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The Preparation of a Non-Membrane Associated PIP Analogue to Inhibit the Localisation of AKT
[Thesis]. Manchester, UK: The University of Manchester; 2020.
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Abstract
Phosphatidylinositols (PI) such as phosphatidylinositol-3,4,5-triphosphate (PIP3) play a vital role in cell signalling through recruiting proteins to the plasma membranes. PIP3 is the natural substrate of phosphatase and tensin homologue deleted from chromosome 10 (PTEN), a tumour suppressor protein which has been discovered to be one of the most frequently mutated proteins in cancers. PIP3 is dephosphorylated to phosphatidylinositol-4,5-biphosphate (PIP2) by PTEN, leading to a decrease in the membrane localisation of AKT, a cell survival kinase. In PTEN-null or mutated cells, this dephosphorylation cannot occur thus leading to an accumulation in the levels of PIP3 at the plasma membrane, resulting in increased activity of AKT and thereby increased cell proliferation and growth. Previous research into small molecule inhibitors showed that the binding of PIP3 can be affected by disrupting the interaction between PIP3 and the PH domain of AKT. The purpose of this project was to prepare analogues of phosphatidylinositol (PI) with varying lipophilic chain lengths in the phosphonate group at the 1-position, in order to establish their effects in cells. Initial synthesis led to the formation of 1,4 myo-inositol diol through the selective protection of the hydroxyl groups. Following on from this, phosphorus chemistry was explored in the hope of identifying a suitable phosphorus fragment, for the selective phosphorylation of the 1-position of the protected myo-inositol headgroup. Hydrolysis and oxidation issues with phosphorus(III) species highlighted issues with this line of chemistry, so attentions turned to phosphorus(V) chemistry. It was revealed that the selective protection of the 1,4-diol was not possible, with phosphorylation reactions leading to the bis-phosphorylated product. This led to the design and synthesis of a penta-protected myo-inositol headgroup which was phosphorylated with a newly synthesised phosphorylating agent with the POM prodrug incorporated. This project also sought to form a PI analogue in which the myo-inositol headgroup is modified, with the 2 and 6 hydroxyl groups removed. The desired stereochemistry of the 1,3,4 and 5 hydroxyl groups were set through a sequence of steps leading to the successful formation of the headgroup which was phosphorylated to yield the desired phosphonate. This synthetic approach was used to synthesis four analogues with varying alkyl chain lengths and three of these phosphonates were evaluated using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and sulforhodamine B (SRB) assays. From these studies, an apparent SAR was established with the longer chain phosphonates displaying higher activity.