Ligand-targeted drug delivery is currently one of the most challenging areas in pharmaceutical research involving the judicious choice of the drug, linker and targeting ligand. A promising new strategy involves conjugating a cytotoxin with a tumour-targeting protein through an acid-labile linker that is stable at physiological pH. Internalisation at the target tumour site via receptor-mediated processes exposes the conjugate to endosomal and lysosomal pH (4.5-6.0), resulting in selective release of the original cytotoxin inside the tumour cell. This thesis describes the synthesis of a new targeted anti-cancer agent and in particular, the development of the drug and linker component.

Drug development: Isatin (11) and its derivatives are present in numerous natural products including the Australian marine mollusc, Dicathais orbita. These derivatives possess a broad spectrum of biological properties including anti-cancer activity. New tricyclic and polycyclic derivatives of isatin (11) were prepared since previous research within our group indicated isatin derivatives exhibit highly potent cytotoxic effects against a variety of human cancer cell lines. To expand the cytotoxic structure-relationship activity of isatin derivatives, the N1-C7 region of isatin (11) was investigated through the addition of a 5- or 6-membered ring (i.e. pyrrolo[3,2,1-hi]indole-1,2-dione and pyrrolo[3,2,1-ij]quinoline-1,2-dione derivatives respectively) to yield 23 analogues, 18 of which were novel. In vitro assays revealed the inclusion of the extra ring reduced cytotoxicity compared to the parent molecules. However, a novel brominated tetracyclic acridine-based isatin, 4-bromo-6H-pyrrolo[3,2,1-de]acridine-1,2-dione (144), displayed an IC50 value of 3.01 μM against U937 lymphoma cells and was the most cytotoxic tricyclic/polycyclic isatin derivative synthesised within this project. In general, thetetracyclic and pentacyclic isatins displayed greater activity than the analogous tricyclic derivatives.

Linker development: The design, synthesis and utility of a series of novel acid-labile, imine-based linkers, which were attached to a potent )-alkylisatinderived cytotoxin, 5,7-dibromo-)-(p-methoxybenzyl)isatin (24) was also investigated. In order to model the conjugation of these acid-labile linkers to the lysine residues of tumour-targeting proteins, the aryl imines (153-157) were coupled via their free carboxylic acids to a protected lysine amino acid residue to produce a novel series of imine-lysine conjugates (158-162). Both the aryl imines (153-157) and the imine-lysine conjugates (158-162) were stable for an extended period at pH 7.4 but readily cleaved in aqueous acidic solutions at physiological temperature, with half-lives ranging from 17.0 to 85.2 min. Observed rates of hydrolysis for the embedded imine-acid moiety were in the order para-phenylpropionic acid > phenylacetic acid (para > meta) > benzoic acid (meta > para).

The 3-iminoisatins (153-157) and the isatin-lysine conjugates (158-162) were also assessed for biological activity. 3-Iminoisatins 153-157 displayed cytotoxicity in the low to sub-micromolar region against U937 cells, while isatin-lysine conjugates 158-162 all exhibited cytotoxicity in the nanomolar region after being exposed to U937 cells for 24 h. The meta-substituted isatin-lysine 161 was the most cytotoxic molecule synthesised within this entire project, with an IC50 value of 165 nM against U937 cells.

Ligand-targeted drug development: Finally, the 3-iminoisatin 155 was conjugated to the tumour-targeting proteins PAI-2 (an endogenous inhibitor of the urokinase plasminogen activation system) and transferrin (Tf) via amide bond formation with free lysine residues. UV-Vis spectrophotometry was used to visualise evidence of the isatin-protein conjugates 165 and 166. Upon treating the transferrin receptor over-expressing breast cancer cell line MDA-MB-231 with the isatin-Tf conjugate (166) for 48 h, a significant cytotoxic response (compared to Tf) was observed. At the highest concentration tested (1.56 μM), the conjugate 166 was 47±13% more cytotoxic than the Tf control. This work suggests that imine-linked isatin-protein conjugates possess the potential to be used as new therapeutic agents in the future.