Antiviral Activity Assay

The in vitro antiviral efficacy of compounds was determined in Vero E6 cells as previously described.¹¹ Briefly, the cells were pretreated with the chemical compound with a concentration of 10 μM for 1 h and then infected with SARS-CoV-2 with multiplicity of infection of 0.01 for 2 h. After this, the virus–drug mixture was wiped out, and the cells were placed in the medium filled with fresh drugs for further cultivation. At 72 h post infection, viral RNA (vRNA) was extracted from the culture supernatant and detected by quantitative real-time PCR (qRT-PCR).

The anti-SARS-CoV-2 activity of selective compounds was determined through the plaque-reduction assay. Compounds in different dilution concentrations were mixed with SARS-CoV-2 (100 plaque-forming units), and 200 μL of mixtures was injected into 1 × 10⁵ monolayer Vero E6 cells lasted for 1 h. Then, the cells were washed twice with a fresh medium; after this, the cells were incubated with 0.9% agarose containing indicated chemical compounds. After infection, at the 4th day, the cells were fixed in 4% polyoxymethylene for 30 min and finally dyed with crystal violet. The plaque-forming units were counted.