Probing new 3-hydrazinyl indole phenacetamide derivatives as multitarget anti-Alzheimer: Synthesis, in vivo, in vitro, and in silico studies.

The development of multi-target directed ligands (MTDLs) amassed great attention to combat the multifactorial nature of Alzheimer's disease (AD). The present study showcases the synthesis of a novel series of 3-hydrazinyl indole phenacetamide derivatives aimed at addressing AD and neuroinflammation by targeting acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and β-secretase (BACE1) enzymes. The primarily in vivo anti-inflammatory screening nominated derivatives 5a-f, 5h, 5j and 5o for the initial in vitro screening against AChE. Compounds 5a-c, 5j, and 5o, exhibited the most potent inhibitory activity against AChE and BChE, were subsequently subjected to further in vivo biological evaluations. Also, 5a-c were inspected for their impact on hallmarks of AD and histopathological changes. N-phenylacetamide indole derivative bearing unsubstituted phenylhydrazinyl side chain 5a depicted the most cognitive enhancement compared to the reference standard donepezil and significantly improved spatial memory capabilities, mitigated histopathological alterations, reduced AD hallmarks, AChE, BACE1, amyloid beta (Aβ), and p-tubulin associated unit (p-Tau), and modulated oxidative and inflammatory markers, GSH and IL-1β. Moreover, in vitro BACE1 enzyme inhibition assay revealed moderate BACE1 inhibitory activity for derivatives 5a-c. Further, in silico docking studies for the most active derivatives 5a-c in AChE and BACE1 binding pockets evidenced interacting with key amino acid residues supporting their remarkable biological activity. Furthermore, molecular dynamics simulations confirmed the stability of derivative 5a within the AChE and BACE1 binding sites throughout the simulation period. Collectively, N-phenylacetamide indole derivative bearing unsubstituted phenylhydrazinyl side chain 5a represents a promising multi-target candidate, combining AChE, BChE and BACE1 inhibition and can be considered as a lead compound for further development in AD therapy.