Design, synthesis, and biological evaluation of pyrrolo[2,3-c]pyridin-7-one derivatives as PKMYT1 inhibitors targeting CCNE1-amplified cancers.

Synthetic lethality has emerged as a transformative therapeutic paradigm in precision oncology, offering unprecedented tumor selectivity while minimizing off-target toxicity-a longstanding limitation of conventional cancer therapies. PKMYT1, a key regulator of CDK1 phosphorylation, represents a promising therapeutic target for cancers with specific DNA damage response deficiencies driven by its synthetic lethal relationship with CCNE1 amplification. Herein, we report the rational design and synthesis of a novel class of PKMYT1 inhibitors via structure-based drug design, integrating three core optimization strategies: proposed water displacement to enhance direct ligand-protein binding, metabolic soft-spot shielding to mitigate metabolic liability, and steric hindrance modulation to preserve critical axial chirality. Through systematic structural refinement of the clinical-stage lead RP-6306, we identified compound 6-A as a favorable candidate featuring enhanced pharmacological profiles and improved in vitro metabolic stability, with no detectable cardiotoxic or genotoxic risks. This compound potently and selectively inhibits PKMYT1, suppresses CDK1 phosphorylation, and exerts robust antiproliferative effects in CCNE1-amplified cell lines. It exhibits favorable metabolic stability and demonstrates significant in vivo antitumor efficacy yet shows only moderate oral exposure in vivo, which warrants further structural optimization. Notably, combination therapy with gemcitabine achieved near-complete inhibition of tumor progression in OVCAR3 xenograft models, with no overt systemic toxicity. Collectively, these results validate compound 6-A as an attractive lead scaffold for further development and underscore the value of the proposed scaffold optimization approach in facilitating targeted treatment of CCNE1-amplified cancers.