Pentacycle derivatives as cannabinoid CB1 receptor ligands

Article abstract of DOI:10.1016/j.bmcl.2009.10.015

Cannabinoid CB-1 receptors have been the focus of extensive studies since the first clinical results of rimonabant (SR141716) for the treatment of obesity and obesity-related metabolic disorders were reported in 2001. To further evaluate the properties of

Full text of DOI:10.1016/j.bmcl.2009.10.015

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6632–6636
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pentacycle derivatives as cannabinoid CB1 receptor ligands
Suk Ho Lee, Hee Jeong Seo, Min Ju Kim, Suk Youn Kang, Sung-Han Lee, Kwangwoo Ahn, MinWoo Lee,
*
Ho-Kyun Han, Jeongmin Kim, Jinhwa Lee
Central Research Laboratories, Green Cross Corporation, 303 Bojeong-Dong, Giheung-Gu, Yongin, Gyeonggi-Do 446-770, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Cannabinoid CB-1 receptors have been the focus of extensive studies since the first clinical results of rimona-
bant (SR141716) for the treatment of obesity and obesity-related metabolic disorders were reported in 2001.
To further evaluate the properties of CB receptors, we have designed and efficiently prepared a series of
pentacycle derivatives. Five of the new compounds which displayed high in vitro rCB1 binding affinities were
assayed for binding to hCB2 receptor. Noticeably, 2-(5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-4-(5-
methyl-1,3,4-thiadiazol-2-yl)-1H-pyrazol-3-yl)-5-(1-(trifluoromethyl)cyclopropyl)-1,3,4-oxadiazole (16l)
demonstrated good binding affinity and decent selectivity for rCB1 receptor (IC50 = 1.72 nM, hCB2/
rCB1 = 142).
Received 31 July 2009
Revised 23 September 2009
Accepted 5 October 2009
Available online 8 October 2009
Keywords:
Rimonabant
Antiobesity
Cannabinoid
Antagonist
Pentacycle
Ó 2009 Elsevier Ltd. All rights reserved.
The prevalence of obesity is rapidly increasing globally. Obesity
has reached epidemic proportions especially in developed coun-
tries. Although obesity is associated with the pathogenesis of ma-
jor diseases including diabetes or cardiovascular diseases, no
satisfactorily safe and effective obesity drugs are available at the
moment. Thus, there is a tremendous opportunity to make a signif-
icant impact on the lives of the obese through the discovery and
development of additional pharmacotherapeutic options. Recently,
we reviewed new trends in medicinal chemistry approaches used
to develop drugs for treating obesity.1 Recent development of
obesity drugs reveals that it is possible to control appetite and re-
duce weight by blocking cannabinoid receptors in the brain, liver
or muscle, via cannabinoid (CB1) receptor antagonists or CB1
receptor inverse agonists.2,3 Cannabinoid CB1 receptor antagonist
is designed to block the effects of endogenous cannabinoids. This
type of drug is particularly interesting since it not only causes
weight loss but also reverses the metabolic effects of obesity such
as insulin resistance and hyperlipidemia.4 Another cannabinoid
receptor, CB2 is related to immune regulation and neurodegenera-
tion.5 Therefore, the CB2/CB1 selectivity should be taken into con-
sideration for new drug development of antiobesity agent.
reported to be in various phase of clinical trials.9,10,15d,24 However,
rimonabant was withdrawn from the market in 2008 due to risks
of severe psychiatric problems, including depression, anxiety, and
suicidality. Subsequently, taranabant and otenabant were discon-
tinued from developments at phase III, respectively. However, de-
spite consecutive failures of leading CB1 receptor antagonists,
works continue to identify novel peripherally restricted CB1 antag-
onists that are non-brain penetrant and do not induce serious psy-
chiatric disorders.
Apharmacophoremodelforthebindingofalowenergyconforma-
tion of rimonabant in the CB1 receptor has been well-docu-
mented.10,11 The key receptor–ligand interaction is reported to be a
hydrogen bond between the carbonyl group of rimonabant and the
Lys192-Asp366 residue of the CB1 receptor, thereby exerting a stabi-
lizing effectontheLys192-Asp366saltbridgeasshowninFigure1.2,25
To date, various analogs of rimonabant by replacing the key car-
bonyl group have been designed for the purpose of enhancing
Trp255
Tyr 275
Asp366-Lys192
Cl
Val196
A
The first specific cannabinoid CB1 receptor antagonist, rimona-
bant was discovered in a high throughput screening program at
Sanofi-Synthélabo in 1994.6 Several CB1 receptor antagonists
including SR141716 (rimonabant), SLV319 (ibipinabant),7a CP-
945,598 (otenabant)7b and MK-0364 (taranabant)8 had been
Phe278
O
4
D
3
Phe170
Leu387
5
C
N
HN
N
1
N
2
B
Trp279
Trp356
Cl
Met384
Cl
Phe200
Figure 1. Rimonabant and its receptor–ligand interaction.
* Corresponding author. Tel.: +82 31 260 9892; fax: +82 31 260 9020.
E-mail address: jinhwalee@greencross.com (J. Lee).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.015

S. H. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6632–6636
6633
binding affinity and selectivity for the CB1 receptor. We note that
such approaches were already demonstrated successfully with
imidazole,12 tetrazole.13 Subsequently, we also discovered that
the oxadiazole scaffold14a,b has also been employed for this pur-
pose, even though there are clear differences evident between
our previous works15 and these prior examples.
required carboxylic acid 8 in ‘tens of grams’ amount. Thus, the car-
boxylate 1 was converted to the bromide 2 in 55% yield using NBS
(N-bromosuccinimide) in the presence of a catalytic amount of AIBN
[2,20-azobis(2-methylpropionitrile)],8 and this intermediate was
then treated with silver nitrate in aqueous acetone18 to afford the
corresponding alcohol 3 in 96% yield. Subsequently, alcohol 3 was
protected with TIPSCl (triisopropylsilyl chloride) in the presence of
a suitable base such as imidazole to provide 4. Treatment of the ester
4 with hydrazine efficiently gave rise to hydrazide 5 in 97% yield for
the two steps which was used to couple with an acid in the presence
of appropriate coupling reagents such as EDC {1-[3-(dimethyl-
amino)propyl]-3-ethylcarbodiimide} and HOBt (1-hydroxybenzo-
triazole) to provide acylhydrazide 6 in 75% yield. Cyclization was
then performed smoothly using either Burgess reagent19a,b or
Lawesson’s reagent19c under reflux conditions. These reactions can
be carried out under microwave irradiation as well. Subsequent re-
moval of triisopropylsilyl group with TBAF (tetrabutylammonium
fluoride) was conducted to afford alcohols 7 in high yields. Oxidation
of the alcohols 7 to the corresponding aldehydes was achieved
through the use of Dess–Martin periodinane (80% yield).20 Alde-
hydeswerefurtheroxidizedto acids8 inhighyields byuseof sodium
chlorite and monobasic potassium phosphate in aqueous t-BuOH as
shown in Scheme 1.15a,b
With our efforts to discover and develop a new medicine for the
treatment of obesity, we have reported the diarylpyrazolyl oxadi-
azole derivatives as potent, selective, orally bioavailable cannabi-
noid-1 receptor antagonists for the treatment of obesity.15a
Therein, we demonstrated that incorporation of a 1,2,4-triazole
ring onto the C-4 region of pyrazole scaffold via a methylene linker
improved in vitro binding affinity, in turn leading to excellent
in vivo efficacy on animal model.16 We also reported that the polar
amide groups in the C-4 region of pyrazole scaffold can be accom-
modated based on the observation that this region is capable of
embracing substituents of varying functionality, size, and polar-
ity.15b Along this line, we envisioned that a bioisostere of polar
amide groups in the C-4 region of pyrazole can provide a novel ser-
ies of pentacycle derivatives which act as cannabinoid CB1 recep-
tor antagonists for the treatment of obesity.
Herein, we wish to describe the chemical synthesis, biological
evaluation of a novel series of pentacycle analogues as our addi-
tional research efforts toward discovery of a promising antiobesity
agent.
Alternatively, acids 14 can be prepared by benzylic bromina-
tion-type reaction on pyrazoles 11 as illustrated in Scheme 2.
Synthesis of pentacycle derivatives began with the generic car-
boxylate 1 as shown in Scheme 1.17 This reaction sequence was
developed and reported previously by our laboratory. We were able
to modify and refine some of the previous procedure to provide the
Cl
Cl
Cl
O
a
b
CO2Et
N
N
Cl
Cl
N
NH2
X
HN
N
Cl
Cl
1
a
d
Cl
9
CO2Et
CO2Et
N
N
N
N
Cl
Cl
Cl
Cl
Cl
Cl
O
O
2
3
4
X= Br
1
b
c
N
c
d
N
N
X =OH
NH
HN
N
N
N
X
X = OTIPS
Cl
Cl
Cl
Cl
10
Cl
Cl
Cl
OTIPS
OTIPS
11
O
O
e
f
N
Cl
Cl
N
NH
O
HN
N
Cl
Br
N
NH2
HN
HO
N
R
Cl
Cl
N
X
N
Cl
N
N
f
5
e
6
N
N
X
N
Cl
Cl
Cl
HO
Cl
HO
N
Cl
O
12
13
N
X
g
N
N
N
N
N
R
X
N
Cl
R
HO
N
O
Cl
Cl
Cl
N
X
Cl
N
7
8
N
R =
Cl
Cl
14
Cl
Scheme 1. Reagents and conditions: (a) NBS, AIBN, CCl4, reflux, 55% (b) AgNO3,
acetone–H2O, rt, 96%; (c) TIPSCl, imidazole, DMF, rt; (d) NH2NH2, EtOH, 90 °C, 97%
(two steps); (e) 1-(4-chlorophenyl)cyclopropanecarboxylic acid, EDC, HOBt, NMM,
DMF, rt, 75%; (f) (i) Burgess reagent (X = O) or Lawesson’s reagent (X = S), THF,
reflux, 84% (X = O), 79% (X = S), (ii) TBAF, THF, rt, 96% (X = O), 93% (X = S); (g) (i)
Dess–Martin periodinane, CH2Cl2, rt, 78% (X = O), 81% (X = S), (ii) NaClO2, KH2PO4,
2-methylbut-2-ene, t-BuOH–H2O, rt, 92% (X = O), 95% (X = S).
Scheme 2. Reagents and conditions: (a) NH2NH2, EtOH, 90 °C, 95%; (b) pivalic acid,
EDC, HOBt, NMM, DMF, rt, 87%; (c) Burgess reagent (X = O) or Lawesson’s reagent
(X = S), THF, reflux, 81% (X = O), 83% (X = S); (d) NBS, AIBN, CCl4, reflux, 87% (X = O),
79% (X = S) (e) (i) NaOAc, THF–H2O, rt, 91% (X = O), 88% (X = S), (ii) LiOH, THF–H2O,
rt, 92% (X = O), 93% (X = S); (f) (i) Dess–Martin periodinane, CH2Cl2, rt, 85% (X = O),
79% (X = S), (ii) NaClO2, KH2PO4, 2-methylbut-2-ene, t-BuOH–H2O, rt, 91% (X = O),
96% (X = S).

6634
S. H. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6632–6636
Table 1
The alcohol functionality was then introduced by treating bro-
mides 12 with sodium acetate, and subsequent basic hydrolysis
of the resulting acetates. Next, two-step oxidation reactions of
alcohols 13 to acids 14 were conducted in the same way as de-
scribed previously.15a,b
With requisite acids 8 or 14 in hand, preparation of pentacycle
derivatives was conducted as shown in Scheme 3. Thus, acids 14 were
coupled with a hydrazide such as acetohydrazide in the presence of
suitable coupling reagents such as EDC {1-[3-(dimethylamino)pro-
pyl]-3-ethylcarbodiimide}, HOBt (1-hydroxybenzotriazole), NMM
(4-methylmorpholine) in DMF (N,N-dimethylformamide) to provide
acylhydrazide 15 in 71–87% yields. Cyclization was then performed
using either Burgess reagent19a,b or Lawesson’s reagent19c under re-
flux conditions to generate the target pentacycle derivatives 16 in
67–88% yields as shown in Scheme 3.
The target oxadiazole analogues were evaluated in vitro at a rat
CB1 binding assay,21,23 and the results are shown in Table 1. We fo-
cused on t-butyl, 1-(trifluoromethyl)cyclopropyl, and (4-chloro-
phenyl)cyclopropyl group connected to the oxadiazole, since our
previous findings15a indicate that these lipophilic or fluorine-con-
taining substituents with gem-dimethyl or the corresponding cyclo-
propyl groups demonstrated the favorable biological activity of the
examined diarylpyrazolyl oxadiazoles. Bisoxadiazole 16a, 16b
showed reasonably good binding affinity (16a, IC50 = 6.47 nM; 16b,
IC50 = 3.93 nM) compared with rimonabant (IC50 = 5.0 1.0 nM),
while 1-(trifluoromethyl)cyclopropyl 16c showed rather low bind-
ing affinity for rCB1 receptor (IC50 = 14.7 nM). From the beginning,
it was encouraging to find out that this type of pentacycles acts as
a good rCB1 receptor ligand despite crowded appearances.
Binding affinity of oxadiazoles to rCB1 receptora
N
N
X
Z
N
O
N
N
R1
N
Cl
Cl
16
R1
Compound
rCB1 IC50
b
X
Z
Rimonabant
5.0 1.0c
6.47
3.93
14.7
2.83
10.3
4.14
2.56
4.91
4.70
3.69
2.13
1.72
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
O
O
O
S
t-Bu
16a
16b
16c
16d
16e
16f
16g
16h
16i
(4-Chlorophenyl)cyclopropyl
1-(Trifluoromethyl)cyclopropyl
t-Bu
S
S
O
O
O
S
(4-Chlorophenyl)cyclopropyl
1-(Trifluoromethyl)cyclopropyl
t-Bu
(4-Chlorophenyl)cyclopropyl
1-(Trifluoromethyl)cyclopropyl
t-Bu
16j
16k
16l
S
S
(4-Chlorophenyl)cyclopropyl
1-(Trifluoromethyl)cyclopropyl
a
b
c
rCB1 receptor was collected from brain tissue of SD rat.
These data were obtained by single determinations.
The rCB1R binding affinity for rimonabant has showed a certain number in the
close range (IC50 = 5.0 1.0 nM) in each different assay (>1500 compounds tested).
Next, in order to test the effect of oxadiazole ring at C-4 on pyra-
zole against cannabinoid receptor binding affinities, the oxadiazole
moiety was replaced with its isosteric thiadiazole ring. Unlike oxadi-
azole analogues, increase of rCB1 receptor binding affinity was ob-
served for the t-butyl derivative 16d (IC50 = 2.83 nM) by
replacement of oxadiazole 16a (IC50 = 6.47 nM). This phenomenon
was even more pronounced with 1-(trifluoromethyl)cyclopropyl
(16f, IC50 = 4.14 nM vs 16c, IC50 = 14.7 nM). In order to test the effect
of substituents of diphenylpyrazole, a chlorine atom (X = Cl) was re-
placed with a bromine atom (X = Br). Chlorine at X on 5-phenyl can
be displaced by bromine without exacerbating rCB1 receptor bind-
ing affinity. Rather, binding affinities for rCB1 receptor were im-
proved up to fivefold as exemplified by a pair of compounds
involving (4-chlorophenyl)cyclopropyl 16e (IC50 = 10.3 nM) and
16k (IC50 = 2.13 nM). Up to date, the best rCB1 receptor binding
affinity in the pentacycle series was obtained when X = bromine,
Z = S, and R1 = 1-(trifluoromethyl)cyclopropyl group were intro-
duced as in 16l (IC50 = 1.72 nM). The analogue 16l was also shown
to be potent in the CHO-hCB1R-Luciferase assay,26,27 with IC50 value
being 38.5 nM (for comparison, IC50 = 92.5 nM for rimonabant), thus
demonstrating inverse agonism activity of this series.
Subsequently, the effect of oxadiazole ring at C-3 on pyrazole
against cannabinoid receptor binding affinities was examined
through replacement of oxadiazole ring with its isosteric thiadiazole
ring. The binding affinity data of key diarylpyrazolyl thiadiazoles for
the rCB1 receptor are shown in Table 2. Compared with the corre-
sponding oxadiazoles, slight decreases of rCB1 receptor binding
affinity were observed, especially for the 1-(trifluoromethyl)cyclo-
propyl thiadiazole derivatives (approximately twofold decrease).
O
NH
HN
X
X
CO2H
O
N
N
N
Y
N
a
N
R1
Y
N
N
R1
N
Cl
Cl
Cl
Cl
14
15
N
N
X
Z
N
Y
X = Cl or Br; Y = O or S; Z = O or S;
1 = t-Bu, 1-(trifluoromethyl)cyclopropyl,
(4-chlorophenyl)cyclopropyl
b
N
R
N
R1
N
Cl
Cl
16
Scheme 3. Reagents and conditions: (a) acetohydrazide, EDC, HOBt, DMF, rt, 71–87%; (b) Burgess reagent or Lawesson’s reagent, THF, reflux, 67–88%.

S. H. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6632–6636
6635
Table 2
In conclusion, we investigated a series of pentacycle derivatives
for their binding affinity for cannabinoid rCB1 and hCB2 receptors.
We have identified a novel series of small molecule rCB1 ligands
that demonstrate binding affinity superior to that of known rCB1
antagonists. Several compounds in this series showed potent
rCB1 receptor binding affinities, validating the hypothesis that a
bioisostere of polar amide groups in the C-4 region of pyrazole
can provide a novel series of pentacycle derivatives which act as
rCB1 receptor ligands. Of note is 2-(5-(4-bromophenyl)-1-(2,4-
dichlorophenyl)-4-(5-methyl-1,3,4-thiadiazol-2-yl)-1H-pyrazol-3-
yl)-5-(1-(trifluoromethyl)cyclopropyl)-1,3,4-oxadiazole (16l) was
shown to possess the highest binding affinity in this pentacycle
series prepared to date. The analogue 16l was also shown to be po-
tent in the CHO-hCB1R-Luciferase assay, with IC50 value being
38.5 nM, thus demonstrating inverse agonism activity of this ser-
ies. The information obtained from the SAR studies in this series
might help to design more active CB1 antagonists or inverse ago-
nists that are structurally related to this series.
Binding affinity of thiadiazoles to rCB1 receptora
N
N
X
Z
N
S
N
N
R1
N
Cl
Cl
16
R1
Rimonabant
(4-Chlorophenyl)cyclopropyl
1-(Trifluoromethyl)cyclopropyl
(4-Chlorophenyl)cyclopropyl
1-(Trifluoromethyl)cyclopropyl
t-Bu
Compound
rCB1 IC50
b
X
Z
5.0 1.0c
3.23
26.9
3.03
7.39
4.93
4.44
4.86
2.56
3.70
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
O
O
S
16m
16n
16o
16p
16q
16r
16s
16t
S
O
O
O
S
(4-Chlorophenyl)cyclopropyl
1-(Trifluoromethyl)cyclopropyl
t-Bu
Acknowledgments
S
S
(4-Chlorophenyl)cyclopropyl
1-(Trifluoromethyl)cyclopropyl
16u
16v
We thank Dr. Chong-Hwan Chang for his leadership throughout
small molecule programs at GCC. Also we are grateful to Dr. Eun
Chul Huh, Mr. Jung Ho Kim and Mr. Jung-Won Jeon at GCC Office
of R&D planning and coordination for their supports and services.
4.21
a
b
c
rCB1 receptor was collected from brain tissue of SD rat.
These data were obtained by single determinations.
The rCB1 R binding affinity for rimonabant has showed a certain number in the
close range (IC50 = 5.0 1.0 nM) in each different assay (>1500 compounds tested).
Supplementary data
This observation was made in two pairs of compounds 16n
(IC50 = 26.9 nM) and 16p (IC50 = 7.39 nM) by replacement of oxadi-
azoles 16c (IC50 = 14.7 nM) and 16f (IC50 = 4.14 nM), respectively.
This phenomenon was even more pronounced with bis-thiadiazole
(16v, IC50 = 4.21 nM vs 16l, IC50 = 1.72 nM), indicating bis-thiadia-
zole rings make the molecule somewhat bulkier overall, thereby
slightly reducing binding potency against rCB1 receptor.
The interesting compounds were further evaluated with obser-
vation of the hCB2 receptor binding affinity. 22 The IC50 values were
measured for the recombinant human CB2 receptor expressed in
CHO cells and employing [3H]WIN-55,212-2 as a radio-ligand.21
These results are shown in Table 3. The hCB2/rCB1 selectivity
turned out to be modest, ranging from 142 to 167 among the com-
pounds tested. However, compound with (4-chlorophenyl)cyclo-
propyl appears to deactivate hCB2 receptor binding affinity while
maintaining their favorable binding affinity against rCB1 receptor,
thereby improving hCB2/rCB1 selectivity. In order to further eval-
uate this series, pharmacokinetic (PK) properties of 16l have been
measured in rats. After oral administration of a 5-mg/kg dose of 16l
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.10.015.
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to rats, a Cmax of 0.03 lg/mL was obtained at 1.3 h with a moderate
systemic clearance rate of 12.6 mL/mg/Kg. The elimination half-life
for 16l following oral administration was 18.4 h in rats. 16l showed
relatively low oral bioavailability (F = 7.2%) in rats, implying its
probable solubility-limited absorption.
Table 3
Binding affinity of selected pentacycles to rCB1 and hCB2 receptorsa,b
c
c
Compound
rCB1 IC50
hCB2 IC50
hCB2/rCB1 selectivity
Rimonabant
16g
16l
5.0 1.0d
2.56
1.72
1760c
414
245
352
162
142
16t
16u
2.56
3.70
427
>10,000
167
>2703
a
b
c
rCB1 receptor was collected from brain tissue of SD rat.
hCB2 receptor was recombinant human receptor expressed in CHO cell.
These data were obtained by single determinations.
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d
This data was obtained by multiple determinations.

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in 96-well plate with TME buffer containing 0.5% essentially fatty acid free
bovine serum albumin (BSA), 3 nM [3H]WIN55,212-2 (for CB2 receptor, NEN;
specific activity 50-80 Ci/mmol) or 3 nM ([3H]CP55,940, [3H]2-[(1S,2R,5S)-5-
hydroxy-2-(3-hydroxypropyl) cyclohexyl]-5-(2-methyloctan-2-yl)phenol, for
rCB1 receptor, NEN; specific activity 120–190 Ci/mmol) and various
concentrations of the synthesized cannabinoid ligands in a final volume of
200 lL. The assays were incubated for 1 h at 30 °C and then immediately
filtered over GF/B glass fiber filter (Perkin–Elmer Life and Analytical Sciences,
Boston, MA) that had been soaked in 0.1% PEI for 1 h by a cell harvester
(Perkin–Elmer Life and Analytical Sciences, Boston, MA). Filters were washed
five times with ice-cold TBE buffer containing 0.1% essentially fatty acid free
BSA, followed by oven-dried for 60 min and then placed in 5 mL of scintillation
fluid (Ultima Gold XR; Perkin–Elmer Life and Analytical Sciences, Boston, MA),
and radioactivity was quantitated by liquid scintillation spectrometry. In rCB1
and hCB2 receptor competitive binding assay, non-specific binding was
assessed using
Specific binding was defined as the difference between the binding that
occurred in the presence and absence of 1 M concentrations of rimonabant or
1 lM rimonabant and 1 lM WIN55,212-2, respectively.
20. Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.
l
21. Kuster, J. E.; Stevenson, J. I.; Ward, S. J.; D’Ambra, T. E.; Haycock, D. A. J.
Pharmacol. Exp. Ther. 1993, 264, 1352.
WIN55,212-2 and was 70–80% of the total binding. IC50 was determined by
non-linear regression analysis using Graph-Pad PRISM. All data were collected
in triplicate and IC50 was determined from three independent experiments.
24. As of November 5, 2008, Sanofi-Aventis, Merck, and Pfizer announced that they
have decided to discontinue their ongoing clinical development programs
about rimonabant (SR141716), taranabant (MK-0364), and otenabant (CP-
945,598), respectively, based on changing regulatory perspectives on the risk/
benefit profile of the CB1 class and likely new regulatory requirements for
approval.
22. Murphy, J. W.; Kendall, D. A. Biochem. Pharmacol. 2003, 65, 1623.
23. CB1 and CB2 receptor binding assay. For the rCB1 receptor binding studies, rat
cerebellar membranes were prepared as previously described by the methods
of Kuster et al.20 Male Sprague-Dawley rats (200–300 g) were sacrificed by
decapitation and the cerebella rapidly removed. The tissue was homogenized
in 30 vol of TME buffer (50 mM Tris–HCl, 1 mM EDTA, 3 mM MgCl2, pH 7.4)
using
a Dounce homogenizer. The crude homogenates were immediately
centrifuged (48,000g) for 30 min at 4 °C. The resultant pellets were
resuspended in 30 vol of TME buffer, and protein concentration was
determined by the method of Bradford and stored at ꢀ70 °C until use. For
the hCB2 receptor binding studies, CHO K-1 cells were transfected with the
human CB2 receptor as previously described, and cell membranes were
prepared as described above.21 Competitive binding assays were performed as
25. Hurst, D. P.; Lynch, D. L.; Barnett-Norris, J.; Hyatt, S. M.; Seltzman, H. H.; Zhong,
M.; Song, Z.-H.; Nie, J.; Lewis, D.; Reggio, P. H. Mol. Pharmacol. 2002, 62, 1274.
26. Bouaboula, M.; Perrachon, S.; Milligan, L.; Canat, X.; Rinaldi-Carmona, M.;
Portier, M.; Barth, F.; Calandra, B.; Pecceu, F.; Lupker, J.; Maffrand, J. P.; Le Fur,
G.; Casellas, P. J. Biol. Chem. 1997, 272, 22330.
27. Calandra, B.; Portier, M.; Kerne´is, A.; Delpech, M.; Carillon, C.; Le Fur, G.;
described. Briefly, approximately 10
lg of rat cerebella membranes (containing
Ferrara, P.; Shire, D. Eur. J. Pharmacol. 1999, 374, 445.
rCB1 receptor) or cell membranes (containing hCB2 receptor) were incubated