Induced association of μ opioid (MOP) and type 2 cholecystokinin (CCK2) receptors by novel bivalent ligands

Article abstract of DOI:10.1021/jm800174p

Both μ-opioid (MOP) and type 2 cholecystokinin (CCK₂) receptors are present in areas of the central nervous system that are involved in modulation of pain processing. We conducted bioluminescence resonance energy transfer (BRET) studies on COS

Full text of DOI:10.1021/jm800174p

J. Med. Chem. 2009, 52, 247–258
247
Induced Association of µ Opioid (MOP) and Type 2 Cholecystokinin (CCK2) Receptors by
Novel Bivalent Ligands
Yaguo Zheng,† Eyup Akgu¨n,† Kaleeckal G. Harikumar,‡ Jessika Hopson,‡ Michael D. Powers,† Mary M. Lunzer,†
Laurence J. Miller,*,‡ and Philip S. Portoghese*,†
Department of Medicinal Chemistry, College of Pharmacy, UniVersity of Minnesota, Minneapolis, Minnesota 55455, and Department of
Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259
ReceiVed February 19, 2008
Both µ-opioid (MOP) and type 2 cholecystokinin (CCK2) receptors are present in areas of the central nervous
system that are involved in modulation of pain processing. We conducted bioluminescence resonance energy
transfer (BRET) studies on COS cells coexpressing MOP and CCK2 receptors to determine whether receptor
heterodimerization is involved in such modulation. These studies revealed the absence of constitutive or
monovalent ligand-induced heterodimerization. Heterodimerization of MOP and CCK2 receptors therefore
is unlikely to be responsible for the opposing effects between morphine and CCK in the CNS. However,
association was induced, as indicated by a positive BRET signal, on exposure of the cells to bivalent ligands
containing µ-opioid agonist and CCK2 receptor antagonist pharmacophores linked through spacers containing
16-22 atoms but not with a shorter (9-atom) spacer. These studies demonstrate for the first time that an
appropriately designed bivalent ligand is capable of inducing association of G-protein-coupled receptors.
The finding that opioid tolerance studies with these ligands in mice showed no correlation with the BRET
data is consistent with the absence of association of MOP and CCK2 receptors in vivo.
Introduction
Activation of MOP or DOP receptors promotes the release of
Both opioid (MOPa) and cholecystokinin (CCK) receptors
are members of the family-A G-protein-coupled receptor
(GPCR) superfamily. Opioid receptors constitute a family of
four receptor types, namely, MOP (µ), DOP (δ), KOP (κ), and
NOP (nociceptin), having ∼60% sequence homology. They are
present in the central nervous system (CNS) and peripherally.
Interaction of members of the opioid peptide family with opioid
receptors triggers activation mainly of Gi or Go, which mediate
neuro- or immunomodulation.1,2
CCK which in turn acts to reduce opioid receptor-mediated
analgesia through interaction with the CCK2 receptor. CCK2
receptor knockout mice display enhanced opioid-induced an-
algesia and up-regulation of the endogenous opioid receptor
system.13,14 The rostral ventromedial medulla (RVM) is the key
area where this effect takes place, and in this regard, increased
CCK activity in the RVM during exposure to morphine leads
to morphine tolerance.15,16
Since antagonism of CCK2 receptors is known to block
morphine tolerance, the combined use of a CCK2 antagonist
and opioid agonist represents a potentially important strategy
for the treatment of chronic pain states.17 In this regard, given
the overlapping distribution of MOP and CCK2 receptors in the
brain, an important question concerning the opposing roles of
these receptors relates to their physical organization, particularly
in view of the relatively large number of opioid receptor
heterodimers that are already known to exist.18-20
We have investigated the possible existence of heterodimeric
MOP/CCK2 receptors through the careful application of biolu-
minescence resonance energy transfer (BRET) technology in
cultured cells that contain both MOP and CCK2 receptors. Our
BRET data indicate the absence of constitutive or monovalent
ligand-stimulated association of MOP and CCK2 receptors.
However, in the presence of bivalent ligands that contain a
µ-opioid agonist pharmacophore linked through an appropriate
length spacer to a CCK2 antagonist pharmacophore, the as-
sociation of MOP and CCK2 receptors was observed.
There are two types of CCK receptors,3 CCK1 and CCK2,
having sequence homology of approximately 50%.4-8 CCK1
receptors are distributed principally in the peripheral alimentary
tract, while CCK2 receptors are found mainly in the CNS.9
Activation of both CCK receptors appears to be coupled
primarily to Gq, resulting in the stimulation phospholipase C
and intracellular calcium, which leads to stimulation of gall-
bladder contraction, pancreatic exocrine secretion, enteric motil-
ity, gastric acid secretion, and modulation of neurotransmission.3-9
Areas of the CNS that are involved in pain processing contain
overlapping populations of CCK2 and opioid receptors.10-12
* To whom correspondence should be addressed. For L.J.M.: phone,
(480)-301-6650; fax, (480)-301-6969; e-mail, miller@mayo.edu. For P.S.P.:
phone, (612)-624-9174; fax, (202)-513-8609; e-mail, porto001@umn.edu.
†
University of Minnesota.
Mayo Clinic.
‡
a Abbreviations: BRET, bioluminescence energy transfer; CNS, central
nervous system; CCK, cholecystokinin; CCK1R, cholecystokinin-1 receptor;
CCK2R, cholecystokinin-2 receptor; DCC, N,N′-dicyclohexylcarbodiimide;
CDCl3, chloroform-d; CHCl3, chloroform; CH2Cl2, dichloromethane; DAM-
GO, [D-Ala2,NMePHe4,Gly5-ol]enkephalin; DMEM, Dulbecco’s modified
Eagle’s medium; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfox-
ide; DOP, δ-opioid peptide; ESI, electrospray ionization; EtOAc, ethyl
acetate; FLIPR, fluorometric imaging plate reader; GPCR, G-protein-coupled
receptor; HPLC, high performance liquid chromatography; HOBt, 1-hy-
droxybenzotriazole; KOP, κ-opioid peptide; MeOH, methanol; MOP,
µ-opioid; MOPR, µ-opioid receptor; NOP, nociceptin; Pd/C, palladium on
carbon; Rlu, Renilla luciferase; RVM, rostral ventromedial medulla; THF,
tetrahydrofuran; TFA, trifluoroacetic acid; YFP, yellow fluorescent protein.
Design Considerations for Bivalent Ligands
The design approach for the bivalent ligands 3a-c and 4
(Figure 1) employed in this study consisted of the tethering of
a µ-opioid agonist pharmacophore with a CCK2 antagonist
pharmacophore using a spacer. Nonpeptidic pharmacophores
were employed because it has been established that they bind
within the cavity of the heptahelical (seven transmembrane
10.1021/jm800174p CCC: $40.75
2009 American Chemical Society
Published on Web 12/29/2008

248 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Zheng et al.
Figure 1. Opioid agonist 1 and CCK2 receptor antagonist 2 pharmacophores and related bivalent 3a-c and 4 and monovalent 5 and 6 ligands.
segments) domain of opioid and CCK receptors and would
permit a better estimate of the spacer length required for bridging
the two recognition sites in a putative MOP/CCK2 receptor
heterodimer.21-24 Spacers of varied length ranging from 9 to
22 atoms were employed in order to optimize the length for
bridging of these receptors, and in this regard, efforts were
focused on 16-22 atoms because other reported bivalent ligands
that target dimeric opioid receptors are believed to bridge in
this range.25 The µ-opioid-selective agonist pharmacophore was
derived from 1 (oxymorphone),26 whereas the CCK2 antagonist
pharmacophore is closely related to 2 (L-365,260).27 The
combination of agonist and antagonist pharmacophores in the
bivalent ligand was based on reports that such pharmacologic
interaction results in enhancement of analgesia with concomitant
loss of tolerance.28,29 If this effect were to arise from the
association of receptors as heterodimers, such a bivalent ligand
could potentially serve as a valuable pharmacological tool.
Monovalent ligands 530 and 6 served as controls in our studies.
Reaction of 9 with mono-Boc protected hexamethylene-1,6-
diamine was mediated by HOBt/DCC in anhydrous DMF and
gave the intermediate 10 which was converted in TFA/CH2Cl2
(1:1) at room temperature to the amine 11 (35%). Coupling of
11 to nitrophenyldiglycolamide 12 was mediated by HOBt/DCC
in anhydrous DMF to give the intermediate nitrocompound 13
which was hydrogenated in methanol at 50 psi at room
temperature to amine 14 using Pd/C (10%) catalyst. The reaction
of amine 14 with isocyanate 8 in anhydrous DMF gave the
monovalent ligand 6. This sequence is illustrated in Scheme 2.
The intermediate 16 was obtained by condensation of 15
(R-oxmorphamine)26 with diglycolic anhydride in methylene
chloride and coupled to mono-Boc protected diamines in
anhydrous DMF using HOBt/DCC. These intermediates 17b,c
were deprotected by TFA in methylene chloride, and
intermediate amines 18b,c were obtained in fair to good
yields. The amines were coupled to 12 in DMF using HOBt/
DCC. For the synthesis of 19a (n ) 0), the hydrazine
derivative 12a (X ) NHNH2) was first coupled to 16 in DMF
mediated by HOBt/DCC. The intermediate nitro compounds
19a,c (n ) 0, 2, 6) were hydrogenated at 50 psi to amines
20a-c in the presence of Pd/C (10%) in methanol. The
reaction of 20a-c with isocyanate 8 gave the bivalent ligands
3a-c (n ) 0, 2, 6). The reaction sequences of these reactions
are illustrated in Scheme 3.
Chemistry
The precursor to the CCK2 antagonist pharmacophore 7 was
synthesized using the method of Bock et al.31 Resolution of 7
was accomplished according to the procedure of Reider et al.32
Treatment of R(+)-7 with triphosgene gave the isocyanate 8
(Scheme 1).
N-Methyldiglycolamide 9 was prepared by reaction of N-
methylamine with diglycolic anhydride in anhydrous THF.
The synthetic sequence for the preparation of bivalent ligand
4 started with the HOBt/DCC-mediated coupling in DMF of

Association of MOP and CCK2 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 249
Scheme 1a
a (a) (i) (1S)-(+)-10-camphorsulfonic acid, CH3CN/diethylether; (ii) 10% NaOH, CH2Cl2; (b) mother liquor from first step taken; (i) (1R)-(-)-10-
camphorsulfonic acid, CH3CN/diethyl ether; (ii) 10% NaOH, CH2Cl2; (c) triphosgene, aqueous saturated Na2CO3/CH2Cl2, 0 °C.
Scheme 2a
a (a) HOBt/DCC, DMF, Boc-hexadiamine1,6, room temp; (b) TFA/CH2Cl2, room temp; (c) only 12 used, HOBt/DCC, DMF, room temp; (d) Pd/C (10%),
methanol, 50 psi; (e) 8, DMF, 5 days.
2-(3 nitrophenoxy)ethanolamine 2133 with the glycolic acid
derivative 16. The intermediate nitro compound 22 was reduced
to amine 23 via catalytic hydrogenolysis in methanol using Pd/C
(10%) at 50 psi. The reaction of 23 with 8 in DMSO gave
bivalent ligand 4 (Scheme 4).
antagonist 6 and 3a-c was reflected by a reduction in the BRET
ratio, as shown in the middle panel. The bottom panel illustrates
that no BRET signal was produced with monovalent ligands 5
and 6 individually or together. Thus, heterodimerization of these
receptors was not induced by the occupation of the receptors
with ligands that do not include a linking domain. It is
noteworthy that neither the BRET ratios of MOP or CCK2
receptor homodimers were affected by either bivalent ligand
3c or monovalent ligands 5 and 6 utilized in this work (Figure
4).
Saturation BRET assays were performed to distinguish
specific molecular interactions from random interactions be-
tween donor and acceptor that can occur in a concentration-
dependent manner. Figure 5 shows COS cells expressing Rlu-
tagged MOP receptor and YFP-tagged CCK2 receptor after
transfection using a fixed amount of donor construct (1.0 µg
DNA/dish) and increasing amounts of acceptor construct (0.3
µg to 6 µg DNA/dish). This produced the expected increase in
BRET ratio that then reached a plateau for bivalent ligands 3b,c.
These data support the saturable nature of these molecular
interactions. The plateau of the BRET signal was typically
reached with 1.5-2.0 µg acceptor DNA/dish, representing
approximate molar ratios of acceptor/donor of 1.5-2.0:1. This
supports the specific interactions between MOP and CCK2
receptors stimulated by the bivalent ligands that were suggested
by the static BRET studies described above. The presence of
the combination of monovalent ligands, 5 and 6, or the absence
Biological Results
BRET Studies. BRET technology has been utilized previ-
ously to demonstrate that CCK2 receptors exist as constitutive
homodimers whose BRET signal is unaffected by the binding
of endogenous agonist.34 The literature supports similar con-
stitutive homodimerization of MOP receptors.19,35 This was
confirmed in control studies with cells expressing only MOP
or CCK2 receptors in that a positive homologous receptor BRET
signal was observed (Figure 2). Background nonspecific BRET
signals were determined by expressing the complementary
fluorescent donor or acceptor in a soluble form or attached to
a structurally unrelated family B GPCR, the secretin receptor.
However, receptor association could be induced with bivalent
ligands that were capable of bridging the CCK2 and MOP
receptors, as reflected by a positive heterologous receptor BRET
signal (Figure 3). The top panels show that significant BRET
signals were produced by 3a (16-atom spacer), 3b (18-atom
spacer), and 3c (22-atom spacer) while the spacer in 4 (9 atoms)
is apparently too short to generate such a signal. The highest
BRET signal was generated with the bivalent ligand 3b having
the 18-atom spacer. Competition between the monovalent CCK2

250 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Zheng et al.
Scheme 3a
a (a) Diglycolic anhydride, CH2Cl2, room temp; (b) HOBt/DCC, mono-Boc-diamines (n ) 2; n ) 6), DMF; (c) TAF/CH2Cl2; (d) 12, HOBt/DCC; (e)
Pd/C (10%), methanol, 50 psi; (f) 8, DMF, ∼24 h, room temp.
Scheme 4a
a (a) HOBt/DCC, DMF; (b) Pd/C (10%), methanol, 50 psi; (c) 8, DMSO, 24 h, room temp.
These data are consistent with random interactions between these
receptors taking place in the plasma membrane.
Binding studies of the bivalent ligands were conducted in
singly expressed and coexpressed CHO cells. Only 3a and 3b
shoved significantly higher affinity for CCK2 receptors in
coexpressed cells relative to singly expressed cells, whereas
little, if any, affinity differences for MOP receptors were
observed between singly and doubly expressed cells. The reason
for these results is not clear, but one possibility is that the
conditions employed in binding interfered with association of
CCK2 and MOP receptors.
Given the results of the present study that demonstrate that
MOP and CCK2 receptors do not constitutively form het-
erodimers but can be induced to associate in the presence of
appropriate bivalent ligands, we evaluated the functional proper-
ties of bivalent ligand 3b and monovalent ligands 5 and 6 in
CHO cells coexpressed with MOP and CCK2 receptors using
the calcium mobilization assay.36 The data (Table 2) revealed
that 3b and the MOP receptor agonist 5 promoted Ca2+ release
whereas the monovalent CCK antagonist 6 did not. Interestingly,
both ligands were capable of activating opioid receptors,
although 3b was 4.8-fold more potent than monovalent agonist
5 in this regard. These studies suggest that opioid receptor
activation can occur in the induced association state or as
homomers.
Figure 2. BRET controls. Shown are the BRET ratios for COS cells
expressing various constructs in the combinations noted. The shaded
area represents the intensity of BRET signals felt to be nonspecific,
reflecting a signal that can be generated between Rlu-tagged receptor
and soluble YFP protein or between YFP-tagged receptor and soluble
Rlu protein. BRET signals above this are considered to be significant.
Both CCK2 receptors and MOP receptors exist as constitutive ho-
modimers with significant homologous receptor BRET signals when
expressed in these cells. Values presented represent the mean ( SEM
of data from five independent experiments. Values marked with //
represent BRET signals significantly above nonspecific values at the p
< 0.001 level.
of ligand yielded only a low level signal that was not saturable,
with best fit of the data not different from a linear regression.
Experiments in mice were conducted to evaluate the tolerance
of the bivalent ligands with 9-, 16-, 18-, and 22-atom spacers

Association of MOP and CCK2 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 251
Figure 3. Effects of bivalent ligands on heterodimerization of CCK2 and MOP receptors. Shown are BRET ratios for COS cells expressing both
tagged MOP and CCK2 receptor constructs with or without incubation of various bivalent ligands (10-6 M, 90 min at room temperature). The
panels on the left reflect Rlu-tagged µ-opioid receptor expressed with YFP-tagged CCK2 receptor, while those on the right represent the opposite.
The panels in the top row illustrate BRET ratios for these cells in the absence of ligand and in the presence of bivalent ligands of various spacer
lengths. The panels in the second row reflect the saturability of the BRET ratios in the presence of the bivalent ligands that normally give a
significant signal, that are reduced in the presence of competing monovalent CCK antagonist ligand 6. The panels in the third row demonstrate that
monovalent ligands that recognize only the CCK2 receptor or the opioid receptor, even when mixed together, failed to produce the BRET signal
observed with the bivalent ligand. The shaded area represents the nonspecific BRET signal as described in Figure 2. Values represent the mean (
SEM of data from five independent experiments: (/) p < 0.05; (//) p < 0.001.
4, 3a-c relative to that of the control monovalent 5. These data
are summarized in Table 3. With the exception of 5, none of
the bivalents exhibited tolerance via the icv route. Given that
bivalent ligand 4 with the shortest spacer did not show tolerance
and the quantitative BRET data suggesting the absence of
bridging between MOP and CCK2 receptors for 4, there appears
to be no relationship between ligand-induced association of
receptors and blockage of tolerance.
than their association to heterodimers. However, association of
these receptors could be induced in the presence of bivalent
ligands 3a-c that contain both µ-opioid agonist and CCK2
receptor antagonist pharmacophores connected through spacers
whose length is in the range of 16-22 atoms. In this regard,
the BRET signal for 3b,c plateaued close to a BRET ratio of
∼2, supporting specific saturable interaction between MOP and
CCK2 receptors that was suggested by the BRET ratio enhance-
ment. It is noteworthy that the BRET ratio plateau for 3a was
approximately half that of 3b and 3c. This could reflect the low
efficiency of induction of association by this ligand and different
alignment of donor and acceptor fluorophores attached to their
receptors as a consequence of this relatively short spacer.
Significantly, the bivalent ligand 4 with the shortest spacer (9
atoms) as well as the simultaneous use of the monovalent ligands
Discussion
Careful and systematic BRET analysis shows that MOP and
CCK2 receptors coexpressed in COS cells are present as
homodimers, with no evidence of spontaneous receptor het-
erodimerization. Evidently, the association of either MOP
receptors or CCK2 receptors as homodimers is more favorable

252 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Zheng et al.
not our ligands produce tolerance. The in vivo data (Table 3)
revealed that only monovalent ligand 5 produced tolerance,
either alone or in equimolar combination with monovalent CCK2
antagonist 6. On the other hand, none of the bivalent ligands
3a-c, 4 gave rise to significant tolerance, despite our finding
that 4 does not induce association of MOP and CCK2 receptors
in COS cells. Given our evidence for lack of constitutive
association of MOP and CCK2 receptors in COS cells, it would
be expected that 4 would produce tolerance in mice if a
heterodimer were involved in the modulation process. When
taken together with the results of the BRET data, the tolerance
studies therefore suggest that the modulation of morphine‘s
effects is possibly due to circuitry rather than to physical
association of MOP and CCK2 receptors as heterodimers.
In conclusion, this study demonstrates proof of principle
concerning the ability of bivalent ligands to induce association
of receptors through a bridging mechanism. In this regard, it
has not escaped our attention that other bivalent ligands21-25
whose pharmacologic profiles suggest bridging of dimeric opioid
receptors have spacer lengths in the same range as the effective
bivalent ligands in the present series. We believe this similarity
may reflect a common structural organization of GPCR dimers.
Thus, pharmacophores that bind within the helical bundle and
are linked by linear spacers greater than 16 atoms in length
with optimal lengths in the range of 18-21 atoms have been
reported to most effectively bridge dimeric GPCRs.21-25
Figure 4. Effects of ligands on receptor homodimerzation. Shown are
the BRET signals stimulated by bivalent or monovalent ligands in COS
cells expressing Rlu- and YFP-tagged CCK2 receptor constructs or MOP
receptor constructs or structurally unrelated tagged receptor constructs.
None of the ligands studied had any effect on the constitutive
homodimers of the CCK2 or MOP receptors and did not lead to
heterodimerization of the unrelated receptors. Values represent the mean
( SEM of data from six independent experiments: (/) p < 0.05.
5 and 6 did not induce any recognizable association of MOP
and CCK2 receptors. These data suggest that only bivalent
ligands 3a-c were capable of inducing association because the
spacers are of sufficient length to permit binding of each of the
pharmacophores to their respective receptors through a bridging
mechanism.
Experimental Section
The association of MOP and CCK2 receptors induced by
3a-c, as reflected by the heterologous receptor BRET ratios,
appears to be most favorable with 3b which contains the 18-
atom spacer. The finding that no significant difference of BRET
ratio was observed with the monovalent ligands 5, 6, or bivalent
ligand 4 in cells that contain either homodimerized MOP or
CCK2 receptors indicates that these ligands do not affect the
degree of constitutive homodimerization.
In the presence of the CCK2 receptor monovalent antagonist
ligand 6, the BRET ratios of bivalent ligands 3a-c were
reduced. These data suggest that competition of 6 with the CCK2
receptor antagonist pharmacophore of the bivalent ligands
involved in bridging associated MOP/CCK2 receptors promotes
a transition from the bridged binding mode to univalent binding.
In the absence of constraints due to bridging, the heteromers
dissociate and form the more stable homodimers.
On the basis of the present work, a schematic for induction
of association of MOP and CCK2 receptors is illustrated in
Figure 6. Thus, GPCRs that do not easily associate to form
heterodimers can be associated using an appropriately designed
bivalent ligand. On the other hand, if coexpressed GPCRs
exhibit greater ability to associate as heterodimers, as opposed
to homodimers, it might be expected that they would exist as
constitutive heterodimers. In this connection, the presence of
constitutive heterodimers in cultured cells has been reported for
a number of opioid37-39 and CCK40 receptors. Of significance
is the finding that interacting ligands that selectively target or
activate putative heterodimers in vivo may have pharmacological
Chemistry. General. Oxymorphone was obtained from Mallinck-
rodt & Co. All other chemicals and solvents were purchased from
Aldrich or Fisher without further purification. Some dry solvents
were made by MBRAUN MB-SPS Solvent Dispensing.
1H and 13C NMR spectroscopy were obtained on 300 MHz on
an Oxford Varian VXR 300 MHz NMR spectrometer. Polarities
were obtained on AUTOPOL III automatic polarimeter. Mass
spectroscopy was obtained on Bruker BioTOF II mass spectrometer.
Purities of the monovalent ligands 6 and bivalent ligands 3a-c
and 4 were over 98% based on analysis on HPLC column
(Phenomenex Luna SB-C18 (2) 5 µm, 4.6 mm × 250 mm) which
was eluted with MeOH/buffer (60:40) at a flow rate of 1 mL/min.
3(S)-(-)-Amino-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-
benzodiazepin-2-one (S(-)-7). A solution of 7 (1.0 g, 3.9 mmol)
in acetonitrile (4 mL) was treated with 1(S)-(+)-camphorsulfonic
acid (0.91 g, 3.9 mmol) and diethyl ether (12 mL) and aged
overnight at room temperature. The crystals were filtered and
washed with acetonitrile/ether (1:3), yielding 0.92 g of solid as 3(S)-
(-)-amine-1(S)-(+)-camphorsulfonate salt (98.1%). [R]2D2 -88.
The salt (920 mg) was treated with 10% NaOH (6 mL) in CH2Cl2
(6 mL). The phases were separated, and the aqueous layer was
extracted with CH2Cl2 (3 mL × 5). The organic layers were
combined, dried over MgSO4, and concentrated by a rotary-
evaporator to yield 459.4 mg (89.8%) of 3(S)-(-)-amine. [R]D22
-250, [R]2D5 -269.31 1H NMR (CDCl3): δ 7.62-7.69 (m, ArH,
2H), 7.55-7.53 (m, ArH, 1H), 7.43-7.29 (m, ArH, 5H), 7.21-7.16
(m, ArH, 1H), 4.46 (s, CH, 1H), 3.45 (s, CH3, 3H), 2.75 (s, NH2,
2H). 13C NMR (CDCl3): δ 170.14, 165.85, 142.97, 138.03, 131.46,
130.29, 130.01, 129.50 (×2), 129.04, 128.07 (×2), 123.87, 121.14,
70.27, 35.27.
3(R)-(+)-Amino-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-
benzodiazepin-2-one (R(+)-7). The mother liquor from resolution
of 7 with 1(S)-(+)-camphorsulfonic acid was concentrated by a
rotary-evaporator. The residual was treated with 10% NaOH (30
mL) in CH2Cl2 (30 mL) and extracted with CH2Cl2 (10 mL × 5).
The extracts were combined and concentrated by a rotary-
evaporator. The residual was dissolved in acetonitrile (2.2 mL) and
treated with 1(R)-(-)-10-camphorsulfonic acid (0.49 g, 2.1 mmol)
and diethyl ether (6.6 mL). The mixture was stored overnight at
room temperature and the crystals were filtered and washed with
profiles that differ from those that interact with homodimers.
30
Because our results in cultured cells indicated the absence
of constitutive association in MOP and CCK receptors (in
contrast to literature41 which has reported numerous het-
erodimeric opioid receptors in cultured cells), thereby making
it unlikely that this may be a mechanism for the modulation of
morphine tolerance and potency, we decided to determine the
icv ED50 values of the series in order to evaluate whether or

Association of MOP and CCK2 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 253
Figure 5. Effects of bivalent ligands on bystander BRET. Shown are the saturation curves obtained by coexpressing pairs of Rlu-tagged MOP
receptor and YFP-tagged CCK2 receptor constructs after incubation with bivalent ligands (10-6 M), as indicated. Bivalent ligands 3a-c produced
exponential curves that reached a plateau, supporting specific molecular interactions. Cells treated with no ligand or with a combination of monovalent
ligands for the CCK and µ-opioid receptors showed curves not significantly different from linear fits. Values represent the mean ( SEM of data
from five independent experiments.
Table 1. Binding of DAMGO, 2, Bivalent Ligands 3a-c, and Monovalent Ligands 4 and 6 to Singly and Coexpressed CCK2 and MOP Receptors in
CHO Cellsa
[
125I]CCK
[3H]DAMGO
Bmax (pmol/(mg of protein))
ligands
Ki (nM)
Bmax (pmol/(mg of protein))
Ki (nM)
Cells Expressing CCK2 Receptor
2
18 ( 3
356 ( 67
71 ( 5
260 ( 90
223 ( 11
48 ( 6
14.4 ( 3.4
16.0 ( 3.7
10.3 ( 1.8
18.5 ( 1.7
14.0 ( 3.9
16.0 ( 1.1
3a
3b
3c
4
6
Cells Expressing MOP Receptor
DAMGO
6.7 ( 2.9
8.3 ( 2.4
5.7 ( 1.7
29.4 ( 2.1
24.1 ( 0.7
29.8 ( 10.2
9.2 ( 2.7
9.8 ( 3.5
7.7 ( 1.9
7.4 ( 0.5
12.4 ( 1.9
9.3 ( 1.4
3a
3b
3c
4
5
Cells Expressing CCK2 Receptor/MOP Receptor
DAMGO
2
3a
3b
3c
4
11.0 ( 6.2
9.5 ( 3.3
14 ( 2
57 ( 10
33 ( 3
77 ( 11
210 ( 44
9.1 ( 1.7
9.5 ( 1.9
12.4 ( 1.8
10.9 ( 2.8
9.3 ( 2.5
3.9 ( 2.1
7.3 ( 1.5
16.3 ( 0.5
14.2 ( 2.5
40.7 ( 9.5
8.3 ( 1.4
9.7 ( 2.5
9.3 ( 1.4
10.5 ( 12.6
7.4 ( 0.6
5
6
21 ( 3
9.5 ( 3.9
a Values are expressed as the mean ( SEM of data from three to five independent experiments.
Table 2. GRqi4myr-Mediated Ca2+ Release of CHO Cells Stably Expressing MOP and CCK2 Receptors
EC50 (nM)
3b
5
6
DAMGO
2.66 ( 0.70 (4)
MOPR-CCK2R CHO
119 ( 75 (4)
572 ( 71.1(3)
<10000 (3)
Table 3. Comparison of the ED50 Values (pmol/Mouse) of Bivalent Ligands 3a-c and 4, Monovalent Ligand 5, and Simultaneous Administration of
Monovalent Ligands 5 and 6: A 24 h Tolerance Study
ED50 (pmol/mouse)
3a
3b
3c
4
5
5 + 6
96.29
icv
47.41
390
2020
3036
51.93
(37.19-60.44)
83.86 ( 8.76
84.45 ( 7.81
(280-530)
(1420-2870)
83.93 ( 8.05
64.33 ( 13.10
(2390-3857)
75.45 ( 9.84
57.31 ( 12.98
(39.00-69.14)
70.61 ( 9.65
40.74 ( 11.86d
(64.93-142.8)
82.04 ( 12.08
42.94 ( 13.40d
controla
80.67 ( 9.79
24 hb c
71.29 ( 8.55
,
a The control value represents the ED80 dose for each individual agonist on day 1. b Twenty-four hours later the mice were first tested to see that the
percent antinociception was back to baseline and then the ED80 dose was tested to see if there was any tolerance. c Peak time is between 10 and 20 min.
d Tolerance formed.
cold acetonitrile/ether (1:3), yielding 0.81 g (86.4%) of solid as
3(R)-(+)-amine-1(R)-(-)-camphorsulfonate salt. [R]2D2 +88.
The salt (810 mg) was treated with 10% NaOH (6 mL) in CH2Cl2
(6 mL). The phases were separated, and the aqueous layer was
extracted with CH2Cl2 (3 mL × 5). The organic layers were
combined, dried over MgSO4, and concentrated in a rotary-
evaporator to yield 406 mg (97.3%) of 3(R)-(+)-amine. [R]2D2 +262.
1H NMR (CDCl3): δ 7.62-7.60 (m, ArH, 2H), 7.56-7.53 (m, ArH,

254 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Zheng et al.
Figure 6. Schematic illustration of the effect of a MOP agonist/CCK2 antagonist bivalent ligand on the equilibrium between a mixture of MOP
receptor (MOPR) and cholecystokinin receptors (CCK2R). Note that the bivalent ligand induces heterodimerization through constraint imposed by
its spacer.
1H), 7.44-7.30 (m, ArH, 5H), 7.21-7.17 (m, ArH, 1H), 4.46 (s,
CH, 1H), 3.45 (s, CH3, 3H), 2.61 (s, NH2, 2H). 13C NMR (CDCl3):
δ 170.20, 165.85, 143.01, 138.07, 131.49, 130.31, 130.03, 129.52
(×2), 129.08, 128.10 (×2), 123.89, 121.16, 70.32, 35.30.
(100 mL) was added a solution of commercially available 2-(3-
nitrophenoxy)ethanamine (7.6 g, 55 mmol) in dry THF (100 mL)
at 0 °C. The reaction mixture was stirred at room temperature
overnight. Filtration of the precipitate gave 10.97 g (86.3%) of 12.
1H NMR (DMSO-d6): δ 12.87 (s br, CO2H, 1H), 10.37 (s, CONH,
1H), 8.67 (m, ArH, 1H), 8.01 (m, ArH, 1H), 7.92 (m, ArH, 1H),
7.61 (m, ArH, 1H), 4.22 (s, CH2, 4H), 4.21 (s, CH2, 4H). 13C NMR
(DMSO-d3): δ 171.53, 168.56, 147.75, 139.40, 130.03, 125.51,
118.02, 113.55, 70.22, 67.97.
2-(2-Hydrazinyl-2-oxoethoxy)-N-(2(3nitrophenoxy)ethyl)-
acetamide (12a). To a solution of 12 (149 mg, 0.5 mmol) in
anhydrous DMF (1.5 mL) were added DCC (113.5 mg, 0.55 mmol),
HOBt (148.6 mg, 1.1 mmol), and hydrazine hydrate (29 µL, 0.6
mmol) at room temperature. The reaction mixture was stirred 24 h
at room temperature, and the precipitate was removed by filtration.
The filtrate was concentrated and the residual was purified by flash
chromatography (CH2Cl2/MeOH/NH4OH ) 85:14.5:0.5) and gave
54 mg (34.6%) of 12a. 1H NMR (CDCl3): δ 7.86 (dd, J ) 8.1, 2.1
Hz, 1H), 7.75 (dd, J ) 2.1, 2.1 Hz, 1H), 7.68 (s, 1H), 7.46 (dd, J
) 8.1, 8.1 Hz, 1H), 7.27 (dd, J ) 8.1, 21 Hz, 1H), 6.95 (m, 1H),
4.17 (t, J ) 5.4, 2H), 4.15 (s, 2H), 4.09 (s, 2H), 3.78 (dt, J ) 5.4,
2H), 1.72 (s br, 2H). MS 335.32 (M+ + Na).
1,3-Dihydro-3(R)-(+)-isocyanate-1-methyl-5-phenyl-2H-
1,4-benzodiazepin-2-one (8). To the biphasic mixture of R(+)-7
(600 mg, 2.26 mmol) in CH2Cl2(10 mL)/NaHCO3 (saturated
aqueous solution) cooled in an ice bath was added triphosgene (224
mg, 0.753 mmol). The reaction mixture was stirred in the ice bath
for 20 min and then poured into a separator funnel. The organic
layer was collected, and the aqueous layer was extracted with
CH2Cl2 (3 × 1.5 mL). The combined organic layers were dried
over MgSO4 and concentrated in vacuum to give 553.9 mg (84.1%)
1
of 8. H NMR (CDCl3): δ 7.67-7.4 (m, ArH, 9H), 4.94 (m, CH,
1H), 3.52 (s, CH3, 3H). MS 314.21 (M+ + Na).
N-Methyldiglycolamide (9). To a cold (0 °C) 2 M solution of
methylamine in THF (10 mL, 20 mmol) was added commercially
available diglycolic anhydride (2.32 g, 20 mmol). The reaction
solution was stirred overnight at room temperature and concentrated
by a rotary-evaporator. The residue was dried by a vacuum pump
1
without further purification to give 2.82 g (95.8%) as an oil. H
NMR (DMSO-d6): δ 12.76 (s, CO2H, 1H), 7.78 (s, NH, 1H), 4.07
(s, CH2, 2H), 3.93 (s, CH2, 2H), 2.61 (s, CH3, 3H). 13C NMR
(DMSO-d6): δ 171.23, 168.95, 70.08, 67.75, 25.23.
N-Methyl-2-(13-(3-nitrophenoxy)-2,6,10-trioxo-8-oxa-3,5,11-
triazatridecyloxy)acetamide (13). To a solution of 12 (0.714 g,
2.81 mmol), DCC (0.58 g, 2.81 mmol), and HOBt (0.38 g, 2.81
mmol) in dry DMF (2.5 mL) was added a 2.5 mL dry DMF solution
of 11 (0.625 g, 2.55 mmol). The reaction mixture was stirred
overnight at room temperature, and the precipitate was filtered. The
filtrate was concentrated in a rotary-evaporator and purified by flash
chromatography (CH2Cl2/CH3OH/NH3H2O ) 90:9.5:0.5) to give
N-(BOC-hexamethylamino)-N′-methyldiglycoldiamide (10).
To a solution (0 °C) of 9 (0.37 g, 2.5 mmol), DCC (0.57 g, 2.75
mmol), and HOBt (0.37 g, 2.75 mmol) in dry DMF (2 mL) was
added a solution of mono-Boc protected hexamethylene amine (1,6)
(0.54 g, 2.5 mmol) in DMF (1.5 mL). The reaction mixture was
stirred for 2 days at room temperature, and then the precipitate
was filtered. The filtrate was concentrated and the residue was
purified by flash chromatography (CH3OH/CH2Cl2/NH3H2O ) 5:95:
1
0.48 g (39.1%) of 13. H NMR (DMSO-d6): δ 10.53 (s, CONH,
1H), 8.67 (m, CONH, 1H), 8.08 (m, ArH, 1H), 8.00 (m, ArH +
CONH, 3H), 7.94 (m, ArH, 1H), 7.62 (m, ArH, 1H), 4.18 (s, CH2,
2H), 4.05 (s, CH2, 2H), 3.89 (s, CH2, 4H), 3.10 (m, CH2, 4H), 2.63
(d, J ) 4.8 Hz, CH3, 3H), 1.42 (m, CH2, 4H), 1.25 (m, CH2, 4H).
MS 482.4 (M+ + H), 503.3 (M+ + Na).
1
0.5) to give 0.42 g (61.0%) of 10. H NMR (CDCl3): δ 6.91 (s,
CONH, 2H), 4.61 (s, CONH, 1H), 4.05 (s, diglCH2, 2H), 4.04 (s,
diglCH2, 2H), 3.29 (m, hexCH2, 2H), 3.14 (m, hexCH2, 2H), 2.85
(m, CH3, 3H), 1.57-1.34 (m, hexCH2, 8H), 1.44 (s, CH3, 9H). MS
346.28 (M+ + H), 368.27 (M+ + Na).
2-(13-(3-Aminophenoxy)-2,6,10-trioxo-8-oxa-3,5,11-tria-
zatridecyloxy)-N-methylacetamide (14). A mixture of 13 (90 mg,
0.171 mmol) and Pd/C of 10 wt % (30 mg) in MeOH (5 mL) was
hydrogenated at 50 psi for 4 h at room temperature. The catalyst
was removed by filtration over a Celite pad, and the filtrate was
concentrated by a rotary-evaporator to give 75 mg (88.5%) of
N-(Aminomethyl)-2-(2-(methylamino)-2-oxoethoxy)-
acetamide (11). N-(BOC-hexamethylamino)-N′-methyldiglycold-
iamide 10 (0.4 g, 1.16 mmol) was dissolved in TFA/CH2Cl2 (1:1,
20 mL) and stirred at room temperature until no more starting
materials could be detected on TLC. Solvent was evaporated and
the residue was purified by flash chromatography (CH3OH/CH2Cl2/
1
product 14. H NMR (DMSO-d6): δ 8.21 (m, CONH, 1H), 8.01
1
NH4OH) to give 100 mg (35.1%) of 11. H NMR (DMSO-d6): δ
(m, CONH, 3H), 6.86 (t, J ) 8.1 Hz, ArH, 1H), 6.14-6.11 (m,
ArH, 2H), 6.06 (m, ArH, 1H), 5.02 (m, NH2, 1H), 3.94 (s, CH2,
2H), 3.91 (s, CH2, 2H), 3.89 (m, CH2, 6H), 3.45 (m, CH2, 2H),
3.09 (m, CH2, 4H), 2.63 (d, J ) 4.5 Hz, CH3, 3H), 1.41 (m, CH2,
8.03 (m, CONH, 2H), 7.71 (s br, CONH, 1H), 7.16 (t br, NH2,
2H), 3.89 (s, diglCH2, 4H), 3.09 (m, hexCH2, 2H), 2.75 (m, hexCH2,
2H), 2.63 (d, J ) 7.8 Hz, CH3, 3H), 1.50 (m, hexCH2, 2H), 1.41
(m, hexCH2, 2H), 1.26 (m, hexCH2, 4H). MS 246.20 (M+ + H),
268.19 (M+ + Na).
4H), 1.24 (m, CH2, 4H). MS 496.61 (M+ + H), 518.59 (M+
Na).
+
2-(2-(3-Nitrophenoxyamino)-2-oxoethoxy)acetic Acid (12). To
a suspension of diglycolic anhydride (5.8 g, 50 mmol) in dry CH2Cl2
(R)-Z-N-Methyl-2-(13-(3-(3-(1-methyl-2-oxo-5-phenyl-2,3-
dihydro-1H-benzo[e][1,4]diazepin-3-yl)ureido)phenoxy)-

Association of MOP and CCK2 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 255
2,6,10-trioxo-8-oxa-3,5,11-triazatridecyloxy)acetamide (6). To
a solution of 14 (75 mg, 0.151 mmol) and isocyanate derivative 8
(71.7 mg, 0.166 mmol) in anhydrous DMF (4 mL) was added
triethylamine (23.2 µL, 0.166 mmol). The reaction solution was
stirred at room temperature for 5 days and concentrated. The residue
was purified by flash chromatography to give 23 mg (19.4) of
N-(m-Aminophenoxy)ethyldiglycolamidehydrazide-N′-
oxymorphyldiglycolamidedihydrazide (20a). A mixture of 19a
(66 mg, 0.093 mmol) and Pd/C of 10 wt % (15 mg) in MeOH (4
mL) was hydrogenated at 50 psi for 4 h at room temperature. The
catalyst was removed by filtration over a Celite pad, and the filtrate
was concentrated in a rotary-evaporator to give 60 mg (94.5%) of
product 20a. 1H NMR (CDCl3): δ 8.03 (s br, 1H), 7.52 (s br, 1H),
6.99 (dd, J ) 7.8 Hz, 1H), 6.69 (d, J ) 7.8, Hz, 1H), 6.53 (d, J )
7.8 Hz, 1H), 6.26 (m, 2H), 4.57 (s, 2H), 4.18-4.01 (m, 8H), 3.63
(m, 2H), 3.12 (d, J ) 18.3 Hz, 1H), 2.73-2.55 (m, 11H), 2.40 (m,
1H), 2.35 (s, 3H), 2.28-2.18 (m, 2H), 1.81-1.70 (m, 1H), 1.55
(m, 2H), 1.44-1.36 (m, 1H), 1.04 (m, 1H). MS 683.35 (M+ + H).
1-(N-Oxymorphyldiglycoldiamide)-2-[1-diglycoldiamide-
2-(m-aminophenoxy)ethane]ethane (20b). A mixture of 19b (120
mg, 0.162 mmol) and Pd/C of 10 wt % (30 mg) in MeOH (6.5
mL) was hydrogenated at 50 psi for 4 h at room temperature. The
catalyst was removed by suction filtration and the filtrate was
concentrated by a rotary-evaporator to give 100 mg (86.8%) of
1
product 6. H NMR (CDCl3): δ 8.11 (m, CONH, 1H), 7.59 (m,
CONH, 3H), 7.37 (m, ArH, 5H), 7.17-6.91 (m, ArH, 6H), 6.52
(m, CONH, 1H), 5.49 (d, J ) 7.8 Hz, CH, 1H), 5.02 (m, CONH,
1H), 4.04 (s, CH2, 2H), 4.01 (m, CH2, 6H), 3.98 (s, CH2, 2H), 3.65
(m, CH2, 2H), 3.46 (s, CH3, 3H), 3.22 (m, CH2, 4H), 2.77 (d, J )
4.5 Hz, CH3, 3H), 1.42 (m, CH2, 4H), 1.23 (m, CH2, 4H). MS
787.61 (M+ + H), 809.65 (M+ + Na).
Oxymorphyldiglycolamide (16). A suspension of diglycolic
anhydride (0.645 g, 5.56 mmol) in anhydrous CH2Cl2 (20 mL) was
added dropwise to oxymorphamine 15 (1.6 g, 5.29 mmol) in
anhydrous CH2Cl2 (10 mL) under N2. The mixture was stirred at
room temperature overnight. The precipitate was collected by
suction filtration and washed with CH2Cl2 to give 2.20 g (99.4%)
of product 16. 1H NMR (DMSO-d6): δ 8.75 (m, CONH, 1H), 6.59
(d, J ) 7.8 Hz, ArH, 1H), 6.51 (d, J ) 7.8 Hz, ArH, 1H), 4.45 (m,
1H), 4.32 (m, 1H), 3.92 (m, 4H), 3.16 (s, 1H), 2.72 (m, 1H), 2.55
(s, 2H), 2.42-2.25 (m, 5H), 1.69-1.61 (m, 1H), 1.49 (m, 1H),
1.33 (m, 2H), 0.88 (m, 1H). MS 417.14 (M- - H).
1
product 20b. H NMR (CDCl3): δ 7.58 (m, 1H), 7.48 (t, J ) 5.7
Hz, 1H), 7.39 (m, 1H), 7.10-7.06 (m, 1H), 7.02 (d, J ) 8.1 Hz,
1H), 6.70 (d, J ) 8.1 Hz, 1H), 6.54 (d, J ) 8.1 Hz, 1H), 6.31-6.23
(m, 3H), 4.59-4.52 (m, 2H), 4.08-3.97 (m, 8H), 3.88 (m, 2H),
3.68 (q, J ) 5.4 Hz, 2H), 3.49 (s, 6H), 3.12 (d, J ) 18.6 Hz, 1H),
2.78 (d, J ) 6.0 Hz, 1H), 2.69-2.53 (m, 10H), 2.40 (m, 1H), 2.35
(s, 3H), 2.28-2.21 (m, 2H), 1.92-1.36 (m, 6H), 1.25 (m, 1H),
1.00 (m, 1H). MS 711.21 (M+ + H).
[N-(m-Nitrophenoxy)ethyldiglycolamidehydrazide-N′-
oxymorphyldiglycolamide]dihydrazide (19a). To a solution of
16 (69.7 mg, 0.167 mmol) in anhydrous DMSO (0.5 mL) were
added DCC (37.8 mg, 0.183 mmol), HOBt (24.8 mg, 0.183 mmol),
and a solution of 12a (52 mg, 0.167 mmol) in anhydrous DMSO
(0.5 mL) at room temperature. The mixture was stirred for 24 h at
room temperature, and the precipitate was removed by filtration.
The filtrate was concentrated and the residual was purified by flash
chromatography (CH2Cl2/MeOH/NH4OH ) 90:9.5:0.5) to give 70
1-(N-Oxymorphyldiglycoldiamide)-6-[1-diglycoldiamide-
2-(m-aminophenoxy)ethane]hexane (20c). A mixture of 19c (250
mg, 0.314 mmol) and Pd/C of 10 wt % (80 mg) in MeOH (13 mL)
was hydrogenated at 50 psi for 4 h at room temperature. The catalyst
was removed by filtration over a Celite pad and the filtrate was
concentrated in a rotary-evaporator to give 110 mg (45.7%) of
product 20c. 1H NMR (DMSO-d6): δ 8.21 (m, 1H), 8.03 (m, 2H),
7.57 (m, 1H), 6.86 (t, J ) 7.5 Hz, 1H), 6.56 (d, J ) 8.1 Hz, 1H),
6.46 (d, J ) 8.1 Hz, 1H), 6.11-6.04 (m, 3H), 5.02 (m, 2H), 4.78
(s, 1H), 4.44 (m, 1H), 3.93-3.88 (m, 8H), 3.44 (q, J ) 6.0 Hz,
2H), 3.32 (s, 2H), 3.06 (m, 4H), 2.71 (d, J ) 6.0 Hz, 1H), 2.52 (m,
6H), 2.36 (m, 1H), 2.27 (s, 3H), 2.13 (m, 2H), 1.56 (m, 1H), 1.40
(m, 5H), 1.23 (m, 3H), 0.92 (m, 1H). MS 767.69 (M+ + H), 789.68
(M+ + Na).
1
mg (58.8%) of 19a. H NMR (CDCl3): δ 7.80 (d, J ) 8.1, 1H),
7.70 (m, 1H), 7.41 (t, J ) 8.1 Hz, 2H), 7.21 (m, 2H), 6.69 (d, J )
8.1, 1H), 6.52 (d, J ) 8.1 Hz, 1H), 4.58 (s, 2H), 4.23-4.10 (m,
8H), 3.75 (m, 2H), 3.10 (d, J ) 18.6 Hz, 1H), 2.77 (m, 1H),
2.62-251 (m, 6H), 2.38 (m, 1H), 2.34 (s, 3H), 2.22 (m, 2H),
1.77-1.34 (m, 6H), 1.03 (m, 1H). MS 713.84 (M+ + H).
1-(N-Oxymorphyldiglycoldiamide)-2-[1-diglycoldiamide-
2-(m-nitrophenoxy)ethane]ethane (19b). To a solution of 12 (141
mg, 0.473 mmol) and 18b (198 mg, 0.43 mmol) in anhydrous
DMSO (2.5 mL) were added DCC (97.6 mg, 0.473 mmol) and
HOBt (64 mg, 0.473 mmol). The reaction solution was stirred
overnight at room temperature and concentrated. The residue was
purified by a flash chromatography (CH2Cl2/CH3OH/NH3H2O )
90:10:1) to give 220 mg (69.1%) of 19b. 1H NMR (CDCl3): δ 7.83
(d, J ) 8.1 Hz, 1H), 7.74 (m, 1H), 7.54 (t, J ) 6.0 Hz, 1H), 7.44
(m, 1H), 7.35-7.21 (m, 2H), 6.99 (d, J ) 8.4 Hz, 1H), 6.70 (d, J
) 8.4 Hz, 1H), 6.55 (d, J ) 8.4 Hz, 1H), 4.58-4.53 (m, 2H), 4.19
(t, J ) 5.7 Hz, 2H), 4.07 (m, 8H), 3.76 (q, J ) 5.7 Hz, 2H),
3.61-3.49 (m, 4H), 3.13 (d, J ) 18.0 Hz, 1H), 2.80 (m, 1H),
2.69-2.54 (m, 5H), 2.41 (m, 1H), 2.35 (s, 3H), 2.25 (m, 2H),
1.83-1.69 (m, 8H), 1.55 (d, J ) 9.6 Hz, 1H), 1.44-1.36 (m, 1H),
1.25 (s, 1H), 1.03-0.88 (m, 1H). MS 741.24 (M+ + H).
N-{m-[R(+)-1]Ureaphenoxy}ethyldiglycolamidehydrazide-
N′-oxymorphyldiglycolamidedihydrazide (3a). To a solution of
20a (57 mg, 0.0835 mmol) in anhydrous DMSO (0.5 mL) was
added a solution of 8 (26.8 mg, 0.0916 mmol) in anhydrous DMSO
(0.5 mL). The reaction solution was stirred at room temperature
for 24 h and concentrated. The residue was purified by flash
chromatography (CH2Cl2/MeOH/NH4OH ) 85/15/1.5) to give 10
1
mg (12.3%) of product 3a. H NMR (CDCl3): δ 7.65-7.33 (m,
10H), 7.30-7.22 (m, 2H), 6.97 (dd, J ) 7.8 Hz, 1H), 6.89 (d, J )
7.8, Hz, 1H), 6.66 (d, J ) 7.8 Hz, 1H), 6.31-6.23 (m, 2H), 5.29
(d, J ) 6.0 Hz, 1H), 4.58 (m, 2H), 4.17-3.96 (m, 8H), 3.91-3.75
(m, 3H), 3.61 (m, 3H), 3.49 (s, 3H), 3.16 (d, J ) 18.9 Hz, 1H),
2.81 (m, 1H), 2.41 (s, 3H), 2.22 (m, 2H), 1.97-1.67 (m, 4H),
1.59-1.48 (m, 2H), 1.41-1.38 (m, 2H), 1.00 (m, 1H). MS 974.36
(M+ + H).
1-(N-Oxymorphyldiglycoldiamide)-6-[1-diglycoldiamide-2-
(m-nitrophenoxy)ethane]hexane (19c). To an ice-cold solution
of 12 (109 mg, 0.366 mmol), DCC (75.4 mg, 0.366 mmol), and
HOBt (49.4 mg, 0.366 mmol) in dry DMF (1 mL) was added a
solution of 18c (171.7 mg, 0.332 mmol) in dry DMF (1.5 mL).
The reaction mixture was stirred overnight at room temperature.
The precipitate was filtered, and the filtrate was concentrated by a
rotary-evaporator. The residue was purified by a flash chromatog-
raphy (CH2Cl2/CH3OH/NH3H2O ) 90:10:1) to give 255 mg
1-{1-Diglycoldiamide-2-[m-(R(+)-1)ureaphenoxy]ethane}-
2-(N-oxymorphyldiglycoldiamide)ethane (3b). To a solution of
20b (95.7 mg, 0.135 mmol) in anhydrous DMSO (2 mL) was added
a solution of 8 (39.2 mg, 0.135 mmol) in anhydrous THF (0.5 mL).
The reaction solution was stirred at room temperature for 24 h and
concentrated. The residue was purified by flash chromatography
(CH2Cl2/MeOH/NH4OH ) 90/10/1) to give 24.9 mg (18.4%) of
product 3b. 1H NMR (CDCl3): δ 8.10 (s, 1H), 7.61-7.52 (m, 3H),
7.49-7.42 (m, 2H), 7.39-7.32 (m, 2H), 7.26-7.15 (m, 4H), 6.98
(d, J ) 9.0 Hz, 1H), 6.92 (m, 1H), 6.71-6.61 (m, 2H), 6.53-6.50
(m, 2H), 5.50 (d, J ) 7.8 Hz, 1H), 5.30 (s, 2H), 4.74-4.64 (m,
2H), 4.54-4.49 (m, 2H), 4.10-4.03 (m, 8H), 3.96 (m, 2H), 3.67
(m, 2H), 3.46 (m, 3H), 3.44 (s, 3H), 3.15-3.08 (m, 2H), 2.84-2.76
(m, 2H), 2.65-2.51 (m, 2H), 2.46-2.18 (m, 4H), 2.34 (s, 3H),
1.88-1.36 (m, 9H), 1.25 (s, 1H), 1.02-0.86 (m, 2H). MS 1002.20
(M+ + H).
1
(87.4%) of 19c. H NMR (CDCl3): δ 7.84 (d, J ) 7.8 Hz, 1H),
7.72 (m, 1H), 7.46 (t, J ) 7.8 Hz, 1H), 7.27 (m, 1H), 7.16 (m,
1H), 6.74 (m, 2H), 6.54 (d, J ) 7.2 Hz), 4.59 (m, 2H), 4.16 (m,
2H), 4.08 (m, 4H), 4.05 (m, 4H), 3.76 (m, 2H), 3.33 (m, 3H), 2.62
(s, 8H), 2.35 (s, 3H), 2.25 (m, 2H), 1.71 (m, 5H), 1.56 (m, 3H),
1.36 (m, 4H), 1.01 (m, 1H). MS 797.62 (M+ + H), 819.60 (M+ +
Na).

256 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Zheng et al.
1-{1-Diglycoldiamide-6-[m-(R(+)-1)ureaphenoxy]ethane}-
2-(N-oxymorphyldiglycoldiamide)hexane (3c). To a solution of
20c (194.8 mg, 0.254 mmol) and 8 (120 mg, 0.279 mmol) in
anhydrous DMSO (5 mL) was added triethylamine (50 µL, 0.279
mmol). The reaction solution was stirred at room temperature for
6 h and concentrated. The residue was purified by flash chroma-
tography (CH2Cl2/MeOH/NH4OH ) 90/10/1) to give 21.1 mg
these in frame at the 3′-end of the receptor sequence into NheI/
XbaI sites of pcDNA3.0 vector (Invitrogen, Carlsbad, CA). All
sequences were verified by direct DNA sequencing.
1.2. Receptor-Bearing Cells. Chinese hamster ovary (CHO-
K1) cells stably expressing the CCK2 receptors, the MOP receptors,
and cells coexpressing both of these receptors were prepared and
used as sources of receptor-enriched plasma membranes for receptor
binding studies. CHO cells obtained from the American Type
Culture Collection (Manassas, VA) and receptor-expressing CHO
cell lines were grown in Ham’s F-12 nutrient mixture containing
5% (v/v) Fetal Clone II supplement (Hyclone laboratories, Logan,
UT) in a 5% CO2 environment. Cells were passaged approximately
twice per week. Cell lines stably expressing receptor constructs
were prepared by transfection with appropriate cDNA. Cells were
selected with 1 mg/mL G-418 or 0.5 mg/mL zeocin and then were
subjected to a series of standard limiting dilutions, followed by
screening using radioligand binding to assess receptor expression.
1
(7.9%) of product 3c. H NMR (CDCl3): δ 7.65-7.58 (m, 2H),
7.50-7.32 (m, 6H), 7.24-7.13 (m, 3H), 7.02 (t, J ) 7.8 Hz, 1H),
6.92 (d, J ) 8.1 Hz, 1H), 6.81-6.72 (m, 2H), 6.67 (d, J ) 8.1 Hz,
1H), 6.29-6.20 (m, 2H), 5.30 (d, J ) 7.8 Hz, 1H), 4.58 (d, J )
4.2 Hz, 1H), 4.43 (m, 1H), 4.06-3.99 (m, 8H), 3.72-3.55 (m, 3H),
3.49 (s, 3H), 3.25-3.08 (m, 4H), 2.81 (m, 1H), 2.62 (s, 4H), 2.35
(s, 3H), 2.24 (m, 2H), 1.88-1.56 (m, 8H), 1.41 (m, 6H), 1.25 (m,
6H), 0.95 (m, 1H). MS 1058.44 (M+ + H), 1080.42 (M+ + Na).
N-(m-Nitrophenoxy)ethyl-N′-oxymorphyldiglycoldiamide
(22). To a solution of 16 (209 mg, 0.5 mmol) in anhydrous DMSO
(1.5 mL) were added DCC (113 mg, 0.55 mmol) and HOBt (74.3
mg, 0.55 mmol), and the solution was stirred for 5 min. A solution
of 1-amino-2-(3-nitrophenoxy)ethane 21 (100 mg, 0.55 mmol) in
anhydrous DMSO (1.0 mL) was added to the reaction solution,
and the reaction mixture continued stirring overnight at room
temperature. The precipitate was removed by filtration and the
residue was purified by flash chromatography (CH2Cl2/MeOH/
COS cells transiently expressing receptor constructs were used
for receptor BRET studies. These cells were plated in 10 cm tissue
culture dishes at a density of 0.5 million cells/dish and were
propagated in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 5% Fetal Clone II. Cells were transfected 48 h
before use with approximately 3 µg of total DNA (either a single
construct or a combination of two constructs (donor and acceptor))
1
NH4OH ) 92:7.5:0.5) to give product 22 as oil (223.7 mg). H
42
per dish using the DEAE-dextran method.
NMR (CDCl3): δ 7.84-7.80 (m, 1H), 7.70 (m, 1H), 7.42 (t, J )
8.4 Hz, 1H), 7.22-7.19 (m, 2H), 6.97 (m, 1H), 6.71 (d, J ) 8.4
Hz, 1H), 6.56 (d, J ) 8.4 Hz, 1H), 4.60-4.55 (m, 2H), 4.18-4.14
(m, 4H), 4.07 (d, J ) 6.6 Hz, 1H), 3.84-3.77 (m, 2H), 3.12 (m,
1H), 2.84-2.68 (m, 2H), 2.40 (m, 1H), 2.34 (s, 3H), 2.27-2.21
(m, 1H), 1.79-1.52 (m, 6H), 1.37-1.25 (m, 2H), 0.92 (m, 1H).
MS 583.14 (M+ + H).
1.3. CCK Receptor Binding. Receptor-enriched membranes
were isolated from CHO cell lines using the previously established
density gradient centrifugation procedure.43 The membranes were
suspended in Krebs-Ringers-HEPES (KRH) medium (25 mM
HEPES, pH 7.4, 104 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM
KH2PO4, 1.2 mM MgSO4) containing 0.01% soybean trypsin
inhibitor and 1 mM phenylmethylsulfonyl fluoride and were stored
at -80 °C until use. Radioligand binding studies were performed
as described previously.44 Receptor preparations were mixed with
a constant amount of CCK-like radioligand, 125I-D-Tyr-Gly-
[(Nle28,31)CCK-26-33] (1-5 pM) (specific radioactivity 2000 Ci/
mmol), in the absence or presence of increasing concentrations of
unlabeled ligand for 90 min at room temperature in KRH medium,
pH 7.4, containing 0.01% soybean trypsin inhibitor and 0.2% bovine
serum albumin. Receptor-bound and free radioligand were separated
using a Skatron cell harvester (Molecular Devices, Sunnyvale, CA)
with receptor-binding filtermats. Receptor-bound radioactivity was
quantified using a γ spectrometer. Nonsaturable binding determined
in the presence of 1 µM CCK was consistently less than 10% of
total binding. Saturable radioligand binding data were analyzed
using the LIGAND program45 and were plotted using the nonlinear
least-squares curve-fitting routine in Prism (GraphPad 3.0, San
Diego, CA).
1.4. MOP Receptor Binding. Opioid receptor binding was
performed with membranes isolated from CHO cells (Table 1).
Receptor-enriched membranes were mixed with a constant amount
of specific MOP radioligand [3H]DAMGO (0.4nM) (specific
radioactivity 56.8 Ci/mmol) (Perkin-Elmer, Boston, MA) in the
absence or presence of increasing concentrations of unlabeled ligand
for 2 h at room temperature in KRH medium, pH 7.4, containing
0.01% soybean trypsin inhibitor and 0.2% bovine serum albumin.
The nonspecific binding was determined in the presence of 1 µM
DAMGO (Bachem, Torrace, CA), an agonist ligand for the MOP
receptor. Receptor-bound and free radioligand were separated by
rapid filtration using Whatman GF/C glass microfiber filters
(Whatman Inc., Florham Park, NJ) that were presoaked in 0.3%
polyethylenimine (Sigma, St. Louis, MO). Receptor-bound radio-
activity was quantified in a Beckman-Coulter LS6500 multipurpose
scintillation counter. Saturable radioligand binding data were
analyzed using the LIGAND program45 and were plotted using the
nonlinear least-squares curve-fitting routine in Prism (GraphPad 3.0,
San Diego, CA).
N-(m-Aminophenoxy)ethyl-N′-oxymorphyldiglycoldiamide
(23). A mixture of 22 (220 mg, 0.338 mmol) and Pd/C (10 wt %,
72 mg) in MeOH (13 mL) was hydrogenated at room temperature
and 50 psi for 4 h. The catalyst was removed by filtration over a
Celite pad and the filtrate was concentrated in a rotary-evaporator
to give 196 mg of product 23. 1H NMR (DMSO-d6): δ 8.25 (s br,
1H), 7.57 (m, 1H), 6.85 (t, J ) 8.4 Hz, 1H), 6.56 (d, J ) 8.4 Hz,
1H), 6.45 (d, J ) 8.4 Hz, 1H), 6.12-6.03 (m, 3H), 5.01 (s, 2H),
4.78 (s, 1H), 4.44 (m, 1H), 4.37-4.30 (m, 1H), 3.98 (m, 4H), 3.87
(t, J ) 6.0 Hz, 2H), 3.43 (q, J ) 6.0 Hz, 2H), 3.02 (d, J ) 18.6
Hz, 1H), 2.53 (m, 3H), 2.37-2.32 (m, 1H), 2.27 (m, 3H), 2.17-2.10
(m, 2H), 1.58-1.51 (m, 1H), 1.33 (m, 3H), 0.92 (m, 1H). MS
553.20 (M+ + H).
N-{m-[R(+)-1]Ureaphenoxy}ethyl-N′-oxymorphyldigly-
coldiamide (4). To a solution of 23 (193 mg, 0.349 mmol) in
anhydrous DMSO (4.5 mL) was added a solution of 8 (96.6 mg,
0.332 mmol) in anhydrous DMSO (2 mL). The reaction solution
was stirred at room temperature for 24 h and concentrated. The
residue was purified by flash chromatography (CH2Cl2/MeOH/
NH4OH ) 90:9:1) to give 21 mg (7.5%) of 4 as a solid. 1H NMR
(CDCl3): δ 7.60 (m, 4H), 7.47-7.20 (m, 8H), 7.00 (m, 2H), 6.75
(m, 1H), 6.65 (d, J ) 8.1 Hz, 1H), 6.23 (d, J ) 8.1 Hz, 1H), 6.13
(m, 2H), 5.34 (d, J ) 8.1 1H), 4.56 (m, 1H), 4.39 (m, 1H), 4.03
(m, 1H), 3.88 (t, J ) 5.4 Hz, 1H), 3.77-3.63 (m, 2H), 3.58 (s,
1H), 3.48 (m, 3H), 3.44-3.39 (m, 2H), 3.14 (d, J ) 18.9 Hz, 1H),
2.78 (m, 1H), 2.67-2.58 (m, 1H), 2.41 (m, 1H), 2.34 (s, 3H),
2.25-2.17 (m, 2H), 1.71-1.50 (m, 6H), 1.38-1.20 (m, 3H), 0.89
(m, 1H). MS 844.34 (M+ + H).
Biology. 1. In Vitro Studies. 1.1. Preparation of Receptor
Constructs. Type 2 cholecystokinin (CCK2) and MOP receptor
constructs tagged at their carboxyl-terminal ends with either Renilla
luciferase (Rlu) or yellow fluorescent protein (YFP) were used as
donor and acceptor, respectively, for BRET studies. We previously
characterized the Rlu- and YFP-tagged CCK2 receptor constructs
expressed in COS cells, demonstrating that their binding charac-
teristics and biological activities are similar to those of wild type
CCK2 receptor.34 The Rlu- and YFP-tagged MOP receptor con-
structs were prepared utilizing the Rlu and YFP sequences present
in pBRET and pEYFP-N1 (Stratagene, La Jolla, CA) and cloning
Values are expressed as mean values ( SEM of data from three
to five independent experiments.

Association of MOP and CCK2 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 257
in conscious mice according to the method of Haley and McCor-
mick49 for icv injections. A time course study, including the times
10, 20, 30, and 60 min was used to determine the peak antinoci-
ception. List of compounds and peak times are summarized in the
Table 3. A 24 h tolerance was measured with each compound using
the ED80 - 90 doses measured on day 1 and then repeated 24 h later
on the same mouse.
1.5. BRET Studies. Bioluminescence and fluorescence mea-
surements were performed with aliquots of approximately 25 000
receptor-bearing COS cells in suspension, as described previously.44
For this, the cells were lifted using nonenzymatic cell dissociation
solution (Sigma) 48 h after transient transfection, and they were
washed with KRH medium. The BRET assay was initiated by
adding the cell-permeant Renilla luciferase-specific substrate,
coelenterazine h (Biotium, Hayward, CA), to the cell suspension
to yield a final concentration of 5 µM in a 96-well white Optiplate
(PerkinElmer, Boston, MA). The BRET signal was collected by
using the protocol designed for BRET studies in the 2103 Envision
fluorescence plate reader (Perkin-Elmer, Boston, MA), and the
BRET ratio was calculated based on emission ratios. For ligand
effects, the transfected COS cells were incubated with specified
ligands (1 µM) for 90 min at room temperature before performing
the assay. Saturation BRET experiments were performed as
described previously.44 COS cells were transiently transfected with
a fixed amount of Rlu-tagged µ-opioid receptor construct (1.0 µg
DNA/dish) and with increasing concentrations of YFP-tagged CCK2
receptor construct (0.3-6 µg DNA/dish). Curves were fit to these
data and evaluated for quality-of-fit based on R2 values using Prism
3.0, and the BRET50 and BRETmax values were calculated.
1.6. Calcium Mobilization.36 Calcium mobilization was done
in MOPR-CCK2R coexpressing CHO cells. Stably expressing CHO
CCK2R cells were grown to 90% confluent in HAM F-12 nutrient
media plus 10% bovine calf serum and 1% penicillin/streptomycin
in 75 mm2 tissue culture plates at 37 °C and were transiently
transfected with MOPR construct and chimeric G-protein (Gqi4-
myr) construct using lipofectamine 2000 transfection reagent and
manufactor’s protocol (GIBCO). Ratios were 20 µg of MOPR
construct, 24 µg of Gqi4-myr construct to 80 mL of lipofectamine
reagent. Cells were incubated for 24 h after transfection, seeded
into 96-well plates (Corning 96-well plates half area, black with
clear bottom), and incubated overnight before experiments were
conducted. Calcium mobilization was measured on a FlexStation3
using a FLIPR calcium kit (Molecular Devices) and analyzed using
SoftMax Pro and PRISM software.
2. In Vivo Studies. 2.1. Materials and Methods. All experi-
ments were approved by the Institutional Animal Care and Use
Committee of the University of Minnesota (Minneapolis, MN).
2.2. Subjects. Male ICR mice (17-25 g; Harlan, Madison, WI)
were housed in groups of 8 in a temperature- and humidity-
controlled environment with unlimited access to food and water.
They were maintained on a 12-h light/dark cycle.
2.3. Tail Flick Assay. The tail flick assay was used to test for
antinociception described by D’Amour and Smith46 and modified
by Dewey et al.47 For the measurement the latency of the tail-flick
measurement, the mice were held gently in one hand with the tail
positioned in the apparatus (tail flick analgesia meter, Columbus
Instruments, Columbus, Ohio) for radiant heat stimulus. The tail-
flick response was elicited by applying radiant heat to the dorsal
side of the tail. The intensity of the heat was set at setting 8 so that
the animal flicked its tail within 2-3 s. The test latency was
measured once before drug treatment (control) and again after the
drug treatment (test) at the peak time of the compound; a 10 s
maximum cutoff time was used to prevent damage to the tail.
Antinociception was quantified according to the method of Harris
and Pierson48 as the percent maximal possible effect (% MPE)
which is calculated as
Acknowledgment. This work was supported by NIH Grants
DA 01533 (P.S.P.) and DK 32878 (L.J.M.). We thank Ajay
Yekkirala for assistance with the graphics, and we thank L.-A.
Bruins and R. Happs for technical assistance.
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10 - control
% MPE )
× 100
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