Design and synthesis of thiourea derivatives with sulfur-containing heterocyclic scaffolds as potential tyrosinase inhibitors

Tyrosinase is a key enzyme during the production of melanins in plants and animals. A class of novel N-aryl-N′-substituted phenylthiourea derivatives (3a–i, 6a–k) were designed, synthesized and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed some 4,5,6,7-tetrahydro-2-[[(phenylamino)thioxomethyl]amino]-benzo[b]thiophene-3-carboxylic acid derivatives (3a–i) exhibited moderate inhibitory potency on diphenolase activity of tyrosinase. When the scaffold of 4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid was replaced with 2-(1,3,4-thiadiazol-2-yl)thio acetic acid, the inhibitory activity of compounds (6a–k) against tyrosinase was improved obviously; especially, the inhibitory activity of compound 6h (IC₅₀ = 6.13 μM) is significantly higher than kojic acid (IC₅₀ = 33.3 μM). Moreover, the analysis on inhibition mechanism revealed that compound 6h might plays the role as a noncompetitive inhibitor.     

Development of hydroxylated naphthylchalcones as polyphenol oxidase inhibitors: Synthesis,biochemistry and molecular docking studies

Polyphenol oxidase (Tyrosinase) has received great attention, since it is the key enzyme in melanin biosynthesis. In this study, novel hydroxy naphthylchalcone compounds were synthesized, and their inhibitory effects on mushroom tyrosinase activity were evaluated. The structures of the compounds synthesized were confirmed by ¹H NMR, ¹³C NMR, FTIR and HRMS. Two of the compounds synthesized inhibited the diphenolase activity of tyrosinase in a dose dependent manner and exhibited much higher tyrosinase inhibitory activities (IC₅₀ values of 10.4 μM and 14.4 μM, respectively) than the positive control, kojic acid (IC₅₀: 27.5 μM). Kinetic analysis showed that their inhibition was reversible. Both the novel compounds displayed competitive inhibition with their K_i values of 3.8 μM and 4.5 μM, respectively. Docking results confirmed that the active inhibitors strongly interacted with the mushroom tyrosinase residues. This study suggests hydroxy naphthylchalcone compounds to serve as promising candidates for use as depigmentation agents.     

Synthesis,molecular docking studies of coumarinyl-pyrazolinyl substituted thiazoles as non-competitive inhibitors of mushroom tyrosinase

A series of coumarinyl-pyrazolinyl substituted thiazoles derivatives were synthesized and their inhibitory effects on the DPPH and mushroom tyrosinase were evaluated. The results showed that all of the synthesized compounds exhibited significant mushroom tyrosinase inhibitory activities. In particular, 3-(5-(4-(benzyloxy)-3-methoxyphenyl)-1-(4-(4-bromophenyl)thiazol-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-2-one (7j) exhibited the most potent tyrosinase inhibitory activity with IC₅₀ value 0.00458 ± 0.00022 μM compared with the IC₅₀ value of kojic acid is 16.84 ± 0.052 μM. The inhibition mechanism analyzed by Lineweaver–Burk plots revealed that the type of inhibition of compound 7j on tyrosinase was noncompetitive. The docking study against tyrosinase enzyme was also performed to determine the binding affinity of the compounds. The compound 7a showed the highest binding affinity (−10.20 kcal/mol) with active binding site of tyrosinase. The initial structure activity relationships (SARs) analysis suggested that further development of such compounds might be of interest. The statistics of our results endorses that compound 7j may serve as a structural template for the design and development of novel tyrosinase inhibitors.     

Synthesis of novel azo-resveratrol,azo-oxyresveratrol and their derivatives as potent tyrosinase inhibitors

Ten azo compounds including azo-resveratrol (5) and azo-oxyresveratrol (9) were synthesized using a modified Curtius rearrangement and diazotization followed by coupling reactions with various phenolic analogs. All synthesized compounds were evaluated for their mushroom tyrosinase inhibitory activity. Compounds 4 and 5 exhibited high tyrosinase inhibitory activity (56.25% and 72.75% at 50 μM, respectively). The results of mushroom tyrosinase inhibition assays indicate that the 4-hydroxyphenyl moiety is essential for high inhibition and that 3,5-dihydroxyphenyl and 3,5-dimethoxyphenyl derivatives are better for tyrosinase inhibition than 2,5-dimethoxyphenyl derivatives. Particularly, introduction of hydroxyl or methoxy group into the 4-hydroxyphenyl moiety diminished or significantly reduced mushroom tryosinase inhibition. Among the synthesized azo compounds, azo-resveratrol (5) showed the most potent mushroom tyrosinase inhibition with an IC₅₀ value of IC₅₀ = 36.28 ± 0.72 μM, comparable to that of resveratrol, a well-known tyrosinase inhibitor.     

Design,synthesis and biological evaluation of hydroxy substituted amino chalcone compounds for antityrosinase activity in B16 cells

A series of hydroxy substituted amino chalcone compounds have been synthesized. These compounds were then evaluated for their inhibitory activities on tyrosinase and melanogenesis in murine B16F10 melanoma cell lines. The structures of the compounds synthesized were confirmed by ¹H NMR, ¹³C NMR, FTIR and HRMS. Two novel amino chalcone compounds exhibited higher tyrosinase inhibitory activities (IC₅₀ values of 9.75 μM and 7.82 μM respectively) than the control kojic acid (IC₅₀: 22.83 μM). Kinetic studies revealed them to act as competitive tyrosinase inhibitors with their K_i values of 4.82 μM and 1.89 μM respectively. Both the compounds inhibited melanin production and tyrosinase activity in B16 cells. Docking results confirm that the active inhibitors strongly interact with mushroom tyrosinase residues. This study suggests that the depigmenting effect of novel amino chalcone compounds might be attributable to inhibition of tyrosinase activity, suggesting amino chalcones to be a promising candidate for use as depigmentation agents or as anti-browning food additives.     

Design and synthesis of biphenyl derivatives as mushroom tyrosinase inhibitors

Two new series of biphenyls, analogs of aglycone of natural product fortuneanoside E, were prepared using Suzuki–Miyaura cross-coupling and selective magnesium iodide demethylation/debenzylation, and their mushroom tyrosinase inhibitory activity was evaluated. Most of the 4-hydroxy-3,5-dimethoxyphenyl biphenyl compounds (series II, 20–36) were in general more active than 3,4,5-trimethoxyphenyl biphenyl compounds (series I, 1–19). Structure–activity relationships study showed that monosaccharide substituents, such as glucose, were not necessary and the presence of 4-hydroxy-3,5-dimethoxyphenyl moiety was crucial for inhibitory activity. Among the compounds synthesised, compound 21 (IC₅₀ = 0.02 mM) was found to be the most active one, which exhibited an activity that was 7 times higher than that of fortuneanoside E (IC₅₀ = 0.14 mM) and 10 times higher than that of arbutin (IC₅₀ = 0.21 mM), known as potent tyrosinase inhibitors. The inhibition kinetics analyzed by Lineweaver–Burk plots revealed that compound 21 was a competitive inhibitor (K_i = 0.015 mM).     

2-(4-Fluorophenyl)-quinazolin-4(3H)-one as a novel tyrosinase inhibitor: Synthesis,inhibitory activity,and mechanism

2-(4-Fluorophenyl)-quinazolin-4(3H)-one (FQ) was synthesized, and its structure was identified with ¹H nuclear magnetic resonance (¹H NMR), ¹³C nuclear magnetic resonance (¹³C NMR), fourier transform infrared spectroscopy (FTIR), and high resolution mass spectrometry (HRMS). From the enzyme analysis, the results showed that it could inhibit the diphenolase activity of tyrosinase (IC₅₀ = 120 ± 2 μM). Furthermore, the results of kinetic studies showed that the compound was a reversible mixed-type inhibitor, and that the inhibition constants were determined to be 703.2 (K_I) and 222.1 μM (K_IS). The results of fluorescence quenching experiment showed that the compound could interact with tyrosinase and the substrates (tyrosine and l-DOPA). Molecular docking analysis revealed that the mass transfer rate was affected by FQ blocking the enzyme catalytic center. In brief, current study identified a novel tyrosinase inhibitor which deserved further study for hyperpigmentation drugs.     

Highly potent tyrosinase inhibitor,neorauflavane from Campylotropis hirtella and inhibitory mechanism with molecular docking

Tyrosinase inhibition may be a means to alleviate not only skin hyperpigmentation but also neurodegeneration associated with Parkinson’s disease. In the course of metabolite analysis from tyrosinase inhibitory methanol extract (80% inhibition at 20 μg/ml) of Campylotropis hirtella, we isolated fourteen phenolic compounds, among which neorauflavane 3 emerged as a lead structure for tyrosinase inhibition. Neorauflavane 3 inhibited tyrosinase monophenolase activity with an IC₅₀ of 30 nM. Thus this compound is 400-fold more active than kojic acid. It also inhibited diphenolase (IC₅₀ = 500 nM), significantly. Another potent inhibitor 1 (IC₅₀ = 2.9 μM) was found to be the most abundant metabolite in C. hirtella. In kinetic studies, compounds 3 showed competitive inhibitory behavior against both monophenolase and diphenolase. It manifested simple reversible slow-binding inhibition against monophenolase with the following kinetic parameters: K_i^app = 1.48 nM, k₃ = 0.0033 nM⁻¹ min⁻¹ and k₄ = 0.0049 min⁻¹. Neorauflavane 3 efficiently reduced melanin content in B16 melanoma cells with 12.95 μM of IC₅₀. To develop a pharmacophore model, we explored the binding mode of neuroflavane 3 in the active site of tyrosinase. Docking results show that resorcinol motif of B-ring and methoxy group in A-ring play crucial roles in the binding the enzyme.     

Inhibitory effects of novel synthetic methimazole derivatives on mushroom tyrosinase and melanogenesis

In this study, we synthesized 4 methimazole (2-mercapto-1-methylimidazole, MMI) derivatives. The kinetics of inhibition on mushroom tyrosinase by methimazole and its derivatives were investigated. The results indicated that tert-butyl 3-methyl-2-sulfanylidene-2,3-dihydro-1H-imidazole-1-carboxylate (compound 3; 3), 2-mercaptoimidazole (MI; compound 1; 1) and MMI (compound 2; 2) significantly inhibited tyrosinase activity in a dose-dependent manner, exhibiting an IC₅₀ value of 1.50 mM, 4.11 mM, and 1.43 mM. However, compound 4 (4), compound 5 (5), and compound 6 (6) exerted no inhibitory effect on mushroom tyrosinase activity. Kinetic analysis indicated that 3 was a noncompetitive tyrosinase inhibitor, whereas both 1 and 2 were exhibited as mixed-type tyrosinase inhibitors. Furthermore, 3 exerted a potent inhibitory effect on intracellular melanin formation in the B16/F10 murine melanoma cells and did not cause cytotoxicity, as 1 and 2 did.     

Synthesis and biological activity of oxadiazole and triazolothiadiazole derivatives as tyrosinase inhibitors

A series of 16 oxadiazole and triazolothiadiazole derivatives were designed, synthesized and evaluated as mushroom tyrosinase inhibitors. Five derivatives were found to display high inhibition on the tyrosinase activity ranging from 0.87 to 1.49 μM. Compound 5 exhibited highest tyrosinase inhibitory activity with an IC₅₀ value of 0.87 ± 0.16 μM. The in silico protein–ligand docking using autodock 4.1 was successfully performed on compound 5 with significant binding energy value of ?5.58 kcal/mol. The docking results also showed that the tyrosinase inhibition might be due to the metal chelating effect by the presence of thione functionality in compounds 1–5. Further studies revealed that the presence of hydrophobic group such as cycloamine derivatives played a major role in the inhibition. Piperazine moiety in compound 5 appeared to be involved in an extensive hydrophobic contact and a 2.9 Å hydrogen bonding with residue Glu 182 in the active site.     

Design,synthesis and biological evaluation of hydroxy- or methoxy-substituted 5-benzylidene(thio) barbiturates as novel tyrosinase inhibitors

Here a new class of hydroxy- or methoxy-substituted 5-benzylidene(thio)barbiturates were designed, synthesized and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed that several compounds had more potent tyrosinase inhibitory activities than the widely used tyrosinase inhibitor kojic acid (IC₅₀ = 18.25 μM). In particular, 3′,4′-dihydroxylated 1e was found to be the most potent inhibitor with IC₅₀ value of 1.52 μM. The inhibition mechanism analysis revealed that the potential compounds 1e and 2e exhibited such inhibitory effects on tyrosinase by acting as the irreversible inhibitors. Structure–activity relationships’ (SARs) analysis also suggested that further development of such compounds might be of interest.     

Characterization of a novel laccase from the isolated Coltricia perennis and its application to detoxification of biomass

A highly efficient laccase-producing fungus was isolated from soil and identified as Coltricia perennis SKU0322 by its morphology and by comparison of its internal transcribed spacer (ITS) rDNA gene sequence. Extracellular laccase (Cplac) from C. perennis was purified to homogeneity by anion-exchange and gel filtration chromatography. Cplac is a monomeric glycoprotein with 12% carbohydrate content and a molecular mass of 66 kDa determined by polyacrylamide-gel electrophoresis. Ultraviolet-visible absorption spectroscopy observed type 1 and type 3 copper signals from Cplac. The enzyme acted optimally at pH 3–4 and 75 °C. Its optimal activity was with 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS), it also oxidized various lignin-related phenols. The enzyme was characterized as a multi-copper blue laccase by its substrate specificity and internal amino acid sequence. It showed a higher catalytic efficiency towards ABTS (k_cat/K_m = 18.5 s^?1 μM^?1) and 2,6-dimethoxyphenol (k_cat/K_m = 13.9 s^?1 μM^?1) than any other reported laccase. Its high stability and catalytic efficiency suggest its suitability for industrial applications: it detoxified phenolic compounds in acid-pretreated rice straw and enhanced saccharification yield.     

Discovery of a new type of scaffold for the creation of novel tyrosinase inhibitors

Tyrosinase is known as the key enzyme for melanin biosynthesis, which is effective in preventing skin injury by ultra violet (UV). In past decades, tyrosinase has been well studied in the field of cosmetics, medicine, agriculture and environmental sciences, and a lot of tyrosinase inhibitors have been developed for their needs. Here, we searched for new types of tyrosinase inhibitors and found phenylbenzoic acid (PBA) as a unique scaffold. Among three isomers of PBA, 3-phenylbenzoic acid (3-PBA) was revealed to be the most potent inhibitor against mushroom tyrosinase (IC₅₀ = 6.97 μM, monophenolase activity; IC₅₀ = 36.3 μM, diphenolase activity). The kinetic studies suggested that the apparent inhibition modes for the monophenolase and diphenolase activities were noncompetitive and mixed type inhibition, respectively. Analyses by in silico docking studies using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid group of the 3-PBA could adequately bind to two cupric ions in the tyrosinase. To prove this hypothesis, we examined the effect of modification of the carboxylic acid group of the 3-PBA on its inhibitory activity. As expected, the esterification abrogated the inhibitory activity. These observations suggest that 3-PBA is a useful lead compound for the generation of novel tyrosinase inhibitors and provides a new insight into the molecular basis of tyrosinase catalytic mechanisms.     

Aryl boronic acid inhibition of synthetic melanin polymerization

Inhibitors of melanin formation are sought after for a range of applications. Boronophenylalanine is known to inhibit melanogenesis via boronic acid–catechol interactions. A spectroscopic assay was developed to study the polymerization of l-dopa to synthetic melanin in the presence of para-substituted aryl boronic acids. The best inhibition was observed for aryl boronic acids with electron-withdrawing substituents. The IC₅₀ values exhibit a correlation with the Hammett σ_p parameter (ρ = 0.97, r² = 0.92).     

Tyrosinase inhibitory effects of 1,3-diphenylpropanes from Broussonetia kazinoki

Six 1,3-diphenylpropanes exhibiting inhibitory activities against both the monophenolase and diphenolase actions of tyrosinase were isolated from the methanol (95%) extract of Broussonetia kazinoki. These compounds, 1–6, were identified as kazinol C (1), D (2), F (3), broussonin C (4), kazinol S (5) and kazinol T (6). The latter two species (5 and 6) emerged to be new 1,3-diphenylpropanes which we fully spectroscopically characterized. The IC₅₀ values of compounds (1, 3–5) for monophenolase inhibition were determined to range between 0.43 and 17.9 μM. Compounds 1 and 3–5 also inhibited diphenolase significantly with IC₅₀ values of 22.8, 1.7, 0.57, and 26.9 μM, respectively. All four active tyrosinase inhibitors (1, 3–5) were competitive inhibitors. Interestigly they all mainfested simple reversible slow-binding inhibition against diphenolase. The most potent inhibitor, compound 4 diplayed the following kinetic parameters k₃ = 0.0993 μM^?1 min^?1, k₄ = 0.0048 min^-1, and K_i^app = 0.0485 μM.     

Development of an immunochromatographic assay for diagnosing the production of IMP-type metallo-β-lactamases that mediate carbapenem resistance in Pseudomonas

Rapid and reliable detection of carbapenem-resistant bacteria is an important infection-control measure and a crucial aspect of antimicrobial chemotherapy. IMP-type metallo-β-lactamase (MBL) is an emzyme that mediate carbapenem resistance in bacteria. Here, an immunochromatographic assay was newly developed using novel monoclonal antibodies (mAbs) recognizing IMP-type MBL. Epitope mapping of mAbs and mutational analysis of the epitope region in IMP antigen suggested that the mAbs could react to all known subtypes of IMP-type MBL. Evaluation of the assay using Pseudomonas aeruginosa strains (n = 248) showed that the results of the immunochromatographic detection of the IMP-type MBLs were fully consistent with those of the PCR analysis for bla_IMP genes, showing false positives and negatives. All positive strains were resistant to carbapenem (MIC ≥ 16 μg/ml). The assay also accurately distinguished the production of IMP-type MBLs in Pseudomonas putida, Acinetobacter baumannii, and Alcaligenes xylosoxidans. The detection limit of the assay was 5.7 × 10⁴ cfu per test. Taken together, these data suggest that the developed assay can be used for rapid and reliable diagnosis of the production of IMP-type MBLs in Gram-negative bacteria.     

Effects of boldine on tyrosinase: Inhibition kinetics and computational simulation

Boldine is one of the most potent natural antioxidants and displays some important pharmacological activities, such as cytoprotective and anti-inflammatory activities. Based on its antioxidant properties, we studied the effects of boldine on l-DOPA oxidation by evaluating the inhibitory kinetics and a computational simulation between boldine and tyrosinase. Boldine reversibly inhibited tyrosinase from mushroom (Agaricus bisporus) in a mixed-type manner, with a K_i = 7.203 ± 0.933 mM. To gain insight into the inactivation process, we computed the kinetics via time-interval measurements and continuous substrate reactions. The results indicated that the inactivation induced by boldine was a first-order reaction with biphasic processes and that the substrate can promote the inactivation process. To gain further insight, we performed computational docking and molecular dynamics simulations, and the results showed that boldine can interact with several residues near the tyrosinase active site. Our study provides insight into the inhibition of tyrosinase in response to alkaloids. Based on its tyrosinase-inhibiting effect and low toxicity, boldine is a potential natural anti-pigmentation agent.     

Development of a microtitre plate method for determination of phenol utilization,biofilm formation and respiratory activity by environmental bacterial isolates

Twenty-five aerobic phenol-degrading bacteria, isolated from different environmental samples on phenol agar after several subcultures in phenol broth, utilized phenol (0.2 g l⁻¹) within 24 h, but removal of phenol was more rapid when other carbon sources were also present. A microtitre plate method was developed to determine growth rate, biofilm formation and respiratory activity of the strains isolated. Pseudomonas putida strains C5 and D6 showed maximum growth (as O.D. at 600 nm), P. putida D6 and unidentified bacterial strain M1 were more stable at high concentrations of phenol (0.8 g l⁻¹), and P. putida C5 formed the greatest amount of biofilm in 0.5 g phenol l⁻¹ medium. Measurement of dehydrogenase activity as reduction of triphenyl tetrazolium chloride supported data on growth rate and biofilm formation. The microtitre plate method provided a selective method for detection of the best phenol degrading and biofilm-forming microorganisms, and was also a rapid, convenient means of studying the effect of phenol concentration on growth rate and biofilm formation.