Synthesis and biological evaluation of bivalent ligands for the cannabinoid 1 receptor

Article abstract of DOI:10.1021/jm1006676

Dimerization or oligomerization of many G-protein-coupled receptors (GPCRs), including the cannabinoid 1 (CB1) receptor, is now widely accepted and may have significant implications for medications development targeting these receptor complexes. A library of bivalent ligands composed of two identical CB1 antagonist pharmacophores derived from SR141716 linked by spacers of various lengths were developed. The affinities of these bivalent ligands at CB1 and CB2 receptors were determined using radiolabeled binding assays. Their functional activities were measured using GTP-γ-S accumulation and intracellular calcium mobilization assays. The results suggest that the nature of the linker and its length are crucial factors for optimum interactions of these ligands at CB1 receptor binding sites. Finally, selected bivalent ligands (5d and 7b) were able to attenuate the antinociceptive effects of the cannabinoid agonist CP55,940 (21) in a rodent tail-flick assay. These novel compounds may serve as probes that will enable further characterization of CB1 receptor dimerization and oligomerization and its functional significance and may prove useful in the development of new therapeutic approaches to G-protein-coupled receptor mediated disorders.

Full text of DOI:10.1021/jm1006676

7048 J. Med. Chem. 2010, 53, 7048–7060
DOI: 10.1021/jm1006676
Synthesis and Biological Evaluation of Bivalent Ligands for the Cannabinoid 1 Receptor
Yanan Zhang,*,† Anne Gilliam,† Rangan Maitra,† M. Imad Damaj,‡ Julianne M. Tajuba,† Herbert H. Seltzman,† and
Brian F. Thomas*,†
†Research Triangle Institute, Research Triangle Park, North Carolina 27709, and ‡Department of Pharmacology and Toxicology,
Virginia Commonwealth University, Richmond, Virginia 23298
Received June 2, 2010
Dimerization or oligomerization of many G-protein-coupled receptors (GPCRs), including the cannabinoid
1 (CB1) receptor, is now widely accepted and may have significant implications for medications development
targeting these receptor complexes. A library of bivalent ligands composed of two identical CB1 antagonist
pharmacophores derived from SR141716 linked by spacers of various lengths were developed. The affinities
of these bivalent ligands at CB1 and CB2 receptors were determined using radiolabeled binding assays. Their
functional activities were measured using GTP-γ-S accumulation and intracellular calcium mobilization
assays. The results suggest that the nature of the linker and its length are crucial factors for optimum
interactions of these ligands at CB1 receptor binding sites. Finally, selected bivalent ligands (5d and 7b) were
able to attenuate the antinociceptive effects of the cannabinoid agonist CP55,940 (21) in a rodent tail-flick
assay. These novel compounds may serve as probes that will enable further characterization of CB1 receptor
dimerization and oligomerization and its functional significance and may prove useful in the development of
new therapeutic approaches to G-protein-coupled receptor mediated disorders.
Introduction
activation, signal transduction, and internalization.8-12 For
example, it has been proposed that a μ-δ opioid receptor
heterodimer is the fundamental signaling unit that mediates
opioid tolerance and dependence through specific signal
transducer(s) that recognize and couple to the heterodimer
but not to μ-receptor monomers/homomers.13 In an ana-
logous fashion, modulation of the CB1 receptor dimers or
oligomers may offer novel opportunities to uniquely target
and manipulate function of the endocannabinoid system.
The importance of GPCR dimerization and oligomeriza-
tion in vivo remains to be elucidated and exploited, largely
because of a lack of selective pharmacological tools and
immunological reagents. Among various efforts to modulate
GPCR oligomers, bivalent ligands, which are defined as two
pharmacophores linked by spacers, represent a unique and
promising approach and may provide such a tool.14,15 Bivalent
ligands, provided they have suitable functional affinity at the
monomeric receptor, are expected to selectively bind with
greatly enhanced affinity to ligand recognition sites on hetero-
dimers and oligomers because of the small containment
volume for the second pharmacophore after the binding of
the first one and the formation of thermodynamically more
stable complexes. At the same time, bivalent ligands may
display unique properties, since they interact with more than
one receptor simultaneously. Indeed, bivalent ligands have
been developed for variety of G-protein-coupled receptor
targets, including opioids,14,16 adrenergic,17,18 dopamine,19
serotonin,20,21 and muscarinic receptors.22,23 These bivalent
ligands have been shown to be able to selectively target homo-
or heterodimers and display unique pharmacological proper-
ties compared to their monomeric subunits. However, to the
best of our knowledge, there are no bivalent ligands developed
for the CB1 receptor to date.
The endocannabinoid system (ECS) comprises the cannabi-
noid 1 (CB1a) and cannabinoid 2 (CB2) receptors, their endo-
genous ligands (endocannabinoids), and the proteins involved
in endocannabinoid synthesis and inactivation, as well as the
intracellular signaling pathways affected by endocannabinoids.1
Increasing evidence suggests that the endocannabinoidsystem
is critically involved in a variety of physiological and patho-
logical conditions. More importantly, modulation of the endo-
cannabinoid system may hold therapeutic promise to treat a
wide range of disparate diseases such as pain, inflammatory
diseases, peripheral vascular disease, appetite enhancement or
suppression, and locomotor disorders.2 The CB1 receptor,
which belongs to the G-protein-coupled receptor (GPCR)
superfamily, the largest class of cell surface receptors, is
believed to mediate most of the actions exerted by exogenous
cannabinoids or endocannabinoid in the brain.3
While GPCRs were traditionally considered monomeric,
it is now well accepted that many GPCRs, including the CB1
receptor,4,5 exist on the cell membrane as homo- and hetero-
dimers or higher-order oligomers.6 Moreover, receptor oligo-
merization is often essential for receptor function (e.g., the
GABAB receptor)7 and can also modulate ligand interaction,
*To whom correspondence should be addressed. For Y.Z.: phone,
919-541-1235; fax, 919-541-6499; e-mail, yzhang@rti.org. For B.F.T.:
phone, 919-541-6552; fax, 919-541-6499; e-mail, bft@rti.org.
aAbbreviations: GPCR, G-protein-coupled receptor; CB1, cannabi-
noid 1 receptor; CB2, cannabinoid 2 receptor; SAR, structure-activity
relationship; CNS, central nervous system; BOP, benzotriazole-1-yl-
oxytris(dimethylamino)phosphonium hexafluorophosphate; HOBt,
1-hydroxybenzotriazole; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide; THF, tetrahydrofuran; DMSO, dimethylsulfoxide;
DMF, dimethylformamide; CDI, carbonyldiimidazole; TLC, thin-layer
chromatography; NMR, nuclear magnetic resonance.
r
pubs.acs.org/jmc
Published on Web 09/16/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7049
Figure 1. Compound 1 and 3-substituted analogues with alkyl or polar linkers.
Here we present our efforts in the design and synthesis of
symmetrical bivalent ligands targeting CB1 receptor dimers.
The bivalent ligands contain two identical core structures of
1,5-diarylpyrazole derived from 1 (SR141716, or rimonabant,
Figure 1) joined by a variety of linkers. Compound 1 was
initially reported by Sanofi-Recherche as a highly potent and
selective CB1 receptor antagonist/inverse agonist. It was the
first drug to selectively block both the in vitro and in vivo
effects of cannabinoids that are mediated by the CB1 receptor.
Compound 1 was approved for the treatment of obesity in
Europe before its recent withdrawal from the market because of
undesirable psychological effects. This compound also shows
great promise in many potential therapeutic applications
including smoking addiction, drug and alcohol dependence,
cognitive disorders, inflammation, and arthritis.24,25 By devel-
oping bivalent ligands with 1 as the pharmacophore, we aim to
affect the binding affinities of these ligands to cannabinoid
receptor monomers/dimers and perhaps alter their efficacies or
signal transduction pathways as antagonists/inverse agonists.
We hereby describe the synthesis and preliminary pharmaco-
logical examination of a series of bivalent ligands that possess
linkers of various lengths and describe the results with respect to
the optimal linker length for affinity and their related pharmaco-
logical activity using various pharmacological approaches. For
comparative purposes, corresponding monovalent ligands
were synthesized to evaluate the contribution of the presence of
the linkers to activity.
substituents including alkyl groups and aromatic groups
(2, Figure 1).26-28 Therefore, bivalent ligands linked through
the 3-position were initially developed.
A series of bivalent ligands with 3-position linkers of
varying lengths were synthesized and evaluated in efforts
to optimize the linker length for bridging of the receptors
dimers. The optimal linker lengths, or the distances between
the binding sites on neighboring receptors in receptor dimers
or oligomers, have been reported on a number of GPCRs.
Molecular modeling studies based on the crystal structure of
rhodopsin suggested a distance between the individual re-
˚
ceptors to be ∼35 A, although the receptor dimer was in a
head-to-tail orientation.29 Similarly, molecular modeling on
the opioid receptor suggested that the distance between the
recognition sites of either the interlocking or contact dimers
˚
˚
with a TM5,6-interface is ∼27 A, while it is greater (∼32 A) in
dimers with TM4,5-interface.14 However, during their stud-
ies on opioid bivalent ligands, Portoghese and co-workers
discovered that optimal activity was obtained when spacers
are about 22 A (∼19 atoms).30 On the other hand, Neumeyer
˚
and co-workers found that bivalent ligands for the opioid
receptors having spacers containing 10 methylene units or
less displayed the highest affinities.31,32 More recently, a
series of adenosine A2A antagonist/dopamine D2 agonist
bivalent ligands were developed where linkers ranged between
26 and 66 atoms.33 Interestingly, affinities of the bivalent ligands
to both receptors stayed almost identical with the elongation of
the linkers. The authors indicated that linkers with 26 atoms
were of sufficient length to allow the bivalent ligands to bind to
receptor dimers according to receptor docking experiments and
suggested that the lack of correlation between binding affinity
and linker length might be due to the high flexibility of the
mixed peptide/polyethylene glycol linkers. On the basis of
these findings and others, linkers between 5 and 23 atoms were
initially examined in our laboratory to determine optimal linker
length.
Results
Bivalent Ligand Design. In order to focus our efforts on the
efficient development of bivalent ligands, we selected 1, a
prototypical CB1 receptor antagonist/inverse agonist. In
addition to the high affinity and potency at the CB1 receptor
in vitro and in vivo, the structure-activity relationships on
this class of compounds have been well studied and docu-
mented. This allowed for an informed selection of appro-
priate positions to attach the linkers to the molecules without
likely eliminating their affinity or decreasing their efficacy or
activity significantly. It also permitted efficient synthesis
following known procedures with minimal modifications.
In particular, SAR results on this structure class indicate that
the 3-carboxyamide position generally tolerates the re-
placement of the 1-aminopiperidyl group with a variety of
Three types of linkers have been considered in the design
of the bivalent ligands. The first class investigated was poly-
ethylene glycol linkers. The second category is composed of
small peptides (Figure 1). These two classes of linkers have
been employed in bivalent ligand development by a number
of groups.14,23,34 Not only are these linkers readily available
but they also offer the advantage of gradually increasing the

7050 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Zhang et al.
Figure 2. Bivalent and monovalent ligands with triamine linkers.
Scheme 1. Synthesis of Compounds 3 and 4a
aReagents and conditions: (a) benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1,11-diamino-3,6,9-trioxaundecane,
THF; (b) glycine methyl ester hydrochloride, HOBt, EDCI, Et3N, CH2Cl2; (c) NaOH, MeOH/H2O; (d) LiOH, THF/MeOH/H2O; (e) ethylenediamine,
HOBt, EDCI, Et3N, CH2Cl2.
linker length. However, our preliminary results with these
two types of linkers failed to show promise. Both compounds
3 and 4 had low affinity in radiolabeled binding and were
inactive in GTP-γ-S and calcium assays (data not shown).
This is consistent with literature results that suggest that
hydrophobic groups are generally preferred at this 3-carbox-
amide position of 1.26,27,35 Linkers composed of alkylamines
were also examined (Figure 2). The selection of these hydro-
phobic molecules was based on the SAR studies in our
laboratory and also by Wiley and co-workers that indicate
that substitution of the 3-carboxamide with hydrocarbons
usually retains or sometimes even improves the affinity or
antagonist activity of 1.26,27,35 Accordingly, a series of alkyl-
triamines were selected to construct the bivalent ligands
(5a-f, Figure 2). A protonatable nitrogen atom was intro-
duced in the middle of the chain in order to reduce the
incremental increases in hydrophobicity upon elongation
of the alkyl chains. This nitrogen not only provides sym-
metry of the bivalent ligands but also facilitates the con-
struction of long alkyl linkers. Additionally, the N-methyl
series of analogues (6a-d) were prepared to examine
the possible hydrogen bonding effects of the alkylamine
linker.
Chemistry. Compound 3 was obtained by coupling between
the pyrazole carboxylic acid (9), which was readily prepared
from commercially available 4-chloropropiophenone in three
steps, following the procedure developed in our laboratory,36,37
and 1,11-diamino-3,6,9-trioxaundecane using benzotriazole-
1-yloxytris(dimethylamino)phosphonium hexafluorophosphate
(BOP) as the coupling agent (Scheme 1). In the preparation

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7051
Scheme 2. Synthesis of N-H Triamine Linkers 18c-fa
aReagents and conditions: (a) NaCN, K2CO3, DMSO; (b) benzylamine, K2CO3, 1-butanol or DMF, 100 °C; (c) H2, Raney nickel, ethanol,
2 N NaOH; (d) H2, Pd/C, ethanol.
Scheme 3. Synthesis of Bivalent Ligands 5a-f and Monovalent Ligands 7a-fa
aReagents and conditions: CDI (0.5 equiv for 5a-f, 3 equiv for 7a-f), CH2Cl2.
of 4, acid 9 was coupled to glycine methyl ester hydrochloride
under standard coupling conditions that employed hydro-
xybenzotriazole (HOBt), 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide (EDCI), and triethylamine in tetrahydro-
furan38 to give the methyl ester (10) in almost quantitative
yield. Hydrolysis of 10 in methanolic sodium hydroxide at
room temperature furnished 11 in quantitative yield. Coupling
of 11 with glycine methyl ester hydrochloride under identical
conditions as that of 9, followed by mild hydrolysis (LiOH,
MeOH/THF/H2O), provided 12 in excellent yield. Finally,
reaction of 12 with excess ethylenediamine furnished 4.
The route to bivalent ligands 5a-f and monovalent ligands
7a-f required the use of the N-H triamine linkers 18a-f.
While 18a,b were commercially available, 18c-f were pre-
pared in our laboratory as shown in Scheme 2. For 18c-d,
the starting bromoalkylnitriles (15c-d) were commercially
available. Intermediates 15e (m = 8) and 15f (m = 10) in the
preparation of 18e-f needed to be synthesized from di-
bromides 13 and 14, respectively. This was readily accom-
plished by displacing one of the bromides in these dibro-
moalkanes with a cyano group using sodium cyanide in
dimethylsulfoxide (DMSO). Thereafter, bis-alkylation of
benzylamine with bromides 15c-f in the presence of potas-
sium carbonate in 1-butanol or dimethylformamide (DMF)
furnished amines 16c-f in excellent yields. Reduction of
these nitriles was readily accomplished by hydrogenation
catalyzed with Raney nickel to give 17c-f. Another hydro-
genation using palladium on carbon removed the benzyl
groups to afford triamines 18c-f, which were generally of
sufficient purity and were used in the following step without
further purification. It is worth noting that the sequence
of the hydrogenations was important and debenzylation

7052 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Zhang et al.
followed by reduction of nitriles failed to give the desired
products in satisfactory yields.
one of the primary amino groups could be readily accom-
plished with the employment of excess triamines 18a-f to
provide 7a-f.
The coupling of acid 9 and triamines 18a-f was then
attempted using several methods in order to furnish the
bivalent ligands. Initial trials on the coupling employing
the acid chloride or activation of acid 9 with agents such as
chloroformates or BOP all failed to display selectivity, and
products with acylation at all three amino sites were obtained
as the primary products. Eventually carbonyldiimidazole
(CDI) appeared to provide satisfactory selectivity, and the
desired products 5a-f, where acylation occurred at the two
primary amino sites, were obtained in reasonable yields
(Scheme 3). Under similar conditions, acylation at only
Following a procedure analogous to that of Scheme 2, the
N-methyltriamine linkers were prepared as depicted in
Scheme 4. Bis-alkylation of methylamine in methanol or
methylamine hydrochloride with bromides 15c-f in ethanol
under microwave conditions or heated in sealed pressure
tubes provided 19a-d in almost quantitative yields. Hydro-
genation catalyzed by palladium on carbon provided the
triamines (20a-d) in excellent yields. Similar to Scheme 3,
the N-methyl bivalent ligands 6a-d and the monovalent
controls 8a-d were obtained by coupling reactions between
acid 9 and amines 20a-d using CDI.
Scheme 4. Synthesis of N-Me Bivalent Ligands 6a-d and
Monovalent Ligands 8a-da
Binding Affinities of Synthesized Compounds. All the target
compounds were evaluated in competition binding assays
using both rat whole brain membrane preparations and cells
stably transfected with either the human CB1 or CB2 recep-
tors. The receptor binding affinities were determined in
competitive displacement assays using radioligands [3H]1
and [3H]CP55,940 (21). The results are summarized in Tables
1 and 2.
Most of the bivalent ligands displayed nanomolar affinity at
the CB1 receptor, albeit somewhat lower than the parent
compound 1. Similarly to 1, all bivalent ligands and mono-
valent controls also showed reasonable selectivity for the CB1
receptor over the CB2 receptor, displaying little or no affinity at
the CB2 receptor. Noticeably, all compounds exhibited higher
affinity (2- to 3-fold) for the CB1 receptor in the displacement
of [3H]1 than the structurally different [3H]21. This is in
agreement with observations previously reported by Wiley
and co-workers where derivatives of 21 were usually better
ligands in displacement of radiolabeled 21 than 1.39
aReagents and conditions: (a) methylamine in methanol or methyl-
amine hydrochloride, K2CO3, ethanol, microwave or heated in pressure
tube; (b) H2, Pd/C, ethanol; (c) CDI (0.5 equiv for 6a-d, 3 equiv for
8a-d), CH2Cl2.
Table 1. Binding Affinities of N-H Bivalent Ligands 5a-f and 7a-f against [3H]21 and [3H]1
displacement assay vs tritiated ligands: Ki (nM) in hCB1 (n = 2)
linker
(atoms)
displacement vs
3[3H]21: Ki (nM) in hCB2
compd
n
[3H]21
SEM
[3H]1
SEM
CB1/CB2
1
6.18
229
174
68.1
12.3
54.1
99.3
1225
a
1.2
1.18
94.0
41.9
30.4
4.41
57.4
37.0
506
a
0.1
313
1285
496
451
553
a
50.6
5.6
5a
5b
5c
5d
5e
5f
2
5
75.0
1.0
8.00
5.40
4.40
0.34
44.7
4.55
56.5
3
7
2.9
6.6
5
11
15
19
23
5
12.6
1.10
16.3
35.8
359
7
45.0
>46
>25
>2
9
11
2
a
7a
7b
7c
7d
7e
7f
a
3
7
a
5
11
15
19
23
349
46.7
14.0
4.56
36.5
1.85
2.10
0.83
230
19.5
5.44
2.30
4.50
1.35
0.62
0.20
a
>7
7
622
419
305
13.3
29.9
66.9
9
11
a Ki is greater than the highest standard of 2500 nM.
Table 2. Binding Affinities of N-Me Bivalent Ligands 6a-d and Monovalent Ligands 8a-d against [3H]21 and [3H]1
displacement assay vs tritiated ligands: Ki (nM) in hCB1 (n = 2)
linker
(atoms)
displacement vs
[3H]21: Ki (nM) in hCB2
compd
n
[3H]21
SEM
[3H]1
SEM
CB1/CB2
6a
6b
6c
6d
8a
8b
8c
8d
5
11
15
19
23
11
15
19
23
38.7
17.3
247
4.0
6.35
27.5
94.1
1292
88.8
15.5
6.12
5.51
1.07
1.90
25.9
524
1037
683
a
26.8
39.5
>10
>1.3
>15
51.6
26.5
29.0
7
0.45
29.0
450
9
11
5
1885
162
a
22.5
4.45
1.27
5.27
0.45
0.40
1.06
1.81
a
7
37.5
10.0
14.7
1934
265
426
9
11
a Ki is greater than the highest standard of 2500 nM.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7053
Table 3. Functional Assessment of the Alkyl N-H Series of Bivalent Ligands 5a-f and Monovalent Ligands 6a-f at the CB1 Receptore
GTP-γ-S assay in rat brain GTP-γ-S assay in hCB1 GTP-γ-S assay in hCB1 calcium assay
PA2 in hCB1 (95% confidence limits SEM
compd
linker
EC50 (nM)
Emax (%)
EC50 (nM)
Emax (%)
Ke (nM)
1
56305
b
-37.8
-35.9
-37.2
-29.6
-25.0
10.4
ND
237
84.2
33.0
179
27.9
a
8.59
7.08
7.41
7.53
8.08
7.76
7.56
c
0.08
0.52
0.27
0.28
0.24
0.60
0.35
c
1.1
0.12
411
279
69
5a
5b
5c
5d
5e
5f
5
-25.5
-31.9
8.5
2702
1304
476
567
4165
d
7
718
b
11
15
19
23
5
1193
1.34
b
-38.8
6.5
64
1142
-12.2
-22.9
-34.8
-40.7
-52.8
-65.2
-29.2
7a
7b
7c
7d
7e
7f
7243
b
ND
ND
ND
ND
ND
ND
d
7
c
c
d
11
15
19
23
2222
3279
2393
3616
6.12
7.69
8.25
8.50
0.32
0.46
0.28
0.26
5399
502.6
31.6
146.5
1105
225
3.1
19.1
a EC50 is greater than the highest standard of 25 000 nM. b EC50 is greater than the highest standard of 10 000 nM. c Does not converge, unable to
calculate value. d No shift observed at 10 000 nM. e ND: not done.
Table 4. Functional assessment of the N-Me series of bivalent ligands 6a-d and monovalent ligands 8a-d at the CB1 receptor
GTP-γ-S assay in rat brain
GTP-γ-S assay in hCB1
GTP-γ-S assay in hCB1
PA2 in hCB1 (95% confidence limits
calcium assay
compd
linker
EC50 (nM)
Emax (%)
EC50 (nM)
Emax (%)
Ke (nM)
SEM
6a
6b
6c
6d
8a
8b
8c
8d
11
15
19
23
11
15
19
23
1.78
1460
b
-3.8
-40.8
59.8
2238
161
a
-13.1
-32.7
-23.0
7.96
8.37
7.53
6.30
6.51
7.66
8.34
7.63
0.22
0.24
0.25
0.32
0.77
0.55
0.25
0.65
107.7
478.3
c
20.8
14.3
29.3
3037
4367
3404
8768
17.3
c
-53.3
-62.0
-67.9
-49.3
ND
ND
ND
ND
2873
219.8
140.9
205.5
418
2.8
29.1
2.9
a EC50 is greater than the highest standard of 25 000 nM. b EC50 is greater than the highest standard of 10 000 nM. c No shift observed at 10 000 nM.
Interestingly, the binding affinity of the N-H bivalent
ligand series at the CB1 receptor appeared to be sensitive to
the length of the linkers (Table 1). Specifically, the affinity
initially increased with increasing linker length and then
decreased as the linker was extended. The peak affinity was
obtained with 5d (n = 7), where the linker is composed of
15 atoms, against both radioligands (Ki of 12.3 nM vs [3H]21
and 4.41 nM vs [3H]1). A different pattern was observed for the
corresponding monovalent controls in this series (7a-f, Table 1),
where the affinity increased as the spacer became longer,
with the most potent compound determined to be 7f (n = 11,
23 atoms, Ki of 4.56 nM vs [3H]-21 and 2.30 nM vs [3H]-1).
A similar trend in affinity was also observed in the bivalent
ligands of the N-Me series (6a-d, Table 2) with respect to
their ability to compete for [3H]21 and [3H]1 binding. Affi-
nity initially increased with linker length, with 6a (n = 5) and
6b (n = 7) displaying the greatest ability in displacing either
[3H]1 or [3H]21. Slightly different from the N-H monovalent
ligand series, the binding affinity of N-methyl monovalent
controls (8a-d) at the CB1 receptor initially increased and
then remained relatively constant with the elongation of the
linker, with 8c and 8d displaying almost identical Ki values.
Interestingly, when the linkers are of the same length, the
affinities of the bivalent ligands from both the N-H and
N-Me series are relatively similar, indicating that the pre-
sence of the N-methyl group did not appear to interfere with
the interaction of the bivalent ligands with the receptors.
Inverse Agonist/Antagonist Activity. All compounds were
examined in vitro using both [35S]GTP-γ-S accumulation
and intracellular calcium mobilization assays to characterize
their efficacy, inverse agonist activity, and apparent affinity
(pA2). The results are shown in Tables 3 and 4.
In the [35S]GTP-γ-S assay using whole rat brain, most
dimers and monomers appeared to act as weak inverse
agonists. Similar to 1, most compounds required micromolar
concentrations to show inverse agonist activity in hCB1
transfectants, and the change from basal activity was rela-
tively modest (<25% decrease in basal binding under the
conditions used). However, most compounds potently
shifted the concentration-response curve of the agonist 21,
indicating that they were high affinity antagonists. Signifi-
cantly, the pA2 values against 21 stimulated [35S]GTP-γ-S
binding in hCB1 cells were correlated with their Ki values in
both the N-H and N-Me series. Specifically, the pA2 values
first increased and then decreased for the bivalent ligands
and always increased for the monovalent ligands. The bivalent
ligand with the highest apparent affinity in the N-H series was
5d, whereas 6b showed the highest pA2 value in the N-Me
series. The higher potencies of the bivalent ligand 5d and 6b
than the monovalent ligands 7d and 8b, respectively, is con-
sistent with the binding affinities of these bivalent ligands.
All the compounds were also tested using a calcium
mobilization assay as a measure of CB1 receptor function
and again showed nanomolar potency. The same trend of an
initial increase followed by a subsequent decrease in potency
with increasing linker length was observed for bivalent
ligands in both series (5a-f and 6a-d), with the exception
of 6a, whereas the potency generally increased and stayed
consistent for the monovalent ligands (7a-f and 8a-d).
Compounds 5c and 5d showed the greatest potency in
N-H bivalent ligands, and 6a was the most potent N-Me
bivalent ligand. However, no potency enhancement at the
optimal linker (15 atoms) was observed in this assay between
the bivalent and monovalent ligands (5d vs 7d and 6b vs 8b).

7054 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Zhang et al.
Figure 3. Blockade of the antinociceptive effect of 21 by bivalent and monovalent ligands. Results are expressed as the percent maximal
possible effect (% MPE, where % MPE = [(test_control)/(maximum latency_control) ꢀ 100]) as defined by a 10 s cutoff for the noxious
stimulus. Significant differences (p < 0.05) from vehicle-21 treated controls are denoted with an asterisk (/).
Tail-Flick Studies of Selected Ligands. The bivalent ligands
with the highest affinity and potency, 5d and 6b, and their
monovalent controls, 7d and 8b, were evaluated for their
ability to block the antinociceptive effects of the cannabinoid
agonist 21 in a rodent tail-flick assay. In the experiment, the
tail was exposed to 55 °C warm water and the amount of time
taken for the animal to move (flick) its tail away from the
heat was recorded. Test compounds or vehicle were adminis-
tered at 10 mg/kg ip to male mice 30 min prior to the
administration of vehicle or 1.5 mg/kg 21. Tail-flick times
were measured 30 min after treatment with 21. Antinocicep-
tive response was calculated as percentage of maximum
possible effect. As shown in Figure 3, a single 10 mg/kg ip
dose of the bivalent ligand 6b and its monomeric control 8b
could significantly attenuate the antinociceptive response
to 21. However, the N-H analogues (5d and 7d) were
considerably less active.
result of dimerization and oligomerization of CB1 receptors
are being explored. The availability of high affinity bivalent
ligands for CB1 receptors may provide researchers the neces-
sary probes to identify the physiological importance of such
interactions and further our understanding of the role of
cannabinoid signaling in the context of health and disease.
In the present study, we synthesized a library of symme-
trical bivalent ligands containing two moieties of 1 joined by
aminoalkyl linkers. All the target compounds were evaluated
in radiolabeled binding assays at the CB1 and CB2 receptors,
functional [35S]GTP-γ-S accumulation assay, and functional
calcium mobilization assay. Data from these in vitro assays
displayed subtle differences between Ki, pA2, and Ke values.
This is not surprising, as different end points and biological
systems were experimentally employed for ligand character-
ization. Interestingly, a clear trend could be detected in all
three assays where the bivalent ligands showed initially in-
creased and then decreased affinity/activity with elongation of
the linkers, whereas the monovalent ligands generally con-
tinued increasing or stayed consistent once the linker length
was sufficiently long.
Such an initial increase, followed by a subsequent decrease
in affinity and potency of the bivalent ligands, is consistent
with observations in the bivalent opioid ligands made by the
Portoghese and Neumeyer groups.14,31,42,43 Thus, it is hypo-
thesized that linker length is critical for the ability of the
bivalent ligands to bind the CB1 receptor, possibly two
neighboring CB1 receptors simultaneously, as insufficient
length would not permit bridging and spacers of excessive
length would reduce bridging because of increased confine-
ment volume. This transition suggests that bridging of vicinal
receptors by bivalent ligands may occur most efficiently with
optimal linker length. This hypothesis is also supported by the
different pattern that was observed for the corresponding
monovalent controls in this series (7a-f, Table 1), where the
affinity increased as the spacer became longer, with the most
potent compound determined to be 7f (n = 11, 23 atoms).
Most interestingly, the highest affinity bivalent ligand (5d),
where the linker is composed of 15 atoms, displays higher
affinity (∼4-fold for both radioligands) than the correspond-
ing monovalent control (7d), indicating that the presence
of the second pharmacophore increases the ability for the
Discussions and Conclusions
The concept of homo- and heterodimerization has opened
new potential avenues for the development of drugs targeted
at GPCRs. One emerging approach is to employ bivalent
ligands that specifically bind to these receptor dimers. Ideally,
bivalent ligands with linkers of optimal length will bind to
receptor dimers with greatly enhanced affinity due to the
formation of thermodynamically stable complexes. Indeed,
significant progress has been made in a number of GPCRs
including opioids,14,16 adrenergic,17,18 dopamine,19 serotonin,20,21
and muscarinic receptors.22,23 Most significantly, much suc-
cess has been recently achieved by Portoghese and co-workers
with bivalent opioid ligands in vivo.40,41 In particular, μ-opioid
(MOP) agonist/δ-opioid (DOP) antagonist bivalent ligands
were shown to be potent analgesics after systemic administra-
tion but did not produce the tolerance or dependence seen
with traditional monovalent opioid analgesics.13 However, to
the best of our knowledge, there are no bivalent ligands
developed for the CB1 receptor to date. It is now well
established that this receptor is a viable target to treat various
indications including smoking addiction, drug and alcohol
dependence, metabolic syndrome, cancer, fibrosis, and in-
flammation. Theconsequencesof altered cellularfunction as a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7055
compounds to bind to the receptor, possibly by simultaneous
occupying vicinal recognition sites of neighboring receptors in
the receptor dimer. Again, this affinity enhancement of bivalent
ligands over their corresponding monovalent ligands has been
widely observed in previous studies on the opioid receptors,
although it is often a modest (∼2-fold) difference.31 It is worth
noting that despite the moderate affinity or potency enhance-
ment observed for the bivalent over the monovalent ligands in
the binding or functional assays, additional evidence in sup-
port of the homodimers binding hypothesis has been reported
using techniques such as FRET, as demonstrated by Russo
and co-worker in5-HT4 receptors.20 Theoptimallinker length
of 15 atoms in the present study is consistent with the range
reported for bivalent ligands developed for other GPCRs.30,31,40,44
While these data indicate possible binding of the bivalent
ligands to CB1 receptor homodimers, the fact that monova-
lent ligands display comparable or even higher affinity than
the corresponding bivalent ligands when the linker is suffi-
ciently long (5e,f vs 7e,f, and 6c,d vs 8c,d) raises other
possibilities. Indeed, the observation that monovalent ligands
are more potent than the bivalent ligands has also been
previously reported in bivalent ligands for other GPCRs
including opioids, 5-HT4 and GnRHR.20,31,34,45-47 A single
receptor binding model has been presented in opioid bivalent
ligands which suggests that the linker itself may represent an
additional receptor recognition site and only one pharmaco-
phore is needed when the spacer is of sufficient length.14,48
Thishypothesis may alsobesupported by the observationthat
no relationship between the linker length and binding affinity
was discovered in bivalent opioid ligands where the highest
affinity ligands were at opposite extremes of linker length.46 It
is worth noting that hybrid ligands designed to target single
GPCRs have been developed.49-51 Named bitopic or duals-
teric ligands, these compounds possess pharmacophores for
an orthosteric site and an allosteric site, respectively, and are
capable of binding single receptors with enhanced affinity
and/or selectivity for several GPCRs, including the muscarinic
acetylcholine receptors49,52 and the adenosine A1 receptor.53
Therefore, further studies are clearly needed to elucidate the
binding mechanism of our CB1 bivalent ligands.
Finally, selected bivalent ligands (5d and 7b) and the corres-
ponding monovalent controls (6d and 8b) were able to attenu-
ate the antinociceptive effects of the cannabinoid agonist 21 in
the tail-flick assay. It remains to be determined if the differ-
ences in potencies in vivo can be attributed to differences in
biodistribution or metabolism of the various ligands. Never-
theless, these results suggest that these bivalent compounds,
despite their high molecular weight, are able to attenuate
nociceptive responses by central and or peripheral mechan-
isms by antagonizing the CB1 receptor complexes.
In summary, a series of bivalent ligands featuring two
pharmacophores of 1 and their corresponding monovalent
ligands were designed and synthesized to target CB1 receptor
homodimers. Biological characterization of these compounds
in radioligand binding and functional assays established that
the length and the composition of the linker are crucial for the
affinities and potencies of the bivalent ligands. Selected
bivalent ligands and monovalent ligands were able to attenu-
ate nociceptive effects of CB1 agonist 21. Although the results
suggest possible binding of the bivalent ligands to CB1
receptor homodimers, the possibility of these ligands binding
to different sites on a single receptor cannot be ruled out at the
present time. However, previous studies using saturation
binding experiments with various radioligands do not support
high-affinity secondary binding sites on the CB1 receptor
providing indirect support for these ligands binding to recep-
tor homodimers or oligomers.54 Further evaluation of this
bivalent ligand approach of CB1 receptor dimerization or
oligomerization is clearly needed and may serve as the basis
for development of new medications.
Experimental Section
Chemistry. Reactions were conducted under N2 atmospheres
using oven-dried glassware. All solvents and chemicals used were
reagent grade. Anhydrous tetrahydrofuran, dichloromethane, and
N,N-dimethylformamide (DMF) were purchased from Aldrich
and used as such. Unless otherwise mentioned, all reagents and
chemicals were purchased from commercial vendors and used as
received. Flash column chromatography was carried out on a
Teledyne ISCO CombiFlash Companion system using RediSep Rf
prepacked columns. Purity and characterization of compounds
were established by a combination of HPLC, TLC, gas chromato-
graphy-mass spectrometry (GC-MS), and NMR analytical
1
techniques described below. H and 13CNMR spectra were re-
corded on a Bruker Avance DPX-300 (300 MHz) spectrometer
and were determined in CHCl3-d or MeOH-d4 with tetramethyl-
silane (TMS) (0.00 ppm) or solvent peaks as the internal reference
unless otherwise noted. Chemical shifts are reported in ppm
relative to the solvent signal, and coupling constant (J) values
are reported in hertz (Hz). Thin-layer chromatography (TLC)
was performed on EMD precoated silica gel 60 F254 plates, and
spots were visualized with UV light or I2 detection. Low-resolution
mass spectra were obtained using a Waters Alliance HT/Micro-
mass ZQ system (ESI). High-resolution mass spectra were obtained
in the Mass Spectrometry Laboratory, Department of Chemistry,
University of Michigan. All test compounds were greater than 95%
pure as determined by HPLC on an Agilent 1100 system using an
Agilent Zorbax SB-Phenyl, 2.1 mm ꢀ 150 mm, 5 μm column with
gradient elution using the mobile phases (A) H2O containing 0.1%
CF3COOH and (B) MeCN. A flow rate of 0.5 mL/min was used for
5a-f and 7a-d and 1.0 mL/min for 6a-f and 8a-d.
15c,d and 18a,b were purchased from Aldrich and were used
as such.
5-(4-Chlorophenyl)-N-{13-[5-(4-chlorophenyl)-1-(2,4-dichloro-
phenyl)-4-methyl-1H-pyrazol-3-yl]-13-oxo-3,6,9-trioxa-12-azatridec-
1-yl}-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (3).
Benzotriazole-1-yl-oxytris(dimethylamino)phosphonium hexafluoro-
phosphate (BOP) (116 mg, 0.262 mmol) was added to a solution of
acid 9 (100 mg, 0.262 mmol) in 15 mL of THF. After 5 min, 1,11-
diamino-3,6,9-trioxaundecane (30 mg, 0.157 mmol) was added. The
mixture was stirred at room temperature for 1 h. The solvent was
removed, and the resulting slurry was diluted with saturated aqueous
NaHCO3 and extracted with ethyl acetate (2 ꢀ 30 mL). The combined
organic layers were washed with brine and dried. The residue was
purified on silica using MeOH-CHCl3-NH4OH and EtOAc to give
3(95 mg, 78.9%) as a solid. 1HNMR(CDCl3) δ2.38 (s, 6H), 3.58 (m,
16H), 7.05 (d, J = 9.0, 4H), 7.28 (m, 8H), 7.41 (s, 2H). MS:
C25H29Cl3N4O4, [M þ H]þ 555.2.
Methyl N-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazol-3-yl]carbonyl}glycinate (10). To a solution of acid 9
(2 g, 5.24 mmol) in 60 mL of CH2Cl2 was added sequentially
HOBt (0.78 g, 5.76 mmol), EDCI (1.1 g, 5.76 mmol), and glycine
methyl ester hydrochloride (0.66 g, 5.24 mmol). The mixture was
stirred at room temperature for 15 min before Et3N was added. The
mixture was stirred for 12 h. The mixture was diluted with CH2Cl2
(100 mL) and washed with 1 N HCl, NaHCO3, and then brine. The
organic layer was dried with Na2SO4 and concentrated to give 10 as
a white solid. 1H NMR (CDCl3) δ 2.36 (s, 3H), 3.78 (s, 3H), 4.22
(d, J= 5.7, 2H), 7.05 (d, J=6.6, 2H), 7.30(m, 4H), 7.40(t, J=3.0,
1H), 7.43 (s, 1H). The product was of sufficient purity and was used
in the next step without further purification.
N-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazol-3-yl]carbonyl}glycine (11). A solution of 10 in 30 mL of

7056 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Zhang et al.
MeOH and 30 mL of 2 N NaOH was stirred at room temperature
for 16 h. The solvent was concentrated in vacuo, and the resulting
solution was washed with ether. The aqueous solution was acidified
with 6 N HCl and then extracted with EtOAc (3 ꢀ 100 mL). The
combined organic layers were washed with water, brine and dried
with Na2SO4. The solvent was removed in vacuo to give 11 (2.09 g,
90.9% over both steps). 1H NMR (CDCl3) δ 2.35 (s, 3H), 4.26 (d,
J = 6.0, 2H), 7.06 (d, J = 9.0, 2H), 7.31 (m, 4H), 7.42 (s, 1H), 7.58
(t, 3.0, 1), 10.78 (bs, 1H).
N-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazol-3-yl]carbonyl}glycylglycine (12). Following the proce-
dure for the preparation of 10, 11 was coupled to glycine methyl
ester hydrochloride to provide the methyl ester in 69.5% yield.
1H NMR (CDCl3) δ 2.36 (s, 3H), 3.75 (s, 3H), 4.07 (d, J = 6.0, 2H),
4.16 (d, J = 6.0, 2H), 6.77 (t, J = 3.0, 1H), 7.07 (d, J = 6.0, 2H),
7.29 (m, 4H), 7.43 (s, 1H), 7.53 (t, J = 3.0, 1H).
(m, 5H). The product was of sufficient purity and was used in the
next step without further purification.
7,70-(Benzylimino)diheptanenitrile (16d). 16d (1.43 g, 94.1%)
was obtained from benzylamine (0.5 g, 4.67 mmol) and 7-bro-
moheptanenitrile (1.86 g, 9.80 mmol) as a colorless oil. 1H NMR
(CDCl3) δ 1.20-1.65 (m, 16H), 2.29 (t, J = 6.9, 4H), 2.39 (t, J =
6.9, 4H), 3.52 (s, 2H), 7.29 (m, 5H).
9,90-(Benzylimino)dinonanenitrile (16e). 16e (1.79 g, 100%)
was obtained from benzylamine (0.5 g, 4.67 mmol) and 15e (2.04
g, 9.33 mmol) as an oil. 1H NMR (CDCl3) δ 1.20-1.70 (m, 24H),
2.20-2.45 (m, 8H), 3.51 (s, 2H), 2.24 (m, 5H).
11,110-(Benzylimino)diundecanenitrile (16f). 16f (2.0 g, 84.8%)
was obtained from benzylamine (0.7 g, 6.53 mmol) and 15f (3.38
g, 13.72 mmol). 1H NMR (CDCl3) δ 1.20-1.50 (m, 24H), 1.65
(m, 4H), 1.83 (m, 4H), 2.35 (t, J = 7.2, 4H), 2.90 (m, 4H), 4.16
(s, 2H), 7.43 (m, 3H), 7.64 (m, 2H).
A solution of the above methyl ester (200 mg, 0.40 mmol) and
LiOH (25 mg, 1.2 mmol) in 10 mL of THF-MeOH (3:1) and
2 mL of water was stirred at room temperature for 12 h. The
mixture was acidified with 3 N HCl and extracted with EtOAc
(2 ꢀ 25 mL). The combined organic layers were washed with
water, brine and dried with Na2SO4. The solvent was evaporated
to give 12 (175 mg, 88.4%) as a white solid. 1H NMR (CDCl3) δ
2.31 (s, 3H), 3.78 (s, 2H), 4.09 (s, 2H), 7.19 (d, J = 8.4, 2H), 7.37
(d, J = 8.4, 2H), 7.46-7.56 (m, 3H).
N-(5-Aminopentyl)pentane-1,5-diamine (18c). A suspension
of 16c (0.5 g, 1.86 mmol) and Raney nickel (0.5 g) in ethanol
(40 mL), THF (10 mL), and 2 N sodium hydroxide (8 mL) was
stirred under hydrogen (50 psi) for 20 h. The suspension was
filtered through Celite and concentrated. The resulting slurry
was diluted with water (40 mL) and then extracted with CH2Cl2
(2 ꢀ 50 mL). The combined organic layers were washed with
brine and dried over Na2SO4 to give 17c as an off-white oil. 17c
was used in the next step without purification.
N-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazol-3-yl]carbonyl}glycyl-N-(2-aminoethyl)glycinamide (4).
To a solution of 12 (140 mg, 0.28 mmol) in THF at room
temperature was added BOP (125.0 mg, 0.28 mmol) and ethyl-
enediamine (9.0 μL, 1.41 mmol). The mixture was stirred for
15 min before Et3N was added. The mixture was stirred for 12 h.
The reaction was quenched with water and extracted with
EtOAc (2 ꢀ 40 mL). The combined organic layers were washed
with 1 N HCl, saturated NaHCO3, and brine and then dried with
Na2SO4. The solvent was removed and the residue was purified
on silica gel using MeOH-CHCl3-NH4OH and EtOAc to give
A suspension of 17c (0.35 g, 1.25 mmol), 10% palladium on
carbon (40 mg) in ethanol (15 mL), and acetic acid (5 mL) was
stirred under 50 psi of hydrogen for 3 h. The suspension was
filtered through Celite, and the filtrate was concentrated. To the
resulting slurry was added 2 N NaOH (20 mL), and extraction
was with CH2Cl2 (2 ꢀ 30 mL). The combined organic layers
were washed with water and dried with Na2SO4. The solvent was
evaporated in vacuo to give 18c (228 mg, 91.1% over both steps)
1
as a clear oil. H NMR (CDCl3) δ 1.15-1.55 (m, 12H), 2.53
(t, J = 6.9, 4H), 2.62 (t, J = 6.6, 4H).
N-(7-Aminoheptyl)heptane-1,7-diamine (18d). Following the
procedure for the synthesis of 18c, 18d (0.85 g, 94.6%) was
obtained from 16d (1.2 g, 3.69 mmol). 1H NMR (CDCl3) δ
1.15-1.50 (m, 20H), 2.52 (t, J = 7.2, 4H), 2.61 (t, J = 6.9, 4H).
N-(9-Aminononyl)nonane-1,9-diamine (18e). Following the
procedure for the synthesis of 18c, 18e (0.425 g, 77.5%) was
obtained from 16e (0.7 g, 1.83 mmol). 1H NMR (CDCl3) δ
1.20-1.55 (m, 28H), 2.57 (t, J = 7.2, 4H), 2.66 (t, J= 6.9, 4H).
N-(11-Aminoundecyl)undecane-1,11-diamine (18f). Following
the procedure for the synthesis of 18c, 18f (0.34 g, 84.1%) was
obtained from 16f (0.5 g, 1.14 mmol). 1H NMR (CDCl3) δ
1.10-1.50 (m, 36H), 2.56 (t, J= 7.5, 4H), 2.62 (t, J = 7.2, 4H).
N,N0-(Iminodiethane-2,1-diyl)bis[5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (5a). A
solution of acid 9 (0.2 g, 0.52 mmol) and carbonyldiimidazole
(85 mg, 0.52 mmol) in 5 mL of CH2Cl2 was stirred at room
temperature for 1 h. TLC showed the complete consumption of
the starting material. Triamine 18a (28 μL, 0.26 mmol) was
added, and the mixture was allowed to stir at room tempera-
ture for 3 h. The mixture was diluted with CH2Cl2 and washed
sequentially with NaHCO3, water, and brine. The solution was
dried with Na2SO4 and concentrated. The resulting slurry was
purified on silica using CHCl3-MeOH-NH4OH (80:18:2) and
1
4 (35 mg, 34.5%) as a solid. H NMR (CDCl3) δ 2.31 (s, 3H),
2.87 (d, J = 6.0, 2H), 3.34 (d, J = 6.0, 2H), 3.96 (d, J = 6.0, 2H),
4.08 (d, J = 6.0, 2H), 7.06 (m, 3H), 7.20-7.40 (m, 5H), 7.42
(s, 1H), 7.80 (t, J = 3.0, 1H). MS: C23H23Cl3N6O3, [M þ H]þ
537.4.
9-Bromononanenitrile (15e). Sodium cyanide (3.6 g, 73.5 mmol)
was added in portions to a solution of 1,8-dibromooctane (13)
(20 g, 73.5 mmol) in 50 mL of DMSO at 60 °C. After 30 min, the
reaction was stopped and the mixture was allowed to cool to room
temperature. The mixture was diluted with 200 mL of diethyl ether
and 200 mL of hexane and then washed with water (2 ꢀ 50 mL).
The organic layer was separated, dried with sodium sulfate, and
concentrated. The resulting slurry was purified on silica using
MeOH/CH2Cl2 (1:9) to give 15e (7.16 g, 44.7%) as a colorless
oil. 1H NMR (CDCl3) δ 1.35 (t, J = 7.5, 3H), 1.82 (s, 3H), 4.32 (q,
J=7.5,2H),7.24(d,J= 9.0, 1H), 7.37 (d, J=7.5,1H),7.48(s,1H).
11-Bromoundecanenitrile (15f). 15f (6.95 g, 42.4%) was ob-
tained from 1,10-dibromodecane (14) (20 g, 66.6 mmol) as an
oil. 1H NMR (CDCl3) δ 1.15-1.50 (m, 12H), 1.65 (m, 2H), 1.84
(m, 2H), 3.34 (t, J = 7.5, 2H), 3.41 (t, J = 6.0, 2H).
5,50-(Benzylimino)dipentanenitrile (16c). A mixture of benzyl-
amine (0.5 g, 4.67 mmol), potassium carbonate (1.94 g, 14.0
mmol), and potassium iodide (0.27 g, 1.63 mmol) was heated to
115 °C. A solution of 5-bromopentanenitrile in 1-butanol was
added dropwise. The resulting mixture was kept at 115 °C for
20 h. The mixture was allowed to cool to room temperature and
then filtered. The solid was washed with diethyl ether (2 ꢀ
30 mL). The combined organic layers were extracted with 3 N HCl
(2 ꢀ 20 mL). The aqueous layer was washed with ether and
basified with sodium carbonate. The resulting solution was then
extracted with ether (3 ꢀ 40 mL). The combined organic layers
were dried with Na2SO4 and concentrated to give 16c (1.02 g,
81.1%) as an oil. 1H NMR (CDCl3) δ 1.50-1.70 (m, 8H), 2.26
(t, J = 6.6, 4H), 2.42 (t, J = 6.3, 4H), 3.51 (s, 2H), 7.15-7.32
1
EtOAc to give 5a (0.13 g, 61.1%) as a white solid. H NMR
(CDCl3) δ 2.35 (s, 6H), 2.90 (t, J = 6.0, 4H), 3.52 (dt, J1 = J2 =
6.0, 4H), 7.05 (d, J = 6.0, 4H), 7.22-7.30 (m, 10H), 7.40 (s, 2H).
HRMS: C38H31Cl6N7O2, [M þ H]þ calcd 828.0749, found 828.0768.
N,N0-(Iminodipropane-3,1-diyl)bis[5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (5b).
Following the procedure for the preparation of 5a, 5b was
1
obtained from 18b in 49.5% yield. H NMR (CDCl3) δ 1.75
(m, 4H), 2.35 (s, 6H), 2.72 (t, J = 6.0, 4H), 3.45 (dt, J1 = 9.0,
J2 = 6.0, 4H), 7.05 (d, J = 6.0, 4H), 7.28 (m, 8H), 7.40 (s, 2H),
7.54 (t, J = 3.0, 2H). HRMS: C40H35Cl6N7O2, [M þ H]þ calcd
856.1062, found 856.1076.

Article
N,N0-(Iminodipentane-5,1-diyl)bis[5-(4-chlorophenyl)-1-(2,4-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7057
7.29 (m, 4H), 7.43 (s, 1H). HRMS: C31H42Cl3N5O, [M þ H]þ
calcd 606.2533, found 606.2536.
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (5c).
Following the procedure for the preparation of 5a, 5c was
obtained from 18c in 42.5% yield. 1H NMR (CDCl3) δ
1.37-1.64 (m, 12H), 2.37 (s, 6H), 2.59 (t, J = 6.0, 4H), 3.41
(dt, J1 = 9.0, J2 = 6.0, 4H), 6.96 (t, J = 3.0, 2H), 7.06 (d, J =
6.0, 4H), 7.26 (m, 8H), 7.43 (s, 2H). HRMS: C44H43Cl6N7O2,
[M þ H]þ calcd 912.1688, found 912.1679.
N-{9-[(9-Aminononyl)amino]nonyl}-5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (7e). Fol-
lowing the procedure for the preparation of 7a, 7e was obtained
from 18e in 47.2% yield. 1H NMR (CDCl3) δ 1.18-1.62
(m, 28H), 2.38 (s, 3H), 2.58 (t, J = 7.2, 4H), 2.67 (t, J = 6.9,
2H), 3.40 (dt, J1 = 6.9, J2 = 6.6, 2H), 6.96 (t, J = 3.0, 1H), 7.28
(m, 4H), 7.43 (s, 1H). HRMS: C35H50Cl3N5O, [M þ H]þ calcd
662.3159, found 662.3150.
N,N0-(Iminodiheptane-7,1-diyl)bis[5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (5d).
Following the procedure for the preparation of 5a, 5d was
obtained from 18d in 55.6% yield. 1H NMR (CDCl3) δ
1.34-1.70 (m, 20H) 2.37 (s, 6H), 2.56 (t, J = 6.0, 4H), 3.41
(dt, J1 = 6.9, J2 = 6.6, 4H), 6.95 (t, J = 3.0, 2H), 7.05 (d, J =
6.0, 4H), 7.29 (m, 8H), 7.43 (s, 2H). HRMS: C48H51Cl6N7O2,
[M þ H]þ calcd 968.2314, found 968.2321.
N-{11-[(11-Aminoundecyl)amino]undecyl}-5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (7f).
Following the procedure for the preparation of 7a, 7f was
1
obtained from 18f in 46.7% yield. H NMR (CDCl3) δ 1.20-
1.65 (m, 36H), 2.38 (s, 3H), 2.58 (t, J = 7.2, 4H), 2.67 (t, J = 6.9,
2H), 3.40 (dt, J1 = 6.9, J2 = 6.6, 2H), 6.95 (t, J = 3.0, 1H), 7.05
(d, J = 8.4, 7.28 (m, 4H), 7.43 (s, 1H). HRMS: C39H58Cl3N5O,
[M þ H]þ calcd 718.3785, found 718.3784.
N,N0-(Iminodinonane-9,1-diyl)bis[5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (5e).
Following the procedure for the preparation of 5a, 5e was
obtained from 18e in 42.8% yield. 1H NMR (CDCl3) δ
1.26-1.59 (m, 28H), 2.37 (s, 6H), 2.60 (t, J = 6.0, 4H), 3.40
(dt, J1 = 6.6, J2 = 6.0, 4H), 6.95 (t, J = 3.0, 2H), 7.28 (m,
8H), 7.43 (s, 2H). HRMS: C52H59Cl6N7O2, [M þ H]þ calcd
1024.2940, found 1024.2972.
5,50-(Methylimino)dipentanenitrile (19a). A pressure tube
equipped with a mixture of methylamine hydrochloride (1 g,
16 mmol), 15c (3.7 mL, 32 mmol), potassium carbonate (4.4 g,
32 mmol), and potassium iodide (0.53 g, 3.2 mmol) in 20 mL of
ethanol was heated to 110 °C for 16 h. The mixture was cooled to
room temperature, and the solvent was removed. The resulting
slurry was partitioned between ethyl acetate and water. The
organic layer was washed with brine and dried. The resulting
residue was purified by chromatography on silica gel to give 19a
(1.24 g, 40.1%) as an off-white oil. 1H NMR (CDCl3) δ
1.55-1.85 (m, 8H), 2.19 (s, 3H), 2.38 (m, 8H).
N,N0-(Iminodiundecane-11,1-diyl)bis[5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (5f). Fol-
lowing the procedure for the preparation of 5a, 5f was obtained
1
from 18f in 38.9% yield. H NMR (CDCl3) δ 1.25-1.61 (m,
36H), 2.37 (s, 6H), 2.61 (t, J = 7.5, 4H), 3.40 (dt, J1 = 6.9, J2 =
6.6, 4H), 6.96 (t, J = 6.0, 2H), 7.06 (d, J = 8.4, 4H), 7.29 (m,
8H), 7.42 (s, 2H). HRMS: C56H67Cl6N7O2, [M þ H]þ calcd
1080.3566, found 1080.3590.
7,70-(Methylimino)diheptanenitrile (19b). 19b was synthesized
1
from 15d in 48.8% yield following the procedure for 19a. H
NMR (CDCl3) 1.25-1.70 (m, 16H), 2.18 (s, 3H), 2.33 (m, 8H).
9,90-(Methylimino)dinonanenitrile (19c). 19c was synthesized
from 15e in 55.0% yield following the procedure for 19a. H
N-{2-[(2-Aminoethyl)amino]ethyl}-5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (7a). A
solution of acid 9 (50 mg, 0.13 mmol) and carbonyldiimidazole
(21 mg, 0.13 mmol) in 5 mL of CH2Cl2 was stirred at room
temperature for 30 min. TLC showed the complete consumption
of starting acid. This suspension was then added to a solution of
triamine 18a (41 mg, 0.39 mmol) dropwise. The resulting clear
solution was allowed to stir at room temperature for 3 h. The
mixture was diluted with CH2Cl2 and washed sequentially with
NaHCO3, water, and brine. The solution was dried with Na2SO4
and concentrated. The resulting slurry was purified on silica
using MeOH-CHCl3-NH4OH (80:18:2) and EtOAc to give 5a
(47 mg, 89.0%) as a solid. 1H NMR (CDCl3) δ 2.36 (s, 3H), 2.72
(t, J = 5.7, 2H), 2.80 (t, J = 4.8, 2H), 2.87 (t, J = 6.0, 2H), 3.53
(dt, J1 = J2 = 6.0, 2H), 7.05 (d, J = 6.0, 2H), 7.28 (m, 5H), 7.42
(s, 1H). HRMS: C21H22Cl3N5O, [M þ H]þ calcd 466.0968,
found 466.0971.
N-{3-[(3-Aminopropyl)amino]propyl}-5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (7b). Following
the procedure for the preparation of 7a, 7b was obtained from
18b in 67.9% yield. 1H NMR (CDCl3) δ 1.64 (m, 2H), 1.79 (m,
2H), 2.37 (s, 3H), 2.67-2.77 (m, 6H), 3.53 (dt, J1 = 5.7, J2 =
5.4, 2H), 7.05 (d, J = 8.4, 2H), 7.30 (m, 4H), 7.42 (s, 1H), 7.56
(m, 1H). HRMS: C23H26Cl3N5O, [M þ H]þ calcd 494.1281,
found 494.1284.
1
NMR (CDCl3) 1.25-1.50 (m, 20H), 1.65 (m, 4H), 2.19 (s, 3H),
2.31 (m, 8H).
11,110-(Methylimino)diundecanenitrile (19d). 19d was synthe-
sized from 15f in 42.5% yield following the procedure for 19a.
1H NMR (CDCl3) 1.20-1.50 (m, 24H), 1.66 (m, 4H), 1.85 (m,
4H), 2.36 (t, J = 3.9, 4H), 2.78 (s, 3H), 3.01 (t, J = 8.4, 4H).
N-(5-Aminopentyl)-N-methylpentane-1,5-diamine (20a). 20a
was synthesized from 19a in 59.0% yield following the proce-
1
dure for 17c. H NMR (CDCl3) δ 1.15-1.55 (m, 12H), 2.20
(s, 3H), 2.31 (t, J = 7.5, 4H), 2.69 (t, J = 6.9, 4H).
N-(7-Aminoheptyl)-N-methylheptane-1,7-diamine (20b). 20b
was synthesized from 19b in 73.8% yield following the proce-
1
dure for 17c. H NMR (CDCl3) δ 1.20-1.50 (m, 24H), 2.18
(s, 3H), 2.29 (t, J = 7.5, 4H), 2.67 (t, J = 6.9, 4H).
N-(9-Aminononyl)-N-methylnonane-1,9-diamine (20c). 20c was
synthesized from 19c in 99.0% yield following the procedure for
17c. 1H NMR (CDCl3) δ 1.20-1.55 (m, 28H), 2.19 (s, 3H), 2.29
(t, J = 7.8, 4H), 2.67 (t, J = 6.9, 4H).
N-(11-Aminoundecyl)-N-methylundecane-1,11-diamine (20d).
20d was synthesized from 19d in 79.0% yield following the
1
procedure for 17c. H NMR (CDCl3) δ 1.20-1.50 (m, 36H),
2.19 (s, 3H), 2.29 (t, J = 7.8, 4H), 2.67 (t, J = 6.9, 4H).
N,N0-[(Methylimino)dipentane-5,1-diyl]bis[5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (6a).
Following the procedure for the preparation of 5a, 6a was obtained
from 20a in 50.2% yield. 1H NMR (CDCl3) δ 1.37-1.65 (m, 12H),
2.24 (s, 3H), 2.38 (m, 10H), 3.41 (dt, J1 = 6.6, J2 = 6.0, 4H), 7.00
(t, J = 3.0, 2H), 7.06 (d, J = 6.0, 4H), 7.28 (m, 8H), 4.23 (s, 2H).
HRMS: C45H45Cl6N7O2, [M þ H]þ calcd 926.1844, found
926.1866.
N-{5-[(5-Aminopentyl)amino]pentyl}-5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (7c). Follow-
ing the procedure for the preparation of 7a, 7c was obtained from
18c in 44.9% yield. 1H NMR (CDCl3) δ 1.36-1.60 (m, 12H), 2.37
(s, 3H), 2.59-2.72 (m, 6H), 3.43 (dt, J1 = 9.3, J2 = 6.6, 2H), 6.95
(t, J = 3.0, 1H), 7.06 (d, J = 8.4, 2H), 7.29 (m, 4H), 7.43 (s, 1H).
HRMS: C27H34Cl3N5O, [M þ H]þ calcd 550.1907, found 550.1909.
N-{7-[(7-Aminoheptyl)amino]heptyl}-5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (7d). Following
the procedure for the preparation of 7a, 7d was obtained from
N,N0-[(Methylimino)diheptane-7,1-diyl]bis[5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (6b).
Following the procedure for the preparation of 5a, 6b was obtained
from 20b in 40.3% yield. 1H NMR (CDCl3) δ 1.26-1.60 (m, 20H),
2.18 (s, 3H), 2.28 (t, J = 6.0, 4H), 2.37 (s, 6H), 3.41 (dt, J1 = 6.6,
J2 = 6.0, 4H), 6.95 (t, J = 3.0, 2H), 7.06 (d, J = 6.0, 4H), 7.28
1
18d in 35.2% yield. H NMR (CDCl3) δ 1.26-1.62 (m, 20H),
2.38 (s, 3H), 2.56 (m, 4H), 2.67 (t, J = 6.6, 2H), 3.41 (dt, J1 =
6.9, J2 = 6.6, 2H), 6.96 (t, J = 3.0, 1H), 7.06 (d, J = 6.9, 2H),

7058 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Zhang et al.
(m, 8H), 7.42 (s, 2H). HRMS: C49H53Cl6N7O2, [M þ H]þ calcd
982.2470, found 982.2482.
terminated by vacuum filtration through GF/C glass fiber filter
plates (Packard, Meriden, CT, pretreated in buffer B for at least 1 h)
in a 96-well sampling manifold (Millipore, Bedford, MA). Reaction
vessels were washed twice with 4 mL of ice cold buffer (50 mM
N,N0-[(Methylimino)dinonane-9,1-diyl]bis[5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (6c).
Following the procedure for the preparation of 5a, 6c was obtained
from 20c in53.5%yield. 1H NMR (CDCl3) δ1.28-1.50 (m, 24H),
1.50-1.65 (m, 4H), 2.19 (s, 3H), 2.29 (t, J = 7.8, 4H), 2.38 (s, 6H),
3.40 (dt, J1 = 6.9, J2 = 6.6, 4H), 6.96 (t, J = 3.0, 2H), 7.06 (d, J =
8.4, 4H), 7.29 (m, 8H), 7.43 (s, 2H). HRMS: C53H61Cl6N7O2,
[M þ H]þ calcd 1038.3096, found 1038.3094.
Tris HCl, 1 mg/mL BSA). The filter plates were air-dried and sealed
3
on the bottom. Liquid scintillate was added to the wells and the top
sealed. After incubation of the plates in cocktail for at least 2 h, the
radioactivity present was determined by liquid scintillation spectro-
metry. Assays were done in duplicate, and results represent com-
bined data from three to six independent experiments. Saturation
and displacement data were analyzed by unweighted nonlinear
regression of receptor binding data. For displacement studies,
curve-fitting and IC50 calculation were done with GraphPad Prism
(GraphPad Software, Inc., San Diego, CA), which fits the data to
one- and two-site models and compares the two fits statistically.
GTP-γ-[35S] Assay. GTP-γ-[35S] assays were performed to
determine the ability of target compounds to shift the binding
curves of the agonist 21 or 1. Reaction mixtures consisted of
either 21 (2.5 pM to 25 μM) or 1 (10 pM to 100 μM), 20 μM
N,N0-[(Methylimino)diundecane-11,1-diyl]bis[5-(4-chlorophenyl)-1-
(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (6d).
Following the procedure for the preparation of 5a, 6d was
1
obtained from 20d in 43.8% yield. H NMR (CDCl3) δ 1.20-
1.65(m, 36H), 2.29 (s, 3H), 2.41(m, 10H), 3.42 (dt, J1 =9.0, J2 =
6.0, 4H), 6.94 (t, J = 3.0, 2H), 7.05 (d, J = 9.0, 4H), 7.28 (m, 8H),
7.42 (s, 2H). HRMS: C57H69Cl6N7O2, [M þ H]þ calcd
1094.3722, found 1094.3715.
N-{5-[(5-Aminopentyl)(methyl)amino]pentyl}-5-(4-chlorophenyl)-1-
(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (8a).
Following the procedure for the preparation of 7a, 8a was
GDP, and 100 pM GTP-γ-[35S] in 50 mM Tris HCl, pH 7.4,
3
1 mM EDTA, 5 mM MgCl2, 100 mM NaCl, and 1 mg/mL BSA.
The effects of these compounds on agonist binding were com-
pared at 1, 10, and 100 nM vs reactions with no antagonist in a
final reaction mixture volume of 0.5 mL. Binding was deter-
mined using membrane preparations as previously described.
Data analysis was performed using global nonlinear regression
analysis of the dose-response curves (Prism, GraphPad), and
pA2 values were calculated. The calculations were performed
with the slope of the Schild line constrained to 1, as well as
unconstrained, and an F-test (P < 0.05) was used to determine
the best model.
1
obtained from 20a in 60.8% yield. H NMR (CDCl3) δ 1.22-
1.70 (m, 12H), 2.20 (s, 3H), 2.32 (t, J = 7.5, 4H), 2.37 (s, 3H),
2.69 (t, J = 6.9, 2H), 2.42 (dt, J1 = 6.9, J2 = 6.6, 2H), 6.99 (t,
J = 3.0, 1H), 7.06 (d, J = 6.6, 2H), 7.29 (m, 4H), 7.43 (s, 1H).
HRMS: C28H36Cl3N5O, [M þ H]þ calcd 564.2064, found
564.2073.
N-{7-[(7-Aminoheptyl)(methyl)amino]heptyl}-5-(4-chlorophenyl)-1-
(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (8b).
Following the procedure for the preparation of 7a, 8b was
obtained from 20b in 29.5% yield. 1H NMR (CDCl3) δ
1.20-1.65 (m, 20H), 2.19 (s, 3H), 2.29 (t, J = 7.8, 4H), 2.38
(s, 3H), 2.67 (t, J = 6.6, 2H), 2.41 (dt, J1 = 6.9, J2 = 6.6, 2H),
6.95 (t, J = 3.0, 1H), 7.06 (d, J = 6.9, 2H), 7.28 (m, 4H), 7.43 (s,
1H). HRMS: C32H44Cl3N5O, [M þ H]þ calcd 620.2690, found
620.2687.
Calcium Mobilization Assay. Calcium mobilization was per-
formed in CHO cells coexpressing GR16 protein and the human
CB1 receptor cDNAs. Activation of CB1 receptor leads to
coupling of this receptor to the promiscuous GR16 protein
and consequent mobilization of intracellular calcium. In the
assay, the apparent agonist dissociation equilibrium constant
(Ke) of each compound was determined by running a six-point
half-log 21 concentration response curve in the presence and
absence of a single concentration of antagonist.55 The concen-
tration of antagonist was chosen such that it caused at least a
2-fold increase (shift to the right) in the 21 curve but did not
exceed 10 μM to retain pharmacological relevance. A three-
parameter logistic equation was fit to the concentration response
data with Prism (GraphPad Software; San Diego, CA) to cal-
culate Ke. These values were reported as the mean ( SEM from
at least three independent experiments. 1 was employed as the
positive control (antagonist) for inhibition of CB1 activity.
N-{9-[(9-Aminononyl)(methyl)amino]nonyl}-5-(4-chlorophenyl)-1-
(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (8c).
Following the procedure for the preparation of 7a, 8c was
1
obtained from 20c in 34.9% yield. H NMR (CDCl3) δ 1.20-
1.65 (m, 28H), 2.19 (s, 3H), 2.29 (t, J = 9.0, 4H), 2.38 (s, 3H),
2.67 (s, 3H), 3.41 (dt, J1 = 9.0, J2 = 6.0, 2H), 6.95 (t, J = 3.0,
1H), 7.05 (d, J = 9.0, 2H), 7.28 (m, 4H), 7.43 (s, 1H). HRMS:
C36H52Cl3N5O, [M þ H]þ calcd 676.3316, found 676.3312.
N-{11-[(11-Aminoundecyl)(methyl)amino]undecyl}-5-(4-chloro-
phenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carbox-
amide (8d). Following the procedure for the preparation of 7a,
1
8d was obtained from 20b in 40.6% yield. H NMR (CDCl3)
δ 1.20-1.60 (m, 36H), 2.20 (s, 3H), 2.29 (t, J = 7.8, 4H), 2.38
(s, 3H), 3.40 (dt, J1 = 6.9, J2 = 6.6, 2H), 6.95 (t, J = 3.0, 1H),
7.07 (d, J = 6.6, 2H), 7.29 (m, 4H), 7.43 (s, 1H). HRMS:
C40H60Cl3N5O, [M þ H]þ calcd 732.3942, found 732.3947.
Receptor Binding Assays. CB1 and CB2 Receptor Binding Assays.
The CB1 receptor binding assay involved membranes isolated from
a HEK-293 expression system, whereas the CB2 receptor was
expressed in CHO-K1 cells (Sigma-Aldrich Chemical Co., St. Louis,
MO). The methods used for performing binding assays in trans-
fected cells expressing human CB1 or CB2 receptors were similar to
those previously described for rat brain membrane preparations.26,36
Binding was initiated with the addition of 40 μg of cell membrane
proteins to assay tubes containing [3H]21 (∼130 Ci/mmol) or [3H]1
(∼22.4 Ci/mmol), a test compound (for displacement studies), and a
Acknowledgment. This work was supported by the National
Institutes of Health/National Institute on Drug Abuse
(NIDA) Grants DA019217 (B.F.T.) and DA026582 (Y.Z.)
and NIAAA Grants AA017514 and AA019740 (R.M.).
Supporting Information Available: HPLC data of target
compounds 5a-f, 6a-d, 7a-f, and 8a-d. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) De Petrocellis, L.; Cascio, M. G.; Di Marzo, V. The endocanna-
binoid system: a general view and latest additions. Br. J. Pharmacol.
2004, 141 (5), 765–774.
(2) Pacher, P.; Batkai, S.; Kunos, G. The endocannabinoid system as
an emerging target of pharmacotherapy. Pharmacol. Rev. 2006, 58
(3), 389–462.
(3) Howlett, A. C.; Barth, F.; Bonner, T. I.; Cabral, G.; Casellas, P.;
Devane, W. A.; Felder, C. C.; Herkenham, M.; Mackie, K.;
Martin, B. R.; Mechoulam, R.; Pertwee, R. G. International Union
of Pharmacology. XXVII. Classification of cannabinoid receptors.
Pharmacol. Rev. 2002, 54 (2), 161–202.
sufficient quantity of buffer (50 mM Tris HCl, 1 mM EDTA, 3 mM
3
MgCl2, 5 mg/mL BSA, pH 7.4) to bring the total incubation volume
to 0.5 mL. All assays were performed in polypropylene test tubes. In
the displacement assays, the concentrations of [3H]21 and [3H]1
were 7.2 and 20 nM, respectively. Nonspecific binding was deter-
mined by the inclusion of 10 μM unlabeled 21 or 1. All cannabinoid
analogues were prepared by suspension in buffer A from a 1 mg/mL
ethanol stock. Following incubation at 30 °C for 1 h, binding was

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7059
(4) Mackie, K. Cannabinoid receptor homo- and heterodimerization.
Life Sci. 2005, 77 (14), 1667–1673.
(5) Wager-Miller, J.; Westenbroek, R.; Mackie, K. Dimerization of
G protein-coupled receptors: CB1 cannabinoid receptors as an
example. Chem. Phys. Lipids 2002, 121 (1-2), 83–89.
Synthesis and structure-activity relationships of amide and hy-
drazide analogues of the cannabinoid CB(1) receptor antagonist
N-(piperidinyl)- 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazole-3-carboxami de (SR141716). J. Med. Chem. 2002, 45 (13),
2708–2719.
(27) Thomas, B. F.; Francisco, M. E.; Seltzman, H. H.; Thomas, J. B.;
Fix, S. E.; Schulz, A. K.; Gilliam, A. F.; Pertwee, R. G.; Stevenson,
L. A. Synthesis of long-chain amide analogs of the cannabinoid
CB1 receptor antagonist N-(piperidinyl)-5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-4-methyl-1H-pyra zole-3-carboxamide
(SR141716) with unique binding selectivities and pharmacological
activities. Bioorg. Med. Chem. 2005, 13 (18), 5463–5474.
(28) Dyck, B.; Goodfellow, V. S.; Phillips, T.; Grey, J.; Haddach, M.;
Rowbottom, M.; Naeve, G. S.; Brown, B.; Saunders, J. Potent
imidazole and triazole CB1 receptor antagonists related to
SR141716. Bioorg. Med. Chem. Lett. 2004, 14 (5), 1151–1154.
(29) Palczewski, K.; Kumasaka, T.; Hori, T.; Behnke, C. A.; Motoshima,
H.; Fox, B. A.; Le Trong, I.; Teller, D. C.; Okada, T.; Stenkamp,
R. E.; Yamamoto, M.; Miyano, M. Crystal structure of rhodopsin: a
G protein-coupled receptor. Science 2000, 289 (5480), 739–745.
(30) Portoghese, P. S.; Larson, D. L.; Sayre, L. M.; Yim, C. B.;
Ronsisvalle, G.; Tam, S. W.; Takemori, A. E. Opioid agonist and
antagonist bivalent ligands. The relationship between spacer length
and selectivity at multiple opioid receptors. J. Med. Chem. 1986, 29
(10), 1855–1861.
(31) Neumeyer, J. L.; Zhang, A.; Xiong, W.; Gu, X. H.; Hilbert, J. E.;
Knapp, B. I.; Negus, S. S.; Mello, N. K.; Bidlack, J. M. Design and
synthesis of novel dimeric morphinan ligands for kappa and micro
opioid receptors. J. Med. Chem. 2003, 46 (24), 5162–5170.
(32) Peng, X.; Knapp, B. I.; Bidlack, J. M.; Neumeyer, J. L. Synthesis
and preliminary in vitro investigation of bivalent ligands contain-
ing homo- and heterodimeric pharmacophores at mu, delta, and
kappa opioid receptors. J. Med. Chem. 2006, 49 (1), 256–262.
(33) Soriano, A.; Ventura, R.; Molero, A.; Hoen, R.; Casado, V.;
Cortes, A.; Fanelli, F.; Albericio, F.; Lluis, C.; Franco, R.; Royo,
M. Adenosine A2A receptor-antagonist/dopamine D2 receptor-
agonist bivalent ligands as pharmacological tools to detect A2A-
D2 receptor heteromers. J. Med. Chem. 2009, 52 (18), 5590–5602.
(34) Bonger, K. M.; van den Berg, R. J.; Heitman, L. H.; AP, I. J.;
Oosterom, J.; Timmers, C. M.; Overkleeft, H. S.; van der Marel,
G. A. Synthesis and evaluation of homo-bivalent GnRHR ligands.
Bioorg. Med. Chem. 2007, 15 (14), 4841–4856.
(35) Wiley, J. L.; Jefferson, R. G.; Grier, M. C.; Mahadevan, A.; Razdan,
R. K.; Martin, B. R. Novel pyrazole cannabinoids: insights into
CB(1) receptor recognition and activation. J. Pharmacol. Exp. Ther.
2001, 296 (3), 1013–1022.
(36) Zhang, Y.; Burgess, J. P.; Brackeen, M.; Gilliam, A.; Mascarella,
S. W.; Page, K.; Seltzman, H. H.; Thomas, B. F. Conformationally
constrained analogues of N-(piperidinyl)-5-(4-chlorophenyl)-
1-(2,4- dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide
(SR141716): design, synthesis, computational analysis, and biologi-
cal evaluations. J. Med. Chem. 2008, 51 (12), 3526–3539.
(37) Seltzman, H. H.; Carroll, F. I.; Burgess, J. P.; Wyrick, C. D.; Burch,
D. F. Tritiation of SR141716 by metallation-iodination-reduction:
tritium-proton nOe study. J. Labelled Compd. Radiopharm. 2002,
45 (1), 59–70.
(38) Howes, R.; Barril, X.; Dymock, B. W.; Grant, K.; Northfield, C. J.;
Robertson, A. G.; Surgenor, A.; Wayne, J.; Wright, L.; James, K.;
Matthews, T.; Cheung, K. M.; McDonald, E.; Workman, P.;
Drysdale, M. J. A fluorescence polarization assay for inhibitors
of Hsp90. Anal. Biochem. 2006, 350 (2), 202–213.
(6) George, S. R.; O’Dowd, B. F.; Lee, S. P. G-protein-coupled
receptor oligomerization and its potential for drug discovery.
Nat. Rev. Drug Discovery 2002, 1 (10), 808–820.
(7) Lee, S. P.; O’Dowd, B. F.; Ng, G. Y.; Varghese, G.; Akil, H.;
Mansour, A.; Nguyen, T.; George, S. R. Inhibition of cell surface
expression by mutant receptors demonstrates that D2 dopamine
receptors exist as oligomers in the cell. Mol. Pharmacol. 2000,
58 (1), 120–128.
(8) Franco, R.; Casado, V.; Cortes, A.; Mallol, J.; Ciruela, F.; Ferre,
S.; Lluis, C.; Canela, E. I. G-protein-coupled receptor heteromers:
function and ligand pharmacology. Br. J. Pharmacol. 2008, 153
(Suppl. 1), S90–98.
(9) Rios, C. D.; Jordan, B. A.; Gomes, I.; Devi, L. A. G-protein-
coupled receptor dimerization: modulation of receptor function.
Pharmacol. Ther. 2001, 92 (2-3), 71–87.
(10) Minneman, K. P. Heterodimerization and surface localization of
G protein coupled receptors. Biochem. Pharmacol. 2007, 73 (8),
1043–1050.
(11) Milligan, G. G protein-coupled receptor dimerization: function
and ligand pharmacology. Mol. Pharmacol. 2004, 66 (1), 1–7.
(12) Milligan, G. G-protein-coupled receptor heterodimers: pharma-
cology, function and relevance to drug discovery. Drug Discovery
Today 2006, 11 (11-12), 541–549.
(13) Daniels, D. J.; Lenard, N. R.; Etienne, C. L.; Law, P. Y.; Roerig,
S. C.; Portoghese, P. S. Opioid-induced tolerance and dependence
in mice is modulated by the distance between pharmacophores in a
bivalent ligand series. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (52),
19208–19213.
(14) Portoghese, P. S. From models to molecules: opioid receptor
dimers, bivalent ligands, and selective opioid receptor probes.
J. Med. Chem. 2001, 44 (14), 2259–2269.
(15) Messer, W. S., Jr. Bivalent ligands for G protein-coupled receptors.
Curr. Pharm. Des. 2004, 10 (17), 2015–2020.
(16) Peng, X.; Neumeyer, J. L. Kappa receptor bivalent ligands. Curr.
Top. Med. Chem. 2007, 7 (4), 363–373.
(17) Zheng, W.; Lei, L.; Lalchandani, S.; Sun, G.; Feller, D. R.; Miller,
D. D. Yohimbine dimers exhibiting binding selectivities for human
alpha2a- versus alpha2b-adrenergic receptors. Bioorg. Med. Chem.
Lett. 2000, 10 (7), 627–630.
(18) Lalchandani, S. G.; Lei, L.; Zheng, W.; Suni, M. M.; Moore, B. M.;
Liggett, S. B.; Miller, D. D.; Feller, D. R. Yohimbine dimers
exhibiting selectivity for the human alpha 2C-adrenoceptor sub-
type. J. Pharmacol. Exp. Ther. 2002, 303 (3), 979–984.
(19) Tamiz, A. P.; Bandyopadhyay, B. C.; Zhang, J.; Flippen-Anderson,
J. L.; Zhang, M.; Wang, C. Z.; Johnson, K. M.; Tella, S.; Kozikowski,
A. P. Pharmacological and behavioral analysis of the effects of some
bivalent ligand-based monoamine reuptake inhibitors. J. Med. Chem.
2001, 44 (10), 1615–1622.
(20) Russo, O.; Berthouze, M.; Giner, M.; Soulier, J. L.; Rivail, L.;
Sicsic, S.; Lezoualc’h, F.; Jockers, R.; Berque-Bestel, I. Synthesis of
specific bivalent probes that functionally interact with 5-HT(4)
receptor dimers. J. Med. Chem. 2007, 50 (18), 4482–4492.
(21) Meltzer, P. C.; Kryatova, O.; Pham-Huu, D. P.; Donovan,
P.; Janowsky, A. The synthesis of bivalent 2beta-carbomethoxy-
3beta-(3,4-dichlorophenyl)-8-heterobicyclo[3.2.1]octanes as probes for
proximal binding sites on the dopamine and serotonin transporters.
Bioorg. Med. Chem. 2008, 16 (4), 1832–1841.
(22) Christopoulos, A.; Grant, M. K.; Ayoubzadeh, N.; Kim, O. N.;
Sauerberg, P.; Jeppesen, L.; El-Fakahany, E. E. Synthesis and
pharmacological evaluation of dimeric muscarinic acetylcholine
receptor agonists. J. Pharmacol. Exp. Ther. 2001, 298 (3), 1260–
1268.
(39) Wiley, J. L.; Wang, P.; Mahadevan, A.; Razdan, R. K.; Martin,
B. R. Pyrazole Cannabinoids with Non-CB1, Non-CB2 Activity.
Presented at the 19th Annual Symposium of the International
Cannabinoid Research Society, St. Charles, IL, July 8-11, 2009.
(40) Daniels, D. J.; Kulkarni, A.; Xie, Z.; Bhushan, R. G.; Portoghese,
P. S. A bivalent ligand (KDAN-18) containing delta-antagonist
and kappa-agonist pharmacophores bridges delta2 and kappa1
opioid receptor phenotypes. J. Med. Chem. 2005, 48 (6), 1713–1716.
(41) Xie, Z.; Bhushan, R. G.; Daniels, D. J.; Portoghese, P. S. Interac-
tion of bivalent ligand KDN21 with heterodimeric delta-kappa
opioid receptors in human embryonic kidney 293 cells. Mol.
Pharmacol. 2005, 68 (4), 1079–1086.
(23) Rajeswaran, W. G.; Cao, Y.; Huang, X. P.; Wroblewski, M. E.;
Colclough, T.; Lee, S.; Liu, F.; Nagy, P. I.; Ellis, J.; Levine, B. A.;
Nocka, K. H.; Messer, W. S., Jr. Design, synthesis, and biological
characterization of bivalent 1-methyl-1,2,5,6-tetrahydropyridyl-
1,2,5-thiadiazole derivatives as selective muscarinic agonists.
J. Med. Chem. 2001, 44 (26), 4563–4576.
(42) Portoghese, P. S.; Edward, E. The role of concepts in structure-
activity relationship studies of opioid ligands. J. Med. Chem. 1992,
35 (11), 1927–1937 (Smissman-Bristol-Myers Squibb Award Address) .
(43) Neumeyer, J. L.; Peng, X.; Knapp, B. I.; Bidlack, J. M.; Lazarus,
L. H.; Salvadori, S.; Trapella, C.; Balboni, G. New opioid designed
multiple ligand from Dmt-Tic and morphinan pharmacophores.
J. Med. Chem. 2006, 49 (18), 5640–5643.
(24) Beardsley, P. M.; Thomas, B. F. Current evidence supporting a role
of cannabinoid CB1 receptor (CB1R) antagonists as potential
pharmacotherapies for drug abuse disorders. Behav. Pharmacol.
2005, 16 (5-6), 275–296.
(25) Bifulco, M.; Grimaldi, C.; Gazzerro, P.; Pisanti, S.; Santoro, A.
Rimonabant: just an antiobesity drug? Current evidence on its
pleiotropic effects. Mol. Pharmacol. 2007, 71 (6), 1445–1456.
(26) Francisco, M. E.; Seltzman, H. H.; Gilliam, A. F.; Mitchell, R. A.;
Rider, S. L.; Pertwee, R. G.; Stevenson, L. A.; Thomas, B. F.
(44) Bhushan, R. G.; Sharma, S. K.; Xie, Z.; Daniels, D. J.; Portoghese,
P. S. A bivalent ligand (KDN-21) reveals spinal delta and kappa

7060 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Zhang et al.
opioid receptors are organized as heterodimers that give rise to
delta(1) and kappa(2) phenotypes. Selective targeting of delta-
kappa heterodimers. J. Med. Chem. 2004, 47 (12), 2969–2972.
(45) Bonger, K. M.; van den Berg, R. J.; Knijnenburg, A. D.; Heitman,
L. H.; Ijzerman, A. P.; Oosterom, J.; Timmers, C. M.; Overkleeft,
H. S.; van der Marel, G. A. Synthesis and evaluation of homo-
dimeric GnRHR antagonists having a rigid bis-propargylated
benzene core. Bioorg. Med. Chem. 2008, 16 (7), 3744–3758.
(46) Fulton, B. S.; Knapp, B. L.; Bidlack, J. M.; Neumeyer, J. L. Effect
of linker substitution on the binding of butorphan univalent and
bivalent ligands to opioid receptors. Bioorg. Med. Chem. Lett.
2010, 20 (5), 1507–1509.
(47) Decker, M.; Fulton, B. S.; Zhang, B.; Knapp, B. I.; Bidlack, J. M.;
Neumeyer, J. L. Univalent and bivalent ligands of butorphan:
characteristics of the linking chain determine the affinity and
potency of such opioid ligands. J. Med. Chem. 2009, 52 (23),
7389–7396.
Schrobang, J.; Trankle, C.; Kostenis, E.; Christopoulos, A.; Holtje,
H. D.; Barocelli, E.; De Amici, M.; Holzgrabe, U.; Mohr, K.
Dualsteric GPCR targeting: a novel route to binding and signaling
pathway selectivity. FASEB J. 2009, 23 (2), 442–450.
(50) Valant, C.; Sexton, P. M.; Christopoulos, A. Orthosteric/allosteric
bitopic ligands: going hybrid at GPCRs. Mol. Intervensions 2009,
9 (3), 125–135.
(51) Mohr, K.; Trankle, C.; Kostenis, E.; Barocelli, E.; De Amici, M.;
Holzgrabe, U. Rational design of dualsteric GPCR ligands: quests
and promise. Br. J. Pharmacol. 2010, 159 (5), 997–1008.
(52) Steinfeld, T.; Mammen, M.; Smith, J. A.; Wilson, R. D.; Jasper,
J. R. A novel multivalent ligand that bridges the allosteric and
orthosteric binding sites of the M2 muscarinic receptor. Mol.
Pharmacol. 2007, 72 (2), 291–302.
(53) Narlawar, R.; Lane, J. R.; Doddareddy, M.; Lin, J.; Brussee, J.;
Ijzerman, A. P. Hybrid ortho/allosteric ligands for the adenosine
A(1) receptor. J. Med. Chem. 2010, 53 (8), 3028–3037.
(48) Mathews, J. L.; Fulton, B. S.; Negus, S. S.; Neumeyer,
J. L.; Bidlack, J. M. In vivo characterization of (-)(-)MCL-144
and (þ)(-)MCL-193: isomeric, bivalent ligands with mu/
kappa agonist properties. Neurochem. Res. 2008, 33 (10), 2142–
2150.
(54) Rodriguez-Martin, I.; de Velasco, E. M.; Rodriguez, R. E.
Characterization of cannabinoid-binding sites in zebrafish brain.
Neurosci. Lett. 2007, 413 (3), 249–254.
(55) Kosterlitz, H. W.; Lees, G. M.; Wallis, D. I.; Watt, A. J. Non-
specific inhibitory effects of morphine-like drugs on transmission
in the superior cervical ganglion and guinea-pig isolated ileum.
Br. J. Pharmacol. 1968, 34 (3), 691P–692P.
(49) Antony, J.; Kellershohn, K.; Mohr-Andra, M.; Kebig, A.; Prilla,
S.; Muth, M.; Heller, E.; Disingrini, T.; Dallanoce, C.; Bertoni, S.;