Cytotoxicity Evaluation of Antiviral Compounds

The cytotoxicity of compounds was evaluated by using the neutral red dye-uptake method as previously described [19 (link),20 (link)]. Briefly, the test compounds 1–12, along with 13 (a + b) and acyclovir (Sigma-Aldrich, Germany), used as a positive control were diluted in 0.1% dimethyl sulfoxide (DMSO). Stock solutions were prepared at a concentration of 420 μg/mL using deionized water and then sterilized. Vero cell monolayers cultivated in 96-well microtiter plates with two-fold serial dilutions of the test compounds or the positive control were incubated for 84 h at 37 °C in 5% CO₂ atmosphere. After incubation, the morphological alterations of the treated cells were determined using an inverted optical microscope (Leitz, Berlin, Germany) and the maximum non-toxic concentrations (MNTC) were determined. The CC₅₀ values (concentrations of each drug required to reduce the cell viability by 50%) of compounds 1–12, along with 13 (a + b) and acyclovir were calculated as compared with the untreated control cells. The cytotoxicity of compounds 1–2, 5–6, and 12, active in further anti-HSV tests, was evaluated in duplicate in at least three independent experiments to obtain statistically relevant data for final calculations.