The COVID-19 pandemic caused by the SARS-CoV-2 virus continually poses serious threats to global public health. The main protease (Mpro) of SARS-CoV-2 plays a central role in viral replication. We designed and synthesized 32 new bicycloproline-containing Mpro inhibitors derived from either Boceprevir or Telaprevir, both of which are approved antivirals. All compounds inhibited SARS-CoV-2 Mpro activity in vitro with IC50 values ranging from 7.6 to 748.5 nM. The co-crystal structure of Mpro in complex with MI-23, one of the most potent compounds, revealed its interaction mode. Two compounds (MI-09 and MI-30) showed excellent antiviral activity in cell-based assays. In a SARS-CoV-2 infection transgenic mouse model, oral or intraperitoneal treatment with MI-09 or MI-30 significantly reduced lung viral loads and lung lesions. Both also displayed good pharmacokinetic properties and safety in rats.

A: Schematic of infection experiments. B: Alterations in body weight in rats with (infection group) or without (mock group) SARS-CoV-2 infection. Data are mean±standard error of the mean ( SEM ). C: Viral RNA levels in throat swabs of infected rats at 1 dpi ( n =32), 3 dpi ( n =24), and 5 dpi ( n =16). Each dot indicates log copies of viral genomic N gene (top), E gene (middle), and subgenomic E (sgE) gene (bottom) per swab from an individual rat. D: Viral RNA levels in lungs of infected rats at 1, 3, and 5 dpi. Each red circle (1 dpi, n =8), blue square (3 dpi, n =8), and black triangle (5 dpi, n =8) refers to log viral RNA copies/µg total RNA of lung lobe of an individual rat. E: Gross pathological lung specimens at indicated time points after SARS-CoV-2 infection. F: Hematoxylin/eosin (H&E) staining of the lung sections in SARS-CoV-2-infected rats showing lesions with thickened alveolar septa and infiltration of lymphocytes. Scale bar: 100 µm. G: Immunohistochemical analysis of SARS-CoV-2 N protein in rat lung tissues on 1 dpi. Scale bar: 1 mm for entire lung lobe section (top), 50 µm for enlarged view of boxed areas labeled by numbers in entire section (bottom). H, I: Viral RNA loads in throat swabs (H) and nasal turbinate (I) of rats re-challenged with SARS-CoV-2 for 1 day (1 dpr, n =8) were significantly lower than in initial infection group rats (1 dpi, n =5). * : P <0.05; *** : P <0.001; two-tailed Student’s t -test. J: Viral RNA levels in lungs of infected rats at 1 dpi (initial infection group) and 1 dpr (reinfection group). K: Neutralizing antibody (NAb) titers in SARS-CoV-2-infected rats for protection against re-challenge. Values in D, H, I, and J are presented as mean±standard deviation ( SD ). Dotted lines in C and H represent limit of quantification (250 copies/mL). Dotted lines in D, I, and J represent limit of quantification (8.7 copies/µg total RNA of lung).

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