Rational design, synthesis and biological evaluation of new 1,5-diarylpyrazole derivatives as CB1 receptor antagonists, structurally related to rimonabant

Article abstract of DOI:10.1016/j.ejmech.2008.01.043

Among cannabinoid type-1 (CB₁) receptor antagonists, those developed around the 1,5-diarylpyrazole scaffold of rimonabant (Acomplia) are the most extensively investigated. In recent years, many SAR and QSAR reports on this topic have been published, focusing on the substitution and orientation of the N1 and C5 aryl functionalities and on the substituents at the 3-carboxamide position. In this context, the purpose of our study was to design and synthesize a set of 1-(2,4-dichlorophenyl)-5-arylpyrazoles strictly related to rimonabant, but with the hydrazide/amide group shifted from position 3 to position 4 of the pyrazole scaffold. The synthesized compounds were evaluated in vitro for their affinity on human CB₁ and CB₂ (cannabinoid type-2) receptors. Computational studies, performed both in the design step and after biological assays, contributed to rationalize the obtained results in terms of specific molecular interactions between antagonists and the human CB₁ receptor.

Full text of DOI:10.1016/j.ejmech.2008.01.043

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2627e2638
http://www.elsevier.com/locate/ejmech
Original article
Rational design, synthesis and biological evaluation of new
1,5-diarylpyrazole derivatives as CB1 receptor antagonists,
structurally related to rimonabant
*
Giulia Menozzi, Paola Fossa , Elena Cichero, Andrea Spallarossa, Angelo Ranise, Luisa Mosti
Dipartimento di Scienze Farmaceutiche, Universita` di Genova, Viale Benedetto XV 3, 16132 Genova, Italy
Received 21 November 2007; received in revised form 25 January 2008; accepted 31 January 2008
Available online 8 February 2008
Abstract
Among cannabinoid type-1 (CB1) receptor antagonists, those developed around the 1,5-diarylpyrazole scaffold of rimonabant (AcompliaÔ)
are the most extensively investigated. In recent years, many SAR and QSAR reports on this topic have been published, focusing on the sub-
stitution and orientation of the N1 and C5 aryl functionalities and on the substituents at the 3-carboxamide position. In this context, the purpose
of our study was to design and synthesize a set of 1-(2,4-dichlorophenyl)-5-arylpyrazoles strictly related to rimonabant, but with the hydrazide/
amide group shifted from position 3 to position 4 of the pyrazole scaffold. The synthesized compounds were evaluated in vitro for their affinity
on human CB1 and CB2 (cannabinoid type-2) receptors. Computational studies, performed both in the design step and after biological assays,
contributed to rationalize the obtained results in terms of specific molecular interactions between antagonists and the human CB1 receptor.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: 1,5-Diarylpyrazoles; CB1 antagonists; Rimonabant analogues; Synthesis; Computational studies
1. Introduction
CB1 agonists showed utility as appetite stimulants, anti-
emetics, analgesics, tumour growth inhibitors and neuropro-
tectants [3], while CB1 antagonists proved to be effective for
the treatment of obesity, metabolic disorders [4,5] and drug
abuse, including alcoholism, nicotine dependence and heroine
addiction [6]. The 1,5-diarylpyrazole derivative rimonabant
(SR141716, AcompliaÔ, Fig. 1) is the first therapeutically
relevant CB1 receptor antagonist [7,8], just launched by
Sanofi-Aventis in the European market as an antiobesity
drug. Moreover, recent investigation has shown the ability of
rimonabant to improve memory acquisition and consolidation
in mouse [9] and to prolong spatial memory duration, in com-
bination with donepezil, in cognitive deficits associated with
neurodegenerative disorders in rat [10]. A combination of
a CB1 antagonist with a dopaminergic neurotransmission en-
hancer has been recently patented for the treatment of Parkin-
son disease [11]. Full pharmacological potential of rimonabant
has been discussed in a recent review [12].
Despite the fact that medicinal and cognitive effects of Mar-
ijuana (Cannabis sativa) have been known for thousands of
years, only recent studies have provided convincing information
on the biological mediation of its effects. Currently, two sub-
types of cannabinoid receptor (namely, CB1 and CB2) have
been cloned and pharmacologically characterized. Both CB1
and CB2 belong to the G-protein-coupled receptor family
(GPCRs); CB1 are mainly located within the central nervous
system (CNS) and at presynaptic nerve terminals and they are
present also within the periphery in gut, eye, testis and bladder.
CB2 receptors are mainly associated with immune system cells
(e.g., B-cells, natural killer cells and monocytes) [1], however,
they have recently been found in CNS tissues [2].
The development of CB1 receptor ligands represents un-
doubtedly an interesting field in medicinal chemistry. In fact,
Since its discovery, rimonabant has been extensively stud-
ied to increase its affinity and selectivity toward hCB1
* Corresponding author. Tel.: þ39010 3538238; fax: þ39010 3538358.
E-mail address: fossap@unige.it (P. Fossa).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.01.043

2628
G. Menozzi et al. / European Journal of Medicinal Chemistry 43 (2008) 2627e2638
O
2. Chemistry
N
N
H
CH3
Compounds 4 were obtained through the four-step proce-
dure previously reported (Scheme 1) ([26,27] and literature
cited therein). Dimethylaminomethylene intermediates 1ae
1c,1e and f were known [26,28]; 1d was prepared by treatment
of ethyl 4-iodobenzoylacetate with N,N-dimethylformamide
dimethylacetal, as already reported [26]. Enaminones 1 were
condensed with 2,4-dichlorophenylhydrazine to afford pyra-
N
N
Cl
Cl
Cl
1
zoles 2 as single products, as assessed by H NMR spectra.
Fig. 1. Chemical structure of rimonabant.
The regioselective outcome of the cyclization reaction can
be ascribed to the different nucleophilic properties of the
NHAr and NH2 groups of the 2,4-dichlorophenylhydrazine.
Thus, the highly nucleophilic primary amine group exclusively
reacted with the highly electrophilic extra-chain carbon atom
of intermediates 1ae1f to form pyrazoles 2. Only compound
2a was known [27]. Esters 2 were hydrolyzed in presence of
KOH in 95% ethanol to lead to the carboxylic acids 3 which
were then converted to the corresponding acid chlorides by
treatment with thionyl chloride. The acid chlorides obtained
in this way were reacted with a number of primary and sec-
ondary amines and hydrazines, in the presence of triethyl-
amine, to give the desired carboxamides 4.
receptor. Among the different modifications made on the par-
ent compound, some groups focused on various substitutions
on the 1,5-diphenylpyrazole scaffold [13e19], while others
operated more heavily on the rimonabant structure with isos-
teric replacements of the pyrazole or phenyl rings [20e24].
One of the less explored variations of the parent compound
concerns the position of the carboxamide group, in which all
derivatives have been maintained at position 3, with the excep-
tion of the compounds reported in a patent [25] deposited dur-
ing the development of our research project. Our expertise in
the pyrazole chemistry [26,27] prompted us to design and syn-
thesize a series of 1,5-diarylpyrazoles bearing at 4 position dif-
ferent carboxamide/hydrazide groups (compounds 4, Scheme
1). Our strategy was focused on the variation of the 4-substit-
uent keeping constant the 2,4-dichlorophenyl ring at position 1
and applying limited variations on the phenyl ring at position 5
( para-substitution with groups with different electronic and
steric properties, as halogens, methyl, methoxy). To support
and guide the synthetic efforts, in silico docking studies
were performed. The biological results of the synthesized
compounds provided indirect validation of the calculated
docking models.
3. Molecular modeling
Modeling studies, performed on rimonabant and its 1,5-di-
arylpyrazole analogues [4] predicted that the antagonists bind
to human CB1 (hCB1) receptor in a hydrophobic region lo-
cated within the transmembrane (TM) core region in the crev-
ice formed by TM3, TM5, TM6 and TM7 [29]. The optimal
interaction between rimonabant and CB1 would be amenable
to the double hydrogen bond involving the inhibitor amide
COOC2H5
COOH
NH-NH2 . HCl
COOC2H5
N
N
(CH3)2N
Cl
N
N
a)
b)
c)
+
O
Cl
Cl
R
R
R
Cl
Cl
1a-f
Cl
2a-f
3a-f
CO-R'
d: R = I
e: R = CH3
f: R = OCH3
a: R = Cl
b: R = F
c: R = Br
N
N
d)
Cl
R
4aa-ao,
4ba-bd,
4ca-ce,
4da-df,
4ea-eb,
4fa-fb
Cl
Scheme 1. Synthesis of target rimonabant analogues: (a) absolute EtOH; (b) KOH, EtOH 95%; (c) SOCl2, toluene; (d) hydrazines or amines, Et3N, CH2Cl2.

G. Menozzi et al. / European Journal of Medicinal Chemistry 43 (2008) 2627e2638
2629
function and the key residues Lys192 and Ser383 [30]. In par-
ticular, the interaction with Lys192 is considered essential for
the antagonistic affinity [31], exerting a stabilizing effect on
the Lys192-Asp366 salt bridge at the intracellular end of
TM3 and TM6 helices. RimonabanteCB1 complex would be
further stabilized, according to our binding model, by a number
of hydrophobic contacts between the piperidine ring and
Phe174, Val196, Met384 and Leu387 residues. Moreover,
the two phenyl rings are both involved in pep stacking inter-
actions with Tyr275, Trp279, Trp356 and Phe379 side-chains.
In order to explore any residue and ligand feature involved
in the CB1 antagonism and to analyze the effects of the shift of
the amidic substitution from position 3 (rimonabant) to posi-
tion 4 on receptor affinity, a two step computational protocol
was performed on 1,5-diarylpyrazoles 4.
A three-dimensional model of the hCB1 receptor was built
by homology modeling techniques, starting from the three-di-
mensional structure coordinate file of bovine rhodopsine (PDB
entry 1F88) which is the common template for protein belong-
ing to Class A of GPCRs [32]. Briefly, the amino acid se-
quence of hCB1 was aligned with that of bovine rhodopsine
on the basis of the highly conserved amino acid residues, ac-
cording to the previously reported procedure [33] (seeSection
6). Then rimonabant (assumed as template molecule) was
docked into the putative binding site of the hCB1 receptor
by means of the flexible docking module implemented in
MOE [34]. The results obtained in this first step were in agree-
ment with site-directed mutagenesis data and with other com-
putational studies reported in the literature (Fig. 2) [4,35]. This
evidence indirectly validated our docking protocol which was
therefore used for the in silico screening of a virtual library
created by an exhaustive combination of intermediates 3 and
a set of commercially available amines/hydrazines. The
compounds with the highest predicted hCB1 affinities, accord-
ing to the binding energy evaluation performed by MOE, were
selected and synthesized (compounds 4).
4. Results and discussion
All compounds 4 were preliminary screened by Cerep
(Poitiers, France) at 10 mM concentration in a competitive
binding experiment toward hCB1 and hCB2 receptors, ex-
pressed in membranes of Chinese Hamster Ovarian (CHO)
cells. [3H]-CP 55940 (0.5 nM) and [3H]-WIN 55212-2
(0.8 nM) were used as radioligands in these experiments for
hCB1 and hCB2, respectively. Table 1 summarizes the results
expressed as the percentages of the radioligand displacement
for derivatives 4. In the biological assays, almost all the tested
compounds displayed significant competitive binding (radioli-
gand displacement >50% at 10 mM concentration) but a weak
selectivity toward the hCB1 receptor.
Compound 4aa is the closest rimonabant analogue and
showed a competitive binding of 79% and 37% against
hCB1 and hCB2, respectively. Secondary N-cycloalkyl amides
4afe4ak showed similar or slightly increased affinity toward
CB1 receptor, in comparison with 4aa and the other hydrazides
4abe4ae. The N,N-disubstitution of the carboxamide function
(4ale4an) resulted in a reduction of the binding affinity and
selectivity for hCB1 receptor. The insertion of an ethylene
spacer between the piperidin-1-yl substituent and the carbox-
amide nitrogen atom caused a reduction of both hCB1 and
hCB2 receptors affinities (compare 4aa and 4ao).
Cl/F (4bae4bd) or Cl/CH3 (4ea, 4eb) replacement at the
para-position of 5-phenyl ring lowered affinity for both recep-
tors, while 4-anisoyl derivatives4fa, 4fb showed increased bind-
ing affinity and selectivity for hCB2 receptor. In general, bromo
Fig. 2. Binding mode of rimonabant (yellow) inside hCB1 receptor; H-bonds are displayed as dashed lines.

2630
G. Menozzi et al. / European Journal of Medicinal Chemistry 43 (2008) 2627e2638
Table 1
Binding affinities of compounds 4 toward hCB1 and hCB2 receptors
CO-R'
N
N
Cl
R
Cl
Compd.
R
R0
Radioligand displacement [%] at 10 mM
hCB1 Receptora
hCB2 Receptorb
4aa
Cl
79
37
NH-N
NH-N
NH-N
NH-N
NH-N
4ab
4ac
4ad
4ae
Cl
Cl
Cl
Cl
65
12
O
61 (Ki ¼ 16 mM)
ꢀ23
64
N-CH3
75
86
56
4af
Cl
Cl
87 (Ki ¼ 0.85 mM)
30 (Ki ¼ 21 mM)
NH
NH
4ag
82
61
4ah
4ai
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
68
78
84
72
49
59
58
39
43
30
62
52
50
44
22
43
45
69
14
7
NH
NH
4aj
NH
NH
4ak
4al
N
N
4am
4an
4ao
4ba
4bb
4bc
N
NH-CH2-CH2-N
NH-N
F
ꢀ9
7
NH-N
NH
O
F

G. Menozzi et al. / European Journal of Medicinal Chemistry 43 (2008) 2627e2638
2631
Table 1 (continued)
Compd.
R
R0
Radioligand displacement [%] at 10 mM
hCB1 Receptora
hCB2 Receptorb
4bd
4ca
4cb
4cc
4cd
4ce
4da
4db
4dc
4dd
4de
4df
4ea
4eb
4fa
F
69
18
NH
Br
74
55
NH-N
NH-N
NH
Br
67 (Ki ¼ 2.6 mM)
ꢀ12 (Ki ¼ 30 mM)
N-CH3
Br
84 (Ki ¼ 1.0 mM)
43
Br
81
44
NH
Br
88 (Ki ¼ 1.1 mM)
60
NH
I
94 (Ki ¼ 1.3 mM)
84 (Ki ¼ 1.9 mM)
NH-N
NH-N
NH
I
78
42
60
59
61
44
27
46
70
N-CH3
I
91 (Ki ¼ 0.21 mM)
I
64
NH
I
90 (Ki ¼ 1.1 mM)
NH
NH
I
76
29
48
58
CH3
CH3
OCH3
OCH3
NH-N
NH
NH-N
4fb
Rimonabantc
64
Ki ¼ 0.025 mM
76
Ki ¼ 1.58 mM
NH
Data reported as percentage (n ¼ 2) of displacement of [3H]-CP 55940 (0.5 nM) from hCB1 receptor, at an inhibitor concentration of 10 mM. In brackets the Ki
a
value, when calculated.
b
Data reported as percentage (n ¼ 2) of displacement of [3H]-WIN 55212-2 (0.8 nM) from hCB2 receptor, at an inhibitor concentration of 10 mM. In brackets the
Ki value, when calculated.
c
Ref. [22].
and iodo para-substituted compounds 4cae4ce and 4dae4df
showed a slightly higher binding affinity than their correspond-
ing chlorinated analogues toward both receptor subtypes. For
a number of pyrazole analogues endowed with the highest selec-
tivity/potency toward hCB1 (as assessed by their radioligand
displacements), the affinity constants Ki were evaluated
(Table 1). Derivatives 4ac, 4cb, 4cc, 4ce, 4da and 4de were
endowed with micromolar activity (Ki range: 1.0e16 mM)
whereas 4af showed submicromolar Ki value (0.85 mM). Com-
pound 4dc was identified as the most active compound of the
screened set (Ki ¼ 0.21 mM) but resulted in about 10-fold less
active than rimonabant (Ki ¼ 5.6 nM [7], Ki ¼ 25 nM [22]).
The Ki values for hCB2 of 4af, 4cb and 4da confirmed the
weak selectivity of the synthesized derivatives toward

2632
G. Menozzi et al. / European Journal of Medicinal Chemistry 43 (2008) 2627e2638
Fig. 3. Binding mode of rimonabant (yellow) and its most structurally related analogue 4aa (coloured by atom type), inside hCB1 receptor; H-bonds are displayed
as dashed lines.
cannabinoid receptor type-2, as anticipated by the radioligand
displacement percentages.
for displaying a potent and selective CB1 receptor antagonistic
activity: (a) a 2,4-dichlorophenyl substituent at position 1, (b)
a para-substituted phenyl ring at position 5 and (c) a carboxa-
mide group at position 3 of the pyrazole ring [14e20]. Taking
into account these indications we evaluated in silico the hCB1
binding affinity of a set of 1,5-diarylpyrazole-4-carboxamides
and the best ranked derivatives were synthesized.
The preliminary biological results for compounds 4 sug-
gested that the shift of the carboxamidic function from posi-
tion 3 to position 4 of the pyrazole ring led to CB1 ligands
endowed with micromolar or submicromolar affinity. Docking
studies carried out on rimonabant and its analogues 4, con-
firmed the structural requirements for CB1 interaction and fur-
ther highlighted the importance of a double hydrogen bond
with Lys192 and Ser383 in order to achieve a high affinity to-
ward the receptor. This information will provide the basis for
the synthesis of new 4-substituted 1,5-diarylpyrazoles en-
dowed with improved affinity and selectivity toward hCB1
or hCB2 receptor.
As assessed by computational studies, all compounds 4
share a common binding pose, comparable with that of rimona-
bant. In Fig. 3, as an example, the binding of the closest rimo-
nabant analogue 4aa is reported. The amidic carbonyl is
engaged in a double hydrogen bond with Lys192 and Ser393
(as observed in rimonabandeCB1 binding model) while the
two aromatic rings are involved in pep stacking interactions
with Trp279, Trp356 and Phe379. Differently from rimona-
bant, the 4aa piperidinyl substituent does not interact with
Phe174, Val196, Met384 and Leu387. The loss of these anchor-
ing points inside the receptor could rationalize the lower affin-
ity of pyrazoles 4 in comparison with the parent compound.
Thus, according to our calculations, the double hydrogen bond
with Lys192 and Ser383 seems to be essential for the proper
positioning of ligand within the receptor binding site. However,
this interaction does not appear to be sufficient to switch the Ki
ofthe CB1 antagonist toward nanomolar values. Thisresult seems
to be achieved only by a contemporary hydrophobic interaction
which some specific residues in the surrounding part of the pro-
tein, suggesting that an accurate balance between the hydrophilic
and hydrophobic substituents on the molecule is extremely im-
portant in the case of hCB1 antagonists.
6. Experimental section
6.1. Chemistry
All chemicals and solvents used were commercially avail-
able and of analytical grade or were prepared according to
the procedure described in the literature [26,27]. All com-
pounds were tested for purity by thin layer chromatography
(TLC) on silica gel plates (60 F254, Merck; ethyl acetate/
5. Conclusions
For rimonabant and its 3-substituted analogues, various re-
ports have highlighted three principal structural requirements

G. Menozzi et al. / European Journal of Medicinal Chemistry 43 (2008) 2627e2638
2633
petroleum ether 1:1 as eluant), visualizing with ultraviolet
light. Melting points were determined with a FishereJohns ap-
paratus and are uncorrected. IR spectra were registered on
a PerkineElmer 398 spectrophotometer and are expressed in
2H, ar), 8.22 ppm (s, 1H, pyr H-3). Anal. C18H13BrCl2N2O2
(C, H, N).
6.1.2.3. Ethyl 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-1H-pyr-
azole-4-carboxylate (2d). Yield 4.42 g (91%); mꢀp1 156e
157 ꢂC (from ethyl ether). IR (CHCl3): n ¼ 1713 cm (CO);
1H NMR (CDCl3): d ¼ 1.19 (t, J ¼ 7.2 Hz, 3H, CH3), 4.16
(q, J ¼ 7.2 Hz, 2H, CH2), 6.93 (d, J ¼ 8.6 Hz, 2H, ar), 7.15e
7.25 (m, 2H, ar), 7.35 (s, 1H, ar), 7.57 (d, J ¼ 8.6 Hz, 2H,
ar), 8.13 ppm (s, 1H, pyr H-3). Anal. C18H13Cl2IN2O2 (C,
H, N).
1
cmꢀ1. H NMR spectra were registered on a Varian Gemini
200 (200 MHz) spectrometer; chemical shifts are reported as
d values (ppm) relative to TMS as internal standard; coupling
constants (J ) are expressed in hertz (Hz). The following NMR
abbreviations are used: br (broad), s (singlet), d (doublet), t
(triplet), q (quartet), sex (sextet), m (multiplet), ex (exchange-
able with D2O), ar (aromatic proton), pip (piperidine), pyr
(pyrazole). Analyses indicated by the symbols of the elements
(C, H, N) were performed using a Carlo Erba Elemental Ana-
lyzer Model EA 1110 and were within ꢁ0.4% of the theoret-
ical values.
6.1.2.4. Ethyl 1-(2,4-dichlorophenyl)-5-(4-methylphenyl)-1H-
pyrazole-4-carboxylate (2e). Yield 2.85 g (76%); mp 88e
89 ꢂC (from petroleum ether). IR (CHCl3): n ¼ 1712 cmꢀ1
1
(CO); H NMR (CDCl3): d ¼ 1.27 (t, J ¼ 7.2 Hz, 3H, ethyl
6.1.1. Ethyl 3-(dimethylamino)-2-(4-iodobenzoyl)-acrylate
(1d)
CH3), 2.33 (s, 3H, CH3-Ar), 4.25 (q, J ¼ 7.2 Hz, 2H, CH2),
7.05e7.30 (m, 6H, ar), 7.43 (s, 1H, ar), 8.22 ppm (s, 1H,
pyr H-3). Anal. C19H16Cl2N2O2 (C, H, N).
A solution of ethyl 4-iodobenzoylacetate (6.36 g, 20 mmol)
in N,N-dimethylformamide dimethyl acetal (2.86 g, 24 mmol)
was refluxed for 1 h. The excess acetal was distilled off under
reduced pressure and the residue was chromatographed on
Florisil, using ethyl ether as eluant. The crude product was
then recrystallized from ethyl ether/petroleum ether (5.06 g,
68% yield); mp 89e90 ꢂC. IR (CHCl3): n ¼ 1682 (CO, ester),
6.1.2.5. Ethyl 1-(2,4-dichlorophenyl)-5-(4-methoxyphenyl)-1H-
pyrazole-4-carboxylate (2f). Yield 3.36 g (86%); mp 71e
72 ꢂC (from ethyl ether/petroleum ether). IR (CHCl3):
n ¼ 1709 cmꢀ1 (CO); 1H NMR (CDCl3): d ¼ 1.19 (t,
J ¼ 7.0 Hz, 3H, ethyl CH3), 3.71 (s, 3H, CH3eO), 4.17 (q,
J ¼ 7.0 Hz, 2H, CH2), 7.74 (d, J ¼ 8.8 Hz, 2H, ar), 7.10e
7.20 (m, 4H, ar), 7.35 (s, 1H, ar), 8.13 ppm (s, 1H, pyr H-
3). Anal. C19H16Cl2N2O3 (C, H, N).
1
1628 (CO, ketone) and 1586 cmꢀ1 (aromatic CeC); H NMR
(CDCl3): d ¼ 0.85 (t, J ¼ 7.0 Hz, 3H, ethyl CH3), 2.90 (br s,
6H, N(CH3)2), 3.89 (q, J ¼ 7.0 Hz, 2H, ethyl CH2), 7.40 (d,
J ¼ 8.0 Hz, 2H, ar), 7.67 (d, J ¼ 8.0 Hz, 2H, ar), 7.72 ppm
(s, 1H, ]CH). Anal. C14H16INO3 (C, H, N).
6.1.3. General procedure for the synthesis of compounds
3ae3f
6.1.2. General procedure for the preparation of compounds
2be2f
Potassium hydroxide (1.68 g, 30 mmol) dissolved in 95%
ethanol (10 ml) was added to a solution of the proper ester 2
(10 mmol) in the same solvent (30 ml). The mixture was re-
fluxed under stirring for 5 h, the solvent was evaporated under
reduced pressure and the residue was dissolved with water
(50 ml). The aqueous solution was acidified with 6 N hydro-
chloric acid (pH w 1) and the solid which separated was ex-
tracted with chloroform. The organic layer was separated,
washed with water, dried (magnesium sulphate) and evapo-
rated under reduced pressure to yield a white solid which
was then recrystallized from suitable solvent.
2,4-Dichlorophenylhydrazine
hydrochloride
(2.35 g,
11 mmol) was added to a stirred solution of the proper ethyl
3-(dimethylamino)-2-aroyl-acrylate 1 (10 mmol) in absolute
ethanol (50 ml). The mixture was refluxed for 2 h and evapo-
rated under reduced pressure. The residue was dissolved in
chloroform and the organic solution was washed with water,
dried (magnesium sulphate), filtered and evaporated under re-
duced pressure. The crude product was purified by recrystalli-
zation from proper solvent.
6.1.2.1. Ethyl 1-(2,4-dichlorophenyl)-5-(4-fluorophenyl)-1H-
pyrazole-4-carboxylate (2b). Yield 3.25 g (86%); mp 78e
79 ꢂC (from petroleum ether). IR (CHCl3): n ¼ 1712 cmꢀ1
6.1.3.1. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-1H-pyra-
zole-4-carboxylic acid (3a). Yield 3.60 g (98%); mp 205e
206 ꢂC (from ethyl ether/petroleum ether). IR (CHCl3):
n ¼ 3200e2500 (OH) and 1690 cmꢀ1 (CO); 1H NMR
(CDCl3): d ¼ 7.15e7.35 (m, 6H, ar), 7.44 (s, 1H, ar), 8.28
(s, 1H, pyr H-3), w11.9 ppm (br s, 1H, CO2H, ex). Anal.
C16H9Cl3N2O2 (C, H, N).
1
(CO); H NMR (CDCl3): d ¼ 1.26 (t, J ¼ 7.0 Hz, 3H, CH3),
4.25 (q, J ¼ 7.0 Hz, 2H, CH2), 6.95e7.10 (m, 2H, ar), 7.20e
7.35 (m, 4H, ar), 7.44 (s, 1H, ar), 8.23 ppm (s, 1H, pyr H-
3). Anal. C18H13Cl2FN2O2 (C, H, N).
6.1.2.2. Ethyl 5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1H-
pyrazole-4-carboxylate (2c). Yield 4.05 g (92%); mp 121e
122 ꢂC (from petroleum ether). IR (CHCl3): n ¼ 1714 cmꢀ1
6.1.3.2. 1-(2,4-Dichlorophenyl)-5-(4-fluorophenyl)-1H-pyra-
zole-4-carboxylic acid (3b). Yield 3.30 g (94%); double mp
158e159 and 171e172 ꢂC (from ethyl ether/petroleum ether).
1
(CO); H NMR (CDCl3): d ¼ 1.27 (t, J ¼ 7.2 Hz, 3H, CH3),
1
4.25 (q, J ¼ 7.2 Hz, 2H, CH2), 7.15 (d, J ¼ 8.6 Hz, 2H, ar),
7.20e7.35 (m, 2H, ar), 7.43 (s, 1H, ar), 7.45 (d, J ¼ 8.6 Hz,
IR (CHCl3): n ¼ 3200e2500 (OH) and 1688 cmꢀ1 (CO); H
NMR (CDCl3): d ¼ 6.95e7.10 (m, 2H, ar), 7.20e7.35 (m,

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G. Menozzi et al. / European Journal of Medicinal Chemistry 43 (2008) 2627e2638
4H, ar), 7.44 (s, 1H, ar), 8.28 (s, 1H, pyr H-3), w10.6 ppm (br
s, 1H, CO2H, ex). Anal. C16H9Cl2FN2O2 (C, H, N).
(72%); mp 218e219 ꢂC (from 95% ethanol). IR (CHCl3):
n ¼ 3415 and 3326 (NH) and 1668 cmꢀ1 (CO); 1H NMR
(CDCl3): d ¼ 1.30e1.45 (m, 2H, CH2), 1.55e1.75 (m, 4H,
2CH2), 2.60e2.75 (m, 4H, 2CH2N), 6.40 (br s, 1H, NH, ex),
7.15e7.40 (m, 6H, ar), 7.44 (s, 1H, ar), 8.15 ppm (s, 1H,
pyr H-3). Anal. C21H19Cl3N4O (C, H, N).
6.1.3.3. 5-(4-Bromophenyl)-1-(2,4-dichlorophenyl)-1H-pyra-
zole-4-carboxylic acid (3c). Yield 3.87 g (94%); mp 214e
215 ꢂC (from ethyl ether/petroleum ether). IR (CHCl3):
n ¼ 3200e2500 (OH) and 1691 cmꢀ1 (CO); 1H NMR
(CDCl3): d ¼ 7.15 (d, J ¼ 8.4 Hz, 2H, ar), 7.20e7.40 (m,
2H, ar), 7.44 (s, 1H, ar), 7.46 (d, J ¼ 8.4 Hz, 2H, ar), 8.27
(s, 1H, pyr H-3), w9.3 ppm (br s, 1H, CO2H, ex). Anal.
C16H9BrCl2N2O2 (C, H, N).
6.1.4.2. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-(mor-
pholin-4-yl)-1H-pyrazole-4-carboxamide (4ab). Yield 0.68 g
(50%); mp 232e233 ꢂC (from ethyl acetate). IR (CHCl3):
n ¼ 3415 and 3329 (NH) and 1671 cmꢀ1 (CO); 1H NMR
(CDCl3): d ¼ 2.65e2.85 (m, 4H, 2CH2N), 3.65e3.85 (m,
4H, 2CH2O), 6.67 (s, 1H, NH, ex), 7.10e7.35 (m, 6H, ar),
7.43 (s, 1H, ar), 8.18 ppm (s, 1H, pyr H-3). Anal.
C20H17Cl3N4O2 (C, H, N).
6.1.3.4. 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-1H-pyrazole-
4-carboxylic acid (3d). Yield 4.40 g (96%); mp 232e233 ꢂC
(from ethyl ether). IR (CHCl3): n ¼ 3200e2500 (OH) and
1
1690 cmꢀ1 (CO); H NMR (CDCl3): d ¼ 6.93 (d, J ¼ 8.4 Hz,
2H, ar), 7.10e7.30 (m, 2H, ar), 7.36 (s, 1H, ar), 7.57 (d,
J ¼ 8.4 Hz, 2H, ar), 8.19 (s, 1H, pyr H-3), w10.2 ppm (br s,
1H, CO2H, ex). Anal. C16H9Cl2IN2O2 (C, H, N).
6.1.4.3. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-(4-meth-
ylpiperazin-1-yl)-1H-pyrazole-4-carboxamide
(4ac). Yield
1.03 g (74%); mp 189e190 ꢂC (from ethyl ether). IR
(CHCl3): n ¼ 3413 and 3328 (NH) and 1671 cmꢀ1 (CO); H
1
6.1.3.5. 1-(2,4-Dichlorophenyl)-5-(4-methylphenyl)-1H-pyra-
zole-4-carboxylic acid (3e). Yield 3.36 g (97%); mp 228e
229 ꢂC (from 95% ethanol). IR (CHCl3): n ¼ 3200e2500
NMR (CDCl3): d ¼ 2.18 (s, 3H, CH3), 2.40e2.55 (m, 4H,
2CH2), 2.60e2.75 (m, 4H, 2CH2), 6.21 (s, 1H, NH, ex),
7.05e7.25 (m, 6H, ar), 7.32 (s, 1H, ar), 8.05 ppm (s, 1H,
pyr H-3). Anal. C21H20Cl3N5O (C, H, N).
1
(OH), 1691 cmꢀ1 (CO); H NMR (CDCl3): d ¼ 2.34 (s, 3H,
CH3), 7.05e7.30 (m, 6H, ar), 7.43 (s, 1H, ar), 8.27 (s, 1H,
pyr H-3), w10.5 ppm (br s, 1H, CO2H, ex). Anal.
C17H12Cl2N2O2 (C, H, N).
6.1.4.4.
N-Azepan-1-yl-5-(4-chlorophenyl)-1-(2,4-dichloro-
(4ad). Yield 0.70 g
phenyl)-1H-pyrazole-4-carboxamide
(50%); mp 202e203 ꢂC (from ethyl acetate). IR (CHCl3):
n ¼ 3413 and 3351 (NH) and 1668 cmꢀ1 (CO); 1H NMR
(CDCl3): d ¼ 1.30e1.65 (m, 8H, 2CH2), 2.65e3.05 (m, 4H,
2CH2N), 6.55 (br s, 1H, NH, ex), 7.00e7.35 (m, 6H, ar),
7.37 (s, 1H, ar), 8.09 ppm (s, 1H, pyr H-3). Anal.
C22H21Cl3N4O (C, H, N).
6.1.3.6. 1-(2,4-Dichlorophenyl)-5-(4-methoxyphenyl)-1H-pyra-
zole-4-carboxylic acid (3f). Yield 3.27 g (90%); mp 190e
191 ꢂC (from ethyl ether). IR (CHCl3): n ¼ 3200e2500 (OH)
and 1689 cmꢀ1 (CO); 1H NMR (CDCl3): d ¼ 3.72 (s, 3H, CH3),
6.74 (d, J ¼ 8.0 Hz, 2H, ar), 7.12 (d, J ¼ 8.0 Hz, 2H, ar), 7.15e
7.25 (m, 2H, ar), 7.35 (s, 1H, ar), 8.18 (s, 1H, pyr H-3),
w10.1 ppm (br s, 1H, CO2H, ex). Anal. C17H12Cl2N2O3 (C, H, N).
6.1.4.5. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-(hexa-
hydrocyclopenta[c]pyrrol-2-(1H )yl)-1H-pyrazole-4-carboxa-
mide (4ae). Yield 0.98 g (69%); mp 192e193 ꢂC (from ethyl
acetate). IR (CHCl3): n ¼ 3418 and 3322 (NH) and 1668 cmꢀ1
(CO); 1H NMR (CDCl3): d ¼ 1.20e1.70 (m, 6H, 3CH2),
2.05e2.25 (m, 1H, CH), 2.30e2.60 (m, 4H, 2CH2N), 2.90e
3.10 (m, 1H, CH), 5.95 (s, 1H, NH, ex), 7.00e7.30 (m, 6H,
ar), 7.37 (s, 1H, ar), 8.04 ppm (s, 1H, pyr H-3). Anal.
C23H21Cl3N4O (C, H, N).
6.1.4. General procedure for the synthesis of compounds
4aae4ao, 4bae4bd, 4cae4ce, 4dae4df, 4ea, 4eb, 4fa, 4fb
A mixture of the proper carboxylic acid 3 (3 mmol) and thi-
onyl chloride (0.8 ml, 11 mmol) in 20 ml of toluene was re-
fluxed for 3 h and then evaporated to dryness under reduced
pressure. The residue was taken up in 20 ml of toluene and
the solvent was evaporated again under reduced pressure to
give the crude acid chloride, which was dissolved in 15 ml
of dry dichloromethane. This solution was added dropwise
to a solution of the proper amine/hydrazine (4.5 mmol) and
triethylamine (0.45 g, 4.5 mmol) in 10 ml of dichloromethane,
cooled to 0 ꢂC. After stirring at room temperature for 16 h, the
reaction mixture was added to brine (30 ml) and extracted with
dichloromethane. The organic layer was separated, dried
(magnesium sulphate) and evaporated under reduced pressure.
The residue was purified by chromatography on Florisil using
ethyl acetate as eluant, to give a white solid, which was then
recrystallized from proper solvent.
6.1.4.6.
5-(4-Chlorophenyl)-N-cyclopropyl-1-(2,4-dichloro-
(4af). Yield 1.07 g
phenyl)-1H-pyrazole-4-carboxamide
(88%); mp 210e211 ꢂC (from 95% ethanol). IR (CHCl3):
n ¼ 3433 (NH) and 1659 cmꢀ1 (CO); 1H NMR (CDCl3):
d ¼ 0.25e0.40 (m, 2H, CH2), 0.60e0.75 (m, 2H, CH2),
2.60e2.75 (m, 1H, CHN), 5.79 (br s, 1H, NH, ex), 7.10e
7.25 (m, 6H, ar), 7.33 (s, 1H, ar), 8.02 ppm (s, 1H, pyr H-
3). Anal. C19H14Cl3N3O (C, H, N).
6.1.4.7. 5-(4-Chlorophenyl)-N-cyclopentyl-1-(2,4-dichloro-
phenyl)-1H-pyrazole-4-carboxamide (4ag). Yield 1.22 g
6.1.4.1. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-piperi-
din-1-yl-1H-pyrazole-4-carboxamide (4aa). Yield 0.98 g
(94%); mp 189e190 ꢂC (from ethyl acetate). IR (CHCl3):
n ¼ 3432 (NH) and 1649 cmꢀ1 (CO); 1H NMR (CDCl3):

G. Menozzi et al. / European Journal of Medicinal Chemistry 43 (2008) 2627e2638
2635
d ¼ 1.00e1.25 (m, 2H, CH2), 1.30e1.60 (m, 4H, 2CH2),
1.70e1.95 (m, 2H, CH2N), 4.20 (sex, J ¼ 6.8 Hz, 1H, CHN),
5.34 (d, J ¼ 7.8 Hz, 1H, NH, ex), 7.05e7.30 (m, 6H, ar),
7.34 (s, 1H, ar), 8.06 ppm (s, 1H, pyr H-3). Anal.
C21H18Cl3N3O (C, H, N).
ar), 7.15e7.30 (m, 4H, ar), 7.40 (s, 1H, ar), 7.78 ppm (s,
1H, pyr H-3). Anal. C21H18Cl3N3O (C, H, N).
6.1.4.14. 2-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-1H-
pyrazol-4-yl]carbonyl}-1,2,3,4-tetrahydroisoquinoline (4an).
Yield 1.33 g (92%); mp 163e164 ꢂC (from 95% ethanol). IR
(CHCl3): n ¼ 1615 cmꢀ1 (CO); 1H NMR (CDCl3):
d ¼ 2.40e2.60 (m, 1H, CH), 2.70e2.90 (m, 1H, CH), 3.40e
3.60 (m, 1H, CH), 3.75e3.95 (m, 1H, CH), 4.25e4.45 (m,
1H, CH), 6.65e6.85 (m, 1H, CH), 6.90e7.30 (m, 10H, ar),
7.40 (s, 1H, ar), 7.85 ppm (s, 1H, pyr H-3). Anal.
C25H18Cl3N3O (C, H, N).
6.1.4.8.
5-(4-Chlorophenyl)-N-cyclohexyl-1-(2,4-dichloro-
phenyl)-1H-pyrazole-4-carboxamide (4ah). Yield 1.28 g
(95%); mp 182e183 ꢂC (from 95% ethanol). IR (CHCl3):
n ¼ 3429 (NH) and 1648 cmꢀ1 (CO); 1H NMR (CDCl3):
d ¼ 0.90e1.65 (m, 8H, 4CH2), 1.75e1.95 (m, 2H, CH2),
3.80e4.0 (m, 1H, CHN), 5.38 (d, J ¼ 7.8 Hz, 1H, NH, ex),
7.20e7.40 (m, 6H, ar), 7.42 (s, 1H, ar), 8.14 ppm (s, 1H,
pyr H-3). Anal. C22H20Cl3N3O (C, H, N).
6.1.4.15. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-(2-pi-
peridin-1-ylethyl)-1H-pyrazole-4-carboxamide
(4ao). Yield
6.1.4.9.
5-(4-Chlorophenyl)-N-cycloheptyl-1-(2,4-dichloro-
(4ai). Yield 1.22 g
1.30 g (91%); mp 148e149 ꢂC (from 95% ethanol). IR
(CHCl3): n ¼ 3394 (NH) and 1646 cmꢀ1 (CO); 1H NMR
(CDCl3): d ¼ 1.20e1.40 (m, 6H, 3CH2), 2.10e2.20 (m, 4H,
2 pip CH2N), 2.26 (t, J ¼ 6.0 Hz, 2H, CH2N), 3.30 (q,
J ¼ 6.0 Hz, 2H, CH2NCO), 6.12 (d, J ¼ 6.0 Hz, 1H, NH, ex),
7.10e7.30 (m, 6H, ar), 7.35 (s, 1H, ar), 8.10 ppm (s, 1H,
pyr H-3). Anal. C23H23Cl3N4O (C, H, N).
phenyl)-1H-pyrazole-4-carboxamide
(88%); mp 173e174 ꢂC (from 95% ethanol). IR (CHCl3):
n ¼ 3431 (NH) and 1648 cmꢀ1 (CO); 1H NMR (CDCl3):
d ¼ 1.10e1.55 (m, 10H, 5CH2), 1.65e1.85 (m, 2H, CH2),
3.90e4.10 (m, 1H, CHN), 5.35 (d, J ¼ 7.8 Hz, 1H, NH, ex),
7.10e7.30 (m, 6H, ar), 7.35 (s, 1H, ar), 8.05 ppm (s, 1H,
pyr H-3). Anal. C23H22Cl3N3O (C, H, N).
6.1.4.16. 1-(2,4-Dichlorophenyl)-5-(4-fluorophenyl)-N-piperi-
din-1-yl-1H-pyrazole-4-carboxamide (4ba). Yield 0.80 g
(62%); mp 209e210 ꢂC (from ethyl acetate). IR (CHCl3):
n ¼ 3415 and 3326 (NH) and 1669 cmꢀ1 (CO); 1H NMR
(CDCl3): d ¼ 1.25e1.45 (m, 2H, CH2), 1.55e1.75 (m, 4H,
2CH2), 2.55e2.75 (m, 4H, 2CH2N), 6.30 (br s, 1H, NH, ex),
6.90e7.10 (m, 2H, ar), 7.15e7.35 (m, 4H, ar), 7.43 (s, 1H,
ar), 8.17 ppm (s, 1H, pyr H-3). Anal. C21H19Cl2FN4O (C, H,
N).
6.1.4.10.
5-(4-Chlorophenyl)-N-cyclooctyl-1-(2,4-dichloro-
(4aj). Yield 1.13 g
phenyl)-1H-pyrazole-4-carboxamide
(79%); mp 165e166 ꢂC (from ethyl acetate). IR (CHCl3):
n ¼ 3431 (NH) and 1648 cmꢀ1 (CO); 1H NMR (CDCl3):
d ¼ 1.20e1.55 (m, 12H, 6CH2), 1.60e1.80 (m, 2H, CH2),
3.90e4.10 (m, 1H, CHN), 5.32 (d, J ¼ 7.8 Hz, 1H, NH, ex),
7.10e7.30 (m, 6H, ar), 7.35 (s, 1H, ar), 8.07 ppm (s, 1H,
pyr H-3). Anal. C24H24Cl3N3O (C, H, N).
6.1.4.11.
N-Adamantan-1-yl-5-(4-chlorophenyl)-1-(2,4-di-
(4ak). Yield
6.1.4.17.
1-(2,4-Dichlorophenyl)-5-(4-fluorophenyl)-N-mor-
chlorophenyl)-1H-pyrazole-4-carboxamide
pholin-4-yl-1H-pyrazole-4-carboxamide (4bb). Yield 0.97 g
(75%); mp 210e211 ꢂC (from ethyl acetate). IR (CHCl3):
n ¼ 3417 and 3328 (NH) and 1672 cmꢀ1 (CO); 1H NMR
(CDCl3): d ¼ 2.65e2.85 (m, 4H, 2CH2 N), 3.65e3.85 (m,
4H, 2CH2O), 6.56 (s, 1H, NH, ex), 6.90e7.10 (m, 2H, ar),
7.15e7.35 (m, 4H, ar), 7.42 (s, 1H, ar), 8.17 ppm (s, 1H,
pyr H-3). Anal. C20H17Cl2FN4O2 (C, H, N).
1.30 g (87%); double mp 186e187 and 208e209 ꢂC (from
95% ethanol). IR (CHCl3): n ¼ 3422 (NH) and 1654 cmꢀ1
(CO); 1H NMR (CDCl3): d ¼ 1.50e2.10 (m, 15 H,
6CH2 þ 3CH), 5.08 (br s, 1H, NH, ex), 7.0e7.30 (m, 6H,
ar), 7.35 (s, 1H, ar), 8.03 ppm (s, 1H, pyr H-3). Anal.
C26H24Cl3N3O (C, H, N).
6.1.4.12. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-(pyrro-
lidin-1-ylcarbonyl)-1H-pyrazole (4al). Yield 1.13 g (90%);
double mp 118e119 and 150e151 ꢂC (from ethyl ether/petro-
leum ether). IR (CHCl3): n ¼ 1612 cmꢀ1 (CO); 1H NMR
(CDCl3): d ¼ 1.60e1.90 (m, 4H, 2CH2), 3.15 (t, J ¼ 6.4 Hz,
2H, CH2N), 3.48 (t, J ¼ 6.4 Hz, 2H, CH2N), 7.05e7.25 (m,
6H, ar), 7.37 (s, 1H, ar), 7.85 ppm (s, 1H, pyr H-3). Anal.
C20H16Cl3N3O (C, H, N).
6.1.4.18. N-Cyclopropyl-1-(2,4-dichlorophenyl)-5-(4-fluoro-
phenyl)-1H-pyrazole-4-carboxamide
(4bc). Yield
1.05 g
(90%); mp 177e178 ꢂC (from ethyl ether). IR (CHCl3):
n ¼ 3433 (NH) and 1659 cmꢀ1 (CO); 1H NMR (CDCl3):
d ¼ 0.20e0.35 (m, 2H, CH2), 0.55e0.75 (m, 2H, CH2),
2.55e2.75 (m, 1H, CHN), 5.65 (br s, 1H, NH, ex), 6.85e7.0
(m, 2H, ar), 7.05e7.25 (m, 4H, ar), 7.33 (s, 1H, ar),
8.04 ppm (s, 1H, pyr H-3). Anal. C19H14Cl2FN3O (C, H, N).
6.1.4.13. 1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-1H-
pyrazol-4-yl]carbonyl}piperidine (4am). Yield 1.12 g (86%);
mp 196e197 ꢂC (from 95% ethanol). IR (CHCl3):
6.1.4.19.
N-Cyclohexyl-1-(2,4-dichlorophenyl)-5-(4-fluoro-
(4bd). Yield 0.88 g
phenyl)-1H-pyrazole-4-carboxamide
(68%); mp 170e171 ꢂC (from ethyl acetate). IR (CHCl3):
n ¼ 3428 (NH) and 1648 cmꢀ1 (CO); 1H NMR (CDCl3):
d ¼ 0.90e1.65 (m, 8H, 4CH2), 1.75e1.95 (m, 2H, CH2),
3.75e3.95 (m, 1H, CHN), 5.34 (d, J ¼ 7.8 Hz, 1H, NH, ex),
1
n ¼ 1615 cmꢀ1 (CO); H NMR (CDCl3): d ¼ 1.00e1.25 (m,
2H, CH2), 1.35e1.60 (m, 4H, 2CH2), 3.05e3.25 (m, 2H,
CH2N), 3.45e3.65 (m, 2H, CH2N), 7.06 (d, J ¼ 8.8 Hz, 2H,

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G. Menozzi et al. / European Journal of Medicinal Chemistry 43 (2008) 2627e2638
7.0e7.40 (m, 6H, ar), 7.43 (s, 1H, ar), 8.16 ppm (s, 1H, pyr H-
3). Anal. C22H20Cl2FN3O (C, H, N).
(CDCl3): d ¼ 1.20e1.40 (m, 2H, CH2), 1.50e1.70 (m, 4H,
2CH2), 2.70e2.95 (m, 4H, 2CH2N), 6.55 (br s, 1H, NH, ex),
6.95 (d, J ¼ 8.4 Hz, 2H, ar), 7.10e7.25 (m, 2H, ar), 7.35 (s,
1H, ar), 7.60 (d, J ¼ 8.2 Hz, 2H, ar), 8.18 ppm (s, 1H, pyr
H-3). Anal. C21H19Cl2IN4O (C, H, N).
6.1.4.20. 5-(4-Bromophenyl)-1-(2,4-dichlorophenyl)-N-piperi-
din-1-yl-1H-pyrazole-4-carboxamide (4ca). Yield 0.81 g
(55%); mp 219e220 ꢂC (from 95% ethanol). IR (CHCl3):
n ¼ 3415 and 3326 (NH) and 1668 cmꢀ1 (CO); 1H NMR
(CDCl3): d ¼ 1.30e1.50 (m, 2H, CH2), 1.55e1.80 (m, 2H,
2CH2), 2.60e2.85 (m, 4H, 2CH2N), 6.38 (br s, 1H, NH, ex),
7.05e7.30 (m, 4H, ar), 7.35e7.60 (m, 3H, ar), 7.44 (s, 1H,
ar), 8.12 ppm (s, 1H, pyr H-3). Anal. C21H19BrCl2N4O (C,
H, N).
6.1.4.26. 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-N-(4-meth-
ylpiperazin-1-yl)-1H-pyrazole-4-carboxamide
(4db). Yield
1.22 g (73%); mp 233e234 ꢂC (from 95% ethanol). IR
(CHCl3): n ¼ 3415 and 3327 (NH) and 1671 cmꢀ1 (CO); H
1
NMR (CDCl3): d ¼ 2.23 (s, 3H, CH3), 2.35e2.55 (m, 4H,
2CH2), 2.60e2.80 (m, 4H, 2CH2), 6.20 (s, 1H, NH, ex),
6.95 (d, J ¼ 8.2 Hz, 2H, ar), 7.10e7.25 (m, 2H, ar), 7.35 (s,
1H, ar), 7.60 (d, J ¼ 8.2 Hz, 2H, ar), 8.08 ppm (s, 1H, pyr
H-3). Anal. C21H20Cl2IN5O (C, H, N).
6.1.4.21. 5-(4-Bromophenyl)-1-(2,4-dichlorophenyl)-N-(4-me-
thylpiperazin-1-yl)-1H-pyrazole-4-carboxamide (4cb). Yield
1.60 g (78%); mp 203e204 ꢂC (from ethyl acetate). IR
1
(CHCl3): n ¼ 3417 and 3328 (NH) and 1671 cmꢀ1 (CO); H
6.1.4.27.
N-Cyclopropyl-1-(2,4-dichlorophenyl)-5-(4-iodo-
(4dc). Yield 1.39 g
NMR (CDCl3): d ¼ 2.23 (s, 3H, CH3), 2.35e2.55 (m, 4H,
2CH2), 2.60e2.80 (m, 4H, 2CH2), 6.30 (s, 1H, NH, ex),
7.09 (d, J ¼ 8.0 Hz, 2H, ar), 7.15e7.25 (m, 2H, ar), 7.35 (s,
1H, ar), 7.39 (d, J ¼ 8.0 Hz, 2H, ar), 8.08 ppm (s, 1H, pyr
H-3). Anal. C21H20BrCl2N5O (C, H, N).
phenyl)-1H-pyrazole-4-carboxamide
(93%); mp 228e229 ꢂC (from 95% ethanol). IR (CHCl3):
n ¼ 3433 (NH) and 1658 cmꢀ1 (CO); 1H NMR (CDCl3):
d ¼ 0.25e0.40 (m, 2H, CH2), 0.60e0.75 (m, 2H, CH2),
2.60e2.75 (m, 1H, CHN), 5.65 (br s, 1H, NH, ex), 6.94 (d,
J ¼ 8.6 Hz, 2H, ar), 7.10e7.25 (m, 2H, ar), 7.35 (s, 1H, ar),
7.60 (d, J ¼ 8.6 Hz, 2H, ar), 8.01 ppm (s, 1H, pyr H-3).
Anal. C19H14Cl2IN3O (C, H, N).
6.1.4.22. 5-(4-Bromophenyl)-N-cyclopropyl-1-(2,4-dichloro-
phenyl)-1H-pyrazole-4-carboxamide
(4cc). Yield
1.23 g
(91%); mp 220e221 ꢂC (from 95% ethanol). IR (CHCl3):
n ¼ 3434 (NH) and 1659 cmꢀ1 (CO); 1H NMR (CDCl3):
d ¼ 0.30e0.40 (m, 2H, CH2), 0.65e0.80 (m, 2H, CH2),
2.60e2.75 (m, 1H, CHN), 5.65 (br s, 1H, NH, ex), 7.00e
7.25 (m, 4H, ar), 7.30e7.45 (m, 3H, ar), 8.02 ppm (s, 1H,
pyr H-3). Anal. C19H14BrCl2N3O (C, H, N).
6.1.4.28.
N-Cyclohexyl-1-(2,4-dichlorophenyl)-5-(4-iodo-
(4dd). Yield 1.54 g
phenyl)-1H-pyrazole-4-carboxamide
(95%); mp 228e229 ꢂC (from 95% ethanol). IR (CHCl3):
n ¼ 3429 (NH) and 1647 cmꢀ1 (CO); 1H NMR (CDCl3):
d ¼ 0.85e1.60 (m, 8H, 4CH2), 1.65e1.85 (m, 2H, CH2),
3.70e3.90 (m, 1H, CHN), 5.33 (d, J ¼ 7.8 Hz, 1H, NH, ex),
7.10 (d, J ¼ 7.8 Hz, 2H, ar), 7.15e7.25 (m, 2H, ar), 7.35 (s,
1H, ar), 7.42 (d, J ¼ 7.8 Hz, 2H, ar), 8.05 ppm (s, 1H, pyr
H-3). Anal. C22H20Cl2IN3O (C, H, N).
6.1.4.23.
5-(4-Bromophenyl)-N-cyclohexyl-1-(2,4-dichloro-
(4cd). Yield 1.33 g
phenyl)-1H-pyrazole-4-carboxamide
(90%); mp 206e207 ꢂC (from 95% ethanol). IR (CHCl3):
n ¼ 3429 (NH) and 1648 cmꢀ1 (CO); 1H NMR (CDCl3):
d ¼ 0.85e1.55 (m, 8H, 4CH2), 1.70e1.90 (m, 2H, CH2),
3.70e3.90 (m, 1H, CHN), 5.32 (d, J ¼ 7.8 Hz, 1H, NH, ex),
6.96 (d, J ¼ 8.2 Hz, 2H, ar), 7.10e7.25 (m, 2H, ar), 7.35 (s,
1H, ar), 7.62 (d, J ¼ 8.2 Hz, 2H, ar), 8.04 ppm (s, 1H, pyr
H-3). Anal. C22H20BrCl2N3O (C, H, N).
6.1.4.29.
N-Cycloheptyl-1-(2,4-dichlorophenyl)-5-(4-iodo-
(4de). Yield 1.63 g
phenyl)-1H-pyrazole-4-carboxamide
(98%); mp 199e200 ꢂC (from 95% ethanol). IR (CHCl3):
n ¼ 3431 (NH) and 1648 cmꢀ1 (CO); 1H NMR (CDCl3):
d ¼ 1.10e1.55 (m, 10H, 5CH2), 1.65e1.85 (m, 2H, CH2),
3.90e4.10 (m, 1H, CHN), 5.34 (d, J ¼ 7.8 Hz, 1H, NH, ex),
6.96 (d, J ¼ 8.6 Hz, 2H, ar), 7.10e7.25 (m, 2H, ar), 7.35 (s,
1H, ar), 7.63 (d, J ¼ 8.6 Hz, 2H, ar), 8.05 ppm (s, 1H, pyr
H-3). Anal. C23H22Cl2IN3O (C, H, N).
6.1.4.24. 5-(4-Bromophenyl)-N-cycloheptyl-1-(2,4-dichloro-
phenyl)-1H-pyrazole-4-carboxamide
(4ce). Yield
1.24 g
(81%); double mp 188e189 and 192e193 ꢂC (from 95% eth-
anol). IR (CHCl3): n ¼ 3431 and 3326 (NH) and 1648 cmꢀ1
(CO); 1H NMR (CDCl3): d ¼ 1.10e1.55 (m, 10H, 5CH2),
1.70e1.90 (m, 2H, CH2), 3.90e4.10 (m, 1H, CHN), 5.35 (d,
J ¼ 7.8 Hz, 1H, NH, ex), 7.10 (d, J ¼ 8.6 Hz, 2H, ar), 7.15e
7.25 (m, 2H, ar), 7.35 (s, 1H, ar), 7.42 (d, J ¼ 8.6 Hz, 2H,
ar), 8.05 ppm (s, 1H, pyr H-3). Anal. C23H22BrCl2N3O (C,
H, N).
6.1.4.30. N-Adamantan-1-yl-1-(2,4-dichlorophenyl)-5-(4-iodo-
phenyl)-1H-pyrazole-4-carboxamide
(4df). Yield
1.72 g
(97%); mp 238e239 ꢂC (from 95% ethanol). IR (CHCl3):
n ¼ 3422 (NH) and 1653 cmꢀ1 (CO); 1H NMR (CDCl3):
d ¼ 1.50e2.10 (m, 15 H, 6CH2 þ 3CH), 5.08 (br s, 1H, NH,
ex), 6.95 (d, J ¼ 8.4 Hz, 2H, ar), 7.10e7.25 (m, 2H, ar),
7.35 (s, 1H, ar), 7.62 (d, J ¼ 8.4 Hz, 2H, ar), 8.01 ppm (s,
1H, pyr H-3). Anal. C26H24Cl2IN3O (C, H, N).
6.1.4.25. 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-N-piperi-
din-1-yl-1H-pyrazole-4-carboxamide (4da). Yield 1.08 g
(67%); mp 229e230 ꢂC (from 95% ethanol). IR (CHCl3):
n ¼ 3415 and 3326 (NH) and 1668 cmꢀ1 (CO); 1H NMR
6.1.4.31. 1-(2,4-Dichlorophenyl)-N-(piperidin-1-yl)-5-(4-meth-
ylphenyl)-1H-pyrazole-4-carboxamide (4ea). Yield 0.75 g

G. Menozzi et al. / European Journal of Medicinal Chemistry 43 (2008) 2627e2638
2637
(58%); mp 228e229 ꢂC (from 95% ethanol). IR (CHCl3):
n ¼ 3410 and 3323 (NH) and 1666 cmꢀ1 (CO); 1H NMR
(CDCl3): d ¼ 1.25e1.45 (m, 2H, CH2), 1.55e1.70 (m, 4H,
2CH2), 2.37 (s, 3H, CH3), 2.50e2.65 (m, 4H, 2CH2N), 6.24
(br s, 1H, NH, ex), 7.15e7.30 (m, 6H, ar), 7.42 (s, 1H, ar),
8.22 ppm (s, 1H, pyr H-3). Anal. C22H22Cl2N4O (C, H, N).
Subsequently, a 100 ps molecular dynamics simulation at
300ꢂK (step length: 0.001 ps) involving only the side-chain
atoms was carried out. The structure was then subjected to an-
other 5000 steps of energy minimisation. Structural evaluation
of the overall model was accomplished using the program
PROCHECK. Docking studies were subsequently performed,
according the following protocol. In a first step, a blind dock-
ing procedure was applied on rimonabant so as to identify its
putative binding site inside the protein, then results were com-
pared with site-directed mutagenesis data and with computa-
tional results available in literature. In a second step, the
validated docked procedure was applied to identify the more
promising hCB1 ligands out of a virtual library obtained by
the combinatorial combination of intermediates 3 and a num-
ber of amines/hydrazines available on the market. The docking
poses were evaluated in terms of total estimated binding en-
ergy, internal strain energy of the ligand, van der Waals and
electrostatic interaction energies and for each ligand, 20 best
docked conformations, according to the MOE score, were
saved and analysed.
6.1.4.32.
N-Cyclohexyl-1-(2,4-dichlorophenyl)-5-(4-methyl-
(4eb). Yield 1.10 g
phenyl)-1H-pyrazole-4-carboxamide
(86%); mp 183e184 ꢂC (from ethyl acetate/petroleum ether).
IR (CHCl3): n ¼ 3421 and 3326 (NH) and 1646 cmꢀ1 (CO);
1H NMR (CDCl3): d ¼ 0.80e1.55 (m, 8H, 4CH2), 1.70e
1.90 (m, 2H, CH2), 2.36 (s, 3H, CH3), 3.75e3.95 (m, 1H,
CHN), 5.38 (d, J ¼ 7.8 Hz, 1H, NH, ex), 7.20e7.30 (m, 6H,
ar), 7.42 (s, 1H, ar), 8.21 ppm (s, 1H, pyr H-3). Anal.
C23H23Cl2N3O (C, H, N).
6.1.4.33. 1-(2,4-Dichlorophenyl)-5-(4-methoxyphenyl)-N-(pi-
peridin-1-yl)-1H-pyrazole-4-carboxamide (4fa). Yield 0.80 g
(60%); mp 199e200 ꢂC (from 95% ethanol). IR (CHCl3):
n ¼ 3409 and 3322 (NH) and 1669 cmꢀ1 (CO); 1H NMR
(CDCl3): d ¼ 1.15e1.25 (m, 4H, CH2), 1.45e1.65 (m, 4H,
2CH2), 2.45e2.65 (m, 4H, 2CH2N), 6.24 (br s, 1H, NH, ex),
6.78 (d, J ¼ 8.6 Hz, 2H, ar), 7.0e7.20 (m, 4H, ar), 7.32 (s,
1H, ar), 8.12 ppm (s, 1H, pyr H-3). Anal. C22H22Cl2N4O2
(C, H, N).
All calculations were carried out on a standard personal
computer running under Windows XP.
6.3. Biology
Competitive binding experiments toward the human CB1
and CB2 cannabinoid receptors, expressed in membranes of
Chinese Hamster Ovarian (CHO) cells, were performed by
Cerep (Poitiers, France) at 10 mM concentrations. [3H]-CP
55940 (0.5 nM) and [3H]-WIN 55212-2 (0.8 nM) were used
as radioligands for the hCB1 and hCB2 receptors, respectively.
Further details of the methodologies for each assay can be
found at the following URL: http://www.cerep.fr. The specific
ligand binding to the receptors is defined as the difference be-
tween the total binding and the non-specific binding deter-
mined in the presence of an excess of unlabelled ligand. The
results are expressed as a percentage of control specific bind-
ing obtained in the presence of the test compounds. The affin-
ity constants (Ki) were calculated from the ChengePrusoff
equation (Ki ¼ IC50/(1 þ (L/KD)), where L ¼ concentration of
radioligand in the assay, and KD ¼ affinity of the radioligand
for the receptor).
6.1.4.34. N-Cyclohexyl-1-(2,4-dichlorophenyl)-5-(4-methoxy-
phenyl)-1H-pyrazole-4-carboxamide
(4fb). Yield
0.96 g
(72%); mp 194e195 ꢂC (from 95% ethanol). IR (CHCl3):
n ¼ 3420 (NH) and 1645 cmꢀ1 (CO); 1H NMR (CDCl3):
d ¼ 0.75e1.50 (m, 8H, 4CH2), 1.60e1.80 (m, 2H, 2CH2),
3.74 (s, 3H, CH3), 3.70e3.90 (m, 1H, CHN), 5.30 (d,
J ¼ 7.8 Hz, 1H, NH, ex), 6.80 (d, J ¼ 8.6 Hz, 2H, ar), 7.05e
7.20 (m, 4H, ar), 7.33 (s, 1H, ar), 8.12 ppm (s, 1H, pyr H-
3). Anal. C23H23Cl2N3O2 (C, H, N).
6.2. Computational methods
The studied compounds were sketched and energy mini-
mized within MOE (CCG) [34]. The theoretical three-dimen-
sional model of the hCB1 receptor was built by homology
modeling using bovine rhodopsine coordinates [PDB code
1F88] according to the procedure already described [33].
Briefly, the amino acid sequence of the CB1 receptor was re-
trieved from the SWISSPROT database. The sequence align-
ment was performed with CLUSTALW, results were in
agreement with those obtained by Shim et al. [29]. Secondary
structure predictions were carried out using PSIPRED V2.0.
Fold recognition and sequence to structure alignment was per-
formed using THREADER. The three-dimensional structure
coordinate file of bovine rhodopsin was obtained from the Pro-
tein Data Bank. Only the seven TM receptor domains were
modelled, since the exact conformation of the extracellular
loops is not computable without X ray data of the specific re-
ceptor structure. The final structure was subjected to 5000
steps of energy minimisation using the AMBER force field.
Acknowledgements
Financial support from Italian MIUR (Cofin 2006, prot.
2006030948_002) and Banca Carige S.p.A., Genova, Italy
(E.C. grant) is gratefully acknowledged.
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