In the experiments, we used Vero (green monkey kidney) cell line (RCB 10-87, WHO, Switzerland). Cells were maintained in 2×EMEM medium (Eagle Minimum Essential Medium with doubled amino acids and vitamins), supplemented with 5% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), streptomycin (0.1 mg/mL), and penicillin (100 units/mL) (PanEco, Moscow, Russia).

SARS-CoV-2 strain PIK35 (GISAID ID EPI_ISL_428852) was isolated from a nasopharyngeal swab of a COVID-19 patient [20]. The virus was passaged 5 times in Vero cells and stored as an infected cells suspension at −80 °C.

The antiviral activity was assessed in titer reduction assay, i.e., the inhibitory effect was estimated as compound concentration decreasing viral titer by 50%.

Vero cells were seeded in 96-well plates (approximately 105 cells per well) and incubated at 37 °C in a CO2 incubator for 3 days until a full monolayer was formed. A compound was added to the virus suspension (1000 TCID50) in four concentrations, starting from 5.6 mM. The resulting mixtures were incubated for 1 h at room temperature, then the remaining infectious virus was quantified via titration in Vero cells by its ability to cause cytopathic effect (CPE). The virus titers were calculated according to the Karber method [21]. The antiviral effect was determined by the decrease in the titer of the virus in the samples with the compound in comparison with the negative control (cell culture medium) samples. The EC50 was calculated using the approximation method.

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