1-(4-(芳基乙烯羰基)苯基)-4-羧基-2-吡咯烷酮作為有效抗病毒和抗菌劑的合成、評估和機制

Abstract

通過邁克爾加成、環化、醛醇縮合和去質子化設計並合成了一類1-(4-(芳基乙烯羰基)苯基)-4-羧基-2-吡咯烷酮，以抑制人跨膜蛋白酶絲氨酸2 (TMPRSS2)和弗林蛋白酶， 它們參與修飾 SARS-CoV-2 Spike 以便病毒進入。 發現最有效的抑制劑 2f (81) 可有效抑制 VeroE6 和 Calu-3 細胞中各種 SARS-CoV-2 delta 和 omicron 突變株的複製，通過與病毒預混合，抗病毒的EC50 範圍可達 0.001 至 0.026 µM。 比蛋白酶抑制活性更有效的抗病毒活性顯示，合成的化合物主要以抑制 Spike 受體結合域（RBD）：血管緊張轉化酶 2（ACE2）相互作用作為靶標，並且通過抑制 TMPRSS2 和/或 Furin 增强其抗病毒活性。 為了進一步證實 2f（81）對病毒進入的阻斷作用，使用SARS-CoV-2 Spike假病毒進行進入檢測，結果表明該化合物通過ACE2依賴性途徑抑制假病毒進入，主要是抑制Calu-3 细胞中 RBD：ACE2 相互作用和 TMPRSS2 活性。 最後，在 SARS-CoV-2 感染的體内動物模型中，倉鼠口服 25 mg/kg 2f (81) 可減少體重减輕，感染三天後鼻甲中的病毒 RNA 水平降低了 5 倍。 我們的研究结果證明了先導化合物作為 SARS-CoV-2 治療進一步臨床前研究的潛力。

A class of 1-(4-(arylethylenylcarbonyl)phenyl)-4-carboxy-2-pyrrolidinones were designed and synthesized via Michael addition, cyclization, aldol condensation, and deprotonation to inhibit the human transmembrane protease serine 2 (TMPRSS2) and Furin, which are involved in priming the SARS-CoV-2 Spike for virus entry. The most potent inhibitor 2f (81) was found to efficiently inhibit the replication of various SARS-CoV-2 delta and omicron variants in VeroE6 and Calu-3 cells, with EC50 range of 0.001 to 0.026 µM by pre-incubation with the virus to avoid the virus entry. The more potent antiviral activities than the proteases inhibitory activities led to the discovery that the synthesized compounds also inhibited Spike’s receptor binding domain (RBD):angiotensin converting enzyme 2 (ACE2) interaction as a main target, and their antiviral activities were enhanced by inhibiting TMPRSS2 and/or Furin. To further confirm the blocking effect of 2f (81) on virus entry, SARS-CoV-2 Spike pseudovirus was used in the entry assay and the results showed that the compound inhibited the pseudovirus entry in a ACE2-dependent pathway, via mainly inhibiting RBD:ACE2 interaction and TMPRSS2 activity in Calu-3 cells. Finally, in the in vivo animal model of SARS-CoV-2 infection, the oral administration of 25 mg/kg 2f (81) in hamsters resulted in reduced body weight loss and 5-fold lower viral RNA levels in nasal turbinate three days post-infection. Our findings demonstrated the potential of the lead compound for further preclinical investigation as a potential treatment for SARS-CoV-2.