近日，兰州大学药学院刘映前教授团队与华中科技大学同济药学院徐传瑞教授团队合作，在抗肿瘤新药发现与肿瘤治疗方面取得新进展。相关研究成果“Design and synthesis of Aza-boeravinone derivatives as potential novel topoisomerase I inhibitors”发表于Bioorganic Chemistry期刊（IF：5.275）上。兰州大学药学院2020级硕士研究生周勇和2019级硕士研究生白银鹏为该论文共同第一作者，通讯作者为刘映前教授和徐传瑞教授，兰州大学药学院为该论文的第一单位。

肿瘤是世界上大多数国家的主要死亡原因之一。根据世界卫生组织（WHO）统计，2020年全球估计有1930万新病例的产生和1000万肿瘤病例的死亡，并且全球肿瘤的发病率和死亡率正在迅速增加。因此，研究和开发新的药物和治疗策略以控制肿瘤显得尤为迫切和重要。其中，化疗在肿瘤的治疗中仍然扮演着举足轻重的作用。DNA拓扑异构酶I（Top I）抑制剂是抗肿瘤药物的重要组成部分，在晚期实体瘤和女性癌症中发挥着越来越重要的作用。Top I是真核生物中必需的酶，参与细胞DNA复制、转录、重组和染色体分离等众多过程。多项研究表明，与正常细胞相比，Top I在肿瘤细胞中高度表达并且促进肿瘤生长。因此，Top I已经被证明是抗肿瘤的重要靶点之一。

藏药喜马拉雅紫茉莉为紫茉莉科植物喜马拉雅紫茉莉（Mirabilis himalaica （Edgew.） Heim.）的干燥根。Boeravinones是从喜马拉雅紫茉莉中分离得到的一类天然产物，具有广泛的生物学活性，如抗炎、抗菌、抗氧化、抗病毒和抗肿瘤等。但其自身抗肿瘤谱窄且活性较弱，需进一步进行结构优化或改造而增强活性。因此，基于天然产物Boeravinones的结构骨架，该研究团队采用生物电子等排和“Aza”结构优化策略设计合成了两个系列的6H-色烯并[3,4-b]喹啉衍生物。

Figure 1. Structures of boeravinones from Boerhaavia diffusa and novel structures of 6H-chromeno[3,4-b]quinoline derivatives.

Figure 2. Reagents and conditions: (a) ethyl acetoacetate, AcOH, benzene,reflux; (b) Ph2O, 250 ℃; (c) NBS, AcOH/DCM(1:2), rt; (d) POCl3, reflux; (e)benzoyl peroxide, NBS, CCl4, reflux; (f) NaH, DMF, rt; (g) Pd(OAc)2, PPh3,K2CO3, DMF, 120 ℃; (h) N,N-Dimethylethylenediamine, pridine, 120℃.

作者首先评估了所得化合物对HepG2、A2780、Hela、HCT116、SW1990和MCF7等多种肿瘤细胞株的细胞毒活性。筛选结果发现，化合物ZML-8和ZML-14对HepG2具有显著的抑制效果，IC50值分别为0.58μM和1.94 μM。此外，化合物ZML-8和 ZML-14对HepG2 和 L-02 细胞的选择性高于对照药Topotecan。因此，进一步研究了候选化合物ZML-8和ZML-14的抗肿瘤效果与作用机制。

Table 1. Antiproliferative Activities of Compounds ZML-8, ZML-14, ZML-22, and ZML-23 against Normal Human Liver Cell Line L-02.

通过流式细胞术、Western blotting等实验研究表明，化合物ZML-8 和ZML-14剂量依赖性地诱导HepG2细胞周期G2/M期阻滞。此外，化合物ZML-8和 ZML-14诱导HepG2细胞凋亡并伴随促凋亡蛋白Bax、Bad和Cleaved-caspase3的激活以及抗凋亡蛋白Bcl-2的降低。这些结果表明，化合物ZML-8和 ZML-14诱导Caspase依赖的细胞凋亡。

Figure 4. Compounds ZML-8 and ZML-14 caused G2/M cell cycle arrest of HepG2 cells. (A)Cell cycle distribution HepG2 cells treated with compound ZML-8 and ZML-14 for24 h, Topotecan was used as positive control. Treated cells were then stained with PI/RNase and analyzed using a flow cytometer. (B) Sums of percentages of each cycle were shown as mean ± S.D. for three independent experiments (*P <0.05, **P < 0.01, ***P < 0.001.vs. the control).

Figure 5. Compounds ZML-8 and ZML-14 induced apoptosis of HepG2 cells. (A) Apoptosis in HepG2 cells treated with compound ZML-8 or ZML-14. Cells were exposed to compound ZML-8, ZML-14, or Topotecan for 48 h. The apoptosis rates were then assessed by Annexin V/PI double staining determined by flow cytometry. (B) Apoptosis rates were shown onthe histogram for three independent experiments. Data were shown as mean ± SD.*P < 0.05 or **P < 0.01 or ***P < 0.001 vs. the control.

Figure 6. Western blotting analysis of Caspase-dependent apoptosis proteins in HepG2 cells after compounds ZML-8 and ZML-14 treatment. HepG2 cells were treated with compound ZML-8, ZML-14, or Topotecan for 48 h. The cells were harvested and lysed to detect cleaved Caspase-3, P-53 and Bcl-2 members (Bcl-2 and Bax) using Western blot. α-Tubulin was used as reference. (A) and (B) Representative western blots of proteins in compound ZML-8 or ZML-14 treated cells. (C) and (D) Quantification of western blot. Results are representative of three independent experiments. *p < 0.05,**p < 0.01, and ***p < 0.001 vs control.

根据上述实验结果，化合物ZML-8和ZML-14通过诱导细胞周期阻滞和细胞凋亡来抑制细胞增殖，并且该结果与对照药物Topotecan相似。为此，作者假设化合物ZML-8 和 ZML-14 可能具有与Topotecan相似的作用机制。DNA松弛实验结果表明，化合物ZML-8 和 ZML-14显著抑制Top I的酶活性。化合物ZML-8和ZML-14分别在2.5 μM 和5.0 μM浓度剂量下具有与1.0 μM的 Topotecan抑制效果。此外，Western blotting结果表明，化合物ZML-8 和 ZML-14诱导Top I蛋白水平降解。据报道，抑制 Top I 或捕获 Top1cc 可导致 DNA 损伤。因此，作者进一步检测了DNA损伤的生物标志物γ-H2AX的表达情况。研究发现，化合物ZML-18和ZMl-14处理后γ-H2AX的蛋白表达水平显著增加，表明诱导了DNA损伤。分子对接结果进一步表明，化合物ZML-8和ZML-14可以与Top I-DNA 复合物相互作用，结合能分别为-9.4 kcal/mol和-9.7kcal/mol。

Figure 7. Compounds ZML-8 and ZML-14 down-regulated the Top1 activity and induced DNA damage. (A) The inhibitory activity of compounds ZML-8 and ZML-14 on Top1. Lane 1: relaxed DNA; Lane 2: supercoiled plasmid DNA (pBR322) only; Lane 3: DNA + Top1+DMSO; Lane 4-5: DNA + Top1+ ZML-8; Lane 6-7: DNA + Top1+ ZML-14; Lane 8: DNA + Top1+ Topotecan,were used as positive control. (B) and (C) HepG2 cells were treated with compounds ZML-8 and ZML-14 for 48 h, harvested and lysed for detection of Top1 and γ-H2AX. (D) and (E) Histograms of relative expression of Top1 and γ-H2AX. Data are shown as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control.

Figure 8. Hypothetical binding mode of compounds ZML-8 and ZML-14 in the ternary Top1-DNA-drug complex (PDB ID: 1K4T). Protein is shown as a cartoon, and compounds ZML-8, ZML-14 and Topotecan were shown in stick. Key hydrogen-bonding interactions are indicated with yellow dashes. (A-C) Compounds ZML-8 (pink), ZML-14 (green) and Topotecan (blue) were shown in the ternary complex with a docking energy -9.40, -9.70 and -12.4 kcal/mol, respectively.(D) Overlapping of the binding pose of compounds ZML-8 (pink) and ZML-14(green) with Topotecan (blue).

综上，基于天然产物Boeravinones的结构骨架，团队采用生物电子等排策略设计合成了两个系列的6H-色烯并[3,4-b]喹啉衍生物，并评估了所得化合物对HepG2、A2780、Hela、HCT116、SW1990和MCF7等肿瘤细胞株的细胞毒活性。筛选结果表明，化合物ZML-8和ZML-14对HepG2具有显著的抑制效果，IC50值分别为0.58μM和1.94 μM。此外，化合物ZML-8和ZML-14对HepG2和L-02细胞的选择性高于对照药Topotecan。细胞机制研究表明，化合物ZML-8和ZML-14诱导HepG2细胞周期G2/M期阻滞、细胞凋亡和DNA损伤。此外，化合物ZML-8和ZML-14显著抑制Top I的酶活性，并且诱导Top I蛋白水平的降解。分子对接表明，化合物ZML-8和 ZML-14显著的细胞毒活性是由于与Top1-DNA复合物的良好相互作用。

这项研究的重要进展为后续开发具有完整自主知识产权的抗肿瘤药物奠定了基础。课题组长期致力于天然源抗肿瘤药物的设计合成与创制研究，为临床抗肿瘤药物的开发提供科学依据。研究工作得到了浙江省重点大学优势特色学科开放项目、甘肃省国际科技合作重点项目和国家自然科学基金共同资助。该项研究也是依托甘肃省中藏药功效物质开发与利用行业技术中心，开展源于天然源藏药资源进行新药开发研究取得的又一重要的科研成果，对于推动中藏药功效物质开发与利用具有重要意义。

原文链接：https://doi.org/10.1016/j.bioorg.2022.105747