Development of a suitable PET ligand for the vesicular acetylcholine transporter (vAChT) - an extensive qualitative structure affinity relationship study

Aim: The vesicular acetylcholine transporter (vAChT) is an interesting target for in vivo imaging of cholinergic deficits. Vesamicol (2-(4-phenylpiperidin-1-yl)cyclohexanol) acts as a highly potent inhibitor for this transporter and thus represents the chemical lead for the development of vAChT radioligands. Due to its comparably high affinity towards σ1/σ2 receptors and hence its lack of selectivity it is not suitable for in vivo imaging of cholinergic neurons. The aim of our studies was to develop a selective ligand by varying the vesamicol skeleton in a systematic manner and determining the binding profile of the resulting derivatives. These in vitro data were correlated with the various chemical structures leading to an extensive qualitative structure affinity relationship study.
Materials and methods: Based on suitable epoxide precursors, the derivatives were synthesised by nucleophilic ring opening using structurally varied amines. The regioisomers resulting from asymmetrical epoxides were separated by semi-preparative HPLC and all analogues were characterised using MS, NMR, and HPLC. To obtain the binding profile of all compounds, competitive binding assays were performed in vitro with rvAChT-cDNA stably transfected PC12 cells, rat cortex (σ1), and rat liver (σ2) using (-)-[H-3]vesamicol, (+)-[H-3]pentazocine and [H-3]DTG as radioligands for vAChT, σ1, and σ2 receptor, respectively.
Results: 57 new analogues and seven reference compounds from literature were synthesised and characterised regarding to their in vitro binding profile. Based on different epoxide precursors, the lead structure of vesamicol was modified at the cyclohexyl ring A. Variations on the piperidinyl ring B and phenyl ring C, respectively, were achieved using several amines. In vitro binding studies showed vAChT affinities in the range of low nanomolar to micromolar Ki values. As expected, we found a strong variability regarding affinity and selectivity within each class of derivatives (modified in ring B and C). But surprisingly, also similar analogues from different classes (modified in ring A) varied substantially in their affinities. The resulting selectivity factors (Ki(σ1)/Ki(vAChT) and Ki(σ2)/Ki(vAChT)) ranged from 0.003 to 72.7 indicating none to moderate selectivities towards vAChT.
Conclusion: The results show a great variability of the in vitro binding profile of ligands based on the vesamicol lead structure. Even small structural changes led to unexpected changes in affinities and selectivities towards vAChT and we obtained evidence that an increased vAChT affinity correlated with an increase of σ1/σ2 receptor affinity. Based on our results we conclude that future development of a selective vAChT radioligand might be based on another lead compound.