Identification of an enantiopure monocyclic β-lactam as a potent anti-inflammatory agent via modulation of the NF-κB/JNK signaling pathway.

To develop novel anti-inflammatory agents with improved pharmaceutical profiles, a series of monocyclic β-lactam derivatives were designed and synthesized. Utilizing the Staudinger [2 + 2] cycloaddition, a key 2-azetidinone intermediate was first constructed, followed by C-3 hydroxyl condensation with various carboxylic acids to yield the target analogues. In vitro anti-inflammatory activity was evaluated by screening their inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Compounds 4q, 4r, and 7a exhibited potent inhibitory activity, among which 4r was resolved into its enantiomers 4r-1 and 4r-2via chiral HPLC. Their absolute configurations were unequivocally assigned using circular dichroism spectroscopy and electronic circular dichroism calculations. Among the enantiomers, 4r-1, the non-cytotoxic active enantiomer, demonstrated the most pronounced NO suppression (IC50 = 1.86 ± 0.23 μM) with the lowest cytotoxicity profile, whereas its antipode 4r-2 and the racemate 4r exhibited significant toxicity at the same concentrations. Further assessment revealed that 4r-1 dose-dependently inhibited the release of key inflammatory cytokines, including IL-6, TNF-α, and IL-1β, surpassing the racemate 4r in potency. Importantly, 4r-1 was prioritized for all in-depth mechanistic investigations due to its superior safety profile. Mechanistic studies indicated that 4r-1 significantly suppressed the protein expression of iNOS and COX-2 by modulating the NF-κB/JNK signaling pathway and concurrently down-regulated the mRNA transcription of associated inflammatory genes. Moreover, 4r-1 effectively attenuated intracellular reactive oxygen species (ROS) production. Molecular docking and dynamics simulations revealed a more stable binding mode of 4r-1 with the TNF-α protein (PDB ID: 2AZ5) relative to its antipode 4r-2. In conclusion, the monocyclic β-lactam derivative 4r-1 exhibits remarkable anti-inflammatory efficacy and a favorable safety margin, positioning it as a promising lead candidate for further anti-inflammatory drug development.