The COVID-19 posed a serious threat to human life and health, and SARS-CoV-2 M^pro has been considered as an attractive drug target for the treatment of COVID-19. Herein, we report 2-(furan-2-ylmethylene)hydrazine-1-carbothioamide derivatives as novel inhibitors of SARS-CoV-2 M^pro developed by in-house library screening and biological evaluation. Similarity search led to the identification of compound F8–S43 with the enzymatic IC₅₀ value of 10.76 μM. Further structure-based drug design and synthetic optimization uncovered compounds F8–B6 and F8–B22 as novel non-peptidomimetic inhibitors of M^pro with IC₅₀ values of 1.57 μM and 1.55 μM, respectively. Moreover, enzymatic kinetic assay and mass spectrometry demonstrated that F8–B6 was a reversible covalent inhibitor of M^pro. Besides, F8–B6 showed low cytotoxicity with CC₅₀ values of more than 100 μM in Vero and MDCK cells. Overall, these novel SARS-CoV-2 M^pro non-peptidomimetic inhibitors provide a useful starting point for further structural optimization.

COVID-19 对人类生命和健康构成严重威胁，SARS-CoV-2 M ^pro被认为是治疗 COVID-19 的有吸引力的药物靶点。在此，我们报告了通过内部图书馆筛选和生物学评估开发的2-(furan-2-ylmethylene)hydrazine-1-carbothioamide 衍生物作为 SARS-CoV-2 M ^{pro的新型抑制剂。}相似性搜索导致化合物F8-S43的酶促 IC ₅₀值为 10.76 μM。进一步基于结构的药物设计和合成优化揭示了化合物F8-B6和F8-B22作为具有 IC _{50的 M} ^pro的新型非肽模拟物抑制剂值分别为 1.57 μM 和 1.55 μM。此外，酶动力学分析和质谱分析表明F8-B6是 M ^pro的可逆共价抑制剂。此外，F8-B6在 Vero 和 MDCK 细胞中表现出低细胞毒性，CC ₅₀值超过 100 μM。总体而言，这些新型 SARS-CoV-2 M ^pro非拟肽抑制剂为进一步结构优化提供了有用的起点。