Synthesis and pharmacological in vitro and in vivo profile of 3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (SHA 68), a selective antagonist of the neuropeptide S receptor

Abstract

Neuropeptide S (NPS) has been shown to modulate arousal, sleep wakefulness, anxiety-like behavior, and feeding after central administration of the peptide agonist to mice or rats. We report here the chemical synthesis and pharmacological characterization of SHA 66 (3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid benzylamide) and SHA 68 (3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide), two closely related bicyclic piperazines with antagonistic properties at the NPS receptor (NPSR). The compounds block NPS-induced Ca2+ mobilization, and SHA 68 shows displaceable binding to NPSR in the nanomolar range. The antagonistic activity of SHA 68 seems to be specific because it does not affect signaling at 14 unrelated G protein-coupled receptors. Analysis of pharmacokinetic parameters of SHA 68 demonstrates that the compound reaches pharmacologically relevant levels in plasma and brain after i.p. administration. Furthermore, peripheral administration of SHA 68 in mice (50 mg/kg i.p.) is able to antagonize NPS-induced horizontal and vertical activity as well as stereotypic behavior. Therefore, SHA 68 could be a useful tool to characterize physiological functions and pharmacological parameters of the NPS system in vitro and in vivo.

Fig. 1

Chemical synthesis of SHA 66 and SHA 68. Intermediate compounds are numbered (in bold type) and described in Materials and Methods.

Fig. 2

Pharmacological activity of SHA 66 (A, B) and SHA 68 (C, D) at NPSR Asn¹⁰⁷ (NPSR-N¹⁰⁷) and NPSR Ile¹⁰⁷ (NPSR-I¹⁰⁷). Both compounds were tested in the presence of increasing concentrations of NPS to generate dose-ratios for Schild analysis by measuring mobilization of intracellular Ca²⁺ in HEK 293 cells stably expressing the different NPSR isoforms. Concentrations of antagonist compounds are given in the figure legend of B. Data are shown as means ± SEM. E, Schild analysis of dose-ratios followed by linear regression analysis. F, Displacement of radioligand binding by SHA 68 in cells stably expressing NPSR Ile¹⁰⁷. [¹²⁵I] Tyr¹⁰-NPS (40 pmole) was used as a tracer and incubations were done in triplicate. Data are shown as means ± SEM.

Fig. 3

Pharmacokinetic analysis of SHA 68 in mice. A, Dose-normalized plasma concentrations (in (ng/ml)/(mg/kg)) as a function of time (in h) for i.v. administration of SHA 68 at 1 mg/kg dose (open squares) and i.p. administration at 2.5 mg/kg (triangles) dose. Plasma and brain concentrations of SHA 68 administered i.p. at 5 (B) and 50 mg/kg (C), 0.25, 1, and 2 hours post-dose. D, Brain/plasma ratio (ml/g) calculated from B and C as a function of dose and time. Animal numbers were n = 5 at all time points. Data are shown as means ± S.E.M.

Fig. 4

Effect of SHA 68 on motor activity in mice. Mice had been habituated for 2 h prior to injection. SHA 68 was dissolved in 10% cremophor EL (vehicle) and injected (i.p.) 10 min before NPS or vehicle (PBS, 0.1 % BSA) were injected centrally (i.c.v.). Group sizes: Veh + Veh, n = 12; Veh + SHA 68 (50 mg/kg), n = 12; NPS + Veh, n = 18; NPS + SHA 68 (50 mg/kg), n = 15; NPS + SHA 68 (5 mg/kg), n = 22. A, Time-course of horizontal activity, B, vertical activity, and C, stereotypic behavior over 90 min; D, Cumulative horizontal activity, E, cumulative vertical activity, and F, cumulative stereotypy time during the 90 min observation period; *** p < 0.001 Veh + Veh vs. NPS + Veh; # p < 0.05, ### p < 0.001 NPS + Veh vs. NPS + SHA 68 (50 mg/kg), one-way ANOVA with Bonferroni post-hoc test.