Design, Synthesis, and Biological Activity of Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine Derivatives as Anti-Inflammatory Agents.

We designed and synthesized 26 prototype compounds and studied their anti-inflammatory activity and underlying molecular mechanisms. The inhibitory effects of the compounds on the production of nitric oxide (NO), cytokines, inflammatory-related proteins, and mRNAs in lipopolysaccharide (LPS)-stimulated macrophages were determined by the Griess assay, Enzyme linked immunosorbent assay (ELISA), Western blot analysis, and Reverse transcription-Polymerase Chain Reaction (RT-PCR), respectively. Our results indicated that treatment with A2, A6 and B7 significantly inhibited the secretion of NO and inflammatory cytokines in RAW264.7 cells without demonstrable cytotoxicity. It was also found that A2, A6 and B7 strongly suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase enzyme COX-2, and prevented nuclear translocation of nuclear factor κB (NF-κB) p65 by inhibiting the degradation of p50 and IκBα. Furthermore, the phosphorylation of mitogen-activated protein kinase (MAPKs) in LPS-stimulated RAW264.7 cells was significantly inhibited by A2, A6 and B7. These findings suggest that A2, A6 and B7 may operate as an effective anti-inflammatory agent through inhibiting the activation of NF-κB and MAPK signaling pathways in macrophages. Moreover, rat paw swelling experiments showed that these compounds possess anti-inflammatory activity in vivo, with compound A6 exhibiting similar activities to the reference drug Indomethacin.