Monoacylglycerol lipase (MAGL) represents a key enzyme in the regulation of the endocannabinoid system and arachidonic acid signaling, emerging as a promising target for the treatment of inflammation, neurodegeneration, and cancer. Herein, we report the design, synthesis, and biological evaluation of a novel series of o-hydroxyanilide derivatives as reversible MAGL inhibitors. Starting from a known salicylketoxime scaffold, we introduced strategic modifications to reduce lipophilicity and enhance selectivity and potency. Among the synthesized compounds, 4-phenylbutanamide 40 exhibited the most potent inhibitory activity against human MAGL (IC50 = 0.34 μM), outperforming reference compound 8 (IC50 = 0.68 μM), and showing good selectivity over FAAH and cannabinoid receptors CB1R and CB2R. Biochemical characterization confirmed a reversible and competitive mechanism of inhibition. Molecular modeling studies supported the key interactions of compound 40 with the MAGL oxyanion hole and hydrophobic pocket. In cellular assays, compound 40 activated the Nrf2 antioxidant pathway and significantly inhibited NFκB-mediated inflammation, without inducing cytotoxic effects. ADME profiling revealed good membrane permeability and excellent plasma stability, despite moderate metabolic stability in human liver microsomes. Taken together, these findings highlight compound 40 as a promising reversible MAGL inhibitor endowed with anti-inflammatory and antioxidant properties, representing a valuable lead for the development of therapeutics targeting the endocannabinoid system.
