The interaction of RS 25259‐197, a potent and selective antagonist, with 5‐HT3 receptors, in vitro
Authors: Wong E.H.F. , Clark R. , Leung E. , Loury D. , Bonhaus D.W. , Jakeman L. , Parnes H. , Whiting R.L. , Eglen R.M. 
Published: July 2012

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).

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).

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.

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.

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.

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.

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.