Homologous recombination repair-dependent cytotoxicity of the benzotriazine di-N-oxide CEN-209: comparison with other hypoxia-activated prodrugs

Biochem Pharmacol. 2012 Mar 1;83(5):574-85. doi: 10.1016/j.bcp.2011.12.005. Epub 2011 Dec 13.


CEN-209 (SN30000) is a second-generation benzotriazine di-N-oxide currently in advanced preclinical development as a hypoxia-activated prodrug (HAP). Herein we describe the DNA repair-, hypoxia- and one-electron reductase-dependence of CEN-209 cytotoxicity. We deployed mutant CHO cell lines to generate DNA repair profiles for CEN-209, and compared the profiles with those for other HAPs. Hypoxic selectivity of CEN-209 was significantly greater than PR-104A and the nitro-chloromethylbenzindoline (nCBI/SN29428) and comparable to tirapazamine and TH-302. CEN-209 was selective for homologous recombination (HR) repair-deficient cells (Rad51d⁻/⁻), but less so than nitrogen mustard prodrugs TH-302 and PR-104A. Further, DNA repair profiles for CEN-209 differed under oxic and hypoxic conditions, with oxic cytotoxicity more dependent on HR. This feature was conserved across all three members of the benzotriazine di-N-oxide class examined (tirapazamine, CEN-209 and CEN-309/SN29751). Enhancing one-electron reduction of CEN-209 by forced expression of a soluble form of NADPH:cytochrome P450 oxidoreductase (sPOR) increased CEN-209 cytotoxicity more markedly under oxic than hypoxic conditions. Comparison of oxygen consumption, H₂O₂ production and metabolism of CEN-209 to the corresponding 1-oxide and nor-oxide reduced metabolites suggested that enhanced oxic cytotoxicity in cells with high one-electron reductase activity is due to futile redox cycling. This study supports the hypothesis that both oxic and hypoxic cell killing by CEN-209 is mechanistically analogous to tirapazamine and is dependent on oxidative DNA damage repaired via multiple pathways. However, HAPs that generate DNA interstrand cross-links, such as TH-302 and PR-104, may be more suitable than benzotriazine di-N-oxides for exploiting reported HR repair defects in hypoxic tumour cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antineoplastic Agents / chemistry
  • Antineoplastic Agents / pharmacology
  • Antineoplastic Agents / toxicity*
  • CHO Cells
  • Cricetinae
  • Cyclic N-Oxides / chemistry
  • Cyclic N-Oxides / pharmacology
  • Cyclic N-Oxides / toxicity*
  • DNA Damage / drug effects
  • DNA Repair / drug effects*
  • Genotype
  • Molecular Structure
  • Oxygen
  • Prodrugs / chemistry
  • Prodrugs / pharmacology
  • Prodrugs / toxicity*
  • Triazines / chemistry
  • Triazines / pharmacology
  • Triazines / toxicity*


  • Antineoplastic Agents
  • CEN-209
  • Cyclic N-Oxides
  • Prodrugs
  • Triazines
  • Oxygen