Comparative Pharmacology of 4’-Oxygen And 4’-Sulfur-Substituted Anti-Cancer Thymidine Analogs

Abstract

Pyrimidine antimetabolites such as 5-fluorouracil and Trifluridine have long been used in cancer therapy due to their ability to disrupt nucleotide metabolism, inhibit thymidylate synthase (TS), and incorporate into DNA and RNA. However, the precise impact of structural modifications on their pharmacological behavior and cellular responses remains incompletely understood. Here, we systematically investigated the metabolic fate, DNA/RNA incorporation, and cytotoxic mechanisms of a panel of clinically relevant thymidine analogs and their 4’-sulfur-substituted counterparts across multiple cancer cell lines. Using several liquid chromatography mass spectrometry methods, immunoblotting, and both in vitro and in vivo cancer models, we identify key structure–function relationships that govern analog activity. We find that sulfur substitution enhances metabolic stability by reducing thymidine phosphorylase (TP)–mediated degradation, enabling prolonged TS inhibition, sustained DNA damage, and apoptosis. Specifically, the efficacy of thio-analogs correlates more strongly with the persistence of DNA damage response activation than with peak analog incorporation. Careful kinetic profiling and supplementation studies further reveal that sulfur-modified analogs disrupt both thymidine and uridine biosynthesis pathways, suggesting broader metabolic engagement than their parent compounds. We report that in vitro, 4’-thio-floxuridine did not demonstrate superior efficacy to the parent compound, floxuridine. In contrast, 4’-thio-trifluridine demonstrates superior efficacy to the parent oxygen containing compound, trifluridine, as well as to all other thymidine analogs tested. The compound is well tolerated in vivo and exhibits a longer half-life than conventional pyrimidine analogs. Taken together, our findings establish thio-nucleosides as stabilized derivatives that are mechanistically distinct with unique cellular and metabolic signatures, offering a framework for the design of next-generation antimetabolite therapies.