PSB-24000 (Compound 27) is a selective ecto-5'-nucleotidase (CD73) inhibitor with a Ki value of 563 nM for inhibiting human CD73, and a Ki of 481 nM in membrane-bound CD73 in triple-negative breast cancer cells. It disrupts CD73’s recognition and action on substrate AMP, preventing AMP-induced immunosuppressive and pro-cancer adenosine production. PSB-24000 is promising for cancer research.