Further studies on nociceptin-related peptides: Discovery of a new chemical template with antagonist activity on the nociceptin receptor

Article abstract of DOI:10.1021/jm990075h

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 f

Full text of DOI:10.1021/jm990075h

J . Med. Chem. 2000, 43, 2805-2813
2805
F u r th er Stu d ies on Nocicep tin -Rela ted P ep tid es: Discover y of a New Ch em ica l
Tem p la te w ith An ta gon ist Activity on th e Nocicep tin Recep tor
†
†
†
†
‡
†
Remo Guerrini, Girolamo Calo′, Raffaella Bigoni, Anna Rizzi, Katia Varani, Geza Toth, Stefania Gessi,
#
#
#
†
Eiji Hashiba, Yoshio Hashimoto, David G. Lambert, Pier Andrea Borea, Roberto Tomatis,
Severo Salvadori, and Domenico Regoli*
,
†
Department of Pharmaceutical Sciences and Biotechnology Center and Department of Experimental and Clinical Medicine,
Section of Pharmacology, University of Ferrara, Via Fossato di Mortara 19, 44100 Ferrara, Italy, Isotope Laboratory Institute
of Biochemistry, Biological Research Centre, Szeged H-6701, Hungary, and University Department of Anaesthesia,
Leicester Royal Infirmary, Leicester LE1 5WW, U.K.
Received March 17, 2000
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 accumula-
tion in Chinese hamster ovary cells expressing the human recombinant NC receptor (CHONCR
)
1
2
were investigated. The compounds of the first series (a series) have an ordinary Xaa -Gly
1
2
bond, those of the second series (b series) have a Xaa Ψ(CH
2
-NH)Gly pseudopeptide bond,
1
2
and those of the third series (c series) have a peptoid (Nxaa -Gly ) 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
1
Phe 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,
1
2
1
2
[
Phe Ψ(CH
2
-NH)Gly ]NC(1-17)NH
2
and [Phe Ψ(CH
2
-NH)Gly ]NC(1-13)NH
2
being the most
potent compounds; (III) the compounds of the third series were all inactive both as agonists
1
1
and as antagonists with the exception of [Nphe ]NC(1-17)NH
which behaved as NC receptor antagonists both in the isolated tissue and in CHONCR cells
pK 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 Phe in the NC sequence as obtained with the pseudopeptide
2
2
and [Nphe ]NC(1-13)NH ,
(
B
1
1
2
bond between Phe and Gly or with the displacement of the benzyl side chain by one atom, as
1
1
in Nphe , lead respectively to reduction or elimination of efficacy. Indeed, in contrast to [Phe Ψ-
2
(
CH
assays involving either central or recombinant NC receptors, [Nphe ]NC(1-13)NH
the effect of NC at human recombinant NC receptors and in the mouse tail withdrawal assay.
2
-NH)Gly ]NC(1-13)NH
2
which has been reported to exhibit agonist activity in several
1
2
antagonizes
In tr od u ction 1
Nociceptin (NC)/orphanin FQ is the endogenous ligand
the release of neurotransmitters such as dopamine,
8
9
10
11
glutamate, acetylcholine, noradrenaline, and sero-
1
2,13
2
,3
tonin.
Similarly, in the periphery, NC inhibits the
of the ORL-1 (opioid receptor like 1) receptor, the
G-protein-coupled receptor which inhibits adenylyl cy-
1
4-16
release of neurotransmitters from sympathetic,
1
6,17
18-21
2
,3
4
parasympathetic,
and peptidergic nerves
and
clase,
activates potassium channels, and inhibits
5
22-24
and diuresis.25
calcium channels. The NC receptor is expressed in
different regions of the brain, in the spinal cord, and in
peripheral organs such as intestine, vas deferens, liver,
and spleen (see refs 6, 7 for a review). Sequentially, NC
shows some structural homology with dynorphin A, the
endogenous ligand of the κ opioid receptor. Despite
structural similarities of receptors and ligands between
the opioid and the NC systems, they are pharmacologi-
cally distinct, since NC does not bind opioid receptors
and opioid ligands have no affinity for the NC receptor.
NC exerts several biological effects in the central
nervous system (see refs 6, 7 for a review) and inhibits
causes hypotension, bradycardia,
Structure-activity studies on NC have been per-
formed over the last 2 years: First, we studied the
activities of NC fragments to determine the minimal
sequence (NC(1-13)NH ) that maintains full agonistic
activity and is protected from degradation at the C-
terminus. This tridecapeptide has served as a template
for the identification of further compounds. We were
also able to determine that the active group(s) of NC
are in the N-terminus tetrapeptide Phe-Gly-Gly-Phe
2
2
6
(
message), while the other nine residues (5-13) are
27
required for binding (address) to the NC receptor.
Second, in an attempt to protect the N-terminus from
degradation by aminopeptidases, we discovered that
*
To whom correspondence should be addressed. Tel: +39-0532-
2
91227. Fax: +39-0532-291205. E-mail: d.regoli@unife.it.
†
1
2
Department of Experimental and Clinical Medicine, University of
[
Phe Ψ(CH2-NH)Gly ]NC(1-13)NH2 is an antagonist of
NC receptors expressed in the mouse vas deferens, in
Ferrara.
2
8
‡
Biological Research Centre.
Leicester Royal Infirmary.
#
18,29-32
a variety of in vitro peripheral preparations,
and
1
0.1021/jm990075h CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/07/2000

2
806 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Guerrini et al.
Ch a r t 1. N-Terminus Dipeptide Structures of the a -c
Series Compounds
without further purification to avoid possible epimer-
ization. To test the formation, during the synthesis of
diastereomers of the b series compounds, we adopted
different HPLC strategies. For the compound 2b (the
best analogue of this series), we prepared the relative
diastereomer with a D-Phe residue in position 1,
1
2
28
[D-Phe Ψ(CH2-NH)Gly ]NC(1-13)NH2, and we com-
pared the HPLC behavior of the two peptides, under
the analytical conditions reported in the Experimental
Section. Under these conditions, the two peptides showed
the same retention time. We also adopted an alternative
HPLC solvent including a different ion-pairing system,
e.g. triethylammonium phosphate (pH 6.5) and CH3CN
with various gradients to separate the two diastereo-
meric peptides. Unfortunately, we did not obtain any
separation of the two peptides under these experimental
conditions. In addition, we synthesized two diastereo-
meric tetrapeptides Boc-L- and -D-PheΨ(CH2-NH)Gly-
Gly-Phe-OH, and we compared their HPLC retention
time under the latter HPLC conditions. Once again, the
two diastereomer tetrapeptides were not distinguishable
by HPLC analysis. Finally, we removed the Boc protec-
tion from these tetrapeptides to obtain H-L- and -D-
PheΨ(CH2-NH)Gly-Gly-Phe-OH, and we performed a
new HPLC analysis under the following experimental
conditions: chiral column Daicel Crownpak CR for RP-
HPLC (15 × 0.4 cm, 5 µm) eluted with a solution of
HClO4 (pH 3.1) containing KCl (1 mM) and 10% MeOH
with a flow rate of 0.65 mL/min at 5 °C temperature.
Under these experimental conditions, we obtained two
peaks, not completely baseline resolved, with retention
times of 44.3 and 46.8 min corresponding to H-D- and
also in vivo in the cardiovascular system of the mouse.22
However, this compound maintains agonistic activities,
mimicking the actions of NC especially when tested in
1
1,12,33-37
central nervous system preparations
(see the
review article38 for a detailed analysis of the pharma-
1
2
cological profile of [Phe Ψ(CH2-NH)Gly ]NC(1-13)NH2).
In the present investigation, we have further modified
the N-terminus Phe and identified new antagonists,
which, although of low potency, show an interesting
pharmacological profile.
-
L-PheΨ(CH2-NH)Gly-Gly-Phe-OH, respectively. We
therefore conclude that the application of several HPLC
methods did not result in complete separation of these
diastereoisomeric tridecapeptides and N-terminus tetra-
peptides. However, on the basis of results obtained with
4
0
similar peptides by Ho et al. and on the basis of our
Resu lts a n d Discu ssion
1
2
biological assay results ([D-Phe Ψ(CH2-NH)Gly ]NC(1-
3)NH2 is inactive both as NC receptor agonist and as
Three series of eight compounds were synthesized and
tested. As shown in Chart 1, the compounds of the first
1
28
antagonist ) we feel that isomerization (if it occurs) may
be negligible.
1
2
series (a series) have an ordinary Xaa -Gly bond, those
1
of the second series (b series) have a Xaa Ψ(CH2-NH)-
1
Peptoids 1c-8c with the Xaa side chain N-shift were
2
Gly pseudopeptide bond, and those of the third series
obtained by condensing Boc-Nxaa-OH with the same NC
fragments, obtained by the solid-phase methods re-
ported above (b series), in the presence of DIPCDI and
HOBt in the final acylation step of peptide synthesis.
The syntheses of the peptoid residues, Nxaa-OEt, were
achieved by alkylation of an amine with ethyl bromo-
acetate or by reductive amination of the H-Gly-OEt
amino function in the presence of NaBH3CN. Nxaa-OEt
was protected at the amino function with the Boc group
and the ester function removed by basic hydrolysis to
give Boc-Nxaa-OH.
1
2
(
c series) have a peptoid (Nxaa -Gly ) structure.
Peptides 1-8 of the a and c series were prepared by
solid-phase peptide synthesis performed on a Milligen
050 synthesizer using a Fmoc-PAL-PEG-PS-resin (0.2
9
mmol/g, 0.2 g in all syntheses). Boc-protected amino
aldehydes were prepared by the reduction of the Wein-
reb amide.39 Peptides were assembled using Fmoc-
protected amino acid (4 equiv), DIPCDI (4 equiv), and
HOBt (4 equiv) as coupling agents for 1 h for each
coupling. Side chain protecting groups used were Pmc
for Arg, Trt for Asn and Gln, Boc for Lys, and tBu for
Ser and Thr. Peptides 1b-8b containing modification
of the CO-NH bond were obtained by condensing Boc-
Xaa-CHO with the NC fragments [NC(2-17)-, NC(2-
The chemical features of the new compounds are
presented in Table 1. K′ was determined in two (I and
II) solvent systems to assess the purity of each com-
pound. Mass ion data are also reported to characterize
the synthesized peptides.
1
3)-, NC(2-12)-, and NC(2-9)]-PAL-PEG-PS-resin and
reducing the intermediate imine derivative in situ with
2
8
NaBH3CN in order to minimize racemisation. As
reported by Ho et al.,40 the formation of diastereomeric
peptides due to apparent racemization at the R-carbon
of the Boc-amino aldehyde was observed in several
cases. Protected amino aldehydes were used directly
The peptides of the three series were tested for their
ability to inhibit the electrically evoked contraction
(twitch response) of the mouse vas deferens (mVD), a
pharmacological preparation shown to be sensitive to
1
6
NC. When they were found inactive as agonists, the

Further Studies on Nociceptin-Related Peptides
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2807
Ta ble 1. Abbreviated Names and Analytical Properties of NC Analogues
K′ a
MH+ b
no.
abbreviated names
I
II
calcd
found
1
5
6
7
1
3
4
5
6
7
8
1
2
3
4
5
6
7
8
a
a
a
b
b
b
b
b
b
b
c
c
c
c
c
c
c
c
[Tyr ]NC(1-13)NH2
2.41
2.73
3.56
3.86
3.52
3.81
3.65
3.91
4.15
3.17
3.88
2.14
2.28
2.66
2.37
2.79
3.71
2.32
3.78
4.06
4.87
4.48
4.61
4.38
4.32
4.62
4.81
3.88
4.81
3.72
3.91
4.17
3.83
4.31
4.83
3.61
1398.6
1396.5
1388.6
1795.0
1240.4
926.0
1384.6
1382.5
1374.6
1334.5
1809.0
1382.5
1254.4
940.0
1399.0
1396.5
1388.7
1794.8
1240.1
926.3
1384.5
1381.9
1374.6
1334.3
1808.7
1382.6
1255.0
940.7
1
[(pMe)Phe ]NC(1-13)NH2
1
[Cha ]NC(1-13)NH2
1
2
[Phe Ψ(CH2-NH)Gly ]NC(1-17)NH2
1
2
[Phe Ψ(CH2-NH)Gly ]NC(1-12)NH2
1
2
[Phe Ψ(CH2-NH)Gly ]NC(1-9)NH2
1
2
[Tyr Ψ(CH2-NH)Gly ]NC(1-13)NH2
1
2
[(pMe)Phe Ψ(CH2-NH)Gly ]NC(1-13)NH2
1
2
[Cha Ψ(CH2-NH)Gly ]NC(1-13)NH2
1
2
[Leu Ψ(CH2-NH)Gly ]NC(1-13)NH2
1
[Nphe ]NC(1-17)NH2
1
[Nphe ]NC(1-13)HN2
1
[Nphe ]NC(1-12)NH2
1
[Nphe ]NC(1-9)NH2
1
[Ntyr ]NC(1-13)NH2
1398.6
1396.5
1388.6
1348.5
1398.2
1396.2
1388.1
1348.0
1
[N(pMe)phe ]NC(1-13)NH2
1
[Ncha ]NC(1-13)NH2
1
[Nleu ]NC(1-13)NH2
a
K′ is the capacity factor determined by analytical HPLC. b The mass ion (MH ) was obtained by MALDI-TOF mass spectrometry
+
peptides were assayed as antagonists against the refer-
ence agonist, NC(1-13)NH2, in the same preparation.
Results of biological assays are presented in Table 2 in
terms of pEC50 to describe their agonist potency and in
terms of pKB to indicate their potency as antagonists.
The same compounds were also evaluated in a recently
described NC receptor binding assay performed on
agonist but also as antagonist. In CHONCR cells the
compounds of the b series all behaved as agonists
showing a rank order potency similar to those obtained
in the mVD and in the binding assay.
Compounds of the c series have no agonistic effects;
two of them, compounds 1c and 2c, act as antagonists
of average potency both in the mVD (Table 2) and in
CHONCR cells (Table 3); all other peptides are inactive
also as antagonists. When tested for their ability to
3
mouse forebrain membranes using [ H]NC-NH2 as a
4
1
radioligand. Results of binding experiments, expressed
in terms of pKi, are also presented in Table 2. The
compounds of the three series were also tested in
CHONCR cells for their ability to inhibit forskolin-
stimulated cAMP accumulation. The results of these
experiments are summarized in Table 3.
3
compete with [ H]NC-NH2 for the NC site in mouse
brain membranes, compounds 1c and 2c show average
values of affinity (pKi ≈ 7), compounds 3c and 5c-8c
show pKi ≈ 5.5, and compound 4c has a pKi value below
5.
As reported by us previously,27 position 1 of the ligand
NC(1-13)NH2 can be modulated in different ways, by
replacing Phe with Tyr or with the aliphatic residues
Leu or Cha, which are tolerated with full retention of
agonistic activity, even if the replacement with Tyr gives
Ala-scan studies on NC from different laboratories
1
demonstrated that replacement of Phe appears to be
4
3
44
critical for receptor occupation and activation. The
1
insertion of a pseudopeptide bond between Phe and
Gly , as in compound 2b, maintains affinity but reduces
2
2
6,42
a compound that interacts also with opioid receptors.
the ability to activate the NC receptor. This reduced
efficacy led to antagonism in the mVD but not in
CHONCR cells where the compound behaved as a full
agonist. Reports from various laboratories have shown
that compound 2b may behave as an antagonist, partial
agonist, or full agonist depending on the preparation/
1
Other substitutions of the aromatic Phe , as with
(pCH3)Phe, strongly reduce activity.
Results summarized in Tables 2 and 3 show that
compounds 1a , 2a , 5a , 7a , and 8a are potent agonists,
as active as NC(1-17)NH2, and they are full agonists
in the mVD and in CHONCR cells. In addition they show
high affinities (pKi from 8.4 to 9.1) in the binding assay.
The shorter sequences (compounds 3a and 4a ) show
decreased affinity by approximately 30-fold (compound
1
8,29-32
assay. In several isolated tissues
and in the
cardiovascular system of the mouse in vivo, compound
2b behaves as an antagonist, while this pseudopeptide
is a potent agonist in several tests on central actions of
1
1,12,34-37
3
a ) or by >1000-fold (compound 4a ). Compound 6a is a
NC.
The full agonist activity of compound 2b
low-potency agonist in agreement with its low binding
affinity. Some of the biological data obtained in the mVD
with these compounds have already been communi-
in CHONCR cells has been confirmed in different labo-
3
3,45,46
ratories (present data and
). The reasons for the
dual behavior of compound 2b are at present unknown;
however, recent findings suggest that the intrinsic
activity of the compound may depend on the number of
2
7
cated.
1
2
The compounds of the b series (Xaa Ψ(CH2-NH)Gly )
1
47
are inactive as agonists in the mVD: only the Tyr and
Leu derivatives show weak agonist activities. Affinities
NC receptors expressed in a given preparation (see ref
1
38 for a detailed discussion of this topic). Collectively,
these findings indicate that compound 2b (as well as
the other molecules of the b series) should be viewed
as a low-efficacy agonist.
in the binding assay are quite high for compounds 1b,
2
b, 5b, and 8b, weaker for 3b and 6b, and very weak,
if any, for compound 4b. Some of them, for instance
compounds 1b and 2b, act as antagonists of average
potencies; compounds 3b, 4b, and 6b are weak antago-
nists; compounds 5b and 8b should be considered as
partial agonists. Compound 7b is inactive not only as
Contrary to compound 2b, compound 2c acts as a pure
NC receptor antagonist both in the mVD and in CHONCR
1
cells. In addition, [Nphe ]NC(1-13)NH2 competitively
and selectively antagonized the effects of NC in several

2
808 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Guerrini et al.
isolated tissues (rat vas deferens, guinea pig ileum and
renal pelvis, mouse colon) showing pA2 values ranging
4
8,49
from 6.0 to 6.4.
More importantly, it is also active
in vivo where it antagonizes the pronociceptive and
antimorphine actions of intracerebroventricularly ap-
plied NC, measured in the mouse tail withdrawal
4
9
assay, and the stimulatory effect of NC on food intake
5
0
in the rat.
Some conclusions can be drawn from the data pre-
sented above. The correlation between the values of
apparent affinity (pEC50/pKB) obtained in the mVD and
in the CHONCR assays and the values of actual affinity
(
pKi) obtained in the binding assay for the same
compounds is high, as indicated by the value of the
correlation coefficients (mVD - CHONCR r ) 0.80; mVD
-
mB r ) 0.88; CHONCR - mB r ) 0.90); this suggests
that the same receptor site is expressed in the three
preparations. However, the actual affinity values mea-
sured in the binding assay and the potencies estimated
in the CHONCR assays were always higher, on average
by 10-30-fold, than those of the potencies evaluated by
the bioassay in the mVD. This is quite common in
5
1
peptide pharmacology (see Regoli et al. for the kinin,
5
2
53
Regoli et al. for the neurokinin, and Knapp et al. for
the opioid field) and has been attributed to the different
accessibility of the receptors in the preparations (cell
or cell membranes and intact tissues). It is assumed that
the whole receptor population can be easily reached in
a suspension of cells or plasma membranes, while the
receptors that are present in the various layers of
smooth muscle cells constituting the mVD are not.
Another possible explanation for the different potencies
obtained in the different assays is represented by the
different buffers utilized especially in terms of concen-
+
tration of Na which may affect ligand affinity, particu-
larly for agonists.
Some interesting points have emerged from the
structure-activity study performed with the compounds
of the a -c series. The use of the pseudopeptide bond
1
2
(
CO-NH f CH2-NH) between Xaa and Gly reduces
the efficacy of all compounds. This reduction in efficacy
is evident in the mVD assay (where the compound of
the b series behaves as NCR antagonists) but not in
the CHONCR assays. This different behavior may depend
on the fact that our CHONCR cells express a very high
4
5
number of receptors (Bmax about 1700 fmol/mg protein )
which represents a large receptor reserve. Therefore,
compounds which are actually partial agonists may
elicit maximal effect similar to those evoked by full
4
7
agonists. The results obtained by Toll in CHO express-
ing different levels of NCR support this interpretation.
Compound 2b behaves as a receptor agonist in cells with
high receptor number, while it behaves as an antagonist
4
7
in cells with low receptor number. The fact that
compounds of the b series behave as antagonists in the
mVD suggests that (i) the occupation of the receptor by
1
the antagonist requires the modified message (Xaa Ψ-
(CH2-NH)Gly-Gly-Phe) and a critical C-terminus chain,
whose optimum is the nonapeptide NC(5-13), similar
1
to the requirements for agonists (see a series); (ii) Phe ,
although structurally affected by the presence of the
pseudopeptide bond (b series), contributes to the an-
tagonist affinity better than any other substitution, by
aromatic residues (Tyr, (pCH3)Phe) or aliphatic residues

Further Studies on Nociceptin-Related Peptides
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2809
Ta ble 3. Functional Activities of NC-Related Peptides in CHOhNCR
1
2
1
2
1
2
Xaa -Gly (a series)
Xaa Ψ(CH2-NH)Gly (b series)
Nxaa -Gly (c series)
agonist agonist antag
pEC50 pKB
inactive* 6.17
agonist agonist antag
agonist
pEC50
agonist
antag
compda
Xaa1
Phe
pEC50
Emax (%)
pKB
Emax (%)
pKB
Emax (%)
1
2
3
4
5
6
7
8
, NC(1-17)NH2
9.77
103 ( 1
ND
9.14
(0.20)
8.65
(0.20)
6.59
102 ( 1
104 ( 3
109 ( 3
ND
ND
ND
ND
ND
ND
ND
ND
(
(
(
(
0.20)
(0.35)
6.12
(0.28)
inactive
, NC(1-13)NH2
, NC(1-12)NH2
, NC(1-9)NH2
Phe
Phe
Phe
Tyr
9.49
0.21)
8.54
0.27)
6.74
0.30)
10.05
0.29)
7.98
0.19)
104 ( 1
100 ( 3
97 ( 7
ND
ND
ND
ND
ND
ND
ND
inactive*
inactive
(0.24)
crc incomplete
inactive
inactive
1
, [Tyr ]NC(1-13)NH2
101 ( 1
103 ( 3
104 ( 1
102 ( 1
7.97
(0.22)
6.47
(0.20)
7.20
(0.10)
7.36
(0.55)
102 ( 1
77 ( 2
crc incomplete
crc incomplete
5.28
(0.66)
ND*
(
(
1
, [(pMe)Phe ]NC(1-13)NH2 (pMe)Phe
1
, [Cha ]NC(1-13)NH2
Cha
Leu
10.04
0.34)
9.79
107 ( 1
101 ( 7
6.21
(0.41)
61( 13 ND
(
1
, [Leu ]NC(1-13)NH2
crc incomplete
ND*
(
0.56)
a
The syntheses for compounds 1-4 and 8 of the a series and for compound 2 of the b series have been already published.27,28 The data
of compounds 1-4 of the a series and compounds 2b and 2c are taken from refs 45, 65. The antagonistic properties of these compounds
were tested using NC(1-13)NH2 as agonist. For pEC50 and pKB values the confidence limits 95% are given in parentheses. ND: not
determined because these compounds are full agonists; ND*: not determined as there was >50% inhibition at the highest concentration
used (10 µM); inactive: inactive up to 10 µM; inactive*: small inhibition (<15% at 10 µM); crc incomplete: <50% inhibition at the highest
concentration tested (10 µM). These data are mean of at least 3 separate experiments.
(
Leu or Cha), in contrast with what happens for
peptides were made in distilled water and kept at -20 °C until
use. Krebs solution (gassed with 95% O and 5% CO , pH 7.4)
had the following composition (in mM): NaCl 118.5, KCl 4.7,
KH PO 1.2, NaHCO 25, CaCl 2.5, glucose 10. All other
reagents were from Sigma Chemical Co. (Poole, U.K.) or E.
Merck (Darmstadt, Germany) and were of the highest purity
grade available.
compounds of the a series, which are all agonists of high
potency, with the only exception of compound 6a .
The C f N shift of the Phe1 side chain leads to
complete elimination of efficacy thus giving pure an-
tagonists. In fact the antagonist effects of compounds
2
2
2
4
3
2
1
c and 2c are similar in the mVD and in CHONCR cells.
P ep tid e P u r ifica tion a n d An a lytica l Deter m in a tion s.
Crude peptides were purified by preparative reversed-phase
HPLC using a Water Delta Prep 4000 system with a Waters
PrepLC 40-mm assembly column C18 (30 × 4 cm, 300 Å, 15-
µm spherical particle size column). The column was perfused
at a flow rate of 50 mL/min with a mobile phase containing
solvent A (10%, v/v, acetonitrile in 0.1% TFA), and a linear
gradient from 0% to 50% solvent B (60%, v/v, acetonitrile in
This modification leads however to an important reduc-
tion of affinity. In addition, this C f N shift is inap-
propriate for any other residue than Phe, even in
binding; all compounds with residues other than Phe
in position 1 (compounds 5c-8c) show affinities at least
3
0-fold lower than compounds 1c and 2c. Again, the
whole nonapeptide chain (5-13) at the C-terminus is
needed for receptor occupation, since shorter sequences,
as in compounds 3c and 4c, are inactive.
0
.1% TFA) over 25 min was adopted for the elution of peptides.
Analytical HPLC analyses were performed on a Bruker
liquid chromatography LC 21-C instrument fitted with a
Alltech C18 column (4.6 × 150 mm, 5-µm particle size) and
equipped with a Bruker LC 313 UV variable-wavelength
detector. Recording and quantification were accomplished with
a chromatographic data processor coupled to an Epson com-
puter system (BX-10). Analytical determination and capacity
factors (K′) of the peptides were determinated using HPLC
conditions in the above solvent system (solvents A and B)
programmed at a flow rate of 1 mL/min using the following
linear gradients: (I) from 0% to 50% B in 25 min and (II) from
In conclusion, this study demonstrated that a differ-
1
ent orientation of the aromatic residue Phe of NC(1-
1
Phe and Gly , leads to reduced efficacy. The displace-
ment of the benzyl side chain by one atom, as in Nphe
3)NH2, as obtained with a pseudopeptide bond between
1
2
1
(c series), completely eliminates agonistic activity and
provides a pure antagonist at the NC receptor. While
1
2
[Phe Ψ(CH2-NH)Gly ]NC(1-13)NH2 maintains some
0
% to 20% B in 25 min. All analogues showed less than 1%
residual agonistic activity and is considered to be a
partial agonist with different efficacy depending on the
impurities when monitored at 220 nm.
1
Molecular weights of compounds were determined by a
MALDI-TOF (matrix assisted laser desorption ionization time-
of-flight) analysis using a Hewlett-Packard G2025A LD-TOF
system mass spectrometer and R-cyano-4-hydroxycinnamic
preparations, [Nphe ]NC(1-13)NH2 is completely inac-
tive as an agonist and behaves as a pure NC receptor
antagonist in vitro on native and recombinant NC
receptors (present data) as well as in vivo.4
9,50
Although
+
acid as the matrix. The values are expressed as MH .
weak (pKi ) 7.0; pKB ) 6.3) this new compound provides
a new lead for future development of NCR antagonists.
TLC was performed on precoated plates of silica gel F254
(
Merck, Darmstadt, Germany) using the following solvent
systems: (III) AcOEt/n-hexane (1:1, v/v), (IV) CH Cl /methanol
2
9.5:0.5, v/v), (V) CH Cl /methanol (9:1, v/v), (VI) CH Cl /
2
2
Exp er im en ta l Section
(
2
2
2
methanol/toluene (17:2:1, v/v/v). Ninhydrin (1%) or chlorine
iodine spray reagents were employed to detect the peptides.
Elemental analyses were performed by the Microanalytical
Laboratory of the Chemistry Department of the University of
Ferrara.
Ma ter ia ls. Amino acids, protected amino acids, and chemi-
cals were purchased from Bachem, Novabiochem, or Fluka
Switzerland). N-Benzylglycine was from Aldrich (Milwaukee,
WI). Boc-(pMe)Phe-OH was from RSP (Worcester, MA). The
resin [5-(4′-Fmoc-aminomethyl-3′,5′-dimethoxyphenoxy)valeric
acid]poly(ethylene glycol)/polystyrene suport (Fmoc-PAL-PEG-
PS) was from Millipore (Waltham, MA). Naloxone was from
Tocris Cookson (Bristol, U.K.). Stock solutions (1 mmol) of
(
Optical rotations were determined using a Perkin-Elmer 241
polarimeter with a 10-cm cell using methanol as the solvent
1
and at a peptide concentration of 1%. H NMR spectroscopy

2
810 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Guerrini et al.
was obtained with a 200-MHz Bruker instrument and are
recorded in δ units.
Gen er a l P r oced u r es for th e Solid -P h a se Syn th esis of
a a n d c Ser ies P ep tid es. As an illustrative example the
by treatment of known primary amines: isobutylamine and
cyclohexylmethylamine with ethyl bromoacetate or alterna-
tively by reductive alkylation of 4-hydroxybenzaldehyde or
p-tolualdehyde with glycine ethyl esters in the presence of
1
NaCNBH . The successive protection of the amino function
synthesis of [Nphe ]NC(1-17)NH
2
(1c) is described. Fmoc-
3
with Boc59 and the hydrolysis of ethyl ester gave the Boc-
protected N-alkylglycine employed for the synthesis of com-
pounds 1c-8c.
PAL-PEG-PS resin (0.21 mmol/g, 0.2 g) was treated with 20%
piperine/DMF and linked with N -Fmoc-N -tritylglutamine,
via its N-hydroxybenzotriazole (HOBt) active ester formed in
situ with DIPCDI. The following N -Fmoc amino acids were
sequentially coupled to the growing peptide chain: N -Fmoc-
N -(Trt)-Asn, N -Fmoc-Ala, N -Fmoc-Leu, N -Fmoc-N -(Boc)-
Lys, N -Fmoc-N -(Pmc)-Arg, N -Fmoc-Ala, N -Fmoc-O-(tBu)-
Ser, N -Fmoc-N -(Boc)-Lys, N -Fmoc-N -(Pmc)-Arg, N -Fmoc-
Ala, N -Fmoc-Gly, N -Fmoc-O(tBu)-Thr, N -Fmoc-Phe, N -
R
γ
R
Nleu -OEt. Ethyl bromoacetate (1.1 mL, 10 mmol) in THF
(10 mL) was added dropwise to a chilled (0 °C) solution of
isobutylamine (1 mL, 10 mmol) and Et N (1.4 mL, 10 mmol)
3
in THF (30 mL). After the addition was complete the mixture
was warmed to room temperature and then stirred overnight.
The precipitate Et N‚HBr was filtered and washed with a
3
small amount of THF. The filtrate was concentrated under
reduced pressure and the residue was purified by silica gel
R
γ
R
R
R
ꢀ
R
ω
R
R
R
ꢀ
R
ω
R
R
R
R
R
R
54
Fmoc-Gly, N -Fmoc-Gly, and Boc-Nphe in the final acylation
step of the peptide synthesis. All the N -Fmoc amino acids or
R
Boc-Nphe (4 equiv) were coupled to the growing peptide chain
by using 1,3-diisopropylcarbodiimide (4 equiv) and 1-hydroxy-
benzotriazole (4 equiv) in DMF, and the coupling reaction time
was 1 h. Piperidine (20%)/DMF was used to remove the Fmoc
group at every step. The peptide resin was washed with
column chromatography with solvent system (IV). The product
1
was an oil: yield 0.68 g (43%); TLC R
(CDCl
f
(IV) 0.56; H NMR
3
) δ 0.82 (d, 6H, J ) 6.4 Hz), 1.27 (t, 3H, J ) 7.1 Hz),
1.75 (m, 1H), 3.25 (m, 3H), 3.56 (m, 2H), 3.96 (q, 2H, J ) 7.1
Hz).
1
methanol and dried in vacuo to yield the protected [Nphe ]-
Nch a -OEt. This compound was synthesized as described
NC(1-17)-NH
2
-resin. The other peptides 1-8 in a and c series
for Nleu-OEt. The product was an oil: yield 0.93 g (47%); TLC
1
were synthesized in a similar manner. The protected peptide-
R
f
(IV) 0.51; H NMR (CDCl
3
) δ 1.17 (m, 5H), 1.28 (m, 4H),
5
5
resin was treated with reagent K (TFA/H
dithiol/thioanisole, 82.5:5:5:2.5:5; v/v; 10 mL/0.2 g of resin) for
h at room temperature. After filtration of the exhausted
2
O/phenol/ethane-
1.73 (m, 5H), 2.7 (bs, 1H), 2.96 (m, 2H), 3.7 (m, 2H), 4.15 (q,
2H, J ) 6.8 Hz).
1
Ntyr -OEt. Glycine ethyl ester hydrochloride (1.8 g, 13
mmol) and 4-hydroxybenzaldehyde (1.2 g, 10 mmol) were
resin, the solvent was concentrated in vacuo and the residue
triturated with ether. The crude peptide was purified by
preparative reverse phase HPLC to yield a white powder after
lyophilization.
dissolved in absolute EtOH (50 mL), and then NaCNBH (1.38
3
g, 22 mmol) was added portionwise. The reaction was stirred
at room temperature for 16 h. The EtOH was removed under
reduced pressure and the residue dissolved in EtOAc. The
Gen er a l P r oced u r es for t h e Syn t h esis of b Ser ies
P ep t id es. As an illustrative example the synthesis of
3
organic layer was washed with NaHCO (5%) and brine, dried,
and evaporated to dryness. The residue was purified by silica
1
2
[Phe Ψ(CH
2
NH)Gly ]NC(1-17)NH
2
(1b) is described. Frag-
ment NC(2-17)-PAL-PEG-PS-resin (0.2 g, 0.21 mmol/g, 0.042
mmol) prepared as reported above, was swelled in methanol
containing 1% (v/v) acetic acid (2 mL). After 20 min, a solution
gel column chromatography with solvent system (V). The
1
product was an oil: yield 0.77 g (37%); TLC R
NMR (CDCl
f
(V) 0.53; H
3
) δ 1.17 (t, 3H, J ) 6.9 Hz), 3.82 (m, 2H), 3.89
(m, 2H), 4.16 (q, 2H, J ) 6.9 Hz), 5.27 (bs, 2H), 6.83 (d, 2H, J
of Boc-Phe-CHO (0.016 g, 0.063 mmol) and NaBH
3
CN (0.008
g, 0.13 mmol) dissolved in methanol (0.4 mL) was added and
the reaction mixture stirred for 1 h. After this time, the resin
was washed with methanol and treated with reagent K as for
the synthesis of compound 1c. The other peptides of b series
were prepared in a similar manner.
) 6.5 Hz), 7.15 (d, 2H, J ) 6.5 Hz).
N(p Me)p h e-OEt. This compound was synthesized as de-
scribed for Ntyr-OEt. The product was an oil: yield 0.93 g
1
(45%); TLC R
f
(V) 0.72; H NMR (CDCl
3
) δ 1.15 (t, 3H, J ) 6.9
Hz), 2.25 (s, 3H), 2.8 (bs, 1H), 3.66 (m, 2H), 3.88 (m, 2H), 4.14
Am in o Ald eh yd es. The Boc-protected amino aldehydes
(q, 2H J ) 6.9 Hz), 7.14 (s, 4H).
3
9
1
were prepared as reported, using WSC instead of 1,1′-
carbonyldiimidazole. The Boc-amino acids (Boc-Leu-OH, Boc-
Phe-OH, Boc-Cha-OH, Boc-Tyr(tBu)-OH, and Boc-(pMe)Phe-
OH) (1 mmol) dissolved in DMF (5 mL) were reacted with N,O-
dimethylhydroxylamine hydrochloride (1.5 mmol) in the
presence of WSC (1.1 mmol), HOBt (1.1 mmol) and TEA (1.5
mmol). The reaction was stirred at room-temperature over-
night, diluted with 0.5 N HCl (50 mL) and extracted with
AcOEt (3 × 30 mL). The organic phase was washed with
Boc-Nleu -OEt: yield 87%; oil; TLC R
(CDCl
1.39 and 1.43 (s, 9H, Boc of two conformers), 1.8 (m, 1H), 3.1
f
0.89 (VI); H NMR
3
) δ 0.86 (d, 6H, J ) 6.5 Hz), 1.27 (t, 3H, J ) 6.9 Hz),
(m, 2H), 3.86 and 3.95 (s, 2H, CH
2H, J ) 6.9 Hz).
2
of two conformers), 3.98 (q,
1
Boc-Nch a -OEt: yield 84%; oil; TLC R
(CDCl
Boc of two conformers), 1.79 (m, 5H), 3.15 (m, 2H), 3.92 and
3.96 (s, 2H, CH of two conformers), 4.12 (q, 2H, J ) 6.8 Hz).
f
0.93 (VI); H NMR
3
) δ 1.15 (m, 5H), 1.32 (m, 4H), 1.45 and 1.48 (s, 9H,
2
1
saturated aqueous NaHCO
3
(3 × 15 mL), brine (3 × 15 mL),
Boc-Ntyr -OEt: yield 85%; oil; TLC R 0.81 (VI); H NMR
f
dried over Na SO
2
4
and then filtered and concentrated in vacuo
(CDCl ) δ 1.22 (t, 3H, J ) 7.2 Hz), 1.44 and 1.47 (s, 9H, Boc of
3
to yield the Boc-protected amino acid N,O-dimethylhydrox-
amate. The hydroxamate was then reduced to the correspond-
ing aldehyde with lithium aluminum hydride according to the
published procedure. The crude Boc-amino aldehyde was used
immediately, without further purification, for the synthesis
of compounds 1b-8b. The analytical data of: Boc-Leu-CHO
two conformers), 3.86 and 3.89 (s, 2H, CH of two conformers),
2
4.23 (m, 4H), 6.81 (m, 2H), 7.11 (m, 2H).
1
Boc-N(p Me)p h e-OEt: yield 89%; oil; TLC R
NMR (CDCl
H), 3.83 and 3.92 (s, 2H, CH
f
0.91 (VI); H
3
) δ 1.23 (t, 3H J ) 7.1 Hz), 1.46 (s, 9H), 2.28 (s,
of two conformers), 4.15 (q, 2H,
of two conformers), 7.16
3
2
J ) 7.1 Hz), 4.44 and 4.51 (s, 2H, CH
2
and Boc-Phe-CHO,5 Boc-Cha-CHO, and Boc-Tyr(tBu)-CHO
6
39
57
(s, 4H).
are in accordance with the literature.
Boc-Nleu -OH: yield 93%; mp 71-73 °C; TLC R
f
0.33 (VI);
Boc-(p Me)P h e-N(CH
3
)-OCH
3
: yield 93%; mp 94-96 °C;
4.38; H NMR (CDCl ) δ 1.36 (s, 9H),
.28 (s, 3H), 2.91 (dd, 2H, J ) 14.5, 7.6 Hz), 3.12 (s, 3H), 3.67
s, 3H), 4.72 (m, 1H), 5.12 (bs, 1H), 7.11 (s, 4H). Anal.
) C, H, N.
Boc-(p Me)P h e-CHO: yield 89%; mp 86-88 °C; TLC R
1
H NMR (CDCl ) δ 0.89 (d, 6H, J ) 6.5 Hz), 1.43 and 1.47 (s,
3
2
0
1
TLC R
f
0.43 (III); [R]
D
3
9H, Boc of two conformers), 1.8 (m, 1H), 3.08 (m, 2H), 3.90
and 3.97 (s, 2H CH of two conformers). Anal. (C H NO ) C,
2
(
(
2
11 21
4
H, N.
17 26 2 4
C H N O
Boc-Nch a -OH: yield 89%; mp 64-68; TLC R
f
0.37 (VI);
1
f
H NMR (CDCl ) δ 1.15 (m, 2H), 1.37 (m, 4H), 1.42 and 1.46
3
2
0
1
0
(
9
.84 (III); [R]
s, 3H), 3.09 (m, 2H), 4.42 (m, 1H), 5.03 (bs, 1H), 7.14 (s, 4H),
.62 (s, 1H). Anal. (C15 ) C, H, N.
Boc-N-a lk ylglycin es. The N-substituted glycine ethyl
esters were prepared following the procedure of Skiles et al.58
D
-28.4; H NMR (CDCl
3
) δ 1.43 (s, 9H), 2.32
(s, 9H, Boc of two conformers), 1.75 (m, 5H), 3.12 (m, 2H), 3.89
and 3.97 (s, 2H CH of two conformers). Anal. (C H NO ) C,
2
14 25
4
H
21NO
3
H, N.
Boc-Ntyr -OH: yield 91%; mp 92-95 °C; TLC R
f
0.31 (VI);
1
H NMR (CDCl ) δ 1.46 and 1.48 (s, 9H, Boc of two conform-
3

Further Studies on Nociceptin-Related Peptides
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2811
ers), 3.88 and 3.91 (s, 2H CH
H), 6.79 (m, 2H), 7.13 (m, 2H). Anal. (C14
Boc-N(p Me)p h e-OH: yield 86%; mp 84-86 °C; TLC R
2
of two conformers), 4.43 (m,
) C, H, N.
0.4
) δ 1.48 (s, 9H), 2.33 (s, 3H), 3.80 and
of two conformers), 4.47 and 4.50 (s, 2H CH
) C, H, N.
tides were performed, the maximum concentration tested was
10 µM. All incubations were for 15 min at 37 °C. In antagonist
studies the effects were tested against compound 2a (the
reference agonist). cAMP was extracted and assayed as previ-
2
H19NO
5
f
1
(VI); H NMR (CDCl
3
ously described.45 pK
Gaddum-Schild equation: pK
)/[antagonist]), assuming a slope equal to 1.
Da ta An a lysis a n d Ter m in ology. The data are expressed
as mean of n experiments. For pEC50, pK , and pK values the
confidence limits at 95% (CL95%) are given. A weighted
values were calculated by mean of the
) -log((concentration ratio -
3
.94 (s, 2H CH
2
2
B
of two conformers), 7.13 (s, 4H). Anal. (C15H21NO
4
B
1
m VD stu d ies. Swiss male mice weighing 25-30 g were
used. The bioassay experiments were performed as previously
2
7
B
i
described. Briefly, the vas deferens (mVD) was prepared
6
0
according to Hughes et al. and suspended in 10-mL organ
6
3
2
+
nonlinear least-squares curve-fitting program LIGAND was
used for computer analysis of binding inhibition experiments.
The pharmacological terminology adopted in this study is in
line with the recent IUPHAR recommendations:64 the agonist
apparent affinities are given as pEC50 ) the negative logarithm
to base 10 of the molar concentration of an agonist that
produces 50% of the maximal possible effect; apparent affini-
baths containing Mg -free Krebs solution at 33 °C. The tissues
were stimulated through two platinum ring electrodes with
supramaximal rectangular pulses of 1-ms duration and 0.1-
Hz frequency. The resting tension was maintained at 0.3 g.
The electrically evoked contractions were measured isotoni-
cally by means of a Basile strain gauge transducer and
recorded on a Linseis multichannel chart recorder (model
ties of antagonists are given in terms of pK
calculated using the Gaddum-Schild equation: pK
(CR - 1)/[antagonist]), assuming a slope value equal to unity,
B
which were
2
005). After an equilibration period of about 2 h the contrac-
B
) log-
tions induced by electrical field stimulation were stable. At
this time, cumulative concentration-response curves for NC
and NC-related peptides were performed (0.5 log unit steps).
When required, antagonists (10 µM) were added to the Krebs
solution 15 min before performing concentration-response
(
where CR is the ratio between equieffective concentrations of
agonist in the presence and absence of the antagonist. The
ligand affinities obtained in binding competition experiments
are given as pK ) the negative logarithm to base 10 of the
i
curves for agonists. pK
the Gaddum-Schild equation: pK
1)/[antagonist]), assuming a slope equal to 1.
Bin d in g Stu d ies on Mou se Br a in Mem br a n es. Swiss
male mice weighing 25-30 g were used. The binding experi-
B
values were calculated by mean of
inhibition equilibrium constant.
B
) -log((concentration ratio
-
Ack n ow led gm en t. This work was supported by the
Italian Ministry of the University (Cofin 99 Grant) by
the University of Ferrara (60% grants), by the Consiglio
Nazionale delle Ricerche, and by Prof. G. Smith (Le-
icester, U.K.) funds. We would like to acknowledge the
kind assistance of C. Harrison in performing some of
the experiments on CHONCR.
4
1
ments were performed as previously described. Briefly, the
mice were decapitated and the forebrain was dissected on ice.
The tissue was disrupted in a Polytron homogenizer (setting
5
) in 20 volumes of 50 mM Tris HCl, 2 mM EDTA, 100 µM
phenylmethanesulfonyl fluoride (PMSF) at pH 7.4. The ho-
mogenate was centrifuged at 40000g for 10 min and the pellet
was resuspended in the same buffer. After 30 min of incubation
at 37 °C, the membranes were centrifuged and the resulting
pellets were stored at -80 °C. Prior to freezing, an aliquot of
the homogenate was removed for protein assay according to a
Refer en ces
(
1) Abbreviations follow the IUPAC-IUB J oint Commission on
Biochemical Nomenclature for amino acids and peptides: J . Biol.
Chem. 1985, 260, 1442. Additional abbreviations used herein
are as follows: Boc (tert-butoxycarbonyl), DIPCDI (1,3-diisopro-
pylcarbodiimide), Fmoc ((9-fluorenylmethyl)oxycarbonyl), Fmoc-
PAL-PEG-PS (5-(4′-Fmoc-aminomethyl-3′,5′-dimethoxyphenoxy)-
valeric acid on the poly(ethylene glycol)/polystyrene support),
EtOAc (ethyl acetate), HOBt (1-hydroxybenzotriazole), MALDI-
TOF (matrix assisted laser desorption ionization time-of-flight),
Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl), tBu (tert-butyl),
TEA (triethylamine), TFA (trifluoroacetic acid), THF (terahy-
drofuran), Trt (trityl), WSC (1-ethyl-3-(3′-dimethylaminopropyl)-
carbodiimide‚HCl).
6
1
Bio Rad method, using bovine albumin as reference standard.
The final pellet was resuspended in the same incubation buffer
at a concentration of 200 µg protein/100 µL and this homoge-
nate was used for the binding assay. Inhibition experiments
were carried out in duplicate in a final volume of 250 µL in
3
test tubes containing 0.5 nM [ H]NCNH
2
, 50 mM Tris HCl
buffer, 2 mM EDTA, 100 µM PMSF at pH 7.4, mouse forebrain
membranes (200 µg protein/assay), and different concentra-
tions of the ligand under study. Details about the synthesis of
3
41
the radioligand [ H]NCNH
2
have been already reported.
(2) Meunier, J . C.; Mollereau, C.; Toll, L.; Suaudeau, C.; Moisand,
C.; Alvinerie, P.; Butour, J . L.; Guillemot, J . C.; Ferrara, P.;
Monserrat, B.; Mazarguil, H.; Vassart, G.; Parmentier, M.;
Costentin, J . Isolation and structure of the endogenous agonist
of opioid receptor-like ORL1 receptor. Nature 1995, 377, 532-
Moreover, the inhibitory binding constant, K
i
, was calculated
from the IC50 value according to the Cheng-Prusoff equation.6
2
Nonspecific binding was defined as the binding observed in
2
the presence of 10 µM NC(1-17)NH (compound 1a ) and was
5
35.
about 30% of the total binding. Incubation time was 120 min
at 25 °C, based on the results of previous time-course experi-
(
3) Reinscheid, R. K.; Nothacker, H. P.; Bourson, A.; Ardati, A.;
Henningsen, R. A.; Bunzow, J . R.; Grandy, D. K.; Langen, H.;
Monsma, F. J ., J r.; Civelli, O. Orphanin FQ: a neuropeptide that
activates an opioidlike G protein-coupled receptor. Science 1995,
4
1
ments. Bound and free radioactivities were separated by
filtering the assay mixture through Whatman GF/C glass-fiber
filters using a Brandel cell harvester. The incubation mixture
was diluted with 3 mL of ice-cold incubation buffer and then
vacuum-filtered rapidly and the filters were washed three
times with 3 mL of incubation buffer. The filter-bound
radioactivity was counted in a Beckman LS-1800 spectrometer
2
70, 792-794.
(
4) Connor, M.; Vaughan, C. W.; Chieng, B.; Christie, M. J .
Nociceptin receptor coupling to a potassium conductance in rat
locus coeruleus neurones in vitro. Br. J . Pharmacol. 1996, 119,
1614-1618.
5) Connor, M.; Yeo, A.; Henderson, G. The effect of nociceptin on
Ca2+ channel current and intracellular Ca2+ in the SH-SY5Y
human neuroblastoma cell line. Br. J . Pharmacol. 1996, 118,
(
(efficiency 55%). Under the experimental conditions here
described we demonstrated that the mouse forebrain mem-
2
05-207.
3
branes express a single class of binding sites for [ H]NCNH
with a K value of 0.55 nM and a Bmax value of about 100 fmol/
2
(6) Meunier, J . C. Nociceptin/orphanin FQ and the opioid receptor-
d
like ORL1 receptor. Eur. J . Pharmacol. 1997, 340, 1-15.
(
7) Civelli, O.; Nothacker, H. P.; Reinscheid, R. Reverse physiol-
ogy: discovery of the novel neuropeptide, orphanin FQ/nocice-
ptin. Crit. Rev. Neurobiol. 1998, 12, 163-176.
mg protein. Moreover the pharmacological profile of such a
site was superimposable to that of the NC receptor expressed
4
1
in the mVD.
(
8) Murphy, N. P.; Ly, H. T.; Maidment, N. T. Intracerebroventricu-
lar orphanin FQ/nociceptin suppresses dopamine release in the
nucleus accumbens of anaesthetized rats. Neuroscience 1996, 75,
In h ibition of cAMP Accu m u la tion in CHO Cells. cAMP
accumulation was measured in whole CHONCR cells incubated
in 0.3-mL volumes of Krebs-HEPES buffer containing BSA as
1
-4.
(
9) Nicol, B.; Lambert, D. G.; Rowbotham, D. J .; Smart, D.; McK-
night, A. T. Nociceptin induced inhibition of K+ evoked glutamate
release from rat cerebrocortical slices. Br. J . Pharmacol. 1996,
119, 1081-1083.
4
5
described in detail by Okawa et al. In addition, 1-isobutyl-
-methylxanthine (1 mM) and forskolin (1 µM) were also
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