Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Cancer is one of the leading causes of death in developed countries and rising fast as a cause of death in developing countries. The increase of cancer prevalence in developing countries can be attributed to westernisation trends, with lifestyle cancers such as colorectal and lung cancer being amongst the most commonly reported malignant neoplasms. This means that the development of novel methods of treatment is essential in combatting this disease in the developing world. Combinational chemotherapy is one of the best candidates for treatment, but it is reliant on effective compounds targeting different modes of action. It also means that these compounds should be easily and cheaply available. Makaluvamines have been identified as a class of compounds that may have a novel mode of action on top of being known as topoisomerase II inhibitors. This study attempted to devise a short and concise synthetic strategy, based on reported procedures, to construct makaluvamine C analogues. This involved the introduction of a methyl group to an indole intermediate (7,8-dimethoxy-1,3,4,5-tetrahydropyrrolo[4,3,2-de]quinoline), before oxidation to a quarternized pyrroloiminoquinone (7-methoxy-5-methyl-8-oxo-1,3,4,8- tetrahydropyrrolo[4,3,2-de]quinolin-5-ium chloride). The introduction of this methyl group proved problematic, as the indole substrate proved to be difficult to handle and tended to degrade under reaction conditions. The lack of initial success prompted the deviation from the initial route by quarternizing a quinoline intermediate to form a quinolinium iodide salt (4- (dimethoxymethyl)-6,7-dimethoxy-1-methyl-5-nitroquinolin-1-ium iodide). Upon reduction to give 4-(dimethoxymethyl)-6,7-dimethoxy-1-methyl-1,2,3,4-tetrahydroquinolin-5-amine, it was discovered that the subsequent ring-closing reaction to produce 7,8-dimethoxy-5-methyl- 1,3,4,5-tetrahydropyrrolo[4,3,2-de]quinoline was still problematic. The synthesis of the target compounds has not yet been successfully completed, but will still be pursued so these compounds can be evaluated for their anticancer activity and have their mode of action tested. / AFRIKAANSE OPSOMMING: Kanker lewer van die grootste bydrae tot mortaliteit in ontwikkelde lande en is vining aan die toeneem in ontwikkelende lande. Die toename van kanker voorvalle in ontwikkelende lande kan toegedra word aan die verwesteringstendens, met kankers soos kolo-rektale- en long kanker onder die mees algemene kwaadaardige neoplasmsas wat aangemeld word – kankers wat gekoppel word aan leefstyl keuses. Dit beteken dat daar ‘n dringende nood is aan nuwe metodes van behandeling van die siekte in ontwikkelende lande. Kombinasie chemoterapie is een van die beste kandidate vir behandel, sienende dat dit gebruik maak van middels was verskillende aspekte van die siekte uitbuit. Om effektief te wees, moet die antikanker middels goedkoop en maklik beskikbaar te wees. Makaluvamines is geïdentifiseer as ‘n klas van antikanker middele wat moontlik ‘n nuwe metode van inhibisie het, tesame met hul topoïsomerase II inhibisie. Hierdie study het daarom gepoog om ‘n korter en meer bondige sintetiese roete saam te stel, wat gebaseer is op literatuur prosedures, om analoeë van makaluvamine C te produseer. Dit het die aanhegging van ‘n metiel group aan ‘n indool tussenproduk (7,8-dimetoksie-1,3,4,5- tetrahidropirolo[4,3,2-de]kinolien) behels, gevolg deur die oksidasie tot die kwaternêre piroloiminokwinoon (7-metoksie-5-metiel-8-oxo-1,3,4,8-tetrahidropirolo[4,3,2-de]kinolin-5-ium chloried). Om hierdie metiel groep aan te voeg was, nietemin, problematies, aangesien die indool produk moeilik was om te hanteer sienende dat dit onder reaksie toestande gedegradeer het. Die aanvanklike onsuksesvolle pogings het daartoe gelei dat die sintetiese roete herdink was en is aangepas om eerder gebruik te maak van ‘n kinolinium jodied sout (4-(dimetoksiemetiel)-6,7-dimetoksie-1-metiel-5-nitrokinolin-1-ium jodied). Die reduksie van hierdie sout en agtereenvolgende annulasie reaksie om 7,8-dimetoksie-5-metiel-1,3,4,5- tetrahidropirolo[4,3,2-de]kinolien te vorm was egter steeds problematies. Die sintese van die beoogde produkte was tot dusver nog nie suksesvol nie, maar sal egter steeds aangedurf word om hulle ten einde suksesvol te sintetiseer en dan te stuur om hulle biologiese eienskappe te toets. Dit sluit hulle antikanker aktiwiteit in asook hul metode van inhibisie.

Gold(I)phosphines, nucleoside analogues, and azole derivatives have been identified as promising anticancer compounds. The clinical use of these individual compounds is, however, limited due to non-selectivity associated with adverse effects and developed resistance. This study investigated seven novel gold compounds that contain either a nucleoside analogue or an azole, bound via a gold nitrogen bond, which have been designed and synthesized by Dr. Horvath under the supervision of Prof. Raubenheimer from the University of Stellenbosch. The novel compounds are divided into purinecontaining/ nucleoside analogue compounds (UH 86.2, UH 75.1, UH 58.1, UH 145.1) and azole-containing compounds (UH 107.1, UH 126.1, UH 127.1). The anticancer effects of these novel compounds were compared with that of previously described anticancer compounds [Au(dppe)2]Cl and cisplatin. The octanol/water partition coefficients (PC) of the compounds were measured in order to determine whether a correlation between the lipophilicity of the structures and the cytotoxic potency and selectivity exists. This might provide further insight for structural alterations of the compounds in order to improve their anticancer activity. The results from octanol/water PC determinations, revealed that the purine-containing compounds (UH 86.2, UH 75.1, UH 58.1, and UH 145.1), as well as the azole-containing compound, UH 127.1, exhibited hydrophilic properties, while the azole-containing compounds, UH 107.1 and UH 126.1 are lipophilic. In contrast to results by Berners-Price et al. (1999), that reported a direct proportionality between lipophilicity and cytotoxicity, for the current study, involving HeLa cells, CoLo cells, normal resting and PHA stimulated lymphocytes, no correlation was observed. For the Jurkat cell line, however, an increase in lipophilicity for the series of compounds studied was accompanied by an increase in cytotoxicity. The reason for the exception is not yet fully understood. The in vitro tumour specificity of each compound was established with cytotoxicity assays on various cancer cell lines and normal cell cultures. The cancer cell lines included human cervical cancer (HeLa) cells, human colon cancer (CoLo) cells, and human lymphocytic leukaemia (Jurkat) cells. The normal cell cultures included human resting lymphocytes and human phytohemaglutin (PHA) stimulated lymphocytes. From this data, the four most promising novel compounds were identified. Additional tests were performed by adding these four compounds to cancer cells including human breast cancer (MCF-7) cells, and cisplatin sensitive and resistant human ovarian cancer (A2780 and A2780cis) cells as well as normal chicken embryo fibroblasts. The tumour specificity of each compound was determined from the results obtained via the cytotoxicity assays. The compound is more selective the higher the tumour specificity. Cisplatin exhibited the highest tumour specificity, and [Au(dppe)2]Cl, the lowest. The two most promising novelcompounds were identified as UH 126.1 and UH 127.1, which was evidenced by their high tumour specificities. Further experiments were conducted with these two azolecontaining compounds by using Jurkat cells. The possible mechanism by which the novel compounds induce cytotoxicity was investigated with flow cytometric analysis. The effects of the compounds on the cell death pathway, the mitochondrial membrane potential and the cell cycle were determined. These results indicated that the novel compounds, UH 126.1 and UH 127.1 initiate the apoptotic cell death pathway rather than the necrotic cell death pathway. According to results, UH 126.1 and UH 127.1 influenced the status of the mitochondrial membrane potential (MMP) non-selectively and only at high concentrations. Although involvement of mitochondria in the mechanism of action cannot be excluded, results indicated that it is most likely not the primary target. After investigating the effects of the two novel azole-containing compounds on the cell cycle in Jurkat cells, it was detected that these compounds induce cell accumulation in the G1 phase of the cell cycle. It was concluded that UH 126.1 and UH 127.1 might interfere with the cell cycle indirectly, possibly by inhibition of cyclin-dependent kinases and/or other enzymes necessary for DNA replication. In an acute in vivo toxicity test during this study, results revealed drug induced adverse effects (such as significant weight loss, piloerection and diarrhoea), in the mice that received 3 and 6ìmol/kg of both UH 126.1 and UH 127.1. Evidence also revealed signs of nephrotoxicity and epatotoxicity. Due to minimal adverse effects observed in the groups that received UH 126.1 and UH 127.1 at the concentration of 1,5ìmol/kg, this is the suggested maximum tolerated dose (MTD) for these compounds. Further dose-range studies with UH 126.1 and UH 127.1 are, however, needed in order to evaluate clinicalefficacy. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Pharmacology / unrestricted

The molecular characteristics that affect the activity of various dirhodium complexes are reported. The importance of the axial position in the action of dirhodium compounds was studied. Three dirhodium complexes with increasing number of accessible axial coordination sites were synthesized and characterized. In cis-[Rh2(u-OAc)2(np)2]2+ (np = 1,8- naphthyridine) both axial sites are available for coordination, whereas for cis-[Rh2(u-OAc)2(np)(pynp)]+2 (pynp = 2-(2-pyridyl)1,8-naphthyridine) and cis-[Rh2(u-OAc)2(pynp)2]+2 the pyridyl arm on the ligand pynp blocks one and two axial sites, respectively. The availability of the axial positions affects the in vitro and in cellulo activity of these complexes demonstrating that open axial coordination sites are necessary for biological activity. The inhibitory activity of derivatives of dirhodium-dppz complexes (dppz = dipyrido[3,2-a:2',3'-c]phenazine) has also been investigated. The dppz derivatives included compounds with electron-withdrawing (Cl, CN, and NO2) as well as electro-donating (MeO and Me) substituents. These compounds inhibit transcription of T7-RNA polymerase by reducing accessible cysteine residues. The activity correlates with the electron withdrawing character of the substituent on the dppz ligand. Density functional theory (DFT) calculations reveal that the lowest unoccupied molecular orbitals (LUMOs) in the series are ligand-based pi* orbitals localized on the phenazine ring. These complexes represent the first family of dirhodium complexes whose inhibitory ability can be tuned by controlling their redox properties. The effect of the presence of diimine ligands in the dirhodium core in both in vitro and in cellulo activity is discussed. The presence of one diimine ligand allows for dual binding, intercalation and covalent, as observed by melting temperature and relative viscosity measurements, as well as electrophoretic mobility shift assay (EMSA). The mono-substituted dirhodium complexes are effective against HeLa and COLO-316 cell lines, with [Rh2(u-O2CCH3)2(n1-O2CCH3)(dppz)]+ being the most effective compound of the series. Results of the comet assay indicate that all of the monosubstituted complexes studied damage nuclear DNA, although in different degrees. The cytotoxic effect of these complexes is not affected by the presence of glutathione. The addition of the second diimine ligand hinders the ability of the complexes to damage DNA. The bis-substituted complexes are also slightly less cytotoxic than their mono-substituted congeners. Thus, the number of equatorial positions occupied by diimine ligands play a critical role in the mechanism of cytotoxicity of dirhodium(II,II) complexes. Finally, the results also demonstrate that improving the internalization of the dirhodium complexes can be achieved by co-incubation with cell penetrating peptides. This work provides a foundation for the preparation of new and more effective dirhodium complexes.

Cancer has become a major health problem due to its high rate of morbidity and mortality. Severe side effects associated with most of the available anticancer drugs and the development of the drug resistant cells are the major hurdles limiting their application and therapeutic success. Much attention has thus been focused on natural compounds with minimal or no toxicity in humans and having capacity to suppress proliferation and/or induce apoptosis in cancer cells. Therefore, it is imperative to discover and develop new anticancer drugs. Endophytic fungi though underexploited have remained a rich source of structurally novel and biologically active secondary metabolites. That they are a prolific resource for new compounds in drug discovery is evident from a plethora of reported research findings of the recent past. Those of endophytic fungi especially isolated from medicinal plants with known therapeutic value have especially caught the attention of several research groups worldwide for the production of host associated or novel lead molecules. In this backdrop, the focus of my research work has been centered on exploring the endophytic fungal population associated with Catharanthus roseus in search of production of novel anticancer compounds. Twenty endophytic fungi were isolated from different parts of Catharanthus roseus plant. The ethyl acetate (EA) extracts of three week grown liquid cultures of the individual endophytes were assessed in vitro for their cytotoxic activities in HeLa and HepG2 cancer cells using MTT assay. Of all the isolates thus screened, the EA extract from an isolate identified as Chaetomium nigricolor exhibited significant cytotoxicity in the tested cell lines with a better activity profile against HeLa cells. To understand if the liquid culture filtrate or the mycelium harbored the active cytotoxic principle, the C. nigricolor culture filtrate and mycelia was extracted separately using organic solvents with varying polarity namely ethyl acetate, dichloromethane, chloroform and hexane. These extracts were further tested for cytotoxicity induction in HeLa and MCF-7 cells by MTT assay. The ethyl acetate culture filtrate and mycelial extracts showed best cytotoxic activity on HeLa cells compared to other extracts; therefore, the mycelial and culture filtrate ethyl acetate extracts of C. nigricolor were pooled and was used for further work. Hexane culture filtrate extract of C. nigricolor showed best antiproliferative activity against MCF-7 cells. Thus, the compounds with anticancer potential from both ethyl acetate pooled extracts and hexane culture filtrate extract of C. nigricolor were purified using bioassay guided isolation method. This finally resulted in the isolation of two different anticancer compounds, one each from EA pooled extract and Hexane culture filtrate extracts of C. nigricolor named as compound 1 and compound 2, respectively. Based on various analyses including mass spectrometry (MS and MS-MS) and UV-visible, Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy, the compounds 1 and 2 were identified as 1, 2 Bis (diethylamino) ethene-1, 2 diol (BDED) and Nonacos-4-enoic acid (NA), respectively An improved understanding of the cellular responses to chemotherapeutic agents in cancer cells and the underlying molecular mechanisms gains particular relevance in the efforts to improve the clinical outcome of chemotherapeutic agents. Arresting the growth of cancer cells in one way or the other and induction of apoptosis in a drug specific manner is the hallmark of almost all the clinically used anticancer drugs. Towards this end, evaluation of the cytotoxic effects of the newly identified compounds on various human cancer cells was carried out. BDED was examined for in vitro cytotoxicity of against a panel of five human cancer cell lines namely HeLa, A-431, A549, COLO 205, and MCF-7. Among the cell lines screened, HeLa cells were most vulnerable to BDED treatment with an IC50 value of 27 μM. This cytotoxicity was later identified as resulting from apoptosis induction by BDED which was scored by the characteristic events of membrane externalization of phosphatidylserine, cytoplasmic shrinkage and chromatin condensation which were observed in a large majority of the BDED-treated cells. Further detailed studies were performed to delineate the cellular mechanisms of BDED-induced apoptosis in HeLa cells. Analysis of cell cycle progression by propidium iodide (PI) staining revealed BDED-induced cell cycle arrest in the G1 phase of cell cycle. The results indicated that BDED induced a significant ROS generation and a significant loss of mitochondrial membrane potential which were abrogated upon pretreatment of the cells with an antioxidant, N-acetyl cysteine (NAC). These observations suggested the probable involvement of the initial events of BDED-induced ROS production in causing cell death via mitochondria-based intrinsic pathway of apoptosis. In addition, the results from the expression profile of pro- and anti- apoptotic proteins by western blot analysis in the BDED-treated HeLa cells further corroborated this hypothesis. These included an abnormally deregulated cellular abundance of BAX 1 and BCL 2 proteins, elevated levels of APAF-1, and activated cleaved species of procaspases 9 and 3. In addition, a pronounced abundance of cleaved PARP protein was observed in these experiments. Thus, the results suggest that BDED induced apoptosis in HeLa cells via ROS mediated mitochondrial dependent pathway. In parallel, the in vitro cytotoxic effects of NA was also studied, the second antiproliferative compound identified from hexane extracts of C. nigricolor using a panel of four human cancer cell lines - A-431, A549, COLO 205, and MCF 7. NA was thus found to be most potent against MCF 7 (breast cancer) cells. PI staining-based viability assays and microscopic observations showed a dose dependent cytotoxicity of NA on MCF 7 cells. Our data reveled an IC 50 of 40 μM for NA The NA-induced apoptosis was confirmed by flow cytometric detection of membrane externalization of phosphatidylserine using Annexin V FITC/PI dual staining. NA was also observed to induce cytotoxicity best at 72 h this could be attributed to the fact that it is a derivative of a Nonocosane-(a naturally prevalent molecule found in several vegetables). Similar to our results from BDED-treated HeLa cells, ROS generation and loss of mitochondrial membrane potential which were abrogated on pretreatment with NAC, were also observed in NA-treated MCF-7cells. Similar to several other fatty acids, NA was also observed to induce cell cycle arrest in the G1 phase of cell cycle. Further, apoptotic signature of an altered expression of anti-apoptotic BCL-2 and pro-apoptotic BAX, APAF-1 and procaspase 9 and 3 and a cleaved PARP were observed in NA-treated MCF 7 cells. In conclusion, the results indicate that BDED and NA have cytotoxic and apoptotic effects on HeLa (cervical cancer) and MCF 7 (breast cancer) cell lines, respectively. This leaves open further avenues to evaluate their potential application as anticancer agents for treatment of human cervical and breast cancers.

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