Development of potent and selective ENPP1 inhibitors based on imidazo[1,2-a]pyrazine and pyrazine scaffolds for cancer immunotherapy.

Abstract

In this study, new imidazo[1,2-a]pyrazine derivative 15 (YS3074) and pyrazine derivative 17 (YS3375) are identified and optimized as potent and selective ENPP1 inhibitors. Compounds 15 and 17 both exhibited high potency and selectivity toward ENPP1 (IC50 = 4.23 nM and 19.4 nM, respectively) with negligible ENPP2 inhibition (>10 μM) and weak ENPP3 inhibition (>3 μM). Both enhanced cGAMP-induced expression of STING pathway genes (IFNB1, CXCL10, IL6) in a concentration-dependent manner without cytotoxicity, producing stronger and more sustained activation than MV-658. This effect, dependent on the cGAMP-STING axis, was attributed to inhibition of ENPP1-mediated cGAMP hydrolysis. Additionally, compounds 15 and 17 showed no inhibition of the hERG channel (IC50 > 30 μM), indicating a favorable cardiac safety profile. In the CT-26 colorectal cancer model, compounds 15 and 17, administered at 40 mg/kg in combination with an anti-PD-1 antibody, achieved superior tumor growth inhibition (TGI: 73% and 67%, respectively), significantly prolonged survival, and were well tolerated with no observable body weight loss. These results establish 15 and 17 as promising ENPP1 inhibitors with favorable safety profiles, supporting their potential in combination cancer immunotherapy.