Design, synthesis, and biological evaluation of 6α- and 6β-N-heterocyclic substituted naltrexamine derivatives as μ opioid receptor selective Antagonists

Article abstract of DOI:10.1021/jm801272c

Opioid receptor selective antagonists are important pharmacological probes in opioid receptor structural characterization and opioid agonist functional study. Thus far, a nonpeptidyl, highly selective and reversible μ opioid receptor (MOR) antagonist is u

Full text of DOI:10.1021/jm801272c

1416
J. Med. Chem. 2009, 52, 1416–1427
Design, Synthesis, and Biological Evaluation of 6r- and 6ꢀ-N-Heterocyclic Substituted
Naltrexamine Derivatives as µ Opioid Receptor Selective Antagonists
Guo Li,† Lindsey C. Aschenbach,† Jianyang Chen,†,§ Michael P. Cassidy,‡ David L. Stevens,‡ Bichoy H. Gabra,‡
Dana E. Selley,‡ William L. Dewey,‡ Richard B. Westkaemper,† and Yan Zhang†,*
Department of Medicinal Chemistry, Department of Pharmacology and Toxicology, Virginia Commonwealth UniVersity, 410 North 12th Street,
P.O. Box 980540, Richmond, Virginia 23298-0540
ReceiVed October 7, 2008
Opioid receptor selective antagonists are important pharmacological probes in opioid receptor structural
characterization and opioid agonist functional study. Thus far, a nonpeptidyl, highly selective and reversible
µ opioid receptor (MOR) antagonist is unavailable. On the basis of our modeling studies, a series of novel
naltrexamine derivatives have been designed and synthesized. Among them, two compounds were identified
as leads based on the results of in vitro and in vivo assays. Both of them displayed high binding affinity for
the MOR (Ki ) 0.37 and 0.55 nM). Compound 6 (NAP) showed over 700-fold selectivity for the MOR
over the δ receptor (DOR) and more than 150-fold selectivity over the κ receptor (KOR). Compound 9
(NAQ) showed over 200-fold selectivity for the MOR over the DOR and approximately 50-fold selectivity
over the KOR. Thus these two novel ligands will serve as leads to further develop more potent and selective
antagonists for the MOR.
Introduction
receptor (DOR) (Ki values ratios are µ/δ ≈ 152, κ/δ ≈ 276),10
cyprodime only has a moderate selectivity of the MOR over
the DOR and KOR (Ki values ratios are κ/µ ≈ 45, δ/µ ≈ 40).12
Another drawback of cyprodime is that it has much lower
affinity for the MOR than naloxone and naltrexone,11 which
limits its utility. Further structure-activity relationship studies
of cyprodime derivatives did not generate any additional
antagonist with significantly improved affinity or selectivity for
the MOR.13-17 Although ꢀ-funaltrexamine (ꢀ-FNA), clocin-
namox, and others (Figure 2) have been reported as selective
and irreversible nonpeptide antagonists for MOR,18-21 the fact
that they bind covalently with the receptor largely limits their
utility. In most cases, a reversible antagonist would be preferred
because it can inhibit the receptors temporarily for pharmaco-
logical study and then can be washed out from the binding locus
to “revive” the receptors afterward.
Opioid antagonists have played very important roles in the
study of opioid receptors. In fact, the action of an agonist is
characterized as opioid-receptor-mediated only if it is competi-
tively antagonized by an opioid antagonist.1,2 Receptor-selective
opioid antagonists are important tools to identify the receptor
types that mediate the effects of opioid agonists.3 The charac-
terization of the µ opioid receptor (MORa) is essential because
the analgesic function and addiction/abuse liability of many
clinically available opiates are primarily due to their interaction
with the MOR.1,2,4 Thus, MOR selective antagonists are
essential for the study of MOR function in drug abuse and
addiction. In fact, some antagonists with relatively low selectiv-
ity for MOR, e.g., naltrexone, have been shown to inhibit relapse
and curb drug craving in opiate addicts.5-7
On the basis of the “message-address concept”, highly
selective nonpeptide antagonists for the κ opioid receptor (e.g.,
norbinaltorphimine (norBNI) and 5′-guanidinonaltrindole
(GNTI))8,9 and for the δ receptor (e.g., naltrindole (NTI))10
(Figure 1) were designed and synthesized several years ago.
These compounds are widely used as selective ligands in
pharmacological studies.
Most highly selective and reversible µ opioid receptor
antagonists currently available are conformation-constrained
peptides, e.g., CTOP and CTAP.22-28 They are relatively
metabolically stable and have been used to target the MOR in
in vitro and in vivo studies while their limited bioavailability
may not be suitable for many types of in vivo studies and for
medical applications. Optimal utility of antagonists as pharma-
cological tools requires both in vitro and in vivo activity.
Nonpeptide ligands are preferred due to their ability to penetrate
the CNS and lesser vulnerability to metabolic inactivation
compared to the peptide agents. Therefore, the development of
a nonpeptide, potent, selective, and reversible antagonist for the
µ opioid receptor is highly desirable.
Naltrexone is a promising template for the design of the
opioid receptor selective ligands. The successful modification
of naltrexone in the synthesis of NTI, norBNI, and GNTI are
good examples. While naltrexone has nanomolar affinity for
all three opioid receptors, it also shows moderate selectivity
for the MOR over DOR and KOR. Some chemical structure
features are essential for its high affinity for the opioid receptors
and should not be abolished. For example, the addition of a
3-hydroxyl group onto cyprodime and its derivatives will
Thus far, however, no optimal nonpeptide antagonist has been
developed for the MOR, although some moderately potent
ligands, e.g., cyprodime,11 are available. Compared with the high
selectivity of GNTI for the κ opioid receptor (KOR) (Ki values
ratios are µ/κ ≈ 120, δ/κ ≈ 250)9 and NTI for the δ opioid
* To whom correspondence should be addressed. Phone: 804-828-0021.
Fax: 804-828-7625. E-mail: yzhang2@vcu.edu.
†
Department of Medicinal Chemistry, Virginia Commonwealth University.
Department of Pharmacology and Toxicology, Virginia Commonwealth
‡
University.
§
Current address: Zhejiang Jingxin Pharmaceutical Co. Ltd., 800 Eastern
Dadao Road, Xinchang County, Zhejiang Province, China 312500.
a Abbreviations: GPCRs, G-protein coupled receptors; MOR, µ opioid
receptor; DOR, δ opioid rececptor; KOR, κ opioid receptor; norBNI,
norbinaltorphimine; GNTI, 5′-guanidinonaltrindole; NTI, naltrindole; ꢀ-FNA,
ꢀ-funaltrexamine; EL, extracellular loop; NTX, naltrexone; CHO, Chinese
hamster ovarian.
10.1021/jm801272c CCC: $40.75
2009 American Chemical Society
Published on Web 02/06/2009

µ Opioid Receptor SelectiVe Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1417
Figure 1. The κ opioid receptor selective antagonist norBNI, GNTI, and the δ opioid receptor selective antagonist NTI.
sequence alignment (Figure 3). Molecular dynamics simulations
were conducted to optimize the conformation of the models.
The models contain not only the transmembrane helices but also
the extracellular and intracellular domains so that these models
were integrated and complete. The MOR model was also
optimized in a membrane-aqueous system.39 The DOR and
KOR models were also optimized following the same method.
All amino acid residues in these three models have reasonable
bond lengths and bond angles. The analysis of ꢁ, Ψ, ꢂ1, and ꢂ2
angles of the resulting protein conformations was further
conducted with Procheck 4.1 (see Supporting Information).
Because naltrexone is a universal antagonist at all three opioid
receptors with moderate selectivity for the MOR, we decided
to use it as a probe molecule to identify the antagonist binding
site in all three opioid receptors. By comparison of the
differences among these three binding pockets, we attempted
to identify the amino acid residues that are critical to ligand
selectivity for the MOR. Three steps were involved in the
identification of the critical amino acid residues that differentiate
the binding affinity of naltrexone in the three opioid receptors.
The first step was the interactive docking of naltrexone into
the binding locus of the receptor to form the ligand-receptor
complex. The second step was energy minimization and
molecular dynamics carried out for the ligand-receptor system
to relax and optimize binding interactions between the ligand
and amino acid residues in the binding cavity. The third step is
the identification and comparison of the naltrexone binding locus
in all the three receptors. The ligand-receptor complex structure
obtained after 11 ps of molecular dynamics simulation is
depicted in Figure 4. In these complexes, the distance between
the protonated nitrogen atom in the 17-amino group of NTX
and the carboxyl group of Asp147 (D3.32) was initially
anchored at 4.0 Å and retained at this value by a weak harmonic
restraint (2 kcal/Å) during the molecular dynamics simulation
to represent the putative salt bridge that has been inferred from
experimental studies.53 In the lowest energy conformation of
the complex extracted from the last 5 ps molecular dynamics
simulation, the distance (4.1 Å) was compatible with the initial
setting.
As shown in Figure 4A, in the binding pocket of naltrexone,
the entire molecule in the MOR was mainly composed of
aliphatic amino acid residues. The positively charged amino
moiety of the ligand was within the range of an ionic interaction
with Asp147 (D3.32). We also noticed that the carbonyl group
on C(6) of naltrexone was orienting toward an aromatic binding
pocket formed mainly by amino acid residues from the
extracellular loops (ELs) of the receptor, including Tyr210
(Yxl2.43) and Phe221 (Fxl2.54) from EL2 and Trp318 (W7.35)
at the border of EL3 and Helix 7.
In Figure 4B, the naltrexone’s binding pocket in the DOR
was very similar to that in the MOR except that there existed
no aromatic binding locus formed by multiple amino acid
residues to which the C(6) carbonyl group of naltrexone pointed.
At the conserved region, only Phe202 (Fxl2.54) from EL2 was
in the vicinity while Pro191 (Pxl2.43) and Leu300 (L7.35) are
Figure 2. The µ opioid receptor selective antagonists.
“markedly enhance affinity at all three opioid receptors”.13 In
addition, the chemistry related to the structural modification of
naltrexone has been thoroughly studied. This information will
be beneficial to the synthetic route design of naltrexone
derivatives.
In this paper, we report the design, synthesis, and biological
evaluation of two series of novel naltrexone-derived ligands as
selective MOR antagonists. Molecular modeling of the naltr-
exone binding pocket in the homology models of the three
opioid receptors led to the identification of an alternative
“address” domain in the MOR that may enhance selectivity for
the MOR over the DOR and KOR. Two series of ligands were
designed and synthesized as proof-of-concept. Biological evalu-
ation of these two series of compounds revealed some ligands
with high affinity and selectivity for the MOR. On the basis of
these results, two lead compounds have been identified for future
optimization.
Results and Discussion
Molecular Modeling. To facilitate ligand design, homology
models of all three opioid receptors were constructed. To date,
in the whole superfamily of G-protein coupled receptors
(GPCRs),onlytheX-raycrystalstructuresofbovinerhodopsin,29-32
opsin,33 and the human ꢀ2- and ꢀ1-adrenergic receptor34-38
have been successfully obtained with high resolution. Most
molecular models of other GPCRs have been constructed using
the rhodopsin structure as a template. Therefore, homology
models of the µ, δ, and κ opioid receptors were constructed
based on the X-ray crystal structure of bovine rhodopsin after

1418 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Li et al.
Figure 3. The sequence alignment of the MOR, DOR, and KOR with human ꢀ2AR and bovine rhodposin. The Ballesteros-Weinstein numbering
system was adopted to mark all the conserved amino acid residues among most of the GPCRs and colored in red. The extracellular loop 2 (EL2)
was numbered following the assignment proposed by Johnson et al.61 The MOR protein was numbered accordingly above its sequence. The secondary
structure of the MOR receptor 3D conformation based on bovine rhodopsin crystal structure was marked out below all the sequences. The conserved
aspartate residues (D3.32) among all three opioid receptors were marked out in green. The two nonconserved residues xl2.43 and 7.35 were marked
out in blue.
not aromatic ones. This difference might be applicable in the
design of ligands that are selective for the MOR over the DOR.
Further study of the naltrexone binding pocket in the KOR
(Figure 4C) showed that there was an aromatic/aliphatic binding
pocket formed with the contribution of Phe214 (Fxl2.54) from
EL2, Phe231 (F5.37) on helix 5, and Tyr313 (Y7.35) from helix
7. However, only one residue Tyr313 (Y7.35) may form a
hydrogen bond with the ligand, while in the MOR binding locus,
at least two of them are available to be considered.
selectivity of varies ligands for the MOR.40,41,43,48 These reports
are consistent with the observation from our modeling studies.
Molecular Design. On the basis of the molecular modeling
study, two series of ligands were designed as MOR selective
antagonists (Table 1). None of them have been discussed in
the literature as selective opioid receptor ligands.
In the structure of these ligands, we introduced a heteroaro-
matic moiety onto the 6-position of naltrexone. An amide bond
was adopted as the linkage of the side chain moiety to the
morphinan skeleton. Therefore, these ligands can be considered
derivatives of naltrexamine. The configuration of C(6) will be
either R or ꢀ. Such a stereochemical arrangement could play
an important role for the affinity and the selectivity of the ligand,
as has been demonstrated by ꢀ-FNA and R-FNA.49 The aromatic
character of this side chain was designed to have aromatic
stacking interaction with the aromatic binding locus in the MOR
in order to differentiate from the DOR. The nitrogen atom in
the aromatic system will act as a hydrogen bond acceptor to
probe for the potential formation of a hydrogen bond with
Tyr210 or Trp318 in the binding locus from the ELs of the
MOR in order to possibly differentiate from the KOR. Com-
pounds with phenyl and naphthalenyl substitutions were de-
signed as control compounds to test our hypothesis. These two
series of ligands served as proof-of-concept to test the identi-
fication of the alternate “address” domain in MOR.
Chemical Synthesis. For the synthesis of these 6-substituted
derivatives of naltrexamine, the starting material was naltrexone.
The stereoselective synthesis of R- and ꢀ-naltrexamine has been
applied successfully in the synthesis of their derivatives in the
literature.50
In our case, R-naltrexamine was obtained with a yield of 60%
in three steps, while ꢀ-naltrexamine was obtained with a yield
of 63% in three steps. The amide bond formation between the
Therefore, our molecular modeling study of the MOR
antagonist binding pocket using naltrexone as the probe has
revealed an aromatic binding locus at the extracellular loop
region. Further comparison with the DOR and KOR antagonist
binding pockets indicated that the existence of amino acid
residues acting as potential hydrogen bonding donors and/or
acceptors may be a unique structural feature of this aromatic
binding locus in the MOR. Therefore, this binding domain may
serve as an alternate “address” motif in the MOR that contributes
to ligand recognition of the MOR selectively over DOR and
KOR. Molecular design targeting to this “address” domain could
lead to the identification of selective MOR antagonists. To be
noticed, accumulated evidence has shown that the extracellular
loops of GPCRs may play a critical role in the binding pocket
of their small molecule ligands, including a number of opioid
receptor agonists and antagonists.39-46 It has been found that
EL3 of the MOR is critical for the binding of MOR-selective
agonists by comparing their binding affinities for MOR/DOR
and MOR/KOR chimeric receptors with those for the wild-type
MOR, DOR, and KOR.43,44 Site-directed mutagenesis studies
have revealed that certain amino acid residues in EL3 could be
essential for ligand (including agonist and antagonist) selectivity
for the MOR.45-47 More specifically, Trp318 from EL3 has been
identified as an important residue for the binding affinity and

µ Opioid Receptor SelectiVe Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1419
Biological Evaluation. Biological screening for the synthe-
sized ligands was focused on in vitro radioligand binding assay
and functional assays and in vivo behavioral tests. Basically,
the radioligand binding assay was adopted to characterize the
affinity and selectivity of new ligands for the MOR, DOR, and
KOR, whereas the 35S-GTP[γS]-binding functional assay was
applied to determine whether each new ligand acted as an
agonist, partial agonist, or antagonist of the MOR by determining
its efficacy for G-protein activation relative to a full agonist at
the MOR. Agonist efficacy were measured at the level of
G-protein activation because efficacy is most accurately deter-
mined at this proximal level of signal transduction.51-53 The
use of cell lines heterologously expressing each of the cloned
receptors has become standard practice because it provides a
pure source of each opioid receptor type free of other opioid
receptor types. Furthermore, these systems express the receptor
at high density to provide optimal signal-to-noise ratios in the
radioligand and 35S-GTP[γS] binding assays. The in vivo tests
were focused on the inhibition of morphine’s antinociception
activity and behavioral properties of those compounds showing
high selectivity and low agonist efficacy at the MOR.
In Vitro Pharmacological Study. The primary testing of
these ligands included the competitive radioligand binding assay
using the monocloned opioid receptors expressed in CHO cell
lines. [3H]naloxone, [3H]NTI, and [3H]norBNI were used to label
the MOR, DOR, and KOR, respectively. The binding affinities
of these ligands to the µ, δ, and κ opioid receptors are
summarized in Table 2. Most of these ligands showed subna-
nomolar affinity for the MOR and significant selectivity for the
MOR over DOR and KOR. These results demonstrated that our
primary molecular design was successful.
As shown in Table 2, target compounds 1-6 all have
subnanomolar or nanomolar affinity for the MOR while much
lower affinity for the DOR and KOR. Specifically, compound
4 showed over 1000-fold selectivity for the MOR over DOR,
whereas compound 6 showed over 700-fold selectivity for the
MOR over DOR and over 150-fold selectivity for the MOR
over KOR. The control compounds 7 and 8 showed somewhat
lower affinity for MOR and lower selectivity for MOR over
KOR. These results suggest the possibility of hydrogen bonding
or other polar interactions between the target compounds and
the MOR because the only unique chemical structure in the
target compounds is the nitrogen atom in the pyridine ring.
Similarly, target compounds 9-14 all showed high subna-
nomolar affinity for MOR, whereas compound 9 and 11
exhibited the highest selectivity for the MOR over both the DOR
and KOR. Again we observed significantly lower affinity and
selectivity of the control compounds 15 and 16 for the MOR.
This finding further supported the possibility of hydrogen
bonding or some other polar interaction between the target
compounds and the MOR because of the existence of a quinoline
or isoquinoline ring in the target compounds versus the pure
aromatic ring system of the naphthalene moiety in the control
compounds.
Figure 4. Naltrexone (NTX) docked in the homology models in the
MOR, DOR, and KOR. NTX is in ball and stick, and colored as carbon,
red-orange; hydrogen, cyan; oxygen, red and nitrogen, blue; the amino
acid residues are in stick and colored as carbon, grey; oxygen, red and
nitrogen, blue. The receptor homology models are in ribbon. NTX is
in A) MOR, red; B) DOR, cyan; and C) KOR, yellow.
All of these ligands except the controls (which showed lower
affinity for the MOR) were then tested in the 35S-GTP[γS]
binding functional assay using the MOR-expressing CHO cell
line (Table 3). The 35S-GTP[γS] binding results were analyzed
in such a way as to normalize the stimulation produced by each
novel ligand to that obtained with the full agonist DAMGO,
which provided a measurement of relative efficacy. These results
demonstrated that all of the novel ligands showed partial
agonism.
naltrexamine and the side chain moiety was straightforward.
Depending on the commercial availability of the aromatic
moiety, either in acyl chloride or acid form, condition 1 or 2
was adopted. Under mild basic condition, the intermediate, 3,6-
disubstituted naltrexamine, was converted to the 6-monosub-
stituted target compound (Scheme 1) with reasonable yield. All
the ligands were fully characterized before submitting for
biological studies.

1420 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Li et al.
Table 1. Ligands Designed as the µ Opioid Receptor Selective Antagonists
Scheme 1. Synthetic Route for the Target Compounds Designed
Specifically, compounds 6 and 9 showed the lowest relative
efficacy, with approximately 20% of the maximal stimulation
produced by DAMGO. Compounds 6 and 9 produced stimula-
tion similar to nalbuphine, a ligand with very low efficacy to
activate the MOR. As the main goal of this project is to develop
a neutral antagonist of the MOR, their high affinity and
selectivity for the MOR and very low agonism at the MOR
shed light on our future molecular design. To further characterize
their pharmacological profile, we conducted in vivo study of
these compounds.
In Vivo Pharmacological Evaluation. The potential MOR
selective antagonists were evaluated for acute agonistic and
antagonistic effects in mice. In detail, they were tested for their
ability to produce antinociception and to antagonize the anti-
nociceptive effects of morphine in the mouse tail immersion
test. The data are summarized in Table 4. As shown, both lead
compounds, 6 and 9, were found to be potent antagonists of
morphine. Their antagonist AD50 values were 4.51 and 0.45 nM,
and neither of these ligands produced any agonist effect in this
test at doses up to 100 mg/kg. This is in agreement with our
original molecular design hypothesis as well as the in vitro
functional assays. Therefore, these two compounds may be
applied as leads for our next generation of molecular design
and synthesis to identify pure, potent, and highly selective
antagonists for the MOR. In addition, compounds 1 and 2 had
similar ED50 values to compound 9 and compounds 4, 5, and
13 were equally potent to compound 6 as a morphine antagonist.
On the other hand, compound 12 was more potent than
morphine, and compounds 10 and 11 were equally potent to
morphine in producing antinociception in this test.
Interestingly, some of the target compounds did not show
parallel functional activity between the in vitro and the in vivo
studies. For example, both lead compounds 6 and 9 showed
partial agonism in the 35S-GTP[γS] binding assay while acting
as full antagonists in the warm-water tail immersion test. On
the other hand, compounds 10, 11, and 12 showed only
moderately higher partial agonism in the 35S-GTP[γS] binding
assay but acted as full agonists in the in vivo assays. To our
understanding, there are several factors that might have con-
tributed to these observations. First, it has been reported that
the level of antinociception produced by an opioid is dependent
on both the intrinsic efficacy of the drug and the stimulus
intensity. Some low efficacy MOR partial agonists, such as
butorphanol, produced maximal levels of antinociception at a
lower temperature nociceptive stimulus (50 °C) but not at a
higher temperature (56 °C) stimulus.54 On the other hand,

µ Opioid Receptor SelectiVe Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1421
Table 2. The Binding Affinity and Selectivity of C(6) Naltrexamine Derivatives (n ) 3)a
Ki (nM) ( SEM
selectivity ratio
compd
MOR
DOR
KOR
δ/µ
κ/µ
NTX
ꢀ-FNA
CTAP
1
2
3
4
0.26 ( 0.02
0.41 ( 0.04
2.02 ( 0.71
2.65 ( 0.42
5.85 ( 1.41
0.15 ( 0.07
0.14 ( 0.04
0.48 ( 0.12
0.37 ( 0.07
1.41 ( 0.62
0.92 ( 0.30
0.55 ( 0.15
0.10 ( 0.06
0.21 ( 0.11
0.11 ( 0.03
0.12 ( 0.03
0.07 ( 0.02
8.36 ( 1.71
55.6 ( 3.6
117.06 ( 8.94
27.78 ( 4.60
1441.0 ( 106.1
64.46 ( 15.97
215.18 ( 21.02
40.78 ( 8.39
191.98 ( 15.58
186.64 ( 32.07
277.51 ( 7.97
385.84 ( 83.99
478.82 ( 19.94
132.50 ( 27.01
15.42 ( 9.74
148.20 ( 64.80
3.86 ( 1.21
5.15 ( 0.26
0.94 ( 0.05
1012.7 ( 174.8
222.58 ( 11.97
277.96 ( 67.77
77.23 ( 22.43
5.42 ( 1.18
19.56 ( 9.06
60.72 ( 5.58
41.69 ( 6.06
7.79 ( 1.53
450
68
713
24
20
2
501
84
47
501
40
41
163
30
8
48
16
46
47
16
8
37
265
1413
389
747
274
522
241
156
693
36
5
6 (NAP)
7
8
9 (NAQ)
26.45 ( 5.22
1.58 ( 0.68
10
11
12
13
14
15
16
9.84 ( 0.96
5.04 ( 1.30
32.19 ( 1.01
11.61 ( 2.99
518.32 ( 14.06
29.26 ( 4.74
1.81 ( 0.11
277
157
62
0.57 ( 0.20
608.76 ( 15.77
65.26 ( 17.26
73
1
0.5
a The Ki values for the µ, δ, and κ opioid receptors are n ) 3. The averages are reported along with their standard error of the means, SEM, for each
compound. The comparison to percent stimulation of DAMGO is the Emax of the compound compared to the Emax of DAMGO (normalized to 100%).
Naltrexone, ꢀ-FNA, and CTAP were tested along as positive controls under the same conditions.
Table 3. The Efficacy and Potency of Target Compounds in
35S-GTP[γS]-Binding Functional Assay in the MOR Expressing CHO
Cells (n ) 3)
or KOR while acting as low efficacy partial agonists at the
MOR, which might explain why compounds 10, 11, and 12
acted as full agonists in vivo. The intrinsic efficacy of these
novel ligands at the DOR and KOR are currently under study
to address this hypothesis.
compd
EC50 (nM)
Emax (% stim)
% max of DAMGO
DAMGO
1
2
3
4
5
6 (NAP)
9 (NAQ)
10
11
12
13
14
45.06 ( 6.63
23.90 ( 4.66
26.28 ( 8.05
1.01 ( 0.40
0.33 ( 0.14
1.26 ( 0.67
1.14 ( 0.38
4.36 ( 0.72
0.27 ( 0.06
0.09 ( 0.04
0.69 ( 0.19
2.29 ( 0.72
0.29 ( 0.02
366.5 ( 23.0
133.5 ( 9.8
150.6 ( 15.8
164.3 ( 16.5
106.7 ( 18.3
136.8 ( 17.8
83.3 ( 3.1
58.00 ( 9.30
120.9 ( 9.0
239.6 ( 22.5
149.8 ( 26.1
164.3 ( 14.5
195.8 ( 32.0
100.0 ( 6.2
37.79 ( 2.68
41.09 ( 4.32
44.82 ( 4.50
29.11 ( 5.00
37.32 ( 4.87
22.72 ( 0.84
15.83 ( 2.53
32.99 ( 2.46
65.38 ( 6.13
40.87 ( 7.20
44.83 ( 3.96
53.41 ( 8.74
Further Molecular Modeling Study. To verify that the two
lead compounds that acted as selective MOR ligands utilized
the alternate “address” domain identified from the previous
modeling study, further molecular modeling study was con-
ducted. First, compound 6 and 9 were built using the InsightII/
Discover program and their conformation energy minimization
was conducted. Then, as we have described for the docking
study of naltrexone in the three opioid receptor homology
models, they were docked into the homology model of MOR
interactively. The orientation of the newly introduced C(6) side
chain was not deliberately considered originally. The lowest
energy conformation after the minimization and the dynamics
simulation of the ligand/receptor complex is illustrated in
Figure 5.
Table 4. AD50 Values for Naloxone and the Two Series of
C6-Naltrexamine Derivatives vs Morphine in the Warm-Water Tail
Immersion Test in Vivo
AD50 value (mg/kg (95% CL))
As shown in Figure 5A, the C(6) side chain in lead compound
6 pointed to the aromatic binding locus at the extracellular loop
region of the MOR. The pyridinyl moiety was in the vicinity
of Tyr210 (Yxl2.43) while the distance between the nitrogen
atom in the pyridine ring and the oxygen atom in the hydroxyl
group of Tyr210 (Yxl2.43) was 3.37 Å. Similarly, the C(6) side
chain in lead compound 9 also pointed to the aromatic binding
locus at the extracellular loop region of the MOR while the
isoquinolinyl moiety in 9 was in the vicinity of Trp318 (W7.35)
(Figure 5B). The distance between the nitrogen atom in the
isoquinoline ring and the nitrogen atom in the indole ring of
Trp318 (W7.35) was 3.64 Å. Both distances could be plausible
for hydrogen bonding interaction between the C(6) side chains
in the ligands and the specified amino acid residue.
compd
for blockade of morphine
antinociception
naloxone
0.05 (0.03-0.09)
0.89 (0.75-1.07)
0.33 (0.26-0.43)
36.77 (29.99-44.98)
1.38 (0.78-2.43)
8.65 (5.35-13.97)
4.51 (2.45-8.26)
0.45 (0.27-0.78)
inactive
inactive
inactive
4.45 (2.41-8.15)
42.55 (23.67-76.51)
1
2
3
4
5
6 (NAP)
9 (NAQ)
10a
11b
12c
13
14
a Agonist, ED50 1.19 mg/kg (morphine ED50 2.59 mg/kg). b Agonist, ED50
4.57 mg/kg. c Agonist, ED50 0.04 mg/kg.
Because these two residues (Tyr210 (Yxl2.43) and Trp318
(W7.35)) are not conserved in the DOR and KOR, it seems
that these two residues could act as an alternate “address”
domain in MOR, and this plausible hydrogen bonding could
contribute to the selectivity of lead compounds 6 and 9 for the
MOR. As our molecular models are based on homology
modeling of these opioid receptors and are preliminary, site-
directed mutagenesis, and radioligand binding analysis with the
butorphanol acted as an antagonist and shifted the dose-effect
curve of the high-efficacy opioid alfentanil to the right in a
competitive manner at a higher temperature (55 °C) stimulus.55
This may explain why the two lead compounds did not show
any efficacy at the higher temperature (56 °C) stimulus and
thereby acted as full antagonists in the in vivo study. Second,
some ligands could have significant intrinsic efficacy at the DOR

1422 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Li et al.
activation mechanisms and on structural parameters that affect
ligand efficacy at the MOR.
Experimental Section
Molecular Modeling. A Silicon Graphics Octane 2 workstation,
equipped with two parallel R12000 processors, was used for all
computational studies. InsightII (Accelrys)57 package was used for
modeling. InsightII/Homology module was used to construct the
homology models of three opioid receptors based on the X-ray
crystal structure of bovine rhodopsin, as reported previously.39
InsightII/Discover module was applied to construct all the small
molecules in their nitrogen-protonated form. Minimization with the
steepest descent and then the conjugate gradient algorithm were
performed to generate the lowest energy conformation for each
ligand studied. Then a molecular dynamics simulation was per-
formed (an equilibration phase of 1000 fs at 300 K, followed by a
collection phase of 5000 fs at the same temperature) to further study
the small molecule conformation. The lowest energy conformation
of the molecule from the last 2 ps molecular dynamics simulation
was extracted and applied as the initial configuration for docking
into the proposed binding site of the opioid receptors. The docking
of the small molecule was conducted interactively using InsightII/
Discover. Experimental studies47 suggest that the protonated
nitrogen moiety interacts with the carboxyl group of Asp 147 to
form a putative salt bridge. In detail, the molecule was docked in
the upper level of transmembrane part in each receptor. The
orientation of the molecule skeleton in the binding locus was mainly
decided by: first, the putative ionic interaction between the tertiary
amino group in naltrexone and the carboxylic group of aspartate
on the transmembrane helix 3 in each opioid receptor (Asp147 in
µ, Asp128 in δ, and Asp138 in κ); second, the hydrophobic portion
of the ligand intend to face the hydrophobic transmembrane helices
while the hydrophilic portion to the more polar extracellular loop
region. The ligand-receptor complex was minimized in gas phase
first with the backbone of the receptor fixed, but all the side chain
atoms were left unconstrained. The optimized conformation was
then used as the initial configuration for the molecular dynamics
simulations. A short-term steepest descent energy minimization
(5000 iterations) and dynamics simulation (10000 step, 1 fs each
step) was conducted to validate the docking primarily followed by
a more vigorous minimizations (50000 iterations) and dynamics
simulation (100000 steps) was conducted with 2000 steps equilibra-
tion for the initial dynamics. The total simulation time was 102 ps.
In both processes, the backbone of the receptor was fixed to prevent
the disruption of the R-helical bundle of the receptor and a generic
distance constraint (4-4.2 Å) was applied between the negatively
charged oxygen atom in aspartate on TM3 and the positively
charged nitrogen atom in the ligand. After the dynamics simulation,
the lowest energy conformation of the complex was extracted and
saved for analysis.
Chemical Synthesis. General Methods. All reagents were
purchased from Sigma-Aldrich or as otherwise states. Melting points
were obtained with a Fisher Scientific micro melting point apparatus
and were uncorrected. All IR spectra were recorded on a Nicolet
Avatar 360 FT-IR instrument. Proton (300 MHz) and Carbon-13
(75 MHz) nuclear magnetic resonance (NMR) spectra were recorded
at ambient temperature with tetramethylsilane as the internal
standard on either a Varian Gemini-300 MHz “Tesla” spectrometer
or Varian Mercury-300 MHz NMR spectrometer. GC/MS analysis
was performed on a Hewlett-Packard 6890 (Palo Alto, CA). TLC
analyses were carried out on the Analtech Uniplate F254 plates.
Chromatographic purification was carried out on silica gel columns
(230-400 mesh, Merck). Yields were not maximized. The final
target compounds’ purity was tested by HPLC and elemental
analysis, and a satisfying purity of >95% was achieved from both
methods. Varian ProStar HPLC System was used on Microsorb-
MV 100-5 C18 column (250 mm × 4.6 mm) with injection volume
at 10 µL and sample concentrations at 1-2 mg/0.5 mL in 100%
acetonitrile; the sample was detected at single wavelength of 210
nm with eluent system of acetonitrile:water (75:25) at 1 mL/min
over 50 min. Elemental analysis was conducted in Atlantic
Figure 5. The docking of lead compound 6 and 9 in the µ opioid
receptor model. The ligands are in ball and stick, and colored as carbon,
red-orange; hydrogen, cyan; oxygen, red and nitrogen, blue; the amino
acid residues are in stick and colored as carbon, grey; oxygen, red and
nitrogen, blue. The receptor homology model is in red.
mutated MOR will be conducted in future studies to confirm
this hypothesis.56
Conclusions
In summary, on the basis of the molecular modeling study
of the opioid receptor antagonist binding pocket using naltrexone
as a probe molecule, an alternative “address” binding domain
has been identified in the MOR antagonist binding pocket. Two
series of novel ligands have been designed and synthesized to
target on this “address” domain as proof-of-concept. Competi-
tion binding and in vitro functional assays have identified two
lead compounds with subnanomolar affinity for the MOR and
high selectivity over the DOR and KOR. Both lead compounds
showed partial agonism in the in vitro G-protein activation test
and potent antagonism in the in vivo antinociceptive test. Further
molecular modeling study has implicated that the selectivity of
these two ligands for the MOR could be the result of potential
hydrogen bonding between the ligand and the “address” binding
locus in the MOR. All of these results will inform our future
ligand design in the pursuit of highly selective and pure
antagonists of the MOR. Moreover, we have also observed that
some of the compounds in these series showed a range of
efficacies as MOR partial agonists. These ligands would serve
as pharmacological tools to obtain information on MOR

µ Opioid Receptor SelectiVe Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1423
Microlab, Inc. All spectral data reported here were obtained from
the hydrochloride salt form of the products while compounds 1-6
and 9-14 were dihydrochloride salts, and compounds 7, 8, 15, and
16 monohydrochloride salts.
17-Cyclopropylmethyl-3,14ꢀ-dihydroxy-4,5r-epoxy-6r-[(3′-
pyridyl)acetamido]morphinan (3). Compound 3 was prepared by
following the general procedure 1 in 54% yield; [R]25 -273° (c
D
) 0.06, MeOH); mp 211-214 °C. IR (KBr, cm-1) νmax: 3215, 1672,
1
1531, 1507, 1322. H NMR (300 MHz, DMSO): δ 9.23 (m, 1 H,
General Procedure 1. A solution of 6R-naltrexamine or 6ꢀ-
naltrexamine (1 equiv) in CH2Cl2 was added acyl chloride (2 equiv)
and triethylamine (4 equiv) on an ice-water bath under N2
protection. The mixture was allowed to stir overnight at room
temperature. After concentrated to remove CH2Cl2, the resulting
residue was dissolved in MeOH and potassium carbonate (2 equiv)
added. The reaction mixture was stirred overnight at room tem-
perature. After concentration, the residue was partitioned between
water and CH2Cl2. The water layer was extracted with CH2Cl2. The
combined CH2Cl2 solution was washed with brine and dried over
Na2SO4. After concentration, the residue was purified by silica gel
column with a CH2Cl2/MeOH (100:1) (1% NH3H2O) solvent system
as eluent to give the aim product. The product was then transferred
into the hydrochloride salt using 1.25 M hydrochloride acid
methanol solution at 0 °C.
General Procedure 2. A solution of carboxylic acid (3 equiv)
in DMF was added N-(3-dimethylaminopropyl)-N′-ethylcarbodi-
imide hydrochloride (EDCI, 2.5 equiv), hydrobenzotriazole (HOBt,
2.5 equiv), 4 Å molecular sieve, and triethylamine (5 equiv) on an
ice-water bath under N2 protection. After 15 min, a solution of
6ꢀ-naltrexamine (1 equiv) in DMF was added. The reaction mixture
was filtered over celite after stirring overnight at room temperature.
The filtrate was concentrated in vacuum to remove DMF. The
residue was dissolved in MeOH and added potassium carbonate (2
equiv). The resulting mixture was stirred overnight at room
temperature. After concentration, the residue was partitioned
between water and CH2Cl2. The water layer was extracted with
CH2Cl2. The combined CH2Cl2 solution was washed with H2O,
brine, and dried with Na2SO4. After concentration, the residue was
purified by silica gel column with a CH2Cl2/MeOH (100:1) (1%
NH3H2O) solvent system as eluent to give the aim product. Then
the product was transferred into a hydrochloride salt.
Ar-H), 8.94 (b, 1 H, exchangeable), 8.92 (s, 1 H, amide-H), 8.75
(d, J ) 5.1.Hz, 1 H, Ar-H), 8.66 (d, J ) 7.5 Hz, 1 H, Ar-H),
7.89 (dd, J ) 5.1, 7.5 Hz, 1 H, Ar-H), 6.73 and 6.58 (2 d, 1 H
each, J ) 8.1 Hz, C1-H, C2-H), 4.76 (m, 1 H, C5-H), 4.63 (m,
1 H, C6-H), 3.97 (m, 1 H), 3.43 (m, 2 H), 3.05 (m, 3 H), 2.71 (m,
1 H), 2.45 (m, 2 H), 1.95 (m, 1 H), 1.63 (m, 1 H), 1.53 (m, 1 H),
1.20 (m, 1 H), 1.06 (m, 1 H), 0.64 (m, 2 H), 0.45 (m, 2 H). 13C
NMR (75 MHz, DMSO) δ: 165.25, 151.34, 147.83, 145.94, 138.43,
136.20, 131.16, 130.84, 125.51, 123.82, 119.56, 117.96, 89.49,
69.88, 62.38, 59.89, 47.28, 45.95, 43.45, 33.71, 29.32, 23.08, 21.38,
9.62, 4.20, 4.06. MS (ESI) m/z: 448.9 (M + H)+. Anal.
(C26H29N3O4 ·2HCl·3H2O) C, H.
17-Cyclopropylmethyl-3,14ꢀ-dihydroxy-4,5r-epoxy-6ꢀ-[(3′-
pyridyl)acetamido]morphinan (4). Compound 4 was prepared by
following the general procedure 1 in 56% yield; [R]25 -141° (c
D
) 0.10, MeOH). mp 225-227 °C. IR (KBr, cm-1) νmax: 3207, 3057,
1
1665, 1540, 1326. H NMR (300 MHz, DMSO): δ 9.18 (m, 1 H,
Ar-H), 9.15 (b, 1 H, amide-H), 8.90 (b, 1 H, exchangeable), 8.86
(m, 1 H, Ar-H), 8.51 (m, 1 H, Ar-H), 7.77 (m, 1 H, Ar-H), 6.75
and 6.68 (2 d, 1 H each, J ) 8.4 Hz, C1-H, C2-H), 4.85 (d, J )
8.4 Hz, 1 H, C5-H), 4.47 (s, 1 H, C6-H), 3.89 (m, 1 H), 3.73 (m,
1 H), 3.38 (m, 1 H), 3.12 (m, 2 H), 2.85 (m, 1 H), 2.45 (m, 2 H),
1.93 (m, 1 H), 1.80 (m, 1 H), 1.64 (m, 1 H), 1.47 (m, 2 H), 1.09
(m, 1 H), 0.64 (m, 2 H), 0.46 (m, 2 H). 13C NMR (75 MHz, DMSO)
δ: 161.34, 147.63, 144.05, 139.39, 136.30, 131.90, 126.87, 126.46,
120.29, 119.68, 115.32, 114.28, 88.05, 66.55, 58.39, 55.48, 47.27,
41.88, 40.16, 27.77, 25.51, 19.75, 18.82, 6.98, 5.63, 3.26. MS (ESI)
m/z: 448.9 (M + H)+. Anal. (C26H29N3O4 ·2HCl·3H2O) C, H.
17-Cyclopropylmethyl-3,14ꢀ-dihydroxy-4,5r-epoxy-6r-[(4′-
pyridyl)acetamido]morphinan (5). Compound 5 was prepared by
following the general procedure 1 in 45% yield; [R]25 -213° (c
D
) 0.09, MeOH); mp 215-217 °C. IR (KBr, cm-1) νmax: 3225, 1653,
17-Cyclopropylmethyl-3,14ꢀ-dihydroxy-4,5r-epoxy-6r-[(2′-
pyridyl)acetamido]morphinan (1). Compound 1 was prepared by
1
1542, 1500, 1318. H NMR (300 MHz, DMSO): δ 8.92 (m, 2 H,
Ar-H), 8.88 (b, 1 H, exchangeable), 8.77 (b, 1 H, amide-H), 8.08
(m, 2 H, Ar-H), 6.73 and 6.59 (2 d, 1 H each, J ) 8.1 Hz, C1-H,
C2-H), 4.78 (m, 1 H, C5-H), 4.62 (m, 1 H, C6-H), 3.95 (m, 1
H), 3.40 (m, 2 H), 3.06 (m, 2 H), 2.74 (m, 1 H), 2.46 (m, 2 H),
1.91 (m, 1 H), 1.65 (m, 1 H), 1.52 (m, 1 H), 1.18 (m, 2 H), 1.09
(m, 1 H), 0.69 (m, 2 H), 0.49 (m, 2 H). 13C NMR (75 MHz, DMSO)
δ: 164.17, 149.72, 149.72, 145.15, 142.08, 137.77, 130.25, 124.71,
121.12, 121.12, 119.07, 117.28, 88.82, 69.06, 61.74, 59.26, 46.81,
46.54, 45.27, 42.58, 33.29, 28.70, 22.43, 8.17, 7.55, 3.56. MS (ESI)
m/z: 448.9 (M + H)+. Anal. (C26H29N3O4 ·2HCl·3H2O) C, H.
17-Cyclopropylmethyl-3,14ꢀ-dihydroxy-4,5r-epoxy-6ꢀ-[(4′-
pyridyl)acetamido]morphinan (6, NAP). Compound 6 was pre-
following the general procedure 1 in 58% yield; [R]25 -244° (c
D
) 0.05, MeOH); mp 212-214 °C. IR (KBr, cm-1) νmax: 3225, 1675,
1521, 1320. 1H NMR (300 MHz, DMSO): δ 8.90 (b, 1 H,
exchangeable), 8.71 (b, 1 H, amide-H), 8.39 (m, 1 H, Ar-H), 8.09
(m, 2 H, Ar-H), 7.68 (m, 1 H, Ar-H), 6.77 and 6.62 (2 d, 1 H
each, J ) 8.1 Hz, C1-H, C2-H), 4.77 (m, 1 H, C6-H), 4.67 (m,
1 H, C5-H), 3.12 (d, J ) 6.3 Hz, 1 H), 3.05 (d, J ) 18.6 Hz, 1 H),
2.67 (m, 1 H), 2.63 (m, 1 H), 2.57 (m, 1 H), 2.35 (m, 1 H), 2.27
(m, 1 H), 2.17 (m, 2 H), 1.84 (m, 1 H), 1.74 (m, 1 H), 1.49 (m, 1
H), 1.14 (m, 1 H), 0.86 (m, 1 H), 0.54 (m, 2 H), 0.12 (m, 2 H). 13
C
NMR (75 MHz, DMSO) δ: 163.88, 150.16, 148.34, 145.74, 137.79,
137.61, 130.10, 126.35, 125.99, 122.69, 119.41, 117.61, 90.45,
69.85, 62.36, 59.91, 47.49, 46.49, 43.39, 33.83, 29.45, 23.11, 21.17,
9.60, 4.21, 4.06. MS (ESI) m/z: 447.7 (M+). Anal. (C26H29N3O4 ·
2HCl·1.5H2O) C, H.
pared by following the general procedure 1 in 45% yield; [R]25
D
-176° (c ) 0.01, MeOH); mp 258-61 °C. IR (KBr, cm-1) νmax
:
1
3386, 1666, 1548, 1502, 1326. H NMR (300 MHz, DMSO): δ
8.81 (b, 1 H, amide-H), 8.45 (m, 2 H, Ar-H), 8.22 (b, 1 H,
exchangeable), 7.60 (m, 2 H, Ar-H), 6.32 and 6.27 (2 d, 1 H each,
J ) 7.8 Hz, C1-H, C2-H), 4.84 (s, 1 H, C5-H), 4.46 (m, 1 H,
C6-H), 3.90 (m, 1 H), 3.69 (m, 1 H), 3.30 (m, 2 H), 3.06 (m, 2
H), 2.85 (m, 1 H), 2.45 (m, 2 H), 1.93 (m, 1 H), 1.80 (m, 1 H),
1.59 (m, 1 H), 1.46 (m, 1 H), 1.07 (m, 1 H), 0.63 (m, 2 H), 0.45
(m, 2 H). 13C NMR (75 MHz, DMSO) δ: 164.30, 149.27, 142.72,
139.62, 130.15, 128.61, 127.80, 124.87, 123.73, 120.89, 118.86,
117.84, 91.15, 69.90, 61.75, 58.84, 50.68, 46.91, 43.50, 39.98,
31.10, 28.60, 22.18, 8.99, 3.68, 3.37. MS (ESI) m/z: 448.9 (M +
H)+. Anal. (C26H29N3O4 ·2HCl·3H2O) C, H.
17-Cyclopropylmethyl-3,14ꢀ-dihydroxy-4,5r-epoxy-6ꢀ-[(2′-
pyridyl)acetamido]morphinan (2). Compound 2 was prepared by
following the general procedure 1 in 65% yield; [R]25D -91° (c )
0.07, MeOH); mp 210-212 °C. IR (KBr, cm-1) νmax: 3384 1673,
1526, 1324. 1H NMR (300 MHz, DMSO): δ 9.07 (b, 1 H, amide-
H), 8.86 (b, 1 H, exchangeable), 8.69 (m, 1 H, Ar-H), 8.03 (m, 2
H, Ar-H), 7.65 (m, 1 H, Ar-H), 6.73 and 6.68 (2 d, 1 H each, J
) 8.1 Hz, C1-H, C2-H), 5.02 (m, 1 H, C6-H), 4.62 (m, 1 H,
C5-H), 3.10 (d, J ) 6.3 Hz, 1 H), 3.03 (d, J ) 18.9 Hz, 1 H), 2.65
(m, 1 H), 2.63 (m, 1 H), 2.58 (m, 1 H), 2.37 (m, 2 H), 2.25 (m, 1
H), 2.19 (m, 1 H), 1.95 (m, 1 H), 1.82 (m, 1 H), 1.61 (m, 1 H),
1.45 (m, 1 H), 0.84 (m, 1 H), 0.54 (m, 2 H), 0.12 (m, 2 H). 13C
NMR (75 MHz, DMSO) δ: 160.27, 145.86, 144.30, 138.97, 136.63,
133.54, 127.19, 122.39, 120.28, 118.59, 115.29, 114.35, 89.11,
66.42, 58.54, 55.44, 47.84, 43.91, 41.96, 40.30, 27.00, 26.22, 20.59,
18.88, 6.82, 5.68. MS (ESI) m/z: 448.1 (M + H)+. Anal.
(C26H29N3O4 ·2HCl·3H2O) C, H.
17-Cyclopropylmethyl-3,14ꢀ-dihydroxy-4,5r-epoxy-6r-(ben-
zamido)morphinan (7). Compound 7 was prepared by following
the general procedure 1 in 54% yield; [R]25 -215° (c ) 0.11,
D
MeOH); mp 182-185 °C. IR (KBr, cm-1) νmax: 3353, 2947, 1638,
1540, 1324. 1H NMR (300 MHz, DMSO): δ 7.75 (m, 2 H, Ar-H),
7.45 (m, 3 H, Ar-H), 6.70 and 6.65 (2 d, 1 H each, J ) 8.4 Hz,
C1-H, C2-H), 6.50 (b, 1 H, amide-H), 4.79 (m, 1 H, C6-H), 4.77

1424 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Li et al.
(m, 1 H, C5-H), 3.14 (m, 1 H), 3.04 (m, 1 H), 2.68 (m, 1 H), 2.65
(m, 1 H), 2.60 (m, 1 H), 2.36 (m, 1 H), 2.29 (m, 1 H), 2.27 (m, 2
H), 1.84 (m, 1 H), 1.58 (m, 1 H), 1.42 (m, 1 H), 1.25 (m, 1 H),
0.86 (m, 1 H), 0.54 (m, 2 H), 0.12 (m, 2 H). 13C NMR (75 MHz,
DMSO) δ: 167.72, 145.85, 138.14, 134.53, 131.57, 131.11, 128.60,
127.47, 125.63, 119.38, 117.86, 90.12, 69.91, 62.32, 59.87, 47.38,
46.98, 43.39, 33.69, 29.45, 23.11, 21.17, 9.56, 4.24, 4.08. MS (ESI)
m/z: 447.9 (M + H)+. Anal. (C27H30N2O4 ·HCl·2.75H2O) C, H.
17-Cyclopropylmethyl-3,14ꢀ-dihydroxy-4,5r-epoxy-6ꢀ-(ben-
zamido)morphinan (8). Compound 8 was prepared by following
(m, 2 H). 13C NMR (75 MHz, DMSO) δ: 160.77, 146.72, 146.08,
145.15, 141.05, 139.27, 134.49, 134.49, 130.24, 130.24, 128.88,
123.95, 122.35, 120.10, 119.33, 118.51, 87.79, 69.88, 62.24, 57.95,
47.18, 46.06, 30.51, 29.42, 23.91, 19.68, 19.68, 5.79, 5.22, 2.31.
MS (ESI) m/z: 498.1 (M + H)+. Anal. (C30H31N3O4 ·2HCl·2.5H2O)
C, H.
17-Cyclopropylmethyl-3,14ꢀ-dihydroxy-4,5r-epoxy-6ꢀ-[(2′-
quinolyl)acetamido]morphinan (12). Compound 12 was prepared
by following the general procedure 1 in 83% yield; [R]25 -112°
D
(c ) 0.1, MeOH); mp 227-229 °C. IR (KBr, cm-1) νmax: 3110,
1
the general procedure 1 in 59% yield; [R]25 -157° (c ) 0.07,
1671, 1533, 1329, 770. H NMR (300 MHz, DMSO): δ 9.26 (m,
D
MeOH); mp 220-221 °C. IR (KBr, cm-1) νmax: 3242, 1638, 1540,
1324. 1H NMR (300 MHz, DMSO): δ 7.84 (m, 2 H, Ar-H),
7.50-7.40 (m, 3 H, Ar-H), 7.21 (b, 1H, amide-H), 6.75 and 6.58
(2 d, 1 H each, J ) 8.1 Hz, C1-H, C2-H), 4.52 (m, 1 H, C5-H),
4.26 (m, 1 H, C6-H), 3.87 (m, 1 H), 3.73 (m, 1 H), 3.15 (m, 1 H),
2.69 (m, 1 H), 2.61 (m, 1 H), 2.40 (m, 1 H), 2.23 (m, 2 H), 1.87
(m, 1 H), 1.72 (m, 1 H), 1.55 (m, 1 H), 1.26 (m, 1 H), 1.12 (m, 1
H), 0.86 (m, 1 H), 0.54 (m, 2 H), 0.12 (m, 2 H). 13C NMR (75
MHz, DMSO) δ: 168.79, 142.59, 140.78, 134.52, 131.55, 131.18,
128.38, 127.23, 123.87, 119.04, 117.62, 91.82, 70.59, 62.61, 58.92,
52.14, 44.49, 30.45, 30.11, 29.66, 24.35, 22.52, 8.76, 3.61, 3.05.
MS (ESI) m/z: 447.9 (M + H)+. Anal. (C27H30N2O4 ·HCl·3.25H2O)
C, H.
1 H, Ar-H), 8.94 (b, 1 H, amide-H), 8.61 (m, 1 H, Ar-H), 8.16
(m, 2 H, Ar-H), 8.12 (b, 1 H, exchangeable), 7.92 (m, 1 H, Ar-H),
7.78 (m, 1 H, Ar-H), 6.79 and 6.68 (2 d, 1 H each, J ) 7.8 Hz,
C1-H, C2-H), 5.15 (s, 1 H, C5-H), 5.12 (m, 1 H, C6-H), 3.93
(m, 1 H), 3.77 (m, 1 H), 3.43 (m, 1 H), 3.10 (m, 2 H), 2.90 (m, 1
H), 2.45 (m, 2 H), 2.09 (m, 1 H), 1.83 (m, 1 H), 1.62 (m, 1 H),
1.48 (m, 1 H), 1.08 (m, 1 H), 0.86 (m, 1 H), 0.65 (m, 2 H), 0.50
(m, 2 H). 13C NMR (75 MHz, DMSO) δ: 163.98, 149.09, 145.84,
141.98, 139.75, 136.87, 130.48, 129.60, 129.12, 128.72, 127.40,
127.26, 123.60, 118.64, 118.44, 117.41, 93.02, 69.83, 61.83, 58.76,
51.23, 47.33, 43.66, 30.11, 29.71, 24.20, 22.20, 8.99,3.62, 3.36.
MS (ESI) m/z: 497.8 (M + H)+. Anal. (C30H31N3O4 ·2HCl·0.5H2O)
C, H.
17-Cyclopropylmethyl-3,14ꢀ-dihydroxy-4,5r-epoxy-6r-[(3′-
17-Cyclopropylmethyl-3,14ꢀ-dihydroxy-4,5r-epoxy-6r-[(3′-
isoquinolyl)acetamido]morphinan (9, NAQ). Compound 9 was
quinolyl)acetamido]morphinan (13). Compound 13 was prepared
prepared by following the general procedure 2 in 70% yield; [R]25
by following the general procedure 2 in 61% yield; [R]25 -192°
D
D
-150° (c ) 0.01, MeOH); mp: 210-213 °C. IR (KBr, cm-1) νmax
:
(c ) 0.05, MeOH); mp > 270 °C. IR (KBr, cm-1) νmax: 3221, 1660,
3222, 1666, 1529, 1261, 801. 1H NMR (300 MHz, DMSO): δ 9.44
(s, 1 H, Ar-H), 8.95 (b, 1 H, exchangeable), 8.64 (s, 1 H, Ar-H),
8.58 (b, 1 H, amide-H), 8.27 (m, 2 H, Ar-H), 7.90 (m, 2 H, Ar-H),
6.79 and 6.62 (2 d, 1 H each, J ) 7.8 Hz, C1-H, C2-H), 4.81 (s,
1 H, C5-H), 4.74 (m, 1 H, C6-H), 3.99 (m, 1 H), 3.45 (m, 2 H),
3.14 (m, 2 H), 2.73 (m, 1 H), 2.58 (m, 1 H), 2.23 (m, 2 H), 1.87
(m, 1 H),1.67 (m, 2 H), 1.48 (m, 1 H), 1.08 (m, 1 H), 0.67 (m, 2
H), 0.47 (m, 2 H). 13C NMR (75 MHz, DMSO) δ: 159.97, 148.87,
146.15, 139.18, 138.21, 137.40, 134.48, 132.47, 131.01, 128.93,
128.86, 128.37, 124.16, 122.44, 120.07, 118.39, 87.91, 69.86, 62.26,
57.93, 47.15, 46.05, 30.51, 29.43, 23.88, 19.57, 19.56, 5.75, 5.18,
2.26. MS (ESI) m/z: 498.1 (M + H)+. Anal. (C30H31N3O4 ·
2HCl·0.5H2O) C, H.
1537, 1318, 777. H NMR (300 MHz, DMSO): δ 9.39 (s, 1 H,
1
Ar-H), 9.06 (s, 1 H, Ar-H), 8.90 (b, 1 H, amide-H), 8.66 (b, 1 H,
exchangeable), 8.19 (m, 2 H, Ar-H), 7.97 (m, 1 H, Ar-H), 7.79
(m, 1 H, Ar-H), 6.73 and 6.59 (2 d, 1 H each, J ) 8.1 Hz, C1-H,
C2-H), 4.83 (m, 1 H, C5-H), 4.70 (m, 1 H, C6-H), 3.95 (m, 1
H), 3.45 (m, 2 H), 3.08 (m, 3 H), 2.72 (m, 1 H), 2.52 (m, 2 H),
1.92 (m, 1 H), 1.65 (m, 1 H), 1.51 (m, 1 H), 1.26 (m, 1 H), 1.10
(m, 1 H), 0.68 (m, 2 H), 0.45 (m, 2 H). 13C NMR (75 MHz, DMSO)
δ: 164.04, 147.59, 146.84, 144.48, 141.03, 139.52, 134.19, 130.41,
129.48, 128.03, 127.76, 125.83, 122.91, 119.93, 119.24, 116.95,
87.73, 70.15, 61.65, 57.70, 47.08, 45.99, 39.40, 30.92, 29.83, 24.30,
20.10, 6.50, 5.99, 3.34. MS (ESI) m/z: 498.9 (M + H)+. Anal.
(C30H31N3O4 ·2HCl·3.75H2O) C, H.
17-Cyclopropylmethyl-3,14ꢀ-dihydroxy-4,5r-epoxy-6ꢀ-[(3′-
isoquinolyl)acetamido]morphinan (10). Compound 10 was pre-
17-Cyclopropylmethyl-3,14ꢀ-dihydroxy-4,5r-epoxy-6ꢀ-[(3′-
quinolyl)acetamido]morphinan (14). Compound 14 was prepared
by following the general procedure 2 in 87% yield; [R]25D -86° (c
) 0.07, MeOH); Mp 235-238 °C. IR (KBr, cm-1) νmax: 3072, 1660,
pared by following the general procedure 2 in 50% yield; [R]25
D
-166° (c ) 0.10, MeOH); mp 235-237 °C. IR (KBr, cm-1) νmax
:
3069, 1665, 1537, 1328, 901. 1H NMR (300 MHz, DMSO): δ 9.46
(s, 1H, Ar-H), 9.27 (b, 1 H, amide-H), 9.15 (s, 1 H, Ar-H), 8.92
(b, 1 H, exchangeable), 8.21 (m, 2 H, Ar-H), 8.00 (m, 1 H, Ar-H),
7.81 (m, 1 H, Ar-H), 6.76 and 6.68 (2 d, 1 H each, J ) 8.4 Hz,
C1-H, C2-H), 5.06 (m, 1 H, C6-H), 4.90 (d, J ) 7.8 Hz, 1 H,
C5-H), 3.91 (m, 1 H), 3.78 (m, 1 H), 3.37 (m, 2 H), 3.10 (m, 2
H), 2.89 (m, 1 H), 2.45 (m, 1 H), 2.00 (m, 1 H), 1.83 (m, 1 H),
1.69 (m, 1 H), 1.49 (m, 2 H), 1.12 (m, 1 H), 0.68 (m, 2 H), 0.48
(m, 2 H). 13C NMR (75 MHz, DMSO) δ: 164.63, 151.51, 143.09,
142.94, 140.57, 135.84, 131.37, 131.26, 129.64, 129.01, 128.16,
128.06, 124.62, 120.53, 119.48, 118.30, 92.95, 62.55, 59.43, 52.26,
44.35, 36.71, 31.66, 30.85, 30.42, 24.69, 22.92, 9.68, 4.22, 4.02.
MS (ESI) m/z: 498.8 (M + H)+. Anal. (C30H31N3O4 ·2HCl·3.5H2O)
C, H.
1549, 1324, 777. H NMR (300 MHz, DMSO): δ 9.47 (s, 1 H,
1
Ar-H), 9.35 (b, 1 H, amide-H), 8.92 (b, 1 H, exchangeable), 8.23
(m, 2 H, Ar-H), 8.22 (s, 1 H, Ar-H), 8.03 (m, 2 H, Ar-H), 6.76
and 6.68 (2 d, 1 H each, J ) 8.4 Hz, C1-H, C2-H), 5.11 (s, 1 H,
C5-H) 5.01 (m, 1 H, C6-H), 3.92 (m, 1 H), 3.78 (m, 1 H), 3.33
(m, 2 H), 3.07 (m, 2 H), 2.89 (m, 1 H), 1.83 (m, 1 H), 1.62 (m, 2
H), 1.46 (m, 3 H), 1.11 (m, 1 H), 0.65 (m, 2 H), 0.47 (m, 2 H). 13
C
NMR (75 MHz, DMSO) δ: 165.68, 147.82, 147.72, 141.71, 140.27,
135.74, 131.00, 130.18, 128.40, 127.21, 127.04, 126.69, 126.50,
122.65, 118.42, 116.87, 99.79, 69.75, 61.90, 57.95, 51.63, 46.22,
44.11, 29.40, 29.24, 23.49, 21.82, 7.55, 2.99, 2.10. MS (ESI) m/z:
498.8 (M + H)+. Anal. (C30H31N3O4 ·2HCl·2.5H2O) C, H.
17-Cyclopropylmethyl-3,14ꢀ-dihydroxy-4,5r-epoxy-6r-[(2′-
naphthalyl)acetamido]morphinan (15). Compound 15 was pre-
17-Cyclopropylmethyl-3,14ꢀ-dihydroxy-4,5r-epoxy-6r-[(2′-
quinolyl)acetamido]morphinan (11). Compound 11 was prepared
by following the general procedure 1 in 93% yield. The product
pared by following the general procedure 1 in 46% yield; [R]25
D
-218° (c ) 0.01, MeOH); mp 213-215 °C. IR (KBr, cm-1) νmax
:
1
3399, 1641, 1503, 1460, 1318. H NMR (300 MHz, DMSO): δ
8.15 (s, 1 H, Ar-H), 7.77-7.73 (m, 4 H, Ar-H), 7.45 (m, 2 H,
Ar-H), 6.85 (b, 1 H, amide-H), 6.85 and 6.50 (2 d, 1 H each, J )
8.1 Hz, C1-H, C2-H), 4.80 (m, 1 H, C6-H), 4.71 (m, 1 H, C5-H),
3.69 (m, 1 H), 3.00 (m, 1 H), 2.63 (m, 1 H), 2.53 (m, 1 H), 2.34
(m, 1 H), 2.23 (m, 1 H), 2.15 (m, 2 H), 1.76 (m, 1 H), 1.49 (m, 1
H), 1.36 (m, 1 H), 1.09 (m, 1 H), 1.14 (m, 1 H), 0.83 (m, 1 H),
0.55 (m, 2 H), 0.12 (m, 2 H). 13C NMR (75 MHz, CD3OD) δ:
167.96, 145.46, 138.32, 134.35, 132.03, 130.76, 128.13, 127.44,
127.37, 127.25, 127.17, 127.00, 125.98, 123.30, 121.61, 119.24,
was transferred into a HCl salt; [R]25 -186° (c ) 0.03, MeOH);
D
mp 212-214 °C. IR (KBr, cm-1) νmax: 3199, 1673, 1528, 1321,
785. 1H NMR (300 MHz, DMSO): δ 8.90 (b, 1 H, amide-H), 8.60
(m, 2 H, Ar-H), 8.21 (b, 1 H, exchangeable), 8.13 (m, 2H, Ar-H),
7.92 (m, 1 H, Ar-H), 7.75 (m, 1 H, Ar-H), 6.76 and 6.63 (2 d, 1
H each, J ) 7.8 Hz, C1-H, C2-H), 4.84 (s, 1 H, C5-H), 4.68 (m,
1 H, C6-H), 3.94 (m, 1 H), 3.65 (m, 1 H), 3.35 (m, 1 H), 3.05 (m,
1 H), 2.71 (m, 1 H), 2.45 (m, 2 H), 1.93 (m, 2 H), 1.82 (m, 1 H),
1.65 (m, 1 H), 1.48 (m, 2 H), 1.06 (m, 1 H), 0.63 (m, 2 H), 0.44

µ Opioid Receptor SelectiVe Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1425
117.59, 87.72, 61.56, 57.25, 44.50, 39.95, 37.39, 29.81, 28.96,
23.29, 18.95, 13.60, 9.87, 5.05, 4.55. MS (ESI) m/z: 496.8 (M+).
Anal. (C31H32N2O4 ·HCl·1.5H2O) C, H.
maximal antinociception) or the test compound at increasing doses
and were reassessed for their tail immersion reaction time 20 min
later. To test for antagonist properties, mice with predetermined
tail immersion baseline, were injected sc with either naloxone (1
mg/kg; a dose that totally block the antinociception induce by 10
mg/kg morphine) or the test compound at various doses and 5 min
later they were administered morphine (10 mg/kg; sc). Mice were
reassessed for their tail immersion reaction time 20 min later.
Effective dose-50 (ED50) values were calculated using least-squares
linear regression analysis followed by calculation of 95% confidence
limits (95% CL) by the method of Bliss.60
Data are expressed as mean values ( SEM analysis of variance
(ANOVA) followed by the post hoc “Student-Newman-Keuls”
test were performed to assess significance using the Instat 3.0
software (GraphPad Software, San Diego, CA). P < 0.05 was
considered significant.
17-Cyclopropylmethyl-3,14ꢀ-dihydroxy-4,5r-epoxy-6ꢀ-[(2′-
naphthalyl)acetamido]morphinan (16). Compound 16 was pre-
pared by following the general procedure 1 in 44% yield; [R]25
D
-123° (c ) 0.09, MeOH); mp 212-215 °C. IR (KBr, cm-1) νmax
:
3248, 2964, 1640, 1508, 1319, 716. 1H NMR (300 MHz, DMSO):
δ 8.36 (b, 1 H, amide-H), 7.95-7.86 (m, 5 H, Ar-H), 7.56 (m, 2
H, Ar-H), 6.76 and 6.59 (2 d, 1 H each, J ) 8.4 Hz, C1-H, C2-H),
4.60 (m, 1 H, C5-H), 4.31 (m, 1 H, C6-H), 3.88 (m, 1 H), 3.75
(m, 1 H), 3.19 (m, 1 H), 2.70 (m, 1 H), 2.42 (m, 1 H), 2.27 (m, 2
H), 1.96 (m, 1 H), 1.70 (m, 1 H), 1.54 (m, 1 H), 1.24 (m, 1 H),
1.49 (m, 1 H), 1.14 (m, 1 H), 0.88 (m, 1 H), 0.50 (m, 2 H), 0.12
(m, 2 H). 13C NMR (75 MHz, DMSO) δ:. 166.36, 147.21, 142.86,
134.85, 132.86, 131.80, 129.57, 128.98, 128.57, 128.35, 128.25,
128.24, 127.48, 124.85, 121.75, 119.20, 119.10, 91.30, 70.44, 62.43,
51.74, 42.96, 35.67, 33.08, 32.17, 30.45, 24.70, 20.10, 9.50, 6.80,
4.00. MS (ESI) m/z: 497.8 (M + H)+. Anal. (C31H32N2O4 ·
HCl·H2O) C, H.
Acknowledgment. We thank Dr. Lee-Yuan Liu-Chen at
Temple University and Dr. Ping-Yee Law at the University of
Minnesota for the generous gift of opioid receptor expressed
CHO cell lines. We appreciate the generous help from Mallinck-
rodt, Inc., for the gift of naltrexone sample. The work was
partially supported by PHS grants from NIH/NIDA, DA010770
(DES), DA007027 and DA000480 (WLD), and DA024022
(YZ). The content is solely the responsibility of the authors and
does not necessarily represent the official views of the National
Institute On Drug Abuse or the National Institutes of Health.
In Vitro Competitive Radioligand-Binding and Functional
Assay. Details of the binding assay was conducted to study the
selectivity of the ligands by using monocloned opioid receptor
expressed in Chinese hamster ovarian (CHO) cell lines as described
previously.44,45 [3H]naloxone, [3H]NTI, and [3H]norBNI were used
to label the µ, δ, and κ opioid receptors, respectively. Aliquots of
a membrane preparation were incubated with the radioligands in
the presence of different concentrations of the drug under investiga-
tion at 30 °C for 1 h. Specific (i.e., opioid receptor related) binding
was determined as the difference in binding obtained in the absence
and presence of 10 µM naltrexone. The potency of the drugs in
displacing the specific binding of the radioligand was determined
from data using linear regression analysis of Hill plots. The IC50
values will then be determined and corrected to Ki values using
the Cheng-Prusoff equation. Functional assays, including 35S-
GTP[γS]-binding assay, were conducted in the same cell mem-
branes used for the receptor binding assays. Three µM of DAMGO
was included in the assay for a maximally effective concentration
of a full agonist for the µ opioid receptor.
In Vivo Acute Function Test Procedure. Animals. Male Swiss
Webster mice (Harlan Laboratories, Indianapolis, IN) weighing
25-30 g were housed six to a cage in animal care quarters and
maintained at 22 ( 2 °C on a 12 h light-dark cycle. Food and
water were available ad libitum. The mice were brought to a test
room (22 ( 2 °C, 12 h light-dark cycle), marked for identification
and allowed 18 h to recover from transport and handling. Protocols
and procedures were approved by the Institutional Animal Care
and Use Committee (IACUC) at Virginia Commonwealth Univer-
sity Medical Center and comply with the recommendations of the
IASP (International Association for the Study of Pain).
Tail Immersion Test. The warm-water tail immersion test was
performed according to Coderre and Rollman58 using a water bath
with the temperature maintained at 56 ( 0.1 °C. Before injecting
the mice, a baseline (control) latency was determined. Only mice
with a control reaction time from 2 to 4 s were used. The average
baseline latency for these experiments was 3.0 ( 0.1 s. The test
latency after drug treatment was assessed at the appropriate time,
and a 10 s maximum cutoff time was imposed to prevent tissue
damage. Antinociception was quantified according to the method
of Harris and Pierson59 as the percentage of maximum possible
effect (% MPE), which was calculated as: %MPE ) [(test latency
- control latency)/(10 - control latency)] × 100. Percent MPE
was calculated for each mouse using at least six mice per drug.
Drugs. Morphine sulfate was purchased from Mallinckrodt, St.
Louis, MO. Naloxone was purchased from Sigma-Aldrich (St.
Louis, MO). All drugs and test compounds were dissolved in
pyrogen-free isotonic saline (Baxter Healthcare, Deerfield, IL) and
were administered to mice subcutaneously (sc).
Supporting Information Available: Procheck results of three
homology models and a table listing the HPLC retention times and
mobile phase and method for analysis. This material is available
free of charge via the Internet at http://pubs.acs.org.
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