3.3. FAAH Enzyme Inhibition Assay

The FAAH inhibition assay was performed using a Fatty Acid Amide Hydrolase Inhibitor Screening Assay Kit (Item # 10005196), Cayman (1180 E Ellsworth Rd Ann Arbor, MI, USA). The compounds were tested in the 10-dose IC₅₀ mode, with 3-fold serial dilution at a starting concentration of 100 μM for the compounds (2a–m) and a concentration of 5 μM for JZL195, which was used as a positive control. The manufacturer’s protocol was followed to perform the assay. The assay buffer (125 mM Tri-HCl with 1 mM EDTA, pH 9.0) was used to dilute the FAAH human recombinant enzyme. AMC-Arachidonoyl amide, at a concentration of 400 μM, was then used as the FAAH substrate. The samples were mixed for 30 s and incubated at 37 °C for 30 min. The fluorescent byproduct AMC (7-amino-4-methylcoumarin) released by the FAAH enzyme was detected and quantified at an excitation wavelength of 355 nm and an emission wavelength of 460 nm using an EnVision plate reader.