The Kingston group's engagement in an International Cooperative Biodiversity Group (ICBG) program and a collaborative research project established between Virginia Tech and the Institute for Hepatitis and Virus Research (IHVR) has focused on the search for bioactive natural products from tropical forests in both Madagascar and South Africa. As a part of this research, a total of four antiproliferative extracts were studied, leading to the isolation of fourteen novel compounds with antiproliferative activity against the A2780 human ovarian cancer line. One extract with antimalarial activity was studied, which led to the isolation of two new natural products with antiplasmodial activity against a drug-resistant Dd2 strain of Plasmodium falciparum. The plants and their secondary metabolites are discussed in the following order: two new antiproliferative acetogenins from a Uvaria sp. (Annonaceae); two new antiproliferative calamenene-type sesquiterpenoids from Sterculia tavia (Malvaceae); two new antiproliferative triterpene saponins from Nematostylis anthophylla (Rubiaceae); six new antiproliferative homoisoflavonoids and two new bufatrienolides from Urginea depressa (Asparagaceae); and two new antiplasmodial anthraquinones from Kniphofia ensifolia (Asphodelaceae). The structures of all these compounds were determined by analysis of their mass spectrometric, 1D and 2D NMR, UV and IR spectroscopic and optical rotation data. Other than structural elucidation, this work also involved bioactivity evaluations of all the isolates, as well as total synthesis of the two antiproliferative sesquiterpenoids, and a structure-activity relationship (SAR) studies on the antiplasmodial anthroquinones. / Ph. D.

Cis-diamminedichloroplatinum (II), or cisplatin, is a widely prescribed anticancer compound, currently one of only three platinum (II) complexes FDA approved for cancer treatment. Despite its widespread use, we lack a comprehensive picture of global drug targets, which would lend valuable insights into the molecular mechanisms of action and resistance in different tissues. Drug binding to genomic DNA is an accepted cause of downstream apoptotic signaling, but less than 10% of Pt (in the case of cisplatin) accumulates within genomic DNA. Non-genomic contributions to cisplatin's therapeutic action are also under active investigation. In particular, cisplatin treatment can disrupt RNA-based processes such as splicing and translation. Pt(II) targeting of non-DNA species such as RNA may contribute to or sensitize a cell to the downstream effects of this drug, including the induction of apoptosis. Chapter I summarizes the activity profile of Pt(II) therapeutics, describing cellular uptake, cellular localization, incidences of Pt(II) accumulation within RNA, and RNA processes affected following drug treatment. Chapter II reports our thorough investigation of the distribution of Pt species throughout messenger and ribosomal RNA, with the discovery that Saccharomyces cerevisiae ribosomes act as a de facto cellular Pt sponge. In Chapter III, we report the synthesis of an azide-functionalized platinum (II) species, picazoplatin, for post-treatment click labeling and isolation of drug targets in vivo. Picazoplatin was designed to circumvent mislocalization and misprocessing of Pt typically encountered when trying to track small molecules tethered to large, charged fluorophores. This chapter contains several proof-of-principle studies validating the use of this class of reagents for future purification and sequencing of Pt-bound nucleic acids. Chapter IV describes the first application of the click-capable Pt reagent technology: the demonstration of significant in-gel fluorescent detection of Pt-bound ribosomal RNA and transfer RNA extracted from picazoplatin-treated S. cerevisiae and the first evidence that cellular tRNA is a platinum substrate. Chapter V summarizes these data, which suggest a potential ribotoxic mechanism for cisplatin cytotoxicity and broadly describe a convenient click chemistry methodology that can be applied to identify other metal or covalent modification-based drug targets. This dissertation includes previously published and unpublished co-authored material.

First isolated from the myxobacterium Sorangium cellulosm So Ce12 in 1994, the chivosazoles have been reported to possess antiproliferative activity against human cancer cell lines, as well as antifungal activity. This thesis focuses on studies towards the total synthesis of chivosazole F. Some developments towards the total synthesis of chivosazole A are also discussed. Chapter 1 discusses the isolation, characterisation and biological activity of the chivosazoles, as well as the first total synthesis of chivosazole F reported by the Kalesse group and the previous work towards synthesising chivosazole F in our group. Chapter 2 describes the synthesis of the three key fragments A, B and C, their coupling reactions and subsequent modifications for assembling the backbone of chivosazole F. Paterson boron aldol methodology and Evans-Tishchenko reduction were utilised to construct the 1,4-syn and 1,3-anti stereochemical relationships within both fragment A and fragment B. Di-tert-butyl silyl group was used for the efficient and precise protection of the terminal diol of B. The key stereochemistry of fragment C was defined with a vinylogous Mukaiyama aldol reaction. Site-selective Stille cross-coupling reactions of the three fragments, via a one-pot process, rapidly installed the requisite stereodefined polyene motifs within chivosazole F. Optimised Still-Gennari-type HWE olefination conditions were applied to install the (2Z,4E)-dienoate in D. MnO2-mediated double oxidation of D turned the terminal alcohol into an aldehyde and the oxazoline into an oxazole, followed by a Stork-Zhao olefination transforming the aldehyde to a Z-vinyl iodide for a macro-Stille coupling reaction, which achieved the ring closure to afford macrocycle E. Chapter 3 discusses the developments towards the synthesis of the southern fragment F of chivosazole A. Sugar I was prepared first and conditions were screened for the glycosylation of H and I to afford G. Chapter 4 outlines the achievements of this research and points out some future issues that need to be tackled.

Actualmente, el cáncer es una causa importante de mortalidad en el mundo. El paclitaxel es un fármaco que actúa de forma eficiente frente a un gran número de tipos de cáncer y se obtiene, principalmente, a partir de especies de tejo (Taxus spp.), un árbol de la familia de las taxáceas. / Currently, cancer is a major cause of mortality worldwide. Paclitaxel is a drug that acts efficiently against a great number of cancer types and is mainly obtained from species of yew (Taxus spp.), a tree of the taxaceas family.

Despite the scientific progress in the last decades in terms of therapeutic agents to fight cancer there is still the need of developing safer and more effective drugs. Developing an innovative drug is not only very expensive, but also highly time consuming; furthermore, the number of anticancer agents that fail in clinical trials with high attrition rates mainly caused by unexpected toxicity and lack of efficacy far outweighs those considered effective, which indicates that drug screening processes require further improvements. In this project the application of Fourier transform infrared microspectroscopy is evaluated in order to develop a spectral based model that could be used to describe the drug-cell interaction and also to discriminate between the metabolic modifications due to a particular drug and the inherent cell cycle of a cell. A computational method was built using the FTIR spectra from a highly resistant renal cell carcinoma cell line, Caki-2, in order to discriminate between the phases of the continuous cell cycle that cells undergo while proliferating in vitro. Such model enabled the discrimination of early events of the cell cycle (G0/G1 phase cells) from G2/M phase cells with a prediction accuracy of 90% and 92.9% respectively. On the other hand, when the RMieS-corrected FTIR spectra corresponding to G0/G1, S and G2/M phases were modelled, the algorithm was able to retrieve each stage of proliferation with 82.3%, 71.8% and 84.4% accuracy respectively. Although the average accuracy yielded by the method was relatively low compared with what has previously been reported in the literature, these results emphasize the need to correct the data from physical distortions due to size and prove the principle that it is possible to create a method for identifying different events of the cell cycle based on the data that the FTIR spectroscopy provides, as well as using the scattering profile characteristic of each phase of development. Once the underlying biochemistry of proliferating Caki-2 cells were characterised by FTIR, the cells were treated with 5-Fluorouracil and paclitaxel, two widely used cytotoxic agents known to induce cellular damage at S or G2/M phase of the cell cycle respectively. The FTIR spectra collected were analysed via multivariate and bivariate techniques. Results demonstrated that, after 24 hours of treatment at the IC50 concentration of each drug, Caki-2 cells displayed spectral features consistent with early stages of apoptosis. These spectral characteristics did not appear to be linked either to the drug’s mode of action or the cell’s cycle phase. The cell’s proliferation stage was not the main classification trend among the drug-treated spectra; nevertheless, the cell cycle phase of each drug-treated population was successfully retrieved by an optimized model capable to classify such phases with an average accuracy of 77.98%. Altogether, this study offers a new perspective when analysing FTIR data from single cells as a function of the cell cycle and also when investigating the biochemical response of a cell line to a given anticancer agent.

Derivatives of the quinoline moiety have been shown to exert a range of biological activities, including anti-neoplastic activity. The clinical application of quinoline derivatives in the treatment of malignancies has been limited due to non-selectivity. Four novel hydroxyquinoline derivatives were synthesised as potential anticancer agents. Each of these compounds contains the characteristic quinoline nucleus: a heterocyclic moiety containing a nitrogen atom. A hydroxyl group is present on C-8 and an azo bond links the heterocyclic quinoline core to a monocyclic benzene ring with various substituents. The aim of this study was to investigate the potential anticancer properties of the four hydroxyquinolines as pertains to their in vitro cytotoxicity, ability to circumvent multidrug resistance and possible mechanism of action. The acute in vivo toxicity profile of the two most promising compounds was also investigated. The tumour specificity of a compound is an indicator of the selective cytotoxicity of a compound towards cancer cells, while maintaining minimal toxicity towards normal cells. To this end the four hydroxyquinolines were screened on a range of commercially available cancer cell lines and primary (normal) cultures. The cancer cell lines used included human cervical adenocarcinoma (HeLa), human estrogen receptor positive breast cancer (MCF-7) and several resistant cancer cell lines. Chicken embryo fibroblasts and human lymphocytes were included as primary cell cultures. From the results the cancer cell lines most sensitive to each compound were identified: breast (MCF-7) and leukaemia (Jurkat) cells were most sensitive to HQ5, a resistant colon cancer cell line (COLO 320DM) was most susceptible to HQ6 and HQ7, and HQ10 was most effective against cervical cancer (HeLa). Data indicated that HQ5 and HQ10 displayed the highest tumour specificities and these two promising hydroxyquinoline derivatives were selected for further investigation. As quinoline derivatives have been reported to modulate multidrug resistance through the inhibition of the P-glycoprotein (P-gp) efflux pump, the effect of HQ5 and HQ10 on P-gp was evaluated in two experimental models. Firstly the experimental hydroxyquinolines were used in combination with the P-gp substrates doxorubicin, vinblastine and paclitaxel on three P-gp expressing cell lines to ascertain whether a synergistic combination could be observed. Secondly the direct effect of HQ5 and HQ10 on the function of P-gp was determined through the rhodamine 123 retention assay. According to the results obtained from the combination therapy, the combinations of the experimental compounds and the known chemotherapeutic agents tested were at most additive. Data obtained from the rhodamine 123 accumulation assay revealed that HQ5 and HQ10 did not inhibit P-gp in the three P-gp expressing cell lines tested but appeared to enhance P-gp activity. The mechanism of action of the two selected hydroxyquinolines was further investigated through flow cytometric analysis. The effect of HQ5 and HQ10 on the induction of apoptosis or necrosis in MCF-7 cells was determined. Results indicated that the two experimental compounds induced apoptosis in a dose and time dependent manner. After investigating the effect of the hydroxyquinolines on the cell cycle progression of MCF-7 cells, it was observed that HQ5 and HQ10 arrested the cell cycle at the G1 checkpoint. Results suggest that at higher concentrations of HQ5 inhibition resembled a G2 inhibitor. In an acute in vivo cytotoxicity study the tolerability and safety profile of HQ5 and HQ10 were investigated. After daily intraperitoneal administration of either of the two compounds at two concentrations, no obvious histological signs of toxicity were reported. However a dose of 2 mg/kg per day of HQ10 caused a significant reduction in body weight. Haematological analysis revealed that administration of 0.1 mg/kg of HQ5 resulted in a significant decrease in white cell count. No other haematological parameters studied showed any difference between the animals in the control and experimental groups. It was thus concluded that daily dosing of HQ5 and HQ10 was well-tolerated and caused no severe toxicity. Chronic in vivo toxicity profiles were not determined in this study. The in vitro studies suggested that HQ5 and HQ10 displayed promising anticancer properties. However, further investigation revealed several unfavourable characteristics, with regards to the solubility, purity and stability of these experimental hydroxyquinolines. In addition in vivo studies added further doubt on the success of these compounds in a clinical setting and it was concluded that these compounds were unsuitable for further development. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Pharmacology / unrestricted

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Yes / We report the encapsulation of highly hydrophobic 16-electron organometallic ruthenium and osmium carborane complexes [Ru/Os(p-cymene)(1,2-dicarba-closo-dodecarborane-1,2-dithiolate)] (1 and 2) in Pluronic® triblock copolymer P123 core–shell micelles. The spherical nanoparticles RuMs and OsMs, dispersed in water, were characterized by dynamic light scattering (DLS), cryogenic transmission electron microscopy (cryo-TEM), and synchrotron small-angle X-ray scattering (SAXS; diameter ca. 15 and 19 nm, respectively). Complexes 1 and 2 were highly active towards A2780 human ovarian cancer cells (IC50 0.17 and 2.50 μM, respectively) and the encapsulated complexes, as RuMs and OsMs nanoparticles, were less potent (IC50 6.69 μM and 117.5 μM, respectively), but more selective towards cancer cells compared to normal cells. / We thank the Leverhulme Trust (Early Career Fellowship no. ECF-2013-414 to NPEB), the University of Warwick (Grant no. RDF 2013-14 to NPEB), the Swiss National Science Foundation (Grant no. PA00P2_145308 to NPEB and PBNEP2_142949 to APB), the ERC (Grant no. 247450 to PJS), EPSRC (EP/G004897/ 1 to APB, and EP/F034210/1 to PJS), Institute of Advanced Study (IAS) – University of Warwick (Fellowship to JJSB), and Science City (AWM/ERDF) for support. We thank the Wellcome Trust (055663/Z/98/Z) for funding to the Electron Microscopy Facility, School of Life Sciences, University of Warwick.

yes / The archetypal prodrug of the imidazotetrazine class is the anticancer agent temozolomide (TMZ). The prodrug activation kinetics of TMZ show an unusual pH dependence: it is stable in acid and rapidly hydrolyses in alkali (Denny, B.J., et al. Biochemistry 1994, 33, 9045–9051). The incipient drug MTIC has the opposite properties—relatively stable in alkali but unstable in acid. In this study, the mechanism of prodrug activation was probed in greater detail to determine whether the reactions are specific or general acid or base catalysed. Three prodrugs and drugs were investigated, TMZ, MTIC and the novel dimeric imidazotetrazine EA27. Hydrolysis in a range of citrate-phosphate buffers (pH 8.0, 7.4, 4.0) was measured by UV spectrophotometry. Reaction of TMZ and MTIC obeyed single-phase, pseudo-first order kinetics (Figure 1). EA27 was more complex, showing biphasic but approximately pseudo-first order kinetics, Figure. General acid or base catalysis indicates that protonation or deprotonation is the rate-limiting step (rls). In biological milieu, the nature and concentration of other acidic or basic solutes may affect the prodrug activation reaction. In contrast, specific acid or base catalysis indicates that protonation or deprotonation occurs before the rls, so catalysis depends only on the local concentration of hydroxide or hydronium ion (i.e., pH) so the reaction kinetics are not expected to change appreciably in a biological medium.