Development and evaluation of novel thiosemicarbazone based ligands with anti-neoplastic activity

Abstract

Introduction Cellular iron levels are tightly regulated due to the potential toxic effects of this highly active metal ion. Numerous studies have revealed that tumours possess altered iron homeostasis, which is mediated by dysregulated expression of iron-related proteins (e.g., transferrin receptor 1, ferritin and ferroportin). In addition, alterations in the processing of other biologically important transition metal ions, including copper(II) and zinc(II), have also been observed in malignancies. This dysregulation of metal homeostasis in cancer cells reveals a particular vulnerability to metal-depletion, which can be utilised therapeutically by way of metal chelation. Metal chelators, particularly iron chelators, have traditionally been used for the treatment of iron overload disease (e.g., thalassaemia). However, recently they have been shown to be highly effective at inhibiting tumour growth. One notable example includes the thiosemicarbazone class, particularly di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and Triapine®. These compounds were believed to target ribonucleotide reductase, a key enzyme involved in the rate-limiting step of DNA synthesis, as their mechanism of action. It is now known that they exhibit multiple effects, including the generation of reactive oxygen species, such as hydroxyl radicals, which lead to cellular injury. This thesis aims to extend our knowledge of the factors important for the anti-proliferative activity of this broad class of chelators. Through detailed structure-activity studies, the role of lipophilicity on metal ion mobilisation, the structural features important for redox activity, and hence, reactive oxygen species generation, as well as the intracellular mechanisms of their observed anti-proliferative activity are investigated. Chapter 3 Iron chelators of the 2′-benzoylpyridine thiosemicarbazone (BpT) class show great potential as anti-cancer agents. To explore structure-activity relationships, new BpT analogues were designed that incorporated halogen substituents on the non-coordinating phenyl group (XBpTs). Halogenation was expected to both enhance the lipophilicity of the molecule, but also to subtly alter the electrochemistry of the chelator-iron complex, consequently improving penetration through cell membranes and potentially enhancing redox activity within the tumour cell. The results indicated that these XBpT ligands possessed potent anti-proliferative activity, with some analogues exceeding that of the parent BpT compound. Importantly, we observed an appreciable therapeutic index in vitro, as mortal cells were significantly less affected by these chelators relative to neoplastic cells. The addition of a halogen led to a halogen-specific increase in the redox potential of XBpT-Fe complexes. Probing for chelator-induced intracellular reactive oxygen species (ROS) with the fluorescent probe, 2’,7’-dichlorofluorescein, revealed a 1.5-4.7-fold increase in fluorescence upon incorporation of Cl, Br or I to the parent analogues. Further, an important structure-activity relationship was deduced where addition of halogens led to a positive correlation between intracellular ROS generation and anti-proliferative activity in the more hydrophilic BpT parent compounds. Chapter 4 Thiosemicarbazone chelators show marked potential as anti-cancer agents. Triapine®, for instance, has been investigated in >20 phase I and II clinical trials. However, side-effects associated with Triapine® administration including methaemoglobinemia, resulting from the oxidation of oxyhaemoglobin to methaemoglobin, limited its clinical utility. Considering this problem, novel analogues based on the 2′-benzoylpyridine thiosemicarbazone (BpT) class of iron chelators, were designed bearing hydrophobic, electron-donating substituents at the para position of the phenyl group (RBpT). Their Fe(III/II) redox potentials were all within the range accessible to cellular oxidants and reductants, suggesting they can redox cycle. These RBpT ligands exhibited potent and selective anti-proliferative activity, which was comparable or exceeded their BpT counterparts. Crucially, major findings include that methaemoglobin formation mediated by the lipophilic t-BuBpT series was significantly (p<0.05-0.001) decreased in comparison to Triapine® in intact red blood cells and were generally comparable to the control. These data indicate the t-BuBpT ligands may minimise methaemoglobinemia, which is a marked advantage over Triapine® and other potent thiosemicarbazones. Chapter 5 Bis(thiosemicarbazones) and their copper (Cu) complexes possess unique anti-neoplastic properties. However, their mechanism of action remains unclear. We examined the structure-activity relationships of twelve bis(thiosemicarbazones) to elucidate factors regarding their anti-cancer efficacy. Importantly, the alkyl substitutions at the diimine position of the ligand backbone resulted in two distinct groups, namely, unsubstituted/monosubstituted and disubstituted bis(thiosemicarbazones). This alkyl substitution pattern governed their: (1) Cu(II/I) redox potentials; (2) ability to induce cellular 64Cu release; (3) lipophilicity; and (4) anti-proliferative activity. The potent anti-cancer Cu complex of the unsubstituted bis(thiosemicarbazone) analog, glyoxal bis(4-methyl-3-thiosemicarbazone) (GTSM), generated ROS, which was attenuated by Cu sequestration by a non-toxic Cu chelator, tetrathiomolybdate, and the anti-oxidant, N-acetyl-L-cysteine. Fluorescence microscopy suggested that the anti-cancer activity of Cu(GTSM) was due, in part, to lysosomal membrane permeabilisation (LMP). For the first time, this investigation highlights the role of ROS and LMP in the anti-cancer activity of bis(thiosemicarbazones). In conclusion, the detailed structure-activity studies described in this thesis demonstrate several crucial structural features involved in the targeting of thiosemicarbazone-based ligands for cancer therapy. These findings highlight the importance of lipophilicity and steric and inductive effects on redox cycling, methaemoglobin formation, and anti-proliferative activity of thiosemicarbazones, as well as the role of lysosomes in the anti-proliferative activity of bis(thiosemicarbazones).