Optimization of an essentially inactive 3,4-dihydro-2H-pyrano[3,2-c]quinoline carboxylic ester derivative as acetylcholinesterase (AChE) peripheral anionic site (PAS)-binding motif by double O → NH bioisosteric replacement, combined with molecular hybridization with the AChE catalytic anionic site (CAS) inhibitor 6-chlorotacrine and molecular dynamics-driven optimization of the length of the linker has resulted in the development of the trimethylene-linked 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridine
6-chlorotacrine hybrid 5a as a picomolar inhibitor of human AChE (hAChE). The tetra-, penta-, and octamethylene-linked homologues 5b
d have been also synthesized for comparison purposes, and found to retain the nanomolar hAChE inhibitory potency of the parent 6-chlorotacrine. Further biological profiling of hybrids 5a
d has shown that they are also potent inhibitors of human butyrylcholinesterase and moderately potent Aβ42 and tau anti-aggregating agents, with IC50 values in the submicromolar and low micromolar range, respectively. Also, in vitro studies using an artificial membrane model have predicted a good brain permeability for hybrids 5a
d, and hence, their ability to reach their targets in the central nervous system. The multitarget profile of the novel hybrids makes them promising leads for developing anti-Alzheimer drug candidates with more balanced biological activities.