Methoxy-Substituted Tyramine Derivatives Synthesis, Computational Studies and Tyrosinase Inhibitory Kinetics

Abstract

Targeting tyrosinase for melanogenesis disorders is an established strategy. Hydroxyl-substituted benzoic and cinnamic acid scaffolds were incorporated into new chemotypes that displayed in vitro inhibitory effects against mushroom and human tyrosinase for the purpose of identifying anti-melanogenic ingredients. The most active compound 2-((4-methoxyphenethyl)amino)-2-oxoethyl (E)-3-(2,4-dihydroxyphenyl) acrylate (Ph9), inhibited mushroom tyrosinase with an IC₅₀ of 0.059 nM, while 2-((4-methoxyphenethyl)amino)-2-oxoethyl cinnamate (Ph6) had an IC₅₀ of 2.1 nM compared to the positive control, kojic acid IC₅₀ 16700 nM. Results of human tyrosinase inhibitory activity in A375 human melanoma cells showed that compound (Ph9) and Ph6 exhibited 94.6% and 92.2% inhibitory activity respectively while the positive control kojic acid showed 72.9% inhibition. Enzyme kinetics reflected a mixed type of inhibition for inhibitor Ph9 (K_i 0.093 nM) and non-competitive inhibition for Ph6 (K_i 2.3 nM) revealed from Lineweaver-Burk plots. In silico docking studies with mushroom tyrosinase (PDB ID:2Y9X) predicted possible binding modes in the catalytic site for these active compounds. Ph9 displayed no PAINS (pan-assay interference compounds) alerts. Our results showed that compound Ph9 is a potential candidate for further development of tyrosinase inhibitors.

Keywords: computational studies; enzyme kinetics mechanism; inhibitory activity; tyramine derivatives; tyrosinase inhibitors.

Figure 1

Lineweaver–Burk plots for inhibition of tyrosinase in the presence of 2-((4-methoxyphenethyl)amino)-2-oxoethyl 3,5-dihydroxybenzoate (Ph5). (A) Concentrations of Ph5 were 0, 0.2 and 0.4 µM, respectively. Substrate l-DOPA concentrations were 0.125, 0.25, 0.5, 1 and 2 mM, respectively. (B) The insets represent the plot of the slope and (C) of the vertical intercepts versus inhibitor Ph5 concentrations to determine inhibition constants. The lines were drawn using linear least-squares fit.

Figure 2

Lineweaver–Burk plots for inhibition of tyrosinase in the presence of 2-((4-methoxyphenethyl)amino)-2-oxoethyl cinnamate (Ph6). (A) Concentrations of Ph6 were 0, 0.001 and 0.002 µM, respectively. Substrate l-DOPA concentrations were 0.125, 0.25, 0.5, 1 and 2 mM, respectively. (B) The insets represent the plot of the slope. The lines were drawn using linear least-squares fit.

Figure 3

Lineweaver–Burk plots for inhibition of tyrosinase in the presence of 2-((4-methoxyphenethyl)amino)-2-oxoethyl(E)-3-(2,4-dihydroxyphenyl)acrylate (Ph9). (A) Concentrations of Ph9 were 0, 0.00003, 0.00006 and 0.00012 µM, respectively. Substrate l-DOPA concentrations were 0.125, 0.25, 0.5, 1 and 2 mM, respectively. (B) The insets represent the plot of the slope and (C) of the vertical intercepts versus inhibitor Ph9 concentrations to determine inhibition constants. The lines were drawn using linear least-squares fit.

Figure 4

Ball and stick representation of the docking pose of (a) Kojic acid (a-brick red), 2-((4-methoxyphenethyl)amino)-2-oxoethyl 3,5-dihydroxybenzoate (Ph5), (b)-magenta, 2-((4-methoxyphenethyl)amino)-2-oxoethyl cinnamate (Ph6), (c)-yellow and 2-((4-methoxyphenethyl)amino)-2-oxoethyl(E)-3-(2,4-dihydroxyphenyl)acrylate (Ph9), (d)-green in the active site of mushroom tyrosinase (2Y9X). The inhibitor binds in the active site closer to the Cu²⁺. Amino acid residue forming the binding pocket of the tyrosinase is labeled in stick representation. Cu²⁺ ions are shown in brown and labelled.

Scheme 1

Synthesis of compounds (4a–e); Reagents and conditions. (i) CH₂Cl₂/ (C₂H₅)₃N, 0–−5 ℃, reflux for 5 h; (ii) DMF/(C₂H₅)₃N/KI, r.t. stirring for 24 h.

Scheme 2

Synthesis of compounds (Ph6–Ph10); (ii) DMF/(C₂H₅)₃N/KI, r.t. Stirring for 24 h.

Figure 5

Structure–activity relationship (SAR) for the synthesized compounds (Ph1–Ph10), promising compounds from both series (Ph1–Ph5 and Ph6–Ph10) are represented. Ph1: 2-((4-methoxyphenethyl)amino)-2-oxoethyl 3-hydroxybenzoate; Ph2: 2-((4-methoxyphenethyl)amino)-2-oxoethyl 4-hydroxybenzoate; Ph3: 2-((4-methoxyphenethyl)amino)-2-oxoethyl 2,4-dihydroxybenzoate, Ph4: 2-((4-methoxyphenethyl)amino)-2-oxoethyl 3,4-dihydroxybenzoate; Ph5: 2-((4-methoxyphenethyl)amino)-2-oxoethyl 3,5-dihydroxybenzoate; Ph6: 2-((4-methoxyphenethyl)amino)-2-oxoethyl cinnamate; Ph7: 2-((4-methoxyphenethyl)amino)-2-oxoethyl (E)-3-(2-hydroxyphenyl)acrylate; Ph8: 2-((4-methoxyphenethyl)amino)-2-oxoethyl(E)-3-(4-hydroxyphenyl)acrylate; Ph9: 2-((4-methoxyphenethyl)amino)-2-oxoethyl(E)-3-(2,4-dihydroxyphenyl)acrylate; Ph10: 2-((4-methoxyphenethyl)amino)-2-oxoethyl (E)-3-(4-chlorophenyl)acrylate.