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)
Rosowsky
A
A
Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
Forsch
R A
RA
Reich
V E
VE
Freisheim
J H
JH
Moran
R G
RG
D000217:Acyltransferases / Q000037:antagonists & inhibitors*; D000818:Animals; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases; D007945:Leukemia, Lymphoid / Q000201:enzymology; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D010453:Peptide Synthases / Q000037:antagonists & inhibitors*; D051026:Phosphoribosylglycinamide Formyltransferase; D013763:Tetrahydrofolates / Q000138:chemical synthesis* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D013763:Tetrahydrofolates; C060147:5-deaza-5,6,7,8-tetrahydrofolic acid; C040505:5-deazafolic acid; D005492:Folic Acid; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D000217:Acyltransferases; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase