Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.1.1.45 (thymidylate synthase)
3,600 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)- N-methylamino]-2-thenoyl)-L-glutamic acid (ICI D1694) is an analogue of the thymidylate synthase inhibitor, N10-propargyl-5,8-dideazafolic acid (CB3717). CB3717 was found to be active in early clinical studies, but its use was limited by nephrotoxicity. ICI D1694 is a more potent antitumour agent than CB3717 and is also more water soluble. Previous studies have shown ICI D1694 to be non-toxic to the kidney following a single administration but its renal effects after chronic administration are unknown. To assess these effects, and further define the time course and dose relationship of CB3717-induced renal damage, an assay of glomerular filtration rate (GFR) has been developed which can be used in mice and hence in the screening of novel compounds. The 14C-inulin clearance assay developed was used to show a linear relationship between CB3717 dosage and renal damage (r = - 0.989) following a single bolus dose (50-200 mg kg-1), in mice. CB3717-induced renal damage is persistent (greater than 6 weeks) and renal scarring was noted. ICI D1694 has been shown to be non-nephrotoxic following weekly administration of 250 mg kg-1 for 6 weeks. Measurement of GFR has been shown to be a more sensitive indicator of impaired renal function than plasma urea and creatine concentration, and the measurement of plasma creatinine concentration in particular, appears to be without value in the screening of potential nephrotoxins in certain mouse strains.
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PMID:The renal effects of N10-propargyl-5,8-dideazafolic acid (CB3717) and a non-nephrotoxic analogue ICI D1694, in mice. 193 3

Modification of the potent thymidylate synthase (TS) inhibitor N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl]-N-prop-2- ynylamino]benzoyl]-L-glutamic acid (1a) has led to the synthesis of quinazoline antifolates bearing alkyl, substituted alkyl, and aryl substituents at C2. In general the synthetic route involved the coupling of the appropriate diethyl N-[4-(alkylamino)benzoyl]-L-glutamate with a C2-substituted 6-(bromo-methyl)-3,4-dihydro-4-oxoquinazoline followed by deprotection using mild alkali. Good enzyme inhibition and cytotoxicity were found with compounds containing small nonpolar groups in the C2 position with the 2-desamino-2-methyl analogue 3a being the most potent. Larger C2 substituents were tolerated by the enzyme, but cytotoxicity was reduced. Highly potent series were followed up by the synthesis of a number of analogues in which the N10 substituent was varied. In this manner a number of interesting TS inhibitors have been prepared. Although none of these was more potent than 1a against the isolated enzyme, over half of the compounds prepared were more potent as cytotoxic agents against L1210 cells in culture. The potential of such compounds as useful antitumor agents was further enhanced by the finding that the improved aqueous solubilities of compounds such as 3a over 1a were reflected in vivo in that 3a was at least 5 times less toxic to mice than 1a.
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PMID:Quinazoline antifolate thymidylate synthase inhibitors: alkyl, substituted alkyl, and aryl substituents in the C2 position. 223 6

A new assay for the enzyme folylpoly-gamma-glutamate synthetase (FPGS) that offers significant advantages over other published procedures has been developed. This assay is based on the addition of high specific activity [3H]glutamic acid to (6-S)-tetrahydrofolate followed by trapping of the labeled tetrahydropteroyldiglutamate product as a covalently bound macromolecular complex by the addition of formaldehyde, fluorodeoxyuridylate, and pure bacterial thymidylate synthase. This complex is then separated from excess labeled glutamic acid by centrifugal elution of a 1-ml Sephadex G-50 column. The assay was found to be useful for the measurement of FPGS on small tissue samples and is amenable with the assay of FPGS in cell sonicates. Typically, blank values of 100-200 cpm are seen with a signal normally more than 10 times higher. Analysis of 20-30 samples can be accomplished in less than 90 min. As a result, this assay has proven useful for detection of enzyme in elution fractions from chromatographic columns.
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PMID:A microassay for mammalian folylpolyglutamate synthetase. 235 71

The uptake and metabolism of radiolabeled 5,8-dideazaisofolic acid (IAHQ) (N-[p- ([(2-amino-4-hydroxy-6-quinazolinyl)amino]methyl)benzoyl]-L-glutamic acid), a new antifol targeted to thymidylate synthase, has been investigated in the human colon adenocarcinoma cell line HCT-8. [3H]IAHQ uptake was very slow, requiring days to achieve the intracellular level achieved in minutes by [3H]methotrexate. This slow transport of IAHQ was consistent with the long exposures required to achieve cytotoxicity. Intracellular [3H]IAHQ was converted in a concentration-dependent manner to poly-gamma-glutamate derivatives containing between two and four additional glutamate residues. These results are consistent with our hypothesis that IAHQ is a "pro-drug" which must be converted to polyglutamate derivatives before it is a sufficiently potent inhibitor of thymidylate synthase to induce a pyrimidineless state and cell death.
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PMID:Uptake and metabolism of 5,8-dideazaisofolic acid in human colon carcinoma cells. 245 26

The synthesis is described of four oligo(gamma-glutamyl) conjugates of N10-propargyl-5,8-dideazafolic acid containing a total of two, three, four, and five L-glutamic acid residues. The tert-butyl group was chosen as the carboxyl protecting group in order to obviate the use of alkali and thus the possibility of gamma----alpha transpeptidation. The starting material, di-tert-butyl glutamate, was coupled to N-(benzyloxycarbonyl)-L-glutamic acid alpha-tert-butyl ester via a mixed anhydride with isobutyl chloroformate. Hydrogenolysis of the benzyloxycarbonyl group in the product gave a carboxyl-protected diglutamate, which either was acylated with 4-[(benzyloxycarbonyl)amino] benzoyl chloride to give a protected aminobenzamide or was cycled further by using the above mixed anhydride/hydrogenolysis sequence into tri-, tetra-, and pentaglutamates. Each of the last named was also acylated, as above, to give a benzamide. The benzyloxycarbonyl group in the benzamides was removed by hydrogenolysis and the amino groups thus exposed were N-alkylated with propargyl bromide. The resulting proparglyamines were further alkylated with 2-amino-6-(bromomethyl)-4-hydroxyquinazoline hydrobromide to give the antifolate poly(t-Bu) esters. Deprotection with trifluoroacetic acid in the final step delivered the desired antifolates as their trifluoroacetate salts. The di- to pentaglutamates were, respectively, 31-, 97-, 171-, and 167-fold more inhibitory to WI-L2 human thymidylate synthase than the parent compound.
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PMID:Quinazoline antifolates inhibiting thymidylate synthase: synthesis of four oligo(L-gamma-glutamyl) conjugates of N10-propargyl-5,8-dideazafolic acid and their enzyme inhibition. 290 28

CB3717 (N-(4-(N-((2-amino-4- hydroxy-6-quinazolinyl)methyl)prop-2-ynylamino ) benzoyl)-L-glutamic acid) is an antitumour agent that inhibits thymidylate synthetase (TS). A dose-dependent fall in plasma thymidine (dThd) (1.43 microM to 0.47 microM) occurred in non-tumour-bearing mice following the administration of CB3717. Further, in mice carrying the L1210/CBRI tumour, the drug's antitumour properties were ablated by co-administration of dThd, an effect consistent with TS being the cytotoxic locus. In vitro studies of protection by dThd against CB3717 cytotoxicity were carried out in an attempt to quantify this reversal. The metabolism of [14C]-dThd was measured in cultures of L1210 cells (10(4)/ml) exposed to a completely cytotoxic dose of CB3717 (50 microM). The cytotoxicity of the drug was only expressed when the dThd concentration (0.5-2 microM) had fallen to less than 0.1 microM in the media. This reduction was due to: (1) dThd incorporation into DNA, (2) catabolism of dThd to thymine. By reducing the initial cell concentration to 10(3)/ml the depletion of dThd was substantially reduced and consequently cells continued to grow for a longer period. The critical concentration of dThd, below which growth in the presence of CB3717 could not be supported was estimated to be between 0.026 and 0.1 microM. Thus even the minimum level of dThd achieved in vivo was still in excess of that required for protection from CB3717 toxicity in vitro. There was a small accumulation of deoxyuridine (dUrd) (approximately 2-fold) in mouse plasma 24 hr after completion of a 5-day course of CB3717 (200 mg/kg) but in vitro studies demonstrated that this was unlikely to modulate CB3717 toxicity in the presence of dThd. We caution against the use of rodent tumour models (or human tumour xenografts) for antitumour or toxicity testing of compounds designed to inhibit the de novo synthesis of thymidylate; they may be misleading because the high dThd levels found in these animals compared with man may mask the cytotoxic effects of these drugs.
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PMID:Modulation of anti-metabolite effects. Effects of thymidine on the efficacy of the quinazoline-based thymidylate synthetase inhibitor, CB3717. 648 75

The synthesis of an 8-deazafolate analogue of the intermediate in the methylation of 2'-deoxyuridylate is described. Alkylation of diethyl 5,6,7,8-tetrahydro-8-deazafolate with 3'-O-acetyl-5-(bromomethyl)-2'-deoxyuridine 5'-[bis-(trichlorethyl) phosphate], followed by removal of the trichloroethyl groups with a Zn/Cu couple and mild saponification, gave the target inhibitor N-[4-[[[2-amino-3,4,5,6,7, 8-hexahydro-4-oxo-5-[(2'-deoxyuridin-5-yl)methyl]-pyrido[3,2-d] pyrimidin-6-yl]methyl]amino]benzoyl]-L-glutamic acid 5'-monophosphate. The free nucleoside and the 5'-(methyl phosphate) diester were similarly prepared. Each of these reactions yielded a pair of diastereoisomers about C-6 of the reduced deazafolate in approximately a 1:1 ratio. These diastereoisomeric mixtures were evaluated as inhibitors of thymidylate synthetase derived from human tumor (HeLa) cells. The 5'-monophosphate was a potent inhibitor, competitive with respect to both 2'-deoxyuridylate (Ki = 0.06 microM) and tetrahydrofolate (Ki = 0.25 microM). In contrast, the nucleoside and the nucleotide methyl ester were poorer inhibitors by more than 3 orders of magnitude, attesting to the importance of the anionic function at the nucleoside 5'-position in the affinity of an inhibitor for the enzyme active site.
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PMID:A potent multisubstrate analogue inhibitor of human thymidylate synthetase. 650 2

A variety of quinazoline analogues of folic acid (5,8-dideazafolates) are of interest as potential antineoplastic agents, biochemical probes, and/or affinity ligands for the purification of folate requiring enzymes. Chief among these are 5,8-dideazaisopteroylglutamate, 5,8-dideazaisoPteGlu, (IAHQ), a compound with proven activity against the growth of human colon adenocarcinoma cells in vitro, and 10-formyl-5,8-dideazapteroylglutamic acid, which serves as a substrate for glycinamide ribonucleotide transformylase and is also an effective inhibitor of mammalian thymidylate synthase. New methods for preparing these compounds in excellent purity as determined by high performance liquid chromatography (HPLC) have been developed. In each case the carboxyl groups of L-glutamic acid are protected with t-butyl ester groups, since these can subsequently be removed readily using trifluoroacetic acid without decomposition or racemization of the final product. This approach has proven to be of particular value in the formation of gamma-L-glutamyl derivatives of IAHQ containing 1-3 additional glutamyl residues.
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PMID:Improved synthetic routes to 5,8-dideazapteroylglutamates amenable to the formation of poly-gamma-L-glutamyl derivatives. 661 23

The biochemical effects of the antitumor agent N-(4-(N-(( 2-amino-4-hydroxy-6-quinazolinyl)methyl)prop-2-ynylamino) benzoyl)-L -glutamic acid (CB3717) were studied in WI-L2 cultured human lymphoblastoid cells. CB3717 was a potent inhibitor of human thymidylate synthetase; the inhibition was competitive with 5,10-methylenetetrahydrofolate (Ki = 4.9 X 10(-9) M). CB3717 also inhibited human dihydrofolate reductase, competitively with dihydrofolate (Ki = 2.3 X 10(-8) M). The growth-inhibitory effect of CB3717 could be prevented completely by 10 microM thymidine. Administration of thymidine could be delayed for up to 8 hr after CB3717 treatment without cytotoxicity but, if thymidine was delayed for 24 hr, severe toxicity resulted. Incubation for 16 hr in the presence of a growth-inhibitory concentration of CB3717 did not result in the appearance of dihydrofolate in WI-L2 cells. These results indicate that, in the presence of CB3717, thymidylate synthetase, rather than dihydrofolate reductase, became rate-limiting for the cycle of dihydrofolate oxidation and reduction. Treatment of cells for 16 hr at an IC50 concentration of CB3717 caused a decrease of 88% in cellular dTTP and a 2,300% increase in dUMP. The level of dUDP also increased, and traces of dUTP appeared in treated cells. No large changes were seen in ribonucleotide pools. A kinetic analysis was made, by computer simulation, of predicted consequences of metabolic effects of compounds that inhibit both dihydrofolate reductase and thymidylate synthetase. It was concluded that, even if the Ki of the inhibitor for thymidylate synthetase were 3 orders of magnitude higher (weaker) than the Ki for dihydrofolate reductase, thymidylate synthetase could still become rate-limiting.
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PMID:Biochemical effects of a quinazoline inhibitor of thymidylate synthetase, N-(4-(N-(( 2-amino-4-hydroxy-6-quinazolinyl)methyl)prop-2-ynylamino) benzoyl)-L-glutamic acid (CB3717), on human lymphoblastoid cells. 666 Dec 52

Reported antifolate activity against leukemia L1210 by N-[14-[[(2-amino-4-hydroxy-6-quinazolinyl)methyl]-propargylamino]benzoyl]]-L-glu tamic acid through potent inhibition of thymidylate synthase (EC 2.1.1.45) prompted us to include the propargyl group in a study of the effect on folate metabolism and membrane transport of replacing the 10-methyl group of methotrexate with other groups. Along with the propyl (8a) and octyl (8b) homologues of methotrexate, the propargyl compound 8c was prepared for evaluation. Syntheses of 8a,b were achieved by a standard multistep sequence involving preparation of the side-chain precursors via tosylated intermediates and then their alkylation with 6-(bromomethyl)-2,4-pteridinediamine hydrobromide. The side-chain precursor to 8c was prepared by direct alkylation of diethyl N-(4-aminobenzoyl)-L-glutamate with propargyl bromide and was separated from unchanged amine and dipropargyl coproduct by a combination of methods, including dry-column chromatography and recrystallization. Subsequent steps leading to 8c were like those used to prepare 8a,b. Biological evaluations of the three compounds consisted of studies of their effects on enzyme inhibition [(dihydrofolate reductase (EC 1.5.1.3) and thymidylate synthase)], L1210 cell growth inhibition, cellular membrane transport with various murine cell types (L210, S180, Ehrlich, and epithelial), in vivo (mice) activity vs. L1210 leukemia and S180 ascites, and plasma clearance in mice. The in vivo results vs. S180 ascites offered evidence that 8c might have a better therapeutic index against this tumor than methotrexate, but no other result from either of these compounds suggested significant superiority over methotrexate.
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PMID:10-Propargylaminopterin and alkyl homologues of methotrexate as inhibitors of folate metabolism. 710 7


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