Synthesis, cannabinoid receptor affinity, and molecular modeling studies of substituted 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides

Article abstract of DOI:10.1021/jm070566z

The new 1-phenyl-5-(1H-pyrrol-1-yl)pyrazole-3-carboxamides were compared with the reference compounds AM251 and SR144528 for cannabinoid hCB₁ and hCB₂ receptor affinity. Compounds bearing 2,4-dichlorophenyl or 2,4-difluorophenyl groups at position 1 and 2,5-dimethylpyrrole moiety at position 5 of the pyrazole nucleus were generally more selective for hCB ₁. On the other hand, the N-cyclohexyl group at the 3-carboxamide was the determinant for the hCB₂ selectivity, in particular when a 3,4-dichlorophenyl group was also present at position 1. Compound 26 was the most selective ligand for the hCB₁ receptor (K_i (CB ₂)/K_i (CB₁) = 140.7). Derivative 30, the most potent hCB₁ ligand (K_i = 5.6 nM), was equipotent to AM251 and behaved as an inverse agonist in the cAMP assay (EC₅₀ ～1 nM). The carbonyl oxygen of both 26 and 30 formed a H-bond with K3.28(192), while the substituents at the nitrogen fitted in a pocket formed by lipophilic residues. This H-bonding interaction was proposed to account for the high affinity for receptors' inactive state and the inverse agonist activity.

Full text of DOI:10.1021/jm070566z

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J. Med. Chem. 2008, 51, 1560–1576
Synthesis, Cannabinoid Receptor Affinity, and Molecular Modeling Studies of Substituted
1-Aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides
Romano Silvestri,*,† Maria Grazia Cascio, Giuseppe La Regina,† Francesco Piscitelli,† Antonio Lavecchia,*,§ Antonella Brizzi,‡
Serena Pasquini,‡ Maurizio Botta,‡ Ettore Novellino,§ Vincenzo Di Marzo, and Federico Corelli*,‡
Istituto PasteursFondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, Sapienza UniVersità di Roma, Piazzale Aldo Moro 5,
I-00185 Roma, Italy, Dipartimento Farmaco Chimico Tecnologico, UniVersità di Siena, Polo Scientifico UniVersitario San Miniato, Via Aldo
Moro 2, I-53100 Siena, Italy, Dipartimento di Chimica Farmaceutica e Tossicologica, UniVersità di Napoli Federico II, Via Domenico
Montesano 49, I-80131, Napoli, Italy, and Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34,
Comprensorio OliVetti, I-80078 Pozzuoli, Napoli, Italy
ReceiVed May 15, 2007
The new 1-phenyl-5-(1H-pyrrol-1-yl)pyrazole-3-carboxamides were compared with the reference compounds
AM251 and SR144528 for cannabinoid hCB1 and hCB2 receptor affinity. Compounds bearing 2,4-
dichlorophenyl or 2,4-difluorophenyl groups at position 1 and 2,5-dimethylpyrrole moiety at position 5 of
the pyrazole nucleus were generally more selective for hCB1. On the other hand, the N-cyclohexyl group at
the 3-carboxamide was the determinant for the hCB2 selectivity, in particular when a 3,4-dichlorophenyl
group was also present at position 1. Compound 26 was the most selective ligand for the hCB1 receptor (Ki
(CB2)/Ki (CB1) ) 140.7). Derivative 30, the most potent hCB1 ligand (Ki ) 5.6 nM), was equipotent to
AM251 and behaved as an inverse agonist in the cAMP assay (EC50 ∼1 nM). The carbonyl oxygen of both
26 and 30 formed a H-bond with K3.28(192), while the substituents at the nitrogen fitted in a pocket formed
by lipophilic residues. This H-bonding interaction was proposed to account for the high affinity for receptors’
inactive state and the inverse agonist activity.
Introduction
receptor-expressing malignant gliomas,18 tumors of immune
origin,19 and immunological disorders such as multiple sclero-
sis.20
The endocannabinoid system is formed by three key com-
ponents, the CB1a and CB2 cannabinoid receptors, their endog-
enous ligands (endocannabinoids), and enzymes, proteins, and
transporters involved in endocannabinoid formation and
inactivation.1,2 The endocannabinoid system is correlated to an
ever increasing number of pathological conditions (pain, im-
munosuppression, peripheral vascular disease, appetite enhance-
ment or suppression, and locomotor disorders), which show
excellent targets for the development of new drugs.3–5
Rimonabant (SR141716, 1) is a potent and selective antago-
nist for the CB1 receptor. It was launched in 2006 by Sanofi-
Aventis for the treatment of obesity and associated risk
factors.13,21,22 In clinical trials, 1 showed CNS (depression
anxiety, dizziness, and insomnia) and gastrointestinal (nausea,
diarrhea) side effects.23 Several pyrazole analogues24 of 1 have
been synthesized: 1,5-diarylpyrazoles (AM25125 (2) and
SR14777821a (3)), 1,4-diarylpyrazoles (AM28126 (4)), and 3,4-
diarylpyrazoles (SLV31927 (5) and SLV32627 (6)). On the other
hand, simple chemical modification of the 1,5-diarylpyrazole
scaffold led to selective CB2 ligands, for example SR14452828
(7; Chart 1).
New cannabinoid receptor–ligands were discovered by re-
placing the central pyrazole nucleus with bioisosteric hetero-
cycles. For example, 4,5-diarylimidazoles29 (8), 1,2-diarylim-
idazoles30 (9), and 1,5-diarylpyrroles31 (10) closely related to
1 showed to be potentially useful to treat obesity. CB1
antagonists were also obtained by expanding the pyrazole core
to 6-membered rings, such as benzene,32 pyridine,33 or pyrim-
idine.34
The neuromodulatory action of endocannabinoids through
CB1 receptors regulates locomotor activity, learning, memory,
emotion, motivation, and appetite.1,6 Targeting cannabinoid
receptors may be a useful way to treat motor disorders, such as
Parkinson’s disease,7 Tourette’s syndrome,8 or choreas,9 with
further therapeutic applications in controlling airway responsive-
ness,10 intraocular pressure,11 and intestinal motility.12 Currently,
CB1 receptor antagonists and inverse agonists13 are evaluated
for obesity, metabolic disorders, smoking cessation, and alcohol
abuse.13,14 On the other hand, selective CB2 ligands may be
used to treat pain,15 inflammation,16 osteoporosis,17 CB2-
We decided to pursue a bioisosteric approach entailing not
the replacement of the central pyrazole nucleus, but the
substitution of a pyrrole ring for the 4-chlorophenyl group at
position 5 of 1. Based on our know-how in pyrrole bioisoster-
ism,35 this modification was expected to provide highly potent
ligands.
In this paper we describe the synthesis and hCB1 and hCB2
receptor affinity of potent pyrrole bioisosteres 11–19 and 22–44,
characterized by different substitutions at the 1-phenyl ring and
at the 3-carboxamide nitrogen (Table 1). For comparison
purposes, the two isomeric 4-carboxamides 20 and 21, structur-
ally related to pyrazole derivatives previously reported by us,36
* To whom correspondence should be addressed. Phone: +39 06 4991
3800 (R.S.); +39 081 678 613 (A.L., molecular modeling); +39 0577
234308 (F.C.). Fax: +39 06 491 491 (R.S.); +39 081 678 613 (A.L.); +39
0577234299(F.C.).E-mail:romano.silvestri@uniroma1.it(R.S.);lavecchi@unina.it
(A.L.); corelli@unisi.it (F.C.).
†
Università di Roma.
Università di Siena.
Università di Napoli Federico II.
‡
§
Consiglio Nazionale delle Ricerche di Pozzuoli.
a Abbreviations: CB, cannabinoid; TM, transmembrane; SAR, structure-
activity relationship; MD, molecular dynamics; GPCR, G protein-coupled
receptor; EL, extracellular loop; WT, wild type; S.I., selectivity index; EDC,
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; HOBt, 1-hy-
droxybenzotriazole.
10.1021/jm070566z CCC: $40.75
2008 American Chemical Society
Published on Web 02/23/2008

1-Aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1561
Chart 1. Selective CB1 and CB2 Ligands
were also synthesized. To rationalize the binding results, we
depicted the putative transmembrane (TM) binding motif on
the hCB1 receptor by molecular modeling. Structure–activity
relationships (SARs) were explained by analyzing the three-
dimensional (3-D) structure of the inverse agonist/receptor
models obtained by molecular docking and molecular dynamics
(MD) simulations.
boxamides 11–44 were obtained through parallel solution-phase
synthesis38 by coupling acids 73–86 and 89 with selected amines
in the presence of EDC/HOBt, using morpholinomethyl-
polystyrene and polymer bound p-toluenesulfonic acid as
scavengers for acidic and basic substances, respectively. The
acidic scavenger was not used in the preparation of the
N-piperidin-1-yl amides.
Biology. The binding affinities (Ki values) of compounds
11–44 for human recombinant CB1 (hCB1) and CB2 (hCB2)
receptors are reported in Table 1. Tested compounds were
evaluated in parallel with 2 and 7 as CB1 and CB2 reference
compounds, respectively. The new compounds were evaluated
in CB1 and CB2 receptor binding assays 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-hydroxypropyl)cyclohexanol ([3H]-CP-55,940; Kd )
0.18 nM for CB1 receptor, and Kd ) 0.31 nM for CB2 receptor)
as the high affinity ligand, as described by the manufacturer
(Perkin-Elmer, Italy).39 Displacement curves were generated by
incubating drugs with [3H]CP-55,940 (0.14 nM for CB1 and
0.084 nM for CB2 binding assay). In all cases, Ki values were
calculated by applying the Cheng-Prusoff equation40 to the IC50
values (obtained by GraphPad) for the displacement of the bound
radioligand by increasing concentrations of the test compounds.
The functional activity of some of the compounds with highest
affinity and selectivity at CB1 receptors, that is, compounds 26,
30, and 39, was only assessed at CB1 receptors by studying
their effects on forskolin-induced cAMP formation in mouse
N18TG2 neuroblastoma cells, because this assay is well-
established in our laboratory. Usually, a good agreement is
observed between the affinity of agonists and inverse agonists
at CB1 receptors and their effects in this assay.41
Chemistry. Potassium 1-cyano-3-ethoxy-3-oxoprop-1-en-2-
olate (45) was obtained by treatment of diethyl oxalate with
acetonitrile in the presence of 18-crown-6 and potassium tert-
butoxide (Scheme 1). Reaction of 45 with phenylhydrazine and
37% HCl or 4-chlorophenylhydrazine hydrochloride in refluxing
ethanol furnished ethyl 1-phenyl-5-amino-1H-pyrazole-3-car-
boxylate (49) or its chloroderivative 52, respectively. Similar
reaction of potassium 2-cyano-4-ethoxy-4-oxobut-2-en-2-olate
(46), which was obtained as 45 using proprionitrile, with
phenylhydrazine or substituted phenylhydrazines furnished esters
50, 53, and 55–58.
Ethyl 2-chloro-2-(2-phenylhydrazono)acetate (47) and its
4-chloroderivative 48 were prepared by treating the appropriate
aryldiazonium salt with ethyl 2-chloroacetoacetate. Reaction of
47 or 48 with malononitrile in the presence of sodium ethoxide
gave 51 and 54, respectively. Pyrrole derivatives 59–65, 67,
69, and 71 were prepared by heating the corresponding amines
49–58 with 2,5-dimethoxytetrahydrofuran in glacial acetic acid
according to the Clauson-Kaas procedure.37 Dimethylpyrroles
66, 68, 70, and 72 were obtained by reaction of amines 55–58
with acetonylacetone in refluxing acetic acid. Lithium hydroxide
hydrolysis of esters 59–72 at room temperature afforded the
corresponding acids 73–86.
The pyrazole-4-carboxylic acid 89 for the synthesis of amides
20 and 21 was prepared as depicted in Scheme 2. Reaction of
4-chlorophenylhydrazine with 2-(1-ethoxyethylidene)malono-
nitrile in the presence of sodium ethoxide gave 5-amino-1-(4-
chlorophenyl)-3-methyl-1H-pyrazole-4-carbonitrile (87), which
was transformed into the corresponding pyrrole derivative 88
by reaction with 2,5-dimethoxytetrahydrofuran in glacial acetic
acid. Transformation of 88 into the acid 89 was achieved by
alkaline hydrolysis in refluxing ethylene glycol. Finally, car-
Results and Discussion
Carboxamides 23–31, 33, 39, 40, and 44 showed high hCB1
receptor binding affinity, with Ki values in the nanomolar range.
The most potent hCB1 ligand 30 (Ki ) 5.6 nM) was comparable
to the CB1 reference compound 2 (Ki ) 2.3 nM). Seven
compounds (24, 27, 28, 30, 31, 33, and 37) were high affinity

1562 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
SilVestri et al.
Table 1. Structure, and hCB1 and hCB2 Receptor Affinity (Ki Values) of Derivatives 11–44 and Reference Compounds 1, 2, and 7a

1-Aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1563
Table 1. Continued
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 hCB1: human CB1 receptor. c hCB2: human CB2 receptor. d 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). e Ki: equilibrium dissociation constant, which
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. f 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. IC50 values are shown in Table 3S of Supporting Information. g S.I.: CB1 selectivity index was calculated as Ki (CB2)/ Ki (CB1) ratio.
h Values calculated at the following website: http://www.vcclab.org. I Compound 1: rimonabant, CB1 reference compound. j Compound 2: AM251, CB1
reference compound. k Compound 7: SR144528, CB2 reference compound. l The binding affinities of reference compounds were evaluated in parallel with
compounds 11–44 under the same conditions.
ligands for the CB2 receptor. Compound 30 (Ki ) 1.7 nM) and
33 (Ki ) 1.3 nM) showed Ki values even lower than that of the
CB2 reference compound 7 (Ki ) 5.4 nM). The CB1 selectivity
indexes, calculated as Ki (CB2)/Ki (CB1) ratio, ranged from 140.7
(26) to 0.04 (37).
boxamide 30, the most potent hCB1 ligand (Ki ) 5.6 nM),
showed receptor affinity comparable to that of 2 (Ki ) 2.3 nM).
Among the 1-(2,4-difluorophenyl)- 34–40 and 1-(2,4,6-trichlo-
rophenyl) derivatives 41–44, only 2,5-dimethylpyrroles 39, 40,
and 44 showed high hCB1 affinity.
Affinity of 22–44 for the hCB2 Receptor. The hCB2 receptor
affinity was correlated with the nature of the N-substituent at
the 3-carboxamide. The 3-(N-cyclohexyl)carboxamides 24, 27,
30, 33, 37, 40, and 44 showed higher affinity for the hCB2
subtype compared to compounds bearing different amide
moieties. Conversely, the N-(3,4-dichlorobenzyl)carboxamides
showed weaker hCB2 affinity. Compounds 30 (Ki ) 1.7 nM)
and 33 (Ki ) 1.3 nM) showed higher affinity for hCB2 than the
reference compound 7 (Ki ) 5.4 nM); however, because of the
concomitant high hCB1 affinity, these ligands were poorly
selective.
Affinity of 11–21 for the hCB1 Receptor. Derivatives 11–21
bearing a phenyl (11–14) or 4-chlorophenyl (15–21) group at
position 1 of the pyrazole nucleus displayed low affinity for
both receptor subtypes, independently of the substituent at
position 4 (H, Me or CN). The 4-methyl derivative 17 showed
higher affinity for the hCB1 than 15 and the corresponding
4-cyano derivative 19 (the isomeric carboxamide 20 showed
no affinity). On the other hand, compounds 14 and 16 showed
higher affinity than 15. logP calculations indicated some
correlation between the hCB1 receptor affinity of 11–21 and
their lipophilic nature (Table 1). These preliminary results
prompted us to design new compounds characterized by (i) two
or three halogen atoms at the 1-phenyl ring, (ii) replacement of
N-piperidin-1-yl substituent of 1 with either cyclohexyl or 3,4-
dichlorobenzyl moiety, (iii) a methyl group at position 4 of
pyrazole, and (iv) the 2,5-dimethyl substitution of the 5-pyrrole
nucleus.
Affinity of 22–44 for the hCB1 Receptor. Derivatives 23–33,
bearing two chlorine atoms at the positions 2,4 or 3,4 of the
1-phenyl ring, showed high affinity for the hCB1 receptor. The
1-(2,4-dichlorophenyl)-5-(2,5-dimethylpyrrol-1-yl)pyrazoles 26
and 27 were as potent as the pyrrole counterparts 23 and 24;
25 showed higher affinity (49-fold) than 22. Compound 26 (S.I.
) 140.7) was the most selective ligand for the hCB1 receptor
within the series and was 2.9-fold more selective than 2 for
this receptor subtype. The 1-(3,4-dichlorophenyl)derivatives
28–33 also showed high hCB1 affinity. The N-cyclohexylcar-
The low receptor affinity displayed by the trichlorophenyl
derivatives (with the exception of 44 for hCB1) suggested that
the chlorine atoms at positions 2 and 2′ could force the 1-phenyl
ring to adopt an unfavorable binding conformation.
CB2/CB1 Selectivity. The CB1 selectivity correlated well with
both 1-(2,4-dihalosubstituted)phenyl ring and 2,5-dimethylpyr-
role nucleus. The N-(3,4-dichlorobenzyl)carboxamides 26 and
39 showed the highest CB1 selectivity (CB2/CB1 S.I. ) 140.7
and 112.6, respectively). Also, the CB2 selectivity was affected
by the substituent at the carboxamide nitrogen. In particular,
among the 1-(dihalophenyl)pyrazoles, N-cyclohexylcarboxam-
ides generally exhibited high CB2 selectivity; 33 and 37 were
the most CB2 selective compounds (CB2/CB1 S.I. ) 0.09 and
0.04, respectively; Chart 2).
Functional Activity at Mouse CB1 Receptors. Compounds
26, 39, and, particularly, 30 behaved as inverse agonists

1564 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
SilVestri et al.
Scheme 1. Synthesis of 1-Phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides 11–19 and 22–44a
a Reagents and conditions: (a) 18-crown-6, t-BuOK, THF, 60 °C, 30 min; (b) R1-phenylhydrazine (R1 ) H, 2,4,6-Cl3), 37% HCl, ethanol, reflux, 6 h,
40–50%; (c) R1-phenylhydrazine (R1 ) 2,4-Cl2, 3,4-Cl2, 2,4-F2) hydrochloride, ethanol, reflux, 2 h, 47–89%; (d) MeCOOH/MeCOONa (1:1), ethanol, water,
-5 °C, 4 h; (e) EtONa, anhydrous ethanol, room temp, 3 h, 65–88%; (f) 2,5-dimethoxytetrahydrofuran, glacial acetic acid, reflux 1 h, 60–96%; (g)
acetonylacetone, glacial acetic acid, reflux 1.5 h, 65–90%; (h) LiOH, tetrahydrofuran, water, room temp, overnight, 83–99%; (i) amine, EDC/HOBt,
dichloromethane, room temp, overnight, 30–93%.
Scheme 2. Synthesis of 1-Phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-4-carboxamides 20 and 21a
a Reagents and conditions: (a) EtONa, anhydrous ethanol, reflux, 1 h; (b) 2,5-dimethoxytetrahydrofuran, glacial acetic acid, reflux, 1 h; (c) 3 N NaOH,
ethylene glycole, reflux, 24 h; (d) amine, EDC/HOBt, dichloromethane, room temp, 40–43%.
inasmuch as they stimulated forskolin-induced cAMP formation
by N18TG2 cells, much in the same way as the previously
reported CB1 inverse agonist 1 (Figure 1A,B). The efficacy of
the three compounds seems to reflect their affinity at CB1
receptors (Table 1), because compounds 26 (EC50 ∼25 nM) and
39 (EC50 > 60 nM) were significantly less efficacious than
compound 30 (EC50 ∼1 nM), and their inhibition of forskolin-
induced cAMP formation was only statistically significant at
the highest dose tested (200–300 nM). Indeed, the lack of effect
of these two compounds at the two lowest doses might be
attributed to neutral antagonism or lack of functional activity.
The stimulation of cAMP formation by the most potent CB1
agonist among the presently reported compounds, that is,
compound 30, was (a) dose-related, (b) observed also in the
absence of forskolin, and (c) not observed in the presence of
the CB1 agonist WIN55,212-2, which instead inhibited forskolin-

1-Aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1565
Chart 2. Key Structural Elements for CB1 and CB2 Selectivity
affinity of 1, 26, and 30 for the inactive state of the receptor,
thus explaining their inverse agonist activity.
The H-bonding analysis also showed the presence of an
additional H-bond between the ligand carboxamide NH and the
S7.39(383) OH oxygen, with an average occupancy of 5% for
1, 22% for 26, and 7% for 30. An interesting difference between
the three compounds is that the carboxamide NH of 26 is
moderately (22%) occupied by H-bonding with S7.39(383)
(Figure 2), whereas the same NH of 1 and 30 interacts only
sporadically with S7.39(383) (5% and 7%, respectively). The
latter result is substantiated by the recent site-directed mutation
data from Kapur and co-workers, which showed that the binding
of 1 does not change when amino acid S7.39(383) is mutated
into alanine.48 In contrast, compound 26 interacts weakly with
K3.28(192) (35%) compared to 1 and 30 and, at the same time,
forms a weak interaction with S7.39(383) (22%). Thus, 26
should have a nearly equal preference for inactive and active
state of the receptor and, consequently, should behave as a
neutral antagonist, according with the above-described functional
studies on mouse CB1 receptors.
N-Substituent of carboxamide well fitted in a pocket formed
by the lipophilic N1.31(116), I1.34(119), F2.57(170), F2.61(174),
F2.64(177), and A7.36(380) residues. In particular, the 3,4-
dichlorobenzyl ring of 26 was involved in π-stacking interac-
tions with both F2.61(174) and F2.64(177) aromatic rings, while
the 2,4-dichlorophenyl ring formed hydrophobic interactions
with a pocket made up by the residues L3.29(193), I6.54(362),
M6.55(363), and V6.59(367). The 2,5-dimethylpyrrole ring
directly stacked with both W5.43(279) and W6.48(356) residues
and placed the two methyls in close contact with the hydro-
phobic residues L6.51(359) and V3.32(196), respectively. The
methyl groups at positions 2 and 5 of the pyrrole ring showed
a positive effect on the binding affinity. This result was
consistent with a nice accommodation of the 2,5-dimethylpyrrole
nucleus into a tiny hydrophobic pocket delimited by L6.51(359)
and V3.32(196).
Both the 3,4-dichlorophenyl ring and pyrrole nucleus of 30
had direct aromatic stacking interactions with W5.43(279),
Y5.39(275), and W6.48(356) residues. As depicted in Figure
4, these residues interacted in the same way with the 2,4-
dichlorophenyl and the 4-chlorophenyl rings of 1. The ligand
bound the aromatic residue-rich TM3-4-5-6 region of the CB1
receptor(residuesF3.36(200),Y5.39(275),F5.42(278),W5.43(279),
and W6.48(356)), colored magenta in Figures 2-4, and formed
a large aromatic cluster in the minimized complex. The π-π
interactions were consistent with the affinity trend of the tested
compounds. In fact, the electron-deficient halogenated phenyl
rings would realize favorable aromatic stacking interactions with
the electron-rich W5.43(279), Y5.39(275), and W6.48(356)
residues. These results were in agreement with chimeric and
site-mutagenesis studies. Studies on chimeric CB1/CB2 receptors
demonstrated that the region delineated by TM4 and TM5 was
crucial for the high-affinity binding of 1 (EL2 did not affect its
binding).49 Recently, McAllister reported that mutating the
aromatic residues F3.36(200), W5.43(279), and W6.48(356) of
CB1 receptor with alanine resulted in a significant loss of
affinity.46 This finding highlighted the role of these native
residues for the binding affinity of 1.
induced cAMP formation by N18TG2 cells (Figure 1). Com-
pound 30, at a per se inactive dose (0.1 nM), also antagonized
the effect of WIN55,212-2 on forskolin-induced cAMP forma-
tion (Figure 1A).
To elucidate other factors behind the binding affinity of the
above-discussed derivatives, molecular docking and MD simu-
lations were performed at a homology model of hCB1 receptor
built using bovine rhodopsin crystal structure as a template.42
Details of the model building are given in the Experimental
Section.
Docking Studies and MD Simulations. To investigate the
ligand recognition site and rationalize SARs, we performed
docking studies of the highly active hCB1 ligands 1, 26, and 30
using our hCB1 receptor model. The most favorable binding
locations and orientations of 1, 26, and 30 were determined using
GOLD 3.1.43,44 Consistency with SARs and site-mutagenesis
data were taken into account in the choice of the best ligand
binding mode.
Mutation data indicated that K3.28(192) is involved in a direct
interaction with the carboxamide of 1 in WT CB1 receptor.45
Among the top-ranking docking conformations (fitness GOLD
score range 46–52 kJ/mol), we chose that involving a H-bond
between the carbonyl oxygen of both ligands and the lysine
nitrogen of the receptor. This binding pose placed the 2,4- and
3,4-dichlorophenyl rings at position 1 of the pyrazole close to
F3.36(200), Y5.39(275), and W5.43(279) residues, in agreement
with previous combined mutation/modeling studies performed
by McAllister.46
To take into account the protein flexibility and evaluate the
dynamic stability of the predicted ligand/receptor interactions,
the complexes obtained from the docking procedure were
submitted to MD simulations for 400 ps at 300 K (see
Experimental Section for details). Analysis of the MD trajec-
tories showed that the three ligands assumed stable binding poses
during the simulation time, as confirmed by their low rmsd
ranges (1.2 Å for 1, 1.4 Å for 26, and 1.1 Å for 30). Some
distances between the ligands and receptor key residues were
also stable throughout the whole simulation period and ac-
counted for their high hCB1 affinity. The binding of 1, 26 (Figure
2), and 30 (Figure 3) occurred in the upper region of the helical
bundle, and the ligands occupied the same area (approximately)
of the binding cavity. The structural correspondence between
the receptor-bound conformations of 1 and 30 inside the hCB1
receptor binding domain is shown in Figure 4.
In agreement with the results of Hurst and co-workers,45,47
the carboxamide oxygen of the three ligands accepted a H-bond
from K3.28(192), which in turn was involved in a salt-bridge
with D6.58(366). This H-bond was maintained throughout the
MD simulation with a frequency of formation of 61% for 1,
35% for 26, and 60% for 30. This finding accounts for the high
Conclusions
We have described the synthesis of new 1-aryl-5-(1H-pyrrol-
1-yl)pyrazole-3-carboxamides based on the bioisosteric replace-
ment of the 4-chlorophenyl group of 1 with a pyrrole nucleus.
In hCB1 and hCB2 receptor binding affinity assays, several

1566 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
SilVestri et al.
Figure 1. Forskolin-induced cAMP formation by compounds 30 (A) and 26 and 39 (B). Cyclic AMP assays were performed on intact confluent
N18TG2 cells. The data for compounds 26 and 39 were obtained in the presence of forskolin 1 µM. The data for compound 30 were obtained both
in the absence and presence of forskolin and both in the absence and presence of the CB1 agonist WIN55,212-2 (Win, 100 nM), whose effect is
also shown. The effect of the CB1 receptor inverse agonist/antagonist, SR141716A (SR), is also shown. #P < 0.05 vs forskolin; §P < 0.05 vs Win
+ Forskolin; @P < 0.05 vs 30 + Forskolin (200 nM).
carboxamides (23–31, 33, 39, 40, and 44) showed Ki values
only 1 order of magnitude higher than that of 2, taken in this
study as a reference compound. Compound 26 was the most
selective ligand for the hCB1 receptor (S.I. ) 140.7, 2.9-fold
higher than 2). The N-cyclohexylcarboxamide 30 (Ki ) 5.6 nM),
the most potent hCB1 ligand, showed receptor affinity compa-
rable to that of 2 (Ki ) 2.3 nM) and behaved as an inverse
agonist in the cAMP assay (EC50 ∼1 nM). At CB2 receptors,
compounds 30 (Ki ) 1.7 nM) and 33 (Ki ) 1.3 nM) showed
higher binding affinities than that of the hCB2 reference
compound 7 (Ki ) 5.4 nM). However, because of the concomi-
tant high hCB1 affinity, these ligands were poorly selective.
Compound 37, endowed with low hCB1 affinity, proved to be
the most selective hCB2 ligand (CB1 S.I. ) 0.04). Docking
studies and MD simulations of 1 and the most selective (26)
and potent (30) hCB1 ligands were undertaken using an inactive
state model of hCB1 receptor. The binding of 1, 26, and 30
occurred in the upper region of the helical bundle, where the
1-phenyl and the 5-pyrrole/5-phenyl rings of these ligands
formed aromatic stacking interactions. The 2,5-dimethylpyrrole
moiety of 26 positively affected the hCB1 binding affinity as a
result of a nice accommodation into a tiny hydrophobic pocket.
Carboxamide N-substituent well fitted in a pocket formed by
lipophilic residues. The ability of the carboxamide to form a
H-bond with K3.28(192) accounted for the high affinity for
hCB1 receptor inactive state and the inverse agonist activity of
26 and 30. In conclusion, the bioisosteric replacement of the
4-chlorophenyl group of 1 with a pyrrole ring furnished new
potent hCB1 inverse agonists. Not unexpectedly, fine chemical
modifications of the same molecular scaffold led to potent hCB2
ligands as well. However, the issue of selectivity still has to be
addressed for this new class of cannabinergic agents.
Experimental Section
Chemistry. Melting points (mp) were determined on a Büchi
510 apparatus, a Kofler hot stage apparatus or using a Mettler
FPI apparatus (2 °C/min) and are uncorrected. Infrared spectra
(IR) were run on a SpectrumOne FT spectrophotometer or on a
Perkin-Elmer 1600 spectrophotometer. Band position and ab-
sorption ranges are given in cm-1. Proton nuclear magnetic
resonance (1H NMR) spectra were recorded on Bruker 200 and
400 MHz FT spectrometers in the indicated solvent. Chemical
shifts are expressed in δ units (ppm) from tetramethylsilane.
Mass spectral data were determined with an Agilent 1100
spectrometer LC/MSD (electrospray) VL (G1946C). Column
chromatography was performed on columns packed with alumina
from Merck (70–230 mesh) or silica gel from Merck (70–230
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 by a Spectroline 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 vacuum controller and Büchi

1-Aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1567
Figure 2. Complex of the hCB1 receptor with compound 26. (a) View from the plane of the cell membrane of the 26/hCB1 complex. (b) View
from outside the cell of the 26/hCB1complex. Only amino acids located within 4 Å distance from the ligand (cyan) 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 orange lines.
V-700 and V-710 vacuum pumps. Elemental analyses were found
within (0.4% of the theoretical values. Büchi Syncore reactor
was used for parallel synthesis, filtration, and evaporation.
Synthesis of Amides 11–44. General Procedure. To a solution
of the appropriate acid, 73–86 or 89 (1 mmol), in dichloromethane,
kept at 0 °C, EDC hydrochloride (1.2 mmol), and HOBt (1.0 mmol)
were added followed by the appropriate amine (1.5 mmol). The
solution was allowed to warm up at room temperature and placed
in the Büchi Syncore reactor. The reaction was stirred at 300 rpm
overnight and then morpholinomethyl-polystyrene (3 equiv/mol)
was added. After 1 h at room temperature, polymer bound
p-toluensulfonic acid (3 equiv/mol) was added and the mixture was
stirred for 24 h at room temperature. The mixture was filtered and
the solution was evaporated to dryness under reduced pressure.
Purification of the crude product by crystallization from dichlo-
romethane furnished the carboxamide 11–44.
ppm (broad s, 1H). IR: ν 1681 cm-1. MS m/z: 350 [M + 1]+ (100),
372 [M + 23]+ (20), 721 [2M + 23]+(50). Anal. (C20H23N5O
(349.43)) C, H, N.
N-Piperidin-1-yl 4-Cyano-1-phenyl-5-(1H-pyrrol-1-yl)-1H-
pyrazole-3-carboxamide (13). Yield 31% (white solid), mp
1
242–243 °C. H NMR (CDCl3): δ 1.42–1.48 (m, 2H), 1.77–1.91
(m, 4H), 3.09–3.25 (m, 4H), 6.29–6.31 (m, 2H), 6.66–6.68 (m, 2H),
7.15–7.21 (m, 2H), 7.40–7.42 (m, 3H), 8.25 ppm (broad s, 1H).
IR: ν 1695, 2243 cm-1. MS m/z: 361 [M + 1]+ (100), 383 [M +
23]+ (60). Anal. (C20H20N6O (360.41)) C, H, N.
N-3,4-Dichlorobenzyl 4-Cyano-1-phenyl-5-(1H-pyrrol-1-yl)-
1H-pyrazole-3-carboxamide (14). Yield 41% (white solid), mp
152–154 °C. 1H NMR (CDCl3): δ 4.58 (d, J ) 6.31 Hz, 2H), 6.31
(s, 2H), 6.70 (s, 2H), 7.13–7.21 (m, 3H), 7.37–7.42 ppm (m, 5H).
IR: ν 1686, 2242 cm-1. MS m/z: 436 [M + 1]+ (40), 459 [M +
23]+ (100). Anal. (C22H15Cl2N5O (436.29)) C, H, Cl, N.
N-Piperidin-1-yl 1-Phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-
carboxamide (11). Yield 57% (pink solid), mp 166–168 °C. H
N-Piperidin-1-yl 1-(4-Chlorophenyl)-5-(1H-pyrrol-1-yl)-1H-
pyrazole-3-carboxamide (15). Yield 30% (beige solid), mp
175–176 °C. H NMR (CDCl3): δ 1.51–1.53 (m, 2H), 1.80–1.88
(m, 4H), 3.19–3.65 (m, 4H), 6.25–6.27 (m, 2H), 6.57–6.59 (m, 2H),
6.93 (s, 1H), 7.05 (d, J ) 8.80, 2H), 7.30 (d, J ) 8.80, 2H), 8.31
ppm (broad s, 1H). IR: ν 1686 cm-1. MS m/z: 370 [M + 1]+ (100),
392 [M + 23]+ (30), 761 [2M + 23]+ (50). Anal. (C19H20ClN5O
(369.85)) C, H, Cl, N.
1
1
NMR (CDCl3): δ 1.56–1.58 (m, 2H), 1.80–1.90 (m, 4Η), 3.28–3.32
(m, 4H), 6.23–6.24 (m, 2H), 6.58–6.59 (m, 2H), 6.92 (s, 1H),
7.11–7.16 (m, 2H), 7.33–7.36 (m, 3H), 8.50 ppm (broad s, 1H).
IR: ν 1682 cm-1. MS m/z: 336 [M + 1]+ (100), 358 [M + 23]+
(30), 693 [2M + 23]+ (90). Anal. (C19H21N5O (335.40)) C, H, N.
N-Piperidin-1-yl 4-Methyl-1-phenyl-5-(1H-pyrrol-1-yl)-1H-
pyrazole-3-carboxamide (12). Yield 62% (beige solid), mp
N-Cyclohexyl 1-(4-Chlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyra-
zole-3-carboxamide (16). Yield 68% (white solid), mp 119–122
1
188–190 °C. H NMR (CDCl3): δ 1.48–1.68 (m, 2H), 1.75–1.89
1
(m, 4Η), 2.24 (s, 3H), 3.96–4.11 (m, 4H), 6.28–6.29 (m, 2H),
°C. H NMR (CDCl3): δ 1.17–1.46 (m, 6Η), 1.50–1.78 (m, 2Η),
6.58–6.59 (m, 2H), 7.04–7.09 (m, 2H), 7.27–7.30 (m, 3H), 7.83
1.98–2.03 (m, 2H), 3.93–3.98 (m, 1H), 6.25–6.27 (m, 2H),

1568 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
SilVestri et al.
Figure 3. Complex of the hCB1 receptor with the potent inverse agonist 30. (a) View from the plane of the cell membrane of the 30/hCB1
complex. (b) View from outside the cell of the 30/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 ligand are colored in magenta. H-bonds are indicated by dashed
orange lines.
Figure 4. Structural correspondence of the receptor-bound conformations of 1 (white) and 30 (green) inside the hCB1 receptor binding domain.
Only amino acids located within 4 Å distance from the ligands 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 orange lines.
6.58–6.61 (m, 2H), 6.79 (d, J ) 8.32 Hz, 1H), 6.90 (s, 1H), 7.04
(d, J ) 8.80 Hz, 2H), 7.31 ppm (d, J ) 8.80 Hz, 2H). IR: ν 1664
cm-1. MS m/z: 369 [M + 1]+ (60), 391 [M + 23]+ (80), 759 [2M
+ 23]+. Anal. (C20H21ClN4O (368.86)) C, H, Cl, N.
N-Piperidin-1-yl 1-(4-Chlorophenyl)-4-methyl-5-(1H-pyrrol-
1-yl)-1H-pyrazole-3-carboxamide (17). Yield 65% (brown solid),
mp 193–195 °C. 1H NMR (CDCl3): δ 1.44–1.50 (m, 2Η), 1.68–1.81
(m, 4Η), 2.24 (s, 3H), 2.90–2.92 (m, 4H), 6.29–6.31 (m, 2H),

1-Aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1569
6.56–6.58 (m, 2H), 6.98 (d, J ) 8.80 Hz, 2H), 7.26 (d, J ) 8.80
Hz, 2H), 7.76 ppm (broad s, 1H). IR: ν 1681 cm-1. MS m/z: 384
[M + 1]+ (100), 406 [M + 23]+ (60), 789 [2M + 23]+ (40). Anal.
(C20H22ClN5O (383.87)) C, H, Cl, N.
J ) 8.70 Hz, 1H), 7.16–7.19 (m, 2H), 7.23–7.50 ppm (m, 3H). IR:
ν 1673 cm-1. MS m/z: 545 [M + K]+ (100). Anal. (C24H20Cl4N4O
(522.25)) C, H, Cl, N.
N-Cyclohexyl 1-(2,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-pyr-
rol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (27). Yield 65%
(white solid), mp 128 °C. 1H NMR (CDCl3): δ 1.17–1.44 (m, 6H),
1.71–1.88 (m, 2H), 1.92 (s, 6H), 1.96–2.13 (m, 2H), 2.15 (s, 3H),
3.88–3.97 (m, 1H), 5.77 (s, 2H), 6.83 (broad d, J ) 8.02 Hz, 1H,
disappeared on treatment with D2O), 6.88 (d, J ) 8.10 Hz, 1H),
7.15 (dd, J ) 1.97 and 8.37 Hz, 1H), 7.47 ppm (d, J ) 1.97 Hz,
1H). IR: ν 1667 cm-1. MS m/z: 469 [M + Na]+ (100). Anal.
(C23H26Cl2N4O (445.38)) C, H, Cl, N.
N-Piperidin-1-yl 1-(3,4-Dichlorophenyl)-4-methyl-5-(1H-pyr-
rol-1-yl)-1H-pyrazole-3-carboxamide (28). Yield 71% (white
solid), mp 165 °C. 1H NMR (CDCl3): δ 1.44–1.50 (m, 2H),
1.70–1.81 (m, 4H), 2.25 (s, 3H), 2.89 (t, J ) 5.23 Hz, 4H), 6.36
(s, 2H), 6.59 (s, 2H), 6.71 (dd, J ) 2.83 and 8.80 Hz, 1H), 7.32 (d,
J ) 8.80 Hz, 1H), 7.66 ppm (s, 1H). IR: ν 1683 cm-1. MS m/z:
418 [M + 1]+ (100). Anal. (C20H21Cl2N5O (418.32)) C, H, Cl, N.
N-3,4-Dichlorobenzyl 1-(3,4-Dichlorophenyl)-4-methyl-5-(1H-
pyrrol-1-yl)-1H-pyrazole-3-carboxamide (29). Yield 75% (pale
yellow solid), mp 170 °C. 1H NMR (CDCl3): δ 2.27 (s, 3H), 4.58
(d, J ) 6.16 Hz, 2H), 6.36–6.37 (m, 2H), 6.60–6.62 (m, 2H), 6.71
(dd, J ) 2.18 and 8.76 Hz, 1H), 7.23 (d, J ) 2.66 Hz, 1H), 7.32 (d,
J ) 8.74 Hz, 1H), 7.36–7.45 ppm (m, 3H). IR: ν 1676 cm-1. MS
m/z: 517 [M + Na]+ (100). Anal. (C22H16Cl4N4O (494.20)) C, H, Cl,
N.
N-Cyclohexylmethyl 1-(4-Chlorophenyl)-4-methyl-5-(1H-pyr-
rol-1-yl)-1H-pyrazole-3-carboxamide (18). Yield 73% (white
1
solid), mp 148–150 °C. H NMR (CDCl3): δ 0.96–1.17 (m, 2H),
1.22–1.28 (m, 4Η), 1.57–1.82 (m, 5H), 2.25 (s, 3H), 3.27 (t, J )
6.29 Hz, 2H), 6.30–6.32 (m, 2H), 6.58–6.59 (m, 2H), 7.01 (d, J )
8.80 Hz, 2H), 7.05 (broad s, 1H), 7.26 ppm (d, J ) 8.80 Hz, 2H).
IR: ν 1664 cm-1. MS m/z: 397 [M + 1]+ (100), 419 [M + 23]+
(60), 815 [2M + 23]+ (30). Anal. (C22H25ClN4O (396.91)) C, H,
Cl, N.
N-Piperidin-1-yl 1-(4-Chlorophenyl)-4-cyano-5-(1H-pyrrol-
1-yl)-1H-pyrazole-3-carboxamide (19). Yield 41% (white solid),
mp 300–301 °C. 1H NMR (CDCl3): δ 1.23–1.53 (m, 2H), 1.71–1.77
(m, 4Η), 2.88–2.93 (m, 4H), 6.34 (s, 2H), 6.68 (s, 2H) 7.15 (t, J )
8.80 Hz, 2H), 7.38 (d, J ) 8.80 Hz, 2H), 7.54 ppm (broad s, 1H).
IR: ν 1664, 2243 cm-1. MS m/z: 395 [M + 1]+ (100), 417 [M +
23]+ (80), 811 [2M + 23]+ (90). Anal. (C20H19ClN6O (394.86))
C, H, Cl, N.
N-Piperidin-1-yl 1-(4-Chlorophenyl)-3-methyl-5-(1H-pyrrol-
1-yl)-1H-pyrazole-4-carboxamide (20). Yield 40% (white solid)
mp 118–121 °C. 1H NMR (CDCl3): δ 1.30–1.33 (m, 2H), 1.50–1.60
(m, 4Η), 2.43 (s, 3H), 2.46–2.48 (m, 2H), 2.56–2.88 (m, 2H), 6.42
(s, 2H), 6.70 (s, 2H), 6.96 (broad s, 1H), 7.03 (d, J ) 8.80 Hz,
2H), 7.25 ppm (d, J ) 8.80 Hz, 2H). IR: ν 1617 cm-1. MS m/z:
369 [M + 1]+ (40), 391 [M + 23]+ (100), 759 [2M + 23]+(30).
Anal. (C20H22ClN5O (383.87)) C, H, Cl, N.
N-3,4-Dichlorobenzyl 1-(4-Chlorophenyl)-3-methyl-5-(1H-
pyrrol-1-yl)-1H-pyrazole-4-carboxamide (21). Yield 43% (white
solid) mp 123–124 °C. 1H NMR (CDCl3): δ 2.50 (s, 3H), 4.30 (d,
J ) 5.68 Hz, 2H), 5.36 (broad s, 1H), 6.37 (s, 2H), 6.69 (s, 2H),
6.91–7.15 (m, 3H), 7.23–7.33 ppm (m, 4H). IR: ν 1621 cm-1. MS
m/z: 460 [M + 1]+ (10), 482 [M + 23]+ (100), 498 [M + 39]+(40).
Anal. (C22H17Cl3N4O (459,76)) C, H, Cl, N.
N-Cyclohexyl 1-(3,4-Dichlorophenyl)-4-methyl-5-(1H-pyrrol-
1-yl)-1H-pyrazole-3-carboxamide (30). Yield 67% (white solid),
1
mp 160 °C. H NMR (CDCl3): δ 1.26–1.45 (m, 6H), 1.62–1.73
(m, 2H), 1.99–2.04 (m, 2H), 2.24 (s, 3H), 3.92–3.97 (m, 1H),
6.33–6.35 (m, 2H), 6.57–6.59 (m, 2H), 6.72 (dd, J ) 1.89 and 8.78
Hz, 1H), 6.83 (broad d, J ) 8.23 Hz, 1H, disappeared on treatment
with D2O), 7.24 (d, J ) 1.89 Hz, 1H), 7.31 ppm (d, J ) 8.72 Hz,
1H). IR: ν 1663 cm-1. MS m/z: 857 [2M + Na]+ (100). Anal.
(C21H22Cl2N4O (417.33)) C, H, Cl, N.
N-Piperidin-1-yl 1-(2,4-Dichlorophenyl)-4-methyl-5-(1H-pyr-
rol-1-yl)-1H-pyrazole-3-carboxamide (22). Yield 74% (yellow
solid), mp 171 °C. 1H NMR (CDCl3): δ 1.41–1.47 (m, 2H),
1.68–1.79 (m, 4H), 2.27 (s, 3H), 2.85 (t, J ) 5.30 Hz, 4H),
6.13–6.18 (m, 2H), 6.53–6.55 (m, 2H), 7.13–7.29 (m, 2H), 7.45
(d, J ) 1.65 Hz, 1H), 7.59 ppm (s, 1H, disappeared on treatment
with D2O). IR: ν 1681 cm-1. MS m/z: 418 [M + 1]+ (100). Anal.
(C20H21Cl2N5O (418.32)) C, H, Cl, N.
N-3,4-Dichlorobenzyl 1-(2,4-Dichlorophenyl)-4-methyl-5-(1H-
pyrrol-1-yl)-1H-pyrazole-3-carboxamide (23). Yield 80% (pale
yellow solid), mp 140 °C. 1H NMR (CDCl3): δ 2.30 (s, 3H), 4.54
(d, J ) 6.14 Hz, 2H), 6.17–6.19 (m, 2H), 6.55–6.57 (m, 2H),
7.15–7.28 (m, 4H), 7.36–7.45 ppm (m, 2H). IR: ν 1674 cm-1. MS
m/z: 517 [M + Na]+ (100). Anal. (C22H16Cl4N4O (494.20)) C, H,
Cl, N.
N-Piperidin-1-yl 1-(3,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-
pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (31). Yield
64% (white solid), mp 156 °C. 1H NMR (CDCl3): δ 1.42–1.45 (m,
2H), 1.68–1.77 (m, 4H), 1.93 (s, 6H), 2.16 (s, 3H), 2.87 (t, J )
5.11 Hz, 4H), 5.78 (s, 2H), 6.87 (d, J ) 9.20 Hz, 1H), 7.22 (dd, J
) 1.55 and 9.20 Hz, 1H), 7.49 (s, 1H), 7.66 ppm (broad s, 1H,
disappeared on treatment with D2O). IR: ν 1682 cm-1. MS m/z: 446
[M + 1]+ (100). Anal. (C22H25Cl2N5O (446.37)) C, H, Cl, N.
N-3,4-Dichlorobenzyl 1-(3,4-Dichlorophenyl)-5-(2,5-dimethyl-
1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide
(32).
1
Yield 70% (pale yellow solid), mp 118 °C. H NMR (CDCl3): δ
1.85 (s, 6H), 2.22 (s, 3H), 4.60 (d, J ) 6.13 Hz, 2H), 5.96 (s, 2H),
6.60 (dd, J ) 2.12 and 8.74 Hz, 1H), 7.22–7.29 (m, 2H), 7.33–7.49
ppm (m, 3H). IR: ν 1675 cm-1. MS m/z: 545 [M + Na]+ (100).
Anal. (C24H20Cl4N4O (522.25)) C, H, Cl, N.
N-Cyclohexyl 1-(2,4-Dichlorophenyl)-4-methyl-5-(1H-pyrrol-
1-yl)-1H-pyrazole-3-carboxamide (24). Yield 51% (pale yellow
solid), mp 192 °C. 1H NMR (CDCl3): δ 1.20–1.49 (m, 6H),
1.70–1.77 (m, 2H), 1.96–2.02 (m, 2H), 2.28 (s, 3H), 3.86–4.00 (m,
1H), 6.16–6.17 (m, 2H), 6.54–6.56 (m, 2H), 6.28 (broad d, J )
8.00 Hz, 1H, disappeared on treatment with D2O), 7.17–7.28 (m,
2H), 7.45 ppm (s, 1H). IR: ν 1662 cm-1. MS m/z: 440 [M + Na]+
(100). Anal. (C21H22Cl2N4O (417.33)) C, H, Cl, N.
N-Piperidin-1-yl 1-(2,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-
pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (25). Yield
50% (white solid), mp 144 °C. 1H NMR (CDCl3): δ 1.45–1.48 (m,
2H), 1.71–1.80 (m, 4H), 1.84 (s, 6H), 2.19 (s, 3H), 2.93 (t, J )
5.22 Hz, 4H), 5.95 (s, 2H), 6.60 (d, J ) 8.56 Hz, 1H), 7.23–7.34
(m, 2H), 7.71 ppm (s, 1H, disappeared on treatment with D2O).
IR: ν 1681 cm-1. MS m/z: 469 [M + Na]+ (100). Anal.
(C22H25Cl2N5O (446.37)) C, H, Cl, N.
N-Cyclohexyl 1-(3,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-pyr-
rol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (33). Yield 79%
(white solid), mp 182 °C. 1H NMR (CDCl3): δ 1.19–1.47 (m, 6H),
1.63–1.75 (m, 2H), 1.84 (s, 6H), 2.02–2.07 (m, 2H), 2.20 (s, 3H),
3.95–4.00 (m, 1H), 5.95 (s, 2H), 6.61 (dd, J ) 2.58 and 8.82 Hz,
1H), 6.87 (broad d, J ) 8.07 Hz, 1H, disappeared on treatment
with D2O), 7.22 (s, 1H), 7.32 ppm (d, J ) 8.82 Hz, 1H). IR: ν
1662 cm-1. MS m/z: 445 [M + 1]+ (100). Anal. (C23H26Cl2N4O
(445.38)) C, H, Cl, N.
N-Piperidin-1-yl 1-(2,4-Difluorophenyl)-4-methyl-5-(1H-pyr-
rol-1-yl)-1H-pyrazole-3-carboxamide (34). Yield 78% (yellow
solid), mp 176 °C. 1H NMR (CDCl3): δ 1.39–1.47 (m, 2H),
1.60–1.79 (m, 4H), 2.28 (s, 3H), 2.85 (t, J ) 5.35 Hz, 4H),
6.18–6.20 (m, 2H), 6.53–6.57 (m, 2H), 6.81–6.94 (m, 2H),
7.21–7.29 (m, 1H), 7.73 ppm (broad s, 1H, disappeared on treatment
with D2O). IR: ν 1682 cm-1. MS m/z: 386 [M + 1]+ (100). Anal.
(C20H21F2N5O (385.41)) C, H, F, N.
N-3,4-Dichlorobenzyl 1-(2,4-Dichlorophenyl)-5-(2,5-dimethyl-
1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide
(26).
N-Morpholin-4-yl 1-(2,4-Difluorophenyl)-4-methyl-5-(1H-pyr-
rol-1-yl)-1H-pyrazole-3-carboxamide (35). Yield 93% (white
solid), mp 152–155 °C. 1H NMR (CDCl3): δ 2.12 (s, 3H), 3.65–3.80
Yield 60% (white solid), mp 132 °C. 1H NMR (CDCl3): δ 1.94 (s,
6H), 2.18 (s, 3H), 4.56 (d, J ) 6.17 Hz, 2H), 5.79 (s, 2H), 6.74 (d,

1570 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
SilVestri et al.
(m, 6H), 3.88–3.95 (m, 2H), 6.19–6.21 (m, 2H), 6.56–6.57 (m, 2H),
6.81–6.90 (m, 2H), 7.14–7.21 ppm (m, 1H). IR: ν 1631 cm-1. MS
m/z: 395 [M + Na]+ (100). Anal. (C19H19F2N5O2 (387.38)) C, H,
F, N.
N-3,4-Dichlorobenzyl 1-(2,4-Difluorophenyl)-4-methyl-5-(1H-
pyrrol-1-yl)-1H-pyrazole-3-carboxamide (36). Yield 85% (yellow
solid), mp 124 °C. 1H NMR (CDCl3): δ 2.28 (s, 3H), 4.55 (d, J )
6.22 Hz, 2H), 6.19–6.20 (m, 2H), 6.55–6.56 (m, 2H), 6.82–6.91
(m, 2H), 7.16–7.36 (m, 3H), 7.39–7.43 ppm (m, 1H). IR: ν 1676
cm-1. MS m/z: 484 [M + Na]+ (100). Anal. (C22H16Cl2F2N4O
(461.29)) C, H, Cl, F, N.
N-Cyclohexyl 1-(2,4-Difluorophenyl)-4-methyl-5-(1H-pyrrol-
1-yl)-1H-pyrazole-3-carboxamide (37). Yield 87% (pale yellow
solid), mp 122 °C. 1H NMR (CDCl3): δ 1.20–1.49 (m, 6H),
1.60–1.79 (m, 2H), 1.96–2.02 (m, 2H), 2.26 (s, 3H), 3.91–3.96 (m,
1H), 6.19–6.20 (m, 2H), 6.55–6.56 (m, 2H), 6.78–6.92 (m, 2H),
7.26–7.33 ppm (m, 1H). IR: ν 1660 cm-1. MS m/z: 791 [2M +
Na]+ (100). Anal. (C21H22F2N4O (384.42)) C, H, F, N.
N-Piperidin-1-yl 1-(2,4-Difluorophenyl)-5-(2,5-dimethyl-1H-
pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (38). Yield
60% (white solid), mp 175 °C. 1H NMR (CDCl3): δ 1.43–1.48 (m,
2H), 1.69–1.80 (m, 4H), 1.88 (s, 6H), 2.15 (s, 3H), 2.87 (t, J )
5.30 Hz, 4H), 5.78 (s, 2H), 6.83–6.92 (m, 2H), 7.06–7.23 (m, 1H),
7.65 ppm (broad s, 1H, disappeared on treatment with D2O). IR: ν
1683 cm-1. MS m/z: 414 [M + 1]+ (100). Anal. (C22H25F2N5O
(413.46)) C, H, F, N.
Potassium 1-Cyano-3-ethoxy-3-oxoprop-1-en-2-olate (45). Di-
ethyl oxalate (4.30 g, 4.00 mL, 0.029 mol) was added to a solution
of 18-crown-6 (0.63 g, 0.0024 mol) and potassium tert-butoxide
(3.25 g, 0.029 mol) in anhydrous THF (27.26 mL) at 0 °C. The
mixture was heated at 60 °C while anhydrous acetonitrile (1.19 g,
1.51 mL, 0.029 mol) was added dropwise, and then the reaction
was stirred at 60 °C for 30 min. After cooling, the solid was filtered
and washed with anhydrous diethyl ether to give 45 (3.96 g, yield
76%), mp 107–109 °C dec (lit.50 mp 105 °C dec.).
Potassium 2-Cyano-4-ethoxy-4-oxobut-2-en-2-olate (46). Syn-
thesized as 46 using propionitrile. Yield 84%, mp 113 °C dec. 1H
NMR (DMSO-d6): δ 1.19 (t, J ) 7.10 Hz, 3H), 1.51 (s, 3H), 4.02
ppm (q, J ) 7.11 Hz, 2H). IR: ν 1708, 2174 cm-1. Anal.
(C7H8KNO3 (193.24)) C, H, N.
Ethyl 2-Chloro-2-phenylhydrazonoacetate (47). A solution of
aniline (10.24 g, 10.02 mL, 0.11 mol), 37% HCl (28 mL), ethanol
(14 mL), and water (14 mL) was cooled at -5 °C, and then a
solution of sodium nitrite (8.28 g, 0.12 mol) in water (38 mL) was
added dropwise while the temperature was maintained below 5 °C.
A cold solution of ethyl 2-chloroacetoacetate (18.10 g, 15.2 mL,
0.11 mol), sodium acetate trihydrate (22.45 g, 1.265 mol) in ethanol
(270 mL), and water (30 mL) was added, and the reaction was
stirred at -5 °C for 4 h. The reaction was quenched with water (4
L) and stirred overnight. The solid was collected and recrystallized
from ethanol to furnish 47 (21.32 g, yield 85%), mp 77–79 °C (from
1
ethanol; lit.51 mp 78–79 °C). H NMR (CDCl3): δ 1.43 (t, J )
7.13 Hz, 3H), 4.41 (q, J ) 7.12 Hz, 2H), 7.06 (t, J ) 7.33 Hz,
1H), 7.25 (d, J ) 7.64 Hz, 2H), 7.36 (t, J ) 7.93 Hz, 2H), 8.36
ppm (broad s, 1H, disappeared on treatment with D2O). IR: ν 1712,
3277 cm-1. Anal. (C10H11ClN2O2 (226.66)) C, H, Cl, N.
Ethyl 2-Chloro-2-(4-chlorophenylhydrazono)acetate (48). Syn-
thesized as 48 using 4-chloroaniline. Yield 97%, mp 144–145 °C
(from ethanol; lit.52 mp 144–145 °C). 1H NMR (DMSO-d6): δ 1.42
(t, J ) 7.13 Hz, 3H), 4.40 (q, J ) 7.13 Hz, 2H), 7.18 (d, J ) 8.87
Hz, 2H), 7.30 (d, J ) 8.98 Hz, 2H), 8.37 ppm (broad s, 1H,
disappeared on treatment with D2O). IR: ν 1706, 3264 cm-1. Anal.
(C10H10Cl2N2O7 (261.10)) C, H, Cl, N.
N-3,4-Dichlorobenzyl 1-(2,4-difluorophenyl)-5-(2,5-dimethyl-
1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide
(39).
Yield 60% (white solid), mp 163 °C. 1H NMR (CDCl3): δ 1.89 (s,
6H), 2.18 (s, 3H), 4.56 (d, J ) 6.23 Hz, 2H), 5.79 (s, 2H), 6.82–6.92
(m, 2H), 7.05–7.10 (m, 1H), 7.12–7.19 (m, 1H), 7.33–7.46 ppm
(m, 2H). IR: ν 1674 cm-1. MS m/z: 513 [M + K]+ (100). Anal.
(C24H20Cl2F2N4O (489.34)) C, H, Cl, F, N.
N-Cyclohexyl 1-(2,4-Difluorophenyl)-5-(2,5-dimethyl-1H-pyr-
rol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (40). Yield 55%
(yellow solid), mp 126 °C. 1H NMR (CDCl3): δ 1.19–1.46 (m,
6H), 1.69–1.78 (m, 2H), 1.88 (s, 6H), 1.95–2.04 (m, 2H), 2.15 (s,
3H), 3.93–3.97 (m, 1H), 5.78 (s, 2H), 6.83–6.92 (m, 2H), 7.08–7.19
ppm (m, 1H). IR: ν 1660 cm-1. MS m/z: 413 [M + 1]+ (100).
Anal. (C23H26F2N4O (412.48)) C, H, F, N.
Ethyl
5-Amino-4-methyl-1-phenyl-1H-pyrazole-3-carboxylate
(50). HCl (37%, 0.35 mL) was added to a solution of phenylhy-
drazine (0.5 g, 0.45 mL, 0.0046 mol) and 46 (0.89 g, 0.0046 mol)
in EtOH (10 mL). The reaction mixture was refluxed for 6 h, and
after cooling, water was added and the reaction was extracted with
ethyl acetate. The organic layer was washed with saturated sodium
hydrogencarbonate solution and then with brine and dried. Removal
of the solvent furnished a residue that was purified by silica gel
column chromatography (ethyl acetate as eluent) to give 50 (0.63
g, yield 56%), mp 108–111 °C (from toluene). 1H NMR (DMSO-
d6): δ 1.29 (t, J ) 7.06 Hz, 3H), 2.10 (s, 3H), 4.25 (q, J ) 6.99
Hz, 2H), 5.20 (broad s, 2H, disappeared on treatment with D2O),
7.40–7.42 (m, 1H), 7.43–7.56 (m, 2H), 7.59–7.62 ppm (m, 2H).
IR: ν 1708, 3329 cm-1. Anal. (C13H15N3O2 (245.28)) C, H, N.
Ethyl 5-Amino-1-phenyl-1H-pyrazole-3-carboxylate (49). Syn-
thesized as 50 using 45. Yield 78%, mp 95–97 °C (from toluene).
1H NMR (DMSO-d6): δ 1.28 (t, J ) 7.10 Hz, 3H), 4.25 (q, J )
7.11 Hz, 2H), 5.56 (broad s, 2H, disappeared on treatment with
D2O), 5.91 (s, 1H), 7.43 (t, J ) 7.27 Hz, 1H), 7.54 (t, J ) 7.75
Hz, 2H), 7.60 ppm (d, J ) 7.34 Hz, 2H). IR: ν 1707, 3183, 3301,
3395 cm-1. Anal. (C12H13N3O2 (231.25)) C, H, N.
N-Piperidin-1-yl 1-(2,4,6-Trichlorophenyl)-4-methyl-5-(1H-
pyrrol-1-yl)-1H-pyrazole-3-carboxamide (41). Yield 75% (pale
yellow solid), mp 176–177 °C. 1H NMR (CDCl3): δ 1.38–1.47 (m,
2H), 1.65–1.79 (m, 4H), 2.25 (s, 3H), 2.85 (t, J ) 5.28 Hz, 4H),
6.16–6.17 (m, 2H), 6.60–6.61 (m, 2H), 7.38 (s, 2H), 7.57 ppm
(broad s, 1H, disappeared on treatment with D2O). IR: ν 1684 cm-1
.
MS m/z: 453 [M + 1]+ (100). Anal. (C20H20Cl3N5O (452.76)) C,
H, Cl, N.
N-3,4-Dihlorobenzyl 1-(2,4,6-Trichlorophenyl)-4-methyl-5-
(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (42). Yield 68%
(pale yellow solid), mp 72–74 °C. 1H NMR (Acetone- d6): δ 2.29
(s, 3H), 4.55 (d, J ) 6.12 Hz, 2H), 6.17 (d, J ) 1.87 Hz, 2H), 6.62
(d, J ) 1.86 Hz, 2H), 7.17–7.23 (m, 1H), 7.37–7.44 ppm (m, 4H).
IR: ν 1675 cm-1. MS m/z: 551 [M + Na]+ (100). Anal.
(C22H15Cl5N4O (528.65)) C, H, Cl, N.
N-Piperidin-1-yl 1-(2,4,6-Trichlorophenyl)-5-(2,5-dimethyl-1H-pyr-
rol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (43). Yield 70% (pale
1
yellow solid), mp 80–82 °C. H NMR (CDCl3): δ 1.40–1.45 (m,
Ethyl 5-Amino-4-cyano-1-phenyl-1H-pyrazole-3-carboxylate (51).
Prepared as previously reported.53 Yield 65%, mp 157–161 °C
2H), 1.71–1.79 (m, 4H), 1.94 (s, 6H), 2.11 (s, 3H), 2.87 (t, J )
5.32 Hz, 4H), 5.81 (s, 2H), 7.38 (s, 2H), 7.63 ppm (broad s, 1H,
disappeared on treatment with D2O). IR: ν 1683 cm-1. MS m/z:
481 [M + 1]+ (100). Anal. (C22H24Cl3N5O (480.82)) C, H, Cl, N.
N-Cyclohexyl 1-(2,4,6-Trichlorophenyl)-5-(2,5-dimethyl-1H-
pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (44). Yield
1
(from ethanol; lit.50 mp 157 °C). H NMR (DMSO-d6): δ 1.29 (t,
J ) 7.09 Hz, 3H), 4.31 (q, J ) 7.00 Hz, 2H), 6.40 (broad s, 2H,
disappeared on treatment with D2O), 7.49–7.59 ppm (m, 5H). IR:
ν 1706, 2217, 3222, 3306 cm-1. Anal. (C13H12N4O2 (256.26)) C,
H, N.
1
78% (pale yellow solid), mp 130–131 °C. H NMR (CDCl3): δ
Ethyl 5-Amino-1-(4-chlorophenyl)-1H-pyrazole-3-carboxylate
(52). 4-Chlorophenylhydrazine hydrochloride (0.55 g, 0.0037 mol)
was added to a suspension of 45 (0.50 g, 0.0028 mol) in EtOH (10
mL). The reaction mixture was refluxed for 2 h. After cooling, water
was added and the precipitate was collected, washed with water,
and recrystallized by ethanol to furnish 52 (0.51 g, yield 69%), mp
1.18–1.44 (m, 6H), 1.60–1.71 (m, 2H), 1.94 (s, 6H), 1.97–2.04 (m,
2H), 2.12 (s, 3H), 3.89–3.98 (m, 1H), 5.81 (s, 2H), 6.82 (broad s,
1H, disappeared on treatment with D2O), 7.38 ppm (s, 2H). IR: ν
1662 cm-1. MS m/z: 501 [M + Na]+ (100). Anal. (C23H25Cl3N4O
(479.83)) C, H, Cl, N.

1-Aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1571
1
176–179 °C (from ethanol). H NMR (DMSO-d6): δ 1.29 (t, J )
7.19–7.22 (m, 2H), 7.37–7.43 ppm (m, 3H). IR: ν 1717 cm-1. Anal.
7.10 Hz, 3H), 4.25 (q, J ) 7.09 Hz, 2H), 5.64 (broad s, 2H,
disappeared on treatment with D2O), 5.91 (s, 1H), 7.58 (d, J )
7.30 Hz, 2H), 7.63 ppm (d, J ) 8.94 Hz, 2H). IR: ν 1702, 3225,
3302, 3361 cm-1. Anal. (C12H12ClN3O2 (265.70)) C, H, Cl, N.
(C16H15N3O2 (281.31)) C, H, N.
Ethyl 4-Methyl-1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-
carboxylate (60). Synthesized as 64 using 50. Yield 77%, mp
1
117–120 °C (from cyclohexane). H NMR (DMSO-d6): δ 1.30 (t,
Ethyl 5-Amino-1-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-
carboxylate (53). Synthesized as 52 using 46. Yield 89%, mp
J ) 7.09 Hz, 3H), 2.10 (s, 3H), 4.33 (q, J ) 7.07 Hz, 2H), 6.24 (s,
2H), 6.86 (s, 2H), 7.14–7.16 (m, 2H), 7.34–7.40 ppm (m, 3H). IR:
ν 1723 cm-1. Anal. (C17H17N3O2 (295.34)) C, H, N.
1
184–186 °C (from ethanol). H NMR (DMSO-d6): δ 1.29 (t, J )
7.06 Hz, 3H), 2.10 (s, 3H), 4.25 (q, J ) 6.99 Hz, 2H), 5.28 (broad
s, 2H, disappeared on treatment with D2O), 7.57 (d, J ) 8.57 Hz,
2H), 7.64 ppm (d, J ) 8.81 Hz, 2H). IR: ν 1701, 3231, 3303, 3359
cm-1. Anal. (C13H14ClN3O2 (279.72)) C, H, Cl, N.
Ethyl 5-Amino-1-(4-chlorophenyl)-4-cyano-1H-pyrazole-3-carboxy-
late (54). Synthesized as 51 using 48. Yield 88%, mp 206–207 °C
(from ethanol). 1H NMR (DMSO-d6): δ 1.29 (t, J ) 7.11 Hz, 3H),
4.32 (q, J ) 7.11 Hz, 2H), 7.00 (broad s, 2H, disappeared on
treatment with D2O), 7.57 (d, J ) 8.84 Hz, 2H), 7.63 ppm (d, J )
8.86 Hz, 2H). IR: ν 1727, 2230, 3223, 3303, 3364 cm-1. Anal.
(C13H11ClN4O2 (290.71)) C, H, Cl, N.
Ethyl 4-Cyano-1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-
carboxylate (61). Synthesized as 64 using 51. Yield 71%, mp
190–194 °C (from etanol). H NMR (DMSO-d6): δ 1.35 (t, J )
7.10 Hz, 3H), 4.43 (q, J ) 7.09 Hz, 2H), 6.31 (s, 2H), 6.97 (s,
2H), 7.29–7.32 (m, 2H), 7.46–7.50 ppm (m, 3H). IR: ν 1719, 2242
cm-1. Anal. (C17H14N4O2 (306.32)) C, H, N.
1
Ethyl 1-(4-Chlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-
carboxylate (62). Synthesized as 64 using 52. Yield 77%, yellow
1
oil. H NMR (DMSO-d6): δ 1.33 (t, J ) 7.10 Hz, 3H), 4.35 (q, J
) 7.10 Hz, 2H), 6.24 (s, 2H), 6.89 (s, 2H), 7.10 (s, 1H), 7.22 (d,
J ) 8.77 Hz, 2H), 7.52 ppm (d, J ) 8.78 Hz, 2H). IR: ν 1719
cm-1. Anal. (C16H14ClN3O2 (315.75)) C, H, Cl, N.
Ethyl 5-Amino-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-
3-carboxylate (55). Synthesized as 52 using 2,4-dichlorophenyl-
hydrazine hydrochloride and 46. Yield 47%, mp 179–181 °C (from
Ethyl 1-(4-Chlorophenyl)-4-methyl-5-(1H-pyrrol-1-yl)-1H-
pyrazole-3-carboxylate (63). Synthesized as 64 using 53. Yield
1
ethanol). H NMR (CDCl3): δ 1.40 (t, J ) 7.12 Hz, 3H), 2.20 (s,
1
80%, mp 79–82 °C (from ethanol). H NMR (DMSO-d6): δ 1.34
3H), 3.47 (broad s, 2H, disappeared on treatment with D2O), 4.40
(q, J ) 7.11 Hz, 2H), 7.40 (dd, J ) 2.06 and 8.45 Hz, 1H), 7.43
(d, J ) 8.51 Hz, 1H), 7.56 ppm (d, J ) 1.67 Hz, 1H). IR: ν 1709,
3183, 3383 cm-1. Anal. (C13H13Cl2N3O2 (314.17)) C, H, Cl, N.
(t, J ) 7.09 Hz, 3H), 2.12 (s, 3H), 4.35 (q, J ) 7.10 Hz, 2H), 6.28
(s, 2H), 6.91 (s, 2H), 7.15 (d, J ) 8.19 Hz, 2H), 7.48 ppm (d, J )
8.85 Hz, 2H). IR: ν 1716 cm-1. Anal. (C17H16ClN3O2 (329.78))
C, H, Cl, N.
Ethyl 5-Amino-1-(3,4-dichlorophenyl)-4-methyl-1H-pyrazole-
3-carboxylate (56). Synthesized as 52 using 3,4-dichlorophenyl-
hydrazine hydrochloride and 46. Yield 62%, mp 92–96 °C (from
Ethyl 1-(2,4-Dichlorophenyl)-4-methyl-5-(1H-pyrrol-1-yl)-1H-
pyrazole-3-carboxylate (65). Synthesized as 64 using 55. Yield
91%, mp 179–181 °C (from ethanol). 1H NMR (CDCl3): δ 1.44 (t,
J ) 7.13 Hz, 3H), 2.28 (s, 3H), 4.47 (q, J ) 7.13 Hz, 2H), 6.22 (s,
2H), 6.59 (s, 2H), 7.27–7.29 (m, 2H), 7.44–7.45 ppm (m, 1H). IR:
ν 1716 cm-1. Anal. (C17H15Cl2N3O2 (364.23)) C, H, Cl, N.
1
ethanol). H NMR (DMSO-d6): δ 1.28 (t, J ) 7.06 Hz, 3H), 2.09
(s, 3H), 4.25 (q, J ) 7.05 Hz, 2H), 5.39 (broad s, 2H, disappeared
on treatment with D2O), 7.64 (dd, J ) 2.04 and 8.45 Hz, 1H), 7.77
(d, J ) 8.75 Hz, 1H), 7.89 ppm (d, J ) 2.07 Hz, 1H). IR: ν 1702,
3361 cm-1. Anal. (C13H13Cl2N3O2 (314.17)) C, H, Cl, N.
Ethyl 1-(3,4-Dichlorophenyl)-4-methyl-5-(1H-pyrrol-1-yl)-1H-
pyrazole-3-carboxylate (67). Synthesized as 64 using 56. Yield
61%, mp 114–115 °C (from ethanol). 1H NMR (CDCl3): δ 1.46 (t,
J ) 7.12 Hz, 3H), 2.24 (s, 3H), 4.49 (q, J ) 7.34 Hz, 2H), 6.39 (s,
2H), 6.65 (s, 2H), 6.84 (dd, J ) 2.52 and 8.73 Hz, 1H), 7.33 (d, J
Ethyl 5-Amino-1-(2,4-difluorophenyl)-4-methyl-1H-pyrazole-
3-carboxylate (57). Synthesized as 52 using 2,4-difluorophenyl-
hydrazine hydrochloride and 46. Yield 65%, mp 92–94 °C (from
chloroform). 1H NMR (CDCl3): δ 1.34 (t, J ) 7.18 Hz, 3H), 2.13
(s, 3H), 4.31 (q, J ) 7.05 Hz, 2H), 4.76 (broad s, 2H, disappeared
on treatment with D2O), 6.84–6.99 (m, 2H), 7.07–7.19 ppm (m,
1H). MS m/z: 304 [M + Na]+ (100). Anal. (C13H13F2N3O2 (281.26))
C, H, F, N.
) 2.34 Hz, 1H), 7.35 ppm (d, J ) 8.72 Hz, 1H). IR: ν 1721 cm-1
Anal. (C17H15Cl2N3O2 (364.23)) C, H, Cl, N.
.
Ethyl 1-(2,4-Difluorophenyl)-4-methyl-5-(1H-pyrrol-1-yl)-1H-
pyrazole-3-carboxylate (69). Synthesized as 64 using 57. Yield
1
60%, mp 80–84 °C. H NMR (CDCl3): δ 1.40 (t, J ) 7.27 Hz,
Ethyl 5-Amino-4-methyl-1-(2,4,6-trichlorophenyl)-1H-pyra-
zole-3-carboxylate (58). Synthesized as 50 using 2,4,6-trichlo-
rophenylhydrazine. Yield 40%, mp 209–211 °C (from ethanol). 1H
NMR (CDCl3): δ 1.39 (t, J ) 7.12 Hz, 3H), 2.23 (s, 3H), 3.35
(broad s, 2H, disappeared on treatment with D2O), 4.40 (q, J )
3H), 2.22 (s, 3H), 4.43 (q, J ) 7.07 Hz, 2H), 6.19 (s, 2H), 6.56 (s,
2H), 6.80–6.91 (m, 2H), 7.27–7.39 ppm (m, 1H). IR: ν 1713 cm-1
.
MS m/z: 354 [M + Na]+ (100). Anal. (C17H15F2N3O2 (331.32)) C,
H, F, N.
7.12 Hz, 2H), 7.48 ppm (s, 2H). IR: ν 1702, 3329, 3420 cm-1
Anal. (C13H12Cl3N3O2 (348.61)). C, H, Cl, N.
.
Ethyl 4-Methyl-5-(1H-pyrrol-1-yl)-1-(2,4,6-trichlorophenyl)-
1H-pyrazole-3-carboxylate (71). Synthesized as 64 using 58. Yield
1
96%, brown oil. H NMR (CDCl3): δ 1.43 (t, J ) 7.12 Hz, 3H),
Synthesis of Pyrrole Derivatives 59–65, 67, 69, and 71.
General Procedure. Example: Ethyl 1-(4-Chlorophenyl)-4-
cyano-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxylate (64). 2,5-
Dimethoxytetrahydrofuran (10.97 g, 10.75 mL, 0.083 mol) was
added to a solution of 54 (22.97 g, 0.079 mol) in glacial acetic
acid (561 mL). The reaction was refluxed for 1 h. After cooling,
water was added, and the mixture was concentrated to a small
volume and extracted with saturated ethyl acetate. The organic layer
was washed with saturated sodium hydrogencarbonate solution and
then with brine and dried. Removal of the solvent gave a residue,
which was purified by silica gel column chromatography (chloro-
form as eluent) to furnish 64 (18.80 g, yield 70%), mp 149–152
2.25 (s, 3H), 4.40 (q, J ) 7.14 Hz, 2H), 6.21 (s, 2H), 6.66 (s, 2H),
7.38 ppm (s, 2H). IR: ν 1711 cm-1. Anal. (C17H14Cl3N3O2 (398.67))
C, H, Cl, N.
Synthesis of Dimethylpyrrole Derivatives 66, 68, 70, and
72. General Procedure. Example: Ethyl 1-(3,4-Dichlorophenyl)-
5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-car-
boxylate (68). A solution of 57 (2.01 g, 0.0064 mol) and
acetonylacetone (0.80 g, 0.81 mL, 0.007 mol) in glacial acetic acid
(15 mL) was refluxed for 1.5 h. After cooling, water was added
and the reaction mixture was extracted with ethyl acetate. The
organic layer was washed with saturated sodium hydrogencarbonate
solution and then with brine and dried. Evaporation of the solvent
gave a residue that was purified by silica gel column chromatog-
raphy (chloroform as eluent) to furnish 68 (1.65 g, yield 65%), mp
1
°C (from ethanol). H NMR (DMSO-d6): δ 1.35 (t, J ) 7.08 Hz,
3H), 4.43 (q, J ) 7.06 Hz, 2H), 6.33 (s, 2H), 6.99 (s, 2H), 7.31 (d,
J ) 8.80 Hz, 2H), 7.58 ppm (d, J ) 8.82 Hz, 2H). IR: ν 1702,
2219 cm-1. Anal. (C17H13ClN4O2 (340.76)) C, H, Cl, N.
1
74–78 °C (from ethanol). H NMR (CDCl3): δ 1.48 (t, J ) 7.13
Ethyl 1-Phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxylate
(59). Synthesized as 64 using 49. Yield 82%, mp 53–55 °C (from
Hz, 3H), 1.88 (s, 6H), 2.19 (s, 3H), 4.50 (q, J ) 7.12 Hz, 2H),
5.99 (s, 2H), 6.74 (dd, J ) 2.56 and 8.81 Hz, 1H), 7.30 (d, J )
1
cyclohexane). H NMR (DMSO-d6): δ 1.30 (t, J ) 7.09 Hz, 3H),
2.50 Hz, 1H), 7.35 ppm (d, J ) 8.82 Hz, 1H). IR: ν 1717 cm-1
.
4.33 (q, J ) 7.09 Hz, 2H), 6.19 (s, 2H), 6.84 (s, 2H), 7.06 (s, 1H),
Anal. (C19H19Cl2N3O2 (392.28)) C, H, Cl, N.

1572 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
SilVestri et al.
Ethyl 1-(2,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-
4-methyl-1H-pyrazole-3-carboxylate (66). Synthesized as 68 using
55. Yield 90%, yellow oil. H NMR (DMSO-d6): δ 1.33 (t, J )
7.10 Hz, 3H), 1.92 (s, 6H), 2.03 (s, 3H), 4.34 (q, J ) 7.10 Hz,
2H), 5.79 (s, 2H), 7.26 (d, J ) 8.58 Hz, 1H), 7.51 (dd, J ) 2.33
and 8.57 Hz, 1H), 7.90 ppm (d, J ) 2.29 Hz, 1H). IR: ν 1716
cm-1. Anal. (C19H19Cl2N3O2 (392.28)) C, H, Cl, N.
ppm (broad s, 1H, disappeared on treatment with D2O). IR: ν 1690
cm-1. Anal. (C15H11Cl2N3O2 (336.17)) C, H, Cl, N.
1
1-(2,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-meth-
yl-1H-pyrazole-3-carboxylic Acid (80). Synthesized as 78 using
66. Yield 90%, mp 205–208 °C (from ethanol). 1H NMR (CDCl3):
1.96 (s, 6H), 2.18 (s, 3H), 5.83 (s, 2H), 6.94 (d, J ) 8.59 Hz, 1H),
7.20 (dd, J ) 2.25 and 8.57 Hz, 1H), 7.51 (d, J ) 2.22 Hz, 1H),
13.23 ppm (broad s, 1H, disappeared on treatment with D2O). IR:
ν 1701 cm-1. Anal. (C17H15Cl2N3O2 (364.23)) C, H, Cl, N
1-(3,4-Dichlorophenyl)-4-methyl-5-(1H-pyrrol-1-yl)-1H-pyra-
zole-3-carboxylic Acid (81). Synthesized as 78 using 67. Yield
Ethyl 1-(2,4-Difluorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-
4-methyl-1H-pyrazole-3-carboxylate (70). Synthesized as 68 using
1
57. Yield 71%, mp 98–102 °C. H NMR (CDCl3): δ 1.41 (t, J )
6.98 Hz, 3H), 1.88 (s, 6H), 2.11 (s, 3H), 4.44 (q, J ) 7.13 Hz,
2H), 5.78 (s, 2H), 6.80–6.88 (m, 2H), 7.15–7.26 ppm (m, 1H). IR
ν 1714 cm-1. MS m/z: 382 [M + Na]+ (100). Anal. (C19H19F2N3O2
(359.37)) C, H, F, N.
1
99%, mp 217–220 °C (from ethanol). H NMR (DMSO-d6): 2.10
(s, 3H), 6.33 (s, 2H), 6.94 (s, 2H), 7.10 (dd, J ) 2.51 and 8.75 Hz,
1H), 7.20 (d, J ) 2.48 Hz, 1H), 7.68 (d, J ) 8.76 Hz, 1H), 13.06
ppm (broad s, 1H, disappeared on treatment with D2O). IR: ν 1690
cm-1. Anal. (C15H11Cl2N3O2 (336.17)) C, H, Cl, N.
1-(3,4-Dichlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-meth-
yl-1H-pyrazole-3-carboxylic Acid (82). Synthesized as 78 using
68. Yield 99%, mp 178–172 °C (from ethanol). 1H NMR (DMSO-
d6): 1.83 (s, 6H), 2.08 (s, 3H), 5.98 (s, 2H), 7.01–7.03 (m, 2H),
7.70 (d, J ) 7.31 Hz, 1H), 13.35 ppm (broad s, 1H, disappeared
on treatment with D2O). IR: ν 1694 cm-1. Anal. (C17H15Cl2N3O2
(364.23)) C, H, Cl, N.
1-(2,4-Difluorophenyl)-4-methyl-5-(1H-pyrrol-1-yl)-1H-pyra-
zole-3-carboxylic Acid (83). Synthesized as 78 using 69. Yield
90%, mp 168–170 °C (from chloroform). 1H NMR (CDCl3): δ 2.24
(s, 3H), 6.21 (s, 2H), 6.58 (s, 2H), 6.84–6.92 (m, 2H), 7.34–7.37
(m, 1H), 10.82 ppm (broad s, 1H, disappeared on treatment with
D2O). IR ν 1691 cm-1. Anal. (C15H11F2N3O2 (303.26)) C, H, F,
N.
1-(2,4-Difluorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-4-meth-
yl-1H-pyrazole-3-carboxylic Acid (84). Synthesized as 78 using
70. Yield 90%, mp 168–170 °C (from chloroform). 1H NMR
(CDCl3): δ 2.24 (s, 3H), 6.21 (s, 2H), 6.58 (s, 2H), 6.84–6.92 (m,
2H), 7.34–7.37 (m, 1H), 10.82 ppm (broad s, 1H, disappeared on
treatment with D2O). IR ν 1690 cm-1. Anal. (C15H11F2N3O2
(303.26)) C, H, F, N.
Ethyl 5-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methyl-1-(2,4,6-trichlo-
rophenyl)-1H-pyrazole-3-carboxylate (72). Synthesized as 68
1
using 58. Yield 87%, mp 109–112 °C (from ethanol). H NMR
(CDCl3): δ 1.43 (t, J ) 7.12 Hz, 3H), 1.98 (s, 6H), 2.10 (s, 3H),
4.47 (q, J ) 7.12 Hz, 2H), 5.84 (s, 2H), 7.38 ppm (s, 2H). IR: ν
1712 cm-1. Anal. (C19H18Cl3N3O2 (426.72)) C, H, Cl, N.
Synthesis of Carboxylic Acids 73–86. General Procedure.
Example: 1-(4-Chlorophenyl)-4-cyano-5-(1H-pyrrol-1-yl)-1H-
pyrazole-3-carboxylic Acid (78). Lithium hydroxide monohydrate
(0.15 g, 0.0036 mol) was added to a solution of 64 (0.40 g, 0.0018
mol) in THF (13 mL) and H2O (8.6 mL). The reaction was stirred
at room temperature overnight. The mixture was acidified with 1
N HCl to pH 2 and extracted with ethyl acetate. The organic layer
was washed with brine and dried. After removal of the solvent,
the residue was crystallized from ethanol to give 78 (0.36 g, yield
97%), mp 97–101 °C (from ethanol). 1H NMR (DMSO-d6): δ 6.32
(s, 2H), 6.98 (s, 2H), 7.30 (d, J ) 8.64 Hz, 2H), 7.57 (d, J ) 8.56
Hz, 2H), 13.32 ppm (broad s, 1H, disappeared on treatment with
D2O). IR: ν 1710, 2242 cm-1. Anal. (C15H9ClN4O2 (312.71)) C,
H, Cl, N.
1-Phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxylic Acid
(73). Synthesized as 78 using 59. Yield 83%, mp 218–221 °C (from
1
ethanol). H NMR (DMSO-d6): δ 6.20 (s, 2H), 6.85 (s, 2H), 7.01
(s, 1H), 7.19–7.22 (m, 2H), 7.41–7.43 (m, 3H), 12.99 ppm (broad
s, 1H, disappeared on treatment with D2O). IR: ν 1686 cm-1. Anal.
(C14H11N3O2 (253.26)) C, H, N.
4-Methyl-5-(1H-pyrrol-1-yl)-1-(2,4,6-trichlorophenyl)-1H-
pyrazole-3-carboxylic Acid (85). Synthesized as 78 using 71. Yield
1
93%, mp 234–237 °C (from ethanol). H NMR (CDCl3): δ 2.27
4-Methyl-1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carbox-
(s, 3H), 6.23 (s, 2H), 6.67 (s, 2H), 7.39 (s, 2H), 13.25 ppm (broad
s, 1H, disappeared on treatment with D2O). IR: ν 1696 cm-1. Anal.
(C15H10Cl3N3O2 (370.62)) C, H, Cl, N.
ylic Acid (74). Synthesized as 78 using 60. Yield 96%, mp 195–197
1
°C (from ethanol). H NMR (DMSO-d6): δ 2.08 (s, 3H), 6.24 (s,
2H), 6.87 (s, 2H), 7.12–7.16 (m, 2H), 7.34–7.41 (m, 3H), 13.18
ppm (broad s, 1H, disappeared on treatment with D2O). IR: ν 1690
cm-1. Anal. (C15H13N3O2 (263.28)) C, H, N.
5-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methyl-1-(2,4,6-trichlo-
rophenyl)-1H-pyrazole-3-carboxylic Acid (86). Synthesized as 78
1
using 72. Yield 98%, mp 237–240 °C (from ethanol). H NMR
4-Cyano-1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxy-
(CDCl3): δ 1.99 (s, 6H), 2.14 (s, 3H), 5.86 (s, 2H), 7.40 (s, 2H),
13.12 ppm (broad s, 1H, disappeared on treatment with D2O). IR:
ν 1682, 1703 cm-1. Anal. (C17H14Cl3N3O2 (398.67)) C, H, Cl, N.
5-Amino-1-(4-chlorophenyl)-3-methyl-1H-pyrazole-4-carbo-
nitrile (87). Prepared according to literature.51 Yield 45%, mp
175–178 °C (lit.52 mp 177–178 °C). 1H NMR (CDCl3): δ 2.31 (s,
3H), 4.58 (broad s, 2H, disappeared on treatment with D2O), 7.44
(d, J ) 9.07 Hz, 2H), 7.48 ppm (d, J ) 9.00 Hz, 2H). IR: ν 2215,
3324, 3463 cm-1. Anal. (C11H9N4(232.67)) C, H, Cl, N.
lic Acid (75). Synthesized as 78 using 61. Yield 97%, mp 226–227
1
°C (from ethanol). H NMR (DMSO-d6): δ 6.30 (s, 2H), 6.97 (s,
2H), 7.28–7.30 (m, 2H), 7.45–7.49 (m, 3H) 12.98 ppm (broad s,
1H, disappeared on treatment with D2O). IR: ν 2238, 1709 cm-1
Anal. (C15H10N4O2 (278.27)) C, H, N.
.
1-(4-Chlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carbox-
ylic Acid (76). Synthesized as 78 using 62. Yield 94%, mp 153–155
1
°C (from ethanol). H NMR (DMSO-d6): δ 6.24 (s, 2H), 6.89 (s,
2H), 7.04 (s, 1H), 7.21 (d, J ) 8.78 Hz, 2H), 7.51 (d, J ) 8.79 Hz,
2H), 13.15 ppm (broad s, 1H, disappeared on treatment with D2O).
IR: ν 1709, 2238 cm-1. Anal. (C14H10ClN3O2 (287.70)) C, H, Cl,
N.
1-(4-Chlorophenyl)-3-methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-
4-carbonitrile (88). Synthesized as 64 using 87. Yield 99%, mp
148–152 °C (from ethanol). 1H NMR (CDCl3): δ 2.49 (s, 3H), 6.37
(t, J ) 2.19 Hz, 2H), 6.72 (t, J ) 2.20 Hz, 2H), 7.08 (d, J ) 9.01
Hz, 2H), 7.37 ppm (d, J ) 9.01 Hz, 2H). IR: ν 2232 cm-1. Anal.
(C15H11ClN4(282.73)) C, H, Cl, N.
1-(4-Chlorophenyl)-4-methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-
3-carboxylic Acid (77). Synthesized as 78 using 63. Yield 96%,
1
mp 202–206 °C (from ethanol). H NMR (DMSO-d6): δ 2.10 (s,
1-(4-Chlorophenyl)-3-methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-
4-carboxylic Acid (89). A solution of 88 (0.20 g, 0.00071 mol), 3
N NaOH (1.2 mL), and ethylene glycol (21 mL) was refluxed for
24 h. After cooling, the reaction was quenched with water and made
acidic with 6 N HCl until pH 2. The mixture was extracted with
ethyl acetate. The organic layer was washed with brine and dried.
Evaporation of the solvent furnished a residue that was purified by
silica gel column chromatography (ethyl acetate as eluent) to give
3H), 6.28 (s, 2H), 6.91 (s, 2H), 7.13 (d, J ) 8.80 Hz, 2H), 7.47 (d,
J ) 8.76 Hz, 2H), 13.12 ppm (broad s, 1H, disappeared on treatment
with D2O). IR: ν 1684 cm-1. Anal. (C15H12ClN3O2 (301.73)) C,
H, Cl, N.
1-(2,4-Dichlorophenyl)-4-methyl-5-(1H-pyrrol-1-yl)-1H-pyra-
zole-3-carboxylic Acid (79). Synthesized as 78 using 65. Yield
1
94%, mp 208–214 °C (from ethanol). H NMR (DMSO-d6): 2.11
1
(s, 3H), 6.13 (s, 2H), 6.78 (s, 2H), 7.55 (dd, J ) 2.29 and 8.51 Hz,
1H), 7.70 (d, J ) 8.53 Hz, 1H), 7.81 (d, J ) 2.21 Hz, 1H), 13.41
89 (0.17 g, yield 79%), mp 236–240 °C (from ethanol). H NMR
(DMSO-d6): δ 2.45 (s, 3H), 6.18 (t, J ) 2.18 Hz, 2H), 6.85 (d, J

1-Aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1573
) 2.18 Hz, 2H), 7.05 (d, J ) 9.03 Hz, 2H), 7.43 (d, J ) 9.03 Hz,
2H), 12.56 ppm (broad s, 1H, disappeared on treatment with D2O).
IR: ν 1670 cm-1. Anal. (C15H12ClN3O2 (301.73)) C, H, Cl, N.
CB1 and CB2 Receptor Binding Assays. For both receptor
binding assays, the new compounds were tested as previously
described.39
Cyclic AMP Assay. Cyclic AMP assays were performed on
intact confluent N18TG2 cells plated in six-well dishes and
stimulated for 10 min at 37 °C with 1 µM forskolin in 400 µL of
serum-free Dulbecco’s modified Eagle’s medium containing 20 mM
HEPES, 0.1 mg/mL BSA, and 0.1 mM 1-methyl-3-isobutylxan-
thine.54 Cells were treated with vehicle (methanol, 0.1%), com-
pounds (at various concentrations) or WIN-55,212 (100 nM). After
incubation, 800 µL of ethanol was added, cells were extracted, and
cyclic AMP was determined by means of a cyclic AMP assay kit
(Amersham, U.K.), as advised by the manufacturer.
Computational Methods. Molecular modeling and graphics
manipulations were performed using the INSIGHTII (InsightII
Molecular Modeling Package, Accelrys Inc., San Diego, CA) and
UCSF-CHIMERA software packages,55 running on a Silicon
Graphics Tezro R16000 workstation. Model building and geometry
optimizations of compounds 1, 26, and 30 were accomplished with
the CVFF force field,56 available within INSIGHTII. Energy
minimizations and MD simulations were realized by employing
the AMBER 9 program,57 selecting the parm99 force field.58
Residue Indexing. The convention used for the amino acid
identifiers, according to the approach of Ballesteros and Weinstein,59
facilitates comparison of aligned residues within the family of Group
A GPCRs. To 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.
Construction of the hCB1 Receptor Homology Model. The
structural model of the hCB1 was built using the recently reported
2.8 Å crystal structure of b-Rho42 (PDB entry code 1F88) as a
structural template. Briefly, the hCB1 sequence was retrieved from
the SWISS-PROT database60 and aligned with the sequence of
b-Rho using CLUSTALW software61 with the following settings:
matrix, Blosum series; gap opening penalty, 10; gap extension
penalty, 0.05. Afterward, we checked and, where necessary,
manually corrected this alignment to reflect the known alignment
features of class A GPCRs, such as the highly conserved positions
and gap-free transmembrane regions. The final alignment was in
good agreement with those previously published and consistent with
literature.62,63 Extension of each helix was contemplated by taking
into account the experimental length of the b-Rho helices and the
secondary structure prediction of hCB1 obtained with the PSIPRED
software,64 as well as the sequence conservation in the possible
extensions of the helices. Following Salo’s suggestion,62 we omitted
thehighlyconservedprolineresidueP5.50(215)(Ballesteros-Weinstein’s
nomenclature59) of rhodopsin (which provides the main anchoring).
is likely to be different: EL2 occupies less volume in the upper
part of the binding pocket in CB receptors than in b-Rho.45
Biophysical and mutation studies demonstrated that the inactive
state of the CB1 receptor is stabilized by two salt bridges. The first
one is established between R3.50(214) and D6.30(338) at the
intracellular side and keeps the intracellular ends of TM3 and TM6
close, constraining the receptor in the inactive state.73,74 Upon
activation, this salt bridge is broken with concomitant relaxation
and rotation of TM6 relative to TM3.75–77 The second salt bridge
is formed between the extracellular K3.28(192) and D6.58(366).
This bond might position an inactive state of K3.28(192) for ligand
interaction rather than stabilize the receptor inactive conformation;
this is well-demonstrated by the same level of constitutive activity
exhibited by wild type (WT) CB1 receptor and the CB1 K3.28(192)A
mutant.78 Therefore, the absence of the K3.28(192)/D6.58(366) salt
bridge does not lead to greater ease of activation. Based on this
evidence, receptor construction harmonic distance constraints were
added between R3.50(214) and D6.30(338) and between K3.28(192)
and D6.58(366).
After all connections were made, the whole structure was energy-
minimized using the SANDER module of the AMBER suite of
programs until the rmsd value of the conjugate gradient was 0.001
kcal/mol per Å. To avoid spurious changes in the general fold and
helix packing due to some still unfavorable electrostatic interactions
or steric clashes, an energy penalty force constant of 10 kcal/Å2/
mol on the protein backbone atoms was applied throughout these
calculations. For the conformational refinement of the hCB1, the
minimized structure was then used as the starting point for
subsequent 500 ps of MD simulation, during which the positional
constraints on the protein backbone atoms were gradually released
from 10 to 0.1 kcal/Å2/mol. The options of MD at 300 K with 0.2
ps coupling constant were: time step of 1 fs; update every 25 fs.
The lengths of bonds with hydrogen atoms were constrained
according to the SHAKE algorithm.79
The average structure from the last 100 ps trajectory of MD was
reminimized with backbone constraints in the secondary structure
until a rms of 0.005 kcal/Å ·mol was reached. The stereochemical
quality of the resulting protein structures was evaluated by
inspectionoftheΦ/ꢀRamachandranplotobtainedfromPROCHECK
analysis.80,81 High quality protein models show >90% of the
residues within the most favored regions of the Ramachandran plots.
Analyzing hCB1 model structure, 94% of the residues were within
that region, and only six amino acids occupied disallowed regions,
all far away from the binding site (Figure 5). The rms deviations
between backbone atoms in all helices were compared to the X-ray
structure of rhodopsin as a template. Figure 1S, Supporting
Information, shows the final hCB1 model compared with the b-Rho
crystal structure. As expected, deviations from the ideal R-helical
structure were located in the respective TM regions of both the
receptors. The MD snapshots were obtained through the PTRAJ
module of AMBER.
Individual TM helical segments were built as ideal helices (using
Φ and ꢀ angles of -63.0° and -41.6°, respectively) with side
chains placed in prevalent rotamers and representative proline kink
geometries. Each model helix was capped with an acetyl group at
the N-terminus and an N-methyl group at the C-terminus. These
structures were then grouped by adding one at a time until a helical
bundle (TM region), matching the overall characteristics of the
crystallographic structure of b-Rho, had been obtained. The relative
orientations and interactions between the helices were adjusted on
the basis of incorporated structural inferences from available
experimental data, such as mutation and ligand binding studies,65–67
cysteinescanningdata,68,69 andsite-directedmutationexperiments.70–72
We only modeled receptor TM domains, because the exact
conformation of the extracellular loops (ELs) is not computable
without an experimental receptor structure. In addition, the EL2 of
b-Rho crystal structure42 differs from that of CB1 receptor (it is
arranged to cover the ligand binding site). Besides different lengths
of the loop, the CB receptors lack the conserved disulfide bridge
between TM3 and EL2. As a result, the binding cleft around TM3-6
Docking Simulations. Docking of compounds 1, 26, and 30 to
the energy-minimized hCB1 receptor model was carried out using
GOLD 3.1 version,43,44 a genetic algorithm-based software, select-
ing GOLDScore as a fitness function. GOLDScore is made up of
four components that account for protein–ligand binding energy:
protein–ligand hydrogen bond energy (external H-bond), protein-
–ligand van der Waals energy (external vdw), ligand internal vdw
energy (internal vdw), and ligand torsional strain energy (internal
torsion). Parameters used in the fitness function (H-bond energies,
atom radii and polarizabilities, torsion potentials, H-bond direc-
tionalities, and so forth) are taken from the GOLD parameter file.
The fitness score is taken as the negative of the sum of the energy
terms, so larger fitness scores indicated better bindings. The fitness
function has been optimized for the prediction of ligand binding
positions rather than the prediction of binding affinities, although
some correlation with the latter can also be found.82 The protein
input file may be the entire protein structure or a part of it
comprising only the residues that are in the region of the ligand
binding site. In the present study, GOLD was allowed to calculate

1574 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
SilVestri et al.
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Figure 5. Ramachandran plot of the hCB1 receptor model. The most
favoured and the additional allowed regions are defined by cyan and
magenta lines, respectively. The disallowed regions are indicated as
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interaction energies within a sphere of an 18 Å radius centered on
the side-chain nitrogen of K3.28(192).
The structures of the ligands 1, 26, and 30 were constructed using
standard bond lengths and bond angles of the INSIGHTII fragment
library. A total of 50 independent docking runs were performed
for each docking experiment, using standard default settings with
a population size of 100, a maximum number of 100000 operations,
and a mutation and crossover rate of 95. The best-generated 20
solutions of each ligand were ranked according to their fitness scores
calculated by the GOLDScore function.
MD Simulations. General AMBER force field (GAFF) param-
eters were assigned to ligands, while the partial charges were
calculated using the AM1-BCC method, as implemented in the
ANTECHAMBER suite of AMBER. The initial 1/hCB1, 26/hCB1,
and 30/hCB1 complexes were energy-minimized for 10500 steps
using combined steepest descent and conjugate gradient methods
until a convergence value of 0.001 kcal/Å ·mol. Upon minimization,
an energy penalty force constant of 5 kcal/Å2/mol on the protein
backbone atoms was applied. The geometry-optimized complexes
were then used as the starting point for subsequent 400 ps MD
simulation, during which the positional constraints on the protein
backbone were gradually reduced from 5 to 0.1 kcal/Å2/mol. For
the MD simulations were used the following: time step of 1 fs;
pairlist update every 25 fs. The temperature was maintained at 300
K using the Berendsen algorithm83 with a 0.2 ps coupling constant.
The SHAKE method79 was applied to constrain all of the covalent
bonds involving hydrogen atoms. 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. The PTRAJ module of AMBER was used to check
rmsd and interatomic distances. The H-bond criterion was a
maximum donor–acceptor distance of 3.5 Å and a minimum
donor-proton-acceptor angle of 120°.
Acknowledgment. Authors from the University of Siena
wish to thank the Ministero dell’Università e della Ricerca
(PRIN 2006 - Prot. n 2006030948_002) for financial support.
Authors also thank Mr. Marco Allarà for technical assistance.
Supporting Information Available: Additional chemical and
biological information. This material is available free of charge
via Internet at http://pubs.acs.org.

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