A 5'-(R)-CH3-substituted 5-fluoro-2'-deoxyuridine monophosphate reduces off-target toxicities while maintaining efficacy in a colorectal cancer model

Since its approval in the early 1960s, 5-fluorouracil (5-FU) has remained an important therapeutic for the treatment of late-stage and metastatic colorectal Cancer (CRC). It acts through intracellular conversion to 5-fluoro-2'-deoxyuridine monophosphate (FdUMP) to inhibit Thymidylate Synthase (TYMS), leading to nucleotide pool imbalance, DNA damage, and disruption of tumor cell proliferation. However, 5-FU is limited by rapid clearance and off-target toxicities, which affects a large proportion of patients with CRC. To address these issues, we developed 5'-(R)-CH₃-FdUMP (Me-FdUMP), a 5'-(R)-CH₃-substituted analog of FdUMP that retains inhibitory activity against purified TYMS. Here, we show that Me-FdUMP is resistant to metabolism by phosphatases and kinases, reduces 5-FU formation, and enhances TYMS inhibition in a human CRC cell line. In mice, Me-FdUMP treatment led to markedly lower 5-FU exposure in the heart and bone marrow, 2 key sites of clinical toxicity. Furthermore, in a mouse xenograft model of human CRC, Me-FdUMP maintained antitumor efficacy comparable to FdUMP. Taken together, these results suggest 5'-(R)-CH₃-substituted FdUMP could be a promising new approach for improving the safety of fluoropyrimidine-based therapeutics. SIGNIFICANCE STATEMENT: Current fluoropyrimidine-based therapeutics for colorectal Cancer suffer from metabolic liabilities that can often lead to severe and dose-limiting side-effects. Results reported here highlight a new fluoropyrimidine derivative with enhanced on-target activity in vitro, maintenance of antitumor efficacy in vivo, and impaired metabolism that can reduce exposure of toxic metabolites. This work represents a new strategy to address the shortcomings of current fluoropyrimidine-based therapeutics with the potential to improve patient outcomes.