14β-Chlorocinnamoylamino derivatives of metopon: Long-term μ-opioid receptor antagonists

Article abstract of DOI:10.1016/S0014-2999(96)00904-1

The affinity, selectivity and antinociceptive properties of 5β-methyl-14β-(p-chlorocinnamoylamino)-7,8-dihydromorphinone (MET-Cl-CAMO) and N-cyclopropyl-methyl-5β-methyl-14β-(p-chlorocinnamoylamino)-7,8 -dihydronormorphinone (N-CPM-MET-Cl-CAMO) for the multiple opioid receptors were characterized. In competition binding assays using bovine striatal membranes, both compounds inhibited the binding of 0.25 nM [³H][D-Ala²,(Me)-Phe⁴,Gly(ol)⁵]enkephalin (DAMGO) with IC₅₀ values of less than 2 nM. Preincubation of membranes with MET-Cl-CAMO and N-CPM-MET-Cl-CAMO produced a concentration-dependent, wash-resistant inhibition of μ-opioid receptor binding. Saturation binding experiments with N-CPM-MET-Cl-CAMO showed a reduction in the number of μ-opioid binding sites without a change in affinity. In the mouse 55°C warm-water tail-flick assay, neither MET-Cl-CAMO nor N-CPM-MET-Cl-CAMO at doses up to 100 nmol produced antinociception after intracerebroventricular administration, but morphine-induced antinociception was antagonized in a time- and dose-dependent manner by both compounds. The antagonism produced by 1 nmol of either MET-Cl-CAMO or N-CPM-MET-Cl-CAMO reached a maximal effect after 24 h, and lasted up to 48 h. Analgesia mediated by δ- or κ-opioids was not altered by either compound. In summary, the data suggest that MET-Cl-CAMO and N-CPM-MET-Cl-CAMO are long-term, μ-opioid receptor antagonists, devoid of agonist properties in the mouse tail-flick assay, and that N-CPM-MET-Cl-CAMO may produce its antagonistic effects by binding irreversibly to the μ-opioid receptor.

Full text of DOI:10.1016/S0014-2999(96)00904-1

European Journal of Pharmacology 320 Ž1997. 121–129
1
4b-Chlorocinnamoylamino derivatives of metopon: long-term m-opioid
receptor antagonists
a
b
b,1
a,)
Jay P. McLaughlin , Alice Sebastian , Sydney Archer , Jean M. Bidlack
a
Department of Pharmacology and Physiology, P.O. Box 711, The UniÕersity of Rochester, School of Medicine and Dentistry, 601 Elmwood AÕenue,
Rochester, NY 14642-8711, USA
Department of Chemistry, Cogswell Laboratory, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, USA
b
Received 4 September 1996; revised 5 November 1996; accepted 12 November 1996
Abstract
The affinity, selectivity and antinociceptive properties of 5b-methyl-14b-Ž p-chlorocinnamoylamino.-7,8-dihydromorphinone ŽMET-
Cl-CAMO. and N-cyclopropyl-methyl-5b-methyl-14b-Ž p-chlorocinnamoylamino.-7,8-dihydronormorphinone ŽN-CPM-MET-Cl-CAMO.
for the multiple opioid receptors were characterized. In competition binding assays using bovine striatal membranes, both compounds
3
2
4
5
inhibited the binding of 0.25 nM w HxwD-Ala ,ŽMe.-Phe ,GlyŽol. xenkephalin ŽDAMGO. with IC values of less than 2 nM.
5
0
Preincubation of membranes with MET-Cl-CAMO and N-CPM-MET-Cl-CAMO produced a concentration-dependent, wash-resistant
inhibition of m-opioid receptor binding. Saturation binding experiments with N-CPM-MET-Cl-CAMO showed a reduction in the number
of m-opioid binding sites without a change in affinity. In the mouse 558C warm-water tail-flick assay, neither MET-Cl-CAMO nor
N-CPM-MET-Cl-CAMO at doses up to 100 nmol produced antinociception after intracerebroventricular administration, but morphine-in-
duced antinociception was antagonized in a time- and dose-dependent manner by both compounds. The antagonism produced by 1 nmol
of either MET-Cl-CAMO or N-CPM-MET-Cl-CAMO reached a maximal effect after 24 h, and lasted up to 48 h. Analgesia mediated by
d- or k-opioids was not altered by either compound. In summary, the data suggest that MET-Cl-CAMO and N-CPM-MET-Cl-CAMO are
long-term, m-opioid receptor antagonists, devoid of agonist properties in the mouse tail-flick assay, and that N-CPM-MET-Cl-CAMO
may produce its antagonistic effects by binding irreversibly to the m-opioid receptor.
Keywords: Morphine derivative; Cinnamoylamino group; b-Endorphin receptor; ŽIrreversible antagonist.; Analgesia; ŽMouse.
1
. Introduction
term antagonism by forming irreversible covalent bonds
with the m-opioid receptor ŽWard et al., 1982, 1985;
Liu-Chen and Phillips, 1987.. However, use of the selec-
tive m-opioid receptor antagonistic properties of b-
funaltrexamine is complicated, requiring a 24 h delay
between administration and use to avoid k-opioid
receptor-mediated agonism ŽWard et al., 1982.. This has
Irreversible opioid ligands are valuable tools for the
characterization of opioid receptors, both in vivo and in
vitro ŽPortoghese et al., 1980; Rice et al., 1983; Bidlack et
al., 1990.. Addition of site-directed alkylating groups to
dihydromorphine, dihydromorphinone, naltrexone and nal-
trexamine has yielded increased selectivity and affinity of
irreversible opioid antagonists ŽPasternak and Hahn, 1980;
Jiang et al., 1992; Derrick et al., 1996.. b-Funaltrexamine,
with the structure of naltrexone but containing a fumara-
mate group in the C-6 position, is one such compound
ŽPortoghese et al., 1980.. The fumaramate side chain is an
led to further research for compounds that act as irre-
versible opioid receptor antagonists, but with greater ease
3
of use. Under alkaline conditions, w Hx14b-Žbromoace-
tamido.-7,8-dihydromorphine ŽH BAM., a dihydromor-
2
phine derivative with a bromoacetamido group in the
C-14b position, selectively labeled a m-opioid binding site
in bovine striatal membranes through formation of a cova-
lent bond ŽBidlack et al., 1993.. However, the alkaline
conditions preclude its use in vivo. 14b-Ž p-Chlorocinna-
moylamino.-7,8-dihydro-N-Žcyclopropylmethyl.-normor-
phinone ŽC-CAM. is a derivative of naltrexone with a
a,b-unsaturated carbonyl, and is thought to act as a
Michael acceptor for nucleophilic attack, providing long-
)
Corresponding author. Tel. Ž1-716. 275-5600; Fax: Ž716. 244-9283;
e-mail: bidlackj@pharmacol.rochester.edu
1
In Memoriam, August 22, 1996.
chlorocinnamoylamino group at the C-14 position, replac-
0
014-2999r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.
PII S0014-2999Ž96.00904-1

1
22
J.P. McLaughlin et al.rEuropean Journal of Pharmacology 320 (1997) 121–129
ing the 14b-hydroxyl group ŽLewis et al., 1988.. C-CAM
was characterized as a long-term m-opioid receptor antago-
nist in the mouse warm-water tail-flick assay ŽAceto et al.,
1
989; Comer et al., 1992.. After peripheral administration,
C-CAM shifted the analgesic dose-response curve of mor-
phine rightward and downward, suggesting C-CAM may
act through a nonequilibrium mechanism at opioid recep-
tors to produce its antagonistic activity ŽComer et al.,
Fig. 1. Structures of MET-Cl-CAMO and its N-cyclopropylmethyl ana-
log, N-CPM-MET-Cl-CAMO.
1
992; Burke et al., 1994.. Recently, seeking further in-
creases in m-opioid receptor affinity, cinnamoylamino side
chains were added at the 14b position of metopon, 5b-
methyl-7,8-dihydromorphinone ŽSebastian et al., 1993;
McLaughlin et al., 1995.. Two derivatives containing 14b-
p-nitrocinnamoylamino constituents, 5b-methyl-14b-Ž p-
nitrocinnamoylamino.-7,8-dihydromorphinone ŽMET-
CAMO. and its corresponding N-cyclopropylmethyl ana-
of dihydromorphinone bind covalently to the m-opioid
receptor. Furthermore, it is unclear whether the nitro- or
the chlorocinnamoylamino group is more effective in pro-
ducing long-term opioid receptor antagonism, as all com-
parisons to date have been between opioid ligands with
structural differences aside from the cinnamoyl groups.
Direct comparison of the two cinnamoyl groups would
require synthesis of both nitro- and chlorocinnamoyl
derivatives of the same opioid affinity compound. Finally,
given the difficulties in using the selective m-opioid recep-
tor antagonistic properties of b-funaltrexamine, new irre-
versible opioid receptor antagonists may prove useful in
research if they possess greater efficacy or selectivity for
the opioid receptor.
log, N-CPM-MET-CAMO, demonstrated high affinity and
selectivity for the m-opioid receptor in competition binding
experiments with bovine striatal membranes, and antago-
nized morphine-induced antinociception for up to 72 h in
the mouse 558C warm-water tail-flick assay ŽJiang et al.,
1
993a, 1994.. MET-CAMO and N-CPM-MET-CAMO did
not antagonize antinociception induced by either d- or
k-opioid receptor selective agonists. An intracerebroven-
tricular Ži.c.v.. injection of either compound into mice
shifted the dose-response line for morphine-induced
antinociception rightward and downward, suggesting an
irreversible covalent interaction of the compounds with the
m-opioid receptor ŽJiang et al., 1994.. The cinnamoy-
The present study characterized two derivatives of
metopon, 5b-methyl-14b-Ž p-chlorocinnamoylamino.-7,8-
dihydromorphinone ŽMET-Cl-CAMO. and N-cyclopro-
pylmethyl-5b-methyl-14b-Ž p-chlorocinnamoylamino.-7,8-
dihydronormorphinone ŽN-CPM-MET-Cl-CAMO. ŽFig. 1.,
lamino group in all three affinity ligands may act as a
Michael acceptor, similar to the fumaramate group in
b-funaltrexamine, allowing nucleophilic addition with ei-
ther primary amino groups or thiol groups in the opioid
receptor to confer long-term antagonist effect. Recently,
b-funaltrexamine has been shown to form a covalent bond
with lysine 233 in the cloned rat m-opioid receptor ŽChen
et al., 1996.. Evidence from pharmacological and receptor
for irreversible properties in receptor binding assays and in
the mouse 558C warm-water tail-flick test as a measure of
antinociception. Like MET-CAMO and N-CPM-MET-
CAMO, both derivatives are 5-methyl-7,8-dihydro-
morphinones but, like C-CAM, both possess a chlorocin-
namoylamino group instead of the nitrocinnamoylamino
group in MET-CAMO and N-CPM-MET-CAMO in the
C-14b position, allowing for direct comparison in opioid
receptor antagonist efficacy between the nitro- and
chlorocinnamoylamino groups. A preliminary study sug-
gested both compounds had highest affinity for the m-opioid
receptor, and produced wash-resistant inhibition of m-
opioid receptor binding ŽMcLaughlin et al., 1994..
sequencing studies suggests the opioid receptors possess
an essential sulfhydryl group at or near the opioid binding
site ŽSimon et al., 1973; Pasternak et al., 1975; Childers,
1
984; Thompson et al., 1993; Joseph and Bidlack, 1994..
However, the cinnamoylamino constituent has not been
directly observed to react with thiol groups; when incu-
bated with N-acetylcysteine, N-CPM-MET-CAMO was
recovered unchanged ŽSebastian et al., 1993.. Moreover,
2. Materials and methods
recent experiments incubating mouse brain membranes
3
with w HxC-CAM, followed by acid precipitation of the
2.1. Synthesis of MET-Cl-CAMO and N-CPM-MET-Cl-
CAMO
protein, did not detect specific radiolabelling of receptor
protein ŽZernig et al., 1995., suggesting w HxC-CAM did
not bind covalently to the m-opioid receptor. However,
3
2.1.1. Preparation of MET-Cl-CAMO
3
preliminary studies demonstrated that w Hx14b-Ž p-nitro-
To a solution of 150 mg of 14b-amino-5b-methyl-7,8-
dihydrocodeinone in 20 ml of methylene chloride and 64
ml of triethylamine there was added dropwise with stirring
a solution of 64 ml of p-chlorocinnamoyl chloride in 5 ml
of methylene chloride in an atmosphere of dry nitrogen.
After 3 h, there was added a sodium bicarbonate solution.
cinnamoylamino.-7,8-dihydro-N-cyclopropylmethyl-nor-
3
codeinone Žw HxN-CPM-CACO. bound covalently to the
m-opioid receptor in bovine striatal membranes ŽMc-
Laughlin et al., in press.. Given these conflicting data, it
remains unclear whether 14b-cinnamoylamino derivatives

J.P. McLaughlin et al.rEuropean Journal of Pharmacology 320 (1997) 121–129
123
The layers were separated and the organic layer washed
librium binding with the d-opioid receptor selective pep-
3
2
4
5
with H O and dried. The solvent was evaporated and the
tide w HxwD-Pen , p-Cl-Phe ,D-Pen xenkephalin ŽpCl-
2
residue was chromatographed on a preparative plate using
DPDPE. in the presence of 5 mM MgCl and 0.1 mM
2
methylene chloridermethanol Ž94:6. as the developing
solvent. There was obtained 160 mg of the product, 5b-
methyl-14b-Ž p-chlorocinnamoylamino.-7,8-dihydrocodei-
none MET-Cl-CACO, the codeinone precursor of MET-
Cl-CAMO.
phenylmethylsulfonyl fluoride. To determine the IC50 val-
ues for the inhibition of binding by the compounds, the
3
3
final concentrations of w HxDAMGO, w HxpCl-DPDPE and
3
w HxU69,593 were 0.25 nM, 0.2 nM and 1 nM, respec-
tively. Nonspecific binding was measured with inclusion
of 10 mM naloxone. Binding was terminated by filtering
the samples through Schleicher & Schuell No. 32 glass
fiber filters using a Brandel 48-well cell harvester. Filters
were soaked at least 60 min in 0.25% polyethylenimine for
1
00 mg of this product was demethylated with the aid
of 97 ml of boron tribromide in 5 ml of dry methylene
chloride at y208C. The mixture was allowed to warm to
room temperature and after 1.5 h the reaction mixture was
treated with sodium bicarbonate solution. The separated
organic layer was washed with water, dried and evaporated
to dryness. After purification by thin-layer chromatography
Žchloroformrmethanol, 9:1., 35 mg of MET-Cl-CAMO
ŽFig. 1. was obtained, with a melting point of 180–1868C
3
3
w HxpCl-DPDPE and w HxU69,593 binding experiments.
After filtration, filters were washed three times with 3 ml
of cold 50 mM Tris-HCl, pH 7.5 and were counted in 2 ml
of Ecoscint A scintillation fluid.
To determine the concentration of MET-Cl-CAMO and
N-CPM-MET-Cl-CAMO required to obtain wash-resistant
inhibition of opioid binding, 10 mg of membrane protein
were incubated at 378C for 15 min with seven concentra-
tions of each compound, ranging from 1.6 to 1600 nM, in
a final volume of 2 ml of 50 mM Tris-HCl, pH 7.5. An
incubation time of 15 min was chosen after demonstration
that the corresponding nitrocinnamoylamino compounds
did not increase wash-resistant inhibition of opioid binding
significantly after this point in time course binding experi-
ments ŽJiang et al., 1994.. The contents of the tubes were
1
Ždec.. H nuclear magnetic resonance analysis also con-
firmed the structure of the compound.
2
.1.2. Preparation of N-CPM-MET-Cl-CAMO
0 mg of p-chlorocinnamic acid was added to 3 ml of
7
thionyl chloride and the mixture was refluxed for 1 h. The
thionyl chloride was removed in vacuo and the residue, the
desired acid chloride, was dissolved in 3 ml of dry methy-
lene chloride and added dropwise with stirring to 90 mg of
1
4b-amino-5b-methyl-7,8-dihydrocodeinone and 35 ml of
triethylamine dissolved in 30 ml of methylene chloride
then diluted to 40 ml with cold Tris buffer and centrifuged
at 39 000=g for 15 min at 48C. The washing step was
repeated for a total of four times. Finally, the membranes
were resuspended in 2 ml of 50 mM Tris-HCl, pH 7.5, and
opioid binding to 0.2 ml of membranes was determined as
described above.
kept at y108C under an atmosphere of N . The reaction
2
mixture was stirred at room temperature for 1 h, then
worked up as described above.
The crude product was not purified but was dissolved in
8
ml of dry methylene chloride, cooled to y788C and
treated with 100 ml of boron tribromide. The mixture was
stirred for 2 h as the temperature rose to y108C. After
stirring for another hour at room temperature, the mixture
was worked up as in the case of MET-Cl-CAMO to give
To determine whether the affinity ligands altered the
affinity or reduced the number of opioid binding sites, 10
mg of membranes were incubated with 100 nM MET-Cl-
CAMO for 15 min, with a final volume of 2 ml of 50 mM
Tris-HCl, pH 7.5, followed by four centrifugal washes.
N-CPM-MET-Cl-CAMO at final concentrations of 8 nM
Žfor the study of m-opioid receptor binding. and 100 nM
Žfor the study of k-opioid receptor binding. were incubated
6
0 mg of N-CPM-MET-Cl-CAMO ŽFig. 1., with a melting
point of 160–1708C Ždec..
2
.2. Opioid binding to boÕine striatal membranes
3
with membranes in parallel studies. w HxDAMGO binding
Bovine striatal membranes were prepared and competi-
at concentrations ranging from 0.025 nM to 12.8 nM, and
3
tion binding experiments were performed with MET-Cl-
CAMO and N-CPM-MET-Cl-CAMO to determine affinity
of the compounds for the multiple opioid receptors as
previously described ŽJiang et al., 1994.. Briefly, bovine
w HxU69,593 binding at concentrations ranging from 0.025
nM to 6.4 nM, was measured as described above. The
protein concentration of membranes was determined by the
method of Bradford Ž1976., using bovine serum albumin
striatal membranes were incubated with 12 different con-
centrations of the competitor and the radiolabeled ligands
at 258C in a final volume of 1 ml of 50 mM Tris-HCl, pH
as the standard.
2.3. Animals
7
.5. Incubation times of 60 min were used for the m-opioid
3
2
receptor selective peptide w H xwD-Ala ,ŽM e.-
All experiments used male, ICR mice Ž24–30 g, Harlan
Sprague Dawley ŽIndianapolis, IN, USA... Mice were kept
in groups of eight in a temperature controlled room with a
12-h light-dark cycle. Food and water were available ad
libitum until the time of the experiment.
4
5
Phe ,GlyŽol. xenkephalin ŽDAMGO. and the k-opioid re-
3
ceptor selective ligand w HxŽ5a,7a,8b.-N-methyl-N-w7-Ž1-
pyrrolidinyl.-1-oxaspiroŽ4,5.dec-8-ylx-benzeneacetamide
ŽU69,593., and a 4-h incubation was used to insure equi-

1
24
J.P. McLaughlin et al.rEuropean Journal of Pharmacology 320 (1997) 121–129
2
.4. Injection techniques
compounds were calculated from the equation K s
i
IC rŽ1 q S ., w here S s Žconcentration of
5
0
Intracerebroventricular injections were made directly
radioligand.rŽK of radioligand. ŽCheng and Prusoff,
D
into the lateral ventricle according to the modified method
of Haley and McCormick Ž1957.. The volume of all i.c.v.
injections was 5 ml, using a 10-ml Hamilton microliter
syringe. The mouse was lightly anesthetized with ether, an
incision was made in the scalp, and the injection was made
3
3
1
973.. Saturation w HxDAMGO and w HxU69,593 binding
data were analyzed by nonlinear regression analysis using
the LIGAND program ŽMunson and Rodbard, 1980.. Re-
gression lines, D50 values, the dose producing 50%
antinociception, and 95% confidence limits were deter-
mined with each individual data point using the computer
program by Tallarida and Murray Ž1986.. All data points
2
mm.
mm lateral and 2 mm caudal to bregma at a depth of 3
2
.5. Tail-flick assay
The thermal nociceptive stimulus was 558C water, with
shown are the mean of 7–10 mice, with S.E.M. repre-
sented by error bars.
the latency to tail flick or withdrawal taken as the endpoint
ŽVaught and Takemori, 1979.. After determining control
3
. Results
latencies, the mice received graded i.c.v. doses of opioid
agonists or antagonists. Morphine sulfate, DPDPE and
U50,488 were each given as single i.c.v. injections with
antinociceptive effect measured 20 min after injection
unless otherwise stated. In the antagonistic study, various
doses of either MET-Cl-CAMO or N-CPM-MET-Cl-
CAMO were given as a single pretreatment at 20 min, 4, 8,
3
.1. Affinity and selectiÕity of MET-Cl-CAMO and N-
CPM-MET-Cl-CAMO for the multiple opioid receptors in
boÕine striatal membranes
Both MET-Cl-CAMO and N-CPM-MET-Cl-CAMO
ŽFig. 1. demonstrated higher affinity for the m-opioid
receptor than for the d- or k-opioid receptors, as deter-
1
6, 24, 48, 72 and 96 h before testing. A cut-off time of 15
s was used; if the mouse failed to display a tail flick in that
time, the tail was removed from the water and the animal
assigned a maximal antinociceptive score of 100%. Mice
which showed no response within 5 s in the initial control
test were eliminated from the experiment. At each time
point, antinociception was calculated according to the fol-
lowing formula: % antinociceptions100=Žtest latency
ycontrol latency.rŽ15ycontrol latency..
mined by their IC50 values for the inhibition of m-, d- and
2
.6. Materials
3
3
w HxDAMGO Ž60 Cirmmol. and w HxU69,593 Ž64
Cirmmol. were purchased from Amersham ŽArlington
3
Heights, IL, USA.. w HxpCl-DPDPE Ž48.6 Cirmmol. was
obtained from New England Nuclear ŽBoston, MA, USA..
Morphine sulfate was purchased from Mallinckrodt ŽSt.
Louis, MO, USA.. DPDPE was purchased from Bachem
ŽTorrance, CA, USA.. U50,488 was obtained from Re-
search Biochemicals International ŽNatick, MA, USA..
Ecoscint A was obtained from National Diagnostics
ŽAtlanta, GA, USA.. Schleicher&Schuell No. 32 glass
fiber filters were obtained from Schleicher& Schuell
ŽKeene, NH, USA..
For radioligand binding assays, MET-Cl-CAMO and
N-CPM-MET-Cl-CAMO were dissolved in dimethyl sul-
y3
foxide at a concentration of 10 M. Subsequent dilutions
Fig. 2. Wash-resistant inhibition of opioid binding produced by preincu-
bation of bovine striatal membranes with MET-Cl-CAMO and N-CPM-
MET-Cl-CAMO. Membrane protein at a concentration of 10 mg was
incubated with varying concentrations of MET-Cl-CAMO ŽA. and N-
CPM-MET-Cl-CAMO ŽB. at 378C for 15 min, followed by four centrifu-
were made into 50 mM Tris-HCl, pH 7.5. In the mouse
tail-flick assays, MET-Cl-CAMO and N-CPM-MET-Cl-
CAMO were dissolved in 20% dimethyl sulfoxide, a con-
centration that did not produce any detectable effect.
3
3
gal washes. Binding of 0.25 nM w HxDAMGO, 0.2 nM w HxpCl-DPDPE
3
2
.7. Statistics
w
x
and 1 nM H U69,593 to 0.2 ml of membranes was measured as
described in Section 2. Data are presented as the mean percentage of
control binding"S.E.M. from three or more experiments performed in
triplicate.
IC50 values were calculated by least squares fit to a
logarithm-probit analysis. The Ki values of unlabelled

J.P. McLaughlin et al.rEuropean Journal of Pharmacology 320 (1997) 121–129
125
Table 1
IC50 values for the inhibition of m-, d-, and k-opioid binding to bovine
striatal membranes by MET-Cl-CAMO and N-CPM-MET-Cl-CAMO
Radiolabeled ligands
IC50 ŽnM."S.E.M.
MET-Cl-CAMO
N-CPM-MET-Cl-CAMO
3
w HxDAMGO Žm.
0.44"0.17
48.6"18.0
13.2"0.42
0.91"0.33
23.2"2.44
11.6"3.04
3
w HxpCl-DPDPE Žd.
3
w HxU69,593 Žk.
Bovine striatal membranes were incubated with 12 different concentra-
tions of MET-Cl-CAMO and N-CPM-MET-Cl-CAMO in the presence of
3
3
either 0.25 nM w HxDAMGO, 0.2 nM w HxpCl-DPDPE or 1 nM
3
w HxU69,593 in 50 mM Tris-HCl, pH 7.5, at 258C. After equilibrium
binding was reached, membranes were filtered onto glass-fiber filters.
Data are listed as the mean IC50 values"S.E.M. from three or more
experiments, performed in triplicate.
k-opioid binding to bovine striatal membranes, measured
3
3
3
with w HxDAMGO, w HxpCl-DPDPE and w HxU69,593, re-
spectively ŽTable 1.. MET-Cl-CAMO displayed 2-fold
higher affinity for the m-opioid receptor than N-CPM-
MET-Cl-CAMO in the competition binding assays, but
Fig. 4. Antinociceptive effects of i.c.v. morphine Ž3 nmol, y20 min. in
mice pretreated with a single i.c.v. dose of MET-Cl-CAMO ŽA. or
N-CPM-MET-Cl-CAMO ŽB. Ž1 nmol; 20 min, 4, 8, 16, 24, 72 and 96 h
before testing. in the mouse tail-flick assay. ) Significant from morphine
alone, P-0.05.
N-CPM-MET-Cl-CAMO also displayed high affinity for
the d- and k-opioid receptors.
Bovine striatal membranes were incubated with varying
concentrations of the compounds at 258C for 15 min,
followed by dilution and four centrifugal washes, to detect
if MET-Cl-CAMO and N-CPM-MET-Cl-CAMO could
produce wash-resistant inhibition of opioid binding. Both
MET-Cl-CAMO and N-CPM-MET-Cl-CAMO produced a
concentration-dependent, wash-resistant inhibition of
3
w HxDAMGO binding ŽFig. 2A and B., with a 50% wash-
3
resistant inhibition of the binding of 0.25 nM w HxDAMGO
Fig. 3. Scatchard representation of saturation binding to control and
N-CPM-MET-Cl-CAMO-treated membranes with the receptor-selective
produced with concentrations of 86"18 nM and 13"3
nM, respectively. Interestingly, a N-CPM-MET-Cl-CAMO
concentration of 122"43 nM produced a 50% wash-re-
3
3
opioids w HxDAMGO ŽA. and w HxU69,593 ŽB.. Bovine striatal mem-
branes were incubated in the presence or absence of either 8 or 100 nM
3
3
3
sistant inhibition of the binding of w HxU69,593, while
N-CPM-MET-Cl-CAMO for w HxDAMGO and w HxU69,593 experi-
ments, respectively, for 15 min at 378C. Control samples lacked com-
pound in the preincubation. Membranes were then diluted and submitted
to four centrifugal washes. Binding was then measured as described in
MET-Cl-CAMO produced no wash-resistant inhibition of
k-opioid binding. Neither compound produced wash-re-
sistant inhibition of d-opioid receptor binding.
3
Section 2, with concentrations of w HxDAMGO ŽA. ranging from 0.025 to
3
Saturation w HxDAMGO binding studies demonstrated
3
3
.2 nM, and concentrations of w HxU69,593 ŽB. ranging from 0.05 to 6.4
that membranes pretreated with 100 nM MET-Cl-CAMO
nM. Data are from a representative experiment, which was repeated three
3
times. BrF, boundrfree.
had a Kd value for w HxDAMGO binding of 1.8"0.1

1
26
J.P. McLaughlin et al.rEuropean Journal of Pharmacology 320 (1997) 121–129
nM, a 3-fold increase over the control Kd value of 0.58"
3
0
.05 nM. The Bmax values for w HxDAMGO remained
unchanged compared to control, 94"9 fmolrmg of pro-
tein for control membranes versus 98"11 fmolrmg of
protein for membranes pretreated with MET-Cl-CAMO,
suggesting that inhibition of binding was due to residual
MET-Cl-CAMO.
In contrast, the Kd value for membranes preincubated
with 8 nM N-CPM-MET-Cl-CAMO remained unchanged,
3
but the Bmax value for w HxDAMGO decreased to 41"9
fmolrmg of protein ŽFig. 3A.. Membranes pretreated with
1
4
00 nM N-CPM-MET-Cl-CAMO displayed a K value
d
3
-fold higher for w HxU69,593 than the K value for
d
untreated control membranes Ž1.6"0.7 nM versus 0.40"
Fig. 6. Dose-response lines for i.c.v. morphine in the absence and
presence of i.c.v. MET-Cl-CAMO and N-CPM-MET-Cl-CAMO Ž1 nmol,
administered y24 h before testing. in the mouse tail-flick assay.
0
.01 nM, respectively., while maximal binding of
3
w HxU69,593 was not significantly changed Ž16"5.3
fmolrmg of protein for pretreated membranes versus 13"
0
.5 fmolrmg of protein for control membranes, respec-
3
wash-resistant inhibition of w HxDAMGO binding by alky-
tively. ŽFig. 3B.. The reduction in m-opioid binding sites
without a change in the affinity of the remaining binding
sites suggests that N-CPM-MET-Cl-CAMO produced
lating the m-opioid receptor to form a covalent bond. The
3
unchanged maximal binding of w HxU69,593, accompanied
by a 4-fold increase in the Kd value, suggests that when
membranes were pretreated with N-CPM-MET-Cl-CAMO
at a concentration of 100 nM, there was residual drug
associated with the membranes, which bound to the k-
opioid receptor in a non-covalent manner.
3
.2. Mouse tail-flick assay
No antinociceptive effect was produced by i.c.v. admin-
istration of MET-Cl-CAMO or N-CPM-MET-Cl-CAMO
at doses up to 100 nmol in the mouse 558C warm-water
tail-flick assay. Pretreatment of mice with either MET-Cl-
CAMO or N-CPM-MET-Cl-CAMO produced a time- and
dose-dependent antagonism of morphine-induced antinoci-
ception. Significant antagonism produced by 1 nmol of
MET-Cl-CAMO appeared 16 h after i.c.v. administration
Fig. 5. Antinociceptive effects of i.c.v. morphine sulfate Ž3 nmol, y20
min. in mice pretreated with a single i.c.v. dose of MET-Cl-CAMO ŽA.
and N-CPM-MET-Cl-CAMO ŽB. Ž0.03, 0.1, 0.3 or 1 nmol, administered
y24 h before testing. in the mouse tail-flick assay. Significant from
morphine alone, P-0.01.
Fig. 7. Antinociceptive effects of i.c.v. DPDPE Ž10 nmol, y20 min. or
U50,488 Ž30 nmol, y20 min. in mice pretreated without or with a single
i.c.v. dose of MET-Cl-CAMO and N-CPM-MET-Cl-CAMO Ž100 nmol,
administered y24 h before testing. in the mouse tail-flick assay.
)

J.P. McLaughlin et al.rEuropean Journal of Pharmacology 320 (1997) 121–129
127
and lasted up to 48 h, while antagonism produced by 1
nmol of N-CPM-MET-Cl-CAMO appeared 16 h after
i.c.v. administration and lasted up to 24 h ŽFig. 4A and B..
Maximal antagonism of morphine-induced antinociception
by MET-Cl-CAMO and N-CPM-MET-Cl-CAMO was ob-
served 24 h after injection. The antagonism of morphine-
induced antinociception by MET-Cl-CAMO and N-CPM-
MET-Cl-CAMO was dose-dependent ŽFig. 5A and B..
Pretreatment of mice with 1 nmol of either MET-Cl-CAMO
or N-CPM-MET-Cl-CAMO shifted the dose-response
curve of morphine rightward and downward, demonstrat-
ing insurmountable antagonism ŽFig. 6.. Neither MET-Cl-
further characterized for opioid receptor affinity with re-
ceptor binding assays, and found to bind the m-opioid
receptor with high affinity. Similar to the binding charac-
teristics of the corresponding 14b-p-nitrocinnamoylamino
compounds ŽJiang et al., 1994., the N-methyl compound
was more selective for the m-opioid receptor than the
N-cyclopropylmethyl compound. This finding is consistent
with previous studies that have demonstrated higher selec-
tivity for the m-opioid binding site by morphine derivatives
possessing a N-methyl constituent versus N-cyclopro-
pylmethyl side chains ŽPasternak and Hahn, 1980; Jiang et
al., 1994.. However, N-CPM-MET-Cl-CAMO differed
CAMO nor N-CPM-MET-Cl-CAMO, administered 24 h
before testing in doses up to 100 nmol, antagonized
antinociception produced by the d-opioid receptor agonist
DPDPE or the k-opioid receptor agonist U50,488 ŽFig. 7..
from its nitrocinnamoylamino analog, N-CPM-MET-
CAMO, in proving to be less potent in inhibiting m-opioid
receptor binding in a wash-resistant manner. A 50%
wash-resistant inhibition of the binding of 0.25 nM
3
w HxDAMGO was reported with a N-CPM-MET-CAMO
concentration of 5.2"0.6 nM ŽJiang et al., 1994., a
2.5-fold greater potency in comparison to the chlorocin-
namoylamino analog. These differences in wash-resistant
inhibition of binding between the chloro- and nitrocin-
namoylamino derivatives may be due to the greater elec-
tronegativity of the nitro group over that of the chloro
group, making it more effective at drawing electrons across
the benzene ring of the cinnamoyl constituent. This would
leave the carbonyl double bond more susceptible to
nucleophilic attack, and the nitrocinnamoylamino com-
pounds, therefore, more reactive in forming covalent bonds.
Presumably, this increased reactivity would account for the
4
. Discussion
This study investigated the supraspinal opioid-receptor
mediated effects of two derivatives of metopon, MET-Cl-
CAMO and N-CPM-MET-Cl-CAMO, using the mouse
warm-water tail-flick assay. Neither compound produced
any antinociception in the tail-flick assay, after i.c.v. ad-
ministration of doses up to 100 nmol. This lack of anal-
gesic activity by a N-methyl substituted morphine deriva-
tive is consistent with previous findings using the 14b-
nitrocinnamoylamino analog MET-CAMO ŽJiang et al.,
3
1
994., despite the fact that similarly substituted com-
increased wash-resistant inhibition of w HxDAMGO bind-
pounds in the morphinan and benzomorphan series are
generally agonists ŽHellerbach et al., 1966., such as meto-
pon ŽMcLaughlin et al., 1995.. Like their nitrocinnamoy-
lamino analogs ŽJiang et al., 1994., MET-Cl-CAMO and
ing by the nitrocinnamoylamino derivative in comparison
to its chlorocinnamoylamino analog.
It is not clear why the N-cyclopropylmethyl compound
3
produced wash-resistant inhibition of w HxDAMGO bind-
N-CPM-MET-Cl-CAMO produced long-term antagonism
of antinociception mediated by the m-opioid receptor, and
shifted the dose-response line for morphine-induced
antinociception to the right and downward. This shift in
the dose-response line is characteristic of an irreversible
opioid receptor antagonist. The delay in the appearance of
long-term antagonism of antinociception seen here is con-
sistent with delays reported for most opioid receptor affin-
ity ligands used in vivo. For example, the Michael accep-
tors b-funaltrexamine and N-CPM-MET-CAMO ŽJiang et
al., 1995. both demonstrated at least 8 h delayed antago-
nism of morphine analgesia when the lowest dose produc-
ing maximal antagonism 24 h after administration was
used. This prolonged delay in producing an effect has not
been observed when the compounds were incubated with
isolated membranes ŽJiang et al., 1994.. Therefore, the in
ing at much lower concentrations than the N-methyl
derivative, while both compounds produced similar effects
in the mouse warm-water tail flick test. One possible
explanation could be differences between the in vivo and
in vitro accessibility of the amino acidŽs. on the receptor
that may form a covalent bond with the compounds.
Alternatively, the two compounds may alkylate different
amino acids on the m-opioid receptor. Alkylation of two
different amino acids may produce the same long-term
inhibition of morphine-induced antinociception. For exam-
ple, b-funaltrexamine alkylates lysine-233 on the cloned
rat m-opioid receptor ŽChen et al., 1996.. In contrast, the
X
disulfide-containing affinity ligands 14 a ,14 b-
wdithiobiswŽ2-oxo-2,1-ethanediyl.iminoxxbisŽ7,8-dihydro-
morphinone. ŽTAMO., its N-cyclopropylmethyl derivia-
tive, N-CPM-TAMO, and its N-cyclobutylmethyl deriva-
tive, N-CBM-TAMO, formed a disulfide bond with the
receptor as determined by the finding that the disulfide-
bond reducing reagent, dithiothreitiol, reversed wash-re-
sistant inhibition of m-opioid binding produced by these
compounds ŽJiang et al., 1993b; Archer et al., 1994; Xu et
al., in press.. b-Funaltrexamine, TAMO, N-CPM-TAMO,
vivo delay cannot be easily explained chemically. It may
be due to the downregulation of the opioid receptor, an
event that is known to proceed more slowly than desensiti-
zation of the receptor in mediating opioid-induced inhibi-
tion of adenylyl cyclase activity ŽLaw et al., 1983..
MET-Cl-CAMO and N-CPM-MET-Cl-CAMO were

1
28
J.P. McLaughlin et al.rEuropean Journal of Pharmacology 320 (1997) 121–129
and N-CBM-TAMO produced long-term antagonism of
morphine-induced antinociception ŽJiang et al., 1992, 1995;
Xu et al., in press.. Therefore, despite the fact that b-
receptor in bovine striatal membranes ŽMcLaughlin et al.,
in press..
In conclusion, these results suggest that MET-Cl-CAMO
and N-CPM-MET-Cl-CAMO act like their nitrocinnamoy-
lamino analogs in being devoid of agonist activity but
producing m-opioid receptor selective, long-term antago-
nism of antinociception in the mouse warm-water tail-flick
test. However, variation in potency and k-opioid receptor
binding suggest that there are important differences be-
tween the chlorocinnamoylamino and nitrocinnamoy-
lamino compounds.
funaltrexamine binds to a lysine residue and the disulfide-
containing TAMO derivatives bind to a thiol group, both
types of compounds produced the same long-term antago-
nism of morphine-induced antinociception. With the cin-
namoylamino derivatives, the N-methyl derivative, MET-
Cl-CAMO, and the N-cyclopropylmethyl derivative, N-
CPM-MET-Cl-CAMO, may bind to different amino acids
on the receptor. Alkylation of the receptor produces long-
term antagonism of antinociception mediated by m-opioid
receptors. However, unless the alkylation of the receptor
3
alters the w HxDAMGO binding site, changes in radioli-
Acknowledgements
gand binding would not be observed.
Both the binding and the analgesic studies indicate that
N-CPM-MET-Cl-CAMO forms a covalent bond with the
m-opioid receptor. The suggested covalent bond formed
between N-CPM-MET-Cl-CAMO and the m-opioid recep-
tor may be produced by the chlorocinnamoylamino group
acting as a Michael acceptor, undergoing nucleophilic
addition to eligible substrates in the receptor, such as thiol
or primary amino groups found in cysteine or lysine amino
acids, respectively. The extracellular amino-terminal tail of
the rat m-opioid receptor is reported to have one lysine and
five cysteine amino acids, providing six potential sites for
the formation of a covalent bond with a Michael acceptor
ŽThompson et al., 1993.. Previous studies have suggested
This work was supported by U.S. Public Health Service
Grants DA03742, DA01674 and DA07232 from the Na-
tional Institute on Drug Abuse.
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