Synthesis and biological evaluation of new N-alkyl 1-aryl-5-(1H-pyrrol-1- yl)-1H-pyrazole-3-carboxamides as cannabinoid receptor ligands

Article abstract of DOI:10.1016/j.ejmech.2010.09.053

A series of N-alkyl 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides were synthesized as new ligands of the human recombinant receptor hCB ₁. n-Alkyl carboxamides brought out different SARs from the branched subgroup. Unsubstituted pyrrole derivatives bearing a tert-alkyl chain at the 3-carboxamide nitrogen showed greater hCB₁ receptor affinity than the corresponding unbranched compounds. In particular, the tert-butyl group as a chain terminal moiety strongly improved hCB₁ receptor affinity (compound 24: K_i = 45.6 nM; 29: K_i = 37.5 nM). Acute administration of either compound 12 or 29 resulted in a specific, dose-dependent reduction in food intake in rats. Such results provide an useful basis for the design of new CB₁ ligands.

Full text of DOI:10.1016/j.ejmech.2010.09.053

European Journal of Medicinal Chemistry 45 (2010) 5878e5886
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of new N-alkyl 1-aryl-5-(1H-pyrrol-1-yl)-1H-
pyrazole-3-carboxamides as cannabinoid receptor ligands
,
b
a
a,
Romano Silvestri a , Alessia Ligresti , Giuseppe La Regina , Francesco Piscitelli 1, Valerio Gatti a,
**
*
Antonio Lavecchia c , Antonella Brizzi , Serena Pasquini , Marco Allarà , Noemi Fantini ,
,
d
d
b
e
Mauro Antonio Maria Carai e, Chiara Bigogno f, Marco Giulio Rozio f, Roberta Sinisi f, Ettore Novellino c,
***
Giancarlo Colombo e, Vincenzo Di Marzo b, Giulio Dondio f, Federico Corelli d
,
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 Università di Napoli Federico II, Via Domenico Montesano 49, I-80131 Napoli, Italy
d Dipartimento Farmaco Chimico Tecnologico, Università di Siena, Polo Scientifico Universitario San Miniato, Via Alcide De Gasperi 2, I-53100 Siena, Italy
e Istituto di Neuroscienze, Consiglio Nazionale delle Ricerche, Viale Armando Diaz 182, I-09126 Cagliari, Italy
f NiKem Research Srl, Via Zambeletti 25, 20021 Baranzate, Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-alkyl 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides were synthesized as new
ligands of the human recombinant receptor hCB1. n-Alkyl carboxamides brought out different SARs from
the branched subgroup. Unsubstituted pyrrole derivatives bearing a tert-alkyl chain at the 3-carbox-
amide nitrogen showed greater hCB1 receptor affinity than the corresponding unbranched compounds.
In particular, the tert-butyl group as a chain terminal moiety strongly improved hCB1 receptor affinity
(compound 24: Ki ¼ 45.6 nM; 29: Ki ¼ 37.5 nM). Acute administration of either compound 12 or 29
resulted in a specific, dose-dependent reduction in food intake in rats. Such results provide an useful
basis for the design of new CB1 ligands.
Received 11 June 2010
Received in revised form
20 September 2010
Accepted 21 September 2010
Available online 1 October 2010
Keywords:
Cannabinoid
Human recombinant CB receptor type 1
Pyrrole bioisoteres
Ó 2010 Elsevier Masson SAS. All rights reserved.
Structure-activity relationships
Pharmacological studies
1. Introduction
The endocannabinoid system is involved in various pathological
conditions, such as pain, immunosuppression, peripheral vascular
Obesity is a predisposing factor for the development of type-2
diabetes, hypertension, and cardiovascular disease [1]. Therapeutic
options for morbidly obese patients are limited, as currently only
two drugs have been approved by FDA in the United States for the
long-term treatment of obesity, that is, sibutramine and orlistat.
However, such agents provide modest reduction of the body
weight, and both drugs are associated with adverse effects that
have restricted their therapeutic potential [2]. Alternative treat-
ments are thus desirable [3].
disease, appetite enhancement/suppression, and locomotor disor-
ders [4]. In 2006, SanofieAventis launched Rimonabant (SR141716,
1, Fig. 1) in the European Union as a CB1 receptor inverse agonist for
the treatment of overweight and obesity, and associated cardio-
vascular and metabolic disorders. In 2008, 1 was withdrawn from
all other markets due to gastrointestinal side effects, depression
and anxiety. Despite 1’s failure, many research groups from
academia and industry are still actively searching for novel CB1
receptor antagonists [5], that may provide options for the treat-
ment of obesity, whose prevalence is rapidly increasing globally,
and has reached epidemic proportions in the developed countries.
For example, Taranabant (2), Otenabant (3) Ibipinabant (4) and
AVE1625 (5) are new CB1 receptor antagonists/inverse agonists
which have also been evaluated in clinical studies (2 and 3 have
been recently suspended from clinical development since they
showed unwanted effects similar to 1) [6].
* Corresponding author. Tel.: þ39 06 4991 3800; fax: þ39 06 4969 3268.
** Correponding author. Tel./fax: þ39 081 678 613.
*** Corresponding author. Tel.: þ39 0577 234308; fax: þ39 0577 234299.
E-mail addresses: romano.silvestri@uniroma1.it (R. Silvestri), lavecchi@unina.it
(A. Lavecchia), corelli@unisi.it (F. Corelli).
1
Present address: University of Pennsylvania, Department of Chemistry, 231
South 34th Street, Philadelphia, PA 19104-6323, USA.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.053

R. Silvestri et al. / European Journal of Medicinal Chemistry 45 (2010) 5878e5886
5879
an aliphatic substituent at the 3-carboxamide nitrogen showed the
highest affinity for the CB1 receptor, e.g. 7 [Ki(CB1) ¼ 3.4 nM] [10].
Such findings prompted us to synthesize new carboxamide
derivatives bearing selected aliphatic chains at the nitrogen atom.
Herein, we describe the synthesis and the biological evaluation of
both linear carboxamides 8e20 and isomeric branched carbox-
amides 21e31 (Fig. 1, Table 1).
2. Chemistry
Synthesis of carboxamides 8e31 was performed by coupling
reactions of previously described acids 32e35 [10] with either
unbranched or branched aliphatic amines in the presence of
N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide (EDC) hydro-
chloride and 1-hydroxybenzotriazole (HOBt). Morpholinomethyl
polystyrene and polymer bound para-toluenesulfonic acid were
used as scavengers for acidic and basic substances, respectively
(Scheme 1).
Fig. 1. View from the plane of the cell membrane of the 29/hCB1 complex. Only amino
acids located within 4 Å distance from the ligand (green) are shown in yellow and
labeled. Residues that form part of the aromatic cluster complex with the ligand are
colored in magenta. H-bonds are indicated by dashed yellow lines. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article).
3. Results and discussion
3.1. Binding affinity
The binding affinities (Ki values) of 8e31 for the human
recombinant CB receptors hCB1 and hCB2 were evaluated in parallel
with reference compounds 1e3 (Table 1). The binding to the
receptor was evaluated by means of membranes from HEK cells
transfected with either the hCB1 or 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 [11]. 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 Chenge
Prusoff equation [12].
We first synthesized carboxamides 8e20 bearing C3-C7 normal
alkyl chains at the 3-carboxamide nitrogen. Compounds 8e20
showed affinity for the hCB1 receptor with Ki values ranging from
81.0 (10) to 550.3 (8) nM (Table 1). Compound 17 (CB1 SI ¼ 8.6; CB1
selectivity index (SI) was calculated as Ki(CB2)/Ki(CB1) ratio) had the
In search for new CB1 receptor antagonists/inverse agonists,
several chemical modifications of the template of 1 have been
explored. Mainly, three diverse structural strategies resulted in the
synthesis of: (i) pyrazole analogues of 1, for example AM251 (6),
(ii) bioisosteric compounds containing different heterocyclic rings
in place of the pyrazole nucleus [7], and (iii) compounds repre-
senting structural simplification/complication of 1’s template [8]
(Chart 1).
Replacement of the 4-chlorophenyl group at position 5 of 1 with
a pyrrole nucleus led us to disclose a new class of potent and
selective CB1 inverse agonists [9]. Structural modification of these
prototypical compounds led to new ligands endowed with affinity
and selectivity for the human CB1 (hCB1) receptor comparable to
the reference compounds 1 and 6. In particular, derivatives bearing
N
O
N
O
O
O
CF
3
O
S
NH
O
NH
N
NH
2
NC
N
N
Cl
N
N
NH
NH
N
N
N
N
N
Cl
Cl
Cl
Cl
Cl
Cl
Cl
1
2
3
4
R
NH
2
O
O
O
N
Cl
F
NH
NH
F
N
R
3
N
N
N
N
N
N
N
N
N
Cl
S
O
R
I
1
O
Cl
Cl
8-31
(for R -R see Table 1)
Cl
Cl
1
3
6
5
7
Chart 1. Structures of reference compounds 1e7 and new compounds 8e31.

5880
R. Silvestri et al. / European Journal of Medicinal Chemistry 45 (2010) 5878e5886
Table 1 (continued)
Table 1
Structure, hCB1 and hCB2 receptor affinity ([3H]CP-55,940 radioligand) of carbox-
amides 8e31.a
Ki (nM)b
Compd. R1
R2
R3
H
hCB1
hCB2
SIc
R
2
O
C4
Me
Me
NH
24
25
26
2,4-Cl2
45.6
52.4
1.15
N
Me
R
3
N
N
C7
R
1
Me
2,4-Cl2
2,4-Cl2
H
157.5
237.3
1.51
7.69
Me
8-31
C2
Me
Compd. R1
R2
R3
Ki (nM)b
2,5-Me2
233.5 1796.8
Me
hCB1
hCB2
SIc
C3
Me
C2
Me
8
2,4-Cl2
H
H
H
550.3 1496.2
2.71
27
28
29
30
31
2,4-Cl2
2,4-Cl2
2,4-Cl2
2,4-Cl2
2,4-F2
2,5-Me2
2,5-Me2
2,5-Me2
2,5-Me2
2,5-Me2
150.7
193.5
37.5
20.2
121.5
133.5
135.5
163.4
0.13
0.62
3.56
1.29
1.80
Me
Me
C4
Me
9
2,4-Cl2
2,4-Cl2
272.6
81.0
795.7
205.9
2.91
2.54
Me
Me
C3
Me
C5
Me
10
OH
C4
Me
Me
11
12
2,4-Cl2
2,4-Cl2
H
H
534.4 2344.4
4.38
3.09
C5
Me
C6
Me
124.1
211.0
383.3
255.1
C7
Me
104.7
90.7
C7
Me
Me
13
14
15
16
2,4-Cl2
2,4-Cl2
2,4-Cl2
2,4-Cl2
H
1.21
6.73
3.73
2.72
C4
Me
Me
C3
Me
2,5-Me2
2,5-Me2
2,5-Me2
290.1 1953.3
Me
RId
e
e
e
e
e
e
e
e
e
12
2.3
>2820
790
112
5.4
65.83
48.70
<0.002
C4
Me
123.1
182.1
459.8
495.3
AMe
SRf
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.
C5
Me
b
Ki and IC50 are defined in experimental section.
SI: CB1 selectivity index was calculated as Ki(CB2)/Ki(CB1) ratio.
RI: Rimonabant (1), CB1 reference compound.
AM: AM251 (6), CB1 reference compound.
c
OH
17
2,4-Cl2
2,5-Me2
372.3 3200.5
8.59
d
C5
e
f
SR: SR144528, CB2 reference compound.
C6
18
19
20
2,4-F2
2,4-Cl2
2,4-F2
2,5-Me2
2,5-Me2
2,5-Me2
311.4
97.2
487.3
204.8
365.5
1.56
2.11
0.99
highest CB1 SI value, and derivatives 10 and 19 showed Ki values
<100 nM. Introduction of a hydroxy group at
u position of the
C7
n-pentyl chain of either 10 or 16 provided more selective CB1
Me
C7
R
NH
2
O
O
368.2
Me
OH
C2
Me
N
N
R
R
a
3
3
N
N
N
N
21
2,4-Cl2
H
518.9 1132.4
2.18
Me
R
R
1
1
C2
Me
8-31
32-35
22
23
2,4-Cl2
2,4-Cl2
H
H
89.5
47.6
14.6
0.16
0.11
Me
Me
32: R1 = 2,4-Cl2, R3 = H; 33: R1 = 2,4-Cl2, R3 = 2,5-Me2;
34: R1 = 2,4-F2, R3 = H, 35: R1 = 2,4-F2, R3 = 2,5-Me2;
8-31: for R1-R3 substituents, see Table 1.
Me
Me
C3
Me
5.70
Scheme 1. Synthesis of carboxamides 8e31. Reagents and conditions. a: (i) R2NH2,
HOBt, EDC, dichloromethane, 0 ꢀC to room temp., overnight; (ii) morpholinomethyl
polystyrene and polymer bound p-toluenesulfonic acid, room temp., 24 h; yield
48e98%.

R. Silvestri et al. / European Journal of Medicinal Chemistry 45 (2010) 5878e5886
5881
ligands (11: CB1 SI ¼ 4.4, and0 17: CB1 SI ¼ 8.6) as a result of reduced
affinity for the hCB2 receptor. The CB1 receptor affinity of carbox-
amides 8e20 was not dramatically affected by the length of the
n-alkyl chain, even in the presence of methyl groups at position 2
and 5 of the pyrrole ring (compare 9 with 12 and 13, and 15 with 16
and 19).
In preliminary pharmacokinetic studies in the rat, carboxamide
12 was quickly available in plasma and brain (Tmax ¼ 30 min) after
i.p. administration at 10 mg/kg. The concentration of compound
12 in the two compartments was very similar over time (Cmax
170 ng/mL and 110 ng/g in plasma and brain, respectively). T ½
calculated on the elimination phase and MRT were very similar in
plasma and brain (about 850 min). The brain penetration calculated
as a ratio of the AUCs was about 97% (Table 2).
Introduction of branched alkyl chains at the 3-carboxamide
nitrogen led to derivatives 21e31 which were endowed with higher
receptor affinity for both CB1 and CB2 subtypes. The improvement
of affinity was particularly correlated to the presence of a tert-butyl
moiety. On the other hand, the CB receptor affinity was also greatly
affected by the substitution pattern at position 5 of the pyrrole
nucleus. Derivatives bearing both unsubstituted pyrrole nucleus
and tert-alkyl chain at the amide nitrogen, generally showed
greater hCB1 receptor affinity than the corresponding unbranched
compounds. Such a chemical modification also resulted in signifi-
cant improvement of affinity for the hCB2 receptor of 22, 23 and 24
(compare 22 with 9 (C4), 23 with 10 (C5) and 24 with 12 (C6)). The
tert-butyl moiety strongly affected the receptor affinity when it was
placed as a chain terminator group (24, 29 and 31). Accordingly,
compounds 24 (Ki ¼ 45.6 nM) and 29 (Ki ¼ 37.5 nM) showed the
highest affinity for the hCB1 receptor.
(363), and V6.59(367), and established favourable
p
e
p
stacking
interactions with the indole ring of W5.43(279). Such interactions
involved the aromatic residue-rich TM3-4-5-6 region of hCB1 [14]
(in magenta), and were stable during the MD trajectory (the
electron-withdrawing chlorine atoms contribute to strengthening
the stacking interactions). 29 showed typical binding interactions
previously reported for this class of CB1 ligands [9,10].
3.3. Pharmacological studies
The effects on reduction in food intake were evaluated in rats
after administration of either compound 12 or 29. ANOVA revealed
significant effects of treatment with either compound 12
[Ft(3,28) ¼ 8.47, P < 0.0005] or 29 [Ft(2,20) ¼ 7.62, P < 0.005] on
cumulative food intake over the first 6 h after lights off. Post hoc
analysis (ManneWhitney test) revealed significant differences
(P < 0.05) between the rat group treated with vehicle and (a) the rat
group treated with 12 (1 mg/kg) or 29 (3 mg/kg) at the 2-, 3-, 4-, 5-,
and 6-h time intervals and (b) the rat group treated with 10 mg/kg
12 or 29 at all six time intervals (12: Fig. 2, top panel; 29: Fig. 3, top
panel). Magnitude of reduction, with respect to vehicle-treated
rats, averaged 10e30% and 25e35% in the rat groups treated with 1
and 10 mg/kg 12, respectively. Conversely, the 0.1-mg/kg dose of 12
was virtually ineffective in altering food intake at each time
interval.
At the 24-h time interval, no difference in food intake was
recorded between rats treated with both doses of compound 12 and
rats treated with vehicle [F(3,28) ¼ 2.29, P > 0.05] (Fig. 2, top
panel). Magnitude of reduction, with respect to vehicle-treated
rats, averaged 10e35% and 30e40% in the rat groups treated with 3
and 10 mg/kg 29, respectively (Fig. 3, top panel).
The reducing effect of 29 on food intake was still evident at the
24-h time interval [F(2.20) ¼ 5.88, P < 0.05] (Fig. 3, top panel). Post
hoc analysis indicated that food intake in 3 and 10 mg/kg-treated rat
groups was significantly (P < 0.05) lower than in the vehicle-treated
rat group. Reduction in food intake disappeared in the following 2
days(data notshown). Thereducingeffectof 12 or 29 was specific for
food intake, as such compounds did not affect cumulative water
intake over the first 6 h after lights off [12: Ft(3,28) ¼ 2.47, P > 0.05;
3.2. Molecular modeling
The H-bond with K3.28(192) is recognized to be crucial for the
high affinity of 1 to the inactive receptor state and its inverse ago-
nism activity, by stabilizing a salt bridge between K3.28(192) and
D6.58(366) [13]. In MD experiments using our previously published
homology model of the inactive state of hCB1 [9,10], the carbox-
amide oxygen of 29 formed an H-bond with K3.28(192) during the
whole simulation time (Fig. 1). The N-tert-butyl group fitted into
a pocket formed by the lipophilic residues I1.34(119), F2.57(170),
F2.61(174), F2.64(177), and A7.36(380). The 2,5-dimethylpyrrole
ring formed contacts with the hydrophobic residues V3.32(196) and
C7.42(386), and projected the two methyl groups close to the lipo-
philic residues L6.51(359) and W5.43(279). The 4-methyl group
formed hydrophobic interactions with V3.32(196) and F2.57(170)
residues. Finally, the 2,4-dichlorophenyl ring at position 1 of the
pyrazole nucleus was embedded within a hydrophobic pocket
formed by L3.29(193), Y5.39(275), W5.43(279), L6.51(359), M6.55
Table 2
Rat pharmacokinetic parameters of 12 (10 mg/kg, ip).
Parameter
Plasma
Brain
Cmaxa (ng/mL or ng/g)b
170 ꢁ 61
30
836
889
20414
e
110 ꢁ 13
30
862
860
19801
97
Tmaxc (min)
T1/2d (min)
e
MRTinf (min)
f
AUClast (min ng/mL)
Brain penetration (%)
a
Cmax: maximum concentration.
b
Data represent mean values of three rats ꢁ S.D.
Tmax: time when the maximum concentration is reached.
T1/2: half life.
MRTinf: mean residence time calculated at the infinity.
AUClast: area under the curve calculated up to the last timepoint.
c
d
e
f
Fig. 2. Effect of the acute administration of different doses of compound 12 on food
(top panel) and water (bottom panel) intake in Wistar rats given unlimited access to
regular rodent chow and water. Each bar is the mean ꢁ SEM of n ¼ 8 rats.

5882
R. Silvestri et al. / European Journal of Medicinal Chemistry 45 (2010) 5878e5886
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 Spectrum
One spectrophotometer. Band position and absorption ranges are
given in cmꢂ1. Proton nuclear magnetic resonance (1H NMR)
spectra were recorded on a Bruker Avance 400 MHz FT spectrom-
eter in the indicated solvent. Chemical shifts are expressed in
d
units (ppm) from tetramethylsilane. 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 chromatography
(TLC). Developed plates were visualized with a Spectroline 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 ꢁ0.4% of the theoretical values. Purity of tested compounds
was >95%. Büchi Syncore reactor was used for parallel synthesis,
filtration, and evaporation.
Fig. 3. Effect of the acute administration of different doses of compound 29 on food
(top panel) and water (bottom panel) intake in Wistar rats given unlimited access to
regular rodent chow and water. Each bar is the mean ꢁ SEM of n ¼ 7e8 rats.
29: Ft(2.20) ¼ 0.44, P > 0.05] as well as waterintake at the 124-h time
interval [12: F(3,28) ¼ 0.63, P > 0.05], Fig. 2, bottom panel; [29: F
(2.20) ¼ 2.38, P > 0.05], Fig. 3, bottom panel.
Compounds 12 at 1 and 10 mg/kg and 29 at 3 and 10 mg/kg
reduced food intake in rats, and revealed comparable potency and
efficacy. However, the reducing effect of 29 on food intake seemed
to be longer-lasting than that of 12, as reduction in food intake was
still evident 24 h after drug injection. Although caution has to be
used when comparing data from different sets of experiments,
compounds 12 and 29 seemed to be slightly less effective in
reducing food intake in rats than 1 [15]. The in vivo anorectic effect
of 12 and 29 may be correlated to their antagonist/inverse agonist
activity at the cannabinoid CB1 receptor [15,16] Such a hypothesis
needs to be confirmed by additional evidences.
5.1.1. Parallel synthesis of amides 8e31
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 32e35 [10] (1 mmol) in dichloromethane, and
the selected amines (1.5 mmol) were added at the same
temperature. After warm up to room temperature, the vials were
placed in the Büchi Syncore reactor. Stirring was maintained at
300 rpm overnight and then morpholinomethyl polystyrene
(3 eq/mol) was added. The solutions were kept at room
temperature for 1 h, then polymer bound p-toluenesulfonic acid
(3 eq/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 8e31. Solid products were purified
by crystallization from ethanol or aqueous ethanol (18 and 30).
Oil products were purified by silica gel chromatography column
(dichloromethane as eluent).
4. Conclusion
We have synthesized new N-alkyl 1-aryl-5-(1H-pyrrol-1-yl)-
1H-pyrazole-3-carboxamides as human recombinant receptor hCB1
ligands. SARs of n-alkyl (8e20) and branched (21e31) carboxamide
subgroups highlighted different rules. The CB1 receptor affinity of
carboxamides 8e20 was not dramatically affected by the length of
the C3-C7 n-alkyl chain, even in the presence of methyl groups at
position 2 and 5 of the pyrrole ring. In pharmacokinetic studies,
compound 12 reached significant plasma and brain concentrations,
and supported its centrally mediated anorectic effect after acute
administration in the rat. Unsubstituted pyrrole derivatives bearing
a tert-alkyl chain at the amide nitrogen showed greater hCB1
receptor affinity than the corresponding unbranched compounds.
In particular, the tert-butyl moiety as a chain terminal group,
effectively improved the hCB1 receptor affinity (24: Ki ¼ 45.6 nM;
29: Ki ¼ 37.5 nM). In MD simulations, the carboxamide oxygen of 29
formed an H-bond with K3.28(192) during the whole simulation
time, as it did for 1. Acute administration of 12 and 29 resulted in
a specific, dose-dependent reduction in food intake in rats; 12 at 1
and 10 mg/kg and 29 at 3 and 10 mg/kg showed comparable
potency and effectiveness. On food intake, however, 29 exhibited
longer-lasting reducing effect, as reduction in food intake was still
evident 24 h after drug injection. The results described here provide
a useful basis for the design of new hCB1 ligands.
5.1.1.1. 1-(2,4-Dichlorophenyl)-4-methyl-N-propyl-5-(1H-pyrrol-1-yl)-
1H-pyrazole-3-carboxamide (8). Yield 98% as a white solid, mp
125e127 ꢀC (from ethanol). 1H NMR (CDCl3):
d
0.99 (t, J ¼ 7.4 Hz, 3H),
1.64 (sx, J ¼ 7.3 Hz, 2H), 2.32 (s, 3H), 3.40 (q, J ¼ 6.8 Hz, 2H),
6.20e6.21 (m, 2H), 6.58e6.59 (m, 2H), 6.98 (broad s,1H, disappeared
on treatment with D2O), 7.22 (d, J ¼ 8.2 Hz, 1H), 7.29 (dd, J ¼ 8.5 and
2.2 Hz, 1H), 7.49 ppm (d, J ¼ 2.2 Hz, 1H). IR:
n
1653, 3413 cm-1. Anal.
Calcd. for C18H18Cl2N4O: C, 57.30%; H, 4.81%; N, 14.85%. Found: C,
57.19%; H, 4.78%; N, 14.74%.
5.1.1.2. N-Butyl-1-(2,4-dichlorophenyl)-4-methyl-5-(1H-pyrrol-1-yl)-
1H-pyrazole-3-carboxamide (9). Yield 98% as a white solid, mp
121e122 ꢀC (from ethanol). 1H NMR (CDCl3):
d
0.96 (t, J ¼ 7.3 Hz,
3H), 1.42 (sx, J ¼ 7.7 Hz, 2H), 1.60 (qn, J ¼ 7.5 Hz, 2H), 2.32 (s, 3H),
3.44 (q, J ¼ 6.8 Hz, 2H), 6.21e6.22 (m, 2H), 6.58e6.59 (m, 2H),
6.94 (broad s, 1H, disappeared on treatment with D2O), 7.22 (d,
J ¼ 8.5 Hz, 1H), 7.29 (dd, J ¼ 8.5 and 2.2 Hz, 1H), 7.48 ppm (d,
J
¼
2.2 Hz, 1H). IR: n . Anal. Calcd. for
1655, 3428, cmꢂ1
C19H20Cl2N4O: C, 58.32%; H, 5.15%; N, 14.32%. Found: C, 58.11%; H,
5.11%; N, 14.16%.

R. Silvestri et al. / European Journal of Medicinal Chemistry 45 (2010) 5878e5886
5883
5.1.1.3. 1-(2,4-Dichlorophenyl)-4-methyl-N-pentyl-5-(1H-pyrrol-1-yl)-
1H-pyrazole-3-carboxamide (10). Yield 91% as a white solid, mp
D2O), 7.20 (dd, J ¼ 8.6 and 2.3 Hz, 1H), 7.53 ppm (d, J ¼ 2.3 Hz, 1H).
IR:
1669, 3415 cm-1. Anal. Calcd. for C22H26Cl2N4O: C, 60.97%; H,
6.05%; N, 12.93%. Found: C, 60.71%; H, 6.00%; N, 12.81%.
n
79e80 ꢀC (from ethanol). 1H NMR (CDCl3):
d
0.93 (t, J ¼ 7.1 Hz, 3H),
1.33e1.39 (m, 4H), 1.59e1.61 (m, 2H), 2.32 (s, 3H), 3.43 (q, J ¼ 6.9 Hz,
2H), 6.20e6.21 (m, 2H), 6.58e6.59 (m, 2H), 6.95 (broad s, 1H, dis-
appeared on treatment with D2O), 7.22 (d, J ¼ 8.5 Hz, 1H), 7.29 (dd,
5.1.1.10. 1-(2,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-N-
(5-hydroxypentyl)-4-methyl-1H-pyrazole-3-carboxamide (17). Yield
J ¼ 8.5 and 2.2 Hz, 1H), 7.48 ppm (d, J ¼ 2.2 Hz, 1H). IR:
n
1666,
98% as a colorless oil. 1H NMR (CDCl3):
d 1.44 (broad s, 1H, dis-
3420 cm-1. Anal. Calcd. for C20H22Cl2N4O: C, 59.27%; H, 5.47%; N,
13.82%. Found: C, 59.08%; H, 5.41%; N, 13.73%.
appeared on treatment with D2O), 1.47e1.53 (m, 2H), 1.61e1.71 (m,
4H), 1.96 (s, 6H), 2.20 (s, 3H), 3.47 (q, J ¼ 6.7 Hz, 2H), 3.68 (t,
J ¼ 6.0 Hz, 2H), 5.82 (s, 2H), 6.91 (d, J ¼ 8.5 Hz,1H), 7.06 (broad s,1H,
disappeared on treatment with D2O), 7.21 (dd, J ¼ 8.5 and 2.3 Hz,
5.1.1.4. 1-(2,4-Dichlorophenyl)-N-(5-hydroxypentyl)-4-methyl-5-(1H-
pyrrol-1-yl)-1H-pyrazole-3-carboxamide (11). Yield 98% as a color-
1H), 7.53 ppm (d, J ¼ 2.3 Hz, 1H). IR:
n
1657, 3412 cm-1. Anal. Calcd.
for C22H26Cl2N4O2: C, 58.80%; H, 5.83%; N, 12.47%. Found: C, 58.63%;
H, 5.78%; N, 12.38%.
less oil. 1H NMR (CDCl3):
d
1.37 (broad s, 1H, disappeared on treat-
ment with D2O), 1.46e1.51 (m, 2H), 1.60e1.68 (m, 4H), 2.31 (s, 3H),
3.45 (q, J ¼ 6.7 Hz, 2H), 3.67 (t, J ¼ 6.5 Hz, 2H), 6.21e6.22 (m, 2H),
6.57e6.58 (m, 2H), 6.99 (broad s,1H, disappeared on treatment with
D2O), 7.22 (d, J ¼ 8.4 Hz, 1H), 7.29 (dd, J ¼ 8.4 and 2.2 Hz, 1H),
5.1.1.11. 1-(2,4-Difluorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-N-
hexyl-4-methyl-1H-pyrazole-3-carboxamide (18). Yield 77% as
7.49 ppm (d, J ¼ 1.9 Hz, 1H). IR:
n
1656, 3410 cm-1. Anal. Calcd. for
cream solid, mp 77e79 ꢀC 1H NMR (CDCl3):
d
0.87 (t, 3H, J ¼ 6.4 Hz),
C20H22Cl2N4O2: C, 57.01%; H, 5.26%; N, 13.30%. Found: C, 56.79%; H,
5.21%; N, 13.19%.
1.44e1.23 (m, 6H), 1.64e1.53 (m, 2H), 1.88 (s, 6H), 2.16 (s, 3H),
3.46e3.36 (m, 2H), 5.78 (s, 2H), 7.06e6.83 (m, 2H), 7.17e7.09 ppm (m,
1H). MS m/z: 415 [M þ 1]þ (20%), 437 [M þ 23]þ (100%).
5.1.1.5. 1-(2,4-Dichlorophenyl)-N-hexyl-4-methyl-5-(1H-pyrrol-1-yl)-
1H-pyrazole-3-carboxamide (12). Yield 70% as a colorless oil. 1H
5.1.1.12. 1-(2,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-N-
heptyl-4-methyl-1H-pyrazole-3-carboxamide (19). Yield 75% as
a white solid, mp 51e56 ꢀC (from ethanol-water). 1H NMR (CDCl3):
NMR (200 MHz, CDCl3):
d
0.80 (t, J ¼ 6.0 Hz, 3H), 1.24e1.40 (m, 6H),
1.47e1.54 (m, 2H), 2.22 (s, 3H), 3.27e3.37 (m, 2H), 6.10 (s, 2H), 6.50
(s, 2H), 6.90e6.99 (m, 2H), 7.19 (s, 1H), 7.39 (s, 1H). MS (ESI): m/z:
d
0.89 (t, J ¼ 6.9 Hz, 3H), 1.26e1.33 (m, 8H), 1.61 (qn, J ¼ 7.3 Hz, 2H),
420 [M þ H]þ. IR (CHCl3):
n
1653 cm-1. Anal. Calcd. for C21H24Cl2N4O:
1.96 (s, 6H), 2.20 (s, 3H), 3.44 (q, J ¼ 6.5 Hz, 2H), 5.82 (s, 2H), 6.90 (d,
J ¼ 8.6 Hz, 1H), 7.01 (broad s, 1H, disappeared on treatment with
D2O), 7.20 (dd, J ¼ 8.6 and 2.3 Hz, 1H), 7.53 ppm (d, J ¼ 2.0 Hz, 1H).
C, 60.15%; H, 5.77%; N, 13.36%. Found: C, 59.88%; H, 5.54%; N, 13.17%.
5.1.1.6. 1-(2,4-Dichlorophenyl)-N-heptyl-4-methyl-5-(1H-pyrrol-1-yl)-
IR: n
1670, 3421 cm-1. Anal. Calcd. for C24H30Cl2N4O: C, 62.47%; H,
1H-pyrazole-3-carboxamide (13). Yield 97% as a colorless oil. 1H NMR
6.55%; N, 12.14%. Found: C, 62.19%; H, 6.50%; N, 11.98%.
(CDCl3):
d
0.89 (t, J ¼ 6.7 Hz, 3H), 1.29e1.38 (m, 8H), 1.61 (qn,
J ¼ 7.3 Hz, 2H), 2.31 (s, 3H), 3.42 (q, J ¼ 6.7 Hz, 2H), 6.20e6.21 (m,
2H), 6.58e6.59 (m, 2H), 6.95 (broad s, 1H, disappeared on treatment
with D2O), 7.22 (d, J ¼ 8.5 Hz, 1H), 7.29 (dd, J ¼ 8.5 and 2.2 Hz, 1H),
5.1.1.13. 1-(2,4-Difluorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-N-
heptyl-4-methyl-1H-pyrazole-3-carboxamide (20). Yield 48% as
a white solid, mp 143e145 ꢀC (from ethanol). 1H NMR (CDCl3):
d 0.89
7.48 ppm (d, J ¼ 2.2 Hz, 1H). IR:
n
1668, 3419 cm-1. Anal. Calcd. for
(t, J ¼ 6.8 Hz, 3H), 1.29e1.38 (m, 8H), 1.64 (qn, J ¼ 7.3 Hz, 2H), 1.92 (s,
6H), 2.20 (s, 3H), 3.44 (q, J ¼ 6.2 Hz, 2H), 5.82 (s, 2H), 6.88e6.95 (m,
2H), 6.99 (broad s, 1H, disappeared on treatment with D2O),
C22H26Cl2N4O: C, 60.97%; H, 6.05%; N, 12.93%. Found: C, 60.77%; H,
6.03%; N, 12.83%.
7.14e7.18 ppm (m, 1H). IR:
n
1645, 3335 cm-1. Anal. Calcd. for
5.1.1.7. 1-(2,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-
C24H30F2N4O: C, 67.27%; H, 7.06%; N, 13.07%. Found: C, 67.02%; H,
6.95%; N, 12.79%.
methyl-N-propyl-1H-pyrazole-3-carboxamide (14). Yield 71% as
a white solid, mp 84e86 ꢀC (from ethanol). 1H NMR (CDCl3):
d 1.01
(t, J ¼ 7.4 Hz, 3H), 1.67 (qn, J ¼ 7.3 Hz, 2H), 1.96 (s, 6H), 2.20 (s, 3H),
3.42 (q, J ¼ 7.0 Hz, 2H), 5.82 (s, 2H), 6.91 (d, J ¼ 8.5 Hz, 1H), 7.05
(broad s, 1H, disappeared on treatment with D2O), 7.20 (dd, J ¼ 8.5
5.1.1.14. 1-(2,4-Dichlorophenyl)-N-isopropyl-4-methyl-5-(1H-pyrrol-
1-yl)-1H-pyrazole-3-carboxamide (21). Yield 97% as a white solid,
mp 124e126 ꢀC (from ethanol). 1H NMR (CDCl3):
d
1.27 (d, J ¼ 6.6 Hz,
and 2.9 Hz, 1H), 7.53 ppm (d, J ¼ 2.3 Hz, 1H). IR:
n
1654, 3330 cm-1.
6H), 2.32 (s, 3H), 4.19e4.24 (m, 1H), 6.21e6.22 (m, 2H), 6.58e6.59
(m, 2H), 6.77 (broad s,1H, disappeared on treatment with D2O), 7.24
(d, J ¼ 8.4 Hz, 1H), 7.30 (dd, J ¼ 8.5 and 2.2 Hz, 1H), 7.48 ppm (d,
Anal. Calcd. for C20H22Cl2N4O: C, 59.27%; H, 5.47%; N, 13.82%.
Found: C, 59.06%; H, 5.40%; N, 13.72%.
J ¼ 2.3 Hz, 1H). IR:
n
1672, 3394 cm-1. Anal. Calcd. for C18H18Cl2N4O:
C, 57.30%; H, 4.81%; N, 14.85%. Found: C, 57.23%; H, 4.76%; N, 14.71%.
5.1.1.8. N-Butyl-1-(2,4-dichlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-
1-yl)-4-methyl-1H-pyrazole-3-carboxamide (15). Yield 98% as
a white solid, mp 67e69 ꢀC (from ethanol). 1H NMR (CDCl3):
d
0.97
5.1.1.15. N-tert-Butyl-1-(2,4-dichlorophenyl)-4-methyl-5-(1H-pyrrol-
(t, J ¼ 7.3 Hz, 3H), 1.44 (sx, J ¼ 7.7 Hz, 2H), 1.61 (qn, J ¼ 7.8 Hz, 2H),
1.96 (s, 6H), 2.20 (s, 3H), 3.46 (q, J ¼ 6.6 Hz, 2H), 5.81 (s, 2H), 6.91 (d,
J ¼ 8.5 Hz, 1H), 7.01 (broad s, 1H, disappeared on treatment with
D2O), 7.21 (dd, J ¼ 8.5 and 2.3 Hz, 1H), 7.53 ppm (d, J ¼ 2.2 Hz, 1H).
1-yl)-1H-pyrazole-3-carboxamide (22). Yield 91% as a white solid,
mp 101e103 ꢀC (from ethanol). 1H NMR (CDCl3):
d 1.49 (s, 9H), 2.30
(s, 3H), 6.19e6.21 (m, 2H), 6.57e6.58 (m, 2H), 6.82 (broad s, 1H,
disappeared on treatment with D2O), 7.23 (d, J ¼ 8.8 Hz, 1H), 7.29
IR:
n
1669, 3419 cm-1. Anal. Calcd. for C21H24Cl2N4O: C, 60.15%; H,
(dd, J ¼ 8.5 and 2.2 Hz, 1H), 7.48 ppm (d, J ¼ 1.9 Hz, 1H). IR:
n 1672,
5.77%; N, 13.36%. Found: C, 59.87%; H, 5.72%; N, 13.24%.
3407 cm-1. Anal. Calcd. for C19H20Cl2N4O: C, 58.32%; H, 5.15%; N,
14.32. Found: C, 58.20%; H, 5.13%; N, 14.21%.
5.1.1.9. 1-(2,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-
methyl-N-pentyl-1H-pyrazole-3-carboxamide (16). Yield 71% as
a white solid, mp 54e56 ꢀC (from ethanol/water). 1H NMR (CDCl3):
5.1.1.16. 1-(2,4-Dichlorophenyl)-4-methyl-N-tert-pentyl-5-(1H-pyr-
rol-1-yl)-1H-pyrazole-3-carboxamide (23). Yield 98% as a white
d
0.92 (t, J ¼ 7.1 Hz, 3H), 1.37e1.40 (m, 4H), 1.59e1.64 (m, 2H), 1.96
solid, mp 114e116 ꢀC (from ethanol). 1H NMR (CDCl3):
d 0.93 (t,
(s, 6H), 2.20 (s, 3H), 3.43 (q, J ¼ 6.9 Hz, 2H), 5.82 (s, 2H), 6.91 (d,
J ¼ 8.6 Hz, 1H), 7.01 (broad s, 1H, disappeared on treatment with
J ¼ 7.5 Hz, 3H), 1.43 (s, 6H), 1.85 (q, J ¼ 7.5 Hz, 2H), 2.32 (s, 3H),
6.20e6.22 (m, 2H), 6.57e6.58 (m, 2H), 6.75 (broad s, 1H,

5884
R. Silvestri et al. / European Journal of Medicinal Chemistry 45 (2010) 5878e5886
disappeared on treatment with D2O), 7.23 (d, J ¼ 8.5 Hz, 1H), 7.29
as a white solid, mp 59e61 ꢀC (from ethanol-water). 1H NMR
(CDCl3):
(dd, J ¼ 8.5 and 2.2 Hz, 1H), 7.48 ppm (d, J ¼ 2.2 Hz, 1H). IR:
n
1663,
d
0.88 (t, J ¼ 6.7 Hz, 3H), 1.25 (d, J ¼ 6.6 Hz, 3H), 1.27e1.35
3400 cm-1. Anal. Calcd. for C20H22Cl2N4O: C, 59.27%; H, 5.47%; N,
13.82%. Found: C, 59.01%; H, 5.41%; N, 13.73%.
(m, 9H), 1.54e1.58 (m, 1H), 1.96 (s, 6H), 2.20 (s, 3H), 4.14e4.21 (m,
1H), 6.82 (s, 2H), 6.79 (broad s, 1H, disappeared on treatment with
D2O), 6.92 (d, J ¼ 8.6 Hz, 1H), 7.20 (dd, J ¼ 8.5 and 3.0 Hz, 1H),
5.1.1.17. 1-(2,4-Dichlorophenyl)-N-(3,3-dimethylbutyl)-4-methyl-5-
(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (24). Yield 97% as
a white solid, mp 154e156 ꢀC (from ethanol). 1H NMR (CDCl3):
7.53 ppm (d, J ¼ 2.2 Hz, 1H). IR:
n
1671, 3408 cm-1. Anal. Calcd. for
C25H32Cl2N4O: C, 63.15%; H, 6.78%; N, 11.78%. Found: C, 62.79%; H,
6.74%; N, 11.66%.
d
0.97 (s, 9H), 1.52e1.57 (m, 2H), 2.32 (s, 3H), 3.43e3.49 (m, 2H),
6.21e6.22 (m, 2H), 6.58e6.59 (m, 2H), 6.88 (broad s, 1H, dis-
appeared on treatment with D2O), 7.21 (d, J ¼ 8.8 Hz, 1H), 7.29 (dd,
5.1.1.24. 1-(2,4-Difluorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-N-
(3,3-dimethylbutyl)-4-methyl-1H-pyrazole-3-carboxamide (31). Yield
89% as a white solid, mp 109e110 ꢀC (from ethanol). 1H NMR (CDCl3):
J ¼ 8.5 and 2.2 Hz, 1H), 7.49 ppm (d, J ¼ 2.2 Hz, 1H). IR:
n 1676,
3393 cm-1. Anal. Calcd. for C21H24Cl2N4O: C, 60.15%; H, 5.77%; N,
13.36%. Found: C, 59.98%; H, 5.73%; N, 13.27%.
d 0.99 (s, 9H), 1.55e1.59 (m, 2H), 1.91 (s, 6H), 2.19 (s, 3H), 3.45e3.48
(m, 2H), 5.82 (s, 2H), 6.87e694 (m, 3H), 7.11e7.17 ppm (m, 1H). IR:
n
3323,1654 cm-1. Anal. Calcd. for C23H28F2N4O: C, 66.65%; H, 6.81%; N,
13.52%. Found: C, 66.32%; H, 6.70%; N, 13.33%.
5.1.1.18. 1-(2,4-Dichlorophenyl)-4-methyl-N-(octan-2-yl)-5-(1H-pyr-
rol-1-yl)-1H-pyrazole-3-carboxamide (25). Yield 70% as a colorless
oil. 1H NMR (CDCl3):
d
0.88 (t, J ¼ 6.7 Hz, 3H), 1.24 (d, J ¼ 6.6 Hz, 3H),
5.2. Molecular modeling
1.27e1.33 (m, 9H), 1.40e1.57 (m, 1H), 2.32 (s, 3H), 4.13e4.20 (m,
1H), 6.20e6.22 (m, 2H), 6.58e6.59 (m, 2H), 6.73 (broad s, 1H, dis-
appeared on treatment with D2O), 7.24 (d, J ¼ 8.8 Hz, 1H), 7.30 (dd,
5.2.1. Computational chemistry
Molecular modeling and graphics manipulations were per-
formed using the molecular operating environment (MOE) [17] and
UCSF-CHIMERA software packages [18], running on a 2 CPU (PIV
2.0e3.0 GHZ) Linux workstation. Energy minimizations and MD
simulations were realized by employing the AMBER 9 [19] program
selecting the Cornell force field [20].
J ¼ 8.5 and 2.2 Hz, 1H), 7.48 ppm (d, J ¼ 1.9 Hz, 1H). IR:
n 1667,
3408 cm-1. Anal. Calcd. for C23H28Cl2N4O: C, 61.74%; H, 6.31%; N,
12.52%. Found: C, 61.65%; H, 6.27%; N, 12.46%.
5.1.1.19. 1-(2,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-N-
isopropyl-4-methyl-1H-pyrazole-3-carboxamide (26). Yield 97% as
a white solid, mp 114e116 ꢀC (from ethanol). 1H NMR (CDCl3):
d
1.29
5.2.2. Residue indexing
(d, J ¼ 6.6 Hz, 6H), 1.96 (s, 6H), 2.20 (s, 3H), 4.26e4.34 (m, 1H), 5.82
(s, 2H), 6.81 (broad s, 1H, disappeared on treatment with D2O), 6.92
(d, J ¼ 8.5 Hz, 1H), 7.20 (dd, J ¼ 8.5 and 3.3 Hz, 1H), 7.53 ppm (d,
The convention used for the amino acid identifiers, according to
the approach of Ballesteros and Weinstein [21] and van Rhee and
Jacobson [22], facilitates comparison of aligned residues within the
family of Group A GPCRs. The most conserved residue in a given TM
(TMX, where X is the TM number) is assigned the number X.50, and
residues within a given TM are then indexed relative to the 50
position.
J ¼ 2.1 Hz, 1H). IR:
n
1649, 3327 cm-1. Anal. Calcd. for C20H22Cl2N4O:
C, 59.27%; H, 5.47%; N, 13.82%. Found: C, 59.09%; H, 5.41%; N, 13.71%.
5.1.1.20. N-tert-Butyl-1-(2,4-dichlorophenyl)-5-(2,5-dimethyl-1H-pyr-
rol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (27). Yield 98% as
a white solid, mp 126e128 ꢀC (from ethanol). 1H NMR (CDCl3):
d
1.51
5.2.3. Docking simulations
(s, 9H), 1.96 (s, 6H), 2.19 (s, 3H), 5.81 (s, 2H), 6.90 (broad s, 1H, dis-
The core structure of compound 29 was constructed using
standard bond lengths and bond angles of the MOE fragment library.
Geometry optimizations were accomplished with the MMFF94X
force field, available within MOE. Docking simulations were carried
out starting from the previously published inactive state of hCB1
receptor model [9,10] which was built using the 2.8 Å crystal
structure of bovine rhodopsin (PDB entry code 1F88) [23] as
a structural template. Additional details regarding the receptor
structure, site-directed mutagenesis data and methods applied in
developingthe hCB1 receptor modelareshown in Ref. [6]Compound
29 was docked into the energy-minimized receptor model by means
of GOLD, 4.0 version, [24] a genetic algorithm-based software, taking
the binding orientation of Rimonabant in the hCB1 bundle as
a starting point. The region of interest used by GOLD was defined in
order to contain the residues within 15 Å from the original position
of Rimonabant in the hCB1 model [9]. The “allow early termination”
option was deactivated while the remaining GOLD default param-
eters were used. The ligand was submitted to 100 genetic algorithm
runs by selecting GOLD Score as a fitness function, without any other
constraints. The best docked conformation for GOLD Score was then
compared with the binding conformation of Rimonabant in the hCB1
bundle [9] and the root mean square deviation between the posi-
tions of the heavy atoms was calculated, this parameter being
considered as a measure of the docking accuracy.
appeared on treatment with D2O), 6.91 (d, J ¼ 8.5 Hz, 1H), 7.20 (dd,
J ¼ 8.5 and 2.3 Hz, 1H), 7.52 ppm (d, J ¼ 2.3 Hz, 1H). IR:
n 1676,
3410 cm-1. Anal. Calcd. for C21H24Cl2N4O: C, 60.15%; H, 5.77%; N,
13.36%. Found: C, 59.99%; H, 5.73%; N, 13.29%.
5.1.1.21. 1-(2,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-
methyl-N-tert-pentyl-1H-pyrazole-3-carboxamide (28). Yield 98%
as a colorless oil. 1H NMR (CDCl3):
d
0.95 (t, J ¼ 7.5 Hz, 3H), 1.45
(s, 6H), 1.86 (q, J ¼ 7.5 Hz, 2H), 1.96 (s, 6H), 2.18 (s, 3H), 5.81 (s,
2H), 6.83 (broad s, 1H, disappeared on treatment with D2O), 6.90
(d, J ¼ 8.5 Hz, 1H), 7.20 (dd, J ¼ 8.5 and 3.1 Hz, 1H), 7.52 ppm
(d, J ¼ 2.3 Hz, 1H). IR:
n
1672, 3406 cm-1. Anal. Calcd. for
C22H26Cl2N4O: C, 60.97%; H, 6.05%; N, 12.93%. Found: C, 60.71%;
H, 6.01%; N, 12.80%.
5.1.1.22. 1-(2,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-N-
(3,3-dimethylbutyl)-4-methyl-1H-pyrazole-3-carboxamide (29). Yield
82% as a white solid, mp 143e145 ꢀC (from ethanol).1H NMR (CDCl3):
d
0.99 (s, 9H), 1.54e1.58 (m, 2H), 1.96 (s, 6H), 2.20 (s, 3H), 3.46e3.51
(m, 2H), 5.82 (s, 2H), 6.90 (d, J ¼ 8.5 Hz, 1H), 6.96 (broad s, 1H, dis-
appeared on treatment with D2O), 7.20 (dd, J ¼ 8.5 and 2.3 Hz, 1H),
7.53 ppm (d, J ¼ 3.1 Hz, 1H). IR:
n
1655, 3332 cm-1. Anal. Calcd. for
C23H28Cl2N4O: C, 61.74%; H, 6.31%; N, 12.52%. Found: C, 61.59%; H,
6.27%; N, 12.40%.
5.2.4. MD simulations
5.1.1.23. 1-(2,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-
methyl-N-(octan-2-yl)-1H-pyrazole-3-carboxamide (30). Yield 68%
Refinement of the ligand/receptor complexes was achieved by in
vacuo energy minimization with the SANDER module of AMBER,

R. Silvestri et al. / European Journal of Medicinal Chemistry 45 (2010) 5878e5886
5885
applying an energy penalty force constant of 10 kcal molꢂ1 Åꢂ2 on
the protein backbone atoms. The geometry-optimized complexes
were then used as the starting point for subsequent 1 ns MD
simulation, during which the protein backbone atoms were con-
strained by means of decreasing force constants; moreover, also the
salt bridge between K3.28(192) and D6.58(366) as well as the H-
bond between the ligand carboxamide oxygen and K3.28(192)
were restrained. More specifically, an initial restraint with a force
constant of 10 kcal molꢂ1 Åꢂ2 was applied on all the alpha carbons;
this force constant decreased during the whole MD, and in the last
200 ps, its value was 0.01 kcal molꢂ1 Åꢂ2. As regards the intra-helix
K3.28(192)/D6.58(366) H-bonds and the ligand/K3.28(192) inter-
actions, a restraint of 10 and 50 kcal molꢂ1 Åꢂ2 were applied for
600 ps of MD simulation and, in the last 400 ps, the restraint was
removed. General AMBER force field (GAFF) parameters were
assigned to ligands, while the partial charges were calculated using
the AM1-BCC method as implemented in the ANTECHAMBER suite
of AMBER. A time step of 1 fs and a nonbonded pairlist updated
every 25 fs were used for the MD simulations. The temperature was
regulated by way of Langevin dynamics, with a collision frequency
240, 300, 360, and 1440 min after lights off (1440 min correspond
to 24 h) by weighing food pellets and bottles with a 0.1-g accuracy.
5.4.3. Statistical analysis
Data on the effect of compounds 12 or 29 on cumulative food
and water intake over the first 360 min were analyzed by separate
2-way (treatment; time) ANOVAs with repeated measures on the
factor ‘time’, followed by the NewmaneKeuls test for post hoc
comparisons. Data on the effect of compounds 12 or 29 on food and
water intake at the 1440-min time interval were analyzed by
separate 1-way ANOVAs, followed by the NewmaneKeuls test for
post hoc comparisons.
5.5. Pharmacokinetics
Pharmacokinetics and brain distribution studies were per-
formed in Wistar rats (Charles River Laboratories, Calco, Italy).
Animals were quarantined for approximately 1 week with an
inverted day-night period prior the study. They were housed under
standard conditions and had free access to water and standard
laboratory rodent diet. Compound 12 or 29 was administered
intraperitoneally at the dose of 10 mg/kg (formulation: 2% tween 80
and 98% saline at 1 mL/kg). Plasma and brains were collected from
each rat (n ¼ 18) at the following timepoints: 30, 60, 120, 240,
480 min and 24 h. Plasma and brain samples were kept frozen
(ꢂ80 ꢀC) until submission to extraction and LC-MS/MS analysis.
g
¼ 1.0 psꢂ1. An average structure was calculated from the last
100 ps trajectory and energy-minimized using the steepest descent
and conjugate gradient methods as specified above. Root mean
square (rms) deviations from the initial structures and interatomic
distances were monitored using the PTRAJ module in AMBER.
5.3. CB1 and CB2 receptor binding assays
Plasma samples (100
dard (IS) (100 ng/mL of compound 49) and treated on a Sirocco filter
plate (Waters) containing 400 L of a solution of 3% ammonia (3.2%
in water) in MeOH. The plate was shaken for 20 min and the filtered
samples (350 L) were analyzed by LC-MS/MS. Brain samples
(100 L of fresh brain homogenate made with 20 mM ammonium
formate solution) were spiked with 10 L of IS (100 ng/mL of 4) and
mL) were spiked with 10 mL of internal stan-
For both receptor binding assays, the new compounds were
tested as previously described [11]. Binding affinities of reference
compounds were evaluated in parallel with compounds 8e31. Ki
means concentration of the competing ligand that will bind to half
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).
m
m
m
m
treated on a Sirocco filter plate (Waters) likewise the plasma
samples. The filtered samples were analyzed by LC-MS/MS. Sample
analysis was performed on an Agilent HPLC (column: Synergy
fusion RP 20 ꢃ 2 mm 2.5
HCOOH gradient from 2%B to 100%B in 1.7 min flow 0.6 mL/min;
injected volume 25
L, T column 40 ꢀC) coupled with a CTC PAL
mm; eluent: water, acetonitrile with 0.1%
5.4. Pharmacological studies
m
sample organizer and interfaced to a triple quadrupole API2000
(Applied Biosystem, Ontario, Canada). The mass spectrometer was
operated using an APCI interface in positive mode (Gas1 60, Gas2
40, Source Temp. 450; NC 3). Three different Q1/Q3 transitions were
applied 418.9/317.7 (DP 32, CE 30); 418.9/118.7 (DP 28, CE 54);
418.9/253.5 (DP 23, CE 59); Internal Standard. 520.7/158.6. Area of
the samples was interpolated on 9 points calibration curves from
250 pg/mL (LLQ) to 100 ng/mL in brain and on a 10 points cali-
bration curve from 500 pg/mL (LLQ) to 250 ng/mL in plasma.
Pharmacokinetic parameters were calculated by a non-compart-
mental method using WinNoLin 5.1 software (Pharsight, Mountain
View, USA).
The experimental procedure used here was in accordance with
the Italian law on the ‘Protection of animals used for experimental
and other scientific reasons’.
5.4.1. Animals
Adult male Wistar rats (Charles River Laboratories, Calco, Italy),
weighting approximately 500 g at the time of the tests, 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 ꢁ 2 ꢀC and relative humidity of approximately 60%. Rats were
extensively habituated to handling and intraperitoneal injection.
Food pellets (standard rat chow; Mucedola, Settimo Milanese, Italy)
and water were available 24 h/day.
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