Discovery of Covalent and Cell‐Active ALKBH5 Inhibitors with Potent Antileukemia Effects In Vivo

The N6‐methyladenosine (m6A) demethylase ALKBH5 is the only other identified m6A eraser except for FTO, and dysregulated ALKBH5 functions were closely associated with leukemogenesis. However, the development of ALKBH5 inhibitors is slow compared to FTO inhibitors. Inspired by a non‐catalytic C200‐covalent strategy, a series of maleimide derivatives were designed and synthesized as potent and covalent ALKBH5 inhibitors in this work. The analog 18l exhibited excellent inhibitory effects on ALKBH5 (IC50 = 0.62 μM), and exerted a strong antiproliferative effect on NB4 cells with IC50 of 0.63 μM. The Kd value of 18l binding to ALKBH5 was 804 nM, while no binding was observed with FTO. This result indicated that 18l was a highly selective inhibitor of ALKBH5 rather than FTO. Additionally, proteomic experiments showed that 18l directly targeted ALKBH5 in cells and altered m6A levels on mRNA, blocked the related downstream signal pathways, promoted differentiation, and induced apoptosis. Furthermore, 18l exerted excellent in vivo antitumor activity with TGITV values of 66.3% at 1 mg/kg in NB4 tumor xenograft models.