Optimisation of analytical, biological, and synthetic methods relevant to the pre-clinical development of orellanine as a targeted therapeutic for renal cell carcinoma

Abstract

The work outlined in this thesis addresses key analytical, biological, and synthetic challenges associated with the development of the fungal nephrotoxin orellanine as a potential targeted therapeutic for renal cell carcinoma. By optimising chromatographic methods, refining assay strategies through testing of a broad series of non-orellanine targeted conjugates, and developing a tunable orelline scaffold, this work establishes a framework to support the future pre-clinical development of orellanine-based conjugates. Against the backdrop of increasingly popular targeted therapeutics incorporating naturally derived cytotoxic payloads, orellanine is introduced as a fungal nephrotoxin with remarkable intrinsic tissue selectivity, making it a promising candidate for further refinement within such targeting strategies. Its unique bipyridine N-oxide structure underpins both its biological activity and pronounced photolability, complicating analytical detection and synthetic manipulation. Existing methods for the analysis and synthesis of orellanine and its photodegradants orellinine and orelline are therefore critically assessed, identifying key barriers to successful pre-clinical development. To address these challenges, chromatographic methods for the qualitative and quantitative analysis of orellanine were developed and optimised. A novel cellulose-based thin layerchromatography (TLC) method enabled clear resolution of orellanine from its photodegradants, representing a significant improvement on previously reported approaches. Translation to column-based methods led to the development of an optimise disocratic high performance liquid chromatography (HPLC) protocol, and subsequently a liquid chromatography-mass spectrometry/mass spectrometry selective reaction monitoring (LC-MS/MS SRM) method capable of detecting orellanine at concentrations as low as 0.05 μg/mL. In parallel, assay conditions for the evaluation of targeted drug conjugates were all investigated using a series of colchicine-site-binding MTAs and their targeting peptide conjugates as a model system. In vitro invasion and migration assays, along with ex vivo aortic ring studies revealed substantial variability in conjugate activity relative to their unconjugated payloads. Conjugates sharing a common cytotoxic payload exhibited differing anti-migratory activity between cell lines, and the relative ranking of conjugates by anti-migratory activity was not consistent. These differences were further amplified in an ex vivo context, where the identity of the targeting peptide was shown to modulate the activity of conjugates from minimal effect to inhibition of angiogenesis comparable to the known anti-angiogenic agent combretastatin A-4. Together, these results indicate that linker cleavage efficiency is a key determinant of observed conjugate activity, and that this varies depending on the biological context of the assay. These findings underscore important considerations for the future biological assessment of orellanine conjugates. Finally, the synthetic challenges posed by orellanine’s symmetrical hydroxylation pattern were addressed through the development of an orthogonally protected orelline scaffold. The introduction of the hydroxyl moiety at the 4-position was optimised, and a range of protecting group strategies are reported for the first time in the context of orellanine synthesis. Several Pd-catalysed coupling strategies are outlined, culminating with the successful synthesis of the novel compound 1.6C, the first reported orelline derivative with flexible orthogonality between the 3,3’ and 4,4’ positions. This scaffold provides a foundation for the regiospecific synthesis of defined orellanine conjugates. Taken together, this work advances the analytical, biological, and synthetic understanding required to support the rational development of orellanine-based targeted therapeutics for use in the treatment of renal cell carcinoma.