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.

ABSTRACT INVESTIGATON OF CHEMOPREVENTIVE PROPERTIES OF URTICA DIOICA L., IN MCF-7 AND MDA231 BREAST CANCER CELL LINES. G&uuml / ler, Elif Ph.D., Biological Sciences Department Supervisor : Prof. Dr. Mesude

The tetrahydroisoquinoline alkaloids have been studied thoroughly about their biological and chemical significance over the past 30 years. These natural products show great biological activity, especially ET-743 and saframycin A, makes them promising therapeutics, while their structural complexity and particularity provide challenging synthetic targets. These alkaloids or derivatives show interesting biological activity, but the most important drawback as potential market therapeutics is the minute amount of them available from nature, and the synthetic methods published are inconvenient, difficult, and hard to handle. Herein is described our researches about the tetrahydroisoquinoline alkaloids. Chapter 1 describes relevant background related to the biological significance of these alkaloids, and the currently synthetic studies toward these natural products. Chapter 2 describes our design and synthesis of the analogues based on the anticancer mechanism of the tetrahydroisoquinoline alkaloids, and the biological activities of these analogues. Chapter 3 describes a rapid synthetic route for the common structure of the bis-tetrahydroisoquinoline alkaloids via a controlled mono-Pictet-Spengler cyclization.

The main goal of this research is to understand molecular mechanisms of GAA/TTC-associated genetic instability in a model eukaryotic organism, S. cerevisiae. We demonstrate that expanded GAA/TTC repeats represent a threat to eukaryotic genome integrity by triggering double-strand breaks and gross chromosomal rearrangements. The fragility potential strongly depends on the length of the tracts and orientation of the repeats relative to the replication origin and to block replication fork progression. MutSbeta complex and endonuclease activity of MutLalpha play an important role in facilitation of fragility. In addition to GAA/TTC triplex forming repeats, non-GAA polypurine polypyrimidine mirror repeats that are prone to the formation of similar structures were found to be hotspots for rearrangements in humans and other model organisms. These include H-DNA forming sequences located in the major breakpoint cluster region at BCL2, intron 21 of PKD1, and promoter region of C-MYC. Lastly, we have investigated the effect of the triplex-binding small molecules, azacyanines, on GAA-mediated fragility using the chromosomal arm loss assay. We have found that in vivo, azacyanines stimulate (GAA/TTC)-mediated arm loss in a dose dependent manner in actively dividing cells. Azacyanines treatment enhances the GAA-induced replication arrest. We discovered that also, azacyanines at concentrations that induce fragility also inhibit cell growth. Over 60% of yeast cells are arrested at G2/M stage of the cell cycle. This implies an activation of DNA-damage checkpoint response.

A novel approach of using near infrared region (NIR) dyes is applied to elucidate the binding interaction between human serum albumin (HSA) and mangiferin (MGF). HSA is a blood carrier protein used for drug delivery, while mangiferin is a natural polyphenol found in mangoes that possesses numerous beneficial health properties. The NIR dyes are used as a probe to investigate MGF binding interaction with HSA via monitoring fluorescence of Trp-214 residue. Molecular modeling is used for docking and semi-empirical analysis. The investigation of the binding interaction between Trp-214 and MGF is significant, for it may offer broader pharmacological insight and applications for the polyphenol. Mangiferin in proposed to bind with a 2:1 stoichiometric ratio with HSA to the Trp-214 residue in subdomain IIA and another possible binding site to be determined in future studies. Spectral changes suggest a stabilized protein conformation upon mangiferin binding with the addition of NIR dye E-06 and MHI-06.

Vitamin E is a group of naturally occurring fat soluble compounds which consists of eight distinct forms of tocopherols and tocotrienols. Although a well-defined physiological function of vitamin E is as an antioxidant, beneficial effects of individual vitamin E compounds on chronic human diseases such as cancer need to be better understood. Studies in this dissertation investigated potential application of gamma-tocopherol (gamma-T), gamma-tocotrienol (gamma-T3) or synthetic derivatives of tocotrienols as anticancer agents in comparison to alpha-tocopherol (alpha-T), its redox-silent acetic acid derivative (alpha-TEA) or alpha-tocotrienol (alpha-T3). Redox-silent derivatives of alpha- and gamma-T3; namely alpha-T3EA and gamma-T3EA exhibited potent anti-proliferative and proapoptotic activities in a murine mammary cancer cell line as well as in human breast cancer cell lines. Moreover, studies using human vascular endothelial cells in cell culture showed that the tocotrienol derivatives exhibited strong antiangiogenic activities which were markedly improved over those of the parent compounds. An antitumor efficacy study using the 66cl-4-GFP syngeneic mouse mammary tumor model showed that each tocotrienol derivative, when delivered in the diet, significantly suppressed mammary tumor growth; however serum and tissue concentrations of these novel compounds were lower than those of alpha-TEA, suggesting that the next generation of vitamin E derivatives will need to be modified to improve bioavailability. On the other hand, some natural-source vitamin E forms, especially gamma-forms, display anticancer activities without any chemical modification in both in vitro cell culture studies and in vivo animal models. Dietary delivery of gamma-T3 suppressed tumor growth in a syngeneic implantation mouse mammary cancer model by inhibiting cell proliferation and inducing apoptosis. Cell culture studies using human breast cancer cells showed that gamma-T3 triggered apoptosis by inducing endoplasmic reticulum (ER)-stress mediated by acid sphingomyelinase (ASMase) action. Activation of stress-activated mitogen-activated protein kinases (MAPKs), JNK and p38, was associated with gamma-T3-induced ER stress followed by upregulation of extrinsic death receptor-5 (DR5) expression in a CHOP transcription factor dependent manner. Gamma-T also triggered extrinsic apoptosis signaling by increasing DR5 mRNA, protein and cell surface expression levels followed by mitochondria-dependent apoptotic signaling. In agreement with in vitro studies, gamma-T delivered in the diet suppressed the tumor growth of MDA-MB-231-GFP human breast cancer cells in a xenograft model but the antitumor activity of gamma-T was hampered by co-administration of alpha-T. The preferential tissue retention of alpha-T over gamma-T could be overcome by use of sesamin, a dietary source of human cytochrome P450 inhibitor. Based on data presented, gamma-T and gamma-T3 show preclinical potential for cancer treatment either as single agents or in combination with other agents. / text

The problem of finding targeted medicine is a central problem in chemotherapy. From this point of view the ubiquitin-proteasome system is a highly promising object in the pharmaceutical approach. Proteasome plays a critical role in cellular protein degradation, cell cycle and apoptosis regulation. Proteasome inhibitors are substances blocking the actions of proteasome. Cancer cells are more sensitive to inhibition of the ubiquitin-proteasome system than normal cells. Therefore proteasome inhibitors have the potential to be successfully used in the cancer treatment. The study aimed to test various substances to identify possible proteasome inhibitors with the IncuCyteTM FLR image system and fluorometric microculture cytotoxicity assay. Using the IncuCyte FLR method allows for detecting changes in the molecular processes of living cells. To make proteasome inhibition visible the model cell line MelJuSoUbG76V-YFP is used which helps to detect alterations in proteasome activity by means of the yellow fluorescent protein enrichment in cells as a response to proteasome inhibition. Fluorometric microculture cytotoxicity assay is a method for the determination of cytotoxicity in human tumor cells. The study showed that substance #25 possessed a proteasome inhibitory capacity in a dose-dependent manner as demonstrated with the IncuCyte FLR image system. According to the fluorometric microculture cytotoxicity assay, substance #1 was the most stable and toxic. Substances #2 and #185 had selective toxicity against cancer cells and lower effects against normal cells. Combining IncuCyte FLR and fluorometric microculture cytotoxicity assay allows finding substances which act as proteasome inhibitors with high toxic effect.

Cancer is the second leading cause of death in the United States (more than half a million people each year), and even with billions of dollars in medical effort patients are rarely cured. This dissertation research is devoted to meeting this medical need by providing new cancer therapeutics that are more potent and safer than current chemotherapies. This is achieved by using two state of the art anticancer “warheads”: 1) gold nanoparticle (AuNP) technology and 2) a new class of epigenetic anticancer small molecules, histone deacetylase inhibitors (HDACi). These warheads are then selectively delivered to cancer cells via “homing devices” targeted to receptors that are overexpressed in the cancers. This work primarily focuses on the androgen receptor (AR) to target prostate cancer. The 1st chapter sets the stage, providing scientific rationale and background for the central hypothesis: small molecules that engage the AR can, upon conjugation to a therapeutic agent, enable selective delivery of that agent to prostate cancer cells. Chapter 2 delves into the structural molecular biology of the androgen receptor. There is a survey of the crystallographic data for all nuclear receptors, providing structural information which is used to build AR homology models for antagonist and inverse agonist modes of ligand binding. These models are used to design AR targeting ligands (Chapters 3, 5, 6 and 7). The application of the targeting technology is illustrated by attaching them to AuNPs for selective delivery to prostate cancer cells (Chapter 3). Next, in order to appreciate the importance of using targeting agents in HDACi cancer therapeutics, we reviewed this recently emerged field in Chapter 4. In this chapter we argue that the failure of HDACi in solid tumors, despite more than 500 clinical trials in the last decade, is primarily due to an inability of these small molecules to accumulate at effective concentrations in the cancer. We provide an analysis of the paradigms being pursued to overcome this barrier, including HDAC isoform selectivity, localized administration, and targeting cap groups to achieve selective tissue and cell type distribution. In Chapter 5, this last approach (targeting cap groups, or a “homing device”) is illustrated with HDACi targeted to prostate cancer via antiandrogens that bind the AR. The second generation of improved “homing devices” is disclosed in Chapter 6 (for both AuNPs and HDACi), in addition to preliminary ADMET data and safety studies in mice. Excitingly, our three dimensional understanding of binding to the AR allowed design and structure-activity-relationship studies that lead to the first reported examples of AR inverse agonists (Chapter 7) Several points of significance: • AuNP targeted to AR ∙ have the strongest binding affinity ever reported (IC50 ~14 picomolar) ∙ are actively recruited to prostate cancer cells ∙ overcome treatment resistance in advanced prostate cancer cells ∙ exhibit nanomolar anticancer potency ∙ resolved the identity of the “membrane AR” as the GPRC6A • HDACi targeted to AR ∙ have HDACi activity and AR binding affinity superior to their clinical precursors ∙ exhibit potent AR antagonist activity ∙ induce AR translocation to the nucleus in a HDACi dependent fashion ∙ selectively and potently kill prostate cancer cells that express AR ∙ are safer than Tylenol®, as tested in small animals • Pure AR binding ligand studies ∙ resulted in the discovery of the first examples of AR inverse agonists, which are vastly more potent that clinically available antiandrogens for prostate cancer ∙ work via a never-before-seen mechanism of action, by localizing to the nucleus and recruiting corepressors to actively shut off AR genes

Thermodynamic and kinetic analysis of the ligand substitution reactions of different heterometallic Ru(II)-Pt(II) complexes with a series of bio-relevant thiourea nucleophiles of different steric demands and ionic nucleophiles have been investigated as a function of concentration and temperature using UV/visible and stopped-flow spectrophotometric techniques. To achieve this, five different sets of complexes involving mono di and multinuclear homo and heterometallic complexes with tridentate N-donor ligands of different linker ligands were synthesized and characterized by various spectroscopic methods. The substitution reactions of the chloride complexes were studied in methanol in the presence of 0.02 M LiCf3SO3 adjusted with LiCl to prevent possible solvolysis. The aqua complexes were studied in acidic aqueous medium at pH 2.0. All reactions were investigated under pseudo first-order conditions. Density functional theory (DFT) calculations were used to aid further interpretations and understandings of the experimental results. Substitution reactivity of heterometallic Ru(II)-Pt(II) and Co(II)-Pt(II) complexes bridged by tetra-2-pyridyl-1,4-pyrazine (tppz) ligand was investigated for the first time. The reactions proceeded via two steps. The pseudo first-order rate constants, kobs(1st and 2nd) for the substitution of the chloride ligand(s) from the Pt(II) complexes and subsequent displacement of the linker. The dechelation step was confirmed by 1H NMR and 195Pt NMR studies. Incorporation of Ru(tppz) moiety increases the substitution reactivity and is ascribed to the increased π-back donation from the tppz ligand which increases the electrophilicity of the metal centre, overall charge and the global electrophilicity index of the complex. However, when changed the second metal centre from a Ru(II) to a Co(II), the rate of substitution decreased by a factor of four due to the weaker π- backbonding from Co(II). The substitution reactivity of another set of heterometallic Ru(II)-Pt(II) complexes with a semi-rigid linker, 4’-pyridyl-2,2’:6’,2”-terpyridine (qpy) showed that replacing the cis pyridyl group by a (tpy)Ru(qpy) moiety lowers the energy of anti-bonding LUMO (π*) orbitals and increases the metal-metal interactions and electronic transition within the complex whereby enhancing the reactivity of Pt(II) centre. However, when two Pt(II) moieties are linked to a (qpy)Ru(qpy), the orthogonal geometry at the Ru(II) metal centre prevents the extended π-electron density to flow through the three metal centres. The kinetic results obtained were supported by pKa and 195Pt NMR studies. Substitution reactions of the mononuclear Pt(II) complexes revealed that the polyethylene glycoxy pendent units act as a σ-donors including the lone pair electrons on the first oxygen atom thereby decreasing the reactivity of the parent Pt(II) terpyridine complex. However, this σ-donation towards the terpyridine moiety was found to be effective only up to one unit of the ethylene glycoxy pendant, beyond which the reactivity was sterically controlled. The dinuclear Pt(II) complexes bridged by polyehtyleneglycol ether units show that the reactivity of the complexes depend on the Pt···Pt distance and the steric hindrance at the Pt(II) centre. The substitution reactivity of heterometallic Ru(II)-Pt(II) complexes bridged by the same polyehtyleneglycol ether units indicate that the presence of Ru(tpy)2 moiety influences the structural geometry of the complex system which in turn controls the reactivity of the Pt(II) centre. This is further driven by the entrapment effect of the nucleophile due to the V-shape geometry adopted by the heterometallic complexes. In all cases the reactivity was also controlled by steric and electronic effects. However, when two metal centres are bridged by a flexible non-aromatic linker, the electronic transitions and the metal-metal interactions were found to be minor, especially for the longer linkers. The 1H and 195Pt NMR spectroscopic techniques were used to further understand the observed substitution kinetics and to confirm the degradation of the bridging ligand from the metal centre(s). In all cases, the negative activation entropies obtained support the associative mode of substitution This investigation reveals that the length and the nature of the bridging linker plays an important role in controlling the reactivity of the heterometallic complexes. It is envisaged that the findings of this project would offer a significant contribution to the pharmacological design of effective anticancer drugs. / Ph.D. University of KwaZulu-Natal, Pietermaritzburg 2013.

Australia's marine environment covers extended areas, from the warm northern tropical, to the sub tropical central water, the cool temperate water of the south and the cold sub-Antarctic and Antarctic water. Australia has rich area of coral reefs. The marine biodiversity in Australia is enormous. Despite incredible biodiversity, Australian research in the marine anticancer drug discoveries is low in comparison with other countries. In this research we investigated a collection of marine sponges as a source for new anticancer leads. This thesis comprises six chapters. Chapter 1 covers the importance of natural products as a source of new drugs, and an introduction to cancer as a disease, chemotherapy in cancer treatments, and the natural products as a source for anticancer drugs. Also, the basic anticancer drug development process is highlighted. Finally, a thorough review of anticancer alkaloids isolated from marine sponges is presented. Chapter 2 presents the chemical investigation into a southern Australian marine sponge Stelletta sp., which led to the isolation and structure elucidation of bistellettazines A-C the first reported examples of terpenyl-pyrrolizidines conjugate, and bistellettazole A, a unique cyclic terpenyl-imidazole conjugate. Bistellettazines A-C and bistellettazole A feature unprecedented carbon skeletons that are proposed to share a common convergent biosynthetic origin, arising via the biogenic equivalent of a Diels-Alder addition between two hypothetical polyenyl norsesquiterpene precursors. The cytotoxic activity (in vitro) for these new alkaloids is also discussed. Chapter 3 discusses the isolation and structure elucidation of four new discorhabdins analogues namely, dihydrodiscorhabdin A, debromodiscorhabdin A, discorhabdin X and dihydrodiscorhabdin L. In addition, the known compounds discorhabdin A and discorhabdin D, were isolated from two southern Australian marine sponge specimens of the genera Higginsia and Spongosorites. The cytotoxic activity (in vitro) for these new alkaloids was also discussed. Chapter 4 discloses chemical investigation into two southern Australian marine sponge specimens of the genera Clathria and Ptilocaulis. Four new mirabilin analogues (mirabilins H-K) were isolated and characterized along with known mirabilin C, F (for the first time as TFA salt) and mirabilin G. The cytotoxic activity (in vitro) for these new alkaloids was also discussed. Chapter 5 presents the 1H NMR data for the known compounds isolated during this study, and Chapter 6 is covering the experimental part.