Identification of neuropeptide S antagonists: structure-activity relationship studies, X-ray crystallography, and in vivo evaluation

Abstract

Modulation of the neuropeptide S (NPS) system has been linked to a variety of CNS disorders such as panic disorder, anxiety, sleeping disorders, asthma, obesity, PTSD, and substance abuse. In this study, a series of diphenyltetrahydro-1H-oxazolo[3,4-α]pyrazin-3(5H)-ones were synthesized and evaluated for antagonist activity at the neuropeptide S receptor. The absolute configuration was determined by chiral resolution of the key synthetic intermediate, followed by analysis of one of the individual enantiomers by X-ray crystallography. The R isomer was then converted to a biologically active compound (34) that had a Ke of 36 nM. The most potent compound displayed enhanced aqueous solubility compared with the prototypical antagonist SHA-68 and demonstrated favorable pharmacokinetic properties for behavioral assessment. In vivo analysis in mice indicated a significant blockade of NPS induced locomotor activity at an ip dose of 50 mg/kg. This suggests that analogs having improved drug-like properties will facilitate more detailed studies of the neuropeptide S receptor system.

Figure 1

Representative classes of NPS antagonists.

Scheme 1. Synthesis of Key Intermediate 37

Reagents and conditions: (a) PhLi, THF, −78 °C; (b) H₂ (46 psi), 10% Pd/C, 5% Pd/BaSO₄, EtOH; (c) Boc₂O, DMAP, NEt₃, THF; (d) TFA, DCM.

Scheme 2. Synthesis of Target Molecules Derived from Isocyanate or Acid Chloride Addition

Reagents and Conditions: (a) R-isocyanate, CH₂Cl₂, rt; (b) acid chloride, TEA, 0 °C; (c) CH₃NH₂, DMF, KHCO₃, rt; (d) (CH₃)₂NH, DMF, KHCO₃, rt.

Scheme 3. Preparation of Target Molecules Using Triphosgene Conditions

Reagents and conditions: (a) R-amine, (CCl₃O)₂CO, TEA, THF, 0 °C to rt; (b) TFA, DCM; (c) HCOH, NaH(OAc)₃, 1,2-DCE.

Scheme 4. Identification of the Active Enantiomer of Key Intermediate 37

Reagents and Conditions: (a) chiral HPLC; b) R-camphorsulfonic acid, MeOH; (c) 4-fluorobenzyl isocyanate.

Figure 2

X-ray structure of the inactive enantiomer 38a.

Figure 3

Pharmacokinetic analysis of compound 34 in brain and plasma; 5 mg/kg dose. Sample size, five animals per time point. Data points represent the mean, and error bars represent standard deviation.

Figure 4

Effects of 34 on motor activity in mice. Mice had been habituated for 60 min before injection. Compound 34 was dissolved in 10% Cremophor EL (vehicle) and injected (ip) 10 min before NPS or vehicle (PBS, 0.1% BSA) were injected centrally (icv). Group sizes: Veh + Veh, n = 8; Veh + NPS, n = 7; 34 + PBS, n = 7; 34 (50 mg/kg) + NPS (1 nmole), n = 8; 34 (5 mg/kg) + NPS (1 nmole), n = 9. (A) Time course of the distance traveled over 90 min. (B) Total distance traveled during the 90 min observation period. *, p < 0.001, Veh + Veh versus Veh + NPS; #, p < 0.001 Veh + NPS versus NPS + 34 (50 mg/kg) (Students t test).