Meden_2025_Chem.Biol.Interact_15ChEPon__41276059

Many covalent inhibitors of butyryl- and acetylcholinesterase, typically featuring electrophilic carbamoyl, phosphonyl, and sulfonyl groups, were reported in the literature. In this study, we screened these and several other electrophilic moieties for covalent cholinesterase inhibition. The electrophilic warheads were either installed at different positions on a reversible, selective human butyrylcholinesterase inhibitor scaffold or featured as small molecular weight, fragment-sized compounds. Time-dependency of the enzyme inhibition served as an indicator of covalent binding. The 7-substitution pattern proved optimal for generating time-dependent inhibitors, and carbamate chemotype produced most of the time-dependent hit compounds. Interestingly, the carbamates' reactivity was strongly influenced by their leaving groups' nucleofugality and not by steric hindrance, as sufficiently acidic leaving groups (pKa < 10) enabled carbamoylation of the catalytic serine. Notably, an exception to this rule of a thumb was found in a series of chalcogen carbachol analogues. The gathered insights highlight some key structure-reactivity relationships for covalent hBChE inhibitors and may assist in development of novel serine hydrolase inhibitors.