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Based on the hypothesis that the 2-mercaptoacetamide moiety chelates the copper ions of tyrosinase, 2-mercapto- N -arylacetamide (2-MAA) analogs were designed and synthesized as potential tyrosinase inhibitors. Four 2-MAA analogs showed low IC 50 values ranging from 0.95 to 2.0 μM against mushroom tyrosinase, which was 12-26 times lower than that of kojic acid (IC 50 value = 24.3 μM). However, according to a copper ion chelation experiment performed, the 2-MAA analogs did not participate in chelation with copper ions. To identify the mode of inhibition of the 2-MAA analogs, kinetic studies were performed, and the results were supported by docking results. In addition, docking simulation results suggested that the 2-MAA analogs strongly inhibited tyrosinase activity because of the hydrogen bonding of the amide NH group and the hydrophobic interaction of the aryl ring instead of chelation with copper ions. In experiments using B16F10 cells, 2-MAA analogs were shown to inhibit melanin production by inhibiting cellular tyrosinase activity. Western blotting showed that in addition to directly inhibiting tyrosinase activity, analog 7 also has an anti-melanogenic effect by inhibiting the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. The 2-MAA analogs showed no appreciable cytotoxicity against HaCaT and B16F10 cells, making them suitable for dermal applications. In a depigmentation experiment using zebrafish embryos, analogs 1 and 2 showed more potent depigmentation effects than kojic acid even at 1000 times lower concentration than that of kojic acid. These results suggest that the 2-MAA analogs are promising anti-melanogenic agents that can inhibit most tyrosinases in various species.

Inspired by the potent tyrosinase inhibitory activity of phenolic compounds with a 2-phenylbenzo[ d ]thiazole scaffold, we explored phenolic compounds 1 - 15 with 2-phenylbenzo[ d ]oxazole, which is isosterically related to 2-phenylbenzo[ d ]thiazole, as novel tyrosinase inhibitors. Among these, compounds 3 , 8 , and 13 , featuring a resorcinol structure, exhibited significantly stronger mushroom tyrosinase inhibition than kojic acid, with compound 3 showing a nanomolar IC 50 value of 0.51 μM. These results suggest that resorcinol plays an important role in tyrosinase inhibition. Kinetic studies using Lineweaver-Burk plots demonstrated the inhibition mechanisms of compounds 3 , 8 , and 13 , while docking simulation results indicated that the resorcinol structure contributed to tyrosinase binding through hydrophobic and hydrogen bonding interactions. Additionally, these compounds effectively inhibited tyrosinase activity and melanin production in B16F10 cells and inhibited B16F10 tyrosinase activity in situ in a concentration-dependent manner. As these compounds showed no cytotoxicity to epidermal cells, melanocytes, or keratinocytes, they are appropriate for skin applications. Compounds 8 and 13 demonstrated substantially higher depigmentation effects on zebrafish larvae than kojic acid, even at 800- and 400-times lower concentrations than kojic acid, respectively. These findings suggest that 2-phenylbenzo[ d ]oxazole is a promising candidate for tyrosinase inhibition.

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.

A series of ( E )-1-(furan-2-yl)prop-2-en-1-one derivatives (compounds 1 - 8 ) were synthesized and evaluated for their mushroom tyrosinase inhibitory activity. Among these series, compound 8 (2,4-dihydroxy group bearing benzylidene) showed potent tyrosinase inhibitory activity, with respective IC 50 values of 0.0433 µM and 0.28 µM for the monophenolase and diphenolase as substrates in comparison to kojic acid as standard compound 19.97 µM and 33.47 µM. Moreover, the enzyme kinetics of compound 8 were determined to be of the mixed inhibition type and inhibition constant ( K i ) values of 0.012 µM and 0.165 µM using the Lineweaver-Burk plot. Molecular docking results indicated that compound 8 can bind to the catalytic and allosteric sites 1 and 2 of tyrosinase to inhibit enzyme activity. The computational molecular dynamics analysis further revealed that compound 8 interacted with two residues in the tyrosinase active site pocket, such as ASN260 and MET280. In addition, compound 8 attenuated melanin synthesis and cellular tyrosinase activity, simulated by α-melanocyte-stimulating hormone and 1-methyl-3-isobutylxanthine. Compound 8 also decreased tyrosinase expressions in B16F10 cells. Based on in vitro and computational studies, we propose that compound 8 might be a worthy candidate for the development of an antipigmentation agent.

A new biflavonoid, amentoflavone-7-O-β-D-glucoside, and thirteen known flavonoids were isolated from the fruits of Juniperus chinensis using a bioactivity-guided method and their tyrosinase inhibitory effects were tested using a mushroom tyrosinase bioassay. Two isolates, hypolaetin-7-O-β-D-glucoside and quercetin-7-O-α-L-rhamnoside, were found to reduce tyrosinase activity at a concentration of 50 μM. Quercetin-7-O-α-L-rhamnoside attenuated cellular tyrosinase activity and melanogenesis in α-MSH plus IBMX-stimulated B16F10 melanoma cells. Molecular docking simulation revealed that quercetin-7-O-α-L-rhamnoside inhibits tyrosinase activity by hydrogen bonding with residues His85, His244, Thr261, and Gly281 of tyrosinase. Abbreviations: EtOH, ethanol; CH2Cl2, dichloromethane; EtOAc, ethylacetate; n-BuOH, n-butanol; MeOH, metanol; CHCl3,chloroform; DMSO, dimethylsulfoxide; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; α-MSH, α-melanocyte stimulating hormone; L-DOPA, L-3, 4-dihydroxyphenylalanine.

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.

Hyperpigmentation caused by melanin overproduction is a major skin disorder in humans. Inhibition of tyrosinase, a key regulator of melanin production, has been used as an effective strategy to treat hyperpigmentation. In this study, we investigated the use of solid lipid nanoparticles (SLNs) as a highly effective and nontoxic means to deliver a newly synthesized potent tyrosinase inhibitor, MHY498, and to target melanocytes through the skin. MHY498-loaded SLNs (MHY-SLNs) were prepared by an oil-in-water emulsion solvent-evaporation method, and their morphological and physicochemical properties were characterized. MHY-SLNs showed a prolonged drug-release profile and higher skin permeation than that of MHY solution. In an in vivo evaluation of antimelanogenic activity, MHY-SLNs showed a prominent inhibitory effect against ultraviolet B-induced melanogenesis, resulting in no change in the skin color of C57BL/6 mouse, compared with that observed in an MHY solution-treated group and an untreated control group. The antimelanogenic effect of MHY-SLNs was further confirmed through Fontana-Masson staining. Importantly, MHY-SLNs did not induce any toxic effects in the L929 cell line. Overall, these data indicate that MHY-SLNs show promise in the topical treatment of hyperpigmentation., Competing Interests: The authors report no conflicts of interest in this work.

Background. Uncontrolled melanogenesis and wrinkle formation are an indication of photoaging. Our previous studies demonstrated that (Z)-5-(2,4-dihydroxybenzylidene)thiazolidine-2,4-dione (MHY498) inhibited tyrosinase activity and melanogenesis in vitro. Objective. To examine in vivo effects of MHY498 as an antiaging compound on UVB-induced melanogenesis and wrinkle formation, we topically applied MHY498 on dorsal skin of HRM-2 hairless mice. Methods. Using histological analysis, we evaluated effects of MHY498 on melanogenesis and wrinkle formation after UVB exposure. In addition, related molecular signaling pathways were examined using western blotting, fluorometric assay, and enzyme-linked immunosorbent assay. Results. MHY498 suppressed UVB-induced melanogenesis by inhibiting phosphorylation of CREB and translocation of MITF protein into the nucleus, which are key factors for tyrosinase expression. Consistently, tyrosinase protein levels were notably reduced in the dorsal skin of the hairless mice by MHY498 treatment. Furthermore, MHY498 inhibited UVB-induced wrinkle formation and collagen fiber destruction by increasing type 1 procollagen concentration and decreasing protein expression levels of MMPs, which play an essential role in collagen fiber degradation. As a mechanism, MHY498 notably ameliorated UVB-induced oxidative stress and NF-κB activation in the dermal skin of the hairless mice. Conclusion. Our study suggests that MHY498 can be used as a therapeutic or cosmetic agent for preventing uncontrolled melanogenesis and wrinkle formation.

Tyrosinase inhibitors might have potential use in cosmetic and medicinal products for the prevention of pigmentation disorders. However, only a few inhibitors are currently used due to their cytotoxicity, and lack of selectivity and stability. In this study, we synthesized several tyrosinase inhibitors and investigated their activity. To investigate the action of 2-[4-(5-chlorobenzo[d]thiazol-2-yl)phenoxy]-2-methylpropanoic acid (MHY908) specifically in the inhibition of melanogenesis, a mushroom tyrosinase activity assay was performed. We confirmed the inhibitory effect of MHY908 at various melanin concentrations using α-MSH-induced melanoma cells. Our results indicate that MHY908 potently inhibited mushroom tyrosinase activity (IC50 = 8.19 μM) in a dose-dependent manner. Through a docking simulation, we also analyzed its binding mode to inhibit tyrosinase activity. MHY908 also decreased melanin synthesis without inducing cytotoxicity. These results suggest that MHY908 is a good candidate for prevention and treatment of pigmentation disorders.

Inhibiting tyrosinase is an important goal to prevent melanin accumulation in skin and thereby to inhibit pigmentation disorders. Therefore, tyrosinase inhibitors are an attractive target in cosmetics and treatments for pigmentation disorders. However, only a few tyrosinase inhibitors are currently available because of their toxic effects to skin or lack of selectivity and stability. Here, we newly synthesized thirteen (Z)-2-(benzo[d]thiazol-2-ylamino)-5-(substituted benzylidene)thiazol-4(5H)-one derivatives and examined their effect on melanogenesis. Of these compounds, MHY2081 had the strongest inhibitory effect on tyrosinase without cytotoxicity in B16F10 melanoma cells. Consistently, melanogenesis was notably decreased by MHY2081 treatment. As an underlying mechanism, docking simulation showed that compared to kojic acid, a well-known competitive tyrosinase inhibitor which forms a hydrogen bond and aromatic interaction with tyrosinase, MHY2081 has stronger affinity with tyrosinase by forming three hydrogen bonds and a hydrophobic interaction with residues of tyrosinase. In parallel with this, Lineweaver-Burk plot analysis showed that MHY2081 is a strong competitive inhibitor of tyrosinase. In conclusion, MHY2081 may be a novel tyrosinase inhibitor for prevention and treatment of pigmentation disorders.

Background: Excessive melanin production and accumulation are characteristics of a large number of skin diseases, including melasma, and post-inflammatory hyperpigmentation. During our on-going search for new agents with an inhibitory effect on tyrosinase, we synthesized a new type of tyrosinase inhibitor, 4-(thiazolidin-2-yl)benzene-1,2-diol (MHY-794), which directly inhibits mushroom tyrosinase., Methods: The inhibitory effect of MHY-794 on tyrosinase activity and nitric oxide (NO) scavenging activity was evaluated in cell free system. Additional experiments were performed using B16F10 melanoma cells to demonstrate the effects of MHY-794 in vitro. HRM2 hairless mice were used to evaluate anti-melanogenic effects of MHY-794 in vivo., Results: MHY-794 effectively inhibited mushroom tyrosinase activity in cell free system. In silico docking simulation also supported the inhibitory effects of MHY-794 on mushroom tyrosinase. MHY-794 also proved to be effective at scavenging nitric oxide (NO), which serves as an important modulator in the melanogenesis signaling pathway. In addition, MHY-794 effectively inhibited SNP (NO donor)-induced melanogenesis by directly inhibiting tyrosinase and diminishing NO-mediated melanogenesis signaling in B16 melanoma cells. The anti-melanogenic effects of MHY-794 were further confirmed in HRM2 hairless mice. Ultraviolet light (UV) significantly up-regulated NO-mediated melanogenesis signaling in HRM2 hairless mice, but MHY-794 effectively inhibited both melanogenesis and diminished UV-induced NO-signaling., Conclusions: Our results indicate that MHY-794 is highly effective at inhibiting NO-mediated melanogenesis in vitro and in vivo by direct NO scavenging and directly inhibiting tyrosinase activity, and suggest that MHY-794 be considered a new developmental candidate for the treatment of hyper-pigmentation disorders., General Significance: MHY-794, which showed great efficacy on NO-mediated melanogenesis by direct NO scavenging as well as direct inhibition of tyrosinase catalytic activity, might be utilized for the development of a new candidate for treatment of the hyper-pigmentation disorders., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We synthesized (Z)-5-(2,4-dihydroxybenzylidene)thiazolidine-2,4-dione (MHY498) as a potential tyrosinase inhibitor. MHY498 potently inhibited mushroom tyrosinase activity (mean IC50 = 3.55 μM) in a dose-dependent manner. MHY498 was more potent than the well-known tyrosinase inhibitor, kojic acid (mean IC50 = 22.79 μM). When tested in B16F10 melanoma cells treated with α-melanocyte stimulating hormone (α-MSH), MHY498 inhibited murine tyrosinase activity and decreased melanin production without inducing cytotoxicity. Docking models showed that the binding affinity of MHY498 to tyrosinase was higher than that of kojic acid, and docking simulation results indicated that the tyrosinase binding moieties of MHY498 and kojic acid were similar. Western blotting showed that tyrosinase inhibition by MHY498 partly resulted from the expressional modulations of tyrosinase and its transcription factor, microphthalmia-associated transcription factor, via the cAMP-PKA-CREB pathway. These findings suggest that MHY498 could be useful as an antimelanogenic agent for the prevention and treatment of diseases associated with skin pigmentation.

We synthesized a novel series of (E)-2-((substituted phenyl)diazenyl)phenyl 4-methylbenzenesulfonate derivatives (2 and 3) and (E)-2-((substituted phenyl)diazenyl)phenol derivatives (4 and 5), and conducted an evaluation in order to determine their inhibitory effects on mushroom tyrosinase, with the aim of discovering a tyrosinase inhibitor. Most of the compounds (3-5) exhibited higher inhibitory effects than kojic acid (IC(50) = 49.08 µM), a representative tyrosinase inhibitor. A novel synthesized compound, (E)-2-((2,4-dihydroxyphenyl)diazenyl)phenyl 4-methylbenzenesulfonate (3), showed the best results with an IC(50) of 17.85 µM, and showed competitive inhibition on Lineweaver-Burk plots, as further confirmed by the docking results. In addition, active compounds 3-5 were not cytotoxic to cultured B16F10 cells at the concentrations tested, and inhibited both tyrosinase and melanin synthesis. Therefore the active compounds (3-5) might be considered excellent candidates for use in the development of therapeutic agents for diseases associated with hyperpigmentation.

Background: Ultraviolet B (UVB) radiation is the main physiological stimulus for skin pigmentation. Nitric oxide (NO) and the NO/PKG signaling pathway play an important role in UVB-induced melanogenesis, which is related to the induction of expression of tyrosinase. In an attempt to find a novel anti-melanogenic agent, we synthesized a new compound, 2-bromo-4-(5-chloro-benzo[d]thiazol-2-yl) phenol (MHY966)., Objective: The purpose of this study was to investigate the action of MHY966 on NO and the NO-mediated signaling pathway using in vitro and in vivo models of melanogenesis., Methods: NO generation, melanin synthesis, and the expression of tyrosinase and PKG were measured in B16F10 melanoma cells to verify the anti-melanogenic effect of MHY966 in vitro. Next, melanin-possessing hairless mice were pre-treated with MHY966 and then irradiated with UVB repeatedly. Morphological, histological, and biochemical analyses including the expressions of PKG, tryosinase and nuclear MITF, and productions of nitric oxide, peroxynitrite and ROS were conducted., Results: MHY966 effectively inhibited NO generation and subsequent melanin synthesis induced by sodium nitroprusside, an NO donor, and suppressed the expression of tyrosinase and PKG. Topical application of MHY966 dose-dependently attenuated UVB-induced pigmentation in a mouse model. This hypopigmentation effect induced by MHY966 treatment was mediated by the down-regulation of tyrosinase, PKG, and nuclear MITF, which was accompanied by decreased NO and NO-related oxidative stress., Conclusion: The novel compound, MHY966 had an inhibitory effect on NO generation and the NO-mediated signaling pathway leading to the down-regulation of tyrosinase. The significance of the present study is the finding of a promising anti-melanogenic agent targeting the NO/PKG signaling pathway., (Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

We attempted to design and synthesize (E)-N-substituted benzylidene-hydroxy or methoxy-aniline derivatives and to evaluate their inhibitory effect on tyrosinase activity and anti-melanogenesis activity in murine B16F10 melanoma cells. Derivatives with a 4-methoxy- or 4-hydroxy-anilino group exerted more potent inhibition against mushroom tyrosinase than those with a 2-hydroxyanilino group. (E)-4-((4-Hydroxyphenylimino)methyl)benzene-1,2-diol exhibited the most potent and non-competitive inhibition on mushroom tyrosinase showing an IC(50) of 17.22 ± 0.38 μM and being more effective than kojic acid (51.11 ± 1.42 μM). This compound decreased melanin production stimulated by the alpha-melanocyte-stimulating hormone and inhibited murine tyrosinase activity in a dose-dependent manner. Therefore, we propose (E)-4-((4-hydroxyphenylimino)methyl)benzene-1,2-diol as a new candidate of potent tyrosinase inhibitors that could be used as therapeutic agent with safe skin-whitening efficiency., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

We describe the design, synthesis, and biological activities of 5-chloro-2-(substituted phenyl)benzo[d]thiazole derivatives as novel tyrosinase inhibitors. Among them, 4-(5-chloro-2,3-dihydrobenzo[d]thiazol-2-yl)-2,6-dimethoxyphenol (MHY884) and 2-bromo-4-(5-chloro-benzo[d]thiazol-2-yl)phenol (MHY966) showed inhibitory activity higher than or similar to kojic acid, against mushroom tyrosinase. Therefore, we carried out kinetic studies on the two compounds with potent tyrosinase inhibitory effects. Kinetic analysis of tyrosinase inhibition revealed that all of these compounds are competitive inhibitors. MHY884 and MHY966 effectively inhibited tyrosinase activity and reduced melanin levels in B16 cells treated with α-melanocyte stimulating hormone (α-MSH). These data strongly suggest that the newly synthesized compounds MHY884 and MHY966 could suppress production of melanin via inhibition of tyrosinase activity.

Background: Tyrosinase inhibitors have become increasingly important because of their ability to inhibit the synthesis of the pigment melanin. A search for new agents with strong tyrosinase activity led to the synthesis of the tyrosinase inhibitor (E)-3-(2,4-dihydroxybenzylidene)pyrrolidine-2,5-dione (3-DBP)., Methods: The inhibitory effect of 3-DBP on tyrosinase activity and melanin production was examined in murine melanoma B16F10 cells. Additional experiments were performed using HRM2 hairless mice to demonstrate the effects of 3-DBP in vivo., Results: The novel compound, 3-DBP, showed an inhibitory effect against mushroom tyrosinase (IC50=0.53 μM), which indicated that it was more potent than the well-known tyrosinase inhibitor kojic acid (IC50=8.2 μM). When tested in B16F10 melanoma cells treated with α-melanocyte stimulating hormone (α-MSH), 3-DBP also inhibited murine tyrosinase activity, which in turn induced a decrease in melanin production in these cells. The anti-melanogenic effect of 3-DBP was further verified in HRM2 hairless mice. The skin-whitening index (L value) of HRM2 hairless mice treated with 3-DBP before irradiation with UVB was greater than that of UVB-irradiated mice that were not treated with 3-DBP., General Significance: The newly synthesized 3-DBP has a potent inhibitory effect on tyrosinase. In addition to an in vitro investigation of the effects of 3-DBP on tyrosinase, in vivo studies using an HRM2 hairless mouse model demonstrated the anti-melanogenic potency of 3-DBP. Our newly synthesized 3-DBP showed efficient tyrosinase inhibitory effect in vivo and in vitro. Our finding suggests that 3-DBP can be an effective skin-whitening agent., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: We synthesized (2RS,4R)-2-(2,4-dihydroxyphenyl)thiazolidine-4-carboxylic acid (MHY384) as a potential tyrosinase inhibitor and investigated its antityrosinase activity., Methods: The structure of MHY384 was established using (1)H and (13)C NMR spectroscopy and mass spectral analyses. To investigate dual mechanisms of action of MHY384 for the inhibition of melanin synthesis, we confirmed the inhibitory effect of tyrosinase catalytic activity of MHY384. Then, we confirmed the inhibitory effect of MHY384 on transcription of tyrosinase mRNA through alpha-MSH-induced cAMP-PKA-MITF signaling. In addition, we supported the inhibitory mechanism of MHY384 against tyrosinase using a kinetic study and docking programs., Results: To determine how MHY384 regulates melanogenesis, we measured melanin levels and expression of the genes for microphthalmia-associated transcription factor (MITF) and tyrosinase in α-melanocyte-stimulating hormone (α-MSH)-induced B16F10 melanoma cells. MHY384 potently inhibited tyrosinase activity and melanin production in B16F10 melanoma cells. Through docking models, we were able to construct the tertiary structure of mushroom tyrosinase and simulate its docking with MHY384. The result supports that MHY384 strongly interacts with tyrosinase residues in the active site and it can directly inhibit tyrosinase. To investigate additional mechanisms of action of MHY384, we confirmed that the inhibition of tyrosinase activity was found to be due to the modulation of the expression of tyrosinase and its transcription factor, MITF, through cAMP, which regulates protein kinase A., Conclusions: This study strongly indicates that the depigmenting effect of MHY384 results from the down-regulation of MITF and tyrosinase through direct tyrosinase inhibition., General Significance: Our findings suggest that MHY384 can be an effective skin-whitening agent., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In continuing our search for novel tyrosinase inhibitors, a series of 5-(substituted benzylidene)thiazolidine-2,4-diones were rationally designed and synthesized, and their inhibitory effects on mushroom tyrosinase activity were evaluated. Twelve target compounds 2a-2l were designed and synthesized based on the structural characteristics of N-phenylthiourea, a tyrosinase inhibitor, and tyrosine and L-DOPA, the natural substrates of tyrosinase. Among them, (Z)-5-(4-hydroxybenzylidene)thiazolidine-2,4-dione (2a) and (Z)-5-(3-hydroxy-4-methoxybenzylidene)thiazolidine-2,4-dione (2f) exhibited much higher tyrosinase inhibitory activities, with IC(50) values of 13.36 and 9.87 μM, respectively, than kojic acid (IC(50) = 24.72 μM). Kinetic analysis of tyrosinase inhibition revealed that 2a and 2f are competitive inhibitors of mushroom tyrosinase. In addition, through prediction of the potato catechol oxidase tertiary structure and simulation of docking with compounds 2a and 2f using DOCK6, we found that these inhibitors likely bind to the active site of the enzyme. Docking simulation results suggested that 2a and 2f have high binding affinities with potato catechol oxidase. In addition, compounds 2a and 2f effectively inhibited tyrosinase activity and reduced melanin levels in B16 cells treated with α-melanocyte-stimulating hormone (α-MSH). These data strongly suggest that compounds 2a and 2f suppress the production of melanin via the inhibition of tyrosinase activity., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Herein we describe the design, synthesis and biological activities of 2-(substituted phenyl)thiazolidine-4-carboxylic acid derivatives as novel tyrosinase inhibitors. The target compounds 2a-2j were designed and synthesized from the structural characteristics of N-phenylthiourea, tyrosinase inhibitor and tyrosine, and l-DOPA, the natural substrates of tyrosinase. Among them, (2R/S,4R)-2-(2,4-dimethoxyphenyl)thiazolidine-4-carboxylic acid (2g) caused the greatest inhibition 66.47% at 20 μM of l-DOPA oxidase activity of mushroom tyrosinase. Kinetic analysis of tyrosinase inhibition revealed that 2g is a competitive inhibitor. We predicted the tertiary structure of tyrosinase, and simulated the docking of mushroom tyrosinase with 2g. These results suggest that the binding affinity of 2g with tyrosinase is high. Also, 2g effectively inhibited tyrosinase activity and reduced melanin levels in B16 cells treated with α-MSH. These data strongly suggest that 2g can suppress the production of melanin via the inhibition of tyrosinase activity., (Published by Elsevier Masson SAS.)

Background: Many tyrosinase inhibitors find application in cosmetics and pharmaceutical products for the prevention of the overproduction of melanin in the epidermis. A series of 5-(substituted benzylidene)hydantoin derivatives 2a-2k were prepared, and their inhibitory activities toward tyrosinase and melanin formation were evaluated., Methods: The structures of the compounds were established using (1)H and (13)C NMR spectroscopy and mass spectral analyses. All the synthesized compounds were evaluated for their mushroom tyrosinase inhibition activity., Results: The best results were obtained for compound 2e which possessed hydroxyl group at R(2) and methoxy group at R(3), respectively. We predicted the tertiary structure of tyrosinase, simulated its docking with compound 2e and confirmed that this compound interacts strongly with mushroom tyrosinase residues as a competitive tyrosinase inhibitor. In addition, we found that 2e inhibited melanin production and tyrosinase activity in B16 cells., Conclusions: Compound 2e could be considered as a promising candidate for preclinical drug development in skin hyperpigmentation applications., General Significance: This study will enhance understanding of the mechanism of tyrosinase inhibition and will contribute to the development of effective drugs for use hyperpigmentation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

As a part of an ongoing project searching for new skin-lightening agents, the inhibitory property of 6-(3-Hydroxyphenyl)-2-naphthol (HPN) on melanogenesis was investigated. The inhibitory action of HPN (IC(50)=15.2 muM) on mushroom tyrosinase was revealed. To further explore the action of HPN on melanogenesis, the inhibition of tyrosinase and melanin levels were measured in B16 melanoma cells (B16 cells). Results show that HPN inhibited tyrosinase activity and reduced melanin in B16 cells. Therefore, our data indicate HPN as a new candidate for depigmentation reagents.

3,4-Dihydroxyacetophenone (3,4-DHAP) was evaluated for antimelanogenic activity. The tyrosinase inhibitory action by 3,4-DHAP using mushroom tyrosinase revealed a strong inhibitory effect. To further explore this matter, inhibition of tyrosinase and melanin content was measured in B16 melanoma cells (B16 cells). Further, tyrosinase and microphthalmia transcription factor (MITF) protein levels were determined by the Western blot method. Additionally, tyrosinase and MITF protein levels were reduced by 3,4-DHAP. Our data indicate that the antimelanogenic activity of 3,4-DHAP was probably due to its inhibition of tyrosinase activity and the suppression of tyrosinase and MITF protein levels.

In our previous study, we showed that 4,4'-dihydroxybiphenyl (44'-BP) reduced melanin content via the inhibition of tyrosinase. In the current study, we utilized 44'-BP treated B16 melanoma cells (B16 cells) to measure several key cellular parameters known to be involved in melanogenic activity. Included in these measurements were tyrosinase and microphthalmia transcription factor (MITF) protein levels, cyclic AMP levels, protein kinase A (PKA) activation, and reduced glutathione (GSH) and oxidized glutathione (GSSG) levels. Results showed that 44'-BP effectively suppressed the amounts of tyrosinase and MITF proteins, cAMP levels, and PKA activation. In addition, 44'-BP enhanced the GSH/GSSG ratio. In conclusion, our data provide an evidence that 44'-BP suppressed several cellular key parameters in the melanogenic pathway by downregulating the cAMP-dependent PKA signaling pathway and decreasing MITF gene expression (implied from the reduced protein levels), which in turn suppressed tyrosinase. We propose that the antimelanogenic action of 44'-BP is likely carried out by a combined effect of its anti-oxidant property and its ability to enhance intracellular GSH levels.

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]

The rate-determining role of tyrosinase makes it a critical component in the mechanism that is responsible for melanogenesis. Thirteen (Z)-5-(substituted benzylidene)-3-phenyl-2-thioxothiazolidin-4-one ((Z)-BPTT) analogs were designed based on the structural features of two potent tyrosinase inhibitors, viz. (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-thioxothiazolidin-4-one (5-HMT) and (Z)-2-(2,4-dihydroxybenzylidene)benzo[4,5]imidazo[2,1-b]thiazol-3(2H)-one (compound I). The trisubstituted double bond geometry of the (Z)-BPTT analogs that were generated by Knoevenagel condensation was determined using vicinal 1H and 13C coupling constants in 13C NMR spectra. Four analogs, numbers 1–3 and 6, inhibited mushroom tyrosinase 9 to 29 times more potently than kojic acid did. Kinetic study results indicated that these four analogs inhibited mushroom tyrosinase competitively and this was supported by docking simulation. Also, docking results using human tyrosinase suggested that analogs 2 and 3 might be potent human tyrosinase inhibitors. In vitro studies using B16F10 cells (a melanoma cell line) showed that analogs 1, 2, 3, and 6 inhibited cellular tyrosinase and melanin production more than kojic acid did, without perceptible cytotoxicity. In particular, analog 2, which possesses a catechol group, exerted an extremely potent anti-melanogenic effect. In addition, analog 2 showed strong scavenging activity against DPPH and ABTS radicals. Furthermore, analog 2 not only reduced ROS levels, which induce melanogenesis, but it also suppressed tyrosinase and MITF (microphthalamia-associated transcription factor) protein levels and the expressions of melanogenesis-related genes. These results suggest that analog 2 is an efficient tyrosinase inhibitor that alleviates melanogenesis by dual mechanisms of (i) the inhibition of melanogenesis-related proteins and genes and (ii) the direct inhibition of tyrosinase activity. [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]

Abstract: In this study, we synthesized hydroxy and/or alkoxy substituted phenyl-benzo[d]thiazole derivatives using substituted benzaldehydes and 2-aminothiophenol in MeOH. The structures of these compounds were established by 1H and 13CNMR and mass spectral analyzes. All synthesized compounds were evaluated for their mushroom tyrosinase inhibition activity. Out the 12 generated compounds, 2a and 2d exhibited much higher tyrosinase inhibition activity (45.36–73.07% and 49.94–94.17% at 0.01–20μM, respectively) than kojic acid (9.29–50.80% at 1.25–20μM), a positive control. The cytotoxicity of 2a and 2d was evaluated using B16 cells and the compounds were found to be nontoxic. Compounds 2a and 2d were also demonstrated to be potent mushroom tyrosinase inhibitors, displaying IC50 values of 1.14±0.48 and 0.01±0.0002μM, respectively, compared with kojic acid, which has an IC50 value of 18.45±0.17μM. We also predicted the tertiary structure of tyrosinase, simulated the docking with compounds 2a and 2d and confirmed that the compounds strongly interact with mushroom tyrosinase residues. Kinetic plots showed that 2a and 2d are competitive tyrosinase inhibitors. Substitutions with a hydroxy group at R3 or both R3 and R1 of the phenyl ring indicated that these groups play a major role in the high binding affinity to tyrosinase. We further found that compounds 2a and 2d inhibit melanin production and tyrosinase activity in B16 cells. These results may assist in the development of new potent tyrosinase inhibitors against hyperpigmentation. [Copyright &y& Elsevier]

Abstract: In searching for new agents with a depigmenting effect, we synthesized a derivative of resveratrol, 5-(6-hydroxy-2-naphthyl)-1,2,3-benzenetriol (5HNB) with a potent tyrosinase inhibitory activity. 5HNB inhibited mushroom tyrosinase with an IC50 value of 2.95μM, which is more potent than the well-known anti-tyrosinase activity of kojic acid (IC50 =38.24). The results of the enzymatic inhibition kinetics by Lineweaver–Burk analysis indicated 5HNB inhibits tyrosinase non-competitively when l-tyrosine was used as the substrate. Based on the strong inhibitory action of 5HNB, it is expected that 5HNB can suppress melanin production in which tyrosinase plays the essential role. Our expectation was confirmed by the experimentations with B16 melanoma cells in which 5HNB inhibited melanin production. We propose that 5HNB might have skin-whitening effects as well as therapeutic potential for treating skin pigmentation disorders. [ABSTRACT FROM AUTHOR]