Screening of a library of chemical compounds showed that the dichloropyridine-based analogue 9 was a novel P2X₇ receptor antagonist. To optimize its activity, we assessed the structure–activity relationships (SAR) of 9, focusing on the hydrazide linker, the dichloropyridine skeleton, and the hydrophobic acyl (R₂) group. We found that the hydrazide linker and the 3,5-disubstituted chlorides in the pyridine skeleton were critical for P2X₇ antagonistic activity and that the presence of hydrophobic polycycloalkyl groups at the R₂ position optimized antagonistic activity. In the EtBr uptake assay in hP2X₇-expressing HEK293 cells, the optimized antagonists, 51 and 52, had IC₅₀ values of 4.9 and 13 nM, respectively. The antagonistic effects of 51 and 52 were paralleled by their ability to inhibit the release of the pro-inflammatory cytokine, IL-1β, by LPS/IFN-γ/BzATP stimulation of THP-1 cells (IC₅₀ = 1.3 and 9.2 nM, respectively). In addition, 52 strongly inhibited iNOS/COX-2 expression and NO production in THP-1 cells, further indicating that this compound blocks inflammatory signaling and suggesting that the dichloropyridine analogues may be useful in developing P2X₇ receptor targeted anti-inflammatory agents.

中文翻译：

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新型P2X ₇受体拮抗剂3，5-二氯吡啶衍生物的结构-活性关系和优化

化合物库的筛选显示，基于二氯吡啶的类似物9是新型P2X ₇受体拮抗剂。为了优化其活性，我们评估了9的结构-活性关系（SAR），重点是酰肼连接基，二氯吡啶骨架和疏水性酰基（R ₂）基团。我们发现，酰肼连接基和吡啶骨架中的3,5-二取代的氯化物对于P2X ₇拮抗活性至关重要，并且疏水多环烷基在R ₂位置的存在优化了拮抗活性。在hP2X _7中的EtBr摄取测定中表达HEK293细胞的优化拮抗剂51和52的IC ₅₀值分别为4.9和13 nM。51和52的拮抗作用与其通过TPS-1细胞的LPS /IFN-γ/ BzATP刺激抑制促炎性细胞因子IL-1β释放的能力平行（IC ₅₀ = 1.3和9.2 nM，分别）。另外，有52种药物强烈抑制了THP-1细胞中iNOS / COX-2的表达和NO的产生，进一步表明该化合物阻断了炎症信号传导，并表明二氯吡啶类似物可用于开发靶向P2X ₇受体的抗炎剂。