10-Formyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid (10-Formyl-DDACTHF): A potent cytotoxic agent acting by selective inhibition of human GAR Tfase and the de novo purine biosynthetic pathway

Thomas H. Marsilje, Marc A. Labroli, Michael P. Hedrick, Qing Jin, Joel Desharnais, Stephen J. Baker, Lata T. Gooljarsingh, Joseph Ramcharan, Ali Tavassoli, Yan Zhang, Ian A. Wilson, G. Peter Beardsley, Stephen J. Benkovic, Dale L. Boger

Research output: Contribution to journalArticlepeer-review

18 Scopus citations


The synthesis of 10-formyl-DDACTHF (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) is reported. Aldehyde 3, the corresponding γ- and α-pentaglutamates 21 and 25 and related agents were evaluated for inhibition of folate-dependent enzymes including GAR Tfase and AICAR Tfase. The inhibitors were found to exhibit potent cytotoxic activity (CCRF-CEM IC50 for 3=60 nM) that exceeded their enzyme inhibition potency [Ki (3)=6 and 1 μM for Escherichia coli GAR and human AICAR Tfase, respectively]. Cytotoxicity rescue by medium purines, but not pyrimidines, indicated that the potent cytotoxic activity is derived from selective purine biosynthesis inhibition and rescue by AICAR monophosphate established that the activity is derived preferentially from GAR versus AICAR Tfase inhibition. The potent cytotoxic compounds including aldehyde 3 lost activity against CCRF-CEM cell lines deficient in the reduced folate carrier (CCRF-CEM/MTX) or folylpolyglutamate synthase (CCRF-CEM/FPGS-) establishing that their potent activity requires both reduced folate carrier transport and polyglutamation. Unexpectedly, the pentaglutamates displayed surprisingly similar Ki's versus E. coli GAR Tfase and only modestly enhanced Ki's versus human AICAR Tfase. On the surface this initially suggested that the potent cytotoxic activity of 3 and related compounds might be due simply to preferential intracellular accumulation of the inhibitors derived from effective transport and polyglutamation (i.e., ca. 100-fold higher intracellular concentrations). However, a subsequent examination of the inhibitors against recombinant human GAR Tfase revealed they and the corresponding γ-pentaglutamates were unexpectedly much more potent against the human versus E. coli enzyme (Ki for 3, 14 nM against rhGAR Tfase versus 6 μM against E. coli GAR Tfase) which also accounts for their exceptional cytotoxic potency.

Original languageEnglish (US)
Pages (from-to)2739-2749
Number of pages11
JournalBioorganic and Medicinal Chemistry
Issue number8
StatePublished - 2002

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmaceutical Science
  • Drug Discovery
  • Clinical Biochemistry
  • Organic Chemistry


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