The intention of this study was to synthesize new amino substituted tetracyclic derivatives of imidazo[4,5-b]pyridines and to examine their spectroscopic properties as well as potential application as pH sensors. For the preparation of the target compounds, classical methods of organic synthesis as well as microwave-assisted synthesis were used. Cyclic product, 2-(5-chloro-1H-imidazo[4,5-b]pyridine-2-yl)acetonitrile 3, was prepared by condensation of 2,3-diamino-6-chloropyridine 1 with 2-cyanoacetamide 2. Due to the condensation of cyclic compound 3 with 2-chloro-benzaldehyde 4, an acyclic precursor 5 was synthesized. The thermal cyclization of the acyclic precursor 5 provided a mixture of regioisomers 6a and 6b. Amino substituted tetracyclic derivatives of imidazo[4,5-b]pyridine 7 – 14 were obtained via uncatalyzed microwave assisted amination of regioisomers 6a and 6b. Synthesis of compound 10 was not successful. The structures of all newly synthesizes compounds were confirmed by means of 1H NMR and 13C NMR spectroscopy. The structures of the separated regioisomers were confirmed utilizing 2D NMR spectroscopy. Spectroscopic characterization of regioisomers 7 and 8 was performed by using UV/Vis and fluorimetric spectroscopy in 11 organic solvents of different polarities. From the obtained results, significant influence of the solvent on the spectroscopic characteristics of regioisomers was observed. pH titrations were performed to determine the potential application of prepared regioisomers 7 and 8 as optical pH sensors. From the obtained results it can be concluded that the change of pH causes changes in the intensity and wavelengths of the maximum absorbance. Synthesized derivatives were tested also for their antiproliferative activity in vitro on three cancer cell lines and non-tumor cells. Some of the derivatives displayed very strong antiproliferative activity in nanomolar range of inhibitory concentrations.