Studies On Synthesis, Characterization And Action Mechanism Of Antitumor Platinum Complexes Targeting Telomeric G-quadruplex DNA

DNA is one of the classic targets for anticancer drugs discovery. However, there is a large amount of duplex DNA inside the nucleus, drugs may nonspecific interactions with the duplex DNA and cause serious side effects. To reduce the significant toxicity and the side effects, design and discovery of less toxic, more selective, and more effective agents to treat cancer has become a huge challenge for pharmic scientists. Besides the classical double-stranded DNA structure, some of higher-order DNA structures formed from G-rich sequences, such as G-quadruplex structures, have emerged as one of the most important targets for drug discovery. Compared with duplex DNA structure, G-quadruplex DNA have a very different in size, loop, structures, and their structural diversity suggests that small molecules designed to selectively recognize and stabilize G-quadruplex structures over duplex DNA might be possible.This dissertation mainly focuses on finding anticancer complexes targeting G-quadruplex DNA with high antitumor activity and low toxicity. Twenty one new platinum(Ⅱ) complexes were synthesized and the interaction of these compounds to ct-DNA and G-quadruplex DNA were also investigated. In cellular level, antitumor activity of twenty one platinum complexes, and some of them inhibiting telomerase activity, induced cell cycle arrest, inhibits hTERT mRNA expression were examined.This dissertation consists of four sections as follows:Chapter One, it is the introduction, which briefly reviews the characters of G-quadruplex structures, its biological functions and the relationship with tumoregenesis of typical quadrome gene telomere, and the small molecular ligands research and development based on G-quadruplex.Chapter Two, we designed and synthesized twenty one platinum(Ⅱ) complexes based on the phenanthroimidazole derivatives, their structures have been characterized by means of ESI-MS, elemental analysis, IR,1HNMR and UV-Vis.Chapter Three, results of the MTT assay showed that these complexes exhibited considerable antitumor activity against several human cancer cells, most of them with the IC50values in the range of2-20μmol/L, but has no toxicity effect on HL-7702(normal cell). The complexes with methoxy substituented ligands exhibited strong antitumor activity. However, the substitution of the methoxy group by a nitro group strongly decreased the activity. In the flow cytometry analyses, the HeLa cells treated with complexes1-9for72h exhibited DNA damage at the sub-G1phase with a dose-dependent effect resulting in blockage of the cell cycle at the sub-G1phase, which might contribute to the cell apoptosis observed in HeLa cells, and Complexes1-9induce apoptosis in human HeLa cells in a dose dependent manner.The complexes1-9were tested for the telomerase inhibitory activity and the expression of hTERT mRNA. The results showed that complexes can inhibit the telomerase activity in HeLa cell with inhibition rate in the range from62.16%to85.39%, and down-regulate hTERT mRNA expression. Inhibition of telomerase activity by the complexes1-9may be related to suppressing the hTERT mRNA expressing level and arrest the HeLa cell cycle.Chapter Four, the interactions between the platinum(II) complexes and DNA (ct-DNA, G-quadruplex DNA) and basic structure and binding affinity were also analyzed.1. The absorbance spectrum of the platinum(II) complexes was not bathochromic spectral shift while the significant hypochromism was observed at the radio of [SDS]:[complex]=0-2.5, indicating that the strong electrostatic interaction between the complexes and DNA in buffer solution.2. UV-Vis absorption spectrum was employed to investigate the binding mode and binding affinity of complexes toward ct-DNA and G-quadruplex H21T. Upon the addition of an increasing amount of DNA to a solution of the complexes, the absorbance was bathochromic spectral shift and the apparent hypochromicity was observed for the interaction between the complexes and DNA, indicating that the complexes can interact with ct-DNA by classical intercalating mode, and binding to G-quadruplex may not be restricted to stacking interactions and could involve in interactions to DNA’s loops and phosphate backbone. The binding constants (Kb) value for ct-DNA is approximately25-95fold lower than the binding constants of complexes for the G-quadruplex H21T, this data reveal that complexes selectively binds G-quadruplex H2T1over duplex DNA. The interaction between complexes and ct-DNA can be enhanced by the complexes with electron-withdrawing group and the presence of electron donating group benefit to enhance the interactions between the complexes and G-quadruplex H21T.3. The selectivity of the complexes to different G-quadruplex (ckitl, ckit2, H21T, pu22) and duplex DNA (ds26) was further evaluated by Fluorescent Intercalator Displacement (G4-FID) assay. Most of the complexes appear to be good to strong G-quadruplex binders (0.20μmol/L<G4DC50<0.87μmol/L) whereas they exhibit lower binding affinity to ds26(ds26DC50>1.20μmol/L), These data indicate that the complexes display strong interactions between the complexes and G-quadruplex than duplex structure, which agrees with the results obtained from UV-vis absorption titration experiments. FRET experiments showed that eight complexes all can enthance the Tm value of G-quadruplex, which indicate that the platinum(Ⅱ) complexes are able to stabilize G-quadruplex DNA.4. The conformational changes of DNA induced by the complexes were examined by circular dichroism (CD) spectrum. Upon addition of the complexes to ct-DNA, caused a significant variations of both positive and negative bands with a blue shift were observed, which indicates that the complexes may bind to DNA by intercalation mode. A significant change in the CD spectrum was observed after complexes is added into single-stranded telomeric DNA, a new band appear at about263nm, a increasing in the263nm and290nm band were observed, which indicate that the complexes can induce disordered oligonucleotide H21T to form antiparallel G-quadruplex structure. In the presence of K+, when complexes was added to mixed G-quadruplex H21T, the intensity of the positive CD band at260nm decreased gradually from positive to negative, and the positive peak close to292nm had a weak blue shift, this suggests that mixed G-quadruplex structure can be induced into antiparallel G-quadruplex structure in K+solution by the complexes. In conclusion, these platinum(Ⅱ) complexes can induce and stabilize the H21T to form antiparallel G-quadruplex DNA. Combined with the results of MTT assay, CD, UV-Vis and G4-FID, it is found that these complexes that can inhibit the telomerase activity may be via inducing the single-stranded telomeric DNA to form antiparallel G-quadruplex H21T and stabilizing the antiparallel G-quadruplex H21T.In the present paper, possible action mechanisms for the antitumor activity of the platinum(Ⅱ) complexes were investigated in vitro. These results will provide a new strategy to discover and develop novel antitumor drugs targeting G-quadruplex DNA.