Background: Most of the structural modifications to evodiamine (EVO) have focused on the 3- and 10-positions, while structural modifications to the EVO 2-position have not yet been reported. In this study, we investigated the scaffold diversity and bioactivity of EVO from position 2 to gain more insight into the influence of the chemical space around EVO on bioactivity. Objective: The study aims to synthesize two derivatives of EVO with hydroxy groups, 8a and 8b, and to investigate the antitumor activity of EVO derivatives with hydroxy groups compared to EVO. Methods: The synthesized compounds were structurally characterized by 1H NMR, 13C NMR, and mass spectrometry. The effects of compounds 8a, 8b, and EVO on the proliferation of H460, A549, and Eca109 cells in vitro were determined by MTT. The effect of EVO, 8a and 8b on apoptosis of H460 cells was investigated by the annexed V-FITC/propidium iodide (PI) combination assay. The expression of EVO, 8a and 8b on apoptosis-related proteins was examined by Western blot analysis. To simulate the binding ability between small molecules and proteins, molecular docking calculations of EGFRWT and EGFRT790M with 8a and 8b, respectively, were performed using Schrödinger software. Results: In the cytotoxicity assay, compound 8b showed lower IC50 values for the three tumor cell lines (6.69 μM for H460 cells, 20.02 μM for A549 cells, and 16.47 μM for Eca109 cells) compared to compound 8a and EVO, and 8b induced apoptosis by affecting apoptosis-related proteins CRAF, AKT, and ERK in a late apoptotic manner. The molecular docking results showed that 8b has a good binding ability to EGFR upstream of apoptosis-related proteins. Conclusion: These findings suggest that 8b has significantly higher antitumor biological activity than EVO and 8a. This antitumor effect has important implications for the study of EVO derivatives in antitumor models.

中文翻译：

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含羟基吴茱萸碱衍生物的设计、合成与评价

背景：吴茱萸碱 (EVO) 的大部分结构修饰都集中在 3 位和 10 位，而对 EVO 2 位的结构修饰尚未见报道。在这项研究中，我们从位置 2 研究了 EVO 的支架多样性和生物活性，以更深入地了解 EVO 周围的化学空间对生物活性的影响。目的：本研究旨在合成两种带羟基的EVO衍生物8a和8b，并与EVO相比，考察带羟基的EVO衍生物的抗肿瘤活性。方法：通过1H NMR、13C NMR和质谱对合成化合物进行结构表征。MTT法测定化合物8a、8b和EVO对H460、A549和Eca109细胞体外增殖的影响。EVO的效果，图 8a 和 8b 对 H460 细胞凋亡的影响通过附带的 V-FITC/碘化丙啶 (PI) 组合测定进行了研究。通过蛋白质印迹分析检查EVO、8a和8b在凋亡相关蛋白上的表达。为了模拟小分子和蛋白质之间的结合能力，EGFRWT 和 EGFRT790M 分别与 8a 和 8b 进行分子对接计算，使用 Schrödinger 软件进行。结果：在细胞毒性试验中，与化合物 8a 和 EVO 相比，化合物 8b 对三种肿瘤细胞系的 IC50 值较低（H460 细胞为 6.69 μM，A549 细胞为 20.02 μM，Eca109 细胞为 16.47 μM），并且 8b 诱导细胞凋亡通过以晚期凋亡方式影响凋亡相关蛋白 CRAF、AKT 和 ERK。分子对接结果表明8b对细胞凋亡相关蛋白上游的EGFR具有良好的结合能力。结论：这些发现表明 8b 的抗肿瘤生物活性明显高于 EVO 和 8a。这种抗肿瘤作用对于 EVO 衍生物在抗肿瘤模型中的研究具有重要意义。