Design, synthesis, and biological evaluation of clovamide analogues as anti-colitis agents modulating NF-κB/STAT3/iNOS signaling

Clovamide and its derivatives are bioactive phenolic amides with anti-inflammatory potential, but their structure-activity relationships and therapeutic relevance in intestinal inflammation remain insufficiently defined. In this study, we designed, synthesized, and evaluated a focused series of clovamide analogues to identify structural features associated with anti-colitis activity. Screening based on LPS-induced nitrite production and TNF-α-induced monocyte-epithelial adhesion identified 10E as the most active analogue, showing dose-dependent inhibition with minimal cytotoxicity. Mechanistic studies showed that 10E attenuated NF-κB and STAT3 activation in RAW 264.7 macrophages and HT-29 colonic epithelial cells, resulting in reduced inflammatory mediator expression and increased epithelial barrier-associated protein expression. In a DSS-induced murine colitis model, post-onset administration of 10E alleviated disease severity, histopathological damage, and inflammatory signaling while preserving barrier-associated protein expression. These findings identify 10E as a chemically tractable lead compound and support scaffold-level rigidification of the clovamide framework as a strategy for developing anti-inflammatory agents that modulate NF-κB/STAT3-associated pathways in intestinal inflammation.