Oxazolones: new tyrosinase inhibitors; synthesis and their structure-activity relationships

The tyrosinase inhibitory potential of seventeen synthesized oxazolone derivatives has been evaluated and their structure-activity relationships developed in the present work. All the synthesized derivatives, 3-19, demonstrated excellent in vitro tyrosinase inhibitory properties having IC50 values in the range of 1.23+/-0.37-17.73+/-2.69 microM, whereas standard inhibitors l-mimosine and kojic acid have IC50 values 3.68+/-0.02 and 16.67+/-0.52 microM,, respectively. Compounds 4-8 having IC50 values 3.11+/-0.95, 3.51+/-0.25, 3.23+/-0.66, 1.23 +/- 0.37, and 2.15+/-0.75, respectively, were found to be very active members of the series, even better than both the standard inhibitors. However, compounds 3, 9-11, 13, 14, 16, 17, and 19 were found to be better than kojic acid but not l-mimosine. (2-Methyl-4-[E,2Z)-3-phenyl-2-propenyliden]-1,3-oxazol-5(4H)-one (7) bearing a cinnamyol residue at C-4 of oxazolone moiety and an IC50 = 1.23+/-0.37 microM was found to be the most active one among all tested compounds. These studies reveal that the substitution of functional group (s) at C-4 and C-2 positions plays a vital role in the activity of this series of compounds. It is concluded that compound 7 may act as a potential lead molecule to develop new drugs for the treatment of tyrosinase based disorders.     

Inhibitory effects of cis- and trans-isomers of 3,5-dihydroxystilbene on the activity of mushroom tyrosinase

The effects of cis- and trans-isomers of 3,5-dihydroxystilbene on the activity of mushroom tyrosinase have been studied. The results show that both cis- and trans-isomers of 3,5-dihydroxystilbene can inhibit the diphenolase activity of the enzyme and the inhibition type was reversible. The IC(50) values were estimated as 0.405+/-0.013 and 0.705+/-0.017 mM, respectively. Kinetic analysis showed that the inhibition of cis-3,5-dihydroxystilbene and trans-3,5-dihydroxystilbene on the diphenolase activity of the enzyme belonged to competitive type, and the inhibition constants (K(I)) were determined to be 0.232+/-0.015 and 0.395+/-0.020 mM, respectively. In this investigation, the inhibitory effects of cis-3,5-dihydroxystilbene and trans-3,5-dihydroxystilbene on the diphenolase activity of mushroom tyrosinase were compared. The inhibitory capacity of cis-isomer was stronger than that of corresponding trans-isomer. Nevertheless, the trans-3,5-dihydroxystilbene was used more frequently than its corresponding cis-form compound. This research may offer some references for designing and synthesizing some novel and effective tyrosinase inhibitors. Furthermore, it may improve the use of stilbenes on the field of food preservation and depigmentation.     

Studies on the reactions between human tyrosinase, superoxide anion, hydrogen peroxide and thiols.

Human tyrosinase (5.5 mg) has been purified from a single human melanotic melanoma metastasis (50.5 g). In the presence of dioxygen, L-tyrosine proved to be a very poor substrate for this enzyme with barely detectable activity compared to L-dopa. However, saturating superoxide anion (i.e., greater than 5 x 10(-3) M) enhanced the oxidation rate of L-tyrosine to dopachrome 40-fold. Hydrogen peroxide was shown to be a competitive inhibitor of tyrosinase when L-tyrosine was the substrate. This reversible inhibition is based on a slow pseudocatalase activity for tyrosinase. Monothiols and dithiols inhibit tyrosinase by different mechanisms. Reduced human thioredoxin and 2,3-dithiopropanol are allosteric inhibitors of tyrosinase yielding bis-cysteinate complexes with one of the copper atoms in the enzyme active site. Bis-cysteinate tyrosinase activity is down-regulated to 30% of native enzyme activity in the L-dopa assay; suggesting a true regulatory role for dithiols. Monothiols such as reduced glutathione and beta-mercaptoethanol are much less reactive with tyrosinase although 10(-3) M monothiol totally inhibits enzyme activity. Reduced thioredoxin inhibits tyrosinase 23-fold more than reduced glutathione under the same experimental conditions.     

Stabilization of recombinant adenovirus: site-directed mutagenesis of key asparagine residues in the hexon protein

Bioelectrochemical analysis of neuropathy target esterase (NTE) and its inhibitors is based on the combination of the NTE-catalyzed hydrolysis of phenyl valerate and phenol detection by a tyrosinase carbon-paste electrode. The use of the tyrosinase electrode improves 10-fold the sensitivity of NTE detection in comparison with a spectrophotometric method. The tyrosinase electrode was found to be suitable for measurements in whole human blood where spectrophotometric detection is considerably restricted. The specificity of NTE in blood for mipafox and di-2-propyl phosphorofluoridate was close to that for neuronal NTE. The NTE-like activity in blood was determined to be 0.19 +/- 0.02 nmol/min/mg of protein.     

Anti-melanogenesis potential of a new series of Morita-Baylis-Hillman adducts in B16F10 melanoma cell line

Melanin is a natural polymer pigment which provides skin photoprotection against ultraviolet radiation. An excessive synthesis of melanin leads to hyperpigmentation disorders. Tyrosinase catalyzes the rate limiting steps on melanogenesis. Therefore, tyrosinase inhibitors have potential applications in medicine and cosmetic fields. We carried out herein the screening of a family of cyclic Morita-Baylis-Hillman adducts (MBH) to find out their effects on tyrosinase activity and on melanogenesis in murine melanoma B16F10 cell line. Kinetic analysis of tyrosinase inhibition showed that compounds 1a (2-hydroxymethyl) cyclohex-2-enone) and 3f (diethyl (1-(6-oxocyclohex-1-en-1-yl) ethyl-phosphonate) were competitive inhibitors, whereas the compound 2b (6-oxocyclohex-1-en-1-yl) ethyl acetate) was a non-competitive one. Additionally we have found that (1a, 2b and 3f) compounds had a strong melanogenesis inhibition effect in isobutylmethylxanthine (IBMX)-treated murine melanoma B16F10 cells when tested at low and non cytotoxic dose (10–50 µM), by attenuating the melanin production, intracellular tyrosinase activity and tyrosinase expression. Thus, we suggest that these compounds could be used as effective skin-whitening agents.     

盐水球菌Salinicoccus ventosaetal B2-3-5的代谢产物分析及其抑制酪氨酸酶活性的机制

[背景] 酪氨酸酶是黑色素合成过程中的关键酶,也是引起人体色素障碍性疾病和产生果蔬酶促褐变的主要原因。目前,酪氨酸酶抑制剂的开发已引起广泛关注,但一些酪氨酸酶抑制剂如熊果苷、曲酸等均存在一定的安全隐患。微生物资源丰富且具有许多优点,从微生物中寻找特异性强、高效的酪氨酸酶抑制剂已成为该领域研究的热点。[目的] 通过测定分离自新疆乌鲁木齐达坂城盐湖的盐水球菌Salinicoccus ventosaetal B2-3-5和B6-1-4代谢物提取物对酪氨酸酶活性的影响,比较2株菌发酵过程中代谢物的差异,了解所筛选菌株B2-3-5抑制酪氨酸酶活性的机制。[方法] 以曲酸为阳性对照分别测定B2-3-5和B6-1-4这2个菌株发酵产生的代谢物提取物对蘑菇酪氨酸酶的抑制活性;应用LC-MS代谢组学方法检测2株菌在相同条件下产生的所有代谢物质;采用单变量、多元变量、正交偏最小二乘判别分析（Orthogonal Partial Least Squares-Discrimination Analysis,OPLS-DA）法识别差异代谢物;利用层次聚类分析（Hierarchial Cluster Analysis,HCA）法对识别的差异物进行聚类分析;通过Kyoto Encyclopedia of Genes and Genomes （KEGG）代谢通路对比法分析这些差异代谢物主要参与的代谢途径。[结果] 菌株B2-3-5代谢物提取物对蘑菇酪氨酸酶二酚酶活性的抑制率为67%,其IC₅₀为0.277 mg/mL,同属菌株B6-1-4代谢物提取物则对酪氨酸酶无抑制活性。采用代谢组学的检测方法从2株菌的代谢物中筛选出63个差异代谢物,其中氨基酸类化合物、维生素类化合物和羧酸类化合物的种类及相对含量均是B2-3-5菌株明显高于B6-1-4菌株。通过代谢途径分析发现这些差异代谢物主要参与15个代谢通路,其中维生素B6生物合成通路的影响较为显著。[结论] 推测B2-3-5菌株可能是通过增加一些氨基酸类、维生素类及羧酸类等小分子化合物的含量来抑制酪氨酸酶活性。维生素B6代谢途径的上调也表明菌体细胞可通过产生维生素B6与酪氨酸酶中的必需氨基作用或清除酶催化循环过程中产生的活性氧自由基（reactive oxygen species,ROS）来抑制酪氨酸酶活性。     

N-Benzylbenzamides: a new class of potent tyrosinase inhibitors

A series of potent inhibitors of tyrosinase and their structure-activity relationships are described. N-Benzylbenzamide derivatives (1-21) with hydroxyl(s) were synthesized and tested for their tyrosinase inhibitory activity. With this series, compound 15 provided a potent tyrosinase inhibition: it effectively inhibited the oxidation of l-DOPA catalyzed by mushroom tyrosinase with an IC(50) of 2.2microM.     

小菜蛾酪氨酸酶生物化学性质研究

采用时间动力学法对小菜蛾Plutella xylostella酪氨酸酶的最适反应条件、对抑制剂的敏感度以及发育期变化规律进行了研究。结果表明：小菜蛾酪氨酸酶反应的最适pH值为6.4,反应线性时间为0～2 min,在22℃和4℃下其活性分别可保持12 h 和72 h,在-20℃下至少可保持7 天。不同发育阶段小菜蛾酪氨酸酶活性的大小依次为预蛹＞4龄幼虫＞蛹＞1龄、2龄、3龄幼虫＞成虫。苯基硫脲（phenyl thiourea,PTU）对小菜蛾阿维菌素抗性品系和敏感品系酪氨酸酶活性的抑制中浓度（I₅₀）值分别为1.1796 μmol/L和1.2795 μmol/L,二者无明显差异。     

Effects of hydroxystilbene derivatives on tyrosinase activity

Synthesis of melanin starts from the conversion of L-tyrosine to 3,4-dihydroxyphenylalanine (L-dopa) and then the oxidation of L-dopa yields dopaquinone by tyrosinase. Therefore, tyrosinase inhibitors have been established as important constituents of depigmentation agents. Recently, polyhydroxystilbene compounds, which are trans-resveratrol (3,4('),5-trihydroxy-trans-stilbene) analogs, have been demonstrated as potent tyrosinase inhibitors. However, their detailed inhibitory mechanisms are not clearly understood. In the present study, a variety of synthesized hydroxystilbene compounds were tested for their inhibitory effects against murine tyrosinase activity. The inhibitory potencies of the hydroxy-trans-stilbene compounds were remarkably elevated by increasing number of phenolic hydroxy substituents. Methylated hydroxy-trans-stilbene lost the inhibitory activity. Furthermore, hydrogenated hydroxystilbene or hydroxy-cis-stilbene exerted little or no inhibitory effect compared with hydroxy-trans-stilbene on tyrosinase activity. The structure-activity relationships demonstrated in the present study suggest that the phenolic hydroxy groups and trans-olefin structure of the parent stilbene skeleton contribute to the inhibitory potency of hydroxystilbene for tyrosinase activity.     

A spectrophotometric method for the quantification of an enzyme activity producing 4-substituted phenols: determination of toluene-4-monooxygenase activity

A spectrophotometric method for the quantitative determination of an enzyme activity resulting in the accumulation of 4-substituted phenols is described in this article. Toluene-4-monooxygenase (T4MO) activity in whole cells of Pseudomonas mendocina KR1 is used to demonstrate this method. This spectrophotometric assay is based on the coupling of T4MO activity with tyrosinase activity. The 4-substituted phenol, produced by the action of T4MO on the aromatic ring of a substituted arene, is a substrate for tyrosinase, which converts phenols to o-quinones. The latter react with the nucleophile 3-methyl-2-benzothiazolinone hydrazone (MBTH) to produce intensely colored products that absorb light maximally at different wavelengths, depending on the phenolic substrate used. The incubation of whole cells of P. mendocina KRI with fluorobenzene resulted in the accumulation of 4-fluorophenol. The coupling of T4MO activity with tyrosinase activity in the presence of fluorobenzene resulted in the formation of a colored product absorbing maximally at 480 nm. The molar absorptivity (epsilon) value for the o-quinone-MBTH adduct formed from 4-fluorophenol was determined experimentally to be 12,827 M(-1) cm(-1) with a linear range of quantification between 2.5 and 75 microM. The whole cell assay was run as a continuous indirect assay. The initial rates of T4MO activity toward fluorobenzene, as determined spectrophotometrically, were 61.8+/-4.4 nmol/min/mg P. mendocina KR1 protein (using mushroom tyrosinase), 64.9+/-4.6 nmol/min/mg P. mendocina KR1 protein (using cell extracts Pseudomonas putida F6), and, as determined by HPLC analysis, 62.6+/-1.4 nmol/min/mg P. mendocina KR1 protein.     

Chalcones as potent tyrosinase inhibitors: the importance of a 2,4-substituted resorcinol moiety

Compounds, which inhibit tyrosinase, could be effective as depigmenting agents. We have introduced a group of mono-, di-, tri- and tetra-substituted hydroxychalcones as effective tyrosinase inhibitors, showing that the most important factor determining tyrosinase inhibition efficiency is the position of the hydroxyl group(s) rather their number. The aim of the present study was to investigate the contribution of the different functional groups of the tetrahydroxychalcones to their inhibitory potency, with a view to optimizing the design of whitening agents. Four tetrahydroxychalcones were evaluated, the commercially available Butein and other three were synthesized, and their inhibitory effect on tyrosinase was tested. Results showed that a 2,4-substituted resorcinol subunit on ring B contributed the most to inhibitory potency. Changing the resorcinol substitute to position 3,5- or placing it on ring A significantly diminished the inhibitory effect of the compounds. A catechol subunit on ring A acted as a metal chelator (in the presence of copper ions) and as a competitive inhibitor (in the presence of tyrosinase), while a catechol on ring B oxidized to o-quinone (in the presence of both copper ions and tyrosinase). Three of the compounds also demonstrated antioxidant activity, which may contribute to the prevention of pigmentation. An examination of correlations between inhibitory activity and physical properties of the chalcones tested (such as dissociation energy and molecular planarity) showed positive correlation with the moment dipole value in the Y-axis, which may be used as an indicator of the inhibitory potential of new molecules. The present study revealed two very active tyrosinase inhibitors, 2,4,3',4'-hydroxychalcone and 2,4,2',4'-hydroxychalcone (with IC50 of 0.2 and 0.02 microM, respectively). Structure-related activity studies added some understanding of the role and contribution of different functional groups associated with tyrosinase inhibitors.     

Skin whitening agents: medicinal chemistry perspective of tyrosinase inhibitors

Melanogenesis is a process to synthesize melanin, which is a primary responsible for the pigmentation of human skin, eye and hair. Although numerous enzymatic catalyzed and chemical reactions are involved in melanogenesis process, the enzymes such as tyrosinase and tyrosinase-related protein-1 (TRP-1) and TRP-2 played a major role in melanin synthesis. Specifically, tyrosinase is a key enzyme, which catalyzes a rate-limiting step of the melanin synthesis, and the downregulation of tyrosinase is the most prominent approach for the development of melanogenesis inhibitors. Therefore, numerous inhibitors that target tyrosinase have been developed in recent years. The review focuses on the recent discovery of tyrosinase inhibitors that are directly involved in the inhibition of tyrosinase catalytic activity and functionality from all sources, including laboratory synthetic methods, natural products, virtual screening and structure-based molecular docking studies.     

Structural insight into the active site of mushroom tyrosinase using phenylbenzoic acid derivatives

So far, many inhibitors of tyrosinase have been discovered for cosmetic and clinical agents. However, the molecular mechanisms underlying the inhibition in the active site of tyrosinase have not been well understood. To explore this problem, we examined here the inhibitory effects of 4′-hydroxylation and methoxylation of phenylbenzoic acid (PBA) isomers, which have a unique scaffold to inhibit mushroom tyrosinase. The inhibitory effect of 3-PBA, which has the most potent inhibitory activity among the isomers, was slightly decreased by 4′-hydroxylation and further decreased by 4′-methoxylation against mushroom tyrosinase. Surprisingly, 4′-hydroxylation but not methoxylation of 2-PBA appeared inhibitory activity. On the other hand, both 4′-hydroxylation and methoxylation of 4-PBA increased the inhibitory activity against mushroom tyrosinase. In silico docking analyses using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid or 4′-hydroxyl group of PBA derivatives could chelate with cupric ions in the active site of mushroom tyrosinase, and that the interactions of Asn260 and Phe264 in the active site with the adequate-angled biphenyl group are involved in the inhibitory activities of the modified PBAs, by parallel and T-shaped π-π interactions, respectively. Furthermore, Arg268 could fix the angle of the aromatic ring of Phe264, and Val248 is supposed to interact with the inhibitors as a hydrophobic manner. These results may enhance the structural insight into mushroom tyrosinase for the creation of novel tyrosinase inhibitors.     

Molecular design of potent tyrosinase inhibitors having the bibenzyl skeleton

In order to develop water soluble tyrosinase inhibitors, bibenzyl xyloside 1 isolated from Chlorophytum arundinaceum (liliaceae), and its derivatives 2 and 3 were synthesized by using Wittig reaction and trichloroimidate glycosylation procedure as key steps. Xylosides 1-3 showed potent tyrosinase inhibitory activity with IC(50)s of 1.6, 0.43, and 0.73 microM, respectively, although each NMR data of synthetic bibenzyls was not identical to that of naturally occurring xyloside 1.     

Antioxidant,anti-tyrosinase and anti-melanogenic effects of (E)-2,3-diphenylacrylic acid derivatives

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%, IC₅₀ = 20.04 ± 1.91 µM) with than the other 2,3-DPA derivatives or kojic acid (21.56 ± 2.93%, IC₅₀ = 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.     

Evaluation of 3,4-dihydroquinazoline-2(1H)-thiones as inhibitors of α-MSH-induced melanin production in melanoma B16 cells

Novel 3,4-dihydroquinazoline-2(1H)-thiones (QNTs) 1 were found to be potent inhibitors of α-MSH-induced melanin production. The effect of QNTs to inhibit melanin formation in B16 melanoma cells was screened in the presence of α-MSH. In defining the mechanism of activity, the effects on tyrosinase activity, on tyrosinase synthesis and on the depigmentation of melanin were evaluated. QNTs did not affect the catalytic activity of tyrosinase, but rather acted as an inhibitor of tyrosinase synthesis.     

Chalcones as potent tyrosinase inhibitors: the effect of hydroxyl positions and numbers

The inhibition of tyrosinase is one of the major strategies to treat hyperpigmentation. Various limitations are associated with many of these inhibitors, such as high cytotoxicity, poor skin penetration and low stability in formulations. In continuation of our previous study [J. Agric. Food Chem. 51 (2003) 1201], showing that isoliquiritigenin chalcone (ILC) is a potent tyrosinase inhibitor, the present study aims to characterize the chalcone family as new tyrosinase inhibitors, and demonstrate their potential whitening potency. Nine mono-, di-, tri- and tetrahydroxychalcones were tested as inhibitors of tyrosinase mono- and diphenolase activities, showing that the most important factor in their efficacy is the location of the hydroxyl groups on both aromatic rings, with a significant preference to a 4-substituted B ring, rather than a substituted A ring. Neither the number of hydroxyls nor the presence of a catechol moiety on ring B correlated with increasing tyrosinase inhibition potency. 4-Hydroxychalcone (4-HC), ILC and Butein inhibited tyrosinase and shortened the lag period of enzyme monophenolase activity from about 490 min (control) to 30 min (ILC). As pigmentation also results from auto-oxidation, the antioxidant activity of 4-HC, ILC and Butein, were tested. Results showed that chalcones are also potent antioxidants, with Butein the most potent. We may conclude that chalcones are potentially potent new depigmentation agents, with their double effect of reduction and antioxidant activity. A deeper understanding of the relation between their structures to their potency will contribute to designing the optimal agents.     

Signalling pathways evoked by alpha1-adrenoceptors in human melanoma cells

The biological effects of catecholamines in mammalian pigment cells are poorly understood, but in poikilothermic vertebrates they regulate the translocation of pigment granules. We have previously demonstrated in SK-Mel 23-human melanoma cells the presence of low affinity alpha(1)-adrenoceptors, which mediate a decrease in cell proliferation and increase in tyrosinase activity, with no change of tyrosinase expression. In this report, we investigated the signalling pathways involved in these responses. Calcium mobilization in response to phenylephrine (PHE), an alpha(1)-adrenergic agonist, was investigated by confocal microscopy, and no change of fluorescence during the treatment was observed, suggesting that calcium is not involved in the signalling pathway activated by alpha(1)-adrenoceptors in SK-Mel 23 cells. cAMP levels, determined by enzyme-immunoassay, were significantly increased by PHE (10(-5)-10(-4)M), that could be blocked by the alpha(1)-adrenergic antagonist benoxathian (10(-5)-10(-4)M). Several biological assays were then performed with PHE, for 72 h, in the absence or presence of various signalling pathway inhibitors, in an attempt to determine the intracellular messengers involved in the responses of proliferation and tyrosinase activity. Our results suggest the participation of p38 and ERKs in PHE-induced decrease of proliferation, and possibly also of cAMP and protein kinase A. Regarding PHE-induced increase of tyrosinase activity, it is suggested that the following signalling components are involved: cAMP/PKA, PKC, PI3K, p38 and ERKs.