Synthesis and antitumor evaluation of bis[(pivaloyloxy)methyl]2'-deoxy-5- fluorouridine 5'-monophosphate (FdUMP): A strategy to introduce nucleotides into cells

Article abstract of DOI:10.1021/jm00049a009

The bis[(pivaloyloxy)methyl] [PIV₂] derivative of 2'-deoxy-5- fluorouridine 5'-monophosphate (FdUMP) was synthesized as a potential membrane-permeable prodrug of FdUMP. The compound was designed to enter cells by passive diffusion and to revert to FdUMP after removal of the PIV groups by hydrolytic enzymes. The most convenient preparation of PIV₂FdUMP was by condensation of 2'-deoxy-5-fluorouridine (FUdR) with PIV₂ phosphate in the presence of triphenylphosphine and diethyl azodicarboxylate (the Mitsunobo reagent). PIV₂FdUMP was stable in the pH range 1.0-4.0 (t( 1/2 ) > 100 h). It was also fairly stable at pH 7.4 (t( 1/2 ) = 40.2 h). In 0.05 M NaOH solution, however, it was rapidly degraded (t( 1/2 ) < 2 min). In the presence of hog liver carboxylate esterases, PIV₂FdUMP was converted quantitatively to the mono-[(pivaloyloxy)methyl] [PIV₁] analogue PIV₁FdUMP. After a 24 h incubation, only trace amounts of FdUMP (1-3%) were observed, indicating that PIV₁FdUMP is a poor substrate for carboxylate esterases. In mouse plasma, PIV₂FdUMP was rapidly metabolized, first to PIV₁FdUMP and then to FdUMP. With continued incubation, FUdR was formed, presumably due to further catabolism of FdUMP by plasma phosphatases or 5'-nucleotidases. Since PIV₁FdUMP is a poor substrate for carboxylate esterase, the cleavage of the second PIV group is most likely mediated by plasma phosphodiesterases. The rate of degradation of PIV₂FdUMP in the presence of acid and alkaline phosphatase, 5'-nucleotidase, or spleen phosphodiesterase was the same as that in buffer controls, indicating that the compound is not a substrate for these nucleotide catabolizing enzymes. The concentration of PIV₂FdUMP and its 3'-O-acetyl ester (PIV₂ 3'-O-Ac-FdUMP) required to inhibit the growth of Chinese hamster ovary (CHO) cells in vitro to less than 50 cells per colony was 5 x 10^-6 M, the same as that required for 5-fluorouracil (FU). Both nucleotide prodrugs showed the same growth-inhibitory potency against a mutant CHO cell line that was 20-fold resistant to FU (CHO/FU). Administered intraperitoneally at optimal dosage for 5 consecutive days, PIV₂FdUMP and PIV₂ 3'-O-Ac-FdUMP were as effective as FU at prolonging the life spans of mice bearing intraperitoneally implanted P388 leukemia. Both prodrugs retained full therapeutic activity against a P388 subline resistant to FU. Collectively, these data indicate that PIV₂FdUMP and PIV₂ 3'-O-Ac-FdUMP are effective membrane-permeable prodrugs of FdUMP.