Skip to main content

Synthesis and physicochemical properties of novel lophine derivatives as chemiluminescent in vitro activators for detection of free radicals


The overproduction of free radicals and reactive oxygen species (ROS) has been proved as a basic damage mechanism and cause for oxidative stress. Their measurement is often hindered by the low signal. This could be resolved with the application of luminescent probes (lophines, luminol, lucigenin, etc.). The focus of this study is to synthesize and describe the spectral properties and physicochemical characteristics of lophine and its derivatives as new chemiluminescent in vitro activators. The prepared luminophores are analogues of lophine. Their absorption maxima are in the range 329–340 nm, with good-to-high extinction coefficients. Their spectral properties are measured in methanol and buffer solutions with pH 3.5, 7.4 and 8.5. Same conditions were applied in the systems for chemiluminescent assay in vitro: (1) Fenton’s (Fe2++H2O2) for the generation of ·OH and –OH species, (2) Hydrogen peroxide (H2O2), (3) Iron (II) sulfate (FeSO4), (4) Glutathione-peroxidase, monitoring the deactivation of H2O2, (5) Ascorbic acid-Fenton’s reagent: Vit.C appears a strong oxidant, generating free-radical products when applied in higher than physiological concentrations, (6) Reduced α-nicotinamide adenine dinucleotide (NADH)-phenazine methosulfate—for the generation of superoxide radicals (O2 ·−). Lophine and all novel compounds do not alter the kinetics, except of the dimethyl amino substituted derivative (4-(3a,11b-dihydro-1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)-N,N-dimethylaniline) in the glutathione-peroxidase system, at pH 8.5. Same derivative showed a comparable or higher activity than Lucigenin and Rhodamine 6G. In neutral and acidic medium, in the Fenton’s system, Rhodamine 6G was the most appropriate probe. In alkaline pH and oxidant H2O2, Lucigenin induced a signal twice as strong as the signal compared to all other activators.

This is a preview of subscription content, access via your institution.

Scheme 1
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Scheme 2
Fig. 7









  • Armstrong D, Browne R (1994) The analysis of free radicals, lipid peroxides, antioxidant enzymes and compounds related to oxidative stress as applied to the clinical chemistry laboratory. In: Armstrong D (ed) Free radicals in diagnostic medicine. Plenum Press, New York, pp 43–58

    Chapter  Google Scholar 

  • Babu K, Surendhar V (2014) An alternative synthetic approach towards 2,4,5-trisubstituted-1H-imidazoles. Heterocyclic Lett 4(2):235–238

    Google Scholar 

  • Bartosz G (2006) Use of spectroscopic probes for detection of reactive oxygen species. Clin Chim Acta 368:53–76

    Article  CAS  PubMed  Google Scholar 

  • Bлaдимиpoв ЮA (1997) Xeмилyминecцeнция в биoлoгичecкиx cиcтeмax. Пpиpoдa 3:18–28

    Google Scholar 

  • Che GB et al (2006) Hydrothermal syntheses of some derivatives of tetraazatriphenylene. Synth Commun 36:2519

    Article  CAS  Google Scholar 

  • Faulkner K, Fridovich I (1993) Luminol and lucigenin as detectors for O2. Free Radic Biol Med 15:447–451

    Article  CAS  PubMed  Google Scholar 

  • Gale DJ, Wilshire JK (1970) The preparation of some polymethine Astrazon dyes. Aust J Chem 23:1063

    Article  CAS  Google Scholar 

  • Halliwell B (1994) Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? Lancet 344(8924):721–724

    Article  CAS  PubMed  Google Scholar 

  • Kenichiro N (2003) Lophine derivatives as versatile analytical tools. Biomed Chromatogr BMC 17(2–3):83–95

    Google Scholar 

  • Khosropour A (2008) Ultrasound-promoted greener synthesis of 2,4,5-trisubstituted imidazoles catalyzed by Zr (acac)4 under ambient conditions. Ultrason Sonochem 15(5):659–664

    Article  CAS  PubMed  Google Scholar 

  • Krieg B, Manecke G (1967) Synthese und halbleitereigenschaften arylsubstituierter Imidazole. Z Naturforschg 2(22):132–141

    Google Scholar 

  • Lu FJ, Lin JT, Wang HP et al (1996) A simple, sensitive, non-stimulated photon counting system for detection of superoxide anion in whole blood. Experientia 52:141–144

    Article  CAS  PubMed  Google Scholar 

  • Lu C, Song G, Lin JM (2006) Reactive oxygen species and their chemiluminescence-detection methods. Trends Anal Chem 25(10):985–995

    Article  CAS  Google Scholar 

  • Nakashima K, Yamasaki H, Kuroda N, Akiyama S (1995) Evaluation of lophine derivatives as chemiluminogens by a flow-injection method. Anal Chim Acta 303(1):103–107

    Article  CAS  Google Scholar 

  • Nishikimi M, Appaji N, Yagi K (1972) The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem Biophys Res Commun 46(2):849–854

    Article  CAS  PubMed  Google Scholar 

  • Parveen A (2013) Ionic liquid assisted green synthesis of 2-phenylimidizo[4, 5-f] [1, 10]-phenanthroline at room temperature. J Adv Sci Res 4(2):42–47

    CAS  Google Scholar 

  • Siess H (1985) Oxidative stress. Academic Press, London

    Google Scholar 

  • Vladimirov JA, Azizova OA, Deev AI, Kozlov AV, Ossipov AN, Roshtupkin DI (1991) Free radicals in living systems. In: Science and technique reviews. Biophysics Series, Moscow, VINITI 29:1–252

  • Wardman P (2007) Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects. Free Radic Biol Med 43(7):995–1022

    Article  CAS  PubMed  Google Scholar 

  • Xie N, Chen Y (2007) Synthesis and photophysical properties of 1,4-bis(4,5-diarylimidazol) benzene dyes. J Photochem Photobiol A Chem 189:253–257

    Article  CAS  Google Scholar 

  • Yamaguchi S, Kishikawa N, Ohyama K et al (2010) Evaluation of chemiluminescence reagents for selective detection of reactive oxygen species. Anal Chim Acta 665:74–78

    Article  CAS  PubMed  Google Scholar 

Download references


This research was financially supported by Sofia University “St. Kliment Ohridski”, according to the project “Synthesis and study of the physical and chemical characteristics of lophine and its derivatives as novel chemiluminescent probes for medical and biological research”, No 2666—Scientific Research Centre. We are very thankful for the editorial support of Dr. Mihail Mitov—Research Associate and Michael Alstott—Laboratory Technician Senior at the Redox Metabolism Shared Resource Facility (RM SRF), Markey Cancer Center, University of Kentucky: 1095 V.A. Drive Health Sciences Research Building, room 227, Lexington, KY 40536-0305, Phone: (859) 323-1106.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Elitsa Pavlova.

Ethics declarations

Conflict of interest

Each author declares no financial or commercial conflicts of interest.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Pavlova, E., Kaloyanova, S., Deligeorgiev, T. et al. Synthesis and physicochemical properties of novel lophine derivatives as chemiluminescent in vitro activators for detection of free radicals. Eur Biophys J 44, 623–634 (2015).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Lophine
  • ROS
  • Chemiluminescent probes