The aims of the present study were the synthesis, an original spectroscopic characterization, an evaluation of antiproliferative activities and lipophilicity indices along with pharmacokinetic properties of a novel class of fused azaisocytosine-containing congeners. An optimized, well-established and scalable general method for the synthesis of the target molecules (10–18) via the [4+2] annulation strategy was worked out successfully, which enables reacting the molar equivalents of 1-aryl-2-hydrazinylideneimidazolidine hydroiodides (1–9) and ethyl 4-nitrophenylglyoxylate upon gently heating in n-butanol containing triethylamine and further refluxing in a solvent mixture n-butanol/DMF. The synthesis was hypothesized to proceed via ketimine intermediates that undergo intramolecular cyclization. All the synthesized compounds (10–18) revealed antiproliferative effects in four tumour cell lines of the epithelial origin, exerting superior cytotoxic activities in A549, HeLa and T47D cells to that of a reference drug – pemetrexed. Especially the 12, 13, 14, 15 and 18 seem to be the most promising lead structures for designing more selective cytotoxic agents as they have a lower toxicity for normal epithelial cells after 24-h incubation period. In addition, a number of the innovative antimetabolite-type molecules have been preselected that possess the optimum lipophilicity range significantly correlated with some in silico bioactivity descriptors relevant to the satisfactory pharmacokinetic profile in vivo. Thus, these original molecules might be useful as drug discovery leads to an early phase of drug design.