The interaction of RS 25259‐197, a potent and selective antagonist, with 5‐HT3 receptors, in vitro

1 A series of isoquinolines have been identified as 5‐HT3 receptor antagonists. One of these, RS 25259‐197 [(3aS)‐2‐[(S)‐1‐azabicyclo[2.2. 2]oct‐3‐yl]‐2,3,3a,4,5,6‐hexahydro‐1‐oxo‐1H‐benzo[de]isoquinoline‐hydrochloride], has two chiral centres. The remaining three enantiomers are denoted as RS 25259‐198 (R,R), RS 25233‐197 (S,R) and RS 25233‐198 (R,S). 2 At 5‐HT3 receptors mediating contraction of guinea‐pig isolated ileum, RS 25259‐197 antagonized contractile responses to 5‐HT in an unsurmountable fashion and the apparent affinity (pKB), estimated at 10 nm, was 8.8 ± 0.2. In this tissue, the—log KB values for the other three enantiomers were 6.7 ± 0.3 (R,R), 6.7 ± 0.1 (S,R) and 7.4 ± 0.1 (R,S), respectively. The apparent affinities of RS 25259‐197 and RS 25259‐198, RS 25233‐197 and RS 25233‐198 at 5‐HT3 receptors in membranes from NG‐108‐15 cells were evaluated by a [3H]‐quipazine binding assay. The—log Ki values were 10.5 ± 0.2, 8.4 ± 0.1, 8.6 ± 0.1 and 9.5 ± 0.1, respectively, with Hill coefficients not significantly different from unity. Thus, at these 5‐HT3 receptors, the rank order of apparent affinities was (S,S) > (R,S) > (S,R) = (R,R). 3 RS 25259‐197 displaced the binding of the selective 5‐HT3 receptor ligand, [3H]‐RS 42358‐197, in membranes from NG‐108‐15 cells, rat cerebral cortex, rabbit ileal myenteric plexus and guinea‐pig ileal myenteric plexus, with affinity (pKi) values of 10.1 ± 0.1, 10.2 ± 0.1, 10.1 ± 0.1 and 8.3 ± 0.2, respectively. In contrast, it exhibited low affinity (pKi < 6.0) at 28 other receptors in binding assays, including adrenoceptors (α1A, α1B, α2A, α2B, β1, β2), muscarinic (M1—M4), dopamine (D1, D2), opioid and other 5‐HT (5‐HT1A, 5‐HT1D, 5‐HT2C, 5‐HT4) receptors. 4 RS 25259‐197 was tritium labelled (specific activity: 70 Ci mmol−1) and evaluated in pharmacological studies. Saturation studies with [3H]‐RS 25259‐197 in membranes from NG‐108‐15 and cloned homomeric α subunits of the 5‐HT3 receptor from N1E‐115 cells expressed in human kidney 293E1 cells, revealed an equilibrium dissociation constant (Kd) of 0.05 ± 0.02 and 0.07 ± 0.01 nm, and Bmax of 610 ± 60 and 1068 ± 88 fmol mg−1, respectively. Competition studies in NG‐108‐15 cells indicated a pharmacological specificity entirely consistent with labelling a 5‐HT3 receptor, i.e. RS 25259‐197 > granisetron > (S)‐zacopride > tropisetron > (R)‐zacopride > ondansetron > MDL 72222. 5 In contrast to the majority of radioligands available to label 5‐HT3 receptors, [3H]‐RS 25259‐197 labelled a high affinity site in hippocampus from human post‐mortem tissue with an equilibrium dissociation constant (Kd) of 0.15 ± 0.07 nm and density (Bmax) of 6.8 ± 2.4 fmol mg−1 protein. Competition studies in this tissue indicated a pharmacological specificity consistent with labelling of a 5‐HT3 receptor. 6 Quantitative autoradiographic studies in rat brain indicated a differential distribution of 5‐HT3 receptor sites by [3H]‐RS 25259‐197. High densities of sites were seen in nuclear tractus solitaris and area postrema, a medium density in spinal trigeminal tract, ventral dentate gyrus and basal medial amygdala, and a low density of sites in hippocampal CA1, parietal cortex, medium raphe and cerebellum. 7 In conclusion, the functional, binding and distribution studies undertaken with the radiolabelled and non‐radiolabelled RS 25259‐197 (S,S enantiomer) established the profile of a highly potent and selective 5‐HT3 receptor antagonist.