Design and synthesis of a bivalent ligand to explore the putative heterodimerization of the mu opioid receptor and the chemokine receptor CCR5

Article abstract of DOI:10.1039/c2ob06801j

The bivalent ligand approach has been utilized not only to study the underlying mechanism of G protein-coupled receptors dimerization and/or oligomerization, but also to enhance ligand affinity and/or selectivity for potential treatment of a variety of diseases by targeting this process. Substance abuse and addiction have made both the prevention and the treatment of human immunodeficiency virus (HIV) infection more difficult to tackle. Morphine, a mu opioid receptor (MOR) agonist, can accelerate HIV infection through up-regulating the expression of the chemokine receptor CCR5, a well-known co-receptor for HIV invasion to the host cells and this has been extensively studied. Meanwhile, two research groups have described the putative MOR-CCR5 heterodimers in their independent studies. The purpose of this paper is to report the design and synthesis of a bivalent ligand to explore the biological and pharmacological process of the putative MOR-CCR5 dimerization phenomenon. The developed bivalent ligand thus contains two distinct pharmacophores linked through a spacer; ideally one of which will interact with the MOR and the other with the CCR5. Naltrexone and Maraviroc were selected as the pharmacophores to generate such a bivalent probe. The overall reaction route to prepare this bivalent ligand was convergent and efficient, and involved sixteen steps with moderate to good yields. The preliminary biological characterization showed that the bivalent compound 1 retained the pharmacological characteristics of both pharmacophores towards the MOR and the CCR5 respectively with relatively lower binding affinity, which tentatively validated our original molecular design. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob06801j

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PAPER
Design and synthesis of a bivalent ligand to explore the putative
heterodimerization of the mu opioid receptor and the chemokine receptor
CCR5†
Yunyun Yuan,a Christopher K. Arnatt,a Guo Li,a Kendra M. Haney,a Derong Ding,a Joanna C. Jacob,b
Dana E. Selleyb and Yan Zhang*a
Received 26th October 2011, Accepted 12th January 2012
DOI: 10.1039/c2ob06801j
The bivalent ligand approach has been utilized not only to study the underlying mechanism of G protein-
coupled receptors dimerization and/or oligomerization, but also to enhance ligand affinity and/or
selectivity for potential treatment of a variety of diseases by targeting this process. Substance abuse and
addiction have made both the prevention and the treatment of human immunodeficiency virus (HIV)
infection more difficult to tackle. Morphine, a mu opioid receptor (MOR) agonist, can accelerate HIV
infection through up-regulating the expression of the chemokine receptor CCR5, a well-known co-
receptor for HIV invasion to the host cells and this has been extensively studied. Meanwhile, two research
groups have described the putative MOR–CCR5 heterodimers in their independent studies. The purpose
of this paper is to report the design and synthesis of a bivalent ligand to explore the biological and
pharmacological process of the putative MOR–CCR5 dimerization phenomenon. The developed bivalent
ligand thus contains two distinct pharmacophores linked through a spacer; ideally one of which will
interact with the MOR and the other with the CCR5. Naltrexone and Maraviroc were selected as the
pharmacophores to generate such a bivalent probe. The overall reaction route to prepare this bivalent
ligand was convergent and efficient, and involved sixteen steps with moderate to good yields. The
preliminary biological characterization showed that the bivalent compound 1 retained the pharmacological
characteristics of both pharmacophores towards the MOR and the CCR5 respectively with relatively lower
binding affinity, which tentatively validated our original molecular design.
by the Bouvier group for β2-adrenergic receptor.7 The “dimeriza-
tion–oligomerization” concept for G protein-coupled receptors
Introduction
The negative co-operativity among β-adrenergic receptors in frog
erythrocyte membranes1 and occurrence of opioid receptors in
clusters on the neuroblastoma cell surface2 were attributed to be
the earliest reports of receptor dimerization–oligomerization.3,4
The exact term “dimerization” was actually coined by Gregory
and co-workers in 1982,5 yet it took more than another decade
for more convincing evidence to be recognized. Among the evi-
dence, the most significant pieces are the co-expression studies
with mutant muscarinic–adrenergic receptors conducted by
Maggio et al.6 and a co-immunoprecipitation approach utilized
(GPCRs) was widely accepted by the end of 1990s based on the
research on GABAB receptor from several groups,8 and was
further supported by the X-ray crystal structures of some others
later on.9 The dimerization–oligomerization of GPCRs poses a
differentiated pharmacology from the monomers.10 In this
regard, a number of bivalent ligands have been synthesized to
explore the underlying biology and pharmacology mechanisms
of GPCRs dimerization–oligomerization, as well as to develop
prospective agents with enhanced affinity and/or selectivity to
treat different disorders and diseases by targeting this “novel”
mechanism.4a,11
Since acquired immunodeficiency syndrome (AIDS) was
identified three decades ago,12 the global prevalence of AIDS
has become stable at 0.8%, with over 33 million people infected
with human immunodeficiency virus (HIV) in 2007.13 There are
almost 16 million people who are injecting drug users (IDUs)
worldwide and nearly 10% of HIV infection was attributed to
injecting drug use through contaminated needles.14 Statistics
showed IDUs account for approximately 13% of the total HIV
aDepartment of Medicinal Chemistry, Virginia Commonwealth
University, 800 E. Leigh Street, Richmond, VA 23298, USA.
E-mail: yzhang2@vcu.edu; Fax: +1 804 828 7625; Tel: +1 804 828
0021
bDepartment of Pharmacology and Toxicology, Virginia Commonwealth
University, 410 North 12th Street, Richmond, VA 23298, USA
†Electronic supplementary information (ESI) available: The NMR
spectra for all compounds, and HPLC spectra for the bivalent and mono-
valent ligands. See DOI: 10.1039/c2ob06801j
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Fig. 1 Chemical structures of naltrexone, Maraviroc, designed bivalent (1) and monovalent ligands (2, 3).
infection in thirty-four US states during 2004 to 2007.15 Not
only driving HIV transmission among IDUs, the abused sub-
stances, such as opioids, cocaine, and alcohol also accelerate
the progression of AIDS and complicate the treatment of this
disease.16 Moreover, HIV infection seems to increase drug addic-
tion vulnerability as well.17 Needle-exchange programs (NEPs)
have shown appreciable outcome on reducing HIV prevalence
among IDUs for over a decade.18 The current available treatment
for opioid-dependent HIV patients also adopts opioid substi-
tution therapy (OST), i.e. methadone, buprenorphine and bupre-
norphine–naloxone, into HIV management.19 Although opioid
maintenance therapy have shown to improve patients adherence
and promising outcome for HIV treatment, the adverse drug–
drug interaction between methadone, buprenorphine and antire-
troviral agents compromise the overall effects.20 New agents and
remedies are still highly demanded for the treatment of these
patients.
Opiates and alcohol abuse–addiction liability is mainly associ-
ated with the mu opioid receptor (MOR),21 which is also
involved in different immunomodulatory activities induced by
opioids.22 The chemokine receptor CCR5 was identified as a
major co-receptor for HIV in 1996,23 and is largely expressed on
activated memory CD45RO+ T cells, monocyte–macrophages,
dendritic cells, granulocyte precursors, and natural killer cells.24
Both receptors belong to the seven-transmembrane GPCR super-
family. Several research groups have shown that MOR agonists,
such as morphine, methadone, and DAMGO, can increase
CCR5 expression, thus enhance and facilitate HIV infection and
replication both in vitro and in vivo.25 In light of this obser-
vation, as well as the fact that the opioid receptors and the CCR5
are all present on immune cells, Suzuki and co-workers studied
the interactions between these GPCRs through a co-immunopre-
cipitation approach.26 Their research demonstrated for the first
time that CCR5 and opioid receptors form oligomers and the oli-
gomerization modulates the function of such a complex.26 Two
years later, Chen et al. reported the heterodimerization and
cross-desensitization between the MOR and the CCR5 in co-
expressed Chinese hamster ovary (CHO) cells.27 The authors
proposed that the MOR–CCR5 heterodimers may contribute to
the observed cross-desensitization. Despite these fundamental
studies, a chemical probe that is capable of interacting with both
receptors simultaneously has never been developed to facilitate
the study of the biological and pharmacological process of
MOR–CCR5 dimerization. Herein, we report the design and syn-
thesis of a bivalent ligand 1 (Fig. 1) as a lead compound in order
to test our hypothesis and to reveal the underlying mechanism of
MOR–CCR5 heterodimers, eventually.
Bivalent ligand rational design
Receptor antagonists serve as important pharmacological probes
to uncover the probable involvement of a receptor mechanism.28
Therefore, it seemed ideal to build a bivalent ligand containing
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a MOR-antagonist moiety as well as a CCR5-antagonist one,
linked through an appropriate spacer. Naltrexone29 (Fig. 1) was
selected as the moiety to interact with the MOR based on the fol-
lowing considerations: first, naltrexone has been successfully
used to investigate the dimerization of opioid receptors pre-
viously;30 second, it represents an ideal treatment for alcohol and
opiate addiction and has been successfully used to treat alcohol-
ism clinically.31 Maraviroc32 (Fig. 1) is the only CCR5 antagon-
ist that has been approved for HIV treatment by the FDA so
far33 and thus became our first choice as the CCR5 pharmaco-
phore. Meanwhile, both of these two ligands showed high
affinity and reasonable selectivity toward the MOR and the
CCR5 respectively.
It has been proved that the loci for tethering two pharmaco-
phores through a spacer affect the binding affinities of the
resulted bivalent ligands.34 In addition, the overall chemical
modification of these two pharmacophores for spacer attachment
should also be designed from a synthetic point of view, that is,
chemical reactions should be readily accomplished. Thus, based
on the successful cases from Portoghese group,30a,b the C6-
position of naltrexone was selected as the attaching locus after
transforming its carbonyl group to the 6β-amino group (Fig. 1).
Whereas the discovery process of Maraviroc revealed that both
of the difluorocyclohexyl moiety and the exo-1,2,4-triazole sub-
stituted tropane core are essential to its potent antiviral activity
and weak hERG inhibition.35 Additionally, an interactive
docking study of Maraviroc to a rhodopsin-based CCR5
homology model demonstrated the interactions between Glu283
and the tropane core, as well as Ile198 and the difluorocyclo-
hexyl moiety within the proposed binding pocket.36 Hence, the
para-position of the phenyl ring in Maraviroc was first chosen as
the linking site to avoid severe impacts to the above interactions.
Since EDCI–HOBt mediated coupling reaction between car-
boxylic acid and amine can be easily accomplished, an amino
group was then chosen as the functional group on this para-
phenyl ring to hook Maraviroc up with the spacer. Thus, pharma-
cophore 6 (Scheme 1) was designed as the precursor of the
CCR5 antagonist.
from naltrexone following the reported procedure,39 whereas
nucleophilic reaction of 1,7-diaminoheptane with diglycolic
anhydride30b can readily afford the diacid spacer 5. Similarly to
Maraviroc, retrosynthetic analysis of 6 identified three key frag-
ments: a 4,4-difluorocyclohexanecarboxylic acid 8, a β-phenyl-
alanine ester 9, and a triazole-substituted tropane 10 (Scheme 1).
As the preparation of difluoro acid 8 appeared to be challen-
ging,40 a strategy that enables a later introduction of this
fragment was sought. In order to avoid the tedious reduction–
oxidation procedure as well as to improve the overall yields, an
amide coupling strategy instead of reductive amination40,41 was
postulated to generate 7 by coupling 9 with 10. Several papers
have reported the highly stereoselective introduction of an amino
group through Michael addition with lithium (R)-N-benzyl-N-α-
methylbenzylamide in high yields.42 Hence, the same method
was adopted to prepare fragment 9. Two cinnamic acids are
commercially available to synthesize the substrate 11 for a
later Michael addition: 4-nitrocinnacid and 4-bromocinnacid.
However, the conversion of the nitro group to the amino group
poses an issue for the overall synthetic route since other func-
tional groups, such as double bond, ester, benzyl, and amide, are
present in the same molecule and reducing agents such as Na2S
and SnCl2 are not environment-friendly. Therefore, 4-bromocin-
nacid was chosen as the starting material.
The overall synthesis of the precursor 6 is illustrated in
Schemes 2 and 3. The carboxylic group protection was per-
formed by refluxing 4-bromocinnacid in isopropanol with a few
drops of concentrated sulfuric acid to give a moderate yield of
12.43 The bromide 12 was then converted to aniline 13 using
lithium hexamethyldisilazide (LHMDS) catalyzed by Pd2(dba)3
and P(t-Bu)3,44 which upon heating with di-tert-butyl dicarbo-
nate furnished compound 14 in a good yield.45 The diastereose-
lective Michael addition of 14 was achieved with lithium (R)-N-
benzyl-N-α-methylbenzylamide prepared in situ.42e The stereose-
lectivity was confirmed by comparing with the literature reported
data.42f The following saponification of the conjugate adduct 15
gave acid 16, which was then coupled with 3-(3-isopropyl-5-
methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]octane41a
10 through HOBt–EDCI method to yield 17. The reduction of
17 with either BH3·THF or LiAlH4 at ambient temperature did
not give any amide reduced product 18. Heating 17 with
BH3·THF resulted a mixture of complexes, with the loss of Boc
and/or two benzyl groups. The steric hindrance generated by the
two benzyl groups might complicate the reduction process.
Hence, the catalytic hydrogenolysis of 17 with 10% Pd–C was
conducted to produce intermediate 19 instead. Although reaction
of 19 with BH3·THF did afford compound 7, the majority of the
product formed a complex with tetrahydrofuran (1 : 1), which
requires acid to release the free amine.46 However, the Boc
group may be sensitive to such acidic conditions. Replacement
of BH3·THF with LiAlH4, which only needs water to decompose
the intermediate formed after the reaction,47 provided 7 in a
reasonable yield (Scheme 2). Reaction of 7 with 4,4-difluorocy-
clohexanecarboxylic acid48 8 was mediated by HOBt–EDCI and
the coupling product 20 was subsequently converted to the
CCR5 antagonist precursor 6 with TFA/DCM (1 : 10) at ambient
temperature49 (Scheme 3).
Several studies indicated that a spacer with 16 to 22 atoms
might be beneficial for targeting GPCR dimers, ideally with
21 atoms when both pharmacophores are antagonists of their
respective receptors.30a,b,37 Therefore, the 21-atom-spacer was
adopted as an initial lead in the current study. The design ration-
ale of such a spacer is to keep a favorable balance between
hydrophobicity and hydrophilicity as well as to possess a
reasonable rigidity, high stability and low toxicity.38 Hence, one
alkyldiamine moiety and two diglycolic units were employed to
build up the spacer. Monovalent ligands 2 and 3 (Fig. 1) were
also designed as controls to clarify the potential effects of
the spacer to the binding affinity and potency of the bivalent
ligand.
Chemistry and biological studies
The retrosynthetic analysis of bivalent ligand 1 revealed three
major fragments, 6β-naltrexamine 4, diacid spacer 5, and the
CCR5 antagonist precursor 4-NH2-maraviroc 6 (Scheme 1).
Among them, 6β-naltrexamine 4 can be conveniently prepared
Then the bivalent ligand 1 was prepared following a
linear synthetic route as shown in Scheme 4. Reaction of
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Scheme 1 Retrosynthetic analysis of the bivalent ligand 1.
1,7-diaminoheptane with 0.9 equivalent of benzyl chloroformate
under ice-water bath generated mono-Cbz protected intermediate
21,50 which was further condensed with diglycolic anhydride to
give compound 22.30b Intermediate 23 was prepared by coupling
22 with 4 (6β-naltrexamine39) utilizing HOBt–EDCI method.
Hydrogenation–deprotection of 23 with 10% Pd–C catalyst
yielded amine 24. Condensation of 24 with a second molecule
of diglycolic anhydride provided acid 25, which was then
coupled with the CCR5 antagonist precursor 6 via HOBt–EDCI
mediation to furnish bivalent ligand 1.
From an efficient synthesis perspective, monovalent ligand 3
was prepared according to Scheme 6, considering it only
involved three steps and all the reactions can be simply moni-
tored by UV. Thus, HOBt–EDCI-mediated coupling of 22 with
precursor 6 afforded intermediate 27, which underwent catalytic
hydrogenolysis to yield amine 28. Monovalent ligand 3 was then
obtained by coupling 28 with 26 employing the HOBt–EDCI
method.
All three ligands were then preliminarily further characterized
for their binding affinity and functional activity. In a calcium
mobilization assay with CCR5–MOLT-4 cells,51 compound 1
showed no agonism and its antagonist properties are indicated by
its calcium flux inhibition IC50 value of 231 88 nM. Compared
Monovalent ligand 2 was conveniently synthesized by coup-
ling the intermediate 24 with 2630b via HOBt–EDCI peptide
coupling method (Scheme 5).
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Scheme 2 Synthesis of intermediate 7. Regents and conditions: (a) i-PrOH, H2SO4 (conc.), reflux, 80%; (b) i) LHMDS, Pd2(dba)3, P(t-Bu)3,
toluene, rt; ii) 1N HCl, rt, 90%; (c) Boc2O, THF, reflux, 85%; (d) THF, −78 °C; (e) LiOH, MeOH–H2O (2/1), reflux, 85%, two steps; (f) EDCI,
HOBt, TEA, 10, 4Å MS, DCM, 0 °C to rt, 73%; (g) 10% Pd–C, 60 psi, MeOH, 84%; (h) i) LiAlH4, THF, 0 °C to rt; ii) H2O, NaOH, 71%.
Scheme 3 Synthesis of 4-NH2-Maraviroc (6). Regents and conditions: (a) EDCI, HOBt, TEA, 8, 4Å MS, DCM, 0 °C to rt, 85%; (b) CF3COOH,
DCM, 0 °C to rt, 95%.
with the calcium flux inhibition IC50 value of Maraviroc under
the same experimental conditions, which was 1.57 0.32 nM,
apparently the introduction of the long chain spacer seemed to
be influential to the binding affinity of compound 1 to the recep-
tor CCR5, as indicted by its significant decrement of functional
activity of calcium flux inhibition. This was further supported by
the even lower functional property of the control compound 3, of
which the calcium flux inhibition IC50 value was 833 150 nM.
A couple of reasons could lead to such results. First, the bulki-
ness of the spacer might influence the binding affinity to the
receptor directly. Second, the substitution position of the spacer
on the tailing aromatic ring system of Maraviroc might not be
the most suitable one in preventing serious steric hindrance
effect on the binding affinity. Currently the syntheses of new
ligands with spacers attached at different positions of this tailing
ring system are underway.
7.9 nM, which was lower than naltrexone’s binding affinity (Ki
value was 0.71 0.08 nM) under the same experimental con-
dition. Correspondingly the control compound 2 also showed
somewhat lower binding affinity as indicated by the Ki value of
9.18 3.44 nM.
These preliminary biological activity results supported our
original molecular design that the bivalent ligand did reserve the
original antagonist properties from both pharmacophores while
its relatively lower affinity to the each corresponding receptor
compared to the parent pharmacophores, which was not unusual
based on previous reports from others,34c,37e certainly requires
more extensive structural modification and further syntheses
effort.
Conclusions
Similarly, in 35S-GTP[γS] binding assays in MOR–CHO
cells,52 compound 1 showed very little apparent agonism
(Emax = 11.7 1.2%) compared to the full agonist DAMGO
(100 9.2%, EC50 = 13.7 1.6 nM), while its binding affinity
to the mu opioid receptor as indicated by Ki value was 51.8
In conclusion, a bivalent ligand with 21-atom spacer was
designed and synthesized as a molecular probe to study the bio-
logical and pharmacological mechanisms of the putative hetero-
dimerization between the mu opioid receptor and the chemokine
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Scheme 4 Synthesis of bivalent ligand 1. Regents and conditions: (a) CbzCl, DCM, MeOH, 5 °C, 32%; (b) THF, diglycolic anhydride, rt, 85%; (c)
EDCI, HOBt, TEA, 4·2HCl, 4Å MS, DMF, 0 °C to rt, 76%; (d) 10% Pd–C, 60 psi, MeOH, 99%; (e) DMF, diglycolic anhydride, rt, 82%; (f) EDCI,
HOBt, TEA, 6, 4Å MS, DMF, 0 °C to rt, 50%.
Scheme 5 Synthesis of monovalent ligand 2. Regents and conditions: (a) EDCI, HOBt, TEA, 26, 4Å MS, DMF, 0 °C to rt, 65%.
receptor CCR5. The overall 16-step synthetic route was efficient
and convergent with reasonable yields. The preliminary biologi-
cal data from the calcium mobilization assay and MOR–CHO
binding assay showed that the bivalent ligand 1 retained the
characteristics of its pharmacophores, antagonizing MOR and/or
CCR5 respectively, with relatively lower binding affinity. Further
characterization of these ligands and synthesis of ligands with
different length of spacer and linkage at different position are
undergoing right now. Based on the current pilot study, it is
believed that such a bivalent ligand with a favorable length of
spacer and an optimized linkage site may serve as a pharmaco-
logical probe to study the function of the putative MOR–CCR5
dimerization and help to understand the mechanism of such
protein-protein interactions in various neuronal-immuno
diseases, for example, HIV-infected opiate/alcohol abuse and
addiction.
Experimental
Synthesis
General methods. All reagents were purchased from Sigma-
Aldrich or as otherwise stated. TLC analyses were carried out on
Analtech Uniplate F254 plates. Chromatographic purification
was accomplished on silica gel columns (230–400 mesh,
Merck). Melting points were obtained with a Fisher scientific
micro melting point apparatus without further correction. All IR
spectra were recorded on a Nicolet iS10 FT-IR Instrument.
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Scheme 6 Synthesis of monovalent ligand 3. Regents and conditions: (a) EDCI, HOBt, TEA, 6, 4Å MS, DMF, 0 °C to rt, 72%; (b) 5% Pd–C,
60 psi, MeOH, 51%; (c) EDCI, HOBt, TEA, 26, 4Å MS, DMF, 0 °C to rt, 81%.
Proton (400 MHz) and Carbon-13 (100 MHz) nuclear magnetic
resonance (NMR) spectra were acquired at ambient temperature
with tetramethylsilane as the internal standard on a Bruker
Ultrashield 400 Plus spectrometer. MS analysis was performed
on an Applied Bio Systems 3200 Q trap with a turbo
V source for TurbolonSpray. HPLC analysis of the final
compounds was achieved on Varian ProStar 210 system on
Microsorb-MV 100-5 C18 column (250 mm × 4.6 mm) at
254 (1 and 3) or 210 (2) nm eluting with acetonitrile (0.1%
TFA)–water (50 : 50) at 1 mL min−1 over 10 min. Elemental
analysis of the final compounds was conducted in Atlantic
Microlab, Inc.
column chromatography using CH2Cl2–MeOH (10 : 1) as eluent
to give 87 mg white solid, in 50% yield. H NMR (400 MHz,
1
CD3OD): δ 7.60 (d, J = 8.56 Hz, 2H), 7.33 (d, J = 8.56 Hz, 2H),
6.67 (d, J = 8.08 Hz, 1H), 6.62 (d, J = 8.20 Hz, 1H), 5.03
(t, J = 7.38 Hz, 1H), 4.58 (d, J = 7.64 Hz, 1H), 4.39 (m, 1H),
4.20 (s, 2H), 4.12 (s, 2H), 4.05 (s, 2H), 4.04 (s, 2H), 3.74
(m, 1H), 3.43 (m, 2H), 3.28–3.23 (m, 5H), 2.90–2.74 (m, 3H),
2.50 (s, 3H), 2.48–2.41 (m, 2H), 2.41–2.32 (m, 2H), 2.30–2.20
(m, 2H), 2.19–2.05 (m, 4H), 2.04–1.69 (m, 15H), 1.68–1.46
(m, 8H), 1.37 (m, 6H), 1.34 (d, J = 6.84 Hz, 6H), 0.94 (m, 1H),
0.64–0.58 (m, 2H), 0.29 (m, 2H); 13C NMR (100 MHz,
CD3OD): δ 176.63, 171.70, 171.55, 171.50, 170.10, 161.47,
161.43, 143.77, 143.44, 140.34, 138.06, 137.93, 128.22, 121.89,
120.63, 120.43, 119.03, 92.56, 71.99, 71.79, 71.62, 71.58 (×2),
63.99, 60.76, 60.24, 57.25, 52.46, 52.40, 49.51, 49.30, 44.31,
43.69, 40.06, 38.17, 36.74, 36.08, 35.89, 33.88 (J 13C–19F 23
Hz), 31.20, 30.34, 30.31, 29.95, 28.03, 27.84, 27.82, 27.29,
27.19, 27.07, 27.00, 26.76, 25.17, 23.91, 22.06, 15.72, 12.45,
3.93. IR ν (diamond, cm−1): 3275, 1652, 1532, 1128, 1107. mp
158.5–160 °C. Anal. Calcd for C64H92F2N10O11: C 63.24, H
7.63, N 11.52; Found: C 63.15, H 7.57, N 11.28. MS (ESI) m/z
found 1198.4 (M + H)+, 1220.5 (M + Na)+.
General procedure for amide coupling. In an ice-water bath
EDCI (1.5 eq.), HOBt (1.5 eq.), molecular sieves, and TEA
(4.0 eq.) with N2 protection were added to a solution of acid
in either DCM or DMF (3 mL). After 15 min, a solution of
amine (1.0 eq.) in DMF or DCM (1 mL) was added dropwise.
The resulted mixture was allowed to warm up to ambient temp-
erature gradually. After completion of the reaction as monitored
by TLC, the reaction mixture was filtered through celite. When
DMF was used as the reaction solvent, the filtrate was concen-
trated in vacuum to remove DMF and the residue was then
purified with column chromatography to afford the coupling
product, whereas when DCM was the solvent, the filtrate was
washed with brine, dried over Na2SO4, concentrated and the
crude product was purified by either crystallization or column
chromatography.
Monovalent ligand 2. The title compound was prepared
according to the general amide coupling procedure by reacting
acid 26 with amine 24 in DMF for 8 h. The crude product was
purified with chromatography using CH2Cl2–MeOH (20 : 1) as
eluent to give 32 mg white solid, in 65% yield. 1H NMR
(400 MHz, DMSO-d6): δ 9.03 (brs, 1H), 8.22 (d, J = 8.44 Hz,
1H), 8.04–7.96 (m, 3H), 6.59 (d, J = 8.08 Hz, 1H), 6.53 (d, J =
8.12 Hz, 1H), 4.90 (brs, 1H), 4.60 (d, J = 7.80 Hz, 1H), 3.95
Bivalent ligand 1. The title compound was prepared according
to the general amide coupling procedure by reacting acid 25 with
amine 6 in DMF for 7 days. The crude product was purified by
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(s, 2H), 3.94 (s, 2H), 3.91 (m, 4H), 3.57–3.48 (m, 1H),
3.19–3.09 (m, 4H), 3.03–2.99 (m, 2H), 2.66 (d, J = 4.68 Hz,
3H), 2.63–2.58 (m, 2H), 2.40–2.29 (m, 2H), 2.32–2.09 (m, 1H),
1.99 (dt, J1 = 3.43 Hz, J2 = 11.92 Hz, 1H), 1.84–1.75 (m, 1H),
1.48–1.42 (m, 6H), 1.33–1.24 (m, 8H), 0.86 (m, 1H), 0.48
reduced pressure, the residue was crystallized by EtOAc–hexane
to give 3.470 g white solid as first crop, in 96% yield. H NMR
1
(400 MHz, DMSO-d6): δ 12.78 (brs, 2H), 7.81 (t, J = 5.70 Hz,
2H), 4.10 (s, 4H), 3.94 (s, 4H), 3.08 (q, J = 6.76 Hz, 4H), 1.41
(m, 4H), 1.25 (m, 6H). 13C NMR (100 MHz, CD3OD): δ
171.47, 168.60, 70.12, 67.85, 38.07, 28.98, 28.36, 26.24. IR ν
(diamond, cm−1): 3306, 1699, 1646, 1548, 1247, 1151, 1136,
711. mp 64–67 °C. MS (ESI) m/z found 363.5 (M + H)+.
1
(m, 2H), 0.13 (m, 2H); H NMR (400 MHz, CD3OD): δ 6.67
(d, J = 8.12 Hz, 1H), 6.60 (d, J = 8.12 Hz, 1H), 4.56 (d, J =
7.56 Hz, 1H), 4.10 (s, 2H), 4.09 (s, 2H), 4.08 (s, 2H), 4.06
(s, 2H), 3.82–3.78 (m, 1H), 3.30 (q, J = 7.34 Hz, 4H), 3.18–3.13
(m, 2H), 2.82 (s, 3H), 2.73–2.64 (m, 2H), 2.48–2.40 (m, 2H),
2.32–2.25 (m, 1H), 2.18 (dt, J1 = 3.04 Hz, J2 = 11.81 Hz, 1H),
1.94 (m, 1H), 1.60–1.58 (m, 6H), 1.48–1.32 (m, 8H), 0.95
(m, 1H), 0.56 (m, 2H), 0.19 (m, 2H); 13C NMR (100 MHz,
CD3OD): δ 172.10, 171.51, 171.43, 171.38, 143.71, 141.88,
132.49, 125.45, 120.06, 118.55, 92.89, 71.68, 71.57, 71.53,
71.45, 71.41, 63.72, 60.27, 52.45, 48.88, 45.38, 45.24, 40.02,
31.98, 31.22, 30.35, 29.99, 27.84, 25.87, 25.46, 23.52, 10.29,
4.45, 4.21. IR ν (diamond, cm−1): 3291, 1652, 1544, 1124. mp
74–76 °C. Anal. Calad for C36H55N5O10: C 60.23, H 7.72, N
9.76; Found: C 60.22, H 7.74, N 9.57. MS (ESI) m/z found
701.0 (M + H)+, 722.9 (M + Na)+.
4,4-Difluoro-cyclohexanecarboxylic acid {1-(4-amino-phenyl)-3-
[3-(3-isopropyl-5-methyl-[1,2,4]triazol-4-yl)-8-aza-bicyclo[3,2,1]
oct-8-yl]-propyl}-amide (6). On ice-water bath, to the solution of
20 (165 mg, 0.262 mmol) in DCM (5 mL) was added TFA
(0.5 mL) dropwise. The resultant mixture was allowed to warm
to ambient temperature within 15 min and stirred at the same
temperature for 1.5 h. The mixture was cooled to 0 °C, and satu-
rated Na2CO3 was added. The aqueous layer was adjusted to pH
= 12, and taken up with DCM (20 mL × 3). The combined
organic layers were washed with brine, dried over Na2SO4, evap-
orated and dried in vacuum to afford 131 mg white solid, which
is pure enough for the next step, in 95% yield. 1H NMR
(400 MHz, CDCl3): δ 7.05 (d, J = 8.28 Hz, 2H), 6.62 (d, J =
8.24 Hz, 2H), 6.38 (brs, 1H), 4.98 (q, J = 7.17 Hz, 1H), 4.27
(m, 1H), 3.36 (m, 2H), 2.78 (seq, J = 6.84 Hz, 1H), 2.48 (s, 3H),
2.39 (m, 2H), 2.26–2.02 (m, 8H), 1.94–1.73 (m, 6H), 1.70–1.50
(m, 5H), 1.36 (d, J = 6.72 Hz, 6H); 13C NMR (100 MHz,
CDCl3): δ 173.28, 159.23, 150.75, 145.98, 131.65, 127.67,
122.74 (J 13C–19F 239 Hz), 115.34, 58.98, 58.42, 51.59, 48.16,
47.38, 42.95, 35.59, 35.50, 34.84, 32.91 (J 13C–19F 24 Hz),
26.84, 26.78, 26.06 (J 13C–19F 9.7 Hz), 25.93, 21.75, 13.21. mp
109–110 °C. IR ν (diamond, cm−1): 3334, 3230, 1636, 1517,
1105, 1031, 962, 831. MS (ESI) m/z found 529.6 (M + H)+.
(4-{1-Amino-3-[3-(3-isopropyl-5-methyl-[1,2,4]triazol-4-yl)-8-
aza-bicyclo[3,2,1]oct-8-yl]-propyl}-phenyl)-carbamic acid tert-butyl
ester (7). On ice-water bath, a solution of 19 (764 mg,
1.538 mmol) in dry THF (10 mL) was added dropwise to a sus-
pension of LiAlH4 (292 mg, 7.692 mmol) in dry THF (10 mL).
The resultant mixture was stirred at the same temperature for
15 min and then 3 h at ambient temperature. The mixture was
cooled in an ice bath again, and the complex was decomposed
by dropwise addition of 2.4 mL H2O, 2.4 mL 4 N NaOH, and
4.8 mL H2O cautiously. The resulting white suspension was con-
tinued to stir for 1 h at ambient temperature, then filtered. The
filtrate cake was washed with THF (20 mL × 3), diethyl ether
(20 mL × 3). The combined filtrates were concentrated under
reduced pressure and the residue was purified by silica gel using
DCM–MeOH (6 : 1) to give 523 mg white solid, in 71% yield.
1H NMR (400 MHz, CD3OD): δ 7.39 (d, J = 8.52 Hz, 2H), 7.28
(d, J = 8.60 Hz, 2H), 4.38 (m, 1H), 3.95 (t, J = 6.90 Hz, 1H),
3.42 (m, 2H), 3.25 (seq, J = 5.83 Hz, 1H), 2.45 (s, 3H), 2.43
(m, 1H), 2.42–2.32 (m, 1H), 2.24–2.15 (m, 2H), 2.05–1.96
(m, 3H), 1.94–1.81 (m, 1H), 1.73 (m, 4H), 1.51 (s, 9H), 1.33
(d, J = 6.84 Hz, 6H). 13C NMR (100 MHz, CD3OD): δ 161.38,
155.32, 152.56, 140.28, 139.81, 128.02, 120.01, 80.86, 60.17,
60.07, 55.62, 49.97, 38.02, 36.37, 36.30, 28.76, 27.13, 26.98,
26.76, 22.05, 12.39. mp 103–105 °C. IR ν (diamond, cm−1):
3252, 1713, 1522, 1240, 1159, 838. MS (ESI) m/z found 483.7
(M + H)+.
Monovalent ligand 3. The title compound was prepared
according to the general amide coupling procedure by reacting
acid 26 with amine 28 in DMF overnight. The crude product
was purified with chromatography using CH2Cl2–MeOH (8 : 1)
1
as eluent to give 106 mg white solid, in 81% yield. H NMR
(400 MHz, CD3OD): δ 7.60 (d, J = 8.52 Hz, 2H), 7.33 (d, J =
8.56 Hz, 2H), 5.03 (t, J = 7.32 Hz, 1H), 4.44–4.35 (m, 1H), 4.20
(s, 2H), 4.12 (s, 2H), 4.02 (m, 4H), 3.42 (m, 2H), 3.30–3.20
(m, 5H), 2.78 (s, 3H), 2.50 (s, 3H), 2.47–2.40 (m, 2H),
2.39–2.30 (m, 1H), 2.30–2.17 (m, 2H), 2.15–2.03 (m, 4H),
2.02–1.95 (m, 2H), 1.90–1.65 (m, 10H), 1.60–1.45 (m, 4H),
1.40–1.30 (m, 12H); 13C NMR (100 MHz, CD3OD): δ 176.61,
172.13, 171.68, 171.46, 170.08, 161.42, 152.61, 140.33, 138.08,
128.22, 121.89, 71.97, 71.77, 71.48, 71.44, 60.71, 60.22, 52.40,
44.55, 43.70, 40.07, 40.03, 36.75, 36.10, 33.86 (J 13C–19F 23
Hz), 30.38, 30.35, 29.99, 27.86, 27.20 (J 13C–19F 9 Hz), 27.07,
27.02, 26.98, 26.76, 25.89, 22.06, 12.44. IR ν (diamond, cm−1):
3272, 1652, 1532, 1107. mp 79–81 °C. Anal. Calcd for
C45H73F2N9O9: C 58.61, H 7.98, N 13.67; Found: C 59.29, H
7.96, N 13.47. MS (ESI) m/z found 887.2 (M + H)+, 909.3
(M + Na)+.
6′β-Naltrexamine hydrochloride salt (4·2HCl). The title com-
pound was prepared following the reported procedure39 in 62%
1
yield for two steps (lit.,39 71%). H NMR (400 MHz, DMSO-
d6): δ 9.58 (s, 1H, exchangeable), 8.91 (brs, 1H, exchangeable),
8.43 (m, 3H, exchangeable), 6.80 (d, J = 8.0 Hz, 1H), 6.67
(d, J = 8.0 Hz, 1H), 6.40 (brs, 1H), 4.68 (d, J = 7.2 Hz, 1H),
3.90 (d, J = 4.8 Hz, 1H), 3.33 (m, 2H), 3.04 (dd, J1 = 6.0 Hz, J2
= 18.8 Hz, 2H), 2.90–2.70 (m, 2H), 2.50–2.40 (m, 2H), 1.99
(q, J = 12.5 Hz, 1H), 1.82 (d, J = 14.4 Hz, 1H), 1.78–1.70
(m, 1H), 1.46 (d, J = 8.8 Hz, 1H), 1.32 (m, 1H), 1.06 (m, 1H),
0.67 (m, 1H), 0.59 (m, 1H), 0.51 (m, 1H), 0.41 (m, 1H).
5,15-Dioxo-3,17-dioxa-diazanonadecane-1,19-dioic acid (5). To
the solution of 1,7-diaminoheptane (1.3 g, 10 mmol) in THF
(4 mL) at 0 °C was added diglycolic anhydride (2.44 g,
21 mmol) in one portion. The resultant mixture was stirred at the
same temperature for 15 min and allowed to warm to ambient
temperature and stirred overnight. After removal of THF under
4,4-Difluoro-cyclohexanecarboxlic acid (8). The title compound
was prepared as described by Mackenzie et al.,48 except that the
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1
ester was purified by silica gel using EtOAc–hexane (80 : 1) as
eluent. The total yield is 52%. H NMR (400 MHz, CDCl3): δ
solid, in 85% yield. H NMR (400 MHz, CDCl3): δ 7.60 (d, J =
16.0 Hz, 1H), 7.46 (d, J = 8.68 Hz, 2H), 7.38 (d, J = 8.64 Hz,
2H), 6.55 (brs, 1H), 6.32 (d, J = 16.0 Hz, 1H), 5.13 (seq, J =
6.25 Hz, 1H), 1.52 (s, 9H), 1.30 (d, J = 6.24 Hz, 6H); 13C NMR
(100 MHz, CDCl3): δ 166.77, 152.41, 143.81, 140.30, 129.17,
129.00, 118.33, 117.03, 80.95, 67.63, 28.29, 21.95. mp
159–159.5 °C. IR ν (diamond, cm−1): 3307, 1725, 1688, 1586,
1521, 1150, 1106, 981, 830. MS (ESI) m/z found 306.2
(M + H)+.
1
2.48 (m, 1H), 2.16–2.09 (m, 2H), 2.06–2.02 (m, 2H), 1.94–1.83
(m, 4H). 19F NMR (400 MHz, CDCl3): δ −94.45–95.09 (d, 1F),
−99.19–99.80 (d, 1F). mp 98.5–99.5 °C (lit.,48 105.9 °C).
3-(3-Isopropyl-5-methyl-[1,2,4]triazol-4-yl)-exo-8-aza-bicyclo
[3.2.1]octane (10). The title compound and its precursors were
synthesized following the same procedure by Haycock-
Lewandowski et al.41a The crude product (free base) was crystal-
lized from hexane to give 1.2 g 10, in 97% yield. 1H NMR
(400 MHz, CDCl3): δ 4.33 (seq, J = 6.01 Hz, 1H), 3.74 (m, 2H),
3.02 (seq, J = 6.86 Hz, 1H), 2.53 (s, 3H), 2.18 (dt, J1 = 12.6 Hz,
J2 = 2.66 Hz, 2H), 1.94 (m, 2H), 1.76 (m, 4H), 1.39 (d, J = 6.88
Hz, 6H). mp 190–191 °C.
3-[Benzyl-(1-phenyl-ethyl)-amino-3-(4-tert-butoxycarbonylamino-
phenyl)-propionic acid isopropyl ester (15). In an ice-water bath,
under N2 protection, n-butyllithium (3.47 mL, 2.5 M in hexane)
was added dropwise to a solution of R-(+)-N-benzyl-α-methyl-
benzylamine (1.88 mL, 9.02 mmol) in dry THF. The resulting
purple solution was stirred for 30 min, then cooled down to
−78 °C, then the solution of 14 (1.06 g, 3.47 mmol) in dry THF
was added dropwise. Then the dark red solution was stirred for
2 h at −78 °C. Saturated ammonium chloride aqueous was
added to quench the reaction. The resultant yellow solution was
allowed to warm to ambient temperature within 30 min. After
worked up with ethyl acetate, the organic layer was dried, con-
centrated. The residue was crystallized with ethyl acetate to give
20 mg 15 as colorless crystal. The filtrate was then concentrated
and dried on vacuum. Purification of the crude compound
by silica gel column chromatography gave mixtures of the
excess R-(+)-N-benzyl-α-methylbenzylamine and the product.
Hence, the following procedure was performed to transfer the
excess amine to the amide to facilitate the purification process.
Benzyl chloride (644 μL, 5.55 mmol) was added dropwise into a
mixture of the above filtrate and triethylamine (1.543 mL,
11.1 mmol) in 30 mL CH2Cl2 at 0 °C. After stirred for 2 h,
the reaction mixture was washed with brine. The organic layer,
containing the product 15 and N-benzyl-N-(1-phenyl-ethyl)-
benzamide, was dried, concentrated and used for next step
without further purification. 1H NMR (400 MHz, CDCl3): δ 7.40
(d, J = 7.2 Hz, 2H), 7.35–7.15 (m, 12H), 6.43 (brs, 1H), 4.79
(seq, J = 6.25 Hz, 1H), 4.38 (dd, J1 = 5.2 Hz, J2 = 9.66 Hz, 1H),
3.97 (q, J = 6.75 Hz, 1H), 3.66 (s, 2H), 2.56 (dd, J1 = 5.12 Hz,
J2 = 14.68 Hz, 1H), 2.50 (dd, J1 = 14.58 Hz, J2 = 9.82 Hz, 1H),
1.51 (s, 9H), 1.25 (d, J = 6.76 Hz, 3H), 1.05 (d, J = 6.24 Hz,
3H), 1.00 (d, J = 6.20 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ
171.36, 152.77, 144.15, 141.63, 137.37, 136.34, 128.75, 128.13
(×2), 128.00, 127.84, 126.83, 126.55, 118.16, 80.47, 67.51,
59.06, 57.05, 50.82, 37.71, 28.37, 21.62, 16.46. mp
172–174 °C. IR ν (diamond, cm−1): 1724, 1595, 1522, 1154,
1105, 1051, 697. MS (ESI) m/z found 517.5 (M + H)+.
3-(4-Bromo-phenyl)-acrylic acid isopropyl ester (12). To the sol-
ution of 4-bromocinnacid (1.135 g, 5 mmol) in isopropanol
(50 mL) was added several drops of concentrated sulfuric acid.
The mixture was heated to reflux for 48 h. After cooled down,
the residue was worked up with ethyl acetate. The ethyl acetate
layer was washed with sat. NaHCO3 aqueous solution, dried
over Na2SO4. After filtration and concentration, the resulting
crude product was purified by silica column using hexane and
ethyl acetate (from 100 : 1 to 75 : 1 then 50 : 1) as eluent to give
1
1.08 g white solid, in 80% yield. H NMR (400 MHz, CDCl3):
δ 7.59 (d, J = 16.0 Hz, 1H), 7.51 (d, J = 8.48 Hz, 2H), 7.38
(d, J = 8.44 Hz, 2H), 6.40 (d, J = 16.0 Hz, 1H), 5.14 (seq, J =
6.26 Hz, 1H), 1.31 (d, J = 6.24 Hz, 6H); 13C NMR (100 MHz,
CDCl3): δ 166.38, 143.04, 133.63, 132.26, 129.53, 124.51,
119.70, 68.14, 22.07. mp 64–66 °C. IR ν (diamond, cm−1):
1703, 1637, 1304, 1172, 1104, 981, 817. MS (ESI) m/z found
268.9 (M + H)+, 271.1 (M + 2 + H)+.
3-(4-Amino-phenyl)-acrylic acid isopropyl ester (13). The
mixture of 12 (1.53 g, 5.68 mmol), Pd2(dba)3 (260 mg, 5%
mmol), P(t-Bu)3 (0.23 mL, 1 M in toluene, 4% mmol) in dry
toluene (30 mL) was stirred under N2 protection for 15 min.
Then a solution of LHMDS in toluene (6.2 mL, 1 M in toluene,
6.2 mmol) was added dropwise. After stirred at ambient temp-
erature overnight, the resultant dark color suspension was added
1 N hydrochloric acid (8 mL) slowly. The resulting mixture was
stirred at ambient temperature for 2 h. Then the suspension was
filtered through celite and the filtrate was diluted with dichloro-
methane (70 mL). The organic layer was washed with saturated
NaHCO3 aqueous solution, brine, and dried over Na2SO4. After
filtered and concentrated, the crude product was purified by
silica gel column using hexane and ethyl acetate (2 : 1) as eluent
to give 1.03 g light yellow solid, in 90% yield. 1H NMR
(400 MHz, CDCl3): δ 7.58 (d, J = 15.9 Hz, 1H), 7.35 (d, J =
8.48 Hz, 2H), 6.65 (d, J = 8.56 Hz, 2H), 6.22 (d, J = 15.9 Hz,
1H), 5.12 (seq, J = 6.24 Hz, 1H), 3.91 (brs, 2H), 1.30 (d, J =
6.24 Hz, 6H); 13C NMR (100 MHz, CDCl3): δ 167.20, 148.56,
144.55, 129.80, 124.88, 114.84, 114.35, 67.32, 22.00. mp
78–80 °C. IR ν (diamond, cm−1): 3417, 3335, 1673, 1592,
1513, 1264, 1168, 1104, 980, 823. MS (ESI) m/z found 206.2
(M + H)+.
3-[Benzyl-(1-phenyl-ethyl)-amino-3-(4-tert-butoxycarbonylamino-
phenyl)-propionic acid (16). Lithium hydroxide (831 mg,
34.7 mmol) was added to the above mixture of crude product
15 and N-benzyl-N-(1-phenyl-ethyl)-benzamide in methanol
(30 mL) and water (15 mL). The resulting suspension was
heated to reflux for 48 h. After it had cooled down, the mixture
was concentrated to remove methanol. The water layer was taken
up with CH2Cl2 (30 mL × 3). The organic layer was dried and
concentrated. The residue was purified by silica gel column
using hexane and ethyl acetate (2 : 1) as eluent to give 1.406 g
3-(4-tert-Butoxycarbonylamino-phenyl)-acrylic acid isopropyl
ester (14). The solution of 13 (1.351 g, 6.58 mmol) and Boc2O
(1.58 g, 7.24 mmol) in dry tetrahydrofuran (30 mL) was heated
to reflux overnight. After cooled down and concentrated, the
residue was crystallized from DCM/hexane to give 1.711 g white
1
white solid, in 85% yield for two steps. H NMR (400 MHz,
CDCl3): δ 7.44 (d, J = 8.52 Hz, 2H), 7.37–7.22 (m, 12H), 6.55
(brs, 1H), 4.43 (dd, J1 = 4.46 Hz, J2 = 11.32 Hz, 1H), 4.15
This journal is © The Royal Society of Chemistry 2012
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(q, J = 6.87 Hz, 1H), 3.98 (d, J = 13.68 Hz, 1H), 3.64 (d, J =
13.68 Hz, 1H), 2.92 (dd, J1 = 11.34 Hz, J2 = 16.94 Hz, 1H),
2.41 (dd, J1 = 4.48 Hz, J2 = 16.96 Hz, 1H), 1.54 (s, 9H), 1.28
(d, J = 6.88 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 174.39,
152.82, 141.25, 138.44, 137.70, 132.56, 129.22, 128.80, 128.62,
128.60, 128.19, 127.81, 127.53, 118.52, 80.86, 57.96, 57.82,
50.61, 36.29, 28.34, 15.68. mp 93–95 °C. IR ν (diamond,
cm−1): 3307, 1699, 1594, 1081, 698. MS (ESI) m/z found 475.6
(M + H)+.
(4-{1-[Benzyl-(1-phenyl-ethyl)-amino]-3-[3-(3-isopropyl-5-methyl-
[1,2,4]triazol-4-yl)-8-aza-bicyclo[3,2,1]oct-8-yl]-3-oxo-propyl}-
phenyl)-carbamic acid tert-butyl ester (17). The title compound
was prepared according to the general amide coupling procedure
by reacting acid 16 with amine 10 in DCM for 4 h. The crude
product was crystallized using DCM–hexane to give 986 mg
white solid as first crop, in 73% yield. 1H NMR (400 MHz,
CDCl3): δ 7.88 (d, J = 8.32 Hz, 1H), 7.68 (d, J = 8.28 Hz, 1H),
7.46 (d, J = 7.60 Hz, 2H), 7.41–7.35 (m, 10H), 7.33–7.12
(m, 14H), 6.91 (brs, 1H), 6.66 (brs, 1H), 4.68 (m, 1H), 4.62
(m, 1H), 4.53 (dd, J1 = 5.54 Hz, J2 = 8.10 Hz, 1H), 4.40–4.28
(m, 3H), 4.03–3.96 (m, 2H), 3.82–3.62 (m, 6H), 2.76 (m, 2H),
2.54 (m, 4H), 2.21 (s, 3H), 2.11 (s, 3H), 2.06–1.96 (m, 4H),
1.96–1.55 (m, 12H), 1.51 (s, 9H), 1.50 (s, 9H), 1.30 (m, 12H),
1.28–1.24 (m, 6H); 13C NMR (100 MHz, CDCl3): δ 167.31,
166.99, 158.87, 152.80, 150.35, 144.43, 142.17 (142.05),
137.78, 136.61, 136.32, 80.46, 61.06, 59.34, 56.53 (56.40),
53.82 (53.43), 51.11 (50.80), 50.56 (50.49), 46.67, 38.87
(38.27), 37.60 (37.46), 35.67, 28.34, 26.86 (26.61), 25.79, 21.63
(21.56), 14.59, 13.80, 12.99. mp 128–130 °C. IR ν (diamond,
cm−1): 2966, 1721, 1637, 1545, 1436, 1242, 1165, 742, 705.
MS (ESI) m/z found 691.5 (M + H)+.
give 269 mg white solid, in 85% yield. H NMR (400 MHz,
CDCl3): δ 7.34 (d, J = 8.40 Hz, 2H), 7.11 (d, J = 8.48 Hz, 2H),
6.67 (brs, 1H), 6.54 (d, J = 7.56 Hz, 1H), 5.07 (q, J = 7.01 Hz,
1H), 4.29 (m, 1H), 3.37 (m, 2H), 2.98 (seq, J = 6.48 Hz, 1H),
2.49 (s, 3H), 2.40 (t, J = 6.62 Hz, 2H), 2.26–2.13 (m, 5H),
2.06–1.94 (m, 6H), 1.93–1.63 (m, 8H), 1.51 (s, 9H), 1.38 (d, J =
6.76 Hz, 6H); 13C NMR (100 MHz, CDCl3): δ 173.24, 159.12,
152.82, 150.56, 137.75, 136.34, 127.11, 122.51 (J 13C–19F 250
Hz), 118.92, 80.63, 58.87, 58.18, 51.61, 47.78, 47.28, 42.86,
35.43, 35.29, 34.66, 32.79 (J 13C–19F 25.5 Hz), 28.32, 26.80,
26.76, 25.94 (J 13C–19F 8 Hz), 25.85, 21.64, 13.11. mp
234–235 °C. IR ν (diamond, cm−1): 3272, 1716, 1650, 1236,
1159, 1106, 963, 836. MS (ESI) m/z found 629.6 (M + H)+.
(7-Amino-heptyl)-carbamic acid benzyl ester (21). On an ice-
water bath, to the solution of 1,7-diaminoheptane (1.433 g,
11 mmol) in CH2Cl2–MeOH (125 mL : 125 mL) was added the
solution of CbzCl (1.71 g, 10 mmol) in CH2Cl2 (250 mL) drop-
wise within 12 h while keeping the temperature below 5 °C. The
mixture was allowed to stir at the same temperature for another
half of an hour before concentrated under reduced pressure to
remove most of the MeOH. Water (150 mL) was then added, and
the aqueous layer was adjusted to pH = 2 using 6 N HCl. The
layers were separated. The aqueous layer was washed with DCM
(50 mL × 3), then adjusted to pH = 12 with 10 N NaOH and
extracted with DCM (50 mL × 3). The combined organic layers
were dried over Na2SO4, concentrated and purified by flash
column using DCM–MeOH (9 : 1) to give 856 mg white solid in
1
32% yield. H NMR (400 MHz, CDCl3): δ 7.35–7.29 (m, 5H),
5.09 (s, 2H), 4.73 (brs, 1H), 3.18 (q, J = 6.64 Hz, 2H), 2.67
(t, J = 6.94 Hz, 2H), 1.55–1.44 (m, 2H), 1.44–1.40 (m, 2H),
1.32 (m, 6H). 13C NMR (100 MHz, CDCl3): δ 156.40, 136.70,
128.50, 128.09, 128.06, 66.55, 42.18, 41.06, 33.71, 29.91,
29.09, 26.76, 26.68. mp 78–80 °C. IR ν (diamond, cm−1): 3327,
1686, 1532, 1263, 1144. MS (ESI) m/z found 264.8 (M + H)+.
[(7-Benzyloxycarbonylamino-heptylcarbamoyl)-methoxy]-acetic
acid (22). To the solution of 21 (350 mg, 1.324 mmol) in THF
(4 mL) was added diglycolic anhydride (161 mg, 1.39 mmol) in
one portion. The resultant mixture was stirred at ambient temp-
erature for 12 h. After removed THF under reduced pressure, the
residue was crystallized by EtOAc–hexane to give 429 mg white
(4-{1-Amino-3-[3-(3-isopropyl-5-methyl-[1,2,4]triazol-4-yl)-8-
aza-bicyclo[3,2,1]oct-8-yl]-3-oxyl}-phenyl)-carbamic acid tert-butyl
ester (19). A solution of 17 (500 mg, 0.725 mmol) in methanol
(35 mL) was treated with palladium carbon (100 mg, 10 wt%),
and the resultant slurry was shaken under an atmosphere of
hydrogen at 60 psi for 4 days at ambient temperature. The reac-
tion mixture was filtered through celite. The filtrate cake was
washed with methanol and the combined filtrates were concen-
trated and purified by silica gel using DCM–MeOH (20 : 1) as
1
1
eluent to give 304 mg white solid 19, in 84% yield. H NMR
solid as first crop, in 85% yield. H NMR (400 MHz, DMSO-
(400 MHz, CDCl3): δ 7.34 (d, J = 5.88 Hz, 8H), 6.46 (brs, 2H),
4.88 (m, 2H), 4.57–4.47 (m, 4H), 4.34–4.23 (m, 2H), 2.93 (seq,
J = 7.03 Hz, 2H), 2.73–2.50 (m, 4H), 2.46 (s, 3H), 2.36 (s, 3H),
2.33–1.95 (m, 8H), 1.85–1.71 (m, 8H), 1.52 (s, 18H), 1.40–1.37
(m, 12H); 13C NMR (100 MHz, CDCl3): δ 167.71 (167.41),
158.99 (158.93), 152.86, 150.51 (150.38), 139.43, 137.83
(137.73), 126.95 (126.86), 118.94 (118.88), 80.56, 53.89
(53.71), 52.36 (52.07), 50.85 (50.80), 46.85 (46.76), 43.77
(43.58), 37.67, 35.95 (35.88), 28.64 (28.56), 28.36, 26.97
(26.92), 25.92, 21.73, 21.66 (21.63), 13.13 (13.08). mp
121–122.5 °C. IR ν (diamond, cm−1): 3273, 1713, 1609, 1521,
1413, 1158, 1028, 837. MS (ESI) m/z found 497.3 (M + H)+.
(4-{1-[(4,4-Difluoro-cyclohexancarbonyl)-amino-3-[3-(3-isopropyl-
5-methyl-[1,2,4]triazol-4-yl)-8-aza-bicyclo[3,2,1]oct-8-yl]-propyl}-
phenyl)-carbamic acid tert-butyl ester (20). The title compound
was prepared according to the general amide coupling procedure
by reacting acid 8 with amine 7 in DCM for 4 h. The crude
product was purified by silica gel using DCM–MeOH (18 : 1) to
d6): δ 12.79 (brs, 1H), 7.81 (t, J = 5.52 Hz, 1H), 7.34–7.29
(m, 5H), 7.21 (t, J = 5.46 Hz, 1H), 5.00 (s, 2H), 4.10 (s, 2H),
3.94 (s, 2H), 3.08 (q, J = 6.56 Hz, 2H), 2.97 (q, J = 6.28 Hz,
2H), 1.42–1.37 (m, 4H), 1.24 (m, 6H). 13C NMR (100 MHz,
CDCl3): δ 171.43, 168.53, 156.06, 137.30, 128.27, 127.65,
70.18, 67.88, 65.04, 40.22, 38.08, 29.65, 29.00, 28.38, 26.28,
26.14. mp 74–74.5 °C. IR ν (diamond, cm−1): 3374, 3331,
1726, 1688, 1608, 1548, 1249, 1236, 1135, 956, 701. MS (ESI)
m/z found 381.4 (M + H)+.
6′β-(3,13-Dioxo-1-phenyl-2,15-dioxa-4,12-diazaheptadecanamido)
morphinan (23). The title compound was prepared according to
the general amide coupling procedure by reacting acid 22 with
amine 4·2HCl in DMF overnight. The crude product was
purified with chromatography using CH2Cl2–MeOH (40 : 1) as
eluent to give 339 mg white solid, in 76% yield. 1H NMR
(400 MHz, DMSO-d6): δ 9.01 (brs, 1H), 8.21 (d, J = 8.36 Hz,
1H), 8.01 (t, J = 5.60 Hz, 1H), 7.37–7.30 (m, 5H), 7.19 (m, 1H),
6.58 (d, J = 8.04 Hz, 1H), 6.52 (d, J = 8.04 Hz, 1H), 4.99
2642 | Org. Biomol. Chem., 2012, 10, 2633–2646
This journal is © The Royal Society of Chemistry 2012

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(s, 2H), 4.88 (brs, 1H), 4.59 (d, J = 7.64 Hz, 1H), 3.94 (s, 2H),
3.93 (s, 2H), 3.53–3.45 (m, 1H), 3.18–3.07 (m, 2H), 3.01–2.94
(m, 4H), 2.60–2.56 (m, 2H), 2.38–2.28 (m, 2H), 2.15 (dt, J1 =
4.89 Hz, J2 = 12.29 Hz, 1H), 1.98 (m, 1H), 1.79 (m, 1H),
1.46–1.37 (m, 6H), 1.26 (m, 8H), 0.84 (m, 1H), 0.46 (m, 2H),
(m, 2H), 2.65 (m, 1H), 2.33–2.28 (m, 2H), 1.87–1.78 (m, 1H),
1.59 (m, 1H), 1.49–1.40 (m, 6H), 1.27 (m, 8H), 0.98 (m, 1H),
0.58–0.52 (m, 2H), 0.30 (m, 2H); 1H NMR (400 MHz,
CD3OD): δ 6.72 (d, J = 8.20 Hz, 1H), 6.70 (d, J = 8.40 Hz, 1H),
4.65 (d, J = 7.80 Hz, 1H), 4.07 (s, 2H), 4.06 (s, 2H), 3.98
(s, 2H), 3.93 (s, 2H), 3.77–3.71 (m, 2H), 3.31–3.22 (m, 4H),
3.14–2.99 (m, 3H), 2.94–2.89 (m, 1H), 2.78 (dd, J1 = 7.34 Hz,
J2 = 13.30 Hz, 1H), 2.59–2.46 (m, 2H), 2.03–1.92 (m, 1H),
1.73–1.61 (m, 2H), 1.59–1.50 (m, 6H), 1.37 (m, 6H), 1.06
(m, 1H), 0.77–0.63 (m, 2H), 0.42 (m, 2H); 13C NMR
(100 MHz, CD3OD): δ 177.25, 172.58, 171.61, 171.56, 143.81,
142.81, 131.26, 128.73, 120.70, 119.42, 92.23, 72.25, 71.67,
71.58, 71.47, 71.39, 64.21, 59.23, 52.37, 49.30, 47.97, 40.10,
39.96, 31.20, 30.26, 30.21, 29.95, 29.76, 27.81, 27.79, 24.91,
24.24, 7.78, 5.67, 3.68. mp 193 °C dec. IR ν (diamond, cm−1):
3271, 3069, 1732, 1651, 1548, 1125, 1033. MS (ESI) m/z found
687.4 (M + H)+.
1
0.12 (m, 2H); H NMR (400 MHz, CDCl3): δ 7.57 (d, J = 9.24
Hz, 1H), 7.35–7.32 (m, 5H), 6.90 (t, J = 5.66 Hz, 1H), 6.72
(d, J = 8.12 Hz, 1H), 6.55(d, J = 8.16 Hz, 1H), 5.09 (s, 2H),
4.88 (m, 1H), 4.44 (d, J = 5.48 Hz, 1H), 4.02 (m, 4H), 3.24
(m, 2H), 3.18 (AB, J = 6.64 Hz, 2H), 3.10 (d, J = 5.84 Hz, 1H),
3.03 (d, J = 18.48 Hz, 1H), 2.62 (m, 2H), 2.36 (m, 2H), 2.19
(m, 2H), 1.79 (m, 1H), 1.64 (m, 1H), 1.54–1.48 (m, 7H),
1.32–1.26 (m, 7H), 0.80 (m, 1H), 0.53 (m, 2H), 0.13 (m, 2H);
13C NMR (100 MHz, CDCl3): δ 168.63, 168.45, 156.51,
143.19, 139.38, 136.64, 130.56, 128.53 (×2), 128.10, 124.66,
119.23, 117.77, 92.27, 77.23, 70.89, 70.13, 66.63, 62.38, 59.40,
49.41, 47.20, 43.88, 41.02, 39.04, 31.87, 29.85, 29.50, 28.92,
28.81, 26.78, 26.55, 23.17, 22.61, 9.41, 4.00, 3.81. mp >300 °C.
IR ν (diamond, cm−1): 3670, 1700, 1560, 1136. MS (ESI) m/z
found 705.5 (M + H)+.
Methylcarbamoylmethoxy-acetic acid (26). The title compound
was prepared using the same procedure as that described by
Zheng et al.,30b except that white solid instead of oil was
1
6′β-{2-[2-(7-aminoheptylamino)-2-oxoethoxy]acetamido}morphinan
(24). A solution of 23 (120 mg, 0.167 mmol) in methanol
(20 mL) was hydrogenated in the presence of 10% Pd–C
(12 mg) under a H2 atmosphere (60 psi) at room temperature for
48 h. The mixture was filtered, and the filtrate was concentrated
and purified by silica gel with DCM–MeOH (7 : 1) to give 24 as
obtained. H NMR (400 MHz, DMSO-d6): δ 12.77 (brs, 1H),
7.77 (brs, 1H), 4.10 (s, 2H), 3.94 (s, 2H), 2.62 (d, J = 4.8 Hz,
3H). 13C NMR (100 MHz, DMSO-d6): δ 171.32, 169.08, 70.09,
67.77, 25.11. mp 33–33.5 °C.
(7-{2-[(4-{1-[(4,4-Difluoro-cyclohexanecarbonyl)-amino]-3-[3-(3-
isopropyl-5-methyl-[1,2,4]triazol-4-yl)-8-aza-bicyclo[3.2.1]oct-8yl]-
propyl}-phenylcarbamoyl)-methoxy]-acetylamino}-heptyl)-carbamic
acid benzyl ester (27). The title compound was prepared accord-
ing to the general amide coupling procedure by reacting acid 22
with amine 6 in DMF overnight. The crude product was purified
with chromatography using CH2Cl2–MeOH (13 : 1) as eluent to
1
white foam (110 mg, 99% yield). H NMR (400 MHz, DMSO-
d6): δ 8.22 (d, J = 8.40 Hz, 1H), 8.03 (t, J = 5.74 Hz, 1H), 6.58
(d, J = 8.04 Hz, 1H), 6.52 (d, J = 8.12 Hz, 1H), 4.59 (d, J =
7.36 Hz, 1H), 3.94 (s, 2H), 3.93 (s, 2H), 3.56–3.48 (m, 1H),
3.19–3.09 (m, 2H), 3.10 (d, J = 5.52 Hz, 1H), 2.97 (d, J = 18.8
Hz, 1H), 2.70 (m, 2H), 2.61–2.55 (m, 2H), 2.39–2.28 (m, 2H),
2.18–2.10 (m, 1H), 1.98 (dt, J1 = 3.56 Hz, J2 = 11.92 Hz, 1H),
1.84–1.74 (m, 1H), 1.47–1.44 (m, 6H), 1.32–1.23 (m, 8H), 0.86
(m, 1H), 0.47 (m, 2H), 0.11 (m, 2H); 1H NMR (400 MHz,
CD3OD): δ 6.62 (d, J = 8.08 Hz, 1H), 6.55 (d, J = 8.12 Hz, 1H),
4.51 (d, J = 7.56 Hz, 1H), 4.06 (m, 2H), 4.05 (m, 2H), 3.76 (m,
1H), 3.26 (t, J = 7.08 Hz, 2H), 3.10 (d, J = 5.96 Hz, 1H), 3.07
(d, J = 20.64 Hz, 1H), 2.66 (m, 2H), 2.58 (m, 2H), 2.40 (m,
2H), 2.27–2.11 (m, 2H), 1.90 (m, 1H), 1.60–1.51 (m, 6H),
1.49–1.32 (m, 8H), 0.85 (m, 1H), 0.54 (m, 2H), 0.16 (m, 2H);
13C NMR (100 MHz, CD3OD): δ 171.54, 171.38, 143.90,
142.52, 132.41, 125.06, 120.09, 118.88, 92.80, 71.74, 71.60,
71.55, 63.77, 60.32, 52.54, 48.93, 47.95, 45.28, 40.07, 32.05,
31.27, 30.43, 30.28, 28.38, 27.92, 25.51, 23.55, 22.12, 10.36,
4.49, 4.27. mp 83–85 °C. IR ν (diamond, cm−1): 3278, 3075,
1652, 1548, 1128, 1035. MS (ESI) m/z found 571.6 (M + H)+.
19-(6′β-morphinanamino)-5,15-19-trixox-3,17-dioxa-6,14-diaza-
nonadecan-1-oic acid (25). Diglycolic anhydride (23 mg,
0.198 mmol) was added to the solution of 24 (113 mg,
0.198 mmol) in DMF (2 mL) within 15 min. The resultant
mixture was stirred at ambient temperature for 2 h. After removal
of DMF under reduced pressure, the residue was crystallized by
EtOAc/hexane to give 112 mg light yellow solid as first crop, in
1
give 260 mg white foam, in 72% yield. H NMR (400 MHz,
CDCl3): δ 8.96 (s, 1H), 7.57 (d, J = 8.48 Hz, 2H), 7.35–7.27
(m, 5H), 7.24 (d, J = 8.48 Hz, 2H), 6.75 (m, 1H), 6.70 (m, 1H),
5.12–5.08 (m, 3H), 4.91 (m, 1H), 4.29 (m, 1H), 4.14 (s, 2H),
4.09 (s, 2H), 3.40 (m, 1H), 3.37 (m, 1H), 3.30 (q, J = 6.68 Hz,
2H), 3.15 (q, J = 6.71 Hz, 2H), 2.99 (m, 1H), 2.48 (s, 3H), 2.43
(t, J = 6.48 Hz, 2H), 2.30–2.04 (m, 6H), 2.01–1.71 (m, 8H),
1.70–1.46 (m, 9H), 1.376 (d, J = 6.80 Hz, 3H), 1.373 (d, J =
6.84 Hz, 3H), 1.35–1.31 (m, 6H); 13C NMR (100 MHz, CDCl3):
δ 173.50, 168.64, 167.15, 159.17, 156.55, 150.59, 138.31,
136.66, 136.51, 128.54, 128.12, 128.00, 127.12, 120.51, 77.24,
71.66, 71.50, 66.60, 58.90, 58.30, 51.68, 47.77, 47.24, 42.83,
40.98, 39.14, 35.33, 34.59, 32.82 (J 13C–19F 25.7 Hz), 29.82,
29.36, 28.71, 26.75 (J 13C–19F 10 Hz), 26.48, 25.87, 21.65,
13.15. mp 65–67 °C. IR ν (diamond, cm−1): 3273, 1656, 1529,
1515, 1251, 1106, 697. MS (ESI) m/z found 891.9 (M + H)+.
4,4-Difluoro-cyclohexanecarboxylic acid {1-(4-{2-[(7-amino-
heptylcarbamoyl)-meth-oxy]-acetylamino}-phenyl)-3-[3-(3-isopropyl-
5-methyl-[1,2,4]triazol-4-yl)-8-aza-bicyclo[3.2.1]oct-8-yl]-propyl}-
amide (28). A solution of 27 (260 mg, 0.291 mmol) in methanol
(15 mL) was hydrogenated in the presence of 5% Pd–C (26 mg)
under a H2 atmosphere (60 psi) at room temperature for 48 h.
The mixture was filtered, and the filtrate was concentrated to
give 28 as white foaming (113 mg, 51% yield). 1H NMR
(400 MHz, CDCl3): δ 8.80 (s, 1H), 7.58 (d, J = 8.48 Hz, 2H),
7.25 (d, J = 8.48 Hz, 2H), 6.61 (d, J = 7.48 Hz, 1H), 6.47
(m, 1H), 5.10 (q, J = 7.00 Hz, 1H), 4.30 (m, 1H), 4.17 (s, 2H),
4.12 (s, 2H), 3.40–3.30 (m, 4H), 2.98 (seq, J = 6.90 Hz, 1H),
1
82% yield. H NMR (400 MHz, DMSO-d6): δ 9.22 (brs, 1H),
8.28 (d, J = 8.32 Hz, 1H), 8.06 (t, J = 5.62 Hz, 1H), 7.99
(m, 1H), 6.65 (d, J = 8.12 Hz, 1H), 6.59 (d, J = 8.12 Hz, 1H),
4.68 (d, J = 7.80 Hz, 1H), 4.06 (s, 2H), 3.95 (s, 2H), 3.94
(m, 4H), 3.56–3.47 (m, 1H), 3.24–3.06 (m, 6H), 2.89–2.83
This journal is © The Royal Society of Chemistry 2012
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2.68 (t, J = 6.84 Hz, 2H), 2.48 (s, 3H), 2.43 (t, J = 6.62 Hz, 2H),
2.26–2.12 (m, 5H), 2.08–1.53 (m, 16H), 1.44 (qu, J = 6.72 Hz,
2H), 1.383 (d, J = 6.76 Hz, 3H), 1.382 (d, J = 6.80 Hz, 3H);
1.34–1.30 (m, 6H); 1H NMR (400 MHz, CD3OD): δ 7.60
(d, J = 8.52 Hz, 2H), 7.33 (d, J = 8.56 Hz, 2H), 5.03 (t, J = 7.34
Hz, 1H), 4.39 (m, 1H), 4.20 (s, 2H), 4.12 (s, 2H), 3.41 (m, 2H),
3.30–3.21 (m, 3H), 2.69 (t, J = 7.32 Hz, 2H), 2.50 (s, 3H),
2.46–2.41 (m, 2H), 2.40–2.30 (m, 1H), 2.30–2.05 (m, 2H),
2.05–2.03 (m, 4H), 2.02–1.96 (m, 2H), 1.89–1.65 (m, 10H),
1.60–1.45 (m, 4H), 1.36–1.34 (m, 12H); 13C NMR (100 MHz,
CDCl3): δ 176.59, 171.68, 170.07, 161.41, 152.60, 140.39,
138.07, 128.21, 123.91 (J 13C–19F 239 Hz), 121.88, 71.95,
71.75, 60.69, 60.19, 52.41, 43.69, 42.03, 40.07, 36.77, 36.16,
33.90 (J 13C–19F 24 Hz), 33.87 (J 13C–19F 24 Hz), 32.28, 30.38,
30.11, 27.86, 27.78, 27.20(J 13C–19F 9 Hz), 27.05(J 13C–19F
9 Hz), 26.76, 22.07, 12.45. mp 100–102 °C. IR ν (diamond,
cm−1): 3256, 1651, 1538, 1515, 1261, 1103, 743. MS (ESI) m/z
found 757.9 (M + H)+.
filtration as described above. Bound radioactivity was de-
termined by liquid scintillation spectrophotometry at 95%
efficiency.
Data analysis. For competition binding assay (agonists or
antagonists), Hill plots linear regression analysis and the Cheng–
Prusoff equation were applied to determine the IC50 and Ki
values. In [35S]GTPγS binding assays, agonist concentration
effect curves were fit by non-linear regression to obtain Emax and
EC50 values; antagonist inhibition of agonist-stimulated [35S]
GTPγS binding was analyzed by Hill analysis and AD50 values
were corrected to Ki values using the Cheng-Prusoff equation.
All analyses were using Prism 4.0.
Acknowledgements
We thank the funding support from National Institute on Drug
Abuse DA024022. The content is solely the responsibility of the
authors and does not necessarily represent the official views of
the National Institute on Drug Abuse, or the National Institutes
of Health.
Calcium mobilization assay. The ligands were first tested with
various doses (in the range of 0.1 nM to 1 μM) for possible
agonist activity. The protocol was the same for the following
antagonism study, except no CCL5 (RANTES) was added.
MOLT-4–CCR5 cells were plated in black 96-well plates with
transparent bottom (Greinier Bio-one) at 100,000 cells per well
in 50 : 1 HBSS : HEPES assay buffer. They were incubated for
1 h at 37 °C and 5% CO2 with control buffer or varying concen-
tration of ligand for a total volume of 130 μL per well. Cells
were then incubated with 50 μL of Fluo-4-AM loading buffer
(40 μL 2 μM Fluo-4 dye, 100 μL 2.5 mM probenacid, in 5 mL
assay buffer) for an additional hour. Then 20 μL 200 nM
RANTES solution in assay buffer or assay buffer alone were
added to the wells right before changes in Ca2+ concentration
were monitored by RFU for 90 seconds using a microplate
reader (FlexStation3, Molecular Devices). Peak values were
obtained using SoftMaxPro software (Molecular Devices)
and non-linear regression curves were generated using Prism
(GraphPad) to calculate IC50 values.
Notes and references
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The mu opioid receptor binding and functional assay. MOR–
CHO cell culture and membrane homogenate preparation fol-
lowed the literature report.52
Opioid Receptor Binding. Saturation binding was performed by
incubating membranes for 90 min at 30 °C with 0.5–15 nM [3H]
naloxone in assay buffer in a 0.5 mL volume. Non-specific
binding was determined with 5 μM naltrexone. For competition
assays, membranes were incubated as above with 2 nM [3H]
naloxone and various concentrations of unlabeled ligand, to
determine competitor IC50 for MOR. The reaction was termi-
nated by rapid filtration through Whatman GF–B glass fiber
filters, followed by 3 washes with 3 mL ice-cold Tris buffer.
Bound radioactivity will be determined by liquid scintillation
spectrophotometry at 45% efficiency for [3H].
[35S]GTPγS Binding. Membranes (10 μg protein) were incu-
bated in assay buffer at 30 °C for 90 min with various drugs,
10 μM GDP (cells) and 0.1 nM [35S]GTPγS in 0.5 mL total
volume for appropriate times. Basal binding was assessed in the
absence of agonist, and nonspecific binding was measured with
10 μM unlabeled GTPγS. The reaction was terminated by rapid
2644 | Org. Biomol. Chem., 2012, 10, 2633–2646
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