DYRK2 has become a promising therapeutic target due to its involvement in various processes, including regulation of protein stability and phosphorylation events related to neurodegenerative diseases and cancers. The thioacridine derivative LDN-192960 has been widely recognized as a potent inhibitor of DYRK2. However, it also inhibits other kinases, such as DYRK3, Haspin, DYRK1A and CLK1, making it challenging to delineate the biological roles of DYRK2. In this study, we designed and synthesized a series of benzamide derivatives of the thioacridine LDN-192960, to improve selectivity and potency towards DYRK2. Several compounds demonstrated potent inhibition of DYRK2 and DYRK3, with IC50 values in the nanomolar range. Notably, the para-substituted aldehyde derivative 10f exhibited significant selectivity for DYRK2/DYRK3 against its main off-targets DYRK1A, CLK1 and Haspin, thus surpassing the selectivity profile of LDN-192960. Docking studies revealed new interactions between 10f and DYRK2, compared to LDN-192960 / DYRK2 co-crystallized structure and 10f was shown to impair proteasome activity in HEK293 cells. These findings highlight the pharmacophore study of DYRK2/DYRK3 dual inhibitors, and present compound 10f as a selective chemical probe for DYRK2 and DYRK3, that might be an essential tool for elucidating the specific biological roles of these kinases and advancing targeted therapeutic applications.
