Degree Name

Doctor of Philosophy


School of Biological Sciences


The increased incidence of multidrug resistance (MDR) and systemic toxicity to conventional chemotherapeutic agents suggests that alternative avenues need to be explored in the hope of finding new and effective treatments for metastatic disease. Considering natural products have made enormous contributions to many of the anticancer agents used clinically today, the cytotoxic molluscan metabolite tyrindoleninone (1) and its oxidative artifact, 6-bromoisatin (5), were initially used as templates for drug design in this study. Structural modifications to the isatin scaffold afforded a total of 51 isatin-based analogues, 21 of which were new. Cytotoxicity screening of the compounds against a panel of heamatological and epithelial-derived cancer cell lines in vitro, found the di- and tri-bromoisatins to be the most potent, with activity observed in the low micromolar range. Interestingly compound activity was enhanced by up to a factor of 22 after N-alkyl and N-arylalkylation, highlighting the importance of N1 substitution for cytotoxic activity. 5,7-Dibromo-N-(p-methylbenzyl)-isatin (39) was the most active compound overall and exhibited an IC50 value of 490 nM against U937 and Jurkat leukemic cell lines, after 24 h. 5,7-Dibromo-N-(p-trifluoromethylbenzyl)isatin (54) was also of interest, considering the potent cell killing ability displayed against a metastatic breast adenocarcinoma (MDA-MB-231) cell line. Investigation into the molecular mode of action of the N-alkylisatin series of compounds found the p-trifluoromethylbenzyl derivative (54), together with 9 other representative molecules to destabilise microtubules and induce morphological cell shape changes via inhibition of tubulin polymerisation. This resulted in cell cycle arrest at G2/M and activation of the effector caspases 3 and 7, ultimately resulting in apoptotic cell death. Further investigations into the pharmacological profile of compound 54 in vivo, found it to be moderately efficacious (43% reduction in tumour size compared to vehicle control treated mice) in a human breast carcinoma xenograft mouse model. Although histopathological analysis of the bone marrow in situ after acute dosing found only mild haematopoietic suppression, analysis of biodistribution via SPECT imaging found large amounts of activity also in the gut and liver. In an effort to reduce non-target organ up-take and thus increase accumulation of drug in the tumour, the N-benzylisatin 54 was derivatised so as to contain an acid labile imine linker and was conjugated to the targeting protein PAI-2 (a naturally occurring inhibitor of the urokinase plasminogen activation system) via amide bond formation with free lysine residues. The conjugate was found to contain an average of 4 molecules of 54 per protein molecule without affecting PAI-2 activity. Hydrolytic stability of the PAI-2-cytotoxin conjugate at pH 5-7 as determined by UV/Vis spectrophotometry, was directly correlated with the lack of activity observed in vitro, suggesting a need to investigate cleavable linker systems with enhanced lability in the future. Despite this, PAI-2 conjugated to the cytotoxin 5-FUdr through a succinate linker system, showed enhanced and selective uPA-mediated cytotoxicity, in two different breast cancer cell lines which varied in their expression levels of uPA and its receptor. This suggests that PAI-2-cytotoxin based therapies hold potential, in the future, as new therapeutic agents for targeted therapy of uPA positive malignancies, with limited side effects.

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