New Benzimidazothiazolone Derivatives as Tyrosinase Inhibitors with Potential Anti-Melanogenesis and Reactive Oxygen Species Scavenging Activities

Thirteen (Z)-2-(substituted benzylidene)benzimidazothiazolone analogs were synthesized and evaluated for their inhibitory activity against mushroom tyrosinase. Among the compounds synthesized, compounds 1–3 showed greater inhibitory activity than kojic acid (IC50 = 18.27 ± 0.89 μM); IC50 = 3.70 ± 0.51 μM for 1; IC50 = 3.05 ± 0.95 μM for 2; and IC50 = 5.00 ± 0.38 μM for 3, and found to be competitive tyrosinase inhibitors. In silico molecular docking simulations demonstrated that compounds 1–3 could bind to the catalytic sites of tyrosinase. Compounds 1–3 inhibited melanin production and cellular tyrosinase activity in a concentration-dependent manner. Notably, compound 2 dose-dependently scavenged ROS in B16F10 cells. Furthermore, compound 2 downregulated the protein kinase A (PKA)/cAMP response element-binding protein (CREB) and mitogen-activated protein kinase (MAPK) signaling pathways, which led to a reduction in microphthalmia-associated transcription factor (MITF) expression, and decreased tyrosinase, tyrosinase related protein 1 (TRP1), and TRP2 expression, resulting in anti-melanogenesis activity. Hence, compound 2 may serve as an anti-melanogenic agent against hyperpigmentation diseases.


Introduction
Tyrosinase (polyphenol oxidase, EC 1.14.18.1), a binuclear copper-containing monooxygenase, is a critical rate-limiting melanogenic enzyme involved in melanogenesis, the process of melanin synthesis in the skin. Melanogenesis is initiated by the hydroxylation of tyrosine to L-3,4-dihydroxy-phenylalanine (DOPA), which is catalyzed by tyrosinase. Tyrosinase also catalyzes the subsequent enzymatic conversion of DOPA to dopaquinone [1,2]. Tyrosinase is the key factor involved in inducing dermatological disorders, including age spots, freckles, and melasma. Commercial tyrosinase inhibitors, such as hydroquinone, arbutin [3], kojic acid [4], ellagic acid [5], and tranexamic acid, have been used as skin-whitening agents; however, they are associated with certain side effects, including carcinogenicity, chemical instability, and poor bioavailability [6,7]. Thus, novel and efficient, anti-tyrosinase inhibitor with a favorable safety profile are necessary for anti-hyperpigmentation. There is a need to develop safe skin-whitening agents in order to overcome the limitations of established products.
Reactive oxygen species (ROS) have been linked to several disorders, including aging and age-related diseases [17,18]. Importantly, ROS levels can accelerate skin aging and increase both melanocyte proliferation and hyperpigmentation [19,20]. Among the reactive species (RS) derived from melanocytes and keratinocytes, nitric oxide (·NO) and superoxide anion radical (O 2˙− ) stimulate melanin synthesis by enhancing the protein expression of tyrosinase and TRP1 [21][22][23][24]. Therefore, antioxidants, inhibitors of tyrosinase, and scavengers of ROS may suppress melanogenesis in the epidermis.
In the present study, we evaluated the ability of (Z)-2-(substituted benzylidene) benzimidazothiazolone derivatives to inhibit mushroom tyrosinase enzyme, and performed kinetic studies and molecular docking simulations in silico; and investigated the scavenge ROS and the hyperpigmentation mechanism of a potent compound expression levels of melanogenic proteins and mRNA in B16F10 cells. As part of our continued work on potent tyrosinase inhibitors, this study aimed to evaluate the anti-melanogenic effects of benzimidazothiazolone derivatives.

Mushroom Tyrosinase Inhibitory Assay
The tyrosinase inhibitory activity assay was accomplished using mushroom tyrosinase enzyme, as previously described by Hyun et al., 2008 [32], with minor modifications. The IC 50 (concentration necessary for 50% inhibition of enzyme activity) was calculated by constructing a linear regression curve presenting compound concentrations on the x-axis and percentage inhibition on the y-axis. A negative control was obtained by adding dimethyl sulfoxide (DMSO) instead of the compounds. Kojic acid was used as a positive control.  [33,34] were used to calculate the inhibition mode and inhibition constant (K i ) against mushroom tyrosinase. Kinetic parameters for various concentrations of the L-tyrosine substrate (0.5-4 mM) and inhibitors (0.4, 2, and 10 µM for compound 1; and 2, 4, and 8 µM for compound 2; and 2.5, 5, and 10 µM for compound 3, respectively) were achieved.

Mushroom Tyrosinase Molecular Docking Simulations
The AutoDock4.2 software (La Jolla, CA, USA) was used to determine the structure of the enzyme-inhibitor complex and confirm precision, repeatability, and reliability of the docking results. For docking studies, the crystal structure of mushroom tyrosinase protein target was obtained from protein sequence alignment (PDB ID: 2Y9X) (http:// www.rcsb.org/adb, accessed on 10 February 2021) [35]. For the docking procedure, the 2D structure was converted into a 3D structure, charges were planned, and hydrogen atoms were added using the ChemDraw program (ChemDraw Ultra 10.0, Cambridge soft) (http://www.cambridgesoft.com, accessed on 12 February 2021) [36]. The docking results were visualized and analyzed using the UCSF Chimera molecular graphics system (v1.14, University of California, Berkeley, CA, USA) and AutoDockTools (v1.5.6, The Scripps Research Institute, La Jolla, CA, USA).

Cell Culture and Viability Analysis
The mouse melanoma cell line B16F10 was acquired from American Type Culture Collection (ATCC ® ; CCL-6475™) (Manassas, VA, USA) and cultured in DMEM, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, at 37 • C in a humidified 5% CO 2 atmosphere. Cell viability assays were performed with minor modifications, as previously reported [37]. Cell viability was evaluated using the EZ-Cytox assay kit (DaeilLab, Seoul, Korea). The compounds were dissolved in DMSO for the in vitro experiments.

Determination of Intracellular ROS Levels
ROS scavenging activity was determined according to the method reported by Ali et al. [38] and Lebel and Bondy [39]. Briefly, 2 ,7 -Dichlorodihydrofluorescein-diacetate (DCFH-DA) (2.5 mM, Molecular Probes, Eugene, OR, USA) mixed with esterase (1.5 units/mL), was incubated at 37 • C for 30 min and placed on ice in the dark until immediately prior to the study. Phosphate buffer (50 mM) at pH 7.4 was used. To determine intracellular ROS scavenging activity, B16F10 cells (1 × 10 4 /well) were seeded in black 96-well plates. After 24 h, the cells were incubated with DCFH-DA (20 µM) and SIN-1 (200 µM) for 30 min to induce ROS generation. The fluorescence intensity of oxidized DCFH was measured using a fluorescent microplate reader at excitation and emission wavelengths of 485 and 530 nm, respectively (Tecan, Männedorf, Switzerland). Trolox was used as the positive control.

Melanin Contents in B16F10 Cells
The effect of inhibitors on the melanin content in B16F10 cells was investigated as previously described [40,41]. The amount of melanin was determined spectrophotometrically by measuring the absorbance at 492 nm using a microplate reader (Tecan, Männedorf, Switzerland). All results were standardized to the total protein concentration of the cell lysates using the bicinchoninic acid (BCA) reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Cellular Tyrosinase Activity Assay in B16F10 Cells
B16F10 cells were seeded at a density of 3 × 10 3 /well in 6-well plates for 24 h at 37 • C in an atmosphere of 95% air/5% CO 2 . Each test agent was dissolved in DMSO at a final concentration up to 20 µM and pretreated with the indicated concentration of each compound; then, α-MSH (5 µM) and IBMX (200 µM) were added to the medium for 48 h.
After treatment, the cells were washed with ice-PBS and lysed by the addition of 50 mM phosphate buffer containing 1% Triton X-100 and 1% PMSF at −80 • C for 1 h. The lysates were clarified by centrifugation at 5600× g for 10 min at 4 • C; then, 80 µL of the cell lysate was added to 20 µL of L-DOPA (2 mg/mL in distilled water). The mixture was incubated for 10 min at 37 • C, and the absorbance of the reaction mixture at 492 nm was recorded using a microplate reader (Tecan, Männedorf, Switzerland). Kojic acid was used as the positive control. RNA from cell samples was purified using the RiboEx Total RNA solution (GeneAll Biotechnology, Seoul, Korea) according to the manufacturer's instructions. Total RNA (2 µg) treated with ribonuclease (RNase) free deoxyribonuclease (DNase) was reverse-transcribed using Hyperscript TM One-Step RT-PCR (GeneAll Biotechnology, Seoul, Korea). Real-time qPCR was performed using the SensiFAST TM SYBR ® No-ROX dye (Bioline, London, UK.) and a CFX Connect System (Bio-Rad Laboratories, Hercules, CA, USA). Relative gene expression was calculated with the standard curve method using GAPDH as the internal control. Ct values were obtained with qPCR. Three independent tests were performed. The primer sequences are displayed in Table 1.

Western Blot Analysis
Lysed samples were boiled for 10 min with gel-loading buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 10% 2-mercaptoethanol and 0.2% bromophenol blue) at a volume ratio of 1:1. Total protein equivalents were separated with SDS-PAGE using 9-10% acrylamide gels and then transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA) at 25 V for 10 min in a semi-dry transfer system (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were immediately placed in blocking buffer (5% non-fat milk) in 10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween-20. The blots were blocked at room temperature for 1 h. The membranes were incubated with appropriate specific primary antibodies at 4 • C overnight and then treated with horseradish peroxidase-conjugated anti-mouse, anti-rabbit or anti-goat antibodies (1:5000) at 25 • C for 1 h (Santa Cruz Biotechnology, Dallas, TX, USA). Protein bands were visualized using the SuperSignal ® West Pico Chemiluminescent Substrate kit (Advansta, San Jose, CA, USA) and Davinch-Chemi TM (Davinch-K, Seoul, Korea).

Statistical Analysis
Data are expressed as mean ± standard error of the mean (SEM). The statistical significance of the differences between groups was determined with one-way ANOVA followed by Dunnett's test. An associated probability of (p value) < 0.05 was considered significant.

Synthesis of (Z)-2-(Substituted benzylidene)benzimidazothiazolone Derivatives 1-13
The synthetic route for the benzimidazothiazolone derivatives 1-13 is illustrated in Scheme 1. To synthesize the core template benzimidazothiazolone 15, commercially available 2-mercaptobenzimidazole was first condensed with bromoacetic acid. The reaction provided the desired compound 15 at a yield of 8%, with an intermediate 14 (59%). In the presence of acetic anhydride and pyridine, the intermediate 14 could be converted to the core template 15 rapidly and in high yield (91%) under reflux. Heating 15 and various benzaldehydes in the presence of acetic acid and sodium acetate for 15 h to 3 days produced 13 benzimidazothiazolone derivatives 1-13 as solids in yields of 35-82%. The configuration of the newly formed double bond was determined using the vicinal 1 H and 13 C-coupling constants ( 3 J) in the proton-coupled 13 C spectra.
Vögeli et al. [42] reported that the configuration of trisubstituted exocyclic C, C-double bonds in a general structure A ( Figure 1) differentiated the C, H-spin coupling constants over three bonds. The 3 J of C(1) of compounds in which the oxygen of carbonyl and the 3-hydrogen were on the same side was 3.6 to 6.4 Hz, whereas that of the compounds in which the oxygen of carbonyl and 3-hydrogen were on the same side was roughly twice as large (generally >10 Hz). The 3 J of C(1) of compound 11 was 5.3 Hz, confirming that 11 is a (Z)-isomer. Structures of the 13 final compounds were confirmed with 1 H and 13 C NMR spectroscopy and mass spectroscopy. lowed by Dunnett's test. An associated probability of (p value) <0.05 was considered nificant.

Synthesis of (Z)-2-(Substituted benzylidene)benzimidazothiazolone Derivatives 1-13
The synthetic route for the benzimidazothiazolone derivatives 1-13 is illustrated Scheme 1. To synthesize the core template benzimidazothiazolone 15, commercially av able 2-mercaptobenzimidazole was first condensed with bromoacetic acid. The react provided the desired compound 15 at a yield of 8%, with an intermediate 14 (59%). In presence of acetic anhydride and pyridine, the intermediate 14 could be converted to core template 15 rapidly and in high yield (91%) under reflux. Heating 15 and vari benzaldehydes in the presence of acetic acid and sodium acetate for 15 h to 3 days p duced 13 benzimidazothiazolone derivatives 1-13 as solids in yields of 35-82%. The c figuration of the newly formed double bond was determined using the vicinal 1 H and coupling constants ( 3 J) in the proton-coupled 13 C spectra.
Vögeli et al. [42] reported that the configuration of trisubstituted exocyclic C, C-d ble bonds in a general structure A ( Figure 1) differentiated the C, H-spin coupling c stants over three bonds. The 3 J of C(1) of compounds in which the oxygen of carbonyl a the 3-hydrogen were on the same side was 3.6 to 6.4 Hz, whereas that of the compou in which the oxygen of carbonyl and 3-hydrogen were on the same side was roughly tw as large (generally >10 Hz). The 3 J of C(1) of compound 11 was 5.3 Hz, confirming tha is a (Z)-isomer. Structures of the 13 final compounds were confirmed with 1 H and NMR spectroscopy and mass spectroscopy.
The position and number of hydroxyl substituents on the phenyl ring significantly influenced the inhibitory activity against tyrosinase. However, the details of certain chemical structures attached to the one or two hydroxyphenyl moieties for tyrosinase activity remain unknown; this will be the focus of future research. Insertion of an additional hydroxyl group into the β-phenyl ring of compound 1 with a 4-hydroxyphenyl ring increased tyrosinase inhibitory activity (compounds 1 vs. 2 and 3). Furthermore, the introduction of methoxyl, ethoxyl, or bromo groups at the 3-position of the β-phenyl ring of compound 1 with a 4-hydroxyphenyl ring abated tyrosinase inhibitory activity (compounds 1 vs. 4 and 5 and 13). Compounds 7, 8, 9, and 11, without a hydroxyl group on the β-phenyl ring showed weak or no inhibition. The 3-methoxy-4-hydroxy group of the β-phenyl ring increased tyrosinase inhibitory activity compared to the 3hydroxy-4-methoxy group of the β-phenyl ring (compounds 4 vs. 6). Unlike the hydroxyl group, 2,4-dimethoxyphenyl groups act as hydrogen bond acceptors and are very weak or inactive. The present results are in close agreement with those of a previous reported [45].
Based on the results of our previous studies, 3,4-dihydroxyphenyl compound exhibit potent inhibitory activity against mushroom tyrosinase and cell-based cellular tyrosinase assays [46]. As expected, compound 2, with a 3,4-dihydroxyphenyl substituent (catechol moiety), exerted excellent tyrosinase inhibition, and compounds with 4-hydroxyphenyl (1) and 2,4-dihydroxyphenyl (3, resorcinol moiety) substituents also exerted potent tyrosinase inhibition. These results indicate that the 4-substitueted resorcinol moiety importantly confers tyrosinase inhibiting activity. The position and number of hydroxyl substituents on the phenyl ring significantly influenced the inhibitory activity against tyrosinase. However, the details of certain chemical structures attached to the one or two hydroxyphenyl moieties for tyrosinase activity remain unknown; this will be the focus of future research.

Enzyme Kinetic Studies of Compounds 1-3
To elucidate the type of enzymatic inhibition, a kinetic analysis was accomplished at several concentrations of the L-tyrosine substrate and inhibitors (1-3), according to the Lineweaver-Burk and Dixon plot methods (Table 3 and Figure 2). Kojic acid, a competitive tyrosinase inhibitor, was used as the standard [44]. Using the Lineweaver-Burk plot method, the lines representing inhibitors 1-3 intersected at the same point on the y-axis, demonstrating that Km increased with increasing concentrations of inhibitors 1-3, while 1/V max did not change [33]. Compounds 1-3 showed competitive inhibition. These data suggest that compounds 1-3 are effective inhibitors that bind to the active site of the enzyme [7]. Furthermore, the Dixon plot is a graphical method [plot of 1/enzyme velocity (1/V) against inhibitor concentration (I)] used to determine the type of enzymatic inhibition and the dissociation or inhibition constant (K ic ) for the enzyme-inhibitor complex [33].

Enzyme Kinetic Studies of Compounds 1-3
To elucidate the type of enzymatic inhibition, a kinetic analysis was accomplished at several concentrations of the L-tyrosine substrate and inhibitors (1-3), according to the Lineweaver-Burk and Dixon plot methods (Table 3 and Figure 2). Kojic acid, a competitive tyrosinase inhibitor, was used as the standard [44]. Using the Lineweaver-Burk plot method, the lines representing inhibitors 1-3 intersected at the same point on the y-axis, demonstrating that Km increased with increasing concentrations of inhibitors 1-3, while 1/Vmax did not change [33]. Compounds 1-3 showed competitive inhibition. These data suggest that compounds 1-3 are effective inhibitors that bind to the active site of the enzyme [7]. Furthermore, the Dixon plot is a graphical method [plot of 1/enzyme velocity (1/V) against inhibitor concentration (I)] used to determine the type of enzymatic inhibition and the dissociation or inhibition constant (Kic) for the enzyme-inhibitor complex [33].
The Kic (≈Ki) values for compounds 1-3 were 16.55, 3.21, and 3.01 μM, respectively, against mushroom tyrosinase (Table 3 and Figure 2). As the Kic value represents the concentration required to combine the inhibitor with an enzyme, compounds with lower Kic values were generally more effective tyrosinase inhibitors; this is an important requisite for the development of preventive and therapeutic agents.  The K ic (≈K i ) values for compounds 1-3 were 16.55, 3.21, and 3.01 µM, respectively, against mushroom tyrosinase (Table 3 and Figure 2). As the K ic value represents the concentration required to combine the inhibitor with an enzyme, compounds with lower K ic values were generally more effective tyrosinase inhibitors; this is an important requisite for the development of preventive and therapeutic agents.

Docking Simulation and of Ligands against Tyrosinase
Based on the in vitro results, compound 1-3-docking complexes were evaluated to understand their binding conformation within the active site of the tyrosinase. To predict the binding site of the potent compounds 1-3, molecular docking simulations were performed using the open-source programs for AutoDock4.2 [36] and AutoDock Vina [47] along with AutoDockTools, a graphical user interface compliment to the AutoDock software suite. AutoDock 4.0 uses a semi-empirical free energy force field to predict the binding free energies of protein-ligand complexes of a known structure and the binding energy for both bound and unbound states [36]. AutoDock vina was developed as a successor to AutoDock 4.2 and provides more rapid and accurate results with less direct investment required by the end user [48].

Cell Viability of Compounds 1-3
The anti-melanogenic activity of compounds 1-3, which were the most effective in B16F10 cells, was tested in a cell-based in vitro model. The EZ-Cytox assay was used to estimate cell viability. Cells were treated with various concentrations (5-200 µM) of compounds 1-3 and trolox to examine whether these compounds exhibited cytotoxic effects on melanocytes. The results indicated that compounds 1-3 and trolox did not significantly affect cell viability at concentrations up to 20 µM compared with the vehicle control cells for 48 h (Figure 4). Consequently, 20 µM sample concentrations of compounds 1-3 and trolox were used for further experiments.
Kojic acid c -4.21 -5.6 HIS85, HIS259, ASN260, HIS263, MET280, GLY281, SER282, ALA286. a Binding energy indicates the binding affinity and capacity for the active site of mushroom tyrosinase enzyme. b All amino acid residues of the enzyme-inhibitor complex were determined using the AutoDock 4.2 and AutoDock vina programs. c Reported competitive-type inhibitors.

Cell Viability of Compounds 1-3
The anti-melanogenic activity of compounds 1-3, which were the most effective in B16F10 cells, was tested in a cell-based in vitro model. The EZ-Cytox assay was used to estimate cell viability. Cells were treated with various concentrations (5-200 μM) of compounds 1-3 and trolox to examine whether these compounds exhibited cytotoxic effects on melanocytes. The results indicated that compounds 1-3 and trolox did not significantly affect cell viability at concentrations up to 20 μM compared with the vehicle control cells  for 48 h (Figure 4). Consequently, 20 μM sample concentrations of compounds 1-3 and trolox were used for further experiments.

ROS Scavenging Activity
ROS may regulate melanogenesis in melanoma cells [20,54,55]. The principle of the intracellular ROS assay is that DCF-DA spreads through the cell membrane and is enzymatically hydrolyzed to DCF by esterase; DCFH then responses with ROS to yield DCF [56]. The ROS scavenging activity of compounds 1-3 was investigated in vitro by using fluorescence probes, (DCF-DA) for ROS. SIN-1 is used as an NO donor [57,58]. As demonstrated in Figure 5A, compound 2 exerted potent scavenging activity against ROS, with an IC50 value of 2.36 ± 0.48 μM, comparable with that of the reference control, trolox, which had an IC50 value of 13.25 ± 0.73 μM. However, compounds 1 and 3 did not present any activity at the tested concentrations at ≥50 μM. Furthermore, as shown in Figure 5B, treatment with SIN-1 significantly increased ROS generation compared to that in untreated B16F10 cells. Pretreatment with compound 2 reduced SIN-1-induced ROS generation in a dose-dependent manner. These results indicated that compound 2 attenuated the SIN-1induced increase in ROS activity and intracellular ROS levels. Structure comparison of the benzimidaxothiazolone skeleton and ROS scavenging activities reveals that a ortho-dihydroxy groups of the benzylidene plays a crucial role in the observed scavenging activity. The importance of catechol moiety (3,4-dihydroxy group) for ROS scavenging activity was reported in previous studies in the literature [59,60].

ROS Scavenging Activity
ROS may regulate melanogenesis in melanoma cells [20,54,55]. The principle of the intracellular ROS assay is that DCF-DA spreads through the cell membrane and is enzymatically hydrolyzed to DCF by esterase; DCFH then responses with ROS to yield DCF [56]. The ROS scavenging activity of compounds 1-3 was investigated in vitro by using fluorescence probes, (DCF-DA) for ROS. SIN-1 is used as an NO donor [57,58]. As demonstrated in Figure 5A, compound 2 exerted potent scavenging activity against ROS, with an IC 50 value of 2.36 ± 0.48 µM, comparable with that of the reference control, trolox, which had an IC 50 value of 13.25 ± 0.73 µM. However, compounds 1 and 3 did not present any activity at the tested concentrations at ≥50 µM. Furthermore, as shown in Figure 5B, treatment with SIN-1 significantly increased ROS generation compared to that in untreated B16F10 cells. Pretreatment with compound 2 reduced SIN-1-induced ROS generation in a dose-dependent manner. These results indicated that compound 2 attenuated the SIN-1-induced increase in ROS activity and intracellular ROS levels. Structure comparison of the benzimidaxothiazolone skeleton and ROS scavenging activities reveals that a orthodihydroxy groups of the benzylidene plays a crucial role in the observed scavenging activity. The importance of catechol moiety (3,4-dihydroxy group) for ROS scavenging activity was reported in previous studies in the literature [59,60].

Melanin Contents and Cellular Tyrosinase Assay
α-MSH and IBMX increase the levels of intracellular cAMP, which induces melanogenesis [9,61,62]. Melanin contents were measured after pretreating the B16F10 cells with different concentrations (5, 10, and 20 µM) of the compounds 1-3 for 3 h, followed by 48 h of α-MSH and IBMX treatment. The melanin contents increased to 134.78 ± 2.95%, 137.96 ± 3.02%, and 134.78 ± 2.95% ( Figure 6A-C, respectively) following treatment with α-MSH and IBMX. As shown in Figure 6B, compound 2 led to a dose-dependent decrease in the intracellular tyrosinase activity to 116.75 ± 3.52%, 100.52 ± 2.08%, and 78.80 ± 3.66%. Kojic acid at 20 µM reduced the content to 128.53 ± 2.95%. In parallel to the enzyme assay, compound 1 reduced the melanin content at 20 µM ( Figure 6A), and compound 3 increased the melanin contents in a dose-dependent manner ( Figure 6C). To determine the inhibitory potency of compounds 1-3 in the cellular model system, the inhibitory effect on the tyrosinase activity of B16F10 cells treated with 5 µM α-MSH and 200 µM IBMX were examined. The level of intracellular tyrosinase after α-MSH and IBMX treatment was 347.03 ± 7.97%, 356.28 ± 27.70%, and 341.30 ± 26.76% ( Figure 6D-F, respectively). However, cellular tyrosinase activity decreased in a concentration-dependent manner upon exposure to compounds 1-3. In both melanin contents and cellular tyrosinase inhibitory assay, compound 2 exhibited potent inhibitory activities, indicating that the presence of 3,4-dihydroxy (catechol moiety) substituent of benzimidaxothiazolone derivatives influence inhibitory activity. These results suggest that compound 2 is a promising candidate therapeutic anti-melanogenesis agent among benzimidazothiazolone derivatives.

Melanin Contents and Cellular Tyrosinase Assay
α-MSH and IBMX increase the levels of intracellular cAMP, which induces melanogenesis [9,61,62]. Melanin contents were measured after pretreating the B16F10 cells with different concentrations (5, 10, and 20 μM) of the compounds 1-3 for 3 h, followed by 48 h of α-MSH and IBMX treatment. The melanin contents increased to 134.78 ± 2.95%, 137.96 ± 3.02%, and 134.78 ± 2.95% ( Figure 6A-C, respectively) following treatment with α-MSH and IBMX. As shown in Figure 6B, compound 2 led to a dose-dependent decrease in the intracellular tyrosinase activity to 116.75 ± 3.52%, 100.52 ± 2.08%, and 78.80 ± 3.66%. Kojic acid at 20 μM reduced the content to 128.53 ± 2.95%. In parallel to the enzyme assay, compound 1 reduced the melanin content at 20 μM ( Figure 6A), and compound 3 increased the melanin contents in a dose-dependent manner ( Figure 6C). To determine the inhibitory potency of compounds 1-3 in the cellular model system, the inhibitory effect on the tyrosinase activity of B16F10 cells treated with 5 μM α-MSH and 200 μM IBMX were examined. The level of intracellular tyrosinase after α-MSH and IBMX treatment was 347.03 ± 7.97%, 356.28 ± 27.70%, and 341.30 ± 26.76% ( Figure 6D-F, respectively). However, cellular tyrosinase activity decreased in a concentration-dependent manner upon exposure to compounds 1-3. In both melanin contents and cellular tyrosinase inhibitory assay, compound 2 exhibited potent inhibitory activities, indicating that the presence of 3,4-dihydroxy (catechol moiety) substituent of benzimidaxothiazolone derivatives influence inhibitory activity. These results suggest that compound 2 is a promising candidate therapeutic anti-melanogenesis agent among benzimidazothiazolone derivatives.

Effects of Compound 2 on Melanogenesis-Related Signaling
It is important to determine whether the inhibition of melanin synthesis by compound 2 is related to melanogenesis signaling and gene expression. To investigate

Effects of Compound 2 on Melanogenesis-Related Signaling
It is important to determine whether the inhibition of melanin synthesis by compound 2 is related to melanogenesis signaling and gene expression. To investigate whether compound 2 could affect the melanogenic signaling of tyrosinase, the expression of tyrosinase, phosphorylated CREB and PKA was determined with Western blot analysis using cytosolic factions from the cell lysates of α-MSHand IBMX-induced B16F10 cells. As shown in Figure 7A,B, treatment with α-MSH and IBMX significantly upregulated these proteins; however, compound 2 decreased their expression on these proteins in a dosedependent manner. MITF, a leucine zipper transcription factor, activates the expression of multiple genes encoding enzymes involved in the conversion of tyrosine into melanin, resulting in increased levels of these melanin-producing proteins [63,64]. Expression of MITF in α-MSHand IBMX-induced B16F10 cells was upregulated, while compound 2 significantly suppressed MITF levels in the nuclear faction from total cell lysate ( Figure 7C). The skin is site of oxidative stress due to exposure to daylight and environmental oxidizing pollutants. It is known that ROS stimulates α-MSHand IBMX-induced production in melanoma cells leading to melanogenesis by upregulating MITF, a transcription factor that induces tyrosinase gene expression [65,66]. Since oxidative stress has been shown to activate MITF through MAPK, we investigated whether compound 2 controls MAPK signaling [67,68]. The results shown in Figure 7D,E revealed that compound 2 decreased the levels of phosphorylated JNK and ERK in a concentration-dependent manner. However, the levels of phosphorylated p38 were not affected by treatment with compound 2. In our study, both p-JNK and p-ERK were decreased by compound 2. Thus, our results suggest that compound 2, which suppresses the phosphorylated PKA and MAPK signaling pathways and MITF-mediated melanogenic enzyme expression, has potential as an anti-melanogenic agent.

Conclusions
To determine whether benzimidazothiazolone with a (Z)-β-phenyl-α,β-unsaturated carbonyl scaffold plays an important role in tyrosinase inhibition, 13 derivatives were synthesized. The mushroom tyrosinase inhibitory assay revealed that three benzimidazothi- Figure 7. Effects of compound 2 on melanogenic proteins and mRNA levels in α-MSHand IBMX-induced B16F10 cells. Cells were pre-incubated for 24 h and then stimulated with α-MSH (5 µM) and IBMX (200 µM) in the presence of compound 2 (5, 10, and 20 µM). The expression levels of tyrosinase, p-CREB, p-PKA, p-JNK, p-ERK, p-p38, and β-actin were determined with Western blotting in the cytosolic fraction. β-actin was used as the loading control (A,B,D,E). The expression levels of MITF and TFIIB were determined with Western blotting in the nuclear fraction. TFIIB was used as the loading control (C). Relative mRNA levels of the melanogenic genes MITF, Tyrosinase, Trp1, and Trp2 in the cells were quantified using real-time qPCR and normalized to the GAPDH mRNA level (F). The primer sets are shown in Table 1. Data are presented as the mean ± SEM of three independent experiments. ## p < 0.01 and ### p < 0.001 vs. control. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. α-MSH and IBMX-treated cells.
The effects of compound 2 on the expression of mRNA melanogenic factors were evaluated in α-MSHand IBMX-induced B16F10 cells. Total mRNAs were extracted from cells treated with α-MSH and IBMX in the absence or presence of compound 2. Tyrosinase, TRP1 and TRP2 are key enzymes involved in melanin biosynthesis [64]. During melanin synthesis, tyrosine undergoes tyrosinase-dependent conversion, which is catalyzed by dapaquinone and subsequently by dopachrome. TRP2 converts dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA), whereas TRP1 oxidizes DHICA to a carboxylated indole-quinone, which is eventually converted into melanin [12,68,69]. The mRNA levels of tyrosinase, TRP1, TRP2, and MITF were quantified with real-time qPCR ( Figure 7F). Compound 2 significantly inhibited the expression of the MITF and downstream enzymes, such as tyrosinase, TRP1, and TRP2. These results indicate that compound 2 might be an effective hypopigmenting agent with implications in various dermatologic hyperpigmentation disorders, such as freckles and melasma, and has useful effects in whitening cosmetic agents.

Conclusions
To determine whether benzimidazothiazolone with a (Z)-β-phenyl-α,β-unsaturated carbonyl scaffold plays an important role in tyrosinase inhibition, 13 derivatives were synthesized. The mushroom tyrosinase inhibitory assay revealed that three benzimidazothiazolone derivatives (compounds 1-3) with 4-, 3,4-, and 2,4-dihydroxyls on the phenyl ring of the benzimidazothiazolone scaffold, respectively, inhibited tyrosinase significantly more than the positive control (kojic acid). Enzymatic kinetic studies indicated that compounds 1-3 were competitive inhibitors. Docking simulation of compounds 1-3 predicted that they bind more strongly to the active site of tyrosinase than kojic acid. In vitro experiments showed that compounds 1-3 inhibited cellular tyrosinase inhibitory activity in a dose-dependent manner. Compound 2 scavenged ROS and inhibited melanin biosynthesis downregulating the PKA/CREB and MAPK signaling pathways, leading to suppression of MITF expression and tyrosinase ( Figure 8). This is the first report of benzimidazothiazolone with a (Z)-β-phenyl-α,β-unsaturated carbonyl scaffold for anti-tyrosinase and anti-melanogenesis effects to the best our knowledge. In the present study, it was found that compound 2 suppressed intracellular ROS level in a dose-dependent manner. Compound 2 appears to be a potential whitening agent that might protect melanoma cells from oxidative injury. Hence, compound 2 has potential for use as a novel dermatological anti-melanogenesis agent and an effective skin-whitening agent. Although many synthetic inhibitors exhibited remarkable tyrosinase inhibitory activity, only a few of them showed melanogenesis inhibition activity in cell-based or skin models. Therefore, further studies may be required to assess the potential for developing safe products of skin-whitening agents by in vivo studies [70], such as zebra fish, and may have practical applications for humans. In the present study, it was found that compound 2 suppressed intracellular ROS level in a dose-dependent manner. Compound 2 appears to be a potential whitening agent that might protect melanoma cells from oxidative injury. Hence, compound 2 has potential for use as a novel dermatological anti-melanogenesis agent and an effective skin-whitening agent. Although many synthetic inhibitors exhibited remarkable tyrosinase inhibitory activity, only a few of them showed melanogenesis inhibition activity in cell-based or skin models. Therefore, further studies may be required to assess the potential for developing safe products of skin-whitening agents by in vivo studies [70], such as zebra fish, and may have practical applications for humans.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/antiox10071078/s1. The synthetic methods and 1 H and 13 C NMR data for studied compounds are included.