AChE/BChE-IN-23 (Compound 6e) is a dual inhibitor of acetylcholinesterase and butyrylcholinesterase, exhibiting IC50 values of 0.91 μM for AChE, 1.19 μM for eqBChE, and 1.01 μM for hBChE. This compound also demonstrates antioxidant properties and inhibits the aggregation of Aβ1-42 and Tau proteins. Moreover, AChE/BChE-IN-23 prevents the activation of microglial cells by inhibiting the release of reactive oxygen species and mitochondrial damage. Additionally, it reduces the levels of the NLRP3 inflammasome in human microglial cells and reverses memory impairment in mice induced by scopolamine.

AChE (human):0.91μM(IC50), eqBCHE:1.19μM(IC50), NLRP3:1.02μM(IC50)

AChE/BChE-IN-23 demonstrates various pharmacological activities across different concentrations and conditions. The compound induces a shift in iodide-propidium affinity toward AChE enzymes, with displacement percentages increasing at higher concentrations (5 μM, 2.06%; 10 μM, 11.78%; 20 μM, 14.47%; 50 μM, 27.53%). Additionally, AChE/BChE-IN-23 exhibits antioxidant properties, as shown by a 47.30% scavenging rate of DPPH radicals at 20 μM and an IC50 of 15.17 μM. The compound effectively inhibits the aggregation of Aβ 1-42 at a concentration of 3.125 μM over 72 hours, and similarly inhibits Tau aggregation at 1.125 μM over the same duration. At concentrations ranging from 1 to 20 μM over 24 hours, AChE/BChE-IN-23 is non-toxic to PC-12 cells. Furthermore, at 12.5 μM over 24 hours, it prevents activation of microglia by reducing ROS release and restoring mitochondrial membrane potential. It also exerts an anti-inflammatory effect by inhibiting the activation of inflammasomes and NF-κB in microglial cells.

AChE/BChE-IN-23 administered at 10 mg/kg through intraperitoneal injection over a 14-day period demonstrated therapeutic potential by enhancing both spatial and cognitive memory in Alzheimer's disease (AD) mice, thereby reversing scopolamine-induced Alzheimer's symptoms. At a dosage of 2000 mg/kg administered orally for the same duration, AChE/BChE-IN-23 exhibited no toxicity in Swiss albino mice and did not induce symptoms associated with acute toxicity, such as weight gain, behavioral abnormalities, convulsions, tremors, or diarrhea.