Naphthalenediimide-Linked Bisbenzimidazole Derivatives as Telomeric G‑Quadruplex-Stabilizing Ligands with Improved Anticancer Activity

Human telomeric G-quadruplex DNA stabilization has emerged as an exciting novel approach for anticancer drug development. In the present study, we have designed and synthesized three C₂-symmetric bisubstituted bisbenzimidazole naphthalenediimide (NDI) ligands, <b>ALI-C</b>_<b>3</b>, <b>BBZ-ARO</b>, and <b>BBZ-AROCH</b>_<b>2</b>, which stabilize human telomeric G-quadruplex DNA with high affinity. Herein, we have studied the binding affinities and thermodynamic contributions of each of these molecules with G-quadruplex DNA and compared the same to those of the parent NDI analogue, <b>BMSG-SH-3</b>. Results of fluorescence resonance energy transfer and surface plasmon resonance demonstrate that these ligands have a higher affinity for G₄-DNA over duplex DNA and induce the formation of a G-quadruplex. The binding equilibrium constants obtained from the microcalorimetry studies of <b>BBZ-ARO</b>, <b>ALI-C</b>_<b>3</b>, and <b>BBZ-AROCH</b>_<b>2</b> were 8.47, 6.35, and 3.41 μM, respectively, with <i>h-telo</i> 22-mer quadruplex. These showed 10 and 100 times lower binding affinity with <i>h-telo</i> 12-mer and duplex DNA quadruplexes, respectively. Analysis of the thermodynamic parameters obtained from the microcalorimetry study suggests that interactions were most favorable for <b>BBZ-ARO</b> among all of the synthesized compounds. The Δ<i>G</i>_free obtained from molecular mechanics Poisson–Boltzmann surface area calculations of molecular dynamics (MD) simulation studies suggest that <b>BBZ-ARO</b> interacted strongly with G₄-DNA. MD simulation results showed the highest hydrogen bond occupancy and van der Waals interactions were between the side chains of <b>BBZ-ARO</b> and the DNA grooves. A significant inhibition of telomerase activity (IC₅₀ = 4.56 μM) and induced apoptosis in cancer cell lines by <b>BBZ-ARO</b> suggest that this molecule has the potential to be developed as an anticancer agent