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.

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Background: Cassia angustifolia Vahl. (commonly known as senna makkai or cassia senna), native to Saudi Arabia, Egypt, Yemen and also extensively cultivated in Pakistan, is a medicinal herb used traditionally to cure number of diseases like liver diseases, constipation, typhoid, cholera etc. This study was conducted to evaluate the in-vitro antimicrobial, antioxidant and anticancer assays and phytochemical constituents of aqueous and organic extracts of C. angustifolia leaves. Methods: The antimicrobial activities of C. angustifolia aqueous and organic (methanol, ethanol, acetone, ethyl acetate) extracts were investigated by the disk diffusion method. These extracts were further evaluated for antioxidant potential by the DPPH radical scavenging assay. Anticancer activities of the extracts were determined by the MTT colorimetric assay. The total phenolic and flavonoid contents of C. angustifolia extracts were evaluated by the Folin-Ciocalteu method and aluminum chloride colorimetric assay, respectively. The structures of the bioactive compounds were elucidated by NMR and ESI-MS spectrometry. Results: Bioactivity-guided screening of C. angustifolia extracts, led to the isolation and identification of three flavonoids quercimeritrin (1), scutellarein (2), and rutin (3) reported for the first time from this plant, showed significant anticancer activity against MCF-7 (IC50, 4.0 mu g/mu L), HeLa (IC50, 5.45 mu g/mu L), Hep2 (IC50, 7.28 mu g/mu L) and low cytotoxicity against HCEC (IC50, 21.09 mu g/mu L). Significant antioxidant activity was observed with IC50 2.41 mu g/mL against DPPH radical. Moreover, C. angustifolia extracts have the potential to inhibit microbial growth of E. cloacae, P. aeruginosa, S. mercescens and S. typhi. Conclusion: C. angustifolia extracts revealed the presence of quercimeritrin (1), scutellarein (2), and rutin (3), all known to have useful bioactivities including antimicrobial, antioxidant and anticancer activities.

An introduction, background and recent advances in the areas of microtubule-binding agents and heat shock protein 90 (HSP90) inhibitors as anticancer agents are briefly reviewed. The work in this dissertation is centered on the synthesis of substituted pyrrolo[2,3-<italic>d</italic>]pyrimidines as potential anticancer agents that act via microtubule inhibition or HSP90 inhibition.<br>Microtubule-binding agents are effective against a broad range of tumors and lymphomas and have been common components of combination cancer-chemotherapy in the clinic. Despite the unparalleled success, drawbacks among microtubule-binding agents such as multi-drug resistance, dose-limiting toxicity, poor pharmacokinetic profile and high cost have supported the sustaining momentum in searching for novel agents of this class.<br>The research on microtubule-binding agents in this dissertation was initiated by an unexpected discovery. The lead compound, a 4-<italic>N</italic>-methyl-4'-methoxyaniline-substituted pyrrolo[2,3-<italic>d</italic>]pyrimidine, was found to inhibit the majority cancer cell lines in the NCI-60 panel at sub-micromolar concentration. The COMPARE analysis based on the activity profile indicated microtubule inhibition as the main mechanism of action of this compound, and was later confirmed through multiple assays. Further, the lead compound displaced 70% of [<super>3</super>H]colchicine from tubulin at a concentration of 5 μM, and was identified as a colchicine-site binder. The compound has also shown unabated or even increased activities against several drug-resistant cancer cell lines, especially the cell lines overexpressing P-glycoprotein or βIII-tubulin. In addition, the compound has favorable physicochemical properties such as high water solubility as its hydrochloride salt.<br>Based on the preliminary data and molecular modeling, a hypothesis on the relationship between binding affinity and the lowest-energy conformation of pyrrolo[2,3-<italic>d</italic>]pyrimidines was proposed. To test the hypothesis and search for compounds with improved potency, 38 pyrrolo[2,3-<italic>d</italic>]pyrimidine analogs in six series were designed and synthesized. The biological evaluations of these compounds are currently in progress at the time this dissertation is submitted.<br>HSP90 is one the molecular chaperones that assist the proper folding of the newly synthesized polypeptides and proteins. The majority of its client proteins are signal transducers with unstable conformations, which play critical roles in growth control, cell survival and development. The expressions of these proteins in normal cells were much less than cancer cell, making HSP90 a viable target for cancer chemotherapy. As of 2012, there are 16 HSP90 inhibitors in clinical trial, among which four are based on the purine-scaffold. All the compounds in clinical trials bind to or overlap with the ATP site on the N-terminal of HSP90.<br>The pyrrolo[2,3-<italic>d</italic>]pyrimidine scaffold is structurally close to purines. In the design of receptor tyrosine kinase (RTK) inhibitors, Gangjee et al. have shown that properly functionalized pyrrolo[2,3-<italic>d</italic>]pyrimidines bind to the ATP site and achieve high degrees of selectivity. This was partly attributed to the incorporation of substitution patterns that are impossible on the purine scaffold. Based on these previous findings and the established SAR of the two purine derivatives in clinical trials (<bold>PU-H71</bold> and <bold>BIIB021</bold>), 18 substituted pyrrolo[2,3-<italic>d</italic>]pyrimidines in three series (in connection with this dissertation) were designed and synthesized. The biological evaluations of these compounds are currently in progress. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Medicinal Chemistry / PhD; / Dissertation;

Phytochemical composition, antioxidant activity, pigment stability, bioactive properties, and in-vitro absorption of polyphenolics in acai fruit (Euterpe oleracea Mart.) were investigated. Detailed characterization of phenolic compounds present in acai fruit, acai fruit pulp, and a polyphenolic-enriched acai oil were conducted by HPLCESI- MSn analyses and their stability and influence on antioxidant capacity determined. Anthocyanins were predominant in acai fruits, which also contained several flavone and flavonol glycosides, flavanol derivatives, and phenolic acids. In-vitro absorption and antiproliferative effects of phytochemical extracts from acai pulp and acai oil were determined as a function of chemical composition. Polyphenolic mixtures from both acai pulp and acai oil extracts significantly inhibited HT-29 colon cancer cell proliferation, also inducing the generation of reactive oxygen species. In-vitro intestinal absorption using Caco-2 cell models demonstrated that phenolic acids and monomeric flavanol derivatives are readily transported through cell monolayers in-vitro. The influence of polyphenolic cofactors on the stability of anthocyanins in acai fruit under varying conditions of temperature and pH was evaluated. Significant time, temperature, and pH-dependent anthocyanin losses were observed in all models, yet the presence of phenolic acids, procyanidins, and flavone-C-glycosides had a positive influence on anthocyanin stability. External addition of flavone-C-glycosides significantly enhanced visual color, increased anthocyanin stability during exposures to high pH or storage temperatures, and had comparable effects to those of a commercial anthocyanin enhancer. Anthocyanin polymerization reactions occurring during storage of acai fruit juice models were investigated and potential mechanisms and reaction products identified. Polymeric anthocyanin fractions contained several anthocyanin-flavanol adducts based on cyanidin or pelargonidin aglycones and their presence was related to increased anthocyanin sulfite bleaching resistance and to the appearance of large, unresolved peaks in HPLC chromatograms. A reaction mechanism involving the nucleophilic addition of anthocyanins in their hydrated form to flavanol carbocations resulting from cleavage of interflavanic bonds was proposed for the formation of flavanol-anthocyanin adducts in acai fruit juices. Antiproliferative activity and in-vitro absorption of monomeric and polymeric anthocyanin fractions were also evaluated. Both fractions inhibited HT-29 colon cancer cell growth in a similar, concentration-dependent manner, yet in-vitro absorption trials using Caco-2 intestinal cell monolayers indicated the presence of anthocyanin polymers may influence anthocyanin absorption in acai fruit products.

The mode of Ti and Sn action of anticancer compounds is still poorly understood. For each octahedral Ti(bzac)2 X2 and Sn(bzac)2 X2 (Hbzac = 1-benzoylacetone) complexes there are five possible isomers. It is not known which of these isomers is responsible for the anticancer activity. One of the Ti complex Ti(bzac)2(OEt)2 whose trade name is budotitane® is a anticancer drug on clinically trial base. Therefore we use VT-NMR (low temperature to high temperature) studies and their crystal structures (1a, 1b, and 2a) to examine their isomerization behavior, conversion rate constant k, and conversion barrier energy £GG‡. We suspect the antitumor activity of Ti and Sn compounds strongly depends on the unsubstituted phenyl rings of the £]-diketonato ligands in the outer sphere of the molecule. If these phenyl rings are replaced by methyl groups, the activity totally disappears. Therefore we propose that the anticancer activity of budotitane may be determined by a DNA intercalating mechanism. We further changed the ligand from Hbzac to FHnpac (FHnpac = 4, 4, 4-trifluoro-1-(naphthalen-3-yl) butane-1, 3-dione) in order to monitor their isomers exchange via 1H NMR. The related crystal structures (1a-NF, 1b-NF, and 2a-NF) were obtained fortunately. The stereochemistry of Ti and Sn complexes as well as their controlling factors is discussed.

DNA is both the oldest and newest of targets for cancer therapy. While it is already being targeted by many anticancer drugs in the clinic, the development of sequence-specific DNA binders has brought it back to the limelight as a valuable anticancer drug target.My studies on DNA interacting agents was initiated with the DNA intercalator campotothecin, and also included topoisomerase I enzyme. I have evaluated the structure of topoisomerase I C-terminal domain that consists of the active site tyrosine. My data indicate that this domain exists in a molten globule conformation with a fluctuating tertiary structure. These fluctuations are suggested to be important in interaction with the topoisomerase I core domain and DNA. I have also evaluated the DNA interactions of the camptothecin analogue homocamptothecin and have determined that homocamptothecin intercalate with DNA in the absence of topoisomerase I, and that such intercalation results in its lactone stabilization. Subsequently, the mechanism of topoisomerase I mediated inhibition of HIF-1 by camptothecin was explored. I have shown that camptothecin stimulate topoisomerase I cleavage complex formation in the HIF-1 binding site, which is suggested to prevent the DNA binding of HIF-1.The second part of this study was focused on understanding the mechanism of action of another DNA binder, XR5944. Designed as a dual topoisomerase inhibitor, XR5944 was subsequently shown to have a different mechanism of action - inhibition of trancription. The NMR structural analysis, in our lab, of the drug-DNA complex showed that XR5944 bis-intercalate with DNA, while binding in the DNA major groove. Driven by these combined interaction modes, XR5944 is shown to inhibit the DNA binding and the subsequent transcriptional activity of specific transcription factors such as estrogen receptors and AP-1, which are overexpressed in certain cancers.Finally, I have analyzed G-quadruplex structures formed by telomeric DNA. The formation and stabilization of DNA G-quadruplexes in the human telomeric sequence have been shown to inhibit the activity of telomerase. Thus the telomeric DNA G-quadruplex has been considered as an attractive anticancer drug target. Telomeric DNA forms multiple G-quadruplex conformations, and my data reveal the conformations of the major G-quadruplexes formed by human telomeres.

The anticancer properties of dirhodium tetraacetate were discovered in the 1970's, and subsequently motivated the research of several dirhodium paddlewheel derivatives. The promising results of this research led the Dunbar group to investigate the biological properties of dirhodium partial paddlewheel compounds. Previous work in our group has focused on dirhodium carboxylate derivatives with a series of diimine ligands, namely 1,10-phenanthroline (phen), dipyrido[3,2-f:2',3'-h]quinoxaline (dpq), dipyrido[3,2a:2',3'c] phenazine (dppz), and benzo[i]dipyrido[3,2-a:2',3'-c]phenazine) (dppn). Current research has expanded this diimine series by substituting the carobxylate bridging group with p-methoxyphenylphosphine (PMP). This new series of compounds was characterized by several techniques, including: X-Ray crystallography, 1H NMR spectroscopy, and electronic absorption spectroscopy. The cytotoxicity of these compounds towards HeLa cells was investigated in presence and absence of light in an effort to investigate the ability to use these compounds as photodynamic therapy (PDT) agents. Cytotoxicity measurements were carried out using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. It was found that in the dark [Rh2(PMP)2(dppz)2][BF4]2 (the dppz derivative of the dirhodium PMP compound) had no cytotoxicity towards HeLa cells, but experienced a 7 fold increase in cytotoxicity upon irradiation (with lambdai_rr equal to 350 nm). This dramatic increase in cytotoxicity upon irradiation makes this compound a potential PDT agent. Diiridium (II,II) compounds were prepared in a dual attempt to determine how the properties of the dirhodium core effect the biological activities of these compounds, as well as investigate the biological activity of a set of compounds that has yet to be explored. The compound [Ir2(DTolF)2(CH3CN)6][BF4]2 was chosen because it has a well understood dirhodium analogue, and it is a known compound. However, it was discovered that there was a potential silver contamination in the final product, stemming from the silver trifluoroacetate oxidant used during synthesis. Consequently, a new method of preparing this compound was required. The new synthetic pathway for the diiridium compound [Ir2(DTolF)2(CH3CN)6][BF4]2 was devised, and the cytotoxicity and photocytotoxicity studies were performed for the first time (to our knowledge) on a diiridium (II,II) compound. Despite the stability of the compound, it was determined to be highly toxic, both in the dark and upon irradiation.