Tri- and tetra-substituted naphthalene diimides as potent G-quadruplex ligands Francisco Cuenca, a Olga Greciano, a Mekala Gunaratnam, a Shozeb Haider, a Deeksha Munnur, a Rupesh Nanjunda, b W. David Wilson b and Stephen Neidle a, * a CRUK Biomolecular Structure Group, The School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, UK b Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA Received 20 December 2007; revised 10 January 2008; accepted 14 January 2008 Available online 18 January 2008 Abstract—A series of tri- and tetra-substituted naphthalene diimides have been designed and synthesized. Several compounds show exceptional affinity for telomeric G-quadruplex DNA in classical and competition FRET assays and SPR studies. They inhibit tel- omerase in the TRAP assay, and show potent senescence-based short-term anti-proliferative effects on MCF7 and A549 cancer cell lines, and localize in the nucleus and particularly the nucleolus of MCF7 cells. Ó 2008 Elsevier Ltd. All rights reserved. Telomeres are highly specialized DNA-protein struc- tures at the ends of eukaryotic chromosomes 1 whose integrity is required for the cell to avoid end-to-end fu- sions and recombination. 2 Telomere length progres- sively shortens in somatic cells with successive rounds of cell division, leading eventually to senescence and apoptosis. In contrast telomere length is maintained in cancer cells and is a major factor in immortalization and tumorigenesis. 3 The reverse transcriptase enzyme telomerase is over-expressed in ca 80–85% of cancer cell types 4 where its ability to catalyze the synthesis of telo- meric DNA repeats is responsible for telomere homo- statis. Inhibition of telomerase induces cell senescence and cell death in cancer cells and it is a validated target for cancer therapeutics. 5 One approach to telomerase inhibition involves seques- tering its substrate, single-stranded telomeric DNA, by inducing it to form G-quadruplex (G4) structures. 6 Induction of a G-quadruplex conformation can be achieved by small-molecule ligands; a large number have been reported, 7 although none as yet have reached the clinic. It has been shown that the induction of G4-ligand complexes at the 3 0 -single-strand telomeric DNA over- hang displaces telomere-associated proteins such as hPOT1 and telomerase. 8 This unmasking of the 3 0 -over- hang invokes a rapid DNA damage response, which rapidly leads to selective cell death and anti-tumour activity in vivo. 9 A number of naphthalene imide and diimide (ND) monomer and dimer derivatives bind to duplex DNA. 10 Several show in vivo anti-cancer activity and two (Amonafide and Elinafide) have been evaluated in anti-cancer clinical trials in humans, 11 although they have not progressed due to toxicity combined with lim- ited activity. A series of disubstituted NDs have been screened as G4 ligands but showed only low affinity. 12 We hypothesized that the planarity of the ND moiety would nevertheless be an interesting starting point for the development of more complex ligands since recent chemical developments 13 enabled us to envisage a syn- thetic route to tetra-substituted analogues, which we ar- gued, would possess high affinity for G4s such as the parallel telomeric G4 structure which possesses four tar- getable grooves. 14 This supposition was supported by molecular modelling studies 15 (Fig. 1), and more re- cently, by crystallographic analyses on several G4-ND complexes. 16 Furthermore, the synthetic route allows the introduction to the ND core of up to three different side chains, which in principle can be exploited to produce ligand diversity that may discriminate between 0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2008.01.050 Keywords: G-quadruplex; Telomere; Telomerase inhibition. * Corresponding author. Tel.: +44 207 753 5969; fax: +44 207 753 5970; e-mail: bmcl@pharmacy.ac.uk Available online at www.sciencedirect.com Bioorganic & Medicinal Chemistry Letters 18 (2008) 1668–1673