Selective α3ß4 Nicotinic Acetylcholine Receptor Ligand as a Potential Tracer for Drug Addiction.

α3ß4 Nicotinic acetylcholine receptor (nAChR) has been recognized as an emerging biomarker for the early detection of drug addiction. Herein, α3ß4 nAChR ligands were designed and synthesized to improve the binding affinity and selectivity of two lead compounds, (S)-QND8 and (S)-T2, for the development of an α3ß4 nAChR tracer. The structural modification was achieved by retaining the key features and expanding the molecular structure with a benzyloxy group to increase the lipophilicity for blood-brain barrier penetration and to extend the ligand-receptor interaction. The preserved key features are a fluorine atom for radiotracer development and a p-hydroxyl motif for ligand-receptor binding affinity. Four (R)- and (S)-quinuclidine-triazole (AK1-AK4) were synthesized and the binding affinity, together with selectivity to α3ß4 nAChR subtype, were determined by competitive radioligand binding assay using [3H]epibatidine as a radioligand. Among all modified compounds, AK3 showed the highest binding affinity and selectivity to α3ß4 nAChR with a Ki value of 3.18 nM, comparable to (S)-QND8 and (S)-T2 and 3069-fold higher affinity to α3ß4 nAChR in comparison to α7 nAChR. The α3ß4 nAChR selectivity of AK3 was considerably higher than those of (S)-QND8 (11.8-fold) and (S)-T2 (294-fold). AK3 was shown to be a promising α3ß4 nAChR tracer for further development as a radiotracer for drug addiction.

In Silico Finding of Key Interaction Mediated α3ß4 and α7 Nicotinic Acetylcholine Receptor Ligand Selectivity of Quinuclidine-Triazole Chemotype.

The selective binding of six (S)-quinuclidine-triazoles and their (R)-enantiomers to nicotinic acetylcholine receptor (nAChR) subtypes α3ß4 and α7, respectively, were analyzed by in silico docking to provide the insight into the molecular basis for the observed stereospecific subtype discrimination. Homology modeling followed by molecular docking and molecular dynamics (MD) simulations revealed that unique amino acid residues in the complementary subunits of the nAChR subtypes are involved in subtype-specific selectivity profiles. In the complementary ß4-subunit of the α3ß4 nAChR binding pocket, non-conserved AspB173 through a salt bridge was found to be the key determinant for the α3ß4 selectivity of the quinuclidine-triazole chemotype, explaining the 47-327-fold affinity of the (S)-enantiomers as compared to their (R)-enantiomer counterparts. Regarding the α7 nAChR subtype, the amino acids promoting a however significantly lower preference for the (R)-enantiomers were the conserved TyrA93, TrpA149 and TrpB55 residues. The non-conserved amino acid residue in the complementary subunit of nAChR subtypes appeared to play a significant role for the nAChR subtype-selective binding, particularly at the heteropentameric subtype, whereas the conserved amino acid residues in both principal and complementary subunits are essential for ligand potency and efficacy.

Radiosynthesis of (S)-[18F]T1: The first PET radioligand for molecular imaging of α3ß4 nicotinic acetylcholine receptors.

Recent pharmacologic data revealed the implication of α3ß4 nicotinic acetylcholine receptors (nAChRs) in nicotine and drug addiction. To image α3ß4 nAChRs in vivo, we aimed to establish the synthesis of a [18F]-labelled analog of the highly affine and selective α3ß4 ligand (S)-3-(4-(4-fluorophenyl)-1H-1,2,3-triazol-1-yl)quinuclidine ((S)-T1). (S)-[18F]T1 was synthesized from ethynyl-4-[18F]fluorobenzene ([18F]5) and (S)-azidoquinuclidine by click reaction. After a synthesis time of 130min (S)-[18F]T1 was obtained with a radiochemical yield (non-decay corrected) of 4.3±1.3%, a radiochemical purity of >99% and a molar activity of >158 GBq/µmol. The brain uptake and the brain-to-blood ratio of (S)-[18F]T1 in mice at 30min post injection were 2.02 (SUV) and 6.1, respectively. According to an ex-vivo analysis, the tracer remained intact (>99%) in brain. Only one major radiometabolite was detected in plasma and urine samples. In-vitro autoradiography on pig brain slices revealed binding of (S)-[18F]T1 to brain regions associated with the expression of α3ß4 nAChRs, which could be reduced by the α3ß4 nAChR selective drug AT-1001. These findings make (S)-[18F]T1 a potential tool for the non-invasive imaging of α3ß4 nAChRs in the brain by PET.

Varying Chirality Across Nicotinic Acetylcholine Receptor Subtypes: Selective Binding of Quinuclidine Triazole Compounds.

The novel quinuclidine anti-1,2,3-triazole derivatives T1-T6 were designed based on the structure of QND8. The binding studies revealed that the stereochemistry at the C3 position of the quinuclidine scaffold plays an important role in the nAChR subtype selectivity. Whereas the (R)-enantiomers are selective to α7 over α4ß2 (by factors of 44-225) and to a smaller degree over α3ß4 (3-33), their (S)-counterparts prefer α3ß4 over α4ß2 (62-237) as well as over α7 (5-294). The (R)-derivatives were highly selective to α7 over α3ß4 subtypes compared to (RS)- and (R)-QND8. The (S)-enantiomers are 5-10 times more selective to α4ß2 than their (R) forms. The overall strongest affinity is observed for the (S)-enantiomer binding to α3ß4 (Ki, 2.25-19.5 nM) followed by their (R)-counterpart binding to α7 (Ki, 22.5-117 nM), with a significantly weaker (S)-enantiomer binding to α4ß2 (Ki, 414-1980 nM) still above the very weak respective (R)-analogue affinity (Ki, 5059-10436 nM).