Synthesis and biological activity of neuropeptide S receptor ligands

Neuropeptide S is a neurotransmitter constituted by 20 amino acids, which is involved in the modulation of anxiety, arousal, food intake, and insomnia. NPS activates a dehorphanized G protein-coupled receptor (NPSR) which is distributed in many brain areas with high expression in cortex, hypothalamus, amygdale, and multiple midline thalamic nuclei. Many of these areas have been functionally associated with arousal and processing of emotional behaviour and, on this basis, NPSR ligands could have therapeutic potential. While in the past years there was a big effort in the synthesis of modified peptide ligands, limited information are available about non peptide NPSR ligands. First a series of bicyclic piperazine derivatives has been published in a patent. Those compounds had been partially characterized, hence the aim of my work was the synthesis of such derivatives bearing different substituents, to define the structural figures relevant for antagonist activity. The piperazine derivatives were obtained by synthesizing the scaffold 1,1-diphenyltetrahydro-1H-oxazolo[3,4-a]pyrazin-3(5H)-one and reacting it with the respective isocyanates, acids, or pyridine acetamide analogues, to give the bicyclic piperazine derivatives (1 a-c) bearing different substituents in 7-position [...]. Furthermore, a series of modified analogues lacking the di-phenyl substitution in 1 position have been synthesized by reaction of the tetrahydro-1H-oxazolo[3,4-a]pyrazin-3(5H)-one with the suitable benzyl halide, anhydride or acyl chloride [...]. The in vitro pharmacological activity of non peptide compounds was investigated in a calcium mobilization assay performed using HEK293 cells expressing the recombinant mouse NPS receptor (HEK293mNPSR). The effects of the compounds were evaluated in agonist type experiments in comparisonwith NPS, which causes a concentration dependent intracellular calcium increase. All the compound under investigation were not able to stimulate calcium mobilization up to 10 μM, hence they are not devoted of any agonistic activity. The synthesized derivatives were also tested in antagonist type experiments by evaluating the inhibition of 30 nM NPS stimulatory effect. Tested bicyclic piperazine compounds showed to inhibit NPSR with KB activity in the nanomolar range in a concentration dependent manner. Furthermore, looking at a new scaffold for the development of novel ligands, several compounds of our library, with structural feature in common with the pyperazine compound series, have been tested on mNPSR. On the base of the biological data obtained with this library, and of studies performed with molecular modeling a new series of purine derivatives were designed. The synthesis of purine analogues bearing a benzhydryl group in C-8 or N-9 position, was obtained by cyclization of the suitable 4,5-diamine-6-chloropyrimidine derivatives. The final compounds were then obtained by displacement of the 6-chloro atom with the suitable amines [...]. By reaction of the 6-amino-8-benzhydryl-9-methylpurine with different isocyanates or acids, the 6-ureido or amide purine derivatives 5 [...] have been obtained. All the new compounds were tested in the described agonist and antagonist type experiments and the results showed that only the 8-benzhydryl derivatives bearing a benzylamine group at C-6 position and a hydrogen or methyl group at N-9 position slightly inhibited the effect of NPS [...] In conclusion, none of the tested compounds were able to activate the receptor, and some of them were able to inhibit calcium mobilization after activation of the receptor by NPS. Moreover, it has been demonstrated that the CH2 linker in the side chain of benzylic piperazine derivatives is not crucial for the potency, so giving some possibility for the design of new NPSR antagonists endowed with good affinity. The purine derivatives bearing the benzhydryl group in 8-position and a benzylamine group in 6-position have been shown to slightly inhibit the NPS stimulatory effect.