Discovery andOptimization of a Series of Novel Morpholine-ContainingUSP1 Inhibitors

Ubiquitin-specific protease 1 (USP1), a well-known member of the deubiquitinating enzymes, serves as a key regulator in DNA damage repair (DDR) processes. Herein, we utilized ring-opening and cyclization strategies based on KSQ-4279 to design a novel series of USP1 inhibitors featuring a morpholine scaffold. Notably, compound <b>38-P2</b> exhibited a more potent enzymatic and cellular inhibition activity compared to KSQ-4279. Mechanistically, <b>38-P2</b> was characterized as a selective, reversible, and noncompetitive USP1 inhibitor. <b>38-P2</b> efficiently activated the DDR pathway, induced cell cycle arrest and cell apoptosis, and inhibited cell survival. Importantly, it enhanced the sensitivity of olaparib-resistant cells to olaparib and showed a synergetic effect with andrographolide in BRCA-proficient cancer cells. Furthermore, <b>38-P2</b> had favorable pharmacokinetic profiles and good safety properties <i>in vitro</i> and <i>in vivo</i>. In the MDA-MB-436 xenograft model, <b>38-P2</b> displayed significant, dose-dependent antitumor efficacy. Overall, these findings indicate that <b>38-P2</b> is a promising lead compound for further drug development.