A novel series of tetracyclic imidazo[4,5-b]pyridine derivatives was designed and synthesized as potential antiproliferative agents. Their antiproliferative activity against human cancer cells was influenced by the introduction of chosen amino side chains on the different positions on the tetracyclic skeleton and particularly, by the position of N atom in the pyridine nuclei. Thus, the majority of compounds showed improved activity in comparison to standard drug etoposide. Several compounds showed pronounced cytostatic effect in the submicromolar range, especially on HCT116 and MCF-7 cancer cells. The obtained results have confirmed the significant impact of the position of N nitrogen in the pyridine ring on the enhancement of antiproliferative activity, especially for derivatives bearing amino side chains on position 2. Thus, regioisomers 6, 7 and 9 showed noticeable enhancement of activity in comparison to their counterparts 10, 11 and 13 with IC50 values in a nanomolar range of concentration (0.3-0.9 μM). Interactions with DNA (including G-quadruplex structure) and RNA were influenced by the position of amino side chains on the tetracyclic core of imidazo[4,5-b]pyridine derivatives and the ligand charge. Moderate to high binding affinities (logKs = 5-7) obtained for selected imidazo[4,5-b]pyridine derivatives suggest that DNA/RNA are potential cell targets.