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Compounds with sulfhydryl substituents and azole compounds exhibit potent anti-tyrosinase potency. 2-Thiobenzothiazole (2-TBT), a hybrid structure of sulfhydryl and azole, exists in two tautomeric forms, with the thione form being predominant according to several studies. 2-TBT derivatives were synthesized as potential tyrosinase inhibitors as the thione tautomeric form has the same N-CS moiety as phenylthiourea (PTU), which is suitable for chelation with the copper ions present in the tyrosinase active site. Eight of the ten 2-TBT derivatives inhibited the monophenolase and diphenolase activities of mushroom tyrosinase, with IC 50 values of 0.02-0.83 μM. Kinetic studies and molecular dynamics simulations were performed to determine their mode of action and confirm that the 2-TBT derivatives bind to the tyrosinase active site with high stability. Derivatives 3, 4, 8, and 10 strongly inhibited melanogenesis in B16F10 cells in a pattern similar to the results of cellular tyrosinase inhibition, thereby suggesting that their ability to inhibit melanogenesis was due to their tyrosinase inhibitory activity. In a depigmentation experiment using zebrafish embryos, all 2-TBT derivatives showed better potency than kojic acid, even at 400 to 2000 times lower concentration, and 1 and 10 reduced zebrafish larva pigmentation more strongly than PTU even at 20 times lower concentration. Experiments investigating the changes in tyrosinase inhibitory activity of 2-TBT derivatives in the presence and absence of CuSO 4 and their copper chelating ability supported that these derivatives exert their anti-melanogenic effect by chelating the copper ions of tyrosinase. These results suggest that 2-TBT derivatives are promising candidates for the treatment of hyperpigmentation-related disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Based on the fact that substances with a β-phenyl-α,β-unsaturated carbonyl (PUSC) motif confer strong tyrosinase inhibitory activity, benzylidene-3-methyl-2-thioxothiazolidin-4-one (BMTTZD) analogs 1 - 8 were prepared as potential tyrosinase inhibitors. Four analogs ( 1 - 3 and 5 ) inhibited mushroom tyrosinase strongly. Especially, analog 3 showed an inhibitory effect that was 220 and 22 times more powerful than kojic acid in the presence of l-tyrosine and l-dopa, respectively. A kinetic study utilizing mushroom tyrosinase showed that analogs 1 and 3 competitively inhibited tyrosinase, whereas analogs 2 and 5 inhibited tyrosinase in a mixed manner. A docking simulation study indicated that analogs 2 and 5 could bind to both the tyrosinase active and allosteric sites with high binding affinities. In cell-based experiments using B16F10 cells, analogs 1 , 3 , and 5 effectively inhibited melanin production; their anti-melanogenic effects were attributed to their ability to inhibit intracellular tyrosinase activity. Moreover, analogs 1 , 3 , and 5 inhibited in situ B16F10 cellular tyrosinase activity. In three antioxidant experiments, analogs 2 and 3 exhibited strong antioxidant efficacy, similar to that of the positive controls. These results suggest that the BMTTZD analogs are promising tyrosinase inhibitors for the treatment of hyperpigmentation-related disorders.

( Z )-5-Benzylidene-2-phenylthiazol-4(5 H )-one (( Z )-BPT) derivatives were designed by combining the structural characteristics of two tyrosinase inhibitors. The double-bond geometry of trisubstituted alkenes, ( Z )-BPTs 1 - 14 , was determined based on the 3 J C,Hβ coupling constant of 1 H-coupled 13 C NMR spectra. Three ( Z )-BPT derivatives ( 1 - 3 ) showed stronger tyrosinase inhibitory activities than kojic acid; in particular, 2 was to be 189-fold more potent than kojic acid. Kinetic analysis using mushroom tyrosinase indicated that 1 and 2 were competitive inhibitors, whereas 3 was a mixed-type inhibitor. The in silico results revealed that 1 - 3 could strongly bind to the active sites of mushroom and human tyrosinases, supporting the kinetic results. Derivatives 1 and 2 decreased the intracellular melanin contents in a concentration-dependent manner in B16F10 cells, and their anti-melanogenic efficacy exceeded that of kojic acid. The anti-tyrosinase activity of 1 and 2 in B16F10 cells was similar to their anti-melanogenic effects, suggesting that their anti-melanogenic effects were primarily owing to their anti-tyrosinase activity. Western blotting of B16F10 cells revealed that the derivatives 1 and 2 inhibited tyrosinase expression, which partially contributes to their anti-melanogenic ability. Several derivatives, including 2 and 3 , exhibited potent antioxidant activities against ABTS cation radicals, DPPH radicals, ROS, and peroxynitrite. These results suggest that ( Z )-BPT derivatives 1 and 2 have promising potential as novel anti-melanogenic agents.

In this study, ( Z )-2-(benzylamino)-5-benzylidenethiazol-4(5 H )-one (BABT) derivatives were designed as tyrosinase inhibitors based on the structure of MHY2081, using a simplified approach. Of the 14 BABT derivatives synthesized, two derivatives (( Z )-2-(benzylamino)-5-(3-hydroxy-4-methoxybenzylidene)thiazol-4(5 H )-one [ 7 ] and ( Z )-2-(benzylamino)-5-(2,4-dihydroxybenzylidene)thiazol-4(5 H )-one [ 8 ]) showed more potent mushroom tyrosinase inhibitory activities than kojic acid, regardless of the substrate used; in particular, compound 8 was 106-fold more potent than kojic acid when l-tyrosine was used as the substrate. Analysis of Lineweaver-Burk plots for 7 and 8 indicated that they were competitive inhibitors, which was confirmed via in silico docking. In experiments using B16F10 cells, 8 exerted a greater ability to inhibit melanin production than kojic acid, and it inhibited cellular tyrosinase activity in a concentration-dependent manner, indicating that the anti-melanogenic effect of 8 is attributable to its ability to inhibit tyrosinase. In addition, 8 exhibited strong antioxidant activity to scavenge 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals and peroxynitrite and inhibited the expression of melanogenesis-associated proteins (tyrosinase and microphthalmia-associated transcription factor). These results suggest that BABT derivative 8 is a promising candidate for the treatment of hyperpigmentation-related diseases, owing to its inhibition of melanogenesis-associated protein expression, direct tyrosinase inhibition, and antioxidant activity.

Targeting of tyrosinase has proven to be the best means of identifying safe, efficacious, and potent tyrosinase inhibitors for whitening skin. We designed and synthesized ten NAB (N-(acryloyl)benzamide) derivatives (1a-1j) using the Horner-Wadsworth-Emmons olefination of diethyl (2-benzamido-2-oxoethyl)phosphonate and appropriate benzaldehydes. A mushroom tyrosinase inhibitory assay showed compounds 1a (36.71 ± 2.14% inhibition) and 1j (25.99 ± 2.77% inhibition) inhibited tyrosinase more than the other eight NAB derivatives and kojic acid (21.56 ± 2.93% inhibition), and docking studies indicated 1a (-6.9 kcal/mole) and 1j (-7.5 kcal/mole) had stronger binding affinities for tyrosinase than kojic acid (-5.7 kcal/mole). At a concentration of 25 μM, 1a and 1j were nontoxic in B16F10 melanoma cells and exhibited stronger tyrosinase inhibition (59.70% and 76.77%, respectively) than kojic acid (50.30% inhibition) or arbutin (41.78% inhibition at 400 μM). Similarly, in B16F10 melanoma cells, compounds 1a and 1j at 25 μM decreased total melanin content by 47.97% and 61.77%, respectively (kojic acid; 38.98%). Similarities between inhibitions of tyrosinase activity and melanin contents suggested the anti-melanogenic effects of 1a and 1j were due to tyrosinase inhibition. The excellent DPPH scavenging activity of 1j suggests it might enhance in vivo effect on melanin contents. The study suggests compound 1j offers a potential starting point for the development of safe, potent tyrosinase inhibitors., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Abnormal melanogenesis results in excessive production of melanin, leading to pigmentation disorders. As a key and rate-limiting enzyme for melanogenesis, tyrosinase has been considered an important target for developing therapeutic agents of pigment disorders. Despite having an (E)-β-phenyl-α,β-unsaturated carbonyl scaffold, which plays an important role in the potent inhibition of tyrosinase activity, cinnamic acids have not attracted attention as potential tyrosinase inhibitors, due to their low tyrosinase inhibitory activity and relatively high hydrophilicity. Given that cinnamic acids' structure intrinsically features this (E)-scaffold and following our experience that minute changes in the chemical structure can powerfully affect tyrosinase activity, twenty less hydrophilic cinnamamide derivatives were designed as potential tyrosinase inhibitors and synthesised using a Horner-Wadsworth-Emmons reaction. Four of these cinnmamides (4, 9, 14, and 19) exhibited much stronger mushroom tyrosinase inhibition (over 90% inhibition) at 25 µM compared to kojic acid (20.57% inhibition); crucially, all four have a 2,4-dihydroxy group on the β-phenyl ring of the scaffold. A docking simulation using tyrosinase indicated that the four cinnamamides exceeded the binding affinity of kojic acid, and bound more strongly to the active site of tyrosinase. Based on the strength of their tyrosinase inhibition, these four cinnamamides were further evaluated in B16F10 melanoma cells. All four cinnamamides, without cytotoxicity, exhibited higher tyrosinase inhibitory activity (67.33 - 79.67% inhibition) at 25 μM than kojic acid (38.11% inhibition), with the following increasing inhibitory order: morpholino (9) = cyclopentylamino (14) < cyclohexylamino (19) < N-methylpiperazino (4) cinnamamides. Analysis of tyrosinase activity and melanin content in B16F10 cells showed that the four cinnamamides dose-dependently inhibited both cellular tyrosinase activity and melanin content and that their inhibitory activity at 25 μM was much better than that of kojic acid. The results of melanin content analysis well matched those of the cellular tyrosinase activity analysis, indicating that tyrosinase inhibition by the four cinnamamides is a major factor in the reduction of melanin production. These results imply that these four cinnamamides with a 2,4-dihydroxyphenyl group can act as excellent anti-melanogenic agents in the treatment of pigmentation disorders., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Pigmentation disorders are attributed to excessive melanin which can be produced by tyrosinase. Therefore, tyrosinase is supposed to be a vital target for the treatment of disorders associated with overpigmentation. Based on our previous findings that an (E)-β-phenyl-α,β-unsaturated carbonyl scaffold can play a key role in the inhibition of tyrosinase activity, and the fact that cinnamic acid is a safe natural substance with a scaffolded structure, it was speculated that appropriate cinnamic acid derivatives may exhibit potent tyrosinase inhibitory activity. Thus, ten cinnamamides were designed, and synthesized by using a Horner-Emmons olefination as the key step. Cinnamamides 4 (93.72% inhibition), 9 (78.97% inhibition), and 10 (59.09% inhibition) with either a 2,4-dihydroxyphenyl, or 4-hydroxy-3-methoxyphenyl substituent showed much higher mushroom tyrosinase inhibition at 25 µM than kojic acid (18.81% inhibition), used as a positive control. Especially, the two cinnamamides 4 and 9 having a 2,4-dihydroxyphenyl group showed the strongest inhibition. Docking simulation with tyrosinase revealed that these three cinnamamides, 4, 9, and 10, bind to the active site of tyrosinase more strongly than kojic acid. Cell-based experiments carried out using B16F10 murine skin melanoma cells demonstrated that all three cinnamamides effectively inhibited cellular tyrosinase activity and melanin production in the cells without cytotoxicity. There was a close correlation between cellular tyrosinase activity and melanin content, indicating that the inhibitory effect of the three cinnamamides on melanin production is mainly attributed to their capability for cellular tyrosinase inhibition. These results imply that cinnamamides having the (E)-β-phenyl-α,β-unsaturated carbonyl scaffolds are promising candidates for skin-lighting agents., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Ultraviolet (UV) radiation exposure is the primary cause of extrinsic skin aging, which results in skin hyperpigmentation and wrinkling. In this study, we investigated the whitening effect of (2 E ,5 E )-2,5-bis(3-hydroxy-4-methoxybenzylidene)cyclopentanone (BHCP) on B16F10 melanoma and its anti-wrinkle activity on Hs27 fibroblasts cells. BHCP was found to potently inhibit tyrosinase, with 50% inhibition concentration (IC 50 ) values of 1.10 µM and 8.18 µM for monophenolase (l-tyrosine) and diphenolase (l-DOPA), and the enzyme kinetics study revealed that BHCP is a competitive-type tyrosinase inhibitor. Furthermore, BHCP significantly inhibited melanin content and cellular tyrosinase activity, and downregulated the levels of microphthalmia-associated transcription factor (MITF), phosphorylated levels of cAMP response element-binding (CREB) protein, and tyrosinase in α-melanocyte stimulating hormone (α-MSH)-induced B16F10 melanoma cells. Moreover, BHCP inhibited the phosphorylation of p65 and expression of matrix metalloproteinases (MMP-1, MMP-9, MMP-12, and MMP-13) in Hs27 fibroblasts stimulated with UV radiation. Therefore, our results demonstrate that BHCP may be a good candidate for the development of therapeutic agents for diseases associated with hyperpigmentation and wrinkling.

Many compounds containing the β-phenyl-α,β-unsaturated carbonyl (PUSC) scaffold, including cinnamamide derivatives, have been shown to inhibit tyrosinase potently in vitro and in vivo. Structural changes to cinnamamide derivatives were produced by adding a dithionate functional group to provide eight (Z)-5-(substituted benzylidene)-3-cyclohexyl-2-thioxothiazolidin-4-one analogs with high log p values for skin. These analogs were synthesized using a two-step reaction, and their stereochemistry was confirmed using the 3JC4-Hβ values of C4 measured in proton-coupled 13C mode. Analogs 2 (IC50 = 5.21 ± 0.86 µM) and 3 (IC50 = 1.03 ± 0.14 µM) more potently inhibited mushroom tyrosinase than kojic acid (IC50 = 25.26 ± 1.10 µM). Docking results showed 2 binds strongly to the active site of tyrosinase, while 3 binds strongly to an allosteric site. Kinetic studies using l-tyrosine as substrate indicated 2 and 3 competitively and non-competitively inhibit tyrosinase, respectively, which was supported by our docking results. In B16F10 cells, 3 significantly and concentration-dependently reduced α–MSH plus IBMX induced increases in cellular tyrosinase activity and melanin production and the similarity between these inhibitory patterns implied that the anti-melanogenic effect of 3 might be due to its tyrosinase-inhibitory ability. In addition, 2 and 3 exhibited strong antioxidant effects; for example, they reduced ROS and ONOO– levels and exhibited radical scavenging activities, suggesting that these effects might underlie their anti-melanogenic effects. Furthermore, 3 suppressed the expressions of melanogenesis-associated proteins and genes in B16F10 cells. These results suggest (Z)-5-(substituted benzylidene)-3-cyclohexyl-2-thioxothiazolidin-4-one analogs offer a means of producing novel anti-melanogenesis agents. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Recently, 10 2-mercaptobenzo[ d ]imidazole (2-MBI) compounds (1 – 10) were synthesized. Although all 2-MBI compounds are tyrosinase inhibitors that inhibit mushroom tyrosinase at extremely low concentrations (IC 50 values: 20–740 nM) and effectively inhibit the browning of apples, to our knowledge, no studies have determined whether 2-MBI compounds inhibit mammalian tyrosinase. Mammalian tyrosinase is different from mushroom tyrosinase in its distribution within the cell and has structural characteristics that are different from mushroom tyrosinase in amino acid sequence and in the presence of a quaternary structure. Thus, the effect of the 10 2-MBI compounds on mammalian tyrosinase activity was investigated in B16F10 cells. Six compounds (1 – 6) exhibited stronger intracellular tyrosinase inhibition than that of kojic acid and phenylthiourea (PTU), which are known to be the most potent tyrosinase inhibitors; their strong tyrosinase inhibitory activity robustly inhibited intracellular melanin production in B16F10 cells. None of the tested 2-MBI compounds exhibited appreciable cytotoxicity in HaCaT and B16F10 cells. To confirm the anti -melanogenic efficacy of the 2-MBI compounds in vivo , a zebrafish embryo model was used. At concentrations 100 times lower than kojic acid, most 2-MBI compounds demonstrated much stronger depigmentation efficacy than that of kojic acid, and three 2-MBI compounds (2 – 4) showed depigmentation activity similar to or more potent than that of PTU, resulting in nearly pigment-free zebrafish embryos. These results suggest that 2-MBI compounds may be potential therapeutic agents for hyperpigmentation-related disorders. [Display omitted] • 2-Mercaptobenzo[ d ]imidazole (2-MBI) derivatives (1 – 10) greatly inhibited mammalian cell tyrosinase in B16F10 cells. • Four 2-MBI derivatives inhibited intracellular melanin production in B16F10 cells more potently than kojic acid and PTU. • Depigmentation experiments of 2-MBI derivatives using zebrafish embryos resulted in pigment-free zebrafish embryos. • These 2-MBI derivatives did not exhibit significant cytotoxicity in B16F10 cells and HaCaT cells at concentrations ≤20 μM. • These results suggest that 2-MBI derivatives may be potential therapeutic agents for hyperpigmentation-related disorders. [ABSTRACT FROM AUTHOR]

• Fifteen 2,3-DPA derivatives were designed and synthesized via Perkin reaction. • (Z)-2,3-DPA derivative (1l′) was reported for the first time as a major product. • Mushroom tyrosinase and B16F10 cells were used for in vitro studies. • 1c significantly decreased the activity of mushroom and cellular tyrosinase. • In docking studies, 1c strongly binds to tyrosinase than kojic acid. During our continued search for strong skin whitening agents over the past ten years, we have investigated the efficacies of many tyrosinase inhibitors containing a common (E)-β-phenyl-α,β-unsaturated carbonyl scaffold, which we found to be essential for the effective inhibition of mushroom and mammalian tyrosinases. In this study, we explored the tyrosinase inhibitory effects of 2,3-diphenylacrylic acid (2,3-DPA) derivatives, which also possess the (E)-β-phenyl-α,β-unsaturated carbonyl motif. We synthesized fourteen (E)-2,3-DPA derivatives 1a – 1n and one (Z)-2,3-DPA-derivative 1l′ using a Perkin reaction with phenylacetic acid and appropriate substituted benzaldehydes. In our mushroom tyrosinase assay, 1c showed higher tyrosinase inhibitory activity (76.43 ± 3.53%, IC 50 = 20.04 ± 1.91 µM) with than the other 2,3-DPA derivatives or kojic acid (21.56 ± 2.93%, IC 50 = 30.64 ± 1.27 μM). Our mushroom tyrosinase inhibitory results were supported by our docking study, which showed compound 1c (−7.2 kcal/mole) exhibited stronger binding affinity for mushroom tyrosinase than kojic acid (−5.7 kcal/mole). In B16F10 melanoma cells (a murine cell-line), 1c showed no cytotoxic effect up to a concentration of 25 μM and exhibited greater tyrosinase inhibitory activity (68.83%) than kojic acid (49.39%). In these cells, arbutin (a well-known tyrosinase inhibitor used as the positive control) only inhibited tyrosinase by 42.67% even at a concentration of 400 μM. Furthermore, at 25 µM, 1c reduced melanin contents in B16F10 melanoma cells by 24.3% more than kojic acid (62.77% vs. 38.52%). These results indicate 1c is a promising candidate treatment for pigmentation-related diseases and potential skin whitening agents. [ABSTRACT FROM AUTHOR]