Synthesis, cannabinoid receptor affinity, molecular modeling studies and in vivo pharmacological evaluation of new substituted 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides. 2. Effect of the 3-carboxamide substituent on the affinity and selectivity

Article abstract of DOI:10.1016/j.bmc.2009.06.027

New substituted 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides were synthesized by replacing the 2,4-dichlorobenzyl and cyclohexyl moieties at the 3-carboxamide nitrogen of the previously reported CB₁ receptor antagonists/inverse agonists

Full text of DOI:10.1016/j.bmc.2009.06.027

Bioorganic & Medicinal Chemistry 17 (2009) 5549–5564
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, cannabinoid receptor affinity, molecular modeling
studies and in vivo pharmacological evaluation of new substituted
1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides. 2. Effect
of the 3-carboxamide substituent on the affinity and selectivity profile
b
a
a,
Romano Silvestri a, , Alessia Ligresti , Giuseppe La Regina , Francesco Piscitelli , Valerio Gatti a,
*
Antonella Brizzi c, Serena Pasquini c, Antonio Lavecchia d, , Marco Allarà , Noemi Fantini ,
b
e
*
Mauro Antonio Maria Carai e, Ettore Novellino d, Giancarlo Colombo e, Vincenzo Di Marzo b,
Federico Corelli c,
*
a Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, 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 Aldo Moro 2, I-53100 Siena, Italy
d Dipartimento di Chimica Farmaceutica e Tossicologica, Università di Napoli Federico II, Via Domenico Montesano 49, I-80131 Napoli, Italy
e Istituto di Neuroscienze, Consiglio Nazionale delle Ricerche, Viale Diaz 182, I-09126 Cagliari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
New substituted 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides were synthesized by replacing
the 2,4-dichlorobenzyl and cyclohexyl moieties at the 3-carboxamide nitrogen of the previously reported
CB1 receptor antagonists/inverse agonists 4 and 5. Several ligands showed potent affinity for the hCB1
receptor, with Ki concentrations comparable to the reference compounds 1, 4 and 5, and exhibited CB1
selectivity comparable to 1 and 2. Docking experiments and molecular dynamics (MD) simulations
explained the potent hCB1 binding affinity of compounds 31 and 37. According to our previous studies,
31 and 37 formed a H-bond with K3.28(192), which accounted for the high affinity for the receptor inac-
tive state and the inverse agonist activity. The finding of inhibition of food intake following their acute
administration to rats, supported the concept that the CB1 selective compounds 4 and 52 act as antago-
nists/inverse agonists.
Received 8 April 2009
Revised 9 June 2009
Accepted 14 June 2009
Available online 21 June 2009
Keywords:
Cannabinoid
Human recombinant CB receptor type 1
Pyrrole bioisoteres
Structure–activity relationships
Molecular modeling
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
adverse effects associated with 1 were gastrointestinal; however,
depression and anxiety also became apparent at the daily dose of
Cannabinoid CB1 and CB2 receptors have become intriguing tar-
gets for the development of new drugs, because of their involve-
ment in an ever growing number of pathological conditions, such
as pain, immunosuppression, peripheral vascular disease, appetite
enhancement or suppression, and locomotor disorders.1 Rimona-
bant (SR141716, 1) is a potent and selective CB1 receptor inverse
agonist which was launched in 2006 by Sanofi-Aventis for the
treatment of overweight and obesity, and some associated cardio-
vascular and metabolic disorders.2 In clinical trials, the major
20 mg.3 In 2008, Sanofi-aventis suspended the marketing of 1 by
acknowledging EMEA’s recommendation.4
New CB ligands have been actively searched either by synthesis
of pyrazole analogues of 1 (such as the CB1 selective AM251 (2) and
CB2 selective SR144528 (3) ligands) (Chart 1), or replacement of
the central pyrazole nucleus with a bioisosteric heterocyclic ring.5a
More recently, new CB ligands have been developed by following a
non-bioisosteric approach.5b Our studies on pyrrole bioisosteric
analogues6 prompted the design of new CB ligands by replacing
the 4-chlorophenyl group at position 5 of 1 with a pyrrole nucleus.7
Compound 4, bearing a 2,4-dichlorophenyl group at position 1 and
a 2,5-dimethylpyrrole moiety at position 5 of the pyrazole ring,
was highly selective for the human recombinant CB1 receptor
and showed a CB1 selectivity index (SI) calculated as Ki(CB2)/
Ki(CB1) ratio of 140. On the other hand, compound 5 (Ki = 5.6 nM)
was equipotent to 2 as a hCB1 ligand. Both compounds behaved
as inverse agonists in the cAMP assay.7
* Corresponding authors. Tel.: +39 06 4991 3800; fax: +39 06 491 491 (R.S.); tel./
fax: +39 081 678 613 (A.L.); tel.: +39 0577 234308; fax: +39 0577 234299 (F.C.).
E-mail addresses: romano.silvestri@uniroma1.it (R. Silvestri), lavecchi@unina.it
(A. Lavecchia), corelli@unisi.it (F. Corelli).
Present address: University of Pennsylvania, Department of Chemistry, 231 South
34th Street, Philadelphia, PA 19104-6323, USA.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.027

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R. Silvestri et al. / Bioorg. Med. Chem. 17 (2009) 5549–5564
O
N
NH
O
N
NH
O
NH
N
N
N
N
N
N
Cl
Cl
Cl
I
Cl
Cl
Cl
SR141716 (1)
SR144528 (3)
AM251 (2)
Rimonabant
Cl
O
N
R2
NH
O
Cl
O
NH
NH
R3
N
N
N
N
N
N
N
N
Cl
R1
Cl
6-57
Cl
Cl
( for R1-R3, see Table 1)
4
5
Chart 1. Reference and new CB ligands.
The substituent groups at the 3-carboxamide were not exhaus-
tively explored. We modified here the 2,4-dichlorobenzyl at the 3-
carboxamide nitrogen of 4, by changing both number and position
of the substituents at the phenyl ring, and length of the alkyl
spacer. The cyclohexyl group of 5 was replaced by cyclohomo-
logues and some correlated cycloalkyl substituents (compounds
6–57, Table 1). The methyl groups at the positions 2 and 5 of the
pyrrole ring were either maintained or removed, while the 2,4-
and 3,4-dihalophenyl rings at the position 1 of the pyrazole were
not modified.
Docking experiments and molecular dynamics (MD) simula-
tions were carried out using a previously developed model of the
hCB1 receptor in its inactive form.7 The most active compounds
were docked into this receptor model and the observed ligand–
receptor interactions were discussed, also taking into account the
site-directed mutagenesis data available for this receptor subtype.
branes from HEK cells transfected with either the hCB1 or hCB2
receptor and [3H]-(ꢀ)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-
phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol ([3H]-CP-55,940).
As described by the manufacturer (Perkin–Elmer, Italy), the high
affinity ligand Kd values were 0.18 nM for CB1 receptor, and
0.31 nM for CB2 receptor.8 Incubating the drugs with [3H]CP-
55,940 at 0.14 nM for CB1 and 0.084 nM for CB2 binding assay, al-
lowed to derive the displacement curves. Ki values were calculated
by applying the Cheng–Prusoff equation9 to the IC50 values (from
GraphPad) for the displacement of the bound radioligand at
increasing concentrations of tested compound.
Administration of compound 1, as well as other cannabinoid
CB1 receptor antagonists/inverse agonists, dose-dependently sup-
presses food intake and reduces body weight in lean and obese rats
and mice.10,11 Here, we evaluated the effect of the acute adminis-
tration of the highly CB1 selective compounds 4 and 52 on food in-
take in rats given unlimited access to regular food pellets and
water.
2. Results
2.3. Discussion
2.1. Chemistry
The new 5-(1H-pyrrol-1-yl)pyrazole-3-carboxamides elicited
remarkable affinity and/or selectivity characteristics. Several car-
boxamides (18, 23, 27, 29, 30, 35, 36, 50, 52–55, and 57) were high
affinity ligands for the hCB1 receptor, and showed Ki values in the
same range of concentrations of the reference compounds 1 and 4.
Carboxamides 28, 31, and 37 showed the highest affinities for the
hCB1 receptor with Ki values at low nanomolar concentration, and
were equipotent to 2 and 5. CB1 SIs (calculated as Ki(CB2)/Ki(CB1)
ratios) of derivatives 9, 15, 16, 18, 43, 50, 52, and 53 were superior
the reference compounds 1 and 2. Compounds 28, 30, 31, 36, and
38 showed hCB2 receptor binding affinities comparable to the
CB2 reference compound 3 and the previously reported pyrazole
5.7 Moreover, 38 and 30 possess also significant CB2 selectivity,
38 being the most CB2-selective compound identified in this study.
The affinity for the hCB1 receptor was affected by both nature
and position of the substituents on the phenyl ring at position 1
of the pyrazole nucleus. Generally speaking, the chloro derivatives
Carboxamides 6–57 were obtained through parallel synthesis in
62–98% yield by coupling reaction of acids 58–63, in turn prepared
as previously described,7 with selected amines in the presence of
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride
(EDC) and 1-hydroxybenzotriazole (HOBt), using morpholinometh-
yl-polystyrene and polymer bound p-toluensulfonic acid as scav-
engers for acidic and basic substances, respectively (Scheme 1
and Table 1).
2.2. Biology
The binding affinities (Ki values) of compounds 6–57 for human
recombinant CB1 (hCB1) and CB2 (hCB2) receptors, evaluated in
parallel with CB1 (1 and 2) and CB2 (3) reference compounds, are
shown in Table 1. The previously described pyrazoles 4 and 57
are included for comparison purpose. The CB1 and CB2 receptor
binding of tested compounds was evaluated by means of mem-

R. Silvestri et al. / Bioorg. Med. Chem. 17 (2009) 5549–5564
5551
Table 1
Structure, hCB1 and hCB2 receptor affinity (Ki values, nM) of new derivatives 6–57, reference compounds 1–3, and previously described derivatives 4, 5a
O
R
2
NH
N
R
3
N
N
R
1
4-57
Compound
R1
R2
R3
Kib,c (nM)
hCB1d,f
120
hCB2e,f
SIg
6
7
2,4-Cl2
2,4-Cl2
H
H
80
0.67
6.71
Cl
140
227
940
Cl
OMe
OMe
Br
8
2,4-Cl2
H
609
2.68
OMe
9
2,4-Cl2
2,4-Cl2
H
H
70
>3940
>3940
>56.2
Cl
Cl
Cl
10
157
>25.09
Cl
11
12
2,4-Cl2
2,4-Cl2
H
H
368
218
2887
7.84
OMe
Cl
Cl
>3940
>18.07
13
14
15
2,4-Cl2
2,4-Cl2
2,4-Cl2
H
H
84
20
96
170
2.02
20
1.00
2,5-Me2
2,5-Me2
>3940
>41.04
Cl
16
17
2,4-Cl2
2,4-Cl2
90
>3940
553
>43.78
3.84
Cl
Br
OMe
2,5-Me2
144
OMe
OMe
18
19
20
2,4-Cl2
2,4-Cl2
2,4-Cl2
2,5-Me2
2,5-Me2
2,5-Me2
30
>3940
>3940
3483
>131.33
33.36
Cl
Cl
Cl
117
112
Cl
31.09
OMe
(continued on next page)

5552
R. Silvestri et al. / Bioorg. Med. Chem. 17 (2009) 5549–5564
Table 1 (continued)
Compound
R1
R2
R3
Kib,c (nM)
hCB1d,f
156
hCB2e,f
SIg
Cl
21
2,4-Cl2
2,5-Me2
>3940
>25.25
Cl
22
23
2,4-Cl2
2,4-Cl2
2,5-Me2
2,5-Me2
150
56
310
80
2.07
1.43
24
25
26
3,4-Cl2
3,4-Cl2
3,4-Cl2
H
H
H
1300
70
790
0.61
1.43
5.0
Cl
100
Cl
240
1200
Cl
27
28
3,4-Cl2
3,4-Cl2
H
H
50
12
0.24
0.23
5.6
1.3
29
30
3,4-Cl2
3,4-Cl2
H
H
31
14
20
0.65
0.11
Me
1.6
(R)
Me
(S)
31
3,4-Cl2
H
3.4
2.4
0.71
32
33
34
3,4-Cl2
3,4-Cl2
3,4-Cl2
2,5-Me2
2,5-Me2
2,5-Me2
>2820
100
>3940
790
—
Cl
7.90
>10.10
Cl
390
>3940
Cl
35
36
3,4-Cl2
3,4-Cl2
2,5-Me2
2,5-Me2
36
12
12
0.33
0.35
4.2
37
38
3,4-Cl2
3,4-Cl2
2,5-Me2
2,5-Me2
5.6
80
12
2.14
Me
5.7
0.071

R. Silvestri et al. / Bioorg. Med. Chem. 17 (2009) 5549–5564
5553
Table 1 (continued)
Compound
R1
R2
R3
Kib,c (nM)
hCB1d,f
>2820
hCB2e,f
SIg
39
3,4-Cl2
2,5-Me2
>3940
—
Cl
40
41
2,4-F2
2,4-F2
H
H
2100
340
>3940
3400
3.76
10.0
Cl
Cl
OMe
OMe
Br
42
2,4-F2
H
345
739
2.14
OMe
43
44
2,4-F2
2,4-F2
H
H
80
>3940
1291
>49.25
4.16
Cl
Cl
Cl
Cl
310
45
2,4-F2
H
948
1945
2.05
OMe
Cl
Cl
46
47
48
2,4-F2
2,4-F2
2,4-F2
H
H
H
380
667
1.75
0.46
0.36
2400
560
1100
200
49
50
2,4-F2
2,4-F2
2,5-Me2
2,5-Me2
560
56
>3940
>3940
>7.04
Cl
>70.36
Cl
Br
OMe
51
2,4-F2
2,5-Me2
165
373
2.26
OMe
OMe
52
53
2,4-F2
2,4-F2
2,5-Me2
2,5-Me2
50
54
>3940
>3940
>78.80
>72.96
Cl
Cl
Cl
Cl
54
55
2,4-F2
2,4-F2
2,5-Me2
2,5-Me2
99
41
>3940
721
>39.79
17.58
OMe
Cl
Cl
(continued on next page)

5554
R. Silvestri et al. / Bioorg. Med. Chem. 17 (2009) 5549–5564
Table 1 (continued)
Compound
R1
R2
R3
Kib,c (nM)
hCB1d,f
840
hCB2e,f
SIg
56
57
2,4-F2
2,5-Me2
900
110
1.07
2,4-F2
2,5-Me2
56
1.96
1h
2i
3j
—
—
—
—
—
—
—
—
—
12
2.3
>2820
790
112
5.4
65.83
48.70
<0.002
Cl
4k,l
2,4-Cl2
3,4-Cl2
2,5-Me2
28
3940
140.7
Cl
5k,l
H
5.6
1.7
0.30
a
Data represent mean values for at least three separate experiments performed in duplicate and are expressed as Ki (nM). Standard error of means (SEM) are not shown for
the sake of clarity and were never higher than 5% of the means.
b
Ki: equilibrium dissociation constant, that is the concentration of the competing ligand that will bind to half the binding sites at equilibrium, in the absence of radioligand
or other competitors.
c
IC50: The concentration of competitor that competes for half of the specific binding. This is the measure of the competitor’s potency at interacting with the receptor
against the radioligand.
d
hCB1: human CB1 receptor.
hCB2: human CB2 receptor.
e
f
For both receptor binding assays, the new compounds were tested using membranes from HEK cells transfected with either the hCB1 or hCB2 receptor and [3H]-(ꢀ)-cis-3-
[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-trans-4-(3-hydroxy-propyl)-cyclohexanol ([3H]CP-55,940).
g
SI: CB1 selectivity index was calculated as Ki(CB2)/Ki(CB1) ratio.
Compound 1: Rimonabant, CB1 reference compound.
Compound 2: AM251, CB1 reference compound.
Compound 3: SR144528, CB2 reference compound.
h
i
j
k
The binding affinities of reference compounds 1–3 were evaluated in parallel with compounds 6–57 under the same conditions.
l
Lit.7
were more potent than their fluorinated counterparts (for an
example, compare 14 and 28 with 48, and 6 with 40). The 2,4-
dichlorophenyl substituent allowed the ligands to be more selec-
tive for the CB1 receptor than the corresponding 3,4-dichloro-
phenyl analogs (compare 9 with 26, 15 with 32, 16 with 33, and
18 with 34) (Chart 2).
On the other hand, hCB1 receptor affinity was greatly affected
by the substitution pattern of the pyrrole nucleus at position 5.
Derivatives bearing both the 5-(2,5-dimethylpyrrole) and 1-(2,4-
dihalophenyl) groups showed generally greater affinity for the
hCB1 receptor than the corresponding compounds deprived of
the methyl groups. This trend tends to reverse in the 3,4-dichloro-
phenyl series. Most importantly, the 2,5-dimethylpyrroles were
more selective for the hCB1 receptor in both the 1-(2,4-dichloro-
phenyl) and 1-(2,4-difluorophenyl) series (compare 6 with 15, 7
with 16, and 46 with 53).
Affinity and selectivity for the hCB1 receptor were also affected
by the substituent at the 3-carboxamide group. A 4-chloro- or 3,4-
dichlorophenyl ring linked at the nitrogen of the carboxamide pro-
vided ligands endowed with high selectivity (although not with the
strongest affinity) for the hCB1 receptor (compounds 4, 9, 15, 16,
18, 43, 50, 52, and 53). Such an effect was mostly due to the lack
of affinity of these derivatives for the hCB2 receptor. The presence
of methoxy groups resulted in a decrease of both affinity and selec-
tivity (compare 4 with 17, 18 with 20, and 53 with 54).
The spacer group between the phenyl ring and the nitrogen of
the carboxamide also played a crucial role. With the only exception
of 15, derivatives lacking this spacer showed weak affinity (24, 32,
O
O
R
NH
2
OH
a
N
N
R
R
3
3
N
N
N
N
R
R
1
1
6-57
58-63
58: R1 = 2,4-Cl2; R3 = H; 59: R1 = 2,4-Cl2; R3 = 2,5-Me2; 60: R1 = 3,4-Cl2; R3 = H;
61: R1 = 3,4-Cl2; R3 = 2,5-Me2; 62: R1 = 2,4-F2; R3 = H; 63: R1 = 2,4-F2; R3 = 2,5-Me2.
6-57: for R1-R3 substituents, see Table 1.
Scheme 1. Synthesis of derivatives 6-57. Reagents and reaction conditions. (a) (i) R2NH2, HOBt, EDC, dichloromethane, 0 °C to rt, overnight; (ii) morpholinomethyl-
polystyrene and polymer bound p-toluensulfonic acid, rt, 24 h.

R. Silvestri et al. / Bioorg. Med. Chem. 17 (2009) 5549–5564
5555
O
R
NH
2 C
spacer
group
R
N
R
3
N
N
N
HN (CH )
Cl
N
B
2
n
B
>>
C
optimal
R
A
1
R = H, n = 1
R = Cl, n = 1,2
very good
combination
Cl
F
=
>
A
Cl
Cl
Cl
F
Chart 2. SAR summary for high hCB1 receptor affinity and selectivity.
40, 49) or low selectivity (6) for the hCB1 receptor, while the intro-
duction of a methylene spacer resulted in an increase of hCB1
receptor selectivity. Further extension of the spacer substantially
retained the selectivity for the hCB1 receptor (9, 18, 43, 52 and 53).
Because of the potent affinity displayed by 57 for both CB1 and
CB2 receptors, we synthesized new derivatives bearing a cyclopen-
tyl, cyclohexyl or cycloheptyl group at the 3-carboxamide. Among
them, derivatives 28 and 36, exhibiting a 1-(3,4-dichlorophenyl)
ring, showed strong affinity for both receptor subtypes.
As above-mentioned, the high selectivity of derivatives 4, 9, 15,
16, 18, 43, 50, 52 and 53 for the hCB1 receptor was basically due to
the lack of affinity for the CB2 subtype. The presence of a (CH2)nAr
(where n = 0–2) system at the 3-carboxamide nitrogen proved to
be detrimental for the hCB2 receptor affinity. In an attempt to im-
prove the CB2 selectivity, new compounds bearing 1-adamantyl, 1-
cyclohexylethyl or 1-phenylethyl groups as substituents at the car-
boxamide nitrogen were synthesized. Although derivatives 30, 31,
37 and 38 still exhibited high affinity for both receptors, 38 is pres-
ently the second most CB2-selective ligand in our hands. Moreover,
a certain degree of enantioselectivity was also highlighted, with
the (R)-enantiomer 30 showing approximately sixfold higher
selectivity for the hCB2 receptor than the corresponding (S)-isomer
31. This result suggests that a substituent at C1 of the spacer group
might play a positive role for either affinity or selectivity for both
receptor subtypes.
To assess the stability of the complexes, we performed 1 ns MD
simulations at a constant temperature of 300 K. During the MD
simulations, the rmsd of the protein compared to the starting
hCB1 model structure was quite stable (<1 Å rmsd, data not
shown). The rmsd of the ligands remained low (<2 Å, data not
shown) and did not vary significantly between the two com-
pounds. The average rmsd variation of the heavy atoms of selected
amino acid residues within a 15 Å radius from the drug molecule
was 0.98 0.1 Å. Such a result indicates that both orientation and
conformation of the amino acids involved in ligand binding were
also well preserved.
Figures 1 and 2 clearly show that the carboxamide oxygens of
31 and 37 were able to form H-bond with K3.28(192) (this partic-
ular interaction was present for the whole simulation time). Such
H-bond interaction accounted for the high affinity of 1 for the inac-
tive receptor state and its inverse agonism activity.15 According to
our previous modeling studies,7 a H-bond between the carbonyl
oxygen of 1 and the K3.28(192) residue stabilizes a salt bridge be-
tween K3.28(192) and D6.58(366). Formation of this H-bond in-
creases the affinity and shifts the receptor equilibrium toward
the inactive state.16,19
The two docking models displayed: (i) the 3,4-dichlorophenyl
ring at position 1 of the pyrazole system resided in a hydrophobic
pocket formed by L3.29(193), F3.36(200), I4.56(247), W5.43(279),
M6.55(363), V6.59(367), and established favorable
p–p stacking
interactions with the indole ring of W5.43(279) and the phenyl
2.4. Molecular modeling
ring of F3.36(200). Such interactions, which involve the aromatic
residue-rich TM3-4-5-6 region of hCB1 (colored magenta in Figs.
14
The binding mode of 1 into hCB1 receptor was extensively
investigated through SAR, mutagenesis, and molecular modeling
studies.7,12–17 Key features were: (i) an aromatic cluster formed
by F3.25(190), F3.36(200), W4.64(255), W5.43(279), W6.48(356),
and Y5.39(275) that is in contact with the two phenyl rings of 1
through aromatic stacking interactions;14 (ii) K3.28(192) that
forms a H-bond with the amide carbonyl oxygen of 1. It was pro-
posed, this interaction may stabilize the putative inactive state of
hCB1 receptor.13,15 S7.39(383), which resides across from
K3.28(192) in the active site, appears to have no direct interaction
with 1.12 To explain the potent hCB1 binding affinity of compounds
31 and 37, we built a docking model of both hCB1/31 and hCB1/37
complexes using our previously published homology model of the
inactive state of hCB1.7 Initial structures of the hCB1 complexes
1 and 2), were very stable during the MD trajectory (the electron-
withdrawing chlorine substituents contribute to strength the
p-
stacking interactions); (ii) the 5-pyrrole ring of 31 resided in a
pocket formed by F3.36(200), W6.48(356) and L6.51(359). In con-
trast, the 2,5-dimethylpyrrole ring of 37 formed contacts with the
hydrophobic residue V3.32(196), and projected the two methyl
groups close to the lipophilic residues L7.43(387) and L3.29(193);
(iii) the 4-methyl group of 31 formed hydrophobic interactions
with I6.54(362) and L6.51(359) residues, while that of 37 inter-
acted with L7.43(387); (iv) Ser7.39(383), although located near
the amide bond, did not form any H-bond with both ligands; (v)
the N-substituents of the carboxamide were embedded within a
lipophilic pocket made up by the side chains of Q1.31(116),
I1.34(119), F2.57(170), F2.61(174), F2.64(177), F7.35(379),
A7.36(380), C7.42(386) and L7.43(387). Worthy to note, this lipo-
philic pocket can accommodate aromatic groups bearing elec-
tron-withdrawing substituents (as reported in our previous
paper,7 the 3,4-dichlorobenzyl ring of 4 maintained direct aromatic
stacking interactions with both F2.61(174) and F2.64(177).
were generated by means of the automatic docking program GOLD
,
version 4.0,18 defining the bound 1 in the hCB1 bundle7 as a refer-
ence. In this pose, the carboxamide oxygens of both ligands were
H-bonded to K3.28(192), while the 3,4-dichlorophenyl rings at po-
sition 1 of the pyrazole were close to F3.36(200), Y5.39(275), and
W5.43(279) residues, in well agreement with previous combined
mutation/modeling studies.14,15
Accordingly, the electron-withdrawing chlorine atoms decrease
the electron density at the aromatic ring, thereby improving the

5556
R. Silvestri et al. / Bioorg. Med. Chem. 17 (2009) 5549–5564
Figure 1. Complex of the hCB1 receptor with compound 31. View from the plane of the cell membrane of the 31/hCB1 complex (top view). View from outside the cell of the
31/hCB1complex (bottom view). Only amino acids located within 4 Å distance from the ligand (green–blue) are shown in yellow and labeled. Residues that form part of the
aromatic cluster complex with ligand are colored in magenta. H-bonds are indicated by dashed red lines.
affinity of the ligands for the hCB1 receptor; on the contrary, the
electron-donating methoxy groups increase the electron density
at the aromatic ring, thereby decreasing the affinity of the ligands
for the hCB1 receptor. Such findings are consistent with the activity
trend showed by our series of compounds (compare 4 with 17, 18
with 20, and 53 with 54); (vi) consistently with a nice accommoda-
provided new insight for the design of potent and selective hCB1
ligands.
2.5. In vivo pharmacological studies
ANOVA revealed a significant effect of acute treatment with
compound 4 on cumulative food intake over the first 360 min after
lights off [F(3,28) = 15.89, P <0.0001]. Post hoc analysis revealed
that 1 and 10 mg/kg of compound 4 significantly reduced cumula-
tive food intake, with respect to vehicle, at each time interval (
Fig. 3, top panel). Percent reduction in cumulative food intake in
tion of the (S)-a-methyl group into a small cavity delimited by the
aromatic residues F2.64(177) and F7.35(379), carboxamide 31
showed (moderately) greater hCB1 binding affinity than the corre-
sponding (R)-30 (31, Ki = 3.4 nM; 30, Ki = 14 nM). The binding mod-
els of 31 and 37 generated by docking analysis and MD simulations

R. Silvestri et al. / Bioorg. Med. Chem. 17 (2009) 5549–5564
5557
Figure 2. Complex of the hCB1 receptor with compound 37. View from the plane of the cell membrane of the 37/hCB1 complex (top view). View from outside the cell of the
37/hCB1complex (bottom view). Only amino acids located within 4 Å distance from the ligand (green–blue) are shown in yellow and labeled. Residues that form part of the
aromatic cluster complex with ligand are colored in magenta. H-bonds are indicated by dashed red lines.
the rat groups treated with 1 and 10 mg/kg of compound 4 ranged
between 15 and 30 and between 25 and 40, respectively. At the
1440-min time interval, a tendency toward a reduction in food in-
take in the rat groups treated with 1 and 10 mg/kg of compound 4
was still observed [F(3,28) = 2.74, P = 0.061] (Fig. 3, top panel). The
reducing effect of compound 4 was specific for food intake, as
treatment with this compound did not affect cumulative water in-
take over the first 360 min after lights off [F(3,28) = 0.95, P >0.05]
and water intake at the 1440-min time interval [F(3,28) = 1.57, P
>0.05] (Fig. 3, bottom panel).
after lights off [F(3,23) = 3.20, P <0.05]. Post hoc analysis revealed
that 1 mg/kg of compound 52 significantly reduced cumulative
food intake, with respect to vehicle, only at the 360-min time inter-
val (Fig. 4, top panel).
Conversely, 10 mg/kg of compound 52 significantly reduced
cumulative food intake, with respect to vehicle, at each time inter-
val (Fig. 4, top panel). Percent reduction in cumulative food intake
in the rat group treated with 10 mg/kg of compound 52 ranged be-
tween 25 and 45. At the 1440-min time interval, a tendency to-
ward a reduction in food intake in the rat group treated with
10 mg/kg of compound 52 was still observed [F(3,23) = 2.46,
P = 0.088] (Figure ure4, top panel).
ANOVA revealed a significant effect of acute treatment with
compound 52 on cumulative food intake over the first 360 min

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R. Silvestri et al. / Bioorg. Med. Chem. 17 (2009) 5549–5564
Figure 3. Effect of the acute administration of different doses of compound 4 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 S.E.M. of n = 8 rats. *:P <0.05 with respect to vehicle-treated rats at the same time interval (Newman–Keuls test).
The inhibitory effect of compound 52 was specific for food in-
take, as treatment with this compound did not affect cumulative
water intake over the first 360 min after lights off [F(3,23) = 1.90,
with K3.28(192), as for 1.7 Acute administration of compounds 4
and 52 in rats led to appetite suppression and anorectic effect. Such
findings in an in vivo pharmacological set up confirm that 4 and 52
act as antagonists/inverse agonists at CB1 receptors. Although we
have not fully reached the goal to obtain both potent and selective
CB1 ligands, these findings represent a useful basis for the design of
new analogues.
P
>0.05] and water intake at the 1440-min time interval
[F(3,23) = 1.60, P >0.05] (Fig. 4, bottom panel).
These results indicate that doses of compounds 4 and 52 in-
cluded between 1 and 10 mg/kg may reduce food intake in non-ob-
ese rats. Compound 4 appeared to be more potent than compound
52, as 1 mg/kg of compound 4 was able to significantly reduce
cumulative food intake at each time interval between 60 and
360 min; vice versa, 1 mg/kg of compound 52 significantly reduced
cumulative food intake only at the 360-min time interval. Con-
versely, both drugs displayed comparable efficacy, with their high-
est dose (10 mg/kg) exerting a similar effect (40–45% reduction in
comparison to vehicle-treated rats at the 60-min time interval).
Compounds 4 and 52 had also a comparable duration of effect: at
the 1440-min time interval, both drugs produced only a tendency
towards a reduction of food intake.
4. Experimental
4.1. Chemistry
4.1.1. Materials and methods
Melting points (mp) were determined on a Büchi 510 apparatus
and are uncorrected. Infrared spectra (IR) were run on Perkin–El-
mer 1310 and SpectrumOne spectrophotometers. Band position
and absorption ranges are given in cmꢀ1. Proton nuclear magnetic
resonance (1H NMR) spectra were recorded on Bruker AM-200
(200 MHz) and Bruker Avance 400 MHz FT spectrometers in the
indicated solvent. Chemical shifts are expressed in d units (ppm)
from tetramethylsilane. Column chromatographies were packed
with alumina (Merck, 70–230 mesh) and silica gel (Merck, 70–
230 mesh). Aluminum oxide TLC cards (Fluka, aluminum oxide
precoated aluminum cards with fluorescent indicator at 254 nm)
and silica gel TLC cards (Fluka, silica gel precoated aluminum cards
with fluorescent indicator at 254 nm) were used for thinlayer chro-
matography (TLC). Developed plates were visualized with a Spectr-
oline ENF 260C/F UV apparatus. Organic solutions were dried over
anhydrous sodium sulfate. Evaporation of the solvents was carried
out on a Büchi Rotavapor R-210 equipped with a Büchi V-850 vac-
uum controller and Büchi V-700 (ꢁ5 mbar) and V-710 vacuum
(ꢁ2 mbar) pumps. Elemental analyses were found within 0.4%
3. Conclusions
Several ligands showed potent affinity for the hCB1 receptor,
with Ki concentrations comparable to the reference compounds
1, 4 and 5, and exhibited CB1 selectivity comparable to 1 and 2.
The chloro derivatives were more potent ligands than their fluori-
nated counterparts. Both 5-(2,5-dimethylpyrrole) and 1-(2,4-dihal-
ophenyl) groups led to great affinity for the hCB1 receptor. The 1-
(2,4-dichlorophenyl)pyrazoles were more CB1 selective than the
corresponding 3,4-dichlorophenyl analogs. Introduction of a meth-
ylene spacer resulted in an increase of hCB1 receptor selectivity.
Docking experiments and molecular dynamics (MD) simulations
showed that carboxamide oxygens of 31 and 37 formed H-bond

R. Silvestri et al. / Bioorg. Med. Chem. 17 (2009) 5549–5564
5559
Figure 4. Effect of the acute administration of different doses of compound 52 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 S.E.M. of n = 6–7 rats. *: P <0.05 with respect to vehicle-treated rats at the same time interval (Newman–Keuls test).
of the theoretical values. Purity of tested compounds was >95%.
Büchi Syncore reactor was used for parallel synthesis, filtration,
and evaporation.
(m, 6H), 7.39–7.44 (m, 1H). IR:
482 [M+23]+ (100).
m
1673 cmꢀ1. MS m/z: 460 [M+1]+,
4.1.2.3. N-(2-Bromo-3,4,5-trimethoxybenzyl) 1-(2,4-dichloro-
phenyl)-4-methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxam-
ide (8). Yield 95% as a pale yellow oil. 1H NMR (CDCl3): d 2.29 (s,
3H), 3.83 (s, 3H), 3.85 (s, 3H), 3.87 (s, 3H), 4.64 (d, 2H, J = 6.4 Hz),
6.18–6.19 (m, 2H), 6.54–6.56 (m, 2H), 6.87 (s, 1H), 7.19–7.28 (m,
4.1.2. Parallel synthesis of amides 6–57
To a solution of an appropriate acid 58–63 (1 mmol) in dichlo-
romethane, kept at 0 °C, N-(3-dimethylaminopropyl)-N’-ethylcar-
bodiimide (EDC, 1.2 mmol) and 1-hydroxybenzotriazole (HOBt,
1.0 mmol) were added followed by the appropriate amine
(1.5 mmol). The solutions were allowed to warm up to room tem-
perature and placed in the Büchi Syncore reactor. The reactions
were stirred at 300 rpm overnight and then morpholinomethyl-
polystyrene (3 equiv/mol) was added. After 1 h at room tempera-
ture, polymer bound p-toluensulfonic acid (3 equiv/mol) was
added and the mixtures were stirred at room temperature for addi-
tional 24 h. The mixtures were filtered and the resulting solutions
were evaporated to dryness under reduced pressure. Purification of
the crude products by crystallization from dichloromethane (un-
less otherwise stated) gave carboxamides 6–57.
2H), 7.38 (br t, 1H, J = 6.0 Hz), 7.44 (d, 1H, J = 2.3 Hz). IR:
m
1672 cmꢀ1. MS m/z: 595 [M+1]+ (100), 617 [M+23]+.
4.1.2.4. N-[2-(4-Chlorophenyl)ethyl] 1-(2,4-dichlorophenyl)-4-
methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (9). Yield
98% as a colorless oil. 1H NMR (CDCl3): d 2.29 (s, 3H), 2.86 (t, 2H,
J = 7.1 Hz), 3.56–3.67 (m, 2H), 6.17–6.19 (m, 2H), 6.55–6.56 (m,
2H), 7.03 (br t, 1H, J = 5.7 Hz), 7.12–7.25 (m, 6H), 7.43–7.44 (m,
1H). IR:
m
1671 cmꢀ1. MS m/z: 474 [M+1]+ (100), 496 [M+23]+,
969 [2M+23]+.
4.1.2.5. N-[2-(3,4-Dichlorophenyl)ethyl] 1-(2,4-dichlorophenyl)-4-
methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (10). Yield
95% as a colorless oil. 1H NMR (CDCl3): d 2.28 (s, 3H), 2.86 (t, 2H,
J = 7.0 Hz), 3.57–3.67 (m, 2H), 6.17–6.19 (m, 2H), 6.55–6.56 (m,
2H), 7.01 (br t, 1H, J = 5.7 Hz), 7.05 (dd, 1H, J = 1.9 Hz, J = 8.5 Hz),
4.1.2.1. N-(4-Chlorophenyl) 1-(2,4-dichlorophenyl)-4-methyl-5-
(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (6). Yield 83% as a
white solid, mp 180 °C. 1H NMR (CDCl3): d 2.33 (s, 3H), 6.19–6.21
(m, 2H), 6.58–6.59 (m, 2H), 7.19–7.24 (m, 1H), 7.28 (d, 2H,
J = 8.3 Hz), 7.47 (d, 1H, J = 1.6 Hz), 7.61 (d, 2H, J = 8.8 Hz), 8.75 (s,
7.14–7.34 (m, 4H), 7.44 (d, 1H, J = 1.9 Hz). IR:
m
1672 cmꢀ1. MS
1H). IR:
m
1689 cmꢀ1. MS m/z: 467 [M+23]+ (100).
m/z: 509 [M+1]+, 531 [M+23]+ (100).
4.1.2.2. N-(4-Chlorobenzyl) 1-(2,4-dichlorophenyl)-4-methyl-5-
(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (7). Yield 90% as
white solid, mp 135 °C. 1H NMR (CDCl3): d 2.30 (s, 3H), 4.55 (d,
2H, J = 6.8 Hz), 6.18–6.19 (m, 2H), 6.56–6.57 (m, 2H), 7.11–7.33
4.1.2.6. N-[2-(3-Chloro-4-methoxyphenyl)ethyl] 1-(2,4-dichloro-
phenyl)-4-methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxam-
ide (11). Yield 75% as a white solid, mp 140 °C. 1H NMR (CDCl3): d
2.28 (s, 3H), 2.82 (t, 2H, J = 7.1 Hz), 3.55–3.66 (m, 2H), 3.85 (s, 3H),

5560
R. Silvestri et al. / Bioorg. Med. Chem. 17 (2009) 5549–5564
6.18–6.19 (m, 2H), 6.54–6.55 (m, 2H), 6.84 (d, 2H, J = 8.5 Hz), 7.09–
1H, J = 8.3 Hz), 7.49 (d, 1H, J = 2.0 Hz). IR: m
1671 cmꢀ1. MS m/z:
7.28 (m, 3H), 7.44 (d, 1H, J = 1.8 Hz). IR:
m
1670 cmꢀ1. MS m/z: 504
537 [M+1]+, 559 [M+23]+ (100).
[M+1]+, 526 [M+23]+ (100).
4.1.2.15. N-[2-(3-Chloro-4-methoxyphenyl)ethyl] 1-(2,4-dichlo-
rophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methyl-1H-pyra-
zole-3-carboxamide (20). Yield 90% as a yellow oil. 1H NMR
(CDCl3): d 1.93 (s, 6H), 2.17 (s, 3H), 2.84 (t, 2H, J = 7.1 Hz), 3.57–
3.68 (m, 2H), 3.86 (s, 3H), 5.79 (s, 2H), 6.85 (d, 2H, J = 8.3 Hz),
7.04 (br t, 1H, J = 5.2 Hz), 7.07–7.19 (m, 3H), 7.49 (d, 1H,
4.1.2.7. N-[2-(2,4-Dichlorophenyl)ethyl] 1-(2,4-dichlorophenyl)-4-
methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (12). Yield
90% as a white oil. 1H NMR (CDCl3): d 2.29 (s, 3H), 3.02 (t, 2H,
J = 7.1 Hz), 3.60–3.70 (m, 2H), 6.18–6.19 (m, 2H), 6.55–6.56 (m, 2H),
7.14–7.27 (m, 4H), 7.36 (s, 1H), 7.45 (d, 2H, J = 2.0 Hz). IR:
m
1666 cmꢀ1. MS m/z: 509 [M+1]+, 531 [M+23]+ (100).
J = 2.0 Hz). IR:
m
1666 cmꢀ1. MS m/z: 532 [M+1]+, 554 [M+23]+
(100).
4.1.2.8. N-Cyclopentyl 1-(2,4-dichlorophenyl)-4-methyl-5-(1H-
pyrrol-1-yl)-1H-pyrazole-3-carboxamide (13). Yield 98% as
a
4.1.2.16. N-[2-(2,4-Dichlorophenyl)ethyl] 1-(2,4-dichlorophenyl)-
5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carbox-
amide (21). Yield 94% as a white solid, mp 135 °C. 1H NMR (CDCl3):
d 1.93 (s, 6H), 2.17 (s, 3H), 3.03 (t, 2H, J = 7.3 Hz), 3.61–3.72 (m,
2H), 5.79 (s, 2H), 7.06 (br t, 1H, J = 5.7 Hz), 7.14–7.37 (m, 3H),
white solid, mp 63 °C (from diethyl ether). 1H NMR (CDCl3): d
1.32–1.43 (m, 2H), 1.47–1.59 (m, 2H), 1.63–1.71 (m, 2H), 1.97–
2.09 (m, 2H), 2.28 (s, 3H), 4.27–4.44 (m, 1H), 6.15–6.17 (m, 2H),
6.53–6.55 (m, 2H), 6.85 (br d, 1H, J = 7.4 Hz), 7.17–7.28 (m, 2H),
7.43 (d, 1H, J = 1.8 Hz). IR:
m
1662 cmꢀ1. MS m/z: 403 [M+1]+, 425
7.50 (d, 1H, J = 2.7 Hz). IR: m
1659 cmꢀ1. MS m/z: 537 [M+1]+, 559
[M+23]+ (100), 829 [2M+23]+.
[M+23]+ (100).
4.1.2.9. N-Cycloheptyl 1-(2,4-dichlorophenyl)-4-methyl-5-(1H-
pyrrol-1-yl)-1H-pyrazole-3-carboxamide (14). Yield 95% as white
needles, mp 178 °C (from diethyl ether). 1H NMR (CDCl3): d 1.51–
1.65 (mm, 8H), 1.96–2.06 (m, 4H), 2.27 (s, 3H), 4.03–4.19 (m,
1H), 6.16–6.18 (m, 2H), 6.54–6.55 (m, 2H), 6.86 (br d, 1H), 717–
4.1.2.17. N-Cyclopentyl 1-(2,4-dichlorophenyl)-5-(2,5-dimethyl-
1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (22). Yield
84% as colorless needles, mp 121 °C. 1H NMR (CDCl3): d 1.43–1.71
(mm, 6H), 1.92(s, 6H), 1.98–2.10 (m, 2H), 2.16 (s, 3H), 4.32–4.42
(m, 1H), 5.77 (s, 2H), 6.93 (d, 1H, J = 8.5 Hz), 7.16 (dd, 1H,
7.28 (m, 2H), 7.43 (d, 1H, J = 1.6 Hz). IR:
m
1663 cmꢀ1. MS m/z:
J = 1.9 Hz, J = 8.5 Hz), 7.48 (d, 1H, J = 2.1 Hz). IR: m
1661 cmꢀ1. MS
431 [M+1]+, 453 [M+23]+ (100).
m/z: 431 [M+1]+, 453 [M+23]+ (100), 883 [2M+23]+.
4.1.2.10. N-(4-Chlorophenyl) 1-(2,4-dichlorophenyl)-5-(2,5-di-
methyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide
(15). Yield 77% as a pale yellow pasty solid. 1H NMR (CDCl3): d 1.96
(s, 6H), 2.22 (s, 3H), 5.81 (s, 2H), 6.91 (d, 1H, J = 8.7 Hz), 7.20 (dd,
1H, J = 2.0 Hz, J = 8.6 Hz), 7.30 (d, 2H, J = 8.6 Hz), 7.52 (d, 1H,
4.1.2.18. N-Cycloheptyl 1-(2,4-dichlorophenyl)-5-(2,5-dimethyl-
1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (23). Yield
85% as a pale yellow solid, mp 128 °C. 1H NMR (CDCl3): d 1.51–1.76
(mm, 8H), 1.92 (s, 6H), 1.97–2.07 (m, 4H), 2.16 (s, 3H), 4.03–4.17
(m, 1H), 5.78 (s, 2H), 6.88 (d, 1H, J = 8.8 Hz), 6.94 (br s, 1H), 7.15 (dd,
J = 2.0 Hz), 7.64 (d, 2H, J = 8.8 Hz), 8.69 (s, 1H). IR:
m
1687 cmꢀ1
.
1H, J = 2.1 Hz, J = 8.9 Hz), 7.48 (s, 1H). IR: m
1660 cmꢀ1. MS m/z: 459
MS m/z: 473 [M+1]+, 495 [M+23]+ (100).
[M+1]+, 481 [M+23]+ (100).
4.1.2.11. N-(4-Chlorobenzyl) 1-(2,4-dichlorophenyl)-5-(2,5-di-
methyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide
(16). Yield 91% as a white solid, mp 153.6 °C. 1H NMR (CDCl3): d
1.94 (s, 6H), 2.19 (s, 3H), 4.58 (d, 2H, J = 6.3 Hz), 5.80 (s, 2H), 6.87
(d, 2H, J = 8.7 Hz), 7.16 (dd, 1H, J = 1.8 Hz, J = 8.8 Hz), 7.22–7.35
4.1.2.19. N-(4-Chlorophenyl) 1-(3,4-dichlorophenyl)-4-methyl-
5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (24). Yield 96%
as white needles, mp 196 °C (from diethyl ether). 1H NMR (CDCl3):
d 2.30 (s, 3H), 6.36–6.39 (m, 2H), 6.62–6.64 (m, 2H), 6.72 (dd, 1H,
J = 1.9 Hz, J = 8.8 Hz), 7.28–7.38 (m, 4H), 7.76 (d, 2H, J = 8.8 Hz),
(m, 3H), 7.38 (br t, 1H, 6.0 Hz), 7.47 (d, 1H, J = 2.3 Hz),. IR:
m
8.76 (br s, 1H). IR: m
1687 cmꢀ1. MS m/z: 467 [M+23]+ (100).
1672 cmꢀ1. MS m/z: 488 [M+1]+, 510 [M+23]+ (100).
4.1.2.20. N-(4-Chlorobenzyl) 1-(3,4-dichlorophenyl)-4-methyl-
5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (25). Yield 62% as
a white solid, mp 139 °C. 1H NMR (CDCl3): d 2.27 (s, 3H), 4.60 (d, 2H,
J = 5.9 Hz), 6.35–6.36 (m, 2H), 6.60–6.62 (m, 2H), 6.68 (dd, 1H,
J = 1.7 Hz, J = 8.8 Hz), 7.22–7.33 (m, 6H), 7.37 (br t, 1H, J = 5.5 Hz).
4.1.2.12. N-(2-Bromo-3,4,5-trimethoxybenzyl) 1-(2,4-dichloro-
phenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-
3-carboxamide (17). Yield 97% as a white solid, mp 186–187 °C. 1H
NMR (CDCl3): d 1.92 (s, 6H), 2.17 (s, 3H), 3.85 (s, 6H), 3.87 (s, 3H),
4.65 (d, 2H, J = 6.1 Hz), 5.78 (s, 2H), 6.88 (s, 1H), 7.13–7.19 (m, 2H),
IR:
m
1673 cmꢀ1. MS m/z: 482 [M+23]+ (100).
7.49 (d, 1H, J = 1.9 Hz). IR:
m
1671 cmꢀ1. MS m/z: 623 [M+1]+ (100),
645 [M+23]+.
4.1.2.21. N-[2-(4-Chlorophenyl)ethyl] 1-(3,4-dichlorophenyl)-4-
methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (26). Yield
69% as a white solid, mp 127–130 °C. 1H NMR (CDCl3): d 2.25 (s, 3H),
2.90 (t, 2H, J = 7.0 Hz), 3.60–3.71 (m, 2H), 6.35–6.37 (m, 2H), 6.60–
6.61 (m, 2H), 6.64 (dd, 1H, J = 2.1 Hz, J = 8.6 Hz), 7.09 (br t, 1H,
4.1.2.13. N-[2-(4-Chlorophenyl)ethyl] 1-(2,4-dichlorophenyl)-5-
(2,5-dimethyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carbox-
amide (18). Yield 98% as a cream solid, mp 139.6 °C. 1H NMR
(CDCl3): d 1.93 (s, 6H), 2.18 (s, 3H), 2.89 (t, 2H, J = 7.1 Hz), 3.59–
3.69 (m, 2H), 5.79 (s, 2H), 6.84 (d, 2H, J = 8.7 Hz), 7.07 (br t, 1H,
5.9 Hz), 7.15–7.32 (mm, 6H). IR:
m
1671 cmꢀ1. MS m/z: 474 [M+1]+,
496 [M+23]+, 969 [2M+23]+ (100).
J = 6.3 Hz), 7.13–7.27 (m, 4H), 7.50 (d, 1H, J = 2.2 Hz). IR:
m
1666 cmꢀ1. MS m/z: 503 [M+1]+, 525 [M+23]+.
4.1.2.22. N-Cyclopentyl 1-(3,4-dichlorophenyl)-4-methyl-5-(1H-
pyrrol-1-yl)-1H-pyrazole-3-carboxamide (27). Yield 98% as white
needles, mp 161 °C (from diethyl ether). 1H NMR (CDCl3): d 1.52–
1.78 (mm, 6H), 2.01–2.12 (m, 2H), 2.25 (s, 3H), 4.33–4.43 (m,
1H), 6.34 (s, 2H), 6.58–6.59 (m, 2H), 6.72 (dd, 1H, J = 2.0 Hz,
J = 8.7 Hz), 6.90 (br d, J = 7.4 Hz), 7.24 (s, 1H), 7.31 (d, 1H,
4.1.2.14. N-[2-(3,4-Dichlorophenyl)ethyl] 1-(2,4-dichlorophenyl)-
5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carbox-
amide (19). Yield 95% as a yellow solid, mp 121 °C. 1H NMR
(CDCl3): d 1.93 (s, 6H), 2.17 (s, 3H), 2.88 (t, 2H, J = 7.0 Hz), 3.59–
3.69 (m, 2H), 5.79 (s, 2H), 6.85 (d, 2H, J = 8.8 Hz), 7.07 (dd, 1H,
J = 1.7 Hz, J = 7.8 Hz), 7.16 (dd, 1H, J = 1.9 Hz, J = 8.8 Hz), 7.34 (d,
J = 8.8 Hz). IR:
m
1664 cmꢀ1. MS m/z: 403 [M+1]+, 425 [M+23]+
(100), 827 [2M+23]+.

R. Silvestri et al. / Bioorg. Med. Chem. 17 (2009) 5549–5564
5561
4.1.2.23. N-Cycloheptyl 1-(3,4-dichlorophenyl)-4-methyl-5-(1H-
(mm, 8H), 1.84 (s, 6H), 2.05–2.11 (m, 4H), 2.20 (s, 3H), 4.06–4.22
pyrrol-1-yl)-1H-pyrazole-3-carboxamide (28). Yield 93% as
a
(m, 1H), 5.95 (s, 2H), 6.61 (dd, 1H, J = 2.2 Hz, J = 2.8 Hz), 6.95 (br d,
white solid, mp 173 °C (diethyl ether). 1H NMR (CDCl3): d 1.52–
1.67 (mm, 8H), 2.02–2.17 (m, 2H), 2.24 (s,3H), 4.05–4.19 (m, 1H),
6.34 (s, 2H), 6.58 (s, 2H), 6.72 (d, 1H, J = 10.1 Hz), 6.92 (br d, 1H,
1H, J = 8.2 Hz), 7.22 (d, 1H, J = 2.1 Hz), 7.32 (d, 1H, J = 8.8 Hz). IR: m
1667 cmꢀ1. MS m/z: 459 [M+1]+, 481 [M+23]+ (100), 939 [2M+23]+.
J = 8.2 Hz), 7.23 (s, 1H), 7.31 (d, 1H, J = 8.8 Hz). IR:
m
1661 cmꢀ1
.
4.1.2.32. N-(Adamant-1-yl) 1-(3,4-dichlorophenyl)-5-(2,5-di-
methyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide
(37). Yield 90% as a white solid, mp 208–210 °C. 1H NMR (CDCl3): d
1.62–1.74 (mm, 4H), 1.82 (s, 6H), 2.09–2.17 (mm, 7H), 5.92 (s, 2H),
6.60 (dd, 1H, J = 2.7 Hz, J = 8.8 Hz), 6.77 (br s, 1H), 7.19 (d, 1H,
MS m/z: 431 [M+1]+, 453 [M+23]+ (100), 883 [2M+23]+.
4.1.2.24. N-(Adamant-1-yl) 1-(3,4-dichlorophenyl)-4-methyl-5-
(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (29). Yield 97% as a
white solid, mp 179–182 °C. 1H NMR (CDCl3): d 1.71–1.81 (mm,
6H), 2.13–2.19 (mm, 9H), 2.23 (s, 3H), 6.33 (s, 2H), 6.58 (s, 2H), 7.72
(dd, 1H, J = 1.7 Hz, J = 7.9 Hz), 7.22 (d, 1H, J = 1.8 Hz), 7.30 (d, 1H,
J = 2.0 Hz), 7.29 (d, 1H, J = 8.8 Hz). IR:
m
1668 cmꢀ1. MS m/z: 498
[M+1]+, 520 [M+23]+, 1017 [2M+23]+ (100).
J = 8.5 Hz). IR:
m
1673 cmꢀ1. MS m/z: 470 [M+1]+ (100), 961 [2M+23]+.
4.1.2.33. N-[1-(1-Phenyl)ethyl] 1-(3,4-dichlorophenyl)-5-(2,5-
dimethyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide
(38). Yield 95% as a white solid, mp140–142 °C. 1H NMR (CDCl3): d
1.65 (d, 3H, J = 6.9 Hz), 1.84 (s, 3H), 1.86 (s, 3H), 2.21 (s, 3H), 5.29–
5.43 (m, 1H), 6.64 (dd, 1H, J = 1.9 Hz, J = 8.7 Hz), 7.21–7.37 (m, 5H),
4.1.2.25. (R)-N-[1-(1-Cyclohexyl)ethyl] 1-(3,4-dichlorophenyl)-4-
methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (30). Yield
80% as a colorless oil. 1H NMR (CDCl3): d 1.20 (d, 3H, J = 6.6 Hz),
1.08–1.38 (m, 4H), 1.39–1.46 (m, 1H), 1.67–1.85 (m, 6H), 2.25 (s,
3H), 3.98–4.09 (m, 1H), 6.34 (s, 2H), 6.59 (s, 2H), 6.74 (dd, 1H,
J = 1.7 Hz, J = 9.9 Hz), 6.82 (d, 1H, J = 9.2 Hz), 7.31 (d, 1H, J = 8.6 Hz).
7.40–7.47 (m, 2H). IR:
m
1663 cmꢀ1. MS m/z: 468 [M+1]+ (100), 490
[M+23]+, 957 [2M+23]+.
IR:
m
1662 cmꢀ1. MS m/z: 446 [M+1]+ (100), 468 [M+23]+, 913
4.1.2.34. N-(4-Chlorobenzhydryl) 1-(3,4-dichlorophenyl)-5-(2,5-
dimethyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxam-
ide (39). Yield 98% as a white solid, mp 98–100 °C. 1H NMR (CDCl3): d
1.87 (s, 6H), 2.23 (s, 3H), 5.98 (s, 2H), 6.46 /d, 1H, J = 8.2 Hz), 6.68 (dd,
1H, J = 2.3 Hz, J = 8.8 Hz), 7.20 (s, 1H), 7.28–7.42 (m, 8H), 7.63 (d, 1H,
[2M+23]+.
4.1.2.26. (S)-N-[1-(1-Cyclohexyl)ethyl] 1-(3,4-dichlorophenyl)-4-
methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (31). Yield
97% as a colorless oil. 1H NMR (CDCl3): d 1.19 (d, 3H, J = 6.8 Hz),
1.05–1.32 (m, 4H), 1.37–1.45 (m, 1H), 1.66–1.84 (mm, 6H),2.25 (s,
3H), 4.00–4.09 (m, 1H), 6.32–6.33 (m, 2H), 6.58–6.59 (m, 2H), 6.73
(dd, 1H, J = 2.6 Hz, J = 8.8 Hz), 6.82 (br d, 1H, J = 9.4 Hz), 7.23 (d, 1H,
J = 8.2 Hz). IR: m
1673 cmꢀ1. MS m/z: 564 [M+1]+, 586 [M+23]+ (100).
4.1.2.35. N-(4-Chlorophenyl) 1-(2,4-difluorophenyl)-4-methyl-
5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (40). Yield 98%
as colorless needles, mp 203 °C (from diethyl ether/n-hexane). 1H
NMR (CDCl3): d 2.32 (s, 3H), 6.22 (t, 2H, J = 2.0 Hz), 6.58 (t, 2H,
J = 1.8 Hz), 6.84–6.97 (m, 2H), 7.27–7.35 (m, 3H), 7.62 (d, 2H,
J = 2.7 Hz), 7.30 (d, 1H, J = 8.8 Hz). IR:
m
1651 cmꢀ1. MS m/z: 446
[M+1]+ (100), 468 [M+23]+, 913 [2M+23]+.
4.1.2.27. N-(4-Chlorophenyl) 1-(3,4-dichlorophenyl)-5-(2,5-di-
methyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide
(32). Yield 77% as a white solid, mp 190 °C. 1H NMR (CDCl3): d 1.87
(s, 6H), 2.26 (s, 3H), 5.98 (s, 2H), 6.66 (dd, 1H, J = 2.8 Hz, J = 8.8 Hz),
J = 8.8 Hz), 8.74 (br s, 1H). IR: m
1688 cmꢀ1. MS m/z: 413 [M+1]+,
435 [M+23]+ (100).
4.1.2.36. N-(4-Chlorobenzyl) 1-(2,4-difluorophenyl)-4-methyl-
5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (41). Yield 80%
as a white solid, mp 110 °C. 1H NMR (CDCl3): d 2.29 (s, 3H), 4.56
(d, 2H, J = 6.0 Hz), 6.21 (s, 2H), 6.57 (s, 2H), 6.80–6.92 (m, 3H),
7.26–7.37 (m, 4H), 7.68 (d, 2H, J = 8.8 Hz), 8.81 (s, 1H). IR:
m
1694 cmꢀ1. MS m/z: 495 [M+23]+ (100), 969 [2M+23]+.
4.1.2.28. N-(4-Chlorobenzyl) 1-(3,4-dichlorophenyl)-5-(2,5-di-
methyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide
(33). Yield 97% as a white solid, mp 157 °C. 1H NMR (CDCl3): d 1.85
(s, 6H), 2.23 (s, 3H), 4.61 (d, 2H, J = 6.1 Hz), 5.96 (s, 2H), 6.57 (dd,
1H, J = 2.6 Hz, J = 8.8 Hz), 7.21–7.35 (m, 6H), 7.47 (br t, 1H,
7.19–7.36 (m, 4H). IR:
m
1674 cmꢀ1. MS m/z: 449 [M+23]+ (100),
875 [2M+23]+.
4.1.2.37. N-(2-Bromo-3,4,5-trimethoxybenzyl) 1-(2,4-difluoro-
phenyl)-4-methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxam-
ide (42). Yield 95% as a white solid, mp 50–53 °C. 1H NMR (CDCl3):
d 2.72 (s, 3H), 3.84 (s, 6H), 3.87 (s, 3H), 4.64 (d, 2H, J = 6.1 Hz), 6.19
(t, 2H, J = 1.9 Hz), 6.54–6.55 (m, 2H), 6.81–6.94 (m, 2H), 7.19–7.31
J = 6.1 Hz). IR:
m
1673 cmꢀ1. MS m/z: 510 [M+23]+ (100).
4.1.2.29. N-[2-(4-Chlorophenyl)ethyl] 1-(3,4-dichlorophenyl)-5-
(2,5-dimethyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carbox-
amide (34). Yield 90% as a pale yellow solid, mp 135 °C. 1H NMR
(CDCl3): d 1.84 (s, 6H), 2.21 (s, 3H), 2.93 (t, 2H, J = 7.1 Hz), 3.63–
3.73 (m, 2H), 5.96 (s, 2H), 6.53 (dd, 1H, J = 2.2 Hz, J = 8.9 Hz), 7.12
(m, 2H), 7.38 (br t, 1H, J = 6.1 Hz). IR:
m
1671 cmꢀ1. MS m/z: 562
[M+1]+, 584 [M+23]+ (100).
4.1.2.38. N-[2-(4-Chlorophenyl)ethyl] 1-(2,4-difluorophenyl)-4-
methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (43). Yield
87% as a colorless oil. 1H NMR (CDCl3): d 2.27 (s, 3H), 2.87 (t, 2H,
J = 7.3 Hz), 3.57–3.68 (m, 2H), 6.19–6.21 (m, 2H), 6.56 (s, 2H),
6.82–6.93 (m, 3H), 7.03 (br t, 1H, J = 5.8 Hz), 7.15 (d, 2H,
(br t, 1H, J = 5.9 Hz), 7.19–7.30 (m, 6H). IR:
m
1667 cmꢀ1. MS m/z:
503 [M+1]+ (100), 525 [M+23]+.
4.1.2.30. N-Cyclopentyl 1-(3,4-dichlorophenyl)-5-(2,5-dimethyl-
1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (35). Yield
82% as a white solid, mp 168 °C. 1H NMR (CDCl3): d 1.52–1.80 (mm,
6H), 1.84 (s, 6H), 2.04–2.19 (m, 2H), 2.20 (s, 3H), 4.35–4.49 (m, 1H),
5.95 (s, 2H), 6.61 (dd, 1H, J = 2.8 Hz, J = 8.8 Hz), 6.93 (br d, 1H,
J = 8.4 Hz), 7.25 (d, 2H, J = 8.3 Hz). IR:
m
1669 cmꢀ1. MS m/z: 441
[M+1]+, 463 [M+23]+ (100), 903 [2M+23]+.
4.1.2.39. N-[2-(3,4-Dichlorophenyl)ethyl] 1-(2,4-difluorophenyl)-4-
methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (44). Yield
J = 7.4 Hz), 7.22 (d, 1H, J = 2.7 Hz), 7.31 (d, 1H, J = 8.8 Hz). IR:
m
1669 cmꢀ1. MS m/z: 431 [M+1]+, 453 [M+23]+ (100).
94% as a pale yellow solid, mp 78 °C. H NMR (CDCl3): d 2.27 (s, 3H),
1
2.87 (t, 2H, J = 7.1 Hz), 3.58–3.68 (m, 2H), 6.20 (t, 2H, J = 2.0 Hz), 6.56
4.1.2.31. N-Cycloheptyl 1-(3,4-dichlorophenyl)-5-(2,5-dimethyl-
1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (36). Yield
89% as a white solid, mp 166 °C. 1H NMR (CDCl3): d 1.52–1.70
(t, 2H, J = 1.8 Hz), 6.82–6.93 (m, 3H), 6.98 (br t, 1H, J = 5.8 Hz), 7.07 (dd,
1H, J = 1.8 Hz, J = 8.7 Hz), 7.18–7.37 (m, 2H). IR:
m
1672 cmꢀ1. MS m/z:
498 [M+23]+ (100), 973 [2M+23]+.

5562
R. Silvestri et al. / Bioorg. Med. Chem. 17 (2009) 5549–5564
4.1.2.40. N-[2-(3-Chloro-4-methoxyphenyl)ethyl] 1-(2,4-difluo-
rophenyl)-4-methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carbox-
amide (45). Yield 81% as a colorless oil. 1H NMR (CDCl3): d 2.27 (s,
3H), 2.83 (t, 2H, J = 7.2 Hz), 3.56–3.66 (m, 2H), 3.86 (s, 3H),
6.20–6.21 (m, 2H), 6.56–6.57 (m, 2H), 6.81–6.96 (m, 3H), 6.99 (br
t, 1H, J = 5.7 Hz), 7.09 (dd, 1H, J = 1.9 Hz, J = 8.0 Hz), 7.16–7.28 (m,
7.38 (m, 2H). IR:
[M+39]+.
m
1671 cmꢀ1. MS m/z: 526 [M+23]+ (100), 542
4.1.2.49. N-[2-(3-Chloro-4-methoxyphenyl)ethyl] 1-(2,4-difluo-
rophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methyl-1H-pyra-
zole-3-carboxamide (54). Yield 80% as a yellow oil. 1H NMR
(CDCl3): d 1.88 (s, 6H), 2.16 (s, 3H), 2.84 (t, 2H, J = 7.3 Hz), 3.57–
3.68 (m, 2H), 3.86 (s, 3H), 5.79 (s, 2H), 6.82–6.91 (m, 3H), 7.08–
2H). IR:
m
1672 cmꢀ1. MS m/z: 471 [M+1]+, 493 [M+23]+ (100).
4.1.2.41. N-[2-(2,4-Dichlorophenyl)ethyl] 1-(2,4-difluorophenyl)-4-
methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (46). Yield
83% as a white solid, mp 111 °C. 1H NMR (CDCl3): d 2.27 (s, 3H), 3.02
(t, 2H, J = 7.2 Hz), 3.60–3.70 (m, 2H), 6.20–6.21 (m, 2H), 6.55–6.56 (m,
2H), 6.83–6.92 (m, 3H), 7.02 (br t, 1H, J = 6.3 Hz), 7.14–7.29 (m, 2H),
7.16 (m, 2H), 7.25 (d, 1H, J = 2.0 Hz). IR: m
1666 cmꢀ1. MS m/z:
499 [M+1]+, 521 [M+23]+ (100).
4.1.2.50. N-[2-(2,4-Dichlorophenyl)ethyl] 1-(2,4-difluorophenyl)-
5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carbox-
amide (55). Yield 85% as a white solid, mp 121 °C. 1H NMR (CDCl3): d
1.89 (s, 6H), 2.17 (s, 3H), 3.04 (t, 2H, J = 7.3 Hz), 3.61–3.72 (m, 2H),
5.79 (s, 2H), 6.82–6.94 (m, 2H), 7.03–7.10 (m, 2H), 7.15–7.21 (m,
7.36 (s, 1H). IR: m
1666 cmꢀ1. MS m/z: 476 [M+1]+, 498 [M+23]+ (100).
4.1.2.42. N-Cyclopentyl 1-(2,4-difluorophenyl)-4-methyl-5-(1H-
pyrrol-1-yl)-1H-pyrazole-3-carboxamide (47). Yield 85% as white
needles, mp 109 °C (from diethyl ether/n-hexane). 1H NMR
(CDCl3): d 1.44–1.74 (mm, 6H), 1.97–2.21 (m, 2H), 2.26 (s, 3H),
4.31–4.41 (m, 1H), 6.18 (t, 2H, J = 1.8 Hz), 6.54 (t, 2H, J = 1.8 Hz),
2H), 7.37 (d, 1H, J = 1.666 Hz). IR:
(100).
m
16 cmꢀ1. MS m/z: 526 [M+23]+
4.1.2.51. N-Cyclopentyl 1-(2,4-sifluorophenyl)-5-(2,5-dimethyl-
1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (56). Yield
83% as a white solid, mp 121 °C.1H NMR (CDCl3): d 1.46–1.84 (mm,
6H), 1.87 (s, 6H), 1.98–2.10 (m, 2H), 2.15 (s, 3H), 4.32–4.43 (m, 1H),
6.79–6.92 (m, 2H), 7.21–7.32 (m, 1H). IR:
m
1668 cmꢀ1. MS m/z:
371 [M+1]+, 393 [M+23]+ (100), 763 [2M+23]+.
4.1.2.43. N-Cycloheptyl 1-(2,4-difluorophenyl)-4-methyl-5-(1H-
5.77 (s, 2H), 6.81–6.91 (m, 2H), 7.07–7.19 (m, 1H). IR: m .
1661 cmꢀ1
pyrrol-1-yl)-1H-pyrazole-3-carboxamide (48). Yield 73% as
a
MS m/z: 399 [M+1]+, 421 [M+23]+ (100), 819 [2M+23]+.
white solid, mp 142 °C (from diethyl ether/n-hexane). 1H NMR
(CDCl3): d 1.26–1.66 (mm, 8H), 1.97–2.07 (m, 4H), 2.26 (s, 3H),
4.06–4.14 (m, 1H), 6.19–6.20 (m, 2H), 6.54–6.56 (m, 2H), 6.82–
4.1.2.52. N-Cycloheptyl 1-(2,4-difluorophenyl)-5-(2,5-dimethyl-
1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (57). Yield
86% as a white solid, mp 126 °C. 1H NMR (CDCl3): d 1.50–1.82
(mm, 8H), 1.87 (s, 6H), 1.97–2.07 (m, 4H), 2.14 (s, 3H), 4.05–
4.12 (m, 1H), 5.76 (s, 2H), 6.79–6.93 (m, 2H), 7.07–7.18 (m,
6.92 (m, 2H), 7.22–7.33 (m, 1H). IR:
m
1668 cmꢀ1. MS m/z: 399
[M+1]+, 421 [M+23]+ (100), 819 [2M+23]+.
4.1.2.44. N-(4-Chlorophenyl) 1-(2,4-difluorophenyl)-5-(2,5-di-
methyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide
(49). Yield 80% as a pasty yellow material. 1H NMR (CDCl3): d 1.91
(s, 6H), 2.22 (s, 3H), 5.81 (s, 2H), 6.86–6.96 (m, 2H), 7.10–7.21 (m,
1H), 7.29 (d, 2H, J = 8.7 Hz), 7.64 (d, 2H, J = 8.7 Hz), 8.81 (br s, 1H).
1H). IR: m
1660 cmꢀ1. MS m/z: 427 [M+1]+, 449 [M+23]+ (100),
875 [2M+23]+.
4.2. Molecular modeling
IR:
m
1687 cmꢀ1. MS m/z: 463 [M+23]+ (100).
4.2.1. Computational chemistry
Molecular modeling and graphics manipulations were per-
formed using the molecular operating environment (MOE)20 and
UCSF-CHIMERA21 software packages, running on a 2 CPU (PIV
2.0–3.0 GHZ) Linux workstation. Energy minimizations and MD
simulations were performed by means of AMBER 9 software,22
selecting the Cornell force field.23
4.1.2.45. N-(4-Chlorobenzyl) 1-(2,4-difluorophenyl)-5-(2,5-di-
methyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide
(50). Yield 80% as a white solid, mp 148 °C. 1H NMR (CDCl3): d 1.89
(s, 6H), 2.19 (s, 3H), 4.57 (d, 2H, J = 6.1 Hz), 5,79 (s, 2H), 6.81–6.92
(m, 3H), 7.05–7.17 (m, 2H), 7.29–7.34 (m, 2H). IR:
m
1673 cmꢀ1. MS
m/z: 455 [M+1]+, 477 [M+23]+ (100).
4.2.2. Residue indexing
4.1.2.46. N-(2-Bromo-3,4,5-trimethoxybenzyl) 1-(2,4-difluoro-
phenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-
3-carboxamide (51). Yield 92% as a pale yellow oil. 1H NMR
(CDCl3): d 1.87 (s, 6H), 2.16 (s, 3H), 3.84 (s, 6H), 3.87 (s, 3H), 4.65
(d, 2H, J = 6.3 Hz), 5.77 (s, 2H), 6.76–6.89 (m, 3H), 7.04–7.16 (m,
The convention used for the amino acid identifiers, according to
theapproachof BallesterosandWeinstein24 and van Rhee andJacob-
son,25 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 resi-
dues within a given TM are then indexed relative to the 50 position.
1H), 7.43 (br t, 1H, J = 6.1 Hz). IR:
m
1670 cmꢀ1. MS m/z: 590
[M+1]+, 612 [M+23]+, 1201 [2M+23]+ (100).
4.2.3. Docking simulations
4.1.2.47. N-[2-(4-Chlorophenyl)ethyl] 1-(2,4-difluorophenyl)-5-
(2,5-dimethyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carbox-
amide (52). Yield 97% as a yellow oil. 1H NMR (CDCl3): d 1.88 (s,
6H), 2.17 (s, 3H), 2.89 (t, 2H,J = 7.3 Hz), 3.59–3.69 (m, 2H), 5.79
(s, 2H), 6.82–6.93 (m, 2H), 7.03–7.10 (m, 1H), 7.16 (d, 2H,
The core structures of compounds 31 and 37 were constructed
using standard bond lengths and bond angles of the MOE fragment
library. Geometry optimizations were accomplished with the
MMFF94X force field available inside MOE. Docking simulations
were carried out starting from the previously published inactive
state of hCB1 receptor model,7 which was built using the 2.8 Å crys-
tal structure of bovine rhodopsin (PDB entry code 1F88)26 as a
structural template. Additional details regarding the receptor
structure, site-directed mutagenesis data and methods applied in
developing the hCB1 receptor model were also reported.7 Com-
pounds 31 and 37 were docked into the energy-minimized recep-
tor model by means of GOLD, 4.0 version,18 a genetic algorithm-
based software, taking the binding orientation of 1 in the hCB1
J = 8.4 Hz), 7.25 (d, 2H, J = 8.5 Hz). IR:
m
1666 cmꢀ1. MS m/z: 469
[M+1]+, 491 [M+23]+ (100).
4.1.2.48. N-[2-(3,4-Dichlorophenyl)ethyl] 1-(2,4-difluorophenyl)-
5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carbox-
amide (53). Yield 91% as a pale yellow solid, mp 85–87 °C. 1H NMR
(CDCl3): d 1.89 (s, 6H), 2.16 (s, 3H), 2.89 (t, 2H, J = 7.1 Hz), 3.59–3.69
(m, 2H), 5.79 (s, 2H), 6.82–6.94 (m, 2H), 7.02–7.15 (m, 2H), 7.31–

R. Silvestri et al. / Bioorg. Med. Chem. 17 (2009) 5549–5564
5563
bundle7 as a starting point. The region of interest used by GOLD was
defined in order to contain the residues within 15 Å from the ori-
ginal position of 1 in the hCB1 model.7 The ‘allow early termination’
option was deactivated while the remaining GOLD default parame-
ters were used. The ligands were submitted to 100 genetic algo-
rithm runs by selecting GOLDScore as a fitness function, without
any other constraints. The best docked conformation for GOLDScore
was then compared with the binding conformation of 1 in the hCB1
bundle7 and the root-mean-square deviation between the posi-
tions of the heavy atoms was calculated, this parameter being con-
sidered as a measure of the docking accuracy.
of each experiment, rats were divided into four groups of n = 6–8,
matched for body weight and food intake over the 3 days preced-
ing the start of the experiment, and treated with 0, 0.1, 1, and
10 mg/kg of compounds 4 or 52. Compounds 4 and 52 were sus-
pended in saline with a few drops of Tween 80 and administered
intraperitoneally (injection volume: 1 mL/kg) 30 min before lights
off. Food and water intake was recorded 60, 120, 180, 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. Data
on the effect of compounds 4 or 52 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 Newman-Keuls test for post hoc compari-
sons; data on the effect of compounds 4 or 52 on food and water
intake at the 1440-min time interval were analyzed by separate
1-way ANOVAs.
4.2.4. Md simulations
Refinement of the ligand/receptor complexes was achieved by in
vacuo energy minimization with the SANDER module of AMBER,
applying an energy penalty force constant of 10 kcal molꢀ1 Åꢀ2 on
the protein backbone atoms. The geometry-optimized complexes
were then used as the starting point for subsequent 1 ns MD simu-
lation, during which the protein backbone atoms were constrained
by means of decreasing force constants; the salt bridge between
K3.28(192) and D6.58(366) as well as the H-bond between the li-
gand carboxamide oxygen and K3.28(192) were also restrained.
More specifically, an initial restraint with a force constant of
10 kcal molꢀ1 Åꢀ2 was applied on all the alpha carbons; this force
constant decreased during the whole MD, and in the last 200 ps,
Acknowledgements
Authors from the University of Siena wish to thank the Ministe-
ro dell’Università e della Ricerca (PRIN 2006—Prot. n 2006030948_
002) for financial support.
Supplementary data
its value was 0.01 kcal molꢀ1 Åꢀ2
. Regarding the intra-helix
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.06.027.
K3.28(192)/D6.58(366) H-bonds and the ligand/K3.28(192) interac-
tions, restraints of 10 and 50 kcal molꢀ1 Åꢀ2 were applied for 600 ps
of MD simulation; 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 AM-
BER. A time step of 1 fs and a non-bonded pairlist updated every
25 fs were used for the MD simulations. The temperature was reg-
ulated by the way of Langevin dynamics, with a collision frequency
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Adult male Wistar rats (Charles River Laboratories, Calco, Italy),
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