1-Aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide: An effective scaffold for the design of either CB1 or CB2 receptor ligands

Article abstract of DOI:10.1016/j.ejmech.2011.09.037

New 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides were synthesized as cannabinoid (CB) receptor ligands. Compound 11 (CB₁ K_i = 2.3 nM, CB₁ SI = 163.6) showed CB₁ receptor affinity and selectivity superior to Rimonabant and AM251. Acute administration of 2 mg/kg 11 reduced sucrose, but not regular food, intake in rats. On the other hand, compound 23 (CB₂ K_i = 0.51 nM, CB₂ SI = 30.0) showed significant affinity and selectivity for the CB₂ receptor. The results presented here show that the 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3- carboxamide may serve as an effective scaffold for the design of either CB ₁ or CB₂ receptor ligands.

Full text of DOI:10.1016/j.ejmech.2011.09.037

European Journal of Medicinal Chemistry 46 (2011) 5641e5653
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
1-Aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide: An effective scaffold
for the design of either CB1 or CB2 receptor ligands
Francesco Piscitelli a, Alessia Ligresti b, Giuseppe La Regina a, Valerio Gatti a, Antonella Brizzi c,
Serena Pasquini c, Marco Allarà b, Mauro Antonio Maria Carai d, Ettore Novellino e,
**
,
a,
*
Giancarlo Colombo d, Vincenzo Di Marzo b, Federico Corelli c , Romano Silvestri
a Istituto Pasteur e Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185 Roma, Italy
b Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, Comprensorio Olivetti, I-80078 Pozzuoli, Napoli, Italy
c Dipartimento Farmaco Chimico Tecnologico, Università di Siena, Polo Scientifico Universitario San Miniato, Via Alcide De Gasperi 2, I-53100 Siena, Italy
d Istituto di Neuroscienze, Sezione di Cagliari, Consiglio Nazionale delle Ricerche, S.S. 554, km 4.500, I-09042 Cagliari, Italy
e Dipartimento di Chimica Farmaceutica e Tossicologica, Università di Napoli Federico II, Via Domenico Montesano 49, I-80131 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 April 2011
Received in revised form
5 September 2011
Accepted 21 September 2011
Available online 29 September 2011
New 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides were synthesized as cannabinoid (CB)
receptor ligands. Compound 11 (CB1 Ki ¼ 2.3 nM, CB1 SI ¼ 163.6) showed CB1 receptor affinity and
selectivity superior to Rimonabant and AM251. Acute administration of 2 mg/kg 11 reduced sucrose, but
not regular food, intake in rats. On the other hand, compound 23 (CB2 Ki ¼ 0.51 nM, CB2 SI ¼ 30.0)
showed significant affinity and selectivity for the CB2 receptor. The results presented here show that the
1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide may serve as an effective scaffold for the design of
either CB1 or CB2 receptor ligands.
Keywords:
Cannabinoid
Ó 2011 Elsevier Masson SAS. All rights reserved.
Human recombinant CB receptor type 1
Pyrrole bioisosteres
Structureeactivity relationships
Pharmacological studies
1. Introduction
been withdrawn from the European market, again because of an
adverse side effect profile.
The endocannabinoid system is involved in pain, immunosup-
pression, vascular disease, appetite control and locomotor disor-
ders [1]. Rimonabant (SR141716, 1, Chart 1) is a cannabinoid CB1
receptor antagonist/inverse agonist launched in 2006 for the
treatment of overweight, obesity and associated cardiovascular and
metabolic disorders. Two years later, 1 was withdrawn because of
psychiatric adverse events, including depression and anxiety.
Despite the withdrawal of 1, novel CB1 receptor antagonists are
actively searched by many research groups [2,3]. Indeed, there is
a need of effective drugs to treat obesity [4] because of the: (i) rapid
increase of overweight people in the developed countries, and (ii)
restricted therapeutic potential displayed by the FDA approved
drugs sibutramine and orlistat [5], the former of which has now
In search for new CB1 receptor antagonists/inverse agonists,
three synthetic approaches have been explored so far: (i) analogs of
1 which maintain the central pyrazole nucleus (i.e. AM251 (2)), (ii)
replacement of the pyrazole nucleus with different 5-membered
heterocyclic rings [6], and (iii) compounds obtained by either
opening or closure of 1’s template [7].
Our approach consisted of the replacement of the 4-
chlorophenyl group at position 5 of 1 with a pyrrole nucleus [8],
and led to the finding of new potent and selective CB1 inverse
agonists. In particular, the introduction of a lipophilic substituent at
the 3-carboxamide nitrogen provided ligands endowed with either
high affinity (e.g. 4: Ki(CB1) ¼ 5.6 nM) or selectivity (e.g. 5:
SI ¼ 140.7) for the CB1receptor [9,10]. A new series of N-alkyl 1-aryl-
5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides also proved to be
potent CB1 receptor ligands, although they were one order of
magnitude less potent than the parent compounds [11]. Unfortu-
nately, none of the above compounds proved to possess the desir-
able affinity and selectivity for the CB1 receptor [9e11].
* Corresponding author. Tel.: þ39 06 4991 3800; fax: þ39 06 4969 3268.
** Corresponding author. Tel.: þ39 0577 234308; fax: þ39 0577 234299.
E-mail addresses: corelli@unisi.it (F. Corelli), romano.silvestri@uniroma1.it
(R. Silvestri).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.037

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F. Piscitelli et al. / European Journal of Medicinal Chemistry 46 (2011) 5641e5653
Chart 1. Structures of reference compounds 1e5 and new compounds 6e25.
In the present work, we focused on new unexplored chemical
modifications of both pyrazole and pyrrole nuclei, while retaining
the 3,4-dichlorobenzyl and cyclohexyl substituents at the 3-
Lithium aluminum hydride reduction of the hexahydrophthalic
anhydride in tetrahydrofuran at 60 ꢀC gave the corresponding diol
derivative 54, that was converted into the desired cyclohexane-
1,2-dicarbaldehyde (55) by Swern oxidation with oxalyl chloride
in the presence of dimethylsulfoxide and triethylamine in
dichloromethane at ꢁ78 ꢀC. Octane-3,6-dione (57) was achieved
by the conversion of diethyl succinate in the corresponding
Weinreb diamide 56 in benzene at room temperature, and
subsequent treatment of 56 with the ethylmagnesium bromide
(Scheme 3).
carboxamide nitrogen of compounds
4 and 5, respectively.
Herein, we describe the synthesis and the biological evaluation of
carboxamides 6e25 (Chart 1, Table 1). Compound 11, the most
potent and selective ligand, was evaluated for effects on reduction
of food intake in rats (Chart 1).
2. Chemistry
Reaction of diethyl oxalate with butyronitrile in the presence of
potassium tert-butoxide and 18-crown-6 in tetrahydrofuran fur-
nished the potassium salt of 3-cyano-1-ethoxy-1-oxopent-2-en-2-
olate (27), that was converted into the ethyl 5-amino-4-
ethylpyrazole-3-carboxylates 30 or 31 by reaction with 2,4-
dichloro- or 3,4-dichlorophenylhydrazine hydrochloride, respec-
tively. Compounds 28 and 29 were similarly prepared using pro-
pionitrile, as we previously reported [9]. Clauson-Kaas pyrrole
synthesis by heating 29e31 with 2,5-dimethoxytetrahydrofuran in
glacial acetic acid provided pyrrole derivatives 32 [9], 33 [9], 34 and
35. Reaction of 32 or 33 with 1 or 2 equiv of N-chlorosuccinimide in
N,N-dimethylformamide gave in turn the 2-chloropyrroles 36 or 37,
or the 2,5-dichloropyrrole derivatives 38 or 39, respectively.
Compounds 40e43 were obtained by heating the corresponding
amine with cyclohexane-1,2-dicarbaldehyde (40), hexane-2,5-
diones (41, 42) or octane-3,6-dione (43) in glacial acetic acid
(Scheme 1).
Lithium hydroxide hydrolysis of esters 34e43 in aqueous
tetrahydrofuran at room temperature gave the corresponding
carboxylic acids 44e53. The acids were coupled with either 3,4-
dichlorobenzylamine or cyclohexylamine in the presence of N-
ethyl-N0-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochlo-
ride and 1-hydroxybenzotriazole (HOBt) to give amides 6e25.
Morpholinomethylpolystyrene and polymer bound para-toluene-
sulfonic acid were used as scavengers for acidic and basic
substances, respectively (Scheme 2).
3. Results and discussion
3.1. Binding affinity
Compounds 6e25 were evaluated as ligands for the human
recombinant CB receptors hCB1 and hCB2, and were compared to
reference compounds 1e3 and previous lead compounds 4 and 5
[9] (Table 1).
The binding affinities (Ki values) to the receptor were evaluated
by means of membranes from HEK cells transfected with either the
hCB1or hCB2 receptor and [3H]-CP-55,940. The high affinity ligand
Kd values were 0.18 nM for CB1 receptor, and 0.31 nM for CB2
receptor [12]. Displacement curves were derived after incubation of
the drugs with [3H]-CP-55,940 at 0.14 nM for CB1 and 0.084 nM for
CB2 binding assay. The Ki values were calculated from the IC50
values according to the ChengePrusoff equation [13].
Our previous SAR studies [9e11] showed that both 3,4-
dichlorobenzyl (4) and cyclohexyl (5) moieties at the 3-carboxamide
nitrogenwere optimally shaped for the CB1 receptor affinity of 1-aryl-
5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide ligands. Replace-
mentof the3,4-dichlorobenzyl of 4 with 4-chlorobenzyl group aswell
as thecyclohexylof5with apiperidinylnucleus, resulted ina decrease
of CB1 binding affinity. Moreover, the introduction of methyl groupsat
the positions 2 and 5 of the pyrrole led to ligands endowed with
greater selectivity index (SI) for the CB1 receptor (Chart 2). These
observations suggested that the introduction of additional small

F. Piscitelli et al. / European Journal of Medicinal Chemistry 46 (2011) 5641e5653
5643
Table 1
Structure, hCB1 and hCB2 receptor affinity ([3H]CP-55,940 radioligand) of carboxamides 6e25.a
R2
NH
O
R3
N
R4
N
R1
Compd
R1
R2
R3
R4
Ki (nM)b
Ki (nM)b
SIc
SId
hCB1
hCB2
CB1 vs CB2
CB2 vs CB1
6
2,4-Cl2
Me
25.2
98.5
3.9
0.26
7
8
2,4-Cl2
2,4-Cl2
Me
Me
7.7
18.3
2.4
2.0
0.40
0.49
66.1
135.2
9
2,4-Cl2
Me
9.4
19.1
2.0
0.49
10
11
12
2,4-Cl2
2,4-Cl2
2,4-Cl2
Me
Me
Et
45.9
2.3
72.9
376.4
454.6
1.6
163.6
9.3
0.63
0.006
0.11
48.7
13
14
2,4-Cl2
2,4-Cl2
Et
Et
3.0
15.8
56.1
5.3
0.19
0.94
53.1
1.06
15
16
2,4-Cl2
3,4-Cl2
Et
6.7
753.8
112.5
0.009
Me
>10000
>10000
e
e
17
18
3,4-Cl2
3,4-Cl2
Me
Me
136.3
74.1
409.6
53.9
3.0
0.33
1.38
0.73
19
20
3,4-Cl2
3,4-Cl2
Me
Me
8.6
0.9
0.11
2.9
9.56
0.34
55.6
161.7
(continued on next page)

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F. Piscitelli et al. / European Journal of Medicinal Chemistry 46 (2011) 5641e5653
Table 1 (continued )
Compd
R1
R2
R3
R4
Ki (nM)b
Ki (nM)b
SIc
SId
hCB1
hCB2
CB1 vs CB2
CB2 vs CB1
21
22
3,4-Cl2
3,4-Cl2
Me
Et
5.6
1.3
0.23
4.31
15.3
454.0
29.6
0.065
23
24
3,4-Cl2
3,4-Cl2
Et
Et
15.3
0.51
0.033
0.118
30.0
8.49
965.7
113.7
1.7
25
3,4-Cl2
Et
48.0
0.035
28.2
1e
2e
3e
e
e
e
e
e
e
e
e
e
e
e
e
12
2.3
>2820
790
112
5.4
65.8
48.7
<0.002
0.015
0.021
>522.2
4e
2,4-Cl2
Me
28
3940
140.7
0.007
5e
3,4-Cl2
Me
5.6
1.7
0.3
3.3
a
Data represent mean values for at least three separate experiments performed in duplicate. Standard error of means (SEM) are not shown for the sake of clarity and were
never higher than 5% of the means.
b
Ki and IC50 are defined in experimental section.
SI: CB1 selectivity index was calculated as Ki(CB2)/Ki(CB1) ratio.
SI: CB2 selectivity index was calculated as Ki(CB1)/Ki(CB2) ratio.
Lit. [9].
c
d
e
lipophilic substituents, namely chlorine atoms or methyl/methylene
groups, to unexplored positions of either 4 or 5 could potentially lead
to new CB1 ligands endowed with improved affinity and selectivity.
Introduction of a chlorine atom at position 2 of the pyrrole of
N-cyclohexyl 1-(2,4-dichlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyr-
azole-3-carboxamide [9] to give 7 enhanced both CB1 receptor
affinity (7.3 fold) and SI (2.1 fold). In the 1-(3,4-dichlorophenyl)
pyrazole series, such a chemical modification did not produce
significant improvement of the CB1 binding affinity (compare 5
with 19); on the contrary, it resulted in a greater affinity for the
CB2 receptor. Introduction of a second chlorine at position 5 of the
pyrrole did not alter significantly the binding affinities of the
parent compounds (compare 9 with 7, and 21 with 19).
Considering that ligands bearing both 1-(3,4-dichlorophenyl)
and 5-(2,5-dimethylpyrrolyl) moieties showed significant selec-
tivity for the CB1 receptor (mainly due to decrease of CB2 receptor
affinity) [9], we attempted to further improve their selectivity by
replacing the two methyls with ethyl groups. Surprisingly, both
compounds 16 and 17 showed a dramatic reduction of affinity.
Therefore, we constrained the two ethyl groups into a cyclic unit by
replacing the pyrrole with a 4,5,6,7-tetrahydro-2H-isoindol-2-yl
moiety in the most selective 1-(2,4-dihlorophenyl)pyrazole series.
As a result, we discovered compound 11 as a new ligand endowed
with high CB1 affinity and selectivity (CB1 Ki ¼ 2.3 nM, SI ¼ 163.6).
As a CB1 receptor ligand, 11 was 2.4 fold more potent than 5 [9] (the
previous lead compound for affinity), 5.2 fold more potent than 1,
and equipotent to 2. Moreover, 11 was 2.5- and 3.4-fold more
selective than 1 and 2, respectively.
To further evaluate the effect of homologous substituents, we
replaced the methyl at the position 4 of the pyrazole with an ethyl
group. Such a chemical modification led to opposite results,
depending on the position of the chlorine atoms on the 1-phenyl
ring. Replacement of the 4-methyl group of N-cyclohexyl 1-(2,4-
dichlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide
[9] or its 2,5-dimethylpyrrol-1-yl derivative with an ethyl group led
to compounds endowed with greater CB1 receptor affinity. Thus,
compounds 13 (CB1 Ki ¼ 3.0 nM) and 15 (CB1 Ki ¼ 6.7 nM) were
18.7- and 6.7-fold more potent than their parent compounds.
Unexpectedly, compound 14, the superior homologous of 5, was
devoid of selectivity.
More surprisingly, introducing a 4-ethyl in place of methyl
group in the 1-(3,4-dichlorophenyl)pyrazole series, shifted the
affinity from CB1 to CB2 receptor, because compound 23, the cor-
responding superior homologous of 5 (CB2 Ki ¼ 0.51 nM) was 10
fold more selective for the CB2 receptor than the parent compound.
Three compounds, 22, 23 and 25, also showed unexpected selec-
tivity for the CB2 receptor.
These results show that the 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyr-
azole-3-carboxamide scaffold can be chemically modified in order
to obtain either CB1 or CB2 receptor ligands.
3.2. Pharmacological studies
The effects on reduction in food intake were evaluated in rats
after administration of compound 11, the most potent and selective
CB1 receptor ligand of the series. ANOVA revealed a significant

F. Piscitelli et al. / European Journal of Medicinal Chemistry 46 (2011) 5641e5653
5645
Scheme 1. Synthesis of intermediates 28e43. Reagents and reaction conditions. a: Potassium tert-butoxide, 18-crown-6, tetrahydrofuran, 0e60 ꢀC, 1 h, then room temp., 16 h, 64%;
b: ethanol, reflux, 5 h, 31e65%; c: 2,5-dimethoxytetrahydrofuran, acetic acid, reflux, 1 h, 74e87% (34 and 35); d: N-chlorosuccinimide (1 equiv), N,N-dimethylformamide, 80 ꢀC, 1 h,
47e50%; e: N-chlorosuccinimide (2 equiv), N,N-dimethylformamide, 80 ꢀC, 1.5 h, 93e98%; f: cyclohexane-1,2-dicarbaldehyde (55), acetic acid, 110 ꢀC, 1 h, 47%; g: 2,5-hexanedione,
acetic acid, 110 ꢀC, 1 h, 61e71%; h: 3,6-octanedione (57), acetic acid, 110 ꢀC, 1 h, 41%.
effect of acute treatment with 11 on sucrose intake [F(2.42) ¼ 3.531,
P < 0.05]. Post hoc analysis revealed that sucrose intake in the rat
group treated with 2 mg/kg 11 was significantly reduced, by
approximately 25%, compared to that recorded in vehicle-treated
rats (Fig. 1, top panel). Conversely, intake of regular food was
totally unaffected by treatment with 11 [F(2.42) ¼ 0.009, P > 0.05]
(Fig. 1, bottom panel). Treatment with 11 also failed to alter water
intake [F(2.42) ¼ 3.034, P > 0.05] (data not shown).
The results of the in vivo pharmacological study indicate that
acute treatment with 11 preferentially reduced intake of sucrose
rather than intake of regular food in rats. Administration of 2 mg/kg
11 resulted indeed in a significant and marked reduction in sucrose
intake, while intake of food pellets was unaltered.
The selectivity of 11 for sucrose intake is a common feature of
cannabinoid CB1 receptor antagonists/inverse agonists. Accordingly,
1 has been repeatedly reported to be more potent and effective in
Scheme 2. Synthesis of carboxamides 6e25. Reagents and reaction conditions. a: Lithium hydroxide monohydrate, aqueous tetrahydrofuran, room temp., 6 h, 90e92%; b: (i) R2NH2,
HOBt, EDC, dichloromethane, 0 ꢀC to room temp., overnight; (ii) morpholinomethylpolystyrene and polymer bound p-toluenesulfonic acid, room temp., 24 h; yield 91e99%.

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F. Piscitelli et al. / European Journal of Medicinal Chemistry 46 (2011) 5641e5653
Scheme 3. Synthesis of intermediates 54e57. Reagents and reaction conditions. a: Lithium aluminum hydride, tetrahydrofuran, 60 ꢀC, 2 h, 75%; b: oxalyl chloride, dimethylsulf-
oxide, triethylamine, dichloromethane, ꢁ78 ꢀC, 1 h, 67%; c: (i) trimethyl aluminum, benzene, room temp., 2 h, (ii) diethyl succinate, benzene, room temp, 48 h, 72%; d: ethyl-
magnesium bromide, diethyl ether, room temp., 16 h, 58%.
reducing the intake of palatable foods and fluids (including sucrose
solutions, chocolate-flavored beverages, condensed milk, and
butter cookies) than of regular food in laboratory rodents and non-
human primates [14]. As an example, the acute administration of 1
to marmosets markedly reduced the consumption of a palatable
mixture made of sucrose, milk and cereals, while it was much less
effective on the intake of a standard primate food [14e].
The effects of the few, presently available cannabinoid CB1
receptor neutral antagonists (e.g. AM4113) on food intake and
different food-motivated behaviors are very similar to those
produced by 1 and other cannabinoid CB1 receptor antagonists/
inverse agonists [15]. Therefore, the present data are of limited help
to clarify whether 11 may act as neutral antagonist of the
cannabinoid CB1 receptor. Additional studies are warranted to
properly address this point.
Based on both binding data and 1’s literature,11 was expected to
exert a reducing effect of larger magnitude. Doses of 1 ranging
between 1 and 2 mg/kg (as those of 11 tested in the present study)
have indeed been found to reduce intake of regular food and
produce relatively large suppressions in intake of palatable foods in
rats (see [14b]). We hypothesize that this partial divergence
between the in vitro data (suggesting that 11 has more affinity than
1 at the CB1 receptor binding) and the in vivo data (in which 1
appears to be more potent and effective than 11) may be due to
presently unexplored differences in the pharmacokinetic profile
(e.g., rate and extend of absorption, distribution, passage through
Chart 2. SAR of 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide derivatives; Ki values are nanomolar. A: good affinity; B: good selectivity; C: bad affinity [9e11].

F. Piscitelli et al. / European Journal of Medicinal Chemistry 46 (2011) 5641e5653
5647
the elucidation of the structural determinants for the CB1 versus
CB2 selectivity and the pharmacological characterization of this
class of CB receptor ligand are warranted.
5. Experimental protocols
5.1. Chemistry
Melting points (mp) were determined on a Büchi 510 apparatus
and are uncorrected. Infrared spectra (IR) were run on a Spec-
trumOne spectrophotometer. Band position and absorption ranges
are given in cmꢁ1. Proton nuclear magnetic resonance (1H NMR)
spectra were recorded on a Bruker FT spectrometer (200 or
400 MHz) in the indicated solvent. Chemical shifts are expressed in
d
units (ppm) from tetramethylsilane. Mass spectral (MS) data were
obtained using Agilent 1100 LC/MSD VL system (G1946C) with
a 0.4 mL/min flow rate using a binary solvent system of 95:5
methanol/water. UV detection was monitored at 254 nm. Mass
spectra were acquired either in positive or in negative mode
scanning over the mass range of 105e1500. Chromatography
columns were packed with Merck silica gel (70e230 mesh).
Aluminum oxide TLC cards from Fluka (aluminum oxide precoated
aluminum cards with fluorescent indicator at 254 nm) and silica gel
TLC cards from Fluka (silica gel precoated aluminum cards with
fluorescent indicator at 254 nm) were used for thin layer chroma-
tography (TLC). Developed plates were visualized with a Spectro-
line ENF 260C/F UV apparatus. Organic solutions were dried over
anhydrous sodium sulfate. Concentration and evaporation of the
solvents was carried out on a Büchi Rotavapor R-210 equipped with
a Büchi V-850 vacuum controller and Büchi V-700 (approx. 5 mbar)
and V-710 vacuum (approx. 2 mbar) pumps. Compound purity was
determined by combustion analysis. Elemental analyses were
within ꢂ0.4% of the theoretical values. Purity of tested compounds
was >95%. Büchi Syncore reactor was used for parallel synthesis,
filtration, and evaporation.
Fig. 1. Effect of the acute administration of 11 on sucrose (top panel) and food (bottom
panel) intake in Wistar rats given unlimited access to a sucrose solution (1% w/v, in
water), water, and regular rodent chow. Each bar is the mean ꢂ SEM of n ¼ 15 rats.
*P < 0.05 with respect to vehicle-treated rats (NewmaneKeuls test).
5.1.1. Parallel synthesis of amides 6e25
the bloodebrain barrier, biotransformation into metabolites) of the
two compounds, resulting in different drug concentrations in the
target issue.
EDC hydrochloride (1.2 mmol) and HOBt (1.0 mmol) were added
at 0 ꢀC to each parallel vial containing a solution of the appropriate
acid 44e53 (1 mmol) in dichloromethane, and either cyclohexyl-
amine or 2,3-dichlorobenzylamine (1.5 mmol) was added at the
same temperature. After warming up to room temperature, the
vials were placed in the Büchi Syncore reactor. Stirring was main-
tained at 300 rpm overnight and then morpholinomethylpolys-
tyrene (3 equiv/mol) was added. The solutions were kept at room
temperature for 1 h, then polymer bound p-toluenesulfonic acid
(3 equiv/mol) was added to each vial, and the reaction mixtures
were stirred at room temperature for an additional 24 h. The
mixtures were filtered and the solutions were evaporated to
dryness to give carboxamides 6e25. Solid products were purified
by crystallization from ethanol or aqueous ethanol. Oil products
were purified by silica gel column chromatography column
(dichloromethane as eluent).
4. Conclusions
We have synthesized new cannabinoid receptor ligands based
on the 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide
scaffold. Binding assays showed that both affinity and selectivity
were strongly affected by (i) position of the chlorine atoms at the 1-
phenyl ring, (ii) nature of substituents around the pyrrole nucleus,
and (iii) length of the alkyl group at position 4 of the pyrazole. In
particular, we discovered N-cyclohexyl-1-(3,4-dichlorophenyl)-4-
methyl-5-(4,5,6,7-tetrahydroisoindol-2-yl)-1H-pyrazole-3-carbox-
amide (11: CB1 Ki ¼ 2.3 nM, CB1 SI ¼ 163.6), a new CB1 receptor
ligand endowed with affinity and selectivity superior to 1 and 2. On
the other hand, this study led also to the discovery of N-cyclohexyl-
1-(3,4-dichlorophenyl)-4-ethyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-
carboxamide (23: CB2 Ki ¼ 0.51 nM, CB2 SI ¼ 30.0) as a new CB2
receptor ligand endowed with significant affinity and selectivity.
Acute administration of 11 resulted in preferentially reduced
intake of sucrose rather than intake of regular food in rats.
Administration of 2 mg/kg 11 resulted indeed in a significant and
marked reduction in sucrose intake, while intake of food pellets
was completely unaltered.
5.1.1.1. 5-(2-Chloro-1H-pyrrol-1-yl)-N-(3,4-dichlorobenzyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide
97% as a white solid, mp 118e119 ꢀC. 1H NMR (200 MHz, CDCl3):
7.43e7.33 (m, 4H), 7.23e7.14 (m, 3H), 6.54e6.52 (m, 1H),
(6). Yield
d
6.13e6.10 (m, 1H), 6.08e6.06 (m, 1H), 4.52 (d, J ¼ 6.2 Hz, 2H),
2.23 ppm (s, 3H). MS (ESI): m/z: 529 [M þ H]þ. IR (Nujol):
n
1653 cmꢁ1. Anal. Calcd. for C22H15Cl5N4O: C, 49.98%; H, 2.86%; N,
10.60%. Found: C, 49.71%; H, 2.87%; N, 10.78%.
Overall, the results presented here show that the 1-aryl-5-(1H-
pyrrol-1-yl)-1H-pyrazole-3-carboxamide is a valuable scaffold for
the design of either CB1 or CB2 receptor ligands. Further studies on
5.1.1.2. 5-(2-Chloro-1H-pyrrol-1-yl)-N-cyclohexyl-1-(2,4-dichloroph-
enyl)-4-methyl-1H-pyrazole-3-carboxamide (7). Yield 98% as a white

5648
F. Piscitelli et al. / European Journal of Medicinal Chemistry 46 (2011) 5641e5653
solid, mp 157e158 ꢀC. 1H NMR (200 MHz, CDCl3):
d
7.45e7.46 (m,
93% as a white solid, mp 128e129 ꢀC. 1H NMR (200 MHz, CDCl3):
7.48e7.35 (m, 4H), 7.21e7.14 (m, 2H), 6.91e6.87 (m, 1H), 5.79 (s,
1H), 7.28e7.18 (m, 2H), 6.80 (br d, J ¼ 8.3 Hz, 1H), 6.53e6.51 (m, 1H),
6.09e6.04 (m, 2H), 3.94e3.85 (m, 1H), 2.21 (s, 3H), 2.01e1.95 (m,
2H), 1.76e1.58 (m, 4H), 1.49e1.12 ppm (m, 4H). MS (ESI): m/z: 452
d
2H), 4.56 (d, J ¼ 6.2 Hz, 2H), 2.65 (q, J ¼ 7.4 Hz, 2H), 1.96 (s, 6H),
1.04 ppm (t, J ¼ 7.4 Hz, 3H). MS (ESI): m/z: 537 [M þ H]þ. IR (Nujol):
[M þ H]þ, 474 [M þ Na]þ. IR (Nujol):
n
1653 cmꢁ1. Anal. Calcd. for
n
1653 cmꢁ1. Anal. Calcd. for C25H22Cl4N4O: C, 55.99%; H, 4.13%; N,
C21H21Cl3N4O: C, 55.83%; H, 4.69%; N, 12.40%. Found: C, 55.55%; H,
4.68%; N, 12.51%.
10.45%. Found: C, 55.78%; H, 4.14%; N, 10.28%.
5.1.1.10. N-cyclohexyl-1-(2,4-dichlorophenyl)-5-(2,5-dimethyl-1H-py-
5.1.1.3. 5-(2,5-Dichloro-1H-pyrrol-1-yl)-N-(3,4-dichlorobenzyl)-1-(2,
4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (8). Yield
99% as a white solid, mp 137e138 ꢀC. 1H NMR (200 MHz, CDCl3):
rrol-1-yl)-4-ethyl-1H-pyrazole-3-carboxamide (15). Yield96% as
a yellow oil. 1H NMR (200 MHz, CDCl3):
d
7.48 (d, J ¼ 1.8 Hz, 1H), 7.16
(dd, J1 ¼1.8 Hz, J2 ¼ 8.9 Hz, 1H), 6.91e6.85 (m, 2H), 5.77 (s, 2H),
3.95e3.91 (m, 1H), 2.63 (q, J ¼ 7.4 Hz, 2H), 2.03e1.94 (m, 8H),
1.76e1.59 (m, 4H), 1.42e1.21 (m, 4H), 1.06 ppm (t, J ¼ 7.4 Hz, 3H). MS
d
7.49e7.35 (m, 4H), 7.21e7.18 (m, 3H), 6.07 (s, 2H), 4.56 (d,
J ¼ 6.2 Hz, 2H), 2.22 ppm (s, 3H). MS (ESI): m/z: 564 [M þ H]þ. IR
(Nujol):
n
1654 cmꢁ1. Anal. Calcd for C22H14Cl6N4O: C, 46.93%; H,
(ESI): m/z: 460 [M þ H]þ, 482 [M þ Na]þ. IR (Nujol):
n .
1652 cmꢁ1
2.51%; N, 9.95%. Found: C, 46.71%; H, 2.59%; N, 10.07%.
Anal. Calcd. for C24H28Cl2N4O: C, 62.74%; H, 6.14%; N, 12.20%. Found:
C, 62.57%; H, 6.10%; N, 12.06%.
5.1.1.4. N-cyclohexyl-5-(2,5-dichloro-1H-pyrrol-1-yl)-1-(2,4-dichloro-
phenyl)-4-methyl-1H-pyrazole-3-carboxamide (9). Yield 99% as
5.1.1.11. N-(3,4-dichlorobenzyl)-1-(3,4-dichlorophenyl)-5-(2,5-dieth-
yl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (16). Yield
95% as a white solid, mp 115e116 ꢀC. 1H NMR (200 MHz, CDCl3):
a colorless oil. 1H NMR (200 MHz, CDCl3):
d
7.48 (d, J ¼ 1.8 Hz, 1H),
7.21e7.18 (m, 2H), 6.83 (br d, J ¼ 8.2 1H), 6.04 (s, 2H), 3.95e3.90
(m, 1H), 2.20 (s, 3H), 2.01e1.96 (m, 2H), 1.76e1.56 (m, 4H),
1.47e1.12 ppm (m, 4H). MS (ESI): m/z: 487 [M þ H]þ, 509
d
7.60e7.24 (m, 6H), 6.51 (dd, J1 ¼ 2.1 Hz, J2 ¼ 8.8 Hz, 1H), 6.01 (s,
2H), 4.58 (d, J ¼ 6.2 Hz, 2H), 2.29 (s, 3H), 2.11 (q, J ¼ 7.4 Hz, 4H),
[M þ Na]þ. IR (Nujol):
n
1653 cmꢁ1. Anal. Calcd. for C21H20Cl4N4O:
C, 51.87%; H, 4.15%; N, 11.52%. Found: C, 51.61%; H, 4.21%; N,
11.66%.
1.01 ppm (t, J ¼ 7.4 Hz, 6H). MS (ESI): m/z: 551 [M þ H]þ. IR (Nujol):
n
1654 cmꢁ1. Anal. Calcd. for C26H24Cl4N4O: C, 56.75%; H, 4.40%; N,
10.18%. Found: C, 56.47%; H, 4.39%; N, 10.01%.
5.1.1.5. N-(3,4-dichlorobenzyl)-1-(2,4-dichlorophenyl)-4-methyl-5-(4,
5.1.1.12. N-cyclohexyl-1-(3,4-dichlorophenyl)-5-(2,5-diethyl-1H-pyr-
5,6,7-tetrahydroisoindol-2-yl)-1H-pyrazole-3-carboxamide
(10).
7.43e7.13
rol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (17). Yield 91% as
Yield99% as a colorless oil. 1H NMR (200 MHz, CDCl3):
d
a white solid, mp 157e158 ꢀC. 1H NMR (200 MHz, CDCl3):
d 7.28 (d,
(m, 7H), 6.19 (s, 2H), 4.52 (d, J ¼ 6.2 Hz, 2H), 2.45e2.40 (m, 4H),
J ¼ 8.8 Hz,1H), 7.22 (d, J ¼ 1.9 Hz,1H), 6.91 (br d, J ¼ 8.3 Hz,1H), 6.52
(dd, J1 ¼1.9 Hz, J2 ¼ 8.8 Hz, 1H), 5.99 (s, 2H), 3.98e3.93 (m, 1H), 2.18
(s, 3H), 2.13e1.98 (m, 6H), 1.79e1.61 (m, 4H), 1.50e1.15 (m, 4H),
0.99 ppm (t, J ¼ 7.5 Hz, 6H). MS (ESI): m/z: 474 [M þ H]þ, 496
2.27 (s, 3H), 1.65e1.63 ppm (m, 4H). MS (ESI): m/z: 549 [M þ H]þ.
IR (Nujol):
n
1656 cmꢁ1. Anal. Calcd. for C26H22Cl4N4O: C, 56.95%;
H, 4.04%; N, 10.22%. Found: C, 56.72%; H, 4.06%; N, 10.16%.
[M þ Na]þ. IR (Nujol):
n
1653 cmꢁ1. Anal. Calcd. for C25H30Cl2N4O: C,
5.1.1.6. N-cyclohexyl-1-(3,4-dichlorophenyl)-4-methyl-5-(4,5,6,7-
63.42%; H, 6.39%; N, 11.83%. Found: C, 63.30%; H, 6.31%; N, 11.69%.
tetrahydroisoindol-2-yl)-1H-pyrazole-3-carboxamide (11). Yield 97%
as a colorless oil. 1H NMR (200 MHz, CDCl3):
d
7.46 (d, J ¼ 1.9 Hz,
5.1.1.13. 5-(2-Chloro-1H-pyrrol-1-yl)-N-(3,4-dichlorobenzyl)-1-(3,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (18). Yield
91% as a white solid, mp 139e140 ꢀC. 1H NMR (200 MHz, CDCl3):
1H), 7.29e7.17 (m, 2H), 6.78 (br d, J ¼ 8.3 Hz, 1H), 6.17 (s, 2H),
3.98e3.84 (m, 1H), 2.51e2.44 (m, 4H), 2.25 (s, 3H), 2.00e1.94 (m,
2H), 1.75e1.62 (m, 8H), 1.48e1.11 ppm (m, 4H). MS (ESI): m/z: 472
d
7.57e7.52 (m, 1H), 7.42e7.20 (m, 5H), 6.75e6.71 (m, 1H),
[M þ H]þ, 494 [M þ Na]þ. IR (Nujol):
n
1652 cmꢁ1. Anal. Calcd. for
6.59e6.58 (m, 1H), 6.31e6.29 (m, 1H), 6.23e6.22 (m, 1H), 4.57 (d,
C25H28Cl2N4O: C, 63.69%; H, 5.99%; N, 11.88%. Found: C, 63.92%; H,
5.88%; N, 11.75%.
J ¼ 6.2 Hz, 2H), 2.23 ppm (s, 3H). MS (ESI): m/z: 529 [M þ H]þ, 551
[M þ Na]þ. IR (Nujol):
n
1651 cmꢁ1. Anal. Calcd. for C22H15Cl5N4O: C,
49.98%; H, 2.86%; N, 10.60%. Found: C, 49.73%; H, 2.81%; N, 10.46%.
5.1.1.7. N-(3,4-dichlorobenzyl)-1-(2,4-dichlorophenyl)-4-ethyl-5-(1H-
pyrrol-1-yl)-1H-pyrazole-3-carboxamide (12). Yield 98% as a color-
5.1.1.14. 5-(2-Chloro-1H-pyrrol-1-yl)-N-cyclohexyl-1-(3,4-dichloro-
phenyl)-4-methyl-1H-pyrazole-3-carboxamide (19). Yield 92% as
less oil. 1H NMR (200 MHz, CDCl3):
d
7.42e7.34 (m, 4H), 7.23e7.14
(m, 3H), 6.57e6.56 (m, 2H), 6.19e6.18 (m, 2H), 4.53 (d, J ¼ 6.2 Hz,
a
d
white solid, mp 122e123 ꢀC. 1H NMR (200 MHz, CDCl3):
7.35e7.30 (m, 2H), 6.87 (br d, J ¼ 8.3 Hz, 1H), 6.75 (dd, J1 ¼1.9 Hz,
2H), 2.74 (q, J ¼ 7.2 Hz, 2H), 1.15 ppm (t, J ¼ 7.2 Hz, 3H). MS (ESI): m/
z: 509 [M þ H]þ. IR (Nujol):
n
1653 cmꢁ1
.
Anal. Calcd. for
J2 ¼ 8.8 Hz, 1H), 6.57e6.56 (m, 1H), 6.30e6.29 (m, 1H), 6.27e6.26
(m, 1H), 3.96e3.92 (m, 1H), 2.2 (s, 3H), 2.03e1.80 (m, 2H), 1.72e1.60
(m, 4H), 1.43e1.19 ppm (m, 4H). MS (ESI): m/z: 452 [M þ H]þ, 475
C23H18Cl4N4O: C, 54.35%; H, 3.57%; N, 11.02%. Found: C, 54.59%; H,
3.451%; N, 10.96%.
[M þ Na]þ. IR (Nujol):
n
1654 cmꢁ1. Anal. Calcd. for C21H21Cl3N4O: C,
5.1.1.8. N-cyclohexyl-1-(2,4-dichlorophenyl)-4-ethyl-5-(1H-pyrrol-1-
55.83%; H, 4.69%; N, 12.40%. Found: C, 55.61%; H, 4.61%; N, 12.23%.
yl)-1H-pyrazole-3-carboxamide (13). Yield 99% as a colorless oil. 1H
NMR (200 MHz, CDCl3):
d
7.43e7.42 (m,1H), 7.28e7.18 (m, 2H), 6.81
5.1.1.15. 5-(2,5-Dichloro-1H-pyrrol-1-yl)-N-(3,4-dichlorobenzyl)-1-
(br d, J ¼ 8.3 Hz, 1H), 6.56e6.55 (m, 2H), 6.16e6.15 (m, 2H),
3.94e3.90 (m, 1H), 2.70 (q, J ¼ 7.4 Hz, 2H), 2.01e1.96 (m, 2H),
1.77e1.57 (m, 4H),1.45e1.21 (m, 4H),1.14 ppm (t, J ¼ 7.4 Hz, 3H). MS
(3,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (20).
Yield 99% as a yellow oil. 1H NMR (200 MHz, CDCl3):
d 7.49e7.34
(m, 4H), 7.22e7.18 (m, 3H), 6.07 (s, 2H), 4.56 (d, J ¼ 6.2 Hz, 2H),
(ESI): m/z: 432 [M þ H]þ, 454 [M þ Na]þ. IR (Nujol):
n .
1654 cmꢁ1
2.22 ppm (s, 3H). MS (ESI): m/z: 564 [M þ H]þ. IR (Nujol):
Anal. Calcd. for C22H24Cl2N4O: C, 61.26%; H, 5.61%; N,12.99%. Found:
C, 61.52%; H, 5.55%; N, 12.86%.
n
1654 cmꢁ1. Anal. Calcd. for C22H14Cl6N4O: C, 46.93%; H, 2.51%; N,
9.95%. Found: C, 46.66%; H, 2.44%; N, 9.89%.
5.1.1.9. N-(3,4-dichlorobenzyl)-1-(2,4-dichlorophenyl)-5-(2,5-dime-
5.1.1.16. N-cyclohexyl-5-(2,5-dichloro-1H-pyrrol-1-yl)-1-(3,4-dichlo-
thyl-1H-pyrrol-1-yl)-4-ethyl-1H-pyrazole-3-carboxamide (14). Yield
rophenyl)-4-methyl-1H-pyrazole-3-carboxamide (21). Yield 97% as

F. Piscitelli et al. / European Journal of Medicinal Chemistry 46 (2011) 5641e5653
5649
a colorless oil. 1H NMR (200 MHz, CDCl3):
d
7.39e7.35 (m, 2H),
5.1.1.24. Ethyl
5-amino-1-(3,4-dichlorophenyl)-4-methyl-1H-pyr-
6.89e6.82 (m, 2H), 6.17 (s, 2H), 3.99e3.93 (m, 1H), 2.21 (s, 3H),
2.03e1.80 (m, 2H), 1.76e1.60 (m, 4H), 1.49e1.14 ppm (m, 4H). MS
azole-3-carboxylate (29). This compound was prepared as we
previously reported [9].
(ESI): m/z: 487 [M þ H]þ, 509 [M þ Na]þ. IR (Nujol):
n .
1655 cmꢁ1
Anal. Calcd. for C21H20Cl4N4O: C, 51.87%; H, 4.15%; N, 11.52%. Found:
C, 51.59%; H, 4.10%; N, 11.35%.
5.1.1.25. Ethyl 5-amino-1-(2,4-dichlorophenyl)-4-ethyl-1H-pyrazole-
3-carboxylate (30). A mixture of 2,4-dihlorophenylhydrazine
hydrochloride (7.90 g, 37.0 mmol) and 27 (7.61 g, 37.0 mmol) in
ethanol (130 mL) was heated at reflux temperature for 5 h. After
cooling, the solvent was evaporated, and the residue was dissolved
in water and extracted with ethyl acetate. The combined organic
layers were washed with brine, dried, and evaporated. After
removal of the solvent, the crude residue was purified by column
chromatography (silica gel, dichloromethane as eluent) to furnish
30. Yield 59%, mp 155e157 ꢀC (from ethanol). 1H NMR (400 MHz,
5.1.1.17. N-(3,4-dichlorobenzyl)-1-(3,4-dichlorophenyl)-4-ethyl-5-
(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (22). Yield 91% as
a
d
yellow solid, mp 135e136 ꢀC. 1H NMR (200 MHz, CDCl3):
7.51e7.16 (m, 6H), 6.68 (dd, J1 ¼ 2.0 Hz, J2 ¼ 9.5 Hz, 1H),
6.61e6.60 (m, 2H), 6.36e6.35 (m, 2H), 4.56 (d, J ¼ 6.2 Hz, 2H),
2.69 (q, J ¼ 7.3 Hz, 2H), 1.14 ppm (t, J ¼ 7.3 Hz, 6H). MS (ESI):
m/z: 509 [M þ H]þ. IR (Nujol):
n
1651 cmꢁ1. Anal. Calcd. for
C23H18Cl4N4O: C, 54.35%; H, 3.57%; N, 11.02%. Found: C, 54.21%; H,
3.50%; N, 10.82%.
CDCl3):
d
1.18 (t, J ¼ 7.5 Hz, 3H), 1.41 (t, J ¼ 7.1 Hz, 3H), 2.65 (q,
J ¼ 7.5 Hz, 2H), 3.67 (br s, 2H, disappeared on treatment with D2O),
4.41 (q, J ¼ 7.1 Hz, 2H), 7.52 (dd, J ¼ 8.7 and 2.2 Hz, 1H), 7.56 (d,
J ¼ 8.6 Hz, 1H), 7.82 ppm (d, J ¼ 2.2 Hz, 1H). MS (ESI): m/z: 329
5.1.1.18. N-cyclohexyl-1-(3,4-dichlorophenyl)-4-ethyl-5-(1H-pyrrol-
1-yl)-1H-pyrazole-3-carboxamide (23). Yield 93% as
a
slightly
7.31 (d,
[M þ H]þ, 351 [M þ Na]þ. IR:
n
3074, 1712 cmꢁ1. Anal. Calcd. for
brown solid, mp 189e190 ꢀC. 1H NMR (200 MHz, CDCl3):
d
C14H15Cl2N3O2: C, 51.23%; H, 4.61%; N, 12.80%. Found: C, 51.14%; H,
4.59%; N, 12.69%.
J ¼ 8.8 Hz,1H), 7.24 (d, J ¼ 1.9 Hz,1H), 6.87 (br d, J ¼ 8.3 Hz,1H), 6.70
(dd, J1 ¼1.9 Hz, J2 ¼ 8.8 Hz, 1H), 6.59e6.58 (m, 2H), 6.34e6.33 (m,
2H), 3.97e3.94 (m, 1H), 2.68 (q, J ¼ 7.3 Hz, 2H), 2.04e1.99 (m, 2H),
1.79e1.61 (m, 4H), 1.49e1.25 (m, 4H), 1.14 ppm (t, J ¼ 7.3 Hz, 3H).
5.1.1.26. Ethyl 5-amino-1-(3,4-dichlorophenyl)-4-ethyl-1H-pyrazole-
3-carboxylate (31). This compound was prepared as 30 using 3,4-
dichlorophenylhydrazine hydrochloride. Yield 61%, mp 94e96 ꢀC
MS (ESI): m/z: 432 [M þ H]þ, 455 [M þ Na]þ. IR (Nujol):
n
1652 cmꢁ1. Anal. Calcd. for C22H24Cl2N4O: C, 61.26%; H, 5.61%; N,
12.99%. Found: C, 61.10%; H, 5.52%; N, 12.75%.
(from aqueous ethanol). 1H NMR (400 MHz, CDCl3):
d 1.18 (t,
J ¼ 7.5 Hz, 3H),1.41 (t, J ¼ 7.1 Hz, 3H), 2.65 (q, J ¼ 7.5 Hz, 2H), 3.67 (br
s, 2H, disappeared on treatment with D2O), 4.41 (q, J ¼ 7.1 Hz, 2H),
7.52 (dd, J ¼ 8.7 and 2.2 Hz, 1H), 7.56 (d, J ¼ 8.6 Hz, 1H), 7.82 ppm (d,
5.1.1.19. N-(3,4-dichlorobenzyl)-1-(3,4-dichlorophenyl)-5-(2,5-dime-
thyl-1H-pyrrol-1-yl)-4-ethyl-1H-pyrazole-3-carboxamide (24). Yield
95% as a white solid, mp 117e118 ꢀC. 1H NMR (200 MHz, CDCl3):
J ¼ 2.2 Hz, 1H). MS (ESI): m/z: 329 [M þ H]þ, 351 [M þ Na]þ. IR:
n
3074,1712 cmꢁ1. Anal. Calcd. for C14H15Cl2N3O2: C, 51.23%; H, 4.61%;
N, 12.80%. Found: C, 51.09%; H, 4.60%; N, 12.61%.
d
7.55e7.21 (m, 6H), 6.57 (dd, J1 ¼1.8 Hz, J2 ¼ 8.8 Hz, 1H), 5.96 (s,
2H), 4.59 (d, J ¼ 6.2 Hz, 2H), 2.70 (q, J ¼ 7.4 Hz, 4H), 1.86 (s, 3H),
1.10 ppm (t, J ¼ 7.4 Hz, 6H). MS (ESI): m/z: 537 [M þ H]þ, 559
5.1.1.27. Ethyl 1-(2,4-dichlorophenyl)-4-methyl-5-(1H-pyrrol-1-yl)-
1H-pyrazole-3-carboxylate (32). This compound was prepared as
we previously reported [9].
[M þ Na]þ. IR (Nujol):
n
1653 cmꢁ1. Anal. Calcd. for C25H22Cl4N4O: C,
55.99%; H, 4.13%; N, 10.45%. Found: C, 55.75%; H, 4.09%; N, 10.22%.
5.1.1.20. N-cyclohexyl-1-(3,4-dichlorophenyl)-5-(2,5-dimethyl-1H-
pyrrol-1-yl)-4-ethyl-1H-pyrazole-3-carboxamide (25). Yield 93% as
a slightly brown solid, mp 168e169 ꢀC. 1H NMR (200 MHz, CDCl3):
5.1.1.28. Ethyl 1-(3,4-dichlorophenyl)-4-methyl-5-(1H-pyrrol-1-yl)-
1H-pyrazole-3-carboxylate (33). This compound was prepared as
we previously reported [9].
d
7.30 (d, J ¼ 8.7 Hz,1H), 7.22 (d, J ¼ 1.8 Hz,1H), 6.92 (br d, J ¼ 8.3 Hz,
1H), 6.57 (dd, J1 ¼1.8 Hz, J2 ¼ 8.7 Hz, 1H), 5.94 (m, 2H), 4.07e3.91
(m, 1H), 2.65 (q, J ¼ 7.3 Hz, 2H), 2.05e1.99 (m, 2H), 1.84 (s, 6H),
1.74e1.62 (m, 4H), 1.44e1.25 (m, 4H), 1.09 ppm (t, J ¼ 7.3 Hz, 3H).
5.1.1.29. Ethyl 1-(2,4-dichlorophenyl)-4-ethyl-5-(1H-pyrrol-1-yl)-1H
-pyrazole-3-carboxylate (34). A solution of 30 (3.56 g, 10.8 mmol)
and 2,5-dimethoxytetrahydrofuran (1.6 g, 1.57 mL, 12.1 mmol) in
glacial acetic acid (20 mL) was heated at reflux temperature for
1 h. After cooling, the solvent was evaporated to dryness, and the
residue was dissolved in water and extracted with ethyl acetate.
The combined organic layers were washed with a saturated
solution of sodium hydrogen carbonate and with brine, and dried.
After evaporation of the solvent, the residue was purified by
column chromatography (silica gel, dichloromethane as eluent) to
MS (ESI): m/z: 460 [M þ H]þ, 482 [M þ Na]þ. IR (Nujol):
n
1651 cmꢁ1. Anal. Calcd. for C24H28Cl2N4O: C, 62.74%; H, 6.14%; N,
12.20%. Found: C, 62.49%; H, 6.12%; N, 12.07%.
5.1.1.21. Potassium 3-cyano-1-ethoxy-1-oxobut-2-en-2-olate (26).
This compound was prepared as we previously reported [9].
5.1.1.22. Potassium 3-cyano-1-ethoxy-1-oxopent-2-en-2-olate (27).
Diethyl oxalate (15.00 g, 13.95 mL, 0.1 mol) was added dropwise to
a 0 ꢀC cooled mixture of 18-crown-6 (2.25 g, 0.0085 mol) and
potassium tert-butoxide (11.2 g, 0.1 mol) in anhydrous tetrahydro-
furan (95 mL). Butyronitrile (7.1 g, 9.0 mL, 0.1 mol) was added
dropwise, and the reaction mixture was heated at 60 ꢀC for 1 h and
then at room temperature for an additional 1.5 h. The precipitate
was collected to furnish 27. Yield 64%. 1H NMR (400 MHz, DMSO-
provide 34. Yield 87%, as an oil. 1H NMR (400 MHz, CDCl3):
d 1.16
(t, J ¼ 7.4 Hz, 3H), 1.43 (t, J ¼ 7.1 Hz, 3H), 2.68 (q, J ¼ 7.5 Hz, 2H),
4.46 (q, J ¼ 7.1 Hz, 2H), 6.20 (t, J ¼ 4.3 Hz, 2H), 6.59 (t, J ¼ 4.3 Hz,
2H), 7.26e7.29 (m, 2H), 7.43 ppm (d, J ¼ 1.8 Hz, 1H). IR:
n
1716 cmꢁ1
.
5.1.1.30. Ethyl 1-(3,4-dichlorophenyl)-4-ethyl-5-(1H-pyrrol-1-yl)-1H
-pyrazole-3-carboxylate (35). This compound was prepared as 34
from 31. Yield 74%, 88e89 ꢀC (from aqueous ethanol). 1H NMR
d6):
d
0.85 (t, J ¼ 7.4 Hz, 3H), 1.17 (t, J ¼ 7.1 Hz, 3H), 1.95 (q, J ¼ 7.4 Hz,
2H), 3.99 ppm (q, J ¼ 7.1 Hz, 2H). IR:
n
2160, 1716, 1675 cmꢁ1
.
(400 MHz, CDCl3):
d
1.15 (t, J ¼ 7.5 Hz, 3H), 1.45 (t, J ¼ 7.1 Hz, 3H),
2.66 (q, J ¼ 7.5 Hz, 2H), 4.49 (q, J ¼ 7.1 Hz, 2H), 6.38 (t, J ¼ 4.3 Hz,
2H), 6.63 (t, J ¼ 4.3 Hz, 2H), 6.73 (dd, J ¼ 8.7 and 2.5 Hz, 1H), 7.31 (d,
J ¼ 2.5 Hz, 1H), 7.34 ppm (d, J ¼ 8.7 Hz, 1H). MS (ESI): m/z: 379
5.1.1.23. Ethyl 5-amino-1-(2,4-dichlorophenyl)-4-methyl-1H-pyr-
azole-3-carboxylate (28). This compound was prepared as we
previously reported [9].
[M þ H]þ, 401 [M þ Na]þ. IR:
n . Anal. Calcd. for
1725 cmꢁ1

5650
F. Piscitelli et al. / European Journal of Medicinal Chemistry 46 (2011) 5641e5653
C18H17Cl2N3O2: C, 57.16%; H, 4.53%; N, 11.11%. Found: C, 57.05%; H,
4.51%; N, 11.05%.
60.30%; H, 5.06%; N, 10.05%. Found: C, 60.21%; H, 5.05%; N,
10.03%.
5.1.1.31. Ethyl 5-(2-chloro-1H-pyrrol-1-yl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-carboxylate (36). A solution of 32 [9]
(2.00 g, 5.5 mmol) and N-chlorosuccinimide (0.73 g, 5.5 mmol) in
anhydrous N,N-dimethylformamide (30 mL) was heated at 80 ꢀC
for 1.5 h. After cooling, the reaction mixture was diluted with water
and extracted with ethyl acetate. The combined organic layers were
washed with brine, dried and evaporated. The crude residue was
purified by column chromatography (silica gel, dichlor-
omethaneepetroleum ether 1:1 as eluent) to provide 32. Yield 50%,
5.1.1.36. Ethyl 1-(2,4-dichlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-
yl)-4-ethyl-1H-pyrazole-3-carboxylate (41). This compound was
prepared as 34 from 30 and 2,5-hexanedione. Yield 61%, mp
85e86 ꢀC (from n-hexane). 1H NMR (400 MHz, CDCl3):
d 1.09 (t,
J ¼ 7.5 Hz, 3H), 1.45 (t, J ¼ 7.1 Hz, 3H), 1.98 (s, 6H), 2.61 (q, J ¼ 7.5 Hz,
2H), 4.48 (q, J ¼ 7.1 Hz, 2H), 5.82 (s, 2H), 6.98 (d, J ¼ 8.5 Hz, 1H), 7.19
(dd, J ¼ 8.5 and 2.3 Hz,1H), 7.49 ppm (d, J ¼ 2.3 Hz,1H). MS (ESI): m/
z: 407 [M þ H]þ, 429 [M þ Na]þ. IR:
n
1715 cmꢁ1. Anal. Calcd. for
C20H21Cl2N3O2: C, 59.12%; H, 5.21%; N, 10.34%. Found: C, 59.07%; H,
5.05%; N, 10.32%.
mp 135e138 ꢀC (from ethanol). 1H NMR (400 MHz, CDCl3):
d 1.44 (t,
J ¼ 7.5 Hz, 3H), 2.21 (s, 3H), 4.47 (q, J ¼ 7.1 Hz, 2H), 6.10 (dd, J ¼ 3.7
and 1.8 Hz, 1H), 6.15 (t, J ¼ 3.5 Hz, 1H), 6.57 (dd, J ¼ 3.3 and 1.8 Hz,
1H), 7.25e7.31 (m, 2H), 7.45 ppm (d, J ¼ 1.9 Hz, 1H). MS (ESI): m/z:
5.1.1.37. Ethyl 1-(3,4-dichlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-
yl)-4-ethyl-1H-pyrazole-3-carboxylate (42). This compound was
prepared as 34 from 31 and 2,5-hexanedione. Yield 71%, mp
399 [M þ H]þ, 421 [M þ Na]þ. IR:
n
1716 cmꢁ1. Anal. Calcd. for
C17H14Cl3N3O2: C, 51.22%; H, 3.54%; N, 10.54%. Found: C, 51.16%; H,
3.52%; N, 10.46%.
92e94 ꢀC (from aqueous ethanol). 1H NMR (400 MHz, CDCl3):
d 1.11
(t, J ¼ 7.5 Hz, 3H), 1.47 (t, J ¼ 7.1 Hz, 3H), 1.89 (s, 6H), 2.64 (q,
J ¼ 7.5 Hz, 2H), 4.49 (q, J ¼ 7.1 Hz, 2H), 5.98 (s, 2H), 6.73 (dd, J ¼ 8.8
and 2.6 Hz, 1H), 7.28 (d, J ¼ 2.6 Hz, 1H), 7.34 ppm (d, J ¼ 8.8 Hz, 1H).
5.1.1.32. Ethyl 5-(2-chloro-1H-pyrrol-1-yl)-1-(3,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-carboxylate (37). This compound was
prepared as 36 from 33 [9]. Yield 47%, mp 168e171 ꢀC (from
MS (ESI): m/z: 407 [M þ H]þ, 429 [M þ Na]þ. IR:
n
1712 cmꢁ1. Anal.
Calcd. for C20H21Cl2N3O2: C, 59.12%; H, 5.21%; N, 10.34%. Found: C,
59.05%; H, 5.05%; N, 10.31%.
ethanol). 1H NMR (400 MHz, CDCl3):
d
1.46 (t, J ¼ 7.5 Hz, 3H), 2.21 (s,
3H), 4.48 (q, J ¼ 7.1 Hz, 2H), 6.25e6.27 (m, 1H), 6.34 (t, J ¼ 3.5 Hz,
1H), 6.60e6.62 (m, 1H), 6.90 (dd, J ¼ 8.8 and 2.5 Hz, 1H),
7.37e7.39 ppm (m, 2H). MS (ESI): m/z: 399 [M þ H]þ, 421
5.1.1.38. Ethyl
1-(2,4-dichlorophenyl)-5-(2,5-diethyl-1H-pyrrol-1-
yl)-4-ethyl-1H-pyrazole-3-carboxylate (43). This compound was
prepared as 34 from 29 and 3,6-octanedione (57). Yield 41% as an
oil.
[M þ Na]þ. IR:
n
1687 cmꢁ1. Anal. Calcd. for C17H14Cl3N3O2: C,
51.22%; H, 3.54%; N, 10.54%. Found: C, 51.14%; H, 3.53%; N, 10.45%.
5.1.1.33. Ethyl 5-(2,5-dichloro-1H-pyrrol-1-yl)-1-(2,4-dichlorophen-
yl)-4-methyl-1H-pyrazole-3-carboxylate (38). A solution of N-
chlorosuccinimide (0.48 g, 3.6 mmol) in anhydrous N,N-dime-
thylformamide (6.5 mL) was added dropwise to a solution of 32 [9]
(0.65 g, 1.8 mmol) in anhydrous N,N-dimethylformamide (10 mL).
The reaction mixture was heated to 80 ꢀC under argon atmosphere
for 1.5 h. After cooling, water was added and the mixture was
extracted with ethyl acetate. The combined organic layers were
washed with brine, dried and evaporated. The crude residue was
purified by column chromatography (silica gel, dichloromethane as
eluent) to furnish 38. Yield 93% as an oil. 1H NMR (400 MHz, CDCl3):
5.1.1.39. 1-(2,4-Dichlorophenyl)-4-ethyl-5-(1H-pyrrol-1-yl)-1H-pyr-
azole-3-carboxylic acid (44). A mixture of 34 (1.00 g, 2.6 mmol)
and lithium hydroxide monohydrate (0.34 g, 8.1 mmol) in tetra-
hydrofuran (15 mL) and water (10 mL) was stirred at room
temperature for 16 h. 1 N hydrochloric acid was added until pH
w2, and the resulting mixture was extracted with ethyl acetate.
The combined organic layers were washed with brine, dried and
evaporated. The residue was recrystallized in ethanol to provide
44. Yield 90%, mp 120e125 ꢀC (from ethanol). 1H NMR (400 MHz,
CDCl3):
d
1.18 (t, J ¼ 7.5 Hz, 3H), 2.71 (q, J ¼ 7.5 Hz, 2H), 6.22
(t, J ¼ 2.1 Hz, 2H), 6.60 (d, J ¼ 2.1 Hz, 2H), 7.25e7.28 (m, 2H),
d
1.45 (t, J ¼ 7.5 Hz, 3H), 2.20 (s, 3H), 4.48 (q, J ¼ 7.1 Hz, 2H), 6.11 (s,
7.47 ppm (d, J ¼ 1.5 Hz, 1H). MS (ESI): m/z: 351 [M þ H]þ, 373
2H), 7.24e7.25 (m, 2H), 7.49 ppm (s, 1H). MS (ESI): m/z: 434
[M þ Na]þ. IR:
n
2928 (acid band), 1690 cmꢁ1. Anal. Calcd. for
[M þ H]þ, 456 [M þ Na]þ. IR:
n
1716 cmꢁ1
.
Anal. Calcd. for
C16H13Cl2N3O2: C, 54.87%; H, 3.74%; N, 12.00%. Found: C, 54.69%;
H, 3.68%; N, 10.06%.
C17H13Cl4N3O2: C, 47.14%; H, 3.03%; N, 9.70%. Found: C, 47.08%; H,
3.02%; N, 9.66%.
5.1.1.40. 1-(3,4-Dichlorophenyl)-4-ethyl-5-(1H-pyrrol-1-yl)-1H-pyr-
azole-3-carboxylic acid (45). This compound was prepared as 44
from 35. Yield 97%, mp 189e193 ꢀC (from ethanol). 1H NMR
5.1.1.34. Ethyl 5-(2,5-dichloro-1H-pyrrol-1-yl)-1-(3,4-dichlorophe-
nyl)-4-methyl-1H-pyrazole-3-carboxylate (39). This compound
was prepared as 38 from 33 [9]. Yield 98% as an oil. 1H NMR
(400 MHz, CDCl3):
d
1.18 (t, J ¼ 7.5 Hz, 3H), 2.70 (q, J ¼ 7.5 Hz, 2H),
(400 MHz, CDCl3):
d
1.46 (t, J ¼ 7.5 Hz, 3H), 2.20 (s, 3H), 4.49 (q,
6.40 (t, J ¼ 2.1 Hz, 2H), 6.65 (d, J ¼ 2.1 Hz, 2H), 6.85 (dd, J ¼ 8.7 and
J ¼ 7.1 Hz, 2H), 6.21 (s, 2H), 6.97 (dd, J ¼ 8.7 and 2.4 Hz, 1H), 7.41 (d,
2.5 Hz, 1H), 7.30 (d, J ¼ 2.4 Hz, 1H), 7.37 ppm (d, J ¼ 8.8 Hz, 1H). MS
J ¼ 8.7 Hz, 1H), 7.44 ppm (d, J ¼ 2.5 Hz, 1H). IR:
n
1717 cmꢁ1. MS
(ESI): m/z: 351 [M þ H]þ, 373 [M þ Na]þ. IR:
n 2981 (acid band),
(ESI): m/z: 434 [M þ H]þ. Anal. Calcd. for C17H13Cl4N3O2: C, 47.14%;
1687 cmꢁ1. Anal. Calcd. for C16H13Cl2N3O2: C, 54.87%; H, 3.74%; N,
12.00%. Found: C, 54.51%; H, 3.65%; N, 10.11%.
H, 3.03%; N, 9.70%. Found: C, 47.02%; H, 3.02%; N, 9.66%.
5.1.1.35. Ethyl 1-(2,4-dichlorophenyl)-4-methyl-5-(4,5,6,7-tetrahy-
5.1.1.41. 5-(2-Chloro-1H-pyrrol-1-yl)-1-(2,4-dichlorophenyl)-4-met-
hyl-1H-pyrazole-3-carboxylic acid (46). This compound was
prepared as 44 from 36. Yield 99%, mp 173e176 ꢀC (from ethanol).
dro-2H-isoindol-2-yl)-1H-pyrazole-3-carboxylate
(40). This
compound was prepared as 34 from 28 [9] and cyclohexane-1,2-
dicarbaldehyde (55). Yield 47% as an oil. 1H NMR (400 MHz,
1H NMR (400 MHz, CDCl3):
d 2.22 (s, 3H), 6.10e6.11 (m, 1H), 6.16 (t,
CDCl3):
d
1.42 (t, J ¼ 7.5 Hz, 3H), 1.65e1.68 (m, 4H), 2.24 (s, 3H),
J ¼ 3.5 Hz, 1H), 6.56e6.57 (m, 1H), 7.26e7.27 (m, 2H), 7.46 ppm (s,
2.46e2.47 (m, 4H), 4.44 (q, J ¼ 7.1 Hz, 2H), 6.21 (s, 2H), 7.26e7.27
1H). MS (ESI): m/z: 371 [M þ H]þ, 393 [M þ Na]þ. IR:
n 2900 (acid
(m, 2H), 7.45 ppm (d, J ¼ 1.7 Hz, 1H). MS (ESI): m/z: 419 [M þ H]þ,
band), 1685 cmꢁ1. Anal. Calcd. for C15H10Cl3N3O2: C, 48.61%; H,
2.72%; N, 11.34%. Found: C, 48.40%; H, 2.66%; N, 11.17%.
441 [M þ Na]þ. IR:
n
1715 cmꢁ1. Anal. Calcd. for C21H21Cl2N3O2: C,

F. Piscitelli et al. / European Journal of Medicinal Chemistry 46 (2011) 5641e5653
5651
5.1.1.42. 5-(2-Chloro-1H-pyrrol-1-yl)-1-(3,4-dichlorophenyl)-4-met-
hyl-1H-pyrazole-3-carboxylic acid (47). This compound was
prepared as 44 from 37. Yield 96%, mp 187e188 ꢀC (from ethanol).
5.1.1.49. Cyclohexane-1,2-diyldimethanol (54). A solution of hex-
ahydrophthalic anhydride (mixture of isomers, 5.00 g, 32.0 mmol)
in anhydrous tetrahydrofuran (30 mL) was added dropwise to
a 0 ꢀC cooled suspension of lithium aluminium hydride (4.21 g,
110 mol) in anhydrous tetrahydrofuran (200 mL). The reaction
mixture was heated to 60 ꢀC for 2 h. The reaction mixture was
cooled to 0 ꢀC, and crushed ice was carefully added to neutralize the
excess of lithium aluminium hydride. The precipitate was filtered,
and the mixture was extracted with ethyl acetate. The combined
organic layers were washed with brine, dried and evaporated. The
crude residue was triturated in diethyl ether to give 54. Yield 75%.
1H NMR (400 MHz, CDCl3):
d 2.25 (s, 3H), 6.27e6.29 (m, 1H), 6.35 (t,
J ¼ 3.5 Hz, 1H), 6.63e6.64 (m, 1H), 6.91 (dd, J ¼ 8.7 and 2.5 Hz, 1H),
7.36 (d, J ¼ 2.5 Hz, 1H), 7.40 ppm (d, J ¼ 8.7 Hz, 1H). MS (ESI): m/z:
371 [M þ H]þ, 393 [M þ Na]þ. IR:
n .
2987 (acid band), 1712 cmꢁ1
Anal. Calcd. for C15H10Cl3N3O2: C, 48.61%; H, 2.72%; N, 11.34%.
Found: C, 48.50%; H, 2.69%; N, 11.12%.
5.1.1.43. 5-(2,5-Dichloro-1H-pyrrol-1-yl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-carboxylic acid (48). This compound was
prepared as 44 from 38. Yield 99%, mp 170e172 ꢀC (from ethanol).
1H NMR (400 MHz, CDCl3):
d 1.36e1.55 (m, 8H), 1.91e1.96 (m, 2H),
2.76 (br s, 2H, disappeared on treatment with D2O), 3.58 (dd,
1H NMR (400 MHz, CDCl3):
d
2.23 (s, 3H), 6.12 (s, 2H), 7.21e7.25 (m,
J ¼ 11.0 and 4.1 Hz, 2H), 3.77 ppm (dd, J ¼ 11.0 and 8.2 Hz, 2H). MS
2H), 7.51 ppm (m, 1H). MS (ESI): m/z: 406 [M þ H]þ, 428 [M þ Na]þ.
(ESI): m/z: 145 [M þ H]þ, 167 [M þ Na]þ. IR:
n
3389 cmꢁ1. Anal.
Calcd. for C8H16O2: C, 66.63%; H, 11.18%. Found: C, 66.57%; H, 11.16%.
IR:
n
2858 (acid band), 1695 cmꢁ1. Anal. Calcd. for C15H9Cl4N3O2: C,
44.48%; H, 2.24%; N, 10.37%. Found: C, 44.32%; H, 2.16%; N, 10.11%.
5.1.1.50. Cyclohexane-1,2-dicarbaldehyde (55). Oxalyl chloride
(2.10 g, 1.40 mL,16.3 mmol) was added dropwise to a ꢁ78 ꢀC cooled
solution of dimethylsulfoxide (1.39 mL) in anhydrous dichloro-
methane (7 mL). The reaction mixture was stirred at ꢁ78 ꢀC for
5 min, and then a solution of 54 (1.00 g, 6.9 mmol) in anhydrous
dichloromethane (7 mL) was added dropwise. The reaction mixture
was stirred at ꢁ78 ꢀC for 30 min and then triethylamine (9.86 mL)
was added. The mixture was allowed to rise up to room tempera-
ture and the precipitate was filtered. Evaporation of the solvent
gave a residue that was used without further purification. Yield 67%
as an oil. MS (ESI): m/z: 141 [M þ H]þ, 163 [M þ Na]þ.
5.1.1.44. 5-(2,5-Dichloro-1H-pyrrol-1-yl)-1-(3,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-carboxylic acid (49). This compound was
prepared as 44 from 39. Yield 89%, mp 176e179 ꢀC (from ethanol).
1H NMR (400 MHz, CDCl3):
d 2.24 (s, 3H), 6.24 (s, 2H), 6.98 (dd,
J ¼ 8.7 Hz, 1H), 7.42e7.46 ppm (m, 2H). MS (ESI): m/z: 406
[M þ H]þ, 428 [M þ Na]þ. IR:
n
2875 (acid band), 1687 cmꢁ1. Anal.
Calcd. for C15H9Cl4N3O2: C, 44.48%; H, 2.24%; N, 10.37%. Found: C,
44.48%; H, 2.12%; N, 10.19%.
5.1.1.45. 1-(3,4-Dichlorophenyl)-4-methyl-5-(4,5,6,7-tetrahydro-2H-
isoindol-2-yl)-1H-pyrazole-3-carboxylic acid (50). This compound
was prepared as 44 from 40. Yield 94%, mp 165e170 ꢀC (from
5.1.1.51. N1,N4-dimethoxy-N1,N4-dimethylsuccinamide (56). A solu-
tion of trimethylaluminium 2 M in toluene (15 mL) was added
dropwise to a 5 ꢀC cooled suspension of N,O-dimethylhydroxyl-
amine hydrochloride (2.92 g, 30.0 mmol) in anhydrous benzene
(30 mL). The reaction mixture was stirred in atmosphere of argon at
room temperature for 2 h to give a 0.67 M solution of (methox-
y(methyl)amino)(methyl)aluminum chloride (45 mL). This solution
(30 mL, 20.0 mmol) was added dropwise to a solution of dieth-
ylsuccinate (1.00 g, 0.95 mL, 5.7 mmol) in anhydrous benzene
(5 mL). The reaction mixture was stirred at room temperature for
48 h and then quenched over 6% HCl and ice. The organic layer was
washed with brine, dried and evaporated. The residue was tritu-
rated in hexanes to furnish 56. Yield 72%, mp 67e68 ꢀC. 1H NMR
ethanol). 1H NMR (400 MHz, CDCl3):
d 1.64e1.71 (m, 4H), 2.27 (m,
3H), 2.45e2.47 (m, 4H), 6.23 (s, 2H), 7.21e7.31 (m, 2H), 7.51 ppm (s,
1H). MS (ESI): m/z: 391 [M þ H]þ, 413 [M þ Na]þ. IR:
n 2917 (acid
band), 1690 cmꢁ1. Anal. Calcd. for C19H17Cl2N3O2: C, 58.47%; H,
4.39%; N, 10.77%. Found: C, 58.26%; H, 4.35%; N, 10.54%.
5.1.1.46. 1-(2,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-
ethyl-1H-pyrazole-3-carboxylic acid (51). This compound was
prepared as 44 from 41. Yield 94%, mp 179e182 ꢀC (from ethanol).
1H NMR (400 MHz, CDCl3):
d
1.10 (t, J ¼ 7.4 Hz, 3H), 2.05 (s, 6H), 2.63
(q, J ¼ 7.5 Hz, 2H), 5.83 (s, 2H), 6.96 (d, J ¼ 8.5 Hz, 1H), 7.21 (dd,
J ¼ 8.6 and 2.1 Hz, 1H), 7.53 ppm (d, J ¼ 2.0 Hz, 1H). MS (ESI): m/z:
379 [M þ H]þ, 401 [M þ Na]þ. IR:
n
2921 (acid band), 1689 cmꢁ1
.
(400 MHz, CDCl3): d 2.77 (s, 4H), 3.18 (s, 6H), 3.74 ppm (s, 6H). MS
Anal. Calcd. for C18H17Cl2N3O2: C, 57.16%; H, 4.53%; N, 11.11%. Found:
C, 56.95%; H, 4.48%; N, 10.94%.
(ESI): m/z: 205 [M þ H]þ, 227 [M þ Na]þ. IR:
n
1642 cmꢁ1. Anal.
Calcd. for C8H16N2O2: C, 47.05%; H, 7.90%; N, 13.72%. Found: C,
46.98%; H, 7.89%; N, 13.70%.
5.1.1.47. 1-(3,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-
methyl-1H-pyrazole-3-carboxylic acid (52). This compound was
prepared as 44 from 42. Yield 92%, mp 152e154 ꢀC (from ethanol).
5.1.1.52. Octane-3,6-dione (57). Ethylmagnesium bromide (3.0 M
solution in ether, 2.3 mL, 6.8 mol) was added dropwise to a solution
of 56 (0.35 g, 1.7 mmol) in anhydrous diethyl ether (20 mL). The
reaction mixture was stirred at room temperature for 12 h. A
saturated solution of ammonium chloride was added, and the
mixture was extracted with diethyl ether. The combined organic
layers were washed with brine, dried and evaporated to provide 57
that was used without further purification. Yield 58% as an oil. 1H
1H NMR (400 MHz, CDCl3):
d
1.10 (t, J ¼ 7.4 Hz, 6H), 2.09e2.22 (m,
7H), 6.06 (s, 2H), 6.70 (dd, J ¼ 8.8 and 2.6 Hz, 1H), 7.25 (d, J ¼ 2.6 Hz,
1H), 7.36 ppm (d, J ¼ 8.8 Hz, 1H). MS (ESI): m/z: 393 [M þ H]þ, 415
[M þ Na]þ. IR:
n
2974 (acid band), 1686 cmꢁ1. Anal. Calcd. for
C19H19Cl2N3O2: C, 58.17%; H, 4.88%; N, 10.71%. Found: C, 57.92%; H,
4.80%; N, 10.58%.
NMR (400 MHz, CDCl3):
d
1.06 (t, J ¼ 7.3 Hz, 6H), 2.48 (q, J ¼ 7.3 Hz,
5.1.1.48. 1-(3,4-Dichlorophenyl)-5-(2,5-diethyl-1H-pyrrol-1-yl)-4-
methyl-1H-pyrazole-3-carboxylic acid (53). This compound was
prepared as 44 from 42. Yield 92%, mp 152e154 ꢀC (from ethanol).
4H), 3.74 ppm (s, 4H). IR:
n
1630 cmꢁ1. MS (ESI): m/z: 143 [M þ H]þ,
165 [M þ Na]þ.
1H NMR (400 MHz, CDCl3):
d
1.10 (t, J ¼ 7.4 Hz, 6H), 2.09e2.22 (m,
5.2. CB1and CB2 receptor binding assays
7H), 6.06 (s, 2H), 6.70 (dd, J ¼ 8.8 and 2.6 Hz, 1H), 7.25 (d, J ¼ 2.6 Hz,
1H), 7.36 ppm (d, J ¼ 8.8 Hz, 1H). MS (ESI): m/z: 393 [M þ H]þ, 415
For both receptor binding assays, the new compounds were
tested as previously described [12]. Binding affinities of reference
compounds were evaluated in parallel with compounds 6e25. Ki
means concentration of the competing ligand that will bind to half
[M þ Na]þ. IR:
n
2974 (acid band), 1686 cmꢁ1. Anal. Calcd. for
C19H19Cl2N3O2: C, 58.17%; H, 4.88%; N, 10.71%. Found: C, 57.98%; H,
4.83%; N, 10.60%.

5652
F. Piscitelli et al. / European Journal of Medicinal Chemistry 46 (2011) 5641e5653
[3] For example, Taranabant, Otenabant, Ibipinabant and AVE1625 are new CB1
receptor antagonists/inverse agonists which have also been evaluated in
clinical studies. (a) H.J. Seo, M.J. Kim, S.H. Lee, S.-H. Lee, M.E. Jung, M.-S. Kim,
K. Ahn, J. Kim, J. Lee, Synthesis and structureeactivity relationship of 1,2,4-
triazole-containingdiarylpyrazolyl carboxamide as CB1 cannabinoid
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the binding sites at equilibrium, in the absence of radioligand or
other competitors. IC50 means concentration of competitor that
competes for half of the specific binding (a measure of the
competitor’s potency at interacting with the receptor against the
radioligand). Ki is calculated from EC50 using the ChengePrusoff
equation.
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5.3. Pharmacological studies
The experimental procedure used here was in accordance with
the Italian law on the “Protection of animals used for experimental
and other scientific reasons”.
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Y. Feng, D.J. Marsh, D.S. Stribling, L.P. Shearman, A.M. Strack, M.T. Goulet, Furo
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P.C. Verveer, C.G. Kruse, Design, synthesis, biological properties, and molecular
modeling investigations of novel tacrine derivatives with a combination of
acetylcholinesterase inhibition and cannabinoid CB1 receptor antagonism,
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5.3.1. Animals
Adult male Wistar rats (Charles River Laboratories, Calco, Italy),
weighing approximately 250 g at the start of the experiment, were
used. Rats were individually housed in standard plastic cages with
wood chip bedding. The animal facility was under an inverted
12:12 h lightedark cycle (lights on at 11:00 pm), at a constant
temperature of 22 ꢂ 2 ꢀC and relative humidity of approximately
60%. Rats were extensively habituated to handling and intraperi-
toneal injection.
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5.3.2. Experimental procedure
Rats were exposed to the homecage, 2-bottle choice regimen
between sucrose (1% w/v, in water) and water, with unlimited
access for 24 h/day, for 4 consecutive weeks before the start of the
experiment with 11 (during this time period, daily sucrose intake
stabilized). Bottles were refilled every day with fresh solution and
their lefteright positions interchanged daily to avoid development
of position preference. Food pellets (standard rat chow; Mucedola,
Settimo Milanese, Italy) were available 24 h/day. Daily sucrose,
water, and food intake was expressed as mL/kg (of the sucrose
solution), mL/kg, and g/kg, respectively, and monitored by weigh-
ing the bottles and food pellets (0.1 g accuracy) every day imme-
diately before the start of the dark phase.
On the test day, rats were divided into 3 groups of n ¼ 15,
matched for body weight as well as sucrose and food intake over
the 3 days preceding the start of the experiment. Compound 11 was
suspended in saline with 0.05% (v/v) Tween 80 and administered
intraperitoneally (injection volume: 3 mL/kg), at the doses of 0, 1,
and 2 mg/kg, 30 min before lights off. Sucrose, water, and food
intakes were recorded 6 h after lights off.
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5.3.3. Statistical analysis
Data on the effect of compound 11 on sucrose, water, and food
intakes were analyzed by separate 1-way ANOVAs, followed by the
NewmaneKeuls test for post hoc comparisons.
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