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.

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.

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.

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.

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.

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.

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.

Coenzyme function of cobalamin analogues from calf kidney.

Calf kidney has been used as a tissue source for the isolation of cobalamin analogues, which are defined here as cobalt-containing compounds of distinctive chromatographic behavior that are extractable from tissues by methods conventionally used to extract cobalamin and which, in radioisotope dilution assays, are more active with R-protein as binder than intrinsic factor and are relatively less active in microbiologic assays. Preparatory methods employed reverse affinity chromatography or a series of chemical extractions for the isolation of corrin followed by Dowex-50 chromatography. An analogue-containing fraction (peak 2) was eluted by acetate buffers between pH 4 and 5. This material was shown to contain cobalt, to migrate differently than the four cobalamins in Dowex-50 and paper chromatography, and to display a pattern of properties that is compatible with the above definition of cobalamin analogues. These analogues stimulated crude preparations of N5-methyltetrahydrofolate-homocysteine methyltransferase (EC 2.1.1.13) from Escherichia coli and rat liver at far lower concentrations (1-40 nmol/L) than the major cobalamins. No evidence of enzymatic conversion of cobalamin to analogue could be demonstrated.

Differences in coenzyme specificity of the N5-methyltetrahydrofolate-homocysteine methyltransferases of various species: implications for corrin binding loci.

Investigations of the coenzyme specificity of N5-methyltetrahydrofolate-homocysteine methyltransferases of diverse biological origin revealed previously unrecognized differences between Escherichia coli methyltransferase and the corresponding enzymes of other species. Cyanocobalamin (CNCbl) actively supports methyltransferase in extracts of animal tissues and E. coli. Cobinamide is more active than CNCbl with rat liver methyltransferase; however, it is non-competitively inhibitory with E. coli enzyme. E. coli methyltransferase, but not rat liver enzyme, is competitively inhibited by alpha-ribazole 3'-phosphate and 5,6-dimethyl-benzimidazole, two moieties of the nucleotide loop. This suggests that animal enzyme binds its corrinoid coenzyme at a site on the corrin macro-ring, while E. coli enzyme binds to the nucleotide loop as well as the macro-ring.

Serine hydroxymethyltransferase activity and serine incorporation in leukocytes.

Studies of serine hydroxymethyltransferase activity in extracts of leukocytes from normal and leukemic subjects showed that the enzyme is present in lymphocytes and granulocytes but that activity is higher in lymphocytes. It is also higher than normal in lymphocytes from patients with chronic lymphocytic leukemia and to a lesser extent in the leukocytes of patients with acute myelocytic leukemia and acute lymphocytic leukemia. A striking increase in activity occurs in lymphocytes stimulated by phytohemagglutinin to divide in culture. Enzyme activity rises severalfold before cell number increases. Stimulated lymphocytes take up [3-14C]serine from the medium and incorporate its radioactivity into DNA, RNA, and other cell fractions. The rate of incorporation increases sharply before the rise in cell number. Thus, serine hydroxymethyltransferase activity and serine incorporation in vivo show a temporal correlation in stimulated lymphocytes. Inhibitors of DNA synthesis (e.g., fluorodeoxyuridine or high concentrations of adenosine or thymidine) block incorporation of serine radioactivity into DNA and other cell fractions. The results suggest that serine hydroxymethyltransferase activity and cellular uptake of serine have a significant role in proliferating cells.

Production of formaldehyde from N5-methyltetrahydrofolate by normal and leukemic leukocytes.

Extracts of human normal and leukemic leukocytes contain an enzyme that catalyzes a transfer of labeled methyl carbon from N5-[14C]methyltetrahydrofolate to tryptamine. Evidence is presented that this reaction is not attributable to a methyltransferase but to the following reaction sequence: (a) an oxidation of N5-[14C]methyltetrahydrofolate to N5, N10-[14C]methylenetetrahydrofolate that is catalyzed by N5, N10-methylenetetrahydrofolate reductase (EC 1.1.1.68); (b) spontaneous release of [14C]formaldehyde from N5, N10-[14C]methylenetetrahydrofolate; and (c) nonenzymatic condensation of [14C]formaldehyde with tryptamine to form a radioactive carboline derivative. The occurrence of this sequence in leukocytes is suggested by data that show that the enzyme reaction is strongly stimulated by addition of flavin adenine dinucleotide and that the final product is chromatographically identical to the adduct formed in the reaction of [14C]formaldehyde with tryptamine. In the absence of tryptamine, a product accumulates that can react with other HCHO acceptors, i.e., beta-phenylethylamine and dimedone; another reaction product is tetrahydrofolate. Production of formaldehyde is relatively more active in normal lymphocytes than in normal granulocytes, but it is even higher in lymphocytes of chronic lymphocytic leukemia. Activity in granulocytes from a subject with chronic myelocytic leukemia is also elevated but to a lesser extent than activity in lymphocytes of chronic lymphocytic leukemia. Activity in granulocytes from a subject with chronic myelocytic leukemia is also elevated but to a lesser extent than activity in lymphocytes of chronic lymphocytic leukemia. Formaldehyde production in leukocytes is only slightly stimulated by addition of various cobalamins, and activity is normal in leukocytes from a vitamin B12-deficient patient. We conclude that the system is cobalamin independent. Thus, there exists an active pathway from N5-methyltetrahydrofolate to tetrahydrofolate other than the one catalyzed by cobalamin-dependent N5-methyltetrahydrofolate-homocysteine methyltransferase.

Studies on N5-methyltetrahydrofolate-homocystein methyltransferase in normal and leukemia leukocytes.

A cobalamin-dependent N5-methyltetra-hydrofolate-homocysteine methyltransferase (methyl-transferase) was demonstrated in unfractioned extracts of human normal and leukemia leukocytes. Activity was substantially reduced in the absence of an added cobalamin derivative. Presumably, this residual activity reflects the endogeneous level of holoenzyme. Enzyme activity was notably higher in lymphoid cells than in myeloid cells. Thus, mean specific activities (+/-SD) were: chronic lymphocytic leukemia lymphocytes, 2.15+/-1.16; normal lymphocytes, 0.91+/-0.59; normal mature granulocytes, 0.15+/-0.10; chronic myelocytic leukemia granulocytes, barely detectable activity. Properties of leukocytes enzymes resembled those of methyltransferases previously studied in bacteria and other animal cells. Granulocytes and chronic myelocytic leukemia cells contain a factor or factors that inhibits Escherichia coli enzyme. The data suggest that the prominence of this cobalamin-dependent enzyme in lymphocytes and other mononuclear cell types may be related to their potential for cell division.

Complementation studies of lethal alleles in the mouse causing deficiencies of glucose-6-phosphatase, tyrosine aminotransferase, and serine dehydratase.

The pleiotropic effects of six radiation-induced lethal alleles at the albino locus in the mouse include deficiencies of various enzymes and of serum proteins. Closely correlated with the biochemical deficiencies are severe ultrastructural abnormalities of endoplasmic reticulum membranes and the Golgi apparatus in liver and kidney cells. Complementation studies led to the distinction of four different complementation groups among the six lethal alleles. The results are compatible with the interpretation that the six lethal alleles represent overlapping deletions of various sizes at and around the albino locus in the mouse, in a region concerned with the control of biochemical and structural differentiation.