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R L Kisliuk

Publications and source records attributed to R L Kisliuk.

At least 37 records · Page 2Linked to original sources
PubMed1992

Quantitation of folic acid enhancement of antifolate synergism.

Trimetrexate (TMTX), 5,10-dideazatetrahydrofolate (DDATHF), and 10-propargyl-5,8-dideazafolate (PDDF, CB3717) are antifolates whose primary intracellular targets are dihydrofolate reductase, glycinamide ribonucleotide formyltransferase, and thymidylate synthase, respectively. Varying the medium folic acid (PteGlu) concentration over the range of 0.5 to 100 microM increasingly blocks the growth inhibitory effects of the individual antifolates in Manca human lymphoma cells, but increasingly enhances the synergistic interaction of both TMTX + DDATHF and TMTX+ PDDF combinations. Drug interactions were quantitated using the universal response surface approach, which consists of fitting a concentration-effect surface to experimental data with weighted nonlinear regression, enabling the estimation of the synergism parameter, alpha. Estimates for alpha are larger (more intense synergism) for the TMTX + DDATHF combination (alpha = 4.68 +/- 0.66 at 2 microM PteGlu; alpha = 53.6 +/- 3.7 at 40 microM PteGlu) than for the TMTX + PDDF combination (alpha = 0.690 +/- 0.25 at 2 microM PteGlu; alpha = 7.20 +/- 1.8 at 40 microM PteGlu). However, the relative increase brought about by increasing the PteGlu concentration from 2 microM to 40 microM is similar in each instance, 11- and 10-fold, respectively. These experiments suggest that the enhanced cytotoxic interaction brought about by increased PteGlu concentration with the TMTX + DDATHF combination and the TMTX + PDDF combination may share a common mechanism. The dramatic intensity of the synergism between DDATHF and TMTX at 40 microM PteGlu, along with the concept of modulation of antifolate synergism by folates, suggests future in vivo and/or clinical applications of combinations of these (or similar) compounds.

Cell Division↗
PubMed1991

Folate analogues. 34. Synthesis and antitumor activity of non-polyglutamylatable inhibitors of dihydrofolate reductase.

Five analogues of methotrextate (MTX), 10-deazaaminopterin (10-DAM), and 10-ethyl-10-deazaaminopterin (10-EDAM) in which the glutamate moiety was replaced by either a gamma-methyleneglutamate or beta-hydroxyglutamate were synthesized and evaluated for their antifolate activity. These analogous are 4-amino-4-deoxy-N10-methylpteroyl-beta-hydroxyglutamic acid (1), 4-amino-4-deoxy-10-deazapteroyl-beta-hydroxyglutamic acid (2), 4-amino-4-deoxy-N10-methylpteroyl-gamma-methyleneglutamic acid (3, MMTX), 4-amino-4-deoxy-10-deazapteroyl-gamma-methyleneglutamic acid (4, MDAM), and 4-amino-4-deoxy-10-ethyl-10-deazapteroyl-gamma-methyleneglutamic acid (5, MEDAM). None of these compounds were metabolized to the respective polyglutamate derivative as judged by their inability to serve as substrates for CCRF-CEM human leukemia cell folylpolyglutamate synthetase (FPGS) in vitro. All compounds inhibited recombinant human-dihydrofolate reductase (DHFR) at nearly equivalent magnitude as MTX. Growth-inhibition studies with H35 hepatoma, Manca human lymphoma, and CCRF-CEM human leukemia cells established greater cytotoxic effects with compounds 3-5 than with compounds 1 and 2. gamma-Methyleneglutamate derivatives 3-5 were transported to H35 hepatoma cells better than MTX or beta-hydroxyglutamate derivatives 1 and 2. Compound 3 was 2.5 times better than MTX in competing with folinic acid transport in H35 hepatoma cells. Compound 1 did not have a significant inhibitory effect on folinic acid transport even at 50 microM under identical conditions. The IC50 for compound 1 against H35-hepatoma cell growth was 8.5-fold higher than MTX. Compounds with the gamma-methyleneglutamate moiety (3-5) exhibited almost equal or lower IC50 values than MTX against the growth of CCRF-CEM human leukemia cells. These studies show that on continuous exposure, the non-polyglutamylatable inhibitors DHFR (3-5) can exhibit superior antifolate activity compared to the polyglutamylatable methotrexate, presumably due to their enhanced transport to these cell lines. Compounds 3-5 appear to be excellent models to study the role of polyglutamylation of antifolates in antitumor activity and host toxicity.

Animals↗
PubMed1991

Synthesis and biological activity of 5,11-methylenetetrahydro-5- deazahomofolic acid.

The synthesis of 5,11-methylene-5-deazatetrahydrohomofolate (5), a stable, semirigid mimic of 5,10-methylenetetrahydrofolate (4) is reported as a potential inhibitor of thymidylate synthases (TS). The key intermediate 3-amino-1-oxo-tetrahydropyrimido[4,5-c] [2,6]naphthyridine (6) was obtained by the regiospecific cyclocondensation of 2,4,6-triaminopyrimidine with ethyl 1-benzyl-3-oxo-4-piperidinecarboxylate followed by halogenation (of the resulting lactam 9) and catalytic hydrogenolysis. Selective reduction of 6 followed by arylation with tert-butyl p-fluorobenzoate, saponification, and coupling with diethyl L-glutamate followed by saponification afforded the target compound 5. The title compound was tested as an inhibitor of the growth of Manca human lymphoma cells and also as an inhibitor of TS from Manca cells and Lactobacillus casei and was found to be inactive. In addition, compound 5 also failed to inhibit glycinamide ribonucleotide formyltransferase from L. casei and from Manca cells.

Cell Line↗
PubMed1991

Folate analogues. 35. Synthesis and biological evaluation of 1-deaza, 3-deaza, and bridge-elongated analogues of N10-propargyl-5,8-dideazafolic acid.

Structural modifications at the pyrimidine ring and at the C9,N10-bridge region of the thymidylate synthase (TS) inhibitors N10-propargyl-5,8-dideazafolate (1; PDDF; CB 3717), 2-desamino-N10-propargyl-5,8-dideazafolate (2, DPDDF), and 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolate (3, DMPDDF) have been carried out. Methods for the synthesis of 2-desamino-N10-propargyl-1,5,8-trideazafolate (4), 2-desamino-2-methyl-N10-propargyl-3,5,8-trideazafolate (5a), and 2-desamino-2-methyl-N10-propargyl-5,8-dideaza-1,2-dihydrofolate (6) have been developed. The bridge-extended analogues isohomo-PDDF (7) and isohomo-DMPDDF (8) contain an additional methylene group interposed between N10 and the phenyl ring of 1 and 3, respectively. All new compounds were evaluated as inhibitors of TS and the growth of tumor cells in culture. Selected analogues were tested as substrates of folylpolyglutamate synthetase (FPGS) and striking differences in substrate activity were observed among these compounds, indicating that structural modifications at the pyrimidine ring of classical antifolates profoundly influence their polyglutamylation. Enzyme inhibition data established that both N1 and N3-H of the pyrimidine ring are essential for efficient binding of quinazoline-type antifolates to human TS.

Biological Transport↗
PubMed1990

Tetrahydrohomofolate polyglutamates as inhibitors of thymidylate synthase and glycinamide ribonucleotide formyltransferase in Lactobacillus casei.

In order to determine the mechanism for the effects of homofolates on growth of Lactobacillus casei, polyglutamated derivatives of homofolate (HPteGlu), dihydrohomofolate and tetrahydrohomofolate (H4HPteGlu) were synthesized and tested as inhibitors of folate-requiring enzymes. The following L. casei enzymes were examined: thymidylate synthase (TS), glycinamide ribonucleotide formyltransferase (GARFT), aminoimidazolecarboxamide ribonucleotide formyltransferase, serine hydroxymethyltransferase and dihydrofolate reductase. Polyglutamates of (6R,S)-H4HPteGlu are potent inhibitors of TS and GARFT. For example, the IC50 values of (6R,S)-H4HPteGlu6 are 0.7 microM for TS and 0.3 microM for GARFT. By contrast, the value for HPteGlu6 is greater than 10 microM for both TS and GARFT. Inhibition of TS and GARFT by (6R,S)-H4HPteGlu derivatives increases with polyglutamate chain length. For TS, the Glu5 and Glu6 derivatives of (6R,S)-H4HPteGlu are 20 and 30 times more potent than the monoglutamate, respectively. For GARFT, the Glu2-6 derivatives are 2-3 times more potent than Glu1. Inhibition of TS and GARFT by (6R,S)-H4HPteGlu polyglutamates is almost entirely due to the unnatural (6R) diastereomer at C-6. Homofolate derivatives are only weak inhibitors of aminoimidazolecarboxamide ribonucleotide formyltransferase, serine hydroxymethyltransferase, and dihydrofolate reductase. We conclude that both TS and GARFT are potential targets of (6R)-H4HPteGlu polyglutamates.

Acyltransferases↗
PubMed1990

Synthesis and antifolate properties of 9-alkyl-10-deazaminopterins.

Reformatski condensation of benzyl 2-bromopropionate with 4-carbomethoxybenzaldehyde, followed by dehydration afforded benzyl 2-methyl-p-carbomethoxycinnamate (4a). Hydrogenation over a Pd catalyst gave the hydrocinnamic acid 5a. Conversion to the chloromethyl (6a) and azidomethyl ketone (7a) was followed by hydrogenation to the aminomethyl ketone (8a). Direct N-alkylation by 2,4-diamino-5-nitro-6-chloropyrimidine followed by reductive ring closure in Zn-HOAc and subsequent saponification of the benzoate ester yielded 4-amino-4-deoxy-9-methyl-10-deazapteroic acid (11a). Coupling with diethyl L-glutamate and saponification afforded 9-methyl-10-deazaminopterin (13a). The 9-ethyl analogue (13b) was similarly prepared from benzyl 2-bromobutyrate. The 9-methyl analogue (13a) was 21 times more potent than MTX as an inhibitor of cell growth in L1210 cells. The reason for this enhanced cytotoxicity in L1210 is unclear, since enzyme inhibition and transport parameters were similar to those of MTX. In human Manca leukemia cells growth inhibition was not dramatic and paralleled MTX.

Aminopterin↗
PubMed1990

Synthesis and antifolate properties of 10-alkyl-5,10-dideaza analogues of methotrexate and tetrahydrofolic acid.

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.

Acyltransferases↗
PubMed1990

Folate analogues. 33. Synthesis of folate and antifolate poly-gamma-glutamates by [(9-fluorenylmethoxy)oxy]carbonyl chemistry and biological evaluation of certain methotrexate polyglutamate polylysine conjugates as inhibitors of the growth of H35 hepatoma cells.

Representative examples of folate and antifolate poly-gamma-glutamyl metabolites were synthesized via the [(9-fluorenylmethoxy)oxy]carbonyl (Fmoc) chemistry using the KH polyamide resin. Polyglutamate yields were consistently better in all cases compared to the previous Merrifield method, and the crude products were obtained in greater than 85% purity. The symmetrical anhydride (7) derived from alpha-tert-butyl N-Fmoc-L-glutamate (6) was used for the initial coupling of the first glutamate residue to the KH resin and also for subsequent chain elongation. The alpha-tert-butyl protective groups were not labile under the conditions used for the cleavage of the finished peptide from the resin. A series of poly-gamma-glutamyl metabolites of methotrexate (MTX) with a chain length ranging from two to five glutamyl residues were synthesized and coupled with poly(L-lysine) having an average molecular weight of 27,000 and 52,000. Each conjugate was tested for its ability to inhibit the growth of wild type (H35) and MTX transport resistant (H35R) strains of hepatoma cells in culture, the latter having a 100-fold reduced sensitivity to MTX. 4-Amino-4-deoxy-N10-methylpteroylglutamyl-gamma-glutamylpoly (L-lysine) conjugate [MTX(G2)-poly-L-Lys-52000] and MTX(G4)-poly-L-Lys-52000 were among the most active (I50 = 8.0 and 10 nM against H35 cells) MTX-polylysines synthesized to date, and they were somewhat more inhibitory to the transport resistant cells. MTX(G5)-poly-L-Lys-52000 was approximately 1000 times more effective than MTX(G5)-poly-D-Lys-52000 in inhibiting the growth of H35R hepatoma cells in culture, indicating that internal cleavage of the gamma-glutamate chain of the conjugate with subsequent release of MTX or shorter chain polyglutamates of MTX is unlikely to be an important determinant of MTX-polyglutamate polylysine cytotoxicity. The results indicate that MTX-polyglutamate poly(L-lysine) conjugates are taken up by the cells independently of MTX and probably via endocytosis.

Antimetabolites, Antineoplastic↗
PubMed1989

Inhibition of glycinamide ribonucleotide formyltransferase and other folate enzymes by homofolate polyglutamates in human lymphoma and murine leukemia cell extracts.

In order to determine the biochemical basis for the cytotoxicity of homofolates, poly-gamma-glutamyl derivatives of homofolate (HPteGlu) and tetrahydrohomofolate (H4HPteGlu) were synthesized and tested as inhibitors of glycinamide ribonucleotide formyltransferase (GARFT), aminoimidazolecarboxamide ribonucleotide formyltransferase (AICARFT), thymidylate synthase, and serine hydroxymethyltransferase (SHMT) in extracts of Manca human lymphoma and L1210 murine leukemia cells. The most striking inhibitions are that of GARFT by (6R,S)-H4HPteGlu4-6 with IC50 values from 1.3 to 0.3 microM. Both diastereomers, (6R)-H4HPteGlu6 and (6S)-H4HPteGlu6, inhibit GARFT activity. In Manca cell extracts, the (6S) form is more potent than the (6R) form whereas in the murine system the reverse is true. The (6R,S)-H4HPteGlu polyglutamates are weak inhibitors of human AICARFT (IC50, 6-10 microM). Polyglutamates of HPteGlu, however, are more inhibitory to AICARFT, with HPteGlu4-6 having IC50 values close to 2 microM. Polyglutamates of HPteGlu and of H4HPteGlu are weaker inhibitors of thymidylate synthase (IC50, 8 microM for HPteGlu5-6 and greater than 20 microM for H4HPteGlu1-5). Polyglutamates of HPteGlu and of H4HPteGlu are poor inhibitors of SHMT (IC50, greater than 20 microM). Manca cell growth is inhibited 50% by HPteGlu and (6R,S)-5-methyl-H4HPteGlu at 6 and 8 microM, respectively. Both of these effects are reversed by 0.1 mM inosine. Trimetrexate at a subinhibitory concentration, 10 nM, antagonizes growth inhibition by HPteGlu, raising the IC50 from 6 to 64 microM, but enhances inhibition by (6R,S)-5-methyl-H4HPteGlu, lowering the IC50 from 8 to 5 microM. Our results support the view that homofolates become toxic after conversion to H4HPteGlu polyglutamates which block GARFT, a step in purine biosynthesis.

Acyltransferases↗
PubMed1989

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.

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.

Acyltransferases↗
PubMed1989

Folate analogues. 32. Synthesis and biological evaluation of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid and related compounds.

The chemical synthesis of three close analogues (2-4) of N10-propargyl-5,8-dideazafolate (PDDF) is described. The quinazoline ring of 2 and 4 was constructed from the pivotal intermediate 9 in a novel and unambiguous manner during the final step of the synthesis under very mild conditions. 2-Desamino-2-methyl-N10-propargyl-5,8-dideazafolate (DMPDDF) (2) was a strong inhibitor of human and Lactobacillus casei thymidylate synthases, whereas 2-desamino-2-(trifluoromethyl)-N10-propargyl-5,8-didezafolate (3) and 2-desamino-2,3-dimethyl-N10-propargyl-5,8-dideazafolate (4) were only weak inhibitors of this enzyme. DMPDDF exhibited excellent growth inhibition of Manca human lymphoid leukemia and H35 hepatoma cells in culture. The inhibitor activities of 2 were 43- and 65-fold greater than that of PDDF, respectively, in these cell lines. H35R cells that are resistant to methotrexate by virtue of a transport defect were cross resistant to DMPDDF but not to PDDF. H35FF cells which have 70-fold greater amounts of thymidylate synthase compared to H35N cells were 130-fold resistant to DMPDDF. Furthermore, the toxicity of DMPDDF to H35 hepatoma cells could be completely reversed by thymidine, establishing its locus of action as thymidylate synthase. Transport studies in vitro established that DMPDDF effectively inhibits MTX influx into H35 hepatoma cells, whereas PDDF has no effect on MTX transport in this cell line. These data suggest that the greater activity of DMPDDF relative to PDDF is partly due to the ability of the former compound to enter cells via the MTX/reduced folate transport system. Enzyme inhibition data of 4 suggest that the presence of N3H in DMPDDF is essential for binding to thymidylate synthase.

Breast Neoplasms↗
PubMed1988

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.

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.

Acyltransferases↗
PubMed1988

Synthesis and biological evaluation of 8-deazahomofolic acid and its tetrahydro derivative.

The syntheses of 8-deazahomofolic acid and its tetrahydro derivative, potential inhibitors of thymidylate synthase (TS) and other folate related enzymes, are described. Wittig condensation of 2-acetamido-6-formyl-4-pyrimidinol with the triphenylphosphine ylide 3 derived from N-acetyl-4-(p-carbethoxyanilino)-1-chloro-2-butanone, hydrogenation of the enone intermediate 5, introduction of a 5-amino group via diazonium coupling, and reductive ring closure yielded ethyl N11-acetyl-8-deazahomopteroate (8). Alkaline hydrolysis gave 8-deazahomopteroic acid, which was blocked as the 11-trifluoroacetyl derivative, coupled with diethyl L-glutamate, and the blocking groups saponified to afford 8-deazahomofolic acid (12). Hydrogenation of the glutamate diester intermediate and subsequent saponification yielded the tetrahydro-8-deazahomofolate (14). Growth inhibition of Streptococcus faecium, Lactobacillus casei, and L1210 cells in culture by the target compounds was modest. They were also weak inhibitors of thymidylate synthase, dihydrofolate reductase, glycinamide-ribonucleotide transformylase, and aminoimidazolecarboxamide ribonucleotide transformylase. In contrast, 8-deazafolate showed moderate inhibition of aminoimidazolecarboxamide ribonucleotide transformylase, suggesting that inhibition of this enzyme may be related to its cytotoxic action. Tetrahydro-8-deazahomofolate showed low substrate activity with thymidylate synthase.

Acyltransferases↗
PubMed1988

Synthesis and biological evaluation of poly-gamma-glutamyl metabolites of 10-deazaaminopterin and 10-ethyl-10-deazaaminopterin.

The chemical synthesis of a series of poly-gamma-glutamyl metabolites of the experimental anticancer drugs 10-deazaaminopterin (10-DAAM) and 10-ethyl-10-deazaaminopterin (10-EDAAM) has been carried out by the solid-phase procedure. The synthetic products were identical with the poly-gamma-glutamyl metabolites of radiolabeled 10-DAAM and 10-EDAAM produced by normal mouse tissues with regard to elution volume from [(diethylamino)ethyl]cellulose columns and susceptibility to hydrolysis by human plasma folylpolyglutamate hydrolase. Poly-gamma-glutamyl metabolites with a glutamate chain length of up to four glutamate residues were detected in the tissues. The antifolate activity was evaluated with methotrexate (MTX) sensitive and MTX-resistant strains of Lactobacillus casei and Streptococcus faecium. In general, inhibitory potency decreases with increasing Glu chain length. However there are two exceptions. Addition of one Glu residue to 10-DAAM enhances its potency for MTX-resistant L. casei and addition of one Glu residue to 10-EDAAM enhances its potency for the MTX-sensitive L. casei. As shown earlier for MTX polyglutamates, polyglutamylation greatly enhances the inhibitory potency of 10-DAAM and 10-EDAAM for L. casei thymidylate synthase. MTX polyglutamates are 15-30 times more inhibitory than the corresponding 10-DAAM derivatives and 30-60 times more inhibitory than the corresponding 10-EDAAM derivatives. Polyglutamylation of 10-DAAM had little influence on its ability to inhibit L. casei dihydrofolate reductase; however, with 10-EDAAM, addition of one or two Glu residues enhanced its inhibitory potency 2.3-fold.

Aminopterin↗
PubMed1987

Carrier-mediated transport of folate compounds in L1210 cells. Initial rate kinetics and extent of duality of entry routes for folic acid and diastereomers of 5-methyltetrahydrohomofolate in the presence of physiological anions.

Comparison of the kinetic parameters for influx of highly purified [3H]folic acid versus [3H]methotrexate in L1210 cells under anionic buffer conditions showed a marked discordancy. In addition, the kinetics for influx of [3H]folic acid were unchanged in variant L1210 cells defective in [3H]methotrexate transport. In these variant cells, the Vmax for methotrexate was reduced 17-fold and the Km was increased 3-fold. The results show that [3H]folic acid influx is mediated by a system which has a low affinity, but a 20-fold higher capacity, for folate compounds than the classical high-affinity system mediating [3H]methotrexate influx. Since the latter system also exhibits very low affinity for [3H]folic acid, it would not be expected to contribute significantly to the total influx of [3H]folic acid. The high-capacity system for [3H]folic acid influx is different from that believed to mediate pterin influx in L1210 cells since it was not inhibited by adenine, a potent inhibitor of pterin influx. However, exposure of cells to [3H]folic acid in a nonanionic buffer resulted in marked stimulation of initial influx, and a fraction of influx under these conditions was inhibited by methotrexate. These results suggest that anions modulate the extent of multiplicity of [3H]folic acid influx by their known effects on the high-affinity, reduced folate/methotrexate system. The diastereomers, at carbon 6, of [14C]5-methyltetrahydrohomofolate shared both transport systems. The influx Km for the natural diastereomer was one-half that of the unnatural form for both transport systems. Both diastereomers showed a much greater differential in affinity between the two transport systems than did [3H]folic acid. Our results suggest that an analog which could be effectively transported by the low-affinity/high-capacity route may be useful in the treatment of tumors resistant to methotrexate due to a defective high-affinity/low capacity influx system. We also found that incubation of L1210 cells with [3H]folic acid or the natural diastereomer [14C]5-methyltetrahydrohomofolate for 10 min resulted in the formation of a nonexchangeable fraction of radioactivity amounting to 20-40% of the total accumulation. This non-exchangeable fraction may be explained by the accumulation of metabolites other than polyglutamates. Preloading of cells with methotrexate prior to incubation with [3H]folic acid prevented the accumulation of radioactivity as a nonexchangeable fraction.

Animals↗
PubMed1987

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.

Folic Acid↗
PubMed1986

Identification of poly G bound to thymidylate synthase.

Thymidylate synthase activity is increased in some methotrexate-resistant strains of Streptococcus faecium. The purified enzyme is associated with a polynucleotide which is not removed by dialysis. This polynucleotide contains one mole each of purine ribose and phosphate per mole base. Phosphate analyses after incubation with digestive enzymes indicate a tetranucleotide with one terminal phosphate. The constituent nucleosides are recovered quantitatively in a specific assay for guanosine. On HPLC, they are inseparable from authentic guanosine and the UV spectrum after HPLC is identical to that of guanosine. We conclude that poly G (GpGpGpGp) is bound to thymidylate synthase.

Chromatography, High Pressure Liquid↗