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.