Structure–activity studies on the nociceptin/orphanin FQ receptor antagonist 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide

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Abstract

Twelve derivatives of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) antagonist 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide (Comp 24) were synthesized and tested in binding experiments performed on CHOhNOP cell membranes. Among them, a novel interesting NOP receptor antagonist (compound 35) was identified by blending chemical moieties taken from different NOP receptor ligands. In vitro in various assays, Compound 35 consistently behaved as a pure, highly potent (pA2 in the range 8.0–9.9), competitive and NOP selective antagonist. However compound 35 was found inactive when challenged against N/OFQ in vivo in the mouse tail withdrawal assay. Thus, the usefulness of the novel NOP ligand compound 35 is limited to in vitro investigations.

Graphical abstract

Compound 35 is a novel potent NOP receptor antagonist.

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Introduction

Nociceptin/orphanin FQ (N/OFQ)1, 2 modulates different biological functions via activation of the N/OFQ peptide receptor (NOP).3 Few non-peptide molecules have been reported to selectively interact with the NOP receptor: these include the NOP agonist Ro 64-6198,4 and the antagonists J-1133975 and SB-6121116 (see Chart 1). However, the chemical synthesis of such molecules, which are characterized by two chiral centres, is extremely complex, time consuming and of low yield, making the availability of these tools to the scientific community very limited.

Recently, a novel non-peptide NOP antagonist, 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide, has been reported by Banyu researchers (as Comp 24).7 Comp 24 binds with high affinity (pIC50 9.57) to the human recombinant NOP receptor showing an impressive selectivity (>3000-fold) over classical opioid receptors. Comp 24 behaves as a pure NOP antagonist in the [35S]GTPγS assay with very high potency (pIC50 9.82). Moreover, in vivo in mice Comp 24 at 10 mg/kg sc completely reverses the locomotor inhibitory effect elicited by a NOP receptor agonist.7 Recently this molecule was synthesized and characterized in our laboratories confirming its excellent in vitro pharmacological profile in terms of high antagonist potency and selectivity of action over classical opioid receptors.8 Moreover, in the mouse tail withdrawal assay, Comp 24 at 10 mg/kg ip did not modify per se tail withdrawal latencies but prevented the pronociceptive and antinociceptive effects of 1 nmol N/OFQ given supraspinally and spinally, respectively.8 Collectively, these studies demonstrate that Comp 24 behaves as a pure, highly potent, selective and competitive NOP antagonist.

Interestingly, Comp 24 displays some chemical characteristics typical of N/OFQ related peptides (see Chart 1), these include: (i) a spacer of 12 atoms between the two phenyl rings which matches the Phe-Gly-Gly-Phe sequence of the N/OFQ message domain; (ii) an amide bond which is quite uncommon in non-peptide NOP ligands; (iii) a N-benzyl amino acid, a chemical moiety also present in the N-terminal of the NOP receptor peptide antagonists [Nphe1]N/OFQ(1-13)-NH29 and UFP-101.10 Based on these considerations, in the present study, the importance of the N-benzyl d-Pro of Comp 24 was assessed by replacement with l- or d-Phe, and Nphe. In addition, the amide bond of Comp 24 was substituted with other amide bond isosters. Moreover, the spiroisobenzofurane nucleus of Comp 24 was replaced with chemical moieties derived from other non-peptide NOP ligands that is, Ro 64-6198,4 SB-6121116 and J-1133975 (Chart 1). The novel molecules were evaluated for their ability to bind the human recombinant NOP receptor expressed in CHO cell (CHOhNOP) membranes. The molecule with the highest affinity, that is, compound 35 has been further characterized in vitro at the recombinant human NOP in [35S]GTPγS binding and calcium mobilization assays and at native NOP receptors expressed in isolated animal (mouse, rat and guinea-pig) tissues. Finally compound 35 was assayed in vivo against the effects elicited by N/OFQ in the mouse tail withdrawal assay.

Section snippets

Chemistry

Compound 24 was synthesized following procedures reported by Goto et al.7 The key intermediate 5 (Scheme 1) was obtained starting from commercially available benzanilide 1 that reacts with n-butyllithium at −78 °C and subsequently with the N-benzyl-piperidone to give the spiroisobenzofuran-2-one 2. Compound 2 was reduced with borane dimethyl sulfide complex in THF to give the corresponding isobenzofurane 3. The deprotection of the piperidine nitrogen using H2 in the presence of palladium on

Results and discussion

The ability of the reference ligand, Comp 24, and the novel molecules to bind to the NOP receptor was evaluated using CHOhNOP cell membranes (Table 1). Comp 24 produced a concentration dependent inhibition of [3H]N/OFQ binding with a pKi value of 9.62. This result perfectly matches with that previously reported by Goto et al.7 (pIC50 9.57). The substitution of the N-benzyl d-Pro with d or l-Phe (compounds 9 and 10) or Nphe (compound 11) produced a profound (>100-fold) loss of NOP affinity

Conclusions

The present study demonstrated that the N-benzyl d-Pro moiety as well as the amide bond of Comp 24 is essential for biological activity. In contrast the spirobenzoisofurane can be replaced with 2,6-dichlorophenyl moiety without significant loss of ligand potency, antagonist activity and selectivity of action. Thus by blending chemical moieties taken from known molecules (Comp 24 and SB-612111) we were able to identify compound 35 as a novel non-peptide selective NOP antagonist. While the in

Materials

Melting points (uncorrected) were measured with a Buchi-Tottoli apparatus, and 1H, 13C, and NMR spectra were recorded on a VARIAN400 MHz instrument unless otherwise noted. Chemical shifts are given in ppm (δ) relative to TMS and coupling constants are given in hertz. MS analyses were performed on an ESI-Micromass ZMD 2000. Infrared spectra were recorded on a Perkin–Elmer FT-IR Spectrum 100 spectrometer. Flash chromatography was carried out on a silica gel (Merck, 230–400 Mesh). Elemental

Acknowledgements

This work was supported by funds from the University of Ferrara (FAR grants to SS and GC) and the Italian Ministry of the University (PRIN and FIRB grants to RG).

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