Synthesis and biological evaluation of C6-modified Celastrol derivatives as anti-inflammatory agents targeting the...

Celastrol is a potent natural anti-inflammatory agent, while systematic exploration of C6 structural modification and its structure-activity relationship (SAR) remains limited. In this study, twenty C6-modified Celastrol derivatives were synthesized via thiol-mediated Michael addition followed by phenolic acetylation. For the first time, the anti-inflammatory activity of all synthesized C6-substituted derivatives were systematically evaluated in an LPS-induced RAW264.7 cell model. Most derivatives retained comparable NO inhibitory activity to Celastrol and significantly higher than Curcumin. Preliminary structure-activity relationship analysis indicated that alkyl substitutions at the C6 position exhibited more potent NO inhibitory activity than aryl, and this effect was influenced by multiple factors, including substituent chain length, steric hindrance, and electronic effects. Among all derivatives, compound 6e exhibited a favorable balance between NO inhibitory activity and cytotoxicity. 6e effectively suppressed the production of pro-inflammatory cytokines (TNF-α and IL-6) and inhibited NF-κB signaling by reducing the phosphorylation of IκBα and p65, as well as downregulating iNOS expression. Molecular docking studies targeting IKKβ, a key upstream regulator of the NF-κB signaling pathway, demonstrated that 6e could be favorably accommodated within the active site through multiple stabilizing interactions, providing a structural basis for its inhibitory effects on NF-κB signaling pathway. In conclusion, this study provides a systematic structure-activity relationship (SAR) insight into C6 modification of Celastrol and highlights this position as a viable site for structural diversification while maintaining anti-inflammatory activity, offering insights for further structural optimization.