Discovery of 2-[5-(4-chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-1H- pyrazol-3-yl]-1,5,5-trimethyl-1,5-dihydro-imidazol-4-thione (BPR-890) via an active metabolite. A novel, potent and selective cannabinoid-1 receptor inverse agonist with high antiobes

Article abstract of DOI:10.1021/jm900471u

By using the active metabolite 5 as an initial template, further structural modifications led to the identification of the titled compound 24 (BPR-890) as a highly potent CB1 inverse agonist possessing an excellent CB2/1 selectivity and remarkable in vivo

Full text of DOI:10.1021/jm900471u

4496 J. Med. Chem. 2009, 52, 4496–4510
DOI: 10.1021/jm900471u
Discovery of 2-[5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-1H-pyrazol-3-yl]-1,5,5-trimethyl-
1,5-dihydro-imidazol-4-thione (BPR-890) via an Active Metabolite. A Novel, Potent and Selective
Cannabinoid-1 Receptor Inverse Agonist with High Antiobesity Efficacy in DIO Mice
Chien-Huang Wu,† Ming-Shiu Hung,† Jen-Shin Song,† Teng-Kuang Yeh, Ming-Chen Chou, Cheng-Ming Chu, Jiing-Jyh Jan,
Min-Tsang Hsieh, Shi-Liang Tseng, Chun-Ping Chang, Wan-Ping Hsieh, Yinchiu Lin, Yen-Nan Yeh, Wan-Ling Chung,
Chun-Wei Kuo, Chin-Yu Lin, Horng-Shing Shy, Yu-Sheng Chao, and Kak-Shan Shia*
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, No. 35, Keyan Road, Zhunan Town, Miaoli County
35053, Taiwan, R.O.C. † These authors contributed equally to this work.
Received April 13, 2009
By using the active metabolite 5 as an initial template, further structural modifications led to the
identification of the titled compound 24 (BPR-890) as a highly potent CB1 inverse agonist possessing an
excellent CB2/1 selectivity and remarkable in vivo efficacy in diet-induced obese mice with a minimum
effective dose as low as 0.03 mg/kg (po qd) at the end of the 30-day chronic study. Current SAR studies
along with those of many existing rimonabant-mimicking molecules imply that around the pyrazole
C3-position, a rigid and deep binding pocket should exist for CB1 receptor. In addition, relative to the
conventional carboxamide carbonyl, serving as a key hydrogen-bond acceptor during ligand-CB1
receptor interaction, the corresponding polarizable thione carbonyl might play a more critical role in
stabilizing the Asp366-Lys192 salt bridge in the proposed CB1-receptor homology model and inducing
significant selectivity for CB1R over CB2R.
Introduction
rently only two antiobesity agents orlistat,6 a gastrointestinal
lipase inhibitor, and sibutramine,7 a serotonin reuptake in-
hibitor, are available, but both meet with moderate success
owing to their limited weight-loss efficacy and significant
accompanying adverse effects. However, substantial clinical
evidence revealed that CB1 antagonists might result in risks of
severe psychiatric problems, including depression, anxiety,
and stress disorders; these findings, indeed, have made rimo-
nabant 1 (SR141716A),8 the first CB1 inverse agonist ap-
proved and launched in Europe in 2006, withdrawn from the
market in 2008, and thereof, several CB1 target-related can-
didates including taranabant 2 (MK-0364)9 and otenabant
3 (CP-945598)10 were suspended at the late clinical develop-
ment stage (phase III) (Figure 1). In the meanwhile, increased
evidence indicate that CB1 receptors present in the peripheral
tissues, including fat and liver, might regulate food intake and
energy balance as effectively as those appearing in the central
nervous system (CNS).11 It is widely accepted that adverse
effects would be significantly attenuated when compounds act
solely on receptors or enzymes located in the peripheral
system rather than CNS. As such, instead of the conventional
brain-penetrant CB1 antagonists, an alternative to develop
peripherally restricted CB1 antagonists without penetrating
blood-brain barrier (BBB) was then adopted. Recently, this
strategy was found to meet with a certain degree of success in
that less CNS toxic profile and sufficient weight-reduction
efficacy have been observed with rimonabant-mimicking
analogues acting exclusively on peripheral CB1 receptor as
reported by Piomelli, et al.12 and Jenrin Discovery.13 Never-
theless, it is believed that more extensive safety evidence
should be provided to validate the aforementioned concept.
The endocannabinoid system (ECSa), consisting, to date, of
endocannabinoids such as anandamide and 2-arachidonoyl
glycerol (2-AG), two cannabinoid receptors type 1 (CB1) and
type 2 (CB2), and enzymes responsible for endogenous ligand
synthesis (phospholipase D) and degradation (fatty acid
amide hydrolase (FAAH)), is a complicate physiological
system involved in metabolic homeostasis such as modulating
energy and glucose balance, etc.1-4 The CB1 receptor is
predominantly expressed in several brain areas and peripheral
tissues; in contrast, the CB2 receptor is expressed almost
exclusively in peripheral cells of the immune system related
to immune regulation and neurodegeneration.5 Studies on the
ECS along the CB1-cannabinoid axis have disclosed and
validated that blocking CB1 receptor activity could lead to
weight loss through reduction of food intake and increased
energy expenditure. Therefore, CB1 receptor antagonists were
highly anticipated in the past decade to become a new
therapeutic approach to treat obesity, a typical disease result-
ing from a long-term imbalance of the energy intake and
expenditure, and widely recognized as one of major health
concerns in the modern society, particularly given that cur-
*To whom correspondence should be addressed. Phone: þ886-37-
246166, ext. 35709. Fax: þ886-37-586456. E-mail: ksshia@nhri.org.tw.
a Abbreviations: ECS, endocannabinoid system; CB, cannabinoid;
BBB, blood-brain barrier; DIO, diet-induced obese; NADPH, β-nico-
tinamide adenine dinucleotide phosphate; SAR, structure-activity
relationships; LHMDS, lithium bis(trimethylsily)amide; NBS, N-bro-
mosuccinimde; DMF, N,N-dimethylformamide; Eu-GTP, europium
guanosine 50-triphosphate; IA, inverse agonist; FAAH, fatty acid amide
hydrolase; CNS, central nervous system; ESMS, electrospray mass
spectra.
r
pubs.acs.org/jmc
Published on Web 06/16/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4497
Chart 1. Proposed Mechanism of the Metabolite 5 Derived from Imide 4
Figure 1. Chemical structure of compounds 1-5.
Figure 2. Compound 4 was incubated in rat liver microsomes with NADPH; the blue peak stands for the initial compound 4 with a retention
time of 9.64 min; the red and green peaks stand for the growth of metabolite 5 at the expense of 4 after incubation in 15 and 60 min, respectively;
the pink peak stands for the synthetic compound 5, being identical to metabolite 5, with a retention time of 8.61 min. Details of incubation and
analytical method are described in the Experimental Section B1.
Along this line, a novel series of imide derivatives, originally
intended to reduce the ability in blood-brain penetration,14-16
was then designed and experimentally realized in our
laboratories.17 Unfortunately, it was found that during
pharmacokinetic studies in rats, many of these imide CB1
antagonists disappeared in approximately 50 and 15 min
following oral administration and iv injection, respectively.
Instead, a primary metabolite, which proved to be an active
metabolite later, was significantly formed in each case exa-
mined. Similar metabolic profiles were observed and verified

4498 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Wu et al.
Scheme 1. Synthesis of Imidazol-4-one/thione Compounds 5 and 11-24a
a Reagents and conditions: (a) diethyl oxalate, LHMDS, -78 °C to room temp, 16 h, 65-80%; (b) 2,4-dichlorophenylhydrazine hydrochloride,
EtOH, room temp, 20 h, then AcOH, reflux, 24 h, 36-50% over two steps; (c) KOH, MeOH, 60 °C, 4 h, 84-94%; (d) oxalyl chloride, DMF (cat),
toluene, 1 h; 10a-10g, NEt3, THF, 0 °C to room temp, 15 h, then NaOMe, MeOH, 60 °C, 4 h, 30-60% over two steps; (e) Lawesson’s reagent, toluene,
60 °C, 4 h, 58-77%.
in parallel by incubating them in vitro in the rodent or human
liver microsomes in the presence of β-nicotinamide adenine
dinucleotide phosphate (NADPH). As typified by imide 4
(IC50=82.9 nM; CB2/1=35) incubated in the rat microsomes
(Figure 2), an active metabolite 5 (IC50=54.7 nM; CB2/1=9),
unambiguously identified through comparison with the syn-
thetic molecule illustrated in Scheme 1, appeared within 15
min and steadily grew at the expense of 4 during the progress
of reaction. A mechanistic rationale is depicted in Chart 1. It is
conceivable that the metabolic demethylation occurred initi-
ally to convert 4 into the metastable amine 4a, which under-
went intramolecular cyclization rapidly via an enzyme-
mediated dehydration process to generate the final metabolite
5. On the basis of these findings, herein, we wish to report that
using 5 as an initial model, further structural modifications led
to the identification of compounds 24 (BPR-890) (IC50
=
12.0 nM; CB2/1 = 396) and 28 (IC50 = 32.6 nM; CB2/1 =
314) as highly potent and selective CB1 inverse agonists with a
significant increase in weight-reduction efficacy in diet-
induced obese (DIO) mice by 33-fold and 10-fold, respec-
tively, as compared to agent 1 (IC50=13.2 nM; CB2/1=124).
Detailed description of the design, synthesis, and structure-
activity relationships (SAR) of the newly developed imidazol-
4-ones (thiones) as well as the long-term efficacy study on
weight loss in DIO mice for potential compounds will be
presented as follows.
Chemistry. Compounds 5 and 11-24 were prepared ac-
cording to a general synthetic method shown in Scheme 1

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4499
Scheme 2. Synthesis of Imidazol-4-ones 25 and 26a
a Reagents and conditions: (a) NBS, THF, room temp, 72 h, 59%; (b) KOH, MeOH, 60 °C, 4 h, 87%; (c) oxalyl chloride, DMF (cat), toluene, 1 h; 10a,
NEt3, THF, 0 °C to room temp, 15 h, then NaOMe, MeOH, 60 °C, 4 h, 51% over two steps; (d) C3H5B(OH)2, K2CO3, Pd(PPh3)4, toluene, microwave,
80 °C, 2 h, 35%.
Scheme 3. Synthesis of Imidazol-4-ones 27-33a
a Reagents and conditions: (a) oxalyl chloride, DMF (cat), toluene, 1 h; 10h-10k, NEt3, THF, 0 °C to room temp, 15 h, then POCl3,
1,2-dichloroethane, 80 °C, 4 h, 47-52%; (b) NaH, MeI, DMF, room temp, 2 h, 68-69%.
using compound 5 and its corresponding thioketone 23,
respectively, as a typical example. Treatment of 1-(4-chlor-
ophenyl)-propan-1-one (6a) with diethyl oxalate in the pre-
sence of LHMDS as a base gave rise to lithium salt 7a in 80%
yield, which in turn, without purification, was coupled with
2,4-dichlorophenylhydrazine hydrochloride in ethanol fol-
lowed by intramolecular cyclization in acetic acid under
refluxing conditions to provide ester 8a in 49% yield over
two steps. Compound 8a thus obtained was subjected to
basic hydrolysis under standard conditions to afford car-
boxylic acid 9a in 94% yield. The carboxylic group of 9a was
then activated with oxalyl chloride in the presence of DMF
as a catalyst to form the corresponding acyl chloride, which
was allowed to couple with 2-methyl-2-methylamino-pro-
pionamide (10a) to afford an amide intermediate. This
intermediate, without purification, could undergo intramo-
lecular cyclization effectively under treatment with sodium
methoxide in methanol, giving rise to the desired product 5
in 55% yield over two steps. Compound 5 was smoothly
transformed into the corresponding thioketone 23 with
Lawesson’s reagent18 in toluene in 77% yield. On the other
hand, compounds 25 and 26 were provided according to
a synthetic sequence illustrated in Scheme 2 using inter-
mediate 8b as the starting material. Selective bromination

4500 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Wu et al.
Table 1. Biological Evaluation of Novel 2-pyrazolyl Imidazol-4-one/
thione Derivatives on hCB1 and hCB2 Receptors
conditions with NaOMe as employed in Schemes 1 and 2
for compounds 5 and 11-26. Further alkylation of 27 with
methyl iodide in DMF resulted in the corresponding methy-
lated 32 in 69% yield. All tested compounds mentioned
above exhibited more than 95% purity, as evidenced by
elemental analysis outlined in Supporting Information,
prior to submission for biological assays and animal studies.
Results and Discussion
Compounds described above were subjected to in vitro
biological evaluation toward CB1 and CB2 receptors, results
of which are compiled in Tables 1-3, and related SAR
studies are discussed in the following. Using the active meta-
bolite 5 (IC50=54.7 nM; CB2/1=9) as a novel template, a
series of structurally related imidazol-4-ones/thiones were
thus synthesized according to Schemes 1 and 2. As illustrated
in Table1, when themethylsubstituent of5 atR3 was replaced
with a linear alkyl group, the resulting compounds 11-13
exhibited a significant decrease in binding affinity for both
CB1 and CB2 receptor in chain length-ascending order:
methyl > ethyl > n-propyl > n-butyl. In addition, there
was no improvement in CB2/1 selectivity (CB2/1=4-11) by
above linker-elongation modifications as compared to 5
(CB2/1 = 9). Similar results in poor CB1 binding affinity
and selectivity were also observed for compounds 14-16,
wherein R3 was individually equipped with an aliphatic group
in various dimensions, indicating that a hydrocarbon moiety
bulkier than methyl group seems not to be tolerated at this
position. As such, using the methyl motif as a fixed theme for
R3, further structural modifications were extended to explore
R2 substitution. Encouragingly, it was found that when the
methyl group of R2 in 5 (IC50 =54.7 nM; CB2/1=9) was
replaced with an ethyl unit, the resulting compound 18 (IC50=
12.9 nM; CB2/1=213) showed not only a substantial increase
in CB1 binding affinity but also an excellent selectivity for
CB1R over CB2R. In contrast, when the methyl group
switched to a smaller hydrogen atom, compound 17 (IC50=
325.1 nM) thus obtained resulted in a 6-fold decrease in CB1
activityalongwith aslightimprovement in selectivity (CB2/1=
24). However, once a larger group, such as a cyclopropyl ring
or a bromine atom, was installed instead of the hydrogen, the
CB1 activity was recovered as indicated by compounds 25
(IC50 = 42.5 nM) and 26 (IC50 = 43.9 nM), respectively,
relative to the initial model 5 (IC50=54.7 nM). Although a
variety of functional groups could be established for R2
substitution from synthetic point of view,20 both methyl and
ethyl units were found to be extremely successful for rimona-
bant-mimicking molecules in many historical cases.2 Similar
conclusions could also be derived based on an array of
examples outlined in Table 1. Accordingly, R2 was then
restricted to the methyl or ethyl functionality for further
structural exploration with particular emphasis on the latter
in light of metabolic stability.2 As for R1 substitution, as
expected, bromine atom is an excellent bioisosteric surrogate
for the chlorine atom as highlighted by compounds 19 (IC50=
47.4 nM) and 20 (IC50=16.3 nM) in comparison with their
IC50 (nM)a,b
selectivity
CB2/CB1
compd R1
R2
R3
Me
X
hCB1
54.7 ( 6.9
hCB2
5
Cl Me
O
O
O
486.9 ( 129.8
9
11
7
11 Cl Me
12 Cl Me
13 Cl Me
14 Cl Me
15 Cl Me
Et
n-Pr
107.0 ( 17.9 1173.7 ( 235.1
853.6 ( 135.0 5510.1 ( 318.3
n-Bu O 1110.8 ( 52.0 4763.3 ( 349.7
4
allyl
cyclo-
propyl
Bn
O
248.9 ( 75.9 2881.5 ( 252.3
95.2 ( 28.1 1950.4 ( 151.1
12
21
O
16 Cl Me
17 Cl
O
O
O
O
O
O
O
S
197.0 ( 37.3 5126.9 ( 160.1
325.1 ( 20.2 7946.1 ( 658.9
12.9 ( 3.9 2736.6 ( 374.3 213
47.4 ( 18.9 1095.5 ( 358.1
16.3 ( 1.2 768.4 ( 130.9
211.8 ( 93.1 1058.5 ( 38.6
15.9 ( 3.5 3968.0 ( 116.7 250
6.3 ( 2.7 901.4 ( 189.5 143
12.0 ( 4.1 4746.0 ( 481.5 396
26
24
H
Me
Me
18 Cl Et
19 Br Me
20 Br Et
21 OMeMe
22 CF3 Me
23 Cl Me
24 Cl Et
25 Cl Br
Me
Me
23
47
5
Me
Me
Me
Me
S
Me
O
O
42.5 ( 21.2 1570.6 ( 503.9
43.9 ( 9.2 822.0 ( 76.0
37
19
26 Cl cyclo- Me
propyl
1
13.2 ( 2.3 1631.1 ( 208.5 124
a Binding affinity determined by inhibition of [3H] CP-55,940
([3H] 34) binding to hCB1 or hCB2-transfected HEK 293 membrane is
expressed as IC50.
b Data are expressed as the mean ( SD of at least three
independent experiments.
was effected with NBS in THF to afford 8h in 57% yield,
which in turn was hydrolyzed under basic conditions to give
carboxylic acid 9h in high yield (87%). Compound 9h thus
obtained was reacted with oxalyl chloride and DMF (cat.) to
form the corresponding acyl chloride, which was then con-
densed with 10a to give the amide intermediate. This inter-
mediate, without purification, could undergo intramolecular
cyclization in the presence of sodium methoxide in methanol
at 60 °C to afford product 25 in 51% yield over two steps.
Compound 25 was successfully coupled with cyclopropyl-
boronic acid under Suzuki-Miyaura coupling conditions19
to furnish the desired 4-cyclopropyl pyrazole 26 in 35%
yield. Similarly, compounds 27-33 were readily prepared
following a general synthetic approach depicted in Scheme 3
using compound 27 and its methylated derivative 32, respec-
tively, as a typical example. Carboxylic acid 9a was treated
with oxalyl chloride in toluene to form acyl chloride, which
was coupled with 2-amino-2-methyl-propionamide (10h) to
provide the corresponding amide. The crude amide, without
purification, was then treated with POCl3 in 1,2-dichlor-
oethane at elevated temperature to undergo intramolecular
cyclization, affording product 27 in 52% yield over two
steps. Interestingly, it is noteworthy that the cyclization
process for compounds 27-33 must be carried out under
acidic conditions with POCl3 instead of previous basic
chlorine counterparts 5 (IC50 = 54.7 nM) and 18 (IC50
=
12.9 nM), respectively. Also noted was the finding that
compounds containing an electron-withdrawing group at
R1 appeared superior to the corresponding compounds
with an electron-donating group in bestowing CB1 binding
affinity and selectivity as typified by 22 (R1 =CF3; IC50 =
15.9 nM; CB2/1=250) and its counterpart 21 (R1=OCH3;

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4501
Table 2. Biological Evaluation of Novel 2-Pyrazolyl Imidazol-4-one Derivatives on hCB1 and hCB2 Receptors
IC50 (nM)a,b
Selectivity
CB2/CB1
compd
R1
R2
R3
R4
hCB1
hCB2
27
28
29
30
31
32
33
1
Me
Et
Me
Me
Me
Me
H
53.4 ( 14.2
32.6 ( 4.7
22.8 ( 4.3
14.6 ( 5.2
12.3 ( 2.2
33.6 ( 5.1
33.8 ( 4.7
13.2 ( 2.3
3500.6 ( 831.1
10260.0 ( 579.1
3177.2 ( 441.3
1372.2 ( 317.3
496.7 ( 149.4
3249.2 ( 122.8
4326.9 ( 264.2
1631.1 ( 208.5
66
314
139
94
H
Et
Et
-CH2CH2CH2-
-CH2C2H4CH2-
-CH2C3H6CH2-
Me
H
H
Et
H
40
97
Me
Et
Me
Me
Me
Me
Me
128
124
a Binding affinity determined by inhibition of [3H] 34 binding to hCB1 or hCB2-transfected HEK 293 membrane is expressed as IC50
.
b Data are
expressed as the mean ( SD of at least three independent experiments.
IC50=211.8 nM; CB2/1=5). More encouragingly, when the
oxygen atom of the imidazol-4-one moiety at pyrazole C3-
position was replaced with the sulfur atom, the corresponding
thioketone 23 (IC50 = 6.3 nM; CB2/1 = 143) exhibited a
substantial improvement in both potency and selectivity for
CB1 receptor by approximately 10-fold relative to 5. Similar
structural modifications were also extended to compound 18
(IC50=12.9 nM; EC50=9.8 nM; CB2/1=213), leading to the
identification of thioketone 24 (IC50 =12.0 nM; EC50=5.1
nM; CB2/1=396) with a modest enhancement on selectivity
and functional activity for CB1 receptor, presumably due to
the presence of a polarizable thioketone group resulting in an
unusual stabilizing effect on the Asp366-Lys192 salt bridge as
Figure 3. Binding mode of compound 1 in the proposed CB1-
receptor homology model.
highlighted in the proposed CB1-receptor homology model in
Figure 3, where the conventional carboxamide carbonyl is
believed to serve as a key hydrogen-bond acceptor.2
reflected by a sharp drop in CB2/1 selectivity for compounds
28-31. In general, as exemplified by compounds 32 and 33
(Table 2) and their corresponding regioisomers 5 and 18
(Table 1), these two series exhibit very similar CB1/2 recep-
tor-ligand interaction behaviors in terms of binding affinity
and selectivity. As well, the rimonabant-mimicking molecules
developed above along with many other documented mimetics,
wherein a variety of functionalities at the pyrazole C3-position,
including 1,3,4-oxadiazole ring,20,21 1,2,4-oxadiazole ring,22
imide,17,23 and sulfonamide group,23 have been installed and
proven to be effective bioisosteres to the conventional carbox-
amide moiety, one might come to a conclusion that a rigid and
deep cavity should exist around the CB1-receptor pocket
surrounded by a series of lipophilic residues (Val196/Phe170/
Leu387/Met384) as illustrated in Figure 3.
Compounds with excellent CB1 binding affinity and
selectivity were selected for further studies on their func-
tional activity (EC50) and intrinsic property as detailed
in the Experimental Section B4 As indicated in Table 3, all
promising compounds were evaluated and determined to
be inverse agonists as they exhibited a significant decrease in
the induced Eu-GTP binding intensity relative to the basal
On the basis of these promising results, several structurally
closely related analogues 27-33 (Table 2) were further pre-
pared for biological evaluation following a synthetic sequence
in Scheme 3. It was observed that when R1 was fixed with the
ethylgroup, compounds28 (IC50=32.6nM; CB2/1=314)and
33 (IC50=33.8 nM; CB2/1=128) exhibited significant CB2/1
selectivity compared to their R1-methyl counterparts 27
(IC50=53.4 nM; CB2/1=66) and 32 (IC50=33.6 nM; CB2/
1=97), respectively; however, the corresponding CB1 binding
activities (IC50 values) are comparable, indicating that for this
new series the presence of the ethyl moiety mainly weakens
CB2-binding ability. Along this line, an array of compounds
appended with different cyclic rings R to the carbonyl were
also explored. The results disclosed that the binding affinity
toward both CB1 and CB2 increased with the enlargement of
the ring size as demonstrated by compounds 29 (IC50=22.8 nM;
CB2/1=139; with a four-membered ring), 30 (IC50=14.6 nM;
CB2/1=94; with a five-membered ring), and 31 (IC50=12.3 nM;
CB2/1 = 40; with a six-membered ring), indicating that the
increase of lipophilicity at the R position might enhance dual
CB1/2 binding affinity with a preference for CB2 receptor as

4502 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Wu et al.
Table 3. Functional Activity of Compounds 18, 22, 23, 24, 28, 29, and
33 with High CB1 Binding Affinity and CB1/2 Selectivity
selectivity
compd EC50 (nM)a,c IC50 (nM)b,c CB2/CB1 intrinsic propertyd
18
22
23
24
28
29
33
1
9.8 ( 2.0
81.0 ( 22.1
8.5 ( 1.6
5.1 ( 1.0
20.2 ( 5.2
12.3 ( 2.2
65.5 ( 31.6
15.7 ( 4.6
12.9 ( 3.9
15.9 ( 3.5
6.3 ( 2.7
12.0 ( 4.1
32.6 ( 4.7
22.8 ( 4.3
33.8 ( 4.7
13.2 ( 2.3
213
250
143
396
314
139
128
124
IAe
IA
IA
IA
IA
IA
IA
IA
a Functional activity determined by inhibition of Eu-GTP binding to
hCB1-transfected HEK 293 membrane is expressed as EC50. b Binding
affinity determined by inhibition of [3H] 34 binding to hCB1or hCB2-
transfected HEK 293 membrane as indicated in Tables 1 and 2 is
expressed as IC50. c Data are expressed as the mean ( SD of the least
three independent experiments. d The intrinsic property was assessed by
the use of Eu-GTP binding to hCB1-transfected HEK 293 membrane at
a concentration of 10 μM as indicated in Figure 3.28,29 e Inverse agonist.
Figure 5. Six-hour culmulative food intake of compounds 24 (0.1,
0.3, and 1 mg/kg) and 28 (0.3, 1, and 3 mg/kg) was examined in rat
spontaneous feeding model relative to reference 1 at an oral dose of
10 mg/kg and vehicle C. Data are presented as mean of calorie
ingestion normalized by body weight ( standard error (n = 6/
group). Statistical analysis was performed by t test. P < 0.05 (*)
was considered significant. The experimental protocol is detailed in
section B5.
more encouragingly, reduction of body weight (Figure 6B) was
found to proceed in a dose-dependent manner throughout the
30-day period with a relative weight-loss rate of 14.7%, 19.4%,
and 25.4% for 0.03, 0.1, and 0.3 mg/kg groups, respectively,
indicating that this compound is more efficacious than refer-
ence 1 (10 mg/kg, po qd; 23.8% weight reduction) by more than
33-fold in antiobesity efficacy. Similar in vivo responses includ-
ing food intake suppression (Figure 7A) and body weight loss
(Figure 7B) were also observed with compound 28. As com-
pared to 1 (10 mg/kg) with a 23.7% weight reduction, com-
pound 28 displayed a comparable 27.4% reduction at a dose as
low as 1 mg/kg (Figure 7B) and was determined to be more
efficacious than 1 by at least 1 order of magnitude. Although
compounds 24 and 28 have comparable in vitro activities to 1,
they displayed much more potent in vivo efficacy than 1. These
unexpected outcomes might be attributed to their higher con-
centration found in the several areas of the brain including
hypothalamus, where CB1 receptors are predominantly ex-
pressed. As indicated by pharmacokinetic analysis, the brain
concentration of 24, 28, and 1 was found to be 243 ( 47, 1118 (
204, and 113 ( 14 ng/g at an oral dose of 0.3, 1, and 10 mg/kg,
respectively, suggesting that the distribution of 24 and 28 into
the brain is much higher than 1. These data appear to lend
support to the remarkable efficacies for 24 and 28 in DIO
mouse studies. As well, for comparison purposes, chronic
treatment of DIO rats with compound 24 was also carried
out, and preliminary results revealed that when dosed orally
once a day for 29 days, the relative weight loss compared with
vehicle was 5.1% (vs 19.4% in DIO mice) and 9.5% (vs 25.4%
in DIO mice) for the 0.1 and 0.3 mg/kg groups (po qd),
respectively.24 Accordingly, compound 24 is considered to be
as effective as agent 2 in terms of the corresponding efficacy
data observed with DIO rats in the literature.25
Figure 4. Eu-GTP binding assay of selected 2-pyrazolylimidazol-
4-one/thione 18, 22, 24, 28, 29, and 33, as well as reference
compounds 1 and 34 at a concentration of 10 μM on the hCB1
cannabinoid receptor, was conducted.27-29 Data are expressed as
the mean ( SD of at least three experiments performed in duplicate.
Statistical significance is assessed by unpaired two-tailed t test using
the GraphPad Prism program (GraphPad Software, San Diego,
CA). P < 0.05 (*) was considered significant. Compounds with the
induced Eu-GTP binding intensity around the basal level are
defined as neutral antagonists (NA); compounds with a significant
increase in the intensity relative to the basal level are agonists (A)
such as 34; compounds with a significant decrease in the binding
intensity are inverse agonists (IA) such as reference 1. The experi-
mental protocol is detailed in section B4.
level at a concentration of 10 μM (Figure 4). Compounds 24
(EC50=5.1 nM; IC50=12.0 nM; CB2/1=396; inverse agonist)
and 28 (EC50=20.2 nM; IC50=32.6 nM; CB2/1=314; inverse
agonist), each serving as a representative of two different
series in the current studies, possessed satisfactory biological
profiles in all aspects and thus were elected as potential
candidates for further in vivo efficacy studies. As depict-
ed in Figure 5, it was found that as compared to reference 1
(10 mg/kg), compounds 24 and 28 are orally active and able to
suppress food intake effectively in nondeprived rats at a dose
as low as 0.3 and 3 mg/kg, respectively, indicating that both
are well qualified for the further long-term disease animal
studies. As a result, chronic treatment of diet-induced obese
(DIO) mice with compounds 24 and 28 for 29 days led to
significant weight loss with an impressively minimum effective
dose of 0.03 and 0.1 mg/kg (po qd), respectively. As demon-
strated in Figure 6A, the food intake was substantially
reduced at all doses with 24 and appeared to be restored to
the level of vehicle-treated mice approximately after six days;
In summary, using the active metabolite 5 as an initial
template, further structural modifications led to the identifi-
cation of compound 24 as a highly potent CB1 inverse agonist
with an excellent CB2/1 selectivity and a remarkable anti-
obesity efficacy. As indicated above, in the 30-day chronic
study, compound 24 showed an astounding effect on weight
reduction in DIO mouse model with a minimum effective dose
as low as 0.03 mg/kg and was comparable to agent 1 (10 mg/kg)
at a dose of 0.3 mg/kg, indicating that it could be more potent
than 1 by at least 33-fold. Current SAR studies in conjunction

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4503
Figure 6. Antiobesity efficacy in DIO mouse model following oral administration of compound 24 (0.03, 0.1, and 0.3 mg/kg, po qd) compared
to reference 1 (10 mg/kg, po qd) in the 30-day chronic study (n = 8/group). (A) daily food intake; (B) cumulative percentage of body weight
(BW) change. Data are presented as mean ( standard error. The experimental protocol is detailed in section B6.
Figure 7. Antiobesity efficacy in DIO mouse model following oral administration of compound 28 (0.1 and 1 mg/kg, po qd) compared to
reference 1 (10 mg/kg, po qd) in the 30-day chronic study (n = 8/group). (A) daily food intake; (B) cumulative percentage of body weight (BW)
change. Data are presented as mean ( standard error. The experimental protocol is detailed in section B6.
with those of many other rimonabant-mimicking analogues
might imply that around the pyrazole C3-position, a rigid and
deep binding pocket should exist for CB1 receptor. In addition,
as compared to the conventional carboxamide carbonyl, ser-
ving as a key hydrogen-bond acceptor during ligand-CB1
receptor interaction, the corresponding thione carbonyl might
play a more critical role in stabilizing the putative Asp366-
Lys192 salt bridge and inducing significant selectivity for CB1R
over CB2R. Further efficacy and pharmacokinetic studies,
including central nervous system toxicity, behavioral effects,
and blood-brain penetration ability for aforementioned pro-
mising antiobesity agents are under active investigation, and
results will be reported elsewhere in due course.
Experimental Section
A. Chemistry. Unless otherwise stated, all materials used were
commercially available and used as supplied. Reactions requiring

4504 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Wu et al.
anhydrous conditions were performed in flame-dried glassware
and cooled under an argon or nitrogen atmosphere. Unless
otherwise stated, reactions were carried out under argon or
nitrogen and monitored by analytical thin-layer chromato-
graphy performed on glass-backed plates (5 cmꢀ10 cm) pre-
coated with silica gel 60 F254 as supplied by Merck. Visualization
of the resulting chromatograms was done by looking under an
ultraviolet lamp (λ=254 nm), followed by dipping in an ethanol
solution of vanillin (5% w/v) containing sulfuric acid (3% v/v)
or phosphomolybdic acid (2.5% w/v) and charring by heat gun.
Solvents for reactions were dried and distilled under an argon or
nitrogen atmosphere prior to use as follows: THF, diethyl ether
(ether), and DMF from a dark-blue solution of sodium benzo-
phenone ketyl; toluene, dichromethane, and pyridine from
calcium hydride. Flash chromatography was used routinely
for purification and separation of product mixtures using silica
gel 60 of 230-400 mesh size as supplied by Merck. Eluent
systems are given in volume/volume concentrations. 1H and
13C NMR spectra were recorded on Varian Mercury-300
(300 MHz), Varian Mercury-400 (400 MHz), or Varian Mer-
cury-600 (600 MHz). Chloroform-d or dimethyl sulfoxide-d6
was used as the solvent and TMS (δ 0.00 ppm) as an internal
standard. Chemical shift values are reported in ppm relative to
the TMS in delta (δ) units. Multiplicities are recorded as s
(singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet),
dd (doublet of doublet), dt (doublet of triplet), m (multiplet).
Coupling constants (J) are expressed in Hz. Electrospray mass
spectra (ESMS) were recorded using an agilent 1100 MSD mass
spectrometer. Spectral data were recorded as m/z values. Com-
bustion elemental analyses were performed by the microanaly-
tical laboratory at National Chung Hsing University, Taiwan,
ROC. As required, all tested compounds compiled in Tables 1-
3 possessed a purity of higher than 95%, as evidenced by
elemental analysis data (Supporting Information) for each
compound with an accuracy of elements C, H, and N within
(0.4% with the exception of compound 24, possessing a varia-
tion within ( 0.46% for elements C and N, prior to submitting
for functional assay (Eu-GTP), binding affinity assay, and
animal studies.
The combined extracts were washed with water, saturated aqu-
eous sodium bicarbonate, brine, dried over anhydrous sodium
sulfate, filtered, and concentrated. Purification of the crude
residue by flash chromatography on silica gel with n-hexane/
ethyl acetate (9:1) gave the ester 8a (1.89 g, 49%) as a white solid:
mp 120-121 °C. 1H NMR (400 MHz, CDCl3) δ 7.29 (d, J=2.0
Hz, 1H), 7.27 (d, J=8.4 Hz, 1H), 7.22-7.20 (m, 3H), 6.99 (d, J=
8.4 Hz, 2H), 4.36 (q, J=6.8 Hz, 2H), 2.25 (s, 3H), 1.33 (t, J=
6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 162.8, 143.1, 143.0,
136.1, 136.0, 135.1, 133.2, 131.0 (ꢀ2), 130.9, 130.2, 129.0 (ꢀ2),
127.9, 127.2, 119.2, 61.1, 14.6, 9.8. ESMS m/z: 409.1 (M þ 1).
5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-1H-pyrazole-3-car-
boxylic Acid Ethyl Ester (8b). Treatment of 7b (2.29 g, 8.79
mmol) with 2,4-dichlorophenylhydrazine hydrochloride (1.68 g,
7.5 mmol) and acetic acid (30 mL) gave compound 8b (1.73 g,
1
50%) as a white solid: mp 104-106 °C. H NMR (400 MHz,
CDCl3) δ 7.46 (d, J=2.0 Hz, 1H), 7.41 (d, J=8.4 Hz, 1H), 7.35
(dd, J=8.4, 2.0 Hz, 1H), 7.27(d, J=8.4 Hz, 2H), 7.11 (d, J=
8.4 Hz, 2H), 7.06 (s, 1H), 4.45 (q, J=6.8 Hz, 2H), 1.42 (t, J=
6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 162.2, 145.6, 145.2,
136.6, 136.1, 136.0, 133.3, 130.8, 130.6, 129.4 (ꢀ2), 129.3 (ꢀ2),
128.3, 127.6, 109.1, 61.6, 14.6. ESMS m/z: 395.1 (M þ 1).
5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-1H-pyra-
zole-3-carboxylic Acid Ethyl Ester (8c). Treatment of 7c (2.13 g,
7.38 mmol) with 2,4-dichlorophenylhydrazine hydrochloride
(1.41 g, 6.60 mmol) and acetic acid (30 mL) gave compound
8c (1.41 g, 45%) as a white solid: mp 95-96 °C. 1H NMR (400
MHz, CDCl3) δ 7.36 (d, J=2.0 Hz, 1H), 7.33 (d, J=8.4 Hz, 1H),
7.29 (d, J=8.4 Hz, 2H), 7.26 (dd, J=8.4, 2.0 Hz, 1H), 7.07 (d, J=
8.4 Hz, 2H), 4.45 (q, J=6.8 Hz, 2H), 2.73 (q, J=7.2 Hz, 2H), 1.42
(t, J=6.8 Hz, 3H), 1.18 (t, J=7.2 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 162.8, 142.9, 142.7, 136.3, 136.1, 135.4, 133.4, 131.2
(ꢀ2), 130.9, 130.3, 129.1 (ꢀ2), 127.9, 127.4, 126.0, 61.2, 17.4,
15.9, 14.6. ESMS m/z: 423.1 (M þ 1).
5-(4-Bromo-phenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-pyra-
zole-3-carboxylic Acid Ethyl Ester (8d). Treatment of 7d (2.20 g,
6.89 mmol) with 2,4-dichlorophenylhydrazine hydrochlo-
ride (1.35 g, 6.34 mmol) and acetic acid (30 mL) gave comp-
1
ound 8d (1.51 g, 48%) as a white solid: mp 140-141 °C. H
NMR (400 MHz, CDCl3) δ 7.46 (d, J=8.4 Hz, 2H), 7.38 (d, J=
2.0 Hz, 1H), 7.34 (d, J=8.4 Hz, 1H), 7.28 (dd, J=8.4, 2.0 Hz,
1H), 7.00 (d, J=8.4 Hz, 2H), 4.45 (q, J=6.8 Hz, 2H), 2.33
(s, 3H), 1.43 (t, J=6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3)
δ 162.8, 142.9, 142.7, 136.3, 136.2, 133.4, 132.1 (ꢀ2), 131.4 (ꢀ2),
131.0, 130.4, 128.0, 127.7, 123.7, 119.9, 61.2, 14.7, 9.9. ESMS
m/z: 453.0 (M þ 1).
General Procedure for the Synthesis of Compounds 7a-7g.
The general procedure is illustrated immediately below with
compound 7a as a specific example.
Lithium Salt of 4-(4-Chloro-phenyl)-3-methyl-2,4-dioxo-buty-
ric Acid Ethyl Ester (7a). To a magnetically stirred solution of
lithium bis-(trimethylsilyl)amide (16.0 mL, 1.0 M in THF, 16
mmol) in diethyl ether (45 mL) at -78 °C was added a solution
of 1-(4-chloro-phenyl)-propan-1-one (6a) (2.02 g, 11.98 mmol)
in diethyl ether (15 mL) dropwise under an argon atmosphere.
After the mixture was stirred at the same temperature for an
additional period of 45 min, diethyl oxalate (2.03 g, 13.89 mmol)
was added dropwise. The reaction mixture was allowed to warm
to room temperature and stirred for another 16 h. The pre-
cipitate formed was collected by filtration, washed with diethyl
ether, and dried under vacuum to afford the crude lithium salt 7a
(2.60 g, 79%) as a yellowish solid.
5-(4-Bromo-phenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-1H-pyra-
zole-3-carboxylic Acid Ethyl Ester (8e). Treatment of 7e (1.91 g,
5.73 mmol) with 2,4-dichlorophenylhydrazine hydrochloride
(1.11 g, 5.22 mmol) and acetic acid (30 mL) gave compound
1
8e (1.35 g, 50%) as a white solid: mp 103-104 °C. H NMR
(400 MHz, CDCl3) δ 7.45 (d, J=8.4 Hz, 2H), 7.37 (d, J=2.0 Hz,
1H), 7.33 (d, J=8.4 Hz, 1H), 7.26 (dd, J=8.4, 2.0 Hz, 1H), 7.01
(d, J=8.4 Hz, 2H), 4.45 (q, J=6.8 Hz, 2H), 2.73 (q, J=7.2 Hz,
2H), 1.41 (t, J=6.8 Hz, 3H), 1.18 (t, J=7.2 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 162.7, 142.9, 142.7, 136.3, 136.1, 133.4,
132.1 (ꢀ2), 131.4 (ꢀ2), 130.9, 130.3, 127.9, 127.8, 126.0, 123.6,
61.2, 17.4, 15.9, 14.6. ESMS m/z: 467.0 (M þ 1).
General Procedure for the Synthesis of Compounds 8a-8g.
The general procedure is illustrated immediately below with
compound 8a as a specific example.
1-(2,4-Dichloro-phenyl)-5-(4-methoxy-phenyl)-4-methyl-1H-pyr-
azole-3-carboxylic Acid Ethyl Ester (8f). Treatment of 7f (2.60 g,
9.62 mmol) with 2,4-dichlorophenylhydrazine hydrochloride
(1.81 g, 8.47 mmol) and acetic acid (30 mL) gave compound
8f (1.63 g, 42%) as a white solid: mp 96-97 °C. 1H NMR
(400 MHz, CDCl3) δ 7.36 (d, J=2.0 Hz, 1H), 7.32 (d, J=8.4 Hz,
1H), 7.25 (dd, J=8.4, 2.0 Hz, 1H), 7.04 (d, J=8.7 Hz, 2H), 6.82
(d, J=8.7 Hz, 2H), 4.44 (q, J=6.8 Hz, 2H), 3.78 (s, 3H), 2.32
(s, 3H), 1.41 (t, J=6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3)
δ 163.2, 159.9, 144.2, 143.0, 136.5, 135.9, 133.4, 131.1 (ꢀ2),
131.0, 130.2, 127.8, 120.9, 118.8, 114.2 (ꢀ2), 61.1, 55.4, 14.7, 9.9.
ESMS m/z: 405.1 (M þ 1).
5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-pyra-
zole-3-carboxylic Acid Ethyl Ester (8a). To a solution of the
lithium salt 7a (2.60 g, 9.47 mmol) in ethanol (35 mL) was added
2,4-dichlorophenylhydrazine hydrochloride (1.82 g, 8.52 mmol)
in one portion at room temperature. The resulting mixture was
stirred at the same temperature for 20 h. After the reaction was
complete, the precipitate was collected by filtration, washed
with ethanol and diethyl ether, and dried under vacuum to give a
light-yellow solid. This crude solid, without purification, was
dissolved in acetic acid (30 mL) and heated to reflux for 24 h.
The reaction mixture was poured into ice water, and the result-
ing mixture was extracted with ethyl acetate (2 ꢀ 30 mL).

Article
1-(2,4-Dichloro-phenyl)-5-(4-trifluoromethyl-phenyl)-4-meth-
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4505
1H), 7.30 (dd, J=8.4, 2.0 Hz, 1H), 7.01 (d, J=8.4 Hz, 2H), 2.35
(s, 3H). 13C NMR (100 MHz, CDCl3) δ 165.4, 143.6, 142.3,
136.5, 135.9, 133.1, 132.1 (ꢀ2), 131.3 (ꢀ2), 130.7, 130.4, 128.1,
127.4, 123.7, 119.9, 9.8. ESMS m/z: 425.0 (M þ 1).
yl-1H-pyrazole-3-carboxylic Acid Ethyl Ester (8g). Treatment of
7g (2.42 g, 7.85 mmol) with 2,4-dichlorophenylhydrazine hydro-
chloride (1.56 g, 7.31 mmol) and acetic acid (30 mL) gave
compound 8g (1.50 g, 43%) as a white solid: mp 128-129 °C.
1H NMR (400 MHz, CDCl3) δ 7.58 (d, J=8.4 Hz, 2H), 7.37
(d, J=1.8 Hz, 1H), 7.36 (d, J=8.0 Hz, 1H), 7.29 (dd, J=8.0,
1.8 Hz, 1H), 7.26 (d, J=8.4 Hz, 2H), 4.45 (q, J=6.8 Hz, 2H), 2.35
(s, 3H), 1.41 (t, J=6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3)
δ 162.9, 143.3, 142.7, 136.4, 136.0, 133.2, 132.5, 130.9, 130.4,
130.2 (ꢀ2), 128.1, 125.8, 125.7, 125.2, 122.7, 119.8, 61.2, 14.6,
9.9. ESMS m/z: 443.1 (M þ 1).
5-(4-Bromo-phenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-1H-pyra-
zole-3-carboxylic Acid (9e). Treatment of 8e (0.94 g, 2.01 mmol)
with potassium hydroxide (0.91 g, 16.25 mmol) in methanol
(25 mL) gave compound 9e (0.78 g, 88%) as a white solid: mp
214-215 °C. 1H NMR (400 MHz, CDCl3) δ 7.47 (d, J=8.4 Hz,
2H), 7.40 (d, J=2.0 Hz, 1H), 7.33 (d, J=8.4 Hz, 1H), 7.29 (dd,
J=8.4, 2.0 Hz, 1H),7.03 (d, J=8.4 Hz, 2H), 2.75 (q, J=7.2 Hz,
2H), 1.21 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3)
δ 165.4, 143.5, 141.7, 136.5, 135.8, 133.2, 132.2 (ꢀ2), 131.4(ꢀ2),
130.7, 130.5, 128.0, 127.6, 126.5, 123.9, 17.4, 15.8. ESMS m/z:
439.0 (M þ 1).
4-Bromo-5-(4-chloro-phenyl)-1-(2,4-dichloro-phenyl)-1H-pyr-
azole-3-carboxylic Acid Ethyl Ester (8h). To a solution of 8b
(2.22 g, 5.61 mmol) in THF (70 mL) was added NBS (2.21 g,
12.41 mmol) in one portion at room temperature. The resulting
mixture was stirred at the same temperature for 72 h. The
reaction mixture was filtered, and the filtrate was concentrated.
The residue was purified by flash chromatography on silica gel
with n-hexane/ethyl acetate (9:1) to give 8h (1.56 g, 59%) as a
white solid: mp 107-108 °C. 1H NMR (400 MHz, CDCl3) δ 7.39
(d, J=2.0 Hz, 1H), 7.36 (d, J=8.4 Hz, 1H), 7.33-7.30 (m, 3H),
7.19 (d, J=8.4 Hz, 2H), 4.47 (q, J=6.8 Hz, 2H), 1.43 (t, J=
6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 161.0, 144.1, 142.4,
136.9, 136.1, 135.7, 133.1, 131.2 (ꢀ2), 130.7, 130.5, 129.2 (ꢀ2),
128.2, 125.7, 97.5, 61.8, 14.5. ESMS m/z: 473.0 (M þ 1).
General Procedure for the Synthesis of Compounds 9a-9h.
The general procedure is illustrated immediately below with
compound 9a as a specific example.
1-(2,4-Dichloro-phenyl)-5-(4-methoxy-phenyl)-4-methyl-1H-pyr-
azole-3-carboxylic Acid (9f). Treatment of 8f (1.01 g, 2.49 mmol)
with potassium hydroxide (1.06 g, 18.93 mmol) in methanol
(25 mL) gave compound 9f (0.79 g, 84%) as a white solid:
mp 185-186 °C. 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J=2.0
Hz, 1H), 7.31 (d, J=8.4 Hz, 1H), 7.26 (dd, J=8.4, 2.0 Hz, 1H),
7.06 (d, J=8.4 Hz, 2H), 6.83 (d, J=8.4 Hz, 2H), 3.80 (s, 3H), 2.36
(s, 3H). 13C NMR (100 MHz, CDCl3) δ 166.0, 160.1, 144.7,
142.2, 136.3, 136.1, 133.3, 131.1 (ꢀ2), 130.9, 130.3, 127.9, 120.7,
119.3, 114.3 (ꢀ2), 55.4, 9.8. ESMS m/z: 377.1 (M þ 1).
1-(2,4-Dichloro-phenyl)-4-methyl-5-(4-trifluoromethyl-phen-
yl)-1H-pyrazole-3-carboxylic Acid (9g). Treatment of 8g (0.98 g,
2.21 mmol) with potassium hydroxide (0.95 g, 16.96 mmol) in
methanol (25 mL) gave compound 9g (0.84 g, 91%) as a white
solid: mp 163-164 °C. 1H NMR (400 MHz, CDCl3) δ 7.59 (d,
J=8.4 Hz, 2H), 7.39 (d, J=2.0 Hz, 1H), 7.38 (d, J=8.0 Hz, 1H),
7.30 (dd, J=8.0, 2.0 Hz, 1H), 7.27 (d, J=8.4 Hz, 2H), 2.35 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 166.4, 143.2, 142.8, 136.6,
135.8, 132.9, 132.3, 130.8, 130.5, 130.2 (ꢀ2), 128.2, 125.9, 125.8,
125.3, 122.8, 120.2, 9.8. ESMS m/z: 415.1 (M þ 1).
5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-
pyrazole-3-carboxylic Acid (9a). To a solution of the ester 8a
(1.00 g, 2.44 mmol) in methanol (25 mL) was added potassium
hydroxide (1.02 g, 18.21 mmol) in one portion. The resulting
mixture was heated at 60 °C for 4 h. The reaction mixture was
then poured into ice water and acidified with 10% hydrochloric
acid. The precipitate thus formed was collected by filtration,
washed with water, and dried under vacuum to give carboxylic
acid 9a (0.88 g, 94%) as a white solid: mp 202-203 °C. 1H NMR
(400 MHz, CDCl3) δ 7.37 (d, J=2.0 Hz, 1H), 7.31 (d, J=8.4 Hz,
1H), 7.29-7.26 (m, 3H), 7.05 (d, J=8.4 Hz, 2H), 2.31 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 166.3, 143.5, 142.4, 136.4, 135.9,
135.4, 133.1, 131.1 (ꢀ2), 130.8, 130.4, 129.2 (ꢀ2), 128.1, 127.0,
119.9, 9.8. ESMS m/z: 381.0 (M þ 1).
4-Bromo-5-(4-chloro-phenyl)-1-(2,4-dichloro-phenyl)-1H-pyr-
azole-3-carboxylic Acid (9h). Treatment of 8h (1.07 g, 2.25 mmol)
with potassium hydroxide (1.02 g, 18.21 mmol) in methanol
(25 mL) gave compound 9h (0.88 g, 87%) as a white solid: mp
223-224 °C. 1H NMR (400 MHz, CDCl3) δ 7.40 (d, J=2.0 Hz,
1H), 7.37 (d, J=8.4 Hz, 1H), 7.32-7.31 (m, 3H), 7.20 (d, J=
8.4Hz, 2H), 6.78 (br s, 2H). 13C NMR(100 MHz, CDCl3) δ 164.0,
144.4, 141.8, 137.0, 136.2, 135.5, 133.0, 131.2 (ꢀ2), 130.6, 130.5,
129.2 (ꢀ2), 128.2, 125.5, 97.9. ESMS m/z: 446.7 (M þ 1).
2-[5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-
pyrazol-3-yl]-1,5,5-trimethyl-1,5-dihydro-imidazol-4-one (5). To
a solution of 9a (1.01 g, 2.65 mmol) in toluene (30 mL) at 0 °C
was sequentiallyadded DMF (0.2mL) and oxalyl chloride (1.48 g,
11.66 mmol) dropwise. The resulting mixture was allowed to
warm to room temperature for 1 h, at which time toluene was
removed under reduced pressure and the crude residue was
dissolved in THF (10 mL) and transferred slowly to a mixture
of 10a (0.50 g, 4.30 mmol) and triethylamine (0.42 g, 4.16 mmol)
in THF (25 mL) at 0 °C. After the mixture was warmed and
stirred at room temperature for 15 h, the reaction was quenched
with water and extracted with ethyl acetate (2 ꢀ30 mL). The
combined extracts were washed with brine, dried over anhy-
drous sodium sulfate, filtered, and concentrated to give the
crude residue (1.22 g), which without purification underwent
intramolecular cyclization by treatment with sodium methoxide
(270 mg, 5.00 mmol) in methanol (30 mL) at 60 °C for 4 h. After
the reaction was completed, the solution was concentrated,
poured into ice water, and extracted with ethyl acetate (2ꢀ30 mL).
The combined extracts were washed with brine, dried over
anhydrous sodium sulfate, filtered, and concentrated. The
residue was purified by flash chromatography on silica gel
with n-hexane/ethyl acetate (1:1) to afford the desired product
5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-1H-pyrazole-3-
carboxylic Acid (9b). Treatment of 8b (0.97 g, 2.45 mmol) with
potassium hydroxide (1.03 g, 18.39 mmol) in methanol (25
mL) gave compound 9b (0.84 g, 93%) as a white solid: mp
114-115 °C. 1H NMR (400 MHz, CDCl3) δ 7.47 (d, J=2.0 Hz,
1H), 7.43 (d, J=8.2 Hz, 1H), 7.37 (dd, J=8.2, 2.0 Hz, 1H), 7.29
(d, J=8.3 Hz, 2H), 7.13 (d, J=8.3 Hz, 2H). 13C NMR (100
MHz, CDCl3) δ 165.6, 146.1, 144.6, 136.8, 136.0, 135.6, 133.1,
130.7, 130.6, 129.5 (ꢀ2), 129.3 (ꢀ2), 128.3, 127.4, 109.4. ESMS
m/z: 367.0 (M þ 1).
5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-1H-pyra-
zole-3-carboxylic Acid (9c). Treatment of 8c (1.02 g, 2.41 mmol)
with potassium hydroxide (1.02 g, 18.21 mmol) in methanol
(25 mL) gave compound 9c (0.85 g, 89%) as a white solid: mp
215-216 °C. 1H NMR (400 MHz, CDCl3) δ 7.37 (d, J=8.4 Hz,
1H), 7.33 (d, J=2.0 Hz, 1H), 7.28 (d, J=8.4 Hz, 2H), 7.25 (dd,
J=8.4, 2.0 Hz, 1H), 7.05 (d, J=8.4 Hz, 2H), 2.66 (q, J=7.2 Hz,
2H), 1.12 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3)
δ 166.0, 143.2, 136.2, 135.9, 135.4, 133.5, 132.9, 131.1 (ꢀ2),
131.0, 130.2, 129.1 (ꢀ2), 128.1, 127.4, 125.9, 17.3, 15.9. ESMS
m/z: 395.1 (M þ 1).
5-(4-Bromo-phenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-
pyrazole-3-carboxylic Acid (9d). Treatment of 8d (1.02 g, 2.25
mmol) with potassium hydroxide (1.04 g, 18.57 mmol) in
methanol (25 mL) gave compound 9d (0.81 g, 85%) as a white
solid: mp 182-183 °C. 1H NMR (400 MHz, CDCl3) δ 7.47
(d, J=8.4 Hz, 2H), 7.40 (d, J=2.0 Hz, 1H), 7.34 (d, J=8.4 Hz,
1
5 (0.67 g, 55%) as a white solid: mp 123-124 °C. H NMR
(400 MHz, CDCl3) δ 7.41 (d, J=2.1 Hz, 1H), 7.25 (d, J=8.4 Hz,
2H), 7.22 (dd, J=8.4, 2.1 Hz, 1H), 7.12 (d, J=8.4 Hz, 1H), 7.01

4506 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
(d, J=8.4 Hz, 2H), 3.42 (s, 3H), 2.35 (s, 3H), 1.37 (s, 6H). 13
NMR (100 MHz, CDCl3) δ 195.1, 170.1, 142.9, 142.1, 136.4,
136.0, 135.5, 133.1, 131.1 (ꢀ2), 130.7, 130.5, 129.3 (ꢀ2), 128.2,
127.1, 121.1, 65.2, 30.8, 22.4 (ꢀ2), 10.8. ESMS m/z: 461.1
(M þ 1). Anal. (C22H19Cl3N4O) C, H, N.
Wu et al.
C
δ 194.3, 172.9, 142.3, 142.2, 136.1, 135.9, 135.1, 132.9, 130.7
(ꢀ2), 130.5, 130.3, 128.9 (ꢀ2), 128.0, 126.9, 119.1, 66.9, 26.3,
23.9 (ꢀ2), 10.0, 7.8 (ꢀ2). ESMS m/z: 487.0 (M þ 1). Anal.
(C24H21Cl3N4O) C, H, N.
1-Benzyl-2-[5-(4-chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-
methyl-1H-pyrazol-3-yl]-5,5-dimethyl-1,5-dihydro-imidazol-
4-one (16). Compound 16 was synthesized from 9a (250 mg,
0.66 mmol) and 10g (189 mg, 0.99 mmol) following a similar
synthetic procedure for 5 and obtained as a white solid (155 mg,
44%): mp 99-100 °C. 1H NMR (400 MHz, CDCl3) δ 7.38 (d,
J=2.2 Hz, 1H), 7.31 (d, J=8.4 Hz, 2H), 7.26 (m, 5H), 7.20 (dd,
J=8.4, 2.2 Hz, 1H), 7.04 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 8.4 Hz,
1H), 5.29 (s, 2H), 2.45 (s, 3H), 1.31 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ 195.0, 170.6, 142.8, 141.8, 137.5, 136.1, 135.8, 135.3,
132.8, 131.0 (ꢀ2), 130.5, 130.3, 129.1 (ꢀ2), 128.6 (ꢀ2), 128.0,
127.6, 127.5 (ꢀ2), 127.0, 121.0, 65.7, 48.1, 23.5 (ꢀ2), 10.8. ESMS
m/z: 537.0 (M þ 1). Anal. (C28H23Cl3N4O) C, H, N.
2-[5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-py-
razol-3-yl]-1-ethyl-5,5-dimethyl-1,5-dihydro-imidazol-4-one (11).
Compound 11 was synthesized from 9a (251 mg, 0.66 mmol) and
10b (140 mg, 1.08 mmol) following a similar synthetic proce-
dure for 5, and obtained as a white solid (110 mg, 35%):
mp 213-214 °C. 1H NMR (400 MHz, CDCl3) δ 7.43
(d, J=2.1 Hz, 1H), 7.27 (d, J=8.4 Hz, 2H), 7.25 (dd, J=8.4,
2.1 Hz, 1H), 7.14 (d, J=8.4 Hz, 1H), 7.05 (d, J=8.4 Hz, 2H), 3.90
(q, J=6.8 Hz, 2H), 2.37 (s, 3H), 1.41 (s, 6H), 1.27 (t, J=6.8 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 194.6, 169.8, 142.4, 141.5,
135.9, 135.7, 134.9, 132.6, 130.7 (ꢀ2), 130.3, 130.1, 128.8 (ꢀ2),
127.8, 126.7, 120.6, 65.3, 38.9, 23.0 (ꢀ2), 15.9, 10.5. ESMS m/z:
475.0 (M þ 1). Anal. (C23H21Cl3N4O) C, H, N.
2-[5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-1H-pyrazol-3-
yl]-1,5,5-trimethyl-1,5-dihydro-imidazol-4-one (17). Compound
17 was synthesized from 9b (249 mg, 0.68 mmol) and 10a
(125 mg, 1.08 mmol) following a similar synthetic procedure
for 5 and obtained as a white solid (152 mg, 50%): mp 191-
2-[5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-
pyrazol-3-yl]-5,5-dimethyl-1-propyl-1,5-dihydro-imidazol-4-one
(12). Compound 12 was synthesized from 9a (250 mg, 0.66
mmol) and 10c (150 mg, 1.04 mmol) following a similar
synthetic procedure for 5, and obtained as a white solid (97
mg, 30%): mp 200-201 °C. 1H NMR (600 MHz, CDCl3) δ 7.45
(d, J = 2.2 Hz, 1H), 7.28 (d, J = 8.4 Hz, 2H), 7.24 (dd, J = 8.4,
2.2 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 7.05 (d, J = 8.4 Hz, 2H),
3.76-3.74 (m, 2H), 2.37 (s, 3H), 1.72-1.68 (m, 2H), 1.41
(s, 6H), 0.81 (t, J = 7.3 Hz, 3H). 13C NMR (150 MHz, CDCl3)
δ 194.5, 169.8, 142.4, 141.7, 135.9, 135.7, 135.0, 132.6, 130.7
(ꢀ2), 130.3, 130.0, 128.8, 128.7, 127.8, 126.7, 120.6, 65.3, 41.8,
23.1 (ꢀ2), 22.5, 13.6, 10.7. ESMS m/z: 489.1 (M þ 1). Anal.
(C24H23Cl3N4O) C, H, N.
1-Butyl-2-[5-(4-chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-
methyl-1H-pyrazol-3-yl]-5,5-dimethyl-1,5-dihydro-imidazol-
4one (13). Compound 13 was synthesized from 9a (252 mg,
0.66 mmol) and 10d (161 mg, 1.02 mmol) following a similar
synthetic procedure for 5 and obtained as a white solid
(141 mg, 42%): mp 217-218 °C. 1H NMR (600 MHz,
CDCl3) δ 7.45 (d, J = 2.2 Hz, 1H), 7.28 (d, J = 8.4 Hz,
2H), 7.24 (dd, ;J=8.4, 2.2 Hz, 1H), 7.09 (d, J=8.4 Hz, 1H),
7.05 (d, J = 8.4 Hz, 2H), 3.81-3.79 (m, 2H), 2.39 (s, 3H),
1.69-1.64 (m, 2H), 1.42 (s, 6H), 1.26-1.24 (m, 2H), 0.82 (t,
J = 7.3 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 194.8,
170.0, 142.6, 141.8, 136.1, 135.8, 135.1, 132.7, 130.8 (ꢀ2),
130.3, 130.2, 129.0 (ꢀ2), 127.9, 126.8, 120.8, 65.4, 44.2, 32.6,
23.2 (ꢀ2), 20.1, 13.6, 10.7. ESMS m/z: 503.0 (M þ 1). Anal.
(C25H25Cl3N4O) C, H, N.
1-Allyl-2-[5-(4-chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-
methyl-1H-pyrazol-3-yl]-5,5-dimethyl-1,5-dihydro-imidazol-4-
one (14). Compound 14 was synthesized from 9a (253 mg, 0.66
mmol) and 10e (148 mg, 1.04 mmol) following a similar
synthetic procedure for 5 and obtained as a white solid
(171 mg, 53%): mp 203-204 °C. 1H NMR (600 MHz, CDCl3)
δ 7.42 (d, J = 2.2 Hz, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.23 (dd,
J = 8.4, 2.2 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 7.03 (d, J = 8.4
Hz, 2H), 5.92-5.88 (m, 1H), 5.14-5.07 (m, 2H), 4.54 (d, J = 6.0
Hz, 2H), 2.37 (s, 3H), 1.40 (s, 6H). 13C NMR (150 MHz, CDCl3)
δ 194.8, 170.1, 142.5, 141.6, 136.0, 135.7, 135.1, 134.4, 132.6,
130.8 (ꢀ2), 130.4, 130.1, 128.9 (ꢀ2), 127.9, 126.7, 120.7, 117.5,
65.5, 46.9, 23.2 (ꢀ2), 10.5. ESMS m/z: 487.0 (M þ 1). Anal.
(C24H21Cl3N4O) C, H, N.
2-[5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-
pyrazol-3-yl]-1-cyclopropyl-5,5-dimethyl-1,5-dihydro-imidazol-
4-one (15). Compound 15 was synthesized from 9a (248 mg, 0.65
mmol) and 10f (148 mg, 1.04 mmol) following a similar synthetic
procedure for 5 and obtained as a white solid (122 mg, 38%): mp
206-207 °C. 1H NMR (600 MHz, CDCl3) δ 7.42 (d, J = 2.2 Hz,
1H), 7.28-7.26 (m, 3H), 7.21 (d, J = 8.4 Hz, 1H), 7.07 (d, J =
8.4 Hz, 2H), 2.92-2.90 (m, 1H), 2.29 (s, 3H), 1.49 (s, 6H), 0.89-
0.87 (m, 2H), 0.79-0.77 (m, 2H). 13C NMR (150 MHz, CDCl3)
1
192 °C. H NMR (400 MHz, CDCl3) δ 7.45 (br s, 1H), 7.30-
7.28 (m, 3H), 7.21 (d, J=8.0 Hz, 2H), 7.04 (d, J=8.0 Hz, 2H),
3.48 (s, 3H), 1.36 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 194.5,
168.8, 145.2, 144.5, 136.7, 135.9, 135.6, 132.9, 130.8, 130.4, 129.5
(ꢀ2), 129.3 (ꢀ2), 128.4, 127.1, 111.7, 66.5, 30.6, 22.2 (ꢀ2).
ESMS m/z: 447.0 (M þ 1). Anal. (C21H17Cl3N4O) C, H, N.
2-[5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-1H-
pyrazol-3-yl]-1,5,5-trimethyl-1,5-dihydro-imidazol-4-one (18).
Compound 18 was synthesized from 9c (252 mg, 0.64 mmol)
and 10a (125 mg, 1.08 mmol) following a similar synthetic
procedure for 5 and obtained as a white solid (158 mg, 52%):
1
mp 154-155 °C. H NMR (400 MHz, CDCl3) δ 7.46 (d, J=
2.1 Hz, 1H), 7.31 (d, J=8.4 Hz, 2H), 7.28 (dd, J=8.4, 2.1 Hz,
1H), 7.20 (d, J=8.4 Hz, 1H), 7.10 (d, J=8.4 Hz, 2H), 3.46
(s, 3H), 2.88 (q, J=7.0 Hz, 2H), 1.44 (s, 6H), 1.12 (t, J=7.0 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 195.2, 170.2, 142.5, 141.2,
136.2, 135.8, 135.4, 133.0, 131.1 (ꢀ2), 130.5 (ꢀ2), 129.1 (ꢀ2),
128.0, 127.2, 127.1, 65.0, 30.6, 22.2 (ꢀ2), 17.3, 16.0. ESMS m/z:
475.1 (M þ 1). Anal. (C23H21Cl3N4O) C, H, N.
2-[5-(4-Bromo-phenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-
pyrazol-3-yl]-1,5,5-trimethyl-1,5-dihydro-imidazol-4-one (19).
Compound 19 was synthesized from 9d (251 mg, 0.59 mmol)
and 10a (125 mg, 1.08 mmol) following a similar synthetic
procedure for 5, and obtained as a white solid (170 mg, 57%):
mp 220-221 °C. 1H NMR (400 MHz, CDCl3) δ 7.48-7.46
(m, 3H), 7.29 (dd, J=8.4, 2.2 Hz, 1H), 7.19 (d, J=8.4 Hz, 1H),
7.01 (d, J=8.4 Hz, 2H), 3.49 (s, 3H), 2.41 (s, 3H), 1.43 (s, 6H).
13C NMR (100 MHz, CDCl3) δ 195.0, 170.1, 142.8, 142.1, 136.4,
135.9, 133.0, 132.2 (ꢀ2), 131.3 (ꢀ2), 130.6, 130.4, 128.1, 127.5,
123.7, 121.0, 65.1, 30.7, 22.3 (ꢀ2), 10.8. ESMS m/z: 505.0
(M þ 1). Anal. (C22H19BrCl2N4O) C, H, N.
2-[5-(4-Bromo-phenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-1H-
pyrazol-3-yl]-1,5,5-trimethyl-1,5-dihydro-imidazol-4-one (20).
Compound 20 was synthesized from 9e (250 mg, 0.57 mmol)
and 10a (125 mg, 1.08 mmol) following a similar synthetic
procedure for 5 and obtained as a white solid (160 mg, 54%):
mp 161-162 °C. 1H NMR (400 MHz, CDCl3) δ 7.45-7.43
(m, 3H), 7.27 (dd, J=8.4, 2.2 Hz, 1H), 7.20 (d, J=8.4 Hz, 1H),
7.01 (d, J=8.4 Hz, 2H), 3.44 (s, 3H), 2.84 (q, J=7.3 Hz, 2H), 1.41
(s, 6H), 1.09 (t, J=7.3 Hz, 3H). 13C NMR (100 MHz, CDCl3)
δ 195.2, 170.2, 142.6, 141.3, 136.3, 135.8, 133.0, 132.1 (ꢀ2),
131.3 (ꢀ2), 130.5, 130.4, 128.0, 127.6, 127.2, 123.7, 65.1, 30.6,
22.2 (ꢀ2), 17.3, 16.0. ESMS m/z: 519.1 (M þ 1). Anal.
(C23H21BrCl2N4O) C, H, N.
2-[1-(2,4-Dichloro-phenyl)-5-(4-methoxy-phenyl)-4-methyl-
1H-pyrazol-3-yl]-1,5,5-trimethyl-1,5-dihydro-imidazol-4-one (21).
Compound 21 was synthesized from 9f (249 mg, 0.66 mmol) and
10a (125 mg, 1.08 mmol) following a similar synthetic procedure

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4507
for 5 and obtained as a white solid (143 mg, 47%): mp
207-208 °C. 1H NMR (400 MHz, CDCl3) δ 7.47 (d, J=2.2 Hz,
1H), 7.25 (dd, J=8.4, 2.2 Hz, 1H), 7.16 (d, J=8.4 Hz, 1H), 7.06 (d,
J = 8.4 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H), 3.79 (s, 3H), 3.49
(s, 3H), 2.40 (s, 3H), 1.43 (s, 6H). 13C NMR (100 MHz, CDCl3)
δ 195.2, 170.3, 160.1, 143.8, 141.8, 136.4, 136.0, 133.2, 131.2
(ꢀ2), 130.6 (ꢀ2), 128.0, 120.7, 120.5, 114.3 (ꢀ2), 65.1, 55.5,
30.8, 22.4 (ꢀ2), 10.9. ESMS m/z: 457.0 (M þ 1). Anal.
(C23H22Cl2N4O2) C, H, N.
(10 mg, 0.01 mmol), andpotassium carbonate (30 mg, 0.22 mmol)
in toluene (3 mL) was heated at 80 °C for 2 h under microwave
irradiation. After the reaction was complete, the precipitate was
filtered and the resulting solution was concentrated under
reduced pressure. The residue thus obtained was further purified
by flash chromatography withn-hexane/ethyl acetate (9:1) to give
1
compound 26 (16 mg, 35%) as a liquid. H NMR (400 MHz,
CDCl3) δ 7.47 (d, J = 2.4 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H), 7.26
(dd, J = 8.4, 2.4 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.13 (d, J =
8.4 Hz, 2H), 3.41 (s, 3H), 2.19-2.16 (m, 1H), 1.45 (s, 6H), 0.79-
0.76 (m. 2H), 0.20-0.18 (m, 2H). 13C NMR (100 MHz, CDCl3)
δ 193.6, 170.0, 143.0, 142.4, 136.2, 135.5, 135.2, 132.9, 130.6 (ꢀ2),
130.4, 130.3, 128.7 (ꢀ2), 127.9, 126.7, 125.2, 65.1, 53.4, 30.1, 22.0
(ꢀ2), 7.3, 5.7. ESMS m/z: 487.1 (M þ 1). Anal. (C24H21Cl3N4O)
C, H, N.
2-[1-(2,4-Dichloro-phenyl)-4-methyl-5-(4-trifluoromethyl-
phenyl)-1H-pyrazol-3-yl]-1,5,5-trimethyl-1,5-dihydro-imida-
zol-4-one (22). Compound 22 was synthesized from 9g
(251 mg, 0.60 mmol) and 10a (126 mg, 1.08 mmol) following
a similar synthetic procedure for 5 and obtained as a white
1
solid (176 mg, 60%): mp 188-189 °C. H NMR (400 MHz,
CDCl3) δ 7.59 (d, J=8.0 Hz, 2H), 7.46 (d, J=2.1 Hz, 1H),
7.30-7.27 (m, 3H), 7.22 (d, J=8.0 Hz, 1H), 3.49 (s, 3H), 2.42
(s, 3H), 1.42 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 194.4,
169.6, 153.9, 142.3, 141.7, 136.3, 135.5, 132.7, 132.0, 130.4,
130.2, 129.9 (ꢀ2), 128.0, 125.6, 125.5, 121.2, 112.9, 65.0,
30.6, 22.0 (ꢀ2), 10.5. ESMS m/z: 495.1 (M þ 1). Anal.
(C23H19Cl2F3N4O) C, H, N.
2-[5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-
pyrazol-3-yl]-5,5-dimethyl-3,5-dihydro-imidazol-4-one (27). To
a solution of 9a (1.01 g, 2.65 mmol) in toluene (30 mL) at 0 °C
were sequentially added DMF (0.2 mL) and oxalyl chloride
(1.48 g, 11.66 mmol) dropwise. The mixture was allowed to
warm to room temperature and stirred for 2 h, which was then
transferred slowly to a mixture of 10h (0.56 g, 4.07 mmol) and
triethylamine (0.42 g, 4.20 mmol) in THF (120 mL) at 0 °C.
After the mixture was warmed and stirred at room temperature
for 15 h, water was added to quench the reaction. The aqueous
layer was separated and extracted with ethyl acetate (2ꢀ30 mL).
The combined extracts were washed with brine, dried over
anhydrous sodium sulfate, filtered, and concentrated to give
the crude amide (1.02 g), which was further treated with POCl3
(1.25 g, 8.15 mmol) in 1,2-dichloroethane (25 mL) at 80 °C for
4 h. The mixture was cooled to room temperature and poured
into ice water followed by extraction with ethyl acetate (2 ꢀ
50 mL). The combined extracts were washed with brine, dried
over anhydrous sodium sulfate, filtered, and concentrated to
give the crude residue, which was purified by flash chromatog-
raphy on silica gel with n-hexane/ethyl acetate (7:3) to afford
2-[5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-pyra-
zol-3-yl]-1,5,5-trimethyl-1,5-dihydro-imidazole-4-thione (23). To a
solution of compound 5 (98 mg, 0.21 mmol) in toluene (5 mL)
was added Lawesson’s reagent (150 mg, 0.37 mmol) in one portion.
The resulting mixture was heated at 60 °C for 4 h, cooled to room
temperature, and then poured into ice water. The resulting mixture
was extracted with ethyl acetate (2ꢀ30 mL). The combined extracts
were washed with water, saturated aqueous sodium bicarbonate,
and brine, dried over anhydrous sodium sulfate, filtered, and
concentrated. The residue was purified by flash chromatography
on silica gel with n-hexane/ethyl acetate (7:3) to give the desired
thioketone 23 (78 mg, 77%) as a yellow solid: mp 177-178 °C. 1H
NMR (300 MHz, CDCl3) δ 7.48 (d, J=2.1 Hz, 1H), 7.32 (d, J=
8.4 Hz, 2H), 7.28 (dd, J=8.4, 2.1 Hz, 1H), 7.22 (d, J=8.4 Hz, 1H),
7.08 (d, J=8.4 Hz, 2H), 3.62 (s, 3H), 2.48 (s, 3H), 1.55 (s, 6H). 13C
NMR (75 MHz, CDCl3) δ 230.2, 166.5, 143.1, 141.2, 136.5, 135.9,
135.5, 133.0, 131.2 (ꢀ2), 130.7, 130.5, 129.3 (ꢀ2), 128.2, 126.9, 121.5,
79.3, 32.6, 26.8 (ꢀ2), 11.0. ESMS m/z: 477.0 (M þ 1). Anal.
(C22H19Cl3N4S) C, H, N.
1
27 (0.62 g, 52%) as a white solid: mp 185-186 °C. H NMR
(400 MHz, CDCl3) δ 7.43 (br s, 1H), 7.32-7.27 (m, 4H), 7.05
(d, J = 8.0 Hz, 2H), 7.00 (br s, 1H), 2.38 (s, 3H), 1.81 (s, 6H).
13C NMR (100 MHz, CDCl3) δ 162.1, 144.0, 143.7, 136.5,
136.0, 135.4, 133.2, 131.0 (ꢀ2), 130.7, 130.6, 129.2 (ꢀ2), 128.2,
127.1, 121.1, 118.5, 46.4, 27.7 (ꢀ2), 9.6. ESMS m/z: 447.0
(M þ 1). Anal. (C21H17Cl3N4O) C, H, N.
2-[5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-1H-pyra-
zol-3-yl]-1,5,5-trimethyl-1,5-dihydro-imidazol-4-thione (24). Com-
pound 24 was synthesized from 18 (100 mg, 0.21 mmol) following
a similar synthetic procedure for 23, and obtained as a yellow solid
2-[5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-1H-pyr-
azol-3-yl]-5,5-dimethyl-3,5-dihydro-imidazol-4-one (28). Com-
pound 28 was synthesized from 9c (1.02 g, 2.58 mmol) and 10h
(0.56 g, 4.07 mmol) following a similar synthetic procedure for
27 and obtained as a white solid (0.58 g, 49%): mp 164-165 °C.
1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 2.0 Hz, 1H), 7.30
(d, J = 8.0 Hz, 2H), 7.29-7.25 (m, 2H), 7.05 (d, J = 8.0 Hz, 2H),
7.01 (br s, 1H), 2.79 (q, J = 7.4 Hz, 2H), 1.81 (s, 6H), 1.21 (t, J =
7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 161.6, 143.6, 143.2,
136.2, 135.7, 135.3, 133.1, 130.9 (ꢀ2), 130.5, 130.4, 129.0 (ꢀ2),
127.9, 127.1, 125.0, 120.9, 46.2, 27.5 (ꢀ2), 17.0, 15.7. ESMS m/z:
461.0 (Mþ1). Anal. (C22H19Cl3N4O) C, H, N.
1
(62 mg, 58%): mp 138-139 °C. H NMR (300 MHz, CDCl3)
δ 7.43 (d, J=1.8 Hz, 1H), 7.31 (d, J=8.1 Hz, 2H), 7.26-7.23
(m, 2H), 7.09 (d, J=8.1 Hz, 2H), 3.58 (s, 3H), 2.91 (q, J=7.2 Hz,
2H), 1.51 (s, 6H), 1.09 (t, J=7.2 Hz, 3H). 13C NMR (75 MHz,
CDCl3) δ 230.6, 166.7, 142.9, 140.7, 136.5, 135.9, 135.6, 133.1,
131.2 (ꢀ2), 130.6, 130.5, 129.3 (ꢀ2), 128.2, 127.9, 127.1, 79.3, 32.6,
26.8 (ꢀ2), 17.6, 16.2. ESMS m/z: 491.0 (M þ 1). Anal.
(C23H21Cl3N4S) calcd: C 56.16, H 4.30, N 11.39; found: C 56.62,
H 4.43, N 10.97.
2-[4-Bromo-5-(4-chloro-phenyl)-1-(2,4-dichloro-phenyl)-1H-pyr-
azol-3-yl]-1,5,5-trimethyl-1,5-dihydro-imidazol-4-one (25). Com-
pound 25 was synthesized from 9h (251 mg, 0.56 mmol)
following a similar synthetic procedure for 5 and obtained
6-[5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-1H-pyr-
azol-3-yl]-5,7-diaza-spiro[3.4]oct-5-en-8-one (29). Compound 29
was synthesized from 9c (251 mg, 0.63 mmol) and 10i (140 mg,
0.94 mmol) following a similar synthetic procedure for 27 and
1
as a yellow solid (152 mg, 51%): mp 111-112 °C. H NMR
1
(400 MHz, CDCl3) δ 7.50 (d, J = 2.0 Hz, 1H), 7.46 (d, J =
8.4 Hz, 2H), 7.30 (dd, J = 8.4, 2.0 Hz, 1H), 7.27 (d, J = 8.4 Hz,
1H), 7.21 (d, J = 8.4 Hz, 2H), 3.40 (s, 3H), 1.45 (s, 6H). 13C
NMR (100 MHz, CDCl3) δ 194.4, 168.9, 143.9, 141.4, 137.0,
136.3, 135.5, 132.9, 131.1 (ꢀ2), 130.7, 130.4, 129.2 (ꢀ2), 128.4,
125.4, 98.4, 65.2, 30.4, 22.2 (ꢀ2). ESMS m/z: 526.0 (M þ 1).
Anal. (C21H16BrCl3N4O) C, H, N.
obtained as a white solid (141 mg, 47%): mp 193-194 °C. H
NMR (400 MHz, CDCl3) δ 7.41 (d, J = 2.2 Hz, 1H), 7.34 (s, 1H),
7.31-7.25 (m, 4H), 7.06 (d, J = 8.4 Hz, 2H), 2.87-2.82 (m, 2H),
2.75 (q, J = 7.4 Hz, 2H), 2.48 (dt, J = 17.9, 9.5 Hz, 2H), 2.24-
2.21 (m, 1H), 2.14-2.10 (m, 1H), 1.19 (t, J = 7.4 Hz, 3H). 13
C
NMR (100 MHz, CDCl3) δ 161.8, 143.5, 143.3, 136.4, 135.8,
135.4, 133.1, 131.0 (ꢀ2), 130.6, 130.5, 129.1 (ꢀ2), 128.1, 127.1,
125.1, 120.9, 47.6, 34.3 (ꢀ2), 17.1, 16.3, 15.8. ESMS m/z: 473.1
(M þ 1). Anal. (C23H19Cl3N4O) C, H, N.
2-[5-(4-Chloro-phenyl)-4-cyclopropyl-1-(2,4-dichloro-phenyl)-1H-
pyrazol-3-yl]-1,5,5-trimethyl-1,5-dihydro-imidazol-4-one (26). A
mixture ofcompound 25 (50 mg, 0.09 mmol), cyclopropylboronic
acid (10 mg, 0.12 mmol), tetrakis(triphenylphosphine) palladium
2-[5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-1H-pyr-
azol-3-yl]-1,3-diaza-spiro[4.4]non-1-en-4-one (30). Compound 30

4508 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Wu et al.
was synthesized from 9c (253 mg, 0.64 mmol) and 10j (142 mg,
0.86 mmol) following a similar synthetic procedure for 27 and
obtained as a white solid (152 mg, 49%): mp 166-167 °C. H
NMR (400 MHz, CDCl3) δ 7.39 (d, J = 1.3 Hz, 1H), 7.31-7.26
(m, 4H), 7.15 (s, 1H), 7.06 (d, J = 8.4 Hz, 2H), 2.76 (q, J =
7.4 Hz, 2H), 2.51-2.46 (m, 2H), 2.20-2.15 (m, 2H), 1.88-1.83
(m, 4H), 1.18 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3)
δ 162.1, 143.7, 143.3, 136.3, 135.8, 135.3, 133.1, 131.0 (ꢀ2), 130.7,
130.4, 129.1 (ꢀ2), 128.0, 127.2, 125.0, 121.1, 55.0, 39.4 (ꢀ2), 23.3
(ꢀ2), 17.1, 15.8. ESMS m/z: 487.1 (Mþ1). Anal. (C24H21Cl3N4O)
C, H, N.
then transferred to a centrifuge tube and centrifuged at 3000g for
20 min at room temperature. The supernatant was transferred to
a separate tube. Then 10 μL of supernatant was mixed with 40%
acetonitrile and then analyzed by LC/MS. The chromato-
graphic system consisted of an Agilent 1100 series LC system
(Agilent Technologies, Palo Alto, CA) and equipped with an
Agilent G1946C single quadrupole mass spectrometer with an
ESI in the positive scanning mode. The HPLC analysis was
performed on an Agilent Eclipse XDB-C8 guard column (5 μm,
4.6 mmꢀ12.5 mm) and an Agilent ZORBAX Eclipse XDB-C8
column (5 μm, 4.6 mmꢀ150 mm). A gradient HPLC method was
employed for separation. Mobile phase A consisted of acetoni-
trile, and mobile phase B consisted of 10 mM ammonium acetate
aqueous solution containing 0.1% formic acid. The gradient
system was from A:B (30%:70%) to A:B (95%:5%). The flow
rate was set at 0.8 mL/min.
1
2-[5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-1H-pyr-
azol-3-yl]-1,3-diaza-spiro[4.5]dec-1-en-4-one (31). Compound 31
was synthesized from 9c (249 mg, 0.63 mmol) and 10k (142 mg,
0.79 mmol) following a similar synthetic procedure for 27 and
1
obtained as a white solid (147 mg, 47%): mp 196-197 °C. H
NMR (400 MHz, CDCl3) δ 7.42 (d, J = 1.9 Hz, 1H), 7.31-7.26
(m, 4H), 7.07 (d, J = 8.4 Hz, 2H), 6.97 (s, 1H), 2.77 (q, J=7.4 Hz,
2H), 2.50 (m, 2H), 1.76-1.68 (m, 7H), 1.33-1.32 (m, 1H), 1.20
(t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 161.6, 143.9,
143.3, 136.3, 135.9, 135.3, 133.2, 131.0 (ꢀ2), 130.7, 130.5, 129.1
(ꢀ2), 128.0, 127.3, 125.1, 119.9, 51.6, 35.8 (ꢀ2), 25.0, 22.3 (ꢀ2),
17.1, 15.9. ESMS m/z: 501.1 (M þ 1). Anal. (C25H23Cl3N4O)
C, H, N.
B2. Establishment of Human CB1 (hCB1) and CB2 (hCB2)
Stable Cell Lines and Membrane Purification. The hCB1 cDNA
tagged with Flag at the N terminus or hCB2 cDNA was
subcloned into the pIRES2-EGFP vector (Clontech Labora-
tories, Inc., Mountain View, CA). After transfection to HEK
293 cells, clones stably expressed either hCB1 or hCB2 were
selected by GFP and G418 sulfate and maintained in DMEM
supplemented with 10% fetal bovine serum and 0.5 mg/mL
G418 sulfate under 5% CO2 at 37 °C. For membrane purifica-
tion, cells were homogenized in ice-cold buffer A (50 mM Tris,
5 mM MgCl2, 2.5 mM EDTA, pH 7.4, 10% sucrose) with 1 mM
PMSF. The homogenate was centrifuged for 15 min at 2000g at
4 °C. The resulting supernatant was centrifuged for another
30 min at 43000g at 4 °C. The final pellet was resuspended in
buffer A and stored at -80 °C.
2-[5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-pyr-
azol-3-yl]-3,5,5-trimethyl-3,5-dihydro-imidazol-4-one (32). To a so-
lution of sodium hydride (14.4 mg, 0.66 mol) in DMF (5 mL) at
0 °C was added compound 27 (100 mg, 0.22 mmol) in one portion.
The mixture was allowed to warm to room temperature and stirred
for 30 min, at which time methyl iodide (2.02 g, 14.08 mmol) was
added dropwise. After the mixture was stirred for 2 h, water was
added to quench the reaction followed by extraction with ethyl
acetate (2ꢀ50 mL). The combined extracts were washed with brine,
dried over anhydrous sodium sulfate, filtered, and concentrated.
Purification of the crude residue by flash chromatography on silica
gel with n-hexane/ethyl acetate (1:1) gave the product 32 (70 mg,
B3. Radioligand Binding Assay.26 The radioligand binding
assay was performed according to Felder et al. with minor
modification. An amount of 0.2-8 μg of the purified membrane
was incubated with 0.75 nM [3H] CP-55, 940 ([3H] 34), and
compounds of interest in the incubation buffer (50 mM Tris-
HCl, 5 mM MgCl2, 1 mM EDTA, 0.3% BSA, pH 7.4). The
nonspecific binding was defined in the presence of 1 μM of CP-
55,940. The reactions were incubated for 1.5 h at 30 °C in
Multiscreen microplates (Millipore Corp., Billerica, MA). The
reactions were terminated by manifold filtration and washed
with ice-cold wash buffer (50 mM Tris, pH 7.4, 0.25% BSA) for
four times. The radioactivity bound to the filters was measured
by Topcount (PerkinElmer Inc., Waltham, MA). IC50 was
determined by the concentration of compounds required to
inhibit 50% of the binding of [3H] CP-55, 940 and calculated
by nonlinear regression (GraphPad software, San Diego, CA).
B4. Eu-GTP Binding Assay.27-29 The Eu-GTP binding
assay was performed using the DELFIA Eu-GTP binding kit
(Perkin-Elmer Inc., Waltham, MA) based on methods devel-
oped by Frang et al. with minor modifications as described in the
following: 1-4 μg of purified membrane was incubated with
compounds of interest and 20 nM of CP-55,940 in assay buffer
(50 mM HEPES, pH 7.4, 100 mM NaCl, 100 μg/mL saponin,
5 mM MgCl2, 2 μM GDP, 0.5% BSA) at 30 °C for 60 min in
acroplates (Pall Life Sciences, Ann Arbor, Mich.). Following
the addition of Eu-GTP and incubation of 30 min at 30 °C, the
assay was terminated by washing four times in washing buffer
provided in the kit. The fluorescence signal of Eu-GTP was
determined by Victor 2 multilabel reader (Perkin-Elmer Inc.,
Waltham, MA). EC50 values were analyzed by increasing con-
centrations of test compounds after activation with 30 nM of
CP-55,940 and were determined by nonlinear regression analy-
sis using the GraphPad Prism program (GraphPad software,
San Diego, CA). The intrinsic property was assessed by using
test compounds alone in the absence of agonist CP-55,940;
the percentage change of the induced Eu-GTP binding is
defined as: [(Eu-GTP binding in the presence of test compound
at 10 μM - basal Eu-GTP binding)/basal Eu-GTP binding]
1
68%) as a white solid: mp 113-114 °C. H NMR (400 MHz,
CDCl3) δ 7.45 (d, J = 2.2 Hz, 1H), 7.30 (d, J = 8.4 Hz, 2H), 7.26
(dd, J=8.4, 2.2 Hz, 1H), 7.18 (d, J=8.4 Hz, 1H), 7.07 (d, J=8.4 Hz,
2H), 3.28 (s, 3H), 2.24 (s, 3H), 1.87 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ 165.5, 146.3, 142.3, 135.9, 135.8, 135.0, 133.0, 130.7 (ꢀ2),
130.5, 130.3, 128.9 (ꢀ2), 127.9, 127.1, 121.0, 117.8, 52.3, 33.8, 25.7
(ꢀ2), 9.1. ESMSm/z: 461.1 (Mþ1). Anal. (C22H19Cl3N4O) C, H, N.
2-[5-(4-Chloro-phenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-1H-
pyrazol-3-yl]-3,5,5-trimethyl-3,5-dihydro-imidazol-4-one (33).
Compound 33 was synthesized from compound 28 (101 mg,
0.22 mmol) and methyl iodide (2.02 g, 14.08 mmol) following a
similar synthetic procedure for 32 and obtained as a white
solid (72 mg, 69%): mp 124-125 °C. 1H NMR (400 MHz,
CDCl3) δ 7.42 (br s, 1H), 7.28 (d, J = 7.2 Hz, 2H), 7.26-7.18
(m, 2H), 7.07 (d, J = 7.2 Hz, 2H), 3.21 (s, 3H), 2.62 (q, J = 6.9
Hz, 2H), 1.85 (s, 6H), 1.11 (t, J = 6.9 Hz, 3H). 13C NMR (100
MHz, CDCl3) δ 166.0, 146.2, 142.2, 136.1, 135.9, 135.3, 133.4,
131.0 (ꢀ2), 130.8, 130.4, 129.1 (ꢀ2), 128.0, 127.4, 124.1, 121.1,
52.5, 34.0, 25.8 (ꢀ2), 16.9, 15.8. ESMS m/z: 475.1 (M þ 1).
Anal. (C23H21Cl3N4O) C, H, N.
B. Biological Evaluation. The following methods were used to
generate the data in Figure 2 and Figures 4-7 and Tables
1-3.
B1. Rat Microsome Incubation and LC/MS Analysis. The rat
microsome incubation medium contained 1 mg of microsomal
protein, 100 mM potassium phosphate (pH7.4) buffer, 10 mM
MgCl2, 3 mM NADPH, and 100 μM of compound 4. Total
volume of incubation was 500 μL. Incubation was performed
aerobically at 37 °C with constant shaking in a test tube placed
on a temperature-controlled heating block. After reaction mix-
ture was preincubated without NADPH for 5 min at 37 °C, the
reaction was initiated by addition of NADPH. At indicated time
for 0, 15, and 60 min, the incubation mixture was mixed with 1
mL of acetonitrile to terminate the reaction. The sample was

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4509
ꢀ100%. Statistical analysis was performed by t test. A P value
(*) less than 0.05 was considered significant.
(10) Griffith, D. A.; Hadcock, J. R.; Black, S. C.; Iredale, P. A.;
Carpino, P. A.; Da Silva-Jardine, P.; Day, R.; DiBrino, J.; Dow,
R. L.; Landis, M. S.; , R. E.; Scott, D. O. Discovery of 1-[9-(4-
Chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethylamino-
piperidine-4-carboxylic Acid Amide Hydrochloride (CP-945,598),
a novel, potent, and selective cannabinoid type 1 receptor Antago-
nist. J. Med. Chem. 2009, 52, 234–237.
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and gut-brain signaling. Curr. Opin. Pharmacol. 2007, 7, 575–582.
(b) Pagotto, U.; Cervino, C.; Vicennati, V.; Marsicano, G.; Lutz,
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Rivara, S.; Stella, N.; Xu, C.; Tarzia, G.; Piomelli, D. Synthesis and
characterization of a peripherally restricted CB1 cannabinoid
antagonist, URB447, that reduces feeding and body weight gain
in mice. Bioorg. Med. Chem. Lett. 2009, 19, 639–643.
(13) Mcelroy, J. F.; Chorvat, R. J. Cannabinoid receptor antagonists/
inverse agonists useful for treating obesity. WO patent 2007106721,
2007. Results of the study, entitled “Non-brain penetrant CB1 antago-
nists from Jerin Discovery show beneficial effects in treating diabetes
and obesity,” were presented in a plenary session at the 2008 Annual
Scientific Meeting of the Obesity Society, indicating that CB1 antago-
nists developed by the Jenrin Discovery could play a role in the
treatment of metabolic disorders while reducing risks of psychiatric
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B5. Spontaneously Feeding Model.30 Male Wistar rats were
individually housed under 12 h reverse light-dark cycle (light
off at 10 a.m.) for more than a week and weighed 210-270 g at
the start of the study. Drugs at defined dosage or vehicle (20%
DMSO/10% Tween 80/70% H2O) were orally gavaged before
the light was off, and sufficient powder chow diet was supplied
at 11 a.m. The unconsumed food was measured after 1, 2, 3, and
6 h. Food intake was expressed as calorie (cal) ingestion normal-
ized by body weight (g). Statistical analysis was performed by
t test. A P value (*) less than 0.05 was considered significant.
B6. DIO Mouse Model.31 Six-week-old C57BL/6 mice were
given high-fat diet of 4.73 kcal/g energy density (Research Diet
D 12451; 45% fat, 20% protein, and 35% carbohydrate) for 16
weeks before the drug treatment. Mice weight matched were
assigned to different groups and orally gavaged once daily with
vehicle (10% DMSO/10% Tween 80/80% H2O) or compounds
at defined dosage for 29 days. The sum of food taken for each
treatment and body weight was measured daily.
Acknowledgment. We are grateful to the National Health
Research Institutes and National Science Council of the
Republic of China (grant NSC 96-2323-B-400-001) for finan-
cial support.
(15) Hitchcock, S. A.; Pennington, L. D. Structure-brain exposure
relationships. J. Med. Chem. 2006, 49, 7559–7583.
Supporting Information Available: Combustion analysis data
for compounds 5 and 11-33. This material is available free of
charge via the Internet at http://pubs.acs.org.
(16) Mahar Doan, K. M.; Humphreys, J. E.; Webster, L. O.; Wring,
S. A.; Shampine, L. J.; Serabjit-Singh, C. J.; Adkison, K. K.; Polli,
J. W. Passive permeability and P-glycoprotein-mediated efflux
differentiate central nervous system (CNS) and non-CNS marketed
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