Using Light to Regulate the Activity of RET Kinase: Design, synthesis and evaluation of photoswitchable DFG-out RET kinase inhibitors

Abstract: Protein kinases are essential for signal transduction and regulate most cellular processes, such as metabolism, membrane transport, motility, and cell cycle. Although they play a critical role in cells and have a strong association with diseases, details of their physiologic and pathologic mechanisms remain at least partially unclear, especially the spatiotemporal dynamics of the signalling. Reversible photo-regulation of kinase inhibitors could be an approach to gain spatiotemporal control, thereby resolving the above-mentioned issues. This thesis comprises the synthesis and characterisation of novel azoheteroarenes photoswitches and their use in the development of photoswitchable kinase inhibitors. REarranged during Transfection (RET) is a kinase belonging to the receptor tyrosine kinase family. RET was used as a model target in the development of photoswitchable DFG-out kinase inhibitors. A methodology was initially developed to synthesize aromatic N-heterocycle derived azoheteroarenes, using Buchwald-Hartwig coupling and microwave-assisted dehydrogenation as the keys steps. With this methodology as the main strategy, a series of azoheteroarenes was prepared and their photophysical properties were based on UV-vis absorption spectroscopy and 1H-NMR. Among them, quinoline and 7-azaindole based azoheteroarenes showed the efficiency of synthesis and outstanding photophysical properties in all aspects. By integrating the quinoline-based azoheteroarene into the pharmacophore of a reported DFG-out kinase inhibitor, the first photoswitchable DFG-out RET kinase inhibitor was synthesized and evaluated with respect to the photophysical properties and bioactivity. Four different strategies were explored in order to enlarge the difference of biological activity between the E-isomer and light enriched Z-isomer, using a quinolone-based azoheteroarene as a model system: (i) installing substituents on the quinoline ring; (ii) introducing the second azo unit; (iii) introducing fluorine atoms on the ortho-position of the azoheteroarene phenyl ring; and (iv) changing the heterocyclic moiety. In addition, the corresponding stilbene compounds were prepared as more thermally stable model compounds of the azo-based kinase inhibitors. This provided detailed data on the structure-physical properties relationship, as well as structure activity relationship. As a result, the 7-azaindole-derived azo type inhibitor showed excellent thermal stability and resistance to photo-fatigue. Moreover, it displayed a significant difference of inhibition activity between the E-isomer and the light-enriched Z-isomer sample in both enzymatic and living cell assays.