Characterization of Radicals Formed Following Enzymatic Reduction of 3-substituted Analogues of the Hypoxia-Selective Cytotoxin 3-amino-1,2,4-benzotriazine 1,4-dioxide (Tirapazamine)

J Am Chem Soc. 2010 Mar 3;132(8):2591-9. doi: 10.1021/ja908689f.


The mechanism by which the 1,2,4-benzotriazine 1,4-dioxide (BTO) class of bioreductive hypoxia-selective prodrugs (HSPs) form reactive radicals that kill cancer cells has been investigated by steady-state radiolysis, pulse radiolysis (PR), electron paramagnetic resonance (EPR), and density functional theory (DFT) calculations. Tirapazamine (TPZ, 3-amino BTO, 1) and a series of 3-substituted analogues, -H (2), -methyl (3), -ethyl (4), -methoxy (5), -ethoxymethoxy (6), and -phenyl (7), were reduced in aqueous solution under anaerobic steady-state radiolysis conditions, and their radicals were found to remove the substrates by short chain reactions of different lengths in the presence of formate ions. Multiple carbon-centered radical intermediates, produced upon anaerobic incubation of the compounds with cytochrome P(450) reductase enriched microsomes, were trapped by N-tert-butyl-alpha-phenylnitrone and observed using EPR. The highly oxidizing oxymethyl radical, from compound 5, was identified, and experimental spectra obtained for compounds 1, 2, 3, and 7 were well simulated after the inclusion of aryl radicals. The identification of a range of oxidizing radicals in the metabolism of the BTO compounds gives a new insight into the mechanism by which these HSPs can cause a wide variety of damage to biological targets such as DNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / chemistry*
  • Antineoplastic Agents / metabolism*
  • Cytotoxins / chemistry*
  • Cytotoxins / metabolism*
  • Electron Spin Resonance Spectroscopy
  • Microsomes / metabolism
  • NADPH-Ferrihemoprotein Reductase / metabolism
  • Oxidation-Reduction
  • Pulse Radiolysis
  • Quantum Theory
  • Tirapazamine
  • Triazines / chemistry*
  • Triazines / metabolism*


  • Antineoplastic Agents
  • Cytotoxins
  • Triazines
  • Tirapazamine
  • NADPH-Ferrihemoprotein Reductase