A series of hybrid anaplastic lymphoma kinase (ALK) inhibitors (Y1∼Y30) were designed by assembling aminoindazole of Entrectinib onto 2-position of 2,4-diarylaminopyrimidine (DAAP) fragment to serve as ATP dual-mimic agents. Under structure-based optimization, all conjugates were detected moderate to excellent cytotoxicity potency, among which the pyrrolidine analog Y28 exerted optimal antiproliferative effects on ALK-addicted cell lines with IC₅₀ values below 20 nM. As a highly potent ALK inhibitor (ALK^WT, IC₅₀ = 1.6 nM), Y28 was also capable of suppressing ALK-resistant mutations including ALK^L1196M (0.71 nM) and ALK^G1202R (1.3 nM). Intriguingly, Y28 turned out to effectively inhibit colony formation and restrain cell migration of H2228 cells in a dose dependent manner. In addition, flow cytometric analysis indicated that Y28 could induce cell apoptosis and achieve cell cycle arrest in G2 phase. Notably, oral administration of Y28 at 50 mg/kg regressed tumor in the H2228 xenograft model with tumor growth inhibition value of 70.46%. Finally, the binding models of Y28 with ALK^WT & ALK^G1202R within the active site well established its mode of action and accounted for the superior activities as a promising antitumor candidate.

通过将恩曲替尼的氨基吲唑组装到 2,4-二芳基氨基嘧啶 (DAAP) 片段的 2 位上，作为 ATP 双重模拟剂，设计了一系列混合间变性淋巴瘤激酶 (ALK) 抑制剂 ( Y1 ∼ Y30 )。在基于结构的优化下，检测到所有偶联物均具有中等至优异的细胞毒性效力，其中吡咯烷类似物Y28对 ALK 成瘾细胞系发挥最佳抗增殖作用，IC ₅₀值低于 20 nM。作为一种高效的 ALK 抑制剂（ALK ^WT，IC ₅₀  = 1.6 nM），Y28还能够抑制 ALK 抗性突变，包括 ALK ^L1196M (0.71 nM) 和 ALK^G1202R (1.3 纳米)。有趣的是，Y28以剂量依赖性方式有效抑制集落形成并抑制 H2228 细胞的细胞迁移。此外，流式细胞仪分析表明，Y28可以诱导细胞凋亡，使细胞周期停滞在G2期。值得注意的是，在 H2228 异种移植模型中，口服 50 mg/kg 的Y28可使肿瘤消退，肿瘤生长抑制值为 70.46%。最后，Y28与 ALK ^WT和 ALK ^G1202R在活性位点内的结合模型很好地确立了其作用模式，并解释了作为有前途的抗肿瘤候选物的优越活性。