Novel triaryl sulfonamide derivatives as selective cannabinoid receptor 2 inverse agonists and osteoclast inhibitors: Discovery, optimization, and biological evaluation

Article abstract of DOI:10.1021/jm3017464

Cannabinoid receptors have gained increasing attention as drug targets for developing potential therapeutic ligands. Here, we report the discovery and optimization of triaryl sulfonamides as a novel series possessing significant CB₂ receptor affinity and selectivity. Four sets of triaryl ligands were designed and synthesized for further structural modifications and led to the identification of eight compounds as potent and selective CB₂ inverse agonists with high binding affinity (CB₂K_i < 10 nM). Especially, compound 57 exhibited the strongest binding affinity on the CB₂ receptor (CB₂K_i of 0.5 nM) and the best selectivity over the CB₁ receptor (selectivity index of 2594). Importantly, 57 also showed potent inhibitory activity on osteoclast formation, and it was confirmed by a cell viability assay that the inhibition effects were not derived from the cytotoxicity. Finally, 3D QSAR studies confirmed our SAR findings that three bulky groups play an important role for CB₂ receptor binding affinity.

Full text of DOI:10.1021/jm3017464

Article
pubs.acs.org/jmc
Novel Triaryl Sulfonamide Derivatives as Selective Cannabinoid
Receptor 2 Inverse Agonists and Osteoclast Inhibitors: Discovery,
Optimization, and Biological Evaluation
Peng Yang,†,‡ Liping Wang,†,‡ Rentian Feng,†,‡ Abdulrahman A. Almehizia,†,‡ Qin Tong,†,‡
Kyaw-Zeyar Myint,†,‡ Qin Ouyang,†,‡ Mohammed Hamed Alqarni,†,‡ Lirong Wang,†,‡,§
and Xiang-Qun Xie*,†,‡,§,⊥,∥
‡
†Department of Pharmaceutical Sciences and Computational Chemical Genomics Screening Center, School of Pharmacy; Drug
Discovery Institute; §Pittsburgh Chemical Methods and Library Development (CMLD) Center; ⊥Department of Computational and
∥
Systems Biology; and Department of Structural Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States
ABSTRACT: Cannabinoid receptors have gained increasing
attention as drug targets for developing potential therapeutic
ligands. Here, we report the discovery and optimization of triaryl
sulfonamides as a novel series possessing significant CB2 receptor
affinity and selectivity. Four sets of triaryl ligands were designed
and synthesized for further structural modifications and led to the
identification of eight compounds as potent and selective CB2
inverse agonists with high binding affinity (CB2 Ki < 10 nM).
Especially, compound 57 exhibited the strongest binding affinity
on the CB2 receptor (CB2 Ki of 0.5 nM) and the best selectivity over the CB1 receptor (selectivity index of 2594). Importantly,
57 also showed potent inhibitory activity on osteoclast formation, and it was confirmed by a cell viability assay that the inhibition
effects were not derived from the cytotoxicity. Finally, 3D QSAR studies confirmed our SAR findings that three bulky groups play
an important role for CB2 receptor binding affinity.
side effects of CB1 ligands,19 the study of selective CB2 ligands
INTRODUCTION
■
and their therapeutic potentials drew more attention from
Cannabinoids (CB) are a class of diverse chemical compounds,
medicinal chemists. Several reviews,20−24 including the latest
including the endocannabinoids (produced naturally in the
review from our lab,24 summarize the advances of new CB2
ligands from the literature and patents. The most notable CB2-
selective antagonists/inverse agonists are the diarylpyrazole 5
(SR144528)25 and 6-iodopravadoline 6 (AM 630).26 Both
compounds bind with much higher affinity to CB2 than to CB1
receptors and have been extensively used as standards to
measure the specificity of various cannabinoid agonists. Among
these potential and selective CB2 ligands, 7 (JWH-133)27 is a
representative agonist for the nonpsychoactive CB2 receptor
and hence devoid of any psychoactive side effects or abuse
potential, which also showed good activity to decrease
experimental colitis28 and dose-dependently inhibited intra-
venous cocaine self-administration, cocaine-enhanced locomo-
tion, and cocaine-enhanced accumbens extracellular dopa-
mine.29 Another two notable CB2-selective inverse agonists
are 8 (Sch225336)30 and 9 (JTE-907),31 which have
immunomodulatory properties against inflammatory disorders.
Obviously, increasing evidence indicates that the CB2 receptor
is an attractive and promising target for developing potentially
therapeutic ligands.
body by humans and animals, such as 1 and 2, Figure 1),1−3 the
phytocannabinoids (found in cannabis and some other plants,
such as 3),4 and synthetic cannabinoids (produced chemically
by humans, such as 4).5 Before the 1980s, it was often
speculated that cannabinoids produced their physiological and
behavioral effects via nonspecific interaction with cell
membranes, instead of interacting with specific membrane-
bound receptors. The discovery of the first cannabinoid
receptors in the 1980s helped to resolve this debate.6 Two
kinds of cannabinoid receptors have been found to date and are
termed CB1 and CB2. The CB1 receptor is expressed
predominantly in the brain (central receptor for cannabi-
noids),6 and the CB2 receptor in peripheral cells and tissues
derived from the immune system (peripheral receptor for
cannabinoids).7
The endocannabinoid system is known to play a key role in
numerous biological processes and exhibits pharmacological
effects in a large spectrum of diseases and disorders, such as
pain,8 autoimmune and neurodegenerative disorders,9,10
cancer,11,12 osteoporosis,13 stroke,14 inflammation15 and
fibrosis,16 and cardiovascular and gastrointestinal disor-
ders.16−18 Thus, in the past several decades, investigations
were aimed at designing new synthetic molecules that target
cannabinoid receptors. But due to the unfavorable psychiatric
Received: November 27, 2012
Published: February 13, 2013
© 2013 American Chemical Society
2045
dx.doi.org/10.1021/jm3017464 | J. Med. Chem. 2013, 56, 2045−2058

Journal of Medicinal Chemistry
Article
Figure 1. Representative cannabinoids with various chemical scaffolds.
Figure 2. Novel CB2 ligand 10 was discovered within an in vitro high-throughput screening research program and further modified for SAR studies.
(A) Lead compound 10. (B) 10 was validated by [3H]CP-55040 radiometric binding assays showing high CB2 receptor binding affinity: Ki = 192 nM
and CB1/CB2 selectivity (26 fold). (C) Step-by-step medicinal chemistry optimization.
Here we report the identification, optimization, and
therapeutic potential of a novel class of CB2-selective inverse
agonists. Within an in vitro high-throughput screening research
program to discover novel CB2-selective ligands, N-(4-
chlorophenethyl)-4-methyl-N-tosylbenzenesulfonamide (10)
was identified as a novel chemotype with selective CB2 activity
(CB2 Ki = 192 nM, selectivity index of 26-fold, Figure 2). On
the basis of this promising result, we considered 10 as a lead
compound and conducted further medicinal chemistry
structure−activity relationship (SAR) studies. Four series of
compounds were designed, synthesized, and tested in
competition binding activities and effects on both CB2 and
CB1 receptors to define their structure−activity relationships.
The representative compounds were also examined in cAMP
assays on hCB2 CHO cells, with the aim of evaluating their
functionality. Our systematic studies led to the identification of
eight new derivatives (CB2 Ki < 10 nM) as novel CB2 inverse
agonists with improved CB2 binding affinity and selectivity.
Importantly, some showed promising inhibition activity to
osteoclast cells without any sign of toxicity to the osteoclast
precursor.
RESULTS AND DISCUSSION
■
Chemistry. The synthetic routes to obtain the target triaryl
sulfonamide derivatives are outlined in Scheme 1. The
commercially available 4-(diethylamino)benzaldehyde was
reacted with adamantan-1-amine in methanol to give 11 that,
when treated with NaBH4, gave the secondary amine 12.
Finally, the coupling reaction between intermediate 12 and
selected acyl chloride or sulfonyl chloride yielded the
corresponding compounds 20−30. Taking heptan-1-amine, p-
toluidine, or 4-chloroaniline as the starting material, the
synthesis of target compounds 31−44, 45−53, and 54−63
was accomplished using a procedure similar to that utilized for
preparing compounds 20−30. The target compounds were
purified by flash column chromatography.
Pharmacology and SAR Analysis. Taking 10 as the lead
compound, we have carried out structural modification,
designing and synthesizing 46 analogues for further SAR
2046
dx.doi.org/10.1021/jm3017464 | J. Med. Chem. 2013, 56, 2045−2058

Journal of Medicinal Chemistry
Article
a
Scheme 1. General Synthesis of Triaryl Sulfonamide Derivatives
a
Reagents and conditions: (i) adamantan-1-amine, methanol, refluxed, 10 h; (ii) heptan-1-amine, methanol, reflux, 12 h; (iii) p-toluidine or 4-
chloroaniline, methanol, reflux, 12 h; (iv) NaBH4, methanol, rt, 12 h; (v) acyl chloride or sulfonyl chloride, anhydrous DCM, TEA, rt, 12 h.
studies. The CB2 binding affinities of these 46 derivatives were
determined by performing [3H]CP-55,940 radioligand com-
petition binding assays. The CB1 binding assay was also
conducted for those compounds with high CB2 receptor
binding potency (Ki < 200 nM). CB2-selective ligand 1
(SR144528, CB2 inverse agonist) and CB1 ligand 19
(SR141716, CB1 inverse agonist)32 were used as positive
controls along with tested compounds in bioassay experiments.
Among 46 novel triaryl sulfonamide derivatives, 21 compounds
displayed high affinity for the CB2 receptors (Ki < 100 nM) and
10 compounds showed better affinity for the CB2 receptors (Ki
< 10 nM). The chemical structures, binding activities, and
selectivity index are summarized in Tables 1−4.
To get more potent compounds with higher CB2 affinity and
selectivity, we introduced different functional groups to
compound 10. Our SAR strategies to modify the lead
compound included replacing 4-chlorophenethyl with 4-
(diethylamino)benzyl, which has been confirmed as a good
fragment for CB2 ligands in our previous studies,33 retaining
one of the sulfonyl groups, and replacing another p-
toluenesulfonyl with adamantyl, heptyl, p-tolyl, or p-chlor-
obenzyl.
Removal of one p-toluenesulfonyl group (compound 12, CB2
Ki = 19950 nM, Table 1) dramatically decreased the CB2
binding activity, which indicated these three hydrophobic
groups (rings A, B, and C, Figure 2) may be essential for the
activity.
Compared with lead compound 10, replacing 4-chlorophe-
nethyl (ring C) and one p-toluenesulfonyl (ring A) with 4-
(diethylamino)benzyl and adamantyl (compound 24, CB2 Ki =
47 nM, selectivity index = 425) dramatically increased the CB2
binding activity and selectivity. The result indicated this is a
good direction for further modification. Compared to our
previously reported biamide scaffold,32 this new triaryl
sulfonamide scaffold is more stable. Furthermore, several
compounds for which both arylsulfonamide groups had been
replaced showed unsatisfactory results in our high-throughput
screening (data not shown). Thus, the SAR study was first
focused on another p-toluenesulfonyl group (ring B). An initial
set of 11 compounds (20 − 30, Table 1) was synthesized by
replacing p-toluenesulfonyl with different sulfonyl or acyl
groups. Compared with compound 24, removal of the methyl
group (compound 20, CB2 Ki = 84 nM, selectivity index = 130)
slightly decreased the CB2 binding activity and selectivity.
Replacing the methyl group with fluorine (compound 21, CB2
Ki = 25 nM, selectivity index = 170) increased the activity but
decreased the selectivity while replacing the methyl group with
chlorine (compound 22, CB2 Ki = 173 nM, selectivity index =
11) decreased both the activity and the selectivity. To explore
the effect of the substituent size on CB2 binding activity, we
introduced two larger groups, methoxyl (compound 23) and i-
propyl (compound 26), but both compounds showed
decreased activity and selectivity. Interestingly, moving the
methyl group to the meta position gave a promising compound
25 (Figure 3) with increased activity and selectivity (CB2 Ki =
25 nM, selectivity index = 432). Furthermore, we replaced the
p-toluenesulfonyl group with different acyl groups. Replacing p-
toluenesulfonyl with cyclohexanecarbonyl gave another promis-
ing compound 27 (CB2 Ki = 35 nM, selectivity index = 571),
and replacing p-toluenesulfonyl with phenylacetyl or 4-
chlorophenylacetyl showed similar results (compound 29,
CB2 Ki = 38 nM, selectivity index = 526; compound 30, CB2
Ki = 60 nM, selectivity index of >333). Replacing p-
toluenesulfonyl with octanoyl (compound 28, CB2 Ki = 638
nM) showed dramatically decreased activity. These results
(compounds 27−30) indicated that the sulfonyl group may not
be essential for the CB2 binding activity, but a bulky ring group
plays an important role to maintain better binding affinity.
Retaining the 4-(diethylamino)benzyl group, we extended
the SAR studies replacement of two p-toluenesulfonyl groups
2047
dx.doi.org/10.1021/jm3017464 | J. Med. Chem. 2013, 56, 2045−2058

Journal of Medicinal Chemistry
Article
with heptyl and a series of different sulfonyl or acyl groups to
evaluate the effect of a long chain substituent (compounds 31−
44, Table 2). Unfortunately, all of these compounds showed
dramatically decreased CB2 binding activity (CB2 Ki of >212
nM). These results further confirmed that three bulky rings
may be essential for the CB2 binding activity of triaryl
sulfonamide derivatives.
Table 1. Radioligand Competition Binding Affinity (Ki) Data
for Compounds 12, 20−30
Because three bulky ring groups are important for good CB2
binding activity and selectivity, we retained the 4-
(diethylamino)benzyl group and replaced two p-toluenesulfon-
yl groups with p-tolyl or p-chlorobenzyl and a series of different
sulfonyl or acyl groups to further evaluate the effect on receptor
affinity and selectivity of structural modifications on the
benzene ring. Two series of compounds (17, 45−53, Table
3; 54−63, Table 4) were synthesized.
To retain three bulky ring groups and get more potent
compounds with good binding affinity and selectivity, we first
replaced one p-toluenesulfonyl group with p-chlorobenzyl
(compound 49, CB2 Ki = 37 nM, selectivity index = 3.7).
This result indicated that the introduction of p-chlorobenzyl
may enhance the affinity on the CB2 receptor but lose the
selectivity while compound 17, bearing only two bulky ring
groups (CB2 Ki = 6741 nM), showed dramatically decreased
affinity at the CB2 receptor, suggesting the important role of
three bulky ring groups in this series of compounds, as
confirmed above. Compared with compound 49, removing the
methyl group from p-toluenesulfonyl (compound 45, CB2 Ki =
20 nM, selectivity index = 88) improved both the binding
affinity on the CB2 receptor and the selectivity over the CB1
receptor. Replacing methyl with fluorine (compound 46, CB2
Ki = 73 nM, selectivity index = 15) did not lead to significant
effects on affinity and selectivity while better results were
obtained with compound 47 (CB2 Ki = 36 nM, selectivity index
= 183), in which the methyl group was replaced by chlorine,
showing similar binding affinity but increased selectivity.
Interestingly, replacing methyl with methoxyl (compound 48,
CB2 Ki = 14 nM, selectivity index of >1428, Figure 3) showed
a,b
Binding affinities of compounds for CB1 and CB2 receptors were
evaluated using a [3H]CP-55,940 radioligand competition binding
assay. NB = no binding; Ki > 20000 nM. NT = not tested. SI =
selectivity index for CB2, calculated as Ki(CB1)/Ki(CB2) ratio. The
binding affinities of reference compounds were evaluated in parallel
with compounds 12, 20−30 under the same conditions. CB2
reference compound SR144528. CB1 reference compound SR
141716.
c
d
e
f
g
h
Figure 3. Structures of the lead compound 10 and the modified target compounds 25, 48, 50, 56, and 57.
2048
dx.doi.org/10.1021/jm3017464 | J. Med. Chem. 2013, 56, 2045−2058

Journal of Medicinal Chemistry
Article
Table 2. Radioligand Competition Binding Affinity (Ki) Data
for Compounds 31−44
Table 3. Radioligand Competition Binding Affinity (Ki) Data
for Compounds 17, 45−53
a,b
Binding affinities of compounds for CB1 and CB2 receptor were
evaluated using a [3H]CP-55,940 radioligand competition binding
assay. NB = no binding, Ki > 20000 nM. NT = not tested. SI =
selectivity index for CB2, calculated as Ki(CB1)/Ki(CB2) ratio. The
c
d
e
f
binding affinities of reference compounds were evaluated in parallel
g
with compounds 17, 45−53 under the same conditions. CB2
h
reference compound SR144528. CB1 reference compound SR
141716.
a,b
Binding affinities of compounds for CB1 and CB2 receptor were
evaluated using a [3H]CP-55,940 radioligand competition binding
assay. NB = no binding, Ki > 20000 nM. NT = not tested. SI:
selectivity index for CB2, calculated as Ki(CB1)/Ki(CB2) ratio. The
c
d
e
chlorine (compound 56, CB2 Ki = 3.0 nM, selectivity index =
137, Figure 3) resulted in similar affinity and slightly increased
selectivity for the CB2 and CB1 receptors, suggesting that a
variety of substituents on this position was tolerated. Moving
the methyl group to the meta position (compound 59, CB2 Ki
= 5.8 nM, selectivity index = 37) caused a slight decrease in
affinity and selectivity for the CB2 receptor. In contrast,
replacing methyl with a larger group, isopropyl (compound 60,
CB2 Ki = 4.3 nM, selectivity index = 782), led to significant
improvement of selectivity with similar CB2 binding affinity. In
particular, introduction of methoxyl proved to result in the
greatest affinity for the CB2 receptor in this series (compound
57, CB2 Ki = 0.5 nM, Figure 3), with an excellent selectivity
(selectivity index = 2594) while replacing p-toluenesulfonyl
with substituted acyl (compounds 61−63, Table 4) led to a
significant reduction in affinity and selectivity. Together, these
compounds indicate that p-toluenesulfonyl and p-tolyl are a
good combination to improve affinity and selectivity for the
CB2 receptor.
Functional Activity at CB2 Receptors in Vitro. CB2
functional activities of triaryl sulfonamide derivatives were
investigated by using a cell-based LANCE cAMP assay, which is
a useful method to distinguish between agonists, inverse
agonists, and neutral antagonists. A cellular bioassay was carried
out to measure the functional activities of the CB2-selective
compounds, as previously described.34 Briefly, the cell-based
LANCE cAMP assays were performed on 384-well plates using
f
binding affinities of reference compounds were evaluated in parallel
g
with compounds 31−44 under the same conditions. CB2 reference
h
compound SR144528. CB1 reference compound SR 141716.
high affinity and remarkable selectivity at the CB2 receptor.
Similarly to compounds 25 and 26, moving the methyl group
to the meta position gave a promising compound 50 with
greatly increased affinity and selectivity (CB2 Ki = 2.8 nM,
selectivity index = 309, Figure 3), and introduction of a larger
group, isopropyl (compound 51, CB2 Ki = 222 nM), caused a
dramatic loss of affinity for the CB2 receptor. Differently from
compounds 29 and 27, replacing p-toluenesulfonyl with
aromatic 4-chlorophenylacetyl (compound 52, CB2 Ki = 136
nM) or nonaromatic cyclohexanecarbonyl (compound 53, CB2
Ki = 164 nM) showed slightly decreased CB2 binding affinity.
Besides p-chlorobenzyl, we also replaced one p-toluenesul-
fonyl group with p-tolyl and discovered a series of promising
compounds with greatly improved binding affinity (compounds
54−60, CB2 Ki of <10 nM, Table 4). As seen with the p-
chlorobenzyl series, compound 58 (CB2 Ki = 5.4 nM, selectivity
index = 80) shows greater affinity and selectivity for the CB2
receptor than that for lead compound 10. Compared with
compound 58, removing methyl (compound 54, CB2 Ki = 3.4
nM, selectivity index = 151) or replacing methyl with fluorine
(compound 55, CB2 Ki = 5.6 nM, selectivity index = 153) or
2049
dx.doi.org/10.1021/jm3017464 | J. Med. Chem. 2013, 56, 2045−2058

Journal of Medicinal Chemistry
Article
inverse agonists, indicated by increasing forskolin-induced
Table 4. Radioligand Competition Binding Affinity (Ki) Data
for Compounds 54−63
cAMP production, with EC50 values of 268.4
14.5 nM,
16.4 2.84 nM, 608.6 6.06 nM, 42.7 1.35 nM, and 153.8
5.58 nM, respectively. Such a phenomenon was not observed
with agonists CP55940 and HU308, which inhibited cAMP
production with EC50 values of 47.1 3.43 nM and 83.8
5.63 nM, respectively. The results clearly indicated that four top
compounds (21, 48, 54, and 57) indeed behaved as inverse
agonists.
Osteoclast Formation Bioactivity. Osteoporosis is a
degenerative skeletal disease and a serious public health
problem, particularly in postmenopausal women and older
men, which is characterized by reduced bone mass and
increased risk of fractures. Scientists now speculate that the
main physiologic involvement of CB2 receptors is to maintain
bone remodeling in balance, thus protecting the skeleton
against age-related bone loss,35 leading more experts to believe
that cannabinoids may be “a promising target novel target for
antiosteoporotic drug development”.13 On the basis of binding
affinity, selectivity, functionality, and the druglikeness studies
above, three compounds were selected as top candidates to be
evaluated against RANKL-induced osteoclast differentiation on
RAW 264.7 cells. RAW 264.7 is a mouse monocytic cell line
that is used as a standard osteoclast differentiation model. As
shown in Figure 5, we tested the effect of these most promising
CB2 ligands on osteoclast (OCL) formation using RAW 264.7
cells.36 Each ligand tested induced a concentration-dependent
inhibition of osteoclastogenesis, and all of these three
compounds showed strong potency in suppressing OCL
formation at 10 μM, with inhibition rates of >95%. Compounds
48 and 57 also showed good inhibition activity at a low dose of
1 μM (Figure 5). Meanwhile, these results indicated that the
inhibition activities are consistent with the CB2 binding
affinities. Especially, compound 57 showed the strongest
inhibition activity, with inhibition rates of 46%, 97%, and
100% at 0.1, 1, and 10 μM, respectively.
Cytotoxicity Studies of Compounds 48 and 57 Using
RAW 264.7 Cells. Our newly discovered compounds 48 and
57 showed promising inhibition effects on osteoclastogenesis.
To examine whether the impaired osteoclastogenesis in the
presence of compounds 48 and 57 is due to the decrease in
viability of the precursor cells, we investigated the cytotoxicity
profile of these two compounds upon osteoclast precursors
RAW 264.7 by a standard MTT assay. First, RAW 264.7 cells
were plated on 96-well plates and incubated with compounds
48 and 57 for 3 days. The percentage of cell survival was
determined with the MTT assay. The results indicated that the
cell viability was not significantly affected in comparison with
the vehicle control group at 1.25 and 2.5 μM, and only some
effects on cell viability were observed at high concentrations of
5 and 10 μM (Figure 6). The best compound (57) did not
show any cytotoxic effects at 1.25 μM (97% inhibition rate at 1
μM), and only a slight effect on cell viability was observed at a
high concentration of 5 μM. The results suggested that our
compounds possess favorable therapeutic indexes and the
effects of 57 on osteoclast differentiation were not derived from
its cytotoxicity.
a,b
Binding affinities of compounds for CB1 and CB2 receptors were
evaluated using a [3H]CP-55,940 radioligand competition binding
assay. NB = no binding, Ki > 20000 nM. NT = not tested. SI =
selectivity index for CB2, calculated as Ki(CB1)/Ki(CB2) ratio. The
binding affinities of reference compounds were evaluated in parallel
with compounds 54−63 under the same conditions. CB2 reference
compound SR144528. CB1 reference compound SR 141716.
c
d
e
f
g
h
CHO cells stably expressing the CB2 receptors in the presence
of phosphodiesterase inhibitor RO20-1724 and adenyl cyclase
activator forskolin. As shown in Figure 4, reduction of the
LANCE signal occurred with increasing concentrations of
compounds 21, 48, 54, 57, and 5. These ligands acted as
Figure 4. Comparisons of LANCE signal of different CB2 receptor
ligands in stably transfected CHO cells expressing human CB2
receptors in a concentration-dependent fashion. EC50 values of
compounds 21, 48, 54, 57, and 5 are 268.4 14.5 nM, 16.4 2.84
3D QSAR Studies of New CB2 Ligands. 3D QSAR studies
were carried out for the synthesized analogues to correlate
structural and experimental data for further SAR studies by
using our published protocol.37,38 Compound 57 was chosen as
a template compound for QSAR studies given its high CB2
affinity, selectivity, and high inhibition of osteoclastgenesis. In
nM, 608.6
6.06 nM, 42.7
1.35 nM, and 153.8
5.58 nM,
respectively. EC50 for CP-55,940 and HU308 are 47.1 3.43 nM and
83.8 5.63 nM. Data are mean SEM of all experiments of two or
more performed in duplicate or triplicate.
2050
dx.doi.org/10.1021/jm3017464 | J. Med. Chem. 2013, 56, 2045−2058

Journal of Medicinal Chemistry
Article
Figure 5. Antiosteoclastogenesis activity of the top compounds. (A) Compounds 25, 48, and 57 inhibit RANKL-induced osteoclastogenesis in a
dose-dependent manner. RAW 264.7 cells (3 × 103 cells/well) were treated with or without RANKL (15 ng/mL), followed by addition of the
indicated concentrations of 25, 48, and 57 for 5 days and staining for TRAP expression. The data are the mean of three experiments carried out in
triplicate. The bar indicates the SD. (B) Photographs of cells in the test of compound 57 (original magnification 100×).
this study, the 3D QSAR model was built using the comparative
molecular field analysis (CoMFA) method, which is the most
commonly used 3D QSAR technique in lead optimization and
drug discovery. Molecular dynamic and molecular mechanics
(MD/MM) simulations were carried out based on our
published protocol39 to search for preferred conformations of
compound 57 . MD simulations were performed to generate
four families of conformations. These conformations were
compared to the docking pose that resulted from molecular
docking simulations using our in-house 3D CB2 receptor
model.40 One of the conformations that was closest to the
docking pose conformation was used as a template con-
formation. All compounds from the training set (Table 5) and
the test set (Table 6) were aligned to the preferred conformer
of compound 57. The final alignment of the training and test
sets are depicted in Figure 7A,B.
Subsequently, partial least-squares (PLS) analysis was
performed using leave-one-out cross-validation (LOOCV),
which is a cross-validation method in which each training
sample is left out iteratively and systematically in each training
round to determine the optimal number of components, and
the predictive ability of each CoMFA model was determined by
the cross-validated r2:
CoMFA model successfully correlates the structural informa-
tion of synthesized analogues to their CB2 receptor affinity
values. To further evaluate the generated CoMFA model’s
generalization ability, the model was used to predict the binding
affinity values of test set compounds, which were not part of the
training set molecules. The correlation coefficient (r2) for the
test set was 0.947, which demonstrated that the CoMFA model
had a good generalization performance on the test set
molecules. The experimental activities, predicted activities,
and the residuals of the 33 training set molecules and 12 test set
molecules are listed in Tables 5 and 6. The predicted pKi values
are close to the experimental pKi values for molecules in both
the training set and test set. The regression lines of the
experimental and predicted activity of the training and test set
molecules are shown in Figures 8 and 9. The linearity of the
plot indicates a good correlation and the ability of the
developed CoMFA model to predict CB2 receptor binding
affinities of the synthesized analogues.
The steric and electrostatic contour map for the CoMFA
model was then generated to predict the favorable and
unfavorable regions of the new derivatives for CB2 receptor
binding activity. CoMFA contour maps depict the color-coded
steric and electrostatic regions around the molecules that
associate with ligand biological activities. Green regions indicate
favorable steric interactions that enhance binding affinity while
a yellow region indicates unfavorable steric interactions. The
red and blue contours reflect whether electropositive or
electronegative substituents are favored at a particular position.
As shown in Figure 7C, there is a large sterically preferable
region near rings A and B, which means that the introduction of
a bulky hydrophobic group or an aromatic ring in this area will
enhance the CB2 binding affinity. This is consistent with the
structural modification of compounds 50 and 59, which have a
methyl group at the meta position. This is also consistent with
the results of compounds 31−44 bearing an alkyl chain as ring
2
rcv = (SD − PRESS)/SD
where SD is the sum of the squared deviations of between the
biological activity of the molecules and PRESS is the sum of all
the squared deviations between the actual and predicted activity
values. The PLS analysis showed that the optimal number of
components was 4 and rcv2 was 0.577, which was in the range of
generally accepted criterion for statistical validity. Non-cross-
validated PLS analysis was then performed, and the r2 was
0.969 with a standard error of estimate (SEE) of 0.201. Field
contribution of steric and electrostatic fields accounts for 0.502
and 0.498, respectively. Such results indicate that the trained
2051
dx.doi.org/10.1021/jm3017464 | J. Med. Chem. 2013, 56, 2045−2058

Journal of Medicinal Chemistry
Article
Table 5. Experimental (expt) and Predicted (pred) pKi
Values of Triaryl Sulfonamide Derivatives in the Training
Set
compd
pKi (expt)
pKi (pred)
residual
17
20
21
22
24
26
28
29
30
31
32
34
36
38
39
41
42
45
46
47
49
50
51
52
53
54
56
57
58
59
60
61
63
5.171
7.076
7.602
6.762
7.328
6.34
5.135
7.111
7.346
7.017
7.582
6.339
6.114
7.464
7.387
5.765
5.362
5.126
6.095
6.494
6.225
6.027
6.094
8.134
7.435
7.415
7.404
8.448
6.875
6.863
6.574
8.096
8.49
0.0364
0.0348
0.2565
0.2552
0.2535
0.0006
0.0809
0.0439
0.1653
0.2038
0.2755
0.1589
0.1374
0.07
6.195
7.42
7.222
5.561
5.638
5.285
5.958
6.564
6.157
6.157
5.893
7.899
7.137
7.444
7.432
8.553
6.654
6.866
6.785
8.469
8.523
9.301
8.268
8.237
8.367
7.143
6.654
0.0684
0.1298
0.2011
0.2352
0.2975
0.0288
0.0282
0.1055
0.2209
0.0027
0.2113
0.3727
0.0326
0.2077
0.3137
0.1364
0.104
9.093
7.954
8.373
8.471
6.973
6.778
Figure 6. Cytotoxic effect of top compounds 48 (A) and 57 (B) on
osteoclast precursor. RAW 264.7 cells (3 × 103 cells/well) were plated
on 96-well plates. Cells were incubated with the indicated doses of
compounds 48 and 57 for 3 days. The percentage of cell survival was
determined with the MTT assay. The data are the mean SEM of all
experiments carried out in triplicate.
0.17
0.1241
Table 6. Experimental (expt) and Predicted (pred) pKi
Values of Triaryl Sulfonamide Derivatives in the Test Set
B (CB2 Ki of >212 nM) and 54−60 bearing a bulky group as
ring B (CB2 Ki of <6 nM), which indicates that these bulky
groups play an important role to enhance the CB2 binding
affinity. On the contrary, steric interactions are not preferred
near the para position of ring A as highlighted by a yellow
region. This matches the results of compounds 26, 35, and 51
with isopropyl, isopropoxyl, and isopropyl, respectively, which
showed lower or complete loss of CB2 binding activities. On
the other hand, electronegative interactions are preferred near
the para position of ring B as highlighted by a blue region.
However, electropositive interactions are preferred beside the
ortho position of ring C as highlighted by a red region, which is
consistent with our previous CoMFA studies.33 In conclusion,
our CoMFA studies corroborate our SAR results that these
bulky groups play an important role to retain good CB2 binding
activity and selectivity.
compd
pKi (expt)
pKi (pred)
residual
12
23
25
27
33
37
40
43
48
55
57
62
4.7
4.509
6.827
7.541
7.372
5.088
5.578
5.409
6.596
8.137
8.243
9.093
7.587
0.191
6.863
7.721
7.456
4.886
5.171
5.882
6.674
7.854
8.252
9.301
6.971
0.0357
0.18
0.0843
0.2025
0.4071
0.4729
0.0781
0.2828
0.0094
0.2077
0.6157
synthesized 46 analogues and evaluated their binding affinity
and selectivity for the CB2/CB1 receptors in further SAR
studies. Among them, eight compounds exhibited high binding
affinity to the CB2 receptor (CB2 Ki of <10 nM) and good
selectivity over the CB1 receptor (CB1/CB2 of 37- to 2594-
fold). The top four compounds were measured in functional
assays, revealing that the novel series behaves as CB2 receptor
inverse agonists. Compounds 25, 48, and 57 showed potent
CONCLUSION
■
We have discovered N-(4-(diethylamino)benzyl)-4-methoxy-N-
(p-tolyl)benzenesulfonamides as a novel series of CB2 ligands
possessing high binding affinity to the cannabinoid CB2
receptor. To increase selectivity and avoid or reduce potential
CB1-associated CNS adverse effects, we designed and
2052
dx.doi.org/10.1021/jm3017464 | J. Med. Chem. 2013, 56, 2045−2058

Journal of Medicinal Chemistry
Article
Figure 7. Overall alignments of training set molecules (A) and test set molecules (B) to compound 57 as well as CoMFA contour maps of
compound 57 showing steric and electrostatic (C) interactions. Sterically (bulk) favored areas are color-coded in green and sterically unfavored areas
are in yellow. The red and blue contours reflect whether electropositive or electronegative substituents are favored at a particular position.
230−400 mesh). 1H NMR was recorded on a Bruker 400 MHz
spectrometer. Splitting patterns are indicated as follows: s, singlet; d,
doublet; t, triplet; m, multiplet; br, broad peak. Purity of all final
derivatives for biological testing was confirmed to be >95% as
determined using the following conditions: a Shimadzu HPLC
instrument with a Hamilton reversed phase column (HxSil, C18, 3
μm, 2.1 mm × 50 mm (H2)); eluent A consisting of 5% CH3CN in
H2O; eluent B consisting of 90% CH3CN in H2O; flow rate of 0.2
mL/min; UV detection, 254 and 214 nm.
General Procedure for Synthesis of Secondary Amine
Building Blocks. (3s,5s,7s,E)-N-(4-(Diethylamino)benzylidene)-
adamantan-1-amine (11). (3s,5s,7s)-Adamantan-1-amine hydro-
chloride (3.75 g, 20 mmol) was added slowly to a solution of 4-
(diethylamino)benzaldehyde and methanol (50 mL). The mixture was
stirred and refluxed for 12 h. The reaction mixture was cooled to room
temperature, and the solvent was removed by evaporation in vacuum
to give the crude compound 11, which was used in the next step
without further purification.
Figure 8. Plots of CoMFA-calculated and experimental binding affinity
values (pKi) for the training set.
(3s,5s,7s)-N-(4-(Diethylamino)benzyl)adamantan-1-amine (12).
The crude compound 11 was dissolved in methanol (50 mL), and
NaBH4 (1.14 g, 30 mmol) was added. Stirring of the mixture was
continued for 12 h at room temperature. The reaction solution was
poured into water and extracted with EA. The combined organic layers
were washed with water and brine and then dried over Na2SO4. The
mixture was filtered, and the solvent was evaporated in vacuum. The
residue was purified by flash chromatography on silica gel to obtain 12
1
(5.8 g, 88%). H NMR (400 MHz, DMSO-d6) δ 7.13 (d, J = 8.0 Hz,
2H), 6.62 (d, J = 8.0 Hz, 2H), 3.65 (bs, 1H), 3.42−3.48 (m, 2H),
3.28−3.39 (m, 4H), 2.05−2.07 (m, 3H), 1.58−1.71 (m, 12H), 1.07 (t,
J = 6.8 Hz, 6H). LC−MS (ESI): m/z 313.2 (M + H)+.
Figure 9. Plots of CoMFA-calculated and experimental binding affinity
values (pKi) for the test set.
4-(((4-Chlorophenyl)amino)methyl)-N,N-diethylaniline (17).
1
Yield: 78%. H NMR (400 MHz, DMSO-d6) δ 7.03−7.14 (m, 4H),
6.56−6.62 (m, 4H), 6.19−6.22 (m, 1H), 4.06−4.07 (m, 2H), 3.27−
3.34 (m, 4H), 1.06 (t, J = 6.8 Hz, 3H). LC-MS (ESI): m/z 289.0 (M +
H)+.
inhibitory activity on RANKL-induced osteoclast formation.
Especially, 57 showed the strongest inhibition activity even at a
low concentration of 0.1 μM. The cell viability test of 57 on
RAW 264.7 showed very low cytotoxic effects, which confirmed
that the inhibition effects on osteoclasts were not derived from
the cytotoxicity. 3D QSAR studies also confirmed our SAR
findings that three bulky groups are important for CB2 receptor
activity. Overall, this novel series of compounds offers an
attractive starting point for further optimization and represents
a promising lead for the development of a new class of
antiosteoporosis agents.
General Procedure for the Coupling Reaction between
Secondary Amine and Acyl Chloride or Sulfonic Chloride. N-
((3s,5s,7s)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)-
benzenesulfonamide (20). The intermediate 12 (328 mg, 1.0 mmol)
in dichloromethane (DCM, 5 mL) was chilled in ice with the exclusion
of moisture, and then triethylamine (122 mg, 1.2 mmol) was added to
it. The resulting solution was treated dropwise under stirring with
benzenesulfonyl chloride (177 mg, 1.0 mmol), also dissolved in DCM,
over 30 min at 0 °C and then left overnight at room temperature. The
reaction solution was poured into water and extracted with EA. The
combined organic layers were washed with water and brine and then
dried over Na2SO4. The mixture was filtered, and the solvent was
evaporated in vacuum. The residue was purified by flash chromatog-
EXPERIMENTAL SECTION
■
Chemistry. All reagents were purchased from commercial sources
and used without further purification. Analytical thin-layer chromatog-
raphy (TLC) was performed on SiO2 plates on alumina. Visualization
was accomplished by UV irradiation at 254 nm. Flash column
chromatography was performed using the Biotage Isolera flash
purification system with SiO2 60 (particle size 0.040−0.055 mm,
1
raphy on silica gel to obtain 20 (400 mg, 85%). H NMR (400 MHz,
DMSO-d6) δ 7.13 (d, J = 8.0 Hz, 2H), 6.62 (d, J = 8.0 Hz, 2H), 3.65
(bs, 1H), 3.42−3.48 (m, 2H), 3.28−3.39 (m, 4H), 2.05−2.07 (m, 3H),
1.58−1.71 (m, 12H), 1.07 (t, J = 6.8 Hz, 6H). LC−MS (ESI): m/z
453.1 (M + H)+.
2053
dx.doi.org/10.1021/jm3017464 | J. Med. Chem. 2013, 56, 2045−2058

Journal of Medicinal Chemistry
Article
N-((3s,5s,7s)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)-4-flu-
orobenzenesulfonamide (21). Yield: 87%. 1H NMR (400 MHz,
DMSO-d6) δ 7.87−7.89 (m, 2H), 7.38−7.43 (m, 2H), 7.21 (d, J = 8.8
Hz, 2H), 6.64 (d, J = 8.8 Hz, 2H), 4.59 (s, 2H), 3.29−3.36 (m, 4H),
1.88−1.93 (m, 9H), 1.42−1.51 (m, 6H), 1.07−1.10 (m, 6H). LC−MS
(ESI): m/z 471.0 (M + H)+.
2H), 4.14 (s, 2H), 3.28−3.33 (m, 4H), 2.96−3.00 (m, 2H), 2.40 (s,
3H), 1.14−1.24 (m, 4H), 1.04−1.08 (m, 12H), 0.81−0.83 (m, 3H).
LC−MS (ESI): m/z 432.0 (M + H)+.
N-(4-(Diethylamino)benzyl)-N-heptyl-4-isopropylbenzenesulfo-
namide (33). Yield: 81%. 1H NMR (400 MHz, CDCl3) δ 7.77 (d, J =
8.4 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H), 6.61
(d, J = 8.8 Hz, 2H), 4.24−4.27 (m, 2H), 3.34−3.38 (m, 4H), 2.97−
3.32 (m, 3H), 1.10−1.38 (m, 22H), 0.85−0.89 (m, 3H). LC−MS
(ESI): m/z 460.2 (M + H)+.
N-(4-(Diethylamino)benzyl)-N-heptyl-4-methoxybenzenesulfona-
mide (34). Yield: 89%. 1H NMR (400 MHz, CDCl3) δ 7.77−7.80 (m,
2H), 7.08 (d, J = 8.4 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 6.61 (d, J = 8.4
Hz, 2H), 4.22 (s, 2H), 3.90 (s, 3H), 3.33−3.38 (m, 4H), 3.05−3.09
(m, 2H), 1.15−1.40 (m, 16H), 0.83−0.89 (m, 3H). LC−MS (ESI):
m/z 448.2 (M + H)+.
N-(4-(Diethylamino)benzyl)-N-heptyl-4-isopropoxybenzenesulfo-
namide (35). Yield: 61%. 1H NMR (400 MHz, DMSO-d6) δ 7.76 (d, J
= 8.08 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 6.60
(d, J = 8.0 Hz, 2H), 4.10 (s, 2H), 3.86 (s, 3H), 3.28−3.34 (m, 4H),
2.93−2.96 (m, 2H), 1.03−1.23 (m, 20H), 0.79−0.83 (m, 3H). LC−
MS (ESI): m/z 475.4 (M + H)+.
N-(4-(Diethylamino)benzyl)-4-fluoro-N-heptylbenzenesulfona-
mide (36). Yield: 77%. 1H NMR (400 MHz, CDCl3) δ 7.83−7.87 (m,
2H), 7.16−7.21 (m, 2H), 7.05 (d, J = 8.4 Hz, 2H), 6.60 (d, J = 8.4 Hz,
2H), 4.25 (s, 2H), 3.33−3.38 (m, 4H), 3.08−3.13 (m, 2H), 1.02−1.40
(m, 16H), 0.88−0.92 (m, 3H). LC−MS (ESI): m/z 436.0 (M + H)+.
4-Chloro-N-(4-(diethylamino)benzyl)-N-heptylbenzenesulfona-
mide (37). Yield: 83%. 1H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 8.4
Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 8.8 Hz, 2H), 6.60 (d, J
= 8.4 Hz, 2H), 4.25 (s, 2H), 3.33−3.38 (m, 4H), 3.08−3.12 (m, 2H),
1.04−1.42 (m, 16H), 0.88−0.92 (m, 3H). LC−MS (ESI): m/z 450.7
(M + H)+.
N-((3s,5s,7s)-Adamantan-1-yl)-4-chloro-N-(4-(diethylamino)-
1
benzyl)benzenesulfonamide (22). Yield: 92%. H NMR (400 MHz,
DMSO-d6) δ 7.83 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.8 Hz, 2H), 7.21
(d, J = 8.8 Hz, 2H), 6.65 (d, J = 8.8 Hz, 2H), 4.60 (s, 2H), 3.29−3.37
(m, 4H), 1.88−1.93 (m, 9H), 1.42−1.51 (m, 6H), 1.07−1.11 (m, 6H).
LC−MS (ESI): m/z 487.1 (M + H)+.
N-((3r)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)-4-methoxy-
benzenesulfonamide (23). Yield: 89%. 1H NMR (400 MHz, DMSO-
d6) δ 7.72−7.74 (m, 2H), 7.21 (d, J = 8.8 Hz, 2H), 7.07−7.10 (m,
2H), 6.64 (d, J = 8.8 Hz, 2H), 4.56 (s, 2H), 3.85 (s, 3H), 3.29−3.33
(m, 4H), 1.87−1.92 (m, 9H), 1.42−1.50 (m, 6H), 1.07−1.10 (m, 6H).
LC−MS (ESI): m/z 483.0 (M + H)+.
N-((3s,5s,7s)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)-4-
1
methylbenzenesulfonamide (24). Yield: 86%. H NMR (400 MHz,
DMSO-d6) δ 7.70 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 7.23
(d, J = 8.8 Hz, 2H), 6.65 (d, J = 8.8 Hz, 2H), 4.59 (s, 2H), 3.30−3.42
(m, 4H), 2.40 (s, 3H), 1.87−1.92 (m, 9H), 1.40−1.50 (m, 6H), 1.03−
1.12 (m, 6H). LC−MS (ESI): m/z 467.2 (M + H)+.
N-((3s,5s,7s)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)-3-
1
methylbenzenesulfonamide (25). Yield: 84%. H NMR (400 MHz,
DMSO-d6) δ 7.58−7.62 (m, 2H), 7.42−7.48 (m, 2H), 7.23 (d, J = 8.4
Hz, 2H), 6.65 (d, J = 8.4 Hz, 2H), 4.60 (s, 2H), 3.30−3.34 (m, 4H),
2.39 (s, 3H), 1.87−1.92 (m, 9H), 1.41−1.50 (m, 6H), 1.07−1.11 (m,
6H). LC−MS (ESI): m/z 467.1 (M + H)+.
N-((3s,5s,7s)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)-4-iso-
1
propylbenzenesulfonamide (26). Yield: 71%. H NMR (400 MHz,
DMSO-d6) δ 7.72 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.20
(d, J = 8.8 Hz, 2H), 6.64 (d, J = 8.8 Hz, 2H), 4.57 (s, 2H), 3.27−3.29
(m, 4H), 2.97−3.01 (m, 1H), 1.89−1.92 (m, 9H), 1.41−1.51 (m, 6H),
1.19−1.24 (m, 6H), 1.08−1.10 (m, 6H). LC−MS (ESI): m/z 495.2
(M + H)+.
N-((3s,5s,7s)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)-
cyclohexanecarboxamide (27). Yield: 89%. 1H NMR (400 MHz,
CDCl3) δ 7.03 (d, J = 8.8 Hz, 2H), 6.68 (d, J = 8.8 Hz, 2H), 4.49 (s,
2H), 3.35−3.40 (m, 4H), 2.36−2.42 (m, 1H), 2.22 (d, J = 2.4 Hz,
6H), 2.03−2.07 (m, 3H), 1.54−1.76 (m, 14H), 1.18−1.21 (m, 8H).
LC−MS (ESI): m/z 423.5 (M + H)+.
N-((3s,5s,7s)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)-
octanamide (28). Yield: 81%. 1H NMR (400 MHz, CDCl3) δ 7.05 (d,
J = 8.4 Hz, 2H), 6.68 (d, J = 8.8 Hz, 2H), 4.51 (s, 2H), 3.34−3.39 (m,
4H), 2.25−2.33 (m, 8H), 2.05 (s, 3H), 1.60−1.70 (m, 8H), 1.17−1.31
(m, 14H), 0.88 (t, J = 6.8 Hz, 3H). LC−MS (ESI): m/z 439.4 (M +
H)+.
N-((3s,5s,7s)-Adamantan-1-yl)-2-(4-chlorophenyl)-N-(4-
(diethylamino)benzyl)acetamide (29). Yield: 79%. 1H NMR (400
MHz, CDCl3) δ 7.28 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.4 Hz, 2H),
7.09 (d, J = 8.4 Hz, 2H), 6.71 (d, J = 8.8 Hz, 2H), 4.50 (s, 2H), 3.62
(s, 2H), 3.36−3.41 (m, 4H), 2.27 (s, 6H), 2.06−2.07 (m, 3H), 1.60−
1.70 (m, 6H), 1.21 (t, J = 7.2 Hz, 6H). LC−MS (ESI): m/z 465.2 (M
+ H)+.
N-(4-(Diethylamino)benzyl)-N-heptyl-1-phenylmethanesulfona-
mide (38). Yield: 63%. 1H NMR (400 MHz, CDCl3) δ 7.35−7.40 (m,
5H), 7.16 (d, J = 8.4 Hz, 2H), 6.65 (d, J = 8.4 Hz, 2H), 4.05−4.25 (m,
4H), 3.34−3.39 (m, 4H), 2.93−2.96 (m, 2H), 1.03−1.41 (m, 16H),
0.88−0.92 (m, 3H). LC−MS (ESI): m/z 431.2 (M + H)+.
N-(4-(Diethylamino)benzyl)-N-heptylbutane-1-sulfonamide (39).
1
Yield: 60%. H NMR (400 MHz, CDCl3) δ 7.19 (d, J = 8.4 Hz, 2H),
6.66 (d, J = 8.8 Hz, 2H), 4.31 (s, 2H), 3.40−3.40 (m, 4H), 3.14−3.16
(m, 2H), 2.88−2.92 (m, 2H), 1.77−1.81 (m, 2H), 0.88−1.57 (m,
24H). LC−MS (ESI): m/z 398.0 (M + H)+.
N-(4-(Diethylamino)benzyl)-N-heptyl-2-phenylacetamide (40).
1
Yield: 70%. H NMR (400 MHz, DMSO-d6) δ 7.25−7.33 (m, 5H),
7.08 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 6.65−6.69 (m, 2H),
4.47−4.51 (m, 2H), 3.81−3.82 (m, 2H), 3,31−3.40 (m, 5H), 3.24−
3.28 (m, 1H), 1.12−1.29 (m, 16H), 0.88−0.92 (m, 3H). LC−MS
(ESI): m/z 396.1 (M + H)+.
2-(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)-N-heptylaceta-
1
mide (41). Yield: 65%. H NMR (400 MHz, DMSO-d6) δ 7.16−7.35
(m, 4H), 7.17 (d, J = 8.4 Hz, 1H), 7.08 (d, J = 6.4 Hz, 1H), 6.66−6.71
(m, 2H), 4.51 (s, 2H), 3.80−3.81 (m, 2H), 3,35−3.39 (m, 5H), 3.26−
3.28 (m, 1H), 1.14−1.67 (m, 16H), 0.89−0.92 (m, 3H). LC−MS
(ESI): m/z 428.8 (M + H)+.
N-(4-(Diethylamino)benzyl)-4-(dimethylamino)-N-heptylbenza-
mide (42). Yield: 80%. 1H NMR (400 MHz, CDCl3) δ 7.37 (d, J = 8.4
Hz, 2H), 7.09 (bs, 2H), 6.64−6.66 (m, 4H), 4.55 (s, 2H), 3.32−3.37
(m, 6H), 2.98 (s, 6H), 1.57−1.61 (m, 2H), 1.14−1.28 (m, 14H), 0.86
(t, J = 7.2 Hz, 6H). LC−MS (ESI): m/z 424.4 (M + H)+.
N-(4-(Diethylamino)benzyl)-N-heptylcyclohexanecarboxamide
(43). Yield: 91%. 1H NMR (400 MHz, CDCl3) δ 6.97−7.06 (m, 4H),
6.59−6.66 (m, 4H), 4.11−4.46 (m, 2H), 3.14−3.37 (m, 6H), 2.47−
2.51 (m, 1H), 1.51−1.82 (m, 10H), 1.01−1.49 (m, 16H), 0.84−0.90
(m, 3H). LC−MS (ESI): m/z 387.1 (M + H)+.
N-(4-(Diethylamino)benzyl)-N-heptyloctanamide (44). Yield:
95%. 1H NMR (400 MHz, MeOD) δ 7.02−7.09 (m, 2H), 6.67−
6.87 (m, 2H), 4.49 (s, 2H), 3.24−3.41 (m, 6H), 2.43 (t, J = 7.2 Hz,
2H), 1.13−1.68 (m, 26H), 0.89−0.95 (m, 3H). LC−MS (ESI): m/z
402.9 (M + H)+.
N-((3s,5s,7s)-Adamantan-1-yl)-N-(4-(diethylamino)benzyl)-2-
1
phenylacetamide (30). Yield: 70%. H NMR (400 MHz, CDCl3) δ
7.29−7.32 (m, 2H), 7.22−7.26 (m, 3H), 7.10 (d, J = 8.4 Hz, 2H), 6.71
(d, J = 8.8 Hz, 2H), 4.50 (s, 2H), 3.67 (s, 2H), 3.36−3.41 (m, 4H),
2.28 (s, 6H), 2.06−2.07 (m, 3H), 1.60−1.70 (m, 6H), 1.21 (t, J = 7.2
Hz, 6H). LC−MS (ESI): m/z 431.1 (M + H)+.
N-(4-(Diethylamino)benzyl)-N-heptyl-4-methylbenzenesulfona-
mide (31). Yield: 87%. 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 8.4
Hz, 2H), 7.29−7.33 (m, 2H), 7.09 (d, J = 8.8 Hz, 2H), 6.61 (d, J = 8.4
Hz, 2H), 4.23 (s, 2H), 3.33−3.38 (m, 4H), 3.06−3.09 (m, 2H), 2.46
(s, 3H), 1.17−1.39 (m, 16H), 1.10−1.15 (m, 3H). LC−MS (ESI): m/
z 430.7 (M + H)+.
N-(4-(Diethylamino)benzyl)-N-heptyl-3-methylbenzenesulfona-
1
mide (32). Yield: 89%. H NMR (400 MHz, DMSO-d6) δ 7.61 (bs,
N - ( 4 - C h l o r o p h e n y l ) - N - ( 4 -( di e thy la m in o)b e n zy l) -
2H), 7.49−7.50 (m, 2H), 7.06 (d, J = 8.4 Hz, 2H), 6.60 (d, J = 8.4 Hz,
benzenesulfonamide (45). Yield: 73%. 1H NMR (400 MHz, DMSO-
2054
dx.doi.org/10.1021/jm3017464 | J. Med. Chem. 2013, 56, 2045−2058

Journal of Medicinal Chemistry
Article
d6) δ 7.64−7.73 (m, 5H), 7.32−7.34 (m, 2H), 6.96−7.05 (m, 4H),
6.50 (d, J = 7.2 Hz, 2H), 4.63 (s, 2H), 3.23−3.26 (m, 4H), 1.02−1.04
(m, 6H). LC−MS (ESI): m/z 428.9 (M + H)+.
2H), 6.96 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.4 Hz, 2H), 6.49 (d, J = 8.8
Hz, 2H), 4.56 (s, 2H), 3.86 (s, 3H), 3.22−3.27 (m, 4H), 2.23 (s, 3H),
1.02 (t, J = 7.2 Hz, 6H). LC−MS (ESI): m/z 439.1 (M + H)+.
N-(4-(Diethylamino)benzyl)-4-methyl-N-(p-tolyl)-
benzenesulfonamide (58). Yield: 94%. 1H NMR (400 MHz, DMSO-
d6) δ 7.50 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 8.0
Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 6.49 (d, J
= 8.8 Hz, 2H), 4.57 (s, 2H), 3.22−3.27 (m, 4H), 2.42 (s, 3H), 2.22 (s,
3H), 1.02 (t, J = 7.2 Hz, 6H). LC−MS (ESI): m/z 423.0 (M + H)+.
N-(4-(Diethylamino)benzyl)-3-methyl-N-(p-tolyl)-
benzenesulfonamide (59). Yield: 72%. 1H NMR (400 MHz, DMSO-
d6) δ 7.37−7.51 (m, 4H), 7.05 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 8.4 Hz,
2H), 6.86−6.88 (m, 2H), 6.50 (d, J = 8.8 Hz, 2H), 4.59 (s, 2H), 3.22−
3.28 (m, 4H), 2.39 (s, 3H), 2.23 (s, 3H), 1.03 (t, J = 7.2 Hz, 6H). LC−
MS (ESI): m/z 423.4 (M + H)+.
N-(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)-4-fluorobenze-
1
nesulfonamide (46). Yield: 69%. H NMR (400 MHz, DMSO-d6) δ
7.68−7.70 (m, 2H), 7.44−7.49 (m, 2H), 7.33 (d, J = 8.8 Hz, 2H), 7.06
(d, J = 8.4 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 6.50 (d, J = 8.8 Hz, 2H),
4.62 (s, 2H), 3.22−3.28 (m, 4H), 1.02 (t, J = 6.8 Hz, 6H). LC−MS
(ESI): m/z 446.9 (M + H)+.
4-Chloro-N-(4-chlorophenyl)-N-(4-(diethylamino)benzyl)-
benzenesulfonamide (47). Yield: 65%. 1H NMR (400 MHz, DMSO-
d6) δ 7.70 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.8
Hz, 2H), 7.07 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 6.50 (d, J
= 8.8 Hz, 2H), 4.62 (s, 2H), 3.22−3.28 (m, 4H), 1.01−1.06 (m, 6H).
LC−MS (ESI): m/z 463.0 (M + H)+.
N-(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)-4-methoxyben-
zenesulfonamide (48). Yield: 55%. 1H NMR (400 MHz, DMSO-d6) δ
7.56 (d, J = 8.8 Hz, 2H), 7.32 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 8.8 Hz,
2H), 7.05 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 6.50 (d, J = 8.8
Hz, 2H), 4.58 (s, 2H), 3.86 (s, 3H), 3.22−3.27 (m, 4H), 1.02 (t, J =
6.8 Hz, 6H). LC−MS (ESI): m/z 459.1 (M + H)+.
N-(4-(Diethylamino)benzyl)-4-isopropyl-N-(p-tolyl)-
benzenesulfonamide (60). Yield: 86%. 1H NMR (400 MHz, DMSO-
d6) δ 7.54−7.56 (m, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 8.0 Hz,
2H), 6.96 (d, J = 8.4 Hz, 2H), 6.89 (d, J = 8.4 Hz, 2H), 6.49 (d, J = 8.8
Hz, 2H), 4.58 (s, 2H), 3.22−3.28 (m, 4H), 2.98−3.05 (m, 1H), 2.23
(s, 3H), 1.25 (d, J = 7.2 Hz, 6H), 1.02 (t, J = 7.2 Hz, 6H). LC−MS
(ESI): m/z 450.9 (M + H)+.
N-(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)-4-methylbenze-
1
nesulfonamide (49). Yield: 79%. H NMR (400 MHz, DMSO-d6) δ
N-(4-(Diethylamino)benzyl)-2-phenyl-N-(p-tolyl)acetamide (61).
1
7.31−7.51 (m, 6H), 7.03−7.05 (m, 2H), 6.96 (d, J = 8.8 Hz, 2H), 6.49
(d, J = 8.8 Hz, 2H), 4.59 (s, 2H), 3.22−3.27 (m, 4H), 2,42 (s, 3H),
1.02 (t, J = 6.8 Hz, 6H). LC−MS (ESI): m/z 443.2 (M + H)+.
N-(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)-3-methylbenze-
Yield: 86%. H NMR (400 MHz, DMSO-d6) δ 7.17−7.27 (m, 5H),
7.05 (d, J = 7.2 Hz, 2H), 6.97 (d, J = 8.0 Hz, 2H), 6.91 (d, J = 8.8 Hz,
2H), 6.54 (d, J = 8.8 Hz, 2H), 4.69 (s, 2H), 3.35−3.40 (m, 2H), 3.25−
3.31 (m, 4H), 2.30 (s, 3H), 1.05 (t, J = 7.2 Hz, 6H). LC−MS (ESI):
m/z 387.5 (M + H)+.
1
nesulfonamide (50). Yield: 65%. H NMR (400 MHz, DMSO-d6) δ
7.38−7.53 (m, 4H), 7.32 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H),
6.96 (d, J = 8.8 Hz, 2H), 6.50 (d, J = 8.8 Hz, 2H), 4.61 (s, 2H), 3.22−
3.28 (m, 4H), 2,40 (s, 3H), 1.02 (t, J = 6.8 Hz, 6H). LC−MS (ESI):
m/z 443.0 (M + H)+.
N-(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)-4-isopropylben-
zenesulfonamide (51). Yield: 89%. 1H NMR (400 MHz, DMSO-d6) δ
7.56 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.8 Hz,
2H), 7.06 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 6.50 (d, J = 8.8
Hz, 2H), 4.61 (s, 2H), 3.23−3.26 (m, 4H), 2.98−3.05 (m, 1H), 1.25
(d, J = 6.8 Hz, 6H), 1.03 (t, J = 6.4 Hz, 6H). LC−MS (ESI): m/z
471.1 (M + H)+.
2-(4-Chlorophenyl)-N-(4-(diethylamino)benzyl)-N-(p-tolyl)-
1
acetamide (62). Yield: 86%. H NMR (400 MHz, DMSO-d6) δ 7.31
(d, J = 8.4 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 7.08 (d, J = 8.0 Hz, 2H),
6.98 (d, J = 8.0 Hz, 2H), 6.91 (d, J = 8.4 Hz, 2H), 6.54 (d, J = 8.8 Hz,
2H), 4.68 (s, 2H), 3.35−3.38 (m, 2H), 3.25−3.32 (m, 4H), 2.30 (s,
3H), 1.05 (t, J = 7.2 Hz, 6H). LC−MS (ESI): m/z 420.7 (M + H)+.
N-(4-(Diethylamino)benzyl)-N-(p-tolyl)cyclohexanecarboxamide
1
(63). Yield: 84%. H NMR (400 MHz, DMSO-d6) δ 7.18 (d, J = 8.0
Hz, 2H), 6.94 (d, J = 8.0 Hz, 2H), 6.89 (d, J = 8.4 Hz, 2H), 6.54 (d, J
= 8.4 Hz, 2H), 4.63 (s, 2H), 3.26−3.31 (m, 4H), 2.30 (s, 3H), 2.08−
2.14 (m, 1H), 1.35−1.62 (m, 8H), 0.86−1.09 (m, 8H). LC−MS
(ESI): m/z 379.5 (M + H)+.
N,2-Bis(4-chlorophenyl)-N-(4-(diethylamino)benzyl)acetamide
1
(52). Yield: 79%. H NMR (400 MHz, DMSO-d6) δ 7.44 (d, J = 8.4
Radioligand Competition Binding Assays. A CB ligand
competition binding assay was carried out as described previously.33,41
Briefly, nonradioactive ligands were diluted in binding buffer and
supplemented with 10% dimethyl sulfoxide and 0.4% methyl cellulose.
Each assay plate well contained a total of 200 μL of reaction mixture
composed of 5 μg of CB1 (or CB2) membrane protein, labeled
[3H]CP-55,940 ligand at a final concentration of 3 nM, and the
unlabeled ligand at variable dilutions as stated above. Plates were
incubated at 30 °C for 1 h with gentle shaking. The reaction was
terminated by rapid filtration through Unifilter GF/B filter plates using
a Unifilter Cell Harvester (PerkinElmer). After the plate was allowed
to dry overnight, 30 μL of MicroScint-0 cocktail (PerkinElmer) was
added to each well and the radioactivity was counted by using a
PerkinElmer TopCounter. All assays were performed in duplicate and
data points represented as mean SEM. Bound radioactivity data was
analyzed for Ki values using nonlinear regression analysis via GraphPad
Prism 5.0 software.
Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.09−7.14 (m, 4H), 6.91 (d, J = 8.8
Hz, 2H), 6.54 (d, J = 8.8 Hz, 2H), 4.71 (s, 2H), 3.42 (s, 2H), 3.25−
3.31 (m, 4H), 1.05 (t, J = 6.4 Hz, 6H). LC−MS (ESI): m/z 442.8 (M
+ H)+.
N-( 4-Ch lo rop henyl )-N-(4 - (d iet hyl ami no) benz yl) -
cyclohexanecarboxamide (53). Yield: 68%. 1H NMR (400 MHz,
DMSO-d6) δ 7.44 (d, J = 8.4 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H), 6.89
(d, J = 8.4 Hz, 2H), 6.55 (d, J = 8.4 Hz, 2H), 4.65 (s, 2H), 3.26−3.31
(m, 4H), 2.08 (bs, 1H), 1.36−1.63 (m, 7H), 0.93−1.13 (m, 9H). LC−
MS (ESI): m/z 399.4 (M + H)+.
N-(4-(Diethylamino)benzyl)-N-(p-tolyl)benzenesulfonamide (54).
1
Yield: 80%. H NMR (400 MHz, DMSO-d6) δ 7.60−7.72 (m, 5H),
7.04 (d, J = 8.0 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 8.4 Hz,
2H), 6.50 (d, J = 8.8 Hz, 2H), 4.60 (s, 2H), 3.23−3.28 (m, 4H), 2.23
(s, 3H), 1.03 (t, J = 7.2 Hz, 6H). LC−MS (ESI): m/z 408.9 (M + H)+.
N-(4-(Diethylamino)benzyl)-4-fluoro-N-(p-tolyl)-
benzenesulfonamide (55). Yield: 83%. 1H NMR (400 MHz, DMSO-
d6) δ 7.66−7.68 (m, 2H), 7.43−7.47 (m, 2H), 7.06 (d, J = 8.0 Hz,
2H), 6.96 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.4 Hz, 2H), 6.50 (d, J = 8.8
Hz, 2H), 4.60 (s, 2H), 3.22−3.30 (m, 4H), 2.23 (s, 3H), 1.02 (t, J =
7.2 Hz, 6H). LC−MS (ESI): m/z 427.2 (M + H)+.
The saturation binding of [3H]CP-55,940 to the membrane
proteins was performed as described previously.33,34 Briefly, the CB1
(or CB2) membrane fractions (5 μg) were incubated with increasing
concentrations of [3H]CP-55,940 (0.05−4 nM) in 96-well plates at 30
°C with slow shaking for 1 h. The incubation buffer was composed of
50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 2.5 mM EGTA, and 0.1%
(w/v) fatty acid free BSA. Ligand was diluted in incubation buffer
supplemented with 10% dimethyl sulfoxide and 0.4% methyl cellulose.
Nonspecific binding was determined in the presence of unlabeled CP-
55,940 (5000 nM). The reaction was terminated, and the radioactivity
was counted as stated above. Nonlinear regression analysis revealed
the receptor density (Bmax) and the equilibrium dissociation constant
(Kd) values of [3H]CP-55,940 for the CB2 receptor.
4-Chloro-N-(4-(diethylamino)benzyl)-N-(p-tolyl)-
benzenesulfonamide (56). Yield: 85%. 1H NMR (400 MHz, DMSO-
d6) δ 7.67−7.70 (m, 2H), 7.61−7.63 (m, 2H), 7.06 (d, J = 8.0 Hz,
2H), 6.96 (d, J = 8.8 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 6.50 (d, J = 8.8
Hz, 2H), 4.60 (s, 2H), 3.22−3.28 (m, 4H), 2.23 (s, 3H), 1.02 (t, J =
7.2 Hz, 6H). LC−MS (ESI): m/z 442.8 (M + H)+.
N-(4-(Diethylamino)benzyl)-4-methoxy-N-(p-tolyl)-
benzenesulfonamide (57). Yield: 91%. 1H NMR (400 MHz, DMSO-
d6) δ 7.53−7.56 (m, 2H), 7.11−7.13 (m, 2H), 7.04 (d, J = 8.4 Hz,
2055
dx.doi.org/10.1021/jm3017464 | J. Med. Chem. 2013, 56, 2045−2058

Journal of Medicinal Chemistry
Article
cAMP Assays. Cellular cAMP levels were measured according to
reported method with modifications using LANCE cAMP 384 kits
(PerkinElmer).33,34 The assay is based on competition between a
europium-labeled cAMP trace complex and total cAMP for binding
sites on cAMP-specific antibodies labeled with a fluorescent dye. CB2
receptor wild type (WT) transfected CHO cells were seeded in 384-
well white ProxiPlates with a density of 2000 cells per well in 5 μL of
RPMI-1640 medium containing 1% dialyzed FBS, 25 mM HEPES, 100
μg/mL penicillin, 100 U/mL streptomycin, and 200 μg/mL of G-418.
After culture overnight, 2.5 μL of cAMP antibody and RO20-1724
(final concentration, 50 μM) in stimulation buffer (DPBS 1x,
containing 0.1% BSA) was added to each well, followed by addition
of either 2.5 μL of compound or forskolin (final 5 μM) for an agonist-
inhibited adenylate cyclase (AC) activity assay. After incubation at
room temperature for 45 min, 10 μL of detection reagent was added
into each well. The plate was then incubated for 1 h at room
temperature and measured in a Synergy H1 hybrid reader (BioTek)
with excitation at 340 nm and emission at 665 nm. Each cAMP
determination was made via at least two independent experiments,
each in triplicate. EC50 values were determined by nonlinear
regression, dose−response curves (GraphPad Prism 5).
In Vitro Osteoclast Formation Assay. RAW 264.7 cells were
seeded at 3 × 103 cells per well using 96-well multiplates and cultured
for 24 h in αMEM with 10% fetal bovine serum (FBS). Thereafter, the
cell medium was replaced with same medium containing RANKL (15
ng/mL) and various concentrations of tested compounds. After 5 days,
cells were fixed and stained for tartrate-resistant acid phosphate
(TRAP) (Sigma) activity according to the recommendation of the
manufacturer. TRAP+ multinucleated cells with more than three nuclei
were counted as osteoclasts.42,43
Cytotoxicity Assay of Top Compounds on Precursor Osteoclasts.
To study whether the inhibitory effects of our compounds on
osteoclast development are due to their direct cytotoxicity on RAW
264.7 cells, we performed a cell proliferation assay. RAW 264.7 cells (3
× 103 cells) were plated on 96-well plates, treated with the indicated
concentrations of top compounds, and then incubated for 3 days. The
percentage of cell survival was determined with the MTT assay as
described previously.33,44
3D QSAR CoMFA Studies. Out of 46 compounds from Tables 1−4,
44 compounds were used in the CoMFA QSAR studies. Two
compounds that showed no binding were ignored in the analysis.
Approximately 75% (33 compounds) and 25% (11 compounds) were
randomly selected as a training set and test set, respectively. SYBYL-X
1.3 was used for the QSAR studies and analysis. Using our established
protocol, molecular dynamic simulations were carried out for our best
compound 57. Briefly, dynamic simulations were simulated at 300 K
with a time steps of 1 fs for a total duration of 300 ps, and
conformation samples were collected at every 1 ps, resulting in 300
conformers of compound 57.
All conformers were then minimized and converged into five
families. Then we compared these five representative conformers with
the docking pose from the molecular docking experiment using our in-
house 3D CB2 receptor model. The docking experiment was
performed using the Surflex-Dock module from the Tripos modeling
software. The conformer with maximum agreement between these two
experiments was chosen as a preferred conformer for further CoMFA
studies. All structures were built and energy minimized under the
Tripos force field with 0.05 kcal/(mol Å). The Gasteiger−Huckel
method was used to calculate the charges. Energy minimization was
performed by the Powell method with 2000 iterations. Structural
alignments of all molecules in the training and test sets to the preferred
conformer of compound 57 were performed using the MultiFit
program in Sybyl-X1.3. The CoMFA study was then carried out using
the SYBYL/CoMFA module. Leave one-out cross-validation
(LOOCV) partial least-squares (PLS) analysis was then performed
with a minimum σ (column filter) value of 5.0 kcal·mol−1 to improve
the signal-to-noise ratio by omitting those lattice points with an energy
variation below this threshold. The final model (non-cross-validated
analysis) was developed from the LOOCV model with the highest
cross-validated r2, using the optimal number of components
determined by the LOOCV model.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +1-412-383-5276. Fax: +1-412-383-7436. E-mail:
xix15@pitt.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors from the University of Pittsburgh gratefully
acknowledge the financial support for our laboratory from the
NIH R01DA025612 and R21HL109654 and P50 GM067082.
The authors also acknowledge the collaborative support from
the National Natural Science Foundation of China
(NSFC81090410, NSFC90913018).
ABBREVIATIONS USED
■
CB, cannabinoid; GALAHAD, genetic algorithm-based phar-
macophore alignment; HB, H-bond; SAR, structure−activity
relationship; OCL, osteoclast; QSAR, quantitative structure−
activity relationship; CoMFA, comparative molecular field
analysis; MD, molecular dynamic; MM, molecular mechanics;
LOOCV, leave-one-out cross-validation; PLS, partial least-
squares; SEE, standard error of estimate; MTA, material
transfer agreement; TLC, thin-layer chromatography; EA, ethyl
acetate; DCM, dichloromethane; EGTA, ethylene glycol
tetraacetic acid; BSA, bovine serum albumin; WT, wild type;
DPBS, Dulbecco’s phosphate-buffered saline; AC, adenylate
cyclase; RANKL, receptor activator of nuclear factor kappa-B
ligand; TRAP, tartrate-resistant acid phosphate
REFERENCES
■
(1) Devane, W. A.; Hanus, L.; Breuer, A.; Pertwee, R. G.; Stevenson,
L. A.; Griffin, G.; Gibson, D.; Mandelbaum, A.; Etinger, A.;
Mechoulam, R. Isolation and structure of a brain constituent that
binds to the cannabinoid receptor. Science 1992, 258, 1946−1949.
(2) Mechoulam, R.; Ben-Shabat, S.; Hanus, L.; Ligumsky, M.;
Kaminski, N. E.; Schatz, A. R.; Gopher, A.; Almog, S.; Martin, B. R.;
Compton, D. R.; et al. Identification of an endogenous 2-
monoglyceride, present in canine gut, that binds to cannabinoid
receptors. Biochem. Pharmacol. 1995, 50, 83−90.
(3) Sugiura, T.; Kondo, S.; Sukagawa, A.; Nakane, S.; Shinoda, A.;
Itoh, K.; Yamashita, A.; Waku, K. 2-Arachidonoylglycerol: a possible
endogenous cannabinoid receptor ligand in brain. Biochem. Biophys.
Res. Commun. 1995, 215, 89−97.
(4) Bayewitch, M.; Rhee, M. H.; Avidor-Reiss, T.; Breuer, A.;
Mechoulam, R.; Vogel, Z. (−)-Delta9-tetrahydrocannabinol antago-
nizes the peripheral cannabinoid receptor-mediated inhibition of
adenylyl cyclase. J. Biol. Chem. 1996, 271, 9902−9905.
(5) Felder, C. C.; Joyce, K. E.; Briley, E. M.; Mansouri, J.; Mackie, K.;
Blond, O.; Lai, Y.; Ma, A. L.; Mitchell, R. L. Comparison of the
pharmacology and signal transduction of the human cannabinoid CB1
and CB2 receptors. Mol. Pharmacol. 1995, 48, 443−450.
(6) Matsuda, L. A.; Lolait, S. J.; Brownstein, M. J.; Young, A. C.;
Bonner, T. I. Structure of a cannabinoid receptor and functional
expression of the cloned cDNA. Nature 1990, 346, 561−564.
(7) Munro, S.; Thomas, K. L.; Abu-Shaar, M. Molecular character-
ization of a peripheral receptor for cannabinoids. Nature 1993, 365,
61−65.
(8) Hohmann, A. G. Spinal and peripheral mechanisms of
cannabinoid antinociception: behavioral, neurophysiological and
neuroanatomical perspectives. Chem. Phys. Lipids 2002, 121, 173−190.
2056
dx.doi.org/10.1021/jm3017464 | J. Med. Chem. 2013, 56, 2045−2058

Journal of Medicinal Chemistry
Article
(9) Karsak, M.; Gaffal, E.; Date, R.; Wang-Eckhardt, L.; Rehnelt, J.;
Petrosino, S.; Starowicz, K.; Steuder, R.; Schlicker, E.; Cravatt, B.;
Mechoulam, R.; Buettner, R.; Werner, S.; Di Marzo, V.; Tuting, T.;
Zimmer, A. Attenuation of allergic contact dermatitis through the
endocannabinoid system. Science 2007, 316, 1494−1497.
(10) Palazuelos, J.; Davoust, N.; Julien, B.; Hatterer, E.; Aguado, T.;
Mechoulam, R.; Benito, C.; Romero, J.; Silva, A.; Guzman, M.; Nataf,
S.; Galve-Roperh, I. The CB2 cannabinoid receptor controls myeloid
progenitor trafficking. J. Biol. Chem. 2008, 283, 13320−13329.
(11) Guindon, J.; Hohmann, A. G. The endocannabinoid system and
cancer: therapeutic implication. Br. J. Pharmacol. 2011, 163, 1447−
1463.
(12) Oesch, S.; Gertsch, J. Cannabinoid receptor ligands as potential
anticancer agents–high hopes for new therapies? J. Pharm. Pharmacol.
2009, 61, 839−853.
(13) Idris, A. I.; Ralston, S. H. Cannabinoids and Bone: Friend or
Foe? Calcified Tissue Int. 2010, 87, 285−297.
(14) Hillard, C. J. Role of cannabinoids and endocannabinoids in
cerebral ischemia. Curr. Pharm. Des. 2008, 14, 2347−2361.
(15) Hegde, V. L.; Hegde, S.; Cravatt, B. F.; Hofseth, L. J.;
Nagarkatti, M.; Nagarkatti, P. S. Attenuation of experimental
autoimmune hepatitis by exogenous and endogenous cannabinoids:
involvement of regulatory T cells. Mol. Pharmacol. 2008, 74, 20−33.
(16) Munoz-Luque, J.; Ros, J.; Fernandez-Varo, G.; Tugues, S.;
Morales-Ruiz, M.; Alvarez, C. E.; Friedman, S. L.; Arroyo, V.; Jimenez,
W. Regression of fibrosis after chronic stimulation of cannabinoid CB2
receptor in cirrhotic rats. J. Pharmacol. Exp. Ther. 2008, 324, 475−483.
(17) Izzo, A. A.; Camilleri, M. Emerging role of cannabinoids in
gastrointestinal and liver diseases: basic and clinical aspects. Gut 2008,
57, 1140−1155.
(18) Pacher, P.; Batkai, S.; Kunos, G. The endocannabinoid system as
an emerging target of pharmacotherapy. Pharmacol. Rev. 2006, 58,
389−462.
(19) Compton, D. R.; Rice, K. C.; De Costa, B. R.; Razdan, R. K.;
Melvin, L. S.; Johnson, M. R.; Martin, B. R. Cannabinoid structure-
activity relationships: correlation of receptor binding and in vivo
activities. J. Pharmacol. Exp. Ther. 1993, 265, 218−226.
(20) Huffman, J. W. Cannabimimetic indoles, pyrroles and indenes.
Curr. Med. Chem. 1999, 6, 705−720.
(21) Palmer, S. L.; Thakur, G. A.; Makriyannis, A. Cannabinergic
ligands. Chem. Phys. Lipids. 2002, 121, 3−19.
(22) Raitio, K. H.; Salo, O. M.; Nevalainen, T.; Poso, A.; Jarvinen, T.
Targeting the cannabinoid CB2 receptor: mutations, modeling and
development of CB2 selective ligands. Curr. Med. Chem. 2005, 12,
1217−1237.
induced by oil of mustard and by dextran sulfate sodium. Am. J. Physiol.
Gastrointest. Liver Physiol. 2006, 291, G364−371.
(29) Xi, Z.-X.; Peng, X.-Q.; Li, X.; Song, R.; Zhang, H.-Y.; Liu, Q.-R.;
Yang, H.-J.; Bi, G.-H.; Li, J.; Gardner, E. L. Brain cannabinoid CB2
receptors modulate cocaine’s actions in mice. Nat. Neurosci. 2011, 14,
1160−1166.
(30) Lunn, C. A.; Fine, J. S.; Rojas-Triana, A.; Jackson, J. V.; Fan, X.;
Kung, T. T.; Gonsiorek, W.; Schwarz, M. A.; Lavey, B.; Kozlowski, J.
A.; Narula, S. K.; Lundell, D. J.; Hipkin, R. W.; Bober, L. A. A novel
cannabinoid peripheral cannabinoid receptor-selective inverse agonist
blocks leukocyte recruitment in vivo. J. Pharmacol. Exp. Ther. 2006,
316, 780−788.
(31) Iwamura, H.; Suzuki, H.; Ueda, Y.; Kaya, T.; Inaba, T. In vitro
and in vivo pharmacological characterization of JTE-907, a novel
selective ligand for cannabinoid CB2 receptor. J. Pharmacol. Exp. Ther.
2001, 296, 420−425.
(32) Francisco, M. E.; Seltzman, H. H.; Gilliam, A. F.; Mitchell, R. A.;
Rider, S. L.; Pertwee, R. G.; Stevenson, L. A.; Thomas, B. F. Synthesis
and structure-activity relationships of amide and hydrazide analogues
of the cannabinoid CB(1) receptor antagonist N-(piperidinyl)- 5-(4-
chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carbox-
amide (SR141716). J. Med. Chem. 2002, 45, 2708−2719.
(33) Yang, P.; Myint, K. Z.; Tong, Q.; Feng, R.; Cao, H.; Almehizia,
A. A.; Hamed, A. M.; Wang, L.; Bartlow, P.; Gao, Y.; Gertsch, J.;
Teramachi, J.; Kurihara, N.; Roodman, G. D.; Cheng, T.; Xie, X. Q.
Lead Discovery, Chemistry Optimization and Biological Evaluation
Studies of Novel Bi-amide Derivatives as CB2 Receptor Inverse
Agonists and Osteoclast Inhibitors. J. Med. Chem. 2012, 55, 9973−
9987.
(34) Zhang, Y.; Xie, Z.; Wang, L.; Schreiter, B.; Lazo, J. S.; Gertsch,
J.; Xie, X. Q. Mutagenesis and computer modeling studies of a GPCR
conserved residue W5.43(194) in ligand recognition and signal
transduction for CB2 receptor. Int. Immunopharmacol. 2011, 11,
1303−1310.
(35) Bab, I.; Zimmer, A.; Melamed, E. Cannabinoids and the
skeleton: from marijuana to reversal of bone loss. Ann. Med. 2009, 41,
560−567.
(36) Anandarajah, A. P.; Schwarz, E. M.; Totterman, S.; Monu, J.;
Feng, C. Y.; Shao, T.; Haas-Smith, S. A.; Ritchlin, C. T. The effect of
etanercept on osteoclast precursor frequency and enhancing bone
marrow oedema in patients with psoriatic arthritis. Ann. Rheum. Dis.
2008, 67, 296−301.
(37) Myint, K. Z.; Xie, X. Q. Recent advances in fragment-based
QSAR and multi-dimensional QSAR methods. Int. J. Mol. Sci. 2010,
11, 3846−3866.
(38) Chen, J. Z.; Han, X. W.; Liu, Q.; Makriyannis, A.; Wang, J.; Xie,
X. Q. 3D-QSAR studies of arylpyrazole antagonists of cannabinoid
receptor subtypes CB1 and CB2. A combined NMR and CoMFA
approach. J. Med. Chem. 2006, 49, 625−636.
(39) Xie, X. Q.; Melvin, L. S.; Makriyannis, A. The conformational
properties of the highly selective cannabinoid receptor ligand CP-
55,940. J. Biol. Chem. 1996, 271, 10640−10647.
(40) Xie, X. Q.; Chen, J. Z.; Billings, E. M. 3D structural model of the
G-protein-coupled cannabinoid CB2 receptor. Proteins 2003, 53, 307−
319.
(41) Gertsch, J.; Leonti, M.; Raduner, S.; Racz, I.; Chen, J. Z.; Xie, X.
Q.; Altmann, K. H.; Karsak, M.; Zimmer, A. Beta-caryophyllene is a
dietary cannabinoid. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 9099−
9104.
(42) Feng, R.; Anderson, G.; Xiao, G.; Elliott, G.; Leoni, L.; Mapara,
M. Y.; Roodman, G. D.; Lentzsch, S. SDX-308, a nonsteroidal anti-
inflammatory agent, inhibits NF-kappaB activity, resulting in strong
inhibition of osteoclast formation/activity and multiple myeloma cell
growth. Blood 2007, 109, 2130−2138.
(23) Marriott, K. S.; Huffman, J. W. Recent advances in the
development of selective ligands for the cannabinoid CB(2) receptor.
Curr. Top. Med. Chem. 2008, 8, 187−204.
(24) Yang, P.; Wang, L.; Xie, X. Q. Latest advances in novel
cannabinoid CB(2) ligands for drug abuse and their therapeutic
potential. Future Med. Chem. 2012, 4, 187−204.
(25) Rinaldi-Carmona, M.; Barth, F.; Millan, J.; Derocq, J. M.;
Casellas, P.; Congy, C.; Oustric, D.; Sarran, M.; Bouaboula, M.;
Calandra, B.; Portier, M.; Shire, D.; Breliere, J. C.; Le Fur, G. L. SR
144528, the first potent and selective antagonist of the CB2
cannabinoid receptor. J. Pharmacol. Exp. Ther. 1998, 284, 644−650.
(26) Ross, R. A.; Brockie, H. C.; Stevenson, L. A.; Murphy, V. L.;
Templeton, F.; Makriyannis, A.; Pertwee, R. G. Agonist-inverse agonist
characterization at CB1 and CB2 cannabinoid receptors of L759633,
L759656, and AM630. Br. J. Pharmacol. 1999, 126, 665−672.
(27) Huffman, J. W.; Zengin, G.; Wu, M. J.; Lu, J.; Hynd, G.; Bushell,
K.; Thompson, A. L.; Bushell, S.; Tartal, C.; Hurst, D. P.; Reggio, P.
H.; Selley, D. E.; Cassidy, M. P.; Wiley, J. L.; Martin, B. R. Structure-
activity relationships for 1-alkyl-3-(1-naphthoyl)indoles at the
cannabinoid CB(1) and CB(2) receptors: steric and electronic effects
of naphthoyl substituents. New highly selective CB(2) receptor
agonists. Bioorg. Med. Chem. 2005, 13, 89−112.
(43) Xu, J.; Li, Z.; Luo, J.; Yang, F.; Liu, T.; Liu, M.; Qiu, W. W.;
Tang, J. Synthesis and biological evaluation of heterocyclic ring-fused
betulinic acid derivatives as novel inhibitors of osteoclast differ-
entiation and bone resorption. J. Med. Chem. 2012, 55, 3122−3134.
(28) Kimball, E. S.; Schneider, C. R.; Wallace, N. H.; Hornby, P. J.
Agonists of cannabinoid receptor 1 and 2 inhibit experimental colitis
2057
dx.doi.org/10.1021/jm3017464 | J. Med. Chem. 2013, 56, 2045−2058

Journal of Medicinal Chemistry
Article
(44) Feng, R.; Ma, H.; Hassig, C. A.; Payne, J. E.; Smith, N. D.;
Mapara, M. Y.; Hager, J. H.; Lentzsch, S. KD5170, a novel
mercaptoketone-based histone deacetylase inhibitor, exerts antimye-
loma effects by DNA damage and mitochondrial signaling. Mol.
Cancer. Ther. 2008, 7, 1494−1505.
2058
dx.doi.org/10.1021/jm3017464 | J. Med. Chem. 2013, 56, 2045−2058