Synthesis and structure-activity relationship studies of N-benzyl-2-phenylpyrimidin-4-amine derivatives as potent USP1/UAF1 deubiquitinase inhibitors with anticancer activity against nonsmall cell lung cancer.

Deregulation of ubiquitin conjugation or deconjugation has been implicated in the pathogenesis of many human diseases including cancer. The deubiquitinating enzyme USP1 (ubiquitin-specific protease 1), in association with UAF1 (USP1-associated factor 1), is a known regulator of DNA damage response and has been shown as a promising anticancer target. To further evaluate USP1/UAF1 as a therapeutic target, we conducted a quantitative high throughput screen of >400000 compounds and subsequent medicinal chemistry optimization of small molecules that inhibit the deubiquitinating activity of USP1/UAF1. Ultimately, these efforts led to the identification of ML323 (70) and related N-benzyl-2-phenylpyrimidin-4-amine derivatives, which possess nanomolar USP1/UAF1 inhibitory potency. Moreover, we demonstrate a strong correlation between compound IC50 values for USP1/UAF1 inhibition and activity in nonsmall cell lung cancer cells, specifically increased monoubiquitinated PCNA (Ub-PCNA) levels and decreased cell survival. Our results establish the druggability of the USP1/UAF1 deubiquitinase complex and its potential as a molecular target for anticancer therapies.