Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Synthesis of the 10-methyl and 10-ethyl analogues of 5,10-dideazatetrahydrofolic acid (DDTHF), a potent inhibitor of glycinamide ribotide (GAR) formyltransferase, is reported. Key intermediates in the process were 10-methyl- and 10-ethyl-4-amino-4-deoxy-5,10-dideazapteroic acid. Condensation of the piperidine enamines of branched 4-(p-carbomethoxyphenyl)butyraldehydes with (acetoxymethylene)malononitrile afforded 1,1-dicyano-4-piperidinobutadiene 5a,b. Subsequent reaction with alcoholic ammonium hydroxide yielded the appropriately substituted 2-amino-3-cyanopyridines 6a,b. Ring closure with guanidine gave 10-methyl- and 10-ethyl-4-amino-4-deoxy-5,10-dideazapteroic acids (7a,b). Coupling with diethyl glutamate followed by ester hydrolysis afforded 10-alkyl-5,10-dideazaminopterin analogues 9a,b. Hydrolysis of the 4-amino group of 7a,b yielded the 10-alkylpteroic acids, which were coupled with diethyl glutamate, hydrogenated over PtO2, and saponified to afford 10-alkyl-5,10-dideazatetrahydrofolic acids 13a,b. Aminopterin analogues 9a,b were effective inhibitors of DHFR derived from L1210, but were less potent than methotrexate for inhibition of growth of L1210 in culture. The 10-ethyl (13b) analogue of 5,10-DDTHF was about twice as potent an inhibitor of L1210 cell growth as 5,10-DDTHF, but was only 1/7 as potent for inhibition of GAR formyltransferase. 10-Methyl analogue 13a was similar in potency to 5,10-DDTHF. All of the compounds showed moderately improved transport into L1210 cells relative to methotrexate.
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PMID:Synthesis and antifolate properties of 10-alkyl-5,10-dideaza analogues of methotrexate and tetrahydrofolic acid. 229 33

The Boon-Leigh procedure, involving condensation of a 6-chloro-5-nitropyrimidine (22) with an alpha-amino ketone (20 or 21) followed by reduction of the nitro group, cyclization, and L-glutamylation, led to the formation of 11-deazahomofolate (29) and its 10-methyl derivative (30). The corresponding (6R,S)-5,6,7,8-tetrahydro (4, 5) and 7,8-dihydro (31, 32) derivatives were prepared by catalytic hydrogenation. (6S)-11-Deazatetrahydrohomofolate was prepared from 29 by enzymatic reduction. Compounds 29 and 30 had little effect (IC50 greater than 2 x 10(-5) M) on Lactobacillus casei glycinamide ribonucleotide (GAR) formyltransferase but (6R,S)-11-deazatetrahydrohomofolate (4) is a potent inhibitor of this enzyme (IC50 = 5 x 10(-8) M). It is at least 100 times more inhibitory than 33, the 6S compound, indicating that the 6R component of the mixture having the unnatural configuration at C6 (34) is responsible for the potent inhibition. Compound 4 is a much weaker inhibitor of murine (L1210) and human (MOLT-4) leukemia cell GAR formyltransferases (IC50 greater than 1 x 10(-5) M). (6R,S)-11-Deaza-10-methyltetrahydrohomofolate (5) (IC50 = 1.1 x 10(-5) is 200 times weaker than 4 against L. casei GAR formyltransferase. However, 11-deaza-10-methyldihydrohomofolate (32) is more inhibitory (IC50 = 5.5 x 10(-7) M) than 5 or 30. None of the compounds showed inhibition of L. casei aminoimidazolecarboxamide ribonucleotide (AICAR) formyltransferase, dihydrofolate reductase, or thymidylate synthase. The dihydro derivatives 31 and 32 are 5% as active as dihydrofolate as substrates for L. casei dihydrofolate reductase. Compound 4 showed moderate inhibition of the growth of L. casei, Streptococcus faecium, MOLT-4 cells, and MCF-7 human breast adenocarcinoma cells.
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PMID:Folate analogues. 31. Synthesis of the reduced derivatives of 11-deazahomofolic acid, 10-methyl-11-deazahomofolic acid, and their evaluation as inhibitors of glycinamide ribonucleotide formyltransferase. 249 18

The title compounds were prepared in extensions of a general synthetic approach used earlier to prepare 5-alkyl-5-deaza analogues of classical antifolates. Wittig condensation of 2,4-diaminopyrido[2,3-d]pyrimidine-6-carboxaldehyde (2a) and its 5-methyl analogue 2b with [4-(methoxycarbonyl)benzylidene] triphenylphosphorane gave 9,10-ethenyl precursors 3a and 3b. Hydrogenation (DMF, ambient, 5% Pd/C) of the 9,10-ethenyl group of 3b followed by ester hydrolysis led to 4-[2-(2,4-diamino-5-methylpyrido[2,3-d]pyrimidin-6-yl)ethyl]ben zoi c acid (5), which was converted to 5-methyl-5,10-dideazaaminopterin (6) via coupling with dimethyl L-glutamate (mixed-anhydride method using i-BuOCOCl) followed by ester hydrolysis. Standard hydrolytic deamination of 6 gave 5-methyl-5,10-dideazafolic acid (7). Intermediates 3a and 3b were converted through concomitant deamination and ester hydrolysis to 8a and 8b. Peptide coupling of 8a,b (using (EtO)2POCN) with diesters of L-glutamic acid gave intermediate esters 9a and 9b. Hydrogenation of both the 9,10 double bond and the pyrido ring of 9a and 9b (MeOH-0.1 N HCl, 3.5 atm, Pt) was followed by ester hydrolysis to give 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (11a) and the 5-methyl analogue 11b. Biological evaluation of 6, 7, 11a, and 11b for inhibition of dihydrofolate reductase (DHFR) isolated from L1210 cells and for growth inhibition and transport characteristics toward L1210 cells revealed 6 to be less potent than methotrexate in the inhibition of DHFR and cell growth. Compounds 6, 11a, and 11b were transported into cells more efficiently than methotrexate. Growth inhibition IC50 values for 11a and 11b were 57 and 490 nM, respectively; the value for 11a is in good agreement with that previously reported (20-50 nM). In tests against other folate-utilizing enzymes, 11a and 11b were found to be inhibitors of glycinamide ribonucleotide formyltransferase (GAR formyltransferase) from one bacterial (Lactobacillus casei) and two mammalian (Manca and L1210) sources with 11a being decidedly more inhibitory than 11b. Neither 11a nor 11b inhibited aminoimidazolecarboxamide ribonucleotide formyltransferase. These results support reported evidence that 11a owes its observed antitumor activity to interference with the purine de novo pathway with the site of action being GAR formyltransferase.
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PMID:Synthesis and antifolate activity of 5-methyl-5,10-dideaza analogues of aminopterin and folic acid and an alternative synthesis of 5,10-dideazatetrahydrofolic acid, a potent inhibitor of glycinamide ribonucleotide formyltransferase. 318 24

The folate compound 10-formyldihydrofolate (H2folate) has not been found as a component of intracellular folates in normal tissues but has been identified in the cytosol of methotrexate (MTX)-treated MCF-7 breast cancer cells and normal human myeloid precursor cells. Its identity was verified by coelution of this compound with a synthetic marker on high pressure liquid chromatography, its reduction to 10-formyltetrahydrofolate (H4folate) in the presence of dihydrofolate reductase, and its enzymatic deformylation to dihydrofolate in the presence of aminoimidazolecarboxamide ribonucleotide (AICAR) transformylase. Chemically synthesized monoglutamated or pentaglutamated 10-formyl-H2folate was examined for its interaction with three folate-dependent enzymes: AICAR transformylase, glucinamide ribotide (GAR) transformylase, and thymidylatesynthase. 10-Formyl-H2folate-Glu5 was a competitive inhibitor of thymidylate synthase (Ki = 0.16 microM with 5,10-methylene-H4folate-Glu1 as substrate and 1.6 microM with 5,10-methylene-H4folate-Glu5) and inhibited GAR transformylase (Ki = 2.0 microM). It acted as a substrate for AICAR transformylase (Km = 5.3 microM), and its efficiency was equal to that of the natural substrate 10-formyl-H4folate-Glu5. The inhibition of thymidylate synthase by 10-formyl-H2folate was highly dependent on the inhibitor's polyglutamation state, the -Glu5 derivative having a 52-85-fold greater affinity as compared to the affinity of -Glu1. Polyglutamation of 10-formyl-H2folate did not affect its inhibition of GAR transformylase. While the actual role of 10-formyl-H2folate contributing to the cytotoxicity of MTX has not been determined, this compound has the potential to enhance inhibition of GAR transformylase and thymidylate synthase, and at the same time provides additional substrate for AICAR transformylase. The MTX-induced intracellular accumulation of 10-formyl-H2folate and H2folate may play a role in the drug-related cytotoxicity through the contribution of these folates to the inhibition of thymidylate synthase and de novo purine synthesis.
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PMID:Identification and biochemical properties of 10-formyldihydrofolate, a novel folate found in methotrexate-treated cells. 336 69

We previously reported (Matherly et al., J Biol Chem 267: 23253-23260, 1992) that impaired methotrexate transport in a drug-resistant CCRF-CEM variant (CEM/MTX) involved the synthesis of a structurally altered isoform of the "classical" carrier for methotrexate and related derivatives. Although CEM/MTX cells were highly resistant (162- to 300-fold) to assorted antifolate substrates for the classical transporter, including methotrexate, aminopterin, 10-ethyl-10-deazaaminopterin, ICI D1694, and 1843U89, they were only 3.6-fold resistant to (6R)-5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF). These divergent antifolate sensitivities were not associated with appreciable differences in the levels of dihydrofolate reductase, thymidylate synthase, and 5'-phosphoribosylglycinamide (GAR) transformylase, or the expression of a high affinity membrane folate binding protein receptor in either line. The initial rate of [14C]DDATHF influx was increased 2.9-fold over that for [3H]methotrexate in parental cells (at 2 microM). Whereas [14C]DDATHF initial uptake was, likewise, increased over [3H]methotrexate in CEM/MTX cells (5.3-fold), influx of both compounds was impaired substantially (95-97%). For the parent, influx of [14C]DDATHF was inhibited by substrates for the classical transporter including unlabeled DDATHF, methotrexate, (6R,S)-5-formyl tetrahydrofolate, 10-ethyl-10-deazaaminopterin, ICI D1694, 1843U89, and folic acid. The synthesis of a modified transporter in CEM/MTX cells was accompanied by significant changes in the binding of all these transport substrates. In spite of its impaired transport, [14C]DDATHF (at 2 microM), unlike methotrexate, continued to accumulate in CEM/MTX cells, eventually reaching 62% of the parental drug levels after 4 hr. At this time, 53% (parent) and 71% (CEM/MTX) of the intracellular radioactivity from [14C]DDATHF was identified as polyglutamates. DDATHF polyglutamates in CEM/MTX cells after 4 hr reached 90% of the levels measured in parental cells. While significant levels of methotrexate polyglutamates were detected in the parental line, methotrexate polyglutamylation was negligible in intact CEM/MTX cells. The specific activity of folylpolyglutamate synthetase was measured in cell-free extracts from parental and CEM/MTX cells using aminopterin, methotrexate, and DDATHF as substrates; in each case, CEM/MTX cells showed 2-fold higher enzyme activity than parental cells. These data show that even for tumor cells with severely impaired antifolate transport, the extensive conversion of DDATHF to polyglutamyl forms required for GAR transformylase inhibition preserves high levels of antitumor activity.
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PMID:Determinants of the disparate antitumor activities of (6R)-5,10-dideaza-5,6,7,8-tetrahydrofolate and methotrexate toward human lymphoblastic leukemia cells, characterized by severely impaired antifolate membrane transport. 750 26

5,10-dideaza-5,6,7,8-terrahydrofolic acid (DDATHF) is a potent antiproliferative agent in cell culture systems and in vivo in a number of murine and human xenograft tumors. In contrast to classical antifolates, which are dihydrofolate reductase inhibitors, DDATHF primarily inhibits GAR transformylase, the first folate-dependent enzyme along the pathway of de novo purine biosynthesis. The (6R) diastereomer of DDATHF (Lometrexol), currently undergoing clinical investigation, was used to develop CCRF-CEM human leukemia sublines resistant to increasing concentrations of the drug. Three cell lines were selected for ability to grow in medium containing 0.1 microM, 1.0 microM, and 10 microM of (6R)DDATHF, respectively. Impaired polyglutamylation was identified as a common mechanism of resistance in all three cell lines. A progressive decrease in the level of polyglutamylation was associated with diminished folylpolyglutamate synthetase activity and paralleled increasing levels of resistance to the drug. However, the expression of folylpolyglutamate synthetase RNA was not altered in the resistant cell lines compared to the parent cells. The most resistant cell subline also displayed an increased activity of gamma-glutamyl hydrolase. The sublines were scrutinized for other possible mechanisms of resistance. No alterations in drug transport or in purine economy were found. Modest increases were found in the activity of methylene tetrahydrofolate dehydrogenase but no alterations of other folate-dependent enzymes were observed. Increases in accumulation and conversion of folic acid to reduced forms, particularly 10-formyltetrahydrofolate, was also seen. The resistant cell lines were sensitive to dihydrofolate reductase inhibitors, methotrexate and trimetrexate, for a 72-h exposure period but showed cross-resistance to methotrexate for 4 and 24 h exposures. Cross-resistance was also shown toward other deazafolate analogues for both short- and long-term exposures.
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PMID:Multifactorial resistance to 5,10-dideazatetrahydrofolic acid in cell lines derived from human lymphoblastic leukemia CCRF-CEM. 783 26