Discovery of Pyrrolo1′,2′:1,6pyrimido5,4- c pyridazin-6(5 H )-one Derivatives as Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 Inhibitors

ENPP1 is emerging as a potential target for cancer immunotherapy due to its negatively regulatory effect on the STING pathway via hydrolysis of cGAMP. Herein, we report the identification and optimization of compound A25 starting from hit compound A1. A25 is a potent and selective ENPP1 inhibitor featuring a novel pyrrolo1′,2′:1,6pyrimido5,4-cpyridazin-6(5H)-one core scaffold. It exhibited substantial inhibitory activity against ENPP1 with an IC50 value of 9.5 nM, while showing weak inhibition against ENPP2/3. In the cGAMP-mediated STING pathway, this compound effectively enhanced the expression of downstream genes and promoted the phosphorylation of the relevant protein. Moreover, it displayed favorable pharmacokinetic properties and no evident cytotoxicity. In a 4T1 syngeneic mouse model, oral administration of compound A25 demonstrated significant antitumor effects and enhanced the efficacy of both anti-PD-1 antibody and chemotherapy, with good tolerability. Collectively, these results highlight the potential of compound A25 to potentiate STING-mediated antitumor immunity.