Design and Synthesis of Novel Dimeric Morphinan Ligands for κ and μ Opioid Receptors

Article abstract of DOI:10.1021/jm030139v

A novel series of morphinans were synthesized, and their binding affinity at and functional selectivity for μ, δ, and κ opioid receptors were evaluated. These dimeric ligands can be viewed as dimeric morphinans, which were formed by coupling two identical

Full text of DOI:10.1021/jm030139v

5162
J . Med. Chem. 2003, 46, 5162-5170
Design a n d Syn th esis of Novel Dim er ic Mor p h in a n Liga n d s for K a n d µ Op ioid
Recep tor s
J ohn L. Neumeyer,*,† Ao Zhang,† Wennan Xiong,† Xiao-Hui Gu,† J ames E. Hilbert,‡ Brian I. Knapp,‡
S. Stevens Negus,† Nancy K. Mello,† and J ean M. Bidlack‡
Alcohol and Drug Abuse Research Center, McLean Hospital, Harvard Medical School, 115 Mill Street,
Belmont, Massachusetts 02478, and Department of Pharmacology and Physiology, School of Medicine and Dentistry,
University of Rochester, Rochester, New York 14642
Received March 27, 2003
A novel series of morphinans were synthesized, and their binding affinity at and functional
selectivity for µ, δ, and κ opioid receptors were evaluated. These dimeric ligands can be viewed
as dimeric morphinans, which were formed by coupling two identical morphinan pharmaco-
phores (cyclorphan (1) or MCL 101 (2)) with varying connecting spacers. Ligands 6 and 7 with
alkyl spacers on the nitrogen position and ligands 8 and 9 in which the two morphinan
pharmacophores were coupled by ether moieties at the 3-hydroxyl positions showed significant
decrease in affinity at all three opioid receptors. An improvement in the affinity was achieved
by introducing an ester moiety as the spacer in the dimeric morphinans. It was observed that
the affinity of these ligands was sensitive to the character and length of the spacer. Compound
13 (MCL-139) with a 4-carbon ester spacer, compound 17 (MCL-144) containing a 10-carbon
spacer, and compound 19 (MCL-145) with the conformationally constrained fumaryl spacer
were the most potent ligands in this series, displaying excellent affinities at µ and κ receptors
(Ki ) 0.09-0.2 nM at µ and Ki ) 0.078-0.049 nM at κ), which were comparable to the parent
compound 2. Ligand 12, a compound containing only one morphinan pharmacophore and a
long-chain ester group, had affinity at both µ and κ receptors almost identical to that of the
parent ligand 2. In the [35S]GTPγS binding assay, ligands 13, 17, and 19 and their parent
morphinans 1 and 2 stimulated [35S]GTPγS binding mediated by the µ and κ receptors.
Compounds 13 and 17 were full κ agonists and partial µ agonists, while compound 19 was a
partial agonist at both µ and κ receptors. These novel ligands, as well as their interesting
pharmacological properties, will serve as the basis for our continuing investigation of the dimeric
ligands as potential probes for the pharmacotherapy of cocaine abuse and may also open new
avenues for the characterization of opioid receptors.
In tr od u ction
lengths. Compounds that contain two pharmacophores
or one single pharmacophore and a nonpharmacophore
recognition unit linked through a connecting spacer
have been termed “bivalent ligands”.7 So far, only a
limited number of such bivalent ligands have been
synthesized.8-13 For example, bivalent ligands contain-
ing oxymorphine or naltrexamine pharmacophores with
specific spacer lengths have been reported to have
enhanced opioid agonist or antagonist potencies and
selectivities.8-13
The opioid system modulates several physiological
and behavioral processes such as pain perception, the
stress response, the immune response, and neuroendo-
crine function.1 Pharmacological and molecular cloning
studies2 have identified three major types of opioid
receptors (µ, δ, and κ) that belong to the rhodopsin
subfamily in the superfamily of over 100 G-protein-
coupled receptors (GPCRs). They are highly homologous
(∼60% amino acid identity) and recognize structurally
diverse ligands including peptides, opiates, and a variety
of synthetic non-peptides.3
Recent behavioral studies suggested that opioid κ
agonists with varying activity at the µ receptor ef-
fectively reduced cocaine self-administration in nonhu-
man primates and with fewer undesirable side effects
than the highly selective κ agonists.4 These results
encouraged us to continue developing novel ligands
targeting both κ and µ receptors.5,6 This report describes
a series of novel dimeric morphinans that have mixed
κ agonist and µ agonist/antagonist pharmacophores
coupled by varying connecting spacers with different
The morphinan core structures that we employed for
this study included cyclorphan (1)14,15 and its N-cyclobu-
tylmethyl analogue MCL-101 (2).5a,15 Both 1 and 2 were
found to have high affinity at κ and µ receptors and
longer durations of action than ethylketocyclazocine (5)
(Figure 1). They acted as full κ agonists but partial µ
agonists.4,5 Ligands composed of two such mixed κ
agonist and µ agonist/antagonist pharmacophores may
exhibit increased binding affinity and different phar-
macological properties. The purpose of this study was
to develop potent mixed κ and µ opioid ligands by
optimizing the nature and length of the spacers. Such
dimeric morphinans may serve as candidates for our
continuing development of mixed κ and µ agonists/
antagonists and represent intriguing potential targets
for drug development. We speculated that such dimeric
* To whom correspondence should be addressed. Phone: 617-855-
3388. Fax: 617-855-2519. Email: Neumeyer@mclean.harvard.edu.
† Harvard Medical School.
‡ University of Rochester.
10.1021/jm030139v CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/25/2003

Morphinan Ligands for Opioid Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5163
1,2-cyclopropyldicarboxylic acid, which was hydrolyzed
from 3-oxabicyclo[3.1.0]hexane-2,4-dione, in the pres-
ence of DCC and DMAP.17 Monomeric analogue 12
containing a capped spacer was also synthesized as a
control to factor out any possible contribution of the
spacer moiety.
P h a r m a cologica l Resu lts a n d Discu ssion
Affin it y a n d Select ivit y of t h e Syn t h esized
Liga n d s. All the novel dimeric morphinan ligands were
examined for their affinity at and selectivity for µ, δ,
and κ opioid receptors with Chinese hamster ovary
(CHO) cell membranes stably expressing the human
opioid receptors. The data were summarized in Table
1. For comparison purposes, opioid binding affinity data
for enadoline, cyclorphan (1), MCL-101 (2), and levor-
phanol (3) were also included.
F igu r e 1. Selected morphinan core structures.
Sch em e 1
Traditionally, the 3-phenolic hydroxyl group was
recognized as a hydrogen bond donor and was essential
for the metabolism of traditional opiates.1 Therefore, we
examined first morphinans 6 and 7 where the two
morphinan pharmacophores were connected by alkyl
spacers on the basic nitrogen atoms. Compared to
levorphanol (3), their affinities were decreased 10- to
20-fold at κ, 88- to 140-fold at δ, and 228- to 247-fold at
µ receptors. However, a selectivity (7- to 25-fold) for µ
and κ over δ receptors was retained. Recent studies from
our and Wentiand’s laboratories indicated that substi-
tution of the phenolic hydroxyl group in morphinans18
and benzomorphans19 with other functional groups (e.g.,
amino or carboxamido) may retain good opioid receptor
activity. Similar results were also observed in the
clocinnamox series where the 3-deoxy analogue exhib-
ited high µ opioid receptor affinity compared to clocin-
namox, 14â-cinnamoyl epoxymorphinan.20 Thus, we
investigated the effect of attachment of a spacer at the
3-OH position in the morphinans. Compounds 8 and 9,
containing an ether linkage, displayed a dramatic
decrease in affinity (120- to 1500-fold) at all three opioid
receptors compared to the parent morphinan 2. We then
evaluated ligands 10 and 11 containing an ester moiety
between the 3-positions of the two morphinan core
structures. The affinities of ligands 10 and 11 at all
three opioid receptors were remarkably improved com-
pared to the affinities of compounds 6-9. Morphinan
10, which had two methylene units between the two
ester moieties, was 2-fold less potent at all three opioid
receptors, while a longer ester linker (containing eight
methylene units) in ligand 11 resulted in a 10- to 15-
fold decrease of affinity at all three opioid receptors,
compared to the parent compound 1.
ligands may also be recognized as the basis for the
further development of novel bivalent ligands for the
characterization of κ or µ opioid receptors.
Ch em istr y
Several series of dimeric ligands were synthesized
from the monomeric cyclorphan (1) and MCL 101 (2).
In the first series, the pharmacophore used in the
elaboration of such novel morphinan ligands was nor-
levorphanol (4) because it has 17-nitrogen as a point of
attachment for the spacer chain. The spacers employed
in this series were composed of a varying number of
methylene units. In the initial study, ligands 6 and 7
containing three and six methylene units at the 17-
position were synthesized. Thus, the commercially
available (-)-3-hydroxy-N-methylmorphinan (levorpha-
nol, 3) was N-demethylated to give the norlevorphanol
(4) according to the procedure reported by Olfoson.16
Reaction of 4 with propane-1,3-diyl or hexane-1,6-diyl
ditosylate (prepared from propane-1,3-diol or hexane-
1,6-diol) in DMF at 110 °C produced ligands 6 and 7 in
52% and 45% yields, respectively (Scheme 1).
Compounds 8 and 9 with an ether spacer at the
3-hydroxy position of morphinan 2 were prepared by
coupling 2 equiv of 2 with 1,4-dibromobutane or 1,10-
dibromodecane in the presence of sodium hydride in
DMF (Scheme 1). A third series of morphinan deriva-
tives (10, 11, 13-22) linked by ester functions at the
3-hydroxy position were also synthesized (Scheme 2).
Generally, compounds 10, 11, and 13-21 were prepared
from morphinan 1 or 2 with an appropriate diacid
chloride in the presence of triethylamine. Compound 22
was obtained by condensation of morphinan 2 with cis-
Since ligands esterified at the 3-position exhibited
better affinity at opioid receptors than the ligands with
a 3-position ether spacer or with an alkyl linkage at the
basic nitrogen atom, we investigated the role of the
character and length of the ester spacer in such ligands.
Initially, with 2 as the morphinan core structure,
compounds 13-18 with varying ester spacer lengths
were evaluated. All these ligands displayed high affinity
at both µ and κ receptors and a 35- to 120-fold selectivity
for µ/κ over δ receptors. It is noteworthy that the affinity
at opioid receptors was sensitive to the length of the
ester spacer and a good relationship between the affinity
and the number of methylene units in the ester spacer

5164 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Neumeyer et al.
Sch em e 2
Ta ble 1. Ki Inhibition Values of µ, δ, and κ Opioid Binding to CHO Membranes by MCL Compounds
Ki (nM) ( SE
selectivity
compd
[3H]DAMGO (µ)
[3H]naltrindole (δ)
[3H]U69,593 (κ)
κ/µ
80
2
3
0.09
2
1
2
0.5
1
2
3
2
3
1
2
2
2
κ/δ
enadoline
22 ( 3.2
>1 µM
0.27 ( 0.073
0.034 ( 0.002
0.079 ( 0.003
2.3 ( 0.26
>1000
60
1 (cyclorphan)
2 (MCL-101)
3 (levorphanol)
6 (MCL-133)
7 (MCL-134)
8 (MCL-153)
9 (MCL-154)
10 (MCL-135)
11 (MCL-136)
12 (MCL-176)
13 (MCL-139)
14 (MCL-140)
15 (MCL-177)
16 (MCL-179)
17 (MCL-144)
18 (MCL-178)
19 (MCL-145)
20 (MCL-141)
21 (MCL-142)
22 (MCL-143)
0.062 ( 0.003
0.23 ( 0.01
0.21 ( 0.017
48 ( 2.9
1.9 ( 0.072
5.9 ( 0.55
4.2 ( 0.45
600 ( 43
370 ( 23
730 ( 29
2500 ( 91
3.8 ( 0.14
32 ( 2.9
70
2
24 ( 0.69
25
52 ( 1.8
50 ( 2.8
7
29 ( 1.2
18 ( 1.3
40
20
40
80
630
120
70
37
35
90
75
120
100
270
90
66 ( 4.3
120 ( 8.2
0.14 ( 0.01
0.93 ( 0.09
0.23 ( 0.22
0.16 ( 0.01
3.3 ( 0.36
0.89 ( 0.13
0.97 ( 0.033
0.090 ( 0.004
1.8 ( 0.24
0.20 ( 0.032
4.7 ( 0.64
3.3 ( 0.26
0.28 ( 0.003
0.10 ( 0.006
0.41 ( 0.004
0.070 ( 0.03
0.076 ( 0.002
1.2 ( 0.05
44 ( 6.8
9.4 ( 0.44
80 ( 7.9
24 ( 4.3
0.65 ( 0.52
0.59 ( 0.153
0.049 ( 0.001
1.1 ( 0.1
0.078 ( 0.01
4.2 ( 0.81
3.4 ( 0.08
0.17 ( 0.02
21 ( 0.45
4.2 ( 0.44
82 ( 5.5
9.4 ( 0.54
400 ( 46
920 ( 95
16 ( 1.0
3
1
1
2
was observed (Figure 2). Compound 13 with two meth-
ylene units and 17 containing eight methylene units in
the ester spacer were the most potent ligands in this
subseries. The number of methylene units between two
and eight (compounds 14-16) or longer than eight
(compound 18) did not give better results (Table 1).
These observations suggested that such dimeric ligands
with an appropriate spacer (e.g., ester) and spacer
length may have higher affinity (∼2-fold) and selectivity
than the corresponding monomeric congener.
To further elucidate the effect of the ester spacer on
the affinity, we probed another subseries of morphinans,
19-22, with fumaryl, tere- or isophthalyl, and cyclo-
propyldicarboxyl moieties as the spacers, respectively.

Morphinan Ligands for Opioid Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5165
compounds 10-13 and 15-19, together with cyclorphan
(1) and MCL 101 (2), were selected for the [35S]GTPγS
assay. Table 2 showed the agonist and antagonist
properties of these ligands in stimulating [35S]GTPγS
binding mediated by the κ opioid receptor. Ligands 11,
12, 15, 16, 18, and 19 produced similar maximal
stimulation of [35S]GTPγS binding (Emax) comparable to
that of the monomers 1 and 2, but the stimulation was
less than that of κ selective agonist U50,488. The rank
order of the EC50 values substantially correlated with
the Ki values obtained for the compounds in the binding
assays with [3H]U69,593. Among the most potent ligands
(13, 17, and 19), 13 and 17 had lower efficacy and
ligands 17 and 19 had the lowest EC50 values that were
not statistically different from that of the parent com-
pound 2. Similar to the parent compound 2, most of
these ligands except compound 19 exhibited no inhibi-
tory effect on the stimulation of the κ selective agonist
U50,488, which suggested that most of these ligands
were full κ selective agonists. Ligand 19 produced good
maximal stimulation of [35S]GTPγS binding and also
had a good maximal inhibition (Imax) of the U50,488
stimulated [35S]GTPγS binding, although the IC50 was
very high (2400 nM). These data indicated that ligand
19 was a partial κ agonist.
F igu r e 2. Relationship between ligand ester spacer length
and relative potency.
It is also worth noting that compound 19 with a
conformationally constrained fumaryl group (trans con-
formation) has almost identical affinity and selectivity
as the monomeric compound 2. Introduction of the
phthalyl moiety in 20 and 21 produced a 15- to 150-
fold decrease in affinity (compared to the parent com-
pound 2). However, ligand 22, with a conformationally
constrained cis-cyclopropyldicarboxyl ester linkage, was
slightly less potent at µ and κ receptors than 2 and
equally potent at κ receptor as the trans compound 19
(Table 1). Unexpectedly, ligand 12 with a long diester
chain containing only one morphinan function retained
the identical affinity at both µ and κ receptors, compared
to 2. However, a 7-fold decrease in affinity was observed
at the δ receptor, which resulted in a pronounced
enhancement in the selectivity for κ over δ receptors
(630-fold). Thus, of the compounds we evaluated, three
(13, 17, and 19) were identified with high affinity at µ
and κ receptors and 45- to 120-fold selectivity for µ and
κ over δ receptors.
Although having different properties at κ receptor,
ligands 13, 17, and 19 and the monomer 2 produced
similar maximal stimulation of [35S]GTPγS binding
mediated by µ receptor. Ligands 12, 15, and 16 had
slightly higher efficacy, but the stimulation was con-
siderably lower than that of full µ opioid agonist
DAMGO (Table 3). Ligands 10 and 18 displayed a
pronounced lower efficacy than the others, which indi-
cated that they had very weak µ agonist properties.
Compounds 17 and 19 had the lowest EC50 values that
were not statistically different from that of the parent
compound 2. Compared to the morphinans 1 and 2 that
produced moderate inhibition of the full µ opioid agonist
DAMGO, most of the dimeric ligands had much higher
inhibitiory activity, especially ligands 11 and 19, which
produced maximal inhibitory activity (Imax ) 100%)
indicating that they were more efficacious as µ antago-
nists than the others. Ligand 10 and its monomeric
compound 1 had similar low IC50 values (1.7 nM), which
indicated that they were potent µ opioid antagonists.
Effica cy Assa y of Selected Liga n d s. To character-
ize the relative efficacy of these morphinan ligands,
Ta ble 2. Agonist and Antagonist Properties of Compounds in Stimulating [35S]GTPγS Binding Mediated by the κ Opioid Receptora
pharmacological
properties
Emax
EC50
(nM)
Imax
IC50
(nM)
compd
(% maximal stimulation)
(% maximal inhibition)
(-)-U50,488
agonist
agonist
agonist
agonist
agonist
agonist
agonist
agonist
agonist
agonist
agonist
partial agonist
110 ( 2.0
90 ( 10
80 ( 6.8
70 ( 15
90 ( 7.4
90 ( 9.4
70 ( 5.7
80 ( 2.8
80 ( 7.5
60 ( 0.68
90 ( 1.6
90 ( 6.0
46 ( 16
- - -
- - -
- - -
- - -
1 (cyclorphan)
2 (MCL-101)
10 (MCL-135)
11 (MCL-136)
12 (MCL-176)
13 (MCL-139)
15 (MCL-177)
16 (MCL-179)
17 (MCL-144)
18 (MCL-178)
19 (MCL-145)
0.19 ( 0.04
1.3 ( 0.44
3.3 ( 0.63
4.9 ( 0.43
5.7 ( 0.62
4.5 ( 1.7
- - -
no effect
no effect
no effect
no effect
no effect
no effect
100
11 ( 1.1
15 ( 4.4
0.85 ( 0.053
8.1 ( 0.37
2.3 ( 0.34
2400 ( 75
a
Membranes from CHO cells that stably expressed only the κ opioid receptor were incubated with varying concentrations of the
compounds. The stimulation of [35S]GTPγS binding was measured as described in the Experimental Section. To determine the antagonist
properties of a compound, membranes were incubated with 100 nM of the κ agonist U50,488 in the presence of varying concentrations of
the compound. The Imax value is the maximal percent inhibition obtained with the compound. The IC50 value is the concentration of
compound needed to produce half maximal inhibition. Dashed lines indicate that the compound was not tested for antagonist properties
because of its high Emax value.

5166 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Neumeyer et al.
Ta ble 3. Agonist and Antagonist Properties of Compounds in Stimulating [35S]GTPγS Binding Mediated by the µ Opioid Receptora
pharmacological
properties
Emax
EC50
(nM)
Imax
IC50
(nM)
compd
DAMGO
(% maximal stimulation)
(% maximal inhibition)
agonist
120 ( 12
40 ( 2.9
50 ( 2.5
10 ( 0.60
30 ( 3.0
60 ( 2.2
50 ( 4.4
60 ( 4.7
57 ( 2.4
50 ( 6.6
14 ( 4.0
50 ( 2.5
110 ( 9.0
0.80 ( 0.06
1.6 ( 0.15
- - -
- - -
- - -
1 (cyclorphan)
2 (MCL-101)
10 (MCL-135)
11 (MCL-136)
12 (MCL-176)
13 (MCL-139)
15 (MCL-177)
16 (MCL-179)
17 (MCL-144)
18 (MCL-178)
19 (MCL-145)
partial agonist
partial agonist
antagonist
50 ( 1.2
50 ( 2.6
60 ( 3.9
100
50 ( 1.9
50 ( 0.70
100
54 ( 4.2
60 ( 5.2
60 ( 1.7
100
1.7 ( 0.40
20 ( 2.7
1.8 ( 0.14
90 ( 11
130 ( 6.0
170 ( 32
90 ( 11
360 ( 55
16 ( 3.0
100 ( 2.7
610 ( 71
partial agonist
partial agonist
partial agonist
partial agonist
partial agonist
partial agonist
partial agonist
partial agonist
- - -
8.0 ( 0.57
7.4 ( 1.8
10 ( 0.96
11 ( 0.62
1.3 ( 0.15
- - -
2.9 ( 0.50
a
Membranes from CHO cells that stably expressed only the µ opioid receptor were incubated with varying concentrations of the
compounds. The stimulation of [35S]GTPγS binding was measured as described in the Experimental Section. EC50 values were the
concentration of compound needed to produce 50% of the Emax value. When the Emax value was 30% or lower, it was not possible to
calculate an EC50 value. To determine the antagonist properties of a compound, membranes were incubated with 200 nM of the µ agonist
DAMGO in the presence of varying concentrations of the compound. The Imax value is the maximal percent inhibition obtained with the
compound. The IC50 value is the concentration of compound needed to produce half maximal inhibition. Dashed lines indicate that the
compound was not tested.
F igu r e 4. Partial agonist properties of 17 (MCL 144) as
determined in the [35S]GTPγS binding assay. To determine the
agonist properties (A) of 17, membranes from CHO cells that
stably expressed the µ opioid receptor were incubated with
varying concentrations of 17. [35S]GTPγS binding was mea-
F igu r e 3. Partial agonist properties of 17 (MCL 144) as
determined in the [35S]GTPγS binding assay. To determine the
sured as described in the Experimental Section. To determine
the antagonist properties (B), the membranes were incubated
with 200 nM DAMGO in the presence of varying concentra-
tions of 17.
agonist properties (A) of 17, membranes from CHO cells that
stably expressed the κ opioid receptor were incubated with
varying concentrations of 17. [35S]GTPγS binding was mea-
sured as described in the Experimental Section. To determine
the antagonist properties (B), the membranes were incubated
with 100 nM of the κ agonist U50,488 in the presence of
varying concentrations of 17.
illustrated that most of these ligands were κ agonists
and µ partial agonists except for compound 10, which
was a κ agonist and µ antagonist, and compound 19,
which was a partial agonist at both κ and µ receptors.
Compound 12, compared to its parent 2, gave similar
pharmacological properties at both µ and κ receptors.
The different pharmacological properties of the highly
The preliminary assay for agonist and antagonist
properties of these ligands in stimulating [35S]GTPγS
binding mediated by the κ and µ opioid receptors

Morphinan Ligands for Opioid Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5167
was greater than 100 nM. These findings illustrated
that ligand 19 acted as an agonist at both µ and κ
receptors at low concentrations, while at higher con-
centrations, it inhibited its agonist effects and ulti-
mately acted as an inverse agonist as shown by its
inhibition of basal [35S]GTPγS binding to both receptors.
The reason behind this ability of 19 was not clear.
Con clu sion
This study was conducted to evaluate the important
structural features of morphinan ligands with mixed µ
and κ receptor selectivity. By the choice of cyclorphan
(1) and MCL 101 (2) as the critical pharmacophores, a
novel series of dimeric ligands were synthesized by
coupling two identical morphinans with spacers with
varying lengths. Compounds 6 and 7 with alkyl spacers
at the nitrogen position and compounds 8 and 9 in which
the two morphinan pharmacophores were coupled by
ether moieties at the 3-hydroxyl position showed sig-
nificantly decreased affinity at all three opioid receptors.
An appreciable improvement in affinity was observed
by introducing an ester moiety as the spacer. After
optimization of the morphinan core structure, the spacer
character, and the spacer length, three compounds (13,
17, and 19) were identified as the most potent ligands
within this entire series. Compound 17 displayed a
2-fold higher affinity at µ and κ receptors as the
monomeric congener 2. Since the interaction between
the 3-phenolic hydroxyl group and opioid receptor
proteins (µ, δ, and κ) was historically recognized as
crucial for high affinity, the high potency of these
ligands with a 3-position ester spacer may be indicative
of the existence of some opioid receptor complex whose
pharmacological profiles are distinct from classic µ, δ,
and κ receptors. The unexpected high affinity of 12 (the
monomeric morphinan) at µ and κ receptors indicated
that the spacer may also represent a recognition site
and only one pharmacophore was required for such
ligands. This observation is in agreement with our and
others’ recent findings that the phenolic hydroxyl group
could be replaced by other functional groups without
significant decrease in the opioid receptor activity.18-20
Studies to further elucidate the function of the spacer
itself or to explain the pharmacological difference
between monomeric and dimeric ligands are now ongo-
ing.
In the [35S]GTPγS binding assays, ligands 13, 17, and
19 and their parent compounds 1 and 2 stimulated [35S]-
GTPγS binding mediated by the µ and κ receptors. They
produced Emax values lower than that of the κ-selective
agonist arylacetamide (U50,488), suggesting that these
morphinan ligands, like most classic monomeric mor-
phinans, were less efficacious than U50,488 at the κ
receptor as measured by the stimulating [35S]GTPγS
binding. The most potent ligands 13, 17, and 19 behaved
differently at µ and κ receptors. Both 13 and 17 were
full κ agonists and partial µ agonists. 19 exhibited a
unique pharmacological profile; it showed an inverted
“U” concentration effect curve. The different pharma-
cological profile of 19 makes it unlikely that MCL 101
(2), a possible metabolite of this dimeric ligand, was
responsible for the pharmacological properties observed.
The monomeric ligand 12, compared to its parent 2, not
only retained high affinity but also displayed similar
F igu r e 5. Determining the pharmacological properties of 19
(MCL 145) at the κ and µ opioid receptors as measured with
the [35S]GTPγS binding assay. Membranes from CHO cells
that stably expressed the µ opioid receptor (A) or κ opioid
receptor (B) were incubated with increasing concentrations
of 19.
potent ligands 17 and 19 were further demonstrated in
Figures 3-5.
At concentrations from 0.1 to 100 nM, ligand 17
produced an increasing stimulation with a maximal
stimulating [35S]GTPγS binding of 60% and 50% (Emax
)
at κ and µ receptors, respectively (Figures 3A and 4A).
When membranes were incubated with the κ agonist
U50,488 in the presence of varying concentrations of 17,
there was no significant inhibition of U50,488-induced
[35S]GTPγS binding (Figure 3B). However, when mem-
branes were incubated with the µ agonist DAMGO in
the presence of varying concentrations of 17, it signifi-
cantly antagonized DAMGO-induced stimulation (Fig-
ure 4B). Altogether, these findings demonstrated that
ligand 17 exerts agonist properties at κ and µ receptors
and also acts as a µ antagonist.
Pharmacological properties of ligand 19 were deter-
mined by incubating membranes from CHO cells with
increasing concentrations of 19 (Figure 5). Interestingly,
it showed an inverted “U” concentration effect curve.
At lower concentration (less than 100 nM), it produced
a dose-dependent increase in stimulation at both µ and
κ receptors. A strong antagonism of its self-induced
stimulation was observed when the concentration of 19

5168 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Neumeyer et al.
1,4-Bis(N-cyclobu tylm eth ylm or ph in an -3-oxy)decan e (9,
MCL-154) was prepared as a pale-yellow oil in 40% yield from
2 (311 mg, 1 mmol) and dibromodecane (180 mg, 0.6 mmol)
using a similar procedure as in the case of 8: 1H NMR (CDCl3,
300 MHz) δ 7.01 (d, J ) 8.4 Hz, 1H), 6.78 (d, J ) 2.4 Hz, 1H),
6.68 (dd, J ) 8.4, 2.4 Hz, 1H), 3.91 (t, J ) 6.6 Hz, 2H), 3.01-
1.21 (complex, 27H). Anal. (C50H72N2O2) C, H, N.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of Liga n d s
10-21. To a solution of morphinan 1 or 2 (1 mmol) and Et3N
(0.5 mL) in CHCl3 (10 mL) was added dropwise an appropriate
diacid chloride (0.6 mmol) at 0 °C. The mixture was stirred at
room temperature for 48 h and then diluted with dichlo-
romethane. The organic layer was separated, washed with
saturated Na2CO3 and brine, dried with Na2SO4, and then
concentrated to give a dark oil. The crude product was purified
by column chromatography on silica gel (EtOAc/Et3N, 100:1)
to afford the corresponding bivalent ligands.
pharmacological properties at both µ and κ receptors.
To establish if such dimeric ligands act as bivalent
ligands, it is necessary to prepare additional compounds
in this series as well as to undertake further pharma-
cological investigations. Such studies will be necessary
to further rule out the metabolic conversion of these
dimeric ligands to the corresponding monomers with a
free hydroxyl group, and to address the role of the spacer
moiety in compound 12.
Exp er im en ta l Section
Melting points were determined on a Thomas-Hoover capil-
lary tube apparatus and are reported uncorrected. 1H and 13C
NMR spectra were recorded on a Bruker AC300 spectrometer
using tetramethylsilane as an internal reference. Element
analyses, performed by Atlantic Microlabs, Atlanta, GA, were
within (0.4% of theoretical values. Analytical thin-layer
chromatography (TLC) was carried out on 0.2 mm Kieselgel
60F 254 silica gel plastic sheets (EM Science, Newark). Flash
chromatography was used for the routine purification of
reaction products. The column output was monitored by TLC.
1,3-Bis(3-h yd r oxym or p h in a n -17-yl)p r op a n e (6, MCL-
133). A mixture of norlevorphanol 4 (499 mg, 1.9 mmol),
propane-1,3-diyl ditosylate (294 mg, 0.76 mmol), and NaHCO3
(320 mg, 3.8 mmol) in anhydrous DMF (8 mL) was stirred at
110 °C overnight. The solvent was removed in vacuo. The
residue was partitioned between ethyl acetate and water. The
aqueous phase was extracted with ethyl acetate. The combined
extracts were washed with water and brine and then dried
over Na2SO4. After removal of the solvent, a dark residual oil
remained, which was purified by column chromatography on
silica gel (EtOAc/MeOH.Et3N, 5:1:0.1) to give the product 6
as white solid (207 mg, 52%): mp 162-164 °C; 1H NMR
(CDCl3, 300 MHz) δ 6.89 (d, J ) 8.1 Hz, 1H), 6.73 (d, J ) 2.7
Hz, 1H), 6.61 (dd, J ) 2.7, 8.4 Hz, 1H), 2.91-0.89 (complex,
20H). Anal. (C35H46N2O2‚H2O) C, H, N.
Bis(N-cyclopr opylm eth ylm or ph in an -3-yl) su ccin ate 10
(MCL-135): pale-yellow foam (70%); 1H NMR (CDCl3, 300
MHz) δ 7.07 (d, J ) 8.1 Hz, 1H), 6.95 (d, J ) 2.1 Hz, 1H), 6.85
(dd, J ) 2.4, 8.4 Hz, 1H), 3.10-0.11 (complex, 26H). Anal.
(C45H58N2O4‚0.5H2O) C, H, N.
Bis(N-cyclop r op ylm eth ylm or p h in a n -3-yl) seba coyla te
11 (MCL-136): pale-yellow foam (76.2%); 1H NMR (CDCl3,
300 MHz) δ 7.07 (d, J ) 8.4 Hz, 1H), 6.93 (s, 1H), 6.83 (d, J )
8.1 Hz, 1H), 3.09-0.11 (complex, 31H); 13C NMR (CDCl3, 75
MHz) δ 172.5, 149.5, 142.3, 135.4, 128.6, 118.7, 118.3, 60.2,
55.9, 45.8, 45.2, 42.0, 38.1, 36.8, 34.6, 29.2, 27.0, 26.7, 25.0,
24.5, 22.3, 9.7, 4.2, 3.8. Anal. (C50H68N2O4‚1/2H2O) C, H, N.
Meth yl(N-cyclobu tylm eth ylm or p h in a n -3-yl) seba ca te
12 (MCL-176) was prepared as a pale-yellow oil (230 mg, 88%)
from 2 (155 mg, 0.5 mmol), Et3N (0.2 mL), and methyl 10-
chloro-10-oxodecanoate (175.5 mg, 0.75 mmol) by using a
similar procedure as in the preparation of 11: 1H NMR (CDCl3,
300 MHz) δ 7.09 (d, J ) 8.1 Hz, 1H), 6.91 (d, J ) 2.7 Hz, 1H),
6.84 (dd, J ) 8.4, 2.7, 1H), 3.66-1.32 (complex, 46H); 13C NMR
(CDCl3, 75 MHz) δ 174.4, 172.6, 149.6, 142.0, 134.9, 128.7,
118.8, 118.4, 61.1, 55.8, 51.6, 45.7, 44.4, 41.4, 37.7, 36.6, 34.6,
34.5, 34.2, 28.1, 28.0, 26.8, 26.6, 25.1, 25.0, 24.6, 22.2, 19.0.
Anal. (C33H49NO4‚H2O) C, H, N.
Bis(N-cyclobu tylm eth ylm or p h in a n -3-yl) su ccin a te 13
(MCL-139): pale-yellow foam (23%); 1H NMR (CDCl3, 300
MHz) δ 7.06 (d, J ) 8.4 Hz, 1H), 6.91 (s, 1H), 6.83 (dd, J )
1.8, 8.1 Hz, 1H), 3.01-1.28 (complex, 28H); 13C NMR (CDCl3,
75 MHz) δ 171.0, 149.2, 142.3, 135.6, 128.7, 123.7, 118.6, 118.2,
61.8, 56.1, 46.0, 45.1, 42.0, 38.1, 36.8, 35.2, 29.7, 28.2, 27.1,
26.8, 24.7, 22.4, 19.2. Anal. (C47H62N2O4‚0.5H2O) C, H, N.
Bis(N-cyclobu tylm eth ylm or p h in a n -3-yl) glu ta m a te 14
(MCL-140): pale-pink foam (41%); 1H NMR (CDCl3, 300 MHz)
δ 7.09 (d, J ) 8.4 Hz, 1H), 6.94 (d, J ) 2.7 Hz, 1H), 6.84 (dd,
J ) 2.7, 8.4 Hz, 1H), 3.01-1.25 (complex, 29H); 13C NMR
(CDCl3, 75 MHz) δ 171.8, 149.3, 142.4, 135.6, 128.7, 118.6,
118.3, 61.7, 56.0, 45.9, 45.1, 42.0, 38.0, 36.7, 35.1, 33.5, 28.0,
27.0, 26.7, 24.6, 22.3, 20.2, 19.0. Anal. (C48H64N2O4‚1.5H2O)
C, H, N.
Bis(N-cyclobu tylm eth ylm or p h in a n -3-yl) su ber a te 15
(MCL-177): pale-yellow oil (70.9%); 1H NMR (CDCl3, 300
MHz) δ 7.09 (d, J ) 8.4 Hz, 1H), 6.91 (d, J ) 2.1 Hz, 1H), 6.84
(dd, J ) 8.4, 2.1 Hz, 1H), 3.01-1.25 (complex, 32H); 13C NMR
(CDCl3, 75 MHz) δ 172.5, 149.4, 142.3, 135.5, 128.7, 118.6,
118.3, 61.7, 56.0, 45.9, 45.1, 42.0, 38.0, 36.8, 35.2, 34.5, 28.9,
28.0, 27.0, 26.7, 24.9, 24.6, 22.3, 19.0. Anal. (C51H70N2O4) C,
H, N.
Bis(N-cyclobu tylm eth ylm or p h in a n -3-yl) a zela oa te 16
(MCL-179): pale-yellow foam (42%); 1H NMR (CDCl3, 300
MHz) δ 7.08 (d, J ) 8.4 Hz, 1H), 6.92 (d, J ) 2.1 Hz, 1H), 6.83
(dd, J ) 8.4, 2.1 Hz, 1H), 3.01-1.19 (complex, 33H). Anal.
(C52H72N2O4‚0.5H2O) C, H, N.
Bis(N-cyclobu tylm eth ylm or p h in a n -3-yl) seba coyla te
17 (MCL-144): pale-yellow foam (47%); 1H NMR (CDCl3, 300
MHz) δ 7.07 (d, J ) 8.4 Hz, 1H), 6.89 (s, 1H), 6.81 (dd, J )
8.4, 2.1 Hz, 1H), 2.97 (d, J ) 18.3 Hz, 1H), 2.79 (s, 1H), 2.61-
1.25 (complex, 32H); 13C NMR (CDCl3, 75 MHz) δ 172.4, 149.3,
142.2, 135.4, 128.6, 118.6, 118.3, 61.8, 56.1, 46.0, 45.2, 42.1,
1,6-Bis(3-h yd r oxym or p h in a n -17-yl)h exa n e (7, MCL-
134). To a solution of 1,6-hexanediol (2.36 g, 20 mmol) in
anhydrous dichloromethane (100 mL) was added triethylamine
(8.5 mL, 60 mmol) and tosyl chloride (9.55 g, 50 mmol) at 0
°C. The resulting mixture was stirred at room temperature
overnight. After dilution with CHCl3, the layers were sepa-
rated and the organic phase was washed with brine, dried with
Na2SO4, and concentrated to give a pale-yellow oil. The crude
product was purified by column chromatography on silica gel
(EtOAc/MeOH/Et3N, 50:1:0.1) to afford the corresponding
hexane-1,6-diyl ditosylate as a white crystal: mp 72-73 °C;
1H NMR (CDCl3, 300 MHz) δ 7.78 (d, J ) 8.4 Hz, 2H), 7.35 (d,
J ) 8.4 Hz, 2H), 3.98 (t, J ) 6.6 Hz, 2H), 2.45 (s, 3H), 1.63-
1.57 (m, 2H), 1.29-1.24 (m, 2H). Condensation of the ditosylate
(225 mg, 0.53 mmol) with 4 (244 mg, 1 mmol) by using the
same procedure as in the case of 6 gave product 7 as a white
1
solid (128 mg, 45%): mp 156-158 °C; H NMR (CDCl3, 300
MHz) δ 8.96 (s, 1H), 6.88 (d, J ) 8.4 Hz, 1H), 6.63 (d, J ) 2.1
Hz, 1H), 6.50 (dd, J ) 2.4, 8.4 Hz, 1H), 2.84-0.98 (complex,
22H). Anal. (C38H52N2O2‚H2O) C, H, N.
1,4-Bis(N-cyclobu tylm eth ylm or ph in an -3-oxy)bu tan e (8,
MCL-153). To a solution of 2 (311 mg, 1 mmol) in anhydrous
DMF was added NaH (48 mg, 1.2 mmol) at 0 °C. The mixture
was stirred at room temperature for 1 h. Dibromobutane (130
mg, 0.6 mmol) was added dropwise, and the resulting mixture
was stirred at room temperature for 24 h. After removal of
the solvent, the mixture was diluted with CH2Cl2. The organic
phase was separated, washed with brine, dried over Na2SO4,
and evaporated in vacuo. The residue was chromatographed
on silica gel with hexanes/ethyl acetate (1:1) as the eluent to
give product 8 as pale-yellow oil (250 mg, 72.5%): 1H NMR
(CDCl3, 300 MHz) δ 7.00 (d, J ) 8.4 Hz, 1H), 6.78 (d, J ) 2.4
Hz, 1H), 6.68 (dd, J ) 2.4, 8.4 Hz, 1H), 3.98 (t, J ) 6.6 Hz,
2H), 2.97-1.25 (complex, 27H); 13C NMR (CDCl3, 75 MHz) δ
154.3, 138.9, 127.2, 125.3, 108.5, 108.2, 63.6, 58.5, 52.9, 42.8,
42.1, 38.9, 34.7, 33.6, 31.9, 30.5, 26.5, 25.0, 24.8, 23.8, 23.5,
21.0, 19.2. Anal. (C46H64N2O2) C, H, N.

Morphinan Ligands for Opioid Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5169
38.1, 36.9, 35.3, 34.7, 29.4, 29.3, 28.2, 27.1, 26.9, 25.2, 24.8,
22.5, 19.2. Anal. (C53H74N2O4‚0.4Et3N) C, H, N.
were used for the µ-selective peptide [3H]DAMGO and the
κ-selective ligand [3H]U69,593. A 3 h incubation was used with
the δ-selective antagonist [3H]naltrindole. To determine the
IC50 values for the inhibition of binding by the compounds,
the final concentrations of [3H]DAMGO, [3H]naltrindole, and
[3H]U69,593 were 0.25, 0.2, and 1 nM, respectively. Nonspecific
binding was measured by inclusion of 10 µM naloxone. Binding
was terminated by filtering the samples through Schleicher
& Schuell no. 32 glass fiber filters (Keene, NH) using a Brandel
48-well cell harvester. Filters were soaked for at least 60 min
in 0.25% polyethylenimine for [3H]naltrindole and [3H]U69,593
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. The Ki values
of unlabelled compounds were calculated from the equation
Ki ) IC50/(1 + S), where S ) (concentration of radioligand)/
(KD of radioligand).21
Bis(N-cyclobu tylm eth ylm or p h in a n -3-yl) d od eca n ed io-
1
a te 18 (MCL-178): pale-yellow oil (60.3%); H NMR (CDCl3,
300 MHz) δ 7.09 (d, J ) 8.4 Hz, 1H), 6.91 (d, J ) 2.1 Hz, 1H),
6.83 (dd, J ) 7.8, 2.1 Hz, 1H), 3.01-0.86 (complex, 36H); 13C
NMR (CDCl3, 75 MHz) δ 172.4, 149.2, 142.0, 135.2, 128.4,
118.4, 118.1, 61.5, 55.8, 45.6, 44.9, 41.7, 37.7, 36.5, 34.9, 34.4,
29.3, 29.2, 29.1, 27.8, 26.7, 26.5, 24.9, 24.3, 22.1, 18.8. Anal.
(C55H78N2O4) C, H, N.
Bis(N-cyclobu tylm eth ylm or p h in a n -3-yl) fu m a r a te 19
(MCL-145): pale-yellow foam (45%); 1H NMR (CDCl3, 300
MHz) δ 7.18 (s, 1H), 7.12 (d, J ) 8.4, 1H), 6.98 (d, J ) 2.1 Hz,
1H), 6.91 (dd, J ) 2.1, 8.1 Hz, 1H), 3.04-1.25 (complex, 26H);
13C NMR (CDCl3, 75 MHz) δ 163.5, 148.9, 142.5, 136.1, 134.6,
128.8, 118.3, 118.0, 61.8, 56.1, 46.0, 45.1, 42.0, 38.1, 36.8, 35.2,
28.2, 27.1, 26.8, 24.8, 22.4, 19.2. Anal. (C47H60N2O4‚0.5Et3N‚
H2O) C, H, N.
[
35S]GTP γS Bin d in g Stu d ies To Mea su r e Cou p lin g to
G P r otein s. Membranes from CHO cells stably expressing
either the human κ or µ opioid receptor were used in the
experiments. Cells were scraped from tissue culture plates and
then centrifuged at 1000g for 10 min at 4 °C. The cells were
resuspended in phosphate-buffered saline, pH 7.4, containing
0.04% EDTA. After centrifugation at 1000g for 10 min at 4
°C, the cell pellet was resuspended in membrane buffer, which
consisted of 50 mM Tris-HCl, 3 mM MgCl2, and 1 mM EGTA,
pH 7.4. The membranes were homogenized by with a Dounce
homogenizer, followed by centrifugation at 40000g for 20 min
at 4 °C. The membrane pellet was resuspended in membrane
buffer, and the centrifugation step was repeated. The mem-
branes were then resuspended in assay buffer, which consisted
of 50 mM Tris-HCl, 3 mM MgCl2, 100 mM NaCl, and 0.2 mM
EGTA, pH 7.4. The protein concentration was determined by
the Bradford assay22 using bovine serum albumin as the
standard. The membranes were frozen at -80 °C until use.
CHO cell membranes expressing either the human κ opioid
receptor (15 µg of protein per tube) or µ opioid receptor (7.5
µg of protein per tube) were incubated with 12 different
concentrations of the agonist in assay buffer for 60 min at 30
°C in a final volume of 0.5 mL. The reaction mixture contained
3 µM GDP and 80 pmol of [35S]GTPγS. Basal activity was
determined in the presence of 3 µM GDP and in the absence
of an agonist, and nonspecific binding was determined in the
presence of 10 µM unlabeled GTPγS. Then, the membranes
were filtered onto glass fiber filters by vacuum filtration,
followed by three washes with 3 mL of ice-cold 50 mM Tris-
HCl, pH 7.5. Samples were counted in 2 mL of Ecoscint A
scintillation fluid. Data represent the percent of agonist-
stimulation [35S]GTPγS binding over the basal activity, defined
as [(specific binding/basal binding) × 100] - 100. All experi-
ments were repeated at least three times and were performed
in triplicate. To determine antagonist activity of a compound
at the µ opioid receptors, CHO membranes expressing the µ
opioid receptor were incubated with the compound in the
presence of 200 nM of the µ agonist DAMGO. To determine
antagonist activity of a compound at the κ opioid receptors,
CHO membranes expressing the κ opioid receptor were
incubated with the compound in the presence of 100 nM of
the κ agonist U50,488.
Bis(N-eyclobu tylm eth ylm n or p h in a n -3-yl) ter ep h th a te
20 (MCL-141): pale-yellow foam (52%); 1H NMR (CDCl3, 300
MHz) δ 8.32 (s, 2H), 7.17 (d, J ) 8.1 Hz, 1H), 7.09 (d, J ) 2.1
Hz, 1H), 7.00 (dd, J ) 2.4, 8.1 Hz, 1H), 3.03 (d, J ) 18.6 Hz,
1H), 2.83 (s, 1H), 2.83-1.25 (complex, 26H); 13C NMR (CDCl3,
75 MHz) δ 164.7, 149.4, 142.6, 136.3, 136.0, 134.2, 130.4, 128.9,
118.8, 118.3, 61.7, 56.1, 45.9, 45.1, 42.0, 38.1, 36.8, 35.1, 28.0,
27.0, 26.7, 24.7, 22.4, 19.0. Anal. (C51H62N2O4‚0.5H2O) C,
H, N.
Bis(N-cyclobu tylm eth ylm or p h in a n -3-yl) isop h th a la te
21 (MCL-142): pale-yellow foam (60%); 1H NMR (CDCl3, 300
MHz) δ 9.02 (t, J ) 1.8 Hz, 1H), 8.45 (dd, J ) 1.8, 8.1 Hz, 2H),
7.67 (t, J ) 8.1 Hz, 1H), 7.16 (d, J ) 8.4 Hz, 2H), 7.09 (d, J )
2.1 Hz, 2H), 7.00 (dd, J ) 2.1, 8.4 Hz, 2H), 3.03 (d, J ) 18.6
Hz, 2H), 2.83 (s, 2H), 2.66-1.26 (complex, 24H); 13C NMR
(CDCl3, 75 MHz) δ 164.7, 149.4, 142.6, 136.0, 135.0, 131.9,
130.7, 129.2, 128.9, 118.7, 118.4, 61.7, 56.0, 45.9, 45.1, 42.0,
38.1, 36.8, 35.1, 28.0, 27.0, 26.7, 24.7, 22.3, 19.0. Anal.
(C51H62N2O4‚0.5H2O) C, H, N.
cis-1,2-(N-Cyclobu tylm eth ylm or p h in a n -3-yl)cyclop r o-
p yl Dica r boxyla te 22 (MCL-143). 3-Oxabicyclo[3.1.0]hexane-
2,4-dione (500 mg, 4.46 mmol) was refluxed in water (2 mL)
for 1 h. The solvent was evaporated in vacuo to give cis-1,2-
cyclopropyldicarboxylic acid as a white solid (550 mg, 95%):
mp 135-138 °C [lit.17 139 °C]. The prepared 1,2-cyclopropyl-
dicarboxyl acid (65 mg, 0.5 mmol), morphinan 2 (311 mg, 1
mmol), and a catalytic amount of 4-dimethylaminopyridine
were dissolved in a solution of dichloromethane (5 mL) and
cooled to 0 °C. A solution of N,N′-dicyclohexylcarbodiimide (206
mg, 1 mmol) in dichloromethane (2 mL) was added slowly.
After being stirred for 48 h at room temperature, the reaction
mixture was filtered and evaporated to dryness. Purification
of the crude product by column chromatography gave product
22 as pale-yellow foam (210 mg, 57.5%): 1H NMR (CDCl3, 300
MHz) δ 7.03 (d, J ) 8.4 Hz, 1H), 6.91 (t, J ) 2.1 Hz, 1H), 6.82
(m, 1H), 2.96 (d, J ) 18.6 Hz, 1H), 2.80 (dd, J ) 3.0, 5.4 Hz,
1H), 2.59-1.0 (complex, 26H); 13C NMR (CDCl3, 75 MHz) δ
168.8, 168.7, 149.4, 149.3, 142.3, 135.6, 128,7, 118.6, 118.3,
118.2, 61.7, 56.0, 45.9, 45.1, 41.9, 37.9, 36.7, 35.1, 28.0, 26.9,
26.7, 24.6, 22.4, 22.2, 22.1, 19.0, 12.6. Anal. (C48H62N2O4‚H2O)
C, H, N.
Op ioid Bin d in g to th e Hu m a n µ, δ, a n d K Op ioid
Recep tor s. Chinese hamster ovary (CHO) cells stably trans-
fected with the human κ opioid receptor (hKOR-CHO), δ-opioid
receptor (hDOR-CHO), and the µ-opioid receptor (hMOR-CHO)
were obtained from Drs. Larry Toll (SRI International, Palo
Alto, CA) and George Uhl (NIDA Intramural Program, Be-
thesda, MD), respectively. The cells were grown in 100 mm
dishes in Dulbecco’s modified Eagle’s media (DMEM) supple-
mented with 10% fetal bovine serum (FBS) and penicillin-
streptomycin (10 000 units/mL) at 37 °C in a 5% CO2 atmo-
sphere. The affinity and selectivity of the compounds for the
multiple opioid receptors were determined by incubating the
membranes with radiolabeled ligands and 12 different con-
centrations of the compounds at 25 °C in a final volume of 1
mL of 50 mM Tris-HCl, pH 7.5. Incubation times of 60 min
Ack n ow led gm en t. This work was supported in part
by NIDA Grants K05-DA 00360, U-19-DA 11007, K05-
DA00101, and R01-DA14251. Levorphanol tartrate was
generously donated by Mallinckrodt Inc. We also thank
Prof. Philip Portoghese for useful discussions.
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