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The intramolecular styryl Diels – Alder (ISDA) reaction is a rare and unique [4+2] cycloaddition with potential in the syntheses of polycycles. Its utility is based on the formation of two rings and one stereocenter in a single step, making it an efficient method for the construction of lignan-type natural product targets. Detailed mechanistic studies with complex esters and the application to natural product synthesis has been limited due to drawbacks including the loss of aromaticity, producing slow reactivity, a potentially problematic thermal [1,3]-hydrogen shift, and electronic mismatch related to the substituents on the aryl functional groups. In this research, we found conditions that led to the successful application of the ISDA reaction on the total synthesis of the anticancer deoxypodophyllotoxin and the antiviral agent F4-4. Deoxypodophyllotoxin was synthesized in seven steps, which is a very concise synthesis for a complex lignan. Density functional theory was used to analyze the two components of the ISDA reaction, the [4+2] cycloaddition and the [1,3]-hydrogen shift. Several pathways were analyzed, and the rate determining step was determined to be the [4+2] cycloaddition. We also found that the [1,3]-hydrogen shift is assisted by di-tert-butylhydroxytoluene and is lower in energy than the [4+2] cycloaddition.The two targets chosen for this research have important biological activities. Deoxypodophyllotoxin is known as a potent anticancer agent related to podophyllotoxin. Podophyllotoxin is a more abundant lignan which is the precursor of the FDA approved drugs etoposide and teniposide, used for the treatment of lung and testicular cancer. Other biological activities of deoxypodophyllotoxin have been found including antibacterial, antiviral, and anti-inflamatory activity. Also, it was recently discovered that F4-4 possesses antiviral activities against Herpes simplex viruses 1 (HSV-1), 2(HSV-2), and H. zoster. Since both deoxypodophyllotoxin and F4-4 are not available in large quantities from natural sources, chemical synthesis is important for continuing research and drug development of these compounds.

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Thesis (MSc.(Biochemistry)) -- University of Limpopo, 2021 / Drug discovery from medicinal plants continues to play an important role in the development of anticancer agents, this is because medicinal plants are reservoirs of bioactive compounds that exert a plethora of pharmacological effects on human beings. This study aimed to analyse the phytochemical constituents of the Momordica cardiospermoides crude acetone and methanol leaf extracts as well as investigate their potential anti-metastatic effects on the MDA-MB-231 breast cancer cell line. Momordica cardiospermoides leaves were extracted with absolute methanol or acetone to produce crude methanol and acetone extracts, respectively. The extracts were then screened and analysed for phytochemicals using thin layer chromatography, qualitative and quantitative phytochemical tests, and their antioxidant activity was determined using the quantitative 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical scavenging activity assay. The fingerprint profiles of the M. cardiospermoides leaf extracts revealed that compounds of the acetone extracts were optimally separated in the nonpolar mobile phase (TAE), whereas those of the methanol extract separated best in the polar mobile phase (EMW), thereby suggesting that the crude acetone and methanol extracts had more non-polar and polar compounds present, respectively. Furthermore, the qualitative phytochemical analysis indicated the presence of various phytochemicals such as flavonoids, steroids, coumarins, and tannins in both plant extracts, however, saponins were found present in the methanol extract and not in the acetone extract. Moreover, quantification of major phytochemicals revealed that the acetone extract had the highest total phenolic content (23.0683 mg GAE/g), total tannin content (22.0442 mg GAE/g) and total flavonoid (32.6933 mg QE/g) content as compared to the methanol extract (14.2349 mg GAE/g, 11.3164 mg GAE/g and 7.692 mg QE/g respectively). The DPPH free radical scavenging activity assay revealed that the extracts exhibited an increase in percentage inhibition/ DPPH scavenging effect, with an increase in extract concentration. The results also revealed that the acetone extract possessed a higher radical scavenging activity as compared to the methanol extract. These results are in correlation with the quantitative analysis of the extracts, as all the major phytochemicals found in higher amounts in the acetone extract have antioxidant properties. The extracts were then assessed in vitro for their cytotoxic effects on MDA MB-231 breast cancer cells and HEK 293 cells using the cell count and viability assay and the results obtained revealed a concentration-dependent decrease in the viability of MDA-MB-231 cells at 24 hours of treatment with either the acetone or methanol extract. Comparatively, treatment of HEK 293 cells with the acetone extract resulted in a significant decrease in the percentage of viable cells, whereas treatment with the methanol extract had no significant effect on the viability of HEK 293 cells, as the percentage of viable cells was maintained at 85–98% at 24 hours of treatment. These results also revealed that the methanol extract is more selective to cancer cells in comparison to the acetone extract, suggesting that the methanol extract is a better antineoplastic candidate. The mode of cell death induced by the methanol or acetone extracts was assessed using the acridine orange and ethidium bromide dual staining assay and the annexin V and dead cell kit. The results from the acridine orange/ethidium bromide dual staining assay showed that both extracts induced nuclei and cellular morphological changes in a concentration-depended manner, at 24 hours of treatment. Moreover, the annexin V and dead assay kit results revealed that the acetone extract induced necrotic cell death, while the methanol extract induced apoptotic cell death. Since the acetone extract was shown to be non-selective towards normal cells and induced necrotic cell death, it was discontinued for further assays. The effect of the methanol extract on MDA-MB-231 cell migration and attachment was determined using the wound healing assay and the adhesion assay. The results revealed that treatment with 150 or 300 µg/ml significantly suppressed MDA-MB-231 cell migration, associated with serpin E1 downregulation and TIMP-1 upregulation, at 24 hours of treatment. Moreover, treatment with the methanol extract also significant inhibited MDA-MB-231 cell adhesion in a concentration-dependent manner, as evident by the decrease in the number of crystal violet stained cells. The effect of the methanol extract on the expression of matrix metalloproteinase-2 and -9 was assessed using western blotting, and the results revealed that the extract significantly downregulated the expression of both MMP-2 and -9, suggesting that the methanol extract has inhibitory effects on MDA-MB-231 cell invasion. The human angiogenesis antibody array kit was then used to determine the effect of the extract on the expression of angiogenesis-related proteins. Treatment with 150 or 300 µg/ml of the extract significantly upregulated the expression levels of tissue inhibitor of metalloproteinases (TIMP) -1 and thrombospondin-1 in a concentration-dependent manner. The results also revealed a significant downregulation in the expression of serpin E1, in a concentration-dependent manner, in comparison to the untreated control. However, the expression of uPA, VEGF, and IGFBP-1, 2 and -3 was upregulated following treatment with 150 and 300 µg/ml of the extract. In conclusion, the current study demonstrated the potential of M. cardiospermoides crude methanol extract as an effective anti-metastatic agent or a source of compounds with anti-metastatic properties / South African Medical Research Council (SAMRC) Research Capacity Development Initiative and National Research Foundation (NRF)

The complex natural product paclitaxel (Taxol®), first isolated from Taxus brevifolia, is a member of a large family of taxane diterpenoids. Paclitaxel is extensively used for the treatment of solid tumors, particularly those of the breasts and ovaries. In order to obtain additional information about the mechanism of action of paclitaxel and the environment of the paclitaxel-binding site, several fluorescent analogs of paclitaxel were synthesized, and their biological activities have been evaluated. For the investigation of possible synergistic effects, concurrent modifications on selected positions have been performed and their biological evaluation were studied. / Ph. D.