Investigation of 1,4-quinone bioisosteres and the synthesis of bolinaquinone analogues as clathrin inhibitors

Clathrin is a ubiquitous protein primarily acting as the major protein in clathrin mediated endocytosis (CME). CME is a major process by which extracellular cargo can cross the cell wall, allowing for transport of nutrients, receptors, and other material. Misfunctioning CME has been implicated in multiple diseases, acting in cancer, neurological conditions such as Alzheimer’s and epilepsy, and is a major pathway exploited by viruses for cellular entry. A recent secondary non-CME function has been found for clathrin wherein clathrin in a TACC3-clathrin-chTOG-GTSE1 complex crosslinks and stabilises microtubules allowing for mitosis progression and ultimately cell division. Small molecule inhibitors of clathrin, and by extension inhibitors of CME is a potential route to modulate the above diseases. The current scope of reported CME inhibitors is limited to the Pitstop® series of compounds and early lead compounds such as ES9-17. There are, however, disagreements on target specificity and mode of action. A marine sponge metabolite, bolinaquinone (BLQ) 1 containing a 1,4-quinone moiety, was shown to inhibit CME in vitro (IC50: not reported). Structurally, BLQ possesses a complex decalin region which has limited further synthetic development and analogue generation. Previous work in the McCluskey group identified AG1166 2 (CME: 4.6 ± 0.5 µM, Dyn1: 29.7 ± 3.3 µM, ELISA: 2.77 ± 0.9 µM) as a structurally simplified naphthoquinone lead, possessing low micromolar clathrin inhibition which translated well in cell CME studies. Molecular docking with BLQ found a potentially novel fifth site of interaction to the clathrin terminal domain (CTD). This is yet to be experimentally validated. This thesis elaborates on the BLQ and AG1166 scaffolds, with the aim to replace the 1,4-quinone moiety found in these structures, in efforts to reduce potential promiscuity. Quinones are known to redox cycle, producing reactive oxygen species (ROS) which can result in DNA damage and unwanted cytotoxicity. In addition, this moiety is prone to covalent adduct formation, a potentially undesirable off-target effect. These investigations detail the molecular docking and ab initio calculation lead design of non-quinone containing leads through the synthesis and biological testing of five potential quinone bioisosteres. This ultimately resulted in the identification of three new quinone-free leads, 3-(cyclohexylmethylene)isochroman-1,4-dione 240 (CME: 10.9 µM, Dyn1: 20% at 100 µM, ELISA: not tested), octyl 4-((3-bromo-1,1-dioxido-4-oxo-4H-thiochromen-2-yl)amino)benzoate 355 (CME: not tested, Dyn1: not tested, ELISA: 6.09 µM) and 2-cyclohexylethyl 4-((3-bromo-1,1-dioxido-4-oxo-4H-thiochromen-2-yl)amino)benzoate 356 (CME: not tested, Dyn1: not tested, ELISA: 6.63 µM) of unique chemotype, with activities within 2- to 3- fold of the AG1166 lead. DFT ab initio and molecular docking methods were developed in order to guide synthetic elaboration.