Hydroxy substituted benzoic/cinnamic acid derivatives as inhibitors of melanogenesis: synthesis, molecular docking and tyrosinase inhibitory activities

Abstract

A copper-containing metalloenzyme tyrosinase catalyzes the biosynthesis of macromolecular pigments melanin. This enzyme is present in bacteria, fungi, plants and animals and plays multiple roles in pigmentation, wound healing, radiation protection, primary immune responses and the undesirable browning of fruits and vegetables. Till date several tyrosinase inhibitors have been reported from both natural and synthetic sources for treating dermatological disorders. Nowadays tyrosinase inhibitors have found much attraction in the fields of cosmetics, pharmaceuticals and agriculture industries. Currently reported and most widely used tyrosinase inhibitors have a phenolic moiety in common. Therefore, new approaches are needed for safer and more active anti-melanogenic agents, as better solutions for treating dermatological disorders. Most of the research on tyrosinase inhibitors focuses on the design and synthesis of novel core structures to which variable alkyl or alkoxy chains are attached through various ester, amide and ether linkages. These structural moieties play an important role for the tyrosinase inhibitory activities. In the present study, several series of novel 92 compounds with variable side chain alkyl or alkoxy substituents were synthesized by incorporating ester and amide functionalities as linking groups. The structures of these newly synthesized compounds were established by their spectral data using FTIR, 1HNMR, 13CNMR, LCMS, HRMS and elemental analysis. The in vitro melanin and tyrosinase inhibitory effects were also determined by using murine melanoma cells (B16F10), mushroom and human tyrosinases (A375 melanoma cells). The inhibitory effects of variable alkyl or alkoxy chain and ester/amide functionalities on pigmentation was also explored in vivo by using wild type zebrafish. 3-Methoxyphenol, 4-Methoxyphenol, 3,5-Dimethoxyphenol, 4-Tert-butylphenol, 3- Aminoacetophenone, 4-Aminoacetophenone, 4-Methoxyphenylethylamine, 2,4-Dimethoxy benzylamine, 4-Isopropylaniline and 2-Chloromethylbenzimidazole were used as starting materials for the synthesis of novel tyrosinase inhibitors. The hydroxyl (-OH) and amino (- NH2) group in these starting materials were reacted with 2-chloroacetyl chloride to afford intermediates (1a-i). The synthesized intermediates (1a-i) were then finally condensed with isomeric mono and dihydroxyl substituted benzoic acids (3-Hydroxybenzoic acid, 4- Hydroxybenzoic acid, 2,4-Dihydroxybenzoic acid, 3,4-Dihydroxybenzoic acid, 2,5- Dihydroxybenzoic acid, 3,5-Dihydroxybenzoic acid (2a-f) and trans-cinnamic acids, cinnamic acid, 2-Hydroxycinnamic acid, 4-Hydroxycinnamic acid, 2,4-Dihydroxycinnamic acid, 4-Chlorocinnamic acid, 4-Hydroxyphenylpropionic acid (3a-f) to afford the final vii products having different peripheral alkyl and alkoxy substitutions. All the synthesized final products (3-94) were purified by normal phase silica gel column chromatography using n-hexane: ethyl acetate (3:1) as eluents and HPLC using 0.1% trifluoracetic acid (TFA) in 100% acetonitrile and 0.1% trifluoracetic acid (TFA) in 100% water as buffers. The structures of the title compounds (3-94) were confirmed by their spectral data. In silico computational molecular docking studies of the (92) synthesized compounds with mushroom tyrosinase (PDB ID 2Y9X) was performed to predict the possible binding modes for active compounds in the enzymatic pocket of the target protein. Line-weaver Burk and Dixon Plots were used to determine the enzyme inhibitory kinetics of most potent compounds (6,10,55,56,62,63,66). The reversible/irreversible nature of enzyme inhibitor complex was also determined. Analysis of enzyme kinetics revealed that these novel compounds showed competitive (55), non-competitive (6,10,63) and mixed type inhibition (56,62,66). The in vivo and in vitro tyrosinase inhibitory activities and cytotoxicity of these novel compounds were ascertained. These inhibitory studies on melanoma cells, mushroom and human tyrosinases and in zebrafish revealed that most of these synthesized compounds especially those bearing 2,4-Dihydroxycinnamic acid moieties possess more potent inhibitory effect as compared to reference kojic acid, arbutin and N-phenylthiourea (PTU).