Submitted in partial fulfillment of the requirements for the Degree of Master of Technology: Biological Sciences, Technikon Natal, 2000. / This study focussed on the anti-bacterial, anti-fungal and anti-protozoal activity of four plant extracts, maackiain and formononetin from Virgilia oroboides and chlorophorin and lroko from Chlorophora excelsa / M

Cyanobacteria are notorious for their potential to produce toxins with human health effects, particularly the hepatotoxic microcystins (MCs), but cyanobacteria also produce other bioactive compounds. A wide variety of oligopeptides including aeruginosins, cyanopeptolins and cyanobactins may be as toxic as MCs. To investigate the production of these compounds, an UPLC QTOF-MS/MS method was developed to compare the metabolomic profiles of various strains of a common bloom-forming and toxigenic species, Microcystis aeruginosa, as well as those obtained from lakes with mixed cyanobacterial assemblages. Although many compounds could not be confirmed, MCs were rarely the dominant secondary metabolite in any sample. Since the biological role of MCs remains unknown, I tested the hypothesis that MCs provide protection against oxidative stress as induced through exposure to the herbicide atrazine and UV radiation in pure cultures of toxic vs non-toxic strains. Results were inconclusive and varied between strains suggesting other mechanisms exist to counter oxidative stress.

New methodology has been utilized to provoke or increase targeted metabolic pathways in microbes. The low hanging fruit of natural products has been discovered over the last 50 years. To continue finding new metabolites to be used as possible drug candidates, methodology development such as those proposed herein are necessary. This methodology uses extracts from known pathogenic bacteria to elicit production of latent biosynthetic pathways from environmental bacterial isolates that may be active against the original pathogenic strains. A new compound, MAV-1 (1) of the diketopiperazine family (Figure 1) was isolated and identified utilizing these techniques. The structure of MAV-1 (1) was defined by a combination of mass spectroscopy (MS) and nuclear magnetic resonance (NMR) spectroscopy. Discovery of MAV-1 (1), a possible precursor to other known compounds, demonstrates the continuing utility of microbial sources with new chemodiversity.

A new class of drugs has been developed for treatment of muscle and bone mass wasting diseases called non-steroidal selective androgen receptor modulators (SARMs). Because of their positive androgenic effects such as muscle gain, they are desirable as performance enhancers. One of those substances is LGD-4033 (4-[(2R)-2-[(1R)-2,2,2-trifluoro-1-hydroxyethyl]pyrrolidin-1-yl]-2-(trifluoromethyl)- benzonitrile). It has been detected in human samples in routine doping control and another SARM has been detected in an equine blood sample in routine doping control. It is therefore indicated that SARMs need to be screened for in routine testing in equestrian sport. The aim of this project was to identify what metabolites were found in equine plasma after an intra venous administration of LGD-4033 using UHPLC coupled with QToF-MS and determine whether the parent compound or any of its metabolites were most suitable for doping control. With the sample preparation method protein precipitation, six possible metabolites were identified in samples from three horses. Two of the metabolites were identified as phase I-metabolites (monohydroxylated and dihydroxylated). Four of the metabolites were identified as phase II-metabolites, where glucuronidation had occurred. The most suitable species for doping control were determined based on a semi- quantification and were M1a, M2 and M3a.

D.Phil. (Biochemistry) / This thesis concerns a study of the effect of isonitrosoacetophenone on plant metabolism. Three different systems were investigated; cultured tobacco and sorghum cells as well as Arabidopsis thaliana plants, and a metabolomic approach was followed. Unlike most scientific studies, metabolomics is a discipline which is not driven by a specific hypothesis, but rather by the obtained data to add scientific insights to the topic under investigation. As such, the current study lacks a definite overarching hypothesis, but specific objectives were outlined and answered in each experimental chapter. This thesis is therefore presented as a compilation of nine chapters in which experimental/research work is described in Chapter 3- 8. It is important to note that each chapter is presented in accordance with the guidelines for the respective journal in which the corresponding manuscript was published or submitted to.

No description available.

This thesis comprises both a natural product investigation and a synthetic component. The natural product investigations are presented in Chapters Two and Three. In Chapter Two the isolation and spectroscopic identification of the new isocopalane diterpene 12S,13R,14Sisocopalan- 13-ol-12,14-diacetate (2.1) and two known 3-(14S)-isocopal-12-ene-15-oyl-1- acetyl-sn-glycerol (2.2) and 3-(14S)-isocopal-12-ene-15-oyl-2-acetyl-sn-glycerol (2.3) from a single, large, unidentified sub-Antarctic nudibranch, collected near Marion Island, approximately 2000 km south of Cape Town are described. Chapter Three discusses the isolation, spectroscopic structure elucidation and anti-oesophageal cancer activity (3.1-3.4 only) of two known labdane diterpenes 6β,7α-diacetoxylabda-8,13E-dien-15-ol (3.1) and 2α,6β,7α-triacetoxylabda-8,13E-dien-15-ol (3.2) and one new 6β,7α,15-triacetoxylabda 8,13E-diene (3.3), as well as new 3α,11-dihydroxy-9,11-seco-cholest-4,7-dien-6,9-dione (3.4) and cholest 7-en-3,5,7-triol (3.5) from the endemic pulmonate mollusc, Trimusculus costatus. The absolute configuration of 3.2, and hence 3.1 and 3.3 (from biogenetic arguments) was determined through X-ray diffraction of a single crystal of the camphanate ester of 3.2. The absolute configuration of the secondary hydroxyl at C-3 of 3.4 was established using the Modified Mosher’s method. The synthetic component of the thesis commences in Chapter Four with the semi-synthesis of labdane diterpene nitriles 9α-cyano-15,16-epoxy-7β-hydroxylabda-13(16),14-dien-6-one (4.1), 9α-cyano-15,16-epoxy-7-hydroxylabda-7,13(16),14-trien-6-one (4.2) and 9α-cyano-15,16- epoxy-6β,7β dihydroxylabda-13(16),14-diene (4.3) from the terrestrial labdane diterpene, hispanolone (4.4). This work is an extension of previous synthetic studies directed towards the synthesis of T. costatus metabolites. Diterpenes 4.1-4.3 exhibited in planta activity against the economically important crop pathogens, Magnaporthea grisea and Puccinia recondita. Chapter Five describes the successful semi-synthesis of two isomeric marine molluscan labdane diterpene aldehyde metabolites, labd-13E-ene-8β-ol-15-al (5.1) and labd-13Z-ene- 8β-ol-15-al (5.2) from the commercially available, terrestrial plant derived, labdane diterpene manool (5.3). Diterpenes 5.1 and 5.2, originally isolated from the Mediterranean nudibranch,Pleurobranchaea meckelii and selected diterpenes arising from this synthesis were evaluated for their activity against an oesophageal cancer cell line (WHCO1). Chapter Six further develops the research discussed in Chapter Five, where ethyl 17-norabiet-13(15)-E-en-8β-ol- 16-oate (5.49) and ethyl 17-norabiet-13(15)-Z-en-8β-ol-16-oate (5.50) were first semisynthesized serendipitously. Based on their structural relationship to naturally occurring tricyclic diterpenes with anti-plasmodial activity, tricyclic diterpenes, 17-norpimaran-13α- ethoxy-8,16-olactone (6.6), 17-norisopimar-15-ene-8β,13β-diol (6.7), 17-norisopimarane- 8β,16-diol (6.8) and 17-norabiet-13(15)-ene-8β,16-diol (6.9) were semi-synthesized from the terrestrial labdane diterpene, 5.3, and critically evaluated for their antimalarial potential from parasite inhibition and haemolytic studies.

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.

Chromatographic analyses of crude leaf extracts of Flaveria linearis L. revealed the presence of four acetylenic monothiophenes. Three of these metabolites were purified and structurally characterized by UV, NMR, IR, and GC-MS. Germination, growth, and survival/mortality studies with and without UVA, the activating wavelengths of these metabolites, were conducted with the crude leaf extracts and the purified compounds (taken individually and combined) against selected crop species (lettuce, radish, and carrot). Results suggest that acetylenic metabolites are phytotoxic against lettuce, carrot, and radish, but with variability in response among species. These variations in sensitivity and the allelopathic potential of F. linearis is discussed.

In a program aimed at the identification of compounds responsible for the immunosuppressive and antifertility activities of the perennial twining vine, Tripterygium wilfordii. 5 new and 13 known compounds were isolated from the TRP-4a tissue culture cell line developed from Tripterygium wilfordii. The structures of the new compounds were determined by a combination of spectral analysis, chemical correlation and single crystal X-ray diffraction analysis. 22β-Hydroxy-3-oxoolean-12-en-29-oic acid (137), 22α-hydroxy-3-oxoolean- 12- en-29-oic acid (138) and 3β, 22β-dihydroxyolean-12-en-29-oic acid (139) are new triterpenes possessing an oleanene-type skeleton and were chemically correlated with 3β, 22α-dihydroxyolean-12-en-29-oic acid (51), the structure of which was confirmed by single crystal X-ray diffraction analysis. Oleanolic acid (127), β-sitosterol (128) and polpunonic acid (55), were isolated previously from the TRP-4a cell line in earlier studies in this laboratory. α-Amyrin (145), β-amyrin (146), 3β, 29-dihydroxyolean-12-ene (151) and 3β, llα-dihydroxyolean-12-ene (152) are known triterpenes possessing an oleanene-type skeleton and are isolated for the first time from the TRP-4a cell line. Tingenone (148) and 22β-hydroxytingenone (150) are quinone methide triterpenes, also isolated for the first time from the TRP-4a cell line. Similarly, the novel diterpene, 12-methoxyabieta-8, 11, 13- trien-3α-ol (147) and the novel triterpene, methyl-22β-hydroxy-3, 21-dioxo-D:A-friedo-29-noroleanan-24-oate (149), a member of the friedelane family, are isolated for the first time. A biosynthetic pathway, based on the isolation of 149 and its structural similarity to polpunonic acid (55) and 22β-hydroxytingen6ne (150), is postulated for the quinone methides. The cytotoxic diterpenes, tripdiolide (1) and triptolide (2) and the hydroxy acid, 160, isolated as the methyl ester, 124, have been previously reported from this laboratory. Tripdiolide (1) and triptolide (2) have been shown to possess strong antifertility and immunosuppressive activities. In another aspect of our program, biotransformation studies of the synthetic precursors, 19 (4➙3)abeo-abieta-2, 8, 11, 13-tetraen-19-ol (171) and 19-hydroxy-18(4➙3)abeo-abieta-3, 8, 11, 13-tetraen-18-oic acid lactone (91), and the radioactive congeners, 182 and 209, were carried out using the TRP-4a cell line. It was hoped that the data obtained might shed some light on the "late stage" biosynthetic pathway of the diterpene triepoxides, tripdiolide (1) and triptolide (2). Synthesis of 171 was achieved in 5 steps from dehydroabietic acid (80). The radioactive congener, 182, was synthesised using ¹⁴C-paraformaldehyde with 0.4% incorporation of the radiolabel. Biotransformation of 171 using the TRP-4a cell line yielded 19(4➙3)abeo-abieta-2, 8, 11, 13-tetraen-19-al (185) and 19(4➙3)abeo-abieta-2, 8, 11, 13-tetraen-19-oic acid (186) for spectral identification. Biotransformation of 182 yielded the aldehyde, 183 (33.2%) and the acid, 184 (51.9%), the radioactive congeners of 185 and 186 respectively. Synthesis of 91 was achieved in 4 steps from dehydroabietic acid (80). The radioactive congener, 209, was synthesised using ¹⁴C-methyl iodide via ¹⁴C-dimethylsulphonium methylide, with 0.6% incorporation of the radiolabel. Biotransformation of 91 using TRP-4a tissue cultures yielded 19-hydroxy-7-oxo-18(4➙3)abeo-abieta-3, 8, 11, 13-tetraen-18-oic acid lactone (214), 2β, 19-dihydroxy-7-oxo-18(4➙3)abeo-abieta-3, 8, 11, 13-tetraen-18-oic acid lactone (215), 7β, 19-dihydroxy-18(4➙3)abeo-abieta-3, 8, 11, 13-tetraen-18-oic acid lactone (216) and 2β 19-dihydroxy-18(4➙3)abeo-abieta-3, 8, 11, 13-tetraen-18-oic acid lactone (96), for spectral identification. Biotransformation of 209 yielded the ketone, 210 (56.7%), the hydroxy ketone, 211 (5.9%), the benzylic alcohol, 212 (9.6%) and the C2 alcohol, 213 (6.8%), the radioactive congeners of 214,215,216 and 96 respectively. A biosynthetic pathway to the diterpene triepoxides is postulated based on the oxygenated biotransformation products. [formulas omitted] / Science, Faculty of / Chemistry, Department of / Graduate