The development of multi-targeted therapeutic agents has gained significant attention as a promising approach for managing Alzheimer's disease. This study reported the synthesis of a series of novel benzothiazole-piperazine derivatives and assessment of their efficacy against cholinesterase enzymes and anti-β-amyloid (Aβ) aggregation activity. Compound LB05 emerged as the most effective, demonstrating potent inhibition of acetylcholinesterase (AChE) with an IC50 = 0.40 ± 0.01 μM and an inhibition constant (Ki) of 0.28 μM. Enzyme kinetics and PI displacement assays revealed that LB05 acts as a mixed-type AChE inhibitor. LB05 demonstrated significant efficacy in preventing both self-induced and AChE-mediated aggregation of Aβ1-42. The isothermal calorimetric titration results indicated that LB05 exhibits a moderate binding affinity for Aβ. Additionally, compound LB15 demonstrated significant antioxidant activity, showing 50.72 % DPPH radical scavenging at a concentration of 25 μM along with notable metal-chelating capabilities. In-silico assessments suggested that these compounds possess favorable drug-like properties and blood-brain barrier (BBB) permeability confirmed using the PAMPA-BBB assay. Notably, compound LB05 exhibited a strong safety profile in acute toxicity testing. It effectively alleviated cognitive and memory deficits in a scopolamine-induced amnesic model, demonstrating efficacy similar to that of Donepezil. Therefore, we propose that LB05 has the potential to be a promising lead candidate for further development as a therapeutic agent for Alzheimer's disease.
