Chemical and biological investigation of cyclopropyl containing diaryl-pyrazole-3-carboxamides as novel and potent cannabinoid type 1 receptor antagonists

Article abstract of DOI:10.1021/jm900179y

Obesity is a major clinical problem in the western world, and many molecular targets have been explored in the search for effective therapeutic agents. One of these, antagonism of the cannabinoid 1 (CB1) receptor, rose to prominence following reports demo

Full text of DOI:10.1021/jm900179y

J. Med. Chem. 2009, 52, 4329–4337 4329
DOI: 10.1021/jm900179y
Chemical and Biological Investigation of Cyclopropyl Containing Diaryl-pyrazole-3-carboxamides as
Novel and Potent Cannabinoid Type 1 Receptor Antagonists
,†
Gyorgy Szabo,* Balazs Varga, Dora Payer-Lengyel, Attila Szemzo, Peter Erdelyi, Krisztina Vukics, Judit Szikra,
‡
†
†
†
†
‡
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Eva Hegyi, Monika Vastag, Bela Kiss, Judit Laszy, Istvan Gyertyan, and Janos Fischer
‡
‡
‡
‡
‡
†
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
†Medicinal Chemistry Research Laboratory IV, Gedeon Richter Plc, PO Box 27, H-1475 Budapest 10, Hungary, and ‡Division of Drug Safety and
Pharmacology, Gedeon Richter Plc, PO Box 27, H-1475 Budapest 10, Hungary
Received February 12, 2009
Obesity is a major clinical problem in the western world, and many molecular targets have been explored
in the search for effective therapeutic agents. One of these, antagonism of the cannabinoid 1 (CB1)
receptor, rose to prominence following reports demonstrating the positive modulation of food intake by
the CB1 antagonist, rimonabant (3) (SR141716A). In the present study, various diaryl-pyrazole
derivatives containing cycloalkyl building blocks were synthesized and tested for CB1 receptor binding
affinities. Thorough structure-activity relationship (SAR) studies to optimize the pyrazole substituents
led to several novel CB1 antagonists with Ki e 5 nM and with acceptable metabolic stability with human
liver microsomes. Among these analogues, we identified 5-(4-cyclopropylphenyl)-1-(2,4-dichlorophe-
nyl)-4-ethyl-N-pyrrolidin-1-yl-1H-pyrazole-3-carboxamide (11r), which exhibited a favorable pharma-
cological profile with outstanding efficacy in reducing serum lipid parameters of metabolic syndrome
compared to clinical references.
Introduction
the monaminergic reuptake inhibitor sibutramine 1 and the
lipidase inhibitor orlistat 2 (Figure 1).5
According to the World Health Organization (WHOa)
Global InfoBase, 78% of the population of United States
and 73% of the United Kingdom is classified as obese or
overweight. Moreover, it is not only western society that is
affected, as the prevalence of obesity has increased worldwide
by 75% since 1980 and the WHO have consequently defined it
as a global epidemic.1 The importance of the obesity epidemic
is emphasized by the fact that excessive bodyweight is now
recognized as one of the most important risk factors asso-
ciated with the metabolic syndrome.2 Metabolic syndrome
isa dangerouscombinationofrisk factors: abdominalobesity,
glucose intolerance, hypertension, and dyslipidemia (low
high-density lipoprotein cholesterol (HDLc) but high low-
density lipoprotein cholesterol (LDLc) and triglyceride
levels),3 which are responsible for increased prevalence of
cardiovascular diseases, diabetes, and some cancers, so caus-
ing more than 1 million deaths and 12 million life years of ill
health each year in Europe alone.4
Both compounds have only modest efficacy and are accom-
panied by severe adverse effects.
The main side effect of 1 is elevation of blood pressure,
which is highly undesirable in obese patients, with current
hypertension and/or dyslipidemia.
Orlistat (2), a gastrointestinal lipase inhibitor, has a rather
inconvenient side effect profile characterized by steatorrhea
(lipid evacuation) and fecal incontinence, the prevalence of
which can be up to 30% among patients.
Numerous attempts have been made to identify new po-
tential targets and thus create novel types of antiobesity drugs.
One of these, the discovery of the endocannabinoid system, in
the 1990s, has provided one opportunity to develop and
introduce efficient pharmaceuticals for the treatment of
obesity. In general, cannabinoids exert their effects thro-
ugh two known receptors: CB1, a Gi/o protein coupled
receptor that inhibits neurotransmitter release on axon
terminals in the central nervous system (CNS) but also
can be found in peripheral organs, (e.g., gut, liver, and also
on cells of adipose tissue), and cannabinoid 2 (CB2), which
suppresses immounocytes and exerts little if any effects on
the CNS.6
In spite of the growing awareness of the medical problems
that accompany obesity, current treatment strategies (dietary
modifications, lifestyle change, and drug therapy) cannot be
considered to be optimal and sustained weight loss is often
difficult to achieve.
The endocannabinoid system, and the CB1 receptor
(cloned in 1990)7-9 in particular, plays a special role in en-
ergy homeostasis. Blockade of CB1 receptors decreases food
intake, leadingtoareductioninbody weight. Therefore, itwas
hoped that CB1 receptor antagonists/inverse agonists could
provide effective therapies for the treatment of obesity.
Indeed, in phase III clinical studies, Rimonabant10 (3)
(SR141716A), the first potent and selective CB1 receptor
inverse agonist, induced modest weight loss and had
preferential effects in many parameters of dyslipidemia
Besides the highly abusable norepinephrine stimulants,
there are only two antiobesity pharmaceuticals on the market:
*To whom correspondence should be addressed. Phone: þ36-1-431-
5180. Fax: þ36-1-432-6498. E-mail: gy.szabo@richter.hu.
a Abbreviations: BMI, body mass index; WHO, World Health Orga-
nization; CB1, cannabinoid 1; CB2, cannabinoid 2; CLint, intrinsic
clearance; CNS, central nervous system; SAR, structure-activity rela-
tionship; DIO, diet induced obesity; LDLc, low-density lipoprotein
cholesterol; HDLc, high-density lipoprotein cholesterol; EMEA, Eur-
opean Medicines Agency.
r
2009 American Chemical Society
Published on Web 06/15/2009
pubs.acs.org/jmc

4330 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Szaboꢀ et al.
refluxing toluene. Reaction of these intermediates with
commercially available 1-aminopiperidine or 1-aminopyrro-
lidine in dichloromethane at room temperature provided the
target compounds 11a-r.
1-(4-Cyclopropylphenyl)-2-methoxyethanone (7f) cannot
be prepared by a direct Friedel-Crafts reaction as described
in Scheme 1 because of the chloromethylation property of
methoxyacetyl chloride and the formation of diarylmethane.21
7f was synthesized from 2-methoxy-1,1-bis(trimethylsilyloxy)
ethene (140 and 4-cyclopropylbenzenecarboxylic acid chloride
(13) by the method of Wissner (Scheme 2).22
Figure 1. Structures of sibutramine and orlistat.
Results and Discussion
The present synthetic design was based on the principles of
analogue based drug discovery described by Fischer et al.23
Using this approach, we synthesized several diaryl-pyrazoles
11a-r. Our strategy in evaluating our newly synthesized
compounds was first to determine their in vitro binding
affinities and then their metabolic stabilities characterized by
microsomal intrinsic clearance. Considerable information is
available on the mechanisms of action of diaryl-pyrazoles, but
their pharmacokinetic properties are poorly understood. Since
only one reference has been published on the metabolism of
diaryl-pyrazoles,24 we considered that it was necessary to study
the in vitro metabolism of 3 in detail. Our results showed that in
human and mouse liver microsomes 3 has weak/moderate
metabolic stability (for details, see Table 1). We therefore
believed that potent and in vivo effective, metabolically stable
diaryl-pyrazole analogues might achieve a remarkable im-
provement in the effectiveness of these compounds.
First, by replacing the chloro-substituent of 3 at position 5
by a cyclopropyl group, we synthesized its direct analogue 11a
with good affinity (Ki=3 nM). Unfortunately, this had similar
metabolic instability to 3. Subsequently, we found that com-
pounds containing a cyclobutyl 11b, cyclopentyl 11c, or
cyclohexyl 11d moiety instead of the cyclopropyl group
showed both a significant in vitro CB1 affinity and high
metabolic stability. Nevertheless, they were not effective per
os (po) in the WIN 55,212-2 induced hypothermia test (11b,
ED50=4 mg/kg, 11c, ED50=.3 mg/kg, 11c, ED50=>3 mg/
kg) (for details, see Experimental Section). This unfavorable
result is believed to be due to the poor in vivo absorp-
tion of these compounds, possibly arising from their
high lipophilicity (11b, clogP=7.25, 11c, clogP=7.81, 11d,
clogP=8.37).
The best compound 11a from the cycloalkyl series was
further optimized by changing the substituents R2 and R3 at
position 1 and the R1 substituent at position 4 (Table 1).
Removal of the 2-chlorine and both the chlorine atoms from
position 1 resulted in a drastic drop in efficacy for compound
11o and 11m (Table 1). However, by replacing the 4-chlorine
atom by a bromine atom at position 1, 11q enhanced meta-
bolic stability.
We made some changes at position 4 on the pyrazole ring
such as 4-ethyl-derivative 11p and replacement of the piper-
idine ring at position 3 of 11p with a pyrrolidine moiety 11r.
These changes gave compounds whose efficacies and meta-
bolic stabilities were further improved. On the basis of their
favorable in vitro binding and metabolic stability properties,
11p, 11q, and 11r were selected for further in vivo evaluation.
To determine the in vivo CB1 antagonist efficacy, we used
the CB1 agonist induced hypothermia model as described by
Fox et al.25 In this model, 3 inhibited WIN 55,212-2 induced
Figure 2. Structures of CB1R antagonists/inverse agonists.
(it increased HDLc and adiponectin, decreased trigly-
ceride levels, but did not change total cholesterol, LDLc,
and fasting insulin levels).11 However, one year after its
launch in Europe, its marketing has been discontinued
by the European Medicines Agency (EMEA) because
“benefits of Acomplia no longer outweigh its risks”;such
as depression and anxiety.12,13 Other CB1 antagonists/
inverse agonists were also withdrawn from clinical devel-
opment including taranabant14 (4) (MK-0364) and otena-
bant15,16 (5) (CP-945,598) (Figure 2).
Herein, we describe the design strategies and SAR studies
that led to the identification of a series of cyclopropyl contain-
ing diaryl-pyrazole-3-carboxamide derivatives17,18 with sig-
nificant antiobesity effects in the applied animal model. In
addition, in view of the clinical evidence that obesity is often
associated with lipoprotein abnormalities,3 we have investi-
gated the effect of 3 and our best compounds on serum lipids,
i.e., LDLc and HDLc levels, that resulted in the discovery of
11r (Figure 2), a novel, potent CB1 antagonist with a greater
preferential effect on the lipid profile parameters as compared
to known CB1 antagonists.
Chemistry. A series of new compounds 11a-r based on the
structural similarities with 3 were synthesized as shown in
Scheme 1. Reaction of cycloalkyl-benzenes 6a-d with the
appropriate acyl-chloride in trichloroethylene provided phe-
nones 7a-g. Claisen condensation of 7a-g and imidazol-1-
yl-oxo-acetic acid ethyl ester19 then gave diketone esters
8a-g in good yields. The synthesis of acids 10a-g involved
basic hydrolysis of the corresponding esters 9a-g, which in
turn were prepared by condensing diketone esters 8a-g with
suitably substituted phenyl hydrazines. This reaction is a
regioselective transformation and the 1,5-diarylpyrazoles
could be generated almost exclusively by carrying out the
condensation in the presence of the hydrochloride salt of the
phenyl-hydrazines.20 The intermediates 10a-g were then
converted into their acid chlorides with thionyl chloride in

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4331
Scheme 1. Synthesis of Target Compoundsa
a Reagents and conditions: (a) R1CH2COCl, AlCl3, trichloroethylene, -50°C; (b) LiHMDS, THF, Imidazol-1-yl-oxo-acetic acid ethyl ester, -70°C
f rt, 1 N HCl; (c) For 9a-f, 9p, and 9r 2,4-dichlorophenylhydrazine hydrochloride, 9g 2,4-difluorophenylhydrazine hydrochloride, 9h
4-fluorophenylhydrazine hydrochloride, 9i 2-chlorophenylhydrazine hydrochloride, 9j 4-fluorophenylhydrazine hydrochloride, 4-bromo-2-chlorophe-
nylhydrazine hydrochloride, 9k 2-fluorophenylhydrazine hydrochloride, 9l phenylhydrazine hydrochloride, 9n 4-chloro-2-fluorophenylhydrazine
hydrochloride, 9o 4-chlorophenylhydrazine hydrochloride, 9q 4-bromo-2-chlorophenylhydrazine hydrochloride, EtOH, reflux; (d) 2.5 N KOH, MeOH,
reflux; (e) (COCl)2, DMF, CH2Cl2 rt; (f) for 11a-l, 11n-q, 1-aminopiperidine, for 11m, 11r, 1-aminopyrrolidine, CH2Cl2, rt.
Scheme 2. Synthesis of Intermediate 7fa
a Reagents and conditions: (a) CH3COCl, AlCl3, trichloroethylene, -50 to (-25 °C); (b) Br2, NaOH, dioxan, reflux; (c) SOCl2, DMF, toluene, 80°C;
(d) SnCl4, rt.
hypothermia with an ED50 value of 0.41 mg/kg per os.
Compounds 11p, 11r, and 11q also attenuated the hypother-
mia with an ED50 values of 0.84, 0.84, and 0.22 mg/kg,
respectively, confirming that all are potent CB1 antagonists
in vivo (Figure 3).
As decreased food intake is one of the most important
elements of CB1 antagonist induced weight loss,26 we tested
our compounds for appetite suppressive efficacy in the fasting
induced palatable chow intake test.27 All compounds de-
creased food intake at the 10 mg/kg screen dose (11p=47%;
11r=49%; 11q=54%) with efficacy comparable to that of 3
(50%) (Table 2).
The diet-induced obesity (DIO) model is known to be a
pharmacologically relevant model of human obesity.28,29 In
good agreement with the earlier findings of Ravinet-Trillou,27
we have shown that 3 can decrease DIO (Figure 4). Using this
assay, we assessed the weight decreasing efficacy of our
compounds at three doses. All compounds dose dependently
decreased body weight with similar efficacy to 3 (Table 3). In
spite of the lack of differences in weight loss data, we found

4332 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Szaboꢀ et al.
Table 1. Entries, Binding Affinities,a and Metabolic Stabilitiesb of Compounds 11a-r
intrinsic clearance CLint (mL ꢀ min-1 ꢀ g liver-1
)
entry
R
R1
R2
R3
n
CB1R affinity Ki ( SEM (nM)
mouse
human
3
Cl
cyclopropyl
cyclobutyl
Me
Me
Me
Me
Me
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
1
6 ( 0.8
3 ( 0.4
7 ( 0.9
15 ( 4
3.22
3.28
0.41
0.68
0.01
11.60
11.60
0.90
NDc
9.71
3.20
3.81
1.82
ND
1.09
0.87
0.26
0.16
0.01
0.20
0.60
0.41
ND
0.60
0.41
0.20
0.20
ND
0.21
ND
0.60
0.01
1.04
11a
11b
11c
11d
11e
11f
11g
11h
11i
cyclopentyl
cyclohexyl
13 ( 2
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
16 ( 0.7
3 ( 0.2
34 ( 3
190 ( 36
4 ( 0.4
8 ( 0.8
11 ( 0.8
66 ( 4
121 ( 13
14 ( 0.5
79 ( 1
4 ( 0.7
3 ( 0.6
4 ( 0.2
MeO
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
F
H
Cl
Cl
F
H
11j
F
11k
11 L
11m
11n
11o
11p
11q
11r
H
H
H
H
F
H
Cl
Cl
Cl
Br
Cl
3.40
ND
H
Cl
Cl
Cl
1.10
0.80
1.50
Me
Et
a Affinity of compounds for the CB1 receptor was evaluated using rat cerebellum membrane preparation and [3H]SR141716A. Ki values were
obtained from three independent experiments. b Intrinsic clearance (CLint) was determined as a rate of compound consumption under first-order kinetic
conditions using mouse and human liver microsomes. c Not determined
that our compounds were surprisingly able to lower the
obesity-induced elevation of serum cholesterol levels, while
3, in line with the literature data in mouse tests,27,30 was
ineffective on this variable (p=0.99 vs fat control). 11r was
even more effective in this regard as it decreased serum
cholesterol values to the level of the lean control (p=0.49 vs
lean control). For details, see Table 4.
To investigate the importance of the preferential effects of
compound 11r on the lipid profile parameters, we compared it
face to face with three CB1 antagonists 3, 4, and 5, known to
have reached phase III clinical trials (Table 5). Doses were
selected to favor a similar weight loss efficacy. Indeed, all four
compounds induced a similar weight loss compared to fat
control, but there was no statistical difference between them.
DIO was accompanied by a highly significant elevation in
serum glucose, triglyceride, cholesterol, HDLc, and LDLc
levels. Decrease in fasting glucose and triglyceride levels were
nonsignificant. 11r was the only compound that could de-
crease cholesterol (p = 0.037) and LDLc levels (p=0.006).
None of the drugs changed HDLc levels significantly.
the three compounds had remarkable cholesterol-decreasing
efficacy and, importantly, 11r displayed improved effects on
lipid profile parameters compared to the known, clinically
relevant CB1 receptor antagonist/inverse agonist compounds.
Experimental Section
Chemistry. Research chemicals were either purchased from
Aldrich Co. or Fluka and used without further purification in
the reactions or were prepared according to the procedure
described in the literature. Reactions were monitored by thin-
layer chromatography (TLC) on silica gel plates (60 F254
;
Merck), visualizing with ultraviolet light or iodine. Column
chromatography was performed on Silica Gel 60 (0.043-0.060
mm), Merck. The main reference compounds were prepared
according to the literature procedures.10,14-16 The yields of the
products reported here are unoptimized. Melting points were
determined with a Buchi 535 apparatus and are uncorrected.
1H NMR spectra were recorded on a Varian INOVA-500
spectrometer, operating at 500 MHz. Chemical shifts (δ) are
reported in parts per million downfield from tetramethylsilane.
Mass spectra were scanned on a Finnigan MAT 95SQ spectro-
meter. Purity was verified using a Merck-Hitachi Lachrom
Elite HPLC system. A linear gradient using water and 0.1%
TFA (solvent A) and acetonitrile and 0.1% TFA (solvent B);
t=0 min, 10% B, t=18 min, 64% B, t=20 min, 64% B, t=
22 min, 72% B (25 min) was employed on Chromolith RP18e
(4.6 mmꢀ100 mm) column. Flow rate was 2 mL/min, and UV
detection (diode array detector: Merck-Hitachi Lachrom Elite
L-2450) was set to 254 nM. The LC column was maintained at
40 °C temperature. The purity of the compounds showed g95%
purity for all the synthesized compounds.
Conclusion
In the present study, we investigated a series of cyclopropyl
containing diaryl-pyrazole-3-carboxamide derivatives as
antagonists of the CB1 receptor. Several compounds in this
series exceeded the potency of 3. In addition, 11p, 11q, and 11r
achieved good metabolic stability. Further evaluation of these
compounds identified potent CB1 receptor antagonists with
significant antiobesity effects in the applied animal model. All

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4333
Table 2. Effect of 3, 11p, 11q, and 11r on the Food Intake in Fasted
Mice (n=8-10/group)
group
food intake (g ( SEM) difference from control (%)
control
3 (10 mg/kg)
2.06 ( 0.21
1.02 ( 0.12
50
49
48
54
control
11p (10 mg/kg)
1.91 ( 0.22
0.97 ( 0.06
control
11r (10 mg/kg)
1.89 ( 0.17
0.99 ( 0.13
control
11q (10 mg/kg)
2.19 ( 0.16
1.00 ( 0.12
Figure 4. Body weight decreasing effect of 3 at 3-30 mg/kg po dose
range in diet induced obesity model (DIO) in mice (n = 8/group).
dichloromethane (278 mL), oxalyl chloride (3.8 mL, 0.043
mol) was added dropwise with cooling. The mixture was stirred
for 2 h at room temperature and then evaporated in vacuo and
dissolved in dichloromethane (80 mL). The so-obtained sol-
ution was added dropwise to a stirred mixture of N-amino-
piperidine (3.4 mL, 0.032 mol) and triethyl amine (4.5 mL,
0.032 mol) in dichloromethane (150 mL) at 0-5 °C. The
resulting mixture was allowed to warm to ambient temperature
and stirred for 14-16 h then evaporated in vacuo. The residue
was purified by column chromatography using dichloro-
methane/ethyl acetate as eluent. Yield: 8.6 g (80%); 95.7%
1
purity by HPLC. H NMR (300 MHz, DMSO-d6): 0.62-0.66
m (2H), 0.92-1.02 m (2H), 1.31-1.41 m (2H), 1.54-1.64 m
(4H), 1.86-1.96 m (1H), 2.20 s (3H), 2.76-2.83 m (4H), 7.06 s
(4H), 7.54 dd (J=8.6, 2.3 Hz, 1H), 7.71 d (J=8.6 Hz, 1H), 7.78 d
(J=2.3 Hz, 1H), 9.01 s (1H). FIB-MS 468 [M þ H]þ.
5-(4-Cyclobutylphenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-pi-
peridin-1-yl-1-H-pyrazole-3-carboxamide hydrochloride (11b).
This compound was synthesized according to the procedure
described for 11a using 4-cyclobutylbenzene in step “a”, which
can be prepared by a known method31 starting from commer-
cially available 2-phenyl-1,4-butanediol. Yield: 65%; 96.2%
1
purity by HPLC. H NMR (500 MHz, DMSO-d6): 1.90-2.01
m (1H), 1.99-2.11 m (2H), 2.21-2.29 m (1H), 2.27 s (3H), 3.28-
3.41 m (4H), 3.44-3.55 m (1H), 7.13-7.18 m (2H), 7.23-7.27 m
(2H), 7.59 dd (J=8.5, 2.3 Hz 1H), 7.77 d (J=8.5 Hz, 1H), 7.79 d
(J=2.3 Hz, 1H). EI-MS 482 [M þ H]þ.
Figure 3. Effect of 3, 11p, 11q, and 11r on WIN 55,2121-2 (3 mg/kg
ip) induced hypothermia test in mice (n=8/group). *=difference
from VEH/WIN treated andþ= difference from VEH/VEH treated
control. ***/þþþ p < 0.001 **/þþ p < 0.01; */þ p < 0.05.
5-(4-Cyclopentylphenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-pi-
peridin-1-yl-1-H-pyrazole-3-carboxamide (11c). This compound
was synthesized according to the procedure described for 11a
using 4-cyclopentylbenzene in step “a”, which can be prepared
by a known method.32 Yield: 72%; 97.5% purity by HPLC.
5-(4-Cyclopropylphenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-pi-
peridin-1-yl-1-H-pyrazole-3-carboxamide (11a). To a stirred sus-
pension of 10a (8.9 g, 0.021 mol) and DMF (0.36 mL) in

4334 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Szaboꢀ et al.
1H NMR (500 MHz, DMSO-d6):1.44-1.56m (4H), 1.57-1.67 m
(2H), 1.70-1.79 m (2H), 1.84-1.92 m (4H), 1.95-2.04 m (2H),
2.30 s (3H), 2.89-3.01 m (1H), 3.39-3.51 m (4H), 7.13-7.18 m
(2H), 7.26-7.30 m (2H), 7.59 dd (J=8.6, 2.3 Hz, 1H), 7.78 d
(J=8.6 Hz 1H), 7.79 d (J=2.3Hz,1H), 11.60 brs (1H). EI-MS496
[M þ H]þ.
5-(4-Cyclohexylphenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-piper-
idin-1-yl-1-H-pyrazole-3-carboxamide (11d). This compound was
synthesized according to the procedure described for 11a using
commercially available 4-cyclohexylbenzene in step “a”. Yield:
Table 3. Effect of 3, 11p, 11q, and 11r on the Body Weight of DIO Mice
after 2 Weeks Treatment (n = 8/group)
body weight at day14
(g ( SEM)
difference from
fat control (%)a
1
80%; 96.4% purity by HPLC; H NMR (500 MHz, DMSO-d6):
group
1.16-1.26 m (1H), 1.29-1.41 m (4H), 1.46-1.56 m (2H), 1.64-1.73
m (1H), 1.73-1.81 m (4H), 1.82-1.88 m (4H), 2.28 s (3H), 2.43-
2.50 m (1H), 3.34-3.48 m (4H), 7.12-7.17 m (2H), 7.21-7.28 m
(2H), 7.58 dd (J = 8.6, 2.3 Hz, 1H), 7.76 d (J = 8.6 Hz, 1H), 7.79 d
(J=2.3 Hz, 1H), 11.38 br s (1H). EI-MS 510 [M þ H]þ.
b
fat control
40.07 ( 1.41þþþ
3 (3 mg/kg)
3 (10 mg/kg)
3 (30 mg/kg)
lean control
36.46 ( 1.47þþþ
33.87 ( 0.63***;þþþ
30.28 ( 0.81***;þþþ
25.02 ( 0.42***
9
15
24
38
5-(4-Cyclopropylphenyl)-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-
1-H-pyrazole-3-carboxamide Hydrochloride (11e). This compound
was synthesized according to the procedure described for 11a using
acetyl chloride in step “a”. Yield: 56%; 96.7% purity by HPLC. 1H
NMR (500 MHz, DMSO-d6): 0.64-0.69 m (2H), 0.91-0.98 m
(2H), 1.42-1.51 m (2H), 1.76-1.83 m (4H), 1.84-1.92 m (1H),
3.25-3.35 m (4H), 7.03-7.08 m (2H), 7.10-7.15 m (2H), 7.24 s
(1H), 7.65 dd (J=8.6,2.3Hz,1H),7.81d(J=8.6 Hz, 1H), 7.87 d (J=
2.3 Hz, 1H), 11.21 s (1H). EI-MS 454 [M þ H]þ.
fat control
45.31 ( 0.89þþþ
41.55 ( 1.56þþþ
38.47 ( 1.30***;þþþ
35.51 ( 0.85***;þþþ
28.16 ( 0.59***
11p (3 mg/kg)
11p (10 mg/kg)
11p (30 mg/kg)
lean control
8
15
22
38
fat control
44.00 ( 1.13þþþ
40.48 ( 1.65þþþ
37.53 ( 0.76***;þþþ
34.57 ( 1.06***;þþþ
28.75 ( 0.62***
11r (3 mg/kg)
11r (10 mg/kg)
11r (30 mg/kg)
lean control
8
5-(4-Cyclopropylphenyl)-1-(2,4-dichlorophenyl)-4-methoxy-N-
piperidin-1-yl-1-H-pyrazole-3-carboxamide (11f). This compound
was synthesized according to the procedure described for 11a
starting from 7f in step “b”. Yield: 32%; 96.5% purity by HPLC.
1H NMR (500 MHz, DMSO-d6) 0.70-0.63 (m, 2H), 0.97-0.90
(m, 2H), 1.39-1.29 (m, 2H), 1.63-1.52 (m, 4H), 1.91-1.82 (m,
1H),2.84-2.73 (m, 4H), 3.75 (s, 3H), 7.13-7.02 (m, 4H), 7.59 (dd,
J=8.5, 2.3 Hz, 1H), 7.75 (d, J=8.5 Hz, 7.78 (d, J=2.3 Hz, 1H),
9.03 (s, 1H). EI-MS 484 [M þ H]þ.
15
21
35
fat control
43.73 ( 0.83þþþ
39.67 ( 1.28*;0þþþ
35.61 ( 1.09***;þþþ
32.38 ( 0.84***;þþþ
26.22 ( 0.51***
11q (3 mg/kg)
11q (10 mg/kg)
11q (30 mg/kg)
lean control
9
19
26
40
5-(4-Cyclopropylphenyl)-1-(2,4-difluorophenyl)-4-methyl-N-pi-
peridin-1-yl-1H-pyrazole-3-carboxamide (11g). This compound
was synthesized according to the procedure described for 11a
using commercially available 2,4-difluoro-phenylhydrazine hy-
drochloride in step “c”. Yield: 77%; 98.7% purity by HPLC. 1H
NMR (500 MHz, DMSO-d6): 0.65-0.70 m (2H), 0.91-0.98 m
(2H), 1.28-1.37 m (2H), 1.54-1.63 m (4H), 1.84-1.92 m (1H),
2.19 s (3H), 2.72-2.82 m (4H), 7.03-7.08 m (4H), 7.19-7.25 m
(1H), 7.35-7.42 m (1H), 7.66-7.73 m (1H), 9.02 s (1H). EI-MS
436 [M þ H]þ.
a Percent difference from control values were calculated as follows: %
diff = 100 ꢀ (control - groupX)/control. b * = difference from fat and
þ = difference from lean control. ***/þþþ p < 0.001 **/þþ p < 0.01;
*/þ p < 0.05.
Table 4. Effect of 3, 11p, 11q, and 11r on the Serum Cholesterol Level of
DIO Mice after 2 Weeks Treatment (n=5-6/group)
serum cholesterol
(mmol/mL ( SEM)
difference from
fat control (%)
group
a
fat control
3 (30 mg/kg)
lean control
4.47 ( 0.19þþþ
5-(4-Cyclopropylphenyl)-1-(4-fluorophenyl)-4-methyl-N-piperi-
din-1-yl-1H-pyrazole-3-carboxamide (11h). This compound was
synthesized according to the procedure described for 11a using
commercially available 4-fluoro-phenylhydrazine hydrochloride
in step “c”. Yield: 80%; 96.1% purity by HPLC. 1H NMR (400
MHz, CDCl3): 0.68-0.74 m (2H), 0.97-1.04 m (2H), 1.40-1.50
m (2H), 1.74-1.83 m (4H), 1.83-1.93 m (1H), 2.33 s (3H), 2.91-
3.01 m (4H), 6.95-7.09 m (6H), 7.16-7.23 m (2H), 7.83 br s (1H).
EI-MS 418 [M þ H]þ.
4.47 ( 0.18þþþ
2.70 ( 0.17***
0
40
fat control
11p (30 mg/kg)
lean control
5.49 ( 0.10þþþ
4.46 ( 0.33*;þþþ
2.44 ( 0.10***
19
56
fat control
11r (30 mg/kg)
lean control
5.12 ( 0.17þþþ
3.27 ( 0.30***
2.90 ( 0.19***
36
43
5-(4-Cyclopropylphenyl)-1-(2-chlorophenyl)-4-methyl-N-piperi-
din-1-yl-1H-pyrazole-3-carboxamide (11i). This compound was
synthesized according to the procedure described for 11a using
commercially available 2-chloro-phenylhydrazine hydrochlo-
ride in step “c”. Yield: 81%; 98.8% purity by HPLC. 1H NMR
(400 MHz, CDCl3): 0.64-0.69 m (2H), 0.92-0.99 m (2H), 1.38-
1.47 m (2H), 1.71-1.79 m (4H), 1.79-1.87 m (1H), 2.37 s (3H),
fat control
11q (30 mg/kg)
lean control
5.56 ( 0.24þþþ
4.66 ( 0.14*;þþþ
2.87 ( 0.24***
16
48
a * = difference from fat and þ= difference from lean control. ***/
þþþ p < 0.001 **/þþ p < 0.01; */þ p < 0.05
Table 5. Effect of 3, 4, 5, and 11r on the Body Weight, Serum Glucose, and Lipid Levels of DIO Mice after 2 Weeks Treatment (n = 8/group)
serum levels (mmol/mL)
group
body weights at ay 14 (g ( SEM)
glucose
cholesterol
triglyceride
HDLc
LDLc
a
fat control
41.90 ( 1.30þþþ
9.52 ( 0.38þþþ 5.35 ( 0.12þþþ 1.42 ( 0.04þþ
4.42 ( 0.08þþþ 0.84 ( 0.03þþþ
4.4 ( 0.25þþþ 0.82 ( 0.05þþþ
3 (10 mg/kg)
4 (3 mg/kg)
5 (30 mg/kg)
11r (10 mg/kg)
lean control
35.60 ( 1.10***þþþ
33.70 ( 0.70***þþþ
36.60 ( 1.10***þþþ
35.60 ( 0.90***þþþ
26.10 ( 0.90***
8.84 ( 0.65þþ
9.11 ( 0.49þþ
9.45 ( 0.71þþ
8.79 ( 0.51þ
5.35 ( 0.32þþþ 1.18 ( 0.06
5.01 ( 0.13þþþ 1.44 ( 0.05þþþ 4.16 ( 0.10þþþ 0.82 ( 0.03þþþ
5.34 ( 0.13þþþ 1.32 ( 0.04þ
4.56 ( 0.20 þþþ 1.30 ( 0.09
4.40 ( 0.11þþþ 0.85 ( 0.06þþþ
3.90 ( 0.16þþþ 0.68 ( 0.04** þþþ
6.08 ( 0.42***
2.75 ( 0.08***
1.03 ( 0.07***
2.34 ( 0.07***
0.42 ( 0.02***
a * = difference from fat and þ = difference from lean control. ***/þþþ p < 0.001 **/þþ p < 0.01; */þ p < 0.05.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4335
2.83-2.89 m (4H), 6.93-7.02 m (4H), 7.25-7.35 m (3H), 7.37-
7.7.43 m (1H), 7.68 s (1H). EI-MS 434 [M þ H]þ.
was synthesized according to the procedure described for 11a using
commercially available 4-bromo-2-chloro-phenylhydrazine hydro-
chloride in step “c”. Yield: 81%; 99.1% purity by HPLC. 1H NMR
(400 MHz, CDCl3): 0.65-0.73 m (2H), 0.93-1.01 m (2H), 1.39-
1.46 m (2H), 1.70-1.78 m (4H), 1.79-1.87 m (1H), 2.35 s (3H),
2.81-2.91 m (4H), 6.99 m (4H), 7.18 d (J=8.5 Hz, 1H), 7.41 dd (J=
8.5, 2.2 Hz, 1H), 7.57 d (J=2.2 Hz, 1H), 7.63 s (1H). EI-MS 512
[M þ H]þ.
5-(4-Cyclopropylphenyl)-1-(2-chloro-4-fluorophenyl)-4-methyl-
N-piperidin-1-yl-1H-pyrazole-3-carboxamide (11j). This com-
pound was synthesized according to the procedure described
for 11a using commercially available 4-fluoro-2-chloro-phenyl-
hydrazinehydrochloridein step“c”. Yield: 76%; 96.1% purity by
HPLC. 1H NMR (500 MHz, DMSO-d6): 0.64-0.72 m (2H),
0.90-0.98 m (2H), 1.29-1.38 m (2H), 1.52-1.62 m (4H), 1.84-
1.90 m (1H), 2.20 s (3H), 2.74-2.81 m (4H), 7.03-7.09 m (4H),
7.35 ddd (J=8.8, 8.3, 2.8 Hz, 1H), 7.57 dd (J=8.5, 2.8 Hz, 1H),
7.75 dd (J=8.8, 5.6 Hz, 1H), 8.99 s (1H). EI-MS 452 [M þ H]þ.
5-(4-Cyclopropylphenyl)-1-(2-fluorophenyl)-4-methyl-N-piperi-
din-1-yl-1H-pyrazole-3-carboxamide (11k). This compound was
synthesized according to the procedure described for 11a using
commercially available 2-fluoro-phenylhydrazine hydrochloride
in step “c”. Yield: 66%; 94.8% purity by HPLC. 1H NMR (400
MHz, CDCl3): 0.65-0.71 m (2H), 0.93-1.00 m (2H), 1.38-1.48
m (2H), 1.71-1.79 m (4H), 1.80-1.89 m (1H), 2.36 s (3H), 2.83-
2.90 m (4H), 6.95-7.02 m (4H), 7.02-7.08 m(1H), 7.14-7.20 m
(1H), 7.30-7.40 m (2H), 7.68 br s (1H). EI-MS 418 [M þ H]þ.
5-(4-Cyclopropylphenyl)-1-phenyl-4-methyl-N-piperidin-1-yl-1H-
pyrazole-3-carboxamide (11l). This compound was synthesized
according to the procedure described for 11a using commercially
available phenylhydrazine hydrochloride in step “c”. Yield: 91%;
98.1% purity by HPLC. 1H NMR (400 MHz, CDCl3): 0.67-0.74
m (2H), 0.95-1.03 m (2H), 1.41-1.50 m (2H), 1.74-1.84 m (4H),
1.84-1.92 m (1H), 2.34 s (3H), 2.91-3.04 m (4H), 6.98-7.04 m
(4H), 7.19-7.25 m (2H), 7.25-7.34 m (3H), 7.91 br s (1H). EI-MS
400 [M þ H]þ.
5-(4-Cyclopropylphenyl)-1-(2,4-dichlorophenyl)-4-ethyl-N-pyr-
rolidin-1-yl-1-H-pyrazole-3-carboxamide (11r). This compound
was synthesized according to the procedure described for 11a
using commercially available 1-aminopyrrolidine in step “e”.
Yield: 67%; 95.5% purity by HPLC. 1H NMR (500 MHz,
DMSO-d6): 0.64-0.67 m (2H), 0.91-0.97 m (2H), 1.07 t (J =
7.4 Hz, 3H), 1.71-1.79 m (4H), 1.82-1.92 m (1H), 2.62 q (J=
7.4 Hz, 2H), 2.85-2.94 m (4H), 7.03-7.09 m (4H), 7.53 dd (J=
2.3 Hz, 1H), 7.70 d (J = 2.3 Hz, 1H), 7.73 d (J = 2.3 Hz, 1H),
9.03 s (1H). FIB-MS 468 [M þ H]þ.
Preparation of 1-(4-Cyclopropylphenyl)-2-methoxyethanone
(7f). 2-Methoxy-1,1-bis(trimethylsilyloxy)ethene 14 and 4-cy-
clopropylbenzenecarboxylic acid (12) were prepared as
described in the literature.22,33 To a suspension of 17.4 g
(107 mmol) of 12 in 100 mL of anhydrous toluene, 15.6 mL
(213 mmol) of thionyl chloride and a catalytic amount of DMF
were added and the reaction mixture was stirred for 5 h at 80 °C.
After the reaction completed, the solvent and the excess of
thionyl chloride were evaporated and the residue was distillated
under reduced pressure. The corresponding acid chloride 13 as a
main fraction was collected between 42 and 48 °C at 0.032 mbar
(15.9 g, 82%). then 15.9 g of 13 was added to 20.6 g (88.0 mmol)
of 14 containing 3 drops of SnCl4 in one portion and the mixture
was stirred for 2 h and allowed to stand for overnight. The
reaction mixture was poured into 150 mL of 0.5 M aqueous HCl
and extacted with EtOAc. The organic phase was washed with
saturated aqueous NaHCO3 solution and brine, dried over
Na2SO4, and the solvent was removed by evaporation. The oily
residue was purified by flash chromatography, and the desired
compound was obtained in a poor yield: 3.14 g (19%), as a
yellowish, oily liquid. 95.8% purity by HPLC. 1H NMR
(400 MHz, CDCl3): 0.81-0.75 (m, 2H), 1.10-1.03 (m, 2H),
1.98-1.90 (m, 1H), 3.50 (s, 3H), 4.67 (s, 2H), 7.12 (ca.d, J=8.3,
2H), 7.82 (ca.d, J=8.3, 2H). EI-MS 190 [M þ H]þ.
5-(4-Cyclopropylphenyl)-1-phenyl-4-methyl-N-pirrolidin-1-yl-
1H-pyrazole-3-carboxamide (11m). This compound was synthe-
sized according to the procedure described for 11a using
commercially available 1-aminopyrrolidine in step “e”. Yield:
1
87%; 97.2% purity by HPLC. H NMR (400 MHz, CDCl3):
0.67-0.74 m (2H), 0.95-1.03 m (2H), 1.83-1.91 m (1H), 1.91-
1.98 m (4H), 2.35 s (3H), 3.03-3.16 m (4H), 6.98-7.05 m (4H),
7.20-7.25 m (2H), 7.25-7.34 m (3H), 7.86 br s (1H). EI-MS 386
[M þ H]þ.
5-(4-Cyclopropylphenyl)-1-(2-fluoro-4-chlorophenyl)-4-methyl-
N-piperidin-1-yl-1H-pyrazole-3-carboxamide (11n). This com-
pound was synthesized according to the procedure described
for 11a using commercially available 2-fluoro-4-chloro-phenyl-
hydrazinehydrochloridein step“c”. Yield: 84%; 95.4% purity by
HPLC. 1H NMR (400 MHz, CDCl3): 0.63-0.73 m (2H), 0.95-
1.03 m (2H), 1.39-1.46 m (2H), 1.72-1.79 m (4H), 1.80-1.88 m
(1H), 2.35 s (3H), 2.82-2.91 m (4H), 6.96-7.03 m (4H), 7.08 dd
(J=9.6, 2.2 Hz, 1H), 7.17 ddd (J=8.7, 2.2, 1.3 Hz, 1H), 7.31 dd
(J=8.7, 8.3 Hz, 1H), 7.65 s (1H). EI-MS 452 [M þ H]þ.
5-(4-Cyclopropylphenyl)-1-(4-chlorophenyl)-4-methyl-N-piperi-
din-1-yl-1H-pyrazole-3-carboxamide (11o). This compound was
synthesized according to the procedure described for 11a using
commercially available 4-chloro-phenylhydrazine hydrochloride
in step “c”. Yield: 79%; 96.4% purity by HPLC. 1H NMR
(500 MHz, DMSO-d6): 0.67-0.73 m (2H), 0.94-1.04 m (2H),
1.31-1.41 m (2H), 1.54-1.64 m (4H), 1.88-1.97 m (1H), 2.16 s
(3H), 2.75-2.84 m (4H), 7.05-7.14 m (4H), 7.25-7.31 m (2H),
7.42-7.48 m (2H), 9.02 s (1H). EI-MS 434 [M þ H]þ.
Biological Methods. In Vitro [3H]SR141716A Ligand Binding
atCB1 Receptors. Rat cerebellumwas removedandhomogenized
and a membrane preparation was made according to Thomas et
al.34 and Devane et al.35 and then stored at -80 °C until use.
A solution of 0.4 nM [3H]SR-141716A in 50 mM Tris-HCl
buffer complemented with 5 mM MgCl2, 1 mM EDTA, and
containing 20% ethanol was prepared freshly each day from a
stock solution (0.002 mCi/ml, in ethanol).
Incubation was performed in the above-mentioned buffer
complemented with 1 mg/mL bovine serum albumin (BSA) and
1 mM dithiothreitol (DTT) for 60 min at 30 °C in a thermo-
statted shaker. Incubation mixture (total volume of 1 mL)
contained 200 μg rat cerebellar membrane (thoroughly homo-
genized and kept vortexed under the whole preincubation
procedure) while the final radioligand concentration was 0.04
nM. Assay mixture was then filtered through Whatman GF/C
glass fiber filters (Brandel M48 harvester) and washed with 5 ꢀ
5 mL incubation buffer (without DTT). Filters were dried and
placed in scintillation vials, and 3.5 mL HiSafe scintillation
cocktail was added. Samples were left to stand overnight, and
radioactivity was determined in a Wallac 1409 scintillation
spectrometer. Nonspecific binding was determined in the pre-
sence of 1 μM unlabeled SR141716A.
5-(4-Cyclopropylphenyl)-1-(2,4-dichlorophenyl)-4-ethyl-N-pi-
peridin-1-yl-1-H-pyrazole-3-carboxamide (11p). This compound
was synthesized according to the procedure described for 11a
using butyryl chloride in step “a”. Yield: 68%; 95.3% purity by
1
HPLC. H NMR (400 MHz, DMSO-d6): 0.64-0.69 m (2H),
0.91-0.97 m (2H), 1.07 t (J=7.3 Hz, 3H), 1.30-1.38 m (2H),
1.52-1.65 m (4H), 1.82-1.92 m (1H), 2.62 q (J=7.3 Hz, 2H),
2.74-2.81 m (4H), 7.03-7.09 m (4H), 7.51 dd (J=8.6, 2.3 Hz,
1H), 7.71 d (J=8.6 Hz, 1H), 7.73 d (J=2.3 Hz, 1H), 9.01 s (1H).
FIB-MS 482 [M þ H]þ.
IC50 values were determined from displacement curves using
sigmoid fitting by Origin 6.0 software. Ki values were calculated
by the Cheng-Prusoff equation.36
In Vitro Metabolic Stability. Metabolic stability was as-
sessed in vitro using human, rat or mouse liver microsomes.
5-(4-Cyclopropylphenyl)-1-(2-chloro-4-bromophenyl)-4-methyl-N-
piperidin-1-yl-1H-pyrazole-3-carboxamide (11q). This compound

4336 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Szaboꢀ et al.
Test compounds at 2.5 μM were incubated for various lengths of
time (0-40 min) with human (XenoTech, USA) or CD1 mouse
(Richter) liver microsomes. CLint were calculated from the rate
of compound consumption.
WIN 55,212-2 Induced Hypothermia Test. CD1 mice, weigh-
ing 25-30 g, were kept in groups of 5. On the experimental day,
subjects received the test compound (antagonist) or vehicle
orally (WIN 55,212-2 was suspended in the mixture of 2%
Tween 80 and saline. Test compounds were suspended in the
mixture of 5% Tween 80 and distilled water).
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administered intraperitoneally, and rectal temperature was
measured 45 min later. Data were analyzed with One-Way
ANOVA and Tukey post hoc test.
Acute Appetite Suppressant Assay: Food Intake Test. Test
protocol has been derived from Wiley et al.37 but modified in
several ways in order to adapt to our in-house conditions.
CD1 mice weighing >25 g were kept isolated at least for
3 days before the test. Sixteen hours before measurement, chow
was removed from home cages (overnight fasting). The next day,
subjects were treated per os with vehicle (5% Tween 80 solution)
in their home cages. One hour later (pretreatment time), pre-
weighed palatable chow (Test Diet, no. 58 V8) was added to the
feeding boxes of home cages. After one hour, chow was
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excess body weight over lean control group (it takes usually
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habituation phase (daily treatment with tap water), mice were
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minalis or through plexus retroorbitalis. Immediately there-
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Acknowledgment. We are grateful to Dr. Derek R. Buckle
for his prudent proofreading this manuscript. We are also
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grateful to Zoltan Beni and Janos Koti for recording NMR
and MS spectra, Sandor Levai for analytical support, and
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(17) Szabo, Gy.; Fischer, J., Szemzo, A.; Erdelyi, P.; Varga, B.;
Gyertyan, I.; Szikra, J.; Vastag, M. Novel CB1 antagonists and
their preparation. Patent WO2008/075118 A1, 2008.
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Laura Orban, Ilona Simon, Eva Petenyi, and Zsuzsa Bali for
skillful technical assistance.
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(18) Szabo, Gy.; Vukics, K.; Payer-Lengyel, D.; Szemzo, A.; Erdelyi, P.;
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Varga, B.; Szikra, J.; Kurko, D.; Vastag, M.; Fischer, J.; Gyertyan,
I. New CB1 receptor antagonists: synthesis and pharmacological
investigation. Drugs Future 2008, 33, (Suppl. A), XXth Interna-
tional Symposium on Medicinal Chemistry, Aug 31-Sept 4,
Vienna, 2008; Poster 381.
Supporting Information Available: General synthesis and
experimental and spectroscopic (1H NMR and MS) details for
nonkey compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
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