Development of novel phospho-amino acid prodrugs as powerful tools in drug discovery

Acting as protein-protein inhibitors, phosphotyrosine/serine-mimetics have demonstrated potent and diverse therapeutic activities, particularly against cancer. In particular, phosphotyrosine- derived molecules, peptic or peptidomimetic, could be applied to re-establishing control of the hyperactivated STAT/JAK pathway in several cancers. Overexpression of tumour-promoting genes has been associated with abnormal accumulations of signal transducer and activator of transcription (STAT) proteins being phosphorylated at their SH2 domain, by specific Janus Kinases (JAKs), at specific phosphotyrosine residues. Abundancies of phosphorylated STAT proteins can dimerise, allowing translocation to the nucleus where overexpression of survival genes occurs, aiding tumourigenesis. Small, synthetic molecules that mimic the STAT3 phosphotyrosine residue and inhibit STAT3 homodimerisation have been shown to prohibit dimerisation and gene overexpression. Despite their therapeutic promise, phosphotyrosine-mimetic drugs have not progressed to the clinics owing to their biocleavable and charged phosphate moieties. The repulsive charge of the phosphate group makes this class of compounds impermeable to cell membranes. This shortfall is paralleled in phosphoserine-mimetic drug molecules, which also contain charged phosphate functionalities. In this work, we explored how encaging these phosphate groups in a novel prodrug form would overcome their permeability issue, which could translate into improved pharmacological activity. To this end, the research hypothesis was evaluated on a known phosphotyrosine-mimetic STAT3 inhibitor, ISS-610, as a proof-of-concept. A small library of novel ISS-610-Me phosphoramidate prodrugs was synthesised and evaluated for their anti-STAT3 activity and stability. The positive results from this small series validated the proposal and the prodrug technology was thus progressed to a novel STAT3 inhibitor, derived from the anti-STAT3 pharmacophore, S31-201. A larger library of novel phosphoramidate and known phosphate prodrugs was synthesised for this series, alongside a novel phosphate prodrug delivery system, which also displayed improved potency and good stability. The prodrug application was then applied to phosphopeptide-mimetic molecules, specifically to two anticancer molecules, which inhibit STAT5b or 14-3-3 dimerisation. Upon synthesis of these compounds and their prodrugs, the biological activity of the prodrugs was found to be superior to the parent compounds. In conclusion, this work collectively highlights the potential and applicability of phosphoramidate prodrug technology for improving the delivery and, thus, the pharmacological activity of phosphopeptide-mimetic (anticancer) therapeutics.