Ispitivanje mehanizma nukleofilnih supstitucionih reakcija kompleksa zlata(III)

Abstract

Lekovi na bazi platine imaju važnu ulogu u lečenju tumora. Klinički uspeh platinskih jedinjenja izazvao je veliko interesovanje za ispitivanje drugih platinskih i neplatinskih lekova koji bi ispoljavali bolja citotoksična svojstva, dobre antitumorske karakteristike i povoljniji farmakološki profil. Različita jedinjenja jona metala (npr. rutenijuma, kalaja, paladijuma, titana, zlata, bakra) su intenzivno ispitivana tokom poslednje tri decenije kao potencijalni medikamenti u borbi protiv tumora. Tokom poslednjih 10 godina veliko interesovanje je usmereno na Au(III) jedinjenja. To je za rezultat dalo veliki broj novosintetisanih jedinjenja Au(III) sa većom stabilnošću pri fiziološkim uslovima i dobrom citotoksičnošću in vitro na ćelijama tumora. Prvi Au(III) kompleksi nove generacije sintetisani su 1990 godine. Prihvatljiva stabilnost ovih jedinjenja zlata omogućava široku farmakološku primenu in vivo i in vitro. Nakon toga, nova jedinjenja Au(III) su sintetisana u nekoliko svetskih laboratorija, i pokazala su značajnu biološku aktivnost.

Presently, platinum drugs are playing a major role within established medical treatments of cancer. The wide clinical success of platinum compounds has prompted agreat deal of interest in other platinum and non-platinum metallodrugs that might exhibit comparable cytotoxic properties, hopefully accompanied by a different antitumor specificities and by a more favorable pharmacological profile. Thus, various classes of metal compounds were intensively investigated during the last three decades as potential anticancer agents based on several different metals (e.g., ruthenium, tin, palladium, titanium, gold, copper). In particular, during the last 10 years, much interest has focused on Au(III) compounds because a number of newly synthesized Au(III) metallodrugs turned out to display appreciable stability under physiological conditions. They also show a highly cytotoxic effects in vitro toward selected human tumor cell lines. The first Au(III) complexes of the new generation were described in the 1990s. The acceptable solution stability of these gold compounds facilitated extensive pharmacological testing, both in vitro and in vivo, with encouraging results. Subsequently, several other classes of cytotoxic gold(III) compounds were developed in a few laboratories worldwide and were found to exhibit very attractive biological profiles. Cytotoxic effects of novel Au(III) compounds were measured on different tumour cell lines. Recently reported results have shown that several new Au(III) dithiocarbamate complexes in vitro have even better cytotoxic properties than cisplatin toward a series of human tumour cell lines. Also, some Au(III) complexes show a very weak interaction with DNA and slight modifications of the double helix. Some Au(III) complexes could be significantly stabilized, even at neutral pH, with the appropriate choice of the inert ligands, preserving its peculiar biological properties. Thus, the presence of at least two nitrogen donors directly coordinated to the Au(III) centre leads to a significant decrease in the reduction potential of the complex. In an attempt to define preliminary structure-function relationships within the new class of cytotoxic and potentially antitumor compounds, in this theses is presented the interactions between some Au(III) complexes with representative DNA fragments, the classic target of Pt(II) complexes. The obtained results are presented in this order: - The kinetics of the substitution reactions between monofunctional Au(III) complexes, [Au(dien)Cl]2+ and [Au(terpy)Cl]2+, and biologically relevant ligands such as L-histidine (L-His), inosine (Ino), inosine-5′-monophosphate (5′-IMP) and guanosine-5′- monophosphate (5′-GMP) were studied in detail. All kinetic studies were performed in 25 mM Hepes buffer (pH = 7.2) in the presence of NaCl to prevent the spontaneous hydrolysis of the complexes. The reactions were followed under pseudo-first order conditions as a function of ligand concentration and temperature using stopped-flow spectrophotometry. The [Au(terpy)Cl]2+ complex is more reactive than the [Au(dien)Cl]2+ complex, which was confirmed by quantum chemical DFT calculations. A more than 50 % lower activation energy for the terpy than for the dien based complex was found. - The kinetics of the substitution reactions between bifunctional Au(III) complexes, [Au(bipy)Cl2]+ and [Au(dach)Cl2]+, and biologically relevant ligands such as L-histidine (L-His), inosine (Ino), inosine-5′-monophosphate (5′-IMP) and guanosine-5′-monophosphate (5′-GMP) were studied in 25 mM Hepes buffer (pH = 7.2) in the presence of NaCl. The reactions were followed under pseudo-first order conditions as a function of ligand concentration and temperature using stopped-flow spectrophotometry. The bifunctional [Au(bipy)Cl2]+ complex is more reactive than the [Au(dach)Cl2]+ complex. The reactivity of the studied nucleophiles follows the same order for both complexes, viz. L-His > 5′-GMP > 5′-IMP > Ino. According to the measured activation parameters all studied reactions follow an associative substitution mechanism. Quantum chemical calculations DFT calculations suggest that ligand substitution in [Au(terpy)Cl]2+ and [Au(dien)Cl]2+ by imidazole follows an interchange mechanism with a significant degree of associative character. - The kinetics of the complex formation of [Au(en)Cl2]+ and [Au(SMC)Cl2]+ complexes with fragments of DNA were also studied. The [Au(SMC)Cl2]+ complex is more reactive than [Au(en)Cl2]+ what could be explained by trans-effect of coordinated sulphur atom from S-methyl-L-cysteine. L-Histidne reacts faster than DNA constituents in the reaction with [Au(en)Cl2]+, but in the reaction with [Au(SMC)Cl2]+ 5’-GMP is the best nucleopile. The activation parameters for all studied reactions suggest an associative substitution mechanism. - The cytotoxicity of [Au(en)Cl2]+ and [Au(SMC)Cl2]+ was evaluated in vitro against chronic lymphocytic leukemia cells (CLL). All complexes showed significant cytotoxic effects, but [Au(en)Cl2] + showing a potency comparable to cisplatin. - The cytotoxic activity of [Au(en)Cl2]+, [Au(SMC)Cl2]+ and [AuCl2(DMSO)2]+ complexes on 4T1 mouse breast cancer cell line were investigated in vitro and in vivo and compared the results with cisplatin. Among the tested complexes [AuCl2(en)]+ showed the best cytotoxic effects in vitro. The cytotoxic effects of [Au(en)Cl2]+ and cisplatin were similar at all concentrations. The data from the in vivo experiments showed that only [Au(en)Cl2]+ can prevent the primary breast tumor growth. [Au(en)Cl2]+ was tolerated well and much better than [AuCl2(DMSO)2]+, [Au(SMC)Cl2]+ and cisplatin. In addition, mice that received [Au(en)Cl2]+ showed better survival time in comparison with mice that received cisplatin. Complex [Au(en)Cl2]+ seems to be good candidate for future pharmacological evaluation in breast cancer research. - The cytotoxicity of [Au(en)Cl2]+, [Au(dach)Cl2] and [Au(bipy)Cl2]+ was tested on A549 human lung carcinoma epithelial cell line and was evaluated by cytotoxic (MTT and LDH test) and apoptotic assays. The results showed that all tested gold(III) complexes displayed cytotoxic effect on A549 cells. Among the tested Au(III) complexes, [Au(bipy)Cl2]+ showed the best cytotoxic effects. - The influence of the aurothiomalate, [Au(bipy)Cl2]+ and [AuCl2(DMSO)2]+ on spontaneous contractions of isolated women Fallopian tube ampulla and isthmus were investigated. The results showed that just the increase of [AuCl2(DMSO)2]+ concentration increases the spontaneous activity of the isolated Fallopian tube isthmus without effect on the isolated ampulla.