Archives of Toxicology

, Volume 86, Issue 10, pp 1613–1625 | Cite as

7-Nitro-4-(phenylthio)benzofurazan is a potent generator of superoxide and hydrogen peroxide

  • Eric V. PatridgeEmail author
  • Emma S. E. Eriksson
  • Philip G. Penketh
  • Raymond P. Baumann
  • Rui Zhu
  • Krishnamurthy Shyam
  • Leif A. Eriksson
  • Alan C. Sartorelli


Here, we report on 7-nitro-4-(phenylthio)benzofurazan (NBF-SPh), the most potent derivative among a set of patented anticancer 7-nitrobenzofurazans (NBFs), which have been suggested to function by perturbing protein–protein interactions. We demonstrate that NBF-SPh participates in toxic redox-cycling, rapidly generating reactive oxygen species (ROS) in the presence of molecular oxygen, and this is the first report to detail ROS production for any of the anticancer NBFs. Oxygraph studies showed that NBF-SPh consumes molecular oxygen at a substantial rate, rivaling even plumbagin, menadione, and juglone. Biochemical and enzymatic assays identified superoxide and hydrogen peroxide as products of its redox-cycling activity, and the rapid rate of ROS production appears to be sufficient to account for some of the toxicity of NBF-SPh (LC50 = 12.1 μM), possibly explaining why tumor cells exhibit a sharp threshold for tolerating the compound. In cell cultures, lipid peroxidation was enhanced after treatment with NBF-SPh, as measured by 2-thiobarbituric acid-reactive substances, indicating a significant accumulation of ROS. Thioglycerol rescued cell death and increased survival by 15-fold to 20-fold, but pyruvate and uric acid were ineffective protectants. We also observed that the redox-cycling activity of NBF-SPh became exhausted after an average of approximately 19 cycles per NBF-SPh molecule. Electrochemical and computational analyses suggest that partial reduction of NBF-SPh enhances electrophilicity, which appears to encourage scavenging activity and contribute to electrophilic toxicity.


Benzofurazan Reactive oxygen species Oxidative stress Electrochemistry Electrophilic stress 







Reactive oxygen species




Glutathione S-transferases




Dulbecco’s modified Eagle’s medium




Superoxide dismutase


Glucose-6-phosphate dehydrogenase


NADPH:cytochrome P450 reductase


2-Thiobarbituric acid-reactive substances


Differential pulse


Cyclic voltammetry





The authors are grateful to James Blakemore for his assistance with electrochemical studies and to Dr. Tukiet Lam and Edward Voss for their services and help in mass spectroscopy analysis. This work was supported in part by U.S. Public Health Service Grants CA-090671, CA-122112, and CA-129186 from the National Cancer Institute and a Grant from the National Foundation for Cancer Research.


  1. Andrews J, Ghosh P, Ternai B, Whitehouse M (1982) Ammonium 4-chloro-7-sulfobenzofurazan: a new fluorigenic thiol-specific reagent. Arch Biochem Biophys 214(1):386–396PubMedCrossRefGoogle Scholar
  2. Baez S, Segura-Aguilar J (1995) Effects of superoxide dismutase and catalase during reduction of adrenochrome by DT-diaphorase and NADPH-cytochrome P450 reductase. Biochem Mol Med 56(1):37–44PubMedCrossRefGoogle Scholar
  3. Bard A, Faulkner L (2001) Electrochemical methods: fundamentals and applications, 2nd edn. Wiley, HobokenGoogle Scholar
  4. Barone V, Cossi M (1998) Quantum calculation of molecular energies and energy gradients in solution by a conductor solvent model. J Phys Chem 102(11):1995–2001CrossRefGoogle Scholar
  5. Baumann RP, Seow HA, Shyam K, Penketh PG, Sartorelli AC (2005) The antineoplastic efficacy of the prodrug Cloretazine is produced by the synergistic interaction of carbamoylating and alkylating products of its activation. Oncol Res 15(6):313–325PubMedGoogle Scholar
  6. Becke AD (1993) Density-functional thermochemistry. III. The role of exact exchange. J Chem Phys 98(7):5648–5652CrossRefGoogle Scholar
  7. Belton JG (1974) A Novel N → S oxygen migration in 2,1,3-benzoxadiazole systems. Proc R Ir Acad B 74:185–192Google Scholar
  8. Bindoli A, Scutari G, Rigobello MP (1999) The role of adrenochrome in stimulating the oxidation of catecholamines. Neurotox Res 1(2):71–80PubMedCrossRefGoogle Scholar
  9. Birkett DJ, Price NC, Radda GK, Salmon AG (1970) The reactivity of SH groups with a fluorogenic reagent. FEBS Lett 6(4):346–348PubMedCrossRefGoogle Scholar
  10. Caccuri AM, Ricci G (2006) Italy Patent No. EP1615638B1. EP OfficeGoogle Scholar
  11. Castro F, Mariani D, Panek AD, Eleutherio EC, Pereira MD (2008) Cytotoxicity mechanism of two naphthoquinones (menadione and plumbagin) in Saccharomyces cerevisiae. PLoS ONE 3(12):e3999PubMedCrossRefGoogle Scholar
  12. Cenas N, Nemeikaite A, Dickancaite E, Anusevicius Z, Nivinskas H, Bironaite D (1995) The toxicity of aromatic nitrocompounds to bovine leukemia virus-transformed fibroblasts: the role of single-electron reduction. Biochim Biophys Acta 1268(2):159–164PubMedCrossRefGoogle Scholar
  13. Cossi M (2003) Energies, structures, and electronic properties of molecules in solution with the C-PCM solvation model. J Comp Chem 24(6):669–681CrossRefGoogle Scholar
  14. Federici L, Lo Sterzo C, Pezzola S, Di Matteo A, Scaloni F, Federici G, Caccuri AM (2009) Structural basis for the binding of the anticancer compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol to human glutathione s-transferases. Cancer Res 69(20):8025–8034PubMedCrossRefGoogle Scholar
  15. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson GA et al (2009) Gaussian09. Gaussian, Inc., WallingfordGoogle Scholar
  16. Ghosh PB (1968) Preparation and study of some 5- and 7-substituted 4-nitrobenzofurazans and their N-oxides; a retro-Boulton–Katritzky rearrangement. J Chem Soc B 1:334–338CrossRefGoogle Scholar
  17. Ghosh PB, Whitehouse MW (1968) Potential antileukemic and immunosuppressive drugs. Preparation and in vitro pharmacological activity of some benzo-2,1,3-oxadiazoles (benzofurazans) and their N-oxides (benzofuroxans). J Med Chem 11(2):305–311PubMedCrossRefGoogle Scholar
  18. Ghosh PB, Whitehouse MW (1969) Potential antileukemic and immunosuppressive drugs. II. Further studies with benzo-2,1,3-oxadiazoles (benzofurazans) and their N-oxides (benzofuroxans). J Med Chem 12(3):505–507PubMedCrossRefGoogle Scholar
  19. Ghosh PB, Ternai B, Whitehouse MW (1972) Potential antileukemic and immunosuppressive drugs. 3. Effects of homocyclic ring substitution on the in vitro drug activity of 4-nitrobenzo-2,1,3-oxadiazoles (4-nitrobenzofurazans) and their N-oxides (4-nitrobenzofuroxans). J Med Chem 15(3):255–260PubMedCrossRefGoogle Scholar
  20. Ghosh PB, Ternai B, Whitehouse MW (1981) Benzofurazans and benzofuroxans: biochemical and pharmacological properties. Med Res Rev 1(2):159–187PubMedCrossRefGoogle Scholar
  21. Giulivi C, Cadenas E (1994) One- and two-electron reduction of 2-methyl-1,4-naphthoquinone bioreductive alkylating agents: kinetic studies, free-radical production, thiol oxidation and DNA-strand-break formation. Biochem J 301(Pt 1):21–30PubMedGoogle Scholar
  22. Heimbrook DC, Sartorelli AC (1986) Biochemistry of misonidazole reduction by NADPH-cytochrome c (P-450) reductase. Mol Pharmacol 29(2):168–172PubMedGoogle Scholar
  23. Heyne B (2007) Synthesis and characterization of a new fluorescent probe for reactive oxygen species. Org Biomol Chem 5(9):1454–1458PubMedCrossRefGoogle Scholar
  24. Heyne B, Ahmed S, Scaiano JC (2008) Mechanistic studies of fluorescent sensors for the detection of reactive oxygen species. Org Biomol Chem 6(2):354–358PubMedCrossRefGoogle Scholar
  25. Imai K, Fukushima T, Uzu S (1993) Sensitive determination of enantiomers of amino acids derivatized with the fluorogenic reagent, 4-fluoro-7-nitro-2,1,3-benzoxadiazole, separated on a Pirkle-type column, Sumichiral OA 2500(S). Biomed Chromatogr 7(3):177–178PubMedCrossRefGoogle Scholar
  26. Inbaraj JJ, Chignell CF (2004) Cytotoxic action of juglone and plumbagin: a mechanistic study using HaCaT keratinocytes. Chem Res Toxicol 17(1):55–62PubMedCrossRefGoogle Scholar
  27. Johnson SA, Dalton AE, Pardini RS (1998) Time-course of hypericin phototoxicity and effect on mitochondrial energies in EMT6 mouse mammary carcinoma cells. Free Radic Biol Med 25(2):144–152PubMedCrossRefGoogle Scholar
  28. Juchau MR, Fantel AG, Harris C, Beyer BK (1986) The potential role of redox cycling as a mechanism for chemical teratogenesis. Environ Health Perspect 70:131–136PubMedCrossRefGoogle Scholar
  29. Kappus H, Sies H (1981) Toxic drug effects associated with oxygen metabolism: redox cycling and lipid peroxidation. Experientia 37(12):1233–1241PubMedCrossRefGoogle Scholar
  30. Kennedy KA, Teicher BA, Rockwell S, Sartorelli AC (1980) The hypoxic tumor cell: a target for selective cancer chemotherapy. Biochem Pharmacol 29(1):1–8PubMedCrossRefGoogle Scholar
  31. Klamt A (1998) Refinement and parametrization of COSMO-RS. J Phys Chem A 102(26):5074–5085CrossRefGoogle Scholar
  32. Klamt A, Schuurmann G (1993) COSMO: a new approach to dielectric screening in solvents with explicit expressions for the screening energy and its gradient. J Chem Soc Perkin Trans 2(5):799–805Google Scholar
  33. Knox RJ, Knight RC, Edwards DI (1983) Studies on the action of nitroimidazole drugs. The products of nitroimidazole reduction. Biochem Pharmacol 32(14):2149–2156PubMedCrossRefGoogle Scholar
  34. Moreno SN, Docampo R (1985) Mechanism of toxicity of nitro compounds used in the chemotherapy of trichomoniasis. Environ Health Perspect 64:199–208PubMedCrossRefGoogle Scholar
  35. Onoda M, Uchiyama S, Endo A, Tokuyama H, Santa T, Imai K (2003) First fluorescent photoinduced electron transfer (PET) reagent for hydroperoxides. Org Lett 5(9):1459–1461PubMedCrossRefGoogle Scholar
  36. Ricci G, De Maria F, Antonini G, Turella P, Bullo A, Stella L, Filomeni G, Federici G, Caccuri AM (2005) 7-Nitro-2,1,3-benzoxadiazole derivatives, a new class of suicide inhibitors for glutathione S-transferases. Mechanism of action of potential anticancer drugs. J Biol Chem 280(28):26397–26405PubMedCrossRefGoogle Scholar
  37. Santa T, Okamoto T, Uchiyama S, Mitsuhashi K, Imai K (1999) A new fluorogenic reagent for carboxylic acids, 7-acetylamino-4-mercapto-2,1,3-benzoxadiazole (AABD-SH), derived from an empirical method for predicting fluorescence characteristics. Analyst 124(11):1689–1693CrossRefGoogle Scholar
  38. Stradyn YP, Kadysh VP, Giller SA (1974) Polarography of heterocyclic compounds. Chem Heterocycl Comp 10(2):129–141CrossRefGoogle Scholar
  39. Sun M, Zigman S (1978) An improved spectrophotometric assay for superoxide dismutase based on epinephrine autoxidation. Anal Biochem 90(1):81–89PubMedCrossRefGoogle Scholar
  40. Takabatake T, Hasegawa M, Nagano T, Hirobe M (1990) Toxicities of dicyanobenzofurazans with formation of superoxide in Escherichia coli. Chem Pharm Bull (Tokyo) 38(1):128–132CrossRefGoogle Scholar
  41. Takabatake T, Hasegawa M, Nagano T, Hirobe M (1991) Formation of superoxide by benzofurazans in Escherichia coli under aerobic incubation. Chem Pharm Bull (Tokyo) 39(5):1352–1354CrossRefGoogle Scholar
  42. Takabatake T, Hasegawa M, Nagano T, Hirobe M (1992a) Bacteriostatic effect of 4,7-dicyanobenzofurazan due to inactivation of 2,3-dihydroxyisovalerate dehydratase. Chem Pharm Bull (Tokyo) 40(6):1644–1646CrossRefGoogle Scholar
  43. Takabatake T, Hasegawa M, Nagano T, Hirobe M (1992b) Difference in superoxide toxicity between 4,7-dicyanobenzofurazan and paraquat. J Biol Chem 267(7):4613–4618PubMedGoogle Scholar
  44. Toyo’oka T, Imai K (1983) High-performance liquid chromatography and fluorometric detection of biologically important thiols, derivatized with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F). J Chromatogr 282:495–500PubMedCrossRefGoogle Scholar
  45. Toyo’oka T, Ishibashi M, Takeda Y, Nakashima K, Akiyama S, Uzu S, Imai K (1991) Precolumn fluorescence tagging reagent for carboxylic acids in high-performance liquid chromatography: 4-substituted-7-aminoalkylamino-2,1,3-benzoxadiazoles. J Chromatogr 588(1–2):61–71PubMedGoogle Scholar
  46. Tsveniashvili V, Zhdanov SI, Todres ZV (1966) Polarography of piazothiol and piazoselenol in aqueous solutions. Fresen J Anal Chem 224(1):389–406CrossRefGoogle Scholar
  47. Turella P, Cerella C, Filomeni G, Bullo A, De Maria F, Ghibelli L, Ciriolo MR, Cianfriglia M, Mattei M, Federici G et al (2005) Proapoptotic activity of new glutathione S-transferase inhibitors. Cancer Res 65(9):3751–3761PubMedCrossRefGoogle Scholar
  48. Uchiyama M, Mihara M (1978) Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 86(1):271–278PubMedCrossRefGoogle Scholar
  49. Uchiyama S, Santa T, Okiyama N, Fukushima T, Imai K (2001) Fluorogenic and fluorescent labeling reagents with a benzofurazan skeleton. Biomed Chromatogr 15(5):295–318PubMedCrossRefGoogle Scholar
  50. Watanabe Y, Imai K (1981) High-performance liquid chromatography and sensitive detection of amino acids derivatized with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole. Anal Biochem 116(2):471–472PubMedCrossRefGoogle Scholar
  51. Weiss RF (1970) The solubility of nitrogen, oxygen and argon on water and seawater. Deep Sea Res 17:721–735Google Scholar
  52. Whitehouse MW, Ghosh PB (1968) 4-nitrobenzofurazans and 4-nitrobenzofuroxans: a new class of thiol-neutralising agents and potent inhibitors of nucleic acid synthesis in leucocytes. Biochem Pharmacol 17(1):158–161PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Eric V. Patridge
    • 1
    Email author
  • Emma S. E. Eriksson
    • 2
    • 3
  • Philip G. Penketh
    • 1
  • Raymond P. Baumann
    • 1
  • Rui Zhu
    • 1
  • Krishnamurthy Shyam
    • 1
  • Leif A. Eriksson
    • 2
    • 3
  • Alan C. Sartorelli
    • 1
  1. 1.Department of PharmacologyYale University School of MedicineNew HavenUSA
  2. 2.School of ChemistryNational University of Ireland-GalwayGalwayIreland
  3. 3.Department of Chemistry and Molecular BiologyUniversity of GothenburgGothenburgSweden

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