Discovery of Novel Thiophene-Based Baloxavir Derivatives as Potent Cap-Dependent Endonuclease Inhibitors for Influenza Treatment

The genetic recombination and antigenic variation of influenza viruses may decrease the efficacy of antiviral vaccines, highlighting the imperativeness of developing novel anti-influenza agents. Herein, a series of thiophene-based compounds were designed and synthesized as potent anti-influenza agents. Among them, <b>ATV2301</b> exhibited an excellent anti-influenza activity (EC₅₀, H1N1 = 1.88 nM, H3N2 = 4.77 nM), a higher safety index (SI, H1N1 = 18218, H3N2 = 7180), and a remarkably improved oral bioavailability (<i>F</i> = 71.60%). The prodrug <b>ATV2301A</b> demonstrated strong therapeutic efficacy and protection in H1N1-infected BALB/c mice, with low toxicity and broad tissue distribution. <b>ATV2301</b> also exhibited high stability in both human and mouse liver microsomes. Mechanistic studies indicated that <b>ATV2301</b>'s anti-influenza activity was due to its effects on polymerase acid protein (PA), nuclear protein (NP), and RNA-dependent RNA polymerase (RdRp). Additionally, <b>ATV2301</b> showed potent activities against clinical isolates of anti-influenza A virus (IAV) and anti-influenza B virus (IBV), positioning it as a promising cap-dependent endonuclease inhibitor for further clinical research.