Antiobesity designed multiple ligands: Synthesis of pyrazole fatty acid amides and evaluation as hypophagic agents

Article abstract of DOI:10.1016/j.bmc.2008.10.023

Searching for new antiobesity agents, a new series of fatty acid amide derivatives of 1,5-diarylpyrazole have been synthesized as dual peroxisome proliferator activated receptor alpha (PPARα)/cannabinoid receptor ligands. The compounds have been evaluated in vivo and in vitro as PPARα activators and as cannabinoids in two tests of the mouse tetrad. In vivo, food intake studies have been performed with all the compounds. No significant cannabinoid activity has been found but some compounds behaved as potent PPARα activators. Several compounds showed anorexigenic properties reducing food intake in rats.

Full text of DOI:10.1016/j.bmc.2008.10.023

Bioorganic & Medicinal Chemistry 16 (2008) 10098–10105
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antiobesity designed multiple ligands: Synthesis of pyrazole fatty acid
amides and evaluation as hypophagic agents
b
d
d
Mario Alvarado a, Pilar Goya a, , Manuel Macías-González , Francisco Javier Pavón , Antonia Serrano ,
*
Nadine Jagerovic a, Jose Elguero a, Angel Gutiérrez-Rodríguez c, Santiago García-Granda c,
Margarita Suardíaz d, Fernando Rodríguez de Fonseca d,
a Instituto de Química Médica, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain
*
b CIBER Fisiopatología de la Obesidad y Nutrición, CB06/03, Instituto de Salud Carlos III, Madrid, Spain
c Departamento de Química Física y Analítica, Facultad de Química, Universidad de Oviedo, 33006 Oviedo, Spain
d Fundación IMABIS, Hospital Carlos Haya, Avda. Carlos Haya 82, 29010 Málaga, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Searching for new antiobesity agents, a new series of fatty acid amide derivatives of 1,5-diarylpyrazole
Received 13 March 2008
Revised 30 September 2008
Accepted 2 October 2008
Available online 14 October 2008
have been synthesized as dual peroxisome proliferator activated receptor alpha (PPAR
a)/cannabinoid
receptor ligands. The compounds have been evaluated in vivo and in vitro as PPAR activators and as can-
a
nabinoids in two tests of the mouse tetrad. In vivo, food intake studies have been performed with all the
compounds. No significant cannabinoid activity has been found but some compounds behaved as potent
PPAR
a
activators. Several compounds showed anorexigenic properties reducing food intake in rats.
Keywords:
Pyrazole
Ó 2008 Elsevier Ltd. All rights reserved.
Oleylamide
PPARa
DMLs
1. Introduction
us to apply this strategy to design potential hypophagic agents
capable to target not only appetite, but also lipid and carbohydrate
Obesity has significantly increased in the last decades affecting
an important part of the adult and children population of western
countries.1 It is a complex disease involving many physiological
signaling systems and different factors, and it is linked to serious
health problems.2 Nowadays, only three classes of drug treatments
are available, representatives are Orlistat (Xenical)TM a gastrointes-
tinal lipase inhibitor, Sibutramine (Meridia)TM a dual serotonin-nor-
epinephrine reuptake inhibitor, and Rimonabant (Acomplia)TM, the
first cannabinoid CB1 receptor antagonist approved for clinical
use in humans. Although effective in producing weight loss, cur-
rent therapies against obesity may have some tolerability and/or
safety concerns.3 Moreover, since obesity is a complex disease of-
ten associated with high cardiovascular risk, type II diabetes and
dyslipemia, appetite cannot be considered the only target for med-
icines designed to fight obesity. Therefore, the development of no-
vel antiobesity drugs is a priority and a challenge for medicinal
chemists.
metabolism. Taking in consideration the convergent mechanisms
of peroxisome proliferator activated receptors alpha (PPAR ) and
cannabinoid receptor antagonists as modulators of appetite, lipid
metabolism and carbohydrate management by the liver and adi-
a
pose tissue, we decided to explore a dual cannabinoid/PPAR
a li-
gand (Fig. 1). The rationale was to link, in the same molecule, a
cannabinoid antagonist motif with a group capable of activating
PPAR
a
receptors,5 since the combination of both drugs seems to
be additive in terms of controlling appetite.6 The cannabinoid part
was the 1,5-diarylpyrazole structure present in Rimonabant and
other well established cannabinoid ligands7 and the amide chosen
was oleylethanolamide, OEA, a lipid mediator that regulates feed-
ing and lipid metabolism by activating PPARa
receptors.8 This idea
was further supported by a recent finding in our group in which we
have proved that Rimonabant enhances the metabolic benefits of
long term treatment with OEA in Zucker rats, a genetic model of
obesity, dyslipemia and diabetes due to the lack of leptin
signaling.9
The fact that there are different targets for antiobesity therapy
together with a growing interest in multiple ligands4 prompted
2. Chemistry
* Corresponding authors. Tel.: +34 914113155; fax: +34 915644853 (P.G.); tel.:
+34 669426548; fax: +34 951291447 (F.R.F.).
E-mail addresses: iqmg310@iqm.csic.es (P. Goya), fernando.rodriguez@funda
cionimabis.org (F. Rodríguez de Fonseca).
The synthetic route used for the preparation of compounds
4–11 is depicted in Scheme 1 and starts from the 2,4-dicarbonyl
esters obtained through condensation of the corresponding ketone
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.023

M. Alvarado et al. / Bioorg. Med. Chem. 16 (2008) 10098–10105
10099
gene promoter18 fused with the thymidine kinase (tk) minimal
promoter using vector pGL-2 Basic (Promega, Mannheim,
Germany).
O
O
N
N
H
N
H
OH
N
N
3.2. In vitro translation and bacterial overexpression of
proteins
Cl
Cl
In vitro translated wild-type human PPARa were generated by
coupled in vitro transcription/translation (TNTÒ system) using rab-
bit reticulocyte lysate as recommended by the supplier (Promega,
Cl
OEA
SR141716
Mannheim, Germany). Part of PPARa were translated in presence
of [35S]-methionine and taking the individual numbers of methio-
nine residues per receptor into account, the specific concentration
O
of the receptor proteins was adjusted to approximately 4 ng/lL
R3
after taking the individual number of methionine residues per pro-
tein into account. Bacterial overexpression of GST-TIF2 and GST
alone was obtained from the Escherichia coli BL21(DE3)pLysS strain
(Stratagene, Heildelberg, Germany). GST-TIF2 and GST-fusion pro-
N
H
N
N
R2
Cl
tein expression were stimulated with 0.25 mM isopropyl-b-D-thio-
galactopyranoside (IPTG) for 3 h at 37 °C. The fusion proteins were
purified and immobilized by glutathione-Sepharose 4B beads
(Amersham-Pharmacia, Uppsala, Sweden) according to manufac-
turer’s protocol.
R1
Figure 1. Structures of OEA, SR141716 and designed multiple ligands.
3.3. GST pull down assays
and diethyl oxalate in basic medium.10 The pyrazole esters 1a and
1b used for the synthesis of compounds 8–11 have previously been
reported.11,12 The new pyrazole esters 2–3 were prepared from
ethyl 4-(4-chlorophenyl)-2,4-dioxobutanoate and the correspond-
ing arylhydrazine following the usual procedure.13 In general, this
reaction has been described to provide the 1H-pyrazole-3-carbox-
ylate isomer.14
For the preparation of the pyrazole carboxamides, we followed
a procedure which we had previously used for 1,2,4-triazole-3-car-
boxamides.15 Thus, compounds 4–11 were obtained in high yields
in a one-step synthesis by treatment of the carboxylates with an
aluminum complex prepared in situ reacting trimethylaluminum
with the corresponding N-oleyl and N-hexadecylamine.
The structures of the newly synthesized compounds were
established according to analytical and spectroscopic data. The X-
ray crystal structures of compounds 2 and 7 have been deposited
in the Cambridge Crystallographic Data Centre (Deposition nos.:
CCDC 664037 and 664038).
GST pull down assays were performed with 50
l
L of a 50% Se-
g/
L bovine serum albumin) and 20 ng in vitro translated, [35S]-la-
pharose bead slurry of GST or GST-TIF2 (pre-blocked with 1
l
l
beled PPARa in the presence or absence of their respective com-
pounds.19 Proteins were incubated in immunoprecipitation buffer
[20 mM Hepes (pH 7.9), 200 mM KCl, 1 mM EDTA, 4 mM MgCl2,
1 mM dithiothretiol (DTT), 0.1% Nonidet P-40 and 10% glycerol]
for 20 min at 30 °C. In vitro translated proteins that were not
bound to GST-fusion proteins were washed away with immuno-
precipitation buffer. GST-fusion protein bound [35S]-labeled nucle-
ar receptors were resolved by electrophoresis through 10% SDS–
polyacrylamide gels and quantified on a Fuji FLA3000 reader (To-
kyo, Japan) using Image Gauge software (Fuji Photo Film Co., To-
kyo, Japan).
3.4. Cellular transfection and luciferase reporter gene assays
Human breast cancer cells MCF-7 were seeded onto 6-well
plates (105 cells/mL) and grown overnight in phenol red-free
DMEM supplemented with 5% charcoal-treated fetal bovine serum
(FBS). Plasmid DNA containing liposomes were formed by incubat-
3. Biology
The compounds reported in this study were first evaluated for
in vitro GST pull down to see which compounds could induce inter-
action between PPAR
pounds were also screened in the mouse tetrad for cannabinoid
activity. Finally, the compounds were evaluated in vivo on food in-
take and pharmacology studies in rats.
ing 1
human PPAR
pyl]-N,N,N-trimethylammonium methylsulfate (DOTAP, Roche)
for 15 min at room temperature in a total volume of 100 L. After
dilution with 900 L phenol red-free DMEM, the liposomes were
added to the cells. Phenol red-free DMEM supplemented with
500 L of 15% charcoal-treated FBS was added 4 h after transfec-
lg of the reporter plasmid and expression vector wild-type
a
and coactivator into MCF-7 cells. The com-
a
and TIF2 with 10 g N-[1-(2,3-dioleoyloxy)pro-
l
l
l
3.1. Plasmids
l
tion. At this time, cells were treated for 16 h with solvent (DMSO)
and different concentrations (10À9, 10À8, 10À7, 10À6, 10À5 and
10À4 M) of GW7647, OEA and compounds 4–11 as indicated. The
cells were lysed 16 h after onset of stimulation using the reporter
gene lysis buffer (Roche), and the constant light signal luciferase
reporter gene assay was performed as recommended by the
supplier (Roche Diagnostics).
Full length cDNAs for human PPARa16 were subcloned into the
T7/SV40 promoter-driven pSG5 expression vector (Stratagene,
Heildelberg, Germany). The same constructs were used both for
T7 RNA polymerase-driven in vitro transcription/translation of
the respective cDNAs and for viral promoter-driven overexpression
of the respective proteins in mammalian cells. The nuclear receptor
interaction domains of human TIF2 (spanning from a 646 to 926)17
were subcloned into the GST-fusion protein vector pGEX (Amer-
sham-Pharmacia, Uppsala, Sweden). For reporter gene assays in
MCF-7 cells, the luciferase gene was driven by three copies of the
PPAR response element (PPRE) of the human acyl-CoA oxidase
Stimulation of normalized luciferase activity was calculated in
comparison with solvent-induced cells that do not overexpress
any protein. GW7647 (Tocris Bioscience) was included as positive
control because selectively activates PPARa 20
Data were used to
.
calculate the EC50 [nM] for each compound.

10100
M. Alvarado et al. / Bioorg. Med. Chem. 16 (2008) 10098–10105
OH O NHNH2
R2
EtO
+
O
R3
Cl
O
R1
i)
R3
EtO
N
1a:
R1=R2=Cl, R3=H
1b: R1=R2=Cl, R3=CH3
2:
N
R2
Cl
R1=R2=R3=H
3: R1=Cl, R2=R3=H
R1
ii)
O
O
R3
R3
N
H
N
H
N
N
N
N
R2
R2
Cl
Cl
R1
R1
4:
R1=R2=R3=H
5: R1=Cl, R2=R3=H
8:
6:
R1=R2=R3=H
7: R1=Cl, R2=R3=H
10:
R1=R2=Cl, R3=H
9: R1=R2=Cl R3=CH3
R1=R2=Cl, R3=H
11: R1=R2=Cl, R3=CH3
Scheme 1. Synthesis of pyrazole fatty acid amides 4–11. Reagents and conditions: (i) AcOH, reflux; (ii) RNH2, Al(CH3)3, CH2Cl2, reflux.
3.5. Feeding experiments
at 30, 60, 120 and 240 min after starting the test, and the amount
of food eaten was recorded. At the end of the test, the amount of
water consumed was also measured.
All experiments were performed in male Wistar rats, weighing
250–450 g from Animal Resources Centre, University of Málaga
(Spain). Animals were housed in groups of two in standard plexi-
glas cages in a temperature and humidity controlled room (23 °C
and 50% relative humidity) with a 12:12-h yellow light/dark cycle.
Water and standard chow pellets (Prolab. RMH 2500) were avail-
able ad libitum. Animals were handled daily for a week before
starting the experiments. All animal procedures met the National
Institutes of Health guidelines for the care and use of laboratory
animals, and the European Communities directive 86/609/EEC reg-
ulating animal research.
3.6. Cannabinoid activity
3.6.1. Hypothermia
Core temperatures in mice were measured using a P6 thermom-
eter and a lubricated rectal probe (CIBERTEC, Spain) inserted into
the rectum to a constant depth of 3 cm. Data were recorded before
drugs administration and 15 min after ip injection.
3.6.2. Locomotor activity
The acute effects of drugs on feeding behavior were analyzed in
24 h food-deprived Wistar rats, which had been habituated to han-
dling.6,21 To this end, 48 h before testing, the bedding material was
removed from the cage and a small can containing food pellets was
placed inside the cage for 4 h. The animals were then food-de-
prived for 24 h, with free access to water. The drugs were sus-
pended with 2–3 drops of Tween 80 in saline as vehicle and
administered intraperitoneally (ip) at doses of 0.3, 3 mg/kg and/
or 10 mg/kg. 15 min after drug administration, the animals were
returned to their home cage, where a can with a measured amount
of food (usually 30–40 g) and a bottle containing 250 mL of fresh
water were placed. Food pellets and food spillage were weighed
Although initial studies with hypothermia indicated that the
compounds did not exhibit cannabinoid receptor agonist/antago-
nist properties, we decided to further evaluate exploratory and
locomotor activities in mice treated with the drugs. Mice were
moved into the behavioral testing room at least 1 h prior to testing.
The open field consisted of a 40 Â 40 cm arena divided in 25
squares by lines drawn on the floor of the apparatus. The 9 squares
not bounded by the walls of the test were referred to as center
squares. The following parameters were recorded during the
20 min session: distance traveled (cm), number of squares crossed,
number of center squares entered, time in the center of the field
and immobility time. The apparatus was cleaned between mice

M. Alvarado et al. / Bioorg. Med. Chem. 16 (2008) 10098–10105
10101
with a weak acetic acid solution. Illumination of the test room was
the same as the mouse colony room (100 lux). They were moni-
tored by a video-tracking system equipped with a camera (Smart,
Panlab, Barcelona) that records the animal’s horizontal activity.22
Drugs were injected ip 15 min before placing the animal in the
open field. After drug injection, each mouse was placed into the
central square of the arena and allowed to freely explore the field
for 20 min.
3.6.3. Animals
Adult (25–30 g) male wild-type 129S1/SvImJ mice (WT) (Jack-
son Laboratories, USA) were used in all experiments. Experiments
were conducted between 9:00 and 15:00 h.
3.7. Statistics
Statistical significance of behavioral studies was assessed by
analysis of variance (ANOVA). Following a significant F value, post
hoc analysis (Student–Newman–Keuls) was performed to assess
specific comparisons between dose groups.
4. Results and discussion
As can be seen from Figure 2 and Table 1, oleyl derivatives 4, 5,
8 and 9 are capable of activating PPARa receptors, promoting both,
its binding to DNA and transcriptional activity. While compound 4
and 8 activated transcription with a similar potency than that of
either the reference agonist GW7647 or the endogenous ligand
OEA, compounds 5 and 9 were 2–3-fold less potent and the corre-
sponding hexadecyl derivatives 6, 7, 10 and 11 were deprived of
PPAR
a
receptor agonist activity. Therefore, it seems clear that the
receptor is linked to the presence of an
ability to activate PPAR
a
oleylamide rest, all the saturated derivatives being inactive.
Cannabinoid activity was first studied in the temperature as-
say of the mouse tetrad.23 As shown in Figure 3A none of the
compounds were able to modify the body temperature except
compound 5 which slightly increased body temperature in rela-
tion to the control. The study of the antagonist effect of these
compounds is shown in Figure 3B. As expected for a cannabinoid
agonist WIN 55,292-2 (WIN) reduces body temperature signifi-
cantly. Compounds 4, 6 and 7 do not modify the hypothermia in-
duced by WIN. Oleyl derivative
5
significantly inhibits
hypothermia induced by the agonist although the temperature
does not reach the control values. In the locomotor test only
hexadecyl derivative 6 could prevent the decrease in locomotor
activity induced by WIN. The lack of consistent cannabinoid ago-
nist/antagonist effects suggests that the addition of the long acyl
group to the carboxamide restrains cannabinoid activity in vivo of
the diaryl pyrazole structure. However, the compounds displayed
Figure 2. Individual ligand-triggered interaction profiles of hPPAR
a
with coactiva-
tor (TIF2) in solution. GST pull down assays were performed with bacterially
expressed GST-TIF2 and full-length in vitro translated (35S)-labeled human PPAR
in the absence and presence of different compounds: 4, 5, 6, 7, GW7647 and OEA (A)
and compounds 8, 9, 10 and 11 (B). Percentage of precipitated PPAR was
a
,
a
a very interesting ability to activate PPARa receptors that has not
been described for diaryl pyrazole-based cannabinoid antagonists
quantified in respect to input (mean SEM, n: 3–6). (Ã) p < 0.05 (Student’s t-test) vs
DMSO solvent.
already reported in the literature.
All the synthesized compounds were tested in feeding experi-
ments in food-deprived rats and some representative results are
shown in Figure 4. Compounds 8, 9 and 10 were weak short acting
food inhibitors whereas 5 and 11 were devoid of activity. Oleyl
derivative 4 is both a potent PPARa agonist and shows interesting
activity as feeding suppressant. On the other hand, hexadecyl
olamides such as oleoylethanolamide and anandamide. The identi-
fication of these targets may be linked to the growing evidence for
the existence of multiple receptors for these lipid transmitters that
are potentially involved in feeding and metabolic regulation. The
pharmacological characterization of these new anorectic com-
pounds will be the subject of further studies.
derivatives 6 and 7 show anorectic activity but do not activate
PPAR
is very similar to that of sulfamoyl derivatives synthesized as
PPAR
agonists in our laboratory.24 These compounds also exhib-
ited a range of activation of PPAR receptors and induction of
satiety that were often dissociated. This indicates the existence
of multiple targets for regulating feeding and PPAR activation that
share structural determinants with those exhibited by acylethan-
a receptors. This profile is extremely interesting because it
a
5. Conclusions
a
Dual compounds incorporating cannabinoid and PPAR
a fea-
a
tures in their structure have been synthesized and evaluated. None
of the compounds exhibited significant cannabinoid properties. All

10102
M. Alvarado et al. / Bioorg. Med. Chem. 16 (2008) 10098–10105
Table 1
Data of activation PPAR
from estimation of normalized luciferase activity
**
A
a (EC50 [nM]) of pyrazole fatty acid amides 4–11 calculated
38
37.5
37
Compounds
PPARa
activation EC50 [nM]a
4
5
6
7
8
9
10
11
183 43
524 69
>10,000
>10,000
223 52
467 79
>10,000
>10,000
36.5
36
+
35.5
35
***
34.5
34
OEA
GW7647
121 26
178 31
33.5
33
a
EC50 values calculated in the presence of the different compounds by GraphPad
Prism 4. Results are mean SEM of eight experiments.
32.5
32
the chlorophenylpyrazoles bearing oleylamides were capable of
activating the PPARa receptors whereas the corresponding hexa-
decyl derivatives were inactive. Concerning the pyrazole part, the
introduction of a methyl at position 4 (as in Rimonabant) does
+ Vs control
not improve PPARa activity, being the Rimonabant analog 9
Vs vehicule
*
Treatment
slightly less active than the 4H derivative 8. Some compounds re-
duced food intake at doses of 3 mg/Kg. Oleyl derivative 4 is both a
potent PPARa agonist and a feeding suppressant. Hexadecylamides
6 and 7 also reduced food intake through some other mechanism
38
B
***
37.5
37
36.5
36
which will be the subject of further investigation.
+
*
***
6. Experimental
***
35.5
35
***
***
6.1. General methods
34.5
34
All reagents and solvents were used as commercially received
with exception of CH2Cl2 which was distilled from P2O5 prior to
use. TLC: precoated silica-gel 60 F254 plates (Merck), detection
by UV light (254 nm). Flash-column Chromatography (FC): Kiesel-
gel 60 (230–400 mesh; Merck). Melting points (mp) were deter-
mined in open capillaries with a Gallenkamp capillary melting-
points apparatus and are uncorrected. 1H and 13C NMR spectra
were recorded on Bruker Advance 300 spectrometer operating
at 300.13 and 75.47 MHz, respectively, in CDCl3 as solvent and
Me4Si as the internal standard. Chemical shifts are reported in
ppm on the d scale. The mass spectra (EI-MS; 70 eV) were deter-
mined on a MSD 5973 Hewlett Packard instrument. Elemental
analyses were performed on a Heraeus CHN-O Rapid Analysis in
our Analytical Service at Centro de Química Orgánica ‘‘Manuel
Lora Tamayo” (CSIC). The X-ray crystallographic determination
of 2 and 7 was performed using a Nonius Kappa CCD diffractom-
33.5
33
32.5
32
+ Vs control
Vs vehicule
*
Treatment
Figure 3. The bars show the averages of the values obtained in the rectal
temperature (C°) of the mice (A and B). The bar ‘‘control” is the average of the
values obtained of all the animals used in this study, before administering no
compound to them (Tª). The rest of the bars show the average of the values of the
temperature measured after the administrations of different drugs (mg/kg): (A)
WIN, 2 mg/kg; AM, 3 mg/kg; 4, 3 mg/kg; 6, 3 mg/kg; 5, 3 mg/kg; 7, 10 mg/kg. (B)
WIN, 2 mg/kg plus 3 mg/kg of AM and WIN, 2 mg/kg plus 3 mg/kg 4, 6, 5 and 7,
10 mg/kg. Each point corresponds to the average EEM of the obtained values.
(*p < 0.05; ***p < 0.001 vs control; +p < 0.05 vs WIN ; Tª; T-Student).
eter with copper K (k = 1.54184 Å) radiation. The structure was
a
solved by Direct Methods using SIR92, an absorption correction
was applied and the refinements were carried out with
SHELXL97.
6.2. General procedure for the preparation of ethyl 1,5-diaryl-1
H-pyrazole-3-carboxylate (2–3)
6.2.1. Ethyl 5-(4-chlorophenyl)-1-phenyl-1H-pyrazole-3-
carboxylate (2)
To a solution of ethyl 4-(4-chlorophenyl)-2,4-dioxobutanoate in
glacial acetic acid was added an amount equimolar of the arylhydr-
azine. The reaction mixture was refluxed and later poured into
water. The yellowish oil was separated by extraction with ether
(3 Â 50 mL) and the organic extract was washed with NaHCO3
solution (10%) (3 Â 15 mL) and water (3 Â 15 mL). The extract
was dried (Na2SO4) and the solvent was removed under reduced
pressure to give predominantly 1H-pyrazole-3-carboxylate isomer
as a viscous yellow oil. The residue was purified by flash chroma-
tography (FC) and crystallization.
The title compound was prepared according to the previously
described general procedure by refluxing ethyl 4-(4-chloro-
phenyl)-2,4-dioxobutanoate (3.0 g, 11.8 mmol) and phenylhydr-
azine (1.27 g, 11.8 mmol) in glacial acetic acid (10 mL) during
24 h. The product was purified by FC (SiO2; EtOAc/n-hexane 1:9)
and crystallization (79% yield). Yellowish solid, mp: 94–5 °C
(EtOH); MS/EI: m/z (%) = 328 [M+1]+ (34), 326 (91), 254 (100); 1H
NMR (300 MHz, CDCl3): d = 7.29–7.18 (m, 7 H), 7.07 (d, J = 8.5 Hz,
2H), 6.95 (s, 1H), 4.38 (q, J = 7.0 Hz, 2H), 1,34 (t, J = 7.0 Hz, 3H);
13C NMR (75 MHz, CDCl3): d = 162.2 (CO), 144.3 (C), 143.3 (C),

M. Alvarado et al. / Bioorg. Med. Chem. 16 (2008) 10098–10105
10103
4
6
40.0
30.0
20.0
10.0
0.0
40.0
30.0
*
*
20.0
*
*
*
VEHICLE
10.0
VEHICLE
0.3 mg/kg
3.0 mg/Kg
0.3 mg/kg
3.0 mg/Kg
*
0.0
30
60
120
240
30
60
120
240
TIME AFTER INJECTION (min)
TIME AFTER INJECTION (min)
7
9
40.0
30.0
20.0
10.0
0.0
40.0
30.0
20.0
10.0
*
*
*
*
VEHICLE
0.3 mg/kg
3.0 mg/Kg
VEHICLE
0.3 mg/kg
3.0 mg/Kg
0.0
30
60
120
240
30
60
120
240
TIME AFTER INJECTION (min)
TIME AFTER INJECTION (min)
11
40.0
30.0
20.0
10.0
VEHICLE
0.3 mg/kg
3.0 mg/Kg
0.0
30
60
120
240
TIME AFTER INJECTION (min)
Figure 4. Intake grams per kg weight of the rats in food-deprived animals was tested 30, 60, 120 and 240 min after the ip injection of the different synthesized compounds: 4,
6, 7, 9 and 11 at 0.3 and 3 mg/kg doses, ip. Results are mean SEM of at least eight animals per group. (Ã) p < 0.05 vs vehicle, ANOVA.
139.2 (C), 134.8 (C), 129.8 (CH), 129.0 (CH), 128.8 (CH), 128.5 (CH),
127.9 (C), 125.6 (CH), 109.9 (CH), 61.1 (CH2), 14.3 (CH3).
6.3. General procedure for the preparation of 1,5-diaryl-1H-
pyrazole-3-carboxamide (4–11)
6.2.2. Ethyl 1,5-bis(4-chlorophenyl)-1H-pyrazole-3-carboxylate
(3)
To the respective amine (5 equiv) dissolved in dry CH2Cl2 was
added dropwise during 5 min a commercial solution 2 M Al(CH3)3
in heptane (5 equiv). The reaction mixture was stirred during 1 h at
room temperature. Then, a solution of the suitable 1H-pyrazole-3-
carboxylate (1 equiv) dissolved in dry CH2Cl2 was added and the
mixture was heated at reflux. The reaction was quenched by slow
and careful addition of a solution 2 N HCl (50 mL). The organic
layer was then separated and washed with a solution 2 N HCl
(3 Â 15 mL), dried (Na2SO4) and evaporated to dryness. The residue
was purified by flash chromatography and crystallization.
The title compound was prepared according to the previously
described general procedure by refluxing ethyl 4-(4-chloro-
phenyl)-2,4-dioxobutanoate (3.0 g, 11.8 mmol) and 4-chlorophen-
ylhydrazine chlorhydrate (2.1 g, 11.8 mmol) in glacial acetic acid
(10 mL) during 48 h. The product was purified by FC (SiO2;
EtOAc/n-hexane 1:9) and crystallization (60% yield). Yellowish so-
lid, mp: 120–1 °C (EtOH); MS/EI: m/z (%) = 362 [M+1]+ (59), 360
(94), 288 (100); 1H NMR (300 MHz, CDCl3): d = 7.29–7.23 (m,
4H), 7.20 (d, J = 8.5 Hz, 2H,), 7.08 (d, J = 8.3 Hz, 2H), 6.95 (s, 1H),
4.39 (q, J = 7.1 Hz, 2H), 1.35 (t, J = 7.1 Hz, 3H); 13C NMR (75 MHz,
CDCl3): d = 162.0 (CO), 144.7 (C), 143.4 (C), 137.7 (C), 135.1 (C),
134.3 (C), 129.9 (CH), 129.3 (CH), 129.0 (CH), 127.6 (C), 126.7
(CH), 110.2 (CH), 61.2 (CH2), 14.3 (CH3).
6.3.1. N-(1-Oleyl)-5-(4-chlorophenyl)-1-phenyl-1H-pyrazole-3-
carboxamide (4)
The title compound was prepared according to the previously
described general procedure by refluxing N-oleylamine (0.61 g,

10104
M. Alvarado et al. / Bioorg. Med. Chem. 16 (2008) 10098–10105
2.30 mmol),
a
commercial solution 2 M Al(CH3)3 in heptane
3.38 (q, J = 7.0 Hz, 2H), 1.58–1.49 (m, 2H), 1.29–1.17 (m, 26H),
0.82 (t, J = 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3): d = 161.3 (CO),
147.7 (C), 143.7 (C), 137.8 (C), 135.0 (C), 134.2 (C), 129.9 (CH),
129.3 (CH), 129.0 (CH), 127.9 (C), 126.5 (CH), 108.9 (CH), 39.6
(CH2), 32.3 (CH2), 30.1–29.9 (CH2), 29.7 (CH2), 27.3 (CH2), 23.0
(CH2), 14.5 (CH3); elemental analysis calcd (%) for C32H43Cl2N3O
(555.28): C 69.05, H 7.79, N 7.55, found: C: 68.89; H: 7.51; N: 7.73.
(1.15 mL, 2.30 mmol) and 2 (0.15 g, 0.45 mmol) dissolved in dry
CH2Cl2 during 24 h. The product was purified by FC (SiO2; EtOAc/
n-hexane 1:4) (90% yield). Yellowish oil; MS/EI: m/z (%) = 549
[M+1]+ (20), 547 (42), 281 (100); 1H NMR (300 MHz, CDCl3):
d = 7.33–7.31 (m, 3H), 7.23–7.19 (m, 4H), 7.07 (d, J = 8.5 Hz, 2H,),
6.96 (s, 1H), 5.32–5.25 (m, 2H), 3.38 (q, J = 6.8 Hz, 2H), 1.94–1.90
(m, 4H), 1.55–1.49 (m, 2H), 1.22–1.18 (m, 22H), 0.80 (t,
J = 6.8 Hz, 3H); 13C NMR (75 MHz, CDCl3): d = 161.5 (CO), 147.4
(C), 143.6 (C), 139.3 (C), 134.7 (C), 129.8 (CH), 129.7 (CH), 129.1
(CH), 128.8 (CH), 128.3 (CH), 128.1 (C), 125.3 (CH), 108.1 (CH),
39.2 (CH2), 32.5 (CH2), 31.8 (CH2), 29.7–29.1 (CH2), 27.1 (CH2),
26.9 (CH2), 22.6 (CH2), 14.0 (CH3); elemental analysis calcd (%)
for C34H46ClN3O (547.33): C 74.49, H 8.46, N 7.67, found: C:
74.76; H: 8.70; N: 7.91.
6.3.5. N-(1-Oleyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-
1H-pyrazole-3-carboxamide (8)
The title compound was prepared according to the previously
described general procedure by refluxing N-oleylamine (1.01 g,
3.80 mmol),
a commercial solution 2 M Al(CH3)3 in heptane
(1.90 mL, 3.80 mmol) and ethyl 5-(4-chlorophenyl)-1-(2,4-dichlo-
rophenyl)-1H-pyrazole-3-carboxylate (1a)11 (0.30 g, 0.76 mmol)
dissolved in dry CH2Cl2 during 20 h. The product was purified by
FC (SiO2; EtOAc/n-hexane 1:4) (78% yield). Yellowish oil; MS/EI:
m/z (%) = 617 [M+1]+ (8), 615 (8), 349 (100); 1H NMR (300 MHz,
CDCl3): d = 7.41 (d, J = 1.3 Hz, 1H), 7.29–7.18 (m, 4H), 7.04 (d,
J = 8.4 Hz, 2H), 6.99 (s, 1H), 5.26–5.21 (m, 2H), 3.34 (q, J = 6.4 Hz,
2H), 1.94–1.92 (m, 4H), 1.52–1.50 (m, 2H), 1.21–1.19 (m, 22H),
0.80 (t, J = 6.3 Hz, 3H); 13C NMR (100 MHz, CDCl3): d = 161.3 (CO),
148.3 (C), 145.6 (C), 136.2 (C), 135.9 (C), 135.0 (C), 133.0 (C),
130.5 (CH), 130.4 (CH), 129.9 (CH), 129.7 (CH), 129.1 (CH), 129.0
(CH), 128.1 (CH), 127.5 (C), 107.0 (CH), 39.2 (CH2), 32.5 (CH2),
31.7 (CH2), 29.7-29.2 (CH2), 27.1 (CH2), 26.9 (CH2), 22.6 (CH2),
14.0 (CH3); Elemental analysis calcd (%) for C34H44Cl3N3O
(615.25): C 66.18, H 7.19, N 6.81, found: C: 66.28; H: 7.15; N: 7.02.
6.3.2. N-(1-Oleyl)-1,5-bis(4-chlorophenyl)-1H-pyrazole-3-
carboxamide (5)
The title compound was prepared according to the previously
described general procedure by refluxing N-oleylamine (0.92 g,
3.46 mmol),
a commercial solution 2 M Al(CH3)3 in heptane
(1.73 mL, 3.46 mmol) and 3 (0.25 g, 0.69 mmol) dissolved in dry
CH2Cl2 during 20 h. The product was purified by FC (SiO2; EtOAc/
n-hexane 1:4) (88% yield). Yellowish oil; MS/EI: m/z (%) = 583
[M+1]+ (13), 581 (19), 315 (100); 1H NMR (300 MHz, CDCl3):
d = 7.30–7.22 (m, 4H), 7.17 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.4 Hz,
2H), 6.96 (s, 1H), 5.31–5.25 (m, 2H), 3.38 (q, J = 6.7 Hz, 2H), 1.94–
1.90 (m, 4H), 1.54–1.49 (m, 2H), 1.22–1.18 (m, 22H), 0.80 (t,
J = 6.7 Hz, 3H); 13C NMR (75 MHz, CDCl3): d = 161.3 (CO), 147.7
(C), 143.7 (C), 137.8 (C), 135.0 (C), 134.2 (C), 129.9 (CH), 129.7
(CH), 129.3 (CH), 129.0 (CH), 127.9 (C), 126.5 (CH), 108.4 (CH),
39.2 (CH2), 32.6 (CH2), 31.8 (CH2), 29.7–29.2 (CH2), 27.2 (CH2),
26.9 (CH2), 22.6 (CH2), 14.5 (CH3); elemental analysis calcd (%)
for C34H45Cl2N3O (581.29): C 70.09, H 7.78, N 7.21, found: C:
70.23; H: 7.48; N: 7.25.
6.3.6. N-(1-Oleyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-carboxamide (9)
The title compound was prepared according to the previously
described general procedure by refluxing N-oleylamine (0.81 g,
3.06 mmol),
a commercial solution 2 M Al(CH3)3 in heptane
(1.53 mL, 3.06 mmol) and ethyl 5-(4-chlorophenyl)-1-(2,4-dichlo-
rophenyl)-4-methyl-1H-pyrazole-3-carboxylate (1b)12 (0.25 g,
0.61 mmol) dissolved in dry CH2Cl2 during 30 h. The product was
purified by FC (SiO2; EtOAc/n-hexane 1:4) (47% yield). Yellowish
oil; MS/EI: m/z (%) = 631 [M+1]+ (8), 629 (8), 363 (100); 1H NMR
(300 MHz, CDCl3): d = 7.35 (d, J = 1.3 Hz, 1H), 7.23–7.20 (m, 4H),
6.99 (d, J = 8.5 Hz, 2H), 5.29–5.21 (m, 2H), 3.34 (q, J = 6.7 Hz, 2H),
2.30 (s, 3H), 1.96–1.92 (m, 4H), 1.54–1.47 (m, 2H), 1.21–1.17 (m,
22H), 0.80 (t, J = 6.7 Hz, 3H); 13C NMR (75 MHz, CDCl3): d = 162.5
(CO), 145.1 (C), 142.9 (C), 135.9 (C), 135.8 (C), 134.8 (C), 132.9
(C), 130.7 (CH), 130.5 (CH), 130.2 (CH), 129.9 (CH), 129.7 (CH),
128.8 (CH), 127.8 (CH), 127.2 (C), 117.6 (C), 39.0 (CH2), 32.5
(CH2), 31.8 (CH2), 29.7–29.2 (CH2), 27.3 (CH2), 26.9 (CH2), 22.6
(CH2),14.0 (CH3), 9.3 (CH3); Elemental analysis calcd (%) for
C35H46Cl3N3O (629.27): C 66.61, H 7.35, N 6.66, found: C: 66.34;
H: 7.08; N: 6.86.
6.3.3. N-(1-Hexadecyl)-5-(4-chlorophenyl)-1-phenyl-1H-
pyrazole-3-carboxamide (6)
The title compound was prepared according to the previously
described general procedure by refluxing of N-hexadecylamine
(0.92 g, 3.82 mmol), a commercial solution 2 M Al(CH3)3 in hep-
tane (1.91 mL, 3.82 mmol) and 2 (0.25 g, 0.76 mmol) dissolved in
dry CH2Cl2 during 48 h. The product was purified by FC (SiO2;
EtOAc/n-hexane 1:4) and crystallization (93% yield). White solid,
mp: 59–60 °C (n-hexane); MS/EI: m/z (%) = 523 [M+1]+ (5), 521
(13), 281 (100); 1H NMR (300 MHz, CDCl3): d = 7.33–7.31 (m,
3H), 7.23–7.19 (m, 4H), 7.08 (d, J = 8.4 Hz, 2H), 6.97 (s, 1H), 3.38
(q, J = 6.7 Hz, 2H), 1.56–1.51 (m, 2H), 1.30–1.17 (m, 26H), 0.82 (t,
J = 6.7 Hz, 3H); 13C NMR (75 MHz, CDCl3): d = 161.5 (CO), 147.4
(C), 143.6 (C), 139.3 (C), 134.7 (C), 129.9 (CH), 129.1 (CH), 128.8
(CH), 128.3 (CH), 128.1 (C), 125.4 (CH), 108.5 (CH), 39.6 (CH2),
32.3 (CH2), 30.1–29.9 (CH2), 29.7 (CH2), 27.4 (CH2), 23.0 (CH2),
14.5 (CH3); elemental analysis calcd (%) for C32H44ClN3O
(521.32): C 76.31, H 8.49, N 8.05, found: C: 73.90; H: 8.44; N: 7.95.
6.3.7. N-(1-Hexadecyl)-5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-1H-pyrazole-3-carboxamide (10)
The title compound was prepared according to the previously
described general procedure by refluxing N-hexadecylamine
(0.91 g, 3.80 mmol), a commercial solution 2 M Al(CH3)3 in hep-
tane (1.91 mL, 3.80 mmol) and ethyl 5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-1H-pyrazole-3-carboxylate (1a)11 (0.30 g, 0.76
mmol) dissolved in dry CH2Cl2 during 24 h. The product was puri-
fied by FC (SiO2; EtOAc/n-hexane 1:4) (87% yield). White solid; mp:
61–2 °C (n-hexane); MS/EI: m/z (%) = 591 [M+1]+ (8), 589 (8), 240
(100); 1H NMR (300 MHz, CDCl3): d = 7.41 (d, J = 1.3 Hz, 1H),
7.29–7.17 (m, 4H), 7.05–7.00 (d, J = 8.6 Hz, 2H), 6.99 (s, 1H), 3.34
(q, J = 6.8 Hz, 2H), 1.57–1.47 (m, 2H), 1.25–1.17 (m, 26H), 0.89 (t,
J = 6.8 Hz, 3H); 13C NMR (75 MHz, CDCl3): d = 161.2 (CO), 148.2
(C), 145.6 (C), 136.2 (C), 135.9 (C), 135.0 (C), 133.0 (C), 130.5
6.3.4. N-(1-Hexadecyl)-1,5-bis(4-chlorophenyl)-1H-pyrazole-3-
carboxamide (7)
The title compound was prepared according to the previously
described general procedure by refluxing N-hexadecylamine
(0.83 g, 3.46 mmol), a commercial solution 2 M Al(CH3)3 in hep-
tane (1.73 mL, 3.46 mmol) and 3 (0.25 g, 0.69 mmol) dissolved in
dry CH2Cl2 (100 mL) during 20 h. The product was purified by FC
(SiO2; EtOAc/n-hexane 1:4) and crystallization (90% yield). White
solid, mp: 78–9 °C (n-hexane); MS/EI: m/z (%) = 557 [M+1]+ (10),
555 (15), 315 (100); 1H NMR (300 MHz, CDCl3): d = 7.30–7.22 (m,
4H), 7.17 (d, J = 8.7 Hz, 2H), 7.07 (d, J = 7.0 Hz, 2H), 6.96 (s, 1H),

M. Alvarado et al. / Bioorg. Med. Chem. 16 (2008) 10098–10105
10105
(CH), 130.4 (CH), 129.3 (CH), 129.1 (CH), 128.1 (CH), 127.5 (C),
References and notes
107.0 (CH), 39.2 (CH2), 31.8 (CH2), 29.6–29.2 (CH2), 26.9 (CH2),
22.6 (CH2), 14.0 (CH3); Elemental analysis calcd (%) for
C32H42Cl3N3O (589.24): C 65.03, H 7.16, N 7.11, found: C: 65.18;
H: 7.35; N: 7.40.
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The title compound was prepared according to the previously
described general procedure by refluxing N-hexadecylamine
(0.88 g, 3.67 mmol), a commercial solution 2 M Al(CH3)3 in hep-
tane (1.83 mL, 3.67 mmol) and ethyl 5-(4-chlorophenyl)-1-(2,4-
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(0.30 g, 0.73 mmol) dissolved in dry CH2Cl2 during 96 h. The prod-
uct was purified by FC (SiO2; EtOAc/n-hexane 1:4) (79% yield).
White solid; mp: 64–5 °C (n-hexane); MS/EI: m/z (%) = 605
[M+1]+ (8), 603 (8), 240 (100); 1H NMR (300 MHz, CDCl3):
d = 7.43 (d, J = 1.9 Hz, 1H), 7.31–7.26 (m, 4H), 7.06 (d, J = 8.4 Hz,
2H), 3.39 (q, J = 6.7 Hz, 2H), 2.37 (s, 3H), 1.58–1.54 (m, 2H), 1.34–
1.25 (m, 26H), 0.89 (t, J = 6.8 Hz, 3H); 13C NMR (75 MHz, CDCl3):
d = 161.6 (CO), 144.1 (C), 141.9 (C), 134.9 (C), 134.8 (C), 133.8 (C),
132.0 (C), 129.8 (CH), 129.5 (CH), 129.3 (CH), 127.8 (CH), 126.8
(CH), 126.2 (C), 116.6 (C), 38.0 (CH2), 30.9 (CH2), 28.7–28.3 (CH2),
26.0 (CH2), 21.6 (CH2), 13.1 (CH3), 8.4 (CH3); Elemental analysis
calcd (%) for C33H44Cl3N3O (603.25): C 65.50, H 7.33, N 6.94, found:
C: 65.24; H: 7.46; N: 6.93.
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This work has been supported by Grants from Proyectos de
Excelencia de la Consejería de Innovación, Ciencia y Empresa,
Junta de Andalucía, Instituto de Salud Carlos III (FIS PI04/
0834, CP04/0039, FIS 07/1226, Redes Temáticas RD06/001), Fun-
dación Eugenio Rodríguez Pascual and Fundació Marató TV3.
MCyT, proyectos: MAT2006-01997, SAF2006-13391-C03-02 and
‘‘Factoría de Cristalización” Consolider Ingenio 2010. M. Alva-
rado is also grateful to the Xunta de Galicia (Spain) for a post-
doctoral fellowship.