A molecular modeling study of the interaction of 2'-fluoro-substituted analogues of dUMP/FdUMP with thymidylate synthase.

Molecular dynamics simulations and free energy calculations are presented, exploring previously described experimentally studied interactions of a series of 2'-fluoro-substituted dUMP/FdUMP analogues with thymidylate synthase (TS). The results show the inhibitory behaviors of 2'-F-ara-UMP, 2',2''-diF-dUMP and 2',5-diF-ara-UMP to be dependent upon the binding positions and orientations adopted by the molecules of these compounds in the active site of TS. The binding mode of 2',5-diF-ara-UMP suggests a novel role of the active site residue Trp 80, stabilizing through hydrophobic stacking the binding position of the pyrimidine ring in 2',5-diF-ara-UMP.

Sulfonyl-containing nucleoside phosphotriesters and phosphoramidates as novel anticancer prodrugs of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP).

A series of sulfonyl-containing 5-fluoro-2'-deoxyuridine (FdU) phosphotriester and phosphoramidate analogues were designed and synthesized as anticancer prodrugs of FdUMP. Stability studies have demonstrated that these compounds underwent pH dependent beta-elimination to liberate the corresponding nucleotide species with half-lives in the range of 0.33-12.23 h under model physiological conditions in 0.1 M phosphate buffer at pH 7.4 and 37 degrees C. Acceleration of the elimination was observed in the presence of human plasma. Compounds with an FdUMP moiety (4-9) were considerably more potent than those without (1-3) as well as 5-fluorouracil (5-FU) against Chinese hamster lung fibroblasts (V-79 cells) in vitro. Addition of thymidine (10 microM) reversed the growth inhibition activities of only 5-FU and the compounds with an FdUMP moiety, but had no effect on those without. These results are consistent with thymidylate synthase as the target of the prodrugs.

Synthesis and anticancer activities of amphiphilic 5-fluoro-2'-deoxyuridylic acid prodrugs.

Amphiphilic anticancer prodrugs of 5'-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP) were synthesized according to the hydrogen phosphonate method by coupling lipophilic cytosine derivatives or a phospholipid with 5-fluoro-2'-deoxyuridine (5-FdU). Studies within the in vitro Anticancer Screen Program of the National Cancer Institute have demonstrated high anticancer activities of the heterodinucleoside phosphates: N4-palmitoyl-2'-deoxycytidylyl-(3' --> 5')-3'-O-acetyl-5-fluoro-2'-deoxyuridine (dC(pam)-5-FdU(Ac), N4-palmitoyl-2',3'-dideoxycytidylyl-(5' --> 5')-3'-O-acetyl-5-fluoro-2'-deoxyuridine (ddC(pam)-(5' --> 5')-5-FdU(Ac), 5-fluoro-2'-deoxyuridylyl-(3' --> 5')-5-fluoro-N4-hexadecyl-2'-deoxycytidine (5-FdU-5-FdC(hex)), and of the new liponucleotide 1-O-octadecyl-rac-glycerylyl-(3 --> 5')-5-fluoro-2'-deoxyuridine (Oct1Gro-(3 --> 5')-5-FdU). The anticancer activities of these prodrugs are comparable to those of 5-FdU and the tumor specificities are modulated by their structures. The highest cytotoxic activity being even superior to 5-FdU was expressed by the dimer 5-FdU-5-FdC(hex).

Synthesis and biological studies of novel nucleoside phosphoramidate prodrugs.

A novel approach to the intracellular delivery of nucleotides using phosphoramidate-based prodrugs is described. Specifically, we have developed phosphoramidate prodrugs of the anticancer nucleotide 5-fluoro-2'-deoxyuridine-5'monophosphate (FdUMP). These phosphoramidate prodrugs contain an ester group that undergoes intracellular activation liberating phosphoramidate anion, which undergoes spontaneous cyclization and P-N bond cleavage to yield the nucleoside monophosphate quantitatively. In vitro evaluation of 5-fluoro-2'-deoxyuridine phosphoramidate prodrugs 2a and 3b against L1210 mouse leukemia cells show potent inhibition of cell growth (IC(50) 0.5-3 nM). Cell-based thymidylate synthase inhibition studies show that, in contrast to FUdR, the nitrofuran compound 2a is of comparable potency in wild type vs thymidine kinase deficient LM cells. This result indicates that the activation of this novel prodrug occurs via the proposed mechanism of intracellular delivery. However, naphthoquinone 3b has an IC(50) value for thymidylate synthase inhibition that is comparable to FUdR in thymidine kinase deficient cells. Further studies revealed that 3b rapidly decomposes to the nucleotide in cell culture medium, suggesting that the naphthoquinone analogue is not sufficiently stable to function as a nucleotide prodrug.

Targeted drug delivery to chemoresistant cells: folic acid derivatization of FdUMP[10] enhances cytotoxicity toward 5-FU-resistant human colorectal tumor cells.

Current chemotherapy protocols that include fluoropyrimidines, such as 5-fluorouracil (5-FU), are limited by the development of chemoresistance during the course of treatment. Our laboratory has developed a novel class of fluoropyrimidines, FdUMP[N], that are oligodeoxynucleotides (ODNs) composed of some number, N, of 5-fluoro-2'-deoxyuridine-5'-O-monphosphate (FdUMP) nucleotides. Novel synthetic procedures are described that permit conjugation of folic acid to the 5'-OH of FdUMP[10] via a phosphodiester linkage using automated synthesis. The synthetic methods developed are generally applicable for ODN conjugation with folic acid. The folic acid conjugate FA-FdUMP[10] showed improved cytotoxicity toward human colorectal tumor cells (H630), and 5-FU-resistant colorectal tumor cells (H630-10). Enhanced cytotoxicity was observed for FA-FdUMP[10] relative to nonconjugated FdUMP[10] for cells grown under folate-restricted conditions, consistent with cellular uptake being, in part, receptor-mediated. Folate receptor alpha (FRalpha) mRNA was shown by RT-PCR to be overexpressed 26.3-fold in 5-FU-resistant H630-10 cells relative to H630 cells. Thus, FA-FdUMP[N] may prove useful for the treatment of 5-FU-resistant malignancies.

Synthesis and interactions with thymidylate synthase of 2,4-dithio analogues of dUMP and 5-fluoro-dUMP.

The 2,4-dithio analogues of 2'-deoxyuridine and 2'-deoxy-5-fluorouridine have been synthesized by thiation of the previously described 2-thio analogues, and then phosphorylated enzymatically or chemically to yield 2,4-dithio-dUMP and 2,4-dithio-5-fluoro-dUMP. In striking contrast to the 2-thio and 4-thio analogues of dUMP, which are good substrates of thymidylate synthase, 2,4-dithio-dUMP is not a substrate. But, surprisingly, it is a competitive inhibitor, relative to dUMP, of the purified enzymes from both parental and FdUrd-resistant L1210 cells, with K(i) values of 32 microM and 55 microM, respectively. Although 2,4-dithio-5-fluoro-dUMP behaved as a typical slow-binding inhibitor of the enzyme, its K(i) value was 10(3)-10(4)-fold higher than those for the corresponding 2-thio and 4-thio congeners. Similarly, 2,4-dithio-FdUrd was a much weaker inhibitor of tumour cell growth (IC50 approximately 10(-5)M) than FdUrd (IC50 approximately 10(-9)M), 2-thio-FdUrd(IC50 approximately 10(-7)M) or 4-thio-FdUrd (IC50 approximately 5x10(-8)M), while with 2,4-dithio-dUrd no influence on cell growth could be observed. Theoretical considerations, based on calculated aromaticities of the uracil and thiouracil rings, suggest that lack of substrate activity of 2,4-dithio-dUMP may result from increased pyrimidine ring aromaticity of the latter, leading to resistance of C(6) to nucleophilic attack by the enzyme active center cysteine.

5'-[4-(Pivaloyloxy)-1,3,2-dioxaphosphorinan-2-yl]-2'-deoxy-5-fluorouridine: a membrane-permeating prodrug of 5-fluoro-2'-deoxyuridylic acid (FdUMP).

5'-[4-(Pivaloyloxy)-1,3,2-dioxaphosphorinan-2-yl]-2'-deoxy-5 -fluorouridine (1c) was designed as a potential membrane-permeable prodrug of 2'-deoxy-5-fluorouridine 5'-monophosphate (FdUMP), a putative active metabolite of the antitumor drug 5-fluorouracil (FU). It was anticipated that 1c would be hydrolyzed in vivo by carboxylate esterase (E.C. 3.1.1.1) to the labile 4-hydroxy analogue 2a, which should penetrate cells by passive diffusion and ring open to the aldehyde 3a. Spontaneous elimination of acrolein from 3a would then generate the free nucleotide, FdUMP. 1c might also penetrate cells directly and undergo the same degradation sequence after hydrolysis by cellular esterases. 1c was prepared by condensing 2-hydroxy-2-oxo-4-(pivaloyloxy)-1,3,2-dioxaphosphorinane with 2'-deoxy-5-fluorouridine (FUdR) in the presence of triphenylphosphine and diethyl azodicarboxylate. 1c was moderately stable in aqueous buffers over the pH range 1-7.4 (T1/2 > 30 h). In the presence of carboxylate esterase, however, it was degraded, in a concentration-dependent manner, to FdUMP. No intermediates were detected in the incubation mixture. In mouse plasma, 1c was degraded first to FdUMP and then to FUdR. The latter is presumably formed by dephosphorylation of FdUMP by plasma 5'-nucleotidases or phosphatases. 1c and FU inhibited the growth of Chinese hamster ovary (CHO) cells in culture at a concentration of 5 x 10(-6) M. 1c was equally potent against a CHO variant that was 20-fold resistant to FU. Administered intraperitoneally for 5 consecutive days, 1c was as effective as FU at prolonging the life span of mice bearing P-388 leukemia. In the presence of 2-mercaptoehtanesulfonic acid, an acrolein scavenger, 1c was equally effective against a P-388 mutant cell line that was resistant to FU. Collectively, these data suggest that 1c acts as a membrane-permeable prodrug of FdUMP. This prodrug strategy may be generally useful for introducing dianionic phosphates and phosphonates into cells.

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

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.

Interaction of 5-fluoro-4-thio-2'-deoxyuridine 5'-phosphate with mammalian tumour thymidylate synthase: role of the pyrimidine N(3)-H dissociation.

The role of the pyrimidine N(3)-H in binding of dUMP derivatives to thymidylate synthase was evaluated with the aid of a new dUMP analogue, 5-fluoro-4-thio-dUMP, synthesized by an improved thiation and enzymatic phosphorylation. The interaction of this analogue, and of 5-FdUMP, with the enzyme, and the pH-dependence of these interactions, were compared. Both were slow-binding competitive inhibitors of the enzyme from Ehrlich carcinoma, L1210 and CCRF-CEM cells, with Ki an order of magnitude higher for 5-fluoro-4-thio-dUMP than for 5-FdUMP. With both nucleotides, as well as the parent nucleosides, enzyme inactivation increased as the pH was lowered from 8 to 6. Maximum inactivation with 5-FdUrd was at pH 7.0, and with 5-fluoro-4-thio-dUrd at pH 6.0, in agreement with the higher pKa for the N(3)-H dissociation of the former, and pointing to participation of the N(3)-H as a hydrogen donor in binding to the enzyme.

Synthesis and biological evaluation of neutral derivatives of 5-fluoro-2'-deoxyuridine 5'-phosphate.

5-Fluoro-5'-(2-oxo-1,3,2-oxazaphosphorinan-2-yl)-2'-deoxyuridine (1a) and 5-fluoro-5'-(2-oxo-1,3,2-dioxaphosphorinan-2-yl)-2'-deoxyuridine (1b) were prepared by reaction of 5-fluoro-2'-deoxyuridine (7a) and phosphoryl chloride with 3-amino-1-propanol and 1,3-propanediol, respectively. The thymidine analogues, 1c and 1d, were prepared similarly from thymidine. Compound 1b was synthesized in better yield from 13a and trimethylene phosphate with triphenylphosphine/diethyl azodicarboxylate as a condensing agent. Compounds 1a-d were resistant to degradation by 5'-nucleotidase, alkaline phosphatase, venom phosphodiesterase, and crude snake venom. None of these compounds were significantly biotransformed when incubated with mouse hepatic microsomal preparations in the presence of an NADPH-generating system. When administered intraperitoneally (ip) for 5 consecutive days, 1a was nearly as effective as 5-fluorouracil at prolonging the life spans of BDF1 mice implanted intraperitoneally with leukemia P-388. However, much larger dosages of 1a were required for optimal activity. Compound 1b administered similarly was only marginally effective. Neither 1a nor 1b was active against a P-388 mutant resistant to 5-fluorouracil.

Utilization of 5-fluoro-2'-deoxyuridine triphosphate and 5-fluoro-2'-deoxycytidine triphosphate in DNA synthesis by DNA polymerases alpha and beta from calf thymus.

Chemically synthesized 5-fluoro-2'-deoxyuridine 5'-triphosphate and 5-fluoro-2'-deoxycytidine 5'-triphosphate were used efficiently as substitutes for DNA synthesis catalyzed by DNA polymerases alpha or beta from calf thymus. 5-fluoro-2'-deoxyuridine 5'-triphosphate and 5'-fluoro-2'-deoxycytidine 5'-triphosphate were incorporated into DNA in place of deoxythymidine 5'-triphosphate and deoxycytidine 5'-triphosphate, respectively. The incorporated pyrimidine analogs supported further elongation of DNA. The apparent Km's for 5-fluorodeoxyuridine 5'-triphosphate in the reaction of DNA polymerases alpha and beta were 4.3 and 15.4 microM, while those of 5-fluorodeoxycytidine 5'-triphosphate with DNA polymerases alpha and beta were 7.7 and 8.8 microM, respectively, which are comparable to Km's for natural substrates. These results suggest the new possibility that the fluorinated pyrimidines are incorporated into DNA via their triphosphate forms to exhibit their cytostatic actions.

Synthesis and biological activity of 5-fluoro-2'-deoxyuridine 5'-phosphorodiamidates.

Three 5'-phosphorodiamidate derivatives of 5-fluoro-2'-deoxyuridine (FdUrd), 5-fluoro-2'-deoxyuridine 5'-phosphorodiamidate (4a), 5'-phosphorodiimidazolidate (4b), and 5'-phosphorodimorpholidate (4c), were synthesized by aminolysis of 5-fluoro-2'-deoxyuridine 5'-phosphorodichloridate with the respective amine. In culture, these 5'-phosphorodiamidates inhibited the growth of murine leukemia (L5178Y) cells. 5-Fluoro-2'-deoxyuridine 5'-phosphorodiamidate (4a) was the most active derivative and, on a molar basis, produced a cytostatic effect comparable to that of FdUrd and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUrd-5'-P). Compounds 4b and 4c were less active than 4a, with relative rates of activity 4a > 4b > 4c that corresponded to their rates of hydrolysis to FdUrd-5'-P. None of the 5'-phosphorodiamidates inhibited thymidylate synthetase of concentrations up to 1 mM.

5-Fluoro-2'deoxyuridine 5'-(p-azidophenyl phosphate), a potential photoaffinity label of thymidylate synthetase.

5-Fluoro-2'-deoxyuridine 5'-(p-azidophenyl phosphate) (1), a potential photoaffinity labeling reagent for thymidylate synthetase from a methotrexate-resistant strain of Lactobacillus casei, has been synthesized and characterized. UV254 irradiation of mixtures of thymidylate synthetase with 1, containing 14C-labeled phenyl and 3H-labeled pyrimidine rings, in the presence of excess 5,10-methylenetetrahydrofolate, the cofactor for the reaction, produced two complexes, separable from the native enzyme by polyacrylamide gel electrophoresis, in which only the 3H-containing moiety was bound to the protein. When mixtures of enzyme and 1 were irradiated in the absence of cofactor, complexes separable from the native enzyme were not observed. However, the 14C-containing component of 1 was now bound to the protein in the absence of the 3H-containing portion. The results are discussed in terms of the topography of the enzyme active site.

Phosphonate analogue of 2'-deoxy-5-fluorouridylic acid.

The phosphonate analogue (6) of 2'-deoxy-5-fluorouridylic acid has been prepared via a Pfitzner--Moffatt oxidation and Witting reaction. This compound was found to inhibit thymidylate synthetase from three sources and to be cytotoxic to H.Ep.-2 cells in culture.

Fluorescent inhibitors of thymidylate synthetase.

Two fluorescent derivatives of 2'-deoxy-5-fluorouridine 5'-p-aminophenyl phosphate were prepared by treatment of this compound with fluorescein isothiocyanate in dimethyl sulfoxide or 5-(dimethylamino)naphthalene sulfonyl chloride in pyridine. The products of the reactions were isolated by diethylaminoethylcellulose chromatography and were shown to be homogeneous by polyacrylamide electrophoresis and TLC. Confirmation of the structure was provided by elemental analysis, absorption and fluorescence spectra, PMR measurements, and liberation of nucleotide upon hydrolysis with snake venom phosphodiesterase. The fluorescent derivatives are good competitive inhibitors (Ki approximately10(-6) M) of thymidylate synthetase from a methotrexate-resistant strain of Lactobacillus casei.