Submicromolar, selective G-quadruplex ligands from one pot: thermodynamic and structural studies of human telomeric DNA binding by azacyanines

The discovery of G-quadruplex structures in nucleic acid sequences associated with cancers has created intense interest in G quadruplexes as potential drug targets. These fourstranded structures, with planar G tetrads represent appealing DNA targets (Figure 1A), as they are structurally distinct from the Watson–Crick duplex of most genomic DNA. Small molecules with high affinity and high selectivity for G quadruplexes have even begun to show medicinal promise, although the connection between G-quadruplex binding and in vivo activity might not always be obvious. As one promising example, quarfloxin, an antineoplastic that targets the rRNA–nucleolin complex, is presently in phase II clinical trials. Most investigators who seek new ligands for G-quadruplex DNA have followed two common strategies. First, they have focused on heterocycles with a relatively large and planar surface area, which maximizes stacking with the about 1 nm surface of a G tetrad (e.g. , TmPyP4, Figure 1C). Second, many have used multiple charges to increase electrostatic interactions with the high-charge density G quadruplex (e.g. , BRACO-19, Figure 1D) and to enhance the solubility of potential ligands (often necessary for ligands with large hydrophobic surfaces, e.g. , TmPyP4). While these strategies have resulted in several high affinity ligands for G quadruplexes, most ligands do not exhibit high selectivity over duplex DNA. Recently, several metallated quadruplex ligands have been reported with substantial selectivity for quadruplex DNA. As the authors have suggested, this could be, in part, due to ACHTUNGTRENNUNGinteractions between the metal ion and the lone pairs of the carbonyl oxygen atoms of the exterior quartets, which are not accessible in duplex DNA. Higher-throughput, more combinatorial approaches have begun to show promise as well. For example, Balasubmaranian and co-workers have recently demonstrated dynamic combinatorial selection of quadruplex ligands by disulfide bond formation with a thiol-containing scaffold and side chains. We have taken a different approach to targeting the G quadruplex. We previously discovered that a planar molecule larger than a typical DNA intercalator can selectively bind purine–purine base pairs. Based upon this discovery, we hypothesized that planar, monocationic molecules that are marginally too large to intercalate a Watson–Crick duplex, such as bispurine analogues, might selectively bind G quadruplexes. We report a new class of selective, submicromolar quadruplex ligands with a facile synthetic route: the azacyanines (Figure 1B). The synthesis of azacyanines was previously reported by Kurth and co-workers ; the route is one-pot and workup is by filtration. The route is general and succeeds for aminobenzimidazoles and aminobenzothiazoles. The synthetic ease makes the class amenable to library preparation for highthroughput screening. We investigated the binding of azacyanines to a G-quadruplex sequence, based on the human telomeric repeat dACHTUNGTRENNUNG(TTA