Discovery of 2-(Phenylamino)-6 H -pyrimido[5,4- b ][1,4]oxazin-7(8 H )-one Derivatives as Selective ACK1 Inhibitors with Immunomodulatory Effects Enhancing Anti-PD-1 Therapy for Triple-Negative Breast Cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype with limited therapeutic options due to the lack of actionable targets. ACK1 contributes to tumor progression and immune evasion, representing a promising immunomodulatory target whose inhibition can potentiate anti-PD-1 therapy in TNBC. Herein, we report the discovery and structure−activity relationship (SAR) of 2-(phenylamino)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one derivatives as a new class of ACK1 inhibitors. The optimized compound 20l potently inhibits ACK1 (IC50 = 1 nM) and its autophosphorylation in TNBC cells. In vitro, 20l suppresses TNBC cell proliferation, migration, and invasion while inducing cell cycle arrest and apoptosis. In vivo, 20l not only directly inhibits TNBC cell growth but also remodels the immune microenvironment and activates systemic immunity. Combined treatment with 20l and an anti-PD-1 antibody produced a TGI of 95.7% without evident toxicity. Thus, 20l represents a promising ACK1-targeted therapeutic lead that warrants further investigation for the treatment of aggressive TNBC.