Design, Synthesis, and Anti-Tyrosinase, Anti-Melanogenic, and Antioxidant Activities of Novel (Z)-3-Benzyl-5-Benzylidene-2-Thioxothiazolidin-4-One Analogs

Abstract

To discover novel anti-melanogenic compounds with tyrosinase inhibitory activity, (Z)-3-benzyl-5-benzylidene-2-thioxothiazolidin-4-one ((Z)-BBTT) analogs 1-12, designed based on the hybrid structure of a β-phenyl-α,β-unsaturated carbonyl motif and a 3-benzyl-2-thioxothiazolidin-4-one scaffold, were synthesized as novel tyrosinase inhibitors. Of the 12 analogs, 2 (6 and 8) showed mushroom tyrosinase inhibitory activity similar to that of kojic acid, a representative tyrosinase inhibitor, and 3 analogs (1-3) exhibited mushroom tyrosinase inhibitory activity that was more potent than that of kojic acid. In particular, analog 3 revealed highly potent inhibition with an IC₅₀ value of 90 nM, which was 214 times lower than that of kojic acid (IC₅₀ value = 19.22 μM). A kinetic study using mushroom tyrosinase and analogs 1-3 and 6 demonstrated that these analogs were competitive inhibitors, which was further supported by in silico studies. Analogs 1 and 3 have strong anti-melanogenic potency in B16F10 mammalian cells owing to their anti-tyrosinase activity without perceptible cytotoxicity in melanoma cells (B16F10) and the main epidermal cells (HaCaT). Moreover, analog 3 exhibited strong antioxidant capacity, scavenging reactive oxygen species, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical, and 2,2-diphenyl-1-picrylhydrazyl radical, partially contributing to its anti-melanogenic effect. (Z)-BBTT analogs, including analog 3, may be promising candidates for inhibiting melanin production.

Keywords: (Z)-BBTT; B16F10 cells; melanin; tyrosinase; β-phenyl-α,β-unsaturated carbonyl.

Figure 1

Chemical structures of β-phenyl-α,β-unsaturated carbonyl (PUSC) and lipophilic 3-benzyl-2-thioxothiazolidin-4-one, and the final target compounds, (Z)-3-benzyl-5-benzylidene-2-thioxothiazolidin-4-one ((Z)-BBTT) analogs created by the structural hybrid of PUSC and 3-benzyl-2-thioxothiazolidin-4-one.

Scheme 1

Synthetic scheme of (Z)-3-benzyl-5-benzylidene-2-thioxothiazolidin-4-one ((Z)-BBTT) analogs 1–12. The reagents and conditions were as follows: (a) Et₃N, CH₂Cl₂, RT, 3.5 h, and 94% and (b) NaOAc, AcOH, reflux, 2–4 h, and 47–92%.

Figure 2

Lineweaver–Burk plots for 1–3 (A–C) and 6 (D) obtained by measuring the initial dopachrome production rate in the presence of various l-DOPA concentrations (1, 2, 4, 8, and 16 mM) using mushroom tyrosinase. The concentrations of the test analogs were 0, 20, 40, and 80 μM for 1; 0, 15, 30, and 60 μM for 2; 0, 0.1, 0.2, and 0.4 μM for 3; and 0, 12.5, 25, and 50 μM for 6.

Figure 3

Dixon plots for analogs 1–3 (A–C) and 6 (D) obtained by converting Lineweaver–Burk plots. The concentrations of the test analogs were 0, 20, 40, and 80 μM for 1; 0, 15, 30, and 60 μM for 2; 0, 0.1, 0.2, and 0.4 μM for 3; and 0, 12.5, 25, and 50 μM for 6. l-DOPA was used at concentrations of 1, 2, 4, and 8 mM.

Figure 4

Chemical interactions between tyrosinase and ligands (kojic acid [positive control] and analogs 1–3 and 6) obtained from LigandScout after docking simulation using AutoDock Vina. (A) Two- and (B) three-dimensional pictures. Blue arrow, pi–pi stacking; yellow wave, hydrophobic interaction; green arrow, hydrogen bonding (H–B donor from the ligand’s perspective); and red arrow, hydrogen bonding (H–B acceptor from the ligand’s perspective). (B) In molecules, red, blue, and yellow represent oxygen, nitrogen, and sulfur, respectively.

Figure 5

B16F10 cell viability in the presence of (Z)-3-benzyl-5-benzylidene-2-thioxothiazolidin-4-one analogs 1 (A), 2 (B), and 3 (C). *** p < 0.001 and ** p < 0.01 vs. the nontreated group.

Figure 6

Effect of analogs 1 (A) and 3 (B) on melanin production in B16F10 cells. Stimulators α-MSH and IBMX were treated at 1 and 200 μM, respectively, 1 h after treatment of analogs (2, 5, and 10 μM). After 72 h, melanin contents were determined by measuring the optical density at 405 nm. Kojic acid was used as a positive control. α-MSH, α-melanocyte-stimulating hormone; IBMX, 3-isobutyl-1-methylxanthine. *** p < 0.001, ** p < 0.01, and * p < 0.05 vs. stimulator-treated group; ^### p < 0.001 vs. untreated control.

Figure 7

Effects of analogs 1 (A) and 3 (B) on B16F10 cellular tyrosinase activity. Stimulators α-MSH and IBMX were treated at 1 and 200 μM, respectively, 1 h after treatment of analogs (2, 5, and 10 μM). After 72 h, tyrosinase activity was determined by measuring optical density at 475 nm. α-MSH, α-melanocyte-stimulating hormone; IBMX, 3-isobutyl-1-methylxanthine. *** p < 0.001 and ** p < 0.01 vs. stimulator-treated group; ^### p < 0.001 vs. untreated control.

Figure 8

(A) In situ B16F10 cell tyrosinase activity determined using an l-DOPA staining method. Kojic acid (10 μM) was used as a positive reference standard. Test samples (kojic acid and analogs 1 and 3) were administered 1 h before treatment with α-MSH (1 μM) and IBMX (200 μM). The analogs were treated at 2, 5, and 10 μM. The results of in situ cellular tyrosinase activity were obtained 72 h after treatment with the analogs or kojic acid. Arrows indicate stained cells. Scale bar = 100 μm. (B) Relative amounts of stained areas were measured using an ATTO CS analyzer 3.2. α-MSH, α-melanocyte-stimulating hormone; IBMX, 3-isobutyl-1-methylxanthine; KA, kojic acid. ^### p < 0.001 vs. control; *** p < 0.001 vs. α-MSH plus IBMX-treated group.

Figure 9

Antioxidant effects of (Z)-BBTT analogs 1–12 on radicals of ABTS⁺ (A) and DPPH (B) and ROS (C). (A) Analogs 1–12 and Trolox (TR; positive reference standard) were used at 100 μM. (B) Analogs 1–12 and ascorbic acid (AA; positive reference standard) were used at 500 μM. (C) Analogs 1–12, Trolox (TR; positive reference standard), and SIN-1, a ROS generator, were used at 40, 40, and 10 μM, respectively. In (A,B), *** p < 0.001 vs. the control group and in (C), ** p < 0.01 and *** p < 0.001 vs. the SIN-1-treated group; ^### p < 0.001 vs. the control group. (Z)-BBTT, (Z)-3-benzyl-5-benzylidene-2-thioxothiazolidin-4-one; ABTS⁺; 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation; DPPH, 2,2-diphenyl-1-picrylhydrazyl; SIN-1, 3-morpholinosydnonimine; ROS, reactive oxygen species.

Figure 10

HaCaT cell viability in the presence of analogs 1–3 (A–C). All analogs were administered at concentrations of 0, 2, 5, 10, and 20 μM for 72 h.