Biochemical and growth inhibition studies of methotrexate and aminopterin analogues containing a tetrazole ring in place of the gamma-carboxyl group.

The biological activities of novel analogues of methotrexate (MTX) and aminopterin (AMT) in which the gamma-carboxyl was replaced by a 1H-tetrazol-5-yl ring, an isosteric group with acidic properties similar to a carboxyl group, were investigated. The tetrazolyl analogues of MTX and AMT were more potent inhibitors of the growth of CCRF-CEM and K562 human leukemia cell lines during continuous (120 h) and 24-h pulse exposure than were the respective parent drugs; only when the exposure time was reduced to 6 h were the parent drugs more potent. These inhibitory effects on growth correlated with the onset of and recovery from inhibition of de novo thymidylate biosynthesis. Growth inhibition by the analogues was protectable by leucovorin. MTX-resistant CCRF-CEM sublines with decreased transport or increased dihydrofolate reductase (DHFR) levels were cross-resistant to the analogues. The analogues were as potent as their parent drugs in inhibiting DHFR activity in vitro and at displacing [3H]MTX from intracellular DHFR. Each analogue was more effective than its parent drug at inhibiting uptake of [3H]MTX into CCRF-CEM cells. The tetrazole analogue of AMT was a linear competitive inhibitor (Kis = 50 microM) of CCRF-CEM folylpolyglutamate synthetase, while the tetrazole analogue of MTX, unlike all other inhibitors, was linear noncompetitive (Kis = 51 microM, Kii = 321 microM). The data suggest that, compared with MTX or AMT, the tetrazole substituent, in place of the gamma-carboxyl group, allows more efficient transport into cells via the reduced folate/MTX carrier and the resulting greater uptake of the analogues leads to inhibition of DNA synthesis and cell death at lower extracellular concentrations during long exposures. The mechanism of cell death could involve inhibition at folypolyglutamate synthetase, but DHFR is the primary target. The low potency of the analogues during short exposure is presumably related to the inability to form the poly-gamma-glutamyl metabolites required for intracellular retention.

The phenotype of a dihydrofolate reductase mutant of Saccharomyces cerevisiae.

We have constructed a dihydrofolate reductase mutant (dfr1) of Saccharomyces cerevisiae. The mutant has auxotrophic growth requirements for the C1 metabolites dTMP, adenine, histidine and methionine, similar to those of wild-type (wt) strains grown in the presence of methotrexate (MTX). However, unlike wt strains treated with MTX, the growth requirements of the dfr1 mutant are not satisfied by exogenous 5-formyltetrahydrofolic acid (FA; folinic acid) in complex (YEPD) medium. This result is surprising, as yeast cells treated with MTX are expected to be phenocopies of dfr1 mutants. The inability of the mutants to metabolize FA suggests that the DFR1 gene product may have a role in folate metabolism in addition to its well-characterized function in the reduction of dihydrofolate. From dfr1 strains, we have isolated secondary mutants whose growth can be supported by FA in YEPD medium. This FA-utilizing phenotype is attributable to recessive mutations which we have designated fou. In addition to their inability to metabolize FA, the dfr1 strains are unable to grow on medium containing the non-fermentable carbon source glycerol, suggesting that the DFR1 gene product is also required for mitochondrial function. In order to overcome this lack of respiratory activity in the dfr1 mutants, we isolated strains containing a dominant mutation, DIR, which allows growth on glycerol in the presence of antifolate drugs. When crossed into dfr1 strains, the DIR mutation conferred respiratory competence. These strains should be useful in a variety of studies on the genetics and biochemistry of folate metabolism in this simple eukaryote.

Synthesis and biological activity of novel folic acid analogues: pteroyl-S-alkylhomocysteine sulfoximines.

The synthesis of a novel series of gamma-substituted folic acid analogues, pteroyl-S-alkyl-DL-homocysteine (RS)-sulfoximines, and the corresponding S-methylhomocysteine sulfone is described. Side reactions of the sulfoximine groups of the amino acid ester reactants were considered. The correct structures of the isolated target compounds were confirmed by NMR and FAB/MS excluding other alternatives. The replacement of the gamma-COOH of the glutamate moiety of folic acid with S-alkylsulfoximine groups or S-methylsulfone did not affect the substrate activity of the vitamin for dihydrofolate reductase. The resulting tetrahydrofolate analogues could serve as cofactors for the thymidylate synthase cycle of murine leukemia L1210 cells in situ. The analogues inhibited the growth of these cells in culture with 2 orders of magnitude lower IC50 values [(2-4) x 10(-4) M] than the parent folic acid.

Highly water-soluble lipophilic prodrugs of the anti-HIV nucleoside analogue 2',3'-dideoxycytidine and its 3'-fluoro derivative.

The synthesis of a novel series of lipophilic prodrug derivatives of the anti-HIV drugs 2',3'-dideoxycytidine (ddC, 1) and 3'-fluoro-ddC (2), involving N4-substitution with (N,N-dialkylamino)methylene side chains, is described. The increase in the partition coefficients for the prodrug series, compared to those of the parent drugs 1 and 2, ranged from 1.5- to 122-fold and from 1.6- to 175-fold, respectively. At pH 7.4, 37 degrees C, the hydrolytic t1/2 values ranged from 2 to 52 h, the diisopropyl derivatives (3d and 4d) being most stable in the series. 3d and 4d were greater than or equal to 4-fold and 1.7-fold more soluble in water than 1 and 2, respectively. The in vitro antiretroviral activities of 3d, 4a, and 4d were evaluated; the results indicate efficient prodrug-to-drug conversion under the assay conditions. The results of this investigation demonstrate that it is indeed feasible to chemically modify certain nucleoside analogues with inferior solubility properties to simultaneously achieve significantly enhanced lipid and water solubility.

Design and synthesis of histidine analogues of folic acid and methotrexate as potential folylpolyglutamate synthetase inhibitors.

Folypolyglutamate synthetase (FPGS) is responsible for the conversion of naturally occurring folates and antifolates to their poly-gama-glutamyl derivatives, which are the forms required for intracellular retention of folates and are also the preferred substrates (cofactors) for most folate-dependent enzymes. Folate and methotrexate analogues 6 and 4, with L-histidine in place of L-glutamate, were designed and synthesized as potential FPGS inhibitors. Target compound 5, the N tau-(carboxymethyl)-L-histidine derivative of 4, was also prepared. Compounds 4 and 5 inhibited the growth of L1210 cells (IC50 values: 0.091 and 0.15 microM, respectively) and were potent inhibitors of L1210 dihydrofolate reductase. No significant inhibition of FPGS by 4, 5, or 6 was observed at the high pH of the standard enzyme assay. This could be the consequence of a lack of protonation of the basic side chains, which is likely to be required for FPGS inhibitory activity. The observed cytotoxicity indicates that partial protonation of the imidazole ring permits cellular uptake of the analogues.

Synthesis of 5-selenium-substituted uracil derivatives. Inhibition of thymidylate synthetase by 5-hydroseleno-2'-deoxyuridylate.

5-Selenium-substituted derivatives (diselenides) or uracil, 2'-deoxyuridine, and 2'-deoxyuridylic acid were synthesized via the addition of methyl hypobromite to the 5,6 double bond, followed by reaction of the adducts with sodium diselenide. The physical and chemical properties of these compounds (including their facile reduction by dithiothreitol and rapid reoxidation) were similar to those of the corresponding 5-sulfur analogues. 5-Hydroseleno-2'-deoxyuridylic acid was as potent as 5-mercapto-2'-deoxyuridylate in inhibiting thymidylate synthetase from L. casei (ki approximately 6 X 10(-8) M) but the nucleoside III was considerably less active than 5-mercapto-2'-deoxyuridine in the inhibition of growth of the leukemia L1210 cell in culture.

Synthesis and biological activity of 5-phenylselenenyl-substituted pyrimidine nucleosides.

Several 5-phenylselenenyl derivatives of pyrimidine nucleosides were synthesized by electrophilic addition of phenylselenenyl chloride to the nucleosides under basic conditions. With use of this route, 5-(phenylselenenyl)-6-azauracil was also prepared. These compounds may serve as inhibitors of thymidylate synthase, as potential antiviral and anticancer agents, and as versatile intermediates for the synthesis of 5- or 6-substituted nucleosides. 5-(Phenylselenenyl)arabinosyluracil (PSAU, 4) and the corresponding cytosine analogue (PSAC, 5) were poor inhibitors of a promyelocytic leukemia cell line that was arabinosylcytosine-resistant. PSAU and PSAC were significantly less active than ara-C against L1210 cells and were found to selectively interfere with the cellular uptake and/or phosphorylation of 2'-deoxycytidine and 2'-deoxyuridine in intact L1210 cells.

Folate analogues. 25. Synthesis and biological evaluation of N10-propargylfolic acid and its reduced derivatives.

N10-Propargylfolic acid (2), which is the closest pteridine analogue of the thymidylate synthase inhibitor N10-propargyl-5,8-dideazafolic acid (PDDF), was synthesized starting from diethyl [p-(N-propargylamino)benzoyl]-L-glutamate (5) and N-(3-bromo-2-oxopropyl)phthalimide (8). The 7,8-dihydro derivative of propargylfolic acid served as a synthetic substrate of Lactobacillus casei dihydrofolate reductase. Propargylfolic acid and its reduced derivatives were weak inhibitors of L. casei thymidylate synthase compared to PDDF. All derivatives of propargylfolate were active against the growth of Streptococcus faecium, but with the exception of 7,8-dihydropropargylfolic acid, all were inactive against L. casei. Although less potent than PDDF, marked inhibition of thymidylate synthase by 2 was observed in permeabilized L1210 cells.

Synthesis and biological activity of aromatic amino acid phosphoramidates of 5-fluoro-2'-deoxyuridine and 1-beta-arabinofuranosylcytosine: evidence of phosphoramidase activity.

The amino acid phosphoramidate diesters of FUdR (2) and Ara-C (6), 5-fluoro-2'-deoxy-5'-uridyl N-(1-carbomethoxy-2-phenylethyl)phosphoramidate (5a), 5-fluoro-2'-deoxy-5'- uridyl N-(1-carbomethoxy-2-indolylethyl)phosphoramidate (5b), 1-beta-arabinofuranosylcytosine 5'-N-(1-carbomethoxy-2-phenylethyl) phosphoramidate (8a), and 1-beta-arabinofuranosylcytosine 5'-N-(1-carbomethoxy-2-indolylethyl)phosphoramidate (8b), were synthesized and tested for their antitumor activity against L1210 mouse lymphocytic leukemia cells and CCRF-CEM human T-cell lymphoblastic leukemia cells. Ara-C phosphoramidates 8a,b were found to be inactive at a concentration of 100 microM, while the FUdR conjugates 5a,b exhibited IC50 values within a range of 0.30-0.40 microM. Stability studies revealed that > 99% of the phosphoramidates remained intact after incubation for > 2 days in 20% calf or 20% human serum. Intracellular thymidylate synthase (TS) inhibition studies revealed that treatment of L1210 and CCRF-CEM cells with 5a or 5b resulted in significant inhibition of TS in intact and permeabilized cells, while treatment of L929 TK- cells with these compounds did not result in inhibition of TS activity in intact cells. However, permeabilization of L929 TK- cells enhanced the activity of 5a,b toward intracellular TS by 900- and 1500-fold, respectively. In addition, incubation of cell-free extracts of CEM cells with radiolabeled 5b resulted in the rapid production of FUdR 5'-monophosphate and a lag in the generation of FUdR. Consequently, it is proposed that the metabolism of the phosphoramidate diesters of FUdR in proliferating tissue proceeds through two separate enzymatic steps involving P-N bond cleavage by an unknown phosphoramidase followed by P-O bond cleavage by phosphatases such as 5'-nucleotidase.

Nucleosides. 150. Synthesis and some biological properties of 5-monofluoromethyl, 5-difluoromethyl, and 5-trifluoromethyl derivatives of 2'-deoxyuridine and 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyluracil.

A new synthesis of 5-(monofluoromethyl)- and 5-(difluoromethyl)-2'-deoxy-2'-fluoro-beta-D-arabinofuranosyluracil (F-FMAU and F2-FMAU) is reported. 3',5'-Di-O-(tert-butyldiphenyl)silylated thymidine or FMAU was photochemically brominated with NBS to the corresponding alpha-monobromide, which was hydrolyzed to the 5-hydroxymethyl derivative. Further oxidation of the latter with MnO2 afforded the 5-formyluracil nucleoside. Treatment of these nucleosides with DAST in CH2Cl2 gave the protected alpha-fluorinated nucleosides. Desiylation with TBAF afforded the desired free nucleosides. Also, 5-(trifluoromethyl)-2'-deoxy-2'-fluoro-beta-D-arabinofuranosyluracil (F3-FMAU) was synthesized by copper-catalyzed trifluoromethylation of 5-iodo-2'-fluoro-ara-U (FIAU). These new nucleosides were studied in comparison with the corresponding 2'-deoxy-erythro-pentofuranosyl derivatives, for their inhibitory activity against cellular thymidylate synthase (TS) and [3H]TdR incorporation into DNA, cytotoxicity against HL-60 cells, and antiviral activity against herpes simplex types 1 and 2 (HSV-1 and -2). F2-TDR and F3-TDR strongly inhibited TS and were also quite cytotoxic and antiherpetic, whereas FTDR was only active in the antiviral assay. In the 2'-fluoroarabino series, fluorine substitution at the alpha-methyl function did not alter significantly the antiherpetic activity. Although FMAU and F-FMAU did not inhibit TS to any significant extent, F2-FMAU and F3-FMAU were weakly inhibitory. The latter nucleosides did not inhibit [3H]TDR incorporation into DNA, while all the other alpha-fluorinated thymine nucleosides inhibited the incorporation of radioactivity of [3H]TDR into DNA to various extents. F2-FMAU and F3-FMAU were about 2 orders of magnitude less cytotoxic against HL-60 cells than were F2-TDR and F3-TDR. The results strongly suggest that in both the 2'-deoxy-2'-fluoroarabino and the 2'-deoxy-erythro-pentofurano series the cytotoxic action of the alpha,alpha-difluoro and alpha,alpha,alpha-trifluoro derivatives may involve the inhibition of TS. The synthesis of [2-14C]F2-FMAU, as an experimental imaging agent, is also described. Unfortunately, the highly selective uptake of the labeled compound within infected brain regions previously noted with [2-14C]FMAU was not detected with the derivative [2-14C]F2-FMAU.

Folate analogues. 26. Syntheses and antifolate activity of 10-substituted derivatives of 5,8-dideazafolic acid and of the poly-gamma-glutamyl metabolites of N10-propargyl-5,8-dideazafolic acid (PDDF).

The poly-gamma-glutamyl derivatives of n10-propargyl-5,8-dideazafolic acid (PDDF) with a chain length of up to five glutamate residues were synthesized from N10-propargyl-5,8-dideazapteroic acid by the solid-phase procedure. These compounds were evaluated for their antifolate activity using folate-requiring microorganisms and intact and permeabilized L1210 cells and as inhibitors of dihydrofolate reductase and thymidylate synthase derived from L. casei. The polyglutamylated derivatives of PDDF (1) were more active than the parent compound in inhibiting the growth of L. casei, thymidylate synthesis in permeabilized L1210 cells, and L. casei thymidylate synthase. Two analogues of 5,8-dideazafolic acid (2 and 3), one with a 2-butyne and another with a cyclopropylmethyl substituent at N10, were also synthesized and evaluated for their antifolate activities using the above-mentioned test systems. They were considerably less active than PDDF or its polyglutamylated derivatives. N10-Propargyl-5,8-dideazapteroyl tri-, tetra-, and pentaglutamates were equipotent with 5-fluorodeoxyuridylate as inhibitors of thymidylate synthesis in permeabilized L1210 cells. The polyglutamyl metabolites of PDDF were shown to be the most potent antifolate inhibitors of L. casei and L1210 thymidylate synthases yet described.

Folate analogues. 30. Synthesis and biological evaluation of N10-propargyl-5,8-dideaza-5,6,7,8-tetrahydrofolic acid and related compounds.

The 5,6,7,8-tetrahydro derivative (1) of the powerful thymidylate synthase inhibitor N10-propargyl-5,8-dideazafolic acid (PDDF) has been synthesized and evaluated for its antifolate activity. A convenient method for the preparation of the key intermediate 2-amino-6-(bromomethyl)-4-hydroxy-5,6,7,8-tetrahydroquinazoline (18) is described. Two closely related analogues of 1 were also synthesized and evaluated for their antifolate activity and thymidylate synthase inhibition. N10-Propargyl-5,8-dideaza-5,6,7,8-tetrahydrofolate (1) and N10-methyl and N10-hydrogen analogues 2 and 3 were weaker inhibitors of Lactobacillus casei thymidylate synthase compared to PDDF. N10-Methyl-5,8-dideaza-5,6,7,8-tetrahydrofolate (2) exhibited the most potent antifolate activity against L. casei (IC50 = 2.8 nM) and Streptococcus faecium (IC50 = 0.57 nM). In intact and permeabilized murine leukemia L1210 cells, the replacement of the quinazoline moiety with its tetrahydro derivative resulted in a marked decrease in potency and a loss of the contribution of the propargyl substituent to enzyme inhibition, indicating an altered binding mode to thymidylate synthase.

Rapid determination of thymidylate synthase activity and its inhibition in intact L1210 leukemia cells in vitro.

A rapid and convenient tritium release assay for measuring thymidylate (dTMP) synthase activity and its inhibition within intact mammalian cells is described in detail. Short-term incubation of murine leukemia L1210 cells with an appropriately labeled substrate precursor, either deoxyuridine ([5-3H]dUrd) or deoxycytidine ([5-3H]dCyd), allowed for: (1) uptake and intracellular conversion to the substrate deoxyuridylate ([5-3H]dUMP); and (2) the obligatory displacement of tritium from [5-3H]-dUMP during the dTMP synthase catalyzed reaction. Tritium released into the aqueous environment was quantitated after a quick one-step separation of tritiated H2O from other radiolabeled materials and cell debris. The amount of tritium released was evaluated as a function of a number of variables, including the concentration of labeled substrate precursors, cell number, and incubation time. Tritium from [5-3H]dCyd was released significantly faster than from [5-3H]dUrd under a variety of conditions. Both 5-fluorodeoxyuridine (1 microM) and methotrexate (10 microM), which effectively block intracellular dTMP synthesis, completely inhibited the release of tritium from either [5-3H]dCyd or [5-3H]dUrd demonstrating that the release of tritium is mediated exclusively by the dTMP synthase catalyzed reaction. In addition, there was a good correlation between tritium release, cellular uptake, and incorporation of [2-14C]dUrd into DNA. The inhibitory effects of antifolates such as methotrexate were independent of the type of labeled precursor used. In contrast, preferential interference with the release of tritium from [5-3H]-dCyd by dCyd derivatives and from [5-3H]dUrd by dUrd derivatives was observed, suggesting that competition for uptake and/or phosphorylation may contribute to the overall effects of certain nucleoside analogues on cellular dTMP synthase activity measured using the tritium release assay.

Evaluation of pteroyl-S-alkylhomocysteine sulfoximines as inhibitors of mammalian folylpolyglutamate synthetase.

The similarity between the reactions catalyzed by folylpoly-gamma-glutamate synthetase (FPGS), gamma-glutamylcysteine synthetase, and glutamine synthetase, as well as the susceptibility of the latter two enzymes to inhibition by methionine sulfoximine, suggest that folic acid derivatives with methionine sulfoximine or its alkyl homologs in place of the glutamate side chain of folate are good candidates to act as enzyme-generated transition state analog inhibitors of the FPGS reaction. Thus, pteroylmethionine sulfoximine, and the homologous S-ethyl-, S-propyl-, and S-butylhomocysteine sulfoximine derivatives were evaluated as inhibitors of FPGS that was partially purified from mouse liver and from mouse L1210 cells. The related compound, pteroyl-S-methylhomocysteine sulfone, which cannot undergo enzyme-mediated activation, was also investigated. Unexpectedly, none of these compounds showed significant inhibition of FPGS from these sources under a variety of conditions. These results, taken together with previously established structure-activity correlations, imply that a negative charge at the gamma-position of folate analogs may be required for initial binding to FPGS and thus constitutes a prerequisite for activity of potential mechanism-based inhibitors of this enzyme.

Inhibition of cellular thymidylate synthesis by cytotoxic propenal derivatives of pyrimidine bases and deoxynucleosides.

A series of cytotoxic propenal (3-oxoprop-1-enyl) derivatives of pyrimidine bases and deoxynucleosides was evaluated for their ability to block thymidylate synthesis in intact and permeabilized murine leukemia L1210 cells. Several were potent inhibitors of this process, likely contributing to their cytotoxicity. The IC50 values of thymidine-3-propenal, the prototype of this series, in intact and permeabilized L1210, L-M and L-M(TK-) cells were 21, 7.5, and 75 microM and 1.5, 1.7, and 3.5 microM, respectively. The related base analogue, thymine-1-propenal, is a product of bleomycin-induced DNA strand-scission; the results of the present study bear on the mode of action of this antibiotic.

N4-(dialkylamino)methylene derivatives of 2'-deoxycytidine and arabinocytidine: physicochemical studies for potential prodrug applications.

The synthesis and physicochemical parameters of a series of N4-(N,N-dialkyl)formamidine derivatives of 2'-deoxycytidine (dCyd, 1) and arabinocytidine (ara-C, 2), as prodrug prototypes, are described. The lipophilicity of the formamidine derivatives compared to that of the parent nucleosides increased 1.26- to 145-fold. The corresponding half-lives of the derivatives in aqueous solution at pH 7.4 and 37 degrees C ranged from 3.7 to 52 h. The analogues most stable to hydrolysis in both series (3 and 4) were the diisopropyl derivatives (3d and 4d). The substitution effects of the sugar OH groups at the 2'- and 3'-positions on the corresponding partition coefficients gave pi-values of -0.16 and -0.55, respectively. These values are significantly more positive than those generally associated with aliphatic hydroxy substituents indicating that sugar OH groups of nucleosides have a decreased tendency to associate with the aqueous phase. The in vitro growth inhibitory activities of the ara-C derivatives (4) against murine lymphocytic leukemia L1210 cells indicate that prodrug to drug conversion readily occurs. The results confirm our previous findings about the versatility of the formamidine modification of nucleosides bearing exocyclic amino groups. The data facilitate optimization of lipophilicities and hydrolytic stabilities and thus contribute to the design and further development of the formamidine type of prodrug derivatives.

Diazoketone and chloromethylketone analogs of methotrexate as potential antitumor agents.

The synthesis of 4-amino-4-deoxy-N10-methylpteroyl-(6-diazo-5-oxo)-L-norleucine and 4-amino-4-deoxy-N10-methylpteroyl-(6-chloro-5-oxo)-L-norleucine, analogs of methotrexate in which the gamma-carboxyl group is replaced by a diazoketone and a chloromethylketone, respectively, was carried out. The analogs inhibited the growth of leukemia L-1210 cells in culture by 50% at 4 X 10(-7) M and 2 X 10(-7) M, respectively, and were effective inhibitors of the synthesis of thymidylate in L-1210 cells in vitro (I50 = 3 X 10(-6) M), exhibiting significant antifolate activity. The results demonstrated the feasibility of introducing chemically reactive groups at the gamma-position of pteroyl glutamates with retention of biological activity. However, in the systems investigated thus far, there was no evidence of covalent bond formation due to these reactive groups at the active sites of the enzymes.

Conformational analysis of human dihydrofolate reductase inhibitor complexes: crystal structure determination of wild type and F31 mutant binary and ternary inhibitor complexes.

These structural studies reveal unusual intermolecular interactions for the binding of inhibitors and cofactor in ternary complexes with both wild type and F31 mutant recombinant human DHFR and show that these inhibitors have flexibility in occupying the active site. These studies also possibly indicate the first structural data for a ternary complex with a folate inhibitor and a polyglutamate side chain. However, further refinement of this data is necessary before this can be confirmed. In contrast to the ternary complexes of folate and MTX, the lipophilic antifolate PTX binds with its methoxybenzoyl ring oriented toward the cofactor nicotinamide ring, while that of TMQ it is bound closer to the Phe-31 position. Furthermore, the nicotinamide ring makes a close contact to the N10 amine of TMQ, significantly different from its binding site interactions in MTX complexes. These data also reveal that the conserved contacts between the cofactor carboxyamide with the enzyme backbone residues Ala-9 and Ile-16 are dictated by the enzyme and that changes in the orientation of the structural elements requires only subtle changes in the secondary structural units in which they are contained. Therefore, only by careful analysis of a series of enzyme complexes can the mechanisms of binding action be delineated.

Rational design of selective antimetabolites of DNA-thymine biosynthesis: 5-propynylpyrimidine nucleoside derivatives.

A novel series of 5-propynylpyrimidine nucleosides are proposed as potential antimetabolites of DNA-thymine biosynthesis. This proposal is based on the results of detailed mechanistic analyses of the molecular interactions between dTMP synthase and its inhibitors. It is proposed that a propynyl side-chain at the 5-position of dUMP, bearing an appropriate leaving group, would cause irreversible inactivation of dTMP synthase, which would not require the presence of the cofactor, CH2H4folate.

5-propynylpyrimidine nucleoside derivatives: rationally designed mechanism-based inactivators of thymidylate synthase.

A novel series of 5-propynyl-dUMP derivatives, with a variety of leaving groups on the side-chain, was designed as potential mechanism-based inhibitors of thymidylate synthase (TS), and synthesized from 5-iodo-2'-deoxyuridine by Pd(0)-catalyzed coupling, followed by direct phosphorylation with POCl3. All members of the series inhibited TS competitively with Ki-values of 0.015-18 microM. Analogs with fluorine or imidazole-based leaving groups caused rapid, irreversible inactivation of TS.