In the present study, we explored the potential of coumarin-based compounds, known for their potent anticancer properties, by designing and synthesizing a novel category of 8-methoxycoumarin-3-carboxamides. Our aim was to investigate their antiproliferative activity against liver cancer cells. Toward this, we developed a versatile synthetic approach to produce a series of 8-methoxycoumarin-3-carboxamide analogues with meticulous structural features. Assessment of their antiproliferative activity demonstrated their significant inhibitory effects on the growth of HepG2 cells, a widely studied liver cancer cell line. Among screened compounds, compound 5 exhibited the most potent antiproliferative activity among the screened compounds (IC50 = 0.9 µM), outperforming the anticancer drug staurosporine (IC50 = 8.4 µM), while showing minimal impact on normal cells. The flow cytometric analysis revealed that compound 5 induces cell cycle arrest during the G1/S phase and triggers apoptosis in HepG2 cells by increasing the percentage of cells arrested in the G2/M and pre-G1 phases. Annexin V-FITC/PI screening further supported the induction of apoptosis without significant necrosis. Further, compound 5 exhibited the ability to activate caspase3/7 protein and substantially inhibited β-tubulin polymerization activity in HepG2 cells. Finally, molecular modelling analysis further affirmed the high binding affinity of compound 5 toward the active cavity of β-tubulin protein, suggesting its mechanistic involvement. Collectively, our findings highlight the therapeutic potential of the presented class of coumarin analogues, especially compound 5, as promising candidates for the development of effective anti-hepatocellular carcinoma agents.

中文翻译：

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新型 8-Methoxycoumarin-3-Carboxamides 通过靶向 caspase-3/7 和 β-tubulin 聚合对肝癌具有有效的抗癌活性

在本研究中，我们通过设计和合成一类新型 8-甲氧基香豆素-3-甲酰胺，探索了以其有效抗癌特性而闻名的香豆素基化合物的潜力。我们的目的是研究它们对肝癌细胞的抗增殖活性。为此，我们开发了一种多功能合成方法来生产一系列具有精细结构特征的 8-甲氧基香豆素-3-甲酰胺类似物。对它们的抗增殖活性的评估表明它们对 HepG2 细胞（一种广泛研究的肝癌细胞系）的生长具有显着的抑制作用。在筛选的化合物中，化合物5表现出最有效的抗增殖活性（IC50 = 0.9 µM），优于抗癌药物星形孢菌素（IC50 = 8.4 µM），同时对正常细胞的影响最小。流式细胞术分析表明，化合物 5 诱导细胞周期停滞在 G1/S 期，并通过增加停滞在 G2/M 期和前 G1 期的细胞百分比来触发 HepG2 细胞凋亡。膜联蛋白 V-FITC/PI 筛选进一步支持诱导细胞凋亡而无明显坏死。此外，化合物5表现出激活Caspase3/7蛋白的能力并显着抑制HepG2细胞中的β-微管蛋白聚合活性。最后，分子模型分析进一步证实了化合物5对β-微管蛋白活性腔的高结合亲和力，表明其参与机制。总的来说，我们的研究结果强调了所提出的一类香豆素类似物，特别是化合物 5 的治疗潜力，作为开发有效抗肝细胞癌药物的有希望的候选者。