The aim of the presented work was to biologically evaluate a series of synthesized mono- and tri-heterocyclic azole derivatives. Acetylphenyl-piperazin-1-yl-alkyl derivatives of spiro[imidazolidine-naphthalene/indene]-2,5-dione, imidazo[2,1-f]purine-2,4-dione and pyrimido[2,1-f]purine-1,3,9-trione were designed as structural counterparts of tipiracil and 7-deazaxanthine, potent thymidine phosphorylase inhibitors (TPIs). The library of compounds was obtained via a multistep synthesis according to the synthetic route published elsewhere. TP inhibitory activity was determined spectrophotometrically according to a modified method of Krenitsky. To establish their cytotoxic effects, the compounds were screened for their in vitro antiproliferative activity against a panel of different human cancer cell lines, namely prostate (PC3) and colon (SW480, SW620), in contrast to normal cell lines – human endothelial microvascular cells (HMEC) and Chinese hamster fibroblast (V79), by the MTT method, using doxorubicin as a reference compound. The anticancer profile of the tested derivatives, expressed as IC₅₀ values, showed that the series of spiro[imidazolidyne-naphthalene/indene]-2,5-dione derivatives was incomparably more potent than the purine derivatives. The most active compound (ACz 512) exhibited growth-inhibitory potency on SW480 (IC₅₀ = 16.8 ± 2.37 µM), SW620 (IC₅₀ = 12.9 ± 3.16 µM) and PC3 (IC₅₀ = 20.58 ± 3.35 µM). Furthermore, the highest selectivity index (SI) was achieved for compound ACz 512. Cell viability of 50% was observed above 100 µM for ACz 512 with HMEC cells. Evaluation of potential TP inhibitory activity from the selected compounds demonstrated that ACz 512 exhibited low activity (21.21% of inhibition, 100 μM). The data allowed the identification of a lead-like structure for anticancer activity based on TP inhibition.