<p>Seven-dose screening on SW480 and NCM460 cells.</p>

Colorectal cancer (CRC) remains one of the most prevalent and lethal malignancies worldwide, highlighting the urgent need for developing effective treatments. Molecular hybridization is a promising strategy for identifying new bioactive compounds. This study focused on designing and synthesizing a novel series of benzofuran-pterostilbene hybrid molecules. These compounds were successfully obtained, and their structures were elucidated by spectroscopic analysis. In addition, the activity of the hybrids was evaluated against colorectal adenocarcinoma cells. After the treatments, hybrids 6d and 6e exhibited the highest activity, with GI50 values of 11.93 ± 2.38 µM and 4.74 ± 0.38 µM, respectively, suggesting antiproliferative effects and measurable cytotoxicity under the tested conditions. Additionally, Hoechst 33342 fluorescence imaging revealed chromatin condensation and nuclear fragmentation, along with a diffuse DiOC₆ fluorescence pattern relative to the control, suggesting a form of programmed cell death, which was further supported by flow cytometric analysis showing an increased proportion of hypodiploid cells following propidium iodide staining. In parallel, wound-healing assays demonstrated impaired migration and cytotoxic effects that affected cell viability and structural integrity. Molecular docking simulations showed that compounds 6d and 6e bind strongly to mutant p53, CDK4, and PARP-1 proteins, which, in turn, may explain at the molecular level the in vitro cytotoxic effect of these compounds in SW480 colon cancer cells. Lastly, pharmacokinetic and toxicological modelling suggests that hybrids 6d and 6e possess optimal biopharmaceutical profiles with no major safety concerns. All these findings highlight the potential of the benzofuran-pterostilbene scaffold, with compounds 6d and 6e emerging as strong candidates for further evaluation against colorectal cancer.结直肠癌（CRC）仍是全球范围内发病率最高、致死性最强的恶性肿瘤之一，凸显了开发有效治疗手段的迫切需求。分子杂合是发掘新型生物活性化合物的极具潜力的策略。本研究聚焦于设计并合成一系列新型苯并呋喃-紫檀茋（benzofuran-pterostilbene）杂合分子。该系列化合物均成功获得，并通过光谱分析完成了结构确证。此外，本研究评估了这些杂合分子对结直肠腺癌细胞的抗瘤活性。经处理后，杂合分子6d与6e展现出最高的活性，其半数生长抑制浓度（GI50）分别为11.93±2.38 μM与4.74±0.38 μM，表明在受试条件下二者具备抗增殖活性与可检测的细胞毒性。此外，赫斯特33342（Hoechst 33342）荧光成像结果显示，相较于对照组，受试组出现染色质凝集与核碎裂现象，同时呈现弥散性DiOC₆荧光模式，提示存在程序性细胞死亡；碘化丙啶（propidium iodide）染色后的流式细胞术分析进一步验证了这一结论，结果显示亚二倍体细胞比例显著升高。与此同时，划痕愈合实验结果显示，受试细胞的迁移能力受损，且存在影响细胞活力与结构完整性的细胞毒性效应。分子对接模拟结果表明，化合物6d与6e可与突变型p53、细胞周期蛋白依赖性激酶4（CDK4）以及多聚ADP核糖聚合酶1（PARP-1）紧密结合，这可在分子层面解释二者在SW480结肠癌细胞中展现出的体外细胞毒性效应。最后，药代动力学与毒理学建模分析显示，杂合分子6d与6e具备最优的生物制药学特性，且无显著安全隐患。上述所有研究结果均凸显了苯并呋喃-紫檀茋（benzofuran-pterostilbene）骨架的开发潜力，化合物6d与6e也成为结直肠癌后续抗瘤活性评估的优质候选化合物。