Synthesis of cinnamic amide derivatives and their anti-melanogenic effect in α-MSH-stimulated B16F10 melanoma cells

摘要

Of the three enzymes that regulate the biosynthesis of melanin, tyrosinase and its related proteins TYRP-1 and TYRP-2, tyrosinase is the most important because of its ability to limit the rate of melanin production in melanocytes. For treating skin pigmentation disorders caused by an excess of melanin, the inhibition of tyrosinase enzyme is by far the most established strategy. Cinnamic acid is a safe natural product with an (E)-β-phenyl-α,β-unsaturated carbonyl motif that we have previously shown to play an important role in high tyrosinase inhibition. Since cinnamic acid is relatively hydrophilic, which hinders its absorption on the skin, fifteen less hydrophilic cinnamic amide derivatives (1–15) were designed as safe and more potent tyrosinase inhibitors and were synthesized through a Horner-Wadsworth-Emmons reaction. The use ofconc-HCl and acetic acid for debenzylation of theO-benzyl-protected cinnamic amides40–54produced the following three results. 1) Cinnamic amides43,48, and53with a 2,4-dibenzyloxyphenyl group, irrespective of the amine type of the amides, produced complex compounds with high polarity. 2) Cinnamic amides40–42,44,50–52, and54with a benzylamino, or diethylamino group produced the desired debenzylated cinnamic amides1–3,5,10–13, and15. 3) Cinnamic amides45–47, and49with an anilino moiety provided 3,4-dihydroquinolinones16–19through intramolecular Michael addition of the anilide group. Notably, the use of BBr3as an alternative debenzylating agent for debenzylation of cinnamic amides45–49with the anilino moiety provided our desired cinnamic amides6–10without inducing the intramolecular Michael addition. Debenzylation of cinnamic amides43,48, and53with a 2,4-dibenzyloxyphenyl group was also successfully accomplished using BBr3to give4,9, and14. Among the nine compounds that inhibited mushroom tyrosinase more potently at 25?μM than kojic acid, four cinnamic amides4,5,9, and14showed 3-fold greater tyrosinase inhibitory activity than kojic acid. The docking simulation using tyrosinase indicated that these four cinnamic amides (?6.2 to??7.9?kcal/mol) bind to the active site of tyrosinase with stronger binding affinity than kojic acid (?5.7?kcal/mol). All four cinnamic amides inhibited melanogenesis and tyrosinase activity more potently than kojic acid in α-MSH-stimulated B16F10 melanoma cells in a dose-dependent manner without cytotoxicity. The strong correlation between tyrosinase activity and melanin content suggests that the anti-melanogenic effect of cinnamic amides is due to tyrosinase inhibitory activity. Considering that the cinnamic amides4,9, and14, which exhibited strong inhibition on mushroom tyrosinase and potent anti-melanogenic effect in B16F10?cells, commonly have a 2,4-dihydroxyphenyl substituent, the 2,4-dihydroxyphenyl substituent appears to be essential for high anti-melanogenesis. These results support the potential of these four cinnamic amides as novel and potent tyrosinase inhibitors for use as therapeutic agents with safe skin-lightening efficiency.