Mechanistic Investigations On The Reaction Between Platinum Complexes And DNA In The Presence Of Glutathione

Since the discovery of cisplatin by Rosenberg in 1965,the application and research of platinum-based anticancer drugs have achieved rapid development.Cisplatin as a broad-spectrum anticancer agent has presented amazing results in the treatment of some solid tumors,especially in the tumor therapy of cervix,ovary,testicle,neck and head.However,some side effects,acquired or intrinsic drug resistance,serious toxicity and low solubility in water,have greatly limited its extensive use.The interaction of cisplatin with biological molecules has been found to be one of the key reasons for the drug resistance,because of the high affinity of platinum to those containing sulfur binding sites.It is well known that a variety of sulfur containing biomolecules are available in the body,such as glutathione(GSH)which is present in concentrations of 0.5-10 mM in tumor cells and is clearly correlated with the resistance of platinum-based drugs.Moreover,glutathione molecule possesses all kinds of biological donor atoms:two carboxyls,one thiol,one amino,and two pairs of carbonyl andamide donors within two peptide bonds.Thus,the coordination chemistry of glutathione is of vital importance as it serves as a model system for binding platinum complexes of larger peptide and protein molecules.The main content of this dissertation is as follows:Firstly,in view of the steric hindrance effect of 1,2-diaminocyclohexane in oxaliplatin,we have studied the interaction between oxaliplatinand GMP in the presence of GSH to explore the binding modes of oxaliplatin with GMP and GSH by HPLC and LC-MS techniques.In the process,four key intermediates were found,and five adducts of the reaction have been qualitatively and quantitatively determined.Among these intermediates and adducts,six of them were identified for the first time.Unlike cisplatin,oxaliplatin doesn't form the tri-functional complexes by the loss of ammine in the reaction,which may be the key reason for its activity against cisplatin resistant cancers.The result showed that GSH joins the reaction between oxaliplatin and GMP in two ways.One is to react with oxaliplatin by competing with GMP to bind the platinum atom,reducing the percentage of the active platinum unit to approach DNA;the other is to take the place of GMP that has been coordinated to the metal atom.Secondly,analogous reactions between complex 2a with GMP in the presence of GSH were studied in the same way for comparison.Complex 2aof trans-bicyclo[2.2.2]octane-7,8-diamine with bulky dicyclic skeleton did not significantly hinder the binding of GSH along with GMP bound to the platinum atom,but it could restrain GSH to take the place of GMP in the bi-GMP adduct.Consequently,our researchcan provide a view for platinum-based drug design that a carrier ligand with a suitable spatial moietyis needed in its platinum(?)complex to prevent thebinding of GSH fromPt-DNA adducts.Then,a series of novel platinum(?)complexes with N-monosubstituted 1R,2R-diaminocyclohexane bearing methoxy-substituted benzyl groups as carrier ligands were designed and prepared.Biological assay indicated that complex 1 had potent cytotoxic activity against A549,HCT-116,MCF-7,and SGC7901 cancer cell lines,and was less toxic to human normal liver LO2 cell line,compared with cisplatin and oxaliplatin.Meanwhile,another two complexes 2 and 3,with chloride anions as leaving groups,also showed strong anticancer activity comparable or even superior to cisplatin and oxaliplatin,whereas other complexes with dicarboxylates,except individual cases,exhibited moderate or low cytotoxicity.Moreover,the cytotoxicity of complexes 1-3 against cisplatin resistant cell(SGC7901/CDDP)indicated that they all had the potential to overcome cisplatin resistance.Cell cycle study on MCF-7 cell line indicated that the mechanism of cell cycle arrest induced by complex 1 was different from oxaliplatin.Complex 1 could trigger cancer cell death via an apoptotic pathway.And furthermore,western blot analysis indicated that the induced apoptosis by complex 1 was to some extent caused by intrinsic mitochondrial pathway.The HPLC-MS assay demonstrated that complex 1,with a benzyl moiety containing a methoxyl group at ortho position as steric hindrance,had the monofunctional binding ability to DNA,which was significantly different from classical bifunctional cross-linking platinum(?)complexes.In particular,the monofunctional DNA conjugation of complex 1 is not via the traditional metalloinsertion,but the covalent binding between platinum and N7-guanine site.Moreover,HPLC and LC-MS studies showed that complex 1 was insusceptible to nucleophiles,and the linkage of complex 1 with DNA was stable and insensitive to nucleophilic attack as well.The irreversible binding affinities for DNA could make up its lower cellular uptake,compared with cisplatin and oxaliplatin.All these features,including the satisfactory activity toward the tested cancer cell lines especially the cisplatin resistant line,the binding mode versus other oxaliplatin derivatives,cellular uptake and apoptosis properties,bear out that the introduction of the rigid steric hindrance is much useful to promote the anticancer activity of the resulting platinum complexes,and potentially overcome cisplatin drug resistance.Lastly,we extend the study to improve understanding the structure activity realationship of Pt(?)complexes,including stability in water,the interaction with DNA and the redox reaction.The Pt(?)complexes with different carrier ligands were compared,and the stability of these complexes increased when ammine ligand switched to 1,2-diaminocyclohexane ligand.These axial and carrier ligand had an effect on the stability and substitution rates,while the leaving group didn't.The redox rates increased when the axial halo ligand size decreased,which is in same order to the carrier ligand size.The results indicate that the axial halo ligands are essential for redox reaction,and the PtC(?)complexes with larger axial halo ligands have slower rates.The substitution reaction of Pt(?)complexes-with GMP was also studied,and the data indicated that the leaving group might be substituted by GMP.Moreover,the redox rates of Pt(?)complexes with glutathione were faster than the substitution with GMP.