Discovery of pyrrolo[2,3-d]pyrimidine derivatives as novel FLT3/IRAK4 inhibitors.

Starting with the lead compound L-1, a series of pyrrolo[2,3-d]pyrimidine derivatives were developed as FLT3/IRAK4 inhibitors through three rounds of rational structural optimization. Among them, HB-29 had the remarkable activity towards FLT3-WT (IC50 = 1.95 nM) and IRAK4 (IC50 = 53.72 nM), outperforming the positive control, CA-4948. Besides, it exhibited excellent activities in MV4-11, MOLM3, and BaF3 cells with varying FLT3-TKD and FLT3-ITD-TKD mutations, highlighting its potential to overcome acquired resistance. The toxicity of HB-29 to normal bone marrow cell line HS-5 is relatively low (SI > 2000). Mechanistic studies revealed that HB-29 inhibited FLT3 and IRAK4 pathways in a dose-dependent manner, promoting cell apoptosis. Notably, in the cytokine-induced cell model, HB-29 efficiently induced apoptosis, and while also enhancing SOD activity and reducing ROS accumulation, thereby demonstrating its potential to overcome adaptive resistance. Moreover, HB-29 demonstrated an acceptable bioavailability (F = 13.4 %). These findings confirm that FLT3/IRAK4 inhibitor is a promising strategy for the treatment of AML.