Discovery of pyridine formyl hydrazide derivatives as potent non-nucleoside RSV polymerase inhibitors

Respiratory syncytial virus (RSV) is an RNA virus that infects both the upper and lower respiratory tract and is recognized as a major respiratory health threat. In this study, guided by the cryo-EM structure of the MRK-1-RSV polymerase complex, the substituent region at the C4 position of the shared pyridine core was redesigned. A series of 32 pyridine carbohydrazide derivatives bearing structurally diverse N-substituted amino groups was synthesized to explore the structure-activity relationship and substituent tolerance of the adjacent pocket region. LX1 was selected as a representative compound for further investigation and exhibited potent activity against RSV-A Long strain, with an EC₅₀ value of 37 nM. Minigenome assays showed that LX1 reduces polymerase-dependent reporter activity, consistent with inhibiting RSV RNA synthesis. Furthermore, molecular dynamics simulations revealed that LX1 forms stable hydrogen bonds and π-π stacking interactions with key amino acids in the PRNTase domain. Collectively, LX1 represents a promising lead compound for the further development of novel RSV inhibitors.