Year
2011
Recommended Citation
Matesic, Lidia, Development of Isatin-based compounds for use in targeted anti-Cancer therapy, thesis, , University of Wollongong, 2011. https://ro.uow.edu.au/theses/3346
Abstract
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.
Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.