Abstract

The pharmacological properties of cyclohexanecarboxylic acid, {2-[4-(2-bromo-5-methoxybenzyl)piperazin-1-yl]ethyl}-(2-trifluoromethoxyphenyl)amide (Rec 27/0224), and cyclohexanecarboxylic acid, (2-methoxy-phenyl)-{2-[4-(2-methoxyphenyl)-piperazin-1-yl]ethyl}amide (Rec 27/0074), were characterized using radioligand displacement and guanosine 5′-O-(3-[³⁵S]thiotriphosphate) ([³⁵S]GTPγS) binding assays, as well as electrophysiological experiments, in rat hippocampal and dorsal raphe nucleus (DRN) slices. Both compounds showed a high affinity (K_i, ∼1 nM) and selectivity (>70-fold) at human 5-hydroxytryptamine (5-HT)_1A receptors versus other 5-HT receptors. In [³⁵S]GTPγS binding assays on HeLa cells stably expressing human 5-HT_1A receptors, Rec 27/0224 and Rec 27/0074 inhibited basal [³⁵S]GTPγS binding by 44.8 ± 1.7% (pEC₅₀ = 8.58) and 25 ± 2.5% (pEC₅₀ = 8.86), respectively. In intracellularly recorded CA1 pyramidal cells, 5-HT_1A (hetero)receptor-mediated hyperpolarization, elicited by 100 nM 5-carboxamidoytryptamine (5-CT), was partially antagonized by Rec 27/0224 (∼50%; IC₅₀ = 18.0 nM) and Rec 27/0074 (74%; IC₅₀ = 0.8 nM). In extracellularly recorded DRN serotonergic neurons, Rec 27/0224 and Rec 27/0074 fully antagonized the inhibition of firing caused by the activation of 5-HT_1A (auto)receptors by 30 nM 5-CT with an IC₅₀ of 34.9 nM and 16.5 nM, respectively. The antagonism had a slow time course, reaching a steady state within 60 min. Both compounds also antagonized the citalopram-elicited, endogenous 5-HT-mediated inhibition of cell firing. In conclusion, Rec 27/0224 and Rec 27/0074 exhibited inverse agonism in [³⁵S]GTPγS binding assays and differential antagonistic properties on 5-HT_1A receptor-mediated responses in the hippocampus but not in the DRN. Whether this differential effect is causally related to inverse agonist activity is unclear. The qualitatively different nature of the antagonism in the hippocampus versus the DRN clearly distinguishes the compounds from neutral antagonists, such as N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-2-pyridinylcyclo-hexanecarboxamide (WAY-100635).