Discovery of 2-(4-((1H-1,2,4-triazol-1-yl)methyl)-5-(4-bromophenyl)-1-(2- chlorophenyl)-1H-pyrazol-3-yl)-5-tert-butyl-1,3,4-thiadiazole (GCC2680) as a potent, selective and orally efficacious cannabinoid-1 receptor antagonist

Article abstract of DOI:10.1016/j.bmc.2010.07.013

Structure-activity relationship studies in a series of diarylpyrazolyl thiadiazoles identified cannabinoid-1 receptor antagonists with excellent potency and selectivity. Based on its exceptional in vivo efficacy in animal models and its favorable pharmaco

Full text of DOI:10.1016/j.bmc.2010.07.013

Bioorganic & Medicinal Chemistry 18 (2010) 6377–6388
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of 2-(4-((1H-1,2,4-triazol-1-yl)methyl)-5-(4-bromophenyl)-1-
(2-chlorophenyl)-1H-pyrazol-3-yl)-5-tert-butyl-1,3,4-thiadiazole (GCC2680)
as a potent, selective and orally efficacious cannabinoid-1 receptor antagonist
*
Jinhwa Lee , Hee Jeong Seo, Suk Ho Lee, Jeongmin Kim, Myung Eun Jung, Sung-Han Lee, Kwang-Seop Song,
Junwon Lee, Suk Youn Kang, Min Ju Kim, Mi-Soon Kim, Eun-Jung Son, MinWoo Lee, Ho-Kyun Han
Research Center, Green Cross Corporation, 303 Bojeong-dong, Giheung-gu, Yongin 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:
Structure–activity relationship studies in a series of diarylpyrazolyl thiadiazoles identified cannabinoid-1
receptor antagonists with excellent potency and selectivity. Based on its exceptional in vivo efficacy in
animal models and its favorable pharmacokinetic and toxicological profiles, 2-(4-((1H-1,2,4-triazol-1-
yl)methyl)-5-(4-bromophenyl)-1-(2-chlorophenyl)-1H-pyrazol-3-yl)-5-tert-butyl-1,3,4-thiadiazole
(GCC2680) was selected as a preclinical candidate for the treatment of obesity.
Received 6 May 2010
Revised 6 July 2010
Accepted 6 July 2010
Available online 30 July 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Obesity
Thiadiazole
Cannabinoid
Rimonabant
Triazole
1. Introduction
cally blocking the cannabinoid receptor 1 (CB1) in both the brain
and periphery can provide a novel target for the treatment of obes-
The prevalence of obesity has rapidly increased over the last dec-
ade. Obesity is not simply a cosmetic concern, but a serious health
problem. Thus, the World Health Organization (WHO) recently de-
clared that obesity has become a global epidemic.1,2 Obesity is char-
acterized by an excess of body fat and includes a pro-inflammatory
state, eventually resulting in type 2 diabetes, coronary heart dis-
ease, and hypertension. Furthermore, obesity elevates the relative
risk of mortality due to cardiovascular disease,3 and obesity now
ranks as the second leading cause of preventable death after smok-
ing in the United States.4 Treatment of obesity involves a combina-
tion of diet, exercise and pharmacotherapy. However, there is
growing evidence that short-term dietary changes or exercise alone
cannot adequately address obesity as a chronic disease. Only two
drugs are currently approved for chronic weight loss treatment:
orlistat and sibutramine. However, both of these agents have differ-
ent adverse event profiles that limit more widespread use.5
ity.6a The endocannabinoid system includes endogenous ligands
(such as anandamide and 2-AG)6b and two cannabinoid receptor
subtypes, CB1 and CB2. CB1 and CB2 belong to the G-protein cou-
pled receptor superfamily and were first cloned in 1990 and 1993,
respectively.7–9 The CB1 receptors are mainly expressed in several
brain areas including the limbic system (amygdala and hippocam-
pus), hypothalamus, cerebral cortex, cerebellum, and basal ganglia.
It is known that CB1 receptors, especially in the limbic system-
hypothalamus axis cannabinoids, have an important role in the
control of appetite. In contrast, CB2 receptors are almost exclu-
sively expressed in cells of the immune system.10,11 Considering
the important impact of obesity on public health, its increasing
incidence worldwide, and the lack of highly efficient and well-tol-
erated drugs for treatment, it is not surprising that CB1 antagonism
or inverse agonism is a subject of considerable interest.12
Although
a few CB1 receptor inverse agonists/antagonists
Numerous studies on the causes of obesity have been conducted
to identify new potential targets that could be exploited to create
novel types of anti-obesity drugs.14 Eventually, the discovery was
made that modulation of the endocannabinoid system by specifi-
were recently withdrawn from clinical development, including
5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-
1-yl)-1H-pyrazole-3-carboxamide, 1 (rimonabant, SR141716A),22
N-((2S,3S)-4-(4-chlorophenyl)-3-(3-cyanophenyl)butan-2-yl)-
2-methyl-2-(5-(trifluoromethyl)pyridin-2-yloxy)propanamide, 2
(taranabant, MK-0364),23 and 1-(8-(2-chlorophenyl)-9-(4-chloro-
phenyl)-9H-purin-6-yl)-4-(ethylamino)piperidine-4-carboxamide,
3 (otenabant, CP-945,598),24 many research groups and pharmaceu-
* Corresponding author. Tel.: +82 31 260 9892; fax: +82 31 260 9870.
E-mail addresses: jinhwa_2_lee@hotmail.com, jinhwalee@greencross.com
(J. Lee).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.013

6378
J. Lee et al. / Bioorg. Med. Chem. 18 (2010) 6377–6388
N
N
N
N
N
N
Cl
Cl
N
NH
O
A
A
O
N
N
O
N
NC
Cl
N
H
R
S
N
N
N
N
N
N
CF3
B
B
Cl
Cl
Cl
Cl
Cl
Cl
2
6
7
Cl
1
Figure 4. Exploration of 1,2,4-triazole-containing diarylpyrazolyl thiadiazole series
7.
O
NH2
NH
N
N
N
both imidazole19 and tetrazole20 replacements for the amide
functionality have been reported. Subsequently, we introduced a
triazole ring onto the C4 methyl group of the pyrazole scaffold
via a methylene linker (5, Fig. 3) to afford a potent CB1 receptor
antagonist with a significant anti-obesity effect in an animal mod-
el. A subsequent modeling study revealed that the N2 of the tria-
zole substituent forms a bidentate H-bond with the side chain
OH and backbone NH of Thr 197, which is an interaction exclu-
sively observed in the binding model of 5.17,21
N
N
Cl
Cl
3
Figure 1. Structures of CB1 receptor antagonists/inverse agonists.
As shown in Figure 4, we envisioned that incorporation of the
1,2,4-triazolylmethyl moiety onto a diarylpyrazolyl thiadiazole
could lead to more potent CB1 receptor antagonists based on the
encouraging observation made in our previous studies.17,21 Modifi-
cations into thiadiazole could provide more efficacious series of
compounds not to mention securing superior intellectual property
(IP) position to other existing pyrazole series of CB1 receptor
ligands.
Herein, we describe the design, synthesis and biological evalu-
ation of diarylpyrazolyl thiadiazole analogues as novel CB1 recep-
tor antagonists. Through a process of extensive lead optimization,
we successfully identified 2-(4-((1H-1,2,4-triazol-1-yl)methyl)-5-
(4-bromophenyl)-1-(2-chlorophenyl)-1H-pyrazol-3-yl)-5-tert-bu-
tyl-1,3,4-thiadiazole, 17s (GCC2680), as a candidate for development
as an anti-obesity agent.
Trp255
Asp366-Lys192
Tyr 275
Cl
Val196
A
Phe278
D
O
Phe170
Leu387
C
N
N
HN
N
B
Trp279
Cl
Met384
Cl
Trp356
Phe200
Figure 2. Compound 1 and its receptor–ligand interaction.
tical companies are still seeking novel CB1 antagonists that display
improved physicochemical properties and decreased adverse effects
involving depression, anxiety or suicidality (Fig. 1).26
2. Chemistry
The main objective of this study was to find novel CB1
receptor antagonists. A pharmacophore model for the binding of a
low energy conformation of 1 in the CB1 receptor has been well-
documented.11,13 The key receptor–ligand interaction is known to
be a hydrogen bond between the carbonyl group of 1 and the
Lys192-Asp366 residues of the CB1 receptor, which stabilizes the
Lys192-Asp366 salt bridge as shown in Figure 2.11
As shown in Scheme 1, the synthesis of the diarylpyrazolyl thia-
diazole analogs commenced with the generic acid 8.23,25 Com-
pounds of the general structure 11 were prepared by (i) reaction
of carboxylic acid 8 with a hydrazide compound 9 in the presence
of coupling reagents (EDCI, DMAP) and (ii) thionation–cyclization
of the resulting product 10 using Lawesson’s reagent28 to obtain
a 1,3,4-thiadiazole 11. Alternatively, the acylhydrazide intermedi-
ate 10 was also available through the coupling of the hydrazide
13 with a corresponding acid 14 mediated by coupling reagents
such as DMAP, EDCI or EDCI, HOBt, NMM. For this sequence, the
requisite hydrazide 13 was prepared by treating the known ester
12 with hydrazine in refluxing EtOH, as shown in Scheme 1.
In our previous studies,17,18 we investigated a series of diarylpy-
razolyl oxadiazole derivatives as antagonists for the cannabinoid
CB1 and CB2 receptors. A few of the compounds (e.g., 4, Fig. 3) in
this series exhibited better binding affinity than known CB1 antag-
onists, validating the hypothesis that a 1,3,4-oxadiazole15,16 could
act as a CB1 bioisostere of the amide moiety in 1. Along these lines,
N
N
Cl
Cl
N
Cl
A
A
O
N
N
4
4
A
N
4
N
C
CF3
O
N
N
HN
N
N
N
O
N
1
1
2
2
N
1
B
B
2
Cl
Cl
B
Cl
5 (GCC2469)
Figure 3. The conceptual development of antagonist 5 from Rimonabant (1) and its bioisostere 4.
Cl
Cl
Cl
1
4

J. Lee et al. / Bioorg. Med. Chem. 18 (2010) 6377–6388
6379
R1
Cl
N
H
O
O
O
N
OH
N
H
N
S
N
H
R1
R1
Lawesson's reagent
H2N
N
N
O
9
N
N
N
Cl
Cl
N
DMAP, EDCI
Cl
Cl
Cl
8
10
11
Cl
Cl
Cl
DMAP, EDCI
H
N
O
O
O
NH2
N
O
N
NH2NH2
N
N
Cl
Cl
R1
OH
14
Cl
Cl
Cl
12
13
Cl
Scheme 1. Preparation of thiadiazole 11.
N
N
N
N
N
S
N
S
S
Br
N
N
N
NBS
Triazole
NaH
N
N
N
N
N
Cl
Cl
N
Cl
Cl
Cl
Cl
Cl
Cl
17
15
16
Cl
Scheme 2. Preparation of triazole 17.
O
O
O
O
O
O
Br
N
N
N
N
N
N
(c)
(b)
(a)
N
N
N
Cl
Cl
Cl
Cl
Cl
Cl
12
18
19
Cl
Cl
Cl
N
O
N
O
H
N
O
S
NHNH2
Cl
N
H
N
N
N
N
N
N
N
N
N
N
(e)
N
N
(d)
N
N
N
Cl
Cl
Cl
Cl
Cl
21
20
17
Cl
Cl
Cl
Scheme 3. Preparation of thiadiazole 17. Reagents and conditions: (a) NBS, AIBN, CCl4; (b) 1,2,4-triazoleꢀNa, DMF; (c) hydrazineꢀH2O, EtOH, 65 °C; (d) pivalic acid, EDCI, HOBt,
NMM, DMF; (e) Lawesson’s reagent, THF, reflux or microwave.
The preparation of triazole 17 was initiated with the activated
4-pyrazole intermediate 16, which was prepared from 15 via a
benzylic bromination-type reaction27 as illustrated in Scheme 2.
The triazole group of 17 was then introduced by treating bromide
16 with triazole in the presence of a suitable base such as sodium
hydride or cesium carbonate.
The compounds containing the 1,2,4-triazole could also be ob-
tained by a reaction sequence involving the key intermediate bro-
mide 18. Thus, bromide 18, obtained by reaction of pyrazole 12
with NBS in the presence of catalytic AIBN, was reacted with
1,2,4-triazole sodium derivatives to provide 19. Hydrazinolysis of
ester 19 produced the corresponding hydrazide 20, which could

6380
J. Lee et al. / Bioorg. Med. Chem. 18 (2010) 6377–6388
Table 1
Structures and binding affinities of selected ligands to rat CB1 and human CB2 receptors and hCB2/rCB1 selectivity of the ligandsa,b
N
N
N
X
N
S
N
N
N
R
Cl
Cl
Entry
X
R
Compound
Receptor affinity (IC50, nM)
hCB2/rCB1 selectivity
rCB1
hCB2
1
2
Cl
Cl
1
17
4.53
1.73
1760
1140
389
659
t-Bu
3
4
Cl
Cl
17a
17b
2.40
2.29
—
—
CF3
CF3
3631
1586
5
6
Cl
Cl
17c
17d
2.89
1.79
505
—
175
—
7
Cl
17e
5.74
—
—
Cl
8
Cl
17f
2.49
—
—
9
Br
Br
t-Bu
17g
17h
1.54
2.91
430
—
279
—
10
CF3
CF3
11
12
Br
Br
17i
17j
0.991
3.27
—
—
—
—
13
14
Br
Br
17k
17l
2.23
2.74
—
—
—
—
Cl
15
Br
17m
0.877
—
—
a
These data were obtained by single determinations.
Compound 1 was estimated to have CB1 binding affinity via in-house assay.
b
then be coupled with an acid to give acylhydrazide 21. Alterna-
tively, hydrolysis of ester 19 and activation of the acid followed
by coupling with a hydrazide in the presence of triethylamine then
afforded acylhydrazide 21. Thionation–cyclization was then per-
formed using Lawesson’s reagent under microwave irradiation to
give thiadiazole 17, as illustrated in Scheme 3.
the examined diarylpyrazolyl oxadiazoles.17 A methylcyclopropyl
substituent (17a) was chosen for the direct comparison with t-bu-
tyl thiadiazole 17 (IC50 = 1.73 nM). Compound 17a showed a slight
decrease in rCB1 receptor binding affinity (IC50 = 2.40 nM). Trifluo-
romethylcyclopropyl derivative 17b, which should provide lower
lipophilicity and possibly enhanced water solubility, exhibited an
increase relative to 17a in binding affinity against the rCB1 recep-
tor, albeit to a low degree. Importantly, compound 17b also
showed the highest hCB2/rCB1 receptor selectivity (ꢁ1586), as
noted in Table 1. When the chlorine at the 4-position of one of
the aryl substituents (entries 2–8) was replaced with bromine (en-
tries 9–15), most of the resulting compounds displayed improved
binding affinity against the rCB1 receptor, implying that increased
lipophilicity can improve in vitro activity. Notably, a couple of
compounds were shown to reach subnanomolar rCB1 receptor
binding affinity (IC50 = 0.991 nM for 17i; IC50 = 0.877 nM for 17m).
3. Results and discussion
The target analogs were evaluated in vitro in a rat CB1 binding
assay,30 and the results are shown in Table 1. We focused on
branched aliphatic groups or surrogates, including t-butyl, trifluo-
romethylcyclopropyl, trifluoromethylcyclobutyl and phenylcyclo-
propyl, for the groups connected to the thiadiazole because our
previous findings indicated that these lipophilic or fluorine-con-
taining substituents favorably affected the biological activity of

J. Lee et al. / Bioorg. Med. Chem. 18 (2010) 6377–6388
6381
Table 2
Structures and binding affinities of selected ligands to rat CB1 and human CB2 receptors and hCB2/rCB1 selectivity of the ligandsa,b
N
N
N
X
N
S
N
N
N
R
Y
Entry
X
Y
R
Compound
Receptor affinity (IC50, nM)
hCB2/rCB1 selectivity
rCB1
4.53
hCB2
1
2
1
17n
1760
—
389
—
Cl
Cl
Cl
Cl
t-Bu
1.93
CF3
CF3
3
4
17o
17p
1.66
—
—
—
—
Cl
Cl
Cl
1.01
6.48
CF3
5
6
CF3
17q
17r
76.8
11.85
Cl
Cl
Cl
0.966
265
274
7
8
Br
Br
Cl
Cl
t-Bu
17s
17t
0.681
0.992
549
124
807
125
CF3
CF3
9
Br
Br
Cl
17u
17v
0.848
4.38
73.6
87
CF3
10
CF3
27.6
6.30
a
These data were obtained by single determinations.
Compound 1 was estimated to have CB1 binding affinity via in-house assay.
b
Next, in order to test the effect of modifying the substituents on
17 @ 10mg/kg
17g @ 10mg/kg
17c @ 10mg/kg
Ve hicle
1 @ 10mg/kg
2 @ 10mg/kg
the diphenylpyrazole, one of the chlorine atoms on the N1 phenyl
group was removed. As shown in Table 2, removal of the chlorine
at the 4-position of the N1 phenyl ring actually improved the rCB1
receptor binding affinity, as exemplified by compounds 17p
(IC50 = 1.01 nM) and 17r (IC50 = 0.966 nM). This effect is further
boosted by replacement of chlorine with bromine at the position
marked X, as exemplified by compounds 17s–17u in Table 2. It is
17s @ 10mg/kg
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14
Day
0
-2
-4
-6
-8
worth noting that pyrazolyl-t-butyl thiadiazole 17s (IC50
=
0.681 nM) was shown to possess the highest binding affinity
against the rat CB1 receptor of the pyrazolyl thiadiazole com-
pounds prepared to date. Compound 17s also showed high hCB2/
rCB1 receptor selectivity (ꢁ807) due to lower hCB2 receptor bind-
ing affinity. Interestingly, introduction of a trifluoromethyl group
at the position marked Y decreased the CB2/CB1 receptor selectiv-
ity by increasing the hCB2 receptor binding affinity, as exemplified
by 17q (ꢁ11.85) and 17v (ꢁ6.30) in Table 2. Some selected prom-
ising compounds have been tested on animal models for in vivo
efficacy.
In vivo efficacy was evaluated on high fat diet induced obese
(DIO) mice and rats.31 All animals were housed 1 per cage on a
12/12-h light/dark cycle, were fed food (high fat diet) and water,
and were experimentally naive before testing. The samples of ref-
erence compounds 1 and 2 and the newly designed compounds 17,
17c, 17g, and 17s were prepared fresh daily by dissolving them in
de-ionized water containing 10% DMSO at a volume of 10 mg/kg.
Figure 5 shows that all of our tested compounds 17–17s are
substantially more efficacious than 1 in the in vivo efficacy study
using the DIO mice model. The compounds prepared in this study
exhibited reductions in body weight after 14 days ranging from
-10
-12
-14
-16
-18
-20
-22
-24
-26
-28
-30
-32
-34
Figure 5. DIO mouse 2-week efficacy following daily administration of compounds
1, 2, 17, 17c, 17g and 17s at 10 mg/kg. Each value is the mean S.E.M. of 6 mice.
*
**
P <0.05, P <0.01 versus corresponding vehicle (ANOVA with Dunnett’s test).
23.68 2.58% up to 32.55 2.58%, whereas 1 reduced body weight
moderately by comparison (18.02 2.05%). Moreover, 17s turned
out to be more efficacious than 2 (32.55 2.58% vs 29.55 2.54%).

6382
J. Lee et al. / Bioorg. Med. Chem. 18 (2010) 6377–6388
Vehicle
17s @ 20mg/kg
Vehicle
17c @ 10mg/kg
17s @ 10mg/kg
22 @ 5mg/kg
1 @ 10mg/kg
2 @ 3mg/kg
17s @ 10mg/kg
17s @ 3mg/kg
17c @ 3mg/kg
1 @ 10mg/kg
2 @ 10mg/kg
2
0
Day
0
-5
Day
0
1
2
3
4
5
6
7
8
9 10 11 12 13
0
1
2
3
4
5
6
7
8
9
10 11 12 13
-2
-4
-10
-15
-20
-25
-30
-35
-40
-6
-8
-10
-12
-14
-16
-18
Figure 8. Effects of treatment with tested compounds on body weight in DIO rat.
All rats were fed HFD for 12 wk and then treated for 14 days with compound at
10 mg/kg/day. All results are expressed as mean S.E.M. of six rats. The statistical
analysis was performed using a one-way ANOVA followed by Dunnett’s post hoc
Figure 6. DIO mouse 13 day efficacy following daily administration of 17s at 3, 10,
or 20 mg/kg, shown together with data from administration of sibutramine (22) at
5 mg/kg, 1 at 10 mg/kg, 2 at 3 mg/kg, and 17c at 3 mg/kg. Each value is the
*
**
test. P <0.05, P <0.01 versus corresponding vehicle.
*
**
mean S.E.M. of six mice. P <0.05, P <0.01 versus corresponding Vehicle (ANOVA
with Dunnett’s test).
heterogeneous sizes, while adipocytes in 17s-treated animals were
dose-dependently smaller than those in vehicle-treated animals
(Fig. 7).
For comparison of efficacy in a different animal model, chronic
evaluation of 17c and 17s along with reference compounds 1 and 2
was performed by oral administration at 10 mg/kg for 13 days in
DIO rats. Dosing 17c and 17s at 10 mg/kg each significantly re-
duced body weight (14.43 1.56%, 15.59 2.56%) compared with
1 (10.61 1.89%), while 2 demonstrated superior efficacy in DIO
rats (17.16 1.89%).
Chronic evaluation of 17s was carried out with oral administra-
tion for 13 days in a DIO mouse in order to test its dose dependency.
As indicated by the data in Figure 6, 17s showed a dose-dependent
response in its ability to affect body weight. Dosing at 10 or
20 mg/kg significantly reduced body weights by 34.90 1.89 and
38.06 3.01%, respectively. Dosing 17s at 3 mg/kg proved as effec-
tive as reference compound 2 at the same dose. Compound 17c is
also included in Figure 6 for comparison. Interestingly, dosing 17c
at 3 mg/kg reduced body weight (34.45 1.45%) as much as dosing
17s at 10 mg/kg (34.90 1.89%). The mean cell diameter of white
adipocytes in vehicle-treated DIO mice were large and exhibited
Compound 17s also displayed significant anti-diabetic activity
through oral administration at 10 mg/kg in various animal models
including ob/ob mice, db/db mice, and DIO mice (Fig. 8).32
Figure 7. Effects of treatment with tested compounds on white adipocyte (WAT) phenotype. WAT from mice that were treated 10 mg/kg/d or 3 mg/kg/d compounds for
14 days were stained H&E. The magnification is 400X.

J. Lee et al. / Bioorg. Med. Chem. 18 (2010) 6377–6388
6383
Table 3
Pharmacokinetic profiles of 1, 2, and 17s in rats
Pharmacokinetic
1
2
17s
parameters
po (5 mg/kg, n = 3)
iv (1 mg/kg, n = 3)
po (5 mg/kg, n = 3)
iv (1 mg/kg, n = 3)
po (5 mg/kg, n = 3)
iv (1 mg/kg, n = 3)
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
Tmax (h)
0.50 0.00
0.29 0.14
0.00 0.00
8.23 3.29
154.10 36.63
—
—
1.00 0.00
1.53 0.47
0.03 0.00
7.11 0.06
13.70 2.12
—
—
0.50 0.00
0.36 0.01
0.02 0.00
14.80 0.31
59.77 1.00
—
—
Cmax
Clast
(
l
g/mL)
0.30 0.08
0.01 0.00
3.09 0.32
—
82.40 20.47
12.59 2.74
2.50 1.11
0.05 0.04
5.30 1.40
—
10.70 7.07
119.90 79.27
0.47 0.06
0.01 0.00
2.76 0.38
—
40.83 5.26
24.75 3.09
(lg/mL)
T1/2 (h)
CL/F
CL (mL/min/kg)
AUC0–1 (min/
lg/mL)
33.40 7.99
369.72 56.37
83.64 1.44
Bioavailability (%)
53.1
61.7
67.6
Table 4
Pharmacological data of compounds 17g, 17c, 17s along with reference 1, 2
Compound
CYP 450 (IC50, nM)
hERG (IC50, HM)
Ames assay
Comet assay
1A2
2C9
2D6
3A4
1
2
I7g
17c
17s
>20.0
>20.0
2.7
>20.0
>20.0
5.3
0.93
2.6
>20.0
>20.0
>20.0
0.59
>20.0
>20.0
>20.0
>20.0
>20.0
>20.0
>20.0
>20.0
4.8
Non-mutagenic
Non-mutagenic
Non-mutagenic
Non-mutagenic
Non-mutagenic
neg*
neg
+
neg
neg
14.2
>10.0
>10.0
10.5
Table 5
Single-dose toxicity in rats and 7-day repeat toxicity in rats
Compound
Single-dose
7-day repeat
mg/kg, po
2000
Mortality/total animal number
0/5
LD50 (mg/kg)
>2000
mg/kg ꢂ day, po
Mortality/total animal number
NOAEL (mg/kg)
125
17s
1000 ꢂ 7
500 ꢂ 7
250 ꢂ 7
125 ꢂ 7
0/2
0/5
0/5
0/5
1500
0/5
The pharmacokinetic properties of 17s along with 1 and 2 were
measured in male SD rats (Table 3). After oral administration of
5 mg/kg of 1, 2, and 17s, compound 17s had slightly higher expo-
sure (F = 67.6%) than reference compound 1 (F = 53.1%) or 2
(F = 61.7%) in rats with 14.80 0.31 h T1/2. The clearance of 17s
was determined from iv PK studies (CL = 40.83 5.26 mL/min/kg)
and showed a value in between reference compounds 1 and 2.
Compound 17s also showed a similar brain/plasma ratio (58%) to
that of reference 2 (52%).
horts survived on oral administration q.d. for 7 days. NOAEL (No
adverse effect level) is roughly estimated to be 125 mg/kg. Based
on its favorable toxicity studies, its excellent in vivo efficacy in ani-
mal models, and a satisfactory pharmacokinetic profile, compound
17s was selected as a development candidate.
4. Conclusion
Structure–activity relationship studies in a series of diarylpy-
razolyl thiadiazoles identified cannabinoid-1 receptor antagonists
with excellent potency and selectivity. Modifications of previous
oxadiazole ring into the corresponding thiadiazole ring appears
to provide more efficacious series of compounds, not to mention
securing superior intellectual property (IP) position to other
existing pyrazole series of CB1 receptor ligands. Based on its
exceptional in vivo efficacy in animal models and favorable
pharmacokinetic and toxicological profiles, 2-(4-((1H-1,2,4-tria-
zol-1-yl)methyl)-5-(4-bromophenyl)-1-(2-chlorophenyl)-1H-pyra-
zol-3-yl)-5-tert-butyl-1,3,4-thiadiazole (GCC2680) was selected as
a preclinical candidate for the treatment of obesity.
The off-target activity of CB1 ligands including 17s was also
evaluated as shown in Table 4. Both 17c and 17s were demon-
strated not to be significant inhibitors of the cytochrome p450 en-
zymes (IC50 >20
activity shown by reference 1 (IC50 = 5.3
against CYP2C9. The activity of 17s in the hERG potassium channel
lM). This observation is in sharp contrast to the
l
M) and 2 (IC50 = 0.93 M)
l
assay was determined to be 10.5 l
M for IC50. The comet assay33 and
Ames assay34 were used for detection of mutagenicity of the com-
pounds. Neither 17c nor 17s proved to be mutagenic. In addition,
when the cytotoxicity35 of 17c and 17s was tested on a panel of cells
(Huh-7, Vero, HCT116, and HEK-293), both 17c and 17s demon-
strated their safety by showing >100 lM for EC50.
Subsequently, in vivo toxicity studies were performed for com-
pound 17s to examine critical safety issues. Acute toxicity studies
were performed on oral administration of 2000 mg/kg or 1500 mg/
kg in rats (n = 5). As shown in Table 5, there was no mortality of the
animals tested up to 2000 mg/kg. Thus, the estimated LD50 is great-
er than 2000 mg/kg. Table 5 also describes results of preliminary 7-
day repeat toxicity studies. Every animal in 4 different dosing co-
5. Experimental section
All references to ether are to diethyl ether; brine refers to a sat-
urated aqueous solution of NaCl. Unless otherwise indicated, all
temperatures are expressed in °C (degrees Centigrade). All reactions
were conducted under an inert atmosphere at room temperature

6384
J. Lee et al. / Bioorg. Med. Chem. 18 (2010) 6377–6388
unless otherwise noted, and all solvents were of the highest
available purity unless otherwise indicated. Microwave reactions
were conducted with a Biotage microwave reactor. 1H NMR and
13C NMR spectra were recorded on 400 MHz Fourier Transform-
Nuclear Magnetic Resonance; Varian, 400-MR. Chemical shifts
are expressed in parts per million (ppm, d units). Coupling con-
stants are in units of Hertz (Hz). Splitting patterns describe appar-
ent multiplicities and are designated as s (singlet), d (doublet), t
(triplet), q (quartet), quint (quintet), m (multiplet), or br (broad).
Mass spectra were obtained with either a Micromass, Quattro LC
Triple Quadrupole Tandem Mass Spectrometer, ESI or an Agilent,
1200LC/MSD, ESI. High-resolution mass spectra were determined
by a Jeol, JMS-700 Mstation. For preparative HPLC, ca. 100 mg of
a product was injected in 1 mL of DMSO onto a SunFire™ Prep
5.4. 4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-N’-pivaloyl-1H-pyrazole-3-carbohyd-
razide (21)
To a solution of 4-((1H-1,2,4-triazol-1-yl)methyl)-5-(4-chloro-
phenyl)-1-(2,4-dichlorophenyl)-1H-pyrazole-3-carbohydrazide (20,
5.5 g, 11.9 mmol), pivalic acid (1.6 g, 15.5 mmol), EDCI (3.4 g,
17.9 mmol), HOBt (3.2 g, 23.8 mmol) in DMF (100 mL) was added
NMM (4.0 mL, 36.4 mmol). The resulting mixture was concen-
trated, diluted with CH2Cl2 and washed with 1N-HCl solution.
The organic layer was dried over MgSO4, filtered, and concentrated
in vacuo. The crude product was purified by silica column chroma-
tography (Biotage SP1™) and the desired compound was obtained
as a white solid (7.0 g, 99%). ESI-MS m/z: 546 [M+H]+.
C18 OBD 5
l
m 30 ꢂ 100 mm column with a 10-min gradient from
10% CH3CN to 90% CH3CN in H2O. A BiotageÒ SP1 Flash purification
system was used for normal phase column chromatography with
ethyl acetate and hexane. Flash chromatography was carried out
using Merck Silica Gel 60 (230–400 mesh). Most of the reactions
were monitored by thin-layer chromatography on 0.25 mm E.
Merck silica gel plates (60F-254), visualized with UV light using a
5% ethanolic phosphomolybdic acid or p-anisaldehyde solution.
All tested compounds were found to be greater than 95% pure by
LC/MS, except as otherwise indicated.
5.5. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)-5-tert-butyl-1,3,4-
thiadiazole (17)
To a solution of 4-((1H-1,2,4-triazol-1-yl)methyl)-5-(4-chloro-
phenyl)-1-(2,4-dichlorophenyl)-N’-pivaloyl-1H-pyrazole-3-carbo-
hydrazide (21) (244 mg, 0.45 mmol) and Lawesson’s reagent
(453 mg, 1.12 mmol) in THF (10 mL) was irradiated in a microwave
reactor (Biotage Initiator™) for 50 minutes at 150 °C The resulting
mixture was concentrated, diluted with CH2Cl2 and washed with
brine. The organic layer was dried over MgSO4, filtered, and con-
centrated in vacuo. The crude product was purified by silica gel col-
umn chromatography (Biotage SP1™ FLASH Purification System
was used for normal phase column chromatography with ethyl
acetate and hexane) and the desired compound was obtained as
a white solid (167 mg, 68.8%); 1H NMR (400 MHz, CDCl3) d 8.79
(s, 1H), 7.99 (s, 1H), 7.45–7.42 (m, 3H), 7.39–7.34 (m, 4H), 5.64
(s, 2H), 1.52 (s, 9H); 13C NMR (100 MHz, CDCl3) d 178.9, 160.5,
150.7, 145.3, 143.8, 142.9, 136.0, 135.6, 134.8, 132.6, 131.1,
130.1, 130.0, 129.0, 127.7, 125.2, 113.4, 44.4, 37.9, 32.9; ESI-MS
m/z: 544 [M+H]+; positive HR-FAB-MS m/z: 544.0647 [M+H]+
(calcd for C24H20Cl3N7S: 544.0645).
5.1. Ethyl 4-(bromomethyl)-5-(4-chlorophenyl)-1-(2,4-dichloro-
phenyl)-1H-pyrazole-3-carboxylate (18)
To a solution of ethyl 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-
4-methyl-1H-pyrazole-3-carboxylate (12) (10.0 g, 22.0 mmol) in
CCl4 (150 mL) was added N-bromosuccinimide (4.7 g, 26.4 mmol)
and AIBN (36 mg, 0.2 mmol). The reaction mixture was heated for
8 h at 95 °C. Reaction mixture was filtered. Filtrate was washed with
H2O and brine. The organic layer was dried over anhydrous MgSO4,
filtered, and concentrated in vacuo to provide 10.5 g of desired bro-
mide as a pale yellow solid. The obtained bromide was used without
further purification. ESI-MS m/z: 487 [M+H]+.
5.2. Ethyl 4-((1H-1,2,4-triazol-1-yl)methyl)-5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-1H-pyrazole-3-carboxylate (19)
5.5.1. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)-5-(1-methylcyclopro-
pyl)-1,3,4-thiadiazole (17a)
To a solution of ethyl 4-(bromomethyl)-5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-1H-pyrazole-3-carboxylate (18) (10.5 g,
19.7 mmol) in DMF (50 mL) was added 1,2,4-triazole sodium deriv-
atives (1.97 g, 21.7 mmol). The resulting mixture was concen-
trated, diluted with CH2Cl2 and washed with brine. The organic
layer was dried over MgSO4, filtered, and concentrated in vacuo.
The crude product was purified by silica column chromatography
(Biotage SP1™) and the desired compound was obtained as a white
solid (6.7 g, 58% 2-steps). 1H NMR (400 MHz, CDCl3) d 8.76 (s, 1H),
8.09 (s, 1H), 7.41–7.27 (m, 7H), 5.48 (s, 2H), 4.44 (quartet,
J = 7.2 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H); ESI-MS m/z: 476 [M+H]+.
1H NMR (400 MHz, CDCl3) d 9.13 (s, 1H), 8.16 (s, 1H), 7.48–7.29
(m, 7H), 5.68 (s, 2H), 1.65 (s, 3H), 1.45–1.39 (m, 2H), 1.16–1.10 (m,
2H); ESI-MS m/z: 542 [M+H]+; positive HR-FAB-MS m/z: 542.0486
[M+H]+ (calcd for C24H18Cl3N7S: 542.0488).
5.5.2. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)-5-(1-(trifluoromethyl-
)cyclopropyl)-1,3,4-thiadiazole (17b)
1H NMR (400 MHz, CDCl3) d 8.94 (s, 1H), 8.01 (s, 1H), 7.48–7.28
(m, 7H), 5.65 (s, 2H), 1.83–1.61 (m, 4H); 13C NMR (100 MHz, CDCl3)
d 163.5, 162.0, 150.6, 145.5, 143.7, 142.3, 136.1, 135.8, 134.6,
132.6, 131.0, 130.1, 130.0, 128.9, 127.7, 125.0, 124.9 (q, J =
266 Hz), 113.4, 44.4, 27.0, 16.1; ESI-MS m/z: 596 [M+H]+; positive
HR-FAB-MS m/z: 596.0208 [M+H]+ (calcd for C24H15Cl3F3N7S:
596.0206).
5.3. 4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-chlorophenyl)-1-
(2,4-dichlorophenyl)-1H-pyrazole-3-carbohydrazide (20)
To a solution of ethyl 4-((1H-1,2,4-triazol-1-yl)methyl)-5-(4-
chlorophenyl)-1-(2,4-dichlorophenyl)-1H-pyrazole-3-carboxylate
(19) (14.0 g, 29.4 mmol) in EtOH (400 mL) was added hydrazine
monohydrate (40 mL). The reaction mixture was refluxed for 2 h
at 65 °C. The reaction mixture was diluted with EtOAc and
washed with saturated NaCl. The organic layer was dried over
anhydrous MgSO4, filtered, and concentrated in vacuo to provide
13.0 g (28.1 mmol, 95.6%) as a white solid. The obtained hydra-
zide was used without further purification. ESI-MS m/z: 462
[M+H]+.
5.5.3. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)-5-(1-(trifluoromethyl)-
cyclobutyl)-1,3,4-thiadiazole (17c)
1H NMR (400 MHz, CDCl3) d 9.41 (s, 1H), 8.28 (s, 1H), 7.47–7.45
(m, 1H), 7.43–7.31 (m, 6H), 5.72 (s, 2H), 2.84 (d, J = 8.0 Hz, 4H), 2.20
(m, 2H); 13C NMR (100 MHz, CDCl3) d 167.8, 163.9, 151.6, 146.2,
144.5, 143.0, 136.6, 136.2, 135.1, 133.0, 131.4, 130.5, 130.4, 129.2,
128.0, 126.1 (q, J = 277 Hz), 125.3, 113.6, 48.2 (q, J = 30 Hz), 43.0,

J. Lee et al. / Bioorg. Med. Chem. 18 (2010) 6377–6388
6385
28.8, 15.8; ESI-MS m/z: 610 [M+H]+; positive HR-FAB-MS m/z:
130.4, 128.0, 125.7, 125.1 (q, J = 272 Hz), 124.6, 113.5, 42.9, 25.2,
14.1; ESI-MS m/z: 640 [M+H]+; positive HR-FAB-MS m/z:
639.9705 [M+H]+ (calcd for C24H15BrCl2F3N7S: 639.9700).
610.0365 [M+H]+ (calcd for C25H17Cl3F3N7S: 610.0362).
5.5.4. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)-5-(1-phenylcyclopropyl)-
1,3,4-thiadiazole (17d)
5.5.10. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-bromophenyl)-
1-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)-5-(1-(trifluoromethyl)-
cyclobutyl)-1,3,4-thiadiazole (17j)
1H NMR (400 MHz, CDCl3) d 8.91 (s, 1H), 8.05 (s, 1H), 7.50–7.46
(m, 2H), 7.42–7.21 (m, 10H), 5.63 (s, 2H), 1.98–1.90 (m, 2H), 1.61–
1.53 (m, 2H); 13C NMR (100 MHz, CDCl3) d 175.9, 160.1, 150.7,
145.2, 143.8, 142.8, 141.5, 135.9, 135.6, 134.7, 132.6, 131.0,
130.1, 130.0, 129.8, 128.8, 128.7, 127.9, 127.6, 125.2, 113.2, 44.4,
29.1, 21.3; ESI-MS m/z: 604 [M+H]+; positive HR-FAB-MS m/z:
604.0646 [M+H]+ (calcd for C29H20Cl3N7S: 604.0645).
1H NMR (400 MHz, CDCl3) d 9.2 (s, 1H), 8.12 (s, 1H), 7.51 (d,
J = 8.4 Hz, 2H), 7.45–7.33 (m, 7H), 5.63 (s, 2H), 2.87–2.74 (m, 4H),
2.20–2.11 (m, 2H); ESI-MS m/z: 654 [M+H]+; positive HR-FAB-MS
m/z: 653.9851 [M+H]+ (calcd for C25H17BrCl2F3N7S: 653.9857).
5.5.11. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-bromophenyl)-
1-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)-5-(1-phenylcyclopropyl)-
1,3,4-thiadiazole (17k)
5.5.5. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)-5-(1-p-tolylcyclopro-
pyl)-1,3,4-thiadiazole (17e)
1H NMR (400 MHz, CDCl3) d 8.89 (s, 1H), 8.04 (s, 1H), 7.58–7.45
(m, 4H), 7.42–7.23 (m, 8H), 5.62 (s, 2H), 1.95 (dd, J = 6.8 Hz, 4.4 Hz,
2H), 1.57 (dd, J = 6.8 Hz, 4.4 Hz, 2H); ESI-MS m/z: 648 [M+H]+;
positive HR-FAB-MS m/z: 648.0132 [M+H]+ (calcd for
1H NMR (400 MHz, CDCl3) d 8.89 (s, 1H), 8.04 (s, 1H), 7.47–7.22
(m, 9H), 7.18–7.10 (m, 2H), 5.62 (s, 2H), 2.33 (s, 3H), 1.95–1.88 (m,
2H), 1.56–1.49 (m, 2H); 13C NMR (100 MHz, CDCl3) d 176.4, 160.1,
150.7, 145.1, 143.8, 142.8, 138.7, 137.5, 135.9, 135.6, 134.8, 132.6,
131.0, 130.1, 130.0, 129.7, 129.3, 128.8, 127.6, 125.2, 113.2, 44.4,
28.7, 23.1, 21.4; ESI-MS m/z: 618 [M+H]+; positive HR-FAB-MS m/
z: 618.0807 [M+H]+ (calcd for C30H22Cl3N7S: 618.0801).
C29H20BrCl2N7S: 648.0140).
5.5.12. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-bromophenyl)-
1-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)-5-(1-p-tolylcyclopropyl)-
1,3,4-thiadiazole (17l)
1H NMR (400 MHz, CDCl3) d 8.97 (s, 1H), 8.08 (s, 1H), 7.51
(d, J = 8.0 Hz, 2H), 7.42–7.23 (m, 7H), 7.15 (d, J = 8.0 Hz, 2H), 5.63
(s, 2H), 2.33 (s, 3H), 1.92 (dd, J = 6.8 Hz, 4.4 Hz, 2H), 1.54 (dd, J =
6.8 Hz, 4.4 Hz, 2H); 13C NMR (100 MHz, CDCl3) d 177.7, 161.1,
151.5, 145.8, 144.4, 143.5, 139.2, 138.0, 136.4, 135.2, 133.0, 132.1,
131.6, 130.4, 130.3, 130.0, 129.6, 127.9, 125.9, 124.4, 113.1, 42.9,
26.9, 21.1, 19.4; ESI-MS m/z: 662 [M+H]+; positive HR-FAB-MS m/
z: 662.0299 [M+H]+ (calcd for C30H22BrCl2N7S: 662.0296).
5.5.6. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)-5-(1-(4-chlorophenyl)-
cyclopropyl)-1,3,4-thiadiazole (17f)
1H NMR (400 MHz, CDCl3) d 9.07 (s, 1H), 8.13 (s, 1H), 7.44–7.24
(m, 11H), 5.29 (s, 2H), 1.99–1.88 (m, 2H), 1.59–1.51 (m, 2H); 13C
NMR (100 MHz, CDCl3) d 175.2, 160.2, 150.7, 145.2, 143.8, 142.6,
139.9, 136.0, 135.7, 134.7, 133.7, 132.6, 131.2, 131.0, 130.1,
130.0, 128.9, 128.8, 127.7, 125.1, 113.2, 44.4, 28.6, 21.4; ESI-MS
m/z: 638 [M+H]+; positive HR-FAB-MS m/z: 638.0254 [M+H]+
(calcd for C29H19Cl4N7S: 638.0255).
5.5.13. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-bromophenyl)-
1-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)-5-(1-(4-chlorophenyl)-
cyclopropyl)-1,3,4-thiadiazole (17m)
5.5.7. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-bromophenyl)-
1-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)-5-tert-butyl-1,3,4-
thiadiazole (17g)
1H NMR (400 MHz, CDCl3) d 9.15 (s, 1H), 8.18 (s, 1H), 7.53 (d,
J = 8.4 Hz, 1H), 7.46–7.40 (m, 2H), 7.38–7.24 (m, 8H), 5.67 (s, 2H),
2.05–1.93 (m, 2H), 1.59–1.52 (m, 2H); 13C NMR (100 MHz, CDCl3) d
178.0, 162.7, 153.0, 147.4, 145.9, 144.7, 141.9, 137.9, 136.6, 135.6,
134.5, 133.5, 133.1, 133.0, 132.9, 131.8, 130.6, 129.4, 127.3, 125.9,
114.6, 44.3, 28.2, 20.9; ESI-MS m/z: 682 [M+H]+; positive HR-FAB-
MS m/z: 681.9741 [M+H]+ (calcd for C29H19BrCl3N7S: 681.9750).
1H NMR (400 MHz, CDCl3) d 8.77 (s, 1H), 7.96 (s, 1H), 7.53–7.50
(m, 2H), 7.45–7.44 (m, 1H), 7.39–7.36 (m, 2H), 7.35–7.30 (m, 2H),
5.63 (s, 2H), 1.52 (s, 9H); 13C NMR (100 MHz, CDCl3) d 180.3, 161.5,
151.5, 146.0, 144.5, 143.5, 136.5, 135.2, 133.0, 132.1, 131.7, 130.5,
130.4, 128.0, 125.9, 124.5, 113.4, 43.0, 36.3, 31.3; ESI-MS m/z: 588
[M+H]+; positive HR-FAB-MS m/z: 588.0130 [M+H]+ (calcd for
5.5.14. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-1-(2-chlorophenyl)-
5-(4-chlorophenyl)-1H-pyrazol-3-yl)-5-tert-butyl-1,3,4-thiadi-
azole (17n)
C24H20BrCl2N7S: 588.0140).
5.5.8. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-bromophenyl)-
1-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)-5-(1-methylcyclopro-
pyl)-1,3,4-thiadiazole (17h)
1H NMR (400 MHz, CDCl3) d 9.16 (s, 1H), 8.18 (s, 1H), 7.47–7.31
(m, 8H), 5.71 (s, 2H), 1.54 (s, 9H); 13C NMR (100 MHz, CDCl3) d
180.2, 161.7, 151.5, 145.8, 144.5, 143.2, 136.6, 135.9, 132.2,
131.5, 131.0, 130.5, 129.8, 129.0, 127.6, 125.7, 113.1, 43.1, 36.3,
31.1; ESI-MS m/z: 510 [M+H]+; positive HR-FAB-MS m/z:
510.1032 [M+H]+ (calcd for C24H21Cl2N7S: 510.1034).
1H NMR (400 MHz, CDCl3) d 8.37 (s, 1H), 8.02 (s, 1H), 7.54–7.51
(m, 2H), 7.46–7.44 (m, 1H), 7.38–7.33 (m, 2H), 7.32–7.28 (m, 2H),
5.64 (s, 2H), 1.63 (s, 3H), 1.40 (dd, J = 6.8 Hz, 4.4 Hz, 2H), 1.10 (dd,
J = 6.8 Hz, 4.4 Hz, 2H); 13C NMR (100 MHz, CDCl3) d 177.4, 160.4,
151.5, 145.9, 144.5, 143.5, 136.4, 135.2, 133.1, 132.1, 131.6,
130.5, 130.4, 128.0, 125.9, 124.5, 113.3, 42.9, 23.2, 20.3, 17.7;
ESI-MS m/z: 586 [M+H]+; positive HR-FAB-MS m/z: 585.9984
[M+H]+ (calcd for C24H18BrCl2N7S: 585.9983).
5.5.15. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-1-(2-chlorophenyl)-
5-(4-chlorophenyl)-1H-pyrazol-3-yl)-5-(1-(trifluoromethyl)-
cyclopropyl)-1,3,4-thiadiazole (17o)
1H NMR (400 MHz, CDCl3) d 9.14 (s, 1H), 8.18 (s, 1H), 7.45–7.38
(m, 4H), 7.36–7.32 (m, 3H), 5.68 (s, 2H), 1.75–1.67 (m, 2H),
1.65–1.63 (m, 2H); 13C NMR (100 MHz, CDCl3) d 163.4, 162.2,
150.8, 145.4, 143.7, 142.0, 136.0, 135.5, 131.7, 131.1, 130.7,
130.2, 129.4, 128.7, 127.4, 125.3, 124.9 (q, J = 266 Hz), 113.3,
44.5, 26.8 (q, J = 35 Hz), 16.1; ESI-MS m/z: 562 [M+H]+; positive
HR-FAB-MS m/z: 562.0588 [M+H]+ (calcd for C24H16Cl2F3N7S:
562.0595).
5.5.9. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-bromophenyl)-
1-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)-5-(1-(trifluoromethyl)-
cyclopropyl)-1,3,4-thiadiazole (17i)
1H NMR (400 MHz, CDCl3) d 9.03 (s, 1H), 8.25 (s, 1H), 7.58–
7.54(m, 2H), 7.47 (d, J = 1.6 Hz, 1H), 7.39–7.31 (m, 4H), 5.69 (s,
2H), 1.77–1.65 (m, 4H); 13C NMR (100 MHz, CDCl3) d 164.6, 163.0,
151.6, 146.1, 144.4, 142.9, 136.6, 135.1, 133.0, 132.1, 131.6, 130.5,

6386
J. Lee et al. / Bioorg. Med. Chem. 18 (2010) 6377–6388
5.5.16. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-1-(2-chlorophenyl)-
5-(4-chlorophenyl)-1H-pyrazol-3-yl)-5-(1-(trifluoromethyl)-
cyclobutyl)-1,3,4-thiadiazole (17p)
2.23–2.15 (m, 2H); 13C NMR (100 MHz, CDCl3) d 169.2, 165.6,
153.2, 147.6, 146.0, 144.3, 138.0, 133.6, 133.5, 133.2, 132.7,
132.1, 131.2, 129.2, 127.7 (q, J = 277 Hz), 127.5, 125.9, 114.9, 49.5
(q, J = 30 Hz), 44.6, 30.3, 17.4; ESI-MS m/z: 620 [M+H]+; positive
HR-FAB-MS m/z: 620.0259 [M+H]+ (calcd for C25H18BrClF3N7S:
620.0247).
1H NMR (400 MHz, CDCl3) d 9.01 (s, 1H), 8.10 (s, 1H), 7.45–7.31
(m, 7H), 5.69 (s, 2H), 2.83 (t, J = 8.0 Hz, 4H), 2.23–2.14 (m, 2H); 13
C
NMR (100 MHz, CDCl3) d 167.6, 164.1, 151.6, 146.1, 144.5, 142.7,
136.5, 136.0, 132.1, 131.5, 131.1, 130.6, 129.7, 129.0, 127.7, 126.1
(q, J = 278 Hz), 125.5, 113.4, 48.2 (q, J = 30 Hz), 43.0, 28.8, 15.8;
ESI-MS m/z: 576 [M+H]+; positive HR-FAB-MS m/z: 576.0756
[M+H]+ (calcd for C25H18Cl2F3N7S: 576.0752).
5.5.22. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-bromophenyl)-
1-(2-(trifluoromethyl)phenyl)-1H-pyrazol-3-yl)-5-(1-(trifluoro-
methyl)cyclobutyl)-1,3,4-thiadiazole (17v)
1H NMR (400 MHz, CDCl3) d 8.64 (s, 1H), 7.94 (s, 1H), 7.83 (d,
J = 7.6 Hz, 1H), 7.58 (quint, J = 6.8 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H),
7.41 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 7.6 Hz, 1H), 5.64 (s, 2H), 2.86 (t,
J = 8.4 Hz, 4H), 2.24–2.16 (m, 2H); 13C NMR (100 MHz, CDCl3) d
167.8, 164.0, 151.6, 146.0, 144.5, 142.5, 136.1, 132.6, 132.1, 131.9,
130.1, 128.1, 128.0 (q, J = 5.0 Hz), 127.7, 126.1 (q, J = 278 Hz),
125.8, 124.5, 122.7 (q, J = 273 Hz), 113.8, 48.2 (q, J = 30 Hz), 43.0,
28.8, 15.8; ESI-MS m/z: 654 [M+H]+; positive HR-FAB-MS m/z:
654.0499 [M+H]+ (calcd for C26H18BrF6N7S: 654.0510).
5.5.17. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-chlorophenyl)-
1-(2-(trifluoromethyl)phenyl)-1H-pyrazol-3-yl)-5-(1-(trifluoro-
methyl)cyclobutyl)-1,3,4-thiadiazole (17q)
1H NMR (400 MHz, CDCl3) d 8.61 (s, 1H), 7.92 (s, 1H), 7.81 (d, J =
7.6 Hz, 1H), 7.56 (quint, J = 7.2 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.34
(d, J = 8.4 Hz, 2H), 7.22 (d, J = 7.6 Hz, 1H), 5.63 (s, 2H), 2.84
(t, J = 8.4 Hz, 4H), 2.21–2.17 (m, 2H); 13C NMR (100 MHz, CDCl3)
d 167.8, 164.0, 151.6, 146.0, 144.5, 142.5, 136.2, 136.1, 132.6,
131.7, 130.1, 129.2, 128.1, 128.0 (q, J = 5 Hz), 127.7, 126.2 (q,
J = 280 Hz), 125.4, 122.7 (q, J = 273 Hz), 113.8, 48.2 (q, J = 30 Hz),
43.1, 28.8, 15.8; ESI-MS m/z: 610 [M+H]+; positive HR-FAB-MS m/
z: 610.1021 [M+H]+ (calcd for C26H18ClF6N7S: 610.1015).
5.6. CB1 and CB2 receptor binding assay29,30
For CB1 receptor binding studies, rat cerebellar membranes
were prepared as previously described by the methods of Kuster
et al. Male Sprague-Dawley rats (200–300 g) were sacrificed by
decapitation, and the cerebella were rapidly removed. The tissue
was homogenized in 30 volumes of TME buffer (50 mM Tris–HCl,
1 mM EDTA, 3 mM MgCl2, pH 7.4) using a Dounce homogenizer.
The crude homogenates were immediately centrifuged (48,000g)
for 30 min at 4 °C. The resultant pellets were resuspended in 30
volumes of TME buffer, the protein concentration was determined
by the method of Bradford, and the pellets were stored at ꢃ70 °C
until use.31
5.5.18. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-1-(2-chlorophenyl)-
5-(4-chlorophenyl)-1H-pyrazol-3-yl)-5-(1-(4-chlorophenyl)-
cyclopropyl)-1,3,4-thiadiazole (17r)
1H NMR (400 MHz, CDCl3) d 9.18 (s, 1H), 8.20 (s, 1H), 7.44–7.28
(m, 11H), 5.68 (s, 2H), 1.96–1.93 (m, 2H), 1.56–1.53 (m, 2H); 13C
NMR (100 MHz, CDCl3) d 175.1, 160.4, 150.7, 145.1, 143.7, 142.4,
140.0, 136.0, 135.4, 133.7, 131.7, 131.2, 131.0, 130.6, 130.1,
129.4, 128.8, 128.6, 127.3, 125.4, 112.9, 44.5, 28.6, 21.3; ESI-MS
m/z: 604 [M+H]+; positive HR-FAB-MS m/z: 604.0652 [M+H]+
(calcd for C29H20Cl3N7S: 604.0645).
For CB2 receptor binding studies, CHO K-1 cells were transfec-
ted with human CB2 receptor as previously described, and cell
membranes were prepared as described above.32
5.5.19.1. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-bromo-
phenyl)-1-(2-chlorophenyl)-1H-pyrazol-3-yl)-5-tert-butyl-
1,3,4-thiadiazole (17s)
Competitive binding assays were performed as described.33
Briefly, approximately 10 lg of rat cerebellar membranes (contain-
1H NMR (400 MHz, CDCl3) d 9.09 (s, 1H), 8.13 (s, 1H), 7.50 (d,
J = 8.4 Hz, 2H), 7.45–7.31 (m, 8H), 5.69 (s, 2H), 1.52 (s, 9H); 13C
NMR (100 MHz, CDCl3) d 178.8, 160.6, 150.7, 145.2, 143.8, 142.6,
136.1, 131.7, 131.5, 131.3, 130.6, 130.2, 129.4, 127.3, 125.9,
124.0, 113.1, 44.5, 37.9, 32.9; ESI-MS m/z: 554 [M+H]+; positive
HR-FAB-MS m/z: 554.0530 [M+H]+ (calcd for C24H21BrClN7S:
554.0529).
ing CB1 receptor) or cell membranes (containing CB2 receptor)
were incubated in a 96-well plate with TME buffer containing
0.5% essentially fatty acid free bovine serum albumin (BSA).
Three nM [3H]23 (for CB2 receptor, NEN; specific activity 50–
80 Ci/mmol) or 3 nM 24 ([3H]CP55,940, [3H]2-[(1S,2R,5S)-5-hydro-
xy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol,
for CB1 receptor, NEN; specific activity 120–190 Ci/mmol) and var-
ious concentrations of the synthesized cannabinoid ligands in a fi-
nal volume of 200 lL. The assays were incubated for 1 h at 30 °C
5.5.20. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-bromophenyl)-
1-(2-chlorophenyl)-1H-pyrazol-3-yl)-5-(1-(trifluoromethyl)-
cyclopropyl)-1,3,4-thiadiazole (17t)
and then immediately filtered over GF/B glass fiber filter (PerkinEl-
mer Life and Analytical Sciences, Boston, MA) that had been soaked
in 0.1% PEI for 1 h by a cell harvester (PerkinElmer Life and Analyt-
ical Sciences, Boston, MA). Filters were washed five times with ice-
cold TBE buffer containing 0.1% essentially fatty acid free BSA, fol-
lowed by oven-drying for 60 min and then were 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 the CB1 and CB2 receptor competi-
1H NMR (400 MHz, CDCl3) d 9.26 (s, 1H), 8.24 (s, 1H), 7.51 (d,
J = 8.4 Hz, 2H), 7.46–7.38 (m, 3H), 7.36–7.32 (m, 3H), 5.70 (s, 2H),
1.77–1.75 (m, 2H), 1.67–1.64 (m, 2H); 13C NMR (100 MHz, CDCl3)
d 164.5, 163.2, 151.6, 146.1, 144.4, 142.7, 136.5, 132.1, 132.0,
131.7, 131.1, 130.6, 129.7, 127.7, 126.0, 125.1 (q, J = 271 Hz),
124.4, 113.3, 43.0, 25.0 (q, J = 36 Hz), 14.1; ESI-MS m/z: 606
[M+H]+; positive HR-FAB-MS m/z: 606.0097 [M+H]+ (calcd for
tive binding assay, nonspecific binding was assessed using 1
1 and 1 M 23 (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 1 or 23 and
lM
C24H16BrClF3N7S: 606.0090).
l
5.5.21. 2-(4-((1H-1,2,4-Triazol-1-yl)methyl)-5-(4-bromophenyl)-
1-(2-chlorophenyl)-1H-pyrazol-3-yl)-5-(1-(trifluoromethyl)-
cyclobutyl)-1,3,4-thiadiazole (17u)
l
was 70–80% of the total binding. IC50 was determined by nonlinear
regression analysis using GraphPad PRISM. All data were collected
in triplicate, and IC50 was determined from three independent
experiments.
1H NMR (400 MHz, CDCl3) d 9.22 (s, 1H), 8.21 (s, 1H), 7.51 (d,
J = 8.4 Hz, 2H), 7.45–7.30 (m, 6H), 5.71 (s, 2H), 2.87–2.81 (m, 4H),

J. Lee et al. / Bioorg. Med. Chem. 18 (2010) 6377–6388
6387
5.7. Body weight reduction in diet induced obese (DIO) mice and rat
5.11. Comet and Ames assay
Male C57BL/6 mice and Sprague-Dawley rats were purchased by
Charles River Laboratory (Gyeonggi-Do, Korea). All animals were
housed individually with food and water freely available and were
maintained at room temperature under on a 12/12 h light-dark cy-
cle. Seven-week old mice were fed a high-fat diet (Rodent Diet with
45Kcal%Fat, Research Diet), and rats were fed a high-fat diet (Ro-
dent Diet with 32%Kcal and high in sucrose 25%Kcal, Research Diet)
ad libitum for 12 weeks. Thereafter, DIO mice and DIO rats were
treated with either vehicle or compounds orally in distilled water
with 0.1% Tween 80 for 14 days. Drugs were administered at 1 h be-
fore the dark cycle and their body weight was recorded daily before
drug treatment. After sacrifice, white adipose tissue was isolated,
weighed and fixed in formalin-alcohol for 48 h and paraffin-embed-
ded. Plasma glucose levels were determined with a Blood Chemis-
try System (ADIVA 1650, USA). All procedures were performed in
accordance with the experimental guidelines for animal care and
use in the Humane Care and Use of Laboratory Animals (NIH Guide
for the Care and Use of Laboratory Animals).
For the comet assay, 8 ꢂ 105 cells were seeded into 12 well
plates (Falcon 3043) and then treated with the compounds. The
compounds were tested at three concentration points at a range
of concentrations up to 50
pounds for 2 h, cells were centrifuged for 3 min at 100g (about
1200 rpm) and gently resuspended with PBS, and 100 L of the cell
lM. After treatment with the com-
l
suspension was immediately used for the test. Cells were mixed
with 0.1 ml of 1% low melting point agarose (LMPA, Life Technolo-
gies, MD, USA) and added to a fully frosted slide (Fisher Scientific,
PA, USA) that had been covered with a bottom layer of 100 lL of
1% normal melting agarose (Amresco, OH, USA). The cell suspension
was immediately covered with cover glass, and the slides were then
kept at 4 °C for 5 min to allow solidification. After removing the
cover glass gently, the slides were covered with a third layer of
100 lL of 0.5% LMPA by using a cover glass and then the slide were
kept again at 4 °C for 5 min. The Ames assay was performed essen-
tially as described by Ames et al. (1973, 1975). Media and positive
control chemicals were obtained from commercial sources and
were the purest grades available. Strain TA 100 was chosen as the
representative tester strain because of its high spontaneous rever-
sion rate. Spontaneous revertant numbers were counted and plot-
ted against the dose of the test chemical to produce a survival
curve for the his+ genotype. The mutagenicity assay was performed
by mixing one of the tester strains that was cultured overnight with
the test substance in the presence and in the absence of S9 mixture
condition, where sodium phosphate buffer was added instead of S9
mixture both in negative and positive controls in the test tube.
Then, the mixture was incubated in a water bath for 30 min at
37 °C, and after incubation, the mixture was mixed with top agar
containing a minimal amount of histidine and then poured onto
the surface of a r-ray sterile Petri dish (Falcon, USA) containing
25 ml of solidified bottom agar. The finished plates were incubated
for 48 h at 37 °C, and revertant colonies were counted later. Nega-
tive control plates containing no added test chemical, while posi-
tive control plates containing appropriate amounts of chemicals
known to be active were included with each tester strain. Sodium
azide (SA) and 2-aminoanthracene (2-AA) were used as positive
control substances. Compounds were tested at seven concentration
5.8. Histology of white adipose tissue in DIO mice
Paraffin embedded white adipose tissues were stained with
hematoxylin and eosin and processed with routine immunohisto-
logical analysis technique. An adipocyte of these sections was ana-
lyzed using Olympus microscope with a high-power lens (400X).
5.9. Pharmacokinetics and BBB permeability study
Male Sprague-Dawley rats (200–230 g) were purchased from
Orient Bio Inc. (Gyeonggi-Do, Korea). Two days before dosing, the
femoral artery and vein (intravenous [iv] only) were cannulated
using PE-26 tubing (Becton Dickinson, Lincoln Park, NJ, USA). For
intravenous administration, prepared dosing solution was injected
via the femoral vein. The rats were fasted overnight before drug
administration and until 6 h after dosing. For the po experiment, rats
(three in each group) were given a single dose of 5 mg/kg, and
heparinized samples of blood (0.3 ml) were collected at 5, 15, 30,
60, 90, 120, 180, 240, 360, 480, 600 min and 24 h post-dose. For
the iv experiment, rats (three in each group) were given a single
1 mg/kg dose, and blood samples were collected at 5, 10, 20, 40,
60, 90, 120, 180, 240, 360 and 480 min post-dose. Plasma was
harvested after centrifugation and stored frozen at ꢃ20 °C until ana-
lyzed. The concentrations of compounds in plasma were determined
by LC/MS/MS (API3200). The results are shown as the maximum
plasma concentration (Cmax), the time to reach peak plasma concen-
tration (Tmax), terminal half-life (t1/2) and the area under the plasma
concentration-time curve from zero to time infinity (AUC0–1). For
the BBB permeability assay, brains were taken out of the cranium
after heart perfusion with PBS at 30, 60, 120, 240 and 360 min.
points in duplicate at a range of concentrations up to 2500 lg/plate.
A response was considered to be positive in our criteria if there was
a dose-dependent increase in revertants per plate resulting in at
least a doubling of the background reversion rate for strains TA 100.
5.12. hERG assay
A three pulse protocol was applied by stepping from a holding
potential of ꢃ80 mV to +40 mV for 2 s to inactivate hERG channels.
The membrane voltage was then stepped back to ꢃ50 mV for 2 s to
evoke a tail current prior to returning to the holding potential for
1 s. This sequence was repeated a further two times. This voltage
protocol was applied prior to drug treatment (pre-compound)
and after 600 s in the presence of the drug (post-compound).
5.10. Cytochrome P450 inhibition assay
Incubation mixtures consisted of 0.2 mg human liver micro-
somes, GCC2680 (0.2, 1, 2, 10 and 20
lM), substrate mixture and
Acknowledgment
an NADPH-generating system (0.1 M glucose-6-phosphate, 10 mg/
ml b-NADP+ and 1 U/ml glucose-6-phosphate dehydrogenase) in a
total volume of 200 lL potassium phosphate buffer (0.1 M, pH
We are grateful to Dr. Eun Chul Huh for his leadership at
Research Center, Green Cross Corp. (GCC).
7.4). Reactions were initiated by the addition of NADPH-generating
system and continued in a water bath at 37 °C. After the incubation
Supplementary data
for 30 min, the reaction was stopped by addition of 400
acetic acid, and then internal standard was added (16 M terfena-
dine). Two volumes of acetonitrile were added to each sample
lL of 0.1%
l
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.07.013. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
and mixed. After centrifugation, the supernatants (5
injected onto an HPLC column for LC/MS/MS analyses.
lL) were

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J. Lee et al. / Bioorg. Med. Chem. 18 (2010) 6377–6388
20. Kang, S. Y.; Lee, S.-H.; Seo, H. J.; Jung, M. E.; Ahn, K.; Kim, J.; Lee, J. Bioorg. Med.
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