Structure-Activity Relationship Studies on Oxazolo[3,4- a]pyrazine Derivatives Leading to the Discovery of a Novel Neuropeptide S Receptor Antagonist with Potent In Vivo Activity

Abstract

Neuropeptide S modulates important neurobiological functions including locomotion, anxiety, and drug abuse through interaction with its G protein-coupled receptor known as neuropeptide S receptor (NPSR). NPSR antagonists are potentially useful for the treatment of substance abuse disorders against which there is an urgent need for new effective therapeutic approaches. Potent NPSR antagonists in vitro have been discovered which, however, require further optimization of their in vivo pharmacological profile. This work describes a new series of NPSR antagonists of the oxazolo[3,4-a]pyrazine class. The guanidine derivative 16 exhibited nanomolar activity in vitro and 5-fold improved potency in vivo compared to SHA-68, a reference pharmacological tool in this field. Compound 16 can be considered a new tool for research studies on the translational potential of the NPSergic system. An in-depth molecular modeling investigation was also performed to gain new insights into the observed structure-activity relationships and provide an updated model of ligand/NPSR interactions.

Figure 1

SAR extension performed in this work around the oxazolo[3,4-a]pyrazine nucleus of known NPSR antagonists 1 and 2.

Scheme 1. Synthesis of Final Compounds 3–12

Reagents and conditions: (i) BnBr, EtOH, 75 °C, 5 h; (ii) Boc₂O, DMAP, tetraethylammonium, CH₂Cl₂, rt, 0.5 h; (iii) sec-BuLi, TMEDA, THF, −78 °C, 6 h; (iv) FmocCl, MeCN, 90 °C, 5 h; (v) DBU, THF, rt, 2 h.

Scheme 2. Synthesis of Final Compounds 13–16

Reagents and conditions: (i) DBU, THF, rt, 2 h; (ii) CH₂Cl₂–DMF (2:1), 50 °C, 4 h; (iii) HgCl₂, CH₃CN, 90 °C, 24 h; (iv) BrCN,CH₂Cl₂, NaHCO₃, H₂O, 30 min at 0 °C, 24 h rt; (v) benzyl amine or 4-fluoro benzylamine, p-TsOH, DMSO, 60 °C, 18 h; (vi) K₂CO₃, CH₃CN, 90 °C, 4 h.

Scheme 3. Synthesis of Final Compounds 17–21

Reagents and conditions: (i) NaHCO₃, chloroacetyl chloride, toluene, 0 °C to rt, overnight; (ii) Et₃N, benzylamine, dioxane, reflux, 20 h; (iii) LiAlH₄, THF, reflux, 3 h; (iv) Boc₂O, THF, 0 °C to rt, 1 h; (v) benzophenone, sec-BuLi, TMEDA, THF, −78 °C, 3 h; (vi) FmocCl, MeCN, 90 °C, 5 h and then rt, 18 h; (vii) 4-fluorobenzyl isocyanate, DBU, THF, rt, 2 h.

Figure 2

Calcium mobilization assay performed on HEK293_mNPSR cells. Concentration–response curve to NPS [panel (A)] and inhibition–response curves to 1 against the stimulatory effect of 10 nM NPS [panel (B)]. Data are mean ± SEM of at least five separate experiments made in duplicate.

Figure 3

Calcium mobilization assay performed on HEK293_mNPSR cells. Concentration–response curve to NPS in the absence and in the presence of 100 nM of 1, 16, and 21. Data are mean ± SEM of at least five separate experiments made in duplicate.

Figure 4

Energy-minimized docked poses of compound 1 in BM1 and BM2 (panels A and B, respectively) in the model of NPSR constructed starting from the human neuropeptide Y Y1 receptor (hNPY1R, PDB code 5ZBH).1 and the protein are represented as violet sticks and multicolored ribbons, respectively.

Figure 5

Energy-minimized docked poses of compound 1 [panel (A) for BM1 and (D) for BM2], 16 [panel (B) for BM1 and (E) for BM2], and 21 [panel (C) for BM1 and (F) for BM2] in the model of NPSR constructed starting from the human neuropeptide Y Y1 receptor (hNPY1R, PDB code 5ZBH).1, 16, and 21 are represented as violet, orange, and red sticks, respectively. The protein is represented as cyan ribbons and sticks. H-bonds are represented as dashed yellow lines.

Figure 6

Effects of NPS, 1, 16, and 21 on mouse locomotor activity. The cumulative effects exerted on distance traveled are shown in panel (A), while the total time immobile and number of rearings over the 30 min observation period are shown in panels (B,C), respectively. Data are mean ± SEM of four separate experiments (vehicle + saline, 12 mice; 1 + saline, 8 mice; 16 + saline, 8 mice; 21 + saline, 7 mice; vehicle + NPS, 11 mice, 1 + NPS, 8 mice; 16 + NPS, 7 mice; and 21 + NPS, 7 mice). The two-way ANOVA NPS × antagonist revealed for the total distance traveled, an effect of NPS F(1,59) = 6.63 and of the interaction NPS × antagonist F(3,59) = 4.48; for the immobility time, an effect of NPS F(1,59) = 8.09 and of the interaction NPS × antagonist F(3,59) = 4.26; for the number of rearings, an effect of NPS F(1,59) = 7.35, of antagonist F(3,59) = 10.65, and of the interaction NPS × antagonist F(3,59) = 4.32. *p < 0.05 vs saline, #p < 0.05 vs vehicle according to Bonferroni’s test for multiple comparisons.