Description of a bivalent cannabinoid ligand with hypophagic properties

Article abstract of DOI:10.1002/ardp.201200392

A series of bivalent cannabinoid ligands is proposed. The synthesis of double amides based on the rimonabant structure separated by an alkyl chain and the evaluation of their affinities for cannabinoid receptors are reported. The data of 4d confirmed that a bivalent structure is a suitable scaffold for CB₁ cannabinoid receptor binding. The compound 4d was selected for in vitro and in vivo pharmacological evaluations. Moreover, intraperitoneal administration of 4d to food-deprived rats resulted in a dose-dependent inhibition of feeding that was maintained up to 240 min. A series of bivalent cannabinoid ligands was synthesized. The data of 4d confirmed that a bivalent structure is a suitable scaffold for CB₁ cannabinoid receptor binding. The compound 4d was selected for in vitro and in vivo pharmacological evaluations. Moreover, intraperitoneal administration of 4d to food-deprived rats resulted in a dose-dependent inhibition of feeding that was maintained up to 240 min.

Full text of DOI:10.1002/ardp.201200392

Arch. Pharm. Chem. Life Sci. 2013, 346, 171–179
171
Full Paper
Description of a Bivalent Cannabinoid Ligand with
Hypophagic Properties
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Cristina Fernandez-Fernandez , Juan Decara , Francisco J. Bermudez-Silva , Eva Sanchez ,
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Paula Morales , Marıa Gomez-Canas , Marıa Gomez-Ruız , Luis F. Callado , Pilar Goya ,
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Fernando Rodrıguez de Fonseca , M. Isabel Martın , Javier Fernandez-Ruız , J. Javier Meana , and
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Nadine Jagerovic1
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Instituto de Quımica Medica, CSIC, Madrid, Spain
Laboratorio de Medicina Regenerativa, Hospital Carlos Haya, Fundacion IMABIS, Malaga, Spain
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Facultad de Ciencias de la Salud, Dpto de Farmacologıa y Nutricion, Unidad Asociada de IþDþI al CSIC,
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Universidad Rey Juan Carlos, Alcorcon, Madrid, Spain
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Facultad de Medicina, Dpto de Bioquımica y Biologıa Molecular, Centro de Investigacion Biomedica en
Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto Ramon y Cajal de Investigacion
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Sanitaria (IRYCIS), Universidad Complutense de Madrid, Madrid, Spain
Department of Pharmacology, University of the Basque Country (UPV/EHU), Centro de Investigacion
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Biomedica en Red de Salud Mental (CIBERSAM), Leioa, Bizkaia, Spain
A series of bivalent cannabinoid ligands is proposed. The synthesis of double amides based on the
rimonabant structure separated by an alkyl chain and the evaluation of their affinities for
cannabinoid receptors are reported. The data of 4d confirmed that a bivalent structure is a
suitable scaffold for CB1 cannabinoid receptor binding. The compound 4d was selected for in vitro
and in vivo pharmacological evaluations. Moreover, intraperitoneal administration of 4d to food-
deprived rats resulted in a dose-dependent inhibition of feeding that was maintained up to 240 min.
Keywords: Bivalent ligand / Cannabinoid receptors / CB1 / Food intake / Rimonabant
Received: October 10, 2012; Revised: December 20, 2012; Accepted: December 21, 2012
DOI 10.1002/ardp.201200392
Introduction
Within this context and in continuation to our previous
work on multivalent ligands such as dual cannabinoid/
PPARa [9], opioid/I2 imidazoline [10], and cannabinoid/opioid
ligands [11], we report here our attempt to prepare and
evaluate a series of homobivalent cannabinoids, which could
target CB1 homodimer receptors simultaneously. They are
composed of two functional pharmacophores linked by a
spacer [12]. The two structural motives belong to rimonabant
and N-(1-heptyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-carboxamide 8 (Fig. 1). Rimonabant
was the first CB1 antagonist/inverse agonist to be launched
as anti-obesity agent, but which was later withdrawn due to
an increase in psychiatric disorders [13]. The heptylcarbox-
amide derivative 8 was prepared by Thomas et al. [14] in an
extensive study dealing with different amide derivatives of
rimonabant. Therefore, we propose a series of double amides
based on the rimonabant structure separated by an alkyl
chain of several methylene units (4). These rimonabant
related compounds can, in principle, have a different phar-
macokinetic profile and thus reduced side effects which are
The multivalent approach to drug design has proved
successful in the discovery of dimeric or hybrid drug candi-
dates [1]. Opioid [2], adrenergic [3], serotoninergic [4], and
dopaminergic [5] receptors have been the most explored
systems for this approach. One of the pioneer groups to
synthesize bivalent opioid ligands was Portoghese‘s team
in the early 1980s [6]. Considering that CB1 cannabinoid
receptors belong to the superfamily of the G protein-coupled
receptors (GPCRs) and that homo- and heterodimers have
been recently suggested among GPCRs [7], it is not surprising
that evidences for dimerization of CB1 receptors have already
been reported [8].
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Correspondence: Nadine Jagerovic, Instituto de Quımica Medica, CSIC,
c/Juan de la Cierva, 3, E-28006 Madrid, Spain.
E-mail: nadine@iqm.csic.es
Fax: þ34915644853
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Arch. Pharm. Chem. Life Sci. 2013, 346, 171–179
Figure 1. Structures of rimonabant, the heptylcarboxamide analogue 8 and the bivalent compounds 4a–4h.
the main limitation of rimonabant. Thus, in this paper we
report their synthesis together with their preliminary
pharmacological evaluation, which includes in vitro binding
measurements, functional assays, and in vivo food intake
studies and cannabinoid behavioural tetrad tests.
In addition, we synthesized the compounds 6 and 7, which
are the monovalent counterparts of 4d. Compound 6 was
prepared starting from the acid chloride 3 by reacting with
1,7-diaminoheptane that was previously mono-protected by
a Boc group. Thus, the butoxycarbonylaminoheptyl deriva-
tive 5 was treated with TFA followed by basic extraction of
the free amine to afford 6 (Scheme 2). The ethylcarboxamide
derivative 7 was obtained by acylation of 6 with propionyl
chloride in the presence of potassium bicarbonate and TEBA
as phase transfer catalyst.
The present compounds are disclosed in a Spanish patent
[12]. While this patent was extended to PCT, related rimona-
bant bivalent ligands were reported by Thomas and co-
workers [15]. Their structure differs from ours by the nature
of the linker which are aminoalkyl chains. They showed
interesting cannabinoid CB1 receptor affinities, which
encouraged us to complete the pharmacological evaluation
of our new compounds.
All synthesized pyrazole derivatives 4a–4h were tested for
their respective affinities for CB1 and CB2 receptors. This
evaluation was first performed using human CB1 or CB2
receptor transfected cells. Unfortunately, none of the com-
pounds showed significant affinity. Moreover, some of them,
4c and 4d, could not be tested due to their very low solubility
in the preliminary displacement assay at 40 mM.
Results
Compounds 4a–4h were synthesized as shown in Scheme 1.
Ethyl 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazole-3-carboxylate (1) was prepared according to the pro-
cedure described by Krishnamurthy et al. [16]. Basic hydroly-
sis of 1 in ethanol gave the carboxylic acid 2 which by
treatment with thionyl chloride in refluxing toluene gave
the acid chloride 3. Finally, 3 reacted with different di-
aminoalkanes in the presence of TEA to afford the desired
compounds 4a–4h.
We decided to select one of them, 4d, to evaluate its bind-
ing in a different assay. Prefrontal cortex membranes from
post-mortem human brain was used as source of CB1 recep-
tors in competitive displacement assays with [3H]-CP55940 as
radioligand. Compound 4d showed relatively high affinity to
the CB1 receptor with an affinity constant (Ki) value in the
nanomolar range. Cannabinoid binding of 6 and 7, monomer
analogues of 4d, was also carried out in post-mortem human
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Bivalent Cannabinoid Ligand with Hypophagic Properties
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Scheme 1. Reagents and conditions: (a) NaOH, EtOH, H2O; (b) SOCl2, toluene, reflux; (c) corresponding diaminoalkane, TEA, CH2Cl2.
Scheme 2. Reagents and conditions: (a) tert-butyl-7-aminoheptylcarbamate, TEA, CH2Cl2; (b) TFA, CH2Cl2; (c) Et2O, NaOH;
(d) propionyl chloride, K2CO3, TEBA, CH3CN.
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Table 1. CB1 binding affinity constants of 4d, 6, 7, and the
reference CB1 cannabinoid ligand rimonabant determined by
competitive displacement assays in prefrontal cortex membranes
from post-mortem human brain using [3H]-CP55940 as radioligand.
Compound
Ki (nM) CB1
binding [3H]-CP55940
4d
6
7
53 ꢁ 5
1629 ꢁ 75
635 ꢁ 51
8
Rimonabant
291 ꢁ 69.7a)
3.73 ꢁ 2.5
Figure 2. Acute i.p. administration of 4d (0.3 or 10 mg/kg) to 24-h
food-deprived rats resulted in dose dependent reduction in feeding
behaviour. Data are means ꢁ SEM of eight determinations per
group.
Values are expressed as mean ꢁ standard error of the mean
(SEM) of three different experiments.
Ref. [14].
a)
brain to establish preliminary structure–activity relation-
ships (SAR) (Table 1).
mice [18]. Nociception, temperature, and catalepsy are the
tested parameters in this assay. Intraperitoneal adminis-
tration (up to 5 mg/kg) of 4d did not produce significant
modification in any of these parameters discarding agonistic
activity (Fig. 3). It neither did significantly antagonize the
effects produced by the cannabinoid agonist WIN 55,212-2.
A comparison of the binding data of the N-heptyl carbox-
amide pyrazole derivative 8 (Fig. 1) reported by Thomas et al.
[14] and the N-aminoheptyl carboxamide pyrazole derivative
6 suggests that the CB1 receptor binding site does not
tolerate the presence of an amine on the alkyl chain. It is
also interesting to note that the replacement of one pyrazole Discussion
ring of 4d with an ethyl group (7) is not well tolerated in the
CB1 receptor.
A series of double amides based on the rimonabant structure
Since 4d showed to have good affinity for the CB1 canna-
binoid receptor, its functionality was evaluated in isolated
tissue assays. Inhibition of electrically induced contractions
by cannabinoid agonists has been described in mouse vas
deferens [17]. CB1 and CB2-like cannabinoid receptors seem to
be involved in this effect. WIN 55,212-2 is able to induce dose-
dependent inhibition in this assay and its effect is antago-
nized by the cannabinoid AM251. In this isolated tissue, 4d
did not induce significant modification of the contractile
response (10ꢀ7–10ꢀ4 M). These data show that 4d lacks ago-
nist activity on cannabinoid receptors, but also on adrener-
gic, purinergic, and opioid receptors present in this tissue.
Compound 4d did not either induce a significant decrease of
the inhibition evoked by the cannabinoid agonist WIN
55,212-2, suggesting that 4d does not behave as cannabinoid
antagonist in this assay.
separated by an alkyl chain of 3–12 methylenes was synthes-
ized. From the tested compounds 4a, 4b, and 4d–4h, the
bivalent compound 4d showed good nanomolar affinity
for the CB1 receptor in prefrontal cortex membranes from
post-mortem human brain. It suggests that the linker length
of seven methylenes is optimum for the interaction with
receptor binding site. Interestingly, the data of the N-amino-
heptyl carboxamide pyrazole derivative 6 and its acylated
compound 7 show less activity than for the bis-pyrazolecarbox-
amide 4d. Then, it is worth noting that the presence of a second
diarylpyrazole increases the ability of 4d to bind to the CB1
receptor. This fact supports the hypothesis that the bivalent
ligand 4d could be interacting with a CB1 receptor homodimer.
It is worthy to note that among the bivalent rimonabant
derivative ligands with triamine linkers reported by Thomas
and coworkers [15], the peak affinity was obtained for the
bivalent ligand having a 15 atoms linker.
At the same time, we evaluated the acute effect of 4d on the
feeding behaviour in male Wistar rats deprived of food for
24 h. Intraperitoneal administration of 4d at 0.3 and 10 mg/
kg to food-deprived rats resulted in a dose-dependent inhi-
bition of feeding as shown in Fig. 2. For a possible therapeutic
application as appetite suppression, it is worthy to note that
this action was maintained up to 240 min.
Taking into account the CB1 binding data and the hypo-
phagic behaviour of 4d, a potential interpretation of the
nociception, temperature, and catalepsy tests is that this
compound might have a limited permeation through the
blood brain barrier. Even though we have demonstrated that
rimonabant has more permeation [19] through blood brain
barrier than initially expected, it antagonized the effects of
WIN 55,212-2 in these three behavioural tests using similar
Compound 4d was also tested using a multiple in vivo assay
to evaluate the central cannabinoid activity in male CD-1
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Bivalent Cannabinoid Ligand with Hypophagic Properties
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Figure 3. Effect of i.p. administration of WIN 55,212-2 (WIN) (2.5 mg), 4d (5 mg), and WIN 55,212-2 (2.5 mg) in presence of AM251 (AM)
(1 mg) or 4d (5 mg) in antinociception, hypothermia, and catalepsy. Bars show the mean ꢁ SEM (n ¼ 10). (ꢃꢃ) p < 0.01, (ꢃꢃꢃ) p < 0.001
versus vehicle (VEH); (þþ) p < 0.01, (þþþ) p < 0.001 versus WIN 55,212-2.
experimental conditions [20]. It is interesting to note that Experimental
compound 4d differs from rimonabant in nociception,
temperature, and catalepsy tests but not in feeding behaviour
using acute administration [19] in 24 h food-deprived male
Wister rats. On the other hand, 4d that showed anorectic
activity, exhibited good CB1 cannabinoid receptor affinity
but did not significantly modulate this receptor in our assays.
Therefore, 4d might reduce food intake through other mech-
anism which deserves further investigation. Recently, Tam
et al. [21] showed that peripheral CB1 cannabinoid receptor
inverse agonism reduces obesity by reversing leptin resist-
ance. Since 4d is a limited brain-penetrant cannabinoid,
appetite and weight reduction could be mediated by a mech-
anism involving leptin. Thus, further studies will be oriented
to CB1 inverse agonism at peripheral CB1 receptors.
Chemistry
All starting materials were commercially available research
grade chemicals and used without further purification.
Dichloromethane was distilled over CaCl2. Medium pressure
flash column chromatography was carried out on a FlashMaster
Personal system (Biotage1) with prepacked cartridges FlashPack
of 2, 10, 20, or 50 g. Melting points were determined on a
Reichert Jung Thermovar apparatus. 1H NMR spectra were
recorded on a Varian Gemini [200 MHz (1H), 50 MHz (13C)] spec-
trometer, Varian Inova 300 [(300 MHz (1H), 75 MHz (13C)] spec-
trometer, Varian Inova 400 [400 MHz (1H), 100 MHz (13C)]
spectrometer and Varian Unity 500 [500 MHz (1H), 125 MHz
(
13C)] spectrometer with TMS as internal reference. Mass spectra
were determined on a MSD-Serie 1100 Hewlett Packard apparatus.
Microanalyses were performed with a Heraeus CHN–O Rapid
analysis. LC/MS analyses were carried out on a Waters 2695 photo-
diode array with Micromass ZQ positive electrospray unit, using
Waters column C18–3.5 mm (2.1 mm ꢂ 100 mm) with a mixture
of acetonitrile (ACN, 0.08% formic acid) and MilliQ water (0.05%
formic acid) as mobile phase in the corresponding gradient.
Conclusion
In summary, the synthesis and radioligand binding assays of
bivalent cannabinoid ligands with structures referred to
rimonabant led to the discovery of the bis-pyrazolecarbox-
amide 4d. Indeed, the acute effect of 4d in food-deprived rats
resulted in inhibition of behaviour feeding that was main-
tained up to 240 min. Although slightly less potent than
rimonabant itself, the bivalent cannabinoid 4d can be con-
sidered an alternative lead in this field, since it may have
different side effects. This issue together with modifications
in its structure, such as variations in the nature and length of
the linker, deserve further research.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazole-3-carboxylate acid (2)
To a solution of 1 [16] (120 mg, 0.29 mmol) in EtOH (10 mL) and
water (10 mL) was added NaOH (116 mg, 2.9 mmol). The reaction
mixture was stirred at room temperature for 3 h. It was then
acidified with 1 M HCl and extracted with EtOAc (3 ꢂ 10 mL).
The combined organic layers were washed with brine, dried over
MgSO4 and the solvent was evaporated to give the pyrazole (2) as
a yellowish solid (115 mg). Yield 99%. m.p. ¼ 205–2088C. 1H NMR
(CD3OD, 300.13 MHz) d (ppm): 7.76 (d, 1H, J ¼ 2.2 Hz); 7.72 (d, 1H,
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J ¼ 8,5 Hz); 7.63 (dd, 1H, J ¼ 8.5 Hz, J ¼ 2.2 Hz); 7.56 (d, 2H,
J ¼ 8.3 Hz); 7.40 (d, 2H, J ¼ 8.3 Hz); 2.49 (s, 3H). 13C NMR
(CD3OD, 75.46 MHz) d (ppm): 165.4; 144.7; 144.3; 137.6; 137.1;
136.3; 134.2; 132.5; 131.1; 129.9; 129.8; 129.3; 128.4; 120.4;
9.8. MS (ESþ) m/z: 380 (100%) [MþH]þ. LC/MS (ACN/H2O 95:5)
purity 99%.
(m, 4H). 13C NMR (CDCl3, 125.69 MHz) d (ppm): 162.6; 145.0;
142.9; 135.9; 135.8; 134.8; 132.9; 130.8; 130.5; 130.3; 128.8;
127.8; 127.2; 117.6; 38.8; 29.6; 26.6; 9.4. MS (ESþ) m/z: 845
(100%) [M(35Cl6)þH]þ. Anal. calcd. for (C40H34Cl6N6O2): C,
56.96; H: 4.06; N: 9.96. Found: C, 56.70; H, 4.10; N: 9.90.
N,N0-Heptan-1,7-diylbis[5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-1H-pyrazole-3-carboxamide] (4d)
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazole-3-carboxylate acid chloride (3)
4d was prepared from 3 (4 mmol), 1,7-diaminoheptane (260 mg,
2 mmol) and TEA (556 mL, 4 mmol). Yield 1.00 g (60%) as a yellow
oil. 1H NMR (CDCl3, 499.81 MHz) d (ppm): 7.33 (m, 2H); 7.21–7.16
(m, 8H); 6.97 (d, 4H, J ¼ 8.5 Hz); 6.88 (m, 2H, NH); 3.31 (q, 4H,
J ¼ 6.6 Hz); 2.28 (s, 6H); 1.50 (p, 4H, J ¼ 7.3 Hz); 1.28 (m, 6H).
13C NMR (CDCl3, 125.69 MHz) d (ppm): 162.6; 145.0; 143.0; 135.9;
135.8; 134.7; 132.8; 130.7; 130.4; 130.1; 128.7; 127.7; 127.1; 117.5;
38.8; 29.5; 28.9; 26.8; 8.4. MS (ESþ) m/z: 857 (52%) [M(35Cl6)þNa]þ.
LC/MS (ACN/H2O 80:20–100:0, 5 min gradient) tR ¼ 5.86 min,
purity 94%. Anal. calcd. for (C41H36Cl6N6O2): C, 57.43; H: 4.23;
N: 9.80. Found: C, 57.25; H, 4.37; N: 9.61.
Thionyl chloride (2 mL, 27.6 mmol) was added to a solution of 2
(115 mg, 0.29 mmol) in toluene (6 mL). The reaction mixture was
heated to reflux for 3 h. Then the solvent was evaporated under
reduced pressure. The residue was redissolved in toluene (5 mL)
and the solvent was again evaporated to afford the crude com-
pound 3 that was used in the next step without further
purification.
General procedure for the synthesis of 4a–4h
Acid chloride 3 (2 equiv) dissolved in CH2Cl2 (20 mL) was added
dropwise to a stirred solution of the corresponding diamino-
alkane (1 equiv) and TEA (2 equiv) in dry CH2Cl2 (50 mL) at 08C.
The reaction mixture was stirred at room temperature over-
night. The solvent was evaporated under reduced pressure and
the crude product was purified by medium pressure flash
chromatography (CH2Cl2/MeOH/satNH3 100:1).
N,N0-Octan-1,8-diylbis[5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-1H-pyrazole-3-carboxamide] (4e)
4e was prepared from 3 (0.40 mmol), 1,8-diaminooctane (29 mg,
0.20 mmol) and TEA (55 mL, 0.40 mmol). Yield 23 mg (13%) as a
yellow oil. 1H NMR (CDCl3, 499.81 MHz) d (ppm): 7.39 (d, 2H,
J ¼ 1.7 Hz); 7.25 (m, 8H); 7.03 (d, 4H, J ¼ 8.3 Hz); 6.91 (m, 2H);
3.37 (q, 4H, J ¼ 6.8 Hz); 2.30 (s, 6H); 1.51 (p, 4H, J ¼ 6.8 Hz); 1.31
(m, 10H). 13C NMR (CDCl3, 125.69 MHz) d (ppm): 162.6; 145.1;
142.9; 136.0; 135.9; 134.9; 133.0; 130.8; 130.5; 130.3; 128.9; 127.8;
127.2; 117.7; 38.9; 29.7; 29.1; 26.6; 9.4. MS (ESþ) m/z: 895 (100%)
[M(35Cl6)þNa]þ. Anal. calcd. for (C42H38Cl6N6O2 ꢄ 3H2O): C, 54.50;
H: 4.79; N: 9.08. Found: C, 54.71; H, 4.61; N: 8.97.
N,N0-Propan-1,3-diylbis[5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-1H-pyrazole-3-carboxamide] (4a)
4a was prepared from 3 (0.40 mmol), 1,3-diaminopropane
(15 mg, 0.20 mmol) and TEA (55 mL, 0.40 mmol). Yield 106 mg
(66%) as a yellow solid. m.p. ¼ 127–1308C. 1H NMR (CDCl3,
499.81 MHz) d (ppm): 7.40 (m, 2H); 7.28 (m, 8H); 7.21 (m, 2H);
7.05 (d, 4H, J ¼ 8.5 Hz); 3.52 (q, 4H, J ¼ 6.5 Hz); 2.35 (s, 6H); 1.90
(p, 2H, J ¼ 6.5 Hz); 13C NMR (CDCl3, 125.69 MHz) d (ppm): 162.9;
144.9; 142.9; 135.9; 135.8; 134.8; 132.9; 130.7; 130.5; 130.2;
128.8; 127.8; 127.2; 117.6; 36.3; 30.0; 9.3. MS (ESþ) m/z: 801
(100%) [M(35Cl6)þH]þ. Anal. calcd. for (C37H28Cl6N6O2 ꢄ H2O):
C, 54.23; H: 3.69; N: 10.26. Found: C, 54.31; H, 3.57; N: 9.89.
N,N0-Nonan-1,9-diylbis[5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-1H-pyrazole-3-carboxamide] (4f)
4f was prepared from 3 (0.40 mmol), 1,9-diaminononane (32 mg,
0.20 mmol) and TEA (55 mL, 0.40 mmol). Yield 120 mg (70%) as a
yellow oil. 1H NMR (CDCl3, 499.81 MHz) d (ppm): 7.35 (d, 2H,
J ¼ 1.9 Hz); 7.20 (m, 8H); 6.96 (d, 4H, J ¼ 8.5 Hz); 6.87 (m, 2H);
3.32 (q, 4H, J ¼ 6.8 Hz); 2.30 (s, 6H); 1.51 (p, 4H, J ¼ 7.1 Hz); 1.23
(m, 10H). 13C NMR (CDCl3, 125.69 MHz) d (ppm): 162.6; 145.1;
142.9; 135.9; 135.8; 134.8; 132.9; 130.8; 130.5; 130.3; 128.8; 127.8;
127.2; 117.6; 39.0; 29.7; 29.4; 29.2; 26.9; 9.4. Anal. calcd. for
(C43H40Cl6N6O2 ꢄ 4H2O): C, 53.93; H: 5.05; N: 8.78. Found: C,
53.38; H, 4.50; N: 9.01.
N,N0-Butan-1,4-diylbis[5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-1H-pyrazole-3-carboxamide] (4b)
4b was prepared from 3 (0.25 mmol), 1,4-diaminobutane (11 mg,
0.12 mmol) and TEA (17 mL, 0.12 mmol). Yield 52 mg (51%) as a
yellow oil. 1H NMR (CDCl3, 300.13 MHz) d (ppm): 7.35 (m, 2H);
7.22 (m, 8H); 6.98 (d, 4H, J ¼ 8.3 Hz); 6.93 (m, 2H); 3.39 (m, 4H);
2.28 (s, 6H); 1.63 (m, 4H); 13C NMR (CDCl3, 75.46 MHz) d (ppm):
163.1; 145.4; 143.4; 136.3; 135.3; 133.4; 131.2; 130.9;
130.7; 130.2; 129.3; 128.3; 127.6; 118.0; 39.0; 27.7; 9.8. MS
(ESþ) m/z: 817 (100%) [M(35Cl6)þNa]þ. Anal. calcd. for
(C38H30Cl6N6O2 ꢄ 4H2O): C, 51.43; H: 4.32; N: 9.47. Found: C,
51.03; H, 3.65; N: 9.85.
N,N0-Decan-1,10-diylbis[5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-1H-pyrazole-3-carboxamide] (4g)
4g was prepared from 3 (0.40 mmol), 1,10-diaminodecane
(34 mg, 0.20 mmol) and TEA (55 mL, 0.40 mmol). Yield 107 mg
(60%) as a yellow oil. 1H NMR (CDCl3, 499.81 MHz) d (ppm):
7.35 (d, 2H, J ¼ 1.9 Hz); 7.21 (d, 4H, J ¼ 8.5 Hz); 7.20 (m, 8H);
6.87 (m, 2H); 3.32 (q, 4H, J ¼ 6.8 Hz); 2.30 (s, 6H); 1.51 (p, 4H,
J ¼ 7.3 Hz); 1.24 (m, 12H). 13C NMR (CDCl3, 125.69 MHz) d (ppm):
162.6; 145.1; 142.9; 135.9; 135.8; 134.8; 132.9; 130.8; 130.5;
130.3; 128.8; 127.8; 127.2; 117.6; 39.0; 29.7; 29.4; 26.9; 9.4.
MS (ESþ) m/z: 922 (100%) [M(35Cl6)þNa]þ. Anal. calcd. for
(C44H42Cl6N6O2 ꢄ 2H2O): C, 56.49; H: 4.96; N: 8.98. Found: C,
56.28; H, 4.66; N: 9.72.
N,N0-Hexan-1,6-diylbis[5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-1H-pyrazole-3-carboxamide] (4c)
4c was prepared from 3 (0.40 mmol), 1,6-diaminohexane (23 mg,
0.20 mmol) and TEA (55 mL, 0.40 mmol). Yield 144 mg (86%) as a
yellow oil. 1H NMR (CDCl3, 499.81 MHz) d (ppm): 7.34 (d, 2H,
J ¼ 1.7 Hz); 7.20 (m, 8H); 6.98 (d, 4H, J ¼ 8.3 Hz); 6.89 (m, 2H);
3.33 (q, 4H, J ¼ 6.8 Hz); 2.29 (s, 6H); 1.53 (p, 4H, J ¼ 6.8 Hz); 1.34
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Arch. Pharm. Chem. Life Sci. 2013, 346, 171–179
Bivalent Cannabinoid Ligand with Hypophagic Properties
177
N,N0-Dodecan-1,12-diylbis[5-(4-chlorophenyl)-1-(2,4-
7.03 (d, 2H, J ¼ 8.5 Hz); 6.93 (t, 1H, J ¼ 5.7 Hz); 5.28 (brs, 1H); 3.38
(q, 2H, J ¼ 6.6 Hz); 3.21 (q, 2H, J ¼ 6.9 Hz); 2.34 (s, 3H); 2.17 (q, 2H,
J ¼ 7.6 Hz); 1.66–1.55 (m, 2H); 1.51–1.41 (m 2H); 1.32 (brs, 6H);
1.12 (t, 3H, J ¼ 7.6 Hz). 13C RMN (CDCl3, 100.55 MHz) d (ppm):
174.7; 163.5; 145.9; 143.9; 136.7; 135.7; 133.2; 131.6; 131.4; 131.1;
130.4; 129.7; 128.7; 128.0; 118.5; 53.7; 40.2; 39.7; 30.6; 30.4; 29.7;
27.6; 10.8; 9.1. MS (ESþ) m/z: 589 (100%) [M(35Cl3)þK]þ. LC/MS
(ACN/H2O 95:5) tR ¼ 35.6 min, purity 99%.
dichlorophenyl)-1H-pyrazole-3-carboxamide] (4h)
4h was prepared from 3 (0.40 mmol), 1,12-diaminododecane
(40 mg, 0.20 mmol) and TEA (55 mL, 0.40 mmol). Yield 158 mg
(85%) as a yellow oil. 1H NMR (CDCl3, 499.81 MHz) d (ppm):
7.42 (d, 2H, J ¼ 2.1 Hz); 7.27 (m, 8H); 7.06 (d, 4H, J ¼ 8.6 Hz);
6.93 (m, 2H); 3.41 (q, 4H, J ¼ 6.8 Hz); 2.37 (s, 6H); 1.58–1.25
(m, 16H). 13C NMR (CDCl3, 125.69 MHz) d (ppm): 162.8; 145.3;
143.2; 136.2; 136.1; 135.1; 133.2; 131.0; 130.7; 130.5;
129.1; 128.0; 127.5; 117.9; 39.3; 30.0; 29.8; 29.7; 29.5; 27.2; 9.7.
MS (ESþ) m/z: 928 (100%) [M(35Cl6)þH]þ. Anal. calcd. for
(C46H46Cl6N6O2 ꢄ 4H2O): C, 55.75; H: 4.78; N: 8.30. Found: C,
55.72; H, 4.90; N: 8.78.
Pharmacology
Binding assays
Post-mortem human brain binding assays
CB1 binding assay of compounds 4d, 6, and 7 was performed on
post-mortem human brain membranes. [3H]-CP55940 radioli-
gand (specific activity 160.6 Ci/mmol) was purchased from
PerkinElmer, USA. Neural membranes (P2 fractions) were pre-
pared from the prefrontal cortex of human brains obtained at
autopsy in the Instituto Vasco de Medicina Legal, Bilbao, Spain.
Post-mortem human brain samples of each subject (ꢅ1 g) were
homogenized using a Teflon-glass grinder (10 up-and-down
strokes at 1500 rpm) in 30 volumes of homogenization buffer
(1 mM MgCl2 and 5 mM Tris–HCl, pH 7.4) supplemented with
0.25 M sucrose. The crude homogenate was centrifuged for
5 min at 1000 ꢂ g (48C) and the supernatant was centrifuged
again for 10 min at 40 000 ꢂ g (48C). The resultant pellet was
washed twice in 20 volumes of homogenization buffer and
recentrifuged in similar conditions. The resulting pellet was
additionally incubated at 378C for 15 min to remove the
endogenous cannabinoids. Aliquots of 1 mg of protein were
stored at ꢀ708C until assay. Protein content was measured
according to the Bradford method using BSA as standard, and
was similar in the different brain samples. Specific [3H]-CP55940
binding was measured in 0.55-mL aliquots (50 mM Tris–HCl, pH
7.5) of the neural membranes. Membranes were incubated with
[3H]CP55-940 (1 nM) for 60 min at 308C in the absence or pres-
ence of the competing compounds (10ꢀ12–10ꢀ3 M, ten concen-
trations). Incubations were terminated by diluting the samples
with 5 mL of ice-cold Tris incubation buffer (48C). Membrane
bound radioligand was separated by vacuum filtration through
Whatman GF/C glass fibre filters. Then, the filters were rinsed
twice with 5 mL of incubation buffer and transferred to mini-
vials containing 3 mL of OptiPhase ‘‘HiSafe’’ II cocktail and
counted for radioactivity by liquid scintillation spectrometry.
Specific binding was determined and plotted as a function of the
compound concentration. Non-specific binding was determined
in the presence of WIN 55,212-2 (10ꢀ5 M). Analysis of compe-
tition experiments to obtain the inhibition constant (Ki) was
performed by nonlinear regression using the GraphPad Prism
program. All experiments were analysed assuming a one-site
model of radioligand binding.
N-(tert-Butoxycarbonylaminoheptyl)-5-(4-chlorophenyl)-1-
(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (5)
To a solution of tert-butyl 7-aminoheptylcarbamate (103 mg;
0.45 mmol) and TEA (62 mL, 0.45 mmol) in CH2Cl2 (7 mL) at
08C, was added dropwise 3 solved in CH2Cl2 (2 mL). The reaction
mixture was stirred at room temperature for 18 h. Afterwards,
the solvent was removed under reduced pressure and the crude
was purified by medium pressure chromatography to afford (5)
as
a
yellowish oil (106 mg). Yield 80%. 1H NMR (CDCl3,
399.90 MHz) d (ppm): 7.40–7.02 (m, 7H); 6.91 (m, 1H); 4.51
(m, 1H), 3.38 (q, 2H, J ¼ 6.6 Hz); 3.07 (m, 2H); 2.35 (s, 3H); 1.53
(m, 2H); 1.41 (s, 9H); 1.31 (m, 8H). 13C NMR (CDCl3, 100.55 MHz)
d (ppm): 162.6; 145.0; 143.0; 135.9; 134.8; 132.9; 130.8; 130.5;
130.3; 128.8; 127.9; 127.2; 117.6; 79.0; 40.5; 38.9; 30.0; 29.6; 28.9;
28.4; 26.9; 26.5; 9.4. MS (ESþ) m/z: 593 (100%) [M(35Cl3)þH]þ. LC/MS
(ACN/H2O 50:50) tR ¼ 69.6 min, purity 96%. Anal. calcd. for
(C29H35Cl3N4O3 ꢄ 2.5H2O): C, 57.94; H: 6.00; N: 9.32. Found: C,
58.14; H, 6.04; N: 8.86.
N-(Aminoheptyl)-5-(4-chlorophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (6)
A solution of 5 (130 mg, 0.195 mmol) in 50% TFA/CH2Cl2 (60 mL)
was stirred at room temperature for 30 min. Afterwards, the
solvent was evaporated under reduced pressure. The crude was
dissolved in diethylether (8 mL) and the solution was stirred with
10% NaOH (2 mL) at room temperature for 1 h. The organic
phase was dried over Mg2SO4 and evaporated to afford (6)
(100 mg, 0.18 mmol) as a yellowish oil. Yield 93%. 1H NMR
(CDCl3, 200 MHz) d (ppm): 8.07 (brs, 1H); 7.39–7.02 (m, 7H);
3.35 (q, 2H, J ¼ 6.8 Hz); 2.94 (m, 2H); 2.30 (s, 3H); 1.82–
1.23 (m, 10H). MS (ESþ) m/z: 493 (100%) [M(35Cl3)þH]þ. LC/MS
(ACN/H2O 95:5) tR ¼ 8.8 min, purity 96%.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-[7-
(propionylamino)heptyl]-1H-pyrazole-3-carboxamide (7)
To a solution of 6 (55 mg; 0.09 mmol) in ACN (5 mL) was
added K2CO3 (18 mg, 0.13 mmol) and benzyltriethylammonium
chloride (TEBA, 7 mg, 0.03 mmol). The reaction mixture was
stirred at room temperature for 10 min. Then, propionyl
chloride (14 mL, 0.13 mmol) was added and the mixture was
heated to reflux for 16 h. The precipitate was filtered over celite
and washed with EtOAc. The combined liquid layers were
evaporated under reduced pressure and the crude was purified
on silica gel by a medium pressure column chromatography
to afford (7) as a yellowish oil (26 mg). Yield 20%. 1H NMR
(CDCl3, 399.90 MHz) d (ppm): 7.40 (brs, 1H); 7.30–7.23 (m, 4H);
Human transfected cells binding assays
CB1 binding assay of compounds 4a–4c and 4e–4h, and CB2
binding assay of compounds 4a–4h were performed on human
CB1 receptor and CB2 receptor transfected cell membranes,
respectively. Membranes from transfected cells expressing
human CB1 or CB2 cannabinoid receptors (RBHCB1M400UA
and RBXCB2M400UA) were supplied by PerkinElmer Life and
Analytical Sciences (Boston, MA, USA). The protein concentration
for the CB1 receptor membranes was 8.4 mg/mL, whereas for the
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C. Fernandez-Fernandez et al.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 171–179
CB2 receptor membranes this concentration was 3.6 mg/mL.
The commercial membranes were diluted (ꢅ1:20) with binding
buffer (50 mM Tris–Cl, 5 mM MgCl2 ꢄ H2O, 2.5 mM EDTA,
0.5 mg/mL BSA, and pH 7.4 for CB1 binding buffer; 50 mM
Tris–Cl, 5 mM MgCl2 ꢄ H2O, 2.5 mM EGTA, 1 mg/mL BSA,
and pH 7.5 for CB2 binding buffer). The radioligand used was
[3H]-CP55940 (PerkinElmer) at a concentration of membrane
kD ꢂ 0.8 nm, and the final volume was 200 mL for CB1 binding
and 600 mL for CB2 binding; 96-well plates and tubes necessary
for the experiment were previously siliconized with Sigmacote
(Sigma). Membranes were resuspended in the corresponding
buffer and were incubated with the radioligand and test com-
pound at 40 mM for 90 min at 308C. Non-specific binding was
determined with 10ꢀ5 M WIN 55,212-2, and 100% binding of
the radioligand to the membrane was determined by its
incubation with membrane without test compound. Filtration
was performed by a Harvester filtermate (PerkinElmer) with
Filtermat A GF/C filters pretreated with 0.05% polyethylenimine.
After filtering, the filter was washed with binding buffer
(9 ꢂ 0.2 mL for CB1 and 9 ꢂ 0.6 mL for CB2), dried, and a melt-
on scintillation sheet (Meltilex A, PerkinElmer) was melted onto
it. Radioactivity was then quantified by liquid scintillation
spectrophotometry (Wallac MicroBeta Trilux, PerkinElmer).
Radioligand displacement was then determined. When the per-
centage was superior to 60%, the affinity constants (Ki) were then
evaluated using the new compound at eight concentrations
(10ꢀ11–10ꢀ4 M). Competition binding data were analysed by
using GraphPad Prism program; Ki values are expressed as the
mean ꢁ SEM of at least three experiments performed in tripli-
cate for each point.
Cannabinoid behavioural tests
ICR male mice weighing 25–30 g (n ¼ 8–12) were used. WIN
55,212-2 and 4d dissolved in saline solution and Tween 80 were
administered 30 min before starting the cannabinoid behaviou-
ral tests to evaluate their agonistic effects. When 4d was tested as
an antagonist it was administered 20 min before the reference
agonist, WIN 55,212-2, and was compared to the reference
antagonist AM251. Tests were consecutively conducted with
5 min of interval between them by following: 1-Hypothermia:
Rectum temperature; 2-Nociception: Hot plate at 558C as
nociceptive stimulus. The latency time of licking of the front
paw was taken as an index of nociception. Antinociceptive effect
is expressed as maximum possible effect (M.P.E.); 3-Catalepsy:
Modified ‘ring test’ as originally described by Pertwee [22].
Throughout the experimental procedure, the European
Communities Council Directive of 24 November 1986 (86/609
EEC, Nov. 24, 1986) was followed. All animal procedures were
reviewed and approved by the Animal Care and Use Committee
of Rey Juan Carlos University.
These studies were supported by the Spanish Ministry of Economy and
Competitivity: grants SAF 2009-12422 and SAF 2012-40075, Red de
Trastornos Adictivos (RETICS RD06/001/0000 and RD06/001/0014), from
the Madrid Government (CANNAB-CM: S2010/BMD-2308), from the
University of the Basque Country (UFI 11/35), from the Basque
Government (IT-199-07; SAIOTEK S-PE10UN14), and the Instituto de
Salud Carlos III, Centro de Investigacio´n Biome´dica en Red de Salud
Mental, CIBERSAM. P.M. is recipient of JAE-Pre (ref. JAEPre-2010-01119)
postgraduate fellowships from CSIC. We are grateful to Laura Herna´ndez-
Folgado for her help in the writing of this manuscript.
Mouse vas deferens assay
The authors have declared no conflict of interest.
Mouse vas deferens was isolated from male CD-1 mice weighing
25–30 g. It was suspended in an organ bath containing Krebs
solution and was subjected to alternate periods of electrical
stimulation (5 min) and rest (10 min). Concentration–response
curves for the reference agonist (WIN 55,212-2) and for 4d were
registered. The effect of the reference agonist was also tested in
presence of 4d adding the latter to the organ bath 10 min before
the addition of WIN 55,212-2.
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