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.

中文翻译：

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四氢苯并[4,5]噻吩并[2,3-d]嘧啶衍生物作为抗炎药的设计、合成和生物活性

我们设计并合成了 26 种原型化合物，并研究了它们的抗炎活性和潜在的分子机制。这些化合物对脂多糖 (LPS) 刺激的巨噬细胞中一氧化氮 (NO)、细胞因子、炎症相关蛋白和 mRNA 的产生的抑制作用通过 Griess 试验、酶联免疫吸附试验 (ELISA)、蛋白质印迹法测定分析和逆转录聚合酶链反应 (RT-PCR) 分别进行。我们的结果表明，用 A2、A6 和 B7 处理显着抑制了 RAW264.7 细胞中 NO 和炎性细胞因子的分泌，而没有明显的细胞毒性。还发现A2、A6和B7强烈抑制诱导型一氧化氮合酶（iNOS）和环氧合酶COX-2的表达，并通过抑制 p50 和 IκBα 的降解来阻止核因子 κB (NF-κB) p65 的核易位。此外，A2、A6 和 B7 显着抑制了 LPS 刺激的 RAW264.7 细胞中丝裂原活化蛋白激酶 (MAPK) 的磷酸化。这些发现表明 A2、A6 和 B7 可能通过抑制巨噬细胞中 NF-κB 和 MAPK 信号通路的激活而起到有效的抗炎作用。此外，大鼠爪子肿胀实验表明，这些化合物具有体内抗炎活性，化合物 A6 表现出与参考药物吲哚美辛相似的活性。7细胞被A2、A6和B7显着抑制。这些发现表明 A2、A6 和 B7 可能通过抑制巨噬细胞中 NF-κB 和 MAPK 信号通路的激活而起到有效的抗炎作用。此外，大鼠爪子肿胀实验表明，这些化合物具有体内抗炎活性，化合物 A6 表现出与参考药物吲哚美辛相似的活性。7细胞被A2、A6和B7显着抑制。这些发现表明 A2、A6 和 B7 可能通过抑制巨噬细胞中 NF-κB 和 MAPK 信号通路的激活而起到有效的抗炎作用。此外，大鼠爪子肿胀实验表明，这些化合物具有体内抗炎活性，化合物 A6 表现出与参考药物吲哚美辛相似的活性。