N-Benzylated 5-Hydroxybenzothiophene-2-carboxamides as Multi-Targeted Clk/Dyrk Inhibitors and Potential Anticancer Agents

Abstract

Numerous studies have reported that Dyrk1A, Dyrk1B, and Clk1 are overexpressed in multiple cancers, suggesting a role in malignant disease. Here, we introduce a novel class of group-selective kinase inhibitors targeting Dyrk1A, Dyrk1B, and Clk1. This was achieved by modifying our earlier selective Clk1 inhibitors, which were based on the 5-methoxybenzothiophene-2-carboxamide scaffold. By incorporating a 5-hydroxy group, we increased the potential for additional hydrogen bond interactions that broadened the inhibitory effect to include Dyrk1A and Dyrk1B kinases. Within this series, compounds 12 and 17 emerged as the most potent multi-kinase inhibitors against Dyrk1A, Dyrk1B, and Clk1. Furthermore, when assessed against the most closely related kinases also implicated in cancer, the frontrunner compounds revealed additional inhibitory activity against Haspin and Clk2. Compounds 12 and 17 displayed high potency across various cancer cell lines with minimal effect on non-tumor cells. By examining the effect of these inhibitors on cell cycle distribution, compound 17 retained cells in the G2/M phase and induced apoptosis. Compounds 12 and 17 could also increase levels of cleaved caspase-3 and Bax, while decreasing the expression of the antiapoptotic Bcl-2 protein. These findings support the further study and development of these compounds as novel anticancer therapeutics.

Keywords: 5-hyrdoxybenzothiophene; Clk1; Dyrk1A; apoptosis induction; cell cycle analysis; multi-targeting.

Figure 1

Design of a novel, group-selective scaffold by combining the hydroxybenzothiophene motif from our previous Dyrk1A/Clk1 inhibitors (i) and (ii) with the benzylamide moiety from our Clk1-selective scaffold (iii).

Scheme 1

Reagents and conditions: (i) 1 equiv. of 3-hydroxybenzaldehyde, glacial CH₃COOH, 0.15 equiv. anhydrous CH₃COONa, toluene, 170 °C, reflux, 5 h; (ii) 15% NaOH, reflux, 1 h; (iii) 1.5 equiv. of iodine, THF, reflux, 2 days; (iv) 2 equiv. of EDC, 2 equiv. of DMAP, 1.2 equiv. of the corresponding amine, DMF, room temperature, overnight.

Figure 2

Analysis of cell cycle progression upon treatment with compounds 12 and 17. T24 cells were treated with DMSO (0.1%), 12 (30–50 µM), or 17 (30–50 µM) for 24 h. Cell cycle was examined by propidium iodide (PI) staining and flow cytometry. All data are expressed as the mean ± S.E.M. (n = 3).

Figure 3

Annexin V and PI staining in T24 cells to investigate if compounds 12 and 17 could induce apoptotic death. T24 cells were treated with DMSO (0.1%), 12 (30–50 µM), or 17 (30–50 µM) for 24 h and then stained with Annexin V-FITC antibody and PI. The Annexin V-positive apoptotic cells were monitored using flow cytometry. All data are expressed as the mean ± S.E.M. (n = 3). * p < 0.05, ** p < 0.01 compared with the DMSO group.

Figure 4

Examining the effect of 12 and 17 on proapoptotic and antiapoptotic pathways. T24 cells were treated with DMSO (0.1%), 12 (30–50 µM), or 17 (30–50 µM) for 24 h. Expression levels of cleaved caspase-3, Bax, Bcl-2, and GAPDH were analyzed by Western blot by corresponding antibodies. The quantitation of the cleaved caspase-3/GAPDH, Bax/GAPDH, and Bcl-2/GAPDH ratio are shown. All data are expressed as the mean ± S.E.M. (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the DMSO group.