Determination of the Inhibitory Effect of 2,2,4-trimethyl-2,3-dihydro-1-Hbenzo[b][1,4]diazepine on Acetylcholinesterase Enzyme Activity

Benzodiazepines are used for their anxiolytic, antiepileptic, muscle relaxant and hypnotic effects. In vitro, diazepam is predominantly metabolized to temazepam and nordiazepam (N-desmethyldiazepam). Since acetylcholinesterase is involved in the metabolism of diazepam, inhibition of the enzyme activity may have a significant effect on the therapeutic effect of the drug. To determine the inhibitory effect of 2,2,4-trimethyl-2,3-dihydro-1H-benzo[b][1,4]diazepine on acetylcholinesterase enzyme activity by conducting a comprehensive analysis that includes: measuring the enzyme activity in the presence of various concentrations of the inhibitor, determining the type of inhibition through kinetic studies, and assessing the potential therapeutic applications of the inhibitor in conditions associated with acetylcholinesterase dysfunction. In this study, the inhibitory properties of 2,2,4-trimethyl-2,3-dihydro-1-Hbenzo[ b][1,4]diazepine on the activity of the enzyme acetylcholinesterase were tested spectrophotometrically at three different temperatures of 25℃, 30℃, and 37℃. The substance was synthesized by a condensation reaction between o-phenyldiamine and acetone in the presence of phosphorus oxychloride on solid support (MgO). The solid product was obtained by crystallization from n-hexane. Each tested sample contained an appropriate concentration of the substrate acetylcholine iodide (AChI) in the range from 1.00 to 4.00 mmol·L-1; 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) concentration 3 mmolL-1, phosphate buffer (KH2PO4/K2HPO4) pH value 8, tested substance concentration (17.70, 35.40, 53.10 mmol·L-1), and acetylcholinesterase solution (AChE) activity 0.54 UmL-1. Using the spectrophotometric method, it was concluded that the examined diazepine shows a competitive type of inhibition on the enzyme acetylcholinesterase. 30°C was determined to be the optimal assay temperature. The highest inhibition was observed at 25°C using 53.10 mmol·L⁻¹ of the inhibitor. As the temperature increases, the inhibition decreases. Based on the Lineweaver-Burk diagram, we gain insight into the type of inhibition exhibited by the synthesized compound. The intercept on the ordinate remains unchanged; the slope of the line increases, and the intercept on the abscissa decreases, indicating that it is a competitive inhibition. Considering the results obtained by spectrophotometric analysis, it was concluded that the enzyme acetylcholinesterase follows the Michaelis-Menten model. It has been proven that the synthesized compound exhibits inhibitory properties on the activity of acetylcholinesterase.