Nowadays, small-molecule drugs have become an indispensable part of tumor immunotherapy. Accumulating evidence has indicated that specifically blocking PGE2/EP4 signaling to induce robust antitumor immune response represents an attractive immunotherapy strategy. Herein, a 2H-indazole-3-carboxamide containing compound 1 was identified as a EP4 antagonist hit by screening our in-house small-molecule library. Systematic structure–activity relationship exploration leads to the discovery of compound 14, which displayed single-nanomolar EP4 antagonistic activity in a panel of cell functional assays, high subtype selectivity, and favorable drug-like profiles. Moreover, compound 14 profoundly inhibited the up-regulation of multiple immunosuppression-related genes in macrophages. Oral administration of compound 14, either as monotherapy or in combination with an anti-PD-1 antibody, significantly impaired tumor growth via enhancing cytotoxic CD8+ T cell-mediated antitumor immunity in a syngeneic colon cancer model. Thus, these results demonstrate the potential of compound 14 as a candidate for developing novel EP4 antagonists for tumor immunotherapy.

Cancer cells can effectively suppress the natural immune response in humans, and prostaglandin E2 (PGE2) is a key mediator in the development of tumor cell resistance to immunotherapy. As a major contributor to PGE2-elicited immunosuppressive activity, the EP4 receptor promotes tumor development and progression in the tumor microenvironment, and the development of selective and potent EP4 receptor antagonists should have promising potential for tumor immunotherapy. Aiming at improving the drug-like properties, a series of 4,7-dihydro-5H-thieno­[2,3-c]­pyran derivatives were designed and synthesized through a scaffold hopping strategy. The most promising compound 47 exhibited good EP4 antagonistic activity and excellent subtype selectivity, as well as favorable drug-like properties. It effectively suppressed the expression of multiple immunosuppression-related genes in macrophages. Meanwhile, oral administration of compound 47, alone or in combination with anti-PD-1 antibody, significantly enhanced the antitumor immune response and inhibited tumor growth in the mouse CT26 colon carcinoma model.