Synthesis and Bioactivity Study of Benzimidazole Derivatives with Fluorescent Self-Labeling and Tubulin Polymerization Inhibition

doi:10.6023/cjoc202407044

Abstract

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

Cite this article

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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Fig. & Tab. 11

Figure 1. Structures of CA-4 and its derivatives

Figure 2. Design of target compounds

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

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

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

Figure 3. Effects of IV-02 on tubulin polymerization

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

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.

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

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)

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

References 21

[1]	Hamel, E.; Lin, C. M. Biochem. Pharmacol. 1983, 32, 3864. pmid: 6661259
[2]	Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A. A. J. Med. Chem. 2006, 49, 3033.
[3]	Cui, Y. J.; Liu, C.; Ma, C. C.; Ji, Y. T.; Yao, Y. L.; Tang, L. Q.; Zhang, C. M.; Wu, J. D.; Liu, Z. P. J. Med. Chem. 2020, 63, 14840.
[4]	Seddigi, Z. S.; Malik, M. S.; Saraswati, A. P.; Ahmed, S. A.; Babalghith, A. O.; Lamfon, H. A.; Kamal, A. MedChemComm 2017, 8, 1592. doi: 10.1039/c7md00227k pmid: 30108870
[5]	Gaspari, R.; Prota, A. E.; Bargsten, K.; Cavalli, A.; Steinmetz, M. O. Chem 2017, 2, 102.
[6]	Pecnard, S.; Hamze, A.; Bignon, J.; Prost, B.; Deroussent, A.; Gallego-Yerga, L.; Peláez, R.; Paik, J. Y.; Diederich, M.; Alami, M.; Provot, O. Eur. J. Med. Chem. 2021, 223, 113656.
[7]	Bukhari, S. N. A.; Kumar, G. B.; Revankar, H. M.; Qin, H. L. Bioorg. Chem. 2017, 72, 130. doi: S0045-2068(17)30128-1 pmid: 28460355
[8]	Shan, Y.; Zhang, J.; Liu, Z.; Wang, M.; Dong, Y. Curr. Med. Chem. 2011, 18, 523. pmid: 21143124
[9]	Gu, Y. F.; Wu, C. J.; Wang, S. Q.; Zhang, S. L.; Lu, Y.; Si, X. X.; Jiang, B. L. Chin. J. Org. Chem. 2024, 44, 3497. (in Chinese)
	(顾一飞, 吴彩菊, 王思琪, 张世琳, 陆远, 司鑫鑫, 蒋佰玲, 有机化学, 2024, 44, 3497.) doi: 10.6023/cjoc202403049
[10]	Kremmidiotis, G.; Leske, A. F.; Lavranos, T. C.; Beaumont, D. Mol. Cancer Ther. 2010, 9, 1562. doi: 10.1158/1535-7163.MCT-09-0815 pmid: 20515948
[11]	Flynn, B. L.; Gill, G. S.; Grobelny, D. W.; Chaplin, J. H.; Paul, D.; Leske, A. F.; Lavranos, T. C.; Chalmers, D. K.; Charman, S. A.; Kostewicz, E. J. Med. Chem. 2011, 54, 6014.
[12]	Wang, Q. H.; Arnst, K. E.; Wang, Y. X.; Kumar, G.; Ma, D. J.; White, S. W.; Miller, D. D.; Li, W. M.; Li, W. J. Med. Chem. 2019, 62, 6734.
[13]	Kashyap, V. K.; Wang, Q. H.; Setua, S.; Nagesh, P. K. B.; Chauhan, N.; Kumari, S.; Chowdhury, P.; Miller, D. D.; Yallapu, M. M.; Li, W.; Jaggi, M.; Hafeez, B. B.; Chauhan, S. C. J. Exp. Clin. Cancer Res. 2019, 38, 29. doi: 10.1186/s13046-018-1009-7 pmid: 30674344
[14]	Lai, Q. H.; Wang, Y. X.; Wang, R. X.; Lai, W. R.; Tang, L. Z.; Tao, Y. R.; Liu, Y.; Zhang, R. R.; Huang, L. Y.; Xiang, H. T.; Zeng, S. X.; Gou, L. T.; Chen, H.; Yao, Y. Q.; Yang, J. L. Eur. J. Med. Chem. 2018, 156, 162.
[15]	Huo, Z.; Liu, K.; Zhang, X.; Liang, Y.; Sun, X. Eur. J. Med. Chem. 2022, 235, 114271.
[16]	Li, G.; Wu, J. Q.; Cai, X. J.; Guan, W.; Zeng, Z. J.; Ou, Y. H.; Wu, X. Y.; Li, J. Y.; Fang, X. X.; Liu, J. L.; Zhang, Y. L.; Wang, H. M.; Yin, C. Q.; Yao, H. L. Eur. J. Med. Chem. 2023, 252, 115284.
[17]	Karetnikov, G. L.; Vasilyeva, L. A.; Babayeva, G.; Pokrovsky, V. S.; Skvortsov, D. A.; Bondarenko, O. B. ACS Pharmacol. Transl. Sci. 2024, 7, 384. doi: 10.1021/acsptsci.3c00239 pmid: 38357282
[18]	Tan, Y. C.; Hu, H.; Zhu, W. J.; Wang, T.; Gao, T.; Wang, H. Q.; Chen, J.; Xu, J. Y.; Xu, S. T.; Zhu, H. J. Eur. J. Med. Chem. 2023, 262, 115881.
[19]	Li, Y.; Ma, H. Q.; Wang, Y. L. Chin. J. Org. Chem. 2008, 28, 210. (in Chinese)
	(李焱, 马会强, 王玉炉, 有机化学, 2008, 28, 210.)
[20]	Santosh, P. C.; Pandeya, S. N.; Pathak, A. K. Int. J. Res. Ayurveda Pharm. 2001, 2, 1726.
[21]	Dong, H. Y. Ph.D. Dissertation, Henan University, Kaifeng, 2024. (in Chinese)
	(冬海洋. 博士论文, 河南大学, 开封, 2024).

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