Design, synthesis, and biological evaluation of 14-heteroaromatic- substituted naltrexone derivatives: Pharmacological profile switch from Mu opioid receptor selectivity to Mu/Kappa opioid receptor dual selectivity

Article abstract of DOI:10.1021/jm4012214

On the basis of a mu opioid receptor (MOR) homology model and the isosterism concept, three generations of 14-heteroaromatically substituted naltrexone derivatives were designed, synthesized, and evaluated as potential MOR-selective ligands. The first-generation ligands appeared to be MOR-selective, whereas the second and the third generation ones showed MOR/kappa opioid receptor (KOR) dual selectivity. Docking of ligands 2 (MOR selective) and 10 (MOR/KOR dual selective) to the three opioid receptor crystal structures revealed a nonconserved-residue-facilitated hydrogen-bonding network that could be responsible for their distinctive selectivity profiles. The MOR/KOR dual-selective ligand 10 showed no agonism and acted as a potent antagonist in the tail-flick assay. It also produced less severe opioid withdrawal symptoms than naloxone in morphine-dependent mice. In conclusion, ligand 10 may serve as a novel lead compound to develop MOR/KOR dual-selective ligands, which might possess unique therapeutic value for opioid addiction treatment.

Full text of DOI:10.1021/jm4012214

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of 14-Heteroaromatic-
Substituted Naltrexone Derivatives: Pharmacological Profile Switch
from Mu Opioid Receptor Selectivity to Mu/Kappa Opioid Receptor
Dual Selectivity
Yunyun Yuan,† Saheem A. Zaidi,† Orgil Elbegdorj,† Lindsey C. K. Aschenbach,† Guo Li,†
David L. Stevens,‡ Krista L. Scoggins,‡ William L. Dewey,‡ Dana E. Selley,‡ and Yan Zhang*,†
†Department of Medicinal Chemistry, Virginia Commonwealth University, 800 East Leigh Street, Richmond, Virginia 23298, United
States
‡Department of Pharmacology and Toxicology, Virginia Commonwealth University, 410 North 12th Street, Richmond, Virginia
23298, United States
S
* Supporting Information
ABSTRACT: On the basis of a mu opioid receptor (MOR) homology model
and the isosterism concept, three generations of 14-heteroaromatically
substituted naltrexone derivatives were designed, synthesized, and evaluated as
potential MOR-selective ligands. The first-generation ligands appeared to be
MOR-selective, whereas the second and the third generation ones showed
MOR/kappa opioid receptor (KOR) dual selectivity. Docking of ligands 2
(MOR selective) and 10 (MOR/KOR dual selective) to the three opioid
receptor crystal structures revealed a nonconserved-residue-facilitated hydrogen-
bonding network that could be responsible for their distinctive selectivity profiles. The MOR/KOR dual-selective ligand 10
showed no agonism and acted as a potent antagonist in the tail-flick assay. It also produced less severe opioid withdrawal
symptoms than naloxone in morphine-dependent mice. In conclusion, ligand 10 may serve as a novel lead compound to develop
MOR/KOR dual-selective ligands, which might possess unique therapeutic value for opioid addiction treatment.
prescription opioids in 2010 was four times that in 1999.2
Therefore, efficacious medications are still highly desired to
treat acute (overdose) and chronic (abuse/addiction) side
effects of opioids.
INTRODUCTION
■
Opium, the dried latex obtained from the immature seedpod of
a poppy flower (Papaver somniferum), has been used for
medical and euphoric purposes since ancient time. The major
active ingredient was later identified to be morphine (Figure
The current FDA approved pharmacologic treatment for
1).1 Opioids is a generic term referring to alkaloids isolated
long-term opioid dependence/addiction based on the MOR
mechanisms includes methadone (a full agonist), buprenor-
from opium poppy, their synthetic analogues, and endogenous
substances synthesized in the human body.2 Opioids exert their
phine (a partial agonist), and naltrexone (an antagonist)
(Figure 1).6−8 Although methadone has shown great efficacy
function through interacting with one or more of the three
for opioid-addiction maintenance, it could cause lethal
major opioid receptors, designated as the mu opioid receptor
respiratory depression when taken in overdose because of its
(MOR), the kappa opioid receptor (KOR), and the delta
MOR full-agonist property.6 Furthermore, sudden cessation of
methadone would precipitate a longer period of withdrawal
symptoms than that following morphine termination, which has
led to methadone becoming an abused drug itself.9 Similarly,
buprenorphine also suffered the same drawbacks of methadone,
albeit to a lesser extent because of its partial agonism on the
MOR,10 whereas it was associated with fewer drug−drug
interactions compared to methadone.6 In contrast, as a MOR
antagonist, naltrexone did not induce respiratory suppression
and showed no abuse liability. However, it has precipitated
considerable withdrawal syndrome, which has compromised its
opioid receptor (DOR).3,4 Among them, the MOR plays a
major role in mediating the antinociception and other
unwanted adverse effects of opioids, such as abuse/addiction
liability, respiratory depression, sedation, and constipation.5
Among the side effects, the abuse/addiction liability is the
major concern for prescription of opioid analgesics.
According to the 2012 World Drug Report, the global annual
prevalence of illicit opioids use ranged from 0.6 to 0.8% of the
adult population in 2010. North America (3.8−4.2%), Oceania
(2.3−3.4%), and Eastern and South-Eastern Europe (1.2−
1.3%) have a higher prevalence of illicit opioid use than the
global average. The nonmedical use of prescription opioids
plays a dominant and problematic role in North America. For
the United States alone, the overdose deaths involving
Received: August 7, 2013
© XXXX American Chemical Society
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Figure 1. Morphine and the current available drugs for opioid
dependence/addition treatment based on the MOR mechanisms.
therapeutic efficiency.11−14 The extended-release formulation of
naltrexone significantly improved the adherence rate and
treatment outcome.15,16 Nevertheless, its application is
restricted from people who have end-stage liver disease or
who need a long period of chronic pain management.6
Collectively, these three drugs have associated shortcomings,
although they do serve as proof-of-concept that targeting the
MOR could provide effective treatment for opioid dependence
and addiction.
Figure 2. Representatives of the small molecules possessing MOR-
antagonist characteristics.
In this respect, a number of chemical entities have been
developed as MOR ligands. Some of the representatives are
depicted in Figure 2. Among them, β-funaltrexamine (β-FNA),
clocinnamox, and methocinnamox are potential MOR irrever-
sible antagonists. β-FNA binds with equal potency to the MOR
and KOR, whereas clocinnamox and methocinnamox bind to
all three opioid receptors in mouse brain homogenates with
similar affinity.17 β-FNA also possessed reversible KOR agonist
activity.18−20 Cyprodime is another intensively studied MOR
antagonist, which has moderate selectivity and potency at the
MOR (Ki value ratios are delta/mu ≈39 and kappa/mu ≈10; Ki
and significantly potentiated cocaine-conditioned place prefer-
ence.27 Furthermore, 1 effectively reinstated cocaine-seeking
behavior in mice previously conditioned to cocaine.28 Such
effects of 1 were abolished by the KOR-selective antagonist
nor-binaltorphimine (norBNI) in both studies.27,28 Meanwhile,
it has also been reported that blockade of the dynorphin/KOR
system decreased dependence-induced ethanol self-adminis-
tration in male Wistar rats.29 In contrast, the effects of the
dynorphin/KOR system on opioid dependence and addiction
seemed to be dose-, ligand-, and species-specific. For instance,
low doses of 1 first increased (0.1 mg/kg) and then decreased
(0.5 mg/kg) heroin self-administration in mice.30 KOR-
selective agonist (2E)-N-[(5α,6β)-17-(cyclopropylmethyl)-
3,14-dihydroxy-4,5-epoxymorphinan- 6-yl]-3-(3-furyl)-N-meth-
ylacrylamide (TRK-820) but not N-methyl-2-phenyl-N-
[(5R,7S,8S)-7-(pyrrolidin-1-yl)-1-oxaspiro[4.5]dec-8-yl]-
acetamide (U69,593) decreased morphine-induced conditional
place preference.31,32 NorBNI potentiated naloxone-precipi-
tated morphine-withdrawal symptoms in rats,33 whereas KOR
gene disruption in mice showed reduced naloxone-precipitated
morphine-withdrawal syndrome.34 Collectively, these data
implicated that the dynorphin/KOR system may act as an
important modulator in the neurobiology of drug dependence
and addiction, and it could serve as a cotarget along with the
MOR for drug dependence and addiction treatment.35−37
The continuing interest in developing novel, nonpeptidic,
and reversible opioid receptor ligands in our lab led to the
identification of two potent and highly selective MOR ligands,
NAP and NAQ, the C6-heteroaromatic-substituted naltrexone
derivatives based on a MOR homology model and the
“message-address” concept (Figure 2).38 Both compounds
at the MOR is 10.6
0.7 nM).21,22 Sally et al. recently
disclosed (−)-3-cyclopropylmethyl-2,3,4,4α,5,6,7,7α-octahy-
dro-1H-benzofuro[3,2-e]isoquinolin-9-ol (LTC-274)23 (Figure
2) as a novel MOR antagonist, which showed the least inverse
agonist activity at the MOR among 21 ligands tested.
Meanwhile, this compound bound to the KOR as strongly as
to the MOR, and it acted as a KOR partial agonist (EC50 = 2.7
nM, Emax = 23%).23 2-Methyl-N-{[2′-(pyrrolidin-1-ylsulfonyl)-
biphenyl-4-yl]methyl}propan-1-amine (PF-04455242)24 and
c[Ala1-Pro2-Phe3-Trp4]25 (a cyclic peptidyl derivative) have
also been reported to function as MOR/KOR dual antagonists
(Figure 2). However, both of them were more potent and
selective at the KOR.24,25 Therefore, MOR-selective ligands
with high potency are still highly desirable.
From another standpoint, accumulating evidence shows that
the release of dynorphins and the activation of the KOR
mediates dysphoria and anhedonia associated with drug
withdrawal, stress-induced aversion states, and stress-induced
relapselike behavior.26 For example, the KOR-selective agonist
2-(3,4-dichlorophenyl)-N-methyl-N-[(1R,2R)-2-pyrrolidin-1-
ylcyclohexyl]acetamide 1 (U50,488) mimicked stress exposure
B
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Table 1. Opioid Receptor Binding Affinity, Selectivity, and the MOR [35S]-GTPγS Functional Assay Results of the First
a
Generation of 14-Substituted Naltrexone Derivatives
a
The values are the means SEM of three independent experiments. [3H]NLX, [3H]NTI, and [3H]norBNI were used to label MOR, DOR and
KOR, respectively. The percentage stimulation to DAMGO is the Emax of the compound compared to that of 3 μM DAMGO (normalized to 100%).
NA, not applicable. ND, not detectable.
acted as MOR partial agonists with low efficacy in the [35S]-
GTPγS binding assay but antagonized the effects of the MOR
full agonists both in vitro and in vivo.38,39 Docking experiments
of naltrexone to the same MOR homology model led to the
identification of an alternative “address” domain that is located
around extracellular loop III and the upper-level region of
transmemberane VI/VII,40 which might have the potential to
interact with some putative substitutions at the 14-position of
naltrexone to increase the MOR-binding affinity and selectivity.
To test this hypothesis, three generations of 14-heteroaromatic-
substituted naltrexone derivatives were designed and synthe-
sized consecutively. All of the newly synthesized compounds
were tested in the opioid receptor radioligand competition
binding assay and in the [35S]-GTPγS binding assay for their
affinity, selectivity, and function. Selected compounds were
further evaluated for the acute antinociceptive agonistic/
antagonistic effects in the tail-flick assay as well as for the
opioid-withdrawal symptoms in morphine-dependent mice.
Two representative ligands were also docked into the crystal
structures of three opioid receptors to validate further our
original hypothesis.
bond donor/acceptor residues (i.e., Tyr210 and Trp318) in
one of the MOR-naltrexone binding modes,40 which might be
able to act as a unique MOR address domain. We thus
hypothesized that a functionalized moiety that could interact
with these residues (through hydrogen bonding and/or
aromatic stacking) might provide enhanced MOR affinity and
thus increase the MOR selectivity over the KOR and the DOR.
Six heteroaromatic ring side chains containing one nitrogen
atom (a hydrogen-bond donor under physiological conditions)
and two aromatic rings (as the control compounds) were
chosen to test this hypothesis. These functionalized moieties
were introduced to the 14-hydroxyl group of naltrexone via an
ester bond (compounds 2−9) based on a straightforward
chemical synthetic method. Compound 2 was then identified as
the most selective MOR molecule among the first-generation
ligands. However, there have been reports that sterically
hindered tertiary esters (such as compounds 2−9) could
possibly be hydrolyzed by certain esterases and lipases.41,42 To
avoid such potential shortcomings and also to facilitate future in
vivo studies, the isosteres of compounds 2−9, amide analogues
10−17, were then synthesized as the second-generation
compounds to test whether the high MOR selectivity induced
by these heteroaromatic ring would be retained. Interestingly,
the amide derivatives became selective to both the MOR and
the KOR. Among them, ligand 10 appeared to have the highest
selectivity for these two receptors over the DOR. To investigate
RESULTS AND DISCUSSION
■
Molecular Design. A previous molecular-modeling study
revealed that the 14-hydroxyl group of naltrexone might
putatively point to some nonconserved yet potential hydrogen-
C
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a
whether the selectivity-profile change for ligand 10 was due to
different projection of the 2′-pyridyl ring as a result of the
restricted rotation of the amide bond compared to the ester
linkage, different lengths of spacers were introduced between
the epoxymorphinan skeleton and the 2′-pyridyl ring in
compound 10 to yield the third-generation compounds 18−
20. Surprisingly, these ligands with an extended spacer were still
MOR/KOR dual selective. This suggested that the amide bond
itself might be involved in the receptor−ligand interactions,
which was later supported by our molecular-modeling study
based on the crystal structures of three opioid receptors.
Chemistry. Synthesis of 14-Substituted Naltrexone
Derivatives. The 14-O-substituted naltrexone derivatives (2−
9, Table 1) were readily obtained by reacting naltrexone with
the corresponding acyl chloride in TEA/DMF at 100 °C with
subsequent saponification under either acidic (compounds 2−4
and 6−8) or basic (compounds 5 and 9) conditions (Scheme
1).40
Scheme 2. Synthetic Route of 14-Aminonaltrexone 21
Scheme 1. Synthetic Route of the Three Generations of 14-
a
Substituted Naltrexone Derivatives 2−20
a
(a) RCOCl or RCOOH; (b) K2CO3 or H2SO4.
a
(a) DEAD, benzene, reflux; (b) pyridine·HCl, EtOH/H2O, reflux to
rt, 83%, two steps; (c) TEA, DCM, 0 °C to rt, quantitative; (d)
LiAlH4, THF, reflux, 77%; (e) NaIO4, NaOAc, EtOAc, 73%; (f) Pd/C,
H2/60 psi, AcOH, NaOAc, MeOH, 40%; (g) BBr3, DCM, 44%.
An established synthetic route shown in Scheme 2 was
adopted to prepare 14-aminonaltrexone 21.43 Compounds 10−
20 were then obtained by coupling intermediate 21 with an
acid/acyl chloride, commercially available or prepared in house
(see the Supporting Information), followed by saponification
with K2CO3 as described previously (Scheme 1).38,43,44
Biology. In Vitro and in Vivo Pharmacological Studies.
The synthesized three generations of naltrexone derivatives
were first evaluated in the radioligand competition binding
assay and in the [35S]-GTPγS functional assay on opioid
receptor-transfected CHO cell membranes for their binding
affinity, selectivity, and MOR agonism/antagonism (in vitro).
Then, selected compounds were further advanced to behavioral
tail-flick and opioid-withdrawal assays for their functional
activity (in vivo). Naltrexone (NTX) was tested along for
comparison in all of the assays.
As seen in Table 1, all of the first generation of naltrexone
derivatives retained subnanomolar to one-digit nanomolar
binding affinity at the MOR except for compounds 5, 6, and
9. The relatively low binding affinity of control compounds 5
and 9 compared to the rest of the ester congeners indicated
that the nitrogen atom in the functionalized moiety of these
derivatives did play an important role in the MOR−ligand
interactions. In detail, nitrogen atom substitutions with 2′,3′-
pyridyl (compounds 2 and 3, respectively) and 2′,3′-quinolyl
(compounds 7 and 8, respectively) were favorable for MOR
binding (11−40-fold higher compared to other isomers).
Meanwhile, the first generation of naltrexone derivatives 2−9
generally all displayed considerably decreased binding affinities
at the KOR and DOR compared to naltrexone (except for
compounds 2 and 7 at the DOR), and compounds 2, 3, 6, and
8 showed a greater than 30-fold selectivity to the MOR over the
KOR and the DOR. In this sense, the nitrogen atom position
seemed to be critical as well. Compound 2 (ONP) appeared to
be the most selective and potent MOR ligand in this first
generation of naltrexone derivatives. As for the MOR [35S]-
GTPγS binding assay, all of the first-generation compounds,
except for ligand 8, did not show any detectable MOR agonism
under the tested conditions.
In Vitro Radioligand Binding Assay and [35S]-GTPγS
Functional Assay. The competitive radioligand binding assay
was performed on monoclonal opioid receptor-expressed CHO
cell membranes as described previously.38,39,44 [3H]naloxone
(NLX), [3H]naltrindole (NTI), and [3H]norBNI (for com-
pounds 2−9) or [3H]diprenorphine (DPN, for compounds
10−20) were used to label the MOR, DOR, and KOR,
respectively. The MOR [35S]-GTPγS binding assay was
conducted to determine whether each of the new ligands
would act as a full agonist, a partial agonist, or an antagonist at
the MOR, as illustrated before.38,39,44 The results were
interpreted as the relative efficacy of each compound to a
MOR full agonist [D-Ala2-MePhe4-Gly(ol)5]enkephalin
(DAMGO) for MOR activation.
In contrast, the second generation of naltrexone derivatives
(10−17) also bound to the MOR at subnanomolar to one-digit
nanomolar affinity (Table 2). However, the presence/absence
and the position of the nitrogen atom in the functionalized
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Table 2. Opioid Receptor Binding Affinity, Selectivity, and MOR [35S]-GTPγS Functional Assay Results of the Second and
a
Third Generations of 14-Substituted Naltrexone Derivatives
a
The values are the means SEM of three independent experiments. [3H]NLX, [3H]NTI, and [3H]DPN were used to label MOR, DOR, and KOR,
respectively. The percentage stimulation to DAMGO is the Emax of the compound compared to that of 3 μM DAMGO (normalized to 100%). NA,
b
c
not applicable. Percentage stimulation produced at the maximum concentration of 10 μM. These controls were also reported previously in ref 57.
moiety did not seem to have a significant impact on their MOR
binding affinity. In the meantime, increased binding affinities at
the KOR and DOR were observed for these second-generation
compounds 10−17 in comparison to both naltrexone and the
first-generation ligands, which significantly changed their
opioid-receptor-selectivity profile. Some of the ligands were
both MOR and KOR selective over the DOR (compounds 10−
12), whereas others were even more KOR selective over the
MOR and the DOR (compounds 13−15 and 17). Never-
theless, the majority of the second generation of naltrexone
derivatives produced less than 20% MOR stimulation relative to
DAMGO. Compounds 10 (NNP), 11, and 14 showed
relatively lower MOR efficacy among this series of compounds.
Compared to the undetectable MOR agonism of the first-
generation ligands, the isostere replacement of an ester linkage
with an amide bond not only decreased MOR selectivity over
the KOR but also slightly enhanced ligand MOR efficacy.
Compounds 18−20 were then prepared to test whether the
changed opioid receptor selectivity was due to the restricted
rotation of the amide bond compared to the ester linkage. As
shown in Table 2, ligands 18 and 19 were still MOR/KOR dual
selective, whereas ligand 20 bound to all three opioid receptors
with nearly equal potency. It thus seemed that the conven-
tionally restricted rotation of the amide bond might not be the
reason for the ligand opioid receptor-selectivity change from
MOR (ligand 2) to MOR/KOR (ligand 10). Meanwhile, the
relative lower Ki values of ligands 18−20 with an extended
spacer compared to that of ligand 10 suggests that a less
restricted functionalized moiety could facilitate ligand binding
to the MOR. Ligand 18 behaved as a MOR partial agonist with
relatively high efficacy, whereas ligands 19 and 20 acted as low-
efficacy MOR partial agonists.
Collectively, the radioligand competition binding assay
identified three potent MOR selective ligands (2, 3, and 8)
E
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and three potent MOR/KOR dual-selective ligands (10, 11,
and 19). These six novel ligands all displayed marginal MOR
agonism in the [35S]-GTPγS binding assay. We noticed that a
similar opioid-receptor-selectivity profile change upon isoster-
ism has also been reported for methocinamox (amide linkage)
and its ester analogue. Methocinamox has no selectivity among
the three opioid receptors, whereas its ester analogue is
moderately selective to the MOR over the KOR and the DOR.
(The Ki value ratios for methocinamox are delta/mu ≈1 and
kappa/mu ≈3 and for its ester isostere are delta/mu ≈31 and
kappa/mu ≈15.)45 Such findings somewhat coincided with our
observation of the selectivity-profile change reported herein.
Because compounds 10 and 11 showed relatively higher
MOR/KOR dual selectivity over the DOR among all of the
new ligands, the [35S]-GTPγS binding assays of these two
compounds on the monoclonal KOR and DOR-expressed
CHO cell membranes were further conducted. Compound 14
was also included in these assays because of its minimal efficacy
at the MOR (Table 3). Naltrexone and compounds 10 and 14
Opioid-Withdrawal Assays. Because the ultimate goal of
the study is to identify selective opioid receptor ligands for
opioid dependence and addiction treatment, it is necessary to
evaluate whether these new ligands would precipitate severe
withdrawal symptoms in morphine-dependent mice (Figure 3).
Table 3. KOR/DOR [35S]-GTPγS Binding Results for
a
Compounds 10, 11, and 14
KOR [35S]-GTPγS binding
DOR [35S]-GTPγS binding
compound
EC50 (nM)
% Emax of 1
EC50 (nM)
% Emax of 30
NTX
10 (NNP)
11
0.81 0.08
1.74 0.50
0.56 0.06
2.75 0.71
20.8 0.9
24.9 2.0
35.3 6.8
16.8 0.6
4.4 1.6
19.7 7.4
3.2 2.0
14.2 1.9
5.6 0.6
8.6 0.8
10.3 0.6
23.8 0.7
14
a
The values are the means SEM of three independent experiments.
The percentage stimulation to 1 or 30 is the Emax of the compound
compared to that of 1 or 30 (normalized to 100%).
appeared to carry similar efficacy at the KOR, whereas
compound 11 appeared to show the highest efficacy. However,
naltrexone and compounds 10 and 11 exhibited marginal
efficacy at the DOR, whereas compound 14 showed the highest
efficacy, although its potency was relatively low.
Tail-Flick Assay. Because of their minimum MOR
activation, as shown in the [35S]-GTPγS functional assay,
compounds 10, 11, and 14 were chosen for further evaluation
of their acute agonistic and antagonistic effects in the tail-flick
assay in mice. None of the compounds produced any significant
agonist effect up to a 30 mg/kg dose, although they were found
to potently antagonize the analgesic effect of morphine (Table
4). Compound 10 seemed to be the most potent among these
three new ligands. In general, the in vivo assay results were
consistent with the in vitro MOR [35S]-GTPγS functional data
for these compounds.
Figure 3. 14-N-Heteroaromatic-substituted naltrexone derivatives 10,
11, and 14 in withdrawal assays in chronic morphine-exposed mice:
(A) escape jumps and (B) wet dog shakes.
Similar to previous reports,39,46 the well-known opioid
antagonist naltrexone precipitated significant withdrawal
syndrome, which was manifested by the number of escape
jumps (over 20; Figure 3A) and wet dog shakes (7; Figure 3B)
in morphine-pelleted mice, whereas naloxone showed even
more significant syndrome (Figure 3). Interestingly, in the first
assay, compound 10 displayed a clear decrease in escape jumps
as its dose increased (although this was not significant), with
similar jumping numbers at 30 mg/kg to that of 1 mg/kg of
naltrexone (Figure 3A). In contrast, there seemed to be an
increasing dose−response effect for compound 11 and no
significant dose−response effect for compound 14, respectively.
Compounds 11 and 14 at 30 mg/kg yielded a similar number
of jumps as that produced by 1 mg/kg naloxone.
Table 4. AD50 Values of Compounds 10, 11, and 14 for
Antagonizing the Morphine (10 mg/kg) Antinociception
a
Effect in a Warm-Water Tail-Flick Assay
compound
AD50 values (mg/kg (95% CL))
naloxone
naltrexone
10 (NNP)
11
0.05 (0.03−0.09)
0.006 (0.003−0.014)
0.25 (0.18−0.36)
0.55 (0.35−0.87)
0.87 (0.52−1.47)
There was a minimal number of wet dog shakes for
compound 10 at 3 mg/kg, but a dramatic increase was observed
as the dose went up to 10 mg/kg, which remained at the same
level at 30 mg/kg. Nevertheless, the effects at high doses of
compound 10 were still lower than that of 1 mg/kg naloxone
14
a
All drugs and test compounds were administered to a group of six
mice subcutaneously (s.c.).
F
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Figure 4. (A) Superimposed binding mode of ligand 2 (orange balls and stick) in three opioid receptors: conserved residues (cyan), MOR residues
(green), KOR residues (black), and DOR residues (purple). Pink dashes and green dots represent possible hydrogen-bonding interactions. Asn2.63
(MOR), but not Val2.63 (KOR) or Lys2.63 (DOR), facilitated the hydrogen-bonding interaction (green dots) between Gln2.60 and the pyridyl nitrogen
atom. The formed hydrogen-bonding network yields the high MOR selectivity of ligand 2 over the KOR and DOR. (B) Highest-scored binding
mode of ligand 10 (green balls and stick) in the MOR (cyan). Conserved hydrogen-bonding interactions, as seen for ligand 2, are shown in pink
dashes. A potential internal hydrogen bond (orange dots) between the amide NH and the pyridyl nitrogen in ligand 10 disrupts the hydrogen-
bonding network, as observed for ligand 2. Furthermore, a hydrogen-bonding interaction (brown dots) also formed between the amide NH and the
conserved residue Asp3.32 in all three opioid receptors and thus enhanced the binding affinities of ligand 10 in all three opioid receptors. Together, a
reduced MOR selectivity for ligand 10 was observed as compared to that of ligand 2.
(Figure 3B). Both compounds 11 and 14 appeared to show an
increasing dose−response effect with the wet dog shakes,
whereas at their highest doses tested (30 mg/kg) they
resembled the effect of 1 mg/kg naloxone and naltrexone.
Because all three compounds only showed marginal efficacy at
the MOR, their partial agonism at the KOR and DOR could be
one of the factors for their distinct profiles in the withdrawal
assays. Nevertheless, the withdrawal symptoms of these MOR/
KOR dual-selective ligands seemed to be less significant than
naloxone, which sheds some light on their potential application
in drug-abuse/addiction treatment.
Molecular-Modeling Study. As discussed earlier, the
selectivity profile of the first and second generations of
naltrexone derivatives switched from MOR selective (ester
analogues) to MOR/KOR dual selective (amide analogues). To
understand this phenomena in the context of ligand−receptor
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interaction, automated docking of ligands 2 and 10 (the two
compounds that showed the highest selectivity in each
generation) to the crystal structures of three opioid
receptors47−49 was performed by employing GOLD5.1. In the
majority of the obtained docking modes, the known
morphinan-type pose was scored the highest for both ligands,
and the docking poses of ester bond-linked ligand 2 were
similar to those of amide bond-linked ligand 10.
bonding interaction (Figure 4B, brown dots) with the
conserved residue Asp3.32 in all three opioid receptors, which
would enhance the binding affinities of ligand 10 to all three
opioid receptors. Therefore, disruption of the hydrogen-
bonding network and formation of a hydrogen bond with a
conserved residue in all three opioid receptors together might
account for the reduced MOR selectivity for ligand 10
compared to its ester isostere ligand 2.
To identify the possible explanations for the high MOR
selectivity of ligand 2 over the KOR and DOR, amino acid
residues around the binding pocket for each opioid receptor
along with docked ligand 2 were allowed to attain a lower-
energy conformation by a 10 ps NVT dynamic simulation
(moles, N, volume, V, and temperature, T, are conserved) at
300 K under TRIPOS force field (TFF). After averaging the last
1 ps of the simulation, the obtained energy-minimized
structures were then studied. Interestingly, conformational
changes observed for Gln2.60 of the MOR allowed for a possible
hydrogen-bonding interaction with the pyridyl nitrogen of
ligand 2 (Figure 4A, green dots). However, this conserved Gln
residue was directed differently in both the KOR and DOR,
diminishing the possibility of hydrogen-bonding interactions
between the Gln residue and the pyridyl nitrogen of ligand 2 in
these two receptors (Figure 4A). Looking further within the
same docking pocket, it was noticed that the residues at
position 2.63 (directly above Gln2.60) for the three opioid
receptors projected and functioned differently. Asn2.63 in the
MOR presented a helical turn above Gln2.60 and helped to
direct Gln2.60 toward ligand 2 through a hydrogen bond (Figure
4A, pink dashes). However, for the KOR and DOR, the 2.63
position was occupied by Val118 and Lys108, respectively,
which were not able to interact with the Gln2.60 residue through
hydrogen bonds (Figure 4A). On the basis of the receptor
competition binding assay results and the current modeling
studies, we hypothesized that a hydrogen-bonding network
among the pyridyl nitrogen of ligand 2, the conserved Gln
residue at 2.60 position, and the nonconserved residue at 2.63
position of the three opioid receptors is responsible for the
receptor selectivity of this ligand. The presence of such a
hydrogen-bonding network enables ligand 2 to be more MOR
selective over the KOR and DOR.
In contrast, the MOR selectivity over the KOR was lost for
ligand 10 even though it has the same functionalized moiety as
ligand 2. To understand the possible conformational changes in
residues around the binding pocket that might cause the
decreased MOR selectivity for ligand 10, the ester O atom of
ligand 2 was replaced with amide NH atoms to put ligand 10 in
the same docking pose as ligand 2. This was then followed by
the same dynamic simulation experiment as described earlier.
Figure 4B presents the highest-scored binding mode of ligand
10 in the MOR. The model suggested that for ligand 10,
because of a possible internal hydrogen bond between the
amide NH and the pyridyl nitrogen (Figure 4B, orange dots),
the pyridyl nitrogen preferred to stay close to the amide NH
(potential energy under TRIPOS force field calculated for
ligand 10 pose with the internal hydrogen bond was 12.5 kcal/
mol lower than that without the internal hydrogen bond).
Thus, the pyridyl nitrogen was no longer available to form a
hydrogen bond with the Gln2.60, which seemed to be a key
component of the hydrogen-bonding network that decided the
opioid receptor selectivity of ligand 2 according to our
observation. Furthermore, this internal hydrogen bond in
ligand 10 also facilitated the amide NH to form a hydrogen-
Taken together, the current molecular-modeling study based
on the crystal structures of three opioid receptors identified an
alternative MOR address domain composed of Gln2.60 and
Asn2.63 in transmemberane helix II. Interestingly, such finding is
significantly different from our previous studies that applied
opioid receptor homology models based on the crystal
structure of bovine rhodopsin. Similar discrepant results have
also been observed by other research groups.50 We also noticed
that the introduction of a substitution at the 14-position of
morphinan skeleton could be crucial in altering the
pharmacological profile,51 whereas the orientation (in the
manner of rotation freedom) of such a substitution at this
position may lead to high-potency MOR-agonism activity
during the development of some pseudoirreversible opioid
receptor antagonists.52−54 In combination with our observa-
tions, a more vivid picture of the impact of 14-substitution on
the pharmacology of the morphinan skeleton derivatives is now
available.
CONCLUSIONS
■
On the basis of a previous MOR homology model, three
generations of 14-heteroaromatic-substituted naltrexone deriv-
atives (esters 2−9 and amides 10−20) were designed,
synthesized, and biologically evaluated. The majority of these
new ligands bound to the MOR with subnanomolar to
nanomolar affinity. The selectivity profile of the compounds
switched from MOR selective to MOR/KOR dual selective
upon application of the concept of isosterism, with a marginal
increase of the functional activity in the MOR [35S]-GTPγS
binding assay. Further molecular-modeling studies based on the
crystal structures of three opioid receptors revealed that a
hydrogen-bonding network among the functionalized moiety (a
nitrogen atom on an aromatic-ring system), the Gln residue at
the 2.60 position, and the nonconserved residue at the 2.63
position might decide the opioid receptor selectivity of these
ligands. The presence of such a network led to high MOR
selectivity over the KOR and DOR for ligand 2 (ONP). Among
the amide series of naltrexone derivatives, compounds 10, 11,
and 14 showed minimal MOR efficacy in the [35S]-GTPγS
binding assay. These three compounds also had no agonist-like
property and acted as potent MOR antagonists in the tail-flick
assay. Furthermore, compound 10 produced less severe
withdrawal symptoms than naloxone at high doses, and
compounds 11 and 14 behaved similarly to naltrexone in the
opioid-withdrawal assay. Collectively, because of the highest
MOR/KOR selectivity over the DOR, the minimal MOR/DOR
efficacy and low KOR efficacy in the [35S]-GTPγS binding
assay, the lack of agonist property and potent MOR antagonism
in the tail-flick assay, and the less severe withdrawal symptoms
compared to naloxone, ligand 10 (NNP) thus represents a new
lead compound to develop MOR/KOR dual-selective ligands
that might possess unique therapeutic value for opioid-abuse/
dependence treatment.
H
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precipitate was collected by filtration and dried under vacuum to give
the target compound as its hydrochloride salt, which was then used for
further analysis and biological assays.
EXPERIMENTAL SECTION
■
Chemical Synthesis. General Methods. Chemical reagents were
purchased from either Sigma-Aldrich or Alfa Aesar. TLC analyses were
carried out on Analtech Uniplate F254 plates. Chromatographic
purification was accomplished on silica gel columns (230−400 mesh,
Bodman). Melting points were obtained with a Fisher Scientific micro
melting-point apparatus without correction. IR spectra were recorded
on either a Nicolet iS10 or a Nicolet Avatar 360 FT-IR Instrument. 1H
(300, 400 MHz) and 13C (75, 100 MHz) nuclear magnetic resonance
(NMR) spectra were acquired at ambient temperature with
tetramethylsilane as the internal standard on a Varian Gemini
spectrometer or Bruker Ultrashield 400 Plus spectrometer, respec-
tively. MS analysis was performed on a Quattro II triple quadrupole
mass spectrometer, a Waters Micromass QTOF-II instrument (ESI
source), or an Applied Bio Systems 3200 Q trap with a turbo V source
for TurbolonSpray. HPLC analysis was performed with a Varian
ProStar 210 system on Microsorb-MV 100-5 C18 column (250 mm ×
4.6 mm) at 210/254 nm, eluting with acetonitrile (0.1% TFA)/water
at 1 mL/min over 15 to 50 min. All of the above analytical methods
were used to determine the purity of the newly synthesized
compounds, and their purity was confirmed to be ≥95%.
General Procedure 1. 14-O-Substituted Naltrexone Derivatives
Synthesis. The mixture of naltrexone (1 equiv), acyl chloride (3
equiv), and triethylamine (6 equiv) in dry DMF was heated at 100 °C
for 6 h under N2 protection. After cooling, the reaction mixture was
concentrated under vacuum to remove DMF. The resulting crude
intermediate was then dissolved in MeOH and a 4% H2SO4 aqueous
solution (potassium carbonate aqueous, pH ≈ 10, for compounds 5
and 9) and stirred overnight at ambient temperature. After
concentration, the residue was partitioned between water and
CH2Cl2. The water layer was extracted with CH2Cl2 three times.
The combined organic phase was then washed with brine and dried
over Na2SO4. After removal of the solvent under reduced pressure, the
resulting residue was purified using a silica gel column with a CH2Cl2/
MeOH (150:1 → 100:1) (1% NH3·H2O) solvent system as the eluent
to give the target products 2−9 as free base.
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-O-(pyridyl-2′-
1
carboxy)morphinan-6-one (2). Hydrochloride salt: H NMR (300
MHz, CD3OD) δ 8.97 (m, 1H, Ar−H), 8.71 (m, 1H, Ar−H), 8.27 (m,
1H, Ar−H), 8.18 (m, 1H, Ar−H), 6.75 (d, J = 8.1 Hz, 1H, C1−H),
6.73 (d, J = 8.1 Hz, 1H, C2−H), 4.92 (s, 1H, C5−H), 4.10 (d, J = 6.6
Hz, 1H), 3.50−3.27 (m, 4H), 3.07−2.92 (m, 2H), 2.92−2.67 (m, 2H),
2.30 (m, 1H), 2.10 (m, 1H), 1.72 (m, 2H), 1.15 (m, 1H), 0.86 (m,
1H), 0.78 (m, 1H), 0.58 (m, 2H). 13C NMR (75 MHz, CD3OD) δ
207.59, 161.31, 146.38, 142.36, 139.87, 138.89, 129.90, 127.39, 125.40,
119.98, 117.95, 117.50, 88.54, 61.69, 56.92, 52.99, 37.51, 34.17, 30.23,
29.30, 27.89, 26.90, 22.49, 4.97, 4.34, 1.56. MS (ESI) m/z: 447 (M +
H)+, 342. IR (KBr, cm−1) νmax: 3411, 1660, 1259, 794. mp 250 °C
(dec).
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-O-(pyridyl-3′-
carboxy)morphinan-6-one (3). Free base: 1H NMR (300 MHz,
CDCl3) δ 9.57 (d, J = 1.5 Hz, 1H), 8.86 (dd, J = 1.8, 5.1 Hz, 1H), 8.45
(m,1H), 7.52 (dd, J = 4.95, 7.95 Hz, 1H), 6.80 (d, J = 7.8 Hz, 1H),
6.66 (d, J = 8.1 Hz, 1H), 4.82 (s, 1H), 4.68 (d, J = 5.4 Hz, 1H), 3.18
(d, J = 18.3 Hz, 1H), 3.00 (m, 1H), 2.82−2.68 (m, 3H), 2.61 (dd, J =
5.85, 18.45 Hz, 1H), 2.40−2.20 (m, 4H), 1.85 (dt, J = 3.8, 14.32 Hz,
1H), 1.71 (m, 1H), 0.67 (m, 1H), 0.40 (m, 2H), 0.10 (m, 2H). 13C
NMR (75 MHz, CDCl3) δ 207.43, 163.37, 152.17, 149.96, 143.14,
139.31, 137.56, 137.42, 127.42, 123.75, 123.37, 119.76, 118.21, 93.97,
89.35, 58.86, 55.33, 50.85, 43.47, 35.29, 30.30, 26.56, 22.87, 9.00, 3.46,
3.30. MS (ESI) m/z: 447 (M + H)+, 342. IR (KBr, cm−1) νmax: 2946,
1716, 1282, 1108, 737. mp 202 °C (dec).
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-O-(pyridyl-4′-
carboxy)morphinan-6-one (4). Free base: 1H NMR (300 MHz,
CDCl3) δ 8.84 (m, 2H), 8.02 (m, 2H), 6.96 (d, J = 8.1 Hz, 1H), 6.75
(d, J = 8.4 Hz, 1H), 4.73 (s, 1H), 3.24 (d, J = 6.3 Hz, 1H), 3.14 (d, J =
18.9 Hz, 1H), 3.09−2.98 (m, 2H), 2.79−2.60 (m, 3H), 2.49−2.30 (m,
4H), 2.18 (m, 1H), 1.93 (m, 1H), 0.89 (m, 1H), 0.58 (m, 2H), 0.17
(m, 2H). Hydrochloride salt: 13C NMR (75 MHz, CD3OD) δ 207.27,
160.13, 147.69, 145.32, 143.50, 132.56, 129.71, 129.13, 127.53, 127.02,
123.80, 121.02, 120.88, 90.43, 70.16, 69.69, 62.11, 57.76, 49.17, 34.68,
30.97, 27.21, 23.81, 5.80, 5.23, 2.46. MS (ESI) m/z: 447 (M + H)+,
342, 224. IR (KBr, cm−1) νmax: 3385, 1755, 1724, 1270, 1241, 749. mp
190−195 °C.
General Procedure 2. 14-N-Substituted Naltrexone Derivatives
Synthesis with Acyl Chloride. To a solution of 14β-amino-7,8-
dihydro-17-cyclopropylmethyl-normorphinone (21, 1 equiv) in dry
CH2Cl2 was added acyl chloride (2 equiv) and Et3N (4 equiv) on an
ice-water bath under N2 protection. The mixture was then allowed to
stir overnight at ambient temperature and then concentrated under
reduced pressure.
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-O-
1
(benzoyloxy)morphinan-6-one (5). Free base: H NMR (300 MHz,
CDCl3) δ 8.21 (m, 2H), 7.62 (m, 1H), 7.49 (m, 2H), 6.97 (d, J = 8.1
Hz, 1H), 6.73 (d, J = 8.4 Hz, 1H), 4.72 (s, 1H), 3.24 (m, 1H), 3.13 (d,
J = 19.2 Hz, 1H), 3.10−2.95 (m, 1H), 2.80−2.60 (m, 3H), 2.50−2.40
(m, 4H), 2.34 (m, 1H), 2.18 (m, 1H), 1.90 (m, 1H), 0.89 (m, 1H),
0.58 (m, 2H), 0.17 (m, 2H). Hydrochloride salt: 13C NMR (75 MHz,
CD3OD) δ 205.92, 163.75, 147.54, 133.52, 132.66, 132.18, 129.39,
129.31, 123.44, 122.50, 119.74, 96.46, 93.10, 89.12, 69.35, 69.12,
62.11, 61.64, 58.04, 56.98, 33.76, 30.03, 26.89, 22.87, 4.91, 4.34, 1.56.
MS (ESI) m/z: 446 (M + H)+, 342. IR (KBr, cm−1) νmax: 3398, 1730,
1239, 1055, 710. mp 161−165 °C.
General Procedure 3. 14-N-Substituted Naltrexone Derivatives
Synthesis with Acid. On an ice-water bath, to a solution of acid (3
equiv) in anhydrous DMF (3 mL) were added N-(3-dimethylamino-
propyl)-N′-ethylcarbodiimide hydrochloride (EDCI, 3 equiv), hydro-
benzotriazole (HOBt, 3 equiv), 4 Å molecular sieves, and TEA (5.0
equiv) under N2 protection. Fifteen minutes later, 21 (1.0 equiv) in
DMF (1 mL) was added dropwise. The resultant mixture was allowed
to warm to ambient temperature gradually. Upon completion of the
reaction, the mixture was then filtered through Celite. The filtrate was
concentrated to remove DMF.
General Procedure 4. Saponification of 14-N-Substituted Amide
Intermediates. Methanol (5 mL) and K2CO3 (2 equiv) were added to
the residue obtained from procedure 2 or 3, and the mixture was
stirred at ambient temperature overnight. The mixture was then
filtered through Celite. The filtrate was concentrated to remove
methanol. The residue was then partitioned between CH2Cl2 (50 mL)
and brine (50 mL). The organic layer was separated, dried over
anhydrous MgSO4, and concentrated under reduced pressure. The
residue was then purified by column chromatography, eluting with
CH2Cl2/MeOH (1% NH3·H2O) to afford the corresponding
compound as free base.
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-O-(isoquinolyl-
1
3′-carboxy)morphinan-6-one (6). Free base: H NMR (300 MHz,
CDCl3) δ 8.73 (s, 1H), 8.46 (m, 1H), 8.06 (m, 1H), 7.89 (m, 3H),
7.07 (d, J = 8.1 Hz, 1H), 6.78 (d, J = 8.1 Hz, 1H), 4.74 (s, 1H), 3.66
(d, J = 4.5 Hz, 1H), 3.27 (d, J = 5.4 Hz, 1H), 3.16 (d, J = 18.6 Hz,
1H), 2.80−2.64 (m, 2H), 2.54−2.42 (m, 2H), 2.37 (m, 1H), 2.22 (dt,
J = 3.3, 12.08 Hz, 1H), 1.94 (m, 1H), 1.78−1.62 (m, 2H), 1.18 (m,
1H), 0.90 (m, 1H), 0.60 (m, 2H), 0.20 (m, 2H). 13C NMR (75 MHz,
CDCl3) δ 207.23, 161.70, 147.36, 138.94, 136.64, 132.23, 131.66,
130.75, 130.19, 129.85, 128.34, 128.16, 126.34, 125.01, 122.62, 119.05,
90.27, 69.63, 61.48, 58.79, 53.68, 50.25, 43.05, 33.68, 30.79, 30.38,
22.61, 8.97, 3.62, 3.45. MS (ESI) m/z: 497 (M + H)+. IR (KBr, cm−1)
νmax: 3392, 2921, 1725, 1182, 781. mp 201−204 °C.
General Procedure 5. Hydrochloride Salt Formation. Upon
1
confirmation by H NMR, the free base was then transformed into
hydrochloride salt by dissolving the base in MeOH (0.1 mL) and
CH2Cl2 (2 mL). HCl methanol solution (1.25 M, 4 equiv) was then
added with an ice-water bath, and the mixture was stirred for 5 min.
Diethyl ether (10 mL) was then added. Two hours later, the
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-O-(quinolyl-2′-
1
carboxy)morphinan-6-one (7). Hydrochloride salt: H NMR (300
MHz, DMSO-d6) δ 9.17 (brs, 1H, exchangeable), 8.70 (d, J = 8.1 Hz,
1H), 8.27 (d, J = 8.7 Hz, 2H), 8.18 (d, J = 7.5 Hz, 1H), 7.95 (m, 1H),
I
dx.doi.org/10.1021/jm4012214 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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7.83 (m, 1H), 7.24 (d, J = 8.1 Hz, 1H), 6.94 (d, J = 8.7 Hz, 1H), 5.20
(s, 1H), 4.10 (m, 1H), 3.57−3.35 (m, 2H), 3.30−3.14 (m, 2H), 3.10−
2.90 (m, 2H), 2.77 (m, 1H), 2.60 (m, 1H), 2.25−2.08 (m, 2H), 1.70−
1.50 (m, 2H), 1.14 (m, 1H), 0.71 (m, 1H), 0.65 (m, 1H), 0.56 (m,
1H), 0.46 (m, 1H). Free base: 13C NMR (75 MHz, CDCl3) δ 209.92,
163.00, 143.76, 143.11, 140.65, 138.64, 133.72, 131.41, 130.28, 128.51,
127.38, 123.52, 122.34, 121.09, 119.50, 117.66, 115.70, 90.06, 69.90,
61.56, 58.76, 50.60, 43.20, 35.79, 30.94, 30.18, 22.20, 8.96, 3.62, 3.39.
MS (ESI) m/z: 497 (M + H)+, 342. IR (KBr, cm−1) νmax: 3179, 1731,
1660, 1453, 1240, 730. mp 85−88 °C.
MHz, DMSO-d6) δ 9.19 (s, 1H, exchangeable), 8.76 (d, J = 6.0 Hz,
2H), 8.28 (s, 1H, exchangeable), 7.75 (d, J = 6.0 Hz, 2H), 6.60 (d, J =
8.04 Hz, 1H), 6.56 (d, J = 8.12 Hz, 1H), 5.00 (s, 1H), 4.07 (d, J = 5.2
Hz, 1H), 2.97 (d, J = 18.4 Hz, 1H), 2.69 (m, 2H), 2.47 (m, 1H), 2.44−
2.23 (m, 4H), 2.11 (m, 1H), 2.04 (m, 1H), 1.55 (m, 1H), 1.31 (m,
1H), 0.78 (m, 1H), 0.40 (m, 2H), 0.09 (m, 1H), 0.05 (m, 1H). 13C
NMR (100 MHz, DMSO-d6) δ 208.37, 165.68, 150.09 (×2), 143.40,
142.98, 139.06, 128.35, 124.32, 121.59 (×2), 119.04, 117.18, 88.54,
58.88, 57.79, 57.15, 48.61, 43.21, 36.39, 29.38, 28.03, 21.40, 9.62, 3.85,
3.37. MS m/z found 446.5 (M + H)+. IR (diamond, cm−1) νmax: 3270,
1710, 1686, 1604, 1510, 1221. mp 195 °C.
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-O-(quinolyl-3′-
1
carboxy)morphinan-6-one (8). Hydrochloride salt: H NMR (300
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-N-
MHz, CD3OD) δ 9.64 (m, 1H, Ar−H), 9.58 (m, 1H, Ar−H), 8.40−
8.17 (m, 3H, Ar−H), 7.98 (m, 1H, Ar−H), 7.27 (d, J = 8.1 Hz, 1H,
C1−H), 7.08 (d, J = 8.1 Hz, 1H, C2−H), 5.10 (s, 1H), 4.25 (m, 1H),
3.49 (m, 2H), 3.37 (m, 2H), 3.20 (m, 1H), 3.10 (m, 1H), 2.90 (m,
1H), 2.85 (m, 1H), 2.35 (m, 1H), 2.20 (m, 1H), 2.00 (m, 1H), 1.80
(m, 1H), 1.20 (m, 1H), 0.90 (m, 1H), 0.80 (m, 1H), 0.60 (m, 2H).
13C NMR (75 MHz, CD3OD) δ 206.38, 160.69, 147.10, 146.82,
144.14, 143.32, 134.82, 132.07, 129.70, 129.02, 128.44, 128.19, 127.06,
123.67, 123.29, 122.14, 120.07, 89.52, 69.37, 64.98, 61.44, 56.97,
33.87, 30.16, 26.93, 22.96, 13.57, 4.97, 4.42, 1.63. MS (ESI) m/z: 497
(M + H)+, 342. IR (KBr, cm−1) νmax: 3386, 1725, 1189, 762. mp 187
°C (dec).
(benzamido)morphinan-6-one (13). Compound 13 was prepared by
1
following general procedures 3 and 4 in 60% yield. H NMR (400
MHz, DMSO-d6) δ 9.18 (s, 1H, exchangeable), 8.06 (s, 1H,
exchangeable), 7.86 (m, 2H), 7.53 (m, 3H), 6.62 (d, J = 8.08 Hz,
1H), 6.57 (d, J = 8.12 Hz, 1H), 5.00 (s, 1H), 3.95 (d, J = 5.2 Hz, 1H),
2.98 (d, J = 18.5 Hz, 1H), 2.70 (m, 2H), 2.32 (m, 3H), 2.23 (m, 1H),
2.07 (m, 2H), 1.59 (m, 1H), 1.32 (m, 1H), 1.23 (m, 1H), 0.81 (m,
1H), 0.42 (m, 2H), 0.12 (m, 1H), 0.07 (m, 1H). 13C NMR (100 MHz,
DMSO-d6) δ 208.10, 167.23, 143.40, 138.97, 135.79, 131.09, 128.47,
128.28 (×2), 127.29 (×2), 124.31, 118.92, 117.14, 88.59, 58.82, 57.84,
57.04, 48.40, 43.15, 36.38, 29.53, 28.44, 21.32, 9.58, 3.83, 3.30. MS m/
z found 445.3 (M + H)+. IR (diamond, cm−1) νmax: 2958, 1721, 1659,
1508, 1302, 1112. mp 180−185 °C.
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-O-(2-
1
naphthoyloxy)morphinan-6-one (9). Hydrochloride salt: H NMR
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-N-[(3′-
isoquinolyl)carboxamido]-morphinan-6-one (14). Compound 14
was prepared by following general procedures 3 and 4 in 44% yield. 1H
NMR (400 MHz, DMSO-d6) δ 9.43 (s, 1H), 9.42 (s, 1H,
exchangeable), 9.20 (s, 1H, exchangeable), 8.63 (s, 1H), 8.27 (d, J =
8.1 Hz, 1H), 8.19 (d, J = 8.1 Hz, 1H), 7.89 (m, 1H), 7.82 (m, 1H),
6.63 (d, J = 8.0 Hz, 1H), 6.59 (d, J = 8.16 Hz, 1H), 4.93 (s, 1H), 3.68
(d, J = 5.4 Hz, 1H), 3.06 (d, J = 18.4 Hz, 1H), 2.84 (m, 1H), 2.47 (m,
1H), 2.32 (dd, J = 6.78, 12.62 Hz, 1H), 2.39 (m, 2H), 2.19 (m, 1H),
2.05 (m, 3H), 1.78 (m, 1H), 1.36 (d, J = 11.7 Hz, 1H), 0.89 (m, 1H),
0.50 (m, 2H), 0.23 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ
207.39, 164.26, 151.54, 143.41, 138.94, 135.39, 131.53, 129.28, 129.23,
128.42, 127.94, 127,86, 124.23, 119.62, 119.12, 117.36, 88.70, 59.19,
58.33, 55.89, 54.69, 48.01, 43.38, 36.39, 30.13, 29.46, 21.06, 9.32, 3.85,
3.53. MS m/z found 497.7 (M + H)+. IR (diamond, cm−1) νmax: 2953,
1721, 1676, 1508, 1305, 1111. mp 251−253 °C.
(300 MHz, CD3OD) δ 8.97 (m, 1H, Ar−H), 8.10 (m, 4H, Ar−H),
7.70 (m, 2H, Ar−H), 7.21 (d, J = 8.1 Hz, 1H, C1−H), 7.02 (d, J = 8.1
Hz, 1H, C2−H), 4.94 (s, 1H, C5−H), 4.19 (m, 1H), 3.68 (m, 1H),
3.56 (m, 3H), 3.06 (m, 2H), 2.87 (m, 1H), 2.76 (m, 1H), 2.53 (m,
1H), 2.38 (m, 1H), 2.16 (m, 1H), 1.86 (m, 1H), 1.34 (m, 1H), 1.10−
0.75 (m, 2H), 0.61 (m, 2H). 13C NMR (75 MHz, CD3OD) δ 206.04,
164.81, 135.49, 132.78, 132.02, 131.25, 128.70, 128.15, 127.72, 127.36,
127.04, 126.62, 124.37, 123.53, 122.32, 121.53, 119.80, 119.17, 96.52,
93.17, 89.18, 76.69, 69.37, 61.65, 56.96, 33.79, 26.90, 22.86, 4.92, 4.39,
1.50. MS (ESI) m/z: 496 (M + H)+, 342. IR (KBr, cm−1) νmax: 3386,
1732, 1189, 1056, 776. mp 137−140 °C.
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-N-[(2′-pyridyl)-
carboxamido]morphinan-6-one (10). Compound 10 was prepared
1
by following general procedures 3 and 4 in 45% yield. H NMR (400
MHz, DMSO-d6) δ 9.21 (s, 1H, exchangeable), 9.19 (s, 1H,
exchangeable), 8.69 (m, 1H), 8.13−8.01 (m, 2H), 7.65 (m, 1H),
6.63 (d, J = 8.08 Hz, 1H), 6.60 (d, J = 8.12 Hz, 1H), 4.91 (s, 1H), 3.67
(d, J = 5.4 Hz, 1H), 3.04 (d, J = 18.4 Hz, 1H), 2.76 (m, 1H), 2.55 (m,
2H), 2.42 (dd, J = 6.56, 12.72 Hz, 1H), 2.30 (dd, J = 6.70, 12.66 Hz,
1H), 2.15 (m, 1H), 2.10−1.88 (m, 3H), 1.71 (m, 1H), 1.35 (m, 1H),
0.83 (m, 1H), 0.44 (m, 2H), 0.16 (m, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 207.26, 163.73, 149.58, 148.36, 143.36, 138.96, 138.01,
128.31, 126.70, 124.06, 121.56, 119.01, 117.29, 88.59, 58.97, 58.27,
55.85, 47.96, 43.31, 36.34, 30.01, 29.28, 21.03, 9.25, 3.75, 3.46. MS m/
z found 446.3 (M + H)+. IR (diamond, cm−1) νmax: 2949, 1722, 1682,
1505, 1302, 1112. mp 195 °C (dec).
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-N-[(2′-
quinolyl)carboxamido]morphinan-6-one (15). The title compound
was prepared by following general procedures 3 and 4 in 40% yield.
1
Hydrochloride salt: H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H,
exchangeable), 9.12 (b, 1H, exchangeable), 8.65 (d, J = 8.6 Hz, 1H),
8.34 (d, J = 8.4 Hz, 1H), 8.21 (d, J = 8.5 Hz, 1H), 8.14 (d, J = 8.1 Hz,
1H), 7.94 (m, 1H), 7.78 (m, 1H), 6.79 (d, J = 8.1 Hz, 1H), 6.71 (d, J =
8.1 Hz, 1H), 5.57 (s, 1H), 5.49 (d, J = 5.8 Hz, 1H), 3.46 (m, 2H), 3.17
(m, 2H), 2.93 (m, 1H), 2.68 (m, 4H), 2.23 (m, 1H), 1.73 (m, 1H),
1.61 (m, 1H), 1.07 (m, 1H), 0.73 (m, 1H), 0.66 (m, 1H), 0.55 (m,
1H), 0.44 (m, 1H). 13C NMR (100 MHz, DMSO-d 6) δ 204.73,
163.58, 147.50, 143.19, 141.16, 137.77, 135.94, 128.41, 126.94, 126.66,
126.18, 125.74, 124.35, 118.50, 117.89, 116.33, 115.92, 85.60, 55.57
(×2), 54.92, 45.49 (×2), 43.65, 33.12, 24.56, 19.73, 3.22, 3.09. MS m/
z found 496.7 (M + H)+. IR (diamond, cm−1) νmax: 2946, 1722, 1680,
1499, 1320, 1112. mp 200−202 °C.
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-N-[(3′-pyridyl)-
carboxamido]morphinan-6-one (11). Compound 11 was prepared
1
by following general procedures 2 and 4 in 62% yield. H NMR (400
MHz, DMSO-d6) δ 9.19 (s, 1H, exchangeable), 9.03 (d, J = 1.64 Hz,
1H), 8.74 (dd, J = 1.62, 4.78 Hz, 1H), 8.26 (s, 1H, exchangeable), 8.17
(m, 1H), 7.55 (m, 1H), 6.60 (d, J = 8.08 Hz, 1H), 6.56 (d, J = 8.12 Hz,
1H), 5.00 (s, 1H), 4.06 (d, J = 5.4 Hz, 1H), 2.97 (d, J = 18.4 Hz, 1H),
2.71 (m, 2H), 2.47 (m, 1H), 2.44−2.23 (m, 4H), 2.11 (m, 1H) 2.04
(m, 1H), 1.56 (m, 1H), 1.32 (m, 1H), 0.78 (m, 1H), 0.39 (m, 2H),
0.08 (m, 1H), 0.04 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ
208.30, 165.59, 151.61, 148.36, 143.38, 139.02, 135.26, 131.55, 128.37,
124.28, 123.44, 118.94, 117.11, 88.52, 58.86, 57.66, 57.23, 48.57,
43.14, 36.38, 29.36, 28.12, 21.42, 9.60, 3.85, 3.20. MS m/z found 446.5
(M + H)+. IR (diamond, cm−1) νmax: 2924, 1712, 1677, 1523, 1241.
mp 192−195 °C.
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-N-[(3′-
quinolyl)carboxamido]morphinan-6-one (16). Compound 16 was
1
prepared by following general procedures 3 and 4 in 48% yield. H
NMR (400 MHz, DMSO-d6) δ 9.29 (d, J = 2.12 Hz, 1H), 9.20 (s, 1H,
exchangeable), 8.82 (d, J = 1.84 Hz, 1H), 8.42 (brs, 1H,
exchangeable), 8.13 (m, 2H), 7.88 (m, 1H), 7.72 (m, 1H), 6.62 (d,
J = 8.08 Hz, 1H), 6.58 (d, J = 8.12 Hz, 1H), 5.04 (s, 1H), 4.10 (d, J =
5.3 Hz, 1H), 2.99 (d, J = 18.4 Hz, 1H), 2.77 (m, 2H), 2.55 (m, 1H),
2.37 (m, 4H), 2.15 (m, 1H), 2.08 (m, 1H), 1.61 (m, 1H), 1.35 (m,
1H), 0.84 (m, 1H), 0.41 (m, 2H), 0.13 (m, 1H), 0.06 (m, 1H). 13C
NMR (100 MHz, DMSO-d6) δ 208.28, 165.62, 148.96, 148.29, 143.41,
139.04, 135.57, 131.09, 128.93, 128.66, 128.61, 128.41, 127.41, 126.46,
124.31, 118.97, 117.15, 88.57, 58.86, 57.76, 57.42, 48.61, 43.19, 36.42,
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-N-[(4′-pyridyl)-
carboxamido]morphinan-6-one (12). Compound 12 was prepared
1
by following general procedures 2 and 4 in 77% yield. H NMR (400
J
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29.46, 28.22, 21.48, 9.62, 3.88, 3.25. MS m/z found 496.3 (M + H)+.
IR (diamond, cm−1) νmax: 2929, 1715, 1659, 1499, 1305, 1102. mp 185
°C.
saline (Baxter Healthcare, Deerfield, IL) or sterile-filtered distilled/
deionized water.
Animals. Male Swiss-Webster mice (Harlan, Indianapolis, IN)
weighing 25 to 30 g were housed six per cage in animal care quarters 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 at Virginia
Commonwealth University Medical Center and comply with the
recommendations of the International Association for the Study of
Pain.
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-N-[(2′-
naphthamido]morphinan-6-one (17). The title compound was
1
prepared by following general procedures 3 and 4 in 45% yield. H
NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H, exchangeable), 8.46 (s,
1H), 8.24 (s, 1H, exchangeable), 8.03 (m, 3H), 7.94 (m, 1H), 7.62 (m,
2H), 6.61 (d, J = 8.08 Hz, 1H), 6.57 (d, J = 8.12 Hz, 1H), 5.03 (s, 1H,
C5−H), 4.00 (d, J = 5.3 Hz, 1H), 3.01 (d, J = 18.4 Hz, 1H), 2.73 (m,
2H), 2.56 (m, 1H), 2.34 (m, 4H), 2.09 (m, 2H), 1.63 (m, 1H), 1.35
(m, 1H), 0.83 (m, 1H), 0.43 (m, 2H), 0.16 (m, 1H), 0.09 (m, 1H).
13C NMR (100 MHz, DMSO-d6) δ 208.05, 167.19, 143.42, 138.98,
In Vitro Competitive Radioligand Binding Assay. The radioligand
binding assay and the [35S]-GTPγS binding assay were conducted
using monoclonal mice opioid receptor expressed in chinese hamster
ovary (CHO) cell lines as described previously.38,39,44 [3H]NLX,
[3H]NTI, and [3H]norBNI (or [3H]DPN) were used to label the
MOR, DOR, and KOR, respectively. Aliquots of a membrane protein
(30 μg) were incubated with the corresponding radioligand in the
presence of different concentrations of the drug under investigation in
TME buffer (50 mM Tris, 3 mM MgCl2, and 0.2 mM EGTA, pH 7.7)
at 30 °C for 1.5 h. The bound radioactive ligand was separated from
the free radioligand by filtration using the Brandel harvester
(Biomedical Research & Development Laboratories). Specific (i.e.,
opioid receptor-related) binding was determined as the difference in
binding obtained in the absence and presence of 5 μM naltrexone, 5
μM 1, and 5 μM 4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-
yl](3-methoxyphenyl)methyl]-N,N-diethylbenzamide 30 (SNC80)58
for the MOR, KOR, and DOR, respectively. The potency of the drugs
in displacing the specific binding of the radioligand was determined
from the specific binding using linear regression analysis of Hill plots.
The IC50 values were then determined and corrected to Ki values using
the Cheng−Prusoff equation.
138.0, 134.02, 133.05, 132.02, 128.64, 128.48, 127.88, 127.59, 127.52,
127.40, 126.76, 124.30, 118.92, 117.14, 88.62, 58.81, 58.00, 57.15,
48.42, 43.17, 36.41, 34.0, 28.53, 21.36, 9.61, 3.90, 3.31. MS m/z found
495.7 (M + H)+. IR (diamond, cm−1) νmax: 3029, 1717, 1668, 1506,
1302, 1021. mp 192−195 °C.
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-N-[2′-(pyridin-
2″-yl)acetamido]morphinan-6-one (18). The title compound was
1
prepared by following general procedures 3 and 4 in 43% yield. H
NMR (400 MHz, DMSO-d6) δ 9.17 (s, 1H, exchangeable), 8.51 (d, J =
4.16 Hz, 1H), 8.10 (b, 1H, exchangeable), 7.74 (m, 1H), 7.52 (d, J =
7.80 Hz, 1H), 7.25 (m, 1H), 6.58 (d, J = 8.0 Hz, 1H), 6.53 (d, J = 8.0
Hz, 1H), 4.82 (s, 1H), 3.92 (s, 1H), 3.78 (m, 2H), 2.91 (d, J = 18.12
Hz, 1H), 2.71 (m, 1H), 2.59 (m, 1H), 2.41 (m, 1H), 2.25 (m, 4H),
2.02 (m, 2H), 1.47 (m, 1H), 1.24 (m, 1H), 0.70 (m, 1H), 0.41 (m,
2H), 0.05 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 208.02,
169.21, 156.41, 148.85, 143.35, 139.06, 136.60, 128.53, 124.28, 123.67,
121.80, 119.04, 117.14, 88.45, 58.90, 56.83, 56.76, 48.36, 45.79, 43.45,
36.21, 29.04, 27.86, 21.24, 9.12, 3.62, 3.47. MS m/z found 460.2 (M +
H)+. IR (diamond, cm−1) νmax: 3011, 1720, 1689, 1537, 1304, 1015.
mp 195−198 °C.
In Vitro Functional Assay: Stimulation of [35S]-GTPγS Binding.
[35S]-GTPγS functional assays were conducted in the same cell
membranes used for the receptor binding assays. Membrane proteins
(10 μg) were incubated with varying concentrations of drugs, GDP
(10 μM), and 0.1 nM [35S]-GTPγS in assay buffer (50 mM Tris, 3 mM
MgCl2, 100 mM NaCl, and 0.2 mM EGTA, pH 7.7) for 1.5 h at 30 °C.
Nonspecific binding was determined with 20 μM unlabeled GTP[γS].
DAMGO (3 μM), 1 (5 μM), and 30 (5 μM) were included in the
assay for a maximally effective concentration of a full agonist for the
MOR, KOR, and DOR, respectively.
In Vivo Pharmacology. Tail-Flick Test. The warm-water tail-flick
test was performed according to Coderre and Rollman55 using a water
bath with the temperature maintained at 56 0.1 °C. All drugs and
test compounds were administered to mice subcutaneously (s.c.).
Before injecting, the baseline latency (control) of the mice was
determined. Only mice with a reaction time from 2 to 4 s were used.
The average baseline latency for the experiment 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 Pierson56 as the percentage of maximum possible effect (%
MPE), which was calculated as % MPE = [(test latency − control
latency)/(10 − control latency)] × 100. The percent MPE was
calculated for each mouse using at least six mice per drug.
Opioid-Withdrawal Assay. A 75 mg morphine pellet was implanted
into the base of the neck of male Swiss Webster mice following the
reported procedure.39 The animals were allowed to recover in their
home cages before testing. Mice were allowed to habituate for 30 min
in an open-topped, square, clear Plexiglas observation chamber (26 ×
26 × 26 cm3) with lines partitioning the bottom into quadrants before
they were given antagonist. All drugs and test compounds were
administered subcutaneously (s.c.). Withdrawal was precipitated at 72
h from pellet implantation with naloxone (1.0 mg/kg, s.c.), naltrexone
(1.0 mg/kg, s.c.), and the test compounds at indicated doses.
Withdrawal commenced within 1 min after antagonist administration.
Escape jumps and wet dog shakes were quantified by counting their
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-N-[3′-(pyridin-
2″-yl)propanamido]morphinan-6-one (19). The title compound was
1
prepared by following general procedures 3 and 4 in 38% yield. H
NMR (400 MHz, DMSO-d6) δ 9.15 (s, 1H, exchangeable), 8.46 (m,
1H), 7.68 (m, 1H), 7.67 (s, 1H, exchangeable), 7.30 (d, J = 7.8 Hz,
1H), 7.19 (m, 1H), 6.56 (d, J = 8.1 Hz, 1H), 6.51 (d, J = 8.1 Hz, 1H),
4.80 (s, 1H), 3.96 (d, J = 5.1 Hz, 1H), 3.03 (m, 2H), 2.89 (d, J = 18.4
Hz, 1H), 2.68 (m, 2H), 2.55 (m, 2H), 2.38 (m, 1H), 2.20 (m, 4H),
1.96 (m, 2H), 1.41 (m, 1H), 1.18 (m, 1H), 0.76 (m, 1H), 0.42 (m,
2H), 0.09 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 208.44,
171.68, 160.43, 148.76, 143.34, 138.97, 136.44, 128.63, 124.46, 122.82,
121.35, 118.99, 117.03, 88.49, 58.81, 56.64, 56.58, 48.40, 43.39, 36.17,
35.32, 33.18, 28.96, 27.83, 21.25, 9.35, 3.61, 3.52. MS m/z found 474.3
(M + H)+. IR (diamond, cm−1) νmax: 3020, 1721, 1674, 1505, 1304,
1031. mp 210−212 °C.
17-Cyclopropylmethyl-4,5α-epoxy-3-hydroxy-14β-N-{2′-[(pyri-
din-2″-yl)carboxamido]acetamido}morphinan-6-one (20). The title
compound was prepared by following general procedures 3 and 4 in
37% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.15 (s, 1H,
exchangeable), 9.08 (t, J = 5.64 Hz, 1H, exchangeable), 8.66 (m,
1H), 8.07 (m, 1H), 8.00 (m, 1H), 7.84 (s, 1H, exchangeable), 7.62 (m,
1H), 6.58 (d, J = 8.04 Hz, 1H), 6.51 (d, J = 8.08 Hz, 1H), 4.74 (s, 1H),
4.06 (d, J = 5.76 Hz, 2H), 3.69 (d, J = 5.32 Hz, 1H), 2.89 (d, J = 18.4
Hz, 1H), 2.71 (m, 1H), 2.52 (m, 1H), 2.46 (m, 1H), 2.16 (m, 4H),
1.97 (m, 2H), 1.55 (m, 1H), 1.23 (m, 1H), 0.53 (m, 1H), 0.27 (m,
2H), 0.02 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 207.64,
168.78, 164.19, 149.30, 148.52, 143.30, 138.97, 137.81, 128.54, 126.74,
124.20, 121.86, 119.04, 117.24, 88.43, 58.59, 57.58, 56.13, 48.20,
43.47, 42.95, 36.25, 29.31, 28.37, 21.06, 9.06, 3.48, 3.25. MS m/z
found 503.2 (M + H)+. IR (diamond, cm−1) νmax: 3014, 1718, 1691,
1652, 1532, 1319, 1029. mp 205−208 °C.
Biological Evaluation. Drugs. Morphine sulfate was purchased
from Mallinckrodt (St. Louis, MO) or provided by NIDA. Morphine
pellets (75 mg) and placebo pellets were provided by NIDA. Naloxone
and naltrexone was purchased from Sigma-Aldrich (St. Louis, MO).
All drugs and test compounds were dissolved in pyrogen-free isotonic
K
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Journal of Medicinal Chemistry
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occurrences over 20 min for each mouse using at least four mice per
drug.
Statistical Analysis. One-way ANOVA followed by the posthoc
Dunnett test were performed to assess significance using Prism 3.0
software (GraphPad Software, San Diego, CA).
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Molecular Modeling Study. Chemical structures of the ligands
were sketched in SybylX-2.0, and their Gasteiger−Huckel charges were
̈
assigned before energy minimization (10 000 iterations) with the TFF.
The X-ray crystal structures for MOR (4DKL), KOR (4DJH), and
DOR (4EJ4) were retrieved from the Protein Data Bank (PDB).
Automated docking on these retrieved receptor structures was done
utilizing genetic algorithm docking program GOLD 5.1. The binding
site was defined to include all atoms within 10 Å of the γ-carbon atom
of Asp3.32 for the three opioid crystal structures along with a hydrogen-
bond constraint between the 17-N of the ligand’s morphinan skeleton
and the carboxylate group of Asp3.32. The best CHEM-PLP scored
solutions were chosen for further analyses. All molecular dynamic
(MD) simulations were performed in SybylX-2.0 for 10 ps under NVT
ensemble. All of the residues outside the 15 Å sphere radius of 14-C of
the ligand were defined as aggregates, and MD simulations were run
after assigning Gasteiger−Huckel charges and initial temperature at
̈
300 K. The average structure of the last 1 ps of the simulation was
again energy minimized after assigning Gasteiger−Huckel charges for
̈
1000 iterations. Pictures were generated using PyMOL Molecular
Graphics System, version 1.5.0.4.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis and characterization of 14-aminonaltrexone 21, acid
side chain synthesis for ligands 19 and 20, and HPLC spectra of
ligands 2−20. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 804-828-0021. Fax: 804-828-7625. E-mail: yzhang2@
vcu.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Drs. Lee-Yuan Liu-Chen (Temple
University) and Ping-Yee Law (University of Minnesota) for
the generous gift of opioid receptor-expressing CHO cell lines.
Y.Y. and O.E. thank Irma B. Adams, Joanna C. Jacob, and
Jordan O. Cox for technical guidance with the biological assays.
The work was funded by a PHS grant from NIH/NIDA,
DA024022 (Y.Z.). 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.
ABBREVATIONS USED
■
CHO, chinese hamster ovary; DAMGO, [D-Ala2-MePhe4-
Gly(ol)5]enkephalin; DOR, delta opioid receptor; DPN,
diprenorphine; β-FNA, β-funaltrexamine; KOR, kappa opioid
receptor; MOR, mu opioid receptor; NLX, naloxone; NTX,
naltrexone; norBNI, nor-binaltorphimine; NTI, naltrindole;
NAP, 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-
[(4′-pyridyl)carboxamido]morphinan; NAQ, 17-cyclopropyl-
methyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3-
carboxamido)morphinan; TFF, TRIPOS force field
L
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