Docking-guided design, synthesis, and evaluation of nitrazepam-pyridinium aldoxime conjugates for acetylcholinesterase reactivation with predicted blood-brain barrier penetration

We report a docking-guided design, synthesis, and in vitro evaluation of nitrazepam-pyridinium aldoxime conjugates as bifunctional antidotes for organophosphate (paraoxon) poisoning that aim to combine acetylcholinesterase (AChE) reactivation with improved blood-brain barrier (BBB) penetration and intrinsic anticonvulsant activity. A focused library of 21 conjugates varying pyridinium aldoxime position (2, 3, 4) and linker length was screened by molecular docking against paraoxon-inhibited human AChE (PDB 8dt2 ). Docking prioritized propyl- and butyl- linked pyridinium-4-aldoxime derivatives (Nit-Pr-4, Nit-Bu-4) which were synthesized, fully characterized (NMR, IR and elemental analysis), and tested in vitro using paraoxon-inhibited human RBC AChE and Ellman assays. Nit-Pr-4 and Nit-Bu-4 showed measurable reactivation (42.5% and 33.4% at 0.1 mM, respectively) with second-order rate constants (k r ) comparable to the pralidoxime as known antidote, while in silico ADMET profiling predicted enhanced lipophilicity and BBB permeability for the conjugates. Cytotoxicity testing in hADSCs identified Nit-Bu-4 as the more biocompatible lead. These results demonstrate the feasibility of a nitrazepam-conjugation strategy to produce centrally accessible reactivators and support further in vivo efficacy and optimization studies.