The influenza virus hemagglutinin (HA) is an attractive target for antiviral therapy due to its essential role in mediating virus entry into the host cell. We here report the identification of a class of N-benzyl-4,4,-disubstituted piperidines as influenza A virus fusion inhibitors with specific activity against the H1N1 subtype. Using the highly efficient one-step Ugi four-component reaction, diverse library of piperidine-based analogues was synthesized and evaluated to explore the structure-activity relationships (SAR). Mechanistic studies, including resistance selection with the most active compound (2) demonstrated that it acts as an inhibitor of the low pH-induced HA-mediated membrane fusion process. Computational studies identified an as yet unrecognized fusion inhibitor binding site, which is located at the bottom of the HA₂ stem in close proximity to the fusion peptide. A direct π-stacking interaction between the N-benzylpiperidine moiety of 2 and F9_HA2 of the fusion peptide, reinforced with an additional π-stacking interaction with Y119_HA2, and a salt bridge of the protonated piperidine nitrogen with E120_HA2, were identified as important interactions to mediate ligand binding. This site rationalized the observed SAR and provided a structural explanation for the H1N1-specific activity of our inhibitors. Furthermore, the HA₁-S326V mutation resulting in resistance to 2 is close to the proposed new binding pocket. Our findings point to the N-benzyl-4,4,-disubstituted piperidines as an interesting class of influenza virus inhibitors, representing the first example of fusion peptide binders with great potential for anti-influenza drug development.

流感病毒血凝素（HA）是介导病毒进入宿主细胞的重要作用，因此是抗病毒治疗的诱人靶标。我们在这里报告鉴定出一类N-苄基-4,4，-二取代哌啶作为甲型流感病毒融合抑制剂，具有针对H1N1亚型的特异性活性。使用高效的一步式Ugi四组分反应，合成并评估了各种基于哌啶的类似物文库，并探讨了结构-活性关系（SAR）。机理研究，包括选择活性最高的化合物（2）证明它可作为低pH诱导的HA介导的膜融合过程的抑制剂。计算研究确定了尚无法识别的融合抑制剂结合位点，该位点位于HA ₂茎的底部，紧邻融合肽。2的N-苄基哌啶部分与融合肽的F9 _HA2之间直接的π堆积相互作用，与Y119 _HA2的额外π堆积相互作用增强了质子化的哌啶氮与E120 _HA2的盐桥被认为是介导配体结合的重要相互作用。该站点合理化了所观察到的SAR，并为我们的抑制剂的H1N1特异性活性提供了结构性解释。此外，导致对2的抗性的HA ₁ -S326V突变接近拟议的新结合口袋。我们的发现指出，N-苄基-4,4，-二取代哌啶是一类有趣的流感病毒抑制剂，代表了融合肽结合剂具有抗流感药物开发潜力的第一个实例。