Trisubstituted sulfonamides: A new chemotype for development of potent and selective CB2 receptor inverse agonists

Article abstract of DOI:10.1021/ml3004236

An extensive exploration of the structure-activity relationship of a trisubstituted sulfonamide series led to the identification of 39, which is a potent and selective CB₂ receptor inverse agonist [K _i(CB₂) = 5.4 nM, and K_i(CB₁) = 500 nM]. The functional properties measured by cAMP assays indicated that the selected compounds were CB₂ inverse agonists with high potency values (for 34, EC₅₀ = 8.2 nM, and for 39, EC₅₀ = 2.5 nM). Furthermore, an osteoclastogenesis bioassay indicated that trisubstituted sulfonamide compounds showed great inhibition of osteoclast formation.

Full text of DOI:10.1021/ml3004236

Letter
pubs.acs.org/acsmedchemlett
Trisubstituted Sulfonamides: A New Chemotype for Development of
Potent and Selective CB2 Receptor Inverse Agonists
Qin Ouyang,†,‡,@ Qin Tong,†,‡ Rentian Feng,†,‡ Kyaw-Zeyar Myint,†,‡,∥ Peng Yang,†,‡
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 Center, Department of Computational and Systems
⊥
Biology, and Department of Structural Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States
@College of Pharmacy, Third Military Medical University, Chongqing 400038, China
S
* Supporting Information
ABSTRACT: An extensive exploration of the structure−activity relationship of
a trisubstituted sulfonamide series led to the identification of 39, which is a
potent and selective CB2 receptor inverse agonist [Ki(CB2) = 5.4 nM, and
Ki(CB1) = 500 nM]. The functional properties measured by cAMP assays
indicated that the selected compounds were CB2 inverse agonists with high
potency values (for 34, EC50 = 8.2 nM, and for 39, EC50 = 2.5 nM).
Furthermore, an osteoclastogenesis bioassay indicated that trisubstituted
sulfonamide compounds showed great inhibition of osteoclast formation.
KEYWORDS: Cannabinoid receptors, inverse agonists, trisubstituted sulfonamides, osteoclast inhibitors
annabinoid receptors 1 and 2 (CB1 and CB2, respectively)
on the basis of the research in three-dimensional CB2 receptor
structure model29,30 and pharmacophore database searches, our
group also reported the discovery of novel bis-amide derivatives
[1 (Figure 1)] as CB2 receptor inverse agonists and osteoclast
inhibitors.31 However, the optimization of bis-amide derivatives
is limited by the synthesis method and symmetrical scaffold.
C
were identified in the early 1990s as members of the G
protein-coupled receptor (GPCR) superfamily.1,2 While CB1
receptors are primarily found in the central nervous system
(CNS), CB2 receptors are predominantly located in tissues and
cells of the immune system, such as tonsils, spleen, macro-
phages, and lymphocytes.3 Also, there is some evidence of the
presence of the CB2 receptor in the CNS.4,5
Recently, numerous agonists and antagonists of cannabinoid
receptors have been explored because of the important role of
the endocannabinoid system in various diseases and disorders.6
Among these, CB1 receptor ligands have been developed for
pain, appetite stimulation, nausea, neurodegeneration, hyper-
motility, and inflammation.7 However, the CB1 receptor ligands
are known to cause side effects in the CNS such as cognitive
dysfunction, motor incoordination, and sedation.8 Because of
differences in receptor distribution and signal transduction
mechanisms, CB2-selective ligands are considered as medi-
cations without CNS side effects,9 and such ligands are being
actively investigated for use in a multitude of diseases and
pathological conditions,10 such as atherosclerosis,11 myocardial
infarction,12 stroke,13 gastrointestinal inflammatory,14 auto-
immune,15 and neurodegenerative16 disorders, bone disor-
ders,17−19 and cancer.20
The development of CB2 receptor-selective ligands has
attracted significant attention because of the therapeutic
potential of CB2 receptor modulation.21−23 The first CB2
inverse agonist is SR144528, which is extensively used as the
standard to measure the specificity of various cannabinoid
inverse agonists for CB2 in animal models.24 Other notable
examples of CB2 receptor agonists and antagonists include
AM630,25 JTE-907,26 Sch225336,27 and JWH-133.28 Recently,
Figure 1. Structures of CB2 receptor inverse agonists and new scaffold
discovery.
On the basis of continuing virtual screening and QSAR
results,32 we designed and synthesized 2, with a trisubstituted
sulfonamide scaffold, as a novel chemotype with CB2 binding
activity [Ki(CB2) = 750 nM]. Compared with the structure of 1
and considering the QSAR results, we believed that a longer
chain in zone A was important for the CB2 inverse agonist
(Figure 1). Compound 3 with a diethylamino group was
synthesized and confirmed to have a better CB2 binding affinity
[Ki(CB2) = 53 nM] and a good CB2 selectivity index [SI = 43,
Received: November 27, 2012
Accepted: February 22, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
calculated as the Ki(CB1)/Ki(CB2) ratio]. Given this promising
result, 3 was chosen as a prototype for further structure−
activity relationship (SAR) studies. Herein, we reported the
design and synthesis of novel trisubstituted sulfonamide
derivatives as CB2 inverse agonists. Binding activities were
investigated to define their SAR and ligand functionality. After
we modified the groups at zones A−C, some derivatives, such
as 34 [Ki(CB2) = 5.5 nM, and SI = 15], 39 [Ki(CB2) = 5.4 nM,
and SI = 92], and 45 [Ki(CB2) = 4.0 nM, and SI = 120], were
identified as CB2-selective ligands with improved CB2 binding
affinity and high selectivity. These compounds were selected for
the functional property investigation by a cAMP assay, which
showed their high potency (for 34, EC50 = 8.2 nM, and for 39,
EC50 = 2.5 nM) as CB2 inverse agonists. Moreover, these
compounds also showed great inhibition activity with osteoclast
cells.
Table 1. CB1 and CB2 Receptor Affinities of Compounds 3−
12 with a Modified Zone A
Ki (nM)
ab
,
ab
,
c
compd
R1
CB2
53
CB1
SI
3
-N(CH2CH3)2
-N(CH3)2
6
2300 200
NT
43
4
680 10
5
-(CH2CH2Cl2)2
1-piperidinyl
-OCH(CH3)2
-OCH2CH3
-CH2CH2CH3
-CH2CHCH2
-n-butyl
166
44
4
NT
6
8
1300 200
NT
28
7
174
7
8
230 60
130 20
130 40
280 50
230 40
2.1 0.4
NT
NT
9
NT
10
11
12
NT
The trisubstituted sulfonamide derivatives were synthesized
by two general strategies (Scheme 1). In the first strategy, the
NT
-CH(CH3)2
NT
de
,
a
SR144528
SR141716
NT
Scheme 1. Synthesis of Trisubstituted Sulfonamides
df
,
11
1
a
Binding affinities of compounds for CB1 and CB2 receptors were
evaluated using the [3H]CP-55,940 radioligand competition binding
assay. Data are means the standard error of the mean of at least
three experiments performed in duplicate. NT, not tested. SI,
b
c
selectivity index for CB2, calculated as the Ki(CB1)/Ki(CB2) ratio.
d
The binding affinities of reference compounds were evaluated in
e
parallel with compounds 3−12 under the same conditions. CB2
reference compound. CB1 reference compound.
f
the sulfophenyl, such as Cl, F, H, acetylamide, isopropyl,
methoxyl, isopropoxyl, and trifluoromethoxyl. Other than 20
(R2 = 4-methoxyphenyl), which had a binding affinity for the
CB2 receptor (Ki = 46 nM) similar to that of 3, 13−22 did not
show obvious increased binding affinity. Large groups, like
naphthyl and 1,1′-biphenyl, were also used to replace the 4-
methylphenyl. This replacement resulted in a lower binding
affinity. These results indicated that 4-methylphenyl and 4-
methoxylphenyl are the best groups among our modifications
of zone B. Moreover, the CB2 selective indexes of 15, 16, 19,
and 20 were quite low, ranging from 6 to 15.
After investigation of the SAR of R1 and R2 in zones A and B,
the binding affinities were not obviously improved. The
following modification of group R3 in zone C was based on
maintenance of the R1 and R2 groups in 3 (Table 3). As
expected, group R3 is a key substituent for enhancing the CB2
affinity. First, another CH2 group was introduced at the N atom
and the phenyl ring; compound 26 showed a lower CB2
binding affinity (Ki = 120 nM). The same reduced trends
were found when diethylamino and halogen (Cl and F) were
added to the phenyl ring in zone C. The binding affinities for
the CB2 receptor of 27−30 were 540, 210, 390, and 120 nM,
respectively. After substitution of the phenyl in zone C with
cyclohexyl, a small increase in affinity was achieved [for 31,
Ki(CB2) = 34 nM], while replacement by five-membered
heterocyclic moieties (in 32 and 33) enhanced affinity
dramatically, yielding a Ki values of 9.5 and 14 nM at CB2
receptors and 570 and 610 nM at CB1 receptors, respectively. It
seemed that a smaller group in zone C is preferable for CB2
binding activity, so some compounds with smaller group were
synthesized. Compound 34 with the reduced CH2 between the
N atom in the core and phenyl group showed a higher CB2
binding affinity (Ki = 5.5 nM, and SI = 15). However,
replacement with other smaller groups, such as cyclohexyl (35),
a
Reagents and conditions: (a) CH3OH, reflux; (b) CH3OH, NaBH4;
(c) Et3N, CH2Cl2; (d) K2CO3, CH3COCH3, reflux.
different imine intermediates, synthesized from reductive
amination reactions of substituted aryl aldehydes and various
amines, were reacted with different sulfonyl chlorides to
prepare compounds 3−12, 14−25, 31−36, and 39−48, as
well as with benzyl bromide to obtain 13. Compounds 26−30,
37, and 38 were obtained by the reaction of intermediate 25
with various bromides. The structures of all the compounds
were characterized by 1H NMR and ESI-HRMS spectra; purity
was confirmed by HPLC.
Biological data of compounds with different substituents of
zone A are listed in Table 1. Some alkyl chains, whose
substituents had been shown in our previous study to be
favorable for CB2 receptor affinity,31 were introduced at
position 4 of phenyl, such as the bialkyl chain (dimethylamino,
isopropoxyl, and isopropyl), monoalkyl chain substituents
(ethoxyl, propoxyl, and butyl), and cycloalkanes (1-piperidinyl).
Among them, 3 (R1 = diethylamino) and 6 (R1 = 1-piperidinyl)
showed higher affinities for the CB2 receptor (53 and 44 nM,
respectively) with a good CB2 selective index (SI = 43 and 28,
respectively). These results indicated that longer chains and
double chains are necessary for improving binding affinity in
this scaffold.
The modification of zone B was based on maintenance of the
diethylamino group in zone A, as shown in Table 2. Compound
13, in which the sulfo group was replaced with a CH2 group,
showed a slightly lower affinity. A similar result was found when
a CH2 group was added between the sulfo and the phenyl
group (14). Many substituents were introduced at position 4 of
B
dx.doi.org/10.1021/ml3004236 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Table 2. CB1 and CB2 Receptor Affinities of Compounds
13−24 with a Modified Zone B
Table 3. CB1 and CB2 Receptor Affinities of Compounds
25−38 with a Modified Zone C
a−g
Same as the footnotes of Table 1.
5-methylthiazolyl (36), cyclopentyl (37), and allyl (38),
significantly reduced the binding activity [Ki(CB2) = 18, 14,
36, and 66 nM, respectively]. Especially, 25, without any
substituted group except with an H atom at R3, showed quite
low CB2 binding activity [Ki(CB2) = 3600 nM]. This result
suggests that the space of the phenyl group is suitable for zone
C and thus addition of a larger or smaller group results in lower
CB2 binding activity.
a−g
Same as the footnotes of Table 1.
protocol33 to measure the agonistic or antagonistic functional
activities of the CB2-selective compounds. Because CB2 is a Gαi-
coupled receptor, an agonist inhibits the forskolin-induced
cAMP production, resulting in an increase in the magnitude of
the LANCE signal, while an antagonist or inverse agonist
decreases the magnitude of the LANCE signal. In addition to
34, 39, 45, and 47, with respective modifications in zones A−C
for good binding activity, were selected for cAMP assays. As
shown in Figure 2, reduction of the magnitude of the LANCE
signal occurred with increasing concentrations of 34, 39, 45, 47,
and SR144528 with EC50 values of 8.2 3.1, 2.5 1.4, 73 2,
On the basis of the SAR research of zones A−C, we found
that the introduction of some groups at R1−R3 could improve
CB2 binding activity. Further SRA research was based on the
compound library of combinations of these groups: R1
=
diethylamino and 1-piperidinyl, R3 = phenyl, cyclohexyl, furan-
2-ylmethyl, and 5-methylthiazolyl, and R4 = methoxyl, Cl,
isopropyl, and methyl. The results are listed in Table 4 with the
CB2 binding affinity ranging from 4 to 310 nM and the selective
index ranging from 9 to 120. Among them, two compounds
were identified with better CB2 binding activity and selectivity,
39 [Ki(CB2) = 5.4 nM, and SI = 92] and 45 [Ki(CB2) = 4.0
nM, and SI = 120].
49
2, and 14
3 nM, respectively. Such a contrary
phenomenon was observed with agonists CP55,940 and
HU308,34 which showed cAMP production with EC50 values
of 11 2 and 85 5 nM, respectively. On the basis of the
LANCE signal change and the high EC50 value closely
correlated with the high affinity value, it suggests that 34, 39,
45, and 47 behaved as CB2 receptor inverse agonists.
Functional properties were investigated in cAMP assays by
using cell-based LANCE cAMP assays as our published
C
dx.doi.org/10.1021/ml3004236 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
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Table 4. CB1 and CB2 Receptor Affinities of Compounds 39−48
Ki (nM)
SIc
a,b
a,b
compd
R1
R3
R4
CB2
5.4 0.5
CB1
39
40
41
42
43
44
45
46
47
48
-N(CH2CH3)2
-N(CH2CH3)2
-N(CH2CH3)2
-N(CH2CH3)2
-N(CH2CH3)2
-N(CH2CH3)2
-N(CH2CH3)2
1-piperidinyl
phenyl
phenyl
-OCH3
500 60
2000 200
840 80
1300 100
660 60
1400 100
600 10
NT
92
66
42
42
9
-CH(CH3)2
-OCH3
30
20
31
75
55
4
2
4
6
2
furan-2-ylmethyl
furan-2-ylmethyl
cyclohexyl
-CH(CH3)2
-OCH3
cyclohexyl
-CH(CH3)2
-OCH3
25
120
5-methylthiazolyl
phenyl
4.0 0.6
310 20
-CH3
1-piperidinyl
phenyl
-OCH3
32
7
590 50
NT
18
1-piperidinyl
phenyl
-Cl
140 10
2.1 0.4
NT
SR144528d,e
SR141716d,f
NT
11
1
a−f
Same as the footnotes of Table 1.
Figure 2. Comparisons of the 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 34, 39, 45, 47, and SR144528 are 8.2 3.1, 2.5 1.4,
73 2, 49 2, and 14 3 nM, respectively. EC50 values for CP55,940
and HU308 are 11 2 and 85 5 nM, respectively. Data are means
the standard error of the mean of one representative experiment of
two or more performed in duplicate or triplicate.
Figure 3. Inhibition of osteoclastogenesis by CB2 ligands 34, 39, and
45. All experiments were performed in triplicate. Results are means
the standard deviation. Note that the control is vehicle control.
In summary, we have discovered the trisubstituted
sulfonamide chemotype as a novel series possessing significant
cannabinoid CB2 receptors affinity. Some compounds with high
binding affinities and selective indexes of CB2 receptors were
identified by optimization of zones A−C. The potencies of the
novel compounds were measured in functional assays, with high
potency values (represented by EC50) that are closely
correlated with the high affinities (expressed as Ki), revealing
that the novel series behaves as CB2 receptor inverse agonists.
The promising inhibition activity to osteoclast cells of this
novel series of compounds offers an attractive starting point for
further optimization.
Modulating osteoclast function is a well-known activity of
CB2 receptor agonists35 and inverse agonists.19 Three
compounds, 34, 39, and 45, were selected on the basis of the
results of binding affinity and selectivity, as well as their
functionality as candidate inhibitors of osteoclast (OCL)
formation. As shown in Figure 3, we tested the effects of
these most promising CB2 ligands on osteoclast (OCL)
formation using mouse bone marrow mononuclear cells treated
by the mouse receptor activator of NF-κB ligand (RANKL)
plus macrophage-colony-stimulating factor (M-CSF) (see the
Supporting Information). These three compounds exhibited
strong inhibition of osteoclastogenesis. Among them, 34
showed the most favorable activity. At 0.1, 1, and 5 μM, it
suppressed osteoclast formation by 55, 83, and 99.7%,
respectively. To investigate their cell toxicity, 34 was tested in
a cytotoxicity assay without showing any cytotoxic effects at a
concentration of 5 μM. These results indicate that our
compounds possess favorable therapeutic indexes and the
inhibition of osteoclastogenesis is not a result of their
cytotoxicity.
ASSOCIATED CONTENT
* Supporting Information
Synthetic experimental details, analytical data, and biological
assay protocols. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Telephone: (412) 383-5276. Fax: (412) 383-7436. E-mail:
xix15@pitt.edu.
■
D
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(16) Gorantla, S.; Makarov, E.; Roy, D.; Finke-Dwyer, J.; Murrin, L.
C.; Gendelman, H. E.; Poluektova, L. Immunoregulation of a CB2
Receptor Agonist in a Murine Model of NeuroAIDS. Journal of
Neuroimmune Pharmacology 2010, 5 (3), 456−468.
(17) Idris, A. I.; van’t Hof, R. J.; Greig, I. R.; Ridge, S. A.; Baker, D.;
Ross, R. A.; Ralston, S. H. Regulation of bone mass, bone loss and
osteoclast activity by cannabinoid receptors. Nat. Med. 2005, 11 (7),
774−779.
(18) Sophocleous, A.; Landao-Bassonga, E.; van’t Hof, R. J.; Idris, A.
I.; Ralston, S. H. The type 2 cannabinoid receptor regulates bone mass
and ovariectomy-induced bone loss by affecting osteoblast differ-
entiation and bone formation. Endocrinology 2011, 152 (6), 2141−
2149.
(19) Schuehly, W.; Paredes, J. M. V.; Kleyer, J.; Huefner, A.; Anavi-
Goffer, S.; Raduner, S.; Altmann, K. H.; Gertsch, J. Mechanisms of
Osteoclastogenesis Inhibition by a Novel Class of Biphenyl-Type
Cannabinoid CB2 Receptor Inverse Agonists. Chem. Biol. 2011, 18
(8), 1053−1064.
(20) Pisanti, S.; Bifulco, M. Endocannabinoid system modulation in
cancer biology and therapy. Pharmacol. Res. 2009, 60 (2), 107−116.
(21) Yang, P.; Wang, L.; Xie, X. Q. Latest advances in novel
cannabinoid CB2 ligands for drug abuse and their therapeutic
potential. Future Med. Chem. 2012, 4 (2), 187−204.
(22) Raitio, K. H.; Salo, O. M. H.; Nevalainen, T.; Poso, A.; Jarvinen,
T. Targeting the cannabinoid CB2 receptor: Mutations, modeling and
development of CB2 selective ligands. Curr. Med. Chem. 2005, 12
(10), 1217−1237.
(23) Marriott, K. S. C.; Huffman, J. W. Recent advances in the
development of selective ligands for the cannabinoid CB2 receptor.
Curr. Top. Med. Chem. 2008, 8 (3), 187−204.
(24) 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. SR
144528, the first potent and selective antagonist of the CB2
cannabinoid receptor. J. Pharmacol. Exp. Ther. 1998, 284 (2), 644−
650.
(25) 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 (3), 665−672.
(26) Ueda, Y.; Miyagawa, N.; Matsui, T.; Kaya, T.; Wamura, H.;
Iwamura, H. Involvement of cannabinoid CB2 receptor-mediated
response and efficacy of cannabinoid CB2 receptor inverse agonist,
JTE-907, in cutaneous inflammation in mice. Eur. J. Pharmacol. 2005,
520 (1−3), 164−171.
(27) Lunn, C. A.; Fine, J. S.; Rojas-Triana, A.; Jackson, J. V.; Fan, X.
D.; 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 (2), 780−788.
(28) 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
(9), 1160−1166.
(29) 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 (2), 625−636.
(30) Xie, X. Q.; Chen, J. Z.; Billings, E. M. 3D structural model of the
G-protein-coupled cannabinoid CB2 receptor. Proteins: Struct., Funct.,
Genet. 2003, 53 (2), 307−319.
Funding
We authors from the University of Pittsburgh gratefully
acknowledge the financial support of our laboratory by
National Institutes of Health Grants R01DA025612 and
HL109654 (X.-Q.X.). We also acknowledge the collaboration
support from the National Natural Science Foundation of
China (NSFC81090410, NSFC90913018, and
NSFC21202201).
Notes
The authors declare no competing financial interest.
REFERENCES
■
(1) 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 (6284), 561−564.
(2) Munro, S.; Thomas, K. L.; Abushaar, M. Molecular Character-
ization of a Peripheral Receptor for Cannabinoids. Nature 1993, 365
(6441), 61−65.
(3) Svizenska, I.; Dubovy, P.; Sulcova, A. Cannabinoid receptors 1
and 2 (CB1 and CB2), their distribution, ligands and functional
involvement in nervous system structures: A short review. Pharmacol.,
Biochem. Behav. 2008, 90 (4), 501−511.
(4) Van Sickle, M. D.; Duncan, M.; Kingsley, P. J.; Mouihate, A.;
Urbani, P.; Mackie, K.; Stella, N.; Makriyannis, A.; Piomelli, D.;
Davison, J. S.; Marnett, L. J.; Di Marzo, V.; Pittman, Q. J.; Patel, K. D.;
Sharkey, K. A. Identification and functional characterization of
brainstem cannabinoid CB2 receptors. Science 2005, 310 (5746),
329−332.
(5) Atwood, B. K.; Mackie, K. CB2: A cannabinoid receptor with an
identity crisis. Br. J. Pharmacol. 2010, 160 (3), 467−479.
(6) Pertwee, R. G.; Howlett, A. C.; Abood, M. E.; Alexander, S. P. H.;
Di Marzo, V.; Elphick, M. R.; Greasley, P. J.; Hansen, H. S.; Kunos, G.;
Mackie, K.; Mechoulam, R.; Ross, R. A. International Union of Basic
and Clinical Pharmacology. LXXIX. Cannabinoid Receptors and Their
Ligands: Beyond CB1 and CB2. Pharmacol. Rev. 2010, 62 (4), 588−
631.
(7) Talwar, R.; Potluri, V. K. Cannabinoid 1 (CB1) receptor:
Pharmacology, role in pain and recent developments in emerging CB1
agonists. CNS Neurol. Disord.: Drug Targets 2011, 10 (5), 536−544.
(8) Reggio, P. H. The cannabinoid receptors; Humana: New York,
2009.
(9) Malan, T. P., Jr.; Ibrahim, M. M.; Lai, J.; Vanderah, T. W.;
Makriyannis, A.; Porreca, F. CB2 cannabinoid receptor agonists: Pain
relief without psychoactive effects? Curr. Opin. Pharmacol. 2003, 3 (1),
62−67.
(10) Pacher, P.; Mechoulam, R. Is lipid signaling through
cannabinoid 2 receptors part of a protective system? Prog. Lipid Res.
2011, 50 (2), 193−211.
(11) Montecucco, F.; Matias, I.; Lenglet, S.; Petrosino, S.; Burger, F.;
Pelli, G.; Braunersreuther, V.; Mach, F.; Steffens, S.; Di Marzo, V.
Regulation and possible role of endocannabinoids and related
mediators in hypercholesterolemic mice with atherosclerosis. Athero-
sclerosis 2009, 205 (2), 433−441.
(12) Zhang, M.; Adler, M. W.; Abood, M. E.; Ganea, D.; Jallo, J.;
Tuma, R. F. CB2 receptor activation attenuates microcirculatory
dysfunction during cerebral ischemic/reperfusion injury. Microvasc.
Res. 2009, 78 (1), 86−94.
(13) Murikinati, S.; Juttler, E.; Keinert, T.; Ridder, D. A.;
Muhammad, S.; Waibler, Z.; Ledent, C.; Zimmer, A.; Kalinke, U.;
Schwaninger, M. Activation of cannabinoid 2 receptors protects
against cerebral ischemia by inhibiting neutrophil recruitment. FASEB
J. 2010, 24 (3), 788−798.
(14) Izzo, A. A.; Sharkey, K. A. Cannabinoids and the gut: New
developments and emerging concepts. Pharmacol. Ther. 2010, 126 (1),
21−38.
(15) Pandey, R.; Mousawy, K.; Nagarkatti, M.; Nagarkatti, P.
Endocannabinoids and immune regulation. Pharmacol. Res. 2009, 60
(2), 85−92.
(31) 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 (22),
9973−9987.
E
dx.doi.org/10.1021/ml3004236 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(32) Myint, K. Z.; Wang, L.; Tong, Q.; Xie, X. Q. Molecular
fingerprint-based artificial neural networks QSAR for ligand biological
activity predictions. Mol. Pharmaceutics 2012, 9 (10), 2912−2923.
(33) Zhang, Y. X.; Xie, Z. J.; Wang, L. R.; 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
(9), 1303−1310.
(34) Hanus, L.; Breuer, A.; Tchilibon, S.; Shiloah, S.; Goldenberg, D.;
Horowitz, M.; Pertwee, R. G.; Ross, R. A.; Mechoulam, R.; Fride, E.
HU-308: A specific agonist for CB2, a peripheral cannabinoid receptor.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96 (25), 14228−14233.
(35) Ofek, O.; Karsak, M.; Leclerc, N.; Fogel, M.; Frenkel, B.;
Wright, K.; Tam, J.; Attar-Namdar, M.; Kram, V.; Shohami, E.;
Mechoulam, R.; Zimmer, A.; Bab, I. Peripheral cannabinoid receptor,
CB2, regulates bone mass. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (3),
696−701.
F
dx.doi.org/10.1021/ml3004236 | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX