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A total of 32 compounds was prepared to investigate the functional role of Phe⁴ in NC(1−13)-NH2, the minimal sequence maintaining the same activity as the natural peptide nociceptin. These compounds could be divided into three series in which Phe⁴ was replaced with residues that would (i) alter aromaticity or side chain length, (ii) introduce steric constraint, and (iii) modify the phenyl ring. Compounds were tested for biological activity as (a) inhibitors of the electrically stimulated contraction of the mouse vas deferens; (b) competitors of the binding of [³H]-NC-NH2 to mouse brain membranes; and (c) inhibitors of forskolin-stimulated cAMP accumulation in CHO cells expressing the recombinant human OP4 receptor. Results indicate that all compounds of the first and second series were inactive or very weak with the exception of [N(CH3)Phe⁴]NC(1−13)-NH2, which was only 3-fold less potent than NC(1−13)-NH2. Compounds of the third series showed higher, equal, or lower potencies than NC(1−13)-NH2. In particular, [(pF)Phe⁴]NC(1−13)-NH2 (pF) and [(pNO2)Phe⁴]NC(1−13)-NH2 (pNO2) were more active than NC(1−13)-NH2 by a factor of 5. In the mVD, these compounds showed the following order of potency:  (pF) = (pNO2) ≥ (pCN) > (pCl) > (pBr) > (pI) = (pCF3) = (pOCH3) > (pCH3) > (pNH2) = (pOH). (oF) and especially (mF) maintained high potencies but were less active than (pF). Similar orders of potency were observed in binding competition and cAMP accumulation studies. There was a strong (r² ≥ 0.66) correlation between data observed in these assays. Biological activity data of compounds of the third series were plotted against some Hansch parameters that are currently used to quantify physicochemical features of the substituents. In the three biological assays agonist potency/affinity positively correlates with the electron withdrawal properties of the groups in the p-position of Phe⁴ and inversely with their size.

Three series of nociceptin (NC)-related peptides were synthesized and their abilities (i) to bind to the NC sites expressed in mouse forebrain membranes, (ii) to inhibit the electrically evoked contraction of the mouse vas deferens, and (iii) to inhibit forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells expressing the human recombinant NC receptor (CHONCR) were investigated. The compounds of the first series (a series) have an ordinary Xaa¹-Gly² bond, those of the second series (b series) have a Xaa¹Ψ(CH2-NH)Gly² pseudopeptide bond, and those of the third series (c series) have a peptoid (Nxaa¹-Gly²) structure. The affinity values measured in the binding assay and in the two functional assays with the compounds of the three series showed high levels of correlation. Thus, (I) the compounds of the a series in which Phe¹ was substituted with Tyr, Cha, or Leu acted as potent NC receptor agonists; (II) the b series compounds behaved as NC receptor antagonists in the mouse vas deferens and as full agonists in CHONCR cells with different potencies depending on the first amino acid residue, [Phe¹Ψ(CH2-NH)Gly²]NC(1−17)NH2 and [Phe¹Ψ(CH2-NH)Gly²]NC(1−13)NH2 being the most potent compounds; (III) the compounds of the third series were all inactive both as agonists and as antagonists with the exception of [Nphe¹]NC(1−17)NH2 and [Nphe¹]NC(1−13)NH2, which behaved as NC receptor antagonists both in the isolated tissue and in CHONCR cells (pKB 6.1−6.4). In conclusion, this study demonstrates that chemical requirements for NC receptor agonists are different from those of antagonists. Moreover, modifications of the steric orientation of the aromatic residue Phe¹ in the NC sequence as obtained with the pseudopeptide bond between Phe¹ and Gly² or with the displacement of the benzyl side chain by one atom, as in Nphe¹, lead respectively to reduction or elimination of efficacy. Indeed, in contrast to [Phe¹Ψ(CH2-NH)Gly²]NC(1−13)NH2 which has been reported to exhibit agonist activity in several assays involving either central or recombinant NC receptors, [Nphe¹]NC(1−13)NH2 antagonizes the effect of NC at human recombinant NC receptors and in the mouse tail withdrawal assay.

In the present study, the minimal fragment sequence required to fully activate the nociceptin (NC) receptor, namely NC(1−13)-NH2, was used as template for the design of a series of new compounds. Changes were made in the N-terminal tetrapeptide Phe-Gly-Gly-Phe, which has been shown to be essential for receptor occupation and activation. The new compounds were tested for their ability to inhibit the electrically evoked contraction of the mouse vas deferens, a pharmacological preparation sensitive to NC. Results obtained indicate that (a) the replacement of Gly² or Gly³ with an aromatic residue (Phe) of l or d chirality eliminates the ability of the peptide to occupy the NC receptor; (b) the distance between Phe¹ and Phe⁴ of NC appears to be critical, since any alteration of it leads to a marked decrease or a total elimination of biological activity; and (c) the insertion of a pseudopeptide bond between Phe¹ and Gly² maintains affinity but eliminates the ability of the peptide to activate the NC receptor and leads to antagonism. The peptide [Phe¹ψ(CH2−NH)Gly²]-NC(1−13)-NH2 acts as a selective NC receptor antagonist and is inactive on opioid receptors. The results summarized in this paper confirm and extend our previous findings by showing that the structural requirements for NC binding to its receptor are clearly different from those of opioids; in addition, this structure−activity study has led to the identification of the first NC receptor selective antagonist.