, where b is the blank value using phosphate buffer without ChEs. For IC 50 determinations, at least seven different concentrations for each compound were used. The IC 50 values were obtained by plotting the residual ChE activities against the applied inhibitor concentrations, with the experimental data fitted to a four-parameter logistic function (GraphPad Prism 9.4). Tacrine and donepezil were used as positive controls. The progress curves for the time courses of product formation for the hydrolysis of butyrylthiocholine were measured on an Agilent Cary 3500 UV-VIS spectrophotometer with Compact Peltier (thermostatted at 25 °C) at 412 nm using a 0.6 mL 0.5 cm cuvette. 30 mM Tris buffer pH 7.0 with 1 mg/mL BSA and 0.02% NaN 3 was used and all dilutions were done in glass vessels (note: dilute hBChE solutions rapidly lose activity, presumably due to adsorption to plastic, preventing meaningful decarbamoylation assays. In our hands, under the above conditions only, the diluted and stock solutions of hBChE were stable for at least a month at room temperature.). Total volume in the cuvette was always 500 µL, and enzyme solutions that gave upon dilution v 0 = (dA/dt) ≈ 0.5 (for carbamoylation) and ≈ 1 for (for decarbamoylation) were used. For carbamoylation experiments, 0.5-1 µL of enzyme stock, 5 µL of compound solution, and 484 µL of buffer were preincubated for the specified time, then 10 µL of the mixture of 25 mM Ellman's reagent (DTNB) and 20 mM butyrylthiocholine (large excess to prevent substrate depletion) were added, quickly mixed, and measurement started. For decarbamoylation experiments, 50 µL of the enzyme stock solution and 0.25-0.5 µL of the compound solution in MeOH (concentration chosen to achieve 85-95% inhibition, typically 0.5-1 mM) were incubated for 1 h at room temperature, then the mixture was diluted 1:1000 with the buffer, and 500 µL aliquots were drawn at different timepoint, mixed with 10 µL of the mixture of 25 mM Ellman's reagent (DTNB) and 20 mM butyrylthiocholine, and hBChE activity assayed. The data analysis followed the literature. Experimental Gibbs free energy activation barrier ΔG ‡ was calculated from the (de)carbamoylation rates via the Eyring equation (taking reference concentration of 1 M, at 298.15 K).