Development of alkaloid derivatives to probe novel α4β2 nicotinic acetylcholine receptor (nAChR) binding sites

Abstract

Nicotinic acetylcholine receptors (nAChRs) are a complex class of ligand gated ion channels consisting of multiple subtypes with different stoichiometries. Of these, the two most commonly found in the brain are the α7 and α4β2 nAChRs. The binding site of agonists and competitive antagonists at nAChRs is well established. However, the binding site of many allosteric modulators remains unknown. Additionally, despite their importance in brain function, the role of specific subtypes and stoichiometries is largely unknown. This thesis deals with the synthesis of alkaloid derivatives that can be used to establish the binding sites of allosteric modulators and study the role of specific subtypes and stoichiometries in the brain. Such information can be used to develop better drugs and drug targets for the treatment of neurological diseases. Galanthamine and codeine are reported to be positive allosteric modulators at nAChRs. In order to establish their binding site, thiol reactive analogues of galanthamine, codeine and the structurally similar alkaloid, morphine, were synthesised for use as probes in covalent trapping experiments. The α,β-unsaturated ketone derivatives of each alkaloid; narwedine, codeinone and morphinone were synthesised along with the codeine mustard and a protected derivative of the benzyl chloride analogue of codeine. While the chlorinated derivatives were too unstable for use as probes, the α,β-unsaturated ketone derivatives were stable in aqueous solution and their reactivity towards thiols was assessed by monitoring their reaction with a cysteine derivative. All of the α,β-unsaturated ketone derivatives displayed sufficient reactivity for use as thiol reactive probes. Methyllycaconitine (MLA) is an antagonist at nAChRs that is known to bind at the α7–α7 interface of α7 nAChRs and at the α4–α4 and α4–β2 interfaces of α4β2 nAChRs. Small bicyclic ester analogues of MLA were synthesised with functional groups targeting key residues that are unique to the binding sites at the α4–α4 and α4–β2 interfaces of α4β2 nAChRs. Esters with the pyridine moiety were synthesised to target an aspartic acid residue in the α4–β2 binding site via salt bridge interactions. Esters with the acetamide moiety were synthesised to target a tryptophan residue in the α4–α4 binding site via hydrogen bonding interactions. Preliminary screening of the esters with both moieties for inhibition at α7 and α4β2 nAChRs revealed them to be inhibitors at α4β2 nAChRs, particularly (α4)3(β2)2 nAChRs which contain the α4–α4 binding site.