Structure-affinity relationship study of novel vesamicol analogs as basis for the development of a selective PET-Ligand for the vesicular acetylcholine transporter

Objectives: The vesicular acetylcholine transporter (VAChT) is a transmembrane protein located in presynaptic vesicles of cholinergic neurons and is discussed to be an interesting target for PET imaging of neurodegenerative disorders. Previous developments of VAChT radiotracers are based on vesamicol (trans-2-(4-phenylpiperidino)cyclohexanol) and are focused on structural modifications in order to keep the high affinity of vesamicol to VAChT and to lower the affinity to σ₁ and σ₂ receptors, two off-targets which are also addressed by vesamicol. The moderate selectivity of several VAChT-ligands is one of the limiting factors for a successful application as radiotracer. Based on numerous literature data and our own intensive synthetic work in this field, we observed that structural modifications based on single-target structure affinity considerations are rarely successful. Therefore, we synthesized different series of systematically modified vesamicol analogs as basis for the development of a 3D-QSAR model, which will consider for the first time the affinity to VAChT as well as to the σ₁ and σ₂ receptors.

Methods: Nucleophilic ring opening of an epoxide precursor with a secondary amine was used to synthesize a large number of vesamicol derivatives. By varying the epoxide precursor, structural modifications in ring A of the vesamicol skeleton were realized (left part of the figure). Due to the selection of different amines, the rings B and C were sterically or electronically modified (right part of the figure). The binding affinities to VAChT were determined with competitive binding experiments using preparations of PC12 cells stably transfected with ratVAChT cDNA and (-)-[³H]vesamicol. To determine the binding affinity to the sigma receptors, rat cortex membranes and (+)-[³H]pentazocin for σ₁ and rat liver membranes and [³H]DTG for σ₂ receptors were used.

Results: So far we synthesized 55 derivatives belonging to the four classes of vesamicols, F-benzoylvesamicols, benzovesamicols and azavesamicols (Fig.) and determined their binding affinity to VAChT, σ₁ and σ₂ receptors. A few of them such as benzovesamicol or FBT are already known and were used as reference compounds. Interestingly, the VAChT binding data obtained by our test system differ to some extent from the values reported in literature. Therefore, VAChT binding data of selected vesamicol derivatives obtained either with PC12 cells or membranes of pig and/or rat brain are compared and differences of K_i values are discussed. The use of preparations obtained by freezing and thawing of PC12 cells stably transfected with ratVAChT cDNA and (-)-[³H]vesamicol was found to be the most practicable and reliable method. As a preliminary trend we could observe that modifications in ring B and C led to a loss of VAChT affinity, whereas modifications in ring A partly increased VAChT affinity, which was however often accompanied by an increase in σ₁/σ₂ receptor affinity.

Figure. Vesamicol as chemical lead and derived new analogs

Conclusion: Based on our previous results, we have to conclude that VAChT affinity suitable for PET imaging correlates often with significant off-target affinity to the σ₁ and σ₂ receptors.
Acknowledgment: Supported by DFG (WE 2927/4-1).