5-Methoxybenzothiophene-2-Carboxamides as Inhibitors of Clk1/4: Optimization of Selectivity and Cellular Potency.

Clks have been shown by recent studies to be promising targets for cancer therapy, as they are considered key regulators in the process of pre-mRNA splicing, which in turn affects every aspect of tumor biology. In particular, Clk1 and -4 are overexpressed in several human tumors. Most of the potent Clk1 inhibitors reported in the literature are non-selective, mainly showing off-target activity towards Clk2, Dyrk1A and Dyrk1B. Herein, we present new 5-methoxybenzothiophene-2-carboxamide derivatives with unprecedented selectivity. In particular, the introduction of a 3,5-difluoro benzyl extension to the methylated amide led to the discovery of compound (cell-free IC = 12.7 nM), which was four times more selective for Clk1 over Clk2 than the previously published flagship compound . Moreover, showed an improved growth inhibitory activity with T24 cells (GI = 0.43 µM). Furthermore, a new binding model in the ATP pocket of Clk1 was developed based on the structure-activity relationships derived from new rigidified analogues.