Discovery of Naphthyridinone Derivatives as Selective PKMYT1/WEE1 Dual Inhibitors for Cancer Therapy.

Dual inhibition of PKMYT1 and WEE1, key G2/M checkpoint kinases that phosphorylate CDK1 at T14 and Y15, offers a strategy for tumors with abrogated G1/S checkpoint and high replication stress. Building on our prior PKMYT1 chemotype, we designed 1,7-naphthyridinone derivatives by displacing crystallographic water (core 5'-N-Asp251) and adding a 7'-ring nitrogen to retain physicochemical properties. 5'-Site structure fine-tuning enhanced WEE1 engagement while preserving the PKMYT1-preferred hinge flip and superior kinome selectivity. Optimization identified compound 24 with single-digit nM PKMYT1 NanoBRET and sub-μM WEE1 NanoBRET potency, translating to pCDK1 T14 IC50 4.9 nM and pCDK1 Y15 0.186 μM in HCC1569 cells. Kinome profiling confirmed favorable selectivity. In colorectal cancer organoids, 24 outperformed our prior PKMYT1 inhibitor (6), RP-6306, and WEE1 inhibitor AZD1775, with efficacy correlating to improved WEE1 activity. Compound 24 also showed favorable in vitro ADME and early safety profiles, supporting dual checkpoint targeting in checkpoint-deficient cancers.