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 μ Opioid Receptor/δ Opioid Receptor Ligands

Article abstract of DOI:10.1021/acschemneuro.5b00100

We previously reported a small series of mixed-efficacy μ opioid receptor (MOR) agonist/δ opioid receptor (DOR) antagonist peptidomimetics featuring a tetrahydroquinoline scaffold and showed the promise of this series as effective analgesics after intrape

Full text of DOI:10.1021/acschemneuro.5b00100

Research Article
pubs.acs.org/chemneuro
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 μ Opioid Receptor/δ Opioid
Receptor Ligands
†
‡
‡
‡
‡
Aaron M. Bender, Nicholas W. Griggs, Jessica P. Anand, John R. Traynor, Emily M. Jutkiewicz,
,†,§
and Henry I. Mosberg*
†Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109,
United States
‡
Department of Pharmacology, School of Medicine, University of Michigan, Ann Arbor, Michigan 48109, United States
§
Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, United States
*
S Supporting Information
ABSTRACT: We previously reported a small series of mixed-efficacy μ opioid receptor (MOR) agonist/δ opioid receptor
DOR) antagonist peptidomimetics featuring a tetrahydroquinoline scaffold and showed the promise of this series as effective
(
analgesics after intraperitoneal administration in mice. We report here an expanded structure−activity relationship study of the
pendant region of these compounds and focus in particular on the incorporation of heteroatoms into this side chain. These
analogues provide new insight into the binding requirements for this scaffold at MOR, DOR, and the κ opioid receptor (KOR),
and several of them (10j, 10k, 10m, and 10n) significantly improve upon the overall MOR agonist/DOR antagonist profile of
our previous compounds. In vivo data for 10j, 10k, 10m, and 10n are also reported and show the antinociceptive potency and
duration of action of compounds 10j and 10m to be comparable to those of morphine.
KEYWORDS: Opioid, mixed efficacy, intraperitoneal, dependence, tolerance, tetrahydroquinoline
Opioid analgesics have long been the standard for the
treatment of severe pain. Unfortunately, the use of opioids
can lead to the development of a number of undesirable side
effects, such as respiratory depression, constipation, and
antinociceptive activity but with a reduced side-effect profile
compared with a MOR agonist alone.
4
−7
Because of these findings, our group and others have sought
to develop bifunctional ligands that bind to both MOR and
DOR but stimulate only MOR. Several classes of compound8s
have been employed in this pursuit, including peptides,
1
perhaps most problematically, dependence and tolerance.
There is therefore a great unmet need to develop agents that
act as potent analgesics but without the development of these
9
,10
11,12
pseudopeptides,
and small molecules.
The MOR/DOR
2
,3
side effects.
bivalent ligands developed by Portoghese and colleagues have
There is a growing body of evidence to suggest that the δ
opioid receptor (DOR) plays a significant role in the
modulation of the side effects related to the chronic use of
opioid analgesics. Although the analgesic effects of traditional
opioid agents such as morphine are associated with stimulation
of the μ opioid receptor (MOR), the coadministration of DOR
antagonists has been shown to maintain the desired
been demonstrated to be effective analgesics with a diminished
tolerance profile (Figure 1d), and recently the MOR/DOR
heteromer-biased agonist CYM51010 was also shown to display
13
Received: March 25, 2015
Revised: May 1, 2015
Published: May 4, 2015
©
2015 American Chemical Society
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Figure 1. Bifunctional MOR/DOR ligands. Compound a is from ref 17.
a
Scheme 1. Synthesis of Intermediates 4a−c,f−o
a
Reagents and conditions: (a) 3-bromopropionyl chloride, K CO , DCM, r.t.; (b) NaOtBu, DMF, r.t.; (c) TfOH, DCE, r.t.; (d) (Boc) O, DMAP,
2
3
2
DIPEA, DCM, reflux; (e) NBS, benzoyl peroxide, CCl , reflux; (f) boronic acid or pinacol ester, Pd(dppf)Cl , K CO , acetone, water, 100 °C with
4
2
2
3
microwave irradiation; (g) secondary amine, K CO , DMF, r.t.
2
3
reduced antinociceptive tolerance compared with morphine
receptors. Simple heteroatom replacements not only had
profound effects on receptor selectivity but also led to
compounds that improved upon the in vivo profile of our
initial THQ analogue. Compounds 10j and 10m both
produced a maximum antinociceptive response in the mouse
warm water tail withdrawal (WWTW) assay, and both
improved significantly upon the duration of action of our
lead peptidomimetic. These findings highlight the promise of
this scaffold in vivo and show that our SAR strategy focusing on
polar side chains was effective for improving bioavailability. In
vitro data for both binding affinity and efficacy are presented for
all three opioid receptor types (MOR, DOR, and the κ opioid
receptor (KOR)).
1
4
(Figure 1c). MOR agonist/DOR antagonist compounds are
also being developed clinically for the treatment of irritable
15
bowel syndrome. We have recently reported two MOR
agonist/DOR antagonist compounds that are effective and
16
bioavailable analgesics: a glycosylated cyclic pentapeptide and
a small molecule with a tetrahydroquinoline (THQ) core
1
7,18
(
Figure 1a).
Additionally, UMB425, a small-molecule MOR
agonist/DOR antagonist derived from thebaine, was reported
to display analgesia after subcutaneous administration with
19
reduced tolerance compared with morphine (Figure 1b).
The compounds reported in this paper build on the limited
initial structure−activity relationship (SAR) study done on our
bioavailable THQ lead compound (S)-2-amino-N-((R)-6-
benzyl-1,2,3,4-tetrahydroquinolin-4-yl)-3-(4-hydroxy-2,6-
dimethylphenyl)propanamide (a in Figure 1a). Initially, bulky
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of compounds 10a−c,f−o began
with the acylation of p-toluidine with 3-bromopropionyl
chloride (Scheme 1). The resulting alkyl bromide then
underwent an intramolecular cyclization followed by triflic
hydrophobic modifications to the side chain at position R (see
1
17
Table 1) were examined, as these substitutions were directly
comparable to a previously reported series of MOR/DOR
2
0,21
23,24
cyclic peptides
and were hypothesized to interact favorably
acid-mediated β-lactam rearrangement to give ketone 1,
with residues in the MOR active pocket to act as full MOR
agonists while simultaneously behaving as DOR antagonists.
These initial compounds were highly lipophilic, which is
desirable for blood−brain barrier penetration but less than
which was subsequently Boc-protected on the THQ nitrogen to
give 2. Ketone 2 was then brominated on the aryl methyl group
25
as described previously to give 3, onto which could be added
the pendant of choice, through either Suzuki coupling (4a,f−i)
or substitution with the appropriate secondary amine (4b,c,j−
o). It is important to note that 3 could be synthesized on a
multigram scale and that all substitutions on this intermediate
were high-yielding. For the synthesis of compounds 10d and
22
optimal for both aqueous solubility and metabolic stability.
We therefore explored a variety of polar side chains on this
scaffold for the purpose of improving these parameters and
further probing the chemical space in this region of each of the
1
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a
Scheme 2. Synthesis of Intermediates 4d and 4e
a
Reagents and conditions: (a) 3-bromopropionyl chloride, K CO , DCM, r.t.; (b) NaOtBu, DMF, r.t.; (c) TfOH, DCE, r.t.; (d) (Boc) O, DMAP,
2
3
2
DIPEA, DCM, reflux.
a
Scheme 3. Final Steps in the Synthesis of 10a−o
a
Reagents and conditions: (h) (R)-(+)-2-methyl-2-propanesulfinamide, Ti(OEt) , THF, reflux; (i) NaBH , THF; (j) conc. HCl, 1,4-dioxane, r.t.; (k)
4
4
Boc-L-Dmt, PyBOP, DIPEA, HOBt-Cl, DMF, r.t.; (l) TFA, DCM, r.t.; (m) (Ac) O, pyridine, r.t..
2
1
0e, commercially available para-substituted anilines were
carried forward in a similar manner as in the synthesis of
compound 2 to give intermediates 4d and 4e (Scheme 2).
Ketones 4a−o were converted to the corresponding imines
with (R)-(+)-2-methyl-2-propanesulfinamide and Ti(OEt) and
4
could then be reduced asymmetrically with NaBH in situ to
4
give tert-butanesulfinyl-protected amines 9a−o as single
diastereomers (Scheme 3), as previously described for
2
6,27
analogous scaffolds.
Deprotection with concentrated HCl
gave the corresponding primary, enantiomerically pure (R)-
amines as HCl salts. The stereochemistry of the HCl salts was
verified by X-ray crystallography of 6-benzyl-1-(tert-butoxycar-
bonyl)-1,2,3,4-tetrahydroquinolin-4-aminium chloride, which
was prepared by an identical synthetic route (Figure 2).
Boc-protected L-2,6-dimethyltyrosine (Boc-L-Dmt) could
then be coupled to the chiral HCl salt, and subsequent
deprotection with trifluoroacetic acid (TFA) afforded 10a−l
Figure 2. Crystal structure of (R)-6-benzyl-1-(tert-butoxycarbonyl)-
1
,2,3,4-tetrahydroquinolin-4-aminium chloride.
(
Scheme 3). The analogues were then purified by reversed-
was estimated by 19F NMR analysis as described previously
using a fluorinated analogue of 10m (see the Supporting
Information) and was found to be approximately 2.5 TFA
phase (RP) HPLC to provide enough material for in vitro and
in vivo pharmacological evaluation (∼5−10 mg). In the case of
the N-acetylated analogues 10m−o, Boc deprotection of the
THQ nitrogen was performed prior to coupling to Boc-L-Dmt.
After the amide coupling, the acetyl group was introduced by
stirring the crude material in excess pyridine/acetic anhydride
28
molecules per molecule of final compound.
SAR Studies. In previous reports, we described a mixed-
efficacy MOR agonist/DOR antagonist opioid peptidomimetic
featuring a THQ scaffold and a benzyl pendant at ring position
(
1:1) overnight, followed by a second Boc deprotection and
1
7,18
RP-HPLC purification. The TFA content of the final analogues
6
(a in Figure 1a). This compound was shown to be an
1
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effective analgesic in the WWTW assay after intraperitoneal
Table 1. Opioid Receptor Binding Affinities and clogP
Values for Analogues 10a−o
a
administration, with a duration of action slightly shorter than
17
that of morphine. The initial SAR study done on this lead
compound was focused on several additional hydrophobic,
aromatic substitutions at the 6 position, including 1-
methylnaphthyl, 2-methylnaphthyl, 2-methylindanyl, and ethyl-
phenyl. The side chains of these four compounds were chosen
to mirror modifications made in a peptide series upon which
20
the peptidomimetic scaffold was based, and as expected,
modifications featuring a more extended pendant (2-methyl-
naphthyl, 2-methylindanyl, ethylphenyl) were compatible with
the larger DOR inactive binding pocket but not the smaller
DOR active pocket, explaining the observed low efficacy at
DOR. While these compounds displayed the desired MOR
agonist/DOR antagonist efficacy profile, their binding profile
was not optimal. The MOR affinity for all four compounds was
at least an order of magnitude higher than the DOR affinity,
and the 2-methylnaphthyl compound showed an over 2 orders
of magnitude preference for MOR. Ligands with more balanced
binding affinities at MOR and DOR would provide a better
starting point for further development of this type of mixed-
9
,30
efficacy opioid ligand. Additionally, although we showed that
an extended hydrophobic pendant translates to low DOR
efficacy, changes in the electronic characteristics and polarity of
the pendant were left unexplored.
To begin our expanded SAR, we first replaced the phenyl
pendant of our lead compound (Figure 1a) with a 3-pyridine
(10a; Table 1). We observed not only a slight loss in binding
affinity (K ) at both MOR and DOR (Table 1) but also a
i
significant loss in MOR efficacy (EC ) and potency (as
5
0
maximal % stimulation) (Table 2) (see Methods for details of
the in vitro assays). Although 10a adopts a similar
conformation as our lead compound in the MOR active site,
this loss in MOR binding and efficacy can be attributed to loss
of hydrophobic contacts in this region of the receptor binding
pocket (see Figure 3). Although this analogue did not improve
upon the MOR agonist/DOR antagonist profile of our previous
compounds, we were intrigued by the drastic consequences that
a simple change in pendant electronics had on both the binding
and efficacy and wished to explore this further. Compared with
10a and our lead compound, replacement with piperidine in
analogue 10b widened the binding affinity preference for MOR
over DOR even further, although this compound behaved as a
moderately potent full agonist at MOR, improving upon the
MOR efficacy profile of 10a. Expansion of the piperidine ring in
a
Binding affinities (K ) were obtained by competitive displacement of
H]diprenorphine in membrane preparations expressing either MOR,
i
[3
10b to azepane (10c) resulted in improved binding at DOR
and KOR. In contrast, the morpholine analogue 10d displayed
diminished binding affinities at DOR and KOR and also
decreased potency at MOR compared with 10b. We next
turned our attention to smaller aromatic systems, including
DOR, or KOR. All values are mean ± standard error of the mean
b
(SEM) of three separate assays performed in duplicate. Data from ref
c
17. Calculated using ChemBioDraw Ultra, version 14.0.
1
,2,4-triazole (10e) and 3-furan (10f). While the overall
extended pendant and pendant electronic characteristics are
important for maintaining binding at DOR for this series. Using
our previously published models of interactions of opioid
binding profile of 10f was comparable to those for the previous
substitutions, 10e displayed a marked loss in binding affinity for
MOR and KOR and displayed no efficacy at MOR.
2
0,21
ligands with the active states of the three receptors,
we
17
In the initial series, the 2-methylnaphthyl modification
resulted in the highest MOR efficacy, but the MOR/DOR
binding balance favored MOR by over 2 orders of magnitude.
To see whether changes in the electronics of the naphthyl
system could improve DOR binding while maintaining low
DOR efficacy, we first synthesized quinoline analogue 10g.
Interestingly, the binding affinities of 6-quinoline analogue 10g
at all three receptors were considerably lower than those of the
previous bicyclic analogues. This finding suggests that both an
docked 10g into the MOR active binding pocket. The
quinoline nitrogen of 10g was found to extend much deeper
into the hydrophobic pocket of the MOR active pocket,
disrupting important contacts with hydrophobic residues
W133, V143, and I144, as shown in Figure 3.
These initial data suggested that superior MOR efficacy (and
low DOR efficacy) may result from a fused-ring pendant in
which the six-membered non-heteroatom-containing aromatic
moiety is located in the position most distal from the THQ
1
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a
Table 2. Opioid Receptor Efficacies for Analogues 10a−o
EC50 (nM)
% stimulation
DOR
compound
MOR
DOR
KOR
MOR
KOR
b
b
b
b
b
b
a (Fig. 1)
1.6 ± 0.3
93 ± 20
9 ± 1
25 ± 11
60 ± 2
dns
110 ± 6
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
540 ± 72
dns
81 ± 2
37 ± 7
73 ± 8
52 ± 2
82 ± 2
dns
16 ± 2
22 ± 2
dns
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
72 ± 24
23 ± 13
2.2 ± 0.9
14 ± 3
3 ± 1
0.4 ± 0.1
2.0 ± 0.5
6 ± 2
>1000
dns
18 ± 2
34 ± 6
84 ± 6
36 ± 3
96 ± 4
105 ± 6
56 ± 2
91 ± 8
118 ± 5
72 ± 3
>40
0g
0h
0i
dns
dns
dns
dns
dns
0j
15 ± 9
90 ± 65
14 ± 2
25 ± 4
14 ± 1
46 ± 5
32 ± 1
>20
0k
0l
600 ± 400
160 ± 36
400 ± 130
>2000
0m
0n
0o
0.9 ± 0.4
40 ± 20
a
35
Efficacy data were obtained using agonist-induced stimulation of [ S]GTPγS binding in membrane preparations expressing either MOR, DOR, or
KOR. Potencies are represented as EC50 (nM) and efficacies as percent maximal stimulation relative to the standard agonist DAMGO (MOR),
DPDPE (DOR), or U69,593 (KOR) at 10 μM. All values are expressed as the mean ± SEM of three separate assays performed in duplicate. dns:
b
does not stimulate. Data from ref 17.
THQ core improves DOR affinity without increasing DOR
efficacy, so we likewise explored the effect of an acetyl
substituent here, giving the final analogues 10m−o. We were
pleased to see not only that this modification improved DOR
binding relative to the unacetylated counterpart compounds
(
10j−l) but also that 10m showed similarly high affinity for
MOR and DOR and, interestingly, for KOR as well. As
expected from its high binding affinity and lack of stimulation
35
of [ S]GTPγS binding, 10m acted as an antagonist of DPDPE.
0m afforded a 7.8-fold rightward shift in the agonist
1
concentration−response curve for DPDPE, giving an antagonist
affinity constant (K ) of 4.6 nM for 10m.
e
An overlay of 10k docked into the active sites of all three
receptors is shown in Figure 4. The compound fits nicely into
the MOR active site but clashes with M199 and L125 in the
DOR active site. It is interesting to note that 10k and 10m,
both featuring the 1,2,3,4-tetrahydroisoquinoline (THIQ)
pendant, behave as partial KOR agonists. As shown in Figure
Figure 3. Docking of 10g in the MOR active site. Key hydrophobic
contacts (I144, V143, W133) are highlighted in red.
core. To test this hypothesis, analogues 10h−l were
synthesized. 10h showed high efficacy at MOR and
approximately 10-fold improved DOR binding compared with
the lead compound and 10g. 10j and 10k both behaved as
potent full MOR agonists that improved upon the efficacy of
our original lead with no efficacy at DOR. On the other hand,
the MOR efficacy was reduced in the case of 3,4-
(
methylenedioxy)phenyl analogue 10i. This is again consistent
with the observation that distal electron rich substitutions
adversely affect the MOR efficacy. Reduction of the aromatic
ring of 10k to give decahydroisoquinoline analogue 10l
maintained a comparable but slightly inferior in vitro profile
compared with 10k. While 10j and 10k showed potent
stimulation at MOR (while exhibiting no efficacy at DOR), we
still wished to improve the binding affinities of each at DOR.
We reasoned that the THQ aniline was synthetically accessible
and amenable to substitutions and would be the next logical site
for diversification. Preliminary studies on related analogues (to
be published in due course) suggested that N-acetylation at the
Figure 4. Overlay of 10k in the MOR, DOR, and KOR active sites.
Gray, yellow, and purple residues correspond to MOR, DOR, and
KOR.
1
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, 10k fits in the KOR active site but clashes slightly with I294
Research Article
4
(
and thus displays lower efficacy compared with MOR).
Additionally, the THIQ nitrogen of 10k is positioned to make a
polar contact with Y312, a residue unique to the KOR binding
pocket at this position, which may account for the high affinity
of 10k and 10m for KOR. The MOR agonist/KOR agonist
mixed-efficacy profile has shown promise as a treatment for
31−33
drug dependence, specifically cocaine addiction,
and an
additional SAR study on MOR/KOR agonist peptides has
34
recently been reported. Further substitutions on the THIQ
pendant will have to be explored to fully optimize this profile,
particularly for the purpose of improved potency at KOR.
In Vivo Studies. On the basis of their favorable in vitro
profiles, compounds 10j, 10k, 10m, and 10n were chosen for in
vivo studies. The effects of 10j, 10k, 10m, and 10n were
compared with those of the lead compound by two-way
analysis of variance with Tukey’s multiple comparisons post
hoc test. A significant interaction (F(12,76) = 8.7, p < 0.0001)
and significant main effects of dose (F(3,76) = 82.7, p <
Figure 6. Time courses of antinociceptive response for compounds
1
0j and 10m in the mouse WWTW assay after intraperitoneal
administration of a cumulative dose of 10 mg/kg.
were found to be completely stable after 30 min at 37 °C (99.6,
0
.0001) and compound (F(4,76) = 24.6, p < 0.0001) were
found. In the mouse WWTW assay (Figure 5), our benzyl
1
00.3, and 95.3 mean % remaining, respectively, after 30 min
compared with the positive control eucatropine (43.2%)).
There also appears to be no correlation between the predicted
clogP values for these analogues (Table 1) and the observed in
vivo data. While the origin of the disparate in vivo results is
unclear, differences in first-pass metabolism after intraperitoneal
administration may be a contributing factor. To this end,
additional synthetic and pharmacokinetic studies will need to
be performed to shed light on how these subtle structural
differences can have such a profound effect on the
bioavailability of this scaffold.
35
CONCLUSIONS
■
We have described a series of opioid peptidomimetics with
chemically diverse substitutions at the 6 position of the THQ
scaffold and have shown that changes in both the steric and
electronic characteristics of the pendant can have profound
impacts on receptor selectivity for both binding and efficacy.
Intermediate 3, which is relatively simple to synthesize and can
be produced on a multigram scale, represents a valuable
building block for the expedient synthesis of a wide range of
opioid small molecules and makes possible the incorporation of
diverse and readily available side chains (through either Suzuki
Figure 5. Cumulative antinociceptive dose−response curves for
compounds 10j, 10k, 10m, and 10n in the mouse WWTW assay
after intraperitoneal administration (n = 3−6). Data are plotted as
mean ± SEM.
pendant lead compound and compounds 10j and 10m were
fully efficacious and produced dose-dependent increases in
latency to tail flick, with 3.2 mg/kg (at least p < 0.05) and 10
mg/kg (p < 0.001) significantly increasing the latency time
compared with the baseline. 10m was not statistically different
from the lead compound, but 10j produced slightly higher tail
flick latencies at 3.2 mg/kg (p < 0.001) and 10 mg/kg (p <
coupling or S 2 substitution) that have not yet been explored
N
in traditional opioid ligands. Compounds 10j and 10m also
display promise in vivo, with efficacy and duration of action
comparable to those of morphine, and improve upon the
duration of action of our original lead.
0
.05) compared with the lead compound. It is interesting to
METHODS
■
note that 10k (which lacks only the N-acetyl group of 10m)
and 10n (which is the N-acetylated counterpart of 10j) did not
significantly increase the tail flick latency above baseline levels
up to a dose of 10 mg/kg. To determine the duration of action
of compounds 10j and 10m, tail withdrawal latencies were
measured at intervals following the administration of the
cumulative 10 mg/kg dose (Figure 6). Compounds 10j and
General Synthetic Methods. All of the reagents and solvents
were obtained from commercial sources and used without additional
purification. Reactions were carried out in anhydrous solvents under
an inert atmosphere unless otherwise specified. Suzuki couplings were
performed in a Discover S-class (CEM) microwave reactor in a closed
vessel with a maximum power input of 300 W. Flash column
chromatography was carried out using P60 silica gel (230−400 mesh).
Purification of final compounds was performed using a Waters
semipreparative HPLC instrument with a Vydac protein and peptide
C18 RP column, using a linear gradient of 10% solvent B (0.1% TFA
in acetonitrile) in solvent A (0.1% TFA in water) to 60% solvent B in
solvent A at a rate of 1% per minute. UV absorbance was monitored at
1
0m showed a full antinociceptive response for 200 min before
returning to baseline. Compared with the lead compound
Figure 1a), these compounds both displayed a much longer
(
duration of action after intraperitoneal injection.
In an effort to explain the unpredictable in vivo results for
these structurally similar analogues, compounds 10j, 10k, and
2
30 nm. The purities of synthesized compounds were determined on a
Waters Alliance 2690 analytical HPLC instrument with a Vydac
protein and peptide C18 RP column, using a linear gradient of 0%
1
0m were screened to determine their plasma stabilities, and all
1
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solvent B in solvent A to 45% solvent B in solvent A in 45 min,
All of the samples were analyzed by LC−MS/MS using an AB Sciex
API 4000 instrument coupled to a Shimadzu LC-20AD LC pump
system. Analytical samples were separated using a Waters Atlantis T3
dC18 RP-HPLC column (10 mm × 2.1 mm) at a flow rate of 0.5 mL/
min. The mobile phase consisted of 0.1% formic acid in water (solvent
A) and 0.1% formic acid in 100% acetonitrile (solvent B).
measuring the UV absorbance at 230 nm. The purities of the final
1
compounds used for testing were ≥95% as determined by HPLC. H
13
19
NMR, C NMR, and F NMR data were obtained on either a 400 or
5
00 MHz Varian instrument. In CDCl , the chemical shifts were
3
referenced to tetramethylsilane (TMS). If the TMS peak was not
visible in the 13C NMR spectrum, the chemical shifts were referenced
to the solvent peak (δ 77.16). Samples in CD OD were unreferenced.
Mass spectral analysis was performed using an Agilent 6130 LC−MS
instrument in positive mode.
ASSOCIATED CONTENT
3
■
*
S
Supporting Information
1
13
Experimental procedures, H and C NMR data, MS (EI) and
HPLC data, X-ray crystallography data, and copies of NMR
spectra. The Supporting Information is available free of charge
on the ACS Publications website at DOI: 10.1021/
acschemneuro.5b00100.
In Vitro Assays. Binding affinity (K ) was measured by the
competitive displacement of [ H]diprenorphine (a nonselective opioid
antagonist) in C6 cells stably expressing MOR or DOR or in Chinese
hamster ovary (CHO) cells stably expressing KOR. In vitro potencies
i
3
(
EC ) and efficacies (as maximal % stimulation) were obtained by
50
35
agonist-stimulated [ S]GTPγS binding in the same cell types using
previously described protocols.
2
1,29,36
To determine the DOR
AUTHOR INFORMATION
Corresponding Author
■
antagonist activity of 10m, a concentration−response curve for
35
DPDPE was obtained using the [ S]GTPγS binding assay in C6
*
E-mail: him@med.umich.edu. Phone: (734) 764-8117.
cells expressing DOR in the presence or absence of 30 nM compound
21
1
0m, as previously described. The ratio of EC50 values of DPDPE in
Author Contributions
the presence and absence of 10m was determined to provide the dose
Synthesis was carried out by A.M.B. The research project was
designed by A.M.B., H.I.M., and J.R.T. In vitro assays were
carried out by N.W.G. In vivo studies were designed by J.P.A.
and E.M.J. and carried out by J.P.A.
ratio. The antagonist affinity constant K for 10m was calculated using
e
the equation K = [10m]/(DR − 1), where DR is the dose ratio of
e
agonist in the presence and absence of 10m. All of the concentration−
response curves were analyzed using GraphPad Prism (La Jolla, CA).
Animals. Adult male C57BL/6 mice, purchased from Harlan
Laboratories (Indianapolis, IN, USA) and weighing between 20 and 30
g at 8−16 weeks old, were used for the described experiments. 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 9 am and 5 pm. Studies were performed in accordance with
the University of Michigan Committee on the Use and Care of
Funding
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 (T32 DA007267) and N.W.G. by the Pharmacological
Sciences Training Program administered by NIGMS (T32
GM007767).
Notes
3
7
Animals and the Guide for the Care and Use of Laboratory Animals.
The authors declare no competing financial interest.
Antinociception. Each compound was dissolved in sterile saline
and administered by intraperitoneal injection in a volume of 10 mL/kg
of body weight. Antinociceptive effects were evaluated in the WWTW
assay. Tail withdrawal latencies were determined by briefly placing a
mouse into a plastic, cylindrical restrainer and putting 2−3 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 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.
ACKNOWLEDGMENTS
■
We are grateful to Dr. Irina Pogozheva for assistance with the
ligand−receptor docking. We thank Tyler Trask, Evan
Schramm, Aaron Chadderdon, and Chao Gao for additional
in vitro studies, Dylan Kahl for additional synthetic
contributions, and Brittany Van Koevering for in vivo
contributions.
Acute antinociceptive effects were determined using a cumulative
dosing procedure. Each animal received an intraperitoneal injection of
saline, and then 30 min later the baseline withdrawal latencies (3−6 s)
were recorded. Following baseline determinations, increasing cumu-
lative doses of the test compound were given intraperitoneally at 30
min intervals. Thirty minutes after each injection, the tail withdrawal
latency was measured as described above.
Plasma Stability. Plasma stability was assessed by Quintara
Discovery (San Francisco, CA). Mouse plasma (K2 EDTA) was
obtained from BioreclamationIVT. The assay was carried out in 96-
well microtiter plates. Compounds were diluted to 200 μM in DMSO
and then spiked into the plasma. After mixing, samples were
immediately aliquoted into three 96-well plates. The t = 0 plate was
quenched immediately. The other two plates were incubated at 37 °C.
Reaction mixtures (20 μL) contained a test compound final
concentration of 1 μM. The extent of metabolism was calculated as
the disappearance of the test compound compared with the 0 min
control reaction incubations. Eucatropine was included as a positive
control to verify the assay performance.
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