Design, synthesis and biological evaluation of triazolo[1,5-a]pyrimidinone derivatives as potent and orally efficacious WRN helicase inhibitors for MSI cancer therapy.

Microsatellite instability-high (MSI-H) cancers represent an unmet clinical need due to treatment resistance, and WRN helicase is a validated synthetic lethal target for this indication. Herein, we rationally designed, synthesized, and biologically evaluated a novel series of triazolo[1,5-a]pyrimidinone derivatives as potent WRN helicase inhibitors. Systematic structure-activity relationship (SAR) exploration identified optimal pharmacophores: 4-chlorobenzothiophene/2,2-difluoro-4-bromo-1,3-benzodioxole as R1, dihydro-2H-pyran as R2, and 3-hydroxypyridine/4-methyl-5-hydroxypyrimidines as R3. Lead candidate 10r exhibited nanomolar enzymatic inhibition (IC50 = 0.015 ± 0.004 μM) and sub-micromolar cellular potency (IC50 = 0.174 ± 0.036 μM) against HCT116-MSI cells, with high oral bioavailability (71.53 ± 17.96%) and favorable pharmacokinetics in mice. In HCT116-MSI cell-derived xenograft models, 10r achieved robust tumor growth inhibition (TGI = 94.7 ± 15.5%) at 100 mg/kg BID, with durable efficacy after treatment cessation, and a well-tolerated safety profile. This study delivers an attractive lead series worthy of further investigation for MSI cancers and provide a structural framework for further development.