Biological evaluation of N-Ferrocenyl amino acid and dipeptide ester derivatives as potential anti-cancer agents and investigation into their mode of action in preparation for in vivo evaluation.

The aim of this research thesis was to explore the structure-activity relationship (SAR) of ferrocenyl-peptide bioconjugates and to explore methods by which these compounds exert their anti-proliferative on cancer cells. Furthermore, the applicability across different cell types including cancer cells which have acquired resistance to chemotherapeutics and non-cancerous cells was investigated. Finally, we attempted to overcome the insolubility of the ferrocene compounds in a liquid which is suitable for delivery in vivo. We evaluated a panel of novel ferrocenyl-peptide bioconjugates for their anti-cancer potential in lung and melanoma cells. Utilizing structure-activity relationship (SAR) studies we found the anti-proliferative activity of the compounds tested varies greatly depending on the configuration of the compound. Two of the most active compounds N-(6-ferrocenyl-2-naphthoyl)-glycine-glycine ethyl and methyl ester showed IC50 values in the nanomolar range. Cisplatin was used as a comparison and was found to have higher IC50 values. From these SAR studies the following trends were observed: 1. 1-alkyl-1’- N-ortho- derivatives exhibited lower growth inhibition compared to the meta and para derivatives for the dipeptides employed 2. Cytotoxicity of the 1-alkyl-1’-N-para, N-meta and N-ortho-(ferrocenyl) benzoyl dipeptide esters, decreases with the increase of the size of the alkyl group incorporated (propyl < ethyl < methyl) on the ferrocene moiety 3. The order of the amino acids in the dipeptide chain is crucial for activity, the trend being Gly Gly > Gly L-Ala > Gly L-Leu > Gly L-Phe. 4. The presence of the ethynyl moiety does not result in any significant enhancement of the anti-proliferative effect of the N-(ferrocenyl) benzoyl and naphthoyl dipeptide derivatives. 5. Replacing the ethyl ester in the previously most active, N-(6-ferrocenyl-2-naphthoyl) glycine glycine ethyl ester, with a methyl ester further increases its activity. Two of the biggest factors which affect the use of chemotherapeutic drugs in a clinical setting are the side-effects caused by the drug and also resistance of the cancer cells to the drug. We have addressed this issue by testing the most active compounds from the SAR studies in cell lines which have been made resistant to two common chemotherapeutics, cisplatin and temozolomide. We also tested the two most active compounds in normal human dermal fibroblast (NHDF) cells as an indicator of the effect which the compounds would have on healthy tissue. Results were favourable as one of the compounds was able to overcome the resistance and both compounds showed less cytotoxicity against NHDF than cisplatin. We analysed the effect the most active compound had on the cell cycle in lung cancer and melanoma cells. We observed an increase in the sub-G0 fraction which is indicative of apoptosis. Induction of apoptosis was confirmed using the TUNEL assay which detects late stage apoptotic cells. Due to the insolubility it was not possible to test the compounds in vivo. However, we tested a range of approaches to improve their solubility for future in vivo testing. From the results gathered, a number of conclusions can be made. It is clear that chemical structure of the ferrocenyl compounds plays an important role in their activity levels. A key aspect in the mode of action of the compound tested is the induction of apoptosis/necorsis. With resistance to chemotherapeutics being an ongoing problem in cancer treatment, the ability of a compound to overcome resitance to important chemotherapeutics while having a significantly lower activity level in normal human cells is an appealing characterstic of the tested compound. Finally, the issue with solubility has, at the time of writing this research thesis, been unsolved. However, with promising research being undertaken in the field of improving solubility of compounds, the future looks hopeful.