An in depth chemical investigation of the major and minor pyrroloiminoquinone metabolites produced by four species of endemic South African Latrunculid sponges, collected from Algoa Bay and the Tsitsikamma Marine Reserve off the south eastern coast of South Africa, yielded eleven new and twelve known pyrroloiminoquinone metabolites. The structures of the new metabolites were determined using standard spectroscopic techniques. Tsitsikamma pedunculata was shown to contain 7,8-dehydro-3-dihydro-discorhabdin C (2.1), 14-bromo-7,8-dehydro-3-dihydro-discorhabdin C (2.2), discorhabdin S (2.3), 14-bromo-1-hydroxy-discorhabdin S (2.4), 1-bromo-2-hydroxy-4-debromo-discorhabdin S (2.5), and 2,4-debromo-3-dihydro-discorhabdin C (2.6), together with the known compounds 14-bromo-discorhabdin C (1.51), 14-bromo-3-dihydro-discorhabdin C (1.52) and 3-dihydro-discorhabdin C. The metabolites from T. pedunculata were characterised by the presence of a reduced C-3 carbonyl and bromination at C-14. Compounds isolated from a second Latrunculid sponge, Latrunculia lorii, ranged from a substituted bicyclic pyrrolecarboxylic acid, makaluvic acid A (1.47), to the simple tricyclic known pyrroloiminoquinones makaluvamine C (1.33) and damirone B (1.20) and the more complex discorhabdin D type metabolites, discorhabdin M (3.2), 1-amino discorhabdin D (3.3), 1-methoxy discorhabdin D (3.4) and 1-alanyl discorhabdin D (3.5). Discorhabdin G* (3.1) was also isolated and characterised. This is the first reported occurrence of the known compounds 1.20, 1.33 and 1.47 in a Latrunculia sponge. Discorhabdin and bis-pyrroloiminoquinone type compounds predominated in Tsitsikamma favus. Three known, tsitsikammamines A (1.71) and B (1.72), 1.52, and five new pyrroloiminoquinones, tsitsikammamine N-oxime (4.1), tsitsikammamine B N-oxime (4.2), 2.1, 2.4 and 2.6, were isolated from this sponge. A fourth Latrunculid sponge (Strongylodesma sp.) yielded three known compounds, discorhabdins A (1.57), D (1.61) and 1.53, and one new pyrroloiminoquinone 3.3. The dual role of these metabolites as cytotoxic agents and pigments resulted in an attempt to relate the photochemical properties of these metabolites to their cytotoxicity. The pyrroloiminoquinone metabolites studied exhibited moderate singlet oxygen quantum yields, while three compounds (1.57, 4.1 and 4.2) were shown to be capable of producing radicals at a wavelength of 532 nm. The possibility of a correlation between the electrochemical properties and anti-cancer (HCT-116) activity of selected pyrroloiminoquinones was explored. A study of the oesophageal and ovarian cytotoxicities of two pyrroloiminoquinones (1.57 and 1.72), together with an investigation into the intercalation and topoisomerase I inhibitory activity of the bis-pyrroloiminoquinones (1.71, 1.72, 4.1 and 4.2), are presented.

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 investigates the variability occurring in the secondary metabolites produced by three South African marine molluscs. Chapter Two discusses the isolation and spectroscopic structure elucidation of the metabolites isolated from two Siphonaria species. The re-investigation of Siphonaria capensis yielded siphonarienfuranone (2.2) as the only common polypropionate isolated from both the 1998 and 2009 collections of S. capensis from the same areas suggesting possible seasonal or genetic variation in polypropionate production. The sterol cholest-7-en-3,5,7- triol (2.33) was also isolated form the 2009 collection of S. capensis and this is the first time this compound has been isolated from a Siphonaria species. The second species, Siphonaria oculus is closely related to S. capensis and the investigation into the former’s secondary metaboliteproduction revealed 2.2 as a major metabolite suggesting an inter-species overlap in polypropionate production. Three new polypropionate metabolites, 2.35, 2.36 and 2.37 were also isolated from S. oculus. An unsuccessful attempt was made to establish the absolute configuration of 2.37 using the modified Mosher’s method and the limited amount of 2.37 available prevented any further attempts at resolving the absolute configuration of this compound. The 1H NMR analysis of the defensive mucus collected directly from S. oculus revealed the presence of the acyclic polypropionate 2.37 as a minor metabolite. The absence of characteristic signals for the furanone containing compounds 2.2, 2.35 and 2.36, might suggest that these compounds cyclise from a hypothetical acyclic precursor (2.38) during standard work up of bulk acetone extracts of Siphonaria species. Chapter Three discusses the re-isolation and spectroscopic structure elucidation of the metabolites isolated from the nudibranch, Leminda millecra. Three known natural products, millecrone A (3.1), 8-hydroxycalamenene (3.6) and cubebenone (3.8) were re-isolated from our 2010 collection of L. millecra, as well as the new minor metabolite 8-acetoxycalamenene (3.16). The cytotoxic prenylated toluquinones and toluhydroquinones (3.9-3.15) initially isolated from the 1998 collection of L. millecra were not found in the 2010 collection supporting the hypothesis that these compounds may be of fungal origin. L. millecra clearly shows variability in the compounds sequestered by this species with millecrone A (3.1) being the only common metabolite in the three investigations of L. millecra to date. An unsuccessful attempt was made to establish the absolute configuration of 3.1, 3.6 and 3.8 through initial LAH reduction of the ketone moiety contained in 3.1 and 3.8 and esterification of the resultant diastereomeric alcohol mixtures and the phenol functionality in 3.6 with (1S)-camphanic chloride. Crystallisation of the (S)- camphanate esters of 3.6 and 3.8 for X-ray analysis were unsuccessful, while the unexpected conjugate addition of a hydride in 3.1 resulted in complex diastereomeric mixtures which could not be separated by HPLC.

Algoa Bay Latrunculid sponges are well known for their production of cytotoxic pyrroloiminoquinones with speculation that these secondary metabolites may have a microbial origin. This study describes a thorough investigation into the bacterial community associated with Tsitsikamma favus, Tsitsikamma scurra a newly described Latrunculia sp. and a yellow encrusting sponge associated with T. scurra. Molecular and chemical characterisation were used in conjunction with traditional taxonomy in identification of the sponge specimens. The 28S rRNA and COX1 analysis confirmed the traditional taxonomy with T. favus and T. scurra being very closely related. Chemical analysis revealed that T. favus and T. scurra shared the discorhabdins 2,4-debromo-3-dihydrodiscorhabdin C, 7,8-dehydro-3-dihydrodiscorhabdin C and 14-bromo-1-hydroxy-discorhabdin V in common with each other and Tsitsikamma pedunculata indicating that these pyrroloiminoquinones are common to Tsitsikamma sponges in general. The bacterial community associated with T. favus was explored using 16S rRNA molecular techniques including DGGE, clonal libraries of full length 16S rRNA genes, as well as 454 pyrosequencing. DGGE analysis revealed that the bacterial community associated with T. favus appeared to be highly conserved, which was confirmed by both the clone library and 454 pyrosequencing, with the Betaproteobacteria as the most dominant class. Further exploration into T. favus, as well as T. scurra, Latrunculia sp. and the yellow encrusting sponge indicated that the bacterial populations associated with each of these sponge species were conserved and species specific. OTU analysis to the species level revealed that T. favus and T. scurra shared an abundant Spirochaete species in common while the most abundant species in the Latrunculia sp. and the yellow encrusting sponge belonged to the class Betaproteobacteria. The exclusivity of the tsitsikammamines to T. favus precipitated attempts to culture the T. favus associated bacteria, with a focus on the dominant betaproteobacterium as indicated by the 16S rRNA clone library. Actinobacteria associated with the Algoa Bay sponge specimens were also cultured and the actinobacterial isolates were sent for screening against Mycobacterium aurum with two Kocuria kristinae isolates and a Streptomyces albdioflavus isolate showing good antimycobacterial activity.

One of the major impediments in the development of marine natural products is the provision of biologically active natural products in sufficient quantity for complete pharmacological evaluation, clinical trials and eventual commercial production. Marine microorganisms show great promise in providing a renewable source of biologically active natural products. The main aim of this study was to develop and evaluate methods for the isolation, identification and cultivation of marine fungi from the South African marine environment for the production of biologically active secondary metabolites. Twenty-four species of fungi were isolated from marine algae collected from the intertidal zone near Port Alfred, South Africa. The fungi were cultivated in small-scale under static and agitated conditions and their crude intra- and extracellular organic extracts were screened by ¹H NMR and a series of bioassays. Using this as a basis, one isolate was selected for further study. By analyses of the lTS1 region of the ribosomal DNA, the fungal isolate was identified as a marine-derived isolate of Eurotium rubrum (Aspergillus ruber). Although E. rubrum has been isolated from the marine environment, no investigations have been undertaken to determine the adaptation of these isolates to the marine environment. In order to optimise productivity, creativity and incubation time, the fungus was cultivated in small-scale using a variety of carbon (glucose, fructose, lactose, sucrose, marmitol and maltose) and nitrogen sources (ammonium tartrate, urea, peptone and yeast extract). An HPLC-DAD method was developed to assess the metabolic creativity and productivity under different fermentation conditions. Distinctive variations in the range and yield of metabolites produced as well as morphology and growth time were observed. The crude extracts from all fermentations were combined and six known compounds were isolated by reversed-phase chromatography and their structures elucidated by spectroscopic techniques. The known compounds were fIavoglaucin, aspergin, isodihydroauroglaucin, isotetrahydroauroglaucin, neoechinuline A and physcion. Neoechinuline A, isodihydroauroglaucin and isotetrahydroauroglaucin showed activity against oesophageal and cervical cancer cell lines.