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. 2000 Nov;131(5):903-8.
doi: 10.1038/sj.bjp.0703661.

(8-Naphthalen-1-ylmethyl-4-oxo-1-phenyl-1,3,8-triaza-spiro[4. 5]dec-3-yl)-acetic acid methyl ester (NNC 63-0532) is a novel potent nociceptin receptor agonist

C Thomsen  1 , R Hohlweg
Affiliations

(8-Naphthalen-1-ylmethyl-4-oxo-1-phenyl-1,3,8-triaza-spiro[4. 5]dec-3-yl)-acetic acid methyl ester (NNC 63-0532) is a novel potent nociceptin receptor agonist

C Thomsen et al. Br J Pharmacol. 2000 Nov.

Abstract

Spiroxatrine was identified as a moderately potent (K:(i)=118 nM) but non-selective agonist at the human nociceptin/orphanin FQ receptor, ORL1. This compound was subject to chemical modification and one of the resulting compounds, (8-naphthalen-1-ylmethyl-4-oxo-1-phenyl-1,3,8-triaza-s piro[4. 5]dec-3-yl)-acetic acid methyl ester (NNC 63-0532) was shown to have high affinity for ORL1 (K:(i)=7.3 nM). NNC 63-0532 showed only moderate affinity for the following receptors (K:(i) values in parentheses): mu-opioid (140 nM), kappa-opioid (405 nM), dopamine D(2S) (209 nM), dopamine D(3) (133 nM) and dopamine D(4.4) (107 nM) out of 75 different receptors, ion-channels and transporters. In functional assays, NNC 63-0532 was shown to be an agonist at ORL1 (EC(50)=305 nM), a much weaker agonist at the mu-opioid receptor (EC(50)>10 microM) and an antagonist or weak partial agonist at dopamine D(2S) (IC(50)=2830 nM). Thus, NNC 63-0532 is a novel non-peptide agonist with approximately 12 fold selectivity for ORL1 and may be useful for exploring the physiological roles of this receptor owing to its brain-penetrating properties.

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Figures

Figure 1
Figure 1
Chemical structure of spiroxatrine and NNC 63-0532.
Figure 2
Figure 2
Affinity of NNC 63-0532 and reference compounds at (A) the cloned human ORL1 and B) the rat nociceptin receptor in cortical membranes. The data are mean±s.e.mean of 3–4 experiments, which were performed in triplicate. IC50 values were calculated from competition binding experiments by a non-linear regression analysis fitted to a one-site model using the GraphPad Prism program (GraphPad Software, San Diego, U.S.A.). In separate experiments, the dissociation constants (Kd) for [125]-Tyr14-nociceptin binding to ORL1-expressing cells (Kd=0.10±0.02 nM, n= 4) and rat cortical membranes (Kd=0.15±0.04 nM, n= 3) were determined and used for calculation of Ki values according to the equation: Ki=IC50/(1+[L]/Kd) where [L] is the concentration of radioligand.
Figure 3
Figure 3
(A) Selectivity of NNC 63-0532 at human ORL1, μ-opioid and κ-opioid receptors, and (B) Selectivity of NNC 63-0532 for human ORL1 over dopamine D2S, D3 and D4.4 receptors. The resulting IC50's were converted to Ki values (see Results) as described in the legend to Figure 2 using the following parameters: ORL1 (Kd=0.10 nM; [L]=0.05 nM); μ-opioid (Kd=1.2 nM; [L]=1 nM); κ-opioid (Kd=2.1 nM; [L]=1 nM); dopamine D2S (Kd=0.05 nM; [L]=0.1 nM); dopamine D3 (Kd=0.09 nM; [L]=0.3 nM) and dopamine D4.4 (Kd=0.22 nM; [L]=0.3 nM).
Figure 4
Figure 4
Selectivity of NNC 63-0532 for the rat nociceptin receptor over rat opioid receptors as measured by displacement of [3H]-diprenorphine binding by NNC 63-0532, morphine and diprenorphine. The data are the mean±s.e.mean of three experiments, which were performed in triplicate. The displacement curves were compared to, respectively, one- and two-site competition binding models using least square analysis with GraphPad Prism software. Only the displacement curve for morphine was preferentially fitted to a two-site competition model over a one-site model (P<0.001). For Ki values see text.
Figure 5
Figure 5
Functional agonism of NNC 63-0532 at ORL1 when measuring (A) inhibition of forskolin-induced cyclic AMP-formation in BHK cells expressing ORL1, (B) stimulation of [35S]-GTP-γ-S binding to membranes from BHK/ORL1 cells and (C) stimulation of [35S]-GTP-γ-S binding to membranes from human μ-opioid receptor expressing cells. The calculated EC50's were (A) 0.83±0.15 nM (nociceptin), 0.49±0.08 nM (ac-RYYRWK-NH2) and 109±11 nM (NNC 63-0532); (B) 2.0±0.1 nM (nociceptin), 1.9±0.2 nM (ac-RYYRWK-NH2), 305±26 nM (NNC 63-0532) and 3050±210 nM (spiroxatrine) and (C) 44±6 nM (DAMGO), 52±8 nM (morphine) and >10.000 nM (NNC 63-0532). The results are mean±s.e.mean of 3–4 separate experiments that were performed in triplicate.
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