Discovery of 5 '-Substituted 5-Fluoro-2 '-deoxyuridine Monophosphate Analogs: A Novel Class of Thymidylate Synthase Inhibitors

5-Fluorouracil and 5-fluorouracil-based prodrugs have been used clinically for decades to treat cancer. Their anticancer effects are most prominently ascribed to inhibition of thymidylate synthase (TS) by metabolite 5-fluoro-2 '- deoxyuridine 5 '-monophosphate (FdUMP). However, 5-fluorouracil and FdUMP are subject to numerous unfavorable metabolic events that can drive undesired systemic toxicity. Our previous research on antiviral nucleotides suggested that substitution at the nucleoside 5 '-carbon imposes conformational restrictions on the corresponding nucleoside monophosphates, rendering them poor substrates for productive intracellular conversion to viral polymerase-inhibiting triphosphate metabolites. Accordingly, we hypothesized that 5 '-substituted analogs of FdUMP, which is uniquely active at the monophosphate stage, would inhibit TS while preventing undesirable metabolism. Free energy perturbation-derived relative binding energy calculations suggested that 5 '(R)-CH3 and 5 '(S)-CF3 FdUMP analogs would maintain TS potency. Herein, we report our computational design strategy, synthesis of 5 '-substituted FdUMP analogs, and pharmacological assessment of TS inhibitory activity.