In-vitro and in-silico studies of annelated 1,4,7,8-tetrahydroazocine ester derivatives as nanomolar selective inhibitors of human butyrylcholinesterase

Based on previous finding showing 2,3,6,11-tetrahydro-1H-azocino[4,5-b]indole as suitable scaffold of novel inhibitors of acetylcholinesterase (AChE), a main target of drugs for the treatment of Alzheimer's disease and related dementias, herein we investigated diverse newly and previously synthesized beta-enamino esters (and ke-tones) derivatives of 1,4,7,8-tetrahydroazocines (and some azonines) fused with benzene, 1H-indole, 4H-chro-men-4-one and pyrimidin-4(3H)-one. Twenty derivatives of diversely annelated eight-to-nine-membered azaheterocyclic ring, prepared through domino reaction of the respective tetrahydropyridine and azepine with activated alkynes, were assayed for the inhibitory activity against AChE and butyrylcholinesterase (BChE). As a major outcome, compound 7c, an alkylamino derivative of tetrahydropyrimido[4,5-d]azocine, was found to be a highly potent BChE-selective inhibitor, which showed a noncompetitive/mixed-type inhibition mechanism against human BChE with single digit nanomolar inhibition constant (Ki = 7.8 +/- 0.2 nM). The four-order magnitude BChE-selectivity of 7c clearly reflects the effect of lipophilicity upon binding to the BChE binding cavity. The ChEs' inhibition data, interpreted by chemoinformatic tools and an in-depth in-silico study (mo-lecular docking combined with molecular dynamics calculations), not only highlighted key structural factors enhancing inhibition potency and selectivity toward BChE, but also shed light on subtle differences dis-tinguishing the binding sites of equine BChE from the recombinant human BChE. Compound 7c inhibited P-glycoprotein with IC50 of 0.27 mu M, which may support its ability to permeate blood-brain barrier, and proved to be no cytotoxic in human liver cancer cell line (HepG2) at the BChE bioactive concentrations. Overall, the biological profile allows us to envision 7c as a promising template to improve design and development of BChE-selective ligands of pharmaceutical interest, including inhibitors and fluorogenic probes.