DOI: 10.1002/cmdc.201000332 Modeling and Biological Investigations of an Unusual Behavior of Novel Synthesized Acridine-Based Polyamine Ligands in the Binding of Double Helix and G-Quadruplex DNA Carla Bazzicalupi,* [a] Matteo Chioccioli, [b] Claudia Sissi,* [c] Elena Porc, [c] Claudia Bonaccini, [b] Claudia Pivetta, [c] Andrea Bencini, [a] Claudia Giorgi, [a] Barbara Valtancoli, [a] Fabrizio Melani, [b] and Paola Gratteri* [b] Introduction The activity of many antimalarial, antibacterial, and anticancer agents is based on their interaction with helical double-strand- ed DNA (dsDNA). [1–3] As a consequence, much effort has been devoted over the past few decades to the design and synthe- sis of new molecules that can reversibly bind and/or react with dsDNA; the aim of these research efforts has been to use these compounds as novel drugs or as probes to better under- stand the mechanism of action of drugs that are already avail- able. More recently, interest has also focused on non-canonical DNA structures. For example, a great deal of attention has been devoted to clarifying the structural features of telomeric DNA. [4–8] In fact, the telomeric sequence has been found to be closely related to the immortalization process of cancer cells [9–14] and genetic stability. [15–17] Therefore, it represents a potentially suitable target for anticancer therapy. Telomeres consist of guanine-rich sequence repeats (in humans, the hexa- nucleotide motif d(TTAGGG) for example) located at the end of chromosomes, where their function is to preserve chromo- some integrity. Telomeric DNA is gradually shortened in normal cells as a function of the replication cycle; this leads to cell- cycle arrest and eventually apoptosis when a critical length minimum is reached. [18] In contrast, cancer cells have evolved mechanisms to maintain telomere length, the most common of which is based on the activation of a reverse transcriptase called telomerase; this occurs in 80–90 % of tumor cells. [19–21] Novel antitumor strategies are aimed at interfering with the mechanisms of telomere maintenance by targeting telomerase Three novel 2,7-substituted acridine derivatives were designed and synthesized to investigate the effect of this functionaliza- tion on their interaction with double-stranded and G-quadru- plex DNA. Detailed investigations of their ability to bind both forms of DNA were carried out by using spectrophotometric, electrophoretic, and computational approaches. The ligands in this study are characterized by an open-chain (L1) or a macro- cyclic (L2, L3) framework. The aliphatic amine groups in the macrocycles are joined by ethylene (L2) or propylene chains (L3). L1 behaved similarly to the lead compound m-AMSA, effi- ciently intercalating into dsDNA, but stabilizing G-quadruplex structures poorly, probably due to the modest stabilization effect exerted by its protonated polyamine chains. L2 and L3, containing small polyamine macrocyclic frameworks, are known to adopt a rather bent and rigid conformation; thus they are generally expected to be sterically impeded from rec- ognizing dsDNA according to an intercalative binding mode. This was confirmed to be true for L3. Nevertheless, we show that L2 can give rise to efficient p–p and H-bonding interac- tions with dsDNA. Additionally, stacking interactions allowed L2 to stabilize the G-quadruplex structure: using the human te- lomeric sequence, we observed the preferential induction of tetrameric G-quadruplex forms. Thus, the presence of short ethylene spacers seems to be essential for obtaining a correct match between the binding sites of L2 and the nucleobases on both DNA forms investigated. Furthermore, current model- ing methodologies, including docking and MD simulations and free energy calculations, provide structural evidence of an in- teraction mode for L2 that is different from that of L3; this could explain the unusual stabilizing ability of the ligands (L2 > L3 > L1) toward G-quadruplex that was observed in this study. [a] Dr. C. Bazzicalupi, Prof. A. Bencini, Dr. C. Giorgi, Prof. B. Valtancoli Department of Chemistry “Ugo Schiff”, University of Florence Via della Lastruccia 3-13, 50019 Sesto Fiorentino, Florence (Italy) Fax: (+ 39) 055-457-3364 E-mail : carla.bazzicalupi@unifi.it [b] Dr. M. Chioccioli, Dr. C. Bonaccini, Prof. F. Melani, Prof. P. Gratteri Laboratory of Molecular Modeling Cheminformatics & QSAR Department of Pharmaceutical Sciences, University of Florence Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence (Italy) Fax: (+ 39) 055-457-3780 E-mail : paola.gratteri@unifi.it [c] Prof. C. Sissi, Dr. E. Porc, Dr. C. Pivetta Department of Pharmaceutical Sciences, University of Padova Via Marzolo 5, 35100 Padova, (Italy) Fax: (+ 39) 049-827-5366 E-mail : claudia.sissi@unipd.it Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201000332. ChemMedChem 2010, 5, 1995 – 2005  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1995