Synthesis and structure-activity relationships (SARs) of 1,5-diarylpyrazole cannabinoid type-1 (CB1) receptor ligands for potential use in molecular imaging

Article abstract of DOI:10.1016/j.bmc.2006.01.047

Cannabinoid type-1 (CB₁) receptor ligands, derived from the 1,5-diarylpyrazole core template of rimonabant (Acomplia), have been the focus of several studies aimed at examining structure-activity relationships (SARs). The purpose of this study was to design and synthesize a set of compounds based on the 1,5-diarylpyrazole template while focusing on the potential for discovery of CB₁ receptor radioligands that might be used as probes with in vivo molecular imaging. Each synthesized ligand was evaluated for potency as an antagonist at CB₁ and cannabinoid type-2 (CB₂) receptors in vitro using a GTPγ³⁵S-binding assay. c log P values were calculated with Pallas 3.0. The antagonist binding affinities (K_B) at CB₁ receptors ranged from 11 to >16,000 nM, CB₁ versus CB₂ selectivities from 0.6 to 773, and c log Ps from 3.61 to 6.25. An interesting new ligand, namely N-(piperidin-1-yl)-1-(2-bromophenyl)-5-(4-methoxyphenyl)-4-methyl-1H-pyrazole-3-carboxamide (9j), emerged from the synthesized set with appealing properties (K_B = 11 nM; CB₁ selectivity > 773; c log P = 5.85), for labeling with carbon-11 and development as a radioligand for imaging brain CB₁ receptors in vivo with positron emission tomography (PET).

Full text of DOI:10.1016/j.bmc.2006.01.047

Bioorganic & Medicinal Chemistry 14 (2006) 3712–3720
Synthesis and structure–activity relationships (SARs) of
1,5-diarylpyrazole cannabinoid type-1 (CB1) receptor ligands
for potential use in molecular imaging
b
a
Sean R. Donohue,a,b, Christer Halldin and Victor W. Pike
*
aMolecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA
bKarolinska Institutet, Department of Clinical Neuroscience, Psychiatry Section, Karolinska Hospital, S-17176 Stockholm, Sweden
Received 1 December 2005; revised 10 January 2006; accepted 12 January 2006
Available online 8 February 2006
Abstract—Cannabinoid type-1 (CB1) receptor ligands, derived from the 1,5-diarylpyrazole core template of rimonabant (Acomp-
liaꢀ), have been the focus of several studies aimed at examining structure–activity relationships (SARs). The purpose of this study
was to design and synthesize a set of compounds based on the 1,5-diarylpyrazole template while focusing on the potential for dis-
covery of CB1 receptor radioligands that might be used as probes with in vivo molecular imaging. Each synthesized ligand was eval-
uated for potency as an antagonist at CB1 and cannabinoid type-2 (CB2) receptors in vitro using a GTPc35S-binding assay. clogP
values were calculated with Pallas 3.0. The antagonist binding affinities (KB) at CB1 receptors ranged from 11 to >16,000 nM, CB1
versus CB2 selectivities from 0.6 to 773, and clogPs from 3.61 to 6.25. An interesting new ligand, namely N-(piperidin-1-yl)-1-(2-
bromophenyl)-5-(4-methoxyphenyl)-4-methyl-1H-pyrazole-3-carboxamide (9j), emerged from the synthesized set with appealing
properties (KB = 11 nM; CB1 selectivity > 773; clogP = 5.85), for labeling with carbon-11 and development as a radioligand for
imaging brain CB1 receptors in vivo with positron emission tomography (PET).
ꢁ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
CB1 receptors represent an interesting target for the treat-
ment of several psychiatric (e.g., anxiety,11 addiction,12
and depression13) and neurodegenerative disorders
(e.g., Huntington’s disease14 and Tourette’s syndrome15).
The role of the CB1 receptors in these disorders is not well
understood. Positron emission tomography (PET) or sin-
gle-photon emission computed tomography (SPECT)
coupled to an effective radioligand for CB1 receptors
might provide a further means for understanding the
many such disorders linked to this receptor.
The medicinal and cognitive effects of Marijuana
(Cannabis sativa) have been known for centuries.
Despite its long history, only recently have studies pro-
vided convincing information on the biological media-
tion of its effects. Currently, in the endocannabinoid
system, two sub-types of receptor, CB1 and CB2, are rec-
ognized as mediating the biological effects arising from
exposure to receptor ligands, whether endogenous,
plant-derived or synthetic receptor agonists, antagonists
or inverse agonists.1 Both CB1 and CB2 receptors belong
to the G-protein-coupled superclass of receptors.2 CB1
receptors are located throughout the body including
within the central nervous system (CNS)3,4 at presynap-
tic nerve terminals and within the periphery at the intes-
tine,5 eye,6 testis,7 and bladder.8 CB2 receptors are
mainly associated with cells of the immune system9,10
(e.g., B-cells, natural killer cells, and monocytes). Brain
Molecular modification of the 1,5-diarylpyrazole core
template of SR 141716A (N-(piperidin-1-yl)-1-(2,4-
dichlorophenyl)-5-(4-chlorophenyl)-4-methyl-1H-pyra-
zole-3-carboxamide; rimonabant; Acompliaꢀ; 1, Table
1),16 a potent and selective CB1 receptor ligand, has been
the focus of several studies aimed at uncovering the
molecular requirements for CB1 receptor antagonism or
inverse agonism.17–21 The pharmacophore derived from
these studies has been previously reviewed.20,22 This ap-
proach has led to the development of ligands with en-
hanced affinity and selectivity for the CB1 receptor. The
most notable of these ligands is AM 251 (N-(piperidin-
1-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-
Keywords: CB1 receptor; PET; 1,5-Diarylpyrazoles; Carbon-11; NIDA
41087; Rimonabant.
*
Corresponding author. Tel.: +1 301 451 3909; fax: +1 301 480
5112; e-mail: donohues@intra.nimh.nih.gov
0968-0896/$ - see front matter ꢁ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.01.047

S. R. Donohue et al. / Bioorg. Med. Chem. 14 (2006) 3712–3720
3713
Table 1. Antagonist binding affinities, selectivities and calculated lipophilicities of 1,5-diarylpyrazole derivatives
Y
O
N
NH
N
N
R1
R5
X
R2
R3
R4
R1
R2
R3
R4
R5
X
Y
CB1 KB
(nM)
CB2 KB
(nM)
CB1 versus CB2
selectivity
clogP
clogD
at pH 7.4
a
a
Ligand
1
Cl
I
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
Cl
H
Cl
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
—
H
H
H
H
H
C
C
C
C
C
C
C
C
C
C
C
C
C
H
C
C
N
C
C
C
C
C
CH2
CH2
O
6.1
3.8
>6776
904
>1111
238
120
32
6.95
7.36
5.66
6.39
5.69
6.11
4.17
5.69
5.69
5.05
6.24
6.32
5.61
6.15
5.85
6.12
4.39
3.61
5.60
5.34
5.18
5.68
6.95
7.36
5.66
6.39
5.69
6.11
4.17
5.69
5.69
5.05
6.24
6.32
5.61
6.15
5.85
6.12
4.39
3.61
5.60
5.02
4.85
5.68
2
3
I
8.9
26
1,069
831
4
MeO
MeO
MeS
MeSO2
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
H
Cl
H
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
5
25
2091
2091
>5770
2868
84
9a
9b
9c
9d
9e
9f
9g
9h
9i
H
>3360
302
154
>3884
570
140
270
55.9
84.8
11
<0.6
>191
19
H
Cl
H
H
>13,241
3471
510
H
H
6
Cl
Cl
Me
Me
Br
CF3
H
H
3.6
6.6
82
H
1769
4579
H
Me
H
>16,908
>8500
2183
>199
>773
42
9j
9k
9l
H
51.6
>4254
>3884
532
431
630
38
H
>16,908
13,421
2660
9m
9n
9o
9p
9q
H
MeSO2
H
<3.5
5
Cl
Br
Cl
Cl
OH
OH
H
2744
2261
6.4
3.6
419
H
FCH2O
H
15,924
a These are single determinations run on plates with several control compounds. The inherent assay variability is about 3-fold.
1H-pyrazole-3-carboxamide; 2, Table 1).17,23 When com-
pared to 1, ligand 2 differs in the para-substituent on the
C-5 aryl ring (I vs Cl, Table 1). An added benefit of 2 is
its amenability for labeling with iodine-123, making it
potentially useful for molecular imaging with SPECT.
might be used in the molecular imaging of brain CB1
receptors with PET or SPECT. In addition to seeking
desirable properties for molecular imaging in brain,28
such as high affinity and high selectivity for CB1 recep-
tors plus an ability to enter brain, we also designed our
target ligands to be amenable to labeling with a molecu-
lar imaging radionuclide, such as 11C (b+, t1/2 = 20.4 min;
18F (b+, t1/2 = 109.8 min), or 123I (c, t1/2 = 13.2 h). Each
target ligand was evaluated for binding affinity at both
CB1 and CB2 receptors in vitro using GTPc35S-binding
assays. For the purposes of clarification and comparison,
a set of reference compounds (SR 141716A (1), AM 251
(2), AM 281 (3), NIDA 41020 (4), and NIDA 41087 (5),
Table 1) were also evaluated in these assays. clogP, as an
index of lipophilicity of each neutral microspecies, was
calculated in order to provide an indication of each
ligand’s ability to cross the blood–brain barrier (BBB)
without giving high non-specific binding. clogD values
were also calculated at pH 7.4.
Studies23 with 123I-labeled 2 have been disappointing be-
cause this radioligand does not enter the CNS sufficiently
to be useful for SPECT imaging. The low brain entry of
the radioligand has been ascribed23,24 to the molecule’s
high lipophilicity (i.e., clogP = 7.36, Table 1). This
conclusion prompted the development of AM 281 (N-
(morpholin-4-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-
methyl-1H-pyrazole-3-carboxamide; 3, Table 1),24 an
analog of AM 251 with lower lipophilicity (clogP = 5.66,
Table 1). This ligand has also been labeled with iodine-
123 and studied in Tourette’s syndrome patients.25 Other
candidate radioligands that have been investigated are:
[18F]SR 144385,26 [18F]SR 147962,26 [11C]SR 149080,27
[11C]SR 149568,27 and [11C]NIDA 4108721 [11C]5, Table
1). However, none of these analogs has proven to be use-
ful in molecular imaging.
2. Results and discussion
2.1. Chemistry
The purpose of this study was to synthesize and charac-
terize further ligands based on the 1,5-diarylpyrazole
template with a view to discovering radioligands that
Compounds 9a–n were synthesized according to a previ-
ously established procedure with some modifica-

3714
S. R. Donohue et al. / Bioorg. Med. Chem. 14 (2006) 3712–3720
tions.17,21 The pathway for the synthesis of the 1,5-
diarylpyrazole derivatives is outlined in Scheme 1. First,
compound 7a was prepared under Friedel–Crafts acyla-
tion conditions with aluminum chloride and propionyl
chloride. Ketones 7a–c were then condensed with diethyl
oxalate in the presence of lithium diisopropylamide in
ether at ꢀ78 ꢂC. The resulting lithium salts were imme-
diately reacted with various arylhydrazines in aqueous
ethanol (70%) at room temperature (RT). Refluxing
the resultant isolated hydrazones in acetic acid yielded
the pyrazole carboxylic esters 8a, c–n. Compound 8b
was prepared by oxidation of 8a with m-chloroperoxy-
benzoic acid. Hydrolysis, conversion into the acyl chlo-
ride, and addition of the 1-aminopiperidine gave the
desired carboxamides 9a–n. Addition of boron tribro-
mide to solutions of 9j and 5 in dry dichloromethane
adequately formed the phenols, 9o and 9p, respectively.
Treating 9p with fluoromethyl bromide in the presence
of potassium carbonate in acetonitrile under microwave
irradiation yielded the fluoromethoxy compound 9q.
began with modification of 4 and 5. The reported21
high-binding affinity and amenability to labeling of
these compounds were initially intriguing. In order to
gain further insights into the optimal substituents for
the N-1 aryl ring, several compounds were prepared
with variation of substituents in the R2, R3, R4 and X
positions. Table 1 shows the antagonist binding affinities
(KB) and selectivities for each of the synthesized candi-
date radioligands at CB1 and CB2 receptors. The report-
ed KB values for the CB1 receptor ranged from 11 to
>4224 nM and the selectivities ranged from <0.6 to
>733. Most of the investigated compounds had little
to no antagonist binding affinity for the CB2 receptor.
The KB values for the CB2 receptor ranged from 510
to >16,908 nM.
Switching the chlorine atoms from R2 (5) to R3 (9d) and
R4 (9c) reduced antagonist binding affinity (KB = 25,
>3884, and 154 nM, respectively) for the CB1 receptor.
This finding is consistent with results from other stud-
ies.17 When all substituents were removed from the
N-1 aryl ring (9e), there was a moderate loss of CB1
receptor affinity (KB = 570 nM). There was almost no
CB1 or CB2 antagonist binding affinity when the
X-position was replaced by a nitrogen (9l; KB > 4254 nM).
2.2. Structure–affinity relationships (SAR)
Research into the expansion of SAR information on
compounds based on the 1,5-diarylpyrazole template
O
a
R1
R1
7a, R1 = MeS
b, R1 = MeO
c, R1 = H
6a, R1 = MeS
b, c, d
O
N
O
O
N
NH
N
N
e, f
N
R1
R5
X
R1
R5
R2
R3
R2
R3
X
R4
R4
8a, R1 = MeS; R2 = Cl; R3, R4, R5 = H; X = C
b, R1 = MeSO2; R2 = Cl; R3, R4, R5 = H; X = C
c, R1 = MeO; R2, R3 = H; R4 = Cl; R5 = H; X = C
d, R1 = MeO; R2 = H; R3 = Cl; R4, R5 = H; X = C
e, R1 = MeO; R2, R3, R4, R5 = H; X = C
9a, R1 = MeS; R2 = Cl; R3, R4, R5 = H; X = C
b, R1 = MeSO2; R2 = Cl; R3, R4, R5 = H; X = C
c, R1 = MeO; R2, R3 = H; R4 = Cl; R5 = H; X = C
d, R1 = MeO; R2 = H; R3 = Cl; R4, R5 = H; X = C
e, R1 = MeO; R2, R3, R4, R5 = H; X = C
g
f, R1 = MeO; R2, R3 = Cl; R4, R5 = H; X = C
g, R1 = MeO; R2 = Cl; R3, R4 = H; R5 = Cl; X = C
h, R1 = MeO; R2 = Me; R3, R4, R5 = H; X = C
i, R1 = MeO; R2, R4 = Me; R3, R5 = H; X = C
j, R1 = MeO; R2 = Br; R3, R4, R5 = H; X = C
k, R1 = MeO; R2 = CF3; R3, R4, R5 = H; X = C
l, R1 = MeO; R2 = Cl; R3, R4 = H; R5 = -; X = N
m, R1 = MeO; R2, R3, R5 = H; R4 = MeSO2; X = C
n, R1 = H; R2 = Cl; R3, R4, R5 = H; X = C
f, R1 = MeO; R2, R3 = Cl; R4, R5 = H; X = C
g, R1 = MeO; R2 = Cl; R3, R4 = H; R5 = Cl; X = C
h, R1 = MeO; R2 = Me; R3, R4, R5 = H; X = C
i, R1 = MeO; R2, R4 = Me; R3, R5 = H; X = C
j, R1 = MeO; R2 = Br; R3, R4, R5 = H; X = C
k, R1 = MeO; R2 = CF3; R3, R4, R5 = H; X = C
l, R1 = MeO; R2 = Cl; R3, R4 = H; R5 = -; X = N
m, R1 = MeO; R2, R3, R5 = H; R4 = MeSO2; X = C
n, R1 = H; R2 = Cl; R3, R4, R5 = H; X = C
h
o, R1 = HO; R2 = Br; R3, R4, R5 = H; X = C
p, R1 = HO; R2 = Cl, R3, R4, R5 = H; X = C
q, R1 = FCH2O; R2 = Cl; R3, R4, R5 = H; X = C
h
5
i
Scheme 1. Reagents and conditions: (a) AlCl3, DCE; (b) LDA, Et2O, (COEt)2; (c) R2, R3, R4, R5, X-ArN2H3ÆHCl, EtOH, rt; (d) AcOH, reflux;
(e) Na2O2, MeOH; (f) (COCl)2, DMF (cat), DCM, TEA, 1-aminopiperidine; (g) m-CPBA, DCM; (h) BBr3, DCM; (i) FCH2Br, K2CO3, microwave.

S. R. Donohue et al. / Bioorg. Med. Chem. 14 (2006) 3712–3720
3715
Dual chloro atoms in the R2, R3 and R2, R6 positions
gave compounds 9f and 9g with moderate loss of CB1
antagonist binding affinity (KB = 140 and 270 nM,
respectively). However, 9f showed a higher antagonist
binding affinity (KB = 510 nM) for the CB2 receptor. A
total loss of CB1 and CB2 antagonist binding affinity
(KB > 3884 nM) was observed with a MeSO2 group in
the R4-position (9m). These results suggested that the
optimal position for improvement in CB1 antagonist
binding affinity is the R2-position.
may therefore be expected to enter the CNS to an extent
similar to that of [123I]AM281.
2.4. Prospective radioligands
Three ligands (5, 9j, and 9q) stand out as potential can-
didate PET radioligands for molecular imaging of brain
CB1 receptors. The preparation and in vitro autoradiog-
raphy of [11C]5 have been reported previously.31 Howev-
er, the in vivo molecular imaging of this radioligand has
not been reported. Ligand 9j is similar to 5, but has over
a 2-fold greater antagonist binding affinity (KB = 11 vs
25 nM). Ligand 9j is also more selective than 5 (773-
vs 84-fold) for CB1 versus CB2 receptors and is similarly
amenable to labeling with carbon-11. The clogP values
of 5 (clogP = 5.69) and 9j (clogP = 5.89) are close to
AM281 (clogP = 5.66), which represents a marker for
acceptable CNS entry. Ligand 9q benefits from the
introduction of the fluoromethoxy group in the R1-posi-
tion, which provides an opportunity for labeling with
Varying the substituent in R2-position with Me (9h)
and CF3 (9k) gave compounds with only a slight loss
in CB1 antagonist binding affinity (KB = 55.9 and
51.6 nM, respectively) and almost no CB2 receptor
affinity. An additional Me group in the R4-position
(9i) lowered the CB1 antagonist binding affinity relative
to 9h (KB = 84.8 vs 55.9 nM). A 2-fold improvement in
CB1 antagonist binding affinity (KB = 11 nM) was
observed when a bromo group was incorporated into
the R2-position (9j), while CB2 receptor antagonist
binding affinity was virtually abolished (KB >
16,908 nM).
fluorine-18.
(KB = 38 nM) of 9q may however be too low to be useful
The
antagonist
binding
affinity
for molecular imaging.
Variations in the R1-substituent of the 1,5-diarylpyraz-
ole template were explored in order to determine the
optimal SAR requirements in the R1-position of the
C-5 aryl ring. Surprisingly, when an MeS group (9a) re-
placed the MeO group (5) in the R1-position, almost all
of the CB1 antagonist binding affinity (KB > 3360 nM vs
25 nM) for the CB1 receptor was lost. However, some
CB1 antagonist binding affinity (KB = 302 nM) was re-
gained when MeSO2 (9b) replaced MeS (9a). There
was reduced CB1 antagonist binding affinity
(KB = 523 nM) with a hydrogen atom in the R1-position
(9n). There was also reduced antagonist binding affinity
(KB = 630 and 431 nM) with an OH-group in the R1-po-
sition (9o, p, respectively). Placing a FCH2O group (9q)
in the R1-position gave a compound with moderate
antagonist binding affinity (KB = 38 nM).
3. Conclusion
A set of new ligands was synthesized based on the
1,5-diarylpyrazole core template of SR 141716A (1).
Pharmacological and physiochemical parameters (i.e.,
KB values, selectivities, and lipophilicities) show 5, 9j,
and 9q to be interesting candidates for labeling with
cyclotron-produced carbon-11 or fluorine-18. Further
studies are proceeding to assess the suitability of these
radioligands for molecular imaging in vivo.
4. Experimental
4.1. Materials
2.3. clogP and clogD determinations
Ligands 1, 4, and 5 were synthesized according to previ-
ously established procedures.17,21 2 (AM251) and 3
(AM281) were obtained from Tocris (Ballwin, MO).
Fluorobromomethane was purchased from ABCR
GmbH & Co (Germany). All other reagents, including
7b and 7c, and solvents (ACS or HPLC grade) were pur-
chased from commercial sources and used as supplied
unless otherwise stated.
Lipophilicity is an important but not always reliable
parameter predicting a molecule’s ability to pass the
BBB and extent of non-specific binding in brain. Several
studies aimed at relating molecular lipophilicity to BBB
penetration have generated a parabolic curve in which
the window for adequate brain entry lies between logP
values of 1.5 and 3.5.29 Molecules that lie outside this
window are generally not considered to be useful for
molecular imaging. However, the examined 1,5-diary-
lpyrazole radioligand, [123I]AM 281, adequately enters
the CNS with a clogP of 5.66 (Table 1) (measured
logP = 4.3).30 The clogP of the investigated ligands ran-
ged from 3.61 to 7.36. With the exception of the phenols
9o and 9p, the prepared ligands 9a–q have clogD (pH
7.4) values identical to their clogP values (Table 1).
From the prepared set of ligands, the three compounds
with attractive pharmacological properties, 5, 9j, and 9q,
have clogP values of 5.69, 5.89, and 5.68, respectively
(Table 1), which are near or slightly higher than that
of [123I]AM 281. These compounds as radioligands
4.2. General methods
All reactions were carried out in an inert atmosphere
with dry solvents under anhydrous conditions, unless
otherwise stated. Melting points (uncorrected) were
determined using a Melt-Temp apparatus (Dubuque;
1
IA, USA). H- (400 MHz) and 13C NMR (100 MHz)
spectra were recorded on a Bruker instrument (Billerica,
MA, USA) for solution in CDCl3 and DMSO-d6 at
1
300 K, respectively, unless otherwise stated. H NMR
(300 MHz) spectra were recorded on a Varian Gemini
(Palo Alto, CA, USA). Tetramethylsilane (TMS) was
used as an internal standard in all cases and signals

3716
S. R. Donohue et al. / Bioorg. Med. Chem. 14 (2006) 3712–3720
are quoted as s (singlet), d (doublet), t (triplet), q (quar-
tet), p (pentuplet) or m (multiplet). Chemical shift (d)
pended in ethanol (70%; 10 mL) and added to 2-chloro-
phenylhydrazine hydrochloride (616 mg, 3.46 mmol).
The mixture was stirred at rt for 3 h. The precipitate
was filtered off, washed with aqueous ethanol (70%;
5 mL), and dried under vacuum yielding a light yellow
hydrazone. The hydrazone was dissolved in acetic acid
(5 mL) and refluxed for 16 h. The reaction mixture
was poured into ice-cold water (50 mL), neutralized with
Na2CO3, and extracted with dichloromethane (3·
50 mL). The combined extracts were dried over MgSO4,
filtered, and evaporated. Purification with gradient flash
chromatography (hexanes–ethyl acetate) gave the ester
1
data for H and 13C NMR spectra are reported in parts
per million (ppm) downfield from the signal for TMS.
The LC–MS analysis was performed on an LCQ Deca
model LC–MS (Thermo Electron Corporation; San
Jose, CA, USA). The chromatography was performed
on
a
reverse-phase column (Luna C18; 150
(l) · 2 (id) mm; 5 lm; Phenomenex: Torrance, CA,
USA). High-resolution mass spectra were determined
using time-of-flight electrospray (University of Illinois
at Urbana, Champain, IL, USA). Thin-layer chroma-
tography (TLC) was carried out with silica gel 60
F254 plates (EM Science). Gradient flash chromatogra-
phy was performed using a Horizon HPFC system (Bio-
tage; Charlottesville, VA, USA). Elemental analyses
were performed by (Quantitative Technologies Inc.,
Whitehouse, NJ). Compounds are indicated by their
molecular formulae followed by the symbols for the ele-
ments measured (C, H, and N). Results are within 0.4%
of their theoretical values.
1
8a (856 mg; 64%); mp: 134–136 ꢂC; H NMR: 7.37 (d,
J = 8 Hz, ArH), 7.33 (m, ArH), 7.15 (d, J = 8 Hz,
ArH), 7.07 (d, J = 8 Hz, ArH), 4.48 (q, J = 8 Hz,
CH2), 2.45 (s, SCH3), 2.37 (s, CH3), 1.44 (t, J = 4 Hz,
CH3).
4.4.3. 1-(2-Chlorophenyl)-4-methyl-5-(4-methanesulfonyl-
phenyl)-1H-pyrazole-3-carboxylic acid, ethyl ester (8b).
To a stirred solution of 8a (300 mg, 777 lmol) in dichlo-
romethane (20 mL) was added a solution of 77%
3-chloroperoxybenzoic acid (435 mg, 1.94 mmol) in
dichloromethane (5 mL). The mixture was stirred for
18 h and then poured into aqueous NaHCO3 (20 mL).
The organic phase was separated, and the aqueous
phase was extracted with dichloromethane (2· 20 mL).
The combined extracts were dried over MgSO4, filtered
and evaporated. Purification with gradient flash chro-
matography (hexanes–ethyl acetate) gave the ester 8b
(293 mg; 90%); mp: 168–170 ꢂC; 1H NMR: 7.89 (d,
J = 8 Hz, ArH), 7.47 (d, J = 8 Hz, ArH), 7.38 (m,
ArH), 4.49 (q, J = 8 Hz, CH2), 3.07 (s, SO2CH3), 2.39
(s, CH3), 1.4 (t, J = 4 Hz, CH3).
4.3. clogP and clogD calculations
The clogP and clogD of ligands 1–5 and 9a–q were
determined from molecular structure using Pallas 3.0
software for Windows (CompuDrug International Inc.;
South San Francisco, CA, USA).
4.4. Chemistry
4.4.1. 40-(Methylthio)propiophenone (7a). Propionyl
chloride (1.96 mL, 22.5 mmol) was added to a stirred
solution of AlCl3 (5.0 g, 37.5 mmol) in dichloromethane
(50 mL) at 0 ꢂC and stirring was continued for 10 min.
Thioanisole (6a) (3.41 mL, 28.8 mmol) was then slowly
added over 30 min. The mixture was allowed to warm
to rt and stirred for 16 h. The mixture was then carefully
poured onto crushed ice. The aqueous phase was
extracted with dichloromethane (2· 50 mL). The organ-
ic phases were combined and washed with water (50 mL)
and then aqueous NaHCO3 (50 mL). The organic layer
was then dried over MgSO4, filtered, and concentrated
in vacuo. The crude product was purified with silica
gel chromatography (20% EtOAc–hexanes) to give 7a
(5.03 g; 70%); mp: 54–56 ꢂC; 1H NMR: 7.88 (d,
J = 8 Hz, ArH), 7.26 (d, J = 8 Hz, ArH), 2.99 (q,
J = 8 Hz, CH2), 2.51 (s, SCH3), 1.23 (t, J = 4 Hz,
CH3); 13C NMR: 199.9, 145.5, 133.2, 128.4, 124.9,
31.6, 14.78, 8.33.
4.4.4. 1-(3-Chlorophenyl)-4-methyl-5-(4-methoxyphenyl)-
1H-pyrazole-3-carboxylic acid, ethyl ester (8c). The pro-
cedure described for the synthesis of 8a was applied to
7c and 3-chlorophenylhydrazine hydrochloride yielding
1
8c (52%); mp: 112–114 ꢂC; H NMR: 7.45 (s, J = 8 Hz,
ArH), 7.25 (d, J = 8 Hz, ArH), 7.18 (t, J = 8 Hz, ArH),
7.09 (d, J = 8 Hz, ArH), 7.06 (d, J = 8 Hz, ArH), 6.92
(d, J = 8 Hz, ArH) 4.49 (q, J = 8 Hz, CH2), 3.83
(s, OCH3), 2.29 (s, CH3), 1.46 (t, J = 4 Hz, CH3).
4.4.5. 1-Phenyl-4-methyl-5-(4-methoxyphenyl)-1H-pyra-
zole-3-carboxylic acid, ethyl ester (8d). The procedure
described for the synthesis of 8a was applied to 7c and
phenylhydrazine hydrochloride yielding 8d (70%); mp:
164–166 ꢂC; 1H NMR: 7.30 (m, ArH), 7.09
(d, J = 8 Hz, ArH), 6.89 (d, J = 8 Hz, ArH), 4.48
(q, J = 8 Hz, CH2), 3.80 (s, OCH3), 2.31 (s, CH3), 1.45
(t, J = 4 Hz, CH3).
4.4.2. 1-(2-Chlorophenyl)-4-methyl-5-(4-methylthiophe-
nyl)-1H-pyrazole-3-carboxylic acid, ethyl ester (8a). A
solution of 7a (2.0 g, 11.1 mmol) in diethyl ether
(10 mL) was added to a stirred solution of lithium diiso-
propylamide (7.4 mL; 1.8 M solution in THF) in diethyl
ether (50 mL) at ꢀ78 ꢂC. The reaction mixture was
allowed to stir at ꢀ78 ꢂC for a further 45 min. After
the addition of diethyl oxalate (1.65 mL, 12.2 mmol),
the reaction was stirred for 16 h while being allowed
to warm to rt. The precipitate was filtered off, washed
with diethyl ether (50 mL), and dried under vacuum.
A portion of the lithium salt (1.0 g, 3.46 mmol) was sus-
4.4.6. 1-(2,3-Dichlorophenyl)-4-methyl-5-(4-methoxyphe-
nyl)-1H-pyrazole-3-carboxylic acid, ethyl ester (8e). The
procedure described for the synthesis of 8a was applied
to 7c and 2,3-dichlorophenylhydrazine hydrochloride
1
yielding 8e (67%); mp: 148–150 ꢂC; H NMR: 7.99 (d,
J = 8 Hz, ArH), 7.52 (d, J = 8 Hz, ArH), 7.15 (t,
J = 8 Hz, ArH), 7.04 (d, J = 8 Hz, ArH), 6.94 (d,
J = 8 Hz, ArH), 4.23 (q, J = 8 Hz, CH2), 3.77 (s,
OCH3), 1.57 (s, CH3), 1.45 (t, J = 4 Hz, CH3).

S. R. Donohue et al. / Bioorg. Med. Chem. 14 (2006) 3712–3720
3717
4.4.7. 1-(2,6-Dichlorophenyl)-4-methyl-5-(4-methoxyphe-
nyl)-1H-pyrazole-3-carboxylic acid, ethyl ester (8f). The
procedure for the synthesis of 8a was applied to 7c
and 2,6-dichlorophenylhydrazine hydrochloride yielding
8f (67%); mp: 108–110 ꢂC; H NMR: 7.33 (m, ArH),
7.18 (d, J = 8 Hz, ArH), 6.82 (d, J = 8 Hz, ArH), 4.48
(q, J = 8 Hz, CH2), 3.77 (s, OCH3), 2.33 (s, CH3), 1.44
(t, J = 4 Hz, CH3).
(d, J = 8 Hz, ArH), 7.50 (d, J = 8 Hz, ArH), 7.10 (d,
J = 8 Hz, ArH), 6.94 (d, J = 8 Hz, ArH), 4.49 (q,
J = 8 Hz, CH2), 3.86 (s, OCH3), 3.09 (s, SO2CH3), 2.30
(s, CH3), 1.46 (t, J = 4 Hz, CH3).
1
4.4.14. 1-(2-Chlorophenyl)-4-methyl-5-phenyl-1H-pyra-
zole-3-carboxylic acid, ethyl ester (8m). The procedure
for the synthesis of 8a was applied to 7d and 2-chloro-
phenylhydrazine hydrochloride yielding 8m (56%); mp:
123–125 ꢂC; 1H NMR: 7.42 (d, J = 8 Hz, ArH), 7.39
(m, ArH), 7.17 (m, ArH), 6.80 (d, J = 8 Hz, ArH),
4.49 (q, J = 8 Hz, CH2), 2.37 (s, CH3), 1.45 (t,
J = 4 Hz, CH3).
4.4.8. 1-(2-Methylphenyl)-4-methyl-5-(4-methoxyphenyl)-
1H -pyrazole-3-carboxylic acid, ethyl ester (8g). The pro-
cedure for the synthesis of 8a was applied to 7c and
2-methylphenylhydrazine hydrochloride yielding 8g (52
%); mp: 149–150 ꢂC; 1H NMR: 7.24 (m, ArH), 7.04
(d, J = 8 Hz, ArH), 6.81 (d, J = 8 Hz, ArH), 4.47 (q,
J = 8 Hz, CH2), 3.76 (s, OCH3), 2.36 (s, CH3), 1.92 (s,
CH3), 1.44 (t, J = 4 Hz, CH3).
4.4.15.
N-(Piperidin-1-yl)-5-(4-methylthiophenyl)-1-(2-
chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (9a).
To a stirred solution of 8a (400 mg; 1.04 mmol) in meth-
anol (20 mL) was added Na2O2 (242 mg; 3.1 mmol). The
mixture was stirred at rt for 3 h and then concentrated in
vacuo. The residue was neutralized with HCl (10%), fil-
tered, and dried under vacuum. A portion of the crude
acid (200 mg, 558 lmol) was dissolved in dry dichloro-
methane (10 mL). To the stirred solution were added
N,N-dimethylformamide (1 drop) and oxalyl chloride
(73.1 lL, 838 lmol). After bubbling ceased, the reaction
was concentrated in vacuo. A solution of 1-aminopiperi-
dine (67 lL, 614 lmol) and triethylamine (118 lL,
838 lmol) in dichloromethane (10 mL) was slowly add-
ed to the acid chloride and stirred at rt for 2 h. Aqueous
NaHCO3 (50 mL) was added to the reaction flask and
extracted with dichloromethane (3· 50 mL). The com-
bined extracts were dried over MgSO4, filtered, and
evaporated. Purification with gradient flash chromatog-
raphy (hexanes–ethyl acetate) gave the carboxamide 9a
(83%) as a white solid; mp: 206–207 ꢂC; 1H NMR:
7.63 (d, J = 8.1 Hz, 2H, ArH), 7.56–7.50 (m, 2H,
ArH), 7.42–7.40 (m, 2H, ArH), 7.33 (d, J = 8.1 Hz,
2H, ArH), 6.31 (br s, 1H, NH) 2.63 (t, J = 5.3 Hz, 4H,
NCH2), 2.54 (s, 3H, SCH3), 2.43 (s, 3H, CH3), 1.69 (p,
J = 5.6 Hz, 4H, CH2), 1.38–1.36 (m, 2H, CH2); LC–
MS, m/z [M+H]+: 441.1; HRMS Calcd m/z (TOF)
[M+H]+: C23H26N4OClS = 441.1516. Found: 441.1525.
Anal. (C23H25N4OClS) C, H, N.
4.4.9. 1-(2,4-Dimethylphenyl)-4-methyl-5-(4-methoxyphe-
nyl)-1H-pyrazole-3-carboxylic acid, ethyl ester (8h). The
procedure for the synthesis of 8a was applied to 7c
and 2,4-dimethylphenylhydrazine hydrochloride yield-
ing 8h (56%); mp: 88–90 ꢂC; 1H NMR: 7.12 (d,
J = 8 Hz, ArH), 7.04 (d, J = 8 Hz, ArH), 6.97 (m,
ArH), 6.82 (d, J = 8 Hz, ArH), 4.47 (q, J = 8 Hz,
CH2), 3.77 (s, OCH3), 2.36 (s, CH3), 2.29 (s, CH3),
1.86 (s, CH3), 1.44 (t, J = 4 Hz, CH3).
4.4.10. 1-(2-Trifluoromethylphenyl)-4-methyl-5-(4-meth-
oxyphenyl)-1H-pyrazole-3-carboxylic acid, ethyl ester
(8i). The procedure for the synthesis of 8a was applied
to 7c and 2-(trifluoro)phenylhydrazine yielding 8i
(35%); mp: 134–136 ꢂC; 1H NMR: 7.71 (d, J = 8 Hz,
ArH), 7.51 (m, ArH), 7.28 (d, J = 8 Hz, ArH), 7.06
(d, J = 8 Hz, ArH), 6.82 (d, J = 8 Hz, ArH), 4.47
(q, J = 8 Hz, CH2), 3.76 (s, OCH3), 2.33 (s, CH3), 1.44
(t, J = 4 Hz, CH3).
4.4.11.
1-(2-Bromophenyl)-4-methyl-5-(4-methoxyphe-
nyl)-1H-pyrazole-3-carboxylic acid, ethyl ester (8j). The
procedure for the synthesis of 8a was applied to 7c
and 2-bromophenylhydrazine hydrochloride yielding 8j
(49%); mp: 140–142 ꢂC; 1H NMR: 7.55 (d, J = 8 Hz,
1H), 7.39 (d, J = 7.39, 1H,), 7.34 (t, J = 8 Hz, 1H),
7.25 (t, J = 8 Hz, 1H), 7.11 (d, J = 8 Hz, 2H), 6.83 (d,
J = 8 Hz, 2H), 4.48 (q, J = 8 Hz, 2H), 3.77 (s, 3H),
2.35 (s, 3H), 1.44 (t, J = 8 Hz, 3H).
4.4.16. N-(Piperidin-1-yl)-5-(4-methanesulfonylphenyl)-
1-(2-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxam-
ide (9b). The procedure for the synthesis of 9a was applied
to 8b yielding 9b (84%) as a white solid; mp: 248–250; 1H
NMR: 7.06 (d, J = 8 Hz, 2H, ArH), 7.71 (br s, 1H, NH),
7.41–7.37 (m, 4H, ArH), 7.36 (d, J = 8 Hz, 2H, ArH),
3.08 (s, 3H, SO2CH3), 2.89 (t, J = 5.3 Hz, 4H, NCH2),
2.45 (s, 3H, CH3), 1.79 (p, J = 4 Hz, 4H, CH2) 1.46–1.38
(m, 2H, CH2); LC–MS, m/z [M+H]+: 473.1; HRMS Calcd
m/z (TOF) [M+H]+: C23H26N4O3ClS = 473.1414. Found:
473.1422. Anal. (C23H25N4O3ClS) C, H, N.
4.4.12. 1-(Pyridin-2-yl)-4-methyl-5-(4-methoxyphenyl)-
1H-pyrazole-3-carboxylic acid, ethyl ester (8k). The
procedure for the synthesis of 8a was applied to 7c and
2-pyridylphenylhydrazine yielding 8k (26%); mp: 92–
94 ꢂC; 1H NMR: 8.37 (d, J = 8 Hz, ArH), 7.73 (t,
J = 8 Hz, ArH), 7.42 (d, J = 8 Hz, ArH), 7.28
(t, J = 8 Hz, ArH), 7.12 (d, J = 8 Hz, ArH), 6.90 (d,
J = 8 Hz, ArH), 4.47 (q, J = 8 Hz, CH2), 3.82 (s,
OCH3), 2.31 (s, CH3), 1.44 (t, J = 4 Hz, CH3).
4.4.17. N-(Piperidin-1-yl)-5-(4-methoxyphenyl)-1-(4-chlo-
rophenyl)-4-methyl-1H-pyrazole-3-carboxamide (9c). The
procedure for the synthesis of 9a was applied to8c yield-
ing 9c (79%) as a white solid; mp: 179–180 ꢂC; 1H NMR:
7.71 (br s, 1H, NH), 7.28 (d, 2H, J = 8 Hz, ArH), 7.18
(d, J = 8 Hz, 2H, ArH), 7.07 (d, J = 8 Hz, 2H, ArH),
6.9 (d, J = 8 Hz, 2H, ArH), 3.83 (s, 3H, OCH3) 2.91
4.4.13. 1-(4-Methanesulfonylphenyl)-4-methyl-5-(4-meth-
oxyphenyl)-1H-pyrazole-3-carboxylic acid, ethyl ester
(8l). The procedure for the synthesis of 8a was applied
to 7c and 4-methansulfonylphenylhydrazine hydrochlo-
1
ride yielding 8l (57%); mp: 136–138 ꢂC; H NMR: 7.87

3718
S. R. Donohue et al. / Bioorg. Med. Chem. 14 (2006) 3712–3720
(t, J = 5.3 Hz, 4H, NCH2), 2.33 (s, 3H, CH3), 1.80 (p,
J = 5.6 Hz, 4H, CH2) 1.46–1.38 (m, 2H, CH2); LC–
MS, m/z [M+H]+: 425.1; HRMS Calcd m/z (TOF)
[M+H]+: C23H26N4O2Cl 425.1744. Found: 425.1765.
Anal. (C23H25N4O2Cl) C, H, N.
ArH), 3.87 (s, 3H, OCH3) 2.97 (t, J = 5.3 Hz, 4H,
NCH2), 2.50 (s, 3H, CH3), 2.05 (s, 3H, CH3), 1.89 (p,
J = 5.6 Hz, 4H, CH2) 1.55–1.48 (m, 2H, CH2); LC–
MS, m/z [M+H]+: 405.2; HRMS Calcd m/z (TOF)
[M+H]+: C24H29N4O2 405.2291. Found: 405.2304. Anal.
(C24H29N4O2) C, H, N.
4.4.18. N-(Piperidin-1-yl)-5-(4-methoxyphenyl)-1-(3-chlo-
rophenyl)-4-methyl-1H-pyrazole-3-carboxamide
(9d).
4.4.23. N-(Piperidin-1-yl)-5-(4-methoxyphenyl)-1-(2-tri-
fluoromethylphenyl)-4-methyl-1H-pyrazole-3-carbox-
amide (9i). The procedure for the synthesis of 9a was
applied to 8i yielding 9i (84%) as a white solid: mp:
196 ꢂC; 1H NMR: 7.78 (d, J = 8 Hz, 1H, ArH), 7.50
(m, 2H, ArH), 7.14 (d, J = 8 Hz, 1H, ArH), 7.04 (d,
J = 8 Hz, 2H, ArH), 6.81 (d, J = 8 Hz, 2H, ArH), 3.76
(s, 3H, OCH3) 2.86 (t, J = 5.1 Hz, 4H, NCH2), 2.37 (s,
3H, CH3), 1.77 (p, J = 5.4 Hz, 4H, CH2) 1.44–1.38 (m,
2H, CH2); LC–MS, m/z [M+H]+: 459.2. Anal.
(C23H25F3N4O2) C, H, N.
The procedure for the synthesis of 9a was applied to
8d yielding 9d (87%) as a white solid; mp: 61–62 ꢂC;1H
NMR: 7.73 (br s, 1H, NH), 7.41 (t, J = 4 Hz, 1H,
ArH), 7.36 (m, 3H, ArH), 7.08 (d, J = 8 Hz, 2H,
ArH), 6.92 (d, J = 8 Hz, 2H, ArH), 3.86 (s, 3H,
OCH3) 2.89 (t, J = 5.3 Hz, 4H, NCH2), 2.33 (s, 3H,
CH3), 1.77 (p, J = 6 Hz, 4H, CH2) 1.48–1.38 (m, 2H,
CH2); LC–MS, m/z [M+H]+: 425.1; HRMS Calcd m/z
(TOF) [M+H]+: C23H26N4O2Cl 425.1744. Found:
425.1748. Anal. (C23H25N4O2ClÆ0.5H2O) C, H, N.
4.4.19. N-(Piperidin-1-yl)-5-(4-methoxyphenyl)-1-(phen-
yl)-4-methyl-1H-pyrazole-3-carboxamide (9e). The pro-
cedure for the synthesis of 9a was applied to 8e yielding
4.4.24. N-(Piperidin-1-yl)-1-(2-bromophenyl)-5-(4-meth-
oxyphenyl)-4-methyl-1H-pyrazole-3-carboxamide (9j).
The procedure for the synthesis of 9a was applied to 8j
yielding 9j (87%) as a white solid: mp: 204–205 ꢂC; H
1
1
9e (79%) as a white solid; mp: 164–165; H NMR: 7.63
(br s, 1H, NH), 7.31–7.20 (m, 6H, ArH), 7.08 (d,
J = 8 Hz, 2H, ArH), 6.89 (d, J = 8 Hz, 2H, ArH), 3.81
(s, 3H, OCH3) 2.86 (t, J = 5.3 Hz, 4H, NCH2), 2.34 (s,
3H, CH3), 1.79 (p, J = 6 Hz, 4H, CH2) 1.48–1.38 (m,
2H, CH2); LC–MS, m/z [M+H]+: 391.2; HRMS Calcd
m/z (TOF) [M+H]+: C23H27N4O2 391.2134. Found:
391.2143. Anal. (C23H26N4O2) C, H, N.
NMR (300 MHz, CDCl3): 7.7 (br s, 1H, NH), 7.60 (d,
J = 9 Hz, 1H, ArH), 7.33–7.21 (m, 3H, ArH), 7.06 (d,
J = 5.1 Hz, 2H, ArH), 6.81 (d, J = 5.1 Hz, 2H, ArH),
3.76 (s, 3H, OCH3) 2.86 (t, J = 5.1 Hz, 4H, NCH2),
2.37 (s, 3H, CH3), 1.77 (p, J = 5.4 Hz, 4H, CH2) 1.44–
1.38 (m, 2H, CH2); LC–MS, m/z [M+H]+: 469.1; HRMS
Calcd m/z (TOF) [M+H]+: C23H25BrN4O2 469.1239,
Found: 469.1232. Anal. (C23H25BrN4O2) C, H, N.
4.4.20. N-(Piperidin-1-yl)-5-(4-methoxyphenyl)-1-(2,3-
dichlorophenyl)-4-methyl- 1H-pyrazole-3-carboxamide
4.4.25.
N-(Piperidin-1-yl)-5-(4-methoxyphenyl)-1-(2,4-
(9f). The procedure for the synthesis of 9a was applied
to 8f yielding 9f (91%) as a white solid: mp: 194–
195 ꢂC; H NMR: 7.64 (br s, 1H, NH), 7.51–7.27 (t,
J = 7.51, 1H, ArH), 7.3–7.23 (m, 4H, ArH) 7.06 (d,
J = 8 Hz, 2H, ArH), 6.83 (d, J = 8 Hz, 2 H, ArH), 3.78
(s, 3H, OCH3) 2.86 (t, J = 5.3 Hz, 4H, NCH2), 2.36 (s,
3H, CH3), 1.76 (p, J = 5.6 Hz, 4H, CH2) 1.48–1.38 (m,
2H, CH2); LC–MS, m/z [M+H]+: 459.1; HRMS Calcd
m/z (TOF) [M+H]+: C23H25N4O2Cl2 459.1355, Found:
459.1363. Anal. (C23H24N4O2Cl2Æ0.4H2O) C, H, N.
dimethylphenyl)-4-methyl-1H-pyrazole-3-carboxamide
(9k). The procedure for the synthesis of 9a was applied
to 8k yielding 9k (89%) as a white solid: mp: 132–
133 ꢂC; 1H NMR: 7.71 (br s, 1H, NH), 7.10 (d,
J = 8 Hz, 1H, ArH) 7.03–6.99 (m, 4H, ArH), 6.81 (d,
J = 8 Hz, 2H, ArH), 3.77 (s, 3H, OCH3) 2.84 (t,
J = 5.3 Hz, 4H, NCH2), 2.37 (s, 3H, CH3), 2.13 (s, 3H,
CH3), 1.89 (s, 3H, CH3), 1.77 (p, J = 5.6 Hz, 4H, CH2)
1.45–1.38 (m, 2H, CH2); LC–MS, m/z [M+H]+: 419.2;
HRMS Calcd m/z (TOF) [M+H]+: C25H31N4O2
419.2447. Found: 419.2462; Anal. (C25H31N4O2Æ0.5-
H2O) C, H, N.
1
4.4.21.
N-(Piperidin-1-yl)-5-(4-methoxyphenyl)-1-(2,
6-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide
(9g). The procedure for the synthesis of 9a was applied
to 8g yielding 9g (75%) as a white solid: mp: 214–
216 ꢂC; H NMR (300 MHz, CDCl3): 7.63 (br s, 1H,
NH), 7.34–7.24 (m, 3H, ArH), 7.16 (d, J = 8 Hz, 2H,
ArH), 6.84 (d, J = 8 Hz, 2H, ArH), 3.77 (s, 3H,
OCH3), 2.89 (t, J = 5.3 Hz, 4H, NCH2), 2.36 (s, 3H,
CH3), 1.77 (p, J = 6 Hz, 4H, CH2) 1.48–1.38 (m, 2H,
CH2); LC–MS, m/z [M+H]+: 459.1; HRMS Calcd m/z
(TOF) [M+H]+: C23H25N4O2Cl2 459.1355, Found:
459.1369. Anal. (C23H24N4O2Cl2Æ0.4H2O) C, H, N.
4.4.26. N-(Piperidin-1-yl)-5-(4-methoxyphenyl)-1-pyridin-
2-yl-4-methyl-1H-pyrazole-3-carboxamide (9l). The pro-
cedure for the synthesis of 9a was applied to 8l yielding
9l (86%) as a white solid: mp: 170–171 ꢂC; H NMR
(300 MHz, CDCl3): 8.46 (d, J = 8 Hz, 1H, NH), 7.8
(br s, 1H, NH), 6.7 (t, J = 9.6 Hz, 1H, ArH) 7.25–7.16
(m, 4H, ArH), 7.11 (d, J = 8 Hz, 2H, ArH), 6.89 (d,
J = 8 Hz, 2H, ArH), 3.82 (s, 3H, OCH3) 2.90 (t,
J = 5.3 Hz, 4H, NCH2), 2.35 (s, 3H, CH3), 1.79 (p,
J = 5.6 Hz, 4H, CH2) 1.48–1.38 (m, 2H, CH2); LC–
MS, m/z [M+H]+: 392.1; HRMS Calcd m/z (TOF)
[M+H]+: C22H25N5O2 392.2087. Found: 392.2093; Anal.
(C22H24N5O2) C, H, N.
1
1
4.4.22. N-(Piperidin-1-yl)-5-(4-methoxyphenyl)-1-(2-meth-
ylphenyl)-4-methyl-1H-pyrazole-3-carboxamide (9h). The
procedure for the synthesis of 9a was applied to 8h yield-
ing 9h (89%) as a white solid: mp: 204–205 ꢂC; 1H
NMR: 7.80 (br s, 1H, NH), 7.8–7.27 (m, 4H, ArH),
7.13 (d, J = 8 Hz, 2H, ArH), 6.90 (d, J = 8 Hz, 2H,
4.4.27. N-(Piperidin-1-yl)-5-(4-methoxyphenyl)-1-(4-meth-
anesulfonylphenyl)-4-methyl-1H-pyrazole-3-carboxamide
(9m). The procedure for the synthesis of 9a was applied

S. R. Donohue et al. / Bioorg. Med. Chem. 14 (2006) 3712–3720
3719
to 8m yielding 9m (85%) as a white solid: mp: 218–
220 ꢂC; H NMR: 7.88 (d, J = 12 Hz, 2H, ArH), 7.73
cavity for 20 min at 150 ꢂC using 100 W. The reaction
solution was poured in the water (20 mL) and extracted
with dichloromethane (2· 20 mL). The combined ex-
tracts were dried over MgSO4, filtered, and evaporated
in vacuo. Purification with gradient flash chromatogra-
phy (hexanes–ethyl acetate) gave 9q as a white solid
1
(br s, 1H, NH), 7.46 (d, J = 12 Hz, 2H, ArH), 7.10 (d,
J = 8 Hz, 2H, ArH), 6.94 (d, J = 8 Hz, 2H, ArH), 3.85
(s, 3H, OCH3), 3.05 (s, 3H, SO2CH3), 2.9 (t,
J = 5.3 Hz, 4H, NCH2), 2.33 (s, 3H, CH3), 1.77 (p,
J = 6 Hz, 4H, CH2) 1.47–1.38 (m, 2H, CH2); LC–MS,
m/z [M+H]+: 469.2; HRMS Calcd m/z (TOF) [M+H]+:
C24H29N4O4S 469.1910. Found: 469.1915; Anal.
(C24H28N4O4S) C, H, N.
1
(34 mg, 16%); mp: 201–203 ꢂC; H NMR: 7.69 (br s,
1H, NH), 7.48–7.35 (m, 4H, ArH), 7.11 (d, J = 8 Hz,
2H, ArH), 6.81 (d, J = 8 Hz, 2H, ArH), 5.75 (d,
J = 52, 2H, OCH2F) 2.86 (t, J = 5.3 Hz, 4H, NCH2),
2.37 (s, 3H, CH3), 1.77 (p, J = 6 Hz, 4H, CH2) 1.48–
1.38 (m, 2H, CH2); LC–MS m/z [M+H]+: 443.1; HRMS
Calcd m/z (TOF) [M+H]+: C23H25N4O2FCl 443.1650.
Found: 443.1660; Anal. (C23H24N4O2FCl) C, H, N.
4.4.28. N-(Piperidin-1-yl)-5-phenyl-1-(2-chlorophenyl)-4-
methyl-1H-pyrazole-3-carboxamide (9n). The procedure
for the synthesis of 9a was applied to 8n yielding 9n
(89%) as a white solid: mp: 224–225 ꢂC; 1H NMR
(300 MHz, CDCl3): 7.76 (br s, 1H, NH), 7.31 (m, 5H,
ArH), 7.07 (d, J = 5.1 Hz, 2H, ArH), 6.80 (d,
J = 5.1 Hz, 2H, ArH), 3.80 (s, 3H, OCH3) 2.89 (t,
J = 5.1 Hz, 4H, NCH2), 2.35 (s, 3H, CH3), 1.79 (p,
J = 5.4 Hz, 4H, CH2) 1.45 (m, 2H, CH2); LC–MS m/z
[M+H]+: 395.1; HRMS Calcd m/z (TOF) [M+H]+:
C22H24N4O2Cl 395.1639. Found: 365.1649. Anal.
(C22H23N4O2Cl) C, H, N.
4.5. CB1 and CB2 GTPc 35S-binding assays
GTPc35S binding was measured in a 96-well format,
using a modified antibody capture technique previously
described.32 Test compounds were diluted in GTP-bind-
ing assay buffer (20 mM Hepes, 100 mM NaCl, and
5 mM MgCl2, pH 7.4) containing bovine serum albumin
(0.5%). Sf9 cell membranes, expressing human CB1 or
CB2 receptors, and Gai3b1g2 (Perkin Elmer, Boston,
MA, USA) in GTP-binding assay buffer and the test
compound were incubated for 15 min at rt. This was fol-
lowed by the addition of GTPc35S (500 pM; NEN, Bos-
ton, MA) and a 35 min incubation. The labeled
membranes were then solubilized with 0.27% Nonidet
P40 detergent (Roche, Indianapolis, IN) and incubated
for 30 min. Rabbit anti-Gai3 antibody was then added
at a final dilution of 1:300 and the mixture was incubated
for another 30 min. Anti-rabbit antibody-coated scintil-
lation proximity assay beads (Amersham Life Sciences,
Arlington Heights, IL) were added and the plates were
incubated at RT for an hour. The plates were then centri-
fuged at 180g for 10 min using a Beckman GS-6R centri-
fuge and counted for 1 min per well using a Wallac 1450
MicroBeta TriLux scintillation counter (Perkin Elmer
Life Sciences, Wallac Inc., Gaithersburg, MD). Agonist
EC50 values and antagonist IC50 values were calculated
with Activity Base software using a four-parameter fit
and KB values (binding constants) calculated as follows:
KB = IC50/[1 + [Agonist]/EC50].
4.4.29. N-(Piperidin-1-yl)-1-(2-bromophenyl)-5-(4-hydroxy-
phenyl)-4-methyl-1H-pyrazole-3-carboxamide (9o). To a
stirred solution of 9j (500 mg; 1.07 mmol) in dry dichlo-
romethane (20 mL) was added BBr3 (5.89 mL; 1 M solu-
tion in dichloromethane). The reaction was stirred at rt
for 3 h. The reaction was quenched with careful addition
of methanol. The combined extracts were dried over
MgSO4, filtered and evaporated. The product was puri-
fied with flash chromatography (chloroform–methanol
10%) yielding 9o (407 mg, 84%) as a white solid: mp:
260–262 ꢂC; 1H NMR (DMSO-d6): 11.7 (br s, 1H,
NH), 7.71 (m, 4H, ArH), 7.04 (d, J = 8 Hz, 2H, ArH),
6.73 (d, J = 8 Hz, 2H, ArH), 2.49 (t, J = 5.1 Hz, 4H,
NCH2), 2.26 (s, 3H, CH3), 1.85 (p, J = 5.4 Hz, 4H,
CH2) 1.49 (m, 2H, CH2); LC–MS m/z [M+H]+: 455.0;
HRMS Calcd m/z (TOF) [M+H]+: C22H24N4O2Br
455.1083. Found: 455.1104. Anal. (C22H23N4O2Br) C,
H, N.
4.4.30. N-(Piperidin-1-yl)-1-(2-chlorophenyl)-5-(4-hydroxy-
phenyl)-4-methyl-1H-pyrazole-3-carboxamide (9p). The
demethylation procedure applied in the synthesis of 9o
was applied to carboxamide 5 (500 mg; 1.18 mmol) yield-
Acknowledgments
1
ing 9p (92%) as a white solid: mp: 256–257 ꢂC; H NMR
(DMSO-d6): 11.7 (br s, 1H, NH), 7.71 (m, 4H, ArH), 7.05
(d, J = 8 Hz, 2H, ArH), 6.76 (d, J = 8 Hz, 2H, ArH), 2.56
(t, J = 5.1 Hz, 4H, NCH2), 2.30 (s, 3H, CH3), 1.91 (p,
J = 5.4 Hz, 4H, CH2) 1.45 (m, 2H, CH2); LC–MS, m/z
[M+H]+: 411.1; HRMS Calcd m/z (TOF) [M+H]+:
C22H24N4O2Cl 411.1588. Found: 411.1596; Anal.
(C22H23N4O2Cl) C, H.
This work was supported by the Intramural Research
Program of the National Institutes of Health (National
Institute of Mental Health). Mr. S. R. Donohue was
supported as a student in the National Institutes of
Health–Karolinska Institutet Graduate Training Part-
nership in Neuroscience. The authors are grateful
to Dr. John Schaus and Ms. Wendy Gough at Lilly
Research Laboratories (Indianapolis, IN, USA) for
performing binding assays.
4.4.31. N-(Piperidin-1-yl)-5-(4-fluoromethoxyphenyl)-1-
(2-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide
(9q). To a stirred solution of carboxamide 9p (200 mg,
488 lmol) and K2CO3 (134 mg, 975 lmol) in acetonitrile
(2 mL) cooled to 0 ꢂC in a sealed microwave vial was
added fluoromethyl bromide (251 mg, 2.24 mmol). The
reaction was irradiated in a single mode microwave
References and notes
1. Howlett, A. C.; Barth, F.; Bonner, T. I.; Cabral, G.;
Casellas, P.; Devane, W. A.; Felder, C. C.; Herkenham,

3720
S. R. Donohue et al. / Bioorg. Med. Chem. 14 (2006) 3712–3720
M.; Mackie, K.; Martin, B. R.; Mechoulam, R.; Pertwee,
R. G. Pharmacol. Rev. 2002, 54, 161–202.
2. Howlett, A. C.; Qualy, J. M.; Khachatrian, L. L. Mol.
Pharmacol. 1986, 29, 307–313.
3. Herkenham, M.; Lynn, A. B.; Little, M. D.; Johnson, M.
R.; Melvin, L. S.; de Costa, B. R.; Rice, K. C. Proc. Natl.
Acad. Sci. U.S.A. 1990, 87, 1932–1936.
18. Wiley, J. L.; Jefferson, R. G.; Grier, M. C.; Mahadevan,
A.; Razdan, R. K.; Martin, B. R. J. Pharmacol. Exp. Ther.
2001, 296, 1013–1022.
19. Thomas, B. F.; Francisco, M. E.; Seltzman, H. H.;
Thomas, J. B.; Fix, S. E.; Schulz, A. K.; Gilliam, A. F.;
Pertwee, R. G.; Stevenson, L. A. Bioorg. Med. Chem.
2005, 13, 5463–5474.
4. Glass, M.; Dragunow, M.; Faull, R. L. Neuroscience 1997,
77, 299–318.
20. Shim, J. Y.; Welsh, W. J.; Cartier, E.; Edwards, J. L.;
Howlett, A. C. J. Med. Chem. 2002, 45, 1447–1459.
21. Katoch-Rouse, R.; Pavlova, O. A.; Caulder, T.; Hoffman,
A. F.; Mukhin, A. G.; Horti, A. G. J. Med. Chem. 2003,
46, 642–645.
22. Reggio, P. H. Curr. Pharm. Des. 2003, 9, 1607–1633.
23. Gatley, S. J.; Gifford, A. N.; Volkow, N. D.; Lan, R.;
Makriyannis, A. Eur. J. Pharmacol. 1996, 307, 331–338.
24. Gatley, S. J.; Lan, R.; Volkow, N. D.; Pappas, N.; King,
P.; Wong, C. T.; Gifford, A. N.; Pyatt, B.; Dewey, S. L.;
Makriyannis, A. J. Neurochem. 1998, 70, 417–423.
5. Croci, T.; Manara, L.; Aureggi, G.; Guagnini, F.; Rinaldi-
Carmona, M.; Maffrand, J. P.; Le Fur, G.; Mukenge, S.;
Ferla, G. Br. J. Pharmacol. 1998, 125, 1393–1395.
6. Straiker, A. J.; Maguire, G.; Mackie, K.; Lindsey, J.
Invest. Ophthalmol. Vis. Sci. 1999, 40, 2442–2448.
7. Gye, M. C.; Kang, H. H.; Kang, H. J. Arch. Androl. 2005,
51, 247–255.
8. Pertwee, R. G.; Fernando, S. R. Br. J. Pharmacol. 1996,
118, 2053–2058.
9. Klein, T. W.; Newton, C.; Friedman, H. Immunol. Today
1998, 19, 373–381.
25. Berding, G.; Muller-Vahl, K.; Schneider, U.; Gielow, P.;
¨
Fitschen, J.; Stuhrmann, M.; Harke, H.; Buchert, R.;
Donnerstag, F.; Hofmann, M.; Knoop, B. O.; Brooks, D. J.;
Emrich, H. M.; Knapp, W. H. Biol. Psychiatry 2004, 55,
904–915.
`
10. Galiegue, S.; Mary, S.; Marchand, J.; Dussossoy, D.;
Carriere, D.; Carayon, P.; Bouaboula, M.; Shire, D.; Le
`
Fur, G.; Casellas, P. Eur. J. Biochem. 1995, 232, 54–61.
11. Haller, J.; Varga, B.; Ledent, C.; Freund, T. F. Behav.
Pharmacol. 2004, 15, 299–304.
12. Le Foll, B.; Goldberg, S. R. J. Pharmacol. Exp. Ther.
2005, 312, 875–883.
13. Vinod, K. Y.; Arango, V.; Xie, S.; Kassir, S. A.; Mann, J.
J.; Cooper, T. B.; Hungund, B. L. Biol. Psychiatry 2005,
57, 480–486.
26. Mathews, W. B.; Scheffel, U.; Finley, P.; Ravert, H. T.;
Frank, R. A.; Rinaldi-Carmona, M.; Barth, F.; Dannals,
R. F. Nucl. Med. Biol. 2000, 27, 757–762.
27. Mathews, W. B.; Scheffel, U.; Rauseo, P. A.; Ravert, H.
T.; Frank, R. A.; Ellames, G. J.; Herbert, J. M.; Barth, F.;
Rinaldi-Carmona, M.; Dannals, R. F. Nucl. Med. Biol.
2002, 29, 671–677.
14. Lastres-Becker, I.; Berrendero, F.; Lucas, J. J.; Martin-
´
Ruiz, J. J. Brain Res. 2002, 929, 236–242.
15. Muller-Vahl, K. R. Expert Opin. Pharmacother. 2003, 4,
1717–1725.
28. Pike, V. W. J. Psychopharmacol. 1993, 7, 139–158.
29. Waterhouse, R. N. Mol. Imaging Biol. 2003, 5, 376–389.
30. Li, Z.; Gifford, A.; Liu, Q.; Thotapally, R.; Ding, Y. S.;
Makriyannis, A.; Gatley, S. J. Nucl. Med. Biol. 2005, 32,
361–366.
Aparicio, E.; Yamamoto, A.; Ramos, J. A.; Fernandez-
16. Rinaldi-Carmona, M.; Barth, F.; Heaulme, M.; Shire, D.;
Calandra, B.; Congy, C.; Martinez, S.; Maruani, J.;
31. Kumar, J. S.; Prabhakaran, J.; Arango, V.; Parsey, R. V.;
Underwood, M. D.; Simpson, N. R.; Kassir, S. A.; Majo,
V. J.; Van Heertum, R. L.; Mann, J. J. Bioorg. Med.
Chem. Lett. 2004, 14, 2393–2396.
32. DeLapp, N. W.; McKinzie, J. H.; Sawyer, B. D.;
Vandergriff, A.; Falcone, J.; McClure, D.; Felder, C. C.
J. Pharmacol. Exp. Ther. 1999, 289, 946–955.
´
Neliat, G.; Caput, D.; Ferrara, T.; Soubrie, P.; Breliere,
J. C.; Le Fur, G. FEBS Lett. 1994, 350, 240–244.
17. Lan, R. X.; Liu, Q.; Fan, P. S.; Lin, S. Y.; Fernando, S.
R.; McCallion, D.; Pertwee, R.; Makriyannis, A. J. Med.
Chem. 1999, 42, 769–776.