Recombinant human mao a

MAO-Glo assay (Promega, V1401) was applied to determine MAO and SSAO activities respectively according to manufacturer’s instructions. Recombinant human MAO A (Sigma, M7316) was included as the positive control, and the assay was conducted with following the optimized protocol [82 ].

AChE (Type VI-S; from Electrophorus electricus), recombinant human MAO-A, MAO-B, and BChE (from equine serum), acetylthiocholine iodide (ATCI), kynuramine, benzylamine, S-butyrylthiocholine iodide (BTCI), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), tacrine, donepezil, toloxatone, and lazabemide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Clorgyline and pargyline (irreversible reference inhibitors of MAO-A and MAO-B, respectively) were from BioAssay Systems (Hayward, CA, USA)^{40 (link)}.

All solvents and fine chemicals were purchased from Sigma–Aldrich (unless specified otherwise) and were used without further purification. For the MAO inhibition studies, recombinant human MAO-A and human MAO-B (5 mg protein/mL) from Sigma–Aldrich were used. Analytical thin layer chromatography was carried out on precoated silica gel 60 F254 aluminum plates purchased from Merck and visualized by UV. Melting points were measured on a Stuart SMP40 automatic melting point apparatus and are uncorrected. For all of the prepared compounds, infrared (IR) spectra were recorded with a Perkin Elmer Spotlight 400 Fourier transform infrared (FTIR) imaging system. GC-MS was performed on a Thermo instrument (ISQELTL). Proton (¹H) and carbon (¹³C) NMR spectra were recorded on JEOL 600 MHz using CDCl₃ as a solvent at the Central Lab Unit, Qatar University and a Bruker Avance III spectrometer (700 MHz) at the King Abdullah Institute for Research & Consulting Studies, King Saud University, Riyadh, Saudi Arabia. The chemical shifts were reported as parts per million (δ) relative to the solvent peak and coupling constants are given in hertz (Hz). The original spectra are provided in the Supporting information. Purity of compounds were confirmed with C, H and S analysis performed on Thermo Scientific FLASH 2000 CHNS/O analyzer.