Oxadiazole-diarylpyrazole 4-carboxamides as cannabinoid CB1 receptor ligands

Article abstract of DOI:10.1016/j.bmcl.2009.02.063

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.2009.02.063

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1899–1902
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Oxadiazole-diarylpyrazole 4-carboxamides as cannabinoid CB1 receptor ligands
Suk Ho Lee, Hee Jeong Seo, Min Ju Kim, Suk Youn Kang, Kwang-Seop Song, Sung-Han Lee,
*
Myung Eun Jung, 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
rimonabant (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 pre-
pared a series of oxadiazole-diarylpyrazole 4-carboxamides. Six of the new compounds which displayed
high in vitro CB1 binding affinities were assayed for binding to CB2 receptor. Noticeably, 5-(4-bromophe-
nyl)-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-1-(2,4-dichlorophenyl)-N-phenyl-1H-pyrazole-4-carboxamide
(12q) and 5-(4-bromophenyl)-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-1-(2,4-dichlorophenyl)-N-(pyridin-
2-yl)-1H-pyrazole-4-carboxamide (12r) demonstrated good binding affinity and decent selectivity for
CB1 receptor (IC50 = 1.35 nM, CB2/CB1 = 286 for 12q; IC50 = 1.46 nM, CB2/CB1 = 256 for 12r).
Ó 2009 Elsevier Ltd. All rights reserved.
Received 11 November 2008
Revised 4 February 2009
Accepted 16 February 2009
Available online 21 February 2009
Keywords:
Rimonabant
Antiobesity
Cannabinoid receptor antagonist
Oxadiazole
Carboxamide
Trp255
Recent development of obesity drugs reveals that it is possible
to control appetite and reduce weight by blocking cannabinoid
receptors in the brain, liver or muscle, via cannabinoid (CB1) recep-
tor antagonists or CB1 receptor inverse agonists.1,2 A cannabinoid
CB1 receptor antagonist is designed to block the effects of endoge-
nous 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.3
The other cannabinoid receptor, CB2 is related to immune regula-
tion and neurodegeneration.4 Therefore, the CB2/CB1 selectivity
should be taken into consideration for new drug development of
anti-obesity agent.
Typ 275
Phe278
Asp366-Lys192
OD
Cl
Val196
Phe170
A
C
N
HN
N
N
Leu387
B
Trp279
Cl
Met384
Cl
Tr p356
Phe200
Figure 1. Rimonabant and its receptor–ligand interaction.
The first specific cannabinoid CB1 receptor antagonist, rimona-
bant was discovered in a high throughput screening program at
Sanofi-Synthélabo in 1994.5 Several CB1 receptor antagonists
including SR141716 (rimonabant), SLV319 (ibipinabant),6 CP-
945,598 (otenabant) and MK-0364 (taranabant)7 have been re-
ported to be in various phase of clinical trials.8,9,23 A pharmaco-
phore model for the binding of a low energy conformation of
rimonabant in the CB1 receptor has been well-documented.9,10
The key receptor–ligand interaction is known to be a hydrogen
bond between the carbonyl group of rimonabant and the Lys192-
Asp366 residue of the CB1 receptor, thereby exerting a stabilizing
effect on the Lys192-Asp366 salt bridge as shown in Figure 1.1
To date, various analogs of rimonabant by replacing the key car-
bonyl group have been designed for the purpose of enhancing
binding affinity and selectivity for the CB1 receptor. We note that
such approaches were already demonstrated successfully with
imidazole,11 tetrazole.12 Subsequently, we also discovered that
the oxadiazole13 scaffold has also been employed for this purpose,
even though there are clear differences evident between our previ-
ous works14 and these prior examples.
With our efforts to discover and develop a new medicine for the
treatment of obesity, we have recently reported the diarylpyrazolyl
oxadiazole derivatives as potent, selective, orally bioavailable can-
nabinoid-1 receptor antagonists for the treatment of obesity.14a
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.15 We envisioned 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 polarity.
* Corresponding author. Tel.: +82 31 260 9892; fax: +82 31 260 9870.
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.2009.02.063

1900
Cl
S. H. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1899–1902
Herein, we wish to describe the chemical synthesis, biological
Cl
X
evaluation of oxadiazole-diarylpyrazole 4-carboxamide analogs
as our additional research efforts toward discovery of a promising
antiobesity agent.
a
d
CO2Et
CO2Et
N
N
N
N
Synthesis of oxadiazole-biarylpyrazoles commenced with the
generic carboxylate 1.16 he carboxylate 1 was converted to the bro-
mide 2 using NBS in the presence of a catalytic amount of AIBN,8
and this intermediate was then treated with silver nitrate in aque-
ous acetone17 to afford the corresponding alcohol 3. 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 which was used to couple with an acid in the presence
of appropriate coupling reagents such as EDC and HOBt to provide
acyl hydrazide 6. Cyclization was then performed using the Bur-
gess reagent18 under heating conditions, and subsequent TIPS
deprotection with TBAF was conducted to afford alcohol 7. Oxida-
tion of the alcohol 7 to the corresponding aldehyde was achieved
through the use of Dess–Martin periodinane.19 Aldehyde was fur-
ther oxidized to acid 8 by use of sodium chlorite and monobasic
potassium phosphate in aqueous t-BuOH as shown in Scheme 1.14a
Alternatively, acid 8 can be prepared by benzylic bromination-
type reaction on pyrazole 9 as illustrated in Scheme 2.24 The
alcohol functionality was then introduced by treating bromide 10
with sodium acetate, followed by basic hydrolysis of the resulting
acetate. Subsequent two-step oxidation of alcohol 7 to acid 8 was
conducted in the same way as described previously.14a
With requisite acid 8 in hand, the preparation of various amides
was conducted as shown in Scheme 3. The first approach toward
12 using coupling reagents such as EDC, HOBt in the suitable base
was unable to effect the requisite coupling reaction in a satisfac-
tory manner. Subsequently, we found out that the two-step se-
quence via acyl chloride 11 proved to be more efficient in
producing amides 12 in reasonable yields.
The target analogs were evaluated in vitro at a rat CB1 binding
assay,20,22 and the results are shown in Table 1.25 Unsubstituted
carboxamide 12a had modest in vitro activity for rat CB1 receptor
(IC50 = 23.8 nM). As the size of the carbon chain on carboxamide in-
creases, increase in the binding affinity for rat CB1 receptor is ob-
served up to C-3 chain. Thus, N-methyl 12b, N-ethyl 12c, and N-
propyl 12d showed rat binding affinity for CB1 receptor
IC50 = 18.3, 12.7, 5.72 nM, respectively. The binding affinity for
CB1 was decreased when the alkyl chain became more prolongated
(12g, IC50 = 17.8 nM).
Cl
Cl
Cl
Cl
2
3
4
X= Br
1
b
c
X =OH
X = OTIPS
OTIPS
O
Cl
Cl
OTIPS
N
O
e
f
N
N
NH
O
HN
N
HN
NH2
R1
Cl
Cl
Cl
Cl
5
6
Cl
Cl
HO
HO
O
g
N
O
N
N
N
N
N
O
R1
R1
N
N
Cl
Cl
Cl
Cl
7
8
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%
(2 steps); (e) R1CO2H, EDC, HOBt, DMF, rt, 71%; (f) (i) Burgess reagent, THF, reflux,
76%; (ii) TBAF, THF, rt, 95%; (g) (i) Dess–Martin periodinane, CH2Cl2, rt, 78%; (ii)
NaClO2, KH2PO4, 2-methylbut-2-ene, t-BuOH–H2O, rt, 95%.
Cl
Cl
Br
N
O
a
b
N
O
N
N
N
N
N
R1
N
R1
Cl
Cl
Cl
Cl
9
10
Cl
HO
N
Cl
HO
N
Preferable in vitro binding affinity displayed for the carbocycle
(N-cyclopropyl, 12f, IC50 = 3.27 nM) than straight chain (N-propyl,
12d, IC50 = 5.72 nM) or branched chain (N-isopropyl, 12e,
IC50 = 9.31 nM) if the same number of carbons are counted. As
the size of carbocycle increases, the binding affinity for CB1 recep-
tor decreases. For example, N-cyclopentyl, 12h showed the slightly
weaker binding affinity for CB1 receptor (IC50 = 7.25 nM), whereas
N-cyclopropyl, 12f showed the superior binding affinity for CB1
receptor (IC50 = 3.27 nM). N,N-disubstitution on carboxamide such
as N,N-dimethyl 12m, N-ethyl-N-methyl 12n appeared to be toler-
O
c
N
N
N
N
R1
O
N
R1
O
N
Cl
Cl
Cl
Cl
8
7
Scheme 2. Reagents and conditions: (a) NBS, AIBN, CCl4, reflux, 78%; (b) (i) NaOAc,
THF–H2O, rt, 63%; (ii) LiOH, THF–H2O, rt, 95%; (c) (i) Dess–Martin periodinane,
CH2Cl2, rt, 78%; (ii) NaClO2, KH2PO4, 2-methylbut-2-ene, t-BuOH–H2O, rt, 95%.
R3
R2
Cl
Cl
HO
Cl
Cl
O
O
N
O
N
O
N
O
a
b
N
N
N
O
N
N
N
R1
R1
N
N
N
R1
N
Cl
Cl
Cl
Cl
Cl
Cl
8
11
12
Scheme 3. Reagents and conditions: (a) oxalyl chloride, cat. DMF, CH2Cl2, rt (b) NHR2R3, THF, rt, 60–70% (2 steps).

S. H. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1899–1902
1901
Table 1
In order to improve the binding affinity levels for the CB1 recep-
tor, a structural replacement of 4-chlorophenyl moiety to 4-
bromophenyl was undertaken.
Binding affinity of 4-carboxamide diarylpyrazoles to rCB1 receptora
R1
N
R2
Cl
In addition, the surrogate effect of the cyclopropyl group for a
gem-dimethyl group was also studied. A structurally related series
of oxadiazole-diarylpyrazole 4-carboxamide derivatives was pre-
pared in an analogous fashion previously described. The binding
affinity data of these oxadiazole-diarylpyrazole 4-carboxamide
analogs are shown in Table 2. The interesting compounds were fur-
ther evaluated with observation of the CB2 receptor binding affin-
ity. The IC50 values were measured for the recombinant human CB2
receptor expressed in CHO cells and employing [3H]WIN-55,212-2
O
N
N
N
O
N
Cl
Cl
12
R1
R2
Compound
rCB1 IC50
b
a
radio-ligand.21 Replacement of 5-(4-chlorophenyl) 12j
Rimonabant
H
Me
5.0 1.0c
23.8
18.3
12.7
5.72
9.31
3.27
17.8
7.25
17.2
1.66
1.47
3.51
13.4
11.5
62.1
as
H
H
H
H
H
H
12a
12b
12c
12d
12e
12f
(IC50 = 1.66 nM) with 5-(4-bromophenyl) 12q (IC50 = 1.35 nM)
slightly improved CB1 receptor binding affinity. However, this phe-
nomenon is not clearly demonstrated by replacement of 12k
(IC50 = 1.47 nM) with 12r (IC50 = 1.46 nM). Regarding the CB2/CB1
selectivity, there appeared to deteriorate as chlorines were
switched to the corresponding bromides, as exemplified by two
pairs of compounds involving 12j (CB2/CB1 selectivity = 445) to
12q(CB2/CB1 selectivity = 286) and 12k(CB2/CB1 selectivity = 433)
to 12r (CB2/CB1 selectivity = 256). As far as the CB2 receptor activ-
ity is concerned, the best result was obtained when R2 is substi-
Et
n-Propyl
i-Propyl
c-Propyl
n-Butyl
c-Pentyl
N-Piperidinyl
Ph
2-Pyridinyl
CH2(CO)CH3
Me
H
H
H
12g
12h
12i
H
12j
H
H
12k
12l
Me
Me
Et
12m
12n
12o
tuted with bulky (4-chlorophenyl)cyclopropyl (12s, IC50 = >10 lM
Et
Et
for CB2R), indicating the importance of massive groups for the re-
gion for improvement of the CB2/CB1 selectivity.26
12p
93.4
CH2CH2
CH2CH2
In conclusion, we investigated a series of oxadiazole-diarylpy-
razole 4-carboxamide derivatives for their binding affinity for can-
nabinoid CB1 and CB2 receptors. We have identified a novel series
of small molecule cannabinoid CB1 ligands that shows binding
affinity superior to that of known CB1 antagonists. Several com-
pounds in this series exhibited potent CB1 receptor binding affini-
ties, validating the hypothesis that the polar amide groups in the C-
4 region of pyrazole scaffold can be accommodated. Importantly,
these analogs also display good selectivity for CB1R over CB2R.
Additional PK and in vivo efficacy studies in addition to further
SAR studies of the oxadiazole-diarylpyrazole 4-carboxamide deriv-
atives will be the subject of future investigations.
a
CB1 receptor was collected from brain tissue of SD rat.
These data were obtained by single determinations.
The CB1R binding affinity for rimonabant has showed a certain number in the
b
c
close range (IC50 = 5.0 1.0 nM) in each different assay (>1500 compounds tested).
ated for the replacement to a degree, but as the size of substituted
chains become bigger (12o, 12p), none appeared more potent than
the monosubstituted carboxamide (12c). As shown in N-(2-oxo-
propyl)-1H-pyrazole-4-carboxamide 12l, polar carbonyl group ap-
pears to be tolerated, indicating that there might be some SAR
potential for other carbocycles. Among the various rings, or chains
tested on carboxamide, the best result was obtained when nitrogen
on carboxamide has simple phenyl (12j) or 2-pyridinyl (12k). They
showed excellent binding affinity for rat CB1R (IC50 = 1.66 nM for
12j, 1.47 nM for 12k, respectively).
Acknowledgements
Financial support was provided by Green Cross Corporation
(GCC). We thank Dr. Chong-Hwan Chang for their many helpful
Table 2
Binding affinity of 4-carboxamide diarylpyrazoles to rCB1 and hCB2 receptorsa,b
R1
HN
X
O
N
N
N
R2
O
N
Cl
Cl
12
R1
R2
X
Compound
rCB1 IC50
hCB2 IC50
CB2/CB1 selectivity
c
c
Rimonabant
Ph
2-Pyridinyl
Ph
2-Pyridinyl
Ph
2-Pyrininyl
5.0 1.0d
1.66
1.47
1.35
1.46
1760c
738
636
386
374
ꢀ352
445
433
286
256
t-Butyl
t-Butyl
t-Butyl
t-Butyl
Cl
Cl
Br
Br
Br
Cl
12j
12k
12q
12r
12s
12t
(4-Chlorophenyl)cyclopropyl
trifluoromethylcyclopropyl
1.96
1.94
>10,000
488
>5102
252
a
CB1 receptor was collected from brain tissue of SD rat.
CB2 receptor was recombinant human receptor expressed in CHO cell.
These data were obtained by single determinations.
b
c
d
This data was obtained by multiple determinations.

1902
S. H. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1899–1902
receptor) or cell membranes (containing CB2 receptor) were incubated 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 CB1
receptor, NEN; specific activity 120–190 Ci/mmol) and various concentrations
discussions throughout small molecule programs at GCC. Also we
are grateful to Dr. Eun Chul Huh, Mr. Jung Ho Kim and Ms. Jae-
Young Jang at GCC Office of R& D planning and coordination for
their supports and services.
of the synthesized cannabinoid ligands in a final volume of 200 lL. The assays
Supplementary data
were incubated for 1 h at 30 °C and then immediately filtered over GF/B glass
fiber fiber filter (PerkinElmer Life and Analytical Sciences, Boston, MA) that had
been soaked in 0.1% PEI for 1 h by a cell harvester (PerkinElmer 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;
PerkinElmer Life and Analytical Sciences, Boston, MA), and radioactivity was
quantitated by liquid scintillation spectrometry. In CB1 and CB2 receptor
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.02.063.
References and notes
competitive binding assay, nonspecific binding was assessed using
rimonabant and 1 M WIN55,212-2, respectively. Specific binding was defined
as the difference between the binding that occurred in the presence and absence
of 1 M concentrations of rimonabant or WIN55,212-2 and was 70–80% of the
total binding. IC50 was determined by nonlinear regression analysis using
Graph–Pad PRISM. All data were collected in triplicate and IC50 was determined
from three independent experiments.
1 lM
1. Lange, J. H. M.; Kruse, C. G. Drug Discovery Today 2005, 10, 693.
2. Howlett, A. C.; Breivogel, C. S.; Childers, S. R.; Deadwyler, S. A.; Hampson, R. E.;
Porrio, L. J. Neuropharmacology 2004, 47, 345.
3. Lafontan, M.; Piazza, P. V.; Girard, J. Diabetes Metab. 2007, 33, 85. and reference
therein..
4. (a) Carrier, E. J.; Kearn, C. S.; Barkmeier, A. J.; Breese, N. M.; Yang, W.;
Nithipatikom, K.; Pfister, S. L.; Cambell, W. B.; Hillard, C. J. Mol. Pharmacol. 2004,
65, 999; (b) Klein, T. W.; Newton, C.; Larsen, K.; Lu, L.; Perkins, I.; Nong, L.;
Friedman, H. J. Leukocyte Biol. 2003, 74, 486.
5. (a) Rinaldi-Carmona, M.; Barth, F.; Héaulme, M.; Alonso, R.; Shire, D.; Congy, C.;
Soubrié, P.; Breliére, J.-C.; le Fur, G. Life Sci. 1995, 56, 1941; (b) Rinaldi-Carmona,
M.; Barth, F.; Héaulme, M.; Shire, D.; Calandra, B.; Congy, C.; Martinez, S.;
Maruani, J.; Néliat, G.; Caput, D.; Ferrara, P.; Soubrié, P.; Breliére, J.-C.; le Fur, G.
FEBS Lett. 1994, 350, 240.
6. Lange, J. H.; Coolen, H. K.; van Stuivenberg, H. H.; Dijksman, J. A.; Herremans, A.
H.; Ronken, E.; Keizer, H. G.; Tipker, K.; McCreary, A. C.; Veerman, W.; Wals, H.
C.; Stork, B.; Verveer, P. C.; den Hartog, A. P.; de Jong, N. M.; Adolfs, T. J.;
Hoogendoorn, J.; Kruse, C. G. J. Med. Chem. 2004, 47, 627.
7. Lin, L. S.; Lanza, T. J.; Jewell, J. J. P.; Liu, P.; Shah, S. K.; Qi, H.; Tong, X.; Wnag, J.;
Xu, S. S.; Fong, T. M.; Shen, C.-P.; Lao, J.; Xiao, J. C.; Shearman, L. P.; Stribling, D.
S.; Rosko, K.; Strack, A.; Marsh, D. J.; Feng, Y.; Kumar, S.; Samuel, K.; Yin, W.; der
Ploeg, L. V.; Mills, S. G.; MacCoss, M.; Goulet, M. T.; Hagmann, W. K. J. Med.
Chem. 2006, 49, 7584.
8. (a) Barth, F.; Congy, C.; Martinez, S.; Rinaldi, M. WO97/19063, 2000.; (b) Barth,
F.; Casellas, P.; Congy, C.; Martinez, S.; Rinaldi, M. C07D/23114, 1995.
9. Hutst, 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.
10. Shim, J.-Y.; Welsh, W. J.; Cartier, E.; Edwards, J. L.; Howlett, A. C. J. Med. Chem.
2002, 45, 1447.
l
l
23. 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.
24.
Comparison of alternative routes to key intermediates 81,b
Scheme 1
Scheme 2
R1
4-(Chlorophenyl) cyclopropyl
19 (8-steps)
8.119
t-Butyl
15 (8-steps)
4.835
Overall yield (%)
Retention time (min)
Purity (%)
99
99
b
14a
Compound 9 was prepared from compound 1 as described in
.
1
HPLC Purity was determined by Agilent 1200 series high performance liquid
chromatography with UV detection at 254 nm (XterraÒ MS C18 3.5
l
m,
2.1 Â 50 mm, 12 min, 0.3 mL/min flow rate, 50–100% 0.05% TFA in CH3CN/ 100–50%
0.05% TFA in H2O).
11. (a) Dow, L.R. U.S. Patent 2004/0077650 A1, 2004.; (b) Coe, J.W. U.S. Patent
2005/0043327 A1, 2005.; (c) Barth, F.; Congy, C.; Gueule, P.; Rinaldi-Carmona,
M.; Van Broeck, D. PCT Patent Application WO2006/087480A1, 2006.
12. Kang, S. Y.; Lee, S.-H.; Seo, H. J.; Jung, M. E.; Ahn, K.; Kim, J.; Lee, J. Bioorg. Med.
Chem. Lett. 2008, 18, 2385.
25.
Table of HPLC retention time and Purity of final compoundsa
Compound
Retention time (min)
Purity (%)
13. (a) Barth, F.; Congy, C.; Gueule, P.; Ridaldi-Carmona, M.; Van Brodeck, D. PCT
Patent WO 2006/087480 A1, 2006.; (b) Moritani, Y. PCT Patent WO 2007/
046550 A1, 2007.
12a
12b
12c
12d
12e
12f
12g
12h
12i
3.717
4.279
5.483
6.831
6.601
5.705
7.988
8.201
2.953
9.540
6.280
4.703
4.513
5.551
6.594
5.338
9.850
6.597
11.795
6.661
>99
>99
>99
99
14. (a) Lee, S. H.; Seo, H. J.; Lee, S.-H.; Jung, M. E.; Park, J.-H.; Yoo, J.; Yun, H.; Na, J.;
Kang, S. Y.; Song, K.-S.; Kim, M.-a.; Chang, C.-H.; Kim, J.; Lee, J. J. Med. Chem.
2008, 51, 7216; (b) Lee, J.; Kim, J.; Chang, C.-H.; Lee, S.H.; Seo, H.J.; Kang, S.Y.;
Song, K.S.; Kim, J.Y.; Kim, M.-a.; Lee, S.-H.; Ahn, K.; Jung, M.E.; Park, J.-H. PCT
Patent WO 2008/039023 A1, 2008.; (c) Kim, J. Y.; Seo, H. J.; Lee, S.-H.; Jung, M.
E.; Ahn, K.; Kim, J.; Lee, J. Bioorg. Med. Chem. Lett. 2009, 19, 142.
15. Hilderbrandt, A. L.; Kelly-Sullivan, D. M.; Black, S. C. Eur. J. Pharmacol. 2003, 462,
125–132.
16. Lan, R.; Liu, Q.; Fan, P.; Lin, S.; Fernando, S. R.; McCallion, D.; Pertwee, R.;
Makriyannis, A. J. Med. Chem. 1999, 42, 769.
17. Amengual, R.; Marsol, C.; Mayeux, E.; Sierra, M.; Wagner, P. PCT Patent WO
2006/133926 A1, 2006.
18. (a) Brain, C. T.; Paul, J. M.; Loong, Y.; Oakely, P. J. Tetrahedron Lett. 1999, 40,
3275–3278; (b) Brain, C. T.; Brunton, S. A. Synlett 2001, 382.
19. Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.
20. Kuster, J. E.; Stevenson, J. I.; Ward, S. J.; D’Ambra, T. E.; Haycock, D. A. J.
Pharmacol. Exp. Ther. 1993, 264, 1352.
99
>99
>99
>99
>99
99
>99
>99
>99
>99
99
12j
12k
12l
12m
12n
12o
12p
12q
12r
12s
12t
>99
>99
>99
99
>99
21. Murphy, J. W.; Kendall, D. A. Biochem. Pharmacol. 2003, 65, 1623.
22. CB1 and CB2 Receptor Binding Assay. For the CB1 receptor binding studies, rat
cerebellar membranes were prepared aspreviously described by the methods of
Kuster et al.20 MaleSprague–Dawley rats (200–300 g) were sacrificed by
decapitation and the cerebella rapidly removed. The tissue was homogenized
in 30 volumes of TME buffer (50 mM Tris–HCl, 1 mM EDTA, 3 mM MgCl2,
a
HPLC Retention time and Purity were determined by Agilent 1200 series high
performance liquid chromatography with UV detection at 254 nm (XterraÒ MS C18
3.5
lm, 2.1 Â 50 mm, 12 min, 0.3 mL/min flow rate, 50–100% 0.05% TFA in CH3CN/
100–50% 0.05% TFA in H2O).
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 volumes of TME buffer, and protein concentration was
determined by the method of Bradford and stored at À70 °C until use. For the CB2
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 described.
26. As pointed out by
a
reviewer, compound 12s represents the best
diarylpyrazole-oxadiazole to date, and the selectivity is impressive. A bulky
group such as (4-chlorophenyl)cyclopropyl in compound 12 appears to
deactivate CB2 receptor binding affinity while maintaining its activity for
CB1 receptor, thereby improving CB2/CB1 selectivity.
Briefly, approximately 10 lg of rat cerebella membranes (containing CB1