Opioid peptidomimetics: Leads for the design of bioavailable mixed efficacy μ opioid receptor (MOR) agonist/δ opioid receptor (DOR) antagonist ligands

Article abstract of DOI:10.1021/jm400050y

We have previously described opioid peptidomimetic, 1, employing a tetrahydroquinoline scaffold and modeled on a series of cyclic tetrapeptide opioid agonists. We have recently described modifications to these peptides that confer a μ opioid receptor (MOR) agonist, δ opioid receptor (DOR) antagonist profile, which has been shown to reduce the development of tolerance to the analgesic actions of MOR agonists. Several such bifunctional ligands have been reported, but none has been demonstrated to cross the blood-brain barrier. Here we describe the transfer of structural features that evoked MOR agonist/DOR antagonist behavior in the cyclic peptides to the tetrahydroquinoline scaffold and show that the resulting peptidomimetics maintain the desired pharmacological profile. Further, the 4R diastereomer of 1 was fully efficacious and approximately equipotent to morphine in the mouse warm water tail withdrawal assay following intraperitoneal administration and thus a promising lead for the development of opioid analgesics with reduced tolerance.

Full text of DOI:10.1021/jm400050y

Article
pubs.acs.org/jmc
Opioid Peptidomimetics: Leads for the Design of Bioavailable Mixed
Efficacy μ Opioid Receptor (MOR) Agonist/δ Opioid Receptor (DOR)
Antagonist Ligands
Henry I. Mosberg,*,†,‡ Larisa Yeomans,† Aubrie A. Harland,‡ Aaron M. Bender,‡
Katarzyna Sobczyk-Kojiro,† Jessica P. Anand,‡ Mary J. Clark,§ Emily M. Jutkiewicz,§ and John R. Traynor§
†Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, United States
‡Interdepartmental Program in Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
§Department of Pharmacology, Medical School, University of Michigan, Ann Arbor, Michigan 48109, United States
ABSTRACT: We have previously described opioid peptidomi-
metic, 1, employing a tetrahydroquinoline scaffold and modeled
on a series of cyclic tetrapeptide opioid agonists. We have
recently described modifications to these peptides that confer a
μ opioid receptor (MOR) agonist, δ opioid receptor (DOR)
antagonist profile, which has been shown to reduce the
development of tolerance to the analgesic actions of MOR
agonists. Several such bifunctional ligands have been reported,
but none has been demonstrated to cross the blood−brain
barrier. Here we describe the transfer of structural features that
evoked MOR agonist/DOR antagonist behavior in the cyclic peptides to the tetrahydroquinoline scaffold and show that the
resulting peptidomimetics maintain the desired pharmacological profile. Further, the 4R diastereomer of 1 was fully efficacious
and approximately equipotent to morphine in the mouse warm water tail withdrawal assay following intraperitoneal
administration and thus a promising lead for the development of opioid analgesics with reduced tolerance.
side chain sulfurs as an ethylene dithioether. This peptide
exhibits high affinity for MOR and DOR (and low affinity for
the κ opioid receptor, KOR) and is a MOR agonist/DOR
antagonist.7 Follow-up studies8 revealed that steric bulk and
conformational constraint of the Aic or similar substitutions
were key factors for achieving in vitro MOR agonism/DOR
antagonism in this series.
INTRODUCTION
■
The recognition that the simultaneous modulation of multiple
targets may generate a more desirable drug profile has
challenged the long prevailing, intuitive bias toward selectively
targeted drugs as the optimal approach for the discovery and
development of new therapeutics (for recent reviews see refs
1−3). This concept is exemplified in the field of opioid
analgesics by the observation that coadministration of a μ
opioid receptor (MOR) agonist with a δ opioid receptor
(DOR) antagonist retains MOR-mediated analgesia but
displays reduced development of tolerance and dependence,4−6
features that limit the clinical use of opioid analgesics.
For pharmacokinetic simplicity it is preferable to incorporate
all desired activities into a single compound, and the
development of bifunctional opioid ligands has thus become
a topic of increasing interest. For many years, our focus had
been on receptor selective opioids. While structure−activity
efforts toward this aim were quite successful, a number of
“failures” (including ligands that displayed high affinity for both
MOR and DOR) resulted and were not pursued further. The
subsequent accumulation of convincing evidence for the value
of MOR agonist/DOR antagonist ligands encouraged us to
reinvestigate our earlier nonselective peptides. This reinvesti-
gation led to the development of a cyclic tetrapeptide Tyr-
c(SEtS)[D-Cys-Aic-D-Pen]OH (KSK103),7 where Aic is 2-
aminoindane-2-carboxylic acid, Pen is penicillamine, and
c(SEtS) designates cyclization through the D-Cys and D-Pen
Other peptide,9 peptide-like,10,11 and non-peptide12 struc-
tures have been reported that display MOR agonist/DOR
antagonist profiles; however, none of these have demonstrated
centrally mediated in vivo activity after peripheral admin-
istration. This poor bioavailability is typical of peptides, and
while several approaches have been demonstrated to improve
peptide penetration of biological membranes,13−15 the
alternative strategy of incorporating the key pharmacophore
elements of a peptide with desired pharmacological properties
into a more druglike scaffold provides a more direct strategy
toward improved bioavailability. The main hurdle that must be
cleared in the latter approach is ensuring that the resulting
peptidomimetic does indeed demonstrate the desired pharma-
cological profile.
We have previously described the peptidomimetic diastereo-
meric pair of 1, designed to incorporate the key opioid
pharmacophore elements of the parent tetrapeptide Tyr-
Received: January 10, 2013
Published: February 18, 2013
© 2013 American Chemical Society
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c(SS)[D-Cys-Phe-D-Pen]OH, 2 (JOM-13,16 Figure 1), and
related cyclic tetrapeptides, namely, a tyramine moiety and a
a
Scheme 1. Asymmetric Synthesis of Compound 7
a
Reagents and conditions: (a) (Boc)2O, DMAP, DIEA, DCM, reflux;
(b) (S)-2-methyl-CBS-oxazoborolidine, BH3·Me2S, THF; (c) phtha-
limide, DIAD, PPh3, THF; (d) N2H4·H2O, EtOH.
Figure 1. (A) Structure of lead peptide 2 (JOM-13, Tyr-c(SS)[D-Cys-
Phe-D-Pen]OH). (B) Structure of lead peptidomimetic 1(4R). (C)
Superposition of proposed bioactive conformations of JOM-13 and
1(4R) (see ref 16).
tion of this unequivocal 4S diastereomer, HPLC revealed a 9:1
ratio of late eluting to early eluting diastereomer of 1,
confirming that the late eluting diastereomer is 4S and the
(higher affinity) early eluting diastereomer is 4R, depicted in
Figure 1.
second aromatic group, attached to a tetrahydroquinoline
(THQ) scaffold.16 This design strategy proved to be successful,
as the higher affinity diastereomer of 1 displayed high binding
affinity to MOR, DOR, and KOR.14 Our observation that Aic
and other replacements for Phe in cyclic peptides related to 2
confer a MOR agonist/DOR antagonist profile suggested that 1
might be a promising starting point for the development of
related peptidomimetics with similar profiles but with improved
bioavailability compared to the peptides. Here we establish that
the higher affinity diastereomer of 1 is the 4R diastereomer
(1(4R)) and demonstrate that (a) 1(4R) displays a promising
in vitro profile with high MOR efficacy and low DOR efficacy,
(b) 1(4R) is approximately equipotent to morphine in the
antinociceptive mouse warm water tail withdrawal assay after
intraperitoneal administration, thus showing great promise as a
lead for the development of a bioavailable MOR agonist/DOR
antagonist, and (c) modifications that confer high MOR
efficacy/low DOR efficacy in our cyclic peptide series retain this
effect when used as replacements for the benzyl pendant on the
THQ scaffold of 1.
Opioid Receptor Binding and Efficacy. Binding affinities
(Ki) (obtained by competitive displacement of radiolabeled
[3H]diprenorphine in C6 cells stably expressing MOR or DOR
or CHO cells stably expressing KOR, as previously
described20,21) for both diastereomers of 1 and efficacy
(assessed by agonist-stimulated [35S] GTPγS binding in the
same cells22) at MOR, DOR, and KOR of the higher affinity 4R
diastereomer are shown in Tables 1 and 2, respectively. As
Table 1. Opioid Receptor Binding Affinities of
a
Peptidomimetics
binding, Ki (nM)
compd
MOR
DOR
KOR
b
1(4R)
0.22 0.02
0.24 0.02
0.76 0.14
0.078 0.007
0.16 0.04
6.3 2.5
9.4 0.8
8.9 1.5
6.0 0.7
68
25
17
54
2
1
1
7
15a
15b
15c
10
2
15d
4.1 1.6
1.2 0.4
c
morphine
171 18.9
60.9 17.3
220 48
b
1(4S)
2.6 0.3
56
120 15
66
180 14
5
RESULTS
■
15a′
15c′
15d′
16 2.6
1200 290
Synthesis of 1. Our original synthesis of 1 yielded the 4R
and 4S diastereomeric pair, which were easily separated by RP-
HPLC and pharmacologically evaluated individually, with the
observation that the diastereomer that elutes earlier on HPLC
displays 5- to 10-fold higher binding affinity at MOR, DOR,
and KOR than the later eluting diastereomer. In order to
confirm the stereochemistry of 1, we undertook an asymmetric
synthesis (Scheme 1). Briefly, ketone intermediate 3 was Boc
protected to give 4, which was reduced with the (S)-methyl-
CBS catalyst17 to give chiral, 4R alcohol 5 in 80% ee, similar to
previous reports for analogous scaffolds.17,18 The secondary
chiral alcohol was then converted to an amine, with complete
inversion of stereochemistry via a Mitsunobu reaction,19
yielding chiral, 4S amine 7 to which di-Boc protected 2,6-
dimethyl-L-tyrosine (Boc-Dmt) was coupled. After deprotec-
1.6 0.4
1
130
110
9
9
8.0 0.8
a
Binding affinities (Ki) were obtained by competitive displacement of
radiolabeled [3H]diprenorphine in membrane preparations. All values
are expressed as the mean SEM of three separate assays performed
in duplicate. From ref 16. From ref 36.
b
c
shown in Table 1, the now confirmed 4R diastereomer of 1
displays very high MOR affinity and is approximately 40- and
300-fold selective for MOR vs DOR or KOR, respectively. The
4S diastereomer shows significantly lower affinity, especially at
MOR. As seen in Table 2, the 4R diastereomer of 1 shows high
MOR potency and efficacy in the stimulation of GTPγS binding
assay. Consistent with the binding data, the potency of 1(4R) is
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a
Table 2. Opioid Receptor Efficacy of Peptidomimetics
EC50 (nM)
% stimulation
DOR
compd
1(4R)
MOR
DOR
110
dns
KOR
MOR
KOR
1.6 0.3
6
540 72
dns
81
2
5
5
3
1
5
16
2
22
2
15a
1.1 0.3
45
93
96
86
57
dns
15
dns
dns
dns
38
15b
0.84 0.35
0.53 0.08
0.24 0.03
194 21
69 35
dns
dns
1
15c
dns
dns
dns
15d
dns
68 15
2
b
morphine
nt
nt
a
Efficacy data were obtained using agonist induced stimulation of [35S]GTPγS binding assay. Efficacy is represented as EC50 (nM) and percent
maximal stimulation relative to standard agonist DAMGO (MOR), DPDPE (DOR), or U69,593 (KOR) at 10 μM. All values are expressed as the
b
mean SEM of three separate assays performed in duplicate. dns: does not stimulate. nt: not tested. From ref 7.
much lower at DOR and KOR. Of special interest is the
observation that 1(4R) is a partial agonist at DOR and KOR,
displaying only 16% and 22% maximal stimulation compared to
the standards DPDPE and U69,593, respectively, suggesting a
very promising starting point for the development of MOR
agonist/DOR antagonist analogues.
Since, as described above, other MOR agonist/DOR
antagonist ligands have been reported, the promise of THQ
scaffold peptidomimetics like 1 is dependent on the ability of
compounds in such a series to display centrally mediated effects
after peripheral administration, a property not yet demon-
strated for other MOR agonist/DOR antagonist ligands. Thus,
we examined the antinociceptive activity of the lead compound
1(4R) in the mouse warm water tail withdrawal (WWTW)
antinociception assay23 following intraperitoneal (ip) admin-
istration.
The dose−response curve for 1(4R) in the WWTW assay is
presented in Figure 2 which shows that 1(4R) produces a dose
Figure 3. Time-course of antinociception (WWTW assay) of 1(4R)
(filled squares) and morphine (open circles) following ip admin-
istration.
60 min followed by a slow decline. The duration of action of
1(4R) is somewhat shorter than that of morphine.
Encouraged by the antinociceptive activity and bioavailability
of 1(4R), we prepared a small series of analogues in which the
benzyl side chain of 1 was replaced by bulkier or more
constrained aromatic moieties. These replacements were based
on results we had obtained in the tetrapeptide and
pentapeptide series developed from 2. Using computational
models of active and inactive states of MOR and DOR,7,8 we
predicted and confirmed that replacing the Phe3 residue in
these tetra- or pentapeptides with a bulkier or more constrained
aromatic residue would maintain MOR efficacy but reduce
DOR efficacy. In particular, 1-naphthylalanine (1-Nal), 2-
naphthylalanine (2-Nal), and 2-aminoindane-2-carboxylic acid
(Aic), substitution for Phe all resulted in analogues that
displayed MOR agonist/DOR antagonist behavior and
maintained high affinity binding to both receptors.7,8,24 Since
peptidomimetic 1 was designed from 2 (Figure 1), we
examined the effect of transferring the corresponding
modifications to the THQ scaffold of 1 in place of the benzyl
moiety (Figure 4), anticipating that, as in the peptide series,
MOR agonist/DOR antagonist profiles would be observed.
Synthesis of Peptidomimetic Analogues of 1. Com-
pounds 8a,b were prepared by P2O5-catalyzed Friedel−Crafts
acylation of benzene or naphthalene with the corresponding p-
nitrocarboxylic acid (Scheme 2).25 8b was synthesized as a
mixture of the 1-naphthalene and 2-naphthalene products in a
1:4 ratio. 8c was prepared by Suzuki coupling26 between 2-
naphthaleneboronic acid and p-nitrobenzyl bromide, and 8d
was prepared by an aldol condensation between 1-indanone
and p-nitrobenzoic acid. The resulting nitro compounds were
Figure 2. Antinociceptive activity of 1(4R) in mouse warm water tail
withdrawal (WWTW) assay following intraperitoneal (ip) admin-
istration. Data represent response following pretreatment (−30 min)
with saline (open circles) or naltrexone (NTX, filled circles) given by
ip injection.
dependent increase in tail withdrawal latency (up to the 20 s
cutoff) with an ED50 ≈ 3 mg/kg (∼6 μmol/kg). Figure 2
further shows that this antinociceptive effect is opioid receptor
mediated, since the opioid antagonist naltrexone at 1 mg/kg ip
produces a rightward shift in the dose/response curve of 1(4R).
The time course of the antinociceptive activity of 1(4R) in the
WWTW assay, along with that for morphine, is shown in
Figure 3. As seen there, 1(4R) displays a rapid onset of effect
and maximal antinociception is maintained for approximately
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a
Scheme 3. Preparation of Compounds 15a−d
Figure 4. Structures of peptidomimetics.
a
Scheme 2. Preparation of Compounds 9a−d
a
Reagents and conditions: (a) 3-bromopropionyl chloride, K2CO3,
DCM; (b) NaOtBu, DMF; (c) TfOH, DCE; (d) NH2OH·HCl,
NaOAc, EtOH, H2O, reflux; (e) 10% Pd/C, H2, AcOH, MeOH; (f)
Boc-Dmt, PyBOP, HOBt-Cl, DIEA, DMF; (g) TFA, DCM.
moiety on the THQ scaffold, it is very likely that the higher
affinity diastereomer in each of the new analogues is, as was
observed for 1, the 4R diastereomer. This would, of course,
need to be confirmed for any analogue chosen for extensive in
vivo testing.
As seen in Table 1, all peptidomimetics bind with highest
affinity at MOR (by a factor of 8−130) and, except for the
diastereomeric pair 15d and 15d′, all slightly prefer DOR over
KOR. Efficacy data for the higher affinity diastereomers 1(4R)
and 15a−d are summarized in Table 2 as both EC50 and
percent maximal stimulation compared to the standard agonists
DAMGO, DPDPE, and U69,593 for MOR, DOR, and KOR,
respectively. With the exception of 15a, all the peptidomimetics
tested display high efficacy at MOR and all are very potent, with
EC50 values of ∼1 nM. By contrast, none of these ligands
significantly stimulate DOR, consistent with the desired MOR
agonist/DOR antagonist profile, and only 15d displays
significant agonist properties at KOR. To confirm the desired
MOR agonist/DOR antagonist profile, the ability of 15c, a
representative example from the series, to antagonize the
stimulation of GTPγS binding evoked by the DOR agonist
DPDPE was assessed. As expected, compound 15c acted as a
DOR antagonist, producing a rightward shift in the stimulation
curve of DPDPE at DOR with Ke = 34.3 2.7 nM, in good
agreement with the observed binding Ki of 10 nM for 15c at
DOR.
a
Reagents and conditions: (a) benzene or naphthalene, P2O5, Al2O3,
reflux; (b) 2-naphthalenylboronic acid, PdCl2, K2CO3, acetone, H2O,
110 °C, microwave irradiation; (c) 1-indanone, KOH, MeOH; (d)
10% Pd/C, H2, HCl, MeOH.
subjected to hydrogenation to afford amines 9a−d, which were
then acylated with 3-bromopropionyl chloride to afford 10a−d.
Cyclization with NaOtBu formed the four-membered lactams
(11a−d), which were then rearranged under Fries conditions to
give dihydroquinolinones 12a−d.27 Oxime formation (13a−d)
and subsequent hydrogenation gave racemic primary amines
14a−d, which could then be coupled to Boc-Dmt under
standard peptide coupling conditions (Scheme 3).
The synthetic protocol described in Schemes 2 and 3 yields
racemic mixtures of 4R and 4S scaffolds which are then used to
generate the diastereomeric pairs of final peptidomimetics 15a
and 15a′, 15c and 15c′, and 15d and 15d′. The diastereomer of
15b was observed but an insufficient amount for testing was
isolated. In each pair, the “unprimed” number (15a−d)
represents the diastereomer that elutes earlier in RP-HPLC,
which in all cases is also the higher affinity diastereomer, as
discussed below.
DISCUSSION AND CONCLUSIONS
■
Two results obtained with 1(4R) are particularly noteworthy.
First, the demonstration that this peptidomimetic modeled on a
full agonist peptide displays high MOR efficacy but low DOR
efficacy establishes it as strong lead for the development of
MOR agonist/DOR antagonists using the THQ scaffold. The
low DOR efficacy of 1(4R) is consistent with our previously
described models of interactions of opioid ligands with active
and inactive states of the receptors.7,8 The binding pocket in
the region of the Phe3 side chain of the tetrapeptide 2 and the
benzyl substituent of the THQ scaffold of 1(4R) (Figure 1C)
As was observed in our original report of 1,16 the earlier
eluting diastereomer of each tested pair displays 1−2 orders of
magnitude higher MOR and DOR affinity compared with the
later eluting diastereomer. Since the structural modifications
within the series are rather minor and confined to the pendent
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includes Asn125, Thr218, and Lys303 in MOR and the
corresponding, bulkier Lys108, Met199, and Trp254 in DOR.
The inactive state of both receptors can accommodate benzyl
and even bulkier substituents; however, these bulkier
substituents clash with the larger residues of DOR in the
more compact binding pocket found in the active state of the
receptor, reducing efficacy at this receptor. As seen in the
overlay of 1(4R) with 2 in Figure 1C, the phenyl ring of 1(4R)
(red) extends farther (deeper into the DOR pocket) giving rise
to its greater steric clash with the DOR active state and hence
its reduced DOR efficacy compared to 2.
The second critical observation for 1(4R) is its promising
bioavailability. As shown in Figures 2 and 3, 1(4R) displays
antinociceptive activity in the mouse WWTW assay after ip
administration and with similar potency as morphine. To date,
no MOR agonist/DOR antagonist ligands have been reported
that display central activity after peripheral administration;
hence, this represents a key result and promising lead.
which allows favorable interactions with MOR, DOR, and
KOR, to “tune” the pharmacological profile by incorporating
modifications that exploit differences in nearby residues of
MOR, DOR, and KOR in order to more closely equalize MOR
and DOR binding affinity and reduce KOR affinity.
Finally, a MOR agonist/DOR antagonist profile is not
unique in being associated with reduced development of
tolerance and other MOR agonist side effects. Several groups
have described positive results with ligands that act as a MOR
agonist/DOR agonist.31−34 Included among these are ligands
that are active after peripheral administration.31−33 It is unclear
whether DOR agonists and DOR antagonists exert their effects
on MOR agonists in similar ways. For example, we showed that
coadministration of a DOR agonist potentiates the potency and
efficacy of morphine even in mice rendered tolerant to
morphine.35 Thus, it is possible that the reported reduction
in MOR tolerance effected by DOR agonists reflects this
potentiation of MOR agonist potency and/or efficacy.
The data in Tables 1 and 2 support the promise of this
peptidomimetic series, as they demonstrate that features that
confer DOR antagonism in our recently reported peptides7,8,24
act similarly in the peptidomimetic series described here. For
example, the 2-methylnaphthyl substituted 15c reduces DOR
efficacy compared to the 1-methylnaphthyl analogue 15b. This
mirrors observations made in the peptide series.8 While all the
peptidomimetics examined display low DOR efficacy, it appears
that modifications with longer extended R (15a, 15c, 15d in
Figure 1) are less compatible with the DOR active state. This is
consistent with the observed low DOR efficacy of 1(4R)
compared with its DOR full agonist peptide counterpart 2
discussed above.
Compounds 15a−d, like 1(4R), are the higher affinity (and
earlier eluting on HPLC) diastereomers of each pair and, given
the structural similarity to 1, most likely share the 4R
stereochemistry that was unequivocally determined for 1(4R).
Compounds 15a−d all display fairly similar pharmacological
profiles, with 15c exhibiting the most promising combination of
high MOR efficacy and potency, potent DOR antagonism, and
somewhat reduced affinity (and no efficacy) at KOR.
Compound 15d displays a slightly different profile from the
other members of the series in its considerably higher KOR
affinity and smaller range of affinities across the three receptors.
It also exhibits a potentially interesting MOR agonist/KOR
partial agonist profile, a profile that may be useful for
combating cocaine dependence.28−30 Preliminary inspection
of peptidomimetic docking to our receptor models suggests
that the indanyl of 15d can interact favorably with Val108 of
transmembrane helix 2 (TM2) of the active conformation of
KOR while the corresponding R substituent of the other
peptidomimetics either adopt alternative low energy con-
formations or sterically clash with this region of the active state
of KOR. Hence, 15d displays both higher KOR affinity and
efficacy than the other analogues described here.
EXPERIMENTAL SECTION
■
Chemistry. All reagents and solvents were obtained from
commercial sources and used without additional purification. To
prepare P2O5/Al2O3 (w/w 50%), Al2O3 was placed in an oven at 120
°C for 24 h. After it reached room temperature in a desiccator, P2O5
was added in equal amounts. The compounds were mixed and
returned to the desiccator for later use. Suzuki couplings were
performed on a Discover S-class (CEM) microwave in a closed vessel
with maximum power input of 300 W and temperature set at 110 °C
for 10 min under the standard method from their Synergy software.
Hydrogenations were performed on a Parr hydrogenator apparatus
from Parr Instrument Company, model 3916EA, at the pressures
specified using 10% Pd/C as the catalyst. Flash column chromatog-
raphy was carried out using P60 silica gel (230−400 mesh).
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 15% solvent B (0.1% TFA in
acetonitrile) in solvent A (0.1% TFA in water) to 50% solvent B in
solvent A at a rate of either 0.5% or 1% per minute and monitoring UV
absorbance at 230 nm. Purity of synthesized compounds was
determined on a Waters Alliance 2690 analytical HPLC instrument
and a Vydac protein and peptide C18 reverse phase column, using a
linear gradient of 0% solvent B in solvent A to 45%, 70%, or 90%
solvent B in solvent A in 45, 70, or 90 min, respectively, and UV
absorbance at 230 nm (gradient A). Purities of the final compounds
1
used for testing were ≥95% as determined by HPLC. H NMR and
13C NMR data were obtained on either a 400 or 500 MHz Varian
spectrometer using CDCl3 or CD3OD solvents. The identity of each
compound was verified by mass spectrometry using an Agilent 6130
LC−MS mass spectrometer in positive mode.
General Procedure A for (S)-2-Amino-N-((S)-6-benzyl-
1,2,3,4-tetrahydroquinolin-4-yl)-3-(4-hydroxy-2,6-
dimethylphenyl)propanamide (1). The amine intermediate 7 (121
mg, 0.358 mmol) was dissolved in DMF (5 mL) followed by the
addition of the coupling reagents PyBOP (186 mg, 0.358 mmol),
HOBt-Cl (70.0 mg, 0.358 mmol), and DIEA (624 μL, 3.58 mmol).
Boc-DMT (146 mg, 0.358 mmol) was dissolved in DMF (5 mL) and
added to the reaction mixture, which was stirred for 18 h at room
temperature. After concentration under reduced pressure the product
was resuspended in EA (30 mL) and washed with a solution of 5%
citric acid in H2O (30 mL). The aqueous layer was then extracted with
EA (10 mL), and the combined organic extracts were washed with
brine (1 × 10 mL), dried with MgSO4, filtered, and concentrated
under reduced pressure. The crude residue was dissolved in a 1:1
mixture of DCM and TFA (10 mL) and stirred for 3 h. The mixture
was concentrated and purified by semipreparative HPLC to yield the
title compound in a 9:1 ratio of diastereomers. HPLC (gradient A):
retention time = 25.50 (early), 28.85 (late). EI-MS 452.2 [M + Na]+
for both diastereomers.
While the peptidomimetics described here show promise,
their profiles require optimization before moving forward to in
vivo tolerance studies. For example, the preferred MOR
agonist/DOR antagonist ligand would display similar, high
affinity for MOR and DOR, translating into potent, high
efficacy MOR agonism and potent DOR antagonism while
interacting poorly with KOR. These preliminary results do,
however, provide strong support for our structure-based design
approach employing our ligand−receptor models and also lend
confidence to our plans to further employ the THQ scaffold,
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Synthesis of Compounds 4−7. tert-Butyl 6-Benzyl-4-oxo-
3,4-dihydroquinoline-1(2H)-carboxylate (4). Compound 316
(2.57 g, 10.8 mmol) was dissolved in DCM (70 mL), followed by
the addition of (Boc)2O (3.06 g, 14.1 mmol), DMAP (132 mg, 1.08
mmol), and DIEA (2.45 mL, 14.1 mmol). The mixture was stirred at
reflux for 35 h, after which time it was quenched with 1 M HCl (50
mL). The aqueous layer was extracted with DCM. The combined
organic extracts were dried with MgSO4, filtered, and concentrated,
and the residue was chromatographed on silica gel (1:2 EA/hex) to
yield the title compound as a white solid (2.79 g, 77%). Rf (50% EA/
hex): 0.71. 1H NMR (400 MHz, CDCl3) δ 7.83 (d, J = 1.6, 1H), 7.69
(d, J = 1.6, 1H), 7.29 (dd, J = 8.6, 1.9, 1H), 7.26−7.19 (m, 2H), 7.18−
7.10 (m, 3H), 4.08 (t, J = 6.2, 2H), 3.90 (s, 2H), 2.68 (t, J = 6.3, 2H),
1.53 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 194.23, 152.74, 142.37,
140.39, 134.61, 128.80, 128.54, 127.14, 126.26, 124.78, 123.81, 82.07,
44.26, 41.14, 38.97, 28.27.
and the aqueous layer was extracted with EA (3 × 10 mL). Combined
organic extracts were washed with brine (20 mL), dried with MgSO4,
filtered, and concentrated under reduced pressure, and the residue was
chromatographed on silica gel (1:9 MeOH/DCM) to yield the title
compound as a colorless oil (240 mg, 60%, 78−80% ee). Rf (10%
MeOH/DCM): 0.38. 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 8.5,
1H), 7.31−7.22 (m, 2H), 7.21−7.12 (m, 4H), 7.01 (dd, J = 8.5, 1.7,
1H), 3.92 (s, 2H), 3.90 (t, J = 5.8, 1H), 3.86−3.79 (m, 1H), 3.71−3.63
(m, 1H), 2.14−2.02 (m, 1H), 1.77−1.65 (m, 1H), 1.51 (s, 9H); 13C
NMR (101 MHz, CDCl3) δ 153.73, 141.13, 136.11, 135.56, 133.26,
128.87, 128.42, 127.44, 127.16, 126.02, 123.79, 80.89, 47.71, 41.57,
41.34, 33.21. Enantioselectivity determined by HPLC (Chiracel OD-
RH column, 34% acetonitrile/water, 1.0 mL/min, 230 nm, 25 °C):
tmajor = 4.79 min, tminor = 8.87 min.
General Procedure B for Compounds 8a,b. 2-(4-Nitro-
phenyl)-1-phenylethanone (8a). To a mixture of p-nitrophenyl-
acetic acid (19.5 g, 108 mmol) and 150 mL of benzene was added 25 g
of the 1:1 P2O5/Al2O3 mixture. The mixture was stirred overnight at
reflux, and since starting material still could be seen by TLC, fresh
P2O5/Al2O3 (10 g) was added and the mixture was kept under reflux
and monitored by TLC until reaction completion (6 h). The mixture
was then filtered, concentrated, and suspended in 200 mL of EA. The
organic layer was washed with NaHCO3 (3 × 50 mL) and brine (1 ×
50 mL), dried over MgSO4, and filtered. The crude residue was
chromatographed on silica gel (1:1 EA/hex) to yield the title
(R)-tert-Butyl 6-Benzyl-4-hydroxy-3,4-dihydroquinoline-
1(2H)-carboxylate (5). Compound 4 (2.69 g, 7.97 mmol) was
dissolved in THF (30 mL) and stirred at room temperature with 4 Å
molecular sieves (1.0 g) for 1 h. This solution was transferred to the
(S)-(−)-2-methyl-CBS-oxazaborolidine catalyst via cannula, and the
reaction vessel was cooled to −20 °C. BH3·Me2S (3.99 mL, 7.97
mmol) was then added via syringe over a period of 10 min. The
mixture was stirred at −20 °C for 6 h. After the addition of MeOH (7
mL) the mixture was allowed to reach room temperature. The mixture
was partitioned between 1 M HCl (10 mL) and Et2O (30 mL). The
aqueous layer was extracted with Et2O (3 × 20 mL). The combined
organic extracts were washed with brine (20 mL), dried with MgSO4,
filtered, and concentrated, and the residue was chromatographed on
silica gel (1:2 EA/hex) to yield the title compound as a colorless oil
1
compound as a yellow solid (18 g, 70%). Rf (50% EA/hex): 0.70. H
NMR (500 MHz, CDCl3) δ 8.22 (d, J = 8.6, 2H), 8.04 (d, J = 7.4, 2H),
7.64 (t, J = 7.4, 1H), 7.52 (dd, J = 10.5, 5.0, 2H), 7.46 (d, J = 8.4, 2H),
4.44 (s, 2H); 13C NMR (126 MHz, CDCl3) δ 196.01, 142.07, 136.17,
133.76, 130.67, 130.52, 128.90, 128.45, 123.76, 77.32, 77.06, 76.81,
44.95.
1
(2.19 g, 81%, 80% ee). Rf (50% EA/hex): 0.48. H NMR (400 MHz,
CDCl3) δ 7.72 (d, J = 8.5, 1H), 7.30−7.14 (m, 6H), 7.08 (dd, J = 8.16,
1.6, 1H), 4.53 (t, J = 4.8, 1H), 3.92 (s, 2H), 3.53 (ddd, J = 13.1, 9.5,
3.8, 1H), 3.40 (s, 1H), 1.98−1.90 (m, 1H), 1.89−1.79 (m, 1H), 1.56
(s, 9H); 13C NMR (101 MHz, CDCl3) δ 153.73, 141.15, 136.11,
135.71, 131.04, 128.92, 128.49, 128.25, 126.10, 123.57, 81.08, 65.47,
41.33, 40.74, 32.13, 28.43. Enantioselectivity determined by HPLC
(Chiracel OD-RH column, 45% acetonitrile/water, 230 nm, 25 °C):
tmajor = 21.3 min, tminor = 25.2 min.
Naphthalen-1-yl(4-nitrophenyl)methanone (8b). 8b was
synthesized according to general procedure A starting from p-
nitrobenzoic acid (15.6 g, 93.0 mmol) and naphthalene (10.0 g, 78.0
mmol) using DCE (200 mL) as solvent to yield a yellow solid (19.0 g,
90%) as a 4:1 mixture of the title compound and the 2-naphthalene
derivative, respectively. 1H NMR (500 MHz, CDCl3) δ 8.34−8.28 (m,
2H), 8.20−8.15 (m, 1H), 8.07 (d, J = 8.1, 1H), 8.02−7.98 (m, 2H),
7.95 (dd, J = 6.8, 2.5, 1H), 7.63−7.49 (m, 4H); 13C NMR (126 MHz,
CDCl3) δ 195.98, 150.15, 143.30, 134.57, 133.77, 132.60, 131.12,
130.73, 128.95, 128.60, 127.86, 126.81, 125.33, 124.23, 123.60, 77.25,
77.00, 76.75.
Procedure for 2-(4-Nitrobenzyl)naphthalene (8c). 2-Naph-
thalenylboronic acid (1.29 g, 7.50 mmol) and p-nitrobenzyl bromide
(1.25 g, 5.79 mmol) were added to a microwave vessel in a 3:1 mixture
of acetone/H2O (7 mL) that was previously degassed and saturated
with argon gas. PdCl2 (17.4 mg, 1.69 mol %) and K2CO3 (2.40 g, 17.4
mmol) were then added. The mixture was irradiated at 110 °C for 10
min. The suspension was then concentrated, suspended in DCM (50
mL), filtered through Celite, and chromatographed on silica gel (8:92
EA/hex) to yield the title compound as a beige solid (795 mg, 52%).
Rf (8% EA/hex): 0.27. 1H NMR (400 MHz, CDCl3) δ 8.13 (d, J = 8.6
Hz, 2H), 7.85−7.77 (m, 3H), 7.63 (s, 1H), 7.51−7.46 (m, 2H), 7.35
(d, J = 8.5 Hz, 2H), 7.27 (d, J = 8.2 Hz, 1H), 4.21 (s, 2H); 13C NMR
(101 MHz, CDCl3) δ 148.69, 136.64, 133.59, 132.28, 129.72, 128.57,
128.25, 127.71, 127.56, 127.43, 127.21, 126.35, 125.82, 123.75, 77.39,
77.08, 76.76, 41.84.
Procedure for 2-(4-Nitrobenzylidene)-2,3-dihydro-1H-
inden-1-one (8d). To a reaction vessel containing MeOH (375
mL) was added KOH (1.91 g, 34.0 mmol). After dissolution, 1-
indanone (3.00 g, 22.7 mmol) was added and allowed to dissolve. p-
Nitrobenzaldehyde (4.12 g, 27.2 mmol) was then added to the
reaction mixture and allowed to stir for 1 h. Solvent was removed
under reduced pressure and the residual solid was washed with cold
H2O (50 mL) and filtered to yield a homogeneous, tan powder (5.57
g, 93%). Rf (40% EA/hex): 0.42. 1H NMR (400 MHz, CDCl3) δ 8.32
(d, J = 8.3, 2H), 7.94 (d, J = 7.7, 1H), 7.82 (d, J = 8.2, 2H), 7.73−7.63
(m, 2H), 7.59 (d, J = 7.5, 1H), 7.47 (t, J = 7.3, 1H), 4.10 (s, 2H); 13C
NMR (101 MHz, CDCl3) δ 193.63, 149.28, 141.63, 138.43, 137.50,
135.26, 130.95, 130.81, 128.06, 126.24, 124.74, 124.08, 32.33.
(S)-tert-Butyl 6-Benzyl-4-(1,3-dioxoisoindolin-2-yl)-3,4-dihy-
droquinoline-1(2H)-carboxylate (6). Compound 5 (2.12 g, 6.27
mmol) was dissolved in THF (25 mL) and added via syringe to a
mixture of phthalimide (1.38 g, 9.40 mmol) and PPh3 (2.47 g, 9.40
mmol). The reaction mixture was allowed to stir for 10 min and then
cooled to 0 °C. Once cooled, a solution of DIAD (2.48 mL, 12.5
mmol) in THF (10 mL) was added to the reaction mixture over a
period of 30 min. The mixture was allowed to reach room temperature
and was then stirred for 24 h. The mixture was concentrated under
reduced pressure, and the residue was dissolved in Et2O (30 mL). A
solution of 2 M NaOH (15 mL) was added, and the aqueous layer was
extracted with Et2O (3 × 10 mL). The combined organic extracts were
washed with brine (15 mL), dried with MgSO4, filtered, and
concentrated, and the residue was chromatographed on silica gel
(2:3 EA/hexane) to yield the title compound as a colorless oil (1.00 g,
1
35%). Rf (50% EA/hex): 0.65. H NMR (400 MHz, CDCl3) δ 7.79−
7.77 (m, 2H), 7.72−7.60 (m, 3H), 7.27−7.08 (m, 2H), 7.08−6.95 (m,
4H), 6.73 (s, 1H), 5.49 (t, J = 7.8, 1H), 4.16 (dt, J = 10.0, 4.4, 1H),
3.79 (s, 2H), 3.70 (ddd, J = 13.1, 9.8, 3.5, 1H), 2.56−2.44 (m, 1H),
2.26−2.16 (m, 1H), 1.54 (s, 9H); 13C NMR (101 MHz, CDCl3) δ
167.75, 153.57, 140.72, 137.40, 136.31, 134.14, 131.80, 128.78, 128.27,
127.87, 126.79, 126.40, 125.93, 124.50, 123.36, 80.98, 46.48, 43.20,
41.01, 28.51, 28.40.
(S)-tert-Butyl 4-Amino-6-benzyl-3,4-dihydroquinoline-1(2H)-
carboxylate (7). Compound 6 (933 mg, 1.99 mmol) was dissolved in
absolute EtOH (20 mL) followed by the addition of hydrazine
monohydrate (460 μL, 5.97 mmol). The reaction mixture was stirred
at room temperature for 48 h, during which time a white precipitate
formed. The mixture was filtered, and the precipitate was washed with
EtOH (20 mL) and the filtrate concentrated under reduced pressure.
The residue was partitioned between EA (20 mL) and H2O (10 mL),
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General Procedure C for Compounds 9a−d. 4-Phenethylani-
line (9a). To a hydrogenation vessel was added 10% Pd/C catalyst
(1.5 g) followed by the slow addition of MeOH (120 mL). 8a (6.2 g,
25.7 mmol) was dissolved in minimal MeOH and added to the vessel,
followed by concentrated HCl (5.8 mL). The reaction vessel was
placed on the hydrogenator under 50 psi of H2 gas and allowed to
shake for 24 h. The reaction mixture was then filtered through a pad of
Celite, and solvent was removed under reduced pressure. The crude
residue was extracted twice with DCM (150 mL) from 2 M NaOH
(200 mL), and the combined organic layers were subsequently washed
2 × NaHCO3 (100 mL), 1 × brine (100 mL), dried under MgSO4,
filtered, and concentrated. The crude residue was chromatographed on
silica gel (4:1 EA/hex) to yield the title compound as a beige-pinkish
129.20, 128.64, 127.25, 127.18, 125.96, 125.55, 125.50, 124.16, 120.34,
77.32, 77.00, 76.68, 40.50, 38.46, 27.11.
3-Bromo-N-(4-(naphthalen-2-ylmethyl)phenyl)propanamide
(10c). 10c was synthesized according to general procedure D starting
from 9c (2.20 g, 9.00 mmol) to yield the title compound as a beige
1
solid (3.30 g, 90%). Rf (75% EA/hex): 0.71. H NMR (400 MHz,
CDCl3) δ 7.81−7.72 (m, 2H), 7.60 (s, 1H), 7.50−7.34 (m, 4H),
7.31−7.23 (m, 1H), 7.18 (d, J = 8.2, 2H), 7.12 (d, J = 8.1, 1H), 4.09 (s,
2H), 3.68 (t, J = 6.6, 2H), 2.90 (t, J = 6.5, 2H); 13C NMR (101 MHz,
CDCl3) δ 167.85, 138.45, 137.43, 135.52, 133.56, 132.06, 129.57,
128.09, 127.59, 127.51, 127.00, 125.99, 125.36, 120.31, 120.20, 77.32,
77.00, 76.68, 41.48, 40.61, 27.09.
3-Bromo-N-(4-((2,3-dihydro-1H-inden-2-yl)methyl)phenyl)-
propanamide (10d). 10d was synthesized according to general
procedure D starting from 9d (686 mg, 3.07 mmol) to yield the title
compound as a fluffy, white solid (1.07 g, 98%). Rf (40% EA/hex):
0.37. 1H NMR (400 MHz, CDCl3) δ 7.44 (d, J = 8.2, 2H), 7.20−7.06
(m, 6H), 3.71 (t, J = 6.5, 2H), 2.98−2.63 (m, 4H), 2.77−2.60 (m,
5H); 13C NMR (101 MHz, CDCl3) δ 167.74, 143.10, 137.96, 135.21,
129.39, 126.11, 124.48, 120.13, 41.46, 40.93, 40.69, 38.81, 27.16.
General Procedure E for Compounds 11a−d. 1-(4-
Phenethylphenyl)azetidin-2-one (11a). NaOtBu (269 mg, 2.8
mmol) was suspended in DMF (20 mL) followed by the addition of
10a (883 mg, 2.66 mmol) previously dissolved in DMF (10 mL) via
cannula. The resulting mixture was stirred at room temperature for 3
h, quenched with H2O (10 mL), and concentrated under reduced
pressure to an oil which was resuspended in EA (20 mL) and washed
with H2O (2 × 20 mL), brine (1 × 20 mL), dried with MgSO4,
filtered, concentrated, and chromatographed on silica gel (1:1 EA/hex)
to yield the title compound as a white solid (457 mg, 70%). Rf (50%
1
solid (3.70 g, 73%). Rf (75% EA/hex): 0.54. H NMR (400 MHz,
CDCl3) δ 7.27−7.21 (m, 2H), 7.19−7.08 (m, 3H), 6.96−6.89 (m,
2H), 6.59−6.50 (m, 2H), 3.45 (d, J = 18.1, 2H), 2.84 (ddd, J = 8.7, 5.4,
2.2, 2H), 2.78 (ddd, J = 8.7, 5.4, 2.2, 2H); 13C NMR (101 MHz,
CDCl3) δ 144.16, 141.88, 131.64, 129.03, 128.33, 128.11, 125.63,
115.06, 77.32, 77.00, 76.68, 38.11, 36.91.
4-(Naphthalenylmethyl)aniline (9b). 9b was synthesized
according to general procedure C starting from 8b (10.0 g, 36.1
mmol) carrying over the 4:1 mixture of 1-naphthalene and 2-
naphthalene intermediates, respectively, to yield a beige solid (8.00g,
1
95%). Rf (40% EA/hex): 0.40. H NMR (500 MHz, CDCl3) δ 7.98
(dd, J = 8.6, 4.1, 1H), 7.83−7.79 (m, 1H), 7.70 (d, J = 8.5, 1H), 7.44−
7.34 (m, 3H), 7.24 (d, J = 6.9, 1H), 6.94 (d, J = 8.3, 2H), 6.54−6.52
(m, 2H), 4.30 (s, 2H); 13C NMR (126 MHz, CDCl3) δ 144.33,
137.29, 133.79, 132.04, 130.44, 129.47, 128.52, 126.97, 126.82, 125.77,
125.46, 125.40, 124.25, 115.21, 77.25, 77.00, 76.75, 38.08.
4-(Naphthalen-2-ylmethyl)aniline (9c). 9c was synthesized
according to general procedure C starting from 8c (3.60 g, 13.7
mmol) to yield a beige solid (2.30 g, 70%). Rf (40% EA/hex): 0.40. 1H
NMR (400 MHz, CDCl3) δ 7.78−7.69 (m, 3H), 7.58 (s, 1H), 7.44−
7.35 (m, 2H), 7.28 (dd, J = 8.4, 1.6 Hz, 1H), 6.98 (d, J = 8.3 Hz, 2H),
6.59−6.56 (m, 2H), 4.00 (s, 2H), 3.43 (d, J = 42.6 Hz, 2H); 13C NMR
(101 MHz, CDCl3) δ 144.49, 139.34, 133.53, 131.93, 130.86, 129.76,
127.85, 127.57, 127.50, 127.44, 126.73, 125.79, 125.11, 115.22, 77.32,
77.00, 76.68, 41.15.
1
EA/hex): 0.49. H NMR (400 MHz, CDCl3) δ 7.29−7.23 (m, 4H),
7.21−7.09 (m, 5H), 3.59 (t, J = 4.4, 2H), 3.08 (t, J = 4.5, 2H), 2.88 (s,
4H); 13C NMR (101 MHz, CDCl3) δ 164.25, 141.47, 137.27, 136.55,
129.07, 128.43, 128.27, 125.89, 116.08, 77.32, 77.00, 76.68, 37.96,
37.91, 37.30, 36.00.
1-(4-(Naphthalen-1-ylmethyl)phenyl)azetidin-2-one (11b).
11b was synthesized according to general procedure E starting from
10b (1.00 g, 2.71 mmol) to yield the title compound as a yellow solid
(603 mg, 77%). Rf (50% EA/hex): 0.38. 1H NMR (400 MHz, CDCl3)
δ 7.94 (dd, J = 6.3, 3.0, 1H), 7.84 (dd, J = 6.1, 3.2, 1H), 7.74 (dd, J =
8.3, 3.6, 1H), 7.45−7.39 (m, 3H), 7.24 (dd, J = 7.8, 6.0, 3H), 7.14 (d, J
= 8.4, 2H), 4.38 (s, 2H), 3.50 (t, J = 4.5, 2H), 3.02 (t, J = 4.5, 2H); 13C
NMR (101 MHz, CDCl3) δ 164.21, 136.64, 136.49, 136.11, 133.88,
131.95, 129.65, 129.31, 128.62, 127.15, 125.92, 125.52, 125.47, 124.11,
116.20, 77.32, 77.00, 76.68, 38.49, 37.90, 35.97.
1-(4-(Naphthalen-2-ylmethyl)phenyl)azetidin-2-one (11c).
11c was synthesized according to general procedure E starting from
10c (3.20 g, 8.70 mmol) to yield the title compound as a yellow solid
(2.30g, 90%). Rf (50% EA/hex): 0.32. 1H NMR (400 MHz, CDCl3) δ
7.80−7.71 (m, 3H), 7.58 (s, 1H), 7.42 (dt, J = 13.7, 6.8, 2H), 7.26 (dd,
J = 12.8, 6.0, 2H), 7.21−7.09 (m, 3H), 4.09 (s, 2H), 3.56 (t, J = 4.2,
2H), 3.06 (t, J = 4.4, 2H); 13C NMR (101 MHz, CDCl3) δ 164.32,
138.51, 136.73, 136.50, 133.54, 132.03, 129.67, 128.07, 127.57, 127.48,
127.41, 126.92, 125.97, 125.34, 116.28, 77.32, 77.00, 76.68, 41.49,
37.98, 36.00.
4-((2,3-Dihydro-1H-inden-2-yl)methyl)aniline (9d). 9d was
synthesized according to general procedure C starting from 8d (2.13
g, 8.04 mmol) to yield a brown solid (1.70 g, 95%). Rf (5% MeOH/
1
DCM): 0.78. H NMR (400 MHz, CDCl3) δ 7.16 (dd, J = 8.0, 4.5,
2H), 7.14−7.08 (m, 2H), 7.01 (d, J = 8.2, 2H), 6.67 (d, J = 8.2, 2H),
3.31 (s, 2H), 2.97 (dd, J = 13.7, 5.7, 2H), 2.75−2.60 (m, 5H); 13C
NMR (101 MHz, CDCl3) δ 143.91, 143.34, 131.97, 129.67, 126.04,
124.48, 115.51, 41.71, 40.73, 38.86.
General Procedure D for Compounds 10a−d. 3-Bromo-N-(4-
phenethylphenyl)propanamide (10a). Compound 9a (587 mg,
2.90 mmol) was added to a suspension of K2CO3 (820 mg, 5.94
mmol) in DCM (10 mL), followed by the addition of 3-
bromopropionyl chloride (300 μL, 2.99 mmol) via syringe. The
resulting mixture was stirred at room temperature for 2 h and
quenched by the addition of H2O (10 mL). The mixture was extracted
with DCM (25 mL), dried with MgSO4, filtered, and concentrated to
yield the title compound as a tan solid (921 mg, 93%). Rf (75% EA/
1-(4-((2,3-Dihydro-1H-inden-2-yl)methyl)phenyl)azetidin-2-
one (11d). 11d was synthesized according to general procedure E
starting from 10d (1.07 g, 3.00 mmol) to yield the title compound as a
1
hex): 0.70. H NMR (400 MHz, CDCl3) δ 7.48 (s, 1H), 7.43−7.39
(m, 2H), 7.29−7.24 (m, 2H), 7.21−7.09 (m, 5H), 3.68 (t, J = 6.6,
2H), 2.90 (t, J = 6.6, 2H), 2.89 (s, 4H); 13C NMR (101 MHz, CDCl3)
δ 167.91, 141.51, 138.24, 135.27, 128.95, 128.41, 128.29, 125.89,
120.21, 77.32, 77.00, 76.68, 40.55, 37.82, 37.24, 27.15.
1
light tan powder (811 mg, 98%). Rf (40% EA/hex): 0.37. H NMR
(400 MHz, CDCl3) δ 7.31 (d, J = 8.3, 2H), 7.20−7.09 (m, 6H), 3.62
(t, J = 4.4, 2H), 3.11 (t, J = 4.4, 2H), 2.97 (dd, J = 14.6, 5.4, 2H),
2.78−2.61 (m, 5H); 13C NMR (400 MHz, CDCl3) δ 164.26, 143.10,
137.03, 136.61, 129.47, 126.14, 124.49, 116.16, 41.56, 41.00, 38.82,
38.01, 36.07.
General Procedure F for Compounds 12a−d. 6-Phenethyl-
2,3-dihydroquinolin-4(1H)-one (12a). Compound 11a (424 mg,
1.68 mmol) was dissolved in DCE (50 mL), and TfOH (448 μL, 5.06
mmol) was added via syringe. The resulting mixture was stirred at
room temperature for 18 h, then quenched with K2CO3 (1.7 g) and
H2O (86 μL), and allowed to stir for 1 h. The mixture was filtered
3-Bromo-N-(4-(naphthalen-1-ylmethyl)phenyl)propanamide
(10b). 10b was synthesized according to general procedure D starting
from 9b (1.40 g, 6.00 mmol). The crude compound was chromato-
graphed on silica gel (4:1 EA/hex) to yield the title compound as a tan
1
solid (1.00 g, 45%). Rf (75% EA/hex): 0.72. H NMR (400 MHz,
CDCl3) δ 7.96−7.91 (m, 1H), 7.83 (dt, J = 4.1, 2.8, 1H), 7.74 (dd, J =
8.0, 4.4 Hz, 1H), 7.42−7.35 (m, 3H), 7.25 (d, J = 6.9), 7.12 (d, J = 8.5,
1H), 4.38 (s, 2H), 3.63 (t, J = 6.6, 2H), 2.85 (t, J = 6.5, 2H); 13C NMR
(101 MHz, CDCl3) δ 167.92, 137.11, 136.41, 135.38, 133.89, 131.98,
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through a plug of MgSO4 and concentrated under reduced pressure to
title compound as a yellow solid (453 mg, 82%). Rf (50% EA/hex):
0.51. 1H NMR (400 MHz, CDCl3) δ 7.77−7.61 (m, 3H), 7.56 (d, J =
9.2, 1H), 7.42−7.32 (m, 2H), 7.26 (dd, J = 8.4, 1.3, 1H), 6.92 (dt, J =
6.8, 3.5, 1H), 6.50 (d, J = 8.3, 1H), 3.97 (s, 2H), 3.28−3.20 (m, 2H),
2.84 (t, J = 6.5, 2H); 13C NMR (101 MHz, CDCl3) δ 152.40, 145.46,
139.04, 133.58, 132.01, 131.37, 131.08, 127.99, 127.57, 127.55, 126.86,
125.83, 125.18, 124.46, 116.67, 116.06, 115.81, 77.32, 77.00, 76.68,
41.36, 40.64, 23.41.
yield the title compound as a yellow solid (338 mg, 80%). Rf (70%
1
EA/hex): 0.49. H NMR (400 MHz, CDCl3) δ 7.72−7.71 (m, 1H),
7.30−7.25 (m, 2H), 7.21−7.14 (m, 3H), 7.10 (dd, J = 8.4, 2.2, 1H),
6.60 (d, J = 8.3, 1H), 4.65−3.78 (bs, 1H), 3.55 (dd, J = 7.4, 6.5, 2H),
2.91−2.78 (m, 4H), 2.69 (dd, J = 7.4, 6.5, 2H); 13C NMR (101 MHz,
CDCl3) δ 193.83, 150.38, 141.62, 135.78, 131.42, 128.41, 128.30,
126.66, 125.87, 119.33, 115.89, 77.30, 76.99, 76.67, 42.45, 38.18,
37.94, 36.88.
(E/Z)-6-((2,3-Dihydro-1H-inden-2-yl)methyl)-2,3-dihydroqui-
nolin-4(1H)-one oxime (13d). 13d was synthesized according to
general procedure G starting from 12d (772 mg, 2.78 mmol) to yield
the title compound as a yellow solid (662 mg, 81%). Rf (40% EA/hex):
6-(Naphthalen-1-ylmethyl)-2,3-dihydroquinolin-4(1H)-one
(12b). 12b was synthesized according to general procedure F starting
from 11b (603 mg, 2.10 mmol) to yield the title compound as a yellow
1
1
solid (450 mg, 74%). Rf (50% EA/hex): 0.43. H NMR (400 MHz,
0.44. H NMR (400 MHz, CDCl3) 7.63 (d, J = 1.8, 1H), 7.19 −7.05
CDCl3) δ 8.00−7.95 (m, 1H), 7.84−7.79 (m, 1H), 7.77 (d, J = 1.8,
1H), 7.71 (d, J = 7.9, 1H), 7.44−7.35 (m, 3H), 7.26 (d, J = 6.5, 1H),
7.05 (dd, J = 8.4, 2.1, 1H), 6.49 (d, J = 8.4, 1H), 4.29 (s, 2H), 3.46 (t, J
= 7.04, 2H), 2.63 (t, J = 6.88, 2H); 13C NMR (101 MHz, CDCl3) δ
193.78, 150.54, 136.60, 135.66, 133.89, 131.92, 130.10, 128.62, 127.24,
127.13, 127.10, 127.07, 125.92, 125.49, 124.10, 119.16, 116.13, 77.32,
77.00, 76.68, 42.28, 38.10, 38.02.
(m, 5H), 6.60 (dd, J = 21.6, 8.3, 1H), 3.30 (t, J = 6.5, 2H), 2.93 (dt, J =
13.0, 7.6, 3H), 2.76−2.59 (m, 6H); 13C NMR (101 MHz, CDCl3) δ
152.50, 145.41, 143.38, 131.51, 131.34, 129.41, 126.08, 124.52, 124.23,
115.78, 41.57, 40.78, 40.74, 38.86, 23.45.
General Procedure H for Compounds 14a−d. 6-Phenethyl-
1,2,3,4-tetrahydroquinolin-4-amine (14a). 10% Pd/C (50 mg)
was added to a hydrogenation vessel followed by the slow addition of
MeOH (10 mL). 13a (170 mg, 0.64 mmol) was dissolved in minimal
MeOH and added to the vessel, followed by the addition of glacial
AcOH (0.4 mL). The reaction vessel was placed on the hydrogenator
under 40 psi of H2 gas and allowed to shake for 12 h. The reaction
mixture was then filtered through a pad of Celite, and solvent was
removed under reduced pressure. The crude residue was resuspended
in 1 M HCl (30 mL) and extracted with 3 × EA (30 mL). The organic
layer was washed with 3 × 2 M NaOH (30 mL), 1 × brine (30 mL),
dried under MgSO4, filtered, and concentrated to yield the title
compound as a tan oil (120 mg, 75%), which was carried to the next
6-(Naphthalen-2-ylmethyl)-2,3-dihydroquinolin-4(1H)-one
(12c). 12c was synthesized according to general procedure F starting
from 11c (2.30 g, 8.01 mmol) to yield the title compound as a yellow
1
solid (1.40 g, 61%). Rf (50% EA/hex): 0.43. H NMR (400 MHz,
CDCl3) δ 7.80−7.70 (m, 4H), 7.60 (s, 1H), 7.46−7.38 (m, 2H), 7.28
(d, J = 8.4, 1H), 7.14−7.09 (m, 1H), 6.54 (d, J = 8.4, 1H), 4.36 (s,
1H), 4.00 (s, 2H), 3.45 (t, J = 6.9, 2H), 2.64 (t, J = 6.9, 2H); 13C NMR
(101 MHz, CDCl3) δ 193.80, 150.64, 138.65, 136.04, 133.48, 131.96,
130.35, 128.01, 127.50, 127.43, 127.33, 127.13, 126.75, 125.87, 125.23,
118.99, 116.22, 77.32, 77.00, 76.68, 42.16, 41.02, 38.00.
1
6-((2,3-Dihydro-1H-inden-2-yl)methyl)-2,3-dihydroquinolin-
4(1H)-one (12d). 12d was synthesized according to general
procedure F starting from 11d (811 mg, 2.93 mmol) to yield the
title compound as a viscous yellow oil (772 mg, 95%). Rf (40% EA/
hex): 0.32. 1H NMR (400 MHz, CDCl3) δ 7.70 (d, J = 1.9, 1H), 7.22−
7.08 (m, 5H), 6.63 (d, J = 8.3, 1H), 4.30 (br s, 1H), 3.57 (t, J = 6.9,
2H), 2.97 (dd, J = 14.2, 5.9, 2H), 2.78−2.60 (m, 7H); 13C NMR (101
MHz, CDCl3) δ 193.84, 150.42, 143.12, 136.09, 131.00, 127.12,
126.06, 124.45, 119.30, 115.90, 42.47, 41.42, 40.42, 38.75, 38.22.
General Procedure G for Compounds 13a−d. (E/Z)-6-
Phenethyl-2,3-dihydroquinolin-4(1H)-one Oxime (13a). Com-
pound 12a (338 mg, 1.35 mmol) was suspended in 1:1 EtOH/H2O
followed by the addition of NH2OH·HCl (64.5 mg, 1.47 mmol) and
NaOAc·H2O (201 mg, 1.47 mmol). The mixture was stirred at reflux
overnight, after which time solvents were condensed and redissolved
in EA (30 mL). The organic layer was washed with brine (10 mL),
dried with MgSO4, filtered, concentrated under reduced pressure, and
chromatographed on silica gel (1:1 EA/hex) to yield the title
compound as a yellow solid (235 mg, 66%). Rf (50% EA/hex): 0.51.
1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 2.0, 1H), 7.29−7.23 (m,
2H), 7.20−7.14 (m, 3H), 6.95 (dd, J = 8.2, 2.1, 1H), 6.57−6.52 (m,
1H), 3.30 (t, J = 6.5, 2H), 2.93 (t, J = 6.5, 2H), 2.90−2.76 (m, 4H);
13C NMR (101 MHz, CDCl3) δ 152.47, 145.29, 141.92, 131.99,
130.95, 128.42, 128.27, 125.78, 123.71, 116.68, 115.85, 77.32, 77.00,
76.68, 40.71, 38.09, 37.16, 23.58.
(E/Z)-6-(Naphthalen-1-ylmethyl)-2,3-dihydroquinolin-4(1H)-
one Oxime (13b). 13b was synthesized according to general
procedure G starting from 12b (450 mg, 1.57 mmol) and
chromatographed on silica gel (4:1 EA/hex) to yield the title
compound as a yellow solid (313 mg, 66%). Rf (75% EA/hex): 0.73.
1H NMR (400 MHz, CDCl3) δ 8.02−7.97 (m, 1H), 7.83−7.77 (m,
1H), 7.70 (s, 2H), 7.41 (m, 3H), 7.26−7.21 (m, 1H), 6.85 (dt, J =
11.2, 5.6, 1H), 6.42 (d, J = 8.3, 1H), 4.28 (s, 2H), 3.17 (t, J = 6.4, 2H),
2.85 (t, J = 5.2, 2H); 13C NMR (101 MHz, CDCl3) δ 152.20, 145.35,
133.83, 130.94, 130.52, 129.49, 128.53, 127.02, 126.87, 125.85, 125.49,
125.41, 124.24, 124.20, 116.68, 115.98, 115.48, 77.32, 77.00, 76.68,
40.55, 38.18, 23.51.
reaction without further purification. H NMR (400 MHz, CDCl3) δ
8.61 (bs, 2H), 7.28−7.20 (m, 3H), 7.16 (dd, J = 13.2, 7.1, 3H), 6.82
(d, J = 8.3, 1H), 6.38 (d, J = 8.2, 1H), 4.33 (s, 1H), 3.45 (t, J = 11.2,
1H), 3.14 (dd, J = 8.2, 4.0, 1H), 2.86−2.77 (m, 2H), 2.74−2.68 (m,
2H), 2.24 (d, J = 12.2, 1H), 2.12−2.03 (m, 1H); 13C NMR (101 MHz,
CDCl3) δ 143.00, 141.99, 131.01, 130.37, 130.15, 128.49, 128.25,
125.77, 115.30, 115.03, 77.33, 77.01, 76.69, 47.35, 38.16, 36.96, 36.61,
26.38. ESI-MS 236.1 [M − NH3 + H]+. HPLC (gradient A): retention
time = 23.79 min.
6-(Naphthalen-1-ylmethyl)-1,2,3,4-tetrahydroquinolin-4-
amine (14b). 14b was synthesized according to general procedure H
starting from 13b (330 mg, 1.09 mmol) to yield the title compound as
1
a tan oil (253 mg, 84%). H NMR (400 MHz, CDCl3) δ 8.00 (d, J =
7.9, 1H), 7.81 (d, J = 7.5, 1H), 7.69 (d, J = 8.0, 1H), 7.44−7.39 (m,
2H), 7.36 (s, 1H), 7.27−7.21 (m, 1H), 7.19 (s, 1H), 6.79 (d, J = 7.9,
1H), 6.31 (d, J = 8.2, 1H), 5.06 (bs, 3H), 4.24 (s, 2H), 4.08 (s, 1H),
3.44−3.29 (m, 1H), 3.14 (m, 1H), 2.00 (s, 2H); 13C NMR (101 MHz,
CDCl3) δ 142.75, 137.30, 133.83, 132.04, 129.94, 129.58, 129.23,
128.55, 127.53, 127.04, 126.84, 125.91, 125.55, 125.44, 124.31, 115.00,
77.32, 77.00, 76.68, 46.97, 38.00, 37.00, 28.34. ESI-MS 272.1 [M −
NH3 + H]+. HPLC (gradient A): retention time = 31.71 min.
6-(Naphthalen-2-ylmethyl)-1,2,3,4-tetrahydroquinolin-4-
amine (14c). 14c was synthesized according to general procedure H
starting from 13c (618 mg, 2.04 mmol) to yield the title compound as
a tan oil (246 mg, 86%). 1H NMR (400 MHz, CD3OD) δ 7.75 (dd, J
= 15.5, 8.4, 3H), 7.62 (s, 1H), 7.41 (dt, J = 12.8, 6.3, 2H), 7.31 (d, J =
8.2, 1H), 7.14 (s, 1H), 7.07 (d, J = 8.0, 1H), 6.67 (d, J = 8.2, 1H), 4.42
(t, J = 4.5, 1H), 4.02 (s, 2H), 3.40−3.31 (m, 2H), 2.26−2.03 (m, 2H);
13C NMR (101 MHz, CD3OD) δ 143.71, 140.64, 135.10, 133.55,
132.32, 132.09, 130.74, 128.96, 128.56, 128.49, 128.47, 127.75, 126.99,
126.32, 117.45, 49.64, 49.43, 49.21, 49.00, 48.79, 48.57, 48.36, 48.01,
42.09, 37.45, 27.37. ESI-MS 272.1 [M − NH3 + H]+. HPLC (gradient
A): retention time = 34.07 min.
6-((2,3-Dihydro-1H-inden-2-yl)methyl)-1,2,3,4-tetrahydro-
quinolin-4-amine (14d). 14d was synthesized according to general
procedure H starting from 13d (662 mg, 2.26 mmol) to yield the title
compound as a brown oil (527 mg, 84%). 1H NMR (400 MHz,
CDCl3) 7.13 (ddd, J = 8.7, 7.0, 4.2, 5H), 7.05 (s, 1H), 6.87 (dd, J =
8.1, 1.8, 1H), 6.46 (t, J = 6.8, 1H), 4.00 (t, J = 4.8, 1H), 3.43−3.34 (m,
1H), 3.29 (dt, J = 11.0, 5.4, 1H), 3.02−2.94 (m, 3H), 2.77−2.59 (m,
(E/Z)-6-(Naphthalen-2-ylmethyl)-2,3-dihydroquinolin-4(1H)-
one oxime (13c). 13c was synthesized according to general
procedure G starting from 12c (522 mg, 1.81 mmol) to yield the
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6H); 13C NMR (101 MHz, CDCl3) δ 157.04, 150.14, 143.38, 130.14,
129.11, 128.42, 125.98, 124.44, 114.50, 46.89, 41.70, 40.74, 38.88,
37.75, 31.65. ESI-MS 262.1 [M − NH3 + H]+. HPLC (gradient A):
retention time = 31.39 min.
General Procedure for Compounds 15a−d). (2S)-2-Amino-3-
(4-hydroxy-2,6-dimethylphenyl)-N-(6-phenethyl-1,2,3,4-tetra-
hydroquinolin-4-yl)propanamide (15a). 15a was synthesized
according to general procedure A from 14a (94 mg, 0.37 mmol) to
yield 150 mg of crude diastereomers which were isolated by
semipreparative HPLC and lyophilized to give white fluffy powders.
HPLC (gradient A): retention time = 25.00 min (early), 28.19 min
(late). ESI-MS 466.1 [M + Na]+ for both diastereomers.
(2S)-2-Amino-3-(4-hydroxy-2,6-dimethylphenyl)-N-(6-(naph-
thalen-1-ylmethyl)-1,2,3,4-tetrahydroquinolin-4-yl)-
propanamide (15b). 15b was synthesized according to general
procedure A from 14b (253 mg, 0.88 mmol) to yield 350 mg of a
crude mixture of 1-naphthalene and 2-naphthalene derivatives as
diastereomers. Only the earliest eluting peak was isolated as a pure
compound by semipreparative HPLC to give a white powder. HPLC
(gradient A): retention time = 31.42 min (early), 33.93 min (late).
Confirmed by NMR to be the 1-naphthalene derivative. ESI-MS 502.1
[M + Na]+ for both diastereomers.
(2S)-2-Amino-3-(4-hydroxy-2,6-dimethylphenyl)-N-(6-(naph-
thalen-2-ylmethyl)-1,2,3,4-tetrahydroquinolin-4-yl)-
propanamide (15c). 15c was synthesized according to general
procedure A from 14c (127 mg, 0.44 mmol) to yield 200 mg of a
crude mixture of diastereomers which were isolated and purified by
semipreparative HPLC, then lyophilized to give white powders. HPLC
(gradient A): retention time = 32.44 min (early), 34.75 min (late).
ESI-MS 502.2 [M + Na]+ for both diastereomers.
Brandel harvester (Brandel, Gaithersburg, MD, U.S.) and washed three
times with 50 mM Tris-HCl buffer. The radioactivity retained on dried
filters was determined by liquid scintillation counting after saturation
with EcoLume liquid scintillation cocktail in a Wallac 1450 MicroBeta
(Perkin-Elmer, Waltham, MA, U.S.). Nonspecific binding was
determined using 10 μM naloxone. Ki values were calculated using
nonlinear regression analysis to fit a logistic equation to the
competition data using GraphPad Prism, version 5.01, for Windows
(GraphPad Software Inc., La Jolla, CA). The results presented are the
mean standard error from at least three separate assays performed in
duplicate.
Stimulation of [35S]GTPγS Binding. Agonist stimulation of
[35S]guanosine 5′-O-[γ-thio]triphosphate ([35S]GTPγS, 1250 Ci, 46.2
TBq/mmol) binding was measured as described previously.22 Briefly,
membranes (10−20 μg of protein/tube) were incubated 1 h at room
temperature in GTPγS buffer (50 mM Tris-HCl, 100 mM NaCl, 5
mM MgCl2, pH 7.4) containing 0.1 nM [35S]GTPγS, 30 μM
guanosine diphosphate (GDP), and varying concentrations of test
peptides. Peptide stimulation of [35S]GTPγS was compared with 10
μM standard compounds [D-Ala2,N-MePhe4,Gly-ol]enkephalin
(DAMGO) at MOR, D-Pen2,5-enkephalin (DPDPE) at DOR, or
U69,593 at KOR. The reaction was terminated by rapidly filtering
through GF/C filters and washing 10 times with GTPγS buffer, and
retained radioactivity was measured as described above. The results
presented are the mean standard error from at least three separate
assays performed in duplicate; maximal stimulation was determined
using nonlinear regression analysis with GraphPad Prism (GraphPad
Software Inc., La Jolla, CA).
In Vivo Pharmacology. Animals. Adult male C57BL/6 mice
weighing between 20 and 30 g at 8−16 weeks old were used for the
current experiments (obtained from Jackson Laboratories (Bar
Harbor, ME), Harlan (Indianapolis, IN), or bred in-house). Mice
were group-housed and had free access to food and water at all times.
Experiments were conducted in the housing room, which was
maintained on a 12 h light/dark cycle (with lights on at 0700).
Each mouse was used only once and experiments were conducted
between 11 a.m. and 4 p.m. 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. The antinociceptive effects of 1(4R) and
morphine were evaluated in the warm water tail withdrawal assay
using a cumulative dosing procedure.23 To determine tail withdrawal
latencies, each mouse was placed briefly into a plastic, cylindrical
restrainer and 2−3 cm of the tail tip was placed into a water bath
maintained at 50 °C. The latency to withdraw the tail was recorded
with a maximum cutoff time of 20 s. If the mouse did not remove its
tail by the cutoff time, the experimenter removed its tail from the water
to prevent tissue damage. Each animal received an injection of saline
(intraperitoneal, ip) and then 30 min later, the baseline withdrawal
latencies were recorded and ranged between 3 and 6 s. Following
baseline determinations, three increasing doses (1, 2.2, and 6.8 mg/kg)
of 1(4R) were given at 30 min intervals to provide final doses of 1, 3.2,
and 10 mg/kg. Thirty minutes after each injection, the tail withdrawal
latency was measured as described above. To confirm that 1(4R) was
acting at opioid receptors, the cumulative dose response was repeated
over the doses 3.2, 10, and 32 mg/kg following a 30 min pretreatment
with 1 mg/kg naltrexone (ip). To determine the time-course of
antinociceptive action the tail-withdrawal test was performed at
varying times following administration of morphine (10 mg/kg, ip) or
1(4R) (10 mg/kg, ip).
(2S)-1-((6-((2,3-Dihydro-1H-inden-2-yl)methyl)-1,2,3,4-tetra-
hydroquinolin-4-yl)amino)-3-(4-hydroxy-2,6-dimethylphenyl)-
1-oxopropan-2-amide (15d). 15d was synthesized according to
general procedure A from 14d as a mixture of crude diastereomers
which were isolated and purified by semipreparative HPLC and
lyophilized to give a white powder of the early diastereomer and a tan
powder of the late diastereomer. HPLC (gradient A): retention time =
31.09 min (early), 33.76 min (late). ESI-MS 492.2 [M + Na]+ for both
diastereomers.
In Vitro Pharmacology. Cell Lines and Membrane Prepara-
tions. All tissue culture reagents were purchased from Gibco Life
Sciences (Grand Island, NY, U.S.). C6-rat glioma cells stably
transfected with a rat μ (C6-MOR) or rat δ (C6-DOR) opioid
receptor20 and Chinese hamster ovary (CHO) cells stably expressing a
human κ (CHO-KOR) opioid receptor21 were used for all in vitro
assays. Cells were grown to confluence at 37 °C in 5% CO2 in
Dulbecco’s modified Eagle medium 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 200g for 3 min. The cell pellet was
suspended in ice-cold 50 mM Tris-HCl buffer, pH 7.4, and
homogenized with a Tissue Tearor (Biospec Products, Inc., Bartles-
ville, OK, U.S.) for 20 s at setting 4. The homogenate was centrifuged
at 20 000g for 20 min at 4 °C, and the pellet was rehomogenized in 50
mM Tris-HCl with a Tissue Tearor for 10 s at setting 2, followed by
recentrifugation. The final pellet was resuspended in 50 mM Tris-HCl
and frozen in aliquots at 80 °C. Protein concentration was determined
via Bradford assay using bovine serum albumin as the standard.
Radioligand Binding Assays. Radioactive compounds were
purchased from Perkin-Elmer (Waltham, MA, U.S.). Opioid ligand-
binding assays were performed using competitive displacement of 0.2
nM [3H]diprenorphine (250 μCi, 1.85 TBq/mmol) by the test
compound from membrane preparations containing opioid receptors.
The assay mixture, containing membrane suspension (20 μg protein/
tube) in 50 mM Tris-HCl buffer (pH 7.4), [3H]diprenorphine, and
various concentrations of test peptide, was incubated at room
temperature for 1 h to allow binding to reach equilibrium. The
samples were rapidly filtered through Whatman GF/C filters using a
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (734) 764-8117. E-mail: him@umich.edu.
Notes
The authors declare no competing financial interest.
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Article
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ACKNOWLEDGMENTS
■
This work was funded by NIH Grants DA003910 (H.I.M.) and
DA004087 (J.R.T.). J.P.A. was supported by NIH Predoctoral
Training Grants DA007281 and GM007767. L.Y. was
supported by NIH Postdoctoral Training Grant DA007267.
A.A.H. was supported by NIH Predoctoral Training Grant
DA007281 and by a University of Michigan College of
Pharmacy Lyons Fellowship.
ABBREVIATIONS USED
■
MOR, μ opioid receptor; DOR, δ opioid receptor; KOR, κ
opioid receptor; Aic, 2-aminoindane-2-carboxylic acid; Pen,
penicillamine; cSEtS, cyclization through an ethylene di-
thioether linkage; cSS, cyclization through a disulfide linkage;
THQ, tetrahydroquinoline; CHO, Chinese hamster ovary;
GTPγS, guanosine 5′-O-[γ-thio]triphosphate; WWTW, warm
water tail withdrawal; TM, transmembrane; 1-Nal, 1-
naphthylalanine; 2-Nal, 2-naphthylalanine; (Boc)2O, di-tert-
butyl dicarbonate; DIEA, N,N-diisopropylethylamine; EA, ethyl
acetate; hex, hexanes; DIAD, diisopropyl azodicarboxylate;
TfOH, triflic acid; PyBOP, benzotriazol-1-yloxytripyrrolidino-
phosphonium hexafluorophosphate; HOBt-Cl, 6-chlorohydrox-
ybenzotriazole; Boc-Dmt, tert-butyloxycarbonyl-protected 2,6-
dimethyltyrosine
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