Thiazol-4(5H)-one analogs as potent tyrosinase inhibitors: Synthesis, tyrosinase inhibition, antimelanogenic effect, antioxidant activity, and in silico docking simulation.

As the ß-phenyl-α,ß-unsaturated carbonyl (PUSC) structure was previously identified to play a key role in tyrosinase inhibition, 14 analogs with a PUSC structure built on a thiazol-4(5H)-one scaffold were synthesized using Knoevenagel condensation to serve as potential tyrosinase inhibitors. Through mushroom tyrosinase inhibition experiments, two analogs 9 and 11 were identified as potent tyrosinase inhibitors, with 11 exhibiting an IC50 value of 0.4 ± 0.01 µM, which indicates its 26-fold greater potency than kojic acid. Kinetic studies using Lineweaver-Burk plots revealed that 9 and 11 are competitive and mixed-type inhibitors, respectively; these kinetic results were supported by docking simulations. According to the B16F10 cell-based experiments, 9 and 11 inhibited melanogenesis more effectively than kojic acid due to their potent cellular tyrosinase inhibitory activity. In addition, analogs 9 and 11 exhibited moderate-to-strong antioxidant capacity, scavenging ABTS+, DPPH, and ROS radicals. In particular, analog 12 with a catechol moiety exhibited very strong ROS-scavenging activity, similar to Trolox. These results suggest that analogs 9 and 11, which exhibit potent tyrosinase inhibitory activity in mushroom and mammalian cells and anti-melanogenic effects in B16F10 cells, are promising antibrowning agents for crops and skin lightening agents for hyperpigmentation-related diseases.

Highly potent anti-melanogenic effect of 2-thiobenzothiazole derivatives through nanomolar tyrosinase activity inhibition.

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 IC50 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 CuSO4 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.

Investigation of the Efficacy of Benzylidene-3-methyl-2-thioxothiazolidin-4-one Analogs with Antioxidant Activities on the Inhibition of Mushroom and Mammal Tyrosinases.

Based on the fact that substances with a ß-phenyl-α,ß-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.

Identification and molecular mechanism of novel 5-alkenyl-2-benzylaminothiazol-4(5H)-one analogs as anti-melanogenic and antioxidant agents.

Mushroom tyrosinase is a tetramer, whereas mammalian tyrosinase is a monomeric glycoprotein. In addition, the amino acid sequence of mushroom tyrosinases differs from that of mammalian tyrosinases. MHY2081 exhibits potent inhibitory activity against both mushroom and mammalian tyrosinases. Accordingly, based on the MHY2081 structure, 5-alkenyl-2-benzylaminothiazol-4(5H)-one analogs were designed as a novel anti-tyrosinase agent and synthesized using 2-((3,4-dimethoxybenzyl)amino)thiazol-4(5H)-one (16), a key intermediate obtained via the rearrangement of a benzylamino group. Compounds 6 and 9 (IC50 = 1.5-4.6 µM) exhibited higher mushroom tyrosinase inhibitory activity than kojic acid (IC50 = 20-21 µM) in the presence of l-tyrosine and/or l-dopa. Based on kinetic analysis using Lineweaver-Burk plots, 6 was a mixed inhibitor, whereas 9 was a competitive inhibitor, and docking simulation results supported that these compounds could bind to the active site of mushroom tyrosinase. Using B16F10 mammalian cells, we demonstrated that these compounds inhibited melanogenesis more potently than kojic acid, and their anti-melanogenic effects could be attributed to tyrosinase inhibition. All synthesized compounds could scavenge reactive oxygen species (ROS), with five compounds exhibiting mild-to-strong ABTS+ and DPPH radical-scavenging abilities. Compounds 6 and 9 were potent tyrosinase inhibitors with strong antioxidant activities against ROS, ABTS+, and DPPH radicals. Moreover, the compounds significantly suppressed tyrosinase expression in a dose-dependent manner. Collectively, these results suggest that the novel 5-alkenyl-2-benzylaminothiazol-4(5H)-one analogs, especially 6 and 9, are potential anti-melanogenic agents with antioxidant activity.

Anti-tyrosinase flavone derivatives and their anti-melanogenic activities: Importance of the ß-phenyl-α,ß-unsaturated carbonyl scaffold.

Flavone derivatives were designed and synthesized based on the hypothesis that flavones containing the ß-phenyl-α,ß-unsaturated carbonyl (PUSC) scaffold have potential anti-tyrosinase activity. Flavones 1a and 1e inhibited mushroom tyrosinase more potently than kojic acid, and 1e inhibited monophenolase and diphenolase 61- and 28-fold more than kojic acid, respectively. Kinetic studies on mushroom tyrosinase indicated that 1a and 1e competitively inhibit monophenolase and diphenolase, and docking results supported these results. In an in vitro assay using B16F10 murine cells, 1a and 1e inhibited melanin production more potently than kojic acid, and this was attributed to the inhibition of tyrosinase. Furthermore, 1a and 1e strongly scavenged DPPH and ABTS radicals and ROS, which suggested that their antioxidant properties were at least partly responsible for their anti-melanogenic effects. Moreover, flavone 1a also inhibited the gene expressions of the melanogenesis-related genes tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. Our findings that flavone derivatives (i) directly inhibit tyrosinase, (ii) act as antioxidants, and (iii) inhibit the expressions of melanogenesis-related genes suggest their potential use as natural melanogenesis inhibitors. Furthermore, the study confirms that the PUSC scaffold confers anti-tyrosinase activity.

Real world co-prescribing contraindicated drugs with fluconazole and itraconazole.

PURPOSE: This study aimed to investigate co-prescribing of contraindicated drugs with fluconazole and itraconazole using real-world nationwide data. METHODS: This retrospective cross-sectional study was performed using claims data collected by the Health Insurance Review and Assessment Service (HIRA) of Korea during 2019-2020. To determine the drugs that should be avoided in patients taking fluconazole or itraconazole, Lexicomp® and Micromedex® were used. The co-prescribed medications, co-prescription rates, and potential clinical consequences of the contraindicated drug-drug interactions (DDIs) were investigated. RESULTS: Of the 197 118 prescriptions of fluconazole, 2847 co-prescriptions with drugs classified as contraindicated DDI by either Micromedex® or Lexicomp® were identified. Further, of the 74 618 prescriptions of itraconazole, 984 co-prescriptions with contraindicated DDI were identified. Solifenacin (34.9%), clarithromycin (18.1%), alfuzosin (15.1%), and donepezil (10.4%) were frequently found in the co-prescriptions of fluconazole, whereas tamsulosin (40.4%), solifenacin (21.3%), rupatadine (17.8%), and fluconazole (8.8%) were frequently found in the co-prescriptions of itraconazole. In 1105 and 95 co-prescriptions of fluconazole and itraconazole, accounting for 31.3% of all co-prescriptions, potential DDIs were associated with a risk of corrected QT interval (QTc) prolongation. Of the total 3831 co-prescriptions, 2959 (77.2%) and 785 (20.5%) were classified as contraindicated DDI by Micromedex® alone and by Lexicomp® alone, respectively, whereas 87 (2.3%) were classified as contraindicated DDI by both Micromedex® and Lexicomp®. CONCLUSIONS: Many co-prescriptions were associated with the risk of DDI-related QTc prolongation, warranting the attention of healthcare providers. Narrowing the discrepancy between databases that provide information on DDIs is required for optimized medicine usage and patient safety.

Design and Synthesis of (Z)-2-(Benzylamino)-5-benzylidenethiazol-4(5H)-one Derivatives as Tyrosinase Inhibitors and Their Anti-Melanogenic and Antioxidant Effects.

In this study, (Z)-2-(benzylamino)-5-benzylidenethiazol-4(5H)-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(5H)-one [7] and (Z)-2-(benzylamino)-5-(2,4-dihydroxybenzylidene)thiazol-4(5H)-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.

Design, Synthesis, In Vitro, and In Silico Insights of 5-(Substituted benzylidene)-2-phenylthiazol-4(5H)-one Derivatives: A Novel Class of Anti-Melanogenic Compounds.

(Z)-5-Benzylidene-2-phenylthiazol-4(5H)-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 3JC,Hß coupling constant of 1H-coupled 13C 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 vitro anti-melanogenic effects of chimeric compounds, 2-(substituted benzylidene)-1,3-indanedione derivatives with a ß-phenyl-α, ß -unsaturated dicarbonyl scaffold.

Tyrosinase is considered a key contributor to melanogenesis, and safe, potent tyrosinase inhibitors are needed for medical and cosmetic purposes to treat skin hyperpigmentation and prevent fruit and vegetable browning. According to our accumulated SAR data on tyrosinase inhibitors, the ß-phenyl-α,ß-unsaturated carbonyl scaffold in either E or Z configurations, can confer potent tyrosinase inhibitory activity. In this study, twelve indanedione derivatives were synthesized as chimeric compounds with a ß-phenyl-α,ß-unsaturated dicarbonyl scaffold. Two of these derivatives, that is, compounds 2 and 3 (85% and 96% inhibition, respectively), at 50 µM inhibited mushroom tyrosinase markedly more potently than kojic acid (49% inhibition). Docking studies predicted that compounds 2 and 3 both inhibited tyrosinase competitively, and these findings were supported by Lineweaver-Burk plots. In addition, both compounds inhibited tyrosinase activity and reduced melanin contents in B16F10 cells more than kojic acid without perceptible cytotoxicity. These results support the notion that chimeric compounds with the ß-phenyl-α,ß-unsaturated dicarbonyl scaffold represent promising starting points for the development of potent tyrosinase inhibitors.

Urolithin and Reduced Urolithin Derivatives as Potent Inhibitors of Tyrosinase and Melanogenesis: Importance of the 4-Substituted Resorcinol Moiety.

We previously reported (E)-ß-phenyl-α,ß-unsaturated carbonyl scaffold ((E)-PUSC) played an important role in showing high tyrosinase inhibitory activity and that derivatives with a 4-substituted resorcinol moiety as the ß-phenyl group of the scaffold resulted in the greatest tyrosinase inhibitory activity. To examine whether the 4-substituted resorcinol moiety could impart tyrosinase inhibitory activity in the absence of the α,ß-unsaturated carbonyl moiety of the (E)-PUSC scaffold, 10 urolithin derivatives were synthesized. To obtain more candidate samples, the lactone ring in synthesized urolithins was reduced to produce nine reduced urolithins. Compounds 1c (IC50 = 18.09 ± 0.25 µM), 1h (IC50 = 4.14 ± 0.10 µM), and 2a (IC50 = 15.69 ± 0.40 µM) had greater mushroom tyrosinase-inhibitory activities than kojic acid (KA) (IC50 = 48.62 ± 3.38 µM). The SAR results suggest that the 4-substituted resorcinol motif makes an important contribution to tyrosinase inhibition. To investigate whether these compounds bind to human tyrosinase, a human tyrosinase homology model was developed. Docking simulations with mushroom and human tyrosinases showed that 1c, 1h, and 2a bind to the active site of both tyrosinases with higher binding affinities than KA. Pharmacophore analyses showed that two hydroxyl groups of the 4-substituted resorcinol entity act as hydrogen bond donors in both mushroom and human tyrosinases. Kinetic analyses indicated that these compounds were all competitive inhibitors. Compound 2a inhibited cellular tyrosinase activity and melanogenesis in α-MSH plus IBMX-stimulated B16F10 melanoma cells more strongly than KA. These results suggest that 2a is a promising candidate for the treatment of skin pigment disorders, and show the 4-substituted resorcinol entity importantly contributes to tyrosinase inhibition.

PPARα Agonist, MHY3200, Alleviates Renal Inflammation during Aging via Regulating ROS/Akt/FoxO1 Signaling.

PPARα is a ligand-dependent transcription factor and its activation is known to play an important role in cell defense through anti-inflammatory and antioxidant effects. MHY3200 (2-[4-(5-chlorobenzo[d]thiazol-2-yl)phenoxy]-2,2-difluoroacetic acid), a novel benzothiazole-derived peroxisome proliferator-activated receptor α (PPARα) agonist, is a synthesized PPARα activator. This study examined the beneficial effects of MHY3200 on age-associated alterations in reactive oxygen species (ROS)/Akt/forkhead box (FoxO) 1 signaling in rat kidneys. Young (7-month-old) and old (22-month-old) rats were treated with MHY3200 (1 mg/kg body weight/day or 3 mg/kg body weight/day) for two weeks. MHY3200 treatment led to a notable decrease in triglyceride and insulin levels in serum from old rats. The elevated kidney ROS level, serum insulin level, and Akt phosphorylation in old rats were reduced following MHY3200 treatment; moreover, FoxO1 phosphorylation increased. MHY3200 treatment led to the increased level of FoxO1 and its target gene, MnSOD. MHY3200 suppressed cyclooxygenase-2 expression by activating PPARα and inhibiting the activation of nuclear factor-κB (NF-κB) in the kidneys of old rats. Our results suggest that MHY3200 ameliorates age-associated renal inflammation by regulating NF-κB and FoxO1 via ROS/Akt signaling.

Novel Anti-Melanogenic Compounds, (Z)-5-(Substituted Benzylidene)-4-thioxothiazolidin-2-one Derivatives: In Vitro and In Silico Insights.

To confirm that the ß-phenyl-α,ß-unsaturated thiocarbonyl (PUSTC) scaffold, similar to the ß-phenyl-α,ß-unsaturated carbonyl (PUSC) scaffold, acts as a core inhibitory structure for tyrosinase, twelve (Z)-5-(substituted benzylidene)-4-thioxothiazolidin-2-one ((Z)-BTTZ) derivatives were designed and synthesized. Seven of the twelve derivatives showed stronger inhibitory activity than kojic acid against mushroom tyrosinase. Compound 2b (IC50 = 0.47 ± 0.97 µM) exerted a 141-fold higher inhibitory potency than kojic acid. Kinetic studies' results confirmed that compounds 2b and 2f are competitive tyrosinase inhibitors, which was supported by high binding affinities with the active site of tyrosinase by docking simulation. Docking results using a human tyrosinase homology model indicated that 2b and 2f might potently inhibit human tyrosinase. In vitro assays of 2b and 2f were conducted using B16F10 melanoma cells. Compounds 2b and 2f significantly and concentration-dependently inhibited intracellular melanin contents, and the anti-melanogenic effects of 2b at 10 µM and 2f at 25 µM were considerably greater than the inhibitory effect of kojic acid at 25 µM. Compounds 2b and 2f similarly inhibited cellular tyrosinase activity and melanin contents, indicating that the anti-melanogenic effects of both were due to tyrosinase inhibition. A strong binding affinity with the active site of tyrosinase and potent inhibitions of mushroom tyrosinase, cellular tyrosinase activity, and melanin generation in B16F10 cells indicates the PUSTC scaffold offers an attractive platform for the development of novel tyrosinase inhibitors.

(E)-1-(Furan-2-yl)-(substituted phenyl)prop-2-en-1-one Derivatives as Tyrosinase Inhibitors and Melanogenesis Inhibition: An In Vitro and In Silico Study.

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 IC50 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 (Ki) 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.

In vitro and in vivo evidence of tyrosinase inhibitory activity of a synthesized (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-thioxothiazolidin-4-one (5-HMT).

Tyrosinase is a key enzyme that catalyses the initial rate-limiting steps of melanin synthesis. Due to its critical role in melanogenesis, various attempts were made to find potent tyrosinase inhibitors although many were not safe and effective in vivo. We evaluated tyrosinase inhibitory activity of six compounds. Among them, (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-thioxothiazolidin-4-one (5-HMT) had the greatest inhibitory effect and potency as the IC50 value of 5-HMT was lower than that of kojic acid, widely-known tyrosinase inhibitor. Based on in silico docking simulation, 5-HMT had a greater binding affinity than kojic acid with a different binding conformation in the tyrosinase catalytic site. Furthermore, its skin depigmentation effect was confirmed in vivo as 5-HMT topical treatment significantly reduced UVB-induced melanogenesis in HRM2 hairless mice. In conclusion, our study demonstrated that 5-HMT has a greater binding affinity and inhibitory effect on tyrosinase and may be a potential candidate for a therapeutic agent for preventing melanogenesis.

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)-ß-phenyl-α,ß-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)-ß-phenyl-α,ß-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 ±â€¯3.53%, IC50 = 20.04 ±â€¯1.91 µM) with than the other 2,3-DPA derivatives or kojic acid (21.56 ±â€¯2.93%, IC50 = 30.64 ±â€¯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.

Inhibitory effects of N-(acryloyl)benzamide derivatives on tyrosinase and melanogenesis.

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 ±â€¯2.14% inhibition) and 1j (25.99 ±â€¯2.77% inhibition) inhibited tyrosinase more than the other eight NAB derivatives and kojic acid (21.56 ±â€¯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.

Tyrosinase inhibition and anti-melanin generation effect of cinnamamide analogues.

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)-ß-phenyl-α,ß-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 ß-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.

Novel SIRT1 activator MHY2233 improves glucose tolerance and reduces hepatic lipid accumulation in db/db mice.

The NAD+-dependent deacetylase SIRT1, which is associated with the improvement of metabolic syndromes, such as type 2 diabetes, is a well-known longevity-related gene. Several in vitro and in vivo studies have shown the known protective effects of SIRT1 activators, such as resveratrol and SRT1720, on diabetes- or obesity-induced fatty liver and insulin resistance. Here, we newly synthesized 18 benzoxazole hydrochloride derivatives based on the structure of resveratrol and SRT1720. We performed an in vitro SIRT1 activity assay to identify the strongest SIRT1 activator. The assay confirmed MHY2233 to be the strongest SIRT1 activator (1.5-fold more potent than resveratrol), and docking simulation showed that the binding affinity of MHY2233 was higher than that of resveratrol and SRT1720. To investigate its beneficial effects, db/db mice were orally administered MHY2233 for 1 month, and various metabolic parameters were assessed in the serum and liver tissues. MHY2233 markedly ameliorated insulin signaling without affecting body weight in db/db mice. In particular, the mRNA expression of lipogenic genes, such as acetyl CoA carboxylase, fatty acid synthase, and sterol regulatory element-binding protein, which increased in db/db mice, decreased following oral treatment with MHY2233. In conclusion, the novel SIRT1 activator MHY2233 reduced lipid accumulation and improved insulin resistance. This finding may contribute toward therapeutic approaches for fatty liver disease and glucose tolerance.

Design, synthesis and anti-melanogenic effect of cinnamamide derivatives.

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)-ß-phenyl-α,ß-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)-ß-phenyl-α,ß-unsaturated carbonyl scaffolds are promising candidates for skin-lighting agents.

The tyrosinase inhibitory effects of isoxazolone derivatives with a (Z)-ß-phenyl-α, ß-unsaturated carbonyl scaffold.

Thirteen (Z)-4-(substituted benzylidene)-3-phenylisoxazol-5(4H)-ones were designed to confirm the geometric effect of the double bond of the ß-phenyl-α, ß-unsaturated carbonyl scaffold on tyrosinase inhibitory activity. Compounds 1a-1m, which all possessed the (Z)-ß-phenyl-α, ß-unsaturated carbonyl scaffold, were synthesized using a tandem reaction consisting of an isoxazolone ring formation and a Knoevenagel condensation, and three starting materials, ethyl benzoylacetate, hydroxylamine and benzaldehydes. Some of the compounds showed inhibitory activity against mushroom tyrosinase as potent as compounds containing the "(E)"-ß-phenyl-α, ß-unsaturated carbonyl scaffold. Compounds 1c and 1m showed greater inhibitory activity than kojic acid: IC50 = 32.08 ±â€¯2.25 µM for 1c; IC50 = 14.62 ±â€¯1.38 µM for 1m; and IC50 = 37.86 ±â€¯2.21 µM for kojic acid. A kinetic study indicated that 1m inhibited tyrosinase in a competitive manner and that it probably binds to the enzyme's active site. In silico docking simulation supported binding of 1m (-7.6 kcal/mol) to the active site of tyrosinase with stronger affinity than kojic acid (-5.7 kcal/mol). Similar results were obtained using cell-based assays, and in B16F10 cells, compound 1m dose-dependently inhibited tyrosinase activity and melanogenesis. These results indicate the anti-melanogenic effect of compound 1m is due to the inhibition of tyrosinase and (Z)-isomer of the ß-phenyl-α, ß-unsaturated carbonyl scaffold can, like its congener the (E)-isomer, act as an excellent scaffold for tyrosinase inhibition.