Discovery of\nImidazo[1,2‑<i>a</i>]pyrazine Derivatives as Potent\nENPP1 Inhibitors

ENPP1 acts as a negative regulator of the cGAS–STING\npathway\nthrough the hydrolysis of 2′3′-cGAMP. Inhibitors of\nENPP1 are regarded as promising agents for stimulating the immune\nresponse in cancer immunotherapy. This study describes the identification\nand optimization of imidazo[1,2-<i>a</i>]pyrazine derivative <b>7</b> as a highly potent and selective ENPP1 inhibitor. Compound <b>7</b> demonstrated substantial inhibitory activity against ENPP1\nwith an IC<sub>50</sub> value of 5.70 or 9.68 nM while showing weak\ninhibition against ENPP2 and ENPP3. Furthermore, compound <b>7</b> was shown to enhance the mRNA expression of cGAMP-induced STING\npathway downstream target genes, such as <i>IFNB1</i>, <i>CXCL10</i>, and <i>IL</i>6. <i>In vivo</i> pharmacokinetic and antitumor studies showed promising results,\nwith <b>7</b> not only exhibiting efficient pharmacokinetic\nproperties but also enhancing the antitumor efficacy of the anti-PD-1\nantibody. Treatment with <b>7</b> (80 mg/kg) combined with anti-PD-1\nantibody achieved a tumor growth inhibition rate of 77.7% and improved\nsurvival in a murine model.