# Screening of Scaffolds for the Design of G-Quadruplex Ligands
## 3 Results and Discussion
### Cytotoxicity Assay  
The scaffold's effect on cell viability was evaluated by an MTT assay, using A549 lung cancer and NHDF healthy cell lines. The results of IC\({}_{50}\) are presented in Table 3. Overall, the scaffolds were found to be much more active on the cancer cell line. The acridine precursors showed a high potency against the A549 cell line, with IC\({}_{50}\) values of 1.3 uM (C\({}_{8}\)NH\({}_{2}\)) and 3.6 uM (C\({}_{5}\)NH\({}_{2}\)). Most relevantly, C\({}_{5}\)NH\({}_{2}\) shows an IC\({}_{50}\) of 17.8 uM in normal human fibroblasts, indicating a good selectivity for its cellular growth inhibitory activity in human cancer cells over human normal cells. The anticancer activity of C\({}_{8}\)NH\({}_{2}\) is consistent with that previously reported for PC-3 (prostate adenocarcinoma) and PNT1A (normal human prostate cell line) [28]. These results can be correlated with the FRET-melting results, which may indicate that the cytotoxicity effect of the acridine ligands can be related to G4 binding ability. On the contrary, the phenanthroline scaffold containing a pyrazine ring (DPPZ) showed lower IC\({}_{50}\) values in both cell lines (IC\({}_{50}\) of 0.4 uM and 0.2 uM for cancer and normal cell line, respectively), suggesting lower cellular selectivity. Most probably, this effect is not mediated by G4 binding, since DPPZ showed weak G4 stabilizer capacity in FRET-melting experiments. The phenanthroline derivative containing an oxazole ring (Phen-1) presented low and no-selective cytotoxicity effect in cancer (IC\({}_{50}\) = 22.1 uM) and healthy (IC\({}_{50}\) = 20.4 uM) cell lines. Conversely, the introduction of an ester group in the phenanthroline scaffold conferred potency against A549 cells (IC\({}_{50}\) = 6.4 uM) and a weak inhibitory effect on healthy cells (IC\({}_{50}\) = 37.0 uM). Therefore, PhenCOOCH\({}_{3}\) can be a promising candidate for further cellular studies. Similarly, phenyl (Ter-1 and ISO-1) derivatives were shown to be promising anticancer candidates, particularly Ter-1, with a pronounced cytotoxicity effect in cancer cells (IC\({}_{50}\) = 1.9 uM) and weak toxicity in healthy cells (IC\({}_{50}\) = 34.0 uM). These results suggest that the 1,4 disubstituted phenyl ring confers higher anticancer activity, when compared with the 1,3 disubstituted congener (ISO-1; with an IC\({}_{50}\) value of 9.8 and 32.0 uM for cancer and healthy cells lines, respectively). The Quinoline derivative also presents selectivity and cytotoxicity effects (IC\({}_{50}\) = 9.0 uM) in cancer cells. Naphthalene scaffolds exhibited a low inhibitory effect on the cancer cell line, but Napht-3 presented low IC\({}_{50}\) on the fibroblast cell line (NHDF). This trend can be related with the size of the polyamine chain, since Napht-3 has a short polyamine chain and is more toxic in normal cells (IC\({}_{50}\) = 5.3 uM) than the derivative containing the pentamine chain (Napht-5; IC\({}_{50}\) = 24.1 uM). For cancer cells, Napht-5 is more potent (IC\({}_{50}\) = 12.5 uM) than Napht-3 (IC\({}_{50}\) = 19.4 uM).  
\begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Ligand**} & \multicolumn{2}{c}{**IC\({}_{50}\) (uM)**} & \multirow{2}{*}{**Selectivity Factor**} \\ \cline{2-3} \cline{5-5}  & **A549** & & **NHDF** \\ \hline Ter-1 & 1.9 & 34.0 & 17.9 \\ ISO-1 & 9.8 & 32.0 & 3.3 \\ Quin-1 & 9.0 & 32.6 & 3.6 \\ Napht-3 & 19.4 & 5.3 & 0.3 \\ Napht-5 & 12.1 & 24.1 & 2.0 \\ Phen-1 & 22.0 & 20.7 & 0.9 \\ DPZ & 0.40 & 0.21 & 0.5 \\ PhenCOOCH\({}_{3}\) & 6.4 & 37.0 & 5.8 \\ C\({}_{8}\)NH\({}_{2}\) & 1.3 & 3.8 & 2.9 \\ C\({}_{5}\)NH\({}_{2}\) & 3.6 & 17.8 & 4.9 \\ \hline \hline \end{tabular}
\end{table}
Table 3: IC\({}_{50}\) \({}^{a}\) values induced by different scaffolds in A549 and NHDF cell lines.