Dyads of G‐Quadruplex Ligands Triggering DNA Damage Response and Tumour Cell Growth Inhibition at Subnanomolar Concentration

Abstract

Naphthalened iimide (NDI) dyads exhibiting ad ifferent substitution pattern and linker length have been synthesiseda nd evaluateda sG -quadruplex (G4) ligands,b yi nvestigating their cytotoxicity in selected cell lines. The dyads with the long C 7 linker exhibit extremelyl ow IC 50 values, below 10 nm,o nd ifferent cancerc ell lines.C ontrary,t he dyads with the shorter C 4 linker were much less effective, with IC valuesi ncreasing up to 1 mm.A mong the three dyads with the longest linker, small differences in the IC 50 valuese merge, suggesting that the linker length plays a more important role than the substitution pattern. We have further shown that the dyads are able to induce cellular DNA damage response, whichi sn ot limited to the telomeric regions and is likely the origin of their cytotoxicity.B oth absorptiont itration and dynamic light scattering of the most cytotoxic dyads in the presence of hTel22 highlight their abilityt oi nduce effective G4 aggregation,a cting as non-covalentc ross-linking agents.[a] Dr.Scheme1.NDI dyads 5-16 synthesised andevaluated as G4 ligands in comparisontot he reference monomericNDI units 1-4.Scheme2.Synthesis of the water-soluble NDI dyads. a) Amine (3.0 equiv), CH 3 CN, 75 8C, 5h.b )Na 2 S 2 O 4 (2 equiv) in aqueous CH 3 CN (1:1), RT,2h. c) NDI 17 (0.95equiv), DMF,m icrowave-assistedprotocol, sealed reactionv essels (M.W., 150 8C, 200 psi, 200 W, 20 min). d) Neat N 1 ,N 1 -dimethylpropane-1,3-diamine, microwave-assistedprotocol, sealed reactionv essels (M.W., 150 8C, 200 psi, 200 W, 3min). Absorption, CD and fluorescencespectraUV/Vis absorption spectra were recorded on as tandard Perkin-Elmer l650 spectrophotometer.C Ds pectra were recorded on a Jasco polarimeter J-715 accumulating four spectra with as can rate of 100 nm min À1 .F luorescence spectra were measured by using 1nms teps and 0.5-1 sd well time. Slits were kept as narrow as possible to 4-8 nm in excitation and 4-8 nm in emission. Where necessary,acut-off filter was used. Right angle detection was used. All the measurements were carried out at 295 Ki nq uartz cuvettes with ap ath length of 1cm. All fluorescence spectra have been obtained for air-equilibrated solutions absorbing less than 0.1 at all wavelengths to avoid inner filter effects and re-absorption of the emission. Furthermore, they have been corrected for wavelengthdependent response of the monochromator/PMT couple.

Trifunctionalized Naphthalene Diimides and Dimeric Analogues as G-Quadruplex-Targeting Anticancer Agents Selected by Affinity Chromatography

“…Finally, to evaluate if the DNA damage induced by NDI-3, NDI-4 and NDI-5 on the transformed cells was located at telomeres, BJ-EHLT cells were treated for 24 h, with each of the three compounds, and the telomeric damage was evaluated by measuring the co-localization spots (telomere-induced foci, TIFs) between γH2AX and TRF1, an effective marker for interphase telomeres ( Figure 5C). Interestingly, quantitative analysis showed that the effects of the three compounds were quite similar, and all of them induced a percentage of TIF positive cells (cells with at least four co-localization spots) and average number of TIFs per cell ( Figure 5D) comparable with the values recently found for other effective NDI dyads [21]. From these data, it is possible to conclude that the selected NDIs are not only potent and effective inducers of telomere localized DNA damage response, but also show a high grade of specificity against transformed cells.…”

“…All the final products were characterized by ESI-MS and 1 H-and 13 C-NMR. The characterization data for NDI-1 [29], NDI-3 [21], NDI-15 [29] and NDI-17 [21] were previously reported.…”

“…In addition, it exhibited a good G4 vs. duplex DNA selectivity [20], thus proving to be a promising compound in the design of optimized G4-selective ligands. In this perspective, dimeric naphthalene diimides featuring different substitution patterns and linkers have been then synthesized, showing high activity on different cancer cell lines [21,22]. Notably, these compounds displayed a remarkable affinity for G4 DNA, but, unfortunately, they displayed a low ability to discriminate G4 vs. duplex DNA.…”

“…Notably, these compounds displayed a remarkable affinity for G4 DNA, but, unfortunately, they displayed a low ability to discriminate G4 vs. duplex DNA. This poor selectivity also affected their cytotoxic behavior [21,22]. In fact, the best dimeric NDIs as G4 binders showed high cytotoxicity toward cancer cells, with IC 50 values in the nM range, but also produced moderate antiproliferative effects toward healthy cells [21].…”

“…First of all, in the dimeric series, one of the NDI units was decorated with anionic side chains at the imide positions, and the other was decorated with protonable alkylamines, in order to reduce the net positive charge of the new hetero dyads at physiological pH. This design, with respect to the previously synthesized dimeric NDIs containing only alkylamines in their side chains [21], allows for the reduction of the unspecific interactions toward duplex DNA, typically resulting from the electrostatic attractions between the backbone phosphates and the protonated ligands. In this frame, the new NDIs should lose some affinity degrees toward the G4 targets but, in turn, gain enhanced G4 vs. duplex selectivity [23].…”