Improved synthesis of zebularine [1-(beta-D-ribofuranosyl)-dihydropyrimidin-2-one] nucleotides as inhibitors of human deoxycytidylate deaminase

J Enzyme Inhib. 1995;9(2):147-62. doi: 10.3109/14756369509042814.

Abstract

The 2'-deoxy (2a) and 2'-ara-fluoro (3a) derivatives of zebularine [1-(beta-D-ribofuranosyl)-dihydropyrimidin-2-one, 1a] were phosphorylated in high yield to the 5'-nucleotides 2b and 3b, respectively, and characterized by HPLC, enzyme degradation, 1H, 13C and 31P NMR, and high resolution mass spectral analysis. Their inhibitory activity against partially purified MOLT-4 deoxycytidylate deaminase (dCMPD) in the presence of the allosteric effector deoxycytidine triphosphate (dCTP) and Mg+2 ion was examined. Compounds 2b and 3b inhibited dCMPD with Ki values of 2.1 x 10(-8) M and 1.2 x 10(-8) M, respectively. The parent nucleotide, zebularine monophosphate 1b was ineffective at concentrations > 100 mumol. The effect of the nucleosides, 1a-3a, as well as tetrahydrouridine (THU) and 2'-deoxy THU (dTHU), on the cellular production of DNA precursors was examined in human MOLT-4 peripheral lymphoblasts. It was shown that 1a, 2a and 3a all elevated intracellular dCTP and TTP levels in whole cells with the most powerful effect elicited by 1a. The 2'-fluoro derivative 3a was chemically phosphorylated much more cleanly and higher yield than 2a, without the formation of diphosphorylated by-products. This compound was found to be infinitely less sensitive to acid-catalyzed degradation than 2a. Since the substitution of fluorine for hydrogen had a slight potentiating effect on the dCMPD inhibitory activity while stabilizing the compound toward acid-catalyzed and enzymatic depyrimidination, compound 3b emerges as a very attractive tool for the pharmacological modulation of pyrimidine deaminase activity.

MeSH terms

  • Cell Line
  • Chromatography, High Pressure Liquid
  • Cytidine / analogs & derivatives
  • DCMP Deaminase / antagonists & inhibitors*
  • DCMP Deaminase / isolation & purification
  • DCMP Deaminase / metabolism
  • Deoxyribonucleotides / metabolism
  • Drug Stability
  • Enzyme Inhibitors / chemical synthesis*
  • Enzyme Inhibitors / isolation & purification
  • Enzyme Inhibitors / pharmacology*
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Lymphocytes / enzymology
  • Lymphocytes / metabolism
  • Magnetic Resonance Spectroscopy
  • Pyrimidine Nucleosides / chemical synthesis*
  • Pyrimidine Nucleosides / isolation & purification
  • Pyrimidine Nucleosides / pharmacology
  • Pyrimidine Nucleotides / chemical synthesis*
  • Pyrimidine Nucleotides / isolation & purification
  • Pyrimidine Nucleotides / pharmacology*
  • Structure-Activity Relationship

Substances

  • Deoxyribonucleotides
  • Enzyme Inhibitors
  • Pyrimidine Nucleosides
  • Pyrimidine Nucleotides
  • Cytidine
  • pyrimidin-2-one beta-ribofuranoside
  • DCMP Deaminase