P2X7 receptor (P2X7R) has a key role in different pathological conditions, importantly overexpressed and activated in cancers. We explored the structure activity relationship (SAR) of three novel pyrazines, quinoline-carboxamide and oxadiazole series. Their selective inhibitory potency in Ca²⁺ mobilization assay using h-P2X7R-MCF-7 cells improved with phenyl ring substitutions (–OCF₃, –CF₃, and –CH₃) in carboxamide and oxadiazole derivatives, respectively. However, highly electronegative fluoro, chloro, and iodo substitutions enhanced affinity. 1e, 2f, 2e, 1d, 2 g and 3e were most potent and selective toward h-P2X7R (IC₅₀ values 0.457, 0.566, 0.624, 0.682, 0.813 and 0.890 µM, respectively) and were inactive at h-P2X4R, h-P2X₂R, r-P2Y₆R, h-P2Y₂R, t-P2Y₁R expressed in MCF-7 and 1321N1 astrocytoma cells. Cell viability (MTT assay at 100 µM, cell line) for 3e was 62% (HEK-293T), 70% (1321N1 astrocytoma) and 85% (MCF-7). >75% cell viability was noted for 2 g and >80% for 2e and 1d in all non-transfected cell lines. Anti-proliferative effects, compared to control (Bz-ATP), of selective antagonists (10 µM) were 3e (11%) 1d, (19%) 1e, (70%, P = 0.005) and 2f, (24%), indicating involvement of P2X7R. Apoptotic cell death by flow cytometry showed 1e to be most promising, with 35% cell death (PI positive cells), followed by 2e (25%), 2f (20%), and 1d (19%), compared to control. Fluorescence microscopic analysis of apoptotic changes in P2X7R-transfected cell lines was established. 1e and 2f at 1X and 2X IC₅₀ increased cellular shrinkage, nuclear condensation and PI/DAPI fluorescence. In-silico antagonist modeling predicted ligand receptor interactions, and all compounds obeyed Lipinski rules. These results suggest that pyrazine, quinoline-carboxamide and oxadiazole derivatives could be moderately potent P2X7R antagonists for in vivo studies and anti-cancer drug development.

P2X7 受体 (P2X7R) 在不同的病理状况中发挥着关键作用，重要的是在癌症中过度表达和激活。我们探索了三种新型吡嗪、喹啉甲酰胺和恶二唑系列的构效关系（SAR）。它们在使用h -P2X7R-MCF-7 细胞的 Ca ²⁺动员测定中的选择性抑制效力分别通过甲酰胺和恶二唑衍生物中的苯环取代（–OCF ₃、–CF ₃和 –CH ₃）得到改善。然而，高负电性的氟、氯和碘取代增强了亲和力。1e、2f、2e、1d、2 g和3e对h -P2X7R最具效力和选择性（IC ₅₀值分别为 0.457、0.566、0.624、0.682、0.813 和 0.890 µM），并且对h -P2X4R、h -P2X4R 无活性。 P2X ₂ R、r-P2Y ₆ R、h -P2Y ₂ R、t -P2Y ₁ R 在 MCF-7 和 1321N1 星形细胞瘤细胞中表达。3e 的细胞活力（MTT 测定，100 µM，细胞系）为 62% (HEK-293T)、70% (1321N1 星形细胞瘤) 和 85% (MCF-7)。在所有未转染的细胞系中，2 g 的细胞活力 >75% ， 2e和1d的细胞活力 >80% 。与对照 (Bz-ATP) 相比，选择性拮抗剂 (10 µM) 的抗增殖作用分别为3e (11%) 1d、(19%) 1e、(70%，P  = 0.005) 和2f 、(24%) ，表明 P2X7R 的参与。流式细胞术显示，与对照相比， 1e最有希望发生细胞凋亡，细胞死亡率为 35%（PI 阳性细胞），其次是2e (25%)、2f (20%) 和1d (19%)。建立了 P2X7R 转染细胞系中细胞凋亡变化的荧光显微镜分析。1e和2f在 1X 和 2X IC ₅₀下细胞收缩、核凝结和 PI/DAPI 荧光增加。计算机模拟拮抗剂模型预测了配体受体的相互作用，并且所有化合物都遵循利平斯基规则。这些结果表明，吡嗪、喹啉甲酰胺和恶二唑衍生物可能是用于体内研究和抗癌药物开发的中等效力的 P2X7R 拮抗剂。