Biological activity and DNA binding studies of 2- substituted benzimidazo[1,2-a]quinolines bearing different amino side chains Nataˇ sa Perin, a Irena Martin-Kleiner, b Raja Nhili, c William Laine, c Marie-H´ el` ene David- Cordonnier, c Oliver Vugrek, b Grace Karminski-Zamola, a Marijeta Kralj * b and Marijana Hranjec * a This manuscript describes the synthesis and biological activity of 2-substituted benzimidazo[1,2-a]- quinolines substituted with different amino side chains on the quinoline nucleus prepared by microwave assisted amination. The majority of compounds were newly synthesized and active at submicromolar IC 50 concentrations, while the alkylamino substituents, either acyclic or cyclic, increased antitumor activities in comparison with previously published nitro and amino substituted benzimidazo [1,2-a]quinolines. The compound with the longest tertiary amino side chain (16) was the least active. A series of additional experiments, including DNA binding propensities, topoisomerases I and II inhibition, inhibition of recombinant green fluorescent protein in a cell-free translation system, cell cycle perturbances and cellular localization, was performed to shed more light on the mechanisms of action of the most active compounds. The DNA intercalation activity correlates with anti-proliferative effect. Several DNA intercalators (11, 20 and 21) also evidence some sequence selective DNA binding. However, only N,N-dimethylaminopropyl analogue 11 was unequivocally demonstrated to be a strong DNA-binder and intercalative agent, which efficiently targets DNA in the cells, while the activity of compound 10, with a bulky i-butylamino side chain, points to its potential antimitotic activity. Introduction The permanent and growing interest in the synthesis of ben- zannulated benzimidazole derivatives, one of the most exten- sively studied classes of heterocyclic compounds, is a direct consequence of their diverse biological properties. 1–4 Since benzimidazole exhibits structural similarity with some natu- rally occurring compounds, their derivatives play a crucial role in the function of some biologically important molecules or can easily interact with biomolecules like DNA, RNA or different proteins of the living systems. 5,6 DNA still represents one of the principal targets in drug development strategies designed to produce novel therapeutics for diseases such as cancer. 7 An understanding of the molecular basis of interactions of small heteroaromatic organic molecules with DNA is a promising approach, which is of utmost importance in the rational development of novel, more selective anticancer agents. 8 Ben- zannulated benzimidazoles usually possess a highly conju- gated, planar chromophore, which imparts them the ability to intercalate between adjacent DNA base pairs. 9–11 Due to high uorescence intensity and excellent spectroscopic characteris- tics, fused benzimidazoles offer a potential application as uorescent probes for detection of biologically important molecules such as DNA or proteins in biomedical diagnos- tics. 12–14 Recently, as part of our continuous scientic research in the eld of potential biologically active benzimidazoles, we have reported on the synthesis and biological activity of several groups of benzimidazo[1,2-a]quinolines, including positively charged amidino-substituted benzimidazo[1,2-a]quinolines and their heteroaromatic analogues as well as versatile benzi- midazo[1,2-a]quinolines-6-carbonitriles. 2,3,15 Biological studies conrmed the anticancer potential of this class of compounds, especially that of positively charged amidino-substituted analogues of benzimidazo[1,2-a]quinolines, which intercalate into double-stranded DNA or RNA. The most active one, 2-imidazolinyl-substituted benzimidazo[1,2-a]quinoline with pronounced selectivity towards colon carcinoma cells, inhibi- ted topoisomerase II and induced strong G2/M cell cycle arrest. On the other hand, uncharged amino- and positively charged diamino-substituted benzimidazo[1,2-a]quinolines a Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Maruli´ cev trg 20, P. O. Box 177, HR-10000 Zagreb, Croatia. E-mail: mhranjec@it.hr; Fax: +385 14597250; Tel: +385 14597245 b Division of Molecular Medicine, Rud  er Boˇ skovi´ c Institute, Bijeniˇ cka cesta 54, P. O. Box 180, HR-10000 Zagreb, Croatia. E-mail: marijeta.kralj@irb.hr; Fax: +385 1 4561 010; Tel: +385 1 4571 235 c INSERM U837, Jean-Pierre Aubert Research Centre (JPARC), Team “Molecular and Cellular Targeting for Cancer Treatment”, Institut pour la Recherche sur le Cancer de Lille, Universit´ e Lille 2, IMPRT-IFR-114, France Cite this: Med. Chem. Commun., 2013, 4, 1537 Received 10th July 2013 Accepted 18th September 2013 DOI: 10.1039/c3md00193h www.rsc.org/medchemcomm This journal is ª The Royal Society of Chemistry 2013 Med. Chem. Commun. , 2013, 4, 1537–1550 | 1537 MedChemComm CONCISE ARTICLE