5-Fluorouracil (5-FU) is a pyrimidine-based antimetabolite that has been widely used for treating various cancers. It exerts the anti-cancer effects predominantly through inhibition of thymidylate synthase (TS) activity by its monophosphate metabolite known as 5-fluoro-2′-deoxyuridine monophosphate (5-FdUMP). Even though 5-FU has been used as the front-line chemotherapeutic drug, the need to administer it by intravenous infusion has limited its therapeutic utility. To overcome this limitation, orally bioavailable 5-fluorouridine based prodrugs such as capecitabine, floxuridine, and deoxyfluridine were developed and used in the clinic. However, these prodrugs need to be anabolized to their active monophosphate metabolite, that is mediated by thymidine kinase (TK). A ProTide of 5-FdUMP (NUC-3373), currently in clinical trials, exhibits promising oral bioavailability and anti-tumor efficacy. However, the monophosphate of these drugs can undergo further phosphorylation to generate the corresponding triphosphate, and incorporated into growing strands of DNA and RNA and causes undesired toxicity. To address this problem, we have developed novel prodrugs of 5-FdUMP that remain largely at the monophosphate stage, thereby preventing their incorporation into DNA. We hypothesized that selective substitutions at the 5′-carbon of the nucleoside place conformational restrictions on the corresponding nucleoside monophosphates that render them poor substrates for cellular kinases. Along with this 5′-substitution innovation, we have also developed prodrugs featuring different lipophilic moieties on the phosphoester group for the targeted delivery to different tissue types, thereby further limiting systemic toxicity. Relative binding energy studies by free energy perturbation (FEP) calculations carried out using the available crystal structures of TS and 5′-substituted 5-FdUMP analogs suggested that 5′(S)-CF3 or 5′(R)-CH3 substitutions at the 5′-carbon should still exhibit potent TS inhibition, whilst limiting further anabolism. To test this, we synthesized various 5′-substituted 5-FdUMP analogs and assessed their TS inhibition ability in vitro. The results, which correlated well with our FEP predictions, demonstrated high TS inhibition activity for the 5′(S)-CF3 and 5′(R)-methyl substituted 5-FdUMP analogs. Cellular metabolism and cytotoxicity studies are underway to assess the therapeutic potential of the 5′-substituted 5-FdUMP analogs, and prodrugs thereof, in oncology.

Development of 5′-substituted 5-fluoro-2′-deoxyuridine monophosphate prodrugs for cancer therapy

ACS Spring 2022

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