Hmaos

Inhibition assays of hChEs and hMAOs (all from Sigma Aldrich) were performed by using already published protocols.^[24] All compounds were assayed at 10 μM concentration and, for those showing inhibition >60 %, IC₅₀ values was calculated by testing seven concentrations in the range 30–0.01 μM. Briefly, the classical spectrophotometric Ellman's test (for ChEs) and the fluorimetric detection of 4‐hydroxyquinoline (for MAOs) were adapted to a plate reader procedure with 96‐well microtiter plates (Greiner Bio‐One GmbH, Frickenhausen, Germany). Readings were made with Infinite M1000 Pro plate reader (Tecan, Cernusco s.N., Italy) and statistical regressions with Prism software (GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, CA, USA). Kinetics of MAO inhibition for compound 9 were calculated with four concentrations of inhibitor (0, 0.5, 0.8, 1.5 μM) and seven concentrations of kynuramine (from 10 to 250 μM), and data analysed by means of the “Enzyme kinetics” module of Prism.

Compounds 4–6, 15, and 16 were assayed for their inhibition of hChEs and hMAOs (all obtained from Sigma Aldrich). Inhibition assays were performed according to previously published protocols [24 (link)]. All compounds were screened at a concentration of 10 μM, and IC₅₀ values were calculated for those showing inhibition >60%, by testing seven concentrations in the range 30–0.01 μM. Briefly, the classical Ellman spectrophotometric assay (for ChEs) [24 (link)] and fluorimetric detection of 4-hydroxyquinoline (for MAOs) [38 (link)] were adapted to a plate reading procedure using 96-well microtiter plates (Greiner Bio-Frickenhausen, Frickenhausen, Germany). Readings were performed using the Infinite M1000 Pro plate reader (Tecan, Cernusco s.N., Cernusco sul Naviglio, MI, Italy), and statistical regression was performed using Prism software (GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, CA, USA).