Discovery of Novel Triple A1/A2A/A2B Adenosine Receptor Antagonists for Cancer Immunotherapy

The A1, A2A, and A2B adenosine receptors are prime targets for immune cell activation and tumor suppression. Herein, we describe the rationale design, synthesis, and biological evaluation of 6-aminonicotinonitrile derivatives as triple A1/A2A/A2B adenosine receptor antagonists. Compound <b>14a</b> demonstrated potent inhibitory activity (IC₅₀ = 0.8 nM) of cAMP production in A2AR-HEK293 cells and strong binding affinity (<i>K</i>_i = 0.6-21 nM) against A1/A2A/A2B receptors. Compound <b>14a</b> also effectively restored T cell proliferation suppressed by 5'-<i>N</i>-ethylcarboxamidoadenosine (NECA) and exhibited superior T cell-mediated cytotoxicity in coculture systems with A1R- and PD-L1-expressed cancer cells compared with ciforadenant (A2AR antagonist) and etrumadenant (A2AR/A2BR dual antagonist). Moreover, the combination of compound <b>14a</b> with avelumab, an anti-PD-L1 antibody, resulted in enhanced infiltration of effector T cells and significantly increased the CD8+/Treg ratio in the CT26 syngeneic mouse model, substantially inhibiting tumor growth. Therefore, compound <b>14a</b> is a promising candidate for multitargeted immunomodulation in cancer immunotherapy.