Preparation And Biological Evaluation Of Platinum Complexes Based On The Ligands Of Dipicolyamine Bisphosphonate Esters

It is well known that the incidence rate of cancer has increased rapidly year by year in the world, which has become a serious threat to our health and life. One of the widely-used metal complexes drugs in clinic is the platinum-based complexes. However, due to their side effects such as gastrointestinal toxicity, hepatotoxicity, nephrotoxicity and neurotoxicity, as well as the intrinsic resistance and acquired resistance, the therapy efficacy and the clinical application of these compounds have been seriously affected and limited. This is mainly related to the fact that the drug lacks the targeting selectivity for the tumor. As well known, bisphosphonates have been used widely as the therapeutic agents for treating several bone-related diseases because of their bone-targeting properties, such as metabolic bone diseases and bone tumors. Some research have shown that the monofunctional platinum complexes bearing a N-heterocyclic ring have great antitumor activities and they also have different action mechanisms from the typical platinum-based drug cisplatin(CDDP), which has been widely used in the clinic. In order to obtain a new kind of platinum-based anticancer drugs with high efficacy and low toxicity, in this study a series of novel platinum complexes were designed and synthesized based on the dipicolyamine bisphosphonate esters that bear a N-heterocyclic ring. Their bone affinity, antitumor activity, action mechanism and hepatotoxicity were studied preliminary, with the hope to reduce the side effects and resistance of drugs by means of improving their tumor targeting efficiency and changing their action modes.In this study, dipicolyamine pamidronate and dipicolyamine alendronate were synthesized from pamidronate and alendronate with 2-(chloromethyl)pyridine hydrochloride in the hydroxide sodium solution, maintaining the p H of the solution at 12 at room temperature. Then, dipicolyamine pamidronate ester and dipicolyamine alendronate ester were further synthesized from the dipicolyamine pamidronate and the dipicolyamine alendronate with(trimethylsilyl)diazomethane, respectively. Then, two novel platinum complexes based on the ligands of dipicolyamine pamidronate ester and dipicolyamine alendronate ester were synthesized respectively. The synthetic and purification conditions were discussed and optimized. All the structures of the intermediate and final compounds were characterized by ESI-MS, 1H-NMR, 13C-NMR, 31P-NMR, ultraviolet spectra, infrared spectra and elemental analysis. The optimized structure, IR and NMR spectra of the platinum complexes have been calculated by DFT method in quantum chemistry. The biological properties of the complexes were also studied systematically.At first, the bone-targeting ability was studied. The bone-targeting efficiency of these complexes was evaluated by using the high performance liquid chromatography(HPLC) method. The complex-hydroxyapatite(HA) binding assay results showed that the HA have affinity for the two dipicolyamine bisphosphonate ester-platinum complexes. The adsorption quantity increased with the increase of the reaction time. When the reaction time is over 24 h, the adsorption equilibrium can be achieved. And the maximum adsorption percentage of dipicolyamine pamidronate ester-platinum complex and dipicolyamine alendronate ester-platinum complex was 64% and 60%, respectively. The lipid-water partition coefficient showed that dipicolyamine pamidronate ester-platinum complex and dipicolyamine alendronate ester-platinum complex have good hydrophilicity which is conducive to bone-targeting ability.The in vitro antitumor activities and action mechanisms of dipicolyamine pamidronate ester-platinum complex and dipicolyamine alendronate ester-platinum complex have been evaluated by MTT assay, hoechst 33342/propidium iodide double-staining experiment and flow cytometric detection. The MTT assay results showed that two complexes both exhibited an inhibition effect against the human cancer cell lines Hep G2, A549, U2 OS, HCT116 and MDA-MB-231, and the cell viability decreased with the increase of the drug concentration and the treating time, especially on the cancer cell lines A549 and Hep G2. The IC50 values of dipicolyamine pamidronate ester-platinum complex against A549 after the treatment of 48 h and 72 h were 180.23 μmol/L and 108.72 μmol/L, respectively. The IC50 values of dipicolyamine pamidronate ester-platinum complex against Hep G2 after the treatment of 48 h and 72 h were 170.72 μmol/L and 139.79 μmol/L. From the hoechst 33342/propidium iodide double-staining experiments, brilliant blue color cell nucleus and apoptotic body can be observed; the flow cytometric detection results showed that the complexes induced the cell cycle to arrest at the G2/M phase. The complexes inhibit cancer cell lines proliferation by causing the G2/M arrest and inducing cell apoptosis. In addition, the hepatotoxicity of two new complexes was also evaluated against the normal human liver cell lines L02 and compared with that of CDDP in order to provide reference for the clinical application. The results showed that the inhibition effect of both complexes against the normal human liver cell lines L02 is far lower than that against the human liver carcinoma cell lines Hep G2. For instance, the cell viabilities of Hep G2 and L02 treated by dipicolyamine pamidronate ester-platinum complex(400 μmol/L) after 72 h were 4.15% and 42.72%, respectively. That is, they have a good selectivity for inhibiting human hepatocarcinoma cells rather than normal liver cells. Furthermore, the hepatotoxicity of both complexes against the normal human liver cell lines L02 was lower than that of CDDP. For example, the cell viabilities of L02 treated by a low concentration of CDDP(25 μmol/L) after 48 h and 72 h were only 52.01% and 25.02%, respectively.Finally, the binding interaction of dipicolyamine bisphosphonate ester-platinum complexes with calf thymus DNA has been explored by circular dichroism and UV spectrum to elucidate its action mechanism preliminary. The DNA binding ability(Kb value) of dipicolyamine pamidronate ester-platinum complex and dipicolyamine alendronate ester-platinum complex were 1.11×105 M-1and 9.48×104 M-1 respectively. Studies on the circular dichroism spectra of CT-DNA showed that the addition of complexes led the helical structure of DNA to become looser and the base stacking interaction become stronger. The DNA double stranded form structure was changed, which hence affected the further duplication of gene and inhibited the proliferation of cancer cell lines.