兼具荧光自标记和微管蛋白聚合抑制作用的苯并咪唑衍生物的合成及生物活性研究

doi:10.6023/cjoc202407044

摘要/Abstract

以天然产物Combretastatin A-4 (CA-4)为先导化合物, 采用理性药物设计策略, 设计并合成了一系列新型苯并咪唑衍生物. 通过目标化合物对HT-29(人结肠癌细胞)的增殖抑制活性筛选, 发现IV-02具有明显生物活性. 在多种肿瘤细胞上测试其抗肿瘤生物活性, 实验结果显示, IV-02对A549细胞的增殖抑制IC₅₀为0.18 µmol/L, 与阳性对照秋水仙碱(IC₅₀=0.15 µmol/L)相当. 生物活性研究表明, IV-02能够抑制微管蛋白聚合, 诱导G2/M期细胞周期阻滞, 并导致细胞凋亡. 同时, 化合物IV-02具有荧光发射效应, 能够实时成像标记化合物在细胞内的动态过程, 表明IV-02是一种具荧光自标记功能的抗肿瘤化合物.

关键词: 苯并咪唑, 微管蛋白, 抗肿瘤作用, 荧光发射

A series of novel Combretastatin A-4-based benzimidazole derivatives were designed and synthesized according to rational drug design strategies. The antiproliferation activities were preliminarily evaluated against cancer cell line HT-29, and compound IV-02 stood out from the target compounds and exhibited significant antiproliferation potency. Further evaluation on multiple cancer cell lines such as HT-1080, HepG2, A549 and A549/Taxol cells, demonstrated that compound IV-02 showed significant cell growth inhibition on A549 cells with IC₅₀ of 0.18 µmol/L, which was comparable to that of positive control colchicine (IC₅₀=0.15 µmol/L). Further mechanistic studies demonstrated that IV-02 could inhibit tubulin polymerization in a concentration-dependent manner, cause cell cycle arrest in G2/M phase and induce the cell apoptosis. In addition, IV-02 exhibited fluorescence emission effect, which would be beneficial to label the intracellular dynamic changes of IV-02 in real time. The results indicated that compound IV-02 could serve as an anticancer compound with fluorescent self-labeling properties.

Key words: benzimidazole, tubulin, antitumor effect, fluorescence emission

引用此文

冬海洋, 蒲佳欣, 李珂珂, 张昊文, 耿晓晴, 梁停停, 王建红. 兼具荧光自标记和微管蛋白聚合抑制作用的苯并咪唑衍生物的合成及生物活性研究[J]. 有机化学, 2025, 45(4): 1315-1324.

Haiyang Dong, Jiaxin Pu, Keke Li, Haowen Zhang, Xiaoqing Geng, Tingting Liang, Jianhong Wang. Synthesis and Bioactivity Study of Benzimidazole Derivatives with Fluorescent Self-Labeling and Tubulin Polymerization Inhibition[J]. Chinese Journal of Organic Chemistry, 2025, 45(4): 1315-1324.

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图/表 11

图1. CA-4 及其衍生物的结构

Figure 1. Structures of CA-4 and its derivatives

图2. 目标化合物的设计

Figure 2. Design of target compounds

图式1. 目标化合物IV-01~IV-17的合成路线

Scheme 1. Synthesis of target compounds IV-01~IV-17

Compd.	R¹	R²	Inhibition rate/%
Compd.	R¹	R²	5 µmol/L	10 µmol/L
IV-01	3,4,5-(OMe)₃	2-OH-4-OMe	40.08	65.38
IV-02	3,4,5-(OMe)₃	2-OH-3-OMe	70.81	72.70
IV-03	3,4,5-(OMe)₃	2-OH-5-OMe	37.73	37.56
IV-04	3,4,5-(OMe)₃	2-OH	31.87	34.03
IV-05	3,4,5-(OMe)₃	H	36.53	41.95
IV-06	3,4,5-(OMe)₃	4-OMe	34.37	33.79
IV-07	4-OMe	2-OH-5-OMe	38.05	34.42
IV-08	4-OMe	2-OH-4-OMe	36.58	38.45
IV-09	4-OMe	2-OH-3-OMe	29.22	25.67
IV-10	4-OMe	2-OH	34.96	34.79
IV-11	4-OMe	H	41.04	32.92
IV-12	4-OMe	4-OMe	37.26	38.38
IV-13	3,4-(OMe)₂	2-OH-4-OMe	37.91	33.03
IV-14	3,4-(OMe)₂	2-OH-5-OMe	31.47	30.96
IV-15	3,4-(OMe)₂	2-OH	30.01	33.86
IV-16	3,4-(OMe)₂	4-OMe	34.85	32.03
IV-17	3,4-(OMe)₂	H	39.02	40.49
Paclitaxel^b	—	—	—	1.43

表1. 目标化合物IV-01~IV-17 (5, 10 μmol/L)对HT-29细胞的增殖抑制率a

Table 1. Inhibition rates of target compounds IV-01~IV-17 (5, 10 μmol/L) against HT-29 cells

Compd.	IC₅₀^b/(µmol•L^–1)
Compd.	HT-29	HT-1080	HepG2	A549	A549/Taxol
IV-02	8.56±4.14	2.34±1.01	1.10±0.30	0.18±0.08	0.39±0.04

表2. 化合物IV-02对各种癌细胞的体外抗增殖作用a

Table 2. In vivo antiproliferation effects of compound IV-02 on cancer cell lines

图3. 化合物IV-02对微管蛋白聚合的影响

Figure 3. Effects of IV-02 on tubulin polymerization

图4. 化合物IV-02对A549细胞内微管蛋白聚合的影响

Figure 4. Effects of compound IV-02 on tubulin polymerization in A549 cells

图5. 化合物IV-02对A549细胞周期的影响

Figure 5. Effects of compound IV-02 on the cell cycle of A549 cells (A) G2/M phase arrest of A549 cells induced by compound IV-02; (B) Histogram indicated the percentage of cell cycle distribution induced by IV-02 in different concentrations.

图6. 化合物IV-02对A549细胞凋亡的影响

Figure 6. Effects of compound IV-02 on the cell apoptosis of A549 cells (A) Apoptotic A549 cells stained with Annexin V-FITC and PI after incubation of A549 cells with compound IV-02 for 48 h; (B) Histogram indicated the percentages of apoptotic A549 cells induced by IV-02

图7. 化合物IV-02在PBS、DMF、DMF/PBS (V∶V=1∶1) (A)和PBS、DMSO、DMSO/PBS (V∶V=1∶1) (B)的荧光发射光谱图(λex=320 nm, λem=386, 460 nm, 激发/发射狭缝宽度: 2/2 nm)

Figure 7. Fluorescence spectra of compound IV-02 in PBS, DMF, DMF/PBS (V∶V=1∶1) (A), and PBS, DMSO, DMSO/PBS (V∶ V=1∶1) (B) (λex=320 nm, λem=386, 460 nm, excitation/emission slit: 2/2 nm)

图8. 化合物IV-02在HepG2细胞中的荧光成像

Figure 8. Fluorescence images of IV-02 in HepG2 cells (λex=405 nm, λem=450~560 nm)

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