Difluoromethoxy-Substituted Hexahydroquinolines as Multi-Target Anti-Inflammatory Agents: Mechanistic and Metabolomic Insights

In this study, a series of 15 difluoromethoxy-substituted hexahydroquinoline (HHQ) derivatives were synthesized and evaluated for their anti-inflammatory potential. The cytotoxicity profiles of the synthesized compounds were assessed using the MTT assay in LPS-stimulated RAW 264.7 macrophages. At 10 μM, several derivatives (1a, 1b, 1e, 2a, 2d, 2e, and 3a–3e) maintained cell viability above 70%, indicating acceptable cytocompatibility. Among the tested compounds, 3b, 3c, and 3d exhibited low cytotoxicity and were selected for further biological evaluation. Compound 3b showed the highest cytocompatibility, with an IC50 value of 99.33 μM after 48 h exposure. Moreover, it exhibited the most pronounced activity by suppressing NF-κB p65 activation and downregulating the expression of major pro-inflammatory mediators, including TNF-α, IL-6, COX-2, iNOS, and MCP-1. In addition, compound 3b reduced LPS-induced TGF-β1 expression. Molecular docking analysis suggested favorable interactions with multiple inflammation-related targets, consistent with the experimental findings. Furthermore, untargeted metabolomic profiling revealed that compound 3b significantly altered the LPS-induced metabolic phenotype in RAW 264.7 cells, as demonstrated by clear group separation in principal component and hierarchical clustering analyses. Collectively, the biological and metabolomic findings, together with molecular docking analysis, suggest that difluoromethoxy-substituted HHQ derivatives possess promising multi-target anti-inflammatory potential, with compound 3b representing a promising scaffold for further structural optimization.