Bivalent ligands that target μ opioid (MOP) and cannabinoid1 (CB 1) receptors are potent analgesics devoid of tolerance

Article abstract of DOI:10.1021/jm4005219

Given that μ opioid (MOP) and canabinoid (CB₁) receptors are colocalized in various regions of the central nervous system and have been reported to associate as heteromer (MOP-CB₁) in cultured cells, the possibility of functional, endogenous MOP-CB₁ in nociception and other pharmacologic effects has been raised. As a first step in investigating this possibility, we have synthesized a series of bivalent ligands 1-5 that contain both μ agonist and CB₁ antagonist pharmacophores for use as tools to study the functional interaction between MOP and CB₁ receptors in vivo. Immunofluorescent studies on HEK293 cells coexpressing both receptors suggested 5 (20-atom spacer) to be the only member of the series that bridges the protomers of the heteromer. Antinociceptive testing in mice revealed 5 to be the most potent member of the series. As neither a mixture of monovalent ligands 9 + 10 nor bivalents 2-5 produced tolerance in mice, MOR-CB₁ apparently is not an important target for reducing tolerance.

Full text of DOI:10.1021/jm4005219

Article
pubs.acs.org/jmc
Bivalent Ligands That Target μ Opioid (MOP) and Cannabinoid1 (CB1)
Receptors Are Potent Analgesics Devoid of Tolerance
Morgan Le Naour,† Eyup Akgun,† Ajay Yekkirala,† Mary M. Lunzer,† Mike D. Powers,†
̈
Alexander E. Kalyuzhny,‡ and Philip S. Portoghese*,†
†Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minneapolis 55455, United States
‡Department of Neuroscience, Medical School, University of Minnesota, Minneapolis, Minnesota 55455, United States
ABSTRACT: Given that μ opioid (MOP) and canabinoid (CB1)
receptors are colocalized in various regions of the central nervous
system and have been reported to associate as heteromer (MOP-
CB1) in cultured cells, the possibility of functional, endogenous
MOP-CB1 in nociception and other pharmacologic effects has been
raised. As a first step in investigating this possibility, we have
synthesized a series of bivalent ligands 1−5 that contain both μ
agonist and CB1 antagonist pharmacophores for use as tools to
study the functional interaction between MOP and CB1 receptors
in vivo. Immunofluorescent studies on HEK293 cells coexpressing both receptors suggested 5 (20-atom spacer) to be the only
member of the series that bridges the protomers of the heteromer. Antinociceptive testing in mice revealed 5 to be the most
potent member of the series. As neither a mixture of monovalent ligands 9 + 10 nor bivalents 2−5 produced tolerance in mice,
MOR-CB1 apparently is not an important target for reducing tolerance.
Given that resonance energy transfer studies on coexpressing
MOP and CB1 receptors in cultured cells have suggested they
are organized as heteromer,25,26 together with reports that these
receptors are colocalized within neurons in rat nucleus
accumbens27 and dorsal horn,28 the possibility is raised that
heteromeric MOP/CB1 receptors may be involved in some of
the pharmacological interactions that have been reported.
As our laboratory has developed a number of selective
ligands for investigating heteromeric GPCRs,29−35 the present
study was initiated to develop bivalent ligands as pharmacologic
tools to study the MOP-CB1 heteromer. The bivalent ligands
designed for this purpose consist of a selective μ receptor
agonist connected to a CB1 receptor-selective antagonist/
inverse agonist via a spacer of varied length. Here we show in
vitro evidence for bridging of protomers in a MOP-CB1
heteromer by a μ agonist/CB1 antagonist bivalent ligands and
their pharmacologic properties in vivo.
INTRODUCTION
■
The pharmacologic interaction between opioid and cannabi-
noid CB1 receptors has received considerable attention over the
years. Both receptors are coupled to Gi /Go G-protein, and they
are expressed in overlapping areas of the central nervous system
(CNS).1−5 Stimulation of either receptor with respective
agonists inhibits adenylcyclase activity, blocks voltage-depend-
ent calcium channels, activates potassium channels, and
stimulates the mitogen activated protein (MAP) kinase cascade.
Each receptor shares pharmacological properties that include
analgesia, tolerance, and dependence upon chronic admin-
istration.6−8 Because of similar distribution of both receptors in
pain processing areas, their interaction can play an important
role in antinociception.9−11 For example, cross-tolerance
between tetrahydrocannabinol and morphine and the possi-
bility that these receptors interact pharmacologically were
demonstrated through antagonism of antinociception by
naloxone or CB1 antagonist.12 Moreover, synergism between
cannabinoids and opioids at subeffective doses has been
reported.13−18 Additionally, coadministration of morphine
with a CB1 antagonist inhibited the development of both
acute and chronic tolerance to morphine.19 Other evidence for
interaction was obtained from self-administration studies
showing that both receptors are involved in reward processes.
In this regard, both CB1 antagonist 7 (SR 141716)20 and opioid
antagonist naloxone reduced self-administration of morphine or
THC.21,22 Furthermore, studies on CB1 knockout mice have
shown that the dependence and reward properties were
attenuated for morphine but not for other drugs of abuse.23
In mice lacking μ (MOP) and δ (DOP) opioid receptors, the
cannabinoid withdrawal syndrome is reduced.24
DESIGN RATIONALE AND CHEMISTRY
■
The design rationale for targeting neighboring protomers in a
MOP-CB1 heteromer was based on our studies with bivalent
ligands that contain both agonist and antagonist pharmaco-
phores for heteromeric opioid receptors.34−36 The MOP
agonist pharmacophore is derived from α-oxymorphamine37
6, which was employed previously for several bivalent series. In
light of reports38,39 that show that a CB1 antagonist is capable
of eliminating morphine tolerance, dependence, and reinstate-
Received: April 9, 2013
© XXXX American Chemical Society
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ment, the CB1 antagonist 7 was employed as a basis for the
second pharmacophore in the bivalent ligand.20
after 7 with a minor modification of the piperidine ring based
upon reported SAR studies.44
The choice of 16 as a CB1 antagonist pharmacophore and
intermediate in the synthesis was based upon reported SAR
studies of 7.44 This intermediate was prepared from 1145 and
converted to 18 with diglycolic anhydride or activated with
carbonyldiimidazole to afford 17 (Scheme 1). Intermediates
12−15 are described in the literature.35 Intermediate 16 is a
modified analogue of 8 whose binding affinity and CB2/CB1
selectivity are comparable to those of SR141716 (7).
The synthesis of target compounds 1−5 and 10 is outlined in
Scheme 2. Bivalent ligands 1 and 2 were prepared by reacting 6
with CB1 antagonist intermediates 17 and 18, respectively. The
intermediates (19a, 19b, 19c) for the synthesis of 3−5 were
obtained by coupling 6 with the spacer components 15a, 15b,
15c (Scheme 1), respectively. Condensation of 19a with 18
afforded 3. Intermediates 19b and 19c gave rise to 4 and 5,
respectively, via standard coupling procedure. The monovalent
ligand 10 was prepared by coupling 13 (n = 4) with 18. The
monovalent ligand 9 was prepared as previously described.46
In prior studies we have explored the relationship between
spacer length and function of protomers in heteromers, with
spacers ranging from 6 to 21 atoms, given that our data are
consistent with efficient bridging in the range of 18−22
atoms.29−36,40−43 For the present series, we have synthesized
bivalent ligands that contain 2, 6, 15, 19, and 20atoms (1−5,
respectively), with the expectation that 4 and/or 5 would be
capable of bridging the protomers (Figure 1). Monovalent μ
agonist 9 and CB1 antagonist 10 served as controls in our
studies. The bivalent ligands 1−5 consisted of a μ agonist
tethered to a CB1 antagonist pharmacophore that was modeled
BIOLOGICAL STUDIES
■
Immunofluorescence. Transiently coexpressing MOP and
CB1 receptors in HEK-293 cells were treated with immuno-
fluorescent antibodies to image the receptors after treatment
with monovalent (9, 10) and bivalent (2−5) ligand. The
present study was based on prior imaging studies that have
revealed a lack of internalization of coexpressing MOP and
DOP receptors in HEK-293 cells upon treatment with a MOP
agonist/DOP antagonist bivalent ligand (MDAN-21), which is
capable of bridging neighboring protomers. This was was in
contrast to its homologue (MDAN-16) with a short spacer or a
combination of monovalent MOP agonist/DOP antagonist
ligands.48 Thus, the internalization assay represents a valuable
correlate for evaluation of agonist−antagonist bivalent ligands
bridging GPCR heteromers.
Under basal conditions (no treatment) or in the presence of
monovalent CB1 antagonist 10, MOP and CB1 receptors were
localized exclusively on the plasma membrane (Figure 2).
However, treatment with monovalent agonist 9, a mixture of
monovalent agonist and antagonist (9 + 10), or bivalent ligands
(2, 3) with relatively short spacers resulted in robust receptor
clustering and endocytosis. A significant level of fluorescence
also was observed in the cytosol when the combination of both
monovalent ligands 9 and 10 was used, and the internalization
was similar to that observed for control ligand 9 alone.
Treatment with the monovalent CB1 antagonist alone
produced no internalization. Ligand 4 did not produce as
intense a co-internalization as shorter bivalent ligands,
especially at the MOP receptor. Significantly, no co-internal-
ization of μ and CB1 receptors was induced by the longest
bivalent ligand 5, as both receptors remained located on the
plasma membrane. These data are consistent with the idea that
5 with a 20-atom spacer bridges neighboring protomers. The
specific number of atoms required for bridging for a particular
bivalent series is dependent on both the constitution of the
antagonist pharmacophore and the bound protomer.
Pharmacological Evaluation. The in vivo profiles of
bivalent ligands with 2-, 6-, 15-, 19-, and 20-atom spacers (1−5)
and monovalent ligands (9, 10) were determined using the
mouse tail-flick assay. The monovalent ligands were adminis-
tered either singly or in combination. All compounds were
evaluated for antinociception and tolerance after intracere-
Figure 1.
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a
Scheme 1. Intermediates for synthesis of bivalent ligands 1-5 and monovalent ligands 9 and 10
a
i. SOCl2; ii. 4-Amino-1-Boc-piperidine, triethylamine; b) TFA; c) Diglycolic anhydride; d) Carbonyldiimidazole, triethylamine.
a
Scheme 2. Synthesis of target compounds (1-5, 10)
a
Triethylamine; b) DCC/HOBt. c) TFA
broventricular (icv) or intrathecal (i.t.) injection. These data are
summarized in Table 1. For evaluation of tolerance, the ED80−90
dose was measured on day 1 and compared to the same dose
measured 24 h later in the same mice. With the exception of the
monovalent opioid agonist 9 and the bivalent ligand 1, the
bivalent compounds (2−5) and combination of monovalent
ligands 9 and 10 were devoid of tolerance. Tolerance occurred
only when the agonist 9 was applied alone36 or when 1 was
administered icv.
appeared to be no significant difference in potency after acute
coadministration of an equimolar dose ratio of 9 and antagonist
10 when compared to that of 9 alone. However, in contrast to 9
alone, the coadministration with CB1 antagonist did not
produce 24 h tolerance by either the icv or i.t. routes (Table 1).
This was consistent with the absence of tolerance of bivalent
ligands 2−5, presumably because of the presence of the CB1
antagonist pharmacophore.
The icv antinociception SAR profile of bivalent ligands 1−5
was somewhat different from that obtained after i.t.
administration. By the icv route, a mixture of 9 and 10 was
significantly more potent than 1 or 4, whereas this mixture was
The monovalent CB1 antagonist 10, like its parent
compound 7 (SR141716),44a did not produce antinociception
even at a relatively high dose (2.5 nmol/mouse). There
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postpone tolerance measurement to 48 h as mentioned in
Table 1.
DISCUSSION
■
A key side effect of morphine and related μ agonists in the
pharmacotherapy of chronic pain is the development of
tolerance. Prior studies12−18 have shown there is pharmacologic
interaction between MOP and cannabinoid CB1 receptors. In
view of evidence for heteromeric MOP-CB1 receptors in
cultured cells and the report that i.t. coadministration of
morphine with a CB1 antagonist prevents tolerance in
rats,19,25,26 we have investigated whether this effect is mediated
via the physical association of MOP and CB1 receptors or
through other mechanisms.
Our approach to addressing this question involved the design
and synthesis of bivalent ligands that contain μ opioid agonist
and CB1 antagonist pharmacophores. As we have reported that
spacers in the range of 18−22 atoms can lead to bridging of
protomers in a variety of heteromers containing opioid
receptors in cultured cells,32,33,48,49 such a bivalent ligand
could be useful in selectively targeting a MOP-CB1 heteromer
to determine if opioid tolerance is affected.
Accordingly, we have synthesized a series of bivalent ligands
containing both μ agonist and CB1 antagonist pharmacophores
that are connected by shorter spacers (1−3) or longer spacers
(4, 5). Control monovalent ligands 9 and 10 also were
prepared. Bivalent ligands 1−3 would be expected to interact
univalently with either MOP or CB1 receptors, whereas 4 and 5
containing 19- and 20-atom spacers, respectively, have been
reported to be in the bridging range of protomers in other
heteromers.29−36,48,49
An immunocytochemical-derived correlate for establishing
bridging of protomers by a bivalent ligand containing μ agonist
and δ antagonist pharmacophores in MOR-DOR heteromer in
HEK-293 cells has been employed.48 In that particular study, it
was determined in HEK293 cells that bivalent ligands with 21-
and 16-atom spacers (MDAN21 and MDAN16) exhibit
different trafficking in that receptors that are not bridged
undergo agonist-induced endocytosis while those that are
bridged remain on the cell surface.47,48 Thus, MDAN21
prevented internalization whereas MDAN16-treated cells
exhibited endocytosis. Similar studies performed on HEK-293
cells coexpressing MOP and CB1 receptors are consistent with
the view that bridging of protomers takes place only with
compound 5 (20-atom spacer), as no significant receptor
internalization was observed. On the other hand, bivalent
ligands with 6-, 15-, and 19-atom spacers (2, 3, and 4,
respectively) induced robust internalization, as did monovalent
μ agonist 9 or a mixture of 9 and monovalent CB1 antagonist
10 (Figure 2).
The evaluation of antinociception and tolerance in mice
using two routes of administration (icv and i.t.) revealed that
bivalent ligands 2−5 and a mixture of monovalent μ agonist 9
and CB1 antagonist 10 were devoid of tolerance. These data
afforded results that are consistent with pharmacologic
interaction between MOP and CB1 receptors. It is noteworthy
that bivalent ligand 1 with the shortest (two-atom) spacer
possessed substantially lower potency with tolerance (Table 1)
relative to other bivalent ligands 2−5 when administered icv.
Since 1 possesses a two-atom spacer, it is likely that the CB1
pharmacophore’s antagonist activity has been compromised
because of its proximity to the opioid pharmacophore. In the
absence of CB1 antagonist activity, 1 would therefore produce
Figure 2. Single cell high-magnification images of double-labeling
immunofluorescence for coexpressed MOP and CB1 receptors on
HEK-293 cells. The identical cells are shown labeled for MOP (red
fluorescence) and CB1 (green fluorescence) receptors. Arrows depict
profiles double-labeled for MOP and CB1.
clearly less potent than 5. When administered i.t., bivalents 2−5
were significantly more potent than 9 + 10. Upon icv
administration, 5 (20-atom spacer) exhibited a substantial 20-
fold potency increase relative to its lower homologue 4 with a
19-atom spacer. By comparison, there was only a 4-fold
increase by the i.t. route. Significantly, 5 not only is the most
potent member of the series but was still active (47%) 24 h
after icv admininistration. This long duration of action led us to
D
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a
Table 1. Antinociception and 24 h tolerance evaluation after icv and i.t. administration of compounds 1-5 and 9-10 in ICR-
CD1 mice
I.C.V.
I.T.
ED50 (95% C.I.)
pmol/mouse
ED50 (95% C.I.)
pmol/mouse
Ratio
icv/i.t.
Cmpd
24 hr tolerance
24 hr tolerance
1
1219 (859- 1730)
523 (223−1228)
55 (37−82)
Day 1: 64.27 6.55 Day 2: 33.85 11.38
Tolerance
559 (372- 840)
27 (13−54)
42 (31−58)
19 (11−34)
5 (3- 8)
Day 1: 73.55 10.93 Day 2: 66.01 6.81
No Tolerance
2.18
19.48
1.31
9.86
1.8
2
3
Day 1: 77.72 9.78 Day 2: 63.70 16.48 No
Tolerance
Day 1: 81.72 9.48 Day 2: 73.41 11.49
No Tolerance
Day 1: 83.47 11.59 Day 2: 66.99 9.55 No
Tolerance
Day 1: 98.33 1.79 Day 2: 72.5 11.22 No
Tolerance
4
189 (132−270)
9 (6- 12)
Day 1: 95.43 2.93 Day 2: 86.33 7.88 No
Tolerance
Day 1: 78.51 10.44 Day 2: 79.58 9.82
No Tolerance
b
5
Day 1 : 89.75 10.95 Day 3: 72.81 12.69
Day 1: 92.20 8.34 Day 2: 87.91 6.76 No
Tolerance
No Tolerance
9
108 (83 - 141)
Day 1: 82.15 9.67 Day 2: 24.14 11.92
Tolerance
106 (41 - 273)
Day 1: 75.98 12.73 Day 2: 39.54 14.28
Tolerance
1.01
c
d
10
NS
NA
NS
NA
9 +
10
78 (55−110)
Day 1: 68.84 11.94 Day 2: 43.92 15.20
No Tolerance
130 (97−173)
Day 1: 78.00 11.62 Day 2: 74.83 8.45
No Tolerance
0.60
e
a
24 h tolerance was calculated using the highest dose of the dose−response curve on day 1 and repeated on day 2. If there was no significant
difference between the two days the animals were said to be not tolerant. Compounds 9 + 10 (equimolar dose), 1-5 were administered at 2500, 250,
1000, and 125 pmol per mouse. As there was 47% antinociception 24 h after the initial 15 pmol/mouse (icv) dose, a 24 h tolerance test could not
b
be completed. However, after 48 h the antinociception had returned to baseline and, at this time tolerance was measured by injection of a subsequent
c
15 pmol/mouse. NS: not significant; compound 10 did not show any antinociceptive activity (only 20−30%) when given at 250 or 2500 pmol per
d
e
mouse. Not applicable. Doses were based on a 1:1 mixture of compounds 9+10.
tolerance. The reduced antinociception is likely due to steric
hindrance of the opioid pharmacophore at the MOP receptor
by the CB1 pharmacophore.
and the possible benefit of using a CB1 antagonist component
to oppose the development of tolerance induced by opioids.
The finding that bivalent ligands 2−5, whose spacers range
from 6 to 20 atoms, and a mixture of monovalents (9 + 10) are
all devoid of tolerance suggests that the ability of CB1
antagonist to reduce or prevent opioid-induced tolerance is
not necessarily mediated via a MOP-CB1 heteromer. On the
other hand, because the most potent bivalent ligand 5 is
capable of bridging such a heteromer in HEK293 cells, it
cannot be excluded as a target by 5.
It is perhaps significant that 5 is substantially more potent
than its lower homologue 4 by a factor of 20 when given
supraspinally. On the other hand, 5 is more potent than 4 only
by a factor of 4 following the i.t. route. The larger potency
difference icv suggests that there may be more MOP-CB1
heteromer supraspinally. This would be consistent with our
observation that the day 2 tolerance test could not be
conducted because of residual antinociception (∼47%). The
possible supraspinal localization of MOP-CB1 heteromer is a
departure from MOP-DOP36 and DOP-KOP30,50 heteromers
that appear to be localized in the cord.
In conclusion, we have obtained immunofluorescence
evidence in HEK293 cells that support the bridging of MOP
and CB1 protomers in a heteromer by bivalent ligand 5 that
contains a μ agonist linked to a CB1 antagonist via a 20-atom
spacer. Lower homologues with 19 or fewer atoms connecting
the pharmacophores do not share this property. Bivalent ligand
5 possessed the highest antinociceptive potency when
administered either icv or i.t. It was found that 5 and lower
homologue bivalent ligands 2−4 that do not bridge MOP-CB1
heteromer, as well as a mixture of monovalent ligands (μ
agonist 9 and CB1 antagonist 10), all were free of opioid
tolerance. Thus, it appears that MOP-CB1 heteromer may not
be an important exclusive target for inhibition of opioid
tolerance by coadministration. These results further support the
synergestic effects between the opioid and cannabinoid systems
EXPERIMENTAL SECTION
■
All commercial reagents and anhydrous solvents that were purchased
from vendors were used without further purification or distillation.
Analytical thin-layer chromatography (TLC) was performed on EM
Science silica gel 60 F254 (0.25 mm). Plates were visualized by UV
light, iodine vapor, or ninhydrin solution. Flash column chromatog-
raphy was performed on Fisher Scientific silica gel (230−400 mesh).
Melting points were determined on a Thomas-Hoover melting point
apparatus and are uncorrected. 1H NMR spectra and 13C NMR spectra
were taken on a Bruker Avance 400 MHz instrument and calibrated
using an internal reference. Chemical shifts are expressed in ppm, and
coupling constants (J) are in hertz (Hz). Peak multiplicities are
abbreviated as follows: broad, br; singlet, s; doublet, d; triplet, t;
multiplet, m. ESI mode mass spectra were recorded on a
BrukerBioTOF II mass spectrometer. Elemental analyses were
performed by M-H-W Laboratories, Phoenix, AZ. Analytical data
confirmed that the purity of the products was ≥95%.
General Procedure for DCC/HOBt Coupling. DCC (37 mg,
0.18 mmol), carboxylic acid 16 (70 mg, 0.12 mmol), and HOBt (22
mg, 0.16 mmol) were dissolved in 5 mL of anhydrous DMF. The
solution was cooled to 0 °C and placed under a nitrogen atmosphere.
After 15 min at 0 °C, amine (0.11 mmol) was added. The solution was
sealed under a nitrogen atmosphere and was allowed to warm and to
stir at room temperature overnight. The reaction mixture was filtered
in order to remove dicyclohexylurea. The filtrate was poured into
water (10× initial volume of DMF) and extracted with ethyl acetate.
The combined organic layers were dried on magnesium sulfate,
filtered, and concentrated under reduced pressure. The residue was
then purified by flash chromatography. The title compound was then
subsequently converted into the HCl salt for biological testing.
4-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazole-3-carboxamido)-N-((4αS,7S,7αR,12βS)-4α,9-dihy-
droxy-3-methyl-2,3,4,4α,5,6,7,7α-octahydro-1H-4,12-
methanobenzofuro[3,2-e]isoquinolin-7-yl)piperidine-1-car-
boxamide (1). Combining amine 6 (85 mg, 0.28 mmol), compound
17 (172 mg, 0.30 mmol), and triethylamine (48 μL, 0.34 mmol) in
DCM gave the target compound which was purified by flash
chromatography [MeOH/DCM/NH4OH = 3/96/1] to provide 1 as
E
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1
a white solid. Yield: 188 mg, 85%. H NMR (CD3OD) δ 0.97 (m,
1H), 4.60 (m, 1H, CONH), 6.53 (d, 1H, J = 8.2 Hz), 6.72 (d, 1H, J =
8.2 Hz), 7.04 (d, 2H, J = 8.5 Hz), 7.15 (d, 1H, J = 8.4 Hz), 7.25 (dd,
1H, J = 8.4 Hz, J = 2.2 Hz), 7.29 (d, 2H, J = 8.6 Hz), 7.44 (d, 1H, J =
2.2 Hz), 7.49 (m, 1H, CONH). Anal. Calcd for C43H45Cl3N6O7: C,
59.02; H, 5.89; N, 10.39. Found: C, 59.18; H, 5.73; N, 10.24. ESI-TOF
MS calculated for C53H63Cl3N8O10, m/z 1078.474, found 540.171 (M/
2H)+, 1079.366 (MH)+.
1H), 1.34−1.48 (m, 4H), 1.68−1.80 (m, 3H), 1.94 (m, 1H), 2.03 (m,
2H), 2.14 (s, 3H), 2.59 (m, 2H), 2.85 (s, 3H), 3.07−3.13 (m, 2H),
3.16−3.22 (m, 2H), 3.13 (d, 1H, J = 8.9 Hz), 3.23 (m, 1H), 3.83 (m,
1H), 4.14 (m, 1H), 4.35 (m, 1H), 4.49 (m, 1H), 4.64 (m, 1H), 6.64
(d, 1H, J = 8.2 Hz), 6.73 (d, 1H, J = 8.2 Hz), 7.21 (d, 2H, J = 8.5 Hz),
7.38 (d, 2H, J = 8.4 Hz), 7.45 (m, 2H), 7.61 (d, 1H, J = 2.2 Hz). 13C
NMR δ 8.84, 22.02, 30.62, 30.75, 33.57, 42.08, 46.50, 47.19, 48.27,
55.83, 61.54, 64.27, 68.16, 71.05, 82.50, 90.38, 117.08, 119.36, 120.66,
123.34, 126.93, 128.37, 128.57, 129.30, 129.97 (2×), 130.12, 131.18,
132.29 (2×), 132.50, 134.34, 136.27, 137.35, 140.41, 143.86, 147.32,
147.78, 159.57, 165.25. Anal. Calcd for C40H41Cl3N6O7: C, 60.65; H,
5.22; N, 10.61. Found: C, 60.78; H, 5.23; N, 10.57. ESI-TOF MS
calculated for C40H41Cl3N6O5, m/z 792.212, found 793.272 (MH)+.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-(1-(2-(2-
( ( ( 4 α S , 7 S , 7 α R , 1 2 β S ) - 4 α , 9 - d i h y d r o x y - 3 - m e t h y l -
2,3,4,4α,5,6,7,7α-octahydro-1H-4,12-methanobenzofuro[3,2-
e]isoquinolin-7-yl)amino)-2-oxoethoxy)acetyl)piperidin-4-yl)-
4-methyl-1H-pyrazole-3-carboxamide (2). Compound 2 was
prepared in a similar fashion as described above; combining α-
oxymorphamine (6) (33 mg, 0.11 mmol), acid 18 (70 mg, 0.12
mmol), DCC (37 mg, 0.18 mmol), and HOBt (22 mg, 0.16 mmol)
gave the target compound which was purified by flash chromatography
[MeOH/DCM/NH4OH = 2/97/1] to provide 2 as a white solid.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-(1-(19-(((4αS,7-
S,7αR,12βS)-4α,9-dihydroxy-3-methyl-2,3,4,4α,5,6,7,7α-octa-
hydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7-yl)-
amino)-5,15,19-trioxo-3,17-dioxa-6,14-diazanonadecan-1-
oyl)piperidin-4-yl)-4-methyl-1H-pyrazole-3-carboxamide (5).
Compound 5 was prepared in a similar fashion as described above;
combining amine 19c (65 mg, 0.12 mmol), acid 18 (77 mg, 0.13
mmol), DCC (41 mg, 0.20 mmol), and HOBt (23 mg, 0.17 mmol)
gave the target compound which was purified by flash chromatography
[MeOH/DCM/NH4OH = 7/92/1] to provide 5 as a clear oil. Yield:
1
28 mg, 22%. H NMR (CD3OD) δ 0.92 (m, 1H), 1.28−1.43 (m,
11H), 1.47−1.62 (m, 8H), 1.83 (m, 1H), 2.07−2.17 (m, 2H), 2.18 (s,
3H), 2.39−2.42 (m, 2H), 2.46 (s, 3H), 2.51 (m, 1H), 2.57 (m, 1H),
2.89−2.95 (m, 2H), 3.11 (d, 1H, J = 8.8 Hz), 3.15−3.21 (m, 4H),
4.01−4.10 (m, 8H), 4.23 (m, 1H), 4.63 (m, 1H), 4.71 (m, 1H,
CONH), 6.51 (d, 1H, J = 8.2 Hz), 6.69 (d, 1H, J = 8.2 Hz), 7.04 (d,
2H, J = 8.5 Hz), 7.13 (d, 1H, J = 8.4 Hz), 7.19 (dd, 1H, J = 8.4 Hz, J =
2.2 Hz), 7.23 (d, 2H, J = 8.6 Hz), 7.41 (d, 1H, J = 2.2 Hz), 7.57 (m,
1H, CONH). Anal. Calcd for C54H65Cl3N8O10: C, 59.37; H, 6.00; N,
10.26. Found: C, 59.51; H, 5.97; N, 10.31. ESI-TOF MS calculated for
C54H65Cl3N8O10, m/z 1092.501, found 1093.671.
1
Yield: 74 mg, 78%. H NMR (CD3OD) δ 1.01 (m, 1H), 1.25−1.36
(m, 4H), 1.41−1.57 (m, 4H), 1.80 (m, 1H), 2.01 (m, 2H), 2.17 (s,
3H), 2.25 (m, 2H), 2.35 (s, 3H), 2.43 (m, 1H), 2.64 (m, 1H), 2,81 (m,
1H), 2.97 (m, 1H), 3.13 (d, 1H, J = 8.9 Hz), 3.23 (m, 1H), 3.98−4.09
(m, 4H), 4.26 (m, 1H), 4.63 (m, 1H), 4.70 (m, 1H, CONH), 6.66 (d,
1H, J = 8.2 Hz), 6.71 (d, 1H, J = 8.2 Hz), 7.07 (d, 2H, J = 8.5 Hz),
7.11 (d, 1H, J = 8.4 Hz), 7.25 (dd, 1H, J = 8.4 Hz, J = 2.2 Hz), 7.32 (d,
2H, J = 8.6 Hz), 7.45 (d, 1H, J = 2.2 Hz), 7.67 (m, 1H, CONH). Anal.
Calcd for C43H45Cl3N6O7: C, 59.76; H, 5.25; N, 9.72. Found: C,
59.88; H, 5.13; N, 9.77. ESI-TOF MS calculated for C43H45Cl3N6O7,
m/z 864.212, found 865.372 (MH)+.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(1-
(3,7,14-trioxo-5,16-dioxa-2,8,13-triazaoctadecan-18-oyl)-
piperidin-4-yl)-1H-pyrazole-3-carboxamide (10). Compound 10
was prepared in a similar fashion as described above; combining amine
13 (n = 4) (24 mg, 0.11 mmol), acid 18 (70 mg, 0.12 mmol), DCC
(37 mg, 0.18 mmol), and HOBt (22 mg, 0.16 mmol) gave the target
compound which was purified by flash chromatography [MeOH/
1
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-(1-(14-(((4αS,7-
S,7αR,12βS)-4α,9-dihydroxy-3-methyl-2,3,4,4α,5,6,7,7α-octa-
hydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7-yl)-
amino)-5,10,14-trioxo-3,12-dioxa-6,9-diazatetradecan-1-oyl)-
piperidin-4-yl)-4-methyl-1H-pyrazole-3-carboxamide (3).
Compound 3 was prepared in a similar fashion as described above;
combining amine 19a (51 mg, 0.11 mmol), acid 18 (70 mg, 0.12
mmol), DCC (37 mg, 0.18 mmol), and HOBt (22 mg, 0.16 mmol)
gave the target compound which was purified by flash chromatography
[MeOH/DCM/NH4OH = 2/97/1] to provide 3 as a white solid.
DCM= 1/99] to provide 10 as a white solid. Yield. 36 mg, 42%. H
NMR (CD3OD) δ 1.51−1.55 (m, 7H), 1.98 (m, 2H), 2,17 (s, 3H),
2.79 (s, 3H), 2.96 (m, 1H), 3.10 (m, 2H), 3.24−3.30 (m, 4H), 4.00−
4.02 (m, 8H), 4.13 (m, 1H), 4.36 (d, 1H, J = 7.3 Hz), 4.70−4.73 (m,
2H, NH), 7.06 (d, 2H, J = 8.5 Hz), 7.16 (d, 1H, J = 8.4 Hz), 7.25 (dd,
1H, J = 8.4 Hz, J = 2.2 Hz), 7.34 (d, 2H, J = 8.6 Hz), 7.45 (d, 1H, J =
2.2 Hz). Anal. Calcd for C35H42Cl3N7O7: C, 53.96; H, 5.43; N, 12.58.
Found: C, 53.88; H, 5.52; N, 12.67. ESI-TOF MS calculated for
C35H42Cl3N7O7, m/z 779.110, Found 780.284 (MH)+.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(pi-
peridin-4-yl)-1H-pyrazole-3-carboxamide (16). A mixture of the
carboxylic acid 11 (1.14 g, 3 mmol) and thionyl chloride (0.8 mL, 11
mmol) in 20 mL of toluene was refluxed for 3 h and then evaporated
to dryness under reduced pressure. The residue was taken up in 20 mL
of toluene, and the solvent was evaporated again under reduced
pressure to give the crude acid chloride, which was dissolved in 15 mL
of dry dichloromethane. This solution was added dropwise to a
solution of N-Boc-4-aminopiperidine (901 mg, 4.5 mmol) and
triethylamine (620 μL, 4.5 mmol) in 10 mL of dichloromethane,
cooled to 0 °C. After being stirred at room temperature for 16 h, the
reaction mixture was added to brine (30 mL) and extracted with
dichloromethane. The organic layer was separated, dried (magnesium
sulfate), and evaporated under reduced pressure. The residue was then
taken up in DCM, and TFA was added (20% volume). The mixture
was stirred for 16 h at room temperature. The solvent was evaporated
under reduced pressure, and the residue was coevaporated with
toluene (2×). The crude mixture was then purified by flash
chromatography (EA) to afford compound 16 as white foam. Yield:
1
Yield: 73 mg, 65%. H NMR (CD3OD) δ 0.97 (m, 1H), 1.25−1.36
(m, 5H), 1.41−1.57 (m, 4H), 1.80 (m, 1H), 2.01−2.04 (m, 2H), 2.16
(s, 3H), 2.25−2.29 (m, 2H), 2.35 (s, 3H), 2.43 (m, 1H), 2.64 (m, 1H),
2.81 (m, 1H), 2.97 (m, 1H), 3.11 (d, 1H, J = 8.9 Hz), 3.14−3.28 (m,
4H), 4.00−4.08 (m, 8H), 4.24 (m, 1H), 4.59 (m, 1H), 6.54 (d, 1H, J =
8.2 Hz), 6.73 (d, 1H, J = 8.2 Hz), 7.04 (d, 2H, J = 8.5 Hz), 7.14 (d,
1H, J = 8.4 Hz), 7.21 (dd, 1H, J = 8.4 Hz, J = 2.2 Hz), 7.35 (d, 2H, J =
8.6 Hz), 7.41 (d, 1H, J = 2.2 Hz), 7.86 (m, 1H, CONH). Anal. Calcd
for C49H55Cl3N8O10: C, 57.56; H, 5.42; N, 10.96. Found: C, 57.72; H,
5.43; N, 10.87. ESI-TOF MS calculated for C49H55Cl3N8O10, m/z
1022.368, found 1023.276 (MH)+.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-(1-(18-(((4αS,7-
S,7αR,12βS)-4α,9-dihydroxy-3-methyl-2,3,4,4α,5,6,7,7α-octa-
hydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7-yl)-
amino)-5,14,18-trioxo-3,16-dioxa-6,13-diazaoctadecan-1-oyl)-
piperidin-4-yl)-4-methyl-1H-pyrazole-3-carboxamide (4).
Compound 4 was prepared in a similar fashion as described above;
combining amine 19b (57 mg, 0.11 mmol), acid 18 (70 mg, 0.12
mmol), DCC (37 mg, 0.18 mmol), and HOBt (22 mg, 0.16 mmol)
gave the target compound which was purified by flash chromatography
[MeOH/DCM/NH4OH = 2/97/1] to provide 4 as a white solid.
1
1.14 g, 82% (overall). H NMR (CDCl3) δ: 1.41 (m, 2H), 1.52 (m,
2H), 1.93 (m, 2H), 2.18 (s, 3H), 2.95 (m, 2H), 3.20 (m, 1H), 4.29 (d,
1H, NH, J = 7.4 Hz), 4.65 (d, 1H, NH, J = 7.2 Hz), 7.07 (d, 2H, J =
8.5 Hz), 7.15 (d, 1H, J = 8.4 Hz), 7.23 (dd, 1H, J = 8.4 Hz, J = 2.2 Hz),
7.39 (d, 2H, J = 8.6 Hz), 7.45 (d, 1H, J = 2.2 Hz). 13C NMR (CDCl3)
δ 163.1, 143.0, 138.5, 136.0, 135.2, 135.1, 133.1, 130.9 (×2), 130.1,
129.9, 128.7 (×2), 127.8, 12 7.2, 118.2, 56.2, 31.8 (x2), 25.8, 23.5, 8.7.
1
Yield: 67 mg, 57%. H NMR (CD3OD) δ 0.88 (m, 1H), 1.24−1.34
(m, 9H), 1.40−1.59 (m, 8H), 1.79 (m, 1H), 1.99−2.02 (m, 2H), 2.16
(s, 3H), 2.28−2.24 (m, 2H), 2.36 (s, 3H), 2.45 (m, 1H), 2.61 (m, 1H),
2.86−2.91 (m, 2H), 3.09 (d, 1H, J = 8.8 Hz), 3.14−3.18 (m, 4H),
3.99−4.07 (m, 8H), 4.20 (m, 1H), 4.25 (m, 1H, CONH), 4.56 (m,
F
dx.doi.org/10.1021/jm4005219 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Mp 162−163 °C (dec). ESI-TOF MS calculated for C22H21Cl3N4O,
m/z 463.787, found 464.705 (MH)+.
Antinociception is quantified according to the method of Harris and
Pierson53 as the percent maximal possible effect (% MPE) which is
calculated as % MPE = [(test − control)/(10 − control)] × 100. At
least three groups of 8−10 mice were used for each dose−response
curve, and each mouse is used only once. ED50 values with 95%
confidence intervals (CI) are computed with GraphPad Prism 4 by
using nonlinear regression methods.
N-(1-(1H-Imidazole-1-carbonyl)piperidin-4-yl)-5-(4-chloro-
phenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-car-
boxamide (17). To a solution of piperidine 16 (200 mg, 0.43 mmol)
dissolved in DCM with triethylamine (90 μL, 0.64 mmol) was added
dropwise carbonyldiimidazole (83 mg, 0.52 mmol) at room temper-
ature. The mixture was stirred overnight and was concentrated to
dryness. The residue was purified by flash chromatography [MeOH/
DCM = 2/98] to provide 17 as a white solid. Yield: 209 mg, 87%. 1H
NMR (CDCl3) δ 1.80−1.87 (m, 2H), 2.25−2.29 (m, 5H with s, 3H at
2.29), 3.10 (t, 1H, J = 11.9 Hz), 3.41 (t, 1H, J = 12.2 Hz), 4.30 (m,
1H), 4.50 (d, 1H, J = 13.4 Hz), 4.95 (d, 1H, J = 13.3 Hz), 7.04 (d, 2H,
J = 8.5 Hz), 7.15 (d, 1H, J = 8.4 Hz), 7.18 (s, 1H), 7.25 (dd, 1H, J =
8.4 Hz, J= 2.2 Hz), 7.29 (d, 2H, J = 8.6 Hz),7.52 (s, 1H), 8.49 (s, 1H),
8.77 (d, 1H, J = 2.3 Hz). 13C NMR (CDCl3) δ 8.89, 31.50. 32.76,
41.53, 46.52, 48.73, 115.97, 116.72, 121.90 (2×), 126.96, 127.90,
128.99 (2×), 129.33, 129.57, 130.60 (2×), 132.91, 135.69, 135.89,
136.32, 142.12, 146.39, 148.81, 163.78. Mp 189−191 °C. ESI-TOF
MS calculated for C26H23Cl3N6O2, m/z 557.859, found 558.913
(MH)+.
2-(2-(4-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-meth-
yl-1H-pyrazole-3-carboxamido)piperidin-1-yl)-2-oxoethoxy)-
acetic Acid (18). To a solution of piperidine 16 (278 mg, 0.6 mmol)
dissolved in DCM with pyridine (60 mL, 0.72 mmol) was added
dropwise diglycolic anhydride (70 mg, 0.6 mmol). The mixture was
stirred at room temperature overnight. After disappearance of starting
material, the solvent was concentrated to dryness. The residue was
coevaporated with toluene (3×) to afford 18 as a white solid. Yield:
347 mg, quantitative. Compound 18 was used without further
purification. 1H NMR (CDCl3) δ 1.59 (m, 2H), 2.03 (m, 2H), 2.18 (s,
3H), 3.06 (m, 1H), 3.32 (m, 1H), 4.07−4.24 (m, 5H), 4.33 (m, 1H),
4.65 (m, 1H), 7.07 (d, 2H, J = 8.4 Hz), 7.16 (d, 1H, J = 8.3 Hz), 7.24
(dd, 1H, J = 8.4 Hz, J = 2.2 Hz), 7.29 (d, 2H, J = 8.6 Hz), 7.43 (d, 1H,
J = 2.2 Hz). 13C NMR (CDCl3) δ 171.9, 169.5, 163.7, 143.0, 138.8,
136.7, 136.6, 135.3, 133.7, 130.6 (×2), 130.5, 130.3, 128.9 (×2), 127.8,
127.1, 118.2, 72.3, 68.4, 57.2, 46.28 (×2), 32.9, 31.3, 8.9. Mp 227−228
°C. ESI-TOF MS calculated for C26H25Cl3N4O5, m/z 579.860, found
578.673(MH)−.
Biology. Immunofluorescence Protocol. To determine the
effects of the MOP-CB1 bivalent ligands on the trafficking of MOP and
CB1 receptors, we utilized the immunofluorescence technique48 in
HEK-293 cells transiently expressing the receptors. HEK-293 cells
were transfected with cDNA for hMOPR and hCB1 (16 μg/2 million
cells) and seeded 24 h later into eight-well chamber slides. After
allowing for a further 24 h for the cells to settle and equilibrate, they
were incubated with the different ligands (1 μM) for 30 min. The cells
were then washed, fixed with 4% paraformaldehyde, and incubated
with the μ (Neuromics Inc.) and CB1 selective primary antibodies
(R&D Systems) overnight at 4 °C. The cells were then washed the
next day and stained with respective fluorescent secondary antibodies
(red for MOP, green for CB1). Images were acquired using a Olympus
BX10 confocal microscope and analyzed using ImageJ software (NIH).
Animals. Male ICR-CD1 mice (17−25g; Harlan, Madison, WI) are
housed in groups of eight in a temperature- and humidity-controlled
environment with unlimited access to food and water. They are
maintained on a 12 h light/dark cycle. All experiments are approved by
the Institutional Animal Care and Use Committee of the University of
Minnesota (Minneapolis, MN).
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 1-612-624-9174. Fax: 1-612-626-6891. E-mail:
porto001@umn.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by National Institute on Drug
Abuse Research Grant DA01533. We thank Prof. Alex
Makriyannis for preliminary binding data on CB1 antagonist
analogues.
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