3.2. AChE/BChE Activity Assay

All enzymes were obtained from Sigma-Aldrich (Steinheim, Germany). Ellman’s reagent and the substrates were obtained from Fluka (Buchs, Switzerland). The chemicals used to prepare the buffer solution were purchased from Merck (Darmstadt, Germany).

The enzyme inhibitory activities of the synthesized compounds were investigated by applying Ellman’s method [35]. Enzyme solutions were dissolved in gelatin solution (1%; 2.5 units/mL). Compounds 4a–4u and reference agents were prepared in 2% DMSO at concentrations of 10⁻³ M and 10⁻⁴ M. The enzyme solution (20 µL/well) and inhibitor solution (20 µL/well) were mixed with buffer (140 µL/well, pH 8 ± 0.1) and incubated at 25 °C for 5 min. Then, the reaction was initiated by the addition of Ellman’s reagent 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB; 20 µL/well, 10 mM) and the substrate (10 µL/well, 75 mM). The absorbance was measured for 10 min at 412 nm. The enzyme solution was processed as a control. All readings were adjusted with blank-reading. All assays were performed in four independent wells. The same procedure was applied for further concentrations (10⁻⁵–10⁻⁹ M) of reference agents and synthesized compounds displaying ≥50% inhibition at initial concentrations (10⁻³ and 10⁻⁴ M). The IC₅₀ values were calculated by applying regression analyses using Microsoft Excel 2013 [36]. Absorbance differences between the two readings were taken, and percentage inhibition rates were calculated according to the following formula:

Blank (B): the well in which the inhibitor compound and substrate were not added;

Control (C): the well where the inhibitor compound was not added;

A(B): the difference in absorbance reading for the blank;

A(C): the difference in absorbance reading for the control;

A(I): the difference in absorbance reading for the inhibitor compounds.