Synthesis and evaluation of alkoxy-substituted enamides against influenza A virus in vitro and in vivo

[Display omitted] •We have newly discovered the antiviral activity of alkoxy substituted amines against influenza viruses.•We revealed that alkoxy-substituted enamide E-2o can inhibit influenza virus replication both in vivo and in vitro, and reduce the damage caused by immune factor storms.•We demonstrated that alkoxy-substituted enamide E-2o could reduce cell apoptosis and autophagy caused by influenza virus, and alleviate inflammatory response by inhibiting the RIG-I pathway. Alkoxy-substituted enamides are often used as synthetic intermediates due to their special reactivity. To the best our knowledge, the biological activity of alkoxy-substituted amines has never been reported so far. We have synthesized a series of alkoxy-substituted enamides to study their anti-influenza A virus activity in vitro and in vivo. Among these compounds, compound E-2o had the best antiviral activity (EC50 = 2.76 ± 0.67 μM) and low cytotoxicity (CC50 = 662.87 ± 24.85 μM). The mechanism of action of this compound was preliminarily explored by us. It alleviated the cytopathic effects and cell death caused by different subtypes of influenza A virus. Different drug delivery methods and timed dosing experiments had shown that E-2o had the best therapeutic effect and mainly played a role in the early stages of virus replication. The expansion of influenza viruses in cells was inhibited by reducing ROS accumulation, cell apoptosis, and autophagy. Alkoxy-substituted enamide E-2o reduced the production of interferon and other pro-inflammatory factors in the RIG-Ⅰ pathway and its downstream NF-κB was induced by influenza A virus in vitro and in vivo. It avoided damage in the mice which was caused by excessive inflammatory factors. In addition, the weight loss and lung lesion damage in mice caused by influenza virus were improved by compound E-2o. Therefore, Alkoxy-substituted enamide E-2o could inhibit the replication of influenza viruses in vivo and in vitro, and has the potential to be developed into a drug for treating influenza.