3.2. NCI-60 Screening

Compounds 1, 2, and 4–8 were tested at one dose assay (10⁻⁵ M) toward a panel of approximately sixty cancer cell lines representing different cancer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers. Primary anticancer assays were performed according to the NCI protocol as described elsewhere (see, e.g., http://dtp.nci.nih.gov accessed on 16 October 2019) [70,71,72,73]. The compounds were added at a single concentration and the cell cultures were incubated for 48 h. The end point determinations were made with a protein binding dye, sulforhodamine B (SRB). The results for each compound are reported as the percent growth (GP %) of treated cells compared to untreated control cells (negative numbers indicate cell kill). Compounds with considerable activity against all tested human tumor cell lines (6 and 7) were selected for the advanced assay against a panel of approximately sixty tumor cell lines at 10-fold dilutions of five concentrations (100 µM, 10 µM, 1 µM, 0.1 µM, and 0.01 µM) [70,71,72,73]. The percentage of growth was evaluated spectrophotometrically versus controls not treated with test agents after 48-h exposure and using SRB protein assay to estimate cell viability or growth. Three antitumor activity dose–response parameters were calculated for each cell line: GI_50—molar concentration of the compound that inhibits 50% net cell growth; TGI—molar concentration of the compound leading to the total inhibition; and LC₅₀—molar concentration of the compound leading to 50% net cell death (presented in negative logarithm). Furthermore, mean graph midpoints (MG_MID) were calculated for each of the parameters, giving an average activity parameter over all cell lines for the tested compound. For the MG_MID calculation, insensitive cell lines were included with the highest concentration tested.