Design and synthesis of 2-amino-4-(trifluoromethyl)pyrimidine derivatives as potential Werner-dependent antiproliferative agents.

To uncover novel inhibitors of Werner (WRN) helicase, this study adopted the scaffold-hopping strategy to design and synthesize 24 novel 2-amino-4-(trifluoromethyl)pyrimidine derivatives. The MTT assay was employed to evaluate the anticancer activity of target compounds against microsatellite instability-high (MSI-H) cell lines (HCT116 and LNCaP) and microsatellite stability (MSS) cell lines (SW620 and PC3). Some compounds demonstrated significant inhibitory activity against all four cancer cell lines. Specifically, compounds 11c, 11f, 11 g, 11 h, and 11 l exhibited greater inhibitory effect toward MSI-H cells (HCT116 and LNCaP) compared to MSS cells (SW620 and PC3). The most active compound 11 g exhibited excellent cellular selectivity, with IC₅₀ values of 1.52 and 1.72 μM against MSI-H cell lines (HCT116 and LNCaP), respectively, while the IC₅₀ values of 11 g against MSS cell lines (SW620 and PC3) were 4.24 and 2.78 μM, respectively, followed by the compound 11 h, whose IC₅₀ values against HCT116, LNCaP, SW620, and PC3 cell lines were 2.22, 1.6, 2.37, and 3.21 µM, respectively. The results of apoptosis induction and cell cycle arrest experiments indicate that compounds 11 g and 11 h induced early apoptosis in HCT116 cells, and G2/M phase cell cycle arrest. This finding was further validated through molecular docking analysis and cellular thermal migration and enzyme activity experiments. The results of WRN helicase inhibition assays showed that the IC₅₀ value of compound 11 g was 6.61 µM. In summary, our study identifies 2-amino-4-(trifluoromethyl)pyrimidine derivative 11 g as potential WRN-dependent anticancer agents.