Discovering the Cholinesterase Inhibitory Potential of Thiosemicarbazone Derivatives through In vitro, Molecular Docking, Kinetics, and Dynamics Studies

Background: The current study explored the cholinesterase inhibitory activities of some thiosemicarbazone derivatives bearing 2,4-dichloro phenylacetic acid scaffold. Objective: This study aimed to screen the synthesized derivatives for their in vitro acetylcholine and butyrylcholinesterase inhibition. Methods: These compounds were synthesized by refluxing 2,4-dichloro phenylacetic acid with sulfuric acid in ethanol to get the ester, which was further refluxed with thiosemicarbazide in ethanol to get the desired compound (2). Different benzaldehydes were treated with compound (2) in ethanol having a catalytic amount of acetic acid to get thiosemicarbazones. Results: In the series, seven compounds, including compounds 2c, 2a, 2b, 2d, 2g, 2e, and 2f, displayed excellent acetylcholinesterase inhibition activities in the range of IC50 values from 41.51 ± 3.88 to 95.48 ± 0.70 µM, surpassing than the standard galantamine (IC50 = 104.5 ± 1.20 µM). Also, compounds 2a, 2g, 2h, 2f, 2b, and 2d with IC50 values ranging from 64.47 ± 2.74 to 80.62 ± 0.73 µM exhibited potent inhibition against butyrylcholinesterase enzyme, being similar to the standard galantamine (IC50 = 156.8 ± 1.50 µM). The molecular docking investigation was performed to assess the binding affinity of the compounds with the active site of the enzyme. These compounds, along with the docked complexes, specifically AChE-compound 2a and BuChE-compound 2g, were chosen and subjected to 100-nanosecond molecular dynamics simulations. The simulations demonstrated strong stability of the ligands within the active pockets of AChE and BuChE enzymes. Conclusion: These derivatives exhibited superior acetylcholinesterase and butyrylcholinesterase inhibitory activities compared to galantamine, with molecular docking and dynamic simulations confirming their strong binding affinity with the active sites of the enzymes.