Design, synthesis and biological evaluation of amino acid-conjugated tryptanthrin derivatives as novel...

Two series of amino acid-conjugated tryptanthrin derivatives were rationally designed and synthesised as novel anti-inflammatory agents using tryptanthrin as the lead scaffold. All derivatives were evaluated using the Cell Counting Kit-8 assay and nitric oxide (NO) production inhibition assay. Among these derivatives, compound d12 exhibited the most potent inhibitory activity against NO production (IC₅₀ = 3.96 ± 0.3 μM) and demonstrated excellent cellular safety. Further investigations revealed that d12 significantly attenuated the lipopolysaccharide induced inflammatory response, as evidenced by the reduced levels of TNF-α, IL-1β, inducible nitric oxide synthase and cyclooxygenase-2 proteins. To explore the underlying molecular mechanisms of the anti-inflammatory activity of d12, RNA-sequencing (RNA-Seq) was performed on d12-treated RAW264.7 cells. Transcriptomic analysis indicated that the anti-inflammatory effects of d12 were closely associated with the suppression of the mitogen-activated protein kinase (MAPK) and TNF signalling pathways. Western blotting analysis confirmed that d12 downregulated the phosphorylation of c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase in the MAPK pathway, as well as inhibited the activation of the p65/IκB axis in the NF-κB signalling cascade, thereby validating the RNA-Seq results. Molecular docking, MD simulations and the cellular thermal shift assay collectively suggested a potential interaction between d12 and TNF-α. Moreover, in vivo studies demonstrated that d12 markedly alleviated paw swelling in adjuvant-induced arthritis rats and reduced the serum levels of TNF-α and IL-1β. These findings indicate that d12 is a promising candidate for the development of novel anti-inflammatory agents.