Structural-activity relationship study on C-4 carbon atom of the CB 1 antagonist SR141716: Synthesis and pharmacological evaluation of 1,2,4-triazole-3-carboxamides

Article abstract of DOI:10.1016/j.ejmech.2005.06.012

A series of 1,2,4-triazole-3-carboxamides has been prepared from alkyl-1,2,4-triazole-3-carboxylates under mild conditions. The ability of these triazoles to displace [³H]-CP55940 from CB₁ cannabinoid receptor was measured. However,

Full text of DOI:10.1016/j.ejmech.2005.06.012

European Journal of Medicinal Chemistry 41 (2006) 114–120
http://france.elsevier.com/direct/ejmech
Short communication
Structural–activity relationship study on C-4 carbon atom
of the CB1 antagonist SR141716:
synthesis and pharmacological evaluation of 1,2,4-triazole-3-carboxamides
Nadine Jagerovic a,*, Laura Hernandez-Folgado a, Ibon Alkorta a, Pilar Goya a,
María Isabel Martín b, María Teresa Dannert c, Ángela Alsasua c, Jordi Frigola d,
María Rosa Cuberes d, Alberto Dordal d, Jörg Holenz d
a
Instituto de Química Médica, CSIC, Juan de la Cierva 3, E-28006 Madrid, Spain
b
Unidad de Farmacología, Dpto. Ciencias de la Salud, Facultad Ciencias de la Salud,
Universidad Rey Juan Carlos, Avda. Atenas s/n, E-28922 Alcorcón, Madrid, Spain
Dpto. Farmacología, Facultad de Medicina, Universidad Complutense de Madrid, Avda. Complutense s/n, E-28040 Madrid, Spain
c
d
Laboratorios del Dr. Esteve S. A., Av. Mare de Déu de Montserrat, 221, E-08041 Barcelona, Spain
Received 4 March 2005; received in revised form 16 June 2005; accepted 20 June 2005
Available online 11 November 2005
Abstract
A series of 1,2,4-triazole-3-carboxamides has been prepared from alkyl-1,2,4-triazole-3-carboxylates under mild conditions. The ability of
these triazoles to displace [3H]-CP55940 from CB1 cannabinoid receptor was measured. However, they showed only poor to moderate binding
affinities, indicating that substitution of the C-4 pyrazole atom of the CB1 reference compound SR141716 by a nitrogen atom results in loss of
affinity. Further investigations for functionality indicated that the compound 6a exhibited significant cannabinoid antagonistic properties in the
mouse vas deferens functional assay. This leads us to the conclusion that 6a binds at a different CB1 binding site or at a new cannabinoid
receptor subtype.
© 2005 Elsevier SAS. All rights reserved.
Keywords: 1,2,4-Triazole; Cannabinoid; Mouse vas deferens; SR141716
Mots clés: 1,2,4-Triazole; Cannabiroide; Canal déférent de souris; SR141716
1. Introduction
zole-3-carboxamide (SR141716) [8]. Currently, SR141716 is
undergoing advanced clinical trials as an appetite suppressant
[9,10]. As a potent antagonist for the CB1 receptor, SR141716
was the lead compound for studies designed to examine the
SAR of related compounds. Structural modifications around
the aminopiperidine region, 2,4-dichlorophenyl and chlorophe-
nyl substituents of the parent pyrazole SR141716 gave insights
into the CB1 pharmacophore [11–16]. Conformationally re-
stricted SR141716 analogues have also been reported [17].
Among various studies dedicated to SAR for SR141716, only
a few have been devoted to the C-4 pyrazole position.
Cannabinoid receptors belong to the large superfamily of
seven transmembrane domains containing receptors, the G-pro-
tein coupled receptors. They include the CB1 and CB2 sub-
types [1–4] and various classes of compounds act as cannabi-
noid ligands [5,6]. Their structure–activity relationship (SAR)
properties have been of considerable interest these last years
[7]. In 1994, the first high-affinity antagonist of the brain,
CB1 cannabinoid receptor, was identified: N-(piperidin-1-yl)-
5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyra-
As part of our ongoing studies dealing with triazoles as can-
nabinoids [18,19] we synthesized and tested in isolated tissues
1,5-diaryl-1,2,4-triazole-3-carboxamides. Reports have been
* Corresponding author. Tel.: +34 91 562 2900; fax: +34 91 564 4853.
E-mail address: nadine@iqm.csic.es (N. Jagerovic).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.06.012

N. Jagerovic et al. / European Journal of Medicinal Chemistry 41 (2006) 114–120
115
published by Dyck et al. [20] and by Lange et al. [21,22] on
1,2,4-triazole-3-carboxamides. These triazoles were less potent
ligands of CB1 receptors than the corresponding pyrazole
SR141716. These studies have prompted us to report here our
own results on 1,2,4-triazole-3-carboxamides. This report de-
scribes a convenient alternative synthesis for triazolecarboxa-
mides with improved yields. In an effort to study the pharma-
cological properties of these compounds and in addition to
their abilities to displace the radioligand [3H]-CP55940 in
CHO cells expressing the human CB1 receptor, their cannabi-
noid functional activity has been studied in the mouse vas de-
ferens.
ducts have been isolated: 4-chloro-N’-(2,4-dichlorophenyl)ben-
zohydrazide (3), ethyl 2-(4-chlorobenzamido)-2-(2-(2,4-di-
chlorophenyl)hydrazinyl)acetate (4) and the desired ethyl 5-
(4-chlorophenyl)-1-(2,4-dichlorophenyl)-1H-1,2,4-triazolecar-
boxylate (5) in a 5:1:6 ratio. The addition product 4 was al-
lowed to cyclize to give triazole 5 by refluxing in 1,4-dioxane
with excess of pyridine for 2 weeks. The preparation of the
final 1,2,4-triazole-3-carboxamides 6a–d was achieved by
adopting a simple one-pot procedure. In fact, the most common
procedure to prepare carboxamides of heterocycles involves
the three classical steps: hydrolysis of the ester to acid followed
by acid chloride formation, and finally, reaction with the ap-
propriate amine or hydrazine to give the corresponding carbox-
amide or carboxylic acid hydrazide [15–17]. Conversion of
ethyl esters to carboxylic acid hydrazides under mild reaction
conditions, via organoaluminum reagents, have been described
by Benderly and Stavchansky [32]. Applying this procedure to
our 1,2,4-triazole series, the 1,2,4-triazole-3-carboxamides 6a–
d were obtained in good yield in a one-step synthesis by treat-
ment of the triazolecarboxylate 5 with 5 equiv. of an aluminum
complex which was previously prepared in situ reacting tri-
methylaluminum with the corresponding amine or hydrazine.
2. Chemistry
1,2,4-Triazole-3-carboxamides used to be synthesized from
4-arylazo-2-aryl-2-oxazolin-5-ones reacting with the appropri-
ate amine [23–27]. Dyck et al. [20] and Lange et al. [21] pre-
pared 1,2,4-triazole-3-carboxylates as described by Tsuji et al.
[28] and Kudo et al. [29], respectively. They then converted
these esters to the corresponding amides by saponification, to
give carboxylic acid, followed by amination. The 1,2,4-tria-
zole-3-carboxamides 6a–d described here were prepared in a
different synthetic pathway as outlined in Scheme 1. Diazotiza-
tion of commercially available 2,4-dichloroaniline in presence
of tetrafluoroboric acid gave the diazonium salt [30], which
was directly coupled with ethyl acetoacetate to afford the ox-
obutanoate 1. Subsequent treatment with bromine, then with an
aqueous solution of ammonium hydroxide led to the iminoace-
tate 2. The cyclization [31] of 2 with 4-chlorobenzoyl chloride
was achieved in the presence of pyridine. Three reaction pro-
3. Pharmacology
The biological activities of compounds 6a–d were evaluated
for their ability to displace the radioligand [3H]-CP55940 in
CHO cells expressing the human CB1 receptor. Their cannabi-
noid functional activity has been studied in the mouse vas de-
ferens. The effect of compounds 6a–d in isolated tissue was
Scheme 1. (a) i) NaNO2, aq. HBF4, 0 °C.; ii) ethyl acetoacetate, NaOAc, EtOH, H2O, 0 °C; (b) i) Br2, NaOAc, AcOH; ii) NH4OH 30%, acetone; (c) 4-
chlorobenzoylchloride, pyridine, 1,4-dioxane, reflux; (d) pyridine, 1,4-dioxane, reflux; (e) 1-aminopiperidine for 6a, 4-aminomorpholine for 6b, cyclohexylamine for
6c and 1-aminoadamantane for 6d, Al(Me)3, N2 atmosphere, dry CH2Cl2, 40 °C.

116
N. Jagerovic et al. / European Journal of Medicinal Chemistry 41 (2006) 114–120
tested in order to determine whether they behave as cannabi-
noid receptor agonists or antagonists.
4. Results and discussion
4.1. Binding assays
Affinities at CB1 receptors for triazoles 6a–d were deter-
mined by measuring their ability to displace the radioligand [
3H]-CP55940. These data are reported in Table 1 including Ki
values of the reference pyrazole SR141617. Compound 6a,
triazole SR141716 analogue, possesses low CB1 receptor affi-
nity (Ki > 1 μM). The value reported here is higher than the Ki
value (382 nM) reported by Lange et al. [21]. In this com-
pound, the C-4 methyl of SR141716 was replaced by a nitro-
gen. For pyrazole SR141716 analogues, it has been shown [13,
15,33] that replacing the methyl group at the C-4 position by
bromine, iodine, hydrogen, or n-pentyl maintained the affinity
value in a nanomolar range. The 1,2-diarylimidazolecarboxa-
mide analogue to the pyrazole SR141716 has been reported
to have similar CB1 receptor affinity as compared with the pyr-
azole parent [21]. Based on these data, it seems reasonable to
conclude that the low recognition of 6a for CB1 receptor can
be attributed to a different geometry of the molecule 6a com-
pared to the pyrazole SR141716. A conformational analysis of
6a and SR141716 was made and is described below in the
present article.
Fig. 1. Effect of WIN55212-2 (WIN), SR141716 (SR), 6a–d in mouse isolated
vas deferens. Lines show the mean % (n = 6, n = 8 for WIN) ( S.E.M.) of
modification of the electrically induced contraction of the mouse vas deferens
by cumulative addition of WIN55212-2, SR141716, and 6a–d.
** P < 0.01 One-way ANOVA Post-test Bonferroni’s multiple comparison test.
4.2. Isolated tissues assays
The cannabinoid activity of target compounds 6a–d was
functionally determined by in vitro assays using the mouse
vas deferens. The mouse vas deferens is a tissue widely used
to characterize cannabinoid agonists and antagonists [35–37].
CB1 cannabinoid agonists inhibit electrically evoked contrac-
tions of the vas deferens by activating the CB1 receptors ex-
pressed in this tissue. As shown in Fig. 1, the agonist
WIN55212-2 induces dose-dependent inhibition of electrically
induced contractions in this tissue. This effect was antagonized
by the CB1 cannabinoid antagonist SR141716 when added to
the organ bath 15 min before WIN55212-2 addition (Fig. 3).
Experiments with SR141716 and with the triazoles 6a–d indi-
cated that they did not induce significant modification of con-
tractile responses at concentrations of 10−8–10−6 M. We can
conclude from these data that 6a–d do not behave as cannabi-
Concerning the N-carboxamide substitution of derivatives of
1,2,4-triazole-3-carboxamide, it is interesting to note that at
1 μM, piperidino (6a, 31.7%) and morpholino (6b, 28.3%) car-
boxamides have modest affinity while cyclohexyl carboxamide
(6c, 55.9%) exhibits slightly improved CB1 receptor binding
and adamantyl carboxamide (6d, 65.8%) shows the highest af-
finity. However, the affinity constant determined for the latter
compound is still moderate (Ki = 498.2 nM). The lipophilicity
of the N-carboxamide substituents seems to be an important
parameter in cannabinoid receptor recognition. The lipophilic
properties of 6a–d have been predicted using the software LogP
of Interactive Analysis [34]. In general, the displacement values
of 6a–d increase with the lipophilic properties (logP = 4.62
(6a); logP = 3.72 (6b); logP = 5.25 (6c); logP = 6.42 (6d)).
Table 1
Displacement of specific [3H]-CP55940b binding (at 1 μM) in CHO cells stably
transfected with human CB1 receptor, expressed as percentage (%)
Compound
CB1: displacements
(%) at 1 μM
(Ki = 11.5 nM) [13]
31.7a
SR141716
6a
6b
6c
6d
28.3 5.4
55.9a
65.8 11.7
Fig. 2. Antagonistic effect of SR141716 (SR) and 6a–d in mouse isolated vas
deferens. Columns represent mean values S.E.M. (n = 6) of modifications
induced by WIN55212-2 (W) at 10−6 M for 15 min after treatment with
SR141716 and 6a–d at 10−6 M.
*** P < 0.001; * P < 0.05, Student’s t-test. One-way ANOVA Post-test
Bonferroni’s multiple comparison test.
(Ki = 498.2 nM)c
a
Values expressed as mean of three determinations with same internal stan-
dard.
b
Kd = 0.52.
c
Ki value was determined using the method of Cheng and Prusoff [47].

N. Jagerovic et al. / European Journal of Medicinal Chemistry 41 (2006) 114–120
117
minimized conformers of SR141716 led us to confirm a low-
energy s-trans conformer defined by the angle (N2=C3–C=O).
This result is in agreement with previous studies reported in the
literature. We carried out energy minimization of triazoles 6a
and 6d structures. For 6a and 6d the AM1 calculations results
indicated an energetically preferred conformation in which the
amide oxygen was placed on the same side of triazole N2 (s-cis
conformation). Based on superimposing the heterocyclic five
atoms of the pyrazole and triazole cores, the alignments of
SR141716 with 6a and SR141716 with 6d were undertaken
and are represented in Fig. 4. As shown in these superposi-
tions, the carboxamides of 6a and 6d adopt a conformation
different from SR141716. This result disagrees with the recep-
tor-based alignment presented by Lange et al. [21] in which the
carboxamide moieties of the triazole and the pyrazole overlap.
However, the pharmacophoric feature presented here suggests
that this difference in carboxamide spatial orientations due to
electronic repulsion between nitrogen and oxygen electronic
pairs, could contribute to conferring to 1,2,4-triazole-3-carbox-
amides 6a–d a low affinity for CB1 receptor.
Fig. 3. Lines show the mean % (n = 6, n = 8 for control) ( S.E.M.) of
modification of the electrically induced contraction of the mouse vas deferens
by cumulative addition of WIN55212-2 in control tissues or tissues incubated at
15 min with SR141716 (SR) or 6a.
*P < 0.05; **P < 0.01 One-way ANOVA Post-test Bonferroni’s multiple
comparison test.
noid agonists and, at the tested doses, they did not show any
intrinsic activity. The ability of triazoles 6a–d to oppose the
effect of WIN55212-2 in this tissue was investigated. Varia-
tions produced by 6a–d, on the inhibition of electrically in-
duced contractions evoked by WIN55212-2, are shown in
Fig. 2. Compounds 6b, 6c and 6d were devoid of antagonistic
properties when added to the organ bath 15 min before starting
the cumulative addition of WIN55212-2. However, compound
6a antagonized the effect of WIN55212-2 as shown in Fig. 3.
These results indicate that in the mouse vas deferens, 6a be-
haves as cannabinoid antagonist with moderate potency com-
pared with SR141716. This last result does not corroborate the
displacement value of 6a (Ki > 1000 nM) suggesting allosteric
CB1 interactions or new subtype cannabinoid receptor recogni-
tion.
5. Conclusion
A series of 1,2,4-triazole-3-carboxamides (6a–d) have been
prepared by a new efficient synthesis. Results from AM1 en-
ergy minimization and the CB1 cannabinoid receptor binding
assays showed the negative contribution of triazole N4 for re-
cognition of the CB1 binding site. However, 6a was found to
be CB1 cannabinoid antagonist in isolated tissues assays. This
result did not corroborate with the binding data. These results
suggest the possibility for 6a to behave as an antagonist by
acting on a new cannabinoid receptor subtype or by binding
to a different CB1 binding site. These hypotheses are supported
by the fact that, in the literature, there is evidence that some
ligands induce their cannabinoid activity through an allosteric
mechanism [40]. In particular, it has been suggested that the
cannabinoid CB1 receptor antagonist SR141716 A induces in-
verse agonistic effect by binding to an additional site on the
cannabinoid CB1 receptor [41]. There are also convincing evi-
dences that support the existence of additional cannabinoid re-
4.3. Conformational studies
The importance of spatial orientation of the carboxamide
group for CB1 receptor recognition has been pointed out [33,
38,39]. Using AM1 molecular orbital calculations, energy
Fig. 4. Superposition of 6a with SR141716 (right) and of 6d with SR141716 (left).

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N. Jagerovic et al. / European Journal of Medicinal Chemistry 41 (2006) 114–120
ceptor [42–44]. Therefore, there is still a need for additional
pharmacological experiments to establish the mechanism un-
derlying the effect of 6a in the mouse vas deferens.
HCl was washed with toluene. The aqueous layer was basified
with 5 N NaOH to pH 8 precipitating a brown solid, which was
then filtered off. This precipitate was then suspended in boiling
EtOH and filtered. The liquid layer was treated with activated
carbon for 30 min, and filtered over celite. Then, the solvent
was evaporated, obtaining 2 (1.76 g, 77%) as a brown solid: m.
p. = 82–87 °C; 1H NMR (200 MHz, CDCl3) δ 7.42 (d,
J = 8.9 Hz, 1H, H6), 7.21 (brs, 1H, H3), 7.15 (brd,
J = 8.9 Hz, 1H, H5), 6.77 (brs, 1H, NH), 4.65 (brs, 2H, NH),
4.32 (q, J = 7.1 Hz, 2H, CH2), 1.34 (t, J = 7.1 Hz, 3H, CH3);
13C NMR (200 MHz, CDCl3) δ 161.8 (CO2CH2CH3), 140.2
(C=NH), 138.4 (C1), 128.4 (C3), 128.0 (C5), 125.1 (C4), 118.8
(C2), 116.2 (C6), 62.5 (CH2), 14.1 (CH3); MS (ES+) m/z (rel.
intensity %) 276 (M++1, 100); Anal. C10H11Cl2N3O2 (C, N,
H).
6. Experimental protocols
6.1. Chemistry
Dry dichloromethane was distilled over calcium chloride,
and dry pyridine over potassium hydroxide. Melting points
(uncorrected) were determined with a Reichert Jung Thermo-
var apparatus. Mass spectra were recorded using electrospray
positive mode. Flash column chromatographies were run on
silica gel 60 (230–400 mesh) or on medium pressure flash sys-
tem with prepacked silica gel cartridge. Analyses indicated by
the symbols of the elements or functions were within 0.4% of
1
the theoretical values. H and 13C NMR spectra were recorded
6.1.3. Ethyl 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-1H-
1,2,4-triazole-3-carboxylate (5)
on a Gemini 200, Varian 300 and 400 unity spectrometers
using TMS as the internal standard.
A mixture of 2 (0.60 g, 2.2 mmol), pyridine (0.20 ml,
2.5 mmol), and 4-chlorobenzoyl chloride (0.32 ml, 2.5 mmol)
in 1,4-dioxane (50 ml) was heated to reflux for 3 h. The sol-
vent was then evaporated. The residue dissolved in CH2Cl2
(120 ml) was washed successively with 1 M HCl, H2O, sat. aq.
K2CO3 and finally H2O. The organic layer was dried over an-
hydrous Na2SO4 and evaporated under reduced pressure. The
residue was chromatographied on silica gel (cyclohexane/
EtOAc 8:1) to give ethyl 2-(4-chlorobenzamido)-2-(2-(2,4-di-
chlorophenyl)hydrazinyl)acetate (4; 53 mg, 6%, yellow solid),
4-chloro-N′-(2,4-dichlorophenyl)benzohydrazide (3; 198 mg,
29%, yellow solid) and the desired product 5 as a white solid
(0.31 g, 36%).
6.1.1. Ethyl 2-[2-(2,4-dichlorophenyl)hydrazono]-3-
oxobutanoate (1)
A solution of 2,4-dichloroaniline (8.00 g, 49.4 mmol) and
HBF4 (13 ml, 48% wt.) in H2O (13 ml) was cooled to 0 °C.
NaNO2 (3.41 g, 49.4 mmol) in H2O (5 ml) previously cooled
to 0 °C was added drop-wise. The reaction mixture was stirred
at 0 °C for 30 min and then the temperature was allowed to
reach r.t. The formed diazonium salt was filtered off, washed
with HBF4 aq., then with EtOH and finally with Et2O. A solu-
tion of dried diazonium salt in EtOH (250 ml) was stirred with
ethyl acetoacetate (6.25 ml, 49.4 mmol), NaOAc (11.12 g,
135.5 mmol) and H2O (25 ml) at 0 °C for 1 h. The precipitate
was filtered off, washed with H2O, and recrystallized from
cyclohexane to give 4.89 g of 1 (33%) as an orange solid:
3. m.p. = 163–166 °C; 1H NMR (200 MHz, CD3OD) δ 7.88
(d, J = 8.6 Hz, 2H, H2), 7.49 (d, J = 8.6 Hz, 2H, H3), 7.31 (d,
J = 2.3 Hz, 1H, H3′), 7.14 (dd, J = 8.7 Hz, J = 2.3 Hz, 1H, H5′),
6.87 (d, J = 8.7 Hz, 1H, H6′); 13C NMR (200 MHz, CD3COD)
δ 169.0 (CONH), 144.9 (C4), 139.5 (C1′), 132.4 (C1), 130.3
and 130.0 (C2, C3 and C3′), 128.9 (C5′), 125.6 (C4′), 120.5
(C2′), 115.3 (C6′); MS (ES+) (rel. intensity %) 315 (M+ + 1,
100); IR (KBr) 3435 cm−1 NH amide, 1649 cm−1 CO amide.
4. m.p. = 168–171 °C; 1H NMR (200 MHz, CDCl3) δ:
10.10 (brs, 1H, NH), 8.81 (brs, 1H, NH), 7.62 (d, J = 8.5 Hz,
2H, H2), 7.32 (d, J = 8.8 Hz, 2H, Aromatics), 7.24 (d,
J = 8.5 Hz, 2H, H3), 7.00 (t, J = 8.8 Hz, 1H, Aromatics), 4.15
(q, J = 7.1 Hz, 2H, CH2), 1.20 (t, J = 7.1 Hz, 3H, CH3); 13C
NMR (200 MHz, CDCl3) δ 165.3 and 163.6 (CO2CH2CH3,
CON), 140.0 (C = N), 139.8 (C4 and C1′), 131.1 (C1), 129.3
and 129.2 (C2 and C3), 129.0 (C3′), 127.9 (C5′), 126.8 (C4′),
122.9 (C2′), 116.1 (C6′), 63.0 (CH2), 14.2 (CH3); MS (ES+)
(rel. intensity %) 414 (M+ + 1, 80).
1
m.p. = 118–121 °C; H NMR (200 MHz, CDCl3) δ 7.93 (d,
J = 8.8 Hz, 1H, H6), 7.54 (brs, 1H, H3), 7.44 (brd,
J = 8.8 Hz, 1H, H5), 4.50 (q, J = 7.1 Hz, 2H, CH2CH3), 2.76
(s, 3H, CH3), 1.55 (t, J = 7.1 Hz, 3H, CH2CH3); 13C NMR
(200 MHz, CDCl3) δ 197.0 (COCH3), 164.5 (CO2CH2CH3),
137.4 (C1), 130.3 (N=C), 129.3 (C3), 128.4 (C5), 127.8 (C4),
122.0 (C2), 117.5 (C6), 61.2 (CH2CH3), 30.8 (CH3), 14.3
(CH2CH3); MS (ES+) m/z (rel. intensity %) 303 (M++1, 100);
Anal. C12H12Cl2N2O3 (C, H, N).
6.1.2. Ethyl 2-[2-(2,4-dichlorophenyl)hydrazinyl]-2-
iminoacetate (2)
To a solution of 1 (1.44 g, 4.7 mmol) and NaOAc (3.52 g,
42.8 mmol) in AcOH (35 ml) was added drop-wise Br2 (243
μl, 4.7 mmol). After 30 min of stirring at r.t., the solution was
poured into H2O (100 ml) and extracted with CH2Cl2. The or-
ganic layers were dried over anhydrous Na2SO4. The solvent
was then removed under reduced pressure. To the residue dis-
solved in acetone (50 ml) was added drop-wise 30% aq.
NH4OH (4 ml) diluted in acetone (10 ml). This mixture was
stirred 1 h at r.t. Then the solvent was evaporated under re-
duced pressure. The residue dissolved in 100 ml of 10% aq.
1
5. m.p. = 113–118 °C; H NMR (200 MHz, CDCl3) δ 7.53
(d, J = 1.9 Hz, 1H, Aromatics), 7.45 (d, J = 8.6 Hz, 2H, H2″),
7.44 (m, 2H, Aromatics), 7.31 (d, J = 8.6 Hz, 2H, H3″), 4.52
(q, J = 7.2 Hz, 2H, CH2), 1.44 (t, J = 7.2 Hz, 3H, CH3); 13C
NMR (200 MHz, CDCl3) δ 159.4 (CO2CH2CH3), 155.9 (C3
triazole), 154.9 (C5 triazole), 137.3, 137.1, 133.8, and 132.9
(C1′, C2′, C4′ and C4″), 130.5 (C6′), 129.9 (C3′), 129.3 and

N. Jagerovic et al. / European Journal of Medicinal Chemistry 41 (2006) 114–120
119
129.0 (C2″ and C3″), 128.4 (C5′), 124.6 (C1″), 62.1 (CH2), 14.1
(CH3); MS (ES+) m/z (rel. intensity %) 296 (M+ + 1, 90).
The adduct product 4 (30 mg, 0.07 mmol) was cyclized into
5 by refluxing in 1,4-dioxane (3 ml) with pyridine (23.4 μl,
0.3 mmol) for 2 weeks. After evaporation, the residue was dis-
solved in CH2Cl2 (20 ml), washed with 1 M HCl, dried over
anhydrous Na2SO4, and evaporated to give 5 (38 mg, 93%).
(CH2NCH2); MS (ES+) m/z (rel. intensity %) 425 (M+ + 1,
100); Anal. C19H16Cl3N5O2.1/2C7H8 (C, H, N).
6.1.4.3. 5-(4-Chlorophenyl)-N-cyclohexyl-1-(2,4-dichlorophe-
nyl)-1H-1,2,4-triazole-3-carboxamide (6c). Compound 6c was
prepared from 5 (120 mg, 0.3 mmol), cyclohexylamine (0.18
ml, 1.5 mmol), Al(Me)3 (0.76 ml, 1.5 mmol); reaction time:
4.5 h; recrystallization from toluene and flash chromatography
[cyclohexane/EtOAc (2:1)]; yield: 83 mg (61%) as a white so-
6.1.4. General procedure for the synthesis of 1,2,4-triazole-3-
carboxamide (6a–d)
1
lid; m.p. = 181–185 °C; H NMR (300 MHz, CDCl3) δ 7.47
(d, J = 1.7 Hz, 1H, Aromatics), 7.40–7.30 (m, 4H, H2″ and
aromatics), 7.26 (d, J = 8.7 Hz, 2H, H3″), 7.02 (d, J = 8.7 Hz,
1H, NH), 4.15–3.85 (m, 1H, NHCH), 2.20–1.11 (m, 10H,
CH2CH2CH2CH2CH2); 13C NMR (300 MHz, CDCl3) δ 157.7
(CONH), 157.5 (C3 triazole), 155.4 (C5 triazole), 137.4, 137.2,
134.3, and 132.7 (C1′, C2′, C4′ and C4″), 130.7 (C6′), 130.1
(C3′), 129.3 and 129.2 (C2″ and C3″), 128.5 (C5′), 125.2
(C1″), 48.3 (NHCH), 33.1 (CH2CH(NH)CH2), 25.5
(CH2CH2CH2CH(NH)CH2CH2), 24.8 (CH2CH2CH2CH(NH)
CH2CH2); MS (ES+) m/z (rel. intensity %) 449 (M+ + 1,
100); Anal. C21H19Cl3N4O (C, H, N).
To a solution of the corresponding hydrazine or amine (5
equiv.) in dry CH2Cl2 (3–5 ml) was added a solution of Al(Me)
3 in heptane (2 M, 5 equiv.) under N2 atmosphere. The reaction
mixture was stirred at r.t. for 1 h. A solution of 5 (1 equiv.) in
dry CH2Cl2 (3–6 ml) was then added drop-wise. The mixture
was heated to 35–50 °C during the respective time, and then
was carefully poured onto 1 N HCl (20–30 ml). The biphasic
solution was heated to 40 °C for 30 min and cooled to r.t. The
organic layer was separated, dried over anhydrous Na2SO4 and
evaporated. The crude product was purified by recrystallization
or chromatography.
6.1.4.4. 5-(4-Chlorophenyl)-N-(1-adamantyl)-1-(2,4-dichloro-
phenyl)-1H-1,2,4-triazole-3-carboxamide (6d). Compound 6d
was prepared from 5 (76 mg, 0.2 mmol), 1-aminoadamantane
(145 mg, 1.0 mmol), Al(Me)3 (0.48 ml, 1.0 mmol); reaction
time: 26 h; flash chromatography [cyclohexane/EtOAc (4:1)];
6.1.4.1. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-(piperi-
din-1-yl)-1H-1,2,4-triazole-3-carboxamide (6a). Compound 6a
was prepared from 5 (240 mg, 0.6 mmol), 1-aminopiperidine
(0.32 ml, 3.0 mmol), Al(Me)3 (1.50 ml, 3.0 mmol); reaction
time: 21 h; medium pressure chromatography [cyclohexane/
EtOAc (25:1–3:1)]; yield: 214 mg (79%) as a white solid; m.
p. = 115–118 °C; 1H NMR (300 MHz, CDCl3) δ 7.82 (brs, 1H,
NH), 7.46 (d, J = 1.9 Hz, 1H, Aromatics), 7.38 (m, 2H, Aro-
matics), 7.36 (d, J = 8.7 Hz, 2H, H2″), 7.26 (d, J = 8.7 Hz,
H3″), 2.84 (t, J = 5.6 Hz, 4H, CH2CH2CH2NCH2CH2), 1.71
(t, J = 5.6 Hz, 4H, CH2CH2CH2NCH2CH2), 1.39 (m, 2H,
CH2CH2CH2NCH2CH2); 13C NMR (300 MHz, CDCl3) δ
157.0 (CONH), 156.2 and 155.7 (C3 triazole and C5 triazole),
137.8, 137.7, 134.5, and 133.0 (C1′, C2′, C4′ and C4″), 130.7
(C6′), 130.0 (C3′), 129.3 and 129.2 (C2″ and C3″), 128.5 (C5′),
1
yield: 75 mg (78%) as a white solid; m.p. = 185–189 °C; H
NMR (300 MHz, CDCl3) δ 7.50 (d, J = 1.7 Hz, 1H, Aromatics),
7.47–7.44 (m, 2H, Aromatics), 7.42 (d, J = 8.9 Hz, 2H, H2″),
7.30 (d, J = 8.9 Hz, 2H, H3″), 6.91 (s, 1H, NH), 2.14 (brs, 6H,
H2 adamantyl), 2.11 (brs, 3H, H3 adamantyl), 1.75–1.66 (m,
6H, H4 adamantyl); 13C NMR (300 MHz, CDCl3) δ 157.9
(CONH), 157.3 (C3 triazole), 155.1 (C5 triazole), 137.2, 137.0,
134.2 and 132.5 (C1′, C2′, C4′ and C4″), 130.6 (C6′), 130.0 (C3′),
129.2 and 129.1 (C2″ and C3″), 128.5 (C5′), 125.1 (C1″), 52.3
(C1 adamantyl), 41.4 (C2 adamantyl), 36.3 (C4 adamantyl), 29.4
(C3 adamantyl); MS (ES+) m/z (rel. intensity %) 501 (M+ + 1,
100); Anal. C25H23Cl3N4O (C, H, N).
125.4
(C1″),
57.2
(CH2CH2CH2NCH2CH2),
25.2
(CH2CH2CH2NCH2CH2), 23.2 (CH2CH2CH2NCH2CH2); MS
(ES+) m/z (rel. intensity %) 450 (M+ + 1, 99); Anal.
C20H18Cl3N5O (C, H, N).
6.2. Pharmacological materials and methods
6.2.1. Binding assays
6.1.4.2. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-morpho-
lino-1H-1,2,4-triazole-3-carboxamide (6b). Compound 6b was
prepared from 5 (150 mg, 0.4 mmol), 4-aminomorpholine
(0.18 ml, 1.9 mmol), Al(Me)3 (0.95 ml, 1.9 mmol); reaction
time: 2.5 h; recrystallization from toluene; yield: 161 mg
(94%) as a white solid; m.p. = 193–195 °C; 1H NMR
(300 MHz, CDCl3) δ 8.00 (brs, 1H, NH), 7.51 (d, J = 1.3 Hz,
1H, Aromatics), 7.44 (brs, 2H, Aromatics), 7.41 (d, J = 8.6 Hz,
2H, H2″), 7.31 (d, J = 8.6 Hz, H3″), 3.87 (t, J = 4.5 Hz, 4H,
CH2OCH2), 3.01 (t, J = 4.5 Hz, 4H, CH2NCH2); 13C NMR
(300 MHz, CDCl3) δ 156.2 (CONH), 156.0 and 155.5 (C3 tria-
zole and C5 triazole), 137.5, 137.4, 134.0, and 132.5 (C1′, C2′,
C4′ and C4″), 130.7 (C6′), 130.0 (C3′), 129.3 and 129.2 (C2″ and
C3″), 128.6 (C5’), 124.8 (C1″), 66.2 (CH2OCH2), 55.9
Membranes from HEK-293 EBNA cells with human CB1
cannabinoid receptor expressed were supplied by PerkinElmer.
The receptor concentration was 3.5 pmol mg–1 protein and the
protein concentration was 6.4 mg ml–1. The binding assays
were performed as described by Ross et al. [45], with modifi-
cations. The commercial membrane was diluted (1:60) with the
binding buffer (50 mM Tris Cl, 5 mM MgCl2, 2.5 mM EDTA,
0,5 mg ml–1 BSA, pH = 7.4). The radioligand used was [3H]-
CP55940 (PerkinElmer) at 0.135 nM and the final volume was
200 μl. The incubation was initiated with the addition of 160 μl
of membrane and the incubation time was 90 min at 30 °C.
After incubation, the membrane was collected onto pretreated
glass fiber filters (Schleicher and Schnell 3362), with polyethy-

120
N. Jagerovic et al. / European Journal of Medicinal Chemistry 41 (2006) 114–120
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lenimine 0.5%. The filter was washed four times each with 1
ml of washing buffer (50 mM Tris Cl, pH 7.4) and then the
filter sections were transferred to vials and 5 ml of Ecoscint H
liquid scintillation cocktail was added to each vial. The vials
were allowed to settle for several hours and then quantified by
liquid scintillation spectrophotometry (Wallac Winspectral
1414). Nonspecific binding was determined with 10 μM
WIN55212-2. Competition binding data were analyzed by
using the LIGAND program [46] and assays were performed
in triplicate determinations for each point.
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Male CD-1 mice weighing 25–30 g each were used. Inhibi-
tion of electrically evoked contractions of mouse vas deferens
was determined following previously described procedures
[19]. WIN55212-2 and SR141716 were obtained, respectively,
from TOCRIS (Biogen Científica S.L., Madrid, Spain) and
from SANOFI AVENTIS (Paris, France). For experiments,
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This work was supported by the Spanish Grant SAF00-
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I3P fellowship from the C.S.I.C.
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