This thesis is concerned with the application of the conjugate addition of enantiopure lithium amides in the asymmetric syntheses of (−)-martinellic acid. Chapter 1 introduces the importance of the quinoline motif in a wide variety of natural products and pharmaceuticals. The natural products (–)-martinellic acid and (+)-martinelline are introduced and previous methods for their synthesis are described. Chapter 2 introduces the conjugate addition reaction of lithium N-benzyl-N-α-methylbenzylamide as a means of synthesising β-amino esters from α,β-unsaturated esters. Both “tandem” and “stepwise” enolate functionalisation pathways to introduce an α-substituent are discussed, and the products are cyclised to the corresponding quinolin-2-ones. Modification of this strategy allowed the development of a double cyclisation reaction to form the pyrroloquinoline core found within (–)-martinellic acid and (+)-martinelline. Initial attempts at elaborating the tricyclic core to the natural products are described. Chapter 3 addresses the difficulties encountered in the initial synthetic route by the use of lithium (R)-N-allyl-N-(α-methyl-4-methoxy-benzyl)amide as an alternative enantiopure ammonia equivalent. A key Wittig and intramolecular Michael reaction is used to introduce the remaining stereogenic centre, allowing access to either epimeric series. Full optimisation of the synthetic sequence is described resulting in the synthesis of a simplified triamine core, lacking only the ester functionality required for (–)-martinellic acid and (+)-martinelline. Chapter 4 presents an asymmetric synthesis of (–)-martinellic acid and the first asymmetric synthesis of 4-epi-martinellic acid using the methodology developed in chapter 3, by incorporation of an ester functionality into substrate. Chapter 5 contains full experimental procedures and characterisation data for all compounds synthesised in Chapters 2, 3 and 4.

The diversity of biosynthetic pathways in prokaryotes and eukaryotes has led to numerous bioactive natural products (NPs) which occupy a vast chemical space. Despite the current challenges in NP research, these molecules are still relevant today as they are a major source of human medicine as well as being useful biological tools. The elucidation of their biosynthetic pathways has also provided information about the biochemical and biophysical properties of fascinating enzyme families such as the α- oxoamine synthases (AOSs). The AOSs are an expanding group of pyridoxal 5’- phosphate (PLP)-dependent enzymes, which are involved in the biosynthesis of several important NP, including those essential for life. This study reports the characterization and structural analysis of a unique AOS denoted as TamD from Pseudoalteromonas tunicata. This enzyme plays a major role in tambjamine biosynthesis and consists of both an acyl carrier protein (ACP) domain and a PLP-binding catalytic domain. UV/vis spectroscopy and mass spectrometry (MS) of the recombinant TamD purified from E. coli revealed that the enzyme forms a Schiff base with PLP via Lys380, which is responsible for its characteristic yellow colour. It binds L-serine as a natural substrate with a Kd of 5.01 ± 0.64 mM. This thesis also reports structural data for TamD from xray crystallography at a resolution of 4.98 Å, which shows four molecules in the asymmetric unit (ASU) suggesting the enzyme exist as a dimer. The absence of the Nterminal region where the ACP domain is located in the crystal strucuture also suggests intrinsic flexibility and disorder within that region. With the increasing demand for new anti-infective therapies, investigations of the molecular interactions between NPs and their protein targets are vital in understanding the inhibition or activation properties of these molecules. The cysteine transpeptidases known as sortases produced by Gram positive bacteria have been identified as attractive targets for NP inhibitors. In this thesis, the molecular basis for the inhibition of Streptococcus mutans sortase A (SrtA) by the plant flavonoid, trans-chalcone is explored, using a combination of MS, enzyme kinetics, molecular modelling and x-ray crystallography. This study reports the first high resolution crystal structure of the H139A mutant of S. mutans SrtA, which reveals a unique N-terminal α-helix domain. Trans-chalcone was found to inhibit the in vitro activity of S. mutans SrtA in a slow, tight–binding manner, with a half maximal inhibitory concentration (IC50) of 5.0 ± 0.6 μM. The interaction resulted in a covalent adduct with the active site cysteine residue (Cys205) via a Michael addition mechanism. Additionally, trans-chalcone showed evidence of S. mutans anti-biofilm activity in a concentration dependent manner up to 250 μM with an efficacy cut-off point at higher concentations. These results indicate that chalcone flavonoids are worth further investigation as potential antibiofilm inhibitors. A renewed interest in plant NPs has also led to a collaborative investigation on the antimicrobial potential of garlic-derived allicin, against Burkholderia cepacia complex (Bcc), the major bacterial phytopathogen for alliums and an intrinsically multiresistant and life-threatening human pathogen. Allicin is the principal antibacterial agent in fresh preparations of garlic extracts. This investigation reports the first evidence that allicin and allicin-contaning garlic extracts possess inhibitory and bactericidal activities against Bcc. The minimum inhibitory concentrations (MICs) of aqueous garlic extract (AGE) against 38 Bcc isolates ranged from 0.5 to 3% (v/v). An investigation into the possible molecular mechanisms of allicin with a recombinant thiol-dependent peroxiredoxin (BCP) from B. cenocepacia revealed that allicin and AGE modify an essential BCP catalytic cysteine residue and suggests a role for allicin as a general electrophilic reagent that targets protein thiols. Present therapeutic options against these life-threatening pathogens are limited; thus, allicin-containing compounds merit further investigation as adjuncts to existing antibiotics.

Magister Scientiae - MSc / Background and objectives: Apoptosis is a process in which a cell programmes its own death. It is a highly organized physiological mechanism in which injured or damaged cells are destroyed. Apart from physiological stimuli however, exogenous factors can induce apoptosis. Many anti-cancer drugs work by activating apoptosis in cancer cells. Natural substances have been found to have the ability to induce apoptosis in various tumour cells and these substances have been used as templates for the construction of novel lead compounds in anticancer treatment. On the other hand, alkylating agents such as cisplatin, cis- [PtCl2 (NH3) 2] have been widely used as antineoplastic agents for a wide variety of cancers including testicular, ovarian, neck and head cancers, amongst others. However, the use of cisplatin as an anticancer agent is limited due to toxicity and resistance problems. The aim of this present study was to screen the leaves of Rhus laevigata, a South African indigenous plant, for the presence of pro-apoptotic and anti-proliferative natural compounds and also to screen newly synthesised palladium based complexes (15 and 57) and a platinum based complex (58) for their antineoplastic activities tested against a panel of cell lines. Results. The results showed that crude methanol extracts from Rhus laevigata as well as the newly synthesised palladium based complexes (15 and 57) and a platinum based complex (58) induced apoptosis in the cell lines tested, as demonstrated by the externalization of phosphatidylserine, mitochondrial membrane permeabilization,caspase-3 activation, and DNA fragmentation. Caski (cervical cancer) and H157 (non small cell lung carcinoma) cell lines treated with the methanol extract from Rhus laevigata however, were more resistant to apoptosis induction. Among the metallocomplexes, complexes 15 and 57, palladium based complexes, were the most active. Conclusion: The methanol extract from the leaves of Rhus laevigata contain pro-apoptotic and antiproliferative natural compound(s), which need to be characterised and elucidated as they could provide the much-needed lead compounds in the fight against cancer. On the other hand the newly synthesized palladium complexes also need further evaluation to see if they can be used as anticancer agents that can overcome the problems associated with cisplatin. / South Africa

Philosophiae Doctor - PhD / Natural products have been a source of many pharmaceutical drugs and a number of drugs that are currently used in the treatment of cancer are derivatives of compounds originally isolated from natural products. There is evidence that extracts of Tulbaghia violacea can be used to treat cancer. The activation of apoptosis in cancer cells is a target for the development of novel anti-cancer drugs since one of the characteristics of cancer cells is resistance to apoptosis due to the deregulation of biochemical pathways leading to apoptosis. In fact, many current anti-cancer drugs exert their effects through the activation of apoptosis. Previous studies showed that extracts of T.violacea induce apoptosis in cancer cells and one study reported on the isolation of a compound (methyl-ԃ-D-glucopyranoside), which is responsible for the pro-apoptotic activity of the T.violacea extract. Therefore the aim of this study was to investigate the anti-cancer activity of methyl-ԃ-Dglucopyranoside and extracts prepared from T.violacea. In this study the pro-apoptotic activity of methyl-ԃ-D-glucopyranoside and extracts prepared from T.violacea were investigated on a panel of human cancer cell lines, which included HepG2, MCF7, H157, HT29 and the non-cancerous cell line, KMST6. The induction of apoptosis was evaluated by flow cytometry using several bioassays which measures biochemical events (caspase activation, phosphatidylserine externalisation and reactive oxygen species (ROS) production that is associated with the induction of apoptosis. The results demonstrated that the effects of methyl--D-glucopyranoside on cultured cells are transient and that the cells recover from the effects of methyl--D-glucopyranoside. This suggested thatmethyl-ԃ-D-glucopyranoside is not the compound responsible for the pro-apoptotic bioactivity in the T.violacea extract. This study also showed that cytotoxic and pro-apoptotic bioactivity of the leaf-extract was significantly higher in comparison to the tuber-extract. The bioactivity of the organic solvent extracts (dichloromethane, hexane, methanol and 50% methanol/water) of T.violacea leaves was also significantly higher than water extracts of T.violacea leaves. A comparison of the different organic extracts prepared from the T.violacea leaves showed that the highest activity was observed for the dichloromethane and hexane extracts. In an effort to identify the bioactive compound(s) the dichloromethane extract was subjected to Versaflash® column chromatography. However, due to problems experienced with the solubility of the dichloromethane sub-fractions, these compounds could not be tested for their bioactivity. Palmitone (16-hentriacontanone) was identified as one of the major compounds present in the dichloromethane sub-fractions. This compound was previously shown to have anticonvulsant bioactivity but there is no evidence in the literature that it has anti-cancer or pro-apoptotic activities. Fingerprinting of the methanol extract showed the presence of long chain fatty acid derivatives, flavonoids and allicin derivatives in the methanol extract. Although, this study failed to isolate the pro-apoptotic bioactive compound(s) present in the extracts of T.violacea, it confirmed that extracts of this plant induce apoptosis in cultured human cancer cell lines.

Seaweeds of the genus Plocamium are known to produce a variety of halogenated monoterpenes. In addition to their ecological role as feeding deterrents, biological activities reported for these compounds include antibacterial, antialgal, antifungal and anticancer activities. An investigation of the non-polar extracts of the seaweed Plocamium corallorhiza resulted in the isolation of six known halogenated monoterpene compounds, 4-bromo-5-bromomethyl-1-chlorovinyl-2, 5-dichloro-methylcyclohexane (2.68), 1,4,8-tribromo-3 ,7-dichloro-3, 7-dimethyl-1,5-octadiene (2.67), 8-bromo-1 ,3,4,7-tetrachloro-3, 7-dimethyl-1,5-octadiene (2.66), 4,6-dibromo-1,1-dichloro-3,7-dimethyl-2,7-octadiene (2.64), 4,8-dibromo-1,1,7-trichloro-3,7-dimethyl-2,5-octadiene (2.65) and 3,4 ,6,7-tetrachloro-3, 7-dimethyl-1-octene (2.63) as well as eight new compounds, including five halogenated monoterpene aldehydes. The new compounds were identified by 1D and 2D NMR spectroscopic techniques as: 8-Bromo-6,7-dichloro-3,7-dimethyl-octa-2,4-dienal (2.72), 8-Bromo-1,1,2,7-tetrachloro-3,7-dimethyl-octa-3,5-diene (2.70), 4,8-Dichloro-3,7-dimethyl-octa-2,4,6-trienal (2.74), 4-Bromo-8-chloro-3, 7-di methyl-octa-2, 6-dienal (2 76), 8-Bromo-4-chloro-3, 7-dimethyl-octa-2,4 ,6-trienaI (2.75), 4-Bromo-1,3,6,7-tetrachloro-3 ,7-dimethyl-octa-1,4-diene (2.71), 8-Bromo-1,3,4,7-tetrachloro-3,7-dimethyl-octa-1,5-diene (2.69), 4,6-Dibromo-3,7 -dimethyl-octa-2,7-dienal (2.73). All compounds were screened for antimicrobial activity, brine shrimp lethality and cytotoxicity towards oesophageal cancer cells. Compound 2.68 was toxic to brine shrimp larvae at a concentration of 50 μ/mL. It also showed promising activity towards oesophageal cancer cells with an IC₅₀, of 2 μg/mL.

This thesis describes the chemical and biological investigation of the extracts of six different marine invertebrate organisms collected along the South African coastline. The work on these extracts has resulted in the isolation and structural elucidation of twenty-one previously undescribed secondary metabolites; The history of marine natural product chemistry in South Africa has not previously been reviewed and so a comprehensive review covering the literature from the 1940's up until the end of 1995 is presented here. The marine ascidian Pseudodistoma species collected in the Tsitsikamma Marine Reserve was shown to contain four new unsaturated amino alcohols [47], [48], [49] and [50] which were isolated as their acetyl derivatives. These compounds exhibited strong antimicrobial activity. Four new pyrroloiminoquinone alkaloids, the tsitsikammamines A [90] to D [93],were isolated from a new genus of Latrunculid sponge collected in the Tsitsikamma Marine Reserve. These highly pigmented compounds also possessed strong antimicrobial activity. An investigation of two phenotypic colour variants of the soft coral Capnella thyrsoidea resulted in the isolation of the known steroid 5α-pregna-1, 20-dien-3-one [97] and an additional six new metabolites, 16β-hydroxy-5α-pregna-1 ,20-dien-3-one 16-acetate [98], 3α,16β-dihydroxy-5α-pregna-1, 20-diene 3,16-diacetate [99] and four xenicane diterpenes, the tsitsixenicins A [100] to D [103]. This is the first reported isolation of xenicane diterpenes from the soft coral family Nephtheiidae. Tsitsixenicin A and B showed good anti-inflammatory activity by inhibiting superoxide production in both rabbit and human cell neutrophils. A further four new metabolites were isolated from two soft corals which could only be identified to the genus level and were designated Alcyonium species A and species B. Alcyonium species A was collected in the Tsitsikamma Marine Reserve and yielded two new polyhydroxysterols, cholest-5-ene-3β, 7β, 19-triol 19-acetate [121] and cholest-5,24-diene-3β, 7β, 19-triol 19-acetate [122]. The soft coral Alcyonium species B was collected off Aliwal Shoal and was found to contain two known xenicane diterpenes, 9-deacetoxy-14, 15-deepoxyxeniculin [110] and zahavin A [16], and two new xenicane diterpenes, 7 -epoxyzahavin A [123] and xeniolide C [124]. Compounds [110], [16] and [123] exhibited strong anti-inflammatory activity and compounds [110] and [16] showed good antithrombotic activity. The endemic soft coral A/cyanium fauri collected at Riet Point near Port Alfred yielded the new sesquiterpene hydroquinone rietone [141] in high yierd, fogether with the minor compounds 8'-acetoxyrietone [142] and 8'-desoxyrietone [143]. Rietone exhibited moderate activity in the NCl's in-vitro anti-HIV bioassays.

A chemical investigation of six different marine invertebrates, collected along the South African coastline, resulted in the isolation and structural elucidation of fifteen previously undescribed secondary metabolites along with seven known compounds. The structures of the new metabolites were determined by a combination of spectroscopic and chemical methods. The endemic false limpet Siphonaria capensis was shown to contain two unusual polypropionate metabolites capensinone (162) and capensifuranone (163) as well as 2,4,6,8-tetramethyl-2-undecenoic acid (164) and the known polypropionates (E)- and (Z)siphonarienfuranone (149 and 161). Capensinone is the first example of a marine polypropionate containing a cyc1opentenone moiety. An investigation of the endemic South African soft coral Pieterfaurea unilobata yielded six new, highly oxygenated, pregnadiene sterols (180-185) and the known metabolite (169). Compounds 180-185 are the first pregnadienes obtained from the marine environment containing a C-7 substituent. An alternative procedure for the quick assignment of the absolute configuration at C-3 in this series of compounds was proposed. A companson of the pyrroloiminoquinone alkaloids of three undescribed l'}trunculid sponges resulted in the isolation of 3-dih¥drodiscorhabdin C (243), 3-dihydrodiscorhabdin B (244), discorhabdin H (197) and the previously reported alkaloids discorhabdin A (189) and discorhabdin D (192). While all three sponges were found to be morphologically different they all contained discorhabdin A as the major metabolite and discorhabdin H as one of their minor metabolites. It was found that a feature common to most of the South African latrunculid sponges is the reduction of the C-3 carbonyl gr,o up in some of the minor metabolites. The indole alkaloids, dilemmaones A-C (261-263), containing an unusual cyc1opentanone-indole skeleton, were isolated in trace amounts by bioassay guided fractionation of an extract obtained from a mixed collection of sponges collected near Cape Town. In an attempt to acquire more of these novel compounds for further investigation of their biological activity, several synthetic strategies towards their total synthesis were explored. A key feature of these approaches was the exploitation of the regioselective Gassman's artha-alkylation procedure for the introduction of an aromatic methyl substituent.

This thesis is divided into two parts, assessing marine and synthetic compounds active firstly against Plasmodium falciparum (Chapter 3 and 4) and secondly active against methicillin resistant Staphylococcus aureus (MRSA, Chapter 5). In Chapter 3 the synthesis of nine new tricyclic podocarpanes (3.203-3.207 and 3.209-3.212) from the diterpene (+)-manool is described. Initial SAR study of synthetic podocarpanes concluded that the most active compound was a C-13 phenyl substituted podocarpane (3.204, IC₅₀ 6.6 μM). By preparing analogues with varying halogenated substituents on the phenyl ring (3.209-3.212) the antiplasmodial activity was improved (IC₅₀ 1.4 μM), while simultaneously decreasing the haemolysis previously reported for this class of compounds. Inspired by the antiplasmodial activity of Wright and Wattanapiromsakul’s tricycle marine isonitriles (2.16-2.21 and 2.24-2.27) an unsuccessfully attempt was made to convert tertiary alcohol moieties to isonitrile functionalities in compounds 3.188, 3.204-3.207 and 3.209-3.212. Over a decade ago Wright et al. proposed a putative antiplasmodial mechanism of action for marine isonitriles (2.4, 2.9, 2.15, 2.19 and 2.35) and isothiocyanate (2.34) which involved interference in haem detoxification by P. falciparum thus inhibiting the growth of the parasite. In Chapter 4 we describe how we successfully managed to scale down Egan’s β-haematin inhibition assay for the analyses of small quantities of marine natural products as potential β-haematin inhibitors. Our modified assay revealed that the most active antiplasmodial marine isonitrile 2.9 (IC₅₀ 13 nM) showed total β-haematin inhibition while 2.15 (IC₅₀ 81 nM) and 2.19 (IC₅₀ 31 nM) showed partial inhibition at three equivalents relative to haem. Using contempary molecular modelling techniques the charge on the isonitrile functionality was more accurately describe and the modified charge data sets was used to explore docking of marine isonitriles to haem using AutoDock. In Chapter 5 we describe how a lead South African marine bisindole MRSA pyruvate kinase inhibitor (5.8) was discovered in collaboration with colleagues at the University of British Columbia (UBC) and how this discovery inspired us to design a synthetic route to the dibrominated bisindole, isobromotopsentin (5.20) in an attempt to increase the bioactivity displayed by 5.8. We devised a fast and high yielding synthetic route using microwave assited organic synthesis. We first tested this synthesis using simple aryl glyoxals (5.27-5.32) as precursors to synthesize biphenylimidazoles (5.21-5.26), which later allowed us to synthesize the ascidian natural product 5.111. This method was sucessfully extended to the synthesis of deoxytopsentin (5.33) from an N-Boc protected indole methyl ketone (5.89). We subsequently were able to effectively remove the carbamate protection via thermal decomposition by heating the protected bisindole imidazole (5.90) in a microwave reactor for 5 min under argon. The synthesis of 5.20 resulted in an inseparable mixture of monoprotected and totally deprotected topsentin products, and due to time constraints we were not able to optimise this synthesis. Nonetheless our synthesis of the marine natural product 5.33 which was faster and higher yielding than previously reported routes could be extended to the synthesis of other topsentin bisindoles (5.138-5.140). Work towards this goal continues in our laboratory.

No description available.

The sea conceals a mermaid’s grotto of useful chemicals-marine natural products of therapeutic potential. Marine sponges in particular are a rich source of natural products with structural diversity and novel biological activity. In recent times, there has been a growing interest in the synthesis of marine natural products. The present thesis entitled, “Synthesis of bioactive marine meroterpenoids: frondosins and liphagal” is an endeavor along the same lines and is organized under two parts –Part A and Part B. Part A: Studies towards the total synthesis of (±) frondosins A and B Frondosins A-E are IL-8 inhibiting marine meroterpenoids, with novel bicyclo[5.4.0]undecane framework, exhibiting anti-inflammatory and anti HIV-1 activities. A relatively simple and inherently flexible ring-closing metathesis (RCM) based strategy was employed to achieve the total synthesis of frondosins A (formal) and B in only 17 linear steps (total 13 operations) and 5% overall yield. A concise route, based on RCM, to the core structure of bioactive frondosins is amenable to ready appendage diversification and enables implementation of functionalization manoeuvres on all positions in the seven-membered ring of the bicyclic framework was also developed. A Diels-Alder strategy that led to the synthesis of 8-des-methyl norfrondosin A dimethyl ether is also delineated in Part A of the thesis. Part B: A concise synthesis of (±) liphagal Liphagal is a marine meroterpenoid displaying an unprecedented “liphagane” skeleton. It is a selective inhibitor of PI3K  and significantly toxic against a small panel of human tumor cell lines (LoVo, CaCo-human colon and MDA-468-human breast). A concise and straightforward biomimetic strategy towards liphagal and its 14-des-formyl analogue that awarded liphagal dimethyl ether in only eight steps from commercially available building blocks is described in Part B of the thesis.