Inhibition of mammalian tumour thymidylate synthetase by 5-alkylated 2'-deoxyuridine 5'-phosphates

Improved syntheses, based on Lewis acid-catalyzed nucleosidation, are described for the preparation of 5-alkyl-2'-deoxyuridines. These were converted to their 5'-phosphates with the use of wheat shoot phosphotransferase. The dUMP analogues, 5-ethyl-dUMP and 5-propyl-dUMP, were competitive vs dUMP inhibitors of thymidylate synthetase purified from mouse L1210, Ehrlich ascites and HeLa cells, the former being the stronger inhibitor. Both analogues were shown to bind cooperatively to each of the mouse tumour enzymes, two molecules of inhibitor interacting with a single enzyme molecule, as reflected by the parabolic character of the replots of the slope vs inhibitor concentrations. dTMP was a stronger inhibitor of the mouse tumour enzymes than its higher alkyl homologues.     

5-p-benzoquinonyl-2'-deoxyuridine 5'-phosphate: a possible mechanism-based inhibitor of thymidylate synthetase

The title compound (1), designed as a suicide inhibitor of thymidylate synthetase, can be prepared by silver(II) oxide oxidative demethylation of the corresponding dimethoxyphenyl derivative. Compound 1 shows time-dependent inactivation of thymidylate synthetase (methotrexate-resistant Lactobacillus casei) and saturation kinetics, and the inactivation is responsive to substrate protection. The inactivation is not reversible on prolonged dialysis in attempts to remove the inhibitor. The rate constant for inactivation is 0.065 s-1; the dissociation constant (Ki) was estimated to be 2 microM. The kinetics of this inactivation are compared to inactivation caused by model thiol reagents that do not have affinity for the active site of thymidylate synthetase.     

5-Cyano-2'-deoxyuridine 5'-phosphate: a potent competitive inhibitor of thymidylate synthetase.

The 5'-phosphate (1) of the antiviral nucleoside 5-cyano-2'-deoxyuridine was synthesized and evaluated for inhibition of thymidylate synthetase purified from methotrexate-resistant Lactobacillus casei. Compound 1 was a potent competitive inhibitor with a K1 of 0.55 microns. Irreversible enzyme inhibition by this compound could not be detected.     

5-(alpha-Bromoacetyl)-2'-deoxyuridine 5'-phosphate: an affinity label for thymidylate synthetase.

5-(alpha-Bromoacetyl)-2'-deoxyuridine 5'-phosphate (1) is an active-site-directed irreversible inhibitor of thymidylate synthetase from Lactobacillus casei. Analysis of the rate of inactivation of the enzyme in the presence of substrate confirmed the intermediate formation of a reversible enzyme-inhibitor complex.     

Thiol addition to quinones: model reactions for the inactivation of thymidylate synthase by 5-p-benzoquinonyl-2'-deoxyuridine 5'-phosphate

The reaction of methyl mercaptoacetate (5) with phenyl-p-benzoquinone (6) or 5-p-benzoquinonyl-3',5'-di-O-acetyl-2'-deoxyuridine (10) resulted in the formation of the three possible adducts to the quinone rings of 6 and 10; an additional product in the reaction with 10 was the unsubstituted hydroquinone (14). Both reactions were found to be solvent dependent; in buffered aqueous acetonitrile the meta and para adducts of 10 were formed in the ratio of 2:1. In ethyl acetate the ortho adduct and the reduction product of 10 were isolated in a ratio of 2:3. The second-order rate constant for the reaction of 5 with 10 in acetonitrile was 0.53 M-1 s-1; the reaction was accelerated by the addition of water. Although the initially proposed mechanism-based enzyme inactivation cannot be excluded, the results of the model reactions support the alternative mechanism, active-site thiol addition to the quinone ring. If this is true the title compound would be classed as an affinity label, not a mechanism-based inhibitor.     

5-[(4-Methyl-1,2,3,4-tetrahydroquinoxalyl)methyl]-2'-deoxyuridine 5'-phosphate: an analogue of a proposed intermediate in thymidylate synthetase catalysis.

In a study of the sequence steps involved in the mechanism of thymidylate synthetase catalysis, 5-[(N-methyl-piperazinyl)methyl]- (5) and 5-[(4-methyl-1,2,3,4-tetrahydroquinoxalyl)methyl]-2'-deoxyuridine 5'-phosphate (6) were synthesized. Compound 6 has high affinity for the Lactobacillus casei enzyme (Ki = 0.75 microM, KI/Km - 0.23), which is 50 times stronger than that of the piperazinyl derivative 5. Compound 6, a possible multisubstrate inhibitor, is an analogue of a proposed intermediate in the reaction mechanism wherein the enzyme is eliminated from the covalent complex (enzyme--substrate--cofactor) prior to the redox reaction leading to the products 2'-deoxythymidine 5'-phosphate and 7,8-dihydrofolic acid.     

Studies on the interaction with thymidylate synthase of analogues of 2'-deoxyuridine-5'-phosphate and 5-fluoro-2'-deoxyuridine-5'-phosphate with modified phosphate groups

The role of the phosphate moiety of dUMP, and some analogues, in their interaction with mammalian thymidylate synthase, has been investigated. Substrate and inhibitor activities, and the pH-dependence of these activities, of dUMP and 5-FdUMP, as well as analogues with modified phosphate groups, were compared. The methyl ester of dUMP was neither a substrate nor an inhibitor. By contrast, the methyl ester of 5-FdUMP was a slow-binding inhibitor of the enzyme from L1210, Ehrlich ascites carcinoma and CCRF-CEM cells, with Ki values in the micromolar range. Both 5-FdUrd and the newly synthesized 5'-methylphosphonate of 5-FdUrd were also slow-binding inhibitors of the Ehrlich carcinoma enzyme, but with Ki values in the millimolar range. The interaction of dUMP, 5-FdUMP, and the methyl ester of the latter decreased with increase in pH, whereas that of the 5'-methyl-phosphonate of 5-FdUrd remained unchanged. The results are discussed in relation to the role of the phosphate hydroxyls of dUMP in binding to the enzyme. 5-FdUMP and its analogues exhibited differing interactions with two binding sites on the enzyme molecule, consistent with cooperativity of binding. A convenient procedure is described for the synthesis of 5-fluoro-2'-deoxyuridine-5'-methylphosphonate, applicable also to the preparation of other 5'-methylphosphonate 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.     

5-Quinone derivatives of 2'-deoxyuridine 5'-phosphate: inhibition and inactivation of thymidylate synthase, antitumor cell, and antiviral studies

Both photochemical aromatic substitution and palladium (0)-catalyzed biaryl coupling reactions have been employed in the synthesis of 5-substituted 2'-deoxyuridines. The former procedure was useful in the preparation of the 3,4-dimethyl-2,5-dimethoxyphenyl derivative 12a and the 3,4,6-trimethyl-2,5-dimethoxyphenyl derivative 12b. The latter reaction was efficient in the preparation of the 2-(3-methyl-1,4-dimethoxynaphthyl) derivative 14. These compounds and their nucleotides (20a-c) were converted to the corresponding quinone nucleosides 19a-c and nucleotides 6-8 by an oxidative demethylation reaction using ceric ammonium nitrate and silver(II) oxide, respectively. The kinetics and products of the reaction of the quinone nucleosides 19a,b with methyl thioglycolate showed rapid addition to the quinone ring in the trisubstituted derivative 19a and somewhat slower redox reactions with the tetrasubstituted quinones 19b and 19c. All six nucleotides had high affinity for the title enzyme from Lactobacillus casei with Ki values ranging from 0.59 to 3.6 microM; the most effective compounds were the dimethyl quinone 6 and the naphthoquinone 8. Somewhat higher inhibitory constants were observed with the quinones against the L1210 enzyme. The dimethyl quinone nucleotide 6 showed time-dependent inactivation (kinact = 0.015 s-1) against the L. casei enzyme, a rate saturation effect, and substrate protection in accord with the kinetic expression for an active-site-directed alkylating agent. The apparent second-order rate of this reaction (2.5 X 10(4) M-1 s-1) is one-twentieth the rate (kcat.) of the normal enzymatic reaction leading to product. None of the compound exhibited sufficient activity in the antitumor cell or antiviral assays to warrant further study.     

Oxime and dithiolane derivatives of 5-formyl-2'-deoxyuridine and their 5'-phosphates: antiviral effects and thymidylate synthetase inhibition

5-Formyl-2'-deoxyuridine (2a), an effective inhibitor of herpes simplex virus type 1 or 2 (HSV-1, HSV-2) and vaccinia virus, was converted to the oxime (3a) and dithiolane (4a) derivatives. The oxime (3a) was equally as potent as the formyl compound against HSV-1, but one-fifth as active against HSV-2, 100 times less effective against vaccinia, and 25 times less toxic for the host cells. In addition, compound 3a was about 10 times less active than 2a in inhibiting thymidylate synthetase in vivo (as reflected by a differential inhibition of dThd and dUrd incorporation into host cell DNA). The dithiolane (4a) did not exert an appreciable effect on either virus multiplication or dThd or dUrd incorporation, nor was it cytotoxic. All these compounds as their 5'-phosphate derivatives were potent in vitro inhibitors of thymidylate synthetase (Lactobacillus casei). The inhibition was competitive with substrate with Ki/Km ratios of 0.05 for the formyl 2b, 0.5 for the oxime 3b, and 0.2 for the dithiolane 4b. Thus, 3b was 10 times less active than 2b as an in vitro inhibitor of thymidylate synthetase, which appears to corroborate the in vivo findings.     

Antiviral, antitumor, and thymidylate synthetase inhibition studies of 5-substituted styryl derivatives of 2'-deoxyuridine and their 5'-phosphates

2′-Deoxyuridine derivatives containing styryl, 3-nitrostyryl, 4-nitrostyryl, and phenylethyl groups substituted at the 5-position of the pyrimidine ring have been evaluated for their effects on vaccinia and herpes simplex virus replication (in primary rabbit kidney cell cultures) and mouse leukemia L-1210 cell culture growth. 5-Phenylethyl-2′-deoxyuridine inhibited herpes simplex (type 1 and 2) virus-induced cytopathogenicity by 50 per cent at a dose (id₅₀) of 10–30 μg/ml. It was inactive against tumor cell growth. The corresponding styryl derivative showed an id₅₀ of 30–70 μg/ml for herpes simplex virus, 20 μg/ml for vaccinia virus, and 280 μg/ml for L-1210 cell growth. 5(E)-(3-Azidostyryl)-2′-deoxyuridine 5′-phosphate inhibited vaccinia replication with an IC₅₀ of 20 μg/ml and L-1210 cell culture growth with an id₅₀ of 80 μg/ml. The nucleotides of these compounds were all potent reversible inhibitors of thymidylate synthetase (Lactobacillus casei) with the following $\text{K}{}_{\mathrm{i}}\text{K}{}_{\mathrm{m}}$ ratios: 3-nitrostyryl, 0.035; 4-nitrostyryl, 0.05; 3-azidostyryl, 0.06; styryl, 0.08; and phenylethyl, 0.31. The photodecomposition of the azidostyryl derivative, a photoaffinity labeling reagent for thymidylate synthetase, was examined at two wavelengths.     

Synthesis and evaluation of 5-substituted 2'-deoxyuridine monophosphate analogues as inhibitors of flavin-dependent thymidylate synthase in Mycobacterium tuberculosis

A series of 5-substituted 2'-deoxyuridine monophosphate analogues has been synthesized and evaluated as potential inhibitors of mycobacterial ThyX, a novel flavin-dependent thymidylate synthase in Mycobacterium tuberculosis. A systematic SAR study led to the identification of compound 5a, displaying an IC(50) value against mycobacterial ThyX of 0.91 μM. This derivative lacks activity against the classical mycobacterial thymidylate synthase ThyA (IC(50) > 50 μM) and represents the first example of a selective mycobacterial FDTS inhibitor.     

Vanoxonin, a new inhibitor of thymidylate synthetase. III. Inhibition of thymidylate synthetase by vanoxonin-vanadium complex

Quinquevalent vanadium complex with two mol of vanoxonin ligated by the two catechols was shown to be the active structure for inhibition of thymidylate synthetase. The catechol group of vanoxonin as the essential moiety for the inhibition of enzyme was further confirmed by studies of structure-activity relationships using the enzyme obtained from Ehrlich ascites carcinoma cells of mice. Vanoxonin-vanadium complex showed competitive inhibition with respect to deoxyuridylic acid but uncompetitive to 5,10-methylenetetrahydrofolate.     

Interaction of 5-ethynyl-2'-deoxyuridylate with thymidylate synthetase

The interaction of 5-ethynyl-2'-deoxyuridylate (5-ethynyl-dUMP; 1) with thymidylate (dTMP) synthetase has been investigated. The compound was an inhibitor of the enzyme, competitive with 2'-deoxyuridylate (dUMP) when the reaction was initiated by addition of enzyme (Ki = 2.7 X 10(-6) M). However, upon preincubation of 1 with dTMP synthetase, the inhibition pattern became noncompetitive. The time course of the enzyme reaction in the presence of 1 was nonlinear, indicating an increase in binding with time. Irreversible inactivation of the enzyme did not occur. The compound did not appear to become altered structurally as a result of interaction with the enzyme. A ternary complex was formed among dTMP synthetase, compound 1, and 5,10-methylenetetrahydrofolate, which was stable enough to survive Sephadex G-25 filtration but dissociated upon denaturation of the enzyme.