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This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of the neuropeptide S receptor (NPSR) antagonist (9R/S)-3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (SHA 68). The (9R)-3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (compound 10) and (9S)-3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (compound 10a) were synthesized and their purity assessed by chiral chromatography. The absolute configuration of the enantiomer 10has been assigned from the crystal structure of the corresponding (S)-phenyl ethyl amine derivative 8. Calcium mobilization studies performed on cells expressing the recombinant NPSR demonstrated that compound 10is the active enantiomer while the contribution of 10ato the NPSR antagonist properties of the racemic mixture is negligible. [ABSTRACT FROM AUTHOR]

To improve the structure−activity studies of the lead δ opioid agonist H-Dmt-Tic-Asp*-Bid, we synthesized and pharmacologically characterized a series of analogues in which the side chain next to 1H-benzimidazole-2-yl (Bid) was substituted by those endowed with different chemical properties. Interesting results were obtained: (1) only Gly, Ala, and Asp resulted in δ agonism, (2) Phe yielded δ antagonism, (3) and all other residues except Glu (devoid of any activity) gave μ agonism. [ABSTRACT FROM AUTHOR]

Neuropeptide S (NPS) has been identified as the endogenous ligand of a previously orphan receptor now named NPSR. Previous studies demonstrated that the N-terminal sequence Phe 2-Arg 3-Asn 4of the peptide is crucial for biological activity. Here we report on a focused structure–activity study of Phe 2which has been replaced with a series of coded and noncoded amino acids. Thirty-one human NPS analogues were synthesized and pharmacologically tested for intracellular calcium mobilization by using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated the following NPS position 2 structure–activity features: (i) lipophilicity but not aromaticity is crucial, (ii) both the size of the chemical moiety and its distance from the peptide backbone are important for biological activity, and (iii) this position plays a role in both receptor binding and activation, since [4,4′-biphenyl-Ala 2]hNPS behaved as a partial agonist. [ABSTRACT FROM AUTHOR]

Neuropeptide S (NPS), the endogenous ligand of a previously orphan receptor now named NPSR, regulates various biological functions in the brain, including arousal, locomotion, anxiety, and food intake. Here we report on a focused structure−activity study of Gly5, which has been replaced with land damino acids. Fifteen NPS related peptides were synthesized and pharmacologically tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated that peptide potency is inversely related to the side chain size, while peptide efficacy strongly depends on the relative land dconfiguration, with the lamino acids favoring agonist while damino acids display antagonist pharmacological activity. [d-Val5]NPS behaved as NPSR pure antagonist in HEK293mNPSRcells showing the highest potency (pKB7.56) among this series of peptides. The antagonist action of [d-Val5]NPS was confirmed in vivo in mice, where the peptide at a dose of 10 nmol completely blocked the stimulatory effect of 0.1 nmol NPS on locomotor activity. [ABSTRACT FROM AUTHOR]

Some reference opioids containing the Dmt-Tic pharmacophore, especially the δ agonists H-Dmt-Tic-Gly-NH-Ph ( 1) and H-Dmt-Tic-NH-( S)CH(CH 2−COOH)-Bid ( 4) (UFP-512) were evaluated for the influence of the substitution of Gly with aspartic acid, its chirality, and the importance of the −NH-Ph and N 1H-Bid hydrogens in the inductions of δ agonism. The results provide the following conclusions: (i) Asp increases δ selectivity by lowering the μ affinity; (ii) −NH-Ph and N 1H-Bid nitrogens methylation transforms the δ agonists into δ antagonists; (iii) the substitution of Gly with l-Asp/ d-Asp in the δ agonist H-Dmt-Tic-Gly-NH-Ph gave δ antagonists; the same substitution in the δ agonist H-Dmt-Tic-NH-CH 2−Bid yielded more selective agonists, H-Dmt-Tic-NH-( S)CH(CH 2−COOH)-Bid and H-Dmt-Tic-NH-( R)CH(CH 2−COOH)-Bid; (iv) l-Asp seems important only in functional bioactivity, not in receptor affinity; (v) H-Dmt-Tic-NH-( S)CH(CH 2−COOH)-Bid(N 1-Me) ( 10) evidenced analgesia similar to 4, which was reversed by naltrindole only in the tail flick. 4and 10had opposite behaviours in mice; 4caused agitation, 10gave sedation and convulsions. [ABSTRACT FROM AUTHOR]

Neuropeptide S (NPS) is the endogenous ligand of the previously orphan G-protein coupled receptor now named NPSR. The NPS−NPSR receptor system regulates important biological functions such as sleep/waking, locomotion, anxiety and food intake. Recently, exhaustive Ala scan and d-amino acid scan studies, together with systematic N- and C-terminal truncation, led to the identification of key residues for biological activity. Because conformational preferences might also play an important role, we undertook a detailed conformational analysis of NPS and several analogues in solution. We show that helicity induced by substitution of three flexible residues in the 5−13 regulatory region abolishes biological activity. A parallel pharmacological and conformational study of single and multiple substitutions of glycines 5, 7, and 9 showed that helicity can be tolerated in the C-terminal part of the peptide but not around Gly7. The identification of hNPSR partial agonists heralds the possibility of designing pure NPS receptor antagonists. [ABSTRACT FROM AUTHOR]