Abstract：
Thirty-five trifluoromethyl hydrazones and seventeen trifluoromethyl oxime esters were designed and synthesized via molecular hybridization. All the target compounds were initially screened for in vitro anti-inflammatory activity by assessing their inhibitory effect on NO release in LPS-stimulated RAW264.7 cells, and the optimal compound was finally identified as 2-(3-Methoxyphenyl)-N\'-((6Z,9Z,12Z,15Z)-1,1,1-trifluorohenicosa-6,9,12,15-tetraen-2-ylidene)acetohydrazide (F26, IC50 = 4.55 ± 0.92 μM) with no cytotoxicity. Moreover, F26 potently reduced the production of PGE2 in LPS-stimulated RAW264.7 cells compared to indomethacin. The interaction of F26 with COX-2 and cPLA2 was directly verified by the CETSA technique. F26 was found to modulate the phosphorylation levels of p38 MAPK and NF-κB p65, as well as the protein expression of IκB, cPLA2, COX-2, and iNOS in LPS-stimulated rat peritoneal macrophages. Additionally, F26 was observed to prevent the nuclear translocation of NF-κB p65 in LPS-stimulated rat peritoneal macrophages by immunofluorescence localization. Therefore, the aforementioned in vitro experiments demonstrated that F26 blocked the p38 MAPK and NF-κB pathways by binding to COX-2 and cPLA2. In the adjuvant-induced arthritis model, F26 demonstrated a significant effect in preventing arthritis symptoms and inflammatory status in rats, exerting an immunomodulatory role by regulating the homeostasis between Th17 and Treg through inhibition of the p38 MAPK/cPLA2/COX-2/PGE2 and NF-κB pathways. Encouragingly, F26 caused less acute ulcerogenicity in rats at a dose of 50 mg/kg compared to indomethacin. Overall, F26 is a promising candidate worthy of further investigation for treating inflammation and associated pain with lesser gastrointestinal irritation, as well as other symptoms in which cPLA2 and COX-2 are implicated in the pathophysiology.
摘要：
通过分子杂交设计并合成了35个三氟甲基腙和17个三氟甲基肟酯。通过评估其对LPS刺激的RAW264.7细胞中NO释放的抑制作用，初步筛选了所有目标化合物的体外抗炎活性。最终确定最佳化合物为2-（3-甲氧基苯基）-N'-（6Z，9Z,12Z,15Z）-1,1,1-三氟苯基-6,9,12,15-四烯-2-亚基)乙酰酰肼（F26，IC50=4.55±0.92μM），无细胞毒性。此外，与吲哚美辛相比，F26有效降低了LPS刺激的RAW264.7细胞中PGE2的产生。通过CETSA技术直接验证了F26与COX-2和cPLA2的相互作用。发现F26可以调节p38MAPK和NF-κBp65的磷酸化水平以及IκB的蛋白表达，LPS刺激的大鼠腹膜巨噬细胞中的cPLA2，COX-2和iNOS。此外,通过免疫荧光定位观察到F26可以防止LPS刺激的大鼠腹膜巨噬细胞中NF-κBp65的核易位。因此,上述体外实验证明F26通过结合COX-2和cPLA2阻断p38MAPK和NF-κB通路。在佐剂诱导的关节炎模型中，F26在预防大鼠关节炎症状和炎症状态方面表现出显着的作用，通过抑制p38MAPK/cPLA2/COX-2/PGE2和NF-κB途径调节Th17和Treg之间的稳态发挥免疫调节作用。令人鼓舞的是，与吲哚美辛相比，F26在50mg/kg的剂量下在大鼠中引起的急性溃疡性减少。总的来说,F26是一个有希望的候选人，值得进一步研究，用于治疗炎症和相关疼痛，胃肠道刺激较小，以及cPLA2和COX-2与病理生理学有关的其他症状。
