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

Abstract

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.

Figure 1

Metabolic activation of 5-fluorouracil (5-FU) to 5-fluoro-2′-deoxyuridine 5′-monophosphate (FdUMP, TS inhibitor), 5-fluoro-2′-deoxyuridine 5′-triphosphate (FdUTP, DNA polymerase substrate), and 5-fluorouridine 5′-triphosphate (FUTP, RNA polymerase substrate). TP, thymidine phosphorylase; TK, thymidine kinase; OPRT, orotate phosphoribosyl transferase; PRPP, phosphoribosyl pyrophosphate; RNR, ribonucleotide reductase; TS, thymidylate synthase (PDB ID 1HZW).

Figure 2

Early prodrug strategies rely on release of 5-FU to generate FdUMP, whereas other strategies (e.g., NUC-3373) generate FdUMP directly without 5-FU release.

Figure 3

Repurposing the nucleoside/nucleotide 5′-functionalization strategy for FdUMP. R groups of interest include small substituents, such as CH₃, CF₃, etc.

Figure 4

Glide-SP docking poses of (A) the 5′(R)-CH₃ and (B) the 5′(S)-CF₃ analogs of FdUMP in hTS (PDB 6QXG) overlaid with FdUMP (in yellow) from the cocrystal structure. (C) Graphical representation of the “substitution tolerant” zone (green sphere) and the “substitution intolerant” zone (red sphere).

Figure 5

Novel 5′-substituted FdUMP analogs 1–5.

Scheme 1. Synthesis of 5′-Functionalized FdUMP Analogs

Reagents and conditions: (a) TBSCl, imidazole, DMAP, DMF, rt, 87%; (b) PPTS, MeOH, rt, 67%; (c) TEMPO, PhI(OAc)₂, MeCN/THF/H₂O, rt, 85%; (d) Me(OMe)NH-HCl, T3P, EtOAc/pyridine, rt (e) MeMgBr, THF, −78°C, 71%, over 2 steps; (f) 9, HCO₂Na, H₂O/EtOAc, rt, 75%, 98:2 dr; (g) i, POCl₃, pyridine, MeCN/H₂O, 0°C to rt; ii, NH₄F, H₂O, rt; iii, Dowex-Li⁺, rt, 7%; (h) 10, HCO₂Na, H₂O/EtOAc, rt, 83%, 7:3 dr; (i) TBAF, THF, 0°C; (j) Ac₂O, DMAP, pyridine, rt, 47% over 2 steps; (k) 10, HCO₂Na, H₂O/EtOAc, rt, 67%, 99:1 dr; (l) POCl₃, pyridine, MeCN/H₂O, 0°C to rt, 20%; (m) i, NH₃(aq), H₂O, rt; ii, Dowex-Li⁺, rt, 27% over 2 steps; (n) TMSCHN₂, toluene, MeOH, rt, 86%; (o) MeMgBr, THF, 0°C to rt, 72%; (p) i, POCl₃, trimethyl phosphate, 0°C to rt, 14%; ii, Dowex-Li⁺, rt, 12%; (q) TrCl, pyridine, μwave, 100°C, 78%; (r) NaH, BnBr, THF, rt, 67%; (s) 80% AcOH/H₂O, rt, 81%; (t) DMP, DCM, rt; (u) TMSCF₃, TBAF, THF, 0 °C to rt, then 0.5 N HCl, rt, silica gel chromatography (EtOAc/hexanes; 0–80%), 45% over 2 steps, 15 (S), 27%; 16 (R), 18%; (v) POCl₃, pyridine, MeCN/H₂O, 0°C to rt; (w) Pd(OH)₂, H₂, MeOH, rt (4 (S), 12%, over 2 steps; 5 (R), 6%, over 2 steps).

Figure 6

Concentration–response curves of FdUMP (a) and active 5′-functionalized analogs (b and c) against recombinant hTS-mediated conversion of 2′-deoxyuridine monophosphate (dUMP) and 5,10-methylene tetrahydrofolate (mTHF) to deoxythymidine monophosphate (dTMP) and dihydrofolate (DHF), respectively. The final concentrations of hTS, dUMP, and mTHF in the reaction systems were 0.84 μM, 50 μM, and 250 μM, respectively. The reaction rates were determined by following the change in the optical absorbance at 340 nm, which corresponded to the formation of the coproduct, DHF.