Synthesis and biological evaluation of methylene spacer incorporated 4-methylsulfonyl phenyl semicarbazones as dual FAAH-MAGL inhibitors

Building upon our previous work, we investigated the impact of a methylene spacer on the dual fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) inhibition activity of 4-methylsulfonylphenyl semicarbazones by designing and synthesizing a set of extended semicarbazone analogs. The inhibition data demonstrated that several compounds had a good inhibitory activity against both FAAH and MAGL. Compound <b>15h</b>, [1-(4-hydroxyphenyl)ethylidene]-2-{[4-(methylsulfonyl)phenyl]amino}acetohydrazide, exhibited a well-balanced FAAH and MAGL inhibition activity with IC₅₀ values of 48 nM and 40 nM, respectively. The enzyme kinetics studies demonstrated that compound <b>15h</b> inhibits FAAH and MAGL reversibly with noncompetitive and mixed inhibition mode, respectively. Moreover, these experimental results were also supported by molecular modeling and simulation studies. The dual FAAH-MAGL inhibitor <b>15h</b>, exhibited no cytotoxicity on human neuronal SH-SY5Y cell lines and showed significant neuroprotection in LPS-induced toxicity. Further, compound <b>15h</b> showed potent analgesic activity and relieved oxidative stress in rats induced by chronic constriction injury (CCI) surgery. Furthermore, compound <b>15h</b> was non-hepatotoxic up to 2000 mg/kg (po). Overall, the present study identified compound <b>15h</b> as a promising dual FAAH-MAGL inhibitor and anti-nociceptive agent that merits further investigation.