Phase I study of (6R)-5,10-dideazatetrahydrofolate: a folate antimetabolite inhibitory to de novo purine synthesis.

BACKGROUND: Cancer chemotherapy with folate antimetabolites has been traditionally targeted at the enzyme dihydrofolate reductase and is based on the requirement of dividing tumor cells for a supply of thymidylate and purines. However, a new compound, 5,10-dideazatetrahydrofolate (DDATHF, whose 6R diastereomer is also known as Lometrexol), has become available that prevents tumor cell growth by inhibiting the first of the folate-dependent enzymes involved in de novo purine synthesis, glycinamide ribonucleotide formyltransferase. PURPOSE: We investigated the toxicity and therapeutic activity of DDATHF in a phase I clinical trial. METHODS: DDATHF was given at one of the following dose levels to 33 patients (16 females and 17 males) with malignant solid tumors: 3.0 mg/m2 per week (level A) to 10 patients, 4.5 mg/m2 per week (level B) to 13 patients, or 6.0 mg/m2 per week (level C) to 10 patients. Each drug cycle consisted of three weekly injections of DDATHF followed by a 2-week rest prior to redosing in the next cycle. RESULTS: Of 33 patients, 27 received at least one full cycle of DDATHF. Thrombocytopenia was the major dose-limiting toxicity, and it was severe in one of 10 patients during the first cycle and in two of four patients during the second cycle. Because of cumulative toxicity at 6.0 mg/m2, second or later cycles were abbreviated to two weekly doses. Stomatitis was generally mild, but it was dose-limiting in one patient. Neutropenia was infrequent and mild, and normocytic anemia requiring blood transfusion was common with repeat dosing. Leucovorin was given for grade 2 or greater thrombocytopenia and resulted in hematologic recovery within 1 week in all eight patients so treated. Without leucovorin, the thrombocytopenia lasted from 7 to 49 days in three patients. A partial response was noted in one patient with non-small-cell lung cancer and a minor response in one patient with breast cancer. Three patients with colorectal cancer achieved stable disease for greater than 3 months with improvement in carcinoembryonic antigen levels in one patient. CONCLUSIONS: DDATHF has an unusual pattern of toxicity with repetitive dosing, and humans with advanced cancer are considerably more sensitive than would be predicted from previous animal studies. Although doses of 6.0 mg/m2 per week on our schedule have been determined to be safe, repeated cycles require careful monitoring because of cumulative toxic effects. IMPLICATIONS: Additional phase I studies of DDATHF that relate toxicity to folate intake and tissue folate pools appear warranted.

Schedule-dependent enhancement of the cytotoxicity of fluoropyrimidines to human carcinoma cells in the presence of folinic acid.

Previous studies from this laboratory indicated that the cytotoxic effects of the fluoropyrimidines on mouse leukemic cells are substantially augmented by folinic acid but that these effects are underestimated in growth inhibition experiments. These results have now been extended to two human tumor cell lines, the WiDr colorectal and T-24 bladder carcinoma cells. In both cell lines, the presence of folinic acid in the medium substantially enhanced the cytotoxicity of a 72-h exposure to either 5-fluorouracil (FUra) or 5-fluoro-2'-deoxyuridine. Folinic acid concentration-response curves for enhancement of the cytotoxicity of FUra to WiDr cells were broad but indicated that response was not maximal until at least 10 microM. Likewise, increased length of exposure to 10 microM folinic acid continuously enhanced the cytotoxicity of a 72-h treatment with FUra, but substantial enhancement was observed even after a 2-h exposure to folinate, and there was a diminished increment of cytotoxicity after 24-h exposure to folinic acid. Surprisingly, folinic acid augmentation of the cytotoxicity of a brief exposure to FUra (4 h) was minimal but enhancement of FUra cytotoxicity became much more pronounced with intervals of exposure to FUra of greater than or equal to 24 h. If these results can be mimicked in vivo without undue host toxicity, our experiments suggest that a substantial improvement in the therapeutic activity of FUra plus folinate would result from prolonged exposure to both agents.

Folinic acid augmentation of the effects of fluoropyrimidines on murine and human leukemic cells.

The effects of the fluoropyrimidines on leukemic cells of mouse and human origin have been studied in the presence of folinic acid. This reduced folate enhanced the cytotoxicity and the growth inhibitory potency of 5-fluorouracil (5-FUra) and of 5-fluoro-2'-deoxyuridine (FUdR) against all cell lines examined. The human leukemic cell lines used (two T- and two B-cells) were affected by these fluoropyrimidines only at substantially higher concentrations than were found to be inhibitory to mouse L1210 cells; however, the enhancement of the activity of the fluoropyrimidines occurred over the same range of folinic acid concentrations in mouse and human cells. Whereas the total intracellular folate pool increased continuously with every increment of folinic acid added to the medium, the enhancement of the potency of the fluoropyrimidines was limited. Augmentation of the effects of FUdR exceeded that of 5-fluorouracil in the human leukemic cells studied. The cytotoxicity of the fluoropyrimidines (as defined by cloning efficiency) was enhanced to a greater extent than was growth inhibition so that an impressive lethal synergism was noted; for instance, exposure of L1210 cells to nontoxic concentrations of 5-fluorouracil or FUdR in the presence of folinic acid resulted in a 98 or 99.9% cell kill, respectively. In contrast to previous predictions, the fluoropyrimidines were more inhibitory to mouse leukemic cells containing folate pools that were suboptimal for growth than for folate-replete cells. Growth rate experiments showed that cells exposed to moderate concentrations of FUdR were initially inhibited but recovered with time, whereas in cells exposed to both FUdR and folinic acid, the initial growth inhibitory effects were sustained. We conclude that folinic acid stabilizes the effects of the fluoropyrimidines on thymidylate synthase of both mouse and human leukemic cell populations and that this enhancement is reflected in both inhibition of the growth of and the lethality to these cells. We suggest that only doses of the fluoropyrimidines that are capable of initially inhibiting thymidylate synthase to a high degree will be synergistic with excess reduced folates.

Effects of allopurinol on the therapeutic efficacy of methotrexate.

The antitumor effects of methotrexate against early leukemia L1210 were partially reversed by the coadministration of allopurinol in vivo, even though allopurinol did not alter the growth-inhibitory effects of methotrexate against L1210 cells in culture. These data suggest that this alteration in antitumor activity results from a decreased catabolism of preformed systemic purines by allopurinol, a potent inhibitor of xanthine oxidase. On the other hand, the therapeutic effect of methotrexate against the P288 leukemia was not significantly altered by allopurinol did not significantly alter the toxicity of methotrexate that, in the mouse, the antileukemic effects of methotrexate are more related to a purineless rather than a thymineless death.

Cytotoxicity of antifolate inhibitors of thymidylate and purine synthesis to WiDr colonic carcinoma cells.

We have studied the cytotoxicity of 5,10-dideazatetrahydrofolate (DDATHF) and of D-1694 to human WiDr colonic carcinoma cells as a model system for the effects of pure inhibitors of either the de novo purine synthesis pathway or thymidylate synthesis. The growth of this cell line was inhibited by very low concentrations of either agent and the lethality of DDATHF and D-1694 was completely prevented by continuous exposure to either hypoxanthine or thymidine, respectively, indicating that these compounds were very potent metabolic inhibitors, each specific for one of these pathways. D-1694 was highly cytotoxic (> 3 logs of kill) after a 4-h exposure to 1 microM drug, or a 24-h exposure to very low concentrations (0.04 microM). On the other hand, the cytotoxicity of DDATHF was substantially lower, with 2 logs of cell kill requiring >> 100 microM with 4 h of exposure or approximately 40 microM for 72 h of exposure. Maximal cell kill induced by D-1694 was 5-6 logs, consistent with elimination of all viable cells except preexisting mutants. A maximum of 2-3 logs of cell kill was observed with DDATHF. Exposure of WiDr cells to either D-1694 or DDATHF caused striking cellular changes, but the morphologies of cells treated with the two drugs were remarkably different. D-1694-treated cells detached from the dish within 1-2 days after a megaloblastosis, whereas DDATHF-treated cells remained adherent to the dishes for at least 10 days after treatment. The addition of thymidine to D-1694-treated cultures or hypoxanthine to DDATHF-treated cells after up to 20 h of drug exposure completely prevented cytotoxicity of either drug. With longer exposures, cytotoxicity of both drugs progressively increased in spite of such rescue. Our results indicate that substantial (99-99.9%) tumor cell kill can be induced by a pure inhibitor of purine synthesis, but that the rate of commitment to cell death and the extent of cell kill is greater with a pure inhibitor of thymidylate synthesis.

Structural organization of the human folypoly-gamma-glutamate synthetase gene: evidence for a single genomic locus.

The cytotoxicity, and probably the selectivity, of folate antimetabolites depend upon the expression of the enzyme folylpoly-gamma-glutamate synthetase in tumor cells. Evidence for the existence of multiple forms of this enzyme and the need to define the control mechanisms determinant of expression levels in normal and neoplastic cells has focused attention on the gene(s) encoding these forms. The organization of the genomic locus for the human folylpoly-gamma-glutamate synthetase (FPGS) gene has been determined. The complete 2256 nucleotides of cDNA for the 5'-untranslated region, mitochondrial leader sequence, coding region, and 3'-untranslated region were distributed on 15 exons stretching over 11.2 kb of genomic DNA. All of the restriction fragments found in diploid human genomic DNA could be accounted for by fragments contained on the isolated genomic clones. Likewise, Southern analysis of the transfected human genomic DNA that complemented the FPGS- phenotype of a hamster cell line indicated that the same gene had been integrated in all of three independently derived transfectants. We conclude that the genomic locus that we now report appears to be the only gene encoding FPGS-related sequences in the human complement.

In vivo kinetics of thymidylate synthetase inhibition of 5-fluorouracil-sensitive and -resistant murine colon adenocarcinomas.

The predictive utility of several biochemical parameters of 5-fluorouracil (5-FUra) action was evaluated in four murine colonic adenocarcinomas: 5-FUra-sensitive Tumor 38 and 5-FUra-resistant Tumors 07/A, 51 and 06/A. Thymidylate synthetase (TS) was determined by a tritiated 5-fluoro-2'-deoxyuridylate (FdUMP)-binding assay. Bolus 5-FUra (80 mg/kg, i.p.) administrated caused in all tumors a rapid decrease in free TS levels. Only Tumor 38, however, showed inhibition of TS to undetectable (less than 0.05 pmol/g) levels, which lasted up to 6 hr after treatment; correction for dissociation of endogenous TS: FdUMP:folate ternary complex during the TS assay was required. Total TS (free enzyme plus ternary complex) was determined with experimental conditions that achieved quantitative recovery of free TS from ternary complex. By 48 hr after 5-FUra, Tumor 38 showed a decrease in total TS proportional to the estimated log kill/dose of 5-FUra; in contrast, the resistant tumors showed no such decrease from pretreatment levels. Assay of FdUMP showed that the free nucleotide was formed rapidly in all tumors in excess over available TS-binding sites. However, tumor sensitivity did not correlate with peak or residual FdUMP levels or with deoxyuridylate levels, which were low and remained so in all tumors. Tumor sensitivity to 5-FUra also could not be explained by the small differences among the tumors in total perchloric acid-soluble metabolites of 5-FUra or drug incorporation into RNA. We conclude from these data that levels of free TS in the tumor after 5-FUra treatment are predictive of chemotherapeutic response in these murine models of human colonic adenocarcinoma.

Activity of the thymidylate synthase inhibitor 2-desamino-N10-propargyl-5,8-dideazafolic acid and related compounds in murine (L1210) and human (W1L2) systems in vitro and in L1210 in vivo.

We examined the in vitro activity of 2-desamino-5,8-dideazafolate and 2-desamino-N10-propargyl-5,8-dideazafolate (desamino-CB3717), the more water-soluble 2-desamino analogues of 5,8-dideazafolate and N10-propargyl-5,8-dideazafolic acid (CB3717). We report Ki values for the inhibition of L1210 thymidylate synthase (TS) of 2 and 0.027 microM for 2-desamino-5,8-dideazafolate and desamino-CB3717, respectively, indicating a 30- and 10-fold loss in TS-inhibitory activity compared with the corresponding 2-NH2 compounds. The synthetic tri- and tetrapolyglutamate derivatives of desamino-CB3717 were 66- and 101-fold more potent than the monoglutamate form as inhibitors of TS. Both desamino compounds were more potent as inhibitors of L1210 and W1L2 cell growth than were their 2-amino counterparts. 2-Desamino-5,8-dideazafolate retains quite good activity against both the TS-overproducing W1L2:C1 line and the L1210 cell line grown in the presence of thymidine, suggesting that a secondary locus of action may be involved. This other target is a folate-dependent enzyme as evidenced by the protection of the inhibition of cell growth by the addition of hypoxanthine or folinic acid together with thymidine. The methotrexate-resistant, dihydrofolate reductase-overproducing L1210:R7A cell line is cross-resistant to 2-desamino-5,8-dideazafolate, which suggests that dihydrofolate reductase is the other target. An L1210 subline (1565) unable to transport reduced folates is 10-fold resistant to desamino-CB3717 and 2-desamino-5,8-dideazafolate but is not cross-resistant to CB3717 or 5,8-dideazafolate. The removal of the 2-amino function of CB3717 did not affect folylpolyglutamate synthetase substrate activity (CB3717 Km = 48 microM, desamino-CB3717 Km = 40 microM). However, both 5,8-dideazafolate and its desamino analogue were about 10-fold better substrates for folylpolyglutamate synthase than were the N10-propargyl compounds, and this may contribute to their good growth-inhibitory properties. In vivo, desamino-CB3717 cured approximately 75% of mice bearing the L1210:ICR tumor at doses of 50 mg/kg daily for 5 days and above (maximum tolerated dose greater than 1000 mg/kg daily for 5 days).(ABSTRACT TRUNCATED AT 400 WORDS)

Augmentation of the therapeutic activity of lometrexol -(6-R)5,10-dideazatetrahydrofolate- by oral folic acid.

Recent clinical trials with lometrexol [(6R)-5,10-dideazatetrahydrofolate] have revealed a level of toxicity in humans that was not predicted on the basis of previous in vivo preclinical studies. Because standard laboratory animal diets contain high levels of folic acid relative to human folate intake, the toxicity and therapeutic activity of lometrexol was studied in mice under conditions of restricted dietary folate intake. Remarkably, the lethality of this drug increased by three orders of magnitude in mildly folate-deficient mice, mimicking the unexpected toxicity seen in humans. Lometrexol had limited therapeutic activity in folate-deficient mice bearing the C3H mammary adenocarcinoma, compared with the substantial therapeutic index for treatment of this tumor in animals on standard diet. When folic acid was administered p.o. to mice that were mildly folate deficient, antitumor activity was again observed at nontoxic doses of lometrexol, and the range of lometrexol doses that allowed safe therapeutic use of this drug increased at higher dietary folate intake. At a fixed dose of lometrexol, the antitumor effects in animals were dependent on the level of dietary folate and went through a distinct optimum. Excessively high folate intake reversed the antitumor effects of lometrexol. Optimization of the folic acid content in the diet and of the lometrexol dosage are predicted to have substantial impact on the clinical activity of this class of drugs.

Tissue-specific expression of functional isoforms of mouse folypoly-gamma-glutamae synthetase: a basis for targeting folate antimetabolites.

Folates and folate antimetabolites are metabolically trapped in mammalian cells as polyglutamates, a process catalyzed by folylpoly-gamma-glutamate synthetase (FPGS). Using 5'-rapid amplification of cDNA ends, RNase protection assays, transfection of cDNAs into FPGS-deficient cells, and kinetic analysis of recombinant enzymes expressed in insect cells, it was determined that the species of active FPGS in mouse liver and kidney was different from that in mouse tumor cells, bone marrow, and intestine. The NH2-terminal peptide of hepatic enzyme contained 18 amino acids not found in enzyme from dividing tissues, and the specificity of the two isoforms for antifolates also differed, suggesting different architecture of the active sites. In most tissues, the expression of one isozyme or the other was an all-or-nothing event. The exclusive use of one of two alternative sets of initial coding exons in different tissues underlies this phenomenon, suggesting the design of antifolates specific for activation by individual FPGS isoforms and hence tissue-selective targeting of antifolate therapy for cancer, arthritis, or psoriasis.

Mouse cDNAs encoding a trifunctional protein of de novo purine synthesis and a related single-domain glycinamide ribonucleotide synthetase.

Three of the enzymatic activities of de novo purine synthesis, glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS) and glycinamide ribonucleotide formyltransferase (GART), can be catalyzed by a single 110-kDa protein in mouse cells. Western blots using a polyclonal antibody (Ab) to this protein identified two species, the trifunctional 110-kDa protein and a 50-kDa cytosolic protein with GARS, but not GART activity. We used Ab and, subsequently, oligodeoxyribonucleotide screens to isolate cDNAs corresponding to these two proteins from mouse T-cell cDNA expression libraries. The sequence of one class of these cDNAs and the partial sequence of a corresponding genomic clone defined an open reading frame (ORF) encoding a 1010-amino-acid (aa) protein, individual domains of which showed high homology to each of the monofunctional bacterial GARS, AIRS and GART proteins, and to each domain of chicken and human trifunctional GARS-AIRS-GARTs. cDNAs corresponding to the smaller protein contained a 1.3-kb ORF with complete identity to the GARS domain of, but with a 3' untranslated region different from, the trifunctional cDNAs. Hence, both cDNAs appear to derive from the same gene due to either differential splicing or use of an intronic polyadenylation signal. The functional requirement for the expression of both trifunctional protein with GARS activity and monofunctional, catalytically active GARS is unknown.

Roles of folylpoly-gamma-glutamate synthetase in therapeutics with tetrahydrofolate antimetabolites: an overview.

Folylpoly-gamma-glutamate synthetase (FPGS) catalyzes the addition of several equivalents of glutamic acid to the gamma-carboxyl group in the side chain of folate cofactors and analogs. Folylpoly-gamma-glutamate synthetase has three functions in folate homeostasis in mammals: polyglutamation prevents efflux of folate cofactors from the cell, it increases the binding of folate cofactors to some of the enzymes of folate interconversion and biosynthesis, and it appears to allow the accumulation of folates in the mitochondria that are required for glycine synthesis. The efficient substrate activity of the newer generations of tetrahydrofolate analogs results in levels of intracellular accumulation of cytotoxic drug in any cell expressing FPGS in which the enzyme activity is not suppressed by feedback, and the binding of folate inhibitors of thymidylate synthase and glycinamide ribonucleotide formyltransferase is substantially increased by polyglutamation. Resistance to these drugs appears to be most frequently due to mutations that change the level of polyglutamation of parent compound, a clear indication of the centrality of the process to the cytotoxicity of these drugs. Folylpoly-gamma-glutamate synthetase is widely expressed in human tumors and is tightly linked either to proliferation or to a lack of differentiation. The cytotoxicity of both thymidylate synthase and purine inhibitors requires continued inhibition of target for greater than one generation time, so that the integrative function of FPGS adds considerably to the efficiency of folate antimetabolites.

Treatment of colon cancer based on biochemical modulation of fluoropyrimidines by hydroxyurea.

Preclinical studies have indicated that hydroxyurea leads to enhancement of 5-fluorouracil (5-FU) activity when given after 5-FU. This is presumably due to hydroxyurea's actions resulting in maintaining low levels of deoxyuridine monophosphate pools. The combination of hydroxyurea and 5-FU has been tested in several inconclusive clinical trials in patients with advanced colorectal cancer. However, pharmacodynamic studies have been lacking, and the schedules tested have not usually tried to follow preclinical findings. With the current renewed interest in 5-FU, a role for hydroxyurea as part of its biochemical modulation is worthy of further study.

N-[4-[[(3,4-dihydro-4-oxo-1,2,3-benzotriazin-6- yl)methyl]amino]benzoyl]-L-glutamic acid, a novel A-ring analogue of 2-desamino-5,8-dideazafolic acid.

N-[4-[[(3,4-Dihydro-4-oxo-1,2,3-benzotriazin-6- yl)methyl]amino]benzoyl]-L-glutamic acid ("2-aza-2-desamino-5,8- dideazafolic acid", ADDF) was synthesized from 2-amino-5-methylbenzamide via a four-step sequence consisting of diazotization, benzylic bromination, condensation with dimethyl N-(4-aminobenzoyl)-L-glutamate, and ester hydrolysis. ADDF was an inhibitor of recombinant mouse thymidylate synthase; inhibition was competitive with 5,10-methylenetetrahydrofolate as variable substrate (Ki = 2.3 microM). It was a substrate for murine folylpolyglutamate synthetase with kinetic characteristics (Km = 28 microM) comparable to those of aminopterin, and it inhibited the growth of L1210 cells in culture (IC50 = 0.52 microM). The structural modification of the A-ring embodied in ADDF appears to offer a novel, heretofore unexplored approach to the design of TS inhibitors.

(6R,6S)-5,8,10-trideaza-5,6,7,8-tetrahydrofolate and 6(R,6S)-5,8,10-trideaza-5,6,7,8-tetrahydropteroyl-L-ornithine as potential antifolates and antitumor agents.

(6R,6S)-5,8,10-Trideaza-5,6,7,8-tetrahydropteroic acid was synthesized in several steps from 4,4-(ethylenedioxy)-cyclohexanone and [4-(tert-butyloxycarbonyl)benzyl]triphenylphosphonium bromide and was elaborated to (6R,6S)-5,8,10-trideaza-5,6,7,8-tetrahydropteroyl-L-glutamic acid and (6R,6S)-5,8,10-trideaza-5,6,7,8-tetrahydropteroyl-L-ornithin e. Compound 1 was found to be a good substrate for partially purified mouse liver folypolyglutamate synthetase (FPGS), with a Michaelis constant (Km = 15 microM) comparable to that reported for the reduced folate substrate (6S)-5,6,7,8-tetrahydropteroyl-L-glutamic acid and for (6R,6S)-5,10-dideaza-5,6,7,8-tetrahydropteroyl-L-glutamic acid (DDATHF). However, in striking contrast to DDATHF, which is potently cytotoxic, 1 failed to inhibit tumor cell growth in culture at concentrations of up to 100 microM. These results suggested that the NH at position 8 of DDATHF is important for cytotoxic activity but not for polyglutamylation. Just as 1 was a good substrate for FPGS, the ornithine analogue 2 proved to be among the more potent competitive inhibitors of this enzyme discovered to date, with a Ki,s of 10 microM. While the binding affinity of 2 was lower than that reported for 5,6,7,8-tetrahydropteroyl-L-ornithine (H4PteOrn), very substantial FPGS inhibition was observed even though N5,N8, and N10 in H4PteOrn were replaced by carbon. Binding to FPGS thus appears to be tolerant of bioisosteric replacements made simultaneously in ring B and the bridge region. Neither 1 nor 2 was active in preventing cell growth in culture at concentrations of up 100 microM. The N delta-hemiphthaloyl derivative of 2, synthesized as a potential prodrug, was also inactive.

Synthesis and biological activity of the 2-desamino and 2-desamino-2-methyl analogues of aminopterin and methotrexate.

The previously undescribed 2-desamino and 2-desamino-2-methyl analogues of aminopterin (AMT) and methotrexate (MTX) were synthesized from 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile. The AMT analogues were obtained via a three-step sequence consisting of condensation with di-tert-butyl N-(4-aminobenzoyl)-L-glutamate, heating with formamidine or acetamidine acetate, and mild acidolysis with trifluoroacetic acid. The MTX analogues were prepared similarly, except that 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile was condensed with 4-(N-methylamino)benzoic acid and the resulting product was annulated with formamidine or acetamidine acetate to obtain the 2-desamino and 2-desamino-2-methyl analogues, respectively, of 4-amino-4-deoxy-N10-methylpteroic acid. Condensation with di-tert-butyl L-glutamate in the presence of diethyl phosphorocyanidate followed by ester cleavage with trifluoroacetic acid was then carried out. Retention of the L configuration in the glutamate moiety during this synthesis was demonstrated by rapid and essentially complete hydrolysis with carboxypeptidase G1 under conditions that likewise cleaved the L enantiomer of MTX but left the D enantiomer unaffected. The 2-desamino and 2-desamino-2-methyl analogues of AMT and MTX inhibited the growth of tumor cells, but were very poor inhibitors of dihydrofolate reductase (DHFR). These unexpected results suggested that activity in intact cells was due to metabolism of the 2-desamino compounds to polyglutamates.

A mechanism for the addition of multiple moles of glutamate by folylpolyglutamate synthetase.

The role of the alpha-carboxyl group in methotrexate (MeAPA-Glu) and the gamma-glutamate derivative of methotrexate (MeAPA-Glu-Glu) in the reaction catalyzed by folylpolyglutamate synthetase (FPGS) has been investigated. MeAPA-Glu and MeAPA-Glu-Glu were accepted as substrates by the same FPGS species contained in an (NH4)2SO4 precipitate of mouse liver protein, as judged by a lack of additivity of product formation at saturating concentrations of both substrates. MeAPA-Gaba, the MeAPA-Glu analogue lacking an alpha-carboxyl, was inactive as a substrate for this enzyme as was MeAPA-Glu-Gaba, the analogue of MeAPA-Glu-Glu that lacked the alpha-carboxyl of the terminal glutamic acid. However, MeAPA-Gaba-Glu, the analogue of MeAPA-Glu-Glu without an alpha-carboxyl on the first glutamic acid, had activity as a substrate for FPGS that approached that of MeAPA-Glu-Glu. These results suggest that the alpha-carboxyl is essential for the binding of folyl monoglutamates to FPGS in the correct orientation to allow catalysis. Moreover, the binding of the terminal alpha-carboxyl of folyl oligoglutamates to the same residue(s) responsible for the binding of the alpha-carboxyl of folyl monoglutamates would allow correct positioning of the terminal gamma-carboxyl of the chain for reaction. This binding mechanism would be compatible with the utilization of a single enzyme species for the addition of glutamate to the monoglutamate or oligoglutamate forms of folates and folate analogues.

5-deazafolate analogues with a rotationally restricted glutamate or ornithine side chain: synthesis and binding interaction with folylpolyglutamate synthetase.

Rotationally restricted analogues of 5-deazapteroyl-L-glutamate and (6R,6S)-5-deaza-5,6,7,8-tetrahydropteroyl-L-glutamate with a one-carbon bridge between the amide nitrogen and the 6'-position of the p-aminobenzoyl moiety were synthesized and tested as substrates for folylpolyglutamate synthetase (FPGS), a key enzyme in folate metabolism and an important determinant of the therapeutic potency and selectivity of classical antifolates. The corresponding bridged analogues of 5-deazapteroyl-L-ornithine and (6R,6S)-5-deaza-5,6,7, 8-tetrahydropteroyl-L-ornithine were also synthesized as potential inhibitors. Condensation of diethyl L-glutamate with methyl 2-bromomethyl-4-nitrobenzoate followed by catalytic reduction of the nitro group, reductive coupling with 2-acetamido-6-formylpyrido[2, 3-d]pyrimidin-4(3H)-one in the presence of dimethylaminoborane, and acidolysis with HBr/AcOH yielded 2-L-[5-[N-(2-acetamido-4(3H)-oxopyrido[2, 3-d]pyrimidin-6-yl)methylamino]-2, 3-dihydro-1-oxo-2(1H)-isoindolyl]glutaric acid (1). When acidolysis was preceded by catalytic hydrogenation, the final product was the corresponding (6R,6S)-tetrahydro derivative 2. A similar sequence starting from methyl N(delta)-benzyloxycarbonyl-L-ornithine led to 2-L-[5-[N-(2-amino-4(3H)-oxopyrido[2, 3-d]pyrimidin-6-yl)methylamino]-2, 3-dihydro-1-oxo-2(1H)-isoindolyl]-5-aminopentanoic acid (3) and the (6R,6S)-tetrahydro derivative 4. Compounds 3 and 4 were powerful inhibitors of recombinant human FPGS, whereas 1 and 2 were exceptionally efficient FPGS substrates, with the reduced compound 2 giving a K(m) (0.018 microM) lower than that of any other substrate identified to date. (6R,6S)-5-Deazatetrahydrofolate, in which the side chain is free to rotate, was rapidly converted to long-chain polyglutamates. In contrast, the reaction of 1 and 2 was limited to the addition of a single molecule of glutamic acid. Hence rotational restriction of the side chain did not interfere with the initial FPGS-catalyzed reaction and indeed seemed to facilitate it, but the ensuing gamma-glutamyl adduct was no longer an efficient substrate for the enzyme.

Methotrexate analogues. 34. Replacement of the glutamate moiety in methotrexate and aminopterin by long-chain 2-aminoalkanedioic acids.

Eight previously unreported methotrexate (MTX) and aminopterin (AMT) analogues with the L-glutamate moiety replaced by DL-2-aminoalkanedioic acids containing up to 10 CH2 groups were synthesized from 4-amino-4-deoxy-N10-methylpteroic or 4-amino-4-deoxy-N10-formylpteroic acid. All the compounds were potent inhibitors of purified L1210 mouse leukemia dihydrofolate reductase (DHFR), with IC50's of 0.023-0.034 microM for the MTX analogues and 0.054-0.067 microM for the AMT analogues. The compounds were not substrates for, but were inhibitors of, partially purified mouse liver folylpolyglutamate synthetase (FPGS). Activity was correlated with the number of CH2 groups in the side chain. The IC50's for inhibition of cell growth in culture by the chain-extended MTX analogues were 0.016-0.64 microM against CEM human leukemic lymphoblasts and 0.0012-0.026 microM against L1210 mouse leukemia cells. However, the optimal chain length for growth-inhibitory activity was species-dependent. Our results suggested that CEM cells were inhibited most actively by the analogue with nine CH2 groups, while L1210 cells were most sensitive to the analogue with six CH2 groups. Among the AMT analogues, on the other hand, the most active compound against L1210 cells was the one with nine CH2 groups, which had an IC50 of 0.000 65 microM as compared with 0.0046 microM for MTX and 0.002 microM for AMT. A high degree of cross-resistance was observed between MTX and the chain-extended compounds in two MTX-resistant cell lines, CEM/MTX and L1210/R81. All the MTX analogues were active against L1210 leukemia in mice on a qd X 9 schedule, with optimal increases in lifespan (ILS) of 75-140%. Notwithstanding their high in vitro activity, the AMT analogues were more toxic and less therapeutically effective than MTX analogues of the same chain length even though neither series of compounds possessed FPGS substrate activity. These MTX and AMT analogues are an unusual group of compounds in that they retain the dicarboxylic acid structure of classical antifolates yet are more lipophilic than the parent compounds because they have more CH2 groups and are almost equivalent in vivo to MTX on the same schedule even though they do not form polyglutamates.

Methotrexate analogues. 31. Meta and ortho isomers of aminopterin, compounds with a double bond in the side chain, and a novel analogue modified at the alpha-carbon: chemical and in vitro biological studies.

Five heretofore undescribed analogues of methotrexate (MTX) and aminopterin (AMT) were synthesized and tested as dihydrofolate reductase (DHFR) inhibitors and tumor cell growth inhibitors. The meta isomer of AMT was obtained from 2,4-diamino-6-(bromomethyl)pteridine and m-(aminobenzoyl)-L-glutamic acid, while the ortho isomer was obtained via the same route by using alpha-methyl gamma-tert-butyl o-(aminobenzoyl)-L-glutamate instead of the free acid. Analogues of MTX and AMT containing a double bond in the side chain were prepared from dimethyl D,L-2-amino-4-hexenedioate and 4-amino-4-deoxy-N10-methylpteroic acid and 4-amino-4-deoxy-N10-formylpteroic acid, respectively. Finally, a positional isomer of MTX with the CH2CH2COOH moiety moved from the alpha-carbon to the adjacent carboxamide nitrogen was synthesized from 3-[N-(carboxymethyl)amino]propanoic acid diethyl ester and 4-amino-4-deoxy-N10-methylpteroic acid. The positional isomers of AMT were weak DHFR inhibitors and showed very little growth-inhibitory activity against L1210 murine leukemia cells or the MTX-resistant L1210/R81 mutant line in culture. The MTX and AMT analogues with the CH2CH2COOH moiety replaced by a CH2CH = CHCOOH side chain showed anti-DHFR activity similar to that of the previously described saturated compound N-(4-amino-4-deoxy-N10-methylpteroyl)-L-2-aminoadipic acid, but were less potent than the parent drugs. The MTX analogue with the CH2CH2COOH side chain displaced from C to N was weakly bound to DHFR, confirming the importance of an intact CONH moiety, and showed greatly diminished cell growth inhibitory potency relative to MTX. None of the compounds was a substrate for folylpolyglutamate synthetase (FPGS) from mouse liver. Furthermore, inhibition of folic acid polyglutamylation in vitro at equimolar 500 microM concentrations of drug and substrate was negligible. The structural changes embodied in these five novel compounds are therefore too great for binding to the FPGS active site.