Glycinamide Ribonucleotide Formyltransferase Inhibitor Research Articles

The Chemistry of DDATHF (5,10-Dideaza-5,6,7,8-tetrahydrofolic Acid) as Antitumor Agent

Over the past 40 years, big efforts have been devoted to the development of novel folate antimetabolites. All of the potent antifolates have reportedly been inhibitors of dihydrofolate reductase (DHFR). In 1985, Taylor and et al. reported the synthesis of 5,10-dideaza-5,6,7,8-tetrahydrofolic acid, DDATHF, which exhibits broad and selective antitumor activity as an inhibitor of glycinamide ribonucleotide formyltransferase (GARFT). DDATHF is a close analog of tetrahydrofolic acid, differs only by replacement of the 5- and 10- position nitrogen atoms by carbon. It may exist in two diastereomeric forms, differing in configuration at carbon 6. Both diastereomers of DDATHF are potent inhibitors of cell growth in culture. DDATHF is currently in Phase II clinical trials

Effect of a novel antifolate, N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine (PT523), on growth of H35 rat hepatoma and HEPG2 human hepatoma cells.

Structural modifications of folic acid have generated various derivatives that act at different target sites. Well-known examples include the dihydrofolate reductase (DHFR) inhibitor methotrexate (MTX),1–3 the thymidylate synthase inhibitor 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid,4,5 and the glycinamide ribonucleotide formyltransferase inhibitor 5,10-dideaza-5,6,7,8-tetrahydrofolic acid.6,7 More recently, folate analogues containing ornithine in place of glutamate have been synthesized as inhibitors of folylpolyglutamate synthetase.8,9 These ornithine-containing analogues showed weaker activity than the corresponding glutamate-containing analogues on cell growth presumably because of inefficient transport across the cell membrane.9,10 In contrast, a novel derivative of an ornithine-containing antifolate, Nα-(4-amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-Lornithine (PT523), exhibits potent in vitro and in vivo antitumor activity.11

Side chain modified 5-deazafolate and 5-deazatetrahydrofolate analogues as mammalian folylpolyglutamate synthetase and glycinamide ribonucleotide formyltransferase inhibitors: synthesis and in vitro biological evaluation.

5-Deazafolate and 5-deazatetrahydrofolate (DATHF) analogues with the glutamic acid side chain replaced by homocysteic acid (HCysA), 2-amino-4-phosphonobutanoic acid (APBA), and ornithine (Orn) were synthesized as part of a larger program directed toward inhibitors of folylpolyglutamate synthetase (FPGS) as probes of the FPGS active site and as potential therapeutic agents. The tetrahydro compounds were also of interest as non-polyglutamatable inhibitors of the purine biosynthetic enzyme glycinamide ribonucleotide formyltransferase (GARFT). Reductive coupling of N2-acetamido-6-formylpyrido[2,3-d]pyrimidin-4(3H)-one with 4-aminobenzoic acid, followed by N10-formylation, mixed anhydride condensation of the resultant N2-acetyl-N10-formyl-5- deazapteroic acid with L-homocysteic acid, and removal of the N2-acetyl and N10-formyl groups with NaOH, afforded N-(5-deazapteroyl)-L-homocysteic acid (5-dPteHCysA). Mixed anhydride condensation of N2-acetyl-N10-formyl- 5-deazapteroic acid with methyl D,L-2-amino-4-(diethoxyphosphinyl)butanoic acid, followed by consecutive treatment with Me3SiBr and NaOH, yielded D,L-2-[(5-deazapteroyl)amino]-4-phosphonobutanoic acid (5-dPteAPBA). Treatment with NaOH alone led to retention of one ethyl ester group on the phosphonate moiety. Catalytic hydrogenation of N2-acetyl-N10-formyl-5-deazapteroic acid followed by mixed anhydride condensation with methyl L-homocysteate and deprotection with NaOH afforded N-(5,6,7,8-tetrahydro-5-deazapteroyl)-L-homocysteic acid (5-dH4PteHCysA). Similar chemistry starting from methyl D,L-2-amino-4-(diethoxyphosphinyl)butanoic acid and methyl N delta-(benzyloxycarbonyl)-L-ornithinate yielded D,L-2-[(5-deaza-5,6,7,8-tetrahydropteroyl)amino]-4-phosphonobut ano ic acid (5-dH4Pte-APBA) and N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine (5-dH4PteOrn), respectively. The 5-deazafolate analogues were inhibitors of mouse liver FPGS, and the DATHF analogues inhibited both mouse FPGS and mouse leukemic cell GARFT. Analogues with HCysA and monoethyl APBA side chains were less active as FPGS inhibitors than those containing an unesterified gamma-PO(OH)2 group, and their interaction with the enzyme was noncompetitive against variable folyl substrate. In contrast, Orn and APBA analogues obeyed competitive inhibition kinetics and were more potent, with Ki values as low as 30 nM. Comparison of the DATHF analogues as GARFT inhibitors indicated that the Orn side chain diminished activity relative to DATHF, but that the compounds with gamma-sulfonate or gamma-phosphonate substitution retained activity, with Ki values in the submicromolar range. The best GARFT inhibitor was the 5-dH4PteAPBA diastereomer mixture, with a Ki of 47 nM versus 65 nM for DATHF. None of the compounds showed activity against cultured WI-L2 or CEM human leukemic lymphoblasts at concentrations of up to 100 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis and biological activity of nor- and homo-5,10-dideazatetrahydrofolic acid

A palladium(0)-catalyzed Heck coupling reaction was used in the synthesis of nor- and homo-DDATHF which are close analogs of the potent glycinamide ribonucleotide formyltransferase inhibitor, 5,10-dideazatetrahydrofolic acid (DDATHF).

Structural features of 5,10-dideaza-5,6,7,8-tetrahydrofolate that determine inhibition of mammalian glycinamide ribonucleotide formyltransferase

We have investigated the structural features of 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF) that determine the activity of this compound as an inhibitor of glycinamide ribonucleotide formyltransferase (GARFT) purified from mouse L1210 cells. 5-Deazatetrahydrofolate was as good an inhibitor of GARFT as DDATHF, indicating that isosteric replacement of nitrogen by carbon at the 5-position of tetrahydrofolate is sufficient for inhibition of GARFT. 5,10-Dideazafolic acid, 5,8,10-trideazatetrahydrofolate, and 2-desamino-5,10-dideazatetrahydrofolate were poor inhibitors of GARFT, indicating that a reduced pyridopyrimidine ring, N-8, and the 2-amino group of DDATHF, respectively, play an important role in the binding of tetrahydrofolate analogues to this enzyme. DDATHF analogues in which the phenyl ring was replaced either by a cyclohexyl ring or by methylene groups retained activity as inhibitors. 5,10-Dideazatetrahydrohomofolate was about 6 times more potent as an inhibitor of GARFT than DDATHF, but 5,10-dideazatetrahydronorfolate had about one-fifth of the activity of DDATHF. An analogue of DDATHF in which the glutamic acid side chain was replaced by aspartic acid (which was not a substrate for polyglutamation and was only weakly cytotoxic) was equiactive with DDATHF as an inhibitor of purified GARFT. Surprisingly, 5,10-dideazatetrahydropteroic acid was about as active as DDATHF as an inhibitor of GARFT, an indication that the glutamic acid in the side chain of DDATHF does not play a role in this ligand-enzyme interaction. The polyglutamate derivatives of DDATHF bound up to 100 times tighter to GARFT than DDATHF itself; longer chain polyglutamates conformed to Goldstein's zone B behavior under experimental conditions and were projected to be in zone C, i.e., stoichiometric inhibition, in vivo. We conclude that the presence of carbon at the 5-position of tetrahydrofolate analogues is sufficient for inhibition of GARFT, that N-8 and the 2-amino group are involved in binding of DDATHF to GARFT, probably through hydrogen bonds, and that the structures of the phenyl ring and amino acid side chain of DDATHF analogues are not primary determinants of GARFT inhibition by monoglutamate forms of these compounds. We also conclude that polyglutamation plays a major role in the potent cytotoxicity of DDATHF.

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 (H 4HPteGlu) 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 (6 R,S)-H 4HPteGlu are potent inhibitors of TS and GARFT. For example, the IC 50 values of (6 R,S)-H 4HPteGlu 6 are 0.7 μ m for TS and 0.3 μ m for GARFT. By contrast, the value for HPteGlu 6 is > 10 μ m for both TS and GARFT. Inhibition of TS and GARFT by (6 R,S)-H 4HPteGlu derivatives increases with polyglutamate chain length. For TS, the Glu 5 and Glu 6 derivatives of (6 R,S)-H 4HPteGlu are 20 and 30 times more potent than the monoglutamate, respectively. For GARFT, the Glu 2–6 derivatives are 2–3 times more potent than Glu 1. Inhibition of TS and GARFT by (6 R,S)-H 4HPteGlu polyglutamates is almost entirely due to the unnatural (6 R) 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 (6 R)-H 4HPteGlu polyglutamates.

Folate analogs. 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.

ChemInform Abstract: 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.

AbstractOlefination of the carboxaldehydes (II), obtained by reduction of the nitriles (I), with the Wittig reagent (III) gives the key intermediates (IV).

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.