Marakovic_2026_Arh.Hig.Rada.Toksikol_77_1

Based on our previous study on Nsubstituted 2-hydroxyiminoacetamides as cholinesterase-targeting ligands, which highlighted 2-hydroxyimino-N-(3-(4-((2-methyl-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-1-phenylpropyl)acetamide as lead compound because of its inhibition potency, we extended this group of compounds by introducing a new, bulkier peripheral anionic site (PAS)-binding substituent - 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline to achieve an orientation of its three modular elements that would comply with our initial design: the benzyl unit to occupy the choline binding region, the bulky substituent to stabilise binding at the peripheral site, and the 2-hydroxyiminoacetamide moiety to bind at the catalytic serine residue. The obtained N-(3-(4-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-1H-1,2,3-triazol-1-yl)-1-phenylpropyl)-2-(hydroxyimino)acetamide inhibited both human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) non-competitively in a low micromolar range. Molecular docking studies predicted high stability of the 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline group at PAS and of the 2-hydroxyiminoacetamide moiety and the benzyl group inside the catalytic site of BChE, while the closest orientation of the three modular elements to our initial design was predicted for AChE, though different orientations were also predicted. Cytotoxicity was observed in the HepG2 and HEK293 cell lines, but only at concentrations exceeding those used in the reversible inhibition assay. Our study confirms N-substituted 2-hydroxiiminoacetamides with three structural elements as low micromolar cholinesterase ligands, successfully extending the scope of PAS substituent to 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline. Beyond cholinesterase inhibition, our findings contribute to better understanding of the cytotoxic properties of constituting structural elements of the tested N-(3-(4-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-1H-1,2,3-triazol-1-yl)-1-phenylpropyl)-2-(hydroxyimino)acetamide.