show quaternary ligand binding to aromatic residues in the active-site gorge of tyrosinase (PDB ID: 2Y9X) as determined using the RCSB Protein Data Bank (http://www. rcsb.org/adb) [42]. Docking simulations were performed between tyrosinase and kojic acid or 1c. For the docking procedure: (1) the 2D structure of 1c was converted into a 3D structure, (2) charges were calculated, and (3) hydrogen atoms were added using ChemOffice software (http://www. cambridgesoft.com). LigandScout 4.1.5 was used to forecast possible interactions between ligands and tyrosinase and to identify pharmacophores.

Cell Culture
Murine melanoma B16F10 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in DMEM containing 10% FBS and 1% streptomycin, and then incubated at 37 • C with 5% CO 2 . The melanoma cells were plated at 95% confluency for all the experiments.

Assay for Cell Viability
Cell viability was assessed with the EZ-Cytox assay kit (Daeillab Service, Seoul, Korea) as previously described [21]. In brief, B16F10 cells were seeded in 96-well plates (Corning, NY, USA) at a density of 1 × 10 4 cells/well for 24 h. The cells were fed fresh, serum-free DMEM that contained different concentrations (up to 100 µM) of 1c, and incubated for 24 h or 48 h. Then, 10 µL EZ-Cytox solution was added to each well and the cells were incubated for 2-4 h. The absorbance of cells in the absence of treatment was regarded as 100% cell survival. Each treatment was achieved in triplicate and each experiment was repeated three times. The 50% cytotoxic concentration (CC 50 ) was defined as the test compound concentration required for the reduction of cell viability by half. Thus, CC 50 value of 1c were calculated by regression analysis.

Inhibitory Activity of Melanin Contents in B16F10 Melanoma Cells
Cellular melanin content was determined as described previously [43]. Cells were seeded in six-well culture plates (Nunc, Denmark) at 2 × 10 5 cells/well for 24 h. To determine the inhibitory effect of 1c on melanogenesis, fresh medium was replaced with medium containing 1c (0.04, 0.2, and 1 µM) or kojic acid (10 µM) as a positive control for 1 h, and then stimulated with α-MSH (5 µM) and IBMX (200 µM) for 48 h. Cells were washed twice with PBS. After centrifugation, cell pellets were disrupted in 100 µL of 1N NaOH, and incubated at 60 • C for 1 h and mixed to solubilize the melanin. Absorbance at 405 nm was compared with a standard curve for synthetic melanin (TECAN, Salzburg, Austria).

Inhibitory Activity of Cellular Tyrosinase Activity Assay in B16F10 Melanoma Cells
Intracellular tyrosinase inhibitory activity assay was achieved by measuring the rate of oxidation of L-DOPA [44]. Cells at a density of 1 × 10 5 cells/well were plated in 6-well plates (Nunc, Denmark) and incubated overnight. Then, cells were treated with various concentrations of 1c (0.04, 0.2, and 1 µM) or kojic acid (10 µM) for 1 h, then stimulated with α-MSH (5 µM) and IBMX (200 µM) for 48 h. The cells were washed with PBS and lysed in a solution containing 100 µL of 50 mM phosphate buffer (pH 6.5), 0.1 mM phenylmethylsulfonylfluoride (PMSF), and 1% Triton X-100. The cells lysates were employed in a deep freezer (−80 • C) for 1 h. After defrosting the cell lysates, the cellular extracts were purified by centrifugation at 12,000 rpm for 30 min at 4 • C. A total of 80 µL of supernatant and 20 µL of L-DOPA (2 mg/mL) were added to a 96-well plate, and the absorbance at a wavelength of 492 nm was measured every 10 min during 1 h using an ELISA plate reader (TECAN, Salzburg, Austria).
Total protein content was determined using BCA protein assay reagent (Thermo Scientific, Rockford, IL, USA).

Statistical Analysis
All statistical analyses were presented as mean ± S.E.M of at least three separate experiments. The data were analyzed by one-way analysis of variance (ANOVA) for the differences between treatments followed by the Bonferroni post-hoc test (SPSS, Chicago, IL, USA). A value of p < 0.05 indicated considered to indicated a statistically significance.

Conclusions
In conclusion, this study first reports the hyperpigmentary mechanism of thiophene chalcone derivatives in B16F10 melanoma cells. Among these derivatives (1a-h), 1c exerted potent tyrosinase inhibitory effect with IC 50 values (0.013 µM for tyrosine hydroxylase and 0.93 µM for dopa oxidase) significant lower than values achieved for the reference inhibitor kojic acid. 1c was shown as a competitive inhibitor against tyrosinase and fit well into the active sites of tyrosinase according to enzyme kinetic studies and molecular docking results. In addition, 1c inhibited cellular tyrosinase activity. Collectively, 1c could be useful as a tyrosinase inhibiting agent for the purpose of prevention and treatment of skin pigmentation and inhibition of melanogenesis. Further in vivo research and clinical trials should be carried out to clarify the efficacy, safety, and precise molecular mechanism of anti-melanogenesis effects of 1c.