Streamlined Synthesis and Structure–Activity Relationship Analysis of 2‐Amidothiophene‐3‐Carboxamides Targeting Influenza Polymerase PA‐PB1 Heterodimerization

: Influenza viruses remain a major global health threat due to their rapid evolution and ability to evade current therapies. Among viral targets, the PA-PB1 interface of the RNA polymerase complex has emerged as an attractive site for small-molecule inhibition. Based on compound 1, a previously identified PA-PB1 interaction inhibitor featuring a cycloheptathiophene-3-carboxamide scaffold, we designed and synthesized a new series of derivatives to investigate the role of the cycloheptyl moiety in antiviral activity and water solubility. In parallel, we developed an improved three-step synthetic route to access 2-amidothiophene-3-carboxamide analogs more efficiently. The new derivatives (2-16) provided valuable structure-activity relationship insights, highlighting how modifications at C-5 influence both anti-influenza potency and solubility. Among them, the C-5 phenyl analog 9 displayed the strongest antiviral activity, achieving sub-micromolar EC50 values (0.19-1.11 µM) across a panel of influenza strains, along with a CC50 value > 100 µM. Notably, the C-5 methyl analog 5 showed the greatest enhancement in aqueous solubility (75.2 µM) while maintaining low-micromolar potency (EC50 of 2 µM) and no significant toxicity (CC50 > 100 µM). Despite structural divergence from the starting hit 1, both compounds preserved the PA-PB1 interaction inhibition mechanism, as demonstrated by enzyme-linked immunosorbent assay (ELISA) and supported by docking studies within the PAC cavity.