Triazole-Linked Pyrimidine Nucleoside Dimers: Design, Synthesis, and Applications in Medicinal and Nucleic Acid Chemistry. Part IV.

A new class of pyrimidine nucleoside dimers featuring an ultrashort 1,4-disubstituted 1,2,3-triazole linker was designed and synthesized to explore the biological potential of structurally robust nucleoside analogues as anticancer agents. The target compounds were efficiently obtained via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) from appropriately functionalized 3'-azido- and 4'-ethynyl-5'-deoxy-5'-nor nucleoside precursors and fully characterized by multinuclear 1D/2D NMR spectroscopy and ESI-MS. Their antiproliferative activity was evaluated against a panel of human cancer cell lines comprising cervical (HeLa), breast (T47D), ovarian (SK-OV-3), and glioblastoma (U-138 MG and U-251 MG) cells, with MRC-5 human fibroblasts serving as the control. Several dimers displayed pronounced and cell line-selective antiproliferative activity while exhibiting limited toxicity toward normal cells. Dimers 4, 9, 11, and 17 demonstrated the highest activity against HeLa cells, whereas Dimers 7 and 15 were the most potent against U-251 MG glioblastoma cells, with activities comparable to or exceeding that of 5-fluoro-2'-deoxyuridine (5FdU). Mechanistic studies performed on the most active glioblastoma-directed dimers revealed a concentration-dependent reduction in clonogenic survival and cell migration, accompanied by increased cell death and pronounced S-phase cell cycle arrest, closely resembling the effect of the reference compound 5FdU. In contrast, only a modest and transient increase in intracellular reactive oxygen species (ROS) was detected, indicating that oxidative stress is unlikely to represent the primary mechanism responsible for their antiproliferative activity. Preliminary structure-activity relationship analysis demonstrated that both the nature of the nucleobase and the rigid triazole-linked dimeric architecture substantially influence biological activity and selectivity. Moreover, the synthesized dimers exhibited excellent chemical stability in cell culture medium and human serum, highlighting their exceptional resistance to degradation. Taken together, these findings identify triazole-linked pyrimidine nucleoside dimers as highly stable bioactive scaffolds that inhibit glioblastoma cell proliferation through disruption of DNA synthesis rather than oxidative stress and represent promising candidates for further development as anticancer agents or durable nucleic acid-targeting molecular tools.