Synthesis and Pharmacological Evaluation of Novel C-8 Substituted Tetrahydroquinolines as Balanced-Affinity Mu/Delta Opioid Ligands for the Treatment of Pain

Article abstract of DOI:10.1021/acschemneuro.8b00139

The use of opioids for the treatment of pain, while largely effective, is limited by detrimental side effects including analgesic tolerance, physical dependence, and euphoria, which may lead to opioid abuse. Studies have shown that compounds with a μ-opioid receptor (MOR) agonist/δ-opioid receptor (DOR) antagonist profile reduce or eliminate some of these side effects including the development of tolerance and dependence. Herein we report the synthesis and pharmacological evaluation of a series of tetrahydroquinoline-based peptidomimetics with substitutions at the C-8 position. Relative to our lead peptidomimetic with no C-8 substitution, this series affords an increase in DOR affinity and provides greater balance in MOR and DOR binding affinities. Moreover, compounds with carbonyl moieties at C-8 display the desired MOR agonist/DOR antagonist profile whereas alkyl substitutions elicit modest DOR agonism. Several compounds in this series produce a robust antinociceptive effect in vivo and show antinociceptive activity for greater than 2 h after intraperitoneal administration in mice.

Full text of DOI:10.1021/acschemneuro.8b00139

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Synthesis and pharmacological evaluation of novel
C-8 substituted tetrahydroquinolines as balanced-
affinity mu/delta opioid ligands for the treatment of pain
Anthony F Nastase, Nicholas W. Griggs, Jessica P. Anand, Thomas J. Fernandez, Aubrie
A Harland, Tyler J Trask, Emily M. Jutkiewicz, John R. Traynor, and Henry I. Mosberg
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00139 • Publication Date (Web): 20 Apr 2018
Downloaded from http://pubs.acs.org on April 22, 2018
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Synthesis and pharmacological evaluation of novel Cꢀ8 substituted tetrahydroquinolines as
balancedꢀaffinity mu/delta opioid ligands for the treatment of pain
Anthony F. Nastase,a,b Nicholas W. Griggs,c Jessica P. Anand,c, d Thomas J. Fernandez,c Aubrie
A. Harland,a,b Tyler J. Trask,c Emily M. Jutkiewicz,c, d John R. Traynor,c, d Henry I. Mosberg*a,b,d
aDepartment of Medicinal Chemistry, College of Pharmacy, University of Michigan, 428 Church
Street, Ann Arbor, MI 48109, United States
bInterdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of
Michigan, Ann Arbor MI 48109, United States
cDepartment of Pharmacology, Medical School, University of Michigan, Ann Arbor, MI 48109,
United States
dEdward F Domino Research Center, University of Michigan, Ann Arbor, MI 48109
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Abstract
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The use of opioids for the treatment of pain, while largely effective, is limited by detrimental
side effects including analgesic tolerance, physical dependence, and euphoria, which may lead to
opioid abuse. Studies have shown that compounds with a ꢀꢀopioid receptor (MOR) agonist/δꢀ
opioid receptor (DOR) antagonist profile reduce or eliminate some of these side effects including
development of tolerance and dependence. Herein we report the synthesis and pharmacological
evaluation of a series of tetrahydroquinolineꢀbased peptidomimetics with substitutions at the Cꢀ8
position. Relative to our lead peptidomimetic with no Cꢀ8 substitution, this series affords an
increase in DOR affinity and provides greater balance in MOR and DOR binding affinities.
Moreover, compounds with carbonyl moieties at Cꢀ8 display the desired MOR agonist/DOR
antagonist profile whereas alkyl substitutions elicit modest DOR agonism. Several compounds in
this series produce a robust antinociceptive effect in vivo and show antinociceptive activity for
greater than two hours after intraperitoneal administration in mice.
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Keywords: Bifunctional ligands, dependence, drug abuse, opioids, peptidomimetics, tolerance
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Introduction
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Opioid analgesics are among the most effective drugs for the treatment of moderate to severe
pain. Complications from longꢀterm pain management with opioids include gastrointestinal
distress, opioidꢀinduced hyperalgesia and the development of analgesic tolerance and physical
dependence. Nearly all clinically prescribed opioid analgesics exert both their beneficial and
undesirable effects through activation of the ꢀꢀopioid receptor (MOR). Evidence suggests that
the δꢀopioid receptor (DOR) plays a key role in modulating some side effects associated with
opioids including analgesic tolerance and physical dependence.1,2 Classic studies using rodent
models have demonstrated that by coꢀadministering a DOR antagonist with morphine, or by
administering chronic morphine to DOR knockout mice, the development of analgesic tolerance,
physical dependence and drugꢀseeking behavior are attenuated.3–7 By simultaneously activating
MOR and blocking DOR, we aim to develop safer analgesics with reduced tolerance and
dependence profiles for the management of a variety of pain conditions.
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Current reports and reviews on bifunctional MOR/DOR ligands, which span a wide range of
scaffolds and physicochemical properties, have provided support for the development of
improved therapeutics that display favorable pharmacodynamic and pharmacokinetic profiles.6–12
Eluxadoline (Viberzi), an orally bioavailable, peripherallyꢀacting MOR agonist/DOR antagonist,
was FDA approved in 2015 for the treatment of irritable bowel syndrome with diarrhea (IBSꢀD)
and has shown to be effective and wellꢀtolerated in the clinic.13–15 Several labs have utilized
morphineꢀlike scaffolds to develop MOR agonist/DOR antagonist small molecules, which may
show promise as analgesics with reduced antinociceptive tolerance and physical dependence
liabilities.10,16,17 An alternative approach by Portoghese and colleagues targets the proposed
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MORꢀDOR heterodimer with a bivalent ligand, whereby the MOR agonist pharmacophore of
oxymorphone is linked with the DOR antagonist pharmacophore of naltrindole.18,19 Studies using
these bivalent ligands in animal models not only show decreased tolerance and dependence as a
function of linker length,18 but also report attenuated conditioned place preference (CPP) and
reinstatement, suggesting a lower abuse liability.19 We have recently reported on two MOR
agonist/DOR antagonist lead compounds, a glycosylated cyclic peptide7 and a peptidomimetic, 1
(Figure 1),20,21 that produce longꢀlasting and doseꢀdependent antinociception in mice after
peripheral administration. Elaboration of the peptidomimetic series primarily through
modification of the Cꢀ6 pendant and at Nꢀ1 resulted in MOR agonist/DOR antagonist analogues
with a range of relative affinities for MOR and DOR.22–24 Further in vivo evaluation of three
such analogs suggested that balanced MOR/DOR affinity is associated with amelioration of
tolerance, dependence, and reinforcing properties.25
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In order to determine what role, if any, balanced MOR/DOR affinities of MOR
agonist/DOR antagonist ligands plays in eliminating opioid side effects, we continue to
expand the SAR of this series for the development of MOR agonist/DOR antagonist
peptidomimetics with a range of relative MOR/DOR affinities. As noted above, our
SAR has focused primarily on substitutions at C-6 and N-1.
Figure 1. Lead peptidomimetic 1, featuring the THQ core and
numbering convention in blue. Current SAR exploration focuses on
substitutions at Cꢀ8.
To further understand and optimize the SAR for our tetrahydroquinoline (THQ) core, we
synthesized and evaluated a series of Cꢀ8 substitutions while keeping the pharmacophores at Cꢀ6
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and Nꢀ1 constant. We had previously found that ligands with bicyclic Cꢀ6 substitutions on the
unmodified THQ core preferentially bind MOR over DOR at least 10ꢀfold,22 and Nꢀ1
substitutions afford improved balance in MOR and DOR binding affinity, but often elicit partial
DOR agonism.24 The Cꢀ8 modifications presented in this series probe the spatial and electronic
constraints of a binding pocket previously unexplored in this series of peptidomimetics. Here we
report the synthesis and evaluation of a series of THQꢀbased ligands featuring a diverse set of
substitutions at Cꢀ8 as part of our effort to optimize the MOR agonist/DOR antagonist profile
and improve upon our lead peptidomimetic.
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Results/Discussion
Following the development of 1, we have extensively evaluated the SAR of our peptidomimetic
series by incorporating diverse substitutions at the Cꢀ6 position,21,22 also referred to as the
“pendant.” Prior SAR and computational docking studies have shown that a benzyl pendant at Cꢀ
6 is well tolerated by a deep binding pocket in both MOR and DOR.20,21,23 To validate the
importance of the aryl Cꢀ6 pharmacophore in receptor binding and activity, the benzyl pendant
was moved to the Cꢀ8 position (Table 1, Compound 2). Compound 2 showed significantly
decreased binding affinities (higher Ki) for all 3 opioid receptors (MOR, DOR and κꢀopioid
receptor (KOR)), and also showed decreased MOR potency (EC50 = 1200 nM) and efficacy
(37% stimulation compared to the standard MOR agonist DAMGO), as measured by [35S]ꢀ
GTPγS binding. Furthermore, compound 2 showed no appreciable activity at DOR or KOR. We
then questioned whether this reduction in MOR activity was due to the loss of the Cꢀ6
pharmacophore, or to unfavorable ligandꢀreceptor interactions at Cꢀ8. To examine this, we
combined the Cꢀ6 and Cꢀ8 benzyl substitutions, giving us compound 7a. As shown in Table 1,
the binding affinity as well as potency and efficacy of 7a at MOR were restored (Ki = 1 nM;
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EC50 = 4 nM; 96% stimulation), while the DOR binding affinity improved 10ꢀfold and efficacy
2ꢀfold compared to 1, not only validating the importance of the Cꢀ6 pharmacophore for MOR
activity, but identifying a key role for the Cꢀ8 pharmacophore in modulating DOR affinity and
efficacy. This moderate loss in MOR affinity and increase in DOR affinity shifted the
MOR/DOR binding ratio (DOR Ki/MOR Ki) from 43 for compound 1 to a more balanced 1.6 for
compound 7a (Table 1). Consequently, this 6ꢀ,8ꢀdisubstituted THQ analogue established Cꢀ8 as
a region of interest for future SAR with the principal aim of balancing MOR and DOR binding
affinities.
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Table 1. Effects of Benzyl Pendant Position on Binding Affinity, Potency and Efficacy
Table 1. Binding affinities (Ki) were obtained by competitive displacement of radiolabeled [3H]ꢀdiprenorphine in
membrane preparations. Functional data were obtained using agonist induced stimulation of [35S]ꢀGTPγS binding
assay. Potency is represented as EC50 (nM) and efficacy as percent maximal stimulation relative to standard agonist
DAMGO (MOR), DPDPE (DOR), or U69,593 (KOR) at 10 ꢁM. All values are expressed as the mean of three
separate assays performed in duplicate with standard error of the mean (SEM) in parenthesis. dns = does not
stimulate (<10%).
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In vitro Structure-Activity Relationships
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Subsequent compounds in the Cꢀ8 series explored the steric environment and depth of the Cꢀ8
binding pocket with various alkyl substitutions, ranging from methyl to tꢀbutyl (Table 2). We
extended this series to include halogens (F, CF3, Br), which largely fit the same trend as the alkyl
set. The alkyl and halogenated series generally showed potent, efficacious agonism at MOR and
partial agonism at DOR. Additionally, most alkylꢀsubstituted analogues showed no KOR
activation, whereas the halogenated compounds were partial agonists at KOR. In terms of
binding, the smallest Cꢀ8 substitutions (7b, 7c, 7g, 7h, 7q) maintained high affinity for MOR and
increased affinity for DOR relative to the unsubstituted lead peptidomimetic 1, however these
compounds bind MOR over DOR by at least 8:1. Conversely, larger Cꢀ8 substitutions (7d, 7e,
7f) slightly decreased MOR affinity and displayed a modest increase in DOR affinity, leading to
an improved balance of opioid receptor binding affinities.
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Table 2. Effects of Alkyl and Halogen Substitutions on Affinity, Potency and Efficacy
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Table 2. Binding affinities (Ki) were obtained by competitive displacement of radiolabeled [3H]ꢀdiprenorphine in
membrane preparations. Functional data were obtained using agonist induced stimulation of [35S]ꢀGTPγS binding
assay. Potency is represented as EC50 (nM) and efficacy as percent maximal stimulation relative to standard agonist
DAMGO (MOR), DPDPE (DOR), or U69,593 (KOR) at 10 ꢁM. All values are expressed as the mean of three
separate assays (n=3) performed in duplicate unless noted otherwise, with SEM in parenthesis. * indicates n=2; dns
= does not stimulate (<10% stimulation).
Expanding upon the alkyl and halogen subsets, we synthesized a series of analogues featuring
aryl, carbonyl, and amine substitutions, summarized in Table 3. The phenethyl (7l) and ethyl
ester (7n) Cꢀ8 substituted compounds both improved the MOR/DOR binding affinity ratio
relative to 1 (DOR Ki/MOR Ki = 4.1 and 3.7, respectively), however, they were not as balanced
as the benzyl Cꢀ8 substituted compound 7a (DOR Ki/MOR Ki = 1.6). Compounds 7l and 7n
maintained appreciable MOR efficacy (>70% stimulation), yet only modestly stimulated DOR
(≤15% stimulation), whereas 7a produced partial agonism at DOR (EC50 = 380 nM; 42%
stimulation). Evaluation of three amideꢀlinked Cꢀ8 substituted compounds (ethyl amide 7m,
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phenyl amide 7o, and benzyl amide 7p) resulted in binding affinities that favored MOR over
DOR approximately 10:1. Functionally, these compounds produced the desired MOR
agonist/DOR antagonist profile. Additionally, this series of carbonylꢀcontaining compounds
increased KOR affinity relative to 1 and displayed KOR antagonism. The carboxylic acidꢀ
substituted compound (7r) was the outlier in the carbonyl series, showing a significant loss of
KOR binding affinity (Ki = 210 nM), however affinity and activity at MOR and DOR were
comparable to the other compounds in the carbonyl series. The amine substitutions examined in
this series were all cyclic, tertiary amines (e.g. piperidine (7i), morpholine (7j), and piperazine
(7k). These Cꢀ8 substitutions elicited minor differences at MOR, with affinities and efficacies
comparable to 1, however, these compounds diverged markedly at DOR and KOR. Compound
7k displayed the weakest DOR affinity in this series by a considerable margin (Ki = 15 nM),
while 7i and 7j both displayed high affinity and partial agonism for DOR. Furthermore, while 7i
and 7k showed a large increase in KOR binding affinity (Ki = 0.93 and 1.9 nM, respectively) and
produced partial KOR agonism, 7j showed a more modest increase in KOR affinity (Ki = 7.3
nM) and did not stimulate KOR.
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Table 3. Effects of Aryl, Carbonyl and Amino Substitutions on Affinity, Potency and
Efficacy
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Table 3. Binding affinities (Ki) were obtained by competitive displacement of radiolabeled [3H]ꢀdiprenorphine in
membrane preparations. Functional data were obtained using agonist induced stimulation of [35S]ꢀGTPγS binding
assay. Potency is represented as EC50 (nM) and efficacy as percent maximal stimulation relative to standard agonist
DAMGO (MOR), DPDPE (DOR), or U69,593 (KOR) at 10 ꢁM. All values are expressed as the mean of three
separate assays (n=3) performed in duplicate unless noted otherwise, with SEM in parenthesis. * indicates n=2; dns
= does not stimulate (<10% stimulation).
In vivo antinociceptive activity. All final compounds excluding 7k and 7o were evaluated in
vivo for antinociceptive activity using the mouse warm water tail withdrawal (WWTW) assay
(Table 4). In the alkyl series, compounds 7b, c and e were fully efficacious, showing dose
dependent antinociception and reaching the cutoff latency of 20 seconds at 10 mg/kg after
intraperitoneal (ip) administration, whereas 7d showed no significant antinociceptive effect at the
same dose. The tꢀbutyl analogue 7f was partially active in vivo, with a latency of 10 seconds at
10 mg/kg. In the carbonyl series, only the ethyl ester analogue 7n also showed full efficacy.
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Larger substitutions including the cyclic amines 7i and j, aryl rings 7a and l, and amides 7m, o
and p, produced no antinociception at the doses tested. Additionally, the relatively small
carboxylic acid 7r and halogenated analogues 7g, h and q produced no antinociceptive effect at
the doses tested. Results of the in vivo screening are summarized in Table 4. Of the bioactive
analogues 7b, c, e and n, the duration of action for 7e and n proved to be the longest at 2.5 hours.
This is a modest improvement over the lead 1 (2 hours).
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Table 4. Antinociceptive Activity of Compounds 7a-r in Mouse WWTW assay
C-8 substitution
Antinociceptive
activity at 10 mg/kg
Duration of
action
C-8 substitution
Antinociceptive
activity at 10 mg/kg
Duration of
action
7a
7b
7c
7d
7e
7f
benzyl
No activity
Fully efficacious
Fully efficacious
No activity
7j
methylmorpholine
methylpiperazine
phenethyl
No activity
Did not test
No activity
methyl
1.5 hr
1.0 hr
7k
7l
ethyl
n-propyl
n-butyl
7m
7n
7o
7p
7q
7r
ethyl amide
No activity
Fully efficacious
Partially active
No activity
2.5 hrs
ethyl ester
Fully efficacious
Did not test
No activity
2.5 hrs
t-butyl
phenyl amide
benzyl amide
bromo
7g
7h
7i
fluoro
trifluoromethyl
methylpiperidine
No activity
No activity
No activity
carboxylic acid
No activity
Table 4. Results from the mouse WWTW assay after ip administration of compound 7aꢀr at 10 mg/kg.
Antinociceptive activities defined as fully efficacious for 20 s latency to tail withdrawal, partially active for 10 s
above baseline, or no activity for no significant difference from baseline. Duration of action is defined here as the
time it takes to return to baseline after a 10 mg/kg bolus injection of test compound.
All compounds reported in this series maintained a high binding affinity at MOR and
demonstrated partial to full MOR agonism in vitro compared to the standard full agonist
DAMGO. With compound 7k as the sole exception, all compounds had improved DOR affinity
relative to the lead compound 1. Accordingly, both lipophilic and polar Cꢀ8 substitutions
provided compounds with a greater balance in MOR/DOR receptor binding affinity. While most
compounds displayed DOR agonist activity, those with carbonyl Cꢀ8 substitutions (7m, n, o, p
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and r) were consistently found to lack efficacy in the GTPγS binding assay. We have previously
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shown that peptidomimetics that do not produce GTPγS binding are in fact functional
antagonists, as demonstrated by a shift in the concentration response curve of a standard agonist.
Using SNC80 as a standard DOR agonist, we calculated the Ke values of 7m, n, o, p, and r to be
25.4 nM, 42.5 nM, 31.5 nM, 16.7 nM, and 15.7 nM, respectively, confirming their DOR
antagonist proerties.
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Based on our computational models,26 we predict the Cꢀ8 substitutions to primarily interact with
extracellular loop 2 and the Nꢀterminal tail of MOR and DOR, and to a lesser extent with
transmembrane helices 3, 4 and 5. Due to the flexibility in these regions of the receptor,
accurately correlating computationally predicted ligandꢀreceptor interactions with the SAR data
was not feasible. Comprehensive evaluation of this series of compounds suggests that Cꢀ8
carbonyl moieties block DOR activation quite effectively and bulky alkyl and aryl groups, such
as nꢀbutyl and phenethyl, respectively, attenuate DOR activation relative to smaller alkyl, aryl,
and halogenꢀcontaining groups. We have previously shown that a bulky Cꢀ6 pendant interacts
favorably with the activeꢀstate MOR binding pocket, yet there is a steric clash between a large Cꢀ
6 pendant and the analogous amino acid residues in the activeꢀstate DOR.21,26 We propose from
our SAR analysis that the activeꢀstate binding pockets of MOR and DOR likely interact with the
Cꢀ8 substitutions in a similar manner. For preferentially binding the MOR activeꢀstate pocket
and the DOR inactiveꢀstate pocket with a high affinity, a bulky Cꢀ6 pendant and carbonyl moiety
at the Cꢀ8 position are key pharmacophore elements that produce the desired MOR agonist/DOR
antagonist profile with an improved receptor binding affinity balance.
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Moreover, in addition to balancing binding affinty at MOR and DOR and demonstrating that Cꢀ8
carbonyl substitutions produce DOR antagonism in vitro, this series afforded several compounds
with promising in vivo antinociceptive activities. We have shown that four compounds (7b, c, e,
and n) produced a full antinociceptive effect in the WWTW assay in mice after peripheral
administration. Contrary to expectations, 7d, which incorporated the nꢀpropyl substitution,
showed no effect in vivo whereas 7e, having the nꢀbutyl substitution, produced full
antinociceptive activity. 7d has only a single carbon difference from the bioactive 7c and 7e, so it
is surprising that its in vivo effects would be drastically different. Among the carbonyl subset,
only the ethyl ester (7n) was fully efficacious.
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As shown in Table 4, most compounds in this series demonstrated no antinociceptive activity at
the doses tested in vivo. While we cannot definitively attribute a loss of activity to any individual
factor for all compounds in the series, typical physicochemical properties such as high molecular
weight, lipophilicity, polar surface area, and hydrogen bond partners are likely to inhibit
membrane permeability and access to the CNS. Accordingly, our reported in vivo SAR was
constrained to small Cꢀ8 modifications. Small alkyl substitutions (7b-f) showed the best
correlation with in vivo antinociceptive activity. However, the relatively low molecular weight
fluoroꢀsubstituted compounds (7g and h) showed no activity in vivo at the doses tested,
indicating pharmacokinetic obstacles besides molecular weight. Another pair of relatively low
molecular weight analogues with differing in vivo effects, 7m and n, suggest new parameters
affecting bioavailability. Although 7m and n are comparable in size, 7m features an hydrogen
bond donating amide moiety, whereas 7n bears a more lipophilic, hydrogen bond accepting ester
functionality. The added hydrogen bond donating amide may affect specific interactions with
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proteins that impact CNS access (e.g. active transporters, efflux proteins, metabolizing enzymes),
or nonspecific parameters, including polarity, and by extension, passive membrane permeability.
Unsurprisingly the carboxylic acidꢀsubstituted compound (7r) showed no in vivo activity despite
its relatively low molecular weight, likely due to the poor bloodꢀbrain barrier permeability of
carboxylic acid moieties. Lastly, the relatively large aryl rings (7a and l), aryl amides (7o and p),
and amine heterocycles (7i and k) evoked no response in vivo at the doses tested, likely due to a
combination of unfavorable physicochemical parameters.
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In summary, we have shown that modifications at the Cꢀ8 position of the THQ scaffold of our
peptidomimetic series maintain high MOR affinity while improving DOR affinity, thus
achieving our goal of developing more balanced receptor binding affinity compounds.
Additionally, we have identified the Cꢀ8 carbonyl moiety as a key pharmacophore element for
blocking DOR activation, thereby achieving the pharmacologically favorable MOR agonist/DOR
antagonist profile. Although it is not known that a 1:1 MOR/DOR binding affinity ratio is
optimal, preliminary in vivo data for a set of related compounds in our peptidomimetic series
show the greatest reduction in tolerance and dependence when MOR/DOR affinity is relatively
balanced.25 Confirmation of this trend will require evaluation of tolerance and dependence
liabilities of additional analogs with a range of MOR and DOR affinities. The compounds
reported to have in vivo activity here (7b, c, e and n) encompass a range of MOR/DOR affinity
ratios (7.9, 21, 4.7, and 3.7, respectively) and therefore should provide valuable data supporting
or refuting this trend. In conclusion, these bioavailable peptidomimetics serve as promising leads
in the search for future MOR agonist/DOR antagonist analgesics.
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Methods
Synthesis
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All reagents and solvents were obtained commercially and were used without further
purification. Intermediates were purified by flash chromatography using a Biotage Isolera One
instrument. Most purification methods utilized a hexanes/ethyl acetate solvent system, with a
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linear
gradient
between
20
and
100%
ethyl
acetate,
though
dichloromethane/methanol/triethylamine solvent systems were utilized for some polar, amineꢀ
containing intermedites. Purification of final compounds was performed using a Waters
semipreparative HPLC with a Vydac protein and peptide C18 reverse phase column, using a
linear gradient of 0% solvent B (0.1% TFA in acetonitrile) in solvent A (0.1% TFA in water) to
100% solvent B in solvent A at a rate 1% per minute, monitoring UV absorbance at 230 nm. The
purity of final compounds was assessed using a Waters Alliance 2690 analytical HPLC
instrument with a Vydac protein and peptide C18 reverse phase column. A linear gradient
(gradient A) of 0% solvent B in solvent A to 70% solvent B in solvent A in 70 min, measuring
UV absorbance at 230 nm was used to determine purity. All final compounds used for testing
were ≥95% pure, as determined by analytical HPLC. 1H NMR and 13C NMR data were obtained
on a 500 MHz Varian spectrometer using CDCl3 or CD3OD solvents. The identities of final
compounds were verified by mass spectrometry using an Agilent 6130 LC–MS mass
spectrometer in positive ion mode.
Compounds presented in this series were synthesized as described in Schemes 1 and 2, starting
with a commercially available, appropriately substituted aniline. In Scheme 1, anilines 1a-h were
first coupled with 3ꢀbromopropionyl chloride (i), yielding intermediates 2a-h. These
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Scheme 1: (i) 3-bromopropionyl chloride, K2CO3. (ii) NaOtBu. (iii) TfOH. (iv) NBS. (v) see Scheme 1b. (vi) benzyl
(5a-k) or phenethyl (5l) boronic acid, Pd(dppf)Cl2, K2CO3. (vii, viii) see Schemes 1c & 1d.
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1a
1b
1c
1d
1e
1f
R1 = Bn
R1 = H
R1 = Br
R1 = H
R1 = H
R1 = H
R1 = Br
R1 = H
R2 = H
2a
2b
2c
2d
2e
2f
R1 = Bn
R1 = H
R1 = Br
R1 = H
R1 = H
R1 = H
R1 = Br
R1 = H
R2 = H
3a
3b
3c
3d
3e
3f
R1 = Bn
R1 = H
R1 = Br
R1 = H
R1 = H
R1 = H
R1 = Br
R1 = H
R2 = H
4a a
4b a
4c
R1 = Bn
R1 = Br
R1 = Br
R1 = Br
R1 = Br
R1 = Br
R1 = Br
R1 = Br
R1 = Br
R1 = Br
R1 = Br
R2 = Br
R2 = Me
R2 = Et
R2 = Me
R2 = Et
R2 = Me
R2 = Et
R2 = Me
R2 = Et
R2 = n-Pr
R2 = n-Bu
R2 = t-Bu
R2 = F
R2 = n-Pr
R2 = n-Bu
R2 = t-Bu
R2 = F
R2 = n-Pr
R2 = n-Bu
R2 = t-Bu
R2 = F
4d a
4e a
4f a
4g
R2 = n-Pr
R2 = n-Bu
R2 = t-Bu
1g
1h
2g
2h
3g
3h
R2 = F
R2 = CF3
R2 = CF3
4h a
4i b
4j b
4k b
R2 = CF3
R2 = CF3
R2 = methylpiperidine (from 3b)
R2 = methylmorpholine (from 3b)
R2 = methylpiperazine (from 3b)
intermediates then underwent (ii) a baseꢀcatalyzed βꢀlactam formation (3a-h). This was followed
by a triflic acidꢀcatalyzed Fries rearrangement (iii) to produce the THQ core (4c, 4g). In order to
achieve the desired aryl bromide substitution pattern for further functionalization, compounds
denoted as following Scheme 1a were treated with Nꢀbromosuccinimide to install a bromine at
either Cꢀ8 (4a) or Cꢀ6 (4b-h). Compounds 4i-k, shown in Scheme 1b, were derived from 3b.
After cyclization and aromatic bromination, the THQ amine was trifluoroacetylꢀprotected, then
the methyl group at Cꢀ8 underwent benzylic bromination with Nꢀbromosuccinimide and benzoyl
peroxide. The benzylic bromide was
then
substituted
under
basic
conditions with potassium carbonate
and the specified amine (along with
partial loss of the trifluoroacetyl
protecting group), leaving the Cꢀ8
substituted aryl bromides 4i-k.
Complete TFA loss observed in step vi.
Scheme 1b: (iii) TfOH. (iv) NBS. (v) TFAA. (v’) NBS, benzoyl peroxide
(v’’) amine, K2CO3.
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To convert intermediates 4 to 5, the aryl bromide was functionalized via either Suzuki coupling
(step vi, Scheme 1) to give 5a-l, or carbonylation (step vii, Scheme 1c) to give 5n and 4a’
followed by amide coupling (step viii, Scheme 1d) for 5m, o, and p.
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Scheme 1c: (vii) CO, Pd(dppf)Cl , MeOH (5n) or H O (4a’)
Scheme 1d: (viii) amine, PyBOP, DIPEA
2
2
Scheme 2: (ix) (R)-(+)-2-methyl-2-propanesulfinamide, then NaBH4. (x) HCl, then diBocDmt, PyBOP, 6-Cl
HOBt, DIPEA, then TFA. (xi) LiOH (prior to TFA deprotection)
5a
5b
5c
R2 = Bn
6a
6b
6c
6d
6e
6f
R2 = Bn
7a
7b
7c
7d
7e
7f
R2 = Bn
R2 = Me
R2 = Me
R2 = Me
R2 = Et
R2 = Et
R2 = Et
5d
5e
5f
R2 = n-Pr
R2 = n-Pr
R2 = n-Pr
R2 = n-Bu
R2 = n-Bu
R2 = n-Bu
R2 = t-Bu
R2 = t-Bu
R2 = t-Bu
5g
R2 = F
6g
6h
6i
R2 = F
7g
7h
7i
R2 = F
5h
5i
R2 = CF3
R2 = CF3
R2 = CF3
R2 = methylpiperidine
R2 = methylmorpholine
R2 = methylpiperazine
R2 = methylpiperidine
R2 = methylmorpholine
R2 = methylpiperazine
R2 = EtPh
R2 = methylpiperidine
R2 = methylmorpholine
R2 = methylpiperazine
R2 = EtPh
5j
6j
7j
5k
6k
6l
7k
7l
R2 = EtPh
(from 4a)
5l
5m d
5n c
5o d
5p d
4a
6m
6n
6o
6p
6q
R2 = CONHEt
R2 = COOEt
R2 = CONHPh
R2 = CONHBn
7m
7n
7o
7p
7q
7r e
R2 = CONHEt
R2 = COOEt
R2 = CONHPh
R2 = CONHBn
R2 = Br
R2 = CONHEt (from 4a)
R2 = COOMe (from 4a)
R2 = CONHPh (from 4a)
R2 = CONHBn (from 4a)
R2 = Br
R2 = Br
(from 4a)
R2 = COOH
(from 6n)
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In Scheme 2, intermediates 5a-p (and 4a) were carried forward through a reductive amination
step that utilized Ti(OEt)4 and a chiral Ellman sulfinamide, followed by NaBH4 to yield the
desired R stereochemistry at Cꢀ4. During this step, the methyl ester of 5n converted to an ethyl
ester, likely due to nucleophilic attack by ethoxide ions liberated from the titanium complex.
Addition of concentrated HCl (x) cleaved the sulfinamide, leaving a primary amine HCl salt.
This amine then underwent amide coupling with N-,O-diBoc 2’,6’ꢀdimethylꢀLꢀtyrosine
(diBocDmt), followed by Boc deprotection with trifluoroacetic acid, yielding final compounds
7a-q. Compound 7r was produced by hydrolysis (xi) of the ester from 6n, prior to Boc
deprotection. All final compounds were purified by semiꢀpreparative reverseꢀphase HPLC.
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In Vitro Pharmacology
Cell Lines and Membrane Preparations. All tissue culture reagents were purchased from
Gibco Life Sciences (Grand Island, NY, U.S.). C6ꢀrat glioma cells stably transfected with a rat
MOR (C6ꢀMOR) or rat DOR (C6ꢀDOR) and Chinese hamster ovary (CHO) cells stably
expressing a human KOR (CHOꢀKOR) were used for all in vitro assays. Cells were grown to
confluence at 37 °C in 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) containing 10%
fetal bovine serum and 5% penicillin/streptomycin. Membranes were prepared by washing
confluent cells three times with ice cold phosphate buffered saline (0.9% NaCl, 0.61 mM
Na2HPO4, 0.38 mM KH2PO4, pH 7.4). Cells were detached from the plates by incubation in
warm harvesting buffer (20 mM HEPES, 150 mM NaCl, 0.68 mM EDTA, pH 7.4) and pelleted
by centrifugation at 1600 rpm for 3 min. The cell pellet was suspended in iceꢀcold 50 mM Trisꢀ
HCl buffer, pH 7.4, and homogenized with a Tissue Tearor (Biospec Products, Inc., Bartlesville,
OK, U.S.) for 20 s. The homogenate was centrifuged at 15,000 rpm for 20 min at 4°C. The pellet
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was rehomogenized in 50 mM TrisꢀHCl with a Tissue Tearor for 10 s, followed by
recentrifugation. The final pellet was resuspended in 50 mM TrisꢀHCl and frozen in aliquots at
80°C. Protein concentration was determined via a BCA protein assay (Thermo Scientific Pierce,
Waltham, MA, U.S.) using bovine serum albumin as the standard.
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Radioligand Competition Binding Assays. Radiolabeled compounds were purchased from
PerkinꢀElmer (Waltham, MA, U.S.). Opioid ligand binding assays were performed by
competitive displacement of 0.2 nM [3H]ꢀdiprenorphine (250 ꢁCi, 1.85 TBq/mmol) by the
peptidomimetic from membrane preparations containing opioid receptors as described above.
The assay mixture, containing membranes (20 ꢁg protein/tube) in 50 mM TrisꢀHCl buffer (pH
7.4), [3H]ꢀdiprenorphine, and various concentrations of test peptidomimetic, was incubated at
room temperature on a shaker for 1 h to allow binding to reach equilibrium. For several
peptidomimetics, radioligand competition binding assays were performed in high Na+ buffer (50
mM TrisꢀHCl, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, pH 7.4) and samples were incubated
at room temperature for 75 min to allow binding to reach equilibrium. Samples were rapidly
filtered through Whatman GF/C filters using a Brandel harvester (Brandel, Gaithersburg, MD,
U.S.) and washed five times with 50 mM TrisꢀHCl buffer. Bound radioactivity on dried filters
was determined by liquid scintillation counting, after saturation with EcoLume liquid
scintillation cocktail, in a Wallac 1450 MicroBeta (PerkinꢀElmer, Waltham, MA, U.S.).
Nonspecific binding was determined using 10 ꢁM naloxone. The results presented are the mean
± standard error (S.E.M.) from at least three separate assays performed in duplicate. K (nM)
i
values were calculated using nonlinear regression analysis to fit a logistic equation to the
competition data using GraphPad Prism, version 6.0c, for Mac OS X (GraphPad Software Inc.,
La Jolla, CA).
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[35S]-GTPγS Binding Assays. Agonist stimulation of [35S]guanosine 5′ꢀOꢀ[γꢀ thio]triphosphate
[35S]ꢀGTPγS, 1250 Ci, 46.2 TBq/mmol) binding to G protein was measured as described
previously.27 Briefly, membranes (10–20 ꢁg of protein/tube) were incubated for 1 h at 25°C in
GTPγS buffer (50 mM TrisꢀHCl, 100 mM NaCl, 5 mM MgCl , pH 7.4) containing 0.1 nM [35S]ꢀ
2
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GTPγS, 30 ꢁM guanosine diphosphate (GDP), and varying concentrations of test
peptidomimetic. G protein activation following receptor activation with peptidomimetic was
compared with 10 ꢁM of the standard compounds [DꢀAla2,NꢀMePhe4,Glyꢀol]enkephalin
(DAMGO) at MOR, DꢀPen2,5ꢀ enkephalin (DPDPE) at DOR, or U69,593 at KOR. The reaction
was terminated by vacuum filtration of GF/C filters that were washed 10 times with GTPγS
buffer. Bound radioactivity was measured as previously described. The results are presented as
the mean ± standard error (S.E.M.) from at least three separate assays performed in duplicate;
potency (EC50 (nM)) and percent stimulation were determined using nonlinear regression
analysis with GraphPad Prism, as above.
Ke Determination. Agonist stimulation of [35S]ꢀGTPγS binding by the known standard agonist
SNC80 at DOR was measured as described above. This was then compared to [35S]ꢀGTPγS
binding stimulated by SNC80 in the presence of test compound (1000 nM). Both conditions
produced 100% stimulation relative to SNC80. The difference between the EC50 of SNC80 alone
and in the presence of test antagonist is the shift in concentration response. The Ke was then
calculated as Ke = (concentration of compound)/ (concentration response shift – 1). The results
presented are the mean from at three separate assays performed in duplicate.
In Vivo Characterization of Compounds
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Drug preparation. All compounds were administered by intraperitoneal (ip) injection in a
volume of 10 mL/kg of body weight. Test compounds were dissolved in 5% DMSO (v/v) in
sterile saline (0.9% NaCl w/v).
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Animals. Male C57BL/6 wild type mice (Stock number 000664, Jackson Laboratory,
Sacramento CA, USA) bred inꢀhouse from breeding pairs and weighing between 20ꢀ30 g at 8ꢀ16
weeks old, were used for behavioral experiments. Mice were groupꢀhoused with free access to
food and water at all times. Experiments were conducted in the housing room, maintained on a
12 h light/dark cycle with lights on at 7:00 am; all experiments were conducted during the light
cycle. Studies were performed in accordance with the University of Michigan Committee on the
Use and Care of Animals and the Guide for the Care and Use of Laboratory Animals (National
Research Council, 2011 publication).
Antinociception. Antinociceptive effects were evaluated in the mouse WWTW assay.
Withdrawal latencies were determined by briefly placing a mouse into a cylindrical plastic
restrainer and immersing 2ꢀ3 cm of the tail tip into a water bath maintained at 50°C. The latency
to tail withdrawal or rapidly flicking the tail back and forth was recorded with a maximum cutoff
time of 20 sec (50°C) to prevent tissue damage. Antinociceptive effects were determined using a
cumulative dosing procedure. Each mouse received an injection of saline ip and then 30 min later
baseline withdrawal latencies were recorded. Following baseline determinations, cumulative
doses of the test compound (1, 3.2, and 10 mg/kg) were given ip at 30 min intervals. Thirty min
after each injection, the tail withdrawal latency was measured as described above. To determine
the duration of antinociceptive action, baseline latencies were determined as described above.
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Thirty minutes after baseline determination, animals were given a 10 mg/kg bolus injection of
test compound ip. Latency to tail withdrawal was then determined at 5, 15, and 30 min after
injections, and every 30 min thereafter until latencies returned to baseline values.
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Supporting information
Extended synthetic schemes
Molecular formula strings (CSV)
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Abbreviations
MOR, ꢀꢀopioid receptor; DOR, δꢀopioid receptor; KOR, κꢀopioid receptor; DAMGO,
[DꢀAla2, NꢀMePhe4, Glyꢀol]ꢀenkephalin; DPDPE, [DꢀPen2,DꢀPen5] enkephalin; THQ,
tetrahydroquinoline; WWTW, warm water tail withdrawal; diꢀBocꢀDmt, N, O-Boc 2’,6’ꢀ
dimethylꢀLꢀtyrosine; DIPEA, N, Nꢀdiisopropylethylamine; PyBOP, benzotriazolꢀ1ꢀ
yloxytripyrrolidinophosphonium hexafluorophosphate; 6ꢀCl HOBt, 1ꢀhydroxyꢀ6ꢀchloroꢀ
benzotriazole; CPP, conditioned place preference; MIDA, Nꢀmethyliminodiacetic acid.
Corresponding Author Information
Henry Mosberg
College of Pharmacy, University of Michigan
428 Churc St., Ann Arbor, MI 48109ꢀ1065
Phone: 734ꢀ764ꢀ8117
Fax: 734ꢀ763ꢀ5595
Email: him@umich.edu
Author Contributions
A.F.N. conceived of, synthesized and performed chemical characterization of 17 of 18
novel final compounds and their related intermediates. A.A.H. synthesized and
characterized compound 7g and its preceding intermediates. A.F.N. and A.A.H. worked
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under the direction of H.I.M. In vitro assays were performed by N.W.G. with assistance
from T.J.F. and T.J.T. under the direction of J.R.T. Animal assays were performed by
J.P.A. under the direction of E.M.J.
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A.F.N. wrote the manuscript with support from N.W.G. and H.I.M. and feedback from
J.P.A., T.J.F., E.M.J., and J.R.T.
Funding Sources
This study was supported by NIH grant DA003910 (H.I.M, E.M.J and J.R.T). J.P.A. was
supported by the Substance Abuse Interdisciplinary Training Program administered by
NIDA, 5 T32 DA007267, and N.W.G. by the Pharmacological Sciences Training
Program, T32ꢀGM007767.
Conflict of Interest
The authors declare no competing financial interest.
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References
5
6
7
8
(1) Porreca, F., Takemori, A. E., Sultana, M., Portoghese, P. S., Bowen, W. D., and Mosberg, H.
I. (1992) Modulation of muꢀmediated antinociception in the mouse involves opioid deltaꢀ2
receptors. J. Pharmacol. Exp. Ther. 263, 147–152.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(2) Zhu, Y., King, M. a., Schuller, A. G. P., Nitsche, J. F., Reidl, M., Elde, R. P., Unterwald, E.,
Pasternak, G. W., and Pintar, J. E. (1999) Retention of supraspinal deltaꢀlike analgesia and loss
of morphine tolerance in δ opioid receptor knockout mice. Neuron 24, 243–252.
(3) Abdelhamid, E. E., Sultana, M., Portoghese, P. S., and Takemori, A. E. (1991) Selective
blockage of delta opioid receptors prevents the development of morphine tolerance and
dependence in mice. J. Pharmacol. Exp. Ther. 258, 299–303.
(4) Fundytus, M. E., Schiller, P. W., Shapiro, M., Weltrowska, G., and Coderre, T. J. (1995)
Attenuation of morphine tolerance and dependence with the highly selective δꢀopioid receptor
antagonist TIPP[ψ]. Eur. J. Pharmacol. 286, 105–108.
(5) Hepburn, M. J., Little, P. J., Gingras, J., and Kuhn, C. M. (1997) Differential effects of
naltrindole on morphineꢀinduced tolerance and physical dependence in rats. J. Pharmacol. Exp.
Ther. 281, 1350–1356.
(6) Schiller, P. W., Fundytus, M. E., Merovitz, L., Weltrowska, G., Nguyen, T. M., Lemieux, C.,
Chung, N. N., and Coderre, T. J. (1999) The opioid mu agonist/delta antagonist DIPPꢀNH(2)[ψ]
produces a potent analgesic effect, no physical dependence, and less tolerance than morphine in
rats. J. Med. Chem. 42, 3520–3526.
(7) Mosberg, H. I., Yeomans, L., Anand, J. P., Porter, V., SobczykꢀKojiro, K., Traynor, J. R.,
and Jutkiewicz, E. M. (2014) Development of a bioavailable ꢁ opioid receptor (MOPr) agonist, δ
opioid receptor (DOPr) antagonist peptide that evokes antinociception without development of
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Page 26 of 29
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2
3
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acute tolerance. J. Med. Chem. 57, 3148–3153.
5
6
7
8
(8) Dietis, N., Guerrini, R., Calo, G., Salvadori, S., Rowbotham, D. J., and Lambert, D. G.
(2009) Simultaneous targeting of multiple opioid receptors: A strategy to improve sideꢀeffect
profile. Br. J. Anaesth. 103, 38–49.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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33
34
35
36
37
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(9) Schiller, P. W. (2010) Biꢀ or multifunctional opioid peptide drugs. Life Sci. 86, 598–603.
(10) Ananthan, S., Saini, S. K., Dersch, C. M., Xu, H., McGlinchey, N., Giuvelis, D., Bilsky, E.
J., and Rothman, R. B. (2012) 14ꢀAlkoxyꢀ and 14ꢀacyloxypyridomorphinans: ꢁ agonist/ δ
antagonist opioid analgesics with diminished tolerance and dependence side effects. J. Med.
Chem. 55, 8350ꢀ8363.
(11) Salvadori, S., Trapella, C., Fiorini, S., Negri, L., Lattanzi, R., Bryant, S. D., Jinsmaa, Y.,
Lazarus, L. H., and Balboni, G. (2007) A new opioid designed multiple ligand derived from the
ꢁ opioid agonist endomorphinꢀ2 and the δ opioid antagonist pharmacophore DmtꢀTic.
Bioorganic Med. Chem. 15, 6876–6881.
(12) Turnaturi, R., Aricò, G., Ronsisvalle, G., Parenti, C., and Pasquinucci, L. (2016) Multitarget
opioid ligands in pain relief: new players in an old game. Eur. J. Med. Chem. 108, 211–228.
(13) Dove, L. S., Lembo, A., Randall, C. W., Fogel, R., Andrae, D., Davenport, J. M., McIntyre,
G., Almenoff, J. S., and Covington, P. S. (2013) Eluxadoline benefits patients with irritable
bowel syndrome with diarrhea in a phase 2 study. Gastroenterology 145, 329–338.e1.
(14) SobolewskaꢀWłodarczyk, A., Włodarczyk, M., Storr, M., and Fichna, J. (2016) Clinical
potential of eluxadoline in the treatment of diarrheaꢀpredominant irritable bowel syndrome. Ther.
Clin. Risk Manag. 12, 771–775.
(15) Lembo, A. J., Lacy, B. E., Zuckerman, M. J., Schey, R., Dove, L. S., Andrae, D. A.,
Davenport, J. M., McIntyre, G., Lopez, R., Turner, L., and Covington, P. S. (2016) Eluxadoline
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4
5
6
7
8
9
for irritable bowel syndrome with diarrhea. N. Engl. J. Med. 374, 242–253.
(16) Ananthan, S., Kezar, H. S., Carter, R. L., Saini, S. K., Rice, K. C., Wells, J. L., Davis, P.,
Xu, H., Dersch, C. M., Bilsky, E. J., Porreca, F., and Rothman, R. B. (1999) Synthesis, opioid
receptor binding, and biological activities of naltrexoneꢀderived pyridoꢀ and
pyrimidomorphinans. J. Med. Chem. 42, 3527–3538.
10
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(17) Wells, J. L., Bartlett, J. L., Ananthan, S., and Bilsky, E. J. (2001) In vivo pharmacological
characterization of SoRI 9409, a nonpeptidic opioid muꢀagonist/deltaꢀantagonist that produces
limited antinociceptive tolerance and attenuates morphine physical dependence. J. Pharmacol.
Exp. Ther. 297, 597–605.
(18) Daniels, D. J., Lenard, N. R., Etienne, C. L., Law, P.ꢀY., Roerig, S. C., and Portoghese, P. S.
(2005) Opioidꢀinduced tolerance and dependence in mice is modulated by the distance between
pharmacophores in a bivalent ligand series. Proc. Natl. Acad. Sci. U. S. A. 102, 19208–19213.
(19) Lenard, N. R., Daniels, D. J., Portoghese, P. S., and Roerig, S. C. (2007) Absence of
conditioned place preference or reinstatement with bivalent ligands containing muꢀopioid
receptor agonist and deltaꢀopioid receptor antagonist pharmacophores. Eur. J. Pharmacol. 566,
75–82.
(20) Wang, C., McFayden, I., Traynor, J. R., and Mosberg, H. I. (1998) Design of a high affinity
peptidomimetic opioid agonist from peptide pharmacophore models. Bioorganic Med. Chem.
Lett. 8, 2685–2688.
(21) Mosberg, H. I., Yeomans, L., Harland, A. a., Bender, A. M., SobczykꢀKojiro, K., Anand, J.
P., Clark, M. J., Jutkiewicz, E. M., and Traynor, J. R. (2013) Opioid peptidomimetics: Leads for
the design of bioavailable mixed efficacy ꢁ opioid receptor (MOR) agonist/δ opioid receptor
(DOR) antagonist ligands. J. Med. Chem. 56, 2139–2149.
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(22) Bender, A. M., Griggs, N. W., Anand, J. P., Traynor, J. R., Jutkiewicz, E. M., and Mosberg,
H. I. (2015) Asymmetric synthesis and in vitro and in vivo activity of tetrahydroquinolines
featuring a diverse set of polar substitutions at the 6 position as mixed efficacy ꢁ opioid
receptor/δ opioid receptor ligands. ACS Chem. Neurosci.6, 1428ꢀ1435..
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(23) Harland, A. A., Yeomans, L., Griggs, N. W., Anand, J. P., Pogozheva, I. D., Jutkiewicz, E.
M., Traynor, J. R., and Mosberg, H. I. (2015) Further optimization and evaluation of
bioavailable, mixedꢀefficacyꢂ ꢁꢀopioid receptor (mor) agonists/ δ ꢀopioid receptor (dor)
antagonists: balancing mor and dor affinities. J. Med. Chem. 58, 8952–8969.
(24) Harland, A. A., Bender, A. M., Griggs, N. W., Gao, C., Anand, J. P., Pogozheva, I. D.,
Traynor, J. R., Jutkiewicz, E. M., and Mosberg, H. I. (2016) Effects of nꢀsubstitutions on the
tetrahydroquinoline (thq) core of mixedꢀefficacy ꢁꢀopioid receptor (mor)/δꢀopioid receptor (dor)
ligands. J. Med. Chem. 59, 4985–4998.
(25) Anand, J. P., Kochan, K. E., Nastase, A. F., Montgomery, D., Griggs, N. W., Traynor, J. R.,
Mosberg, H. I., and Jutkiewicz, E. M. (in press) In vivo effects of ꢁ opioid receptor agonist/δ
opioid receptor antagonist peptidomimetics following acute and repeated administration. Br. J.
Pharmacol. doi: 10.1111/bph.14148
(26) Anand, J. P., Purington, L. C., Pogozheva, I. D., Traynor, J. R., and Mosberg, H. I. (2012)
Modulation of opioid receptor ligand affinity and efficacy using active and inactive state receptor
models. Chem. Biol. Drug Des. 80, 763–770.
(27) Traynor, J. R., and Nahorski, S. R. (1995) Modulation by muꢀopioid agonists of guanosineꢀ
5’ꢀOꢀ(3ꢀ[35S]thio)triphosphate binding to membranes from human neuroblastoma SHꢀSY5Y
cells. Mol. Pharmacol. 47, 848–854.
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Table of Contents Graphic
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this series:
lead peptidomimetic
balanced MOR/DOR affinity
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O
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HN
HN
NH2
NH2
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OH
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R = alkyl, halo, aryl, H-bond
donors and/or acceptors
For Table of Contents Only
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