Synthesis and Antitumor Evaluation of Bis[(pivaloyloxy)methyl] 2'-Deoxy-5-fluorouridine 5'-Monophosphate (FdUMP): A Strategy To Introduce Nucleotides into Cells

Abstract

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 (t1/2 > 100 h). It was also fairly stable at pH 7.4 (t1/2 = 40.2 h). In 0.05 M NaOH solution, however, it was rapidly degraded (t1/2 < 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.

Nucleotide Delivery fromcycloSaligenyl-3′-azido-3′-deoxythymidine Monophosphates (cycloSal-AZTMP)

“…The major differences between the approaches are the delivery mechanisms of the nucleotides. While almost all approaches based on chemical hydrolysis reported to date have proved to be unable to deliver the nucleotide selectively and consequently serve only as nucleoside "depots", the concepts based on enzymatic activation as in the bis-POM phosphotriesters [14] , the bis-SATE phosphotriesters [15] and the aryloxyphosphoramidates [16] demonstrated the successful intracellular delivery of free nucleotides from highly lipophilic precursors. As part of our ongoing program to develop efficient pronucleotide systems [17] , we have reported on the design and synthesis of an entirely new pro-nucleotide approach based on a pH-driven selective chemical hydrolysis: the cyclosaligenyl nucleoside monophosphates (cycloSal-NMP) [18] .…”

A phosphorylated prodrug for the inhibition of Pin1

“…16, 17 Among the various approaches developed for reversibly masking phosphate compounds, the bisPOM (bis-pivaloyloxymethyl) strategy appears to be quite useful. 16,19,22,23 BisPOM derivatives are generally quite stable in buffer and plasma, and they are readily transformed to free phosphate derivatives inside various cell types. 16 We now describe the design and synthesis of two Pin1 inhibitors containing the pSer-Ψ[(Z) CH=C]-Pro isostere, 1 and 2 ( Figure 1).…”

Cytotoxicity of Pivoxil Esters of Antiviral Acyclic Nucleoside Phosphonates: Adefovir Dipivoxil Versus Adefovir

“…However, the introduction of pivoxil moiety in the molecule of bis-POM-PMEA led to the occurrence of inhibitory effects on NO production gested to contribute to allergic respiratory disease 24 , to be a risk factor for initiation of endothelial injury leading to atherosclerosis 25 , and for neurogenic inflammation 26 . Inasmuch as some drugs including antibiotics [27][28][29] and anticancer agents 30,31 are pharmaceutically formulated as pivoxil prodrugs, careful evaluation of their possibly formaldehyde-related side effects and therapeutic implications warrant special consideration.…”