DNA binding acridine–thiazolidinone agents affecting intracellular glutathione Helena Paulíková a,⇑ , Zuzana Vantová a,b , L’uba Hunáková e , Ly ´ dia C ˇ iz ˇeková a , Mária C ˇ arná a , Mária Koz ˇurková c , Danica Sabolová c , Pavol Kristian d , Slávka Hamul’aková d , Ján Imrich d a Department of Biochemistry and Microbiology, Faculty of Chemical and Food Technology, Faculty of Chemical and Food Technology, Radlinského 9, SK-81237 Bratislava, Slovak Republic b Department of Chemical and Biochemical Engineering, Faculty of Chemical and Food Technology, Faculty of Chemical and Food Technology, Radlinského 9, SK-81237 Bratislava, Slovak Republic c Department of Biochemistry, Institute of Chemistry, Faculty of Science, P.J. Šafárik University, Moyzesova 11, SK-04167 Košice, Slovak Republic d Department of Organic Chemistry, Institute of Chemistry, Faculty of Science, P.J. Šafárik University, Moyzesova 11, SK-04167 Košice, Slovak Republic e Cancer Research Institute, Vlárska 7, SK-81237 Bratislava, Slovak Republic article info Article history: Received 12 July 2012 Revised 21 September 2012 Accepted 26 September 2012 Available online 12 October 2012 Keywords: Acridine DNA-intercalation Glutathione Antitumor abstract Three new acridine–thiazolidinone derivatives (2a–2c) have been synthesized and their interactions with calf thymus DNA and a number of cell lines (leukemic cells HL-60 and L1210 and human epithelial ovar- ian cancer cell lines A2780) were studied. The compounds 2a–2c possessed high affinity to calf thymus DNA and their binding constants determined by spectrofluorimetry were in the range of 1.37 Â 10 6 – 5.89 Â 10 6 M À1 . All of the tested derivatives displayed strong cytotoxic activity in vitro, the highest activ- ity in cytotoxic tests was found for 2c with IC 50 = 1.3 ± 0.2 lM (HL-60), 3.1 ± 0.4 lM (L1210), and 7.7 ± 0.5 lM (A2780) after 72 h incubation. The cancer cells accumulated acridine derivatives very fast and the changes of the glutathione level were confirmed. The compounds inhibited proliferation of the cells and induced an arrest of the cell cycle and cell death. Their influence upon cells was associated with their reactivity towards thiols and DNA binding activity. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Antitumor effects of acridines are associated not only with their interaction with DNA but also with proteins, especially some key enzymes important in cell proliferation as topoisomerases or telo- merases. 1–3 Acridines can interact with DNA either via intercala- tion or groove-binding and may thus block a DNA replication, transcription, or DNA repair. 4 In the meantime, the best known acridine drugs used in antitumor therapy are amsacrine and DACA. Damage of DNA by these compounds is usually strengthened by the oxidative stress. 5 The search for novel anticancer drugs encour- ages also syntheses of new acridine derivatives. 6–8 Several series of novel 9- and 3,6-substituted acridines have recently been prepared in our laboratory and investigated for their DNA binding properties and in vitro antineoplastic activity. 9–13 Besides antitumor effects found at micromolar concentrations, all new substances displayed a potent DNA binding with binding constants in the range of 10 5 – 10 6 M À1 . This affinity as well as UV–vis, spectrofluorimetric and CD studies confirmed an intercalation of our ligands into DNA to be a principal mode of interaction. In search for new pharmacophoric structures we were inspired by acridine–thiazolidinone derivatives such as methyl [2-(acridin- 9-ylimino)-3-substituted-4-oxo-thiazolidin-5-ylidene]acetate which were synthesized recently by our group from 1-alkyl(acyl)- 4-(acridin-9-yl)thiosemicarbazides and dimethyl acetylenedicar- boxylate (DMAD). 14–17 The present study was designed to expand our knowledge of the mechanism of cytotoxicity of related acri- dine–thiourea derivatives which possess also reactivity towards thiols. Three new acridine–thiazolidinone derivatives were pre- pared from N-(acridin-9-yl)-N 0 -substituted thioureas (R = sec-bu- tyl, tert-butyl, and 4-bromophenyl) and DMAD and their DNA binding activity was evaluated. Although, anticancer activity of acridines is mainly related to their capacity to bind to DNA, the cytotoxicity of these may be enhanced by an increase of their elec- trophilicity to facilitate the reaction with essential SH groups pres- ent in living organisms, especially a main low-molecular thiol, glutathione. Reactivity of acridines with sulfhydryls could modify SH-proteins and alter intracellular glutathione redox status, which modulates a lot of cellular events, for example, selective gene expression, DNA synthesis, or regulation of the cell cycle. 18–20 In vi- tro cytotoxic effects of these compounds on murine leukemic cells L1210, human promyelocytic leukemic HL-60 cells, and A2780 hu- man ovarian adenocarcinoma cells were monitored. To investigate the mechanism of cytotoxicity, a cellular accumulation of acridine– thiazolidinone derivatives was studied and their influence on the intracellular glutathione level was evaluated. The cell death and changes in the cell cycle were observed. 0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2012.09.068 ⇑ Corresponding author. E-mail address: helena.paulikova@stuba.sk (H. Paulíková). Bioorganic & Medicinal Chemistry 20 (2012) 7139–7148 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc