Through structural optimization and ring fusion strategy, we designed a series of novel imidazo[1,2–a]pyrazine derivatives as potential tubulin inhibitors. These compounds displayed potent anti-proliferative activities (micromolar to nanomolar) against a panel of cancer cell lines (including HepG-2, HCT-116, A549 and MDA-MB-231 cells). Among them, compound TB-25 exhibited the strongest inhibitory effects against HCT-116 cells with an IC₅₀ of 23 nM. Mechanism studies revealed that TB-25 could effectively inhibit tubulin polymerization in vitro, and destroy the dynamic equilibrium of microtubules in HCT-116 cells. In addition, TB-25 dose-dependently induced G2/M phase cell cycle arrest and apoptosis in HCT-116 cells. Furthermore, TB-25 suppressed HCT-116 cell migration in a concentration-dependent manner. Finally, molecular docking showed that TB-25 fitted well in the colchicine binding site of tubulin and overlapped nicely with CA-4. Collectively, these results suggest that TB-25 represents a promising tubulin inhibitor deserving further investigation.

通过结构优化和环融合策略，我们设计了一系列新型咪唑并[1,2-a]吡嗪衍生物作为潜在的微管蛋白抑制剂。这些化合物对一组癌细胞系（包括 HepG-2、HCT-116、A549 和 MDA-MB-231 细胞）显示出有效的抗增殖活性（微摩尔到纳摩尔）。其中化合物TB-25对HCT-116细胞的抑制作用最强，IC ₅₀为23 nM。作用机制研究表明，TB-25可有效抑制体外微管蛋白聚合，破坏HCT-116细胞微管的动态平衡。此外，TB-25剂量依赖性地诱导 HCT-116 细胞的 G2/M 期细胞周期停滞和细胞凋亡。此外，TB-25以浓度依赖性方式抑制 HCT-116 细胞迁移。最后，分子对接显示TB-25与微管蛋白的秋水仙碱结合位点吻合良好，并与 CA-4 很好地重叠。总的来说，这些结果表明TB-25代表了一种有前途的微管蛋白抑制剂，值得进一步研究。