Pharmaceutical Research

, Volume 34, Issue 2, pp 378–393 | Cite as

Why Have Clinical Trials of Antioxidants to Prevent Neurodegeneration Failed? - A Cellular Investigation of Novel Phenothiazine-Type Antioxidants Reveals Competing Objectives for Pharmaceutical Neuroprotection

  • Maike J. Ohlow
  • Selina Sohre
  • Matthias Granold
  • Mathias Schreckenberger
  • Bernd Moosmann
Research Paper



Only a fraction of the currently established low-molecular weight antioxidants exhibit cytoprotective activity in living cells, which is considered a prerequisite for their potential clinical usefulness in Parkinson’s disease or stroke. Post hoc structure-activity relationship analyses have predicted that increased lipophilicity and enhanced radical stabilization could contribute to such cytoprotective activity.


We have synthesized a series of novel phenothiazine-type antioxidants exhibiting systematic variation in their lipophilicity and radical stabilization. Phenothiazine was chosen as lead structure for its superior activity at baseline. The novel compounds were evaluated for their neuroprotective potency in cell culture, and for their primary molecular targets.


Lipophilicity was associated with enhanced cytoprotective activity, but only to a certain threshold (logP ≈ 7). Benzannulation likewise produced improved cytoprotectants that exhibited very low EC50 values of ~8 nM in cultivated neuronal cells. Inhibition of global protein oxidation was the best molecular predictor of cytoprotective activity, followed by the inhibition of membrane protein autolysis. In contrast, the inhibition of lipid peroxidation in isolated brain lipids and the suppression of intracellular oxidant accumulation were poor predictors of cytoprotective activity, primarily as they misjudged the cellular advantage of high lipophilicity.


Lipophilicity, radical stabilization and molecular weight appear to form an uneasy triangle, in which a slightly faulty selection may readily abolish neuroprotective activity.


neuroprotection oxidative stress phenothiazine protein oxidation structure-activity relationship 



2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)


Ascorbic acid; vitamin C


Bicinchoninic acid


Butylated hydroxytoluene


2′,7′-Dichlorofluorescin diacetate


Dulbecco’s modified Eagle medium








Fetal calf serum


Glutamate-sensitive murine hippocampal cells


Lowest unoccupied molecular orbital of a radical


3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetra-zolium bromide


NXY-059; disufenton sodium


Propidium iodide


Thiobarbituric acid


Thiobarbituric acid-reactive substances


Trolox equivalent antioxidative capacity


α-Tocopherol; vitamin E


Trolox; 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid


Methyl tirilazad



The authors would like to thank Dr. James V. Crivello for his generous contribution of compounds 8, 9 and 10. The authors declare that they have no conflicts of interest. This work was supported by the Neuro Graduate School and the Interdisciplinary Research Centre for Neurosciences of the University of Mainz, which are non-profit entities that had no role in the design, execution, interpretation, or publication of this study.


  1. 1.
    Halliwell B. Oxidative stress and neurodegeneration: where are we now? J Neurochem. 2006;97:1634–58.CrossRefPubMedGoogle Scholar
  2. 2.
    Moosmann B, Behl C. Antioxidants as treatment for neurodegenerative disorders. Expert Opin Investig Drugs. 2002;11:1407–35.CrossRefPubMedGoogle Scholar
  3. 3.
    Margaill I, Plotkine M, Lerouet D. Antioxidant strategies in the treatment of stroke. Free Radic Biol Med. 2005;39:429–43.CrossRefPubMedGoogle Scholar
  4. 4.
    O’Collins VE, Macleod MR, Donnan GA, Horky LL, van der Worp BH, Howells DW. 1,026 experimental treatments in acute stroke. Ann Neurol. 2006;59:467–77.CrossRefPubMedGoogle Scholar
  5. 5.
    Shuaib A, Lees KR, Lyden P, Grotta J, Davalos A, Davis SM, et al. NXY-059 for the treatment of acute ischemic stroke. N Engl J Med. 2007;357:562–71.CrossRefPubMedGoogle Scholar
  6. 6.
    Green AR, Ashwood T. Free radical trapping as a therapeutic approach to neuroprotection in stroke: experimental and clinical studies with NXY-059 and free radical scavengers. Curr Drug Targets CNS Neurol Disord. 2005;4:109–18.CrossRefPubMedGoogle Scholar
  7. 7.
    The RANTTAS Investigators. A randomized trial of tirilazad mesylate in patients with acute stroke (RANTTAS). Stroke. 1996;27:1453–8.CrossRefGoogle Scholar
  8. 8.
    Yamaguchi T, Sano K, Takakura K, Saito I, Shinohara Y, Asano T, et al. Ebselen in acute ischemic stroke: a placebo-controlled, double-blind clinical trial. Ebselen Study Group. Stroke. 1998;29:12–7.CrossRefPubMedGoogle Scholar
  9. 9.
    Toyoda K, Fujii K, Kamouchi M, Nakane H, Arihiro S, Okada Y, et al. Free radical scavenger, edaravone, in stroke with internal carotid artery occlusion. J Neurol Sci. 2004;221:11–7.CrossRefPubMedGoogle Scholar
  10. 10.
    Watanabe T, Tahara M, Todo S. The novel antioxidant edaravone: from bench to bedside. Cardiovasc Ther. 2008;26:101–14.CrossRefPubMedGoogle Scholar
  11. 11.
    Hyslop PA, Zhang Z, Pearson DV, Phebus LA. Measurement of striatal H2O2 by microdialysis following global forebrain ischemia and reperfusion in the rat: correlation with the cytotoxic potential of H2O2 in vitro. Brain Res. 1995;671:181–6.CrossRefPubMedGoogle Scholar
  12. 12.
    Seet RC, Lee CY, Chan BP, Sharma VK, Teoh HL, Venketasubramanian N, et al. Oxidative damage in ischemic stroke revealed using multiple biomarkers. Stroke. 2011;42:2326–9.CrossRefPubMedGoogle Scholar
  13. 13.
    Granold M, Moosmann B, Staib-Lasarzik I, Arendt T, Del Rey A, Engelhard K, et al. High membrane protein oxidation in the human cerebral cortex. Redox Biol. 2015;4:200–7.CrossRefPubMedGoogle Scholar
  14. 14.
    Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, Savitz SI, et al. Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke. 2009;40:2244–50.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Zhang Q, Pi J, Woods CG, Andersen ME. A systems biology perspective on Nrf2-mediated antioxidant response. Toxicol Appl Pharmacol. 2010;244:84–97.CrossRefPubMedGoogle Scholar
  16. 16.
    Müller M, Banning A, Brigelius-Flohé R, Kipp A. Nrf2 target genes are induced under marginal selenium-deficiency. Genes Nutr. 2010;5:297–307.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Ames BN, Gold LS. The prevention of cancer. Drug Metab Rev. 1998;30:201–23.CrossRefPubMedGoogle Scholar
  18. 18.
    Nakamura Y, Miyoshi N. Electrophiles in foods: the current status of isothiocyanates and their chemical biology. Biosci Biotechnol Biochem. 2010;74:242–55.CrossRefPubMedGoogle Scholar
  19. 19.
    Macleod MR, van der Worp HB, Sena ES, Howells DW, Dirnagl U, Donnan GA. Evidence for the efficacy of NXY-059 in experimental focal cerebral ischaemia is confounded by study quality. Stroke. 2008;39:2824–9.Google Scholar
  20. 20.
    Savitz SI. A critical appraisal of the NXY-059 neuroprotection studies for acute stroke: a need for more rigorous testing of neuroprotective agents in animal models of stroke. Exp Neurol. 2007;205:20–5.CrossRefPubMedGoogle Scholar
  21. 21.
    Kuroda S, Tsuchidate R, Smith ML, Maples KR, Siesjö BK. Neuroprotective effects of a novel nitrone, NXY-059, after transient focal cerebral ischemia in the rat. J Cereb Blood Flow Metab. 1999;19:778–87.CrossRefPubMedGoogle Scholar
  22. 22.
    Barclay LR, Vinqvist MR. Do spin traps also act as classical chain-breaking antioxidants? A quantitative kinetic study of phenyl tert-butylnitrone (PBN) in solution and in liposomes. Free Radic Biol Med. 2000;28:1079–90.CrossRefPubMedGoogle Scholar
  23. 23.
    Floyd RA, Kopke RD, Choi CH, Foster SB, Doblas S, Towner RA. Nitrones as therapeutics. Free Radic Biol Med. 2008;45:1361–74.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Behl C, Moosmann B. Oxidative nerve cell death in Alzheimer’s disease and stroke: antioxidants as neuroprotective compounds. Biol Chem. 2002;383:521–36.CrossRefPubMedGoogle Scholar
  25. 25.
    Ohlow MJ, Mocko JB, Behl C, Hajieva P, Moosmann B. Comparative evaluation of biochemical antioxidants as neuroprotective agents. Free Radic Biol Med. 2010;49:S192.CrossRefGoogle Scholar
  26. 26.
    Parkinson Study Group. Effects of tocopherol and deprenyl on the progression of disability in early Parkinson’s disease. N Engl J Med. 1993;328:176–83.CrossRefGoogle Scholar
  27. 27.
    Shults CW, Oakes D, Kieburtz K, Beal MF, Haas R, Plumb S, et al. Effects of coenzyme Q10 in early Parkinson disease: evidence of slowing of the functional decline. Arch Neurol. 2002;59:1541–50.CrossRefPubMedGoogle Scholar
  28. 28.
    Petersen RC, Thomas RG, Grundman M, Bennett D, Doody R, Ferris S, et al. Vitamin E and donepezil for the treatment of mild cognitive impairment. N Engl J Med. 2005;352:2379–88.CrossRefPubMedGoogle Scholar
  29. 29.
    Thal LJ, Grundman M, Berg J, Ernstrom K, Margolin R, Pfeiffer E, et al. Idebenone treatment fails to slow cognitive decline in Alzheimer’s disease. Neurology. 2003;61:1498–502.CrossRefPubMedGoogle Scholar
  30. 30.
    Matthews RT, Yang L, Browne S, Baik M, Beal MF. Coenzyme Q10 administration increases brain mitochondrial concentrations and exerts neuroprotective effects. Proc Natl Acad Sci U S A. 1998;95:8892–7.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Vatassery GT, Fahn S, Kuskowski MA. Alpha tocopherol in CSF of subjects taking high-dose vitamin E in the DATATOP study. Neurology. 1998;50:1900–2.CrossRefPubMedGoogle Scholar
  32. 32.
    Gohil K, Oommen S, Quach HT, Vasu VT, Aung HH, Schock B, et al. Mice lacking alpha-tocopherol transfer protein gene have severe alpha-tocopherol deficiency in multiple regions of the central nervous system. Brain Res. 2008;1201:167–76.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Dallner G, Sindelar PJ. Regulation of ubiquinone metabolism. Free Radic Biol Med. 2000;29:285–94.CrossRefPubMedGoogle Scholar
  34. 34.
    Hajieva P, Mocko JB, Moosmann B, Behl C. Novel imine antioxidants at low nanomolar concentrations protect dopaminergic cells from oxidative neurotoxicity. J Neurochem. 2009;110:118–32.CrossRefPubMedGoogle Scholar
  35. 35.
    Crivello JV. Benzophenothiazine and benzophenoxazine photosensitizers for triarylsulfonium salt cationic photoinitiators. J Polym Sci A Polym Chem. 2008;46:3820–9.CrossRefGoogle Scholar
  36. 36.
    Moosmann B, Skutella T, Beyer K, Behl C. Protective activity of aromatic amines and imines against oxidative nerve cell death. Biol Chem. 2001;382:1601–12.CrossRefPubMedGoogle Scholar
  37. 37.
    Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med. 1999;26:1231–7.CrossRefPubMedGoogle Scholar
  38. 38.
    Triguero D, Buciak J, Pardridge WM. Capillary depletion method for quantification of blood–brain barrier transport of circulating peptides and plasma proteins. J Neurochem. 1990;54:1882–8.CrossRefPubMedGoogle Scholar
  39. 39.
    Seelig A, Gottschlich R, Devant RM. A method to determine the ability of drugs to diffuse through the blood–brain barrier. Proc Natl Acad Sci U S A. 1994;91:68–72.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Lien EJ, Ren S, Bui HH, Wang R. Quantitative structure-activity relationship analysis of phenolic antioxidants. Free Radic Biol Med. 1999;26:285–94.CrossRefPubMedGoogle Scholar
  41. 41.
    Tan S, Sagara Y, Liu Y, Maher P, Schubert D. The regulation of reactive oxygen species production during programmed cell death. J Cell Biol. 1998;141:1423–32.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Schubert D, Piasecki D. Oxidative glutamate toxicity can be a component of the excitotoxicity cascade. J Neurosci. 2001;21:7455–362.PubMedGoogle Scholar
  43. 43.
    Ohlow MJ, Moosmann B. Phenothiazine: the seven lives of pharmacology’s first lead structure. Drug Discov Today. 2011;16:119–31.CrossRefPubMedGoogle Scholar
  44. 44.
    Musiek ES, Yin H, Milne GL, Morrow JD. Recent advances in the biochemistry and clinical relevance of the isoprostane pathway. Lipids. 2005;40:987–94.CrossRefPubMedGoogle Scholar
  45. 45.
    Chevion M, Berenshtein E, Stadtman ER. Human studies related to protein oxidation: protein carbonyl content as a marker of damage. Free Radic Res. 2000;33(Suppl):S99–108.PubMedGoogle Scholar
  46. 46.
    Comellas AP, Dada LA, Lecuona E, Pesce LM, Chandel NS, Quesada N, et al. Hypoxia-mediated degradation of Na,K-ATPase via mitochondrial reactive oxygen species and the ubiquitin-conjugating system. Circ Res. 2006;98:1314–22.CrossRefPubMedGoogle Scholar
  47. 47.
    Sotiriou S, Gispert S, Cheng J, Wang Y, Chen A, Hoogstraten-Miller S, et al. Ascorbic-acid transporter Slc23a1 is essential for vitamin C transport into the brain and for perinatal survival. Nat Med. 2002;8:514–7.CrossRefPubMedGoogle Scholar
  48. 48.
    Huang J, Agus DB, Winfree CJ, Kiss S, Mack WJ, McTaggart RA, et al. Dehydroascorbic acid, a blood–brain barrier transportable form of vitamin C, mediates potent cerebroprotection in experimental stroke. Proc Natl Acad Sci U S A. 2001;98:11720–4.CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Ohlow MJ, Granold M, Schreckenberger M, Moosmann B. Is the chromanol head group of vitamin E nature’s final truth on chain-breaking antioxidants? FEBS Lett. 2012;586:711–6.CrossRefPubMedGoogle Scholar
  50. 50.
    Lanigan RS, Yamarik TA. Final report on the safety assessment of BHT. Int J Toxicol. 2002;21 Suppl 2:S19–94.Google Scholar
  51. 51.
    Mocko JB, Kern A, Moosmann B, Behl C, Hajieva P. Phenothiazines interfere with dopaminergic neurodegeneration in Caenorhabditis elegans models of Parkinson’s disease. Neurobiol Dis. 2010;40:120–9.CrossRefPubMedGoogle Scholar
  52. 52.
    Freyschuss A, Al-Schurbaji A, Björkhem I, Babiker A, Diczfalusy U, Berglund L, et al. On the anti-atherogenic effect of the antioxidant BHT in cholesterol-fed rabbits: inverse relation between serum triglycerides and atheromatous lesions. Biochim Biophys Acta. 2001;1534:129–38.CrossRefPubMedGoogle Scholar
  53. 53.
    Tymianski M. Can molecular and cellular neuroprotection be translated into therapies for patients? Yes, but not the way we tried it before. Stroke. 2010;41 Suppl 10:S87–90.CrossRefPubMedGoogle Scholar
  54. 54.
    Hill MD, Martin RH, Mikulis D, Wong JH, Silver FL, Terbrugge KG, et al. Safety and efficacy of NA-1 in patients with iatrogenic stroke after endovascular aneurysm repair (ENACT): a phase 2, randomised, double-blind, placebo-controlled trial. Lancet Neurol. 2012;11:942–50.CrossRefPubMedGoogle Scholar
  55. 55.
    Kaste M. Is the door open again for neuroprotection trials in stroke? Lancet Neurol. 2012;11:930–1.CrossRefPubMedGoogle Scholar
  56. 56.
    Murphy TH, Miyamoto M, Sastre A, Schnaar RL, Coyle JT. Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress. Neuron. 1989;2:1547–58.CrossRefPubMedGoogle Scholar
  57. 57.
    Li Y, Maher P, Schubert D. A role for 12-lipoxygenase in nerve cell death caused by glutathione depletion. Neuron. 1997;19:453–63.CrossRefPubMedGoogle Scholar
  58. 58.
    Long LH, Hoi A, Halliwell B. Instability of, and generation of hydrogen peroxide by, phenolic compounds in cell culture media. Arch Biochem Biophys. 2010;501:162–9.CrossRefPubMedGoogle Scholar
  59. 59.
    Wijtmans M, Pratt DA, Brinkhorst J, Serwa R, Valgimigli L, Pedulli GF, et al. Synthesis and reactivity of some 6-substituted-2,4-dimethyl-3-pyridinols, a novel class of chain-breaking antioxidants. J Org Chem. 2004;69:9215–23.CrossRefPubMedGoogle Scholar
  60. 60.
    Omata Y, Saito Y, Yoshida Y, Jeong BS, Serwa R, Nam TG, et al. Action of 6-amino-3-pyridinols as novel antioxidants against free radicals and oxidative stress in solution, plasma, and cultured cells. Free Radic Biol Med. 2010;48:1358–65.CrossRefPubMedGoogle Scholar
  61. 61.
    Lasarzik I, Thal SC, Jahn-Eimermacher A, Engelhard K, Moosmann B. NH-Phenothiazine dose-dependently improves neuronal outcome after cerebral ischemia in rats. Proceedings of the 2010 Annual Meeting of the American Society of Anesthesiologists (ASA), San Diego, CA, USA. Abstract A1046.Google Scholar
  62. 62.
    Yu MJ, McCowan JR, Smalstig EB, Bennett DR, Roush ME, Clemens JA. A phenothiazine derivative reduces rat brain damage after global or focal ischemia. Stroke. 1992;23:1287–91.CrossRefPubMedGoogle Scholar
  63. 63.
    Tapias V, Mastroberardino PG, Hu X, Nelson J, Sew T, Greenamyre JT. Unsubstituted Phenothiazine is protective in the rotenone model of Parkinson’s disease. Neuroscience Meeting, New Orleans, LA, USA: Society for Neuroscience, 2012. Program No. 856.11.Google Scholar
  64. 64.
    Songarj P, Luh C, Staib-Lasarzik I, Engelhard K, Moosmann B, Thal SC. The antioxidative, non-psychoactive tricyclic phenothiazine reduces brain damage after experimental traumatic brain injury in mice. Neurosci Lett. 2015;584:253–8.CrossRefPubMedGoogle Scholar
  65. 65.
    Murphy CM, Rawer H, Smith NL. Mode of action of phenothiazine-type antioxidants. Ind Eng Chem. 1950;42:2479–89.CrossRefGoogle Scholar
  66. 66.
    Burton GW, Doba T, Gabe E, Hughes L, Lee FL, Prasad L, et al. Autoxidation of biological molecules. 4. Maximizing the antioxidant activity of phenols. J Am Chem Soc. 1985;107:7053–65.CrossRefGoogle Scholar
  67. 67.
    Shang YJ, Jin XL, Shang XL, Tang JJ, Liu GY, Dai F, et al. Antioxidant capacity of curcumin-directed analogues: Structure-activity relationship and influence of microenvironment. Food Chem. 2010;119:1435–42.CrossRefGoogle Scholar
  68. 68.
    Moosmann B, Behl C. The antioxidant neuroprotective effects of estrogens and phenolic compounds are independent from their estrogenic properties. Proc Natl Acad Sci U S A. 1999;96:8867–72.CrossRefPubMedPubMedCentralGoogle Scholar
  69. 69.
    Shahidi F, Zhong Y. Revisiting the polar paradox theory: a critical overview. J Agric Food Chem. 2011;59:3499–504.CrossRefPubMedGoogle Scholar
  70. 70.
    Ritchie TJ, Macdonald SJ, Young RJ, Pickett SD. The impact of aromatic ring count on compound developability: further insights by examining carbo- and hetero-aromatic and -aliphatic ring types. Drug Discov Today. 2011;16:164–71.CrossRefPubMedGoogle Scholar
  71. 71.
    Colmenarejo G, Alvarez-Pedraglio A, Lavandera JL. Cheminformatic models to predict binding affinities to human serum albumin. J Med Chem. 2001;44:4370–8.CrossRefPubMedGoogle Scholar
  72. 72.
    Wasan KM, Brocks DR, Lee SD, Sachs-Barrable K, Thornton SJ. Impact of lipoproteins on the biological activity and disposition of hydrophobic drugs: implications for drug discovery. Nat Rev Drug Discov. 2008;7:84–99.CrossRefPubMedGoogle Scholar
  73. 73.
    Gilgun-Sherki Y, Melamed E, Offen D. Oxidative stress induced-neurodegenerative diseases: the need for antioxidants that penetrate the blood brain barrier. Neuropharmacology. 2001;40:959–75.CrossRefPubMedGoogle Scholar
  74. 74.
    Gao H, Pang Z, Jiang X. Targeted delivery of nano-therapeutics for major disorders of the central nervous system. Pharm Res. 2013;30:2485–98.CrossRefPubMedGoogle Scholar
  75. 75.
    Hajieva P, Bayatti N, Granold M, Behl C, Moosmann B. Membrane protein oxidation determines neuronal degeneration. J Neurochem. 2015;133:352–67.CrossRefPubMedGoogle Scholar
  76. 76.
    Hajieva P, Moosmann B. Brain protein oxidation: what does it reflect? Neural Regen Res. 2015;10:1729–30.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Institute for PathobiochemistryUniversity Medical Center of the Johannes Gutenberg UniversityMainzGermany
  2. 2.Department of Nuclear MedicineUniversity Medical Center of the Johannes Gutenberg UniversityMainzGermany

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