A nonpeptide oxytocin receptor antagonist radioligand highly selective for human receptors

Article abstract of DOI:10.1016/S0014-2999(02)02048-4

A novel, potent nonpeptide oxytocin receptor antagonist (1-(1-(2-(2,2,2-trifluoroethoxy)-4-(1-methylsulfonyl-4-piperidinyloxy) phenylacetyl)-4-piperidinyl)-3,4-dihydro-2(1H)-quinolinone) has been identified that can be labeled to high specific activity wi

Full text of DOI:10.1016/S0014-2999(02)02048-4

European Journal of Pharmacology 450 (2002) 19–28
www.elsevier.com/locate/ejphar
A nonpeptide oxytocin receptor antagonist radioligand highly
selective for human receptors
Wei Lemaire a,1, Julie A. O’Brien a,1, Maryann Burno a, Ashok G. Chaudhary b, Dennis C. Dean b,
Peter D. Williams c, Roger M. Freidinger c, Douglas J. Pettibone a, David L. Williams Jr. a,
*
aDepartment of Neuroscience, Merck Research Laboratories, West Point, PA 19486 USA
bDepartment of Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065-0900, USA
cDepartment of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
Received 4 April 2002; received in revised form 25 June 2002; accepted 2 July 2002
Abstract
A novel, potent nonpeptide oxytocin receptor antagonist (1-(1-(2-(2,2,2-trifluoroethoxy)-4-(1-methylsulfonyl-4-piperidinyloxy) phenyl-
acetyl)-4-piperidinyl)-3,4-dihydro-2(1H)-quinolinone) has been identified that can be labeled to high specific activity with [35S]. In binding
studies, this compound exhibits sub-nanomolar affinity and a high degree of selectivity (900–1800-fold) for human oxytocin receptors
compared to human vasopressin receptors. This compound appears suitable for studying the pharmacology of oxytocin receptors in human
and nonhuman primate tissues, for which there is currently a paucity of highly selective tools. It may also be useful as a nonlabeled
competitor or as a radioligand in autoradiographic studies of oxytocin receptor localization in these tissues.
D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Oxytocin receptor antagonist; Oxytocin; Radioligand; Oxytocin receptor; Vasopressin
1. Introduction
and the vasopressin receptors V1a, V1b, and V2. These four
receptors have been classified on the basis of the signal
transduction systems to which they are coupled as well as by
differences in their affinities for a variety of peptide ana-
logues (e.g., Peter et al., 1995; Barberis and Tribollet, 1996).
All four members of this receptor family have been cloned
and expressed in cell lines, facilitating the study of the
biochemical pharmacology of each receptor (Kimura et al.,
1992; Morel et al., 1992; Thibonnier et al., 1994; Sugimoto
et al., 1994; De Keyzer et al., 1994; Birnbaumer et al., 1992;
Lolait et al., 1992).
Oxytocin and arginine vasopressin are closely related
nonapeptides that are synthesized and released by hypo-
thalamic neurons, and that exert a variety of hormonal and
neurotransmitter-like effects. Oxytocin has well-established
actions in parturition and lactation, and vasopressin plays a
key role in renal water resorption and vasoconstriction.
Aside from their well-known peripheral roles, both hor-
mones have also been reported to act centrally, with oxy-
tocin playing possible roles in analgesia, drug dependence,
and social, sexual and maternal behaviors, and vasopressin
affecting learning, memory and social and reproductive
behaviors (selected reviews: Argiolas and Gessa, 1991;
Barberis and Tribollet, 1996; Verbalis, 1999; Gimpl and
Fahrenholz, 2001).
Centrally administered oxytocin and vasopressin elicit
behavioral effects in rodents and primates, and in some
cases, oxytocin has the opposite effect of vasopressin. For
example, in studies of avoidance behaviors in rodents,
oxytocin has been reported to decrease learning and mem-
ory functions while vasopressin has been reported to
enhance these functions, which may suggest differential
localization of these receptor subtypes in the central nerv-
ous system (see Argiolas and Gessa, 1991; De Wied et al.,
1993). Localization of these receptors in rat brain has been
studied using a number of different radioligands for auto-
radiography, with antibodies for immunohistochemical
studies and with mRNA probes for in situ hybridization
The effects of these peptides are mediated by a family of
four G-protein-coupled receptors—the oxytocin receptor
*
Corresponding author. WP46-300, Merck Research Laboratories,
West Point, PA 19486 USA. Tel.: +1-215-652-6798; fax: +1-215-652-3811.
_
E-mail address: david williams1@merck.com (D.L. Williams).
1
Both authors contributed equally to this paper.
0014-2999/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S0014-2999(02)02048-4

20
W. Lemaire et al. / European Journal of Pharmacology 450 (2002) 19–28
(recently reviewed by Barberis and Tribollet, 1996). The
results of these studies suggest that the oxytocin and
vasopressin V1a receptors are the predominant receptor
subtypes of this family in rat brain and spinal cord, and
that they are differentially distributed. The pharmacological
tools used to distinguish between the rat oxytocin and
vasopressin V1a receptors have shown good selectivity for
one of these two receptor subtypes. However, the selectiv-
ity of these tools (e.g., [d(CH2)5,Tyr(Me)2,Thr4,Tyr-
Laboratories, (Belmont, CA, USA) or Peptides International
(Louisville, KY, USA). [d(CH2)5,Tyr(Me)2,Thr4,Tyr-
NH29]ornithine vasotocin was from Bachem (King of Prus-
sia, PA, USA), [3H]oxytocin, [3H]arginine vasopressin, and
[
125I][d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH29]ornithine vasotocin
were from New England Nuclear (Boston, MA, USA) and
[3H]myo-inositol was from Amersham-Pharmacia (Piscat-
away, NJ, USA). Fluo-4 acetoxymethyl ester (Fluo-4 AM)
was obtained from Molecular Probes (Eugene, OR, USA).
Other chemicals were from Sigma (St. Louis, MO, USA).
9
NH2 ]ornithine vasotocin (Elands et al., 1987) can be much
lower for human (and other primate) receptors, making the
results of autoradiographic studies in these species difficult
to interpret at present (see Barberis and Tribollet, 1996;
Pettibone et al., 1992).
2.3. Cell culture
Cloned human oxytocin receptors were expressed in
Chinese hamster ovary (CHO) cells (Gordon Ng, Merck
Research Laboratories, Montreal). Cells were grown in
Iscove’s modified Dulbecco’s medium containing 10% fetal
bovine serum; 2 mM L-glutamine (GIBCO, Rockville, MD,
USA); penicillin/streptomycin (0.2 mM hypoxanthine,
0.032 mM thymidine GIBCO); 10 Ag/ml puromycin (Clon-
tech, Palo Alto, CA, USA). Human embryonic kidney
(HEK/293) cells expressing cloned human oxytocin recep-
tors or cloned human vasopressin receptors V1b or V2 (M.
Jacobson/C. Salvatore, Merck Research Laboratories, West
Point) were grown in Dulbecco’s modified Eagle medium
with 10% fetal bovine serum, 250 Ag/ml geneticin, 100 Ag/
ml gentamycin, and 250 Ag/ml hygromycin-B. Cultured
cells in Costar (Cambridge, MA, USA) (CHO cells) or
NUNC (Roskilde, Denmark) (HEK/293 cells) flasks were
incubated at 37 jC in a humidified incubator with 95% O2
and 5% CO2.
We have identified a novel, potent nonpeptide oxytocin
receptor antagonist, (1-(1-(2-(2,2,2-trifluoroethoxy)-4-(1-
methylsulfonyl-4-piperidinyloxy) phenylacetyl)-4-piperi-
dinyl)-3,4-dihydro-2(1H)-quinolinone) (Compound A),
which can be labeled to high specific activity with [35S].
This compound exhibits a high degree of selectivity for
human oxytocin receptors compared to human vasopressin
receptors, and appears to be suitable for studying the
pharmacology of oxytocin receptors in human tissues, as
well as serving as a nonlabeled competitor or as a radio-
ligand in autoradiographic studies of oxytocin receptor
localization in these tissues. In this paper, we report the in
vitro pharmacological characterization of the unlabeled
antagonist, and the radioligand binding properties of the
[35S]-labeled compound.
2. Materials and methods
2.4. Preparation of membranes from cultured cells express-
ing cloned human receptors
2.1. Synthesis of compound A
Compound A was prepared as shown in Fig. 1. Details of
the synthesis are given as Supplementary information.
The preparation of membranes from cultured cells was
essentially the same as described in Pettibone et al. (1991).
Briefly, cells expressing the desired receptors at about 90–
100% confluence were collected in phosphate-buffered sal-
2.2. Materials
Â
ine/1 mM EDTA (pH = 7.4) and centrifuged at 500 g at 4
Oxytocin, arginine vasopressin, desamino [D-arg8]vaso-
pressin, Thr4Gly7oxytocin, Phe2Orn8oxytocin/vasotocin, [1-
(h-mercapto-h, h-cyclo-pentamethylene propionic acid), 2-
(O-methyl)tyrosine]Arg8-vasopressin, were from Peninsula
jC for 5 min. The resulting pellets were homogenized with a
Polytron in 50 mM Tris buffer, 5 mM MgCl2 (pH = 7.4), and
Â
centrifuged at 50,000 g at 4 jC for 20 min. The membrane
pellets were re-homogenized and resuspended in appropriate
Fig. 1. Synthesis of Compound A. Standard peptide coupling conditions using amine 1 (Ogawa et al., 1993) and carboxylic acid 2 (Williams et al., 1999)
readily afforded amide derivative 3. The tert-butyl carbamate protecting group in 3 was cleanly removed with HCl gas in ethyl acetate to give the hydrochloride
salt of compound 4. Mesylation of compound 4 using standard conditions provided Compound A in high yield. Use of [35S]methanesulfonyl chloride in the
mesylation step provided [35S]Compound A (Dean et al., 1995).

W. Lemaire et al. / European Journal of Pharmacology 450 (2002) 19–28
21
volumes of 50 mM Tris, 5 mM MgCl2 (pH = 7.4). Aliquots of
with 8.0 ml/well of ice-cold buffer Tris/HCl (50 mM,
pH = 7.4) containing 5 mM MgCl2. Filtermats were dried
and sealed in a plastic sample bag with 24 ml Wallac
BetaScint scintillation cocktail. The radioactivity on the
filter plates was counted by Wallac Beta Plate reader.
Â
the membrane suspension were centrifuged at 17,000 g and
stored in 10% glycerol in 50 mM Tris buffer, 5 mM MgCl2
(pH = 7.4) at À 70 jC. Membrane concentration was deter-
mined using the method of Lowry et al. (1951).
2.5. Preparation of human platelet membranes
2.8. [3H]oxytocin and [3H]vasopressin binding to rat
tissues
The preparation of vasopressin V1a receptor-rich mem-
branes from human platelets is adapted from Swart et al.
(1985). Briefly, human platelet-rich supernatant was
obtained from the American Red Cross. The units were
All harvesting of animal tissues was carried out using
protocols approved by the West Point Institutional Animal
Care and Use Committee in accordance with the provisions of
the ILAR Guide for the Care and Use of Laboratory Animals.
The preparation of membranes from rat uterus, liver, and
kidney medulla is adapted from Fuchs et al. (1985). Briefly,
female Sprague–Dawley rats were pre-treated for 24 h with
0.3 mg/kg diethylstilbestrol dipropionate administered i.p.
The uterus was excised and fatty tissue was removed. The
liver and kidneys were excised from male Sprague–Dawley
rats and the medulla was dissected out of the kidney. All
tissues were homogenized in hypotonic buffer (10 mM Tris/
HCl, pH 7.4, 1 mM EDTA, 0.5 mM dithiothreitol). The liver
Â
pooled and centrifuged at 150 g for 15 min at 4 jC to
remove erythrocytes. The supernatants were then centri-
Â
fuged at 16,000 g at 4 jC. The pellets were resuspended
and homogenized in 80 ml of hypotonic buffer (10 mM Tris/
HCl, pH 7.4, 5 mM EDTA). The resuspended pellets were
Â
centrifuged at 27,500 g for 25 min at 4 jC. The pellets
were washed two more times by resuspension and high-
speed centrifugation as above. Then the final pellets were
resuspended in binding buffer 10 mM Tris/HCl, pH 7.4, 5
mM MgCl2, divided into aliquots and stored at À 70 jC.
Â
homogenate was centrifuged at 1000 g for 10 min at 4 jC.
The liver supernatant, as well as the uterus and kidney
2.6. Radioligand binding to cloned receptors
Â
medulla homogenates were centrifuged at 48,000 g for 30
Radioligand (1 nM [3H]oxytocin, 0.1 nM [125I][d(CH2)5,
Tyr(Me)2,Thr4,Tyr-NH29]ornithine vasotocin, 0.5 nM
[3H]arginine vasopressin, or 30 pM [35S]Compound A) in
50 mM Tris buffer, 5 mM MgCl2 (pH = 7.4) was mixed with
test compounds in 1% dimethyl sulfoxide or with 1 AM
unlabeled ligand (oxytocin, arginine vasopressin, or Com-
pound A) to estimate nonspecific binding (final [dimethyl
sulfoxide] = 0.17%). Unlabeled oxytocin was used to esti-
mate nonspecific binding in the [125I][d(CH2)5,Tyr(Me)2,
Thr4,Tyr-NH29]ornithine vasotocin assays. Membranes were
then added and mixed, and the reaction was incubated for 60
min at 30 jC. The assay mixture was filtered onto 96-well
glass fiber filter plates (Packard, Meriden, CT, USA) pre-
soaked with 0.2% polyethylenimine. The plates were dried,
scintillation cocktail was added, and the plates were counted
with a Packard TopCount.
min. The resulting pellets were washed two more times by
resuspension and high-speed centrifugation as above. The
final pellets were resuspended in binding buffer and
[3H]radioligand binding was performed as described above
except that filtration was performed on a Skatron cell har-
vester using GF/B filters (presoaked with 0.2% polyethyle-
nimine) purchased from Skatron (Lier, Norway). The filters
were counted with a Packard TriCarb 1900.
2.9. Measurement of Ca2+ flux in CHO and HEK/293 cells
expressing cloned human oxytocin receptors
CHO and HEK/293 cells expressing cloned human oxy-
tocin receptors were grown as described above. The cells
were plated in poly-D-lysine-coated 384-well plates from
Becton-Dickinson (40K cells/well for CHO and 20K cells/
well for HEK/293) using a Labsystems Multidrop (Helsinki,
Finland). The plated cells were grown overnight at 6% CO2
2.7. [3H]arginine vasopressin binding to human platelet
membranes (vasopressin V1a receptors)
Â
and 37 jC. The cells were washed with 3 100 Al assay
buffer (Hanks Balanced Salt Solution (Gibco 14025-076)
containing extra MgCl2 to a final concentration of 5 mM, 20
mM HEPES, 2.5 mM probenicid, and 0.1% bovine serum
albumin) using a Skatron Embla384 cell washer. The cells
were loaded with 1 AM Fluo-4 AM in the presence of 0.02%
pluronic acid and 1% bovine serum albumin for 1 h at 6%
CO2 and 37 jC. The cells were washed as above. Ca2 + flux
was measured using Molecular Devices FLIPR384 fluoro-
metric imaging plate reader (Sunnyvale, CA, USA). For
antagonist potency determinations, the cells were pre-incu-
bated for 5 min with various concentrations of Compound A
and then stimulated with oxytocin for 3 min (5 nM for HEK/
To measure specific [3H]arginine vasopressin binding,
100 Al of human platelet membranes at 1:3 dilution was
added to triplicate tubes containing either buffer (10 mM
Tris/HCl, pH 7.4, 5 mM MgCl2) for total binding, test
compounds or 1 AM arginine vasopressin for determination
of nonspecific binding and 0.5 nM of [3H]arginine vaso-
pressin (final volume = 300 Al). After incubation at 30 jC
for 60 min, the incubation mixtures were filtered through a
Wallac (Gaithersburg, MD, USA) glass fiber filtermat (pre-
soaked in 0.1% bovine serum albumin) using a Tomtec cell
harvester (Gaithersburg, MD, USA), and rapidly washed

22
W. Lemaire et al. / European Journal of Pharmacology 450 (2002) 19–28
293 cells; 15 pM for CHO cells). For Ki determinations, the
cells were pre-incubated with various concentrations of
Compound A and stimulated with various concentrations
of oxytocin for 3 min.
various times by filtration, and the filters counted as
described above. The apparent on-rate, Kapp, for each ligand
concentration was determined by nonlinear curve fitting of
specific binding vs. time (SigmaPlot), and these Kapp values
were plotted against ligand concentration. The off-rate, k2,
was determined indirectly from the ordinate intercept of the
line fit to these data, and the on-rate, k1, was determined
from the slope. Off-rates were also determined directly by
allowing [35S]Compound A to come to equilibrium with the
membranes above (60 min), and adding unlabeled Com-
pound A to a final concentration of 15 AM. The dissociation
reaction was stopped at various time points by filtration and
counted as described above. The specific counts bound were
plotted as a function of time after addition of excess
unlabeled compound, and k2 was determined directly from
nonlinear curve fitting of these data (SigmaPlot, SPSS,
Chicago, IL, USA).
2.10. Phosphatidylinositol hydrolysis assay
CHO cells expressing cloned human oxytocin receptors
were grown in 96-well plates and loaded overnight with 1 ACi
[3H]myo-inositol/well. The following day, the medium was
removed and plates were washed twice with 200 ml warmed,
oxygenated buffer A (127 mM NaCl, 25 mM NaHCO3, 10
mM glucose, 2.5 mM KCl, 1.2 mM KH2PO4, 1.2 mM
MgSO4, and 1.8 mM CaCl2) using a Skatron SkanWasher.
Then 100 Al/well of buffer B (buffer A plus 10 mM LiCl and
0.01% human serum albumin) or an appropriate concentra-
tion of antagonist in buffer B was added and allowed to
incubate for 10 min at room temperature. The buffer B was
then removed, and an appropriate concentration of oxytocin
in buffer B was added to each well. The concentration of
oxytocin used was 10 nM, near its EC50 value in this assay.
The plate was placed in a 37 jC incubator for 30 min. The
reaction was terminated by aspirating off the contents of the
wells and washing the plate once with 200 ml ice-cold
phosphate-buffered saline using the SkanWasher. Then 100
Al ice-cold 5% trichloroacetic acid was added to each well,
and the plate was kept on ice for 15 min. The trichloroacetic
acid was neutralized by the addition of 100-Al 0.5 M Tris to
each well. The plate was mixed by aspiration and dispense
cycles in a Tomtek Quadra 96, and 150 Al of the contents of
each well were transferred from the assay plate to tubes
containing 400 Al water. These tubes were again mixed and
400 Al of the contents was transferred to 96-well large volume
3. Results
3.1. Binding studies
The binding affinity and selectivity of Compound A were
tested with both human and rat receptors. In competition
binding studies using [3H]oxytocin or [3H]arginine vaso-
pressin, the compound was found to be highly selective for
human oxytocin receptors compared to human vasopressin
V1a, V1b, or V2 receptors. Ki values for the binding of
Compound A to these receptors were determined from com-
petition binding curves (examples are shown in Fig. 2) by the
method of Cheng and Prussoff (1973), and are presented in
Table 1. However, in the case of the rat receptors, the se-
lectivity of the compound for the oxytocin receptors com-
pared to vasopressin V1a receptors decreases from 1800-fold
to 3-fold, largely due to a higher affinity for the rat, compared
to the human, vasopressin V1a receptor (Table 1; Fig. 2).
The binding affinities and selectivities of Compound A
were compared to those of unlabeled oxytocin and
[d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH29]ornithine vasotocin.
Both of these peptide ligands bind to the oxytocin receptor,
and are available commercially as radioligands. The data for
this comparison are summarized in Table 1. Oxytocin is
essentially nonselective for the oxytocin receptor compared
to the vasopressin V1a receptor in human, but is about 100-
fold selective in rat. [d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH29]or-
nithine vasotocin is also nonselective in human but is about
1300-fold selective for the oxytocin receptor compared to
the V1a receptor in rat. Thus, the selectivity of these ligands
for the oxytocin receptor and vasopressin V1a receptors is
dependent on species: [d(CH2)5,Tyr(Me)2,Thr4,Tyr-
NH29]ornithine vasotocin shows high selectivity for oxy-
tocin receptors in the rat system, while Compound A shows
high selectivity for human oxytocin receptors. The testing of
Compound A against a panel of 175 receptors, transporters,
ion channels, and enzymes (MDS Panlabs, Bothell, WA,
Â
filter plates filled with washed Dowex 1 8 (formate coun-
ter-ion). The sample-loaded column plates were washed
twice with 1 ml water, followed by a 1.2-ml wash of 60
mM ammonium formate, 5 mM sodium tetraborate solution.
Washing was performed by Quadra and filtered using a
Whatman Polyfiltronics manifold system (Clifton, NJ,
USA). A 2-ml 96-well collection plate was then placed in
the filter chamber, and the [3H]inositol phosphates were
eluted from the column plate into the empty collection plate
with 1.2 ml of 1 M ammonium formate, 0.1 M formic acid
solution. A 1-ml aliquot of the eluate was transferred into 20-
ml scintillation vials, and 10-ml Packard Aquassure scintil-
lation cocktail was added to the vials. Radioactivity in each
sample vial was determined with a Packard CA 1900 liquid
scintillation counter.
2.11. Binding kinetics studies
On-rate studies were performed at 30 jC by incubating
[35S]Compound A (15, 30, or 60 pM) in the presence or
absence of 15 AM unlabeled Compound A with membranes
prepared from CHO cells expressing the cloned human
oxytocin receptor. The binding reactions were stopped at

W. Lemaire et al. / European Journal of Pharmacology 450 (2002) 19–28
23
Table 1
Species dependence of ligand selectivity: inhibition of [3H]oxytocin
binding or [3H]arginine vasopressin binding
Ki (nM)
Oxytocin
OVTAa
Compound A
Oxytocin receptor
Human
Rat
0.21 F 0.08 (7)
0.44 F 0.23 (3)
0.28 F 0.30 (3)
0.02
0.29 F 0.17 (5)
1.1 F 0.73 (3)
Vasopressin V1a receptor
Human
Rat
1.18 F 0.38 (3)
49 F 4 (3)
1.39 F 2.36 (17)
27 F 9 (2)
556 F 216 (14)
3.5 F 1.7 (4)
Vasopressin V1b receptor
Human
Rat
241 F 56 (3)
n/d
> 1000 (3)
n/d
>1000 (2)
n/d
Vasopressin V2 receptor
Human
Rat
303 F 142 (4)
37 F 9 (3)
657 F 269 (4)
44 F 21 (2)
275 F 113 (3)
>1000 (4)
Ki determined from binding competition IC50: Ki = IC50/(1+[L]/Kd) (Cheng
and Prussoff, 1973). Values are group mean F standard deviation (n).
Source of receptors—Human: oxytocin receptor, CHO cells expressing
human oxytocin receptors; vasopressin V1a receptors, human platelets;
vasopressin V1b receptors, HEK/293 cells expressing human V1b receptors;
vasopressin V2 receptors, HEK/293 cells expressing human V2 receptors.
Rat: oxytocin receptor, diethylstilbestrol dipropionate-primed rat uterus;
vasopressin V1a receptors, rat liver; vasopressin V2 receptors, rat kidney
medulla.
a
[d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH92]ornithine vasotocin.
Fig. 2. Top panel: Comparison of Compound A inhibition of [3H]oxytocin
binding to cloned human oxytocin receptors expressed in CHO cells (.),
and [3H]arginine vasopressin binding to human vasopressin V1a receptors
(D) expressed in platelets and to human V2 (5) receptors expressed in
HEK/293 cells. Error bars are S.E.M. (n = 3). Bottom panel: Comparison of
Compound A inhibition of [3H]oxytocin binding to rat uterus (.), and
[3H]arginine vasopressin binding to rat liver (vasopressin V1a receptors)
(D) and to rat kidney (vasopressin V2 receptors) (5). Error bars are S.E.M.
(n = 3).
[3H]oxytocin produced Bmax values for [3H]oxytocin binding
similar to those obtained in the co-incubation studies (data
not shown), indicating that this compound binds reversibly to
the oxytocin receptor.
USA) indicated sub-micromolar activity in only two cases:
the a1 adrenergic receptor (Ki = 437 nM) and at the mono-
amine transporter (Ki = 547 nM), suggesting Compound A is
relatively free of potentially confounding activities.
To confirm the Ki of Compound A for the human oxytocin
receptor, [3H]oxytocin saturation studies were performed
with membranes prepared from CHO cells expressing the
cloned human oxytocin receptor in the presence and absence
of fixed concentrations of Compound A. In the absence of
Compound A, [3H]oxytocin bound to a single class of high
affinity sites with a Kd value of 0.33 F 0.12 nM (n = 4). When
the Kd for [3H]oxytocin binding was plotted as a function of
the fixed concentrations of Compound A (Fig. 3), curve
fitting of the points indicated a Ki value of 0.14 F 0.08 nM
(n = 4) for this compound binding to oxytocin receptor,
similar to the values obtained from competition binding
studies (Table 2). Co-incubation with Compound A did not
alter the apparent Bmax value for [3H]oxytocin (data not
shown). In a parallel study, a 60-min pre-incubation of the
membranes with 0.3 nM Compound A before addition of
Fig. 3. Determination of the Ki value for Compound A from saturation
experiments. Apparent Kd values were determined for [3H]oxytocin binding
to membranes prepared from CHO cells expressing human cloned oxytocin
receptors in the presence and absence of 0.1 and 0.3 nM Compound A. The
Ki for Compound A was determined from the equation Kd(apparent)=(Kd/
Ki)[antagonist] + Kd. A plot of Kd(apparent) vs. [antagonist] has intercepts at
Kd on the ordinate and Ki on the abscissa.

24
W. Lemaire et al. / European Journal of Pharmacology 450 (2002) 19–28
Table 2
Table 3
Comparison of Compound A affinity determinations for human oxytocin
receptors
Comparison of binding saturation results with three different radioligands
for human oxytocin receptors
Compound A
Bmax (fmol/mg)
Kd (nM)
Ki (nM) F S.D. (n)
for oxytocin receptors
[3H]oxytocin
1060 F 223 (3)
1380 F 69 (3)
418 F 38 (4)
1.24 F 0.40 (3)
0.24 F 0.01 (3)
0.042F 0.012 (4)
[
[
125I]OVTAa
35S]Compound A
[3H]oxytocin binding competition
[3H]oxytocin binding saturation
0.29 F 0.17 (5)
0.14 F 0.08 (4)
0.23 F 0.16 (2)
b0.50 F 0.12 (2)
a
[
125I][d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH92]ornithine vasotocin. Values
[
125I]OVTAa binding competition
are group mean F standard deviation (n).
Schild plot analysis FLIPR
Ki (nM) F S.D. (n) for CHO cells expressing the human oxytocin receptor.
a
125I][d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH92]ornithine vasotocin.
with potent antagonist activity observed in the phosphati-
dylinositol hydrolysis assay.
[
b
KDb (nM) estimated from pA2 value obtained from FLIPR experi-
ments performed with HEK cells expressing the human oxytocin receptor.
To further characterize the oxytocin receptor antagonism
by Compound A, Schild analysis was performed using Ca2 +
flux data collected from HEK oxytocin receptor-expressing
cells. The EC50 of the oxytocin concentration–response was
shifted to the right by increasing concentrations of Com-
pound A (Fig. 4). When these data were plotted according to
the method of Schild (inset, Fig. 4), the slope of the plot was
1.13 and the KDb determined was 0.50 F 0.12 nM, consistent
with values determined with other methods (Table 3). The
maximum response to oxytocin was decreased at high
concentrations of Compound A, a phenomenon commonly
observed with very potent antagonists in functional assays.
3.2. Functional studies
The oxytocin receptor is coupled to phospholipase C by
Gaq. To determine if Compound A acts as an agonist or
antagonist at the oxytocin receptor, it was evaluated in
functional assays using CHO cells expressing the cloned
human oxytocin receptor. In phosphatidylinositol hydrolysis
assays, Compound A alone did not stimulate accumulation
of inositol phosphates over basal accumulation, indicating
Compound A was not an agonist of the oxytocin receptor.
However, a 10-min preincubation with Compound A
inhibited oxytocin (10 nM) induced stimulation of phos-
phatidylinositol hydrolysis with an IC50 value of 3.6 F 0.9
nM, indicating Compound A is a potent oxytocin receptor
antagonist (data not shown). In an assay of agonist-induced
increases in intracellular calcium in cultured cells expressing
the cloned human oxytocin receptor, Compound A alone
had no effect, while it inhibited oxytocin-stimulated
increases in intracellular calcium with an IC50 value of
0.54 F 0.066 nM (n = 3) for the oxytocin receptor-express-
ing HEK/293 cells and 1.1 F 0.17 nM (n = 3) for oxytocin
receptor-expressing CHO cells (data not shown), consistent
3.3. Characterization of [35S]Compound A binding
Compound A was synthesized with the methyl sulfonyl
group radiolabeled with [35S], and its properties as a radio-
ligand were characterized. Initial binding studies indicated
that [35S]Compound A binding to oxytocin receptors comes
to equilibrium in 20 min at 30 jC (data not shown). At the
membrane concentration used in subsequent assays (30 Ag
protein/ml), percent nonspecific binding was 27%. At 600
Ag/ml, nonspecific binding was 10% (data not shown).
Fig. 5. Representative Scatchard analysis of binding of [35S]Compound A
to membranes prepared from CHO cells expressing the cloned human
oxytocin receptor. Actual saturation curve is shown in the inset. Kd values
of four determinations was 0.042 nM F 0.012. Bmax was 418 F 38 fmol/mg
protein.
Fig. 4. Schild plot determination of Compound A KDb value using Ca2 +
flux data determined by fluorometry from HEK/293 cells expressing cloned
human oxytocin receptors.

W. Lemaire et al. / European Journal of Pharmacology 450 (2002) 19–28
25
Specific [35S]Compound A binding to membranes prepared
from CHO cells expressing the cloned human oxytocin
receptor was saturable (Fig. 5, inset). Nonlinear curve fitting
indicated a single class of binding site with a mean Kd value
of 0.042 F 0.012 nM (n = 4), about 3.8-fold less than the Ki
value determined in saturation studies with the unlabeled
compound, and about 7-fold less than the Ki value deter-
mined in competition studies. The Bmax value derived from
these studies was 418 F 38 fmol/mg protein, 2–3-fold lower
than that obtained for the other two radioligands studied.
These data are presented as a Scatchard plot (Fig. 5) and are
included in the summary in Table 3. Kinetics of [35S]Com-
pound A binding to cloned human oxytocin receptors were
determined at 30 jC. The on-rate for ComÀpo1und A binding
(agonists and antagonists) were compared in competition
binding assays measuring specific [35S]Compound A,
[3H]oxytocin, and [125I][d(CH2)5,Tyr(Me)2,Thr4,Tyr-
9
NH2 ]ornithine vasotocin binding to membranes prepared
from CHO oxytocin receptor-expressing cells. The Ki values
from these studies are shown in Table 4. The absolute
potencies of these reference compounds are in good agree-
ment with these three radioligands. Ki values for these
reference compounds determined with each radioligand are
within a factor of approximately 3 with the exception of
oxytocin, Phe2Orn8oxytocin and [d(CH2)5,Tyr(Me)2,Thr4,
9
Tyr-NH2 ]ornithine vasotocin (within a factor of 4–5). It
should be noted that in these assays a small component of
[35S]Compound A binding ( < 10%) was not competed by
any of the peptide or nonpeptide ligands, but was competed
by Compound A.
9
min À 1 deter-
Â
to these receptors was 7.4 F 5.8 10 M
mined at three different ligand concentrations (n = 2 at each
concentration). The off-rate, determined indirectly from the
ordinate intercept of a plot of the Kapp values obtained in the
on-rate studies vs. [ligand], as well as directly from studies
using addition of a large excess of competing ligand, was
0.075 F 0.036 min À 1 (n = 4) (data not shown). The Kd
calculated from these rates was 10.1 pM, comparable with
the Kd determined in the saturation binding studies.
4. Discussion
Potent, selective ligands are important in the identifica-
tion, characterization, and localization of G-protein-coupled
receptors, and if the ligand can be radiolabeled to high
specific activity, it holds additional interest for the inves-
tigator. A large number of peptide and peptide-related
ligands have been developed by the laboratories of Manning
and Sawyer, which have proven very useful in understanding
the pharmacology of oxytocin and vasopressin receptors
(e.g., Manning and Sawyer, 1993). For the most part, these
peptide analogues have been characterized and optimized
using three biological assays in the rat: in vitro uterine
contraction, and in vivo vasopressor and antidiuretic activity
(e.g., Chan et al., 1996, 2000). In several cases, peptide
analogue oxytocin agonists or antagonists have been radio-
labeled, further increasing their utility (e.g., Elands et al.,
1987, 1988). In binding studies using membrane prepara-
tions from rat tissues, these compounds have behaved largely
as expected. However, significant species differences have
been observed in the ligand selectivity of oxytocin and
vasopressin receptors (e.g., Pettibone et al., 1992), which
have seriously complicated interpretation of results when
using these ligands in species other than rat. An example is
shown in Table 1 where [d(CH2)5,Tyr(Me)2,Thr4,Tyr-
NH29]ornithine vasotocin is seen to be highly selective for
oxytocin receptors vs. vasopressin V1a receptors in rat, but
not in human. Recently, radiolabeled and photoactivable or
fluorescent peptide analogues have been reported that have
been characterized using human oxytocin and vasopressin
receptors (Durroux et al., 1999; Carnazzi et al., 2001; Breton
et al., 2001). However, where the comparison between
oxytocin receptors and vasopressin V1a receptors was made,
at best only modest (17-fold) selectivity was observed (for
vasopressin V1a receptors (Durroux et al., 1999)).
3.4. Comparison of [35S]Compound A, [3H]oxytocin and
[
125I][d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH29]ornithine vasotocin
in binding competition studies
A number of reference compounds of various structural
classes (peptides and nonpeptides) and functional activities
Table 4
Comparison of reference compound affinities for human oxytocin receptors
from binding competition studies using three different radioligands
Ki (nM)
[
35S]Compound A [3H]oxytocin
[
125I]OVTAb
Oxytocin
Arginine
1.12 F 0.44 (7)
3.60 F 1.51 (3)
0.21 F 0.08 (6) 0.69 F 0.74 (3)
1.90 F 0.90 (4) 3.84 F 2.82 (3)
vasopressin
Thr4Gly7oxytocin 6.57 F 3.07 (3)
6.86 F 2.57 (6) 18.79 F 6.85 (3)
47.7 F 18.9 (6) 156 F 43 (3)
Desamino
[D-arg8]
129 F 10 (3)
vasopressin
Phe2Orn8oxytocin/ 13.7 F 0.5 (3)
vasotocin
4.27 F 2.11 (6) 7.00 F 1.24 (3)
6.11 F 4.16 (5) n/d
Kruszynski
compounda
OVTAb
8.59 F 3.13 (3)
1.39 F 0.01 (2)
0.10 F 0.02 (8)
6.94 F 0.32 (4)
0.28 F 0.30 (3) n/d
0.29 F 0.17 (5) 0.23 F 0.16 (3)
7.20 F 4.15 (4) 7.09 F 3.58 (3)
Compound A
L-368,899c
Ki values determined from IC50 values: Ki = IC50/(1+[L]/Kd) (Cheng and
Prussoff, 1973), and represent group means F standard deviation (n).
a
[1-(h-mercapto-h, h-cyclo-pentamethylene propionic acid), 2-(O-
methyl)tyrosine]-Arg8-Vasopressin (Kruszynski et al., 1980).
b
[d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH29]ornithine vasotocin (Elands et al.,
1987).
c
Compound A was selected from a collection of non-
peptide oxytocin receptor antagonists, and was shown to be
well behaved in competition binding assays using both
1-((7,7-dimethyl-2(S)-(2(S)-amino-4-(methylsulfonyl)butyramido)bi-
cyclo [2.2.1]-heptan-1(S)-yl)methyl)sulfonyl)-4-(2-methylphenyl)pipera-
zine (Williams et al., 1994).

26
W. Lemaire et al. / European Journal of Pharmacology 450 (2002) 19–28
[3H]oxytocin and [3H]arginine vasopressin as radioligands
as well as in [3H]oxytocin saturation binding assays as an
unlabeled competitor. Ki values for Compound A were
similar between these two types of assays, and preincuba-
tion of membranes with Compound A in [3H]oxytocin
saturation binding assays did not alter the Bmax values in
the saturation studies, indicating that Compound A binds
reversibly to the oxytocin receptor. Interestingly, while
Compound A was found to be highly selective (900–
1800-fold) for the oxytocin receptors vs. the vasopressin
receptors in the human system, it is not selective in the rat,
and may not be the ligand of choice in this species. Func-
tional studies indicated that Compound A was an oxytocin
receptor antagonist with no agonist properties.
Radiolabeling of Compound A with [35S] in the final
synthetic step afforded a radioligand of high specific activ-
ity. Saturation binding studies of human oxytocin receptors
with [35S]Compound A indicated that the ligand bound to a
single high affinity site with Kd values comparable to the Ki
values determined for the unlabeled compound. The Bmax
values obtained from these studies with [35S]Compound A
pound A or [35S]Compound A may serve as a novel, highly
specific, nonlabeled competitor or radioligand, respectively,
in autoradiographic studies of oxytocin receptor localization
in tissues from these species.
Appendix A. Supplementary information
A.1. Details of synthesis of Compound A
A.1.1. General
Abbreviations: EtOAc = ethyl acetate, DMF = dimethyl-
formamide, HOBT = 1-hydroxybenzotriazole, DIEA= diiso-
propylethylamine, EDC = 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride.
Thin layer chromatographic analysis was accomplished
using Analtech Uniplate silica gel GF (250 Am) TLC plates.
For preparative silica gel column chromatography, EM
Science Silica Gel 60 (230–400 mesh) was utilized.
A Thermo Separations Spectra System P4000 HPLC
instrument was utilized. For the stationary phase, a Hew-
Â
Â
were somewhat lower (2–3 ) than those observed in similar
lett-Packard Zorbax SB-C8 column (75 4.6 mm, 3.5 Am)
studies with [3H]oxytocin or [125I][d(CH2)5,Tyr(Me)2,Thr4,
Tyr-NH29]ornithine vasotocin. The reason for this difference
is not clear, but it does indicate that [35S]Compound A is not
binding to membrane sites in addition to the oxytocin
receptors. In support of this, the Ki values of oxytocin
receptor agonists and antagonists from a variety of structural
classes, determined in competition binding studies using
[35S]Compound A, were very similar to those obtained with
two other well-behaved and widely used radioligands,
[3H]oxytocin and [125I][d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH29]or-
nithine vasotocin.
was employed. The mobile phase consisted of a binary
gradient, 95:5 A/B to 0:100 A/B over 4.5 min, where A is
water containing 0.1% by volume trifluoroacetic acid and B
is acetonitrile containing 0.1% by volume trifluoroacetic
acid, using a flow rate of 3.0 ml/min. Peaks were detected
by UV absorption at 215 nm.
A Waters 2690 HPLC and a ZMD quadrupole mass
spectrometer were used to record LC-MS information. For
the HPLC stationary phase, a YMC PRO column was
Â
employed (3 mm 5 cm, C18 stationary phase, 5 AM
particle size, 120 A pore size). The mobile phase consisted
˚
The data presented above indicate that Compound A is a
potent oxytocin receptor antagonist with a high degree of
selectivity for human oxytocin receptor compared to the
human vasopressin receptors. In addition, the data also
indicate that [35S]Compound A is a well-behaved, highly
selective radioligand for human oxytocin receptors. The use
of a methane [35S]sulfonamide as the radiolabeled function-
ality (Dean et al., 1996) has the benefit of affording a very
high specific radioactivity without the drawbacks due to
structural perturbations (lower affinity, higher lipophilicity)
often observed with [125I] radiolabeling of small molecule
ligands. In preliminary studies, competition binding data
indicate Compound A binding to membranes prepared from
rhesus monkey tissues (uterus, liver, and kidney are highly
enriched in oxytocin, vasopressin V1a and V2 receptors,
respectively) are similar to those observed for human
membranes (data not shown). This is not surprising, con-
sidering the high degree of identity (97%) between rhesus
and human oxytocin receptors (Salvatore et al., 1998). The
high selectivity for human oxytocin receptors suggests that
Compound A may be useful as a novel, highly specific, tool
in studying the pharmacology of oxytocin receptors in
human (and nonhuman primate) tissues. In addition, Com-
of a binary gradient, 92:8 A/B to 0:100 A/B over 3.4 min,
then 0:100 A/B for 0.3 min, where A is water containing
0.05% by volume trifluoroacetic acid and B is acetonitrile
containing 0.05% by volume trifluoroacetic acid, using a
flow rate of 2.0 ml/min. The ionization method for the mass
spectrometer was electrospray, with positive ion detection.
Proton NMR spectra were recorded at 400 MHz using a
Varian spectrometer. Tetramethylsilane was employed an
internal standard (0.00 ppm).
A.1.2. Procedure
Refer to Fig. 1. To a stirred solution of compound 1 (0.20
g, 0.87 mmol), compound 2 (0.37 g, 0.85 mmol), EDC (0.21
g, 1.1 mmol), HOBT hydrate (0.14 g, 0.92 mmol) in DMF
(10 ml) was added enough DIEA (approximately 0.2 ml) to
obtain a reading of pH 7 on wetted E. Merck pH indicator
strips. The mixture was stirred at ambient temperature for 16
h. The solvent was removed under reduced pressure and the
residue was partitioned between EtOAc (50 ml) and satu-
rated aqueous NaHCO3 (50 ml). The organic phase was
dried over anhydrous MgSO4, filtered, and the solvent was
removed under reduced pressure. The residue was purified
using silica gel column chromatography with 3:1 EtOAc/

W. Lemaire et al. / European Journal of Pharmacology 450 (2002) 19–28
27
hexanes as eluant. Compound 3 was obtained as a gum
(0.53 g, 94%). TLC Rf = 0.7 (3:1 EtOAc/hexanes); HPLC
RT = 3.91 min; LC-MS m/z = 646; NMR (CDCl3) 7.24 ppm
(d, J = 8.4 Hz, 1H), 7.15–7.21 ppm (m, 2H), 6.98–7.02
ppm (m, 2H), 6.61 ppm (dd, J = 2.2, 8.4 Hz, 1H), 6.42 ppm
(d, J = 2.2 Hz, 1H), 4.81 ppm (br d, J f 13 Hz, 1H), 4.45
ppm (m, 2H), 4.34 ppm (q, J = 8.2 Hz, 2H), 4.08 ppm (br d,
J f 14 Hz, 1H), 3.64–3.52 (m, 4H), 3.33 ppm (m, 2H),
3.09 ppm (br t, J f 14 Hz, 1H), 2.81 ppm (br t, J f 7 Hz,
CH3OH); HPLC retention time = 3.49 min; LC-MS m/
z = 624; NMR (CDCl3), 7.25 ppm (d, J = 8 Hz, 1H), 7.15–
7.22 ppm (m, 2H), 7.01 ppm (t, J = 8 Hz, 2H), 6.60 ppm (dd,
J = 2.4, 8.3 Hz, 1H), 6.42 ppm (d, J = 2.4 Hz, 1H), 4.81 ppm
(br d, J f 10 Hz, 1H), 4.51 ppm (m, 1H), 4.42 ppm (m, 1H),
4.35 ppm (q, J = 8.0 Hz, 2H), 4.08 ppm (br d, J f 10 Hz, 1H),
3.69 ppm (AB quartet, J f 10 Hz, 2H), 3.3–3.4 ppm (m,
4H), 3.10 ppm (br t, J f 12 Hz, 1H), 2.82 ppm (s and
overlapping m, 5H), 2.64 ppm (br t, J f 12 Hz, 1H), 2.
2H), 2.64 ppm (br t, J f 12 Hz, 1H), 2.56 ppm (dd, J = 5.1,
7.8 Hz, 2H), 2.35–2.52 ppm (m, 2H), 1.90 ppm (m, 2H),
1.74 ppm (m, 4H), 1.47 ppm (s, 9H).
[35S]Methanesulfonyl chloride (Dean et al. (1995)) in
anhydrous dichloromethane (5 mCi, SA= f 1100 Ci/mmol)
was transferred in to a 15-ml pear-shaped flask. The solution
was concentrated by distillation at atmospheric pressure (oil
bath temperature 60–64 jC) to a volume of 50–100 Al. The
flask was removed from the oil bath and briefly allowed to
cool. This solution was then transferred via blunt-end syringe
to a solution containing compound 4 (free base, f 5 mg) in
50 Al of anhydrous dichloromethane. The distillation flask
A stirred solution of compound 3 (0.50 g, 0.78 mmol) in
EtOAc (25 ml) was cooled to 0 jC. HCl gas was bubbled
through the cold solution for 10 min. The mixture was stirred
at 0 jC for 10 min and then at ambient temperature for 45
min. The solvent was removed under reduced pressure and
the resulting solid was triturated in a small volume of EtOAc
and collected by filtration to give the hydrochloride salt of
compound 4 as an amorphous white powder (0.43 g, 93%).
TLC Rf = 0.3 (90:10:0.5 CH2Cl2/CH3OH/NH4OH); HPLC
RT = 2.93 min; LC-MS m/z = 546; NMR (DMSO-d6) 8.9
ppm (br s, 2H), 7.18-7.25 (m, 3H), 7.12 ppm (d, J = 8.2 Hz,
1H), 7.01 ppm (t, J = 7.0 Hz, 1H), 6.76 ppm (d, J = 2.2 Hz,
1H), 6.68 ppm (dd, J = 2.2, 8.4 Hz, 1H), 4.74 ppm (q, J = 8.8
Hz, 2H), 4.66 ppm (m, 1H), 4.50 ppm (br d, J f 12 Hz, 1H),
4.28 ppm (m, 1H), 4.00 ppm (br d, J f 13 Hz, 1H), 3.58
ppm (AB q, J f 12 Hz, 2H), 3.22 ppm (m, 2H), 3.0–3.18
ppm (m, 3H), 2.77 ppm (br t, J f 7 Hz, 2H), 2.64 ppm (br t,
J f 11 Hz, 1H), 2.43 ppm (br t, J f 8 Hz, 2H), 2.33 ppm
(dq, Jd = 4.0 Hz, Jq = 12.3 Hz, 2H), 2.30 ppm (m, 2H), 1.84
ppm (m, 2H), 1.66 ppm (m, 2H).
Â
was rinsed with anhydrous dichloromethane (2 50 Al) and
transferred to the reaction mixture. To this solution was
added 10 Al of anhydrous triethylamine (stored over molec-
ular sieves 4 A). The reaction mixture was stirred at room
˚
temperature for 2 h, then diluted with 1 ml dichloromethane
and 200 Al of water. The dichloromethane layer was sepa-
Â
rated, washed with saturated sodium bicarbonate (2 2 ml).
The dichloromethane solution was dried over anhydrous
sodium sulfate twice and then evaporated under a nitrogen
stream at 30 jC. The resulting residue was dissolved in 75 Al
of acetonitrile/water (1:1 v/v) and purified by using prepa-
Â
rative HPLC (Zorbax-SB-C8, 9.4 250 mm semi-prep col-
umn, 60% water containing 0.1% TFA–40% acetonitrile,
UV = 230 nm, flow rate = 4 ml/min). The pure product-
containing fractions were combined and neutralized with
dilute sodium bicarbonate solution. The acetonitrile was
removed by evaporation and the aqueous solution passed
through a Sep-Pak (Waters, C-18) cartridge. The cartridge
was washed with water, followed by elution of [35S]Com-
pound A with ethanol. The total activity of the ethanol
fraction (10 ml) collected was 2.9 mCi (58% radiochemical
yield). The radiochemical purity was determined to be >99%
A stirred solution of compound 4 (0.20 g, 0.34 mmol)
and triethylamine (0.51 mmol) in CH2Cl2 (15 ml) was
cooled to 0 jC. To the solution was added methanesulfonyl
chloride (0.36 mmol). The cooling bath was removed and
the mixture was stirred at ambient temperature for 1 h. The
reaction was diluted with CH2Cl2 (20 ml) and extracted with
saturated aqueous NaHCO3 (20 ml). The organic phase was
dried over anhydrous MgSO4, filtered, and evaporated
under reduced pressure. The residue was purified using
silica gel column chromatography with 97:3 CH2Cl2/
CH3OH as eluant. Compound A was obtained as an amor-
phous solid (0.20 g, 92% yield). TLC Rf = 0.4 (97:3 CH2Cl2/
Â
by HPLC analysis (Zorbax-SB-C8, 4.6 250 mm column,
55% water containing 0.1% TFA –45% acetonitrile,
UV = 230 nm, flow rate = 1 ml/min) and the specific activity
obtained was 1116 Ci/mmol as determined by LC/MS.

28
W. Lemaire et al. / European Journal of Pharmacology 450 (2002) 19–28
2-(O-methyl)tyrosine]arginine-vasopressin and [1-(h-mercapto-h, h-cy-
References
clo-pentamethylene propionic acid)]arginine-vasopressin, two highly
potent antagonists of the vasopressor response to arginine-vasopressin.
J. Med. Chem. 23, 364–368.
Argiolas, A., Gessa, G.L., 1991. Central functions of oxytocin. Neurosci.
Biobehav. Rev. 15, 217–231.
Lolait, S.J., O’Carroll, A.-M., McBride, O.W., Konig, M., Morel, A.,
Brownstein, M.J., 1992. Cloning and characterization of a vasopressin
V2 receptor and possible link to nephrogenic diabetes insipidus. Nature
357, 336–339.
Barberis, C., Tribollet, E., 1996. Vasopressin and oxytocin receptors in the
central nervous system. Crit. Rev. Neurobiol. 10, 119–154.
Birnbaumer, M., Seibold, A., Gilbert, S., Ishido, M., Barbersi, C., Antar-
amian, A., Brabet, P., Rosenthal, W., 1992. Molecular cloning of the
receptor for human antidiuretic hormone. Nature 357, 333–336.
Breton, C., Chellil, H., Kabbaj-Benmansour, M., Carnazzi, E., Seyer, R.,
Phalipou, S., Morin, D., Durroux, T., Zingg, H., Barberis, C., Mouillac,
B., 2001. Direct identification of human oxytocin receptor-binding do-
mains using a photoactivatable cyclic peptide antagonist. J. Biol. Chem.
276, 26931–26941.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein
measurements with the Folin phenol reagent. J. Biol. Chem. 193,
265–275.
Manning, M., Sawyer, W.H., 1993. Design, synthesis and some uses of
receptor-specific agonists and antagonists of vasopressin and oxytocin.
J. Recept. Res. 13, 195–214.
Morel, A., O’Carroll, A.-M., Brownstein, M.J., Lolait, S.J., 1992. Molec-
ular cloning and expression of a rat V1a arginine vasopressin receptor.
Nature 356, 523–526.
Carnazzi, E., Aumelas, A., Mouillac, B., Breton, C., Guillou, L., Barberis,
C., Seyer, R., 2001. Design, synthesis and pharmacological character-
ization of a potent radioiodinated and photoactivatable peptidic oxy-
tocin antagonist. J. Med. Chem. 44, 3022–3030.
Ogawa, H., Yamamura, Y., Miyamoto, H., Kondo, K., Yamashita, H., Na-
kaya, K., Chihara, T., Mori, T., Tominaga, M., Yabuuchi, Y., 1993.
Orally active, nonpeptide vasopressin V1 antagonists. A novel series
of 1-(substituted 4-piperidinyl)-3,4-dihydro-2(1H)-quinolone. J. Med.
Chem. 36, 2011–2017.
Chan, W.Y., Wo, N.C., Cheng, L.L., Manning, M., 1996. Isosteric substi-
tution of Asn5 in antagonists of oxytocin and vasopressin leads to
highly selective and potent oxytocin and V1a receptor antagonists:
new approaches for the design of potential tocolytics for preterm labor.
J. Pharmacol. Exp. Ther. 277, 999–1003.
Peter, J., Burbach, H., Adan, R.A, Lolait, S.J., van Leeuwen, F.W., Mezey,
E., Palkovits, M., Barberis, C., 1995. Molecular neurobiology and phar-
macology of the vasopressin/oxytocin receptor family. Cell. Mol. Neu-
robiol. 15, 573–595.
Chan, W.Y., Wo, N.C., Stoev, S., Cheng, L.L., Manning, M., 2000. Dis-
covery and design of novel and selective vasopressin and oxytocin
agonists and antagonists: the role of bioassays. Exp. Physiol. 85S,
7S–18S.
Pettibone, D.J., Clineschmidt, B.V., Lis, E.V., Reiss, D.R., Totaro, J.A.,
Woyden, C.J., Bock, M.G., Freidinger, R.M., Tung, R.D., Veber,
D.F., Williams, J.M., Lowensohn, R.I., 1991. In vitro pharmacological
profile of a novel structural class of oxytocin antagonists. J. Pharmacol.
Exp. Ther. 256, 304–308.
Cheng, Y.C., Prussoff, W.H., 1973. Relationship between the inhibition
constant (Ki) and the concentration of inhibitor which causes 50 percent
inhibition (IC50) of an enzymatic reaction. Biochem. Pharmacol. 22,
3099–3107.
Pettibone, D.J., Kishel, M.T., Woyden, C.J., Clineschmidt, B.V., Bock,
M.G., Freidinger, R.M., Veber, D.F., Williams, P.D., 1992. Radioligand
binding studies reveal marked species differences in the vasopressin V1
receptor of rat, rhesus and human tissues. Life Sci. 50, 1953–1958.
Salvatore, C.A., Woyden, C.J., Guidotti, M.T., Pettibone, D.J., Jacobson,
M.A., 1998. Cloning and expression of the rhesus monkey oxytocin
receptor. J. Recept. Signal Transduct. Res. 18, 15–24.
Dean, D.C., Staskiewicz, S.J., Ellsworth, R.L., Melillo, D.G., Shaw, D.R.,
1995. Synthesis of new high-specific activity sulfur-35 compounds. In:
Allen, J., Voges, R. (Eds.), Synthesis and Applications of Isotopically
Labelled Compounds, 1994. Wiley, New York, NY, pp. 795–801.
Dean, D.C., Nargund, R.P., Pong, S.S., Chaung, L.Y.P., Griffin, P., Melillo,
D.G., Ellsworth, R.L., VanDer Ploeg, L.H.T., Patchett, A.A., Smith,
R.G., 1996. Development of a high specific activity sulfur-35-labeled
sulfonamide radioligand that allowed the identification of a new growth
hormone secretagogue receptor. J. Med. Chem. 39, 1767–1770.
De Keyzer, Y., Auzan, C., Lemne, F., Beldjord, C., Thibonnier, M., Barta-
gna, X., Clauser, E., 1994. Cloning and characterization of the human
V1b pituitary vasopressin receptor. FEBS Lett. 356, 215–220.
De Wied, D., Diamant, M., Fodor, M., 1993. Central nervous system effects
of the neurohypophyseal hormones and related peptides. Front. Neuro-
endocrinol. 14, 251–302.
Sugimoto, T., Saito, M., Mochizuki, S., Watanabe, Y., Hashimoto, S., Ka-
washima, H., 1994. Molecular cloning and functional expression of a
cDNA encoding the human V1b vasopressin receptor. J. Biol. Chem.
269, 27088–27092.
Swart, S.S., Maguire, M., Wood, J.K., Barnett, D.B., 1985. Alpha-2 adre-
noreceptor coupling to adenylate cyclase in adrenaline insensitive hu-
man platelets. Eur. J. Pharmacol. 116, 113–119.
Thibonnier, M., Auzan, C., Madhun, Z., Wilkins, P., Berti-Mattera, L.,
Clauser, E., 1994. Molecular cloning, sequencing and functional ex-
pression of a cDNA encoding the human V1a vasopressin receptor. J.
Biol. Chem. 269, 3304–3310.
Durroux, T., Peter, M., Turcatti, G., Chollet, A., Balestre, M.-N., Barberis,
C., Seyer, R., 1999. Fluorescent pseudo-peptide linear vasopressin an-
tagonists: design, synthesis and applications. J. Med. Chem. 42,
13119–13122.
Verbalis, J.G., 1999. The brain oxytocin receptor(s?). Frontiers in Neuro-
endocrinology 20, 146–156.
Elands, J., Barberis, C., Jard, S., Tribollet, E., Dreifuss, J.-J., Bankowski,
K., Manning, M., Sawyer, W.H., 1987. 125I-labelled d(CH2)5[Tyr
(Me)2,Thr4,Tyr-NH92]OVT: a selective oxytocin receptor ligand. Eur. J.
Pharmacol. 147, 197–207.
Elands, J., Barberis, C., Jard, S., 1988. [3H]-[Thr4,Gly7]OT: a highly selec-
tive ligand for central and peripheral OT receptors. Am. J. Physiol:
Endocrinol. Metab. 254 (17), E31–E38.
Williams, P.D., Anderson, P.S., Ball, R.G., Bock, M.G., Carroll, L., Chiu,
S.H., Clineschmidt, B.V., Culberson, J.C., Erb, J.M., Evans, B.E., Fitz-
patrick, S.L., Freidinger, R.M., Kaufman, M.J., Lundell, G.F., Murphy,
J.S., Pawluczyk, J.M., Perlow, D.S., Pettibone, D.J., Pitzenberger, S.M.,
Thompson, K.L., Veber, D.F., 1994. 1-((7,7-dimethyl-2(S)-(2(S)-
amino-4-(methylsulfonyl)butyramido)bicyclo [2.2.1]-heptan-1(S)-yl)-
methyl)sulfonyl)-4-(2-methylphenyl)piperazine (L-368,899): an orally
bioavailable, non-peptide oxytocin antagonist with potential utility for
managing preterm labor. J. Med. Chem. 37, 565–571.
Fuchs, A.R., Fuchs, F., Soloff, M.S., 1985. Oxytocin receptors in nonpreg-
nant human uterus. J. Clin. Endocrinol. Metab. 60, 37–41.
Gimpl, G., Fahrenholz, F., 2001. The oxytocin receptor system: structure,
function, and regulation. Physiol. Rev. 81, 629–683.
Williams, P.D., Bock, M.G., Evans, B.E., Freidinger, R.M., Gallicchio,
S.N., Guidotti, M.T., Jacobson, M.A., Kuo, M.S., Levy, M.R., Lis,
E.V., Michelson, S.R., Pawluczyk, J.M., Perlow, D.S., Pettibone, D.J.,
Quigley, A.G., Reiss, D.R., Salvatore, C., Stauffer, K.J., Woyden, C.J.,
1999. Nonpeptide oxytocin antagonists: analogs of L-371,257 with
improved potency. Bioorg. Med. Chem. Lett. 9, 1311–1316.
Kimura, T., Tanizawa, O., Mori, K., Brownstein, M.J., Okayama, H., 1992.
Structure and expression of a human oxytocin receptor. Nature 356,
526–529.
Kruszynski, M., Lammek, B., Manning, M., Seto, J., Haldar, J., Sawyer,
W.H., 1980. [1-(h-mercapto-h, h-cyclopentamethylene propionic acid),