AChE/BChE-IN-27 is a brain-permeable dual inhibitor of AChE and BChE, with IC50 values of 3.72 μM and 9.65 μM, respectively, and a permeability coefficient (Pe) of 4.12. It exhibits significant antioxidant activity with an IC50 of 6.32 μM in DPPH assays and also demonstrates strong in vitro antioxidant capabilities. The compound shows metal-chelating properties and has neuroprotective potential against oxidative stress by significantly reducing intracellular reactive oxygen species (ROS). In vivo experiments reveal that AChE/BChE-IN-27 effectively restores AChE and BChE levels and improves cognitive function, indicating its potential application for Alzheimer’s disease (AD).

AChE (human):3.72 μM

AChE/BChE-IN-27 (compound 12d) demonstrates significant inhibitory effects against AChE and BChE, with IC 50 values of 3.72 μM and 9.65 μM, respectively, in the concentration range of 10 nM-100 μM over 12 days. In enzyme kinetic assays, AChE/BChE-IN-27 (1-30 μM, 37 ℃, 30 minutes) exhibits mixed inhibition on hAChE and eqBChE. During PAS inhibition studies, it shows a PI displacement rate of 23.58% at 5-50 μM, 25 ℃, for 6 hours. The compound demonstrates the strongest antioxidant activity, with an IC 50 of 6.32 μM, in DPPH assays at concentrations of 1.25-20 μM, 37 ℃, 30 minutes. AChE/BChE-IN-27 displays moderate BBB permeability (Pe > 2.0) at 500 μM, 37 ℃, for 18 hours, suggesting potential as a CNS agent. It acts as a potential therapeutic for metal ion imbalance at 18 μM for 5 minutes. AChE/BChE-IN-27 (5 μM, 37 ℃, 72 h) inhibits amyloid β1-42 aggregation and fibril formation, indicating its role as an aggregation modulator. The compound shows no cytotoxicity to SH-SY5Y cells at 5-80 μM, 24 h, and effectively protects these cells from H 2 O 2-induced neurotoxicity. Additionally, at concentrations of 1-80 μM for 24 hours, it reduces ROS levels, safeguarding SH-SY5Y cells.

AChE/BChE-IN-27 (compound 12d) demonstrates no toxicity at a dosage of 500 mg/kg administered orally to male Swiss albino mice. Administered via intraperitoneal injection at 1-10 mg/kg daily for 7 days, it shows significant anti-cholinesterase activity and improves cognitive function in scopolamine-induced amnesia models, possessing both cholinesterase inhibitory and potent antioxidant pharmacological properties.