Fifteen new 1,10-phenanthrolines disubstituted at positions 2 and 9 via amide bonds with different heterocycles have been designed and synthesized as G-quadruplex DNA stabilizers. Ten compounds were evaluated for the in vitro anticancer activity against 60 human tumor cell lines panel, four of them showing a very good inhibitory activity on several cell lines. To assess the ability of the most active compounds to interact with G-quadruplex DNA (G4-DNA), circular dichroism experiments were performed. The potency of the compounds to stabilize the G4-DNA has been shown from the thermal denaturation experiments. The mechanism of compounds binding to DNA and to G4-DNA was theoretically investigated by molecular docking studies. The experimental results demonstrated excellent capacity of the two compounds bearing two pyridin-3-yl residues (methylated and non-methylated) to act as selective G-quadruplex binders with promising anticancer activity.