Carbamoyl phosphate synthetase 1 (CPS1) catalyzes the initial step within the ammonia-detoxifying urea cycle, converting ammonia to carbamoyl phosphate under physiologic conditions. In cancer, CPS1 overexpression supports pyrimidine synthesis to advertise tumor development in some cancer types, during others CPS1 activity prevents the buildup of toxic amounts of intratumoral ammonia to match sustained tumor growth. Targeted CPS1 inhibitors may, therefore, give a therapeutic benefit for cancer patients with tumors overexpressing CPS1. Herein, we describe the invention of small-molecule CPS1 inhibitors that bind to some formerly unknown allosteric pocket to bar ATP hydrolysis in the initial step of carbamoyl phosphate synthesis. CPS1 inhibitors are involved in cellular assays, blocking both urea synthesis and CPS1 support from the pyrimidine biosynthetic path, while getting no activity against CPS2. These recently discovered CPS1 inhibitors really are a foundation supplying researchers with valuable tools for probing CPS1 cancer biology.H3B-120