Total Synthesis of Anmindenol A and Its Application to the Design, Synthesis, and Biological Evaluation of Derivatives Thereof.

The first total synthesis of anmindenol A is described in four steps. A notable feature of the synthetic route includes the efficient construction of the 3,10-dialkylsubstituted benzofulvene core via a stereoselective vinylogous Stork enamine aldol condensation. The strategy provided a blueprint for the practical preparation of derivatives with modifications in the C-10 alkyl substituents. The novel derivatives inhibited nitric oxide production in stimulated RAW 264.7 macrophage cells.

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.

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.

Selective carbon-hydrogen bond hydroxylation using an engineered cytochrome P450 peroxygenase.

The cytochrome P450 enzyme CYP102A1 (P450BM3) is a versatile monooxygenase enzyme which has been adapted and engineered for multiple applications in chemical synthesis. Mutation of threonine 268 to glutamate (Thr268Glu) converted the heme domain of this enzyme into a H2O2 utilizing peroxygenase. This variant displayed significantly increased peroxide driven hydroxylation activity towards the saturated linear fatty acids tested (undecanoic through to hexadecenoic acid) when compared to the wild-type heme domain. The product distributions arising from fatty acid oxidation using this peroxygenase variant were broadly similar to those obtained with the wild-type monooxygenase holoenzyme, with oxidation occurring predominantly at the ω-1 through to ω-3 positions. 10-Undecenoic acid was regioselectively hydroxylated at the allylic ω-2 carbon by the Thr268Glu peroxygenase. The effect of isotopic substitution were measured using [9,9,10,10-d4]-dodecanoic acid. The kinetic isotope effect for both the monooxygenase and peroxygenase systems ranged between 7.9 and 9.5, with that of the peroxygenase enzyme being marginally lower. This highlights that carbon­hydrogen bond abstraction is important in the mechanism of both the monooxygenase and peroxygenase systems. This would infer that the ferryl-oxo radical cation intermediate, compound I, is the likely reactive intermediate in both systems. The peroxygenase variant offers the possibility of simpler cytochrome P450 systems for selective oxidations. To demonstrate this we used this system to oxidize tetradecanoic acid using light driven generation of H2O2 by a flavin.