新型氨基糖类席夫碱铜配合物的合成及抗肿瘤活性研究

doi:10.6023/A25090314

摘要/Abstract

将生物活性相关的有机部分引入过渡金属配合物的配位领域已成为提高金属药物选择性和生物相容性的有效策略. 本工作设计并合成了2种具有叠氮乙酰化葡萄糖糖胺席夫碱结构的配体L¹和L², 进而与铜金属盐反应合成了3种新型铜配合物. 通过X射线单晶衍射技术解析了配体L¹和配合物3的单晶结构, 通过噻唑蓝(MTT)法测定了化合物对人恶性肿瘤细胞系和正常细胞系的体外细胞毒性. 其中配合物2对肝癌细胞(HepG-2)和宫颈癌细胞(HeLa)细胞表现出高的细胞毒性, 接近或优于阳性对照的值, 但对正常细胞的毒性则远低于阳性对照. 对肿瘤细胞毒性较强的配合物 2 进行了后续抗肿瘤活性研究, 包括细胞凋亡评估、细胞内活性氧生成、线粒体膜电位、细胞周期、细胞摄取、迁移和侵袭及血管生成实验, 进一步证实了其对肿瘤细胞的抑制作用机制. 由于糖骨架在配体中具有优异的水溶性, 使得配合物2表现出良好的溶解性和稳定性. 通过细胞内活性氧(ROS)产生、赫斯特(Hoechst 33342)染色实验、线粒体膜电位实验和膜联蛋白Ⅴ-红色荧光蛋白(Annexin V-mCherry)进一步证明, 配合物2能提高癌症细胞内活性氧的水平, 通过线粒体途径诱导细胞凋亡. 细胞周期实验证明配合物2可以延迟或抑制细胞周期通过S期和G2/M期的进展. 另外, 配合物2能有效抑制癌症细胞的迁移和侵袭, 且有效抑制正常细胞系脐静脉内皮细胞(HUVEC)的血管生成, 分子对接结果显示配合物2与血管内皮细胞生长因子受体2 (VEGFR-2)具有良好的结合能力.

关键词: 晶体结构, 氨基葡萄糖, 细胞凋亡, 细胞摄取, 迁移和侵袭

Introducing biologically active organic moieties into the coordination field of transition metal complexes has become an effective strategy for improving the selectivity and biocompatibility of metal drugs. In this paper, two ligands L¹and L² with azidoacetylated glucosamine Schiff base structure were designed and synthesized, and then reacted with copper metal salts to synthesize three novel copper complexes. The single crystal structures of ligand L¹and complex 3 were analyzed by X-ray single crystal diffraction technology, and the in vitro cytotoxicity of the compounds on human malignant tumor cell lines and normal cell lines was determined by methyl thiazolyl tetrazolium (MTT) assay. Among them, complex 2 showed high cytotoxicity against HepG-2 and HeLa cells, approaching or exceeding the value of the positive control, but the toxicity to normal cells was much lower than that of the positive control. The subsequent anti-tumor activity studies of complex 2 with strong cytotoxicity to tumor cells, including apoptosis assessment, intracellular reactive oxygen species generation, mitochondrial membrane potential, cell cycle, cell uptake, migration and invasion, and angiogenesis experiments, further confirmed its inhibitory mechanism on tumor cells. Due to the excellent water solubility of the sugar skeleton in the ligand, complex 2 exhibited good solubility and stability. We further demonstrated through intracellular reactive oxygen species (ROS) production, Hoechst 33342 staining assay, mitochondrial membrane potential assay and Annexin V-mCherry assay that complex 2 can increase the level of ROS in cancer cells and induce apoptosis through the mitochondrial pathway. Cell cycle experiments have shown that complex 2 can delay or inhibit the progression of the cell cycle through the S and G2/M phases. The significant cell uptake results indicated that the high cytotoxicity of complex 2 might be due to its extensive accumulation in HepG-2 cells. In addition, complex 2 can effectively inhibit the migration and invasion of cancer cells, as well as the angiogenesis of normal cell line umbilical vein endothelial cells (HUVEC). Molecular docking results showed that complex 2 has good binding ability with vascular endothelial growth factor receptor 2 (VEGFR-2).

Key words: crystal structures, glucosamine, apoptosis, cellular uptake, migration and invasion

引用此文

高春艳, 崔馨月, 王凯月, 石雪怡, 贺卿, 赵晋忠, 岳爱琴, 杜维俊, 张永坡. 新型氨基糖类席夫碱铜配合物的合成及抗肿瘤活性研究[J]. 化学学报, 2026, 84(3): 353-363.

Gao Chunyan, Cui Xinyue, Wang Kaiyue, Shi Xueyi, He Qing, Zhao Jinzhong, Yue Aiqin, Du Weijun, Zhang Yongpo. Synthesis and Antitumor Activity of Novel Amino Glucose Derivative Schiff Base Copper(II) Complexes[J]. Acta Chimica Sinica, 2026, 84(3): 353-363.

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图/表 13

图式1. 配体L1、L2和铜配合物1~3的合成路线

Scheme 1. Synthesis route of ligands L1, L2 and Cu(II) complexes 1~3

图1. L1和配合物3的晶体结构图. 为了清晰可见, 所有的氢原子及外界甲醇和水溶剂已省略

Figure 1. Crystal structure diagrams of L1 and complex 3, all hydrogen atoms and external methanol and water solvents were omitted for clarity

Compound	IC₅₀^a/(μmol•L⁻¹)
Compound	HeLa	NCI-H460	BGC-823	HepG-2	HUVEC
Oxaliplatin	48.48±0.22	35.10±0.74	9.16±0.19	12.21±3.17	10.64±3.68
1	29.96±3.11	28.88±4.49	26.34±4.54	28.72±1.98	23.15±2.09
2	22.36±3.96	31.26±0.78	19.20±2.47	13.59±1.00	20.08±1.45
3	48.88±1.12	44.43±2.41	44.83±2.12	30.54±1.24	72.436
L¹	>100	>100	>100	>100	—
L²	>100	>100	>100	>100	—
CuBr₂	161.5	106.0	84.7	117.7	—
CuCl₂	142.0	106.8	88.9	102.3	—

表1. 化合物作用测试细胞系48 h的IC50值

Table 1. IC50 values of the compounds against the tested cell lines for 48 h

图2. 作用时间分别在24、48、72 h时, 不同浓度配合物2作用HeLa (a)和HepG-2 (b)细胞毒性的影响

Figure 2. The effect of time on the cytotoxicity of the complex 2 against HeLa (a) and HepG-2 (b) cells for 24, 48 and 72 h, respectively

图3. Hoechst 33342染色检测配合物2 (20 μmol/L)对HeLa和HepG-2细胞形态24 h的影响以及相对荧光强度的光密度值(10×10). 注: 数据表示为三组独立实验的平均值±标准差. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=非显著性差异(non-significant)

Figure 3. Hoechst 33342 staining to detect the effect of complex 2 (20 μmol/L) on the morphology of HeLa and HepG-2 cells for 24 h and the optical density value of the relative fluorescence intensity (10×10). Note: Data are expressed as mean ± standard deviation of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=non-significant

图4. DCFH-DA探针染色, 测定配合物2对HeLa、HepG-2细胞24 h ROS产生的影响, 以及相对荧光强度的光密度值(10×10)

Figure 4. DCFH-DA probe staining to determine the effect of complex 2 on ROS production in HeLa, HepG-2 cells for 24 h, and the optical density value of the relative fluorescence intensity (10×10)

图5. JC-1测定配合物2对HeLa、HepG-2细胞线粒体膜电位24 h的影响, 及相对荧光强度的光密度值(10×10) (a: HeLa细胞, b: HepG-2细胞)

Figure 5. The effect of complex 2 on mitochondrial membrane potentials of HeLa, HepG-2 cells for 24 h were determined with JC-1 and the optical density value of the relative fluorescence intensity (10×10) (a: HeLa cells, b: HepG-2 cells)

图6. Annexin V-mCherry染色检测配合物2诱导的HepG-2细胞凋亡

Figure 6. Annexin V-mCherry staining detects complex 2 induced apoptosis in HepG-2 cells

图7. 流式细胞术检测配合物2对HepG-2细胞作用24 h细胞周期的影响

Figure 7. Flow cytometry detection of the effect of complex 2 on HepG-2 cell cycle for 24 h

图8. 配合物2作用HepG-2细胞24 h后铜摄取柱状图, **p<0.01 (左); 铜摄取统计数据(右)

Figure 8. Histogram of copper uptake in HepG-2 cells treated with complex 2 for 24 h, **p<0.01 (left); Statistical data of copper uptake (right)

图9. 配合物2对HeLa和HepG-2细胞迁移和侵袭的影响以及通过腔室的细胞数量(10×10). 注: 数据表示为三组独立实验的平均值±标准差. **p<0.01, ##p<0.01 (a: HeLa细胞, 20 μmol/L Oxaliplatin, 20 μmol/L 2; b: HepG-2细胞, 15 μmol/L Oxaliplatin, 15 μmol/L 2)

Figure 9. The effect of complex 2 on HeLa and HepG-2 cells migration and invasion and the number of cells passing through the chamber (10×10). Note: The data is represented as mean ± standard deviation of three independent experiments. **p<0.01, ##p<0.01 (a: HeLa cells, 20 μmol/L Oxaliplatin, 20 μmol/L 2; b: HepG-2 cells, 15 μmol/L Oxaliplatin, 15 μmol/L 2)

图10. 配合物2 (20 μmol/L)对HUVEC细胞血管生成的影响(10×4), 血管分支数量和血管总长度的统计. 注: 数据表示为三组独立实验的平均值±标准差. *p<0.05, **p<0.01

Figure 10. The effect of complex 2 (20 μmol/L) on the angiogenesis of HUVEC cells and the number of vascular branches and the total length of vessels (10×4). Note: Data are expressed as mean ± standard deviation of three independent experiments. *p<0.05, **p<0.01

图11. 配合物2与VEGFR2结合的分子对接图

Figure 11. Docking analysis visualization of VEGFR2 binding with complex 2

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