Analogs of JHU75528, a PET ligand for imaging of cerebral cannabinoid receptors (CB1): Development of ligands with optimized lipophilicity and binding affinity

Article abstract of DOI:10.1016/j.ejmech.2008.03.040

Cyano analogs of Rimonabant with high binding affinity for the cerebral cannabinoid receptor (CB1) and with optimized lipophilicity have been synthesized as potential positron emission tomography (PET) ligands. The best ligands of the series are optimal t

Full text of DOI:10.1016/j.ejmech.2008.03.040

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European Journal of Medicinal Chemistry 44 (2009) 593e608
http://www.elsevier.com/locate/ejmech
Original article
Analogs of JHU75528, a PET ligand for imaging of cerebral
cannabinoid receptors (CB1): Development of ligands
with optimized lipophilicity and binding affinity
Hong Fan a, Evangelia Kotsikorou b, Alexander F. Hoffman c, Hayden T. Ravert a,
Daniel Holt a, Dow P. Hurst b, Carl R. Lupica c, Patricia H. Reggio b,
Robert F. Dannals a, Andrew G. Horti a,
*
a PET Center, Division of Nuclear Medicine, Department of Radiology,
Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
b Center for Drug Discovery, Chemistry and Biochemistry Department, University of North Carolina, Greensboro, NC 27402-6170, USA
c National Institute on Drug Abuse Intramural Research Program, Cellular Neurobiology Branch,
Electrophysiology Research Unit, Baltimore, MD 21224, USA
Received 19 November 2007; received in revised form 21 March 2008; accepted 27 March 2008
Available online 18 April 2008
Abstract
Cyano analogs of Rimonabant with high binding affinity for the cerebral cannabinoid receptor (CB1) and with optimized lipophilicity have
been synthesized as potential positron emission tomography (PET) ligands. The best ligands of the series are optimal targets for the future radio-
labeling with PET isotopes and in vivo evaluation as radioligands with enhanced properties for PET imaging of CB1 receptors in human subjects.
Extracellular electrophysiological recordings in rodent brain slices demonstrated that JHU75528, 4, the lead compound of the new series, has
functional CB antagonist properties that are consistent with its structural relationship to Rimonabant. Molecular modeling analysis revealed an
important role of the binding of the cyano group with the CB1 binding pocket.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Cannabinoid receptor; Carbon-11; JHU75528; Rimonabant; Positron emission tomography; Molecular modeling; Receptor docking
1. Introduction
psychoactive constituent in marijuana (Cannabis sativa L.),
the non-classical cannabinoids including (ꢀ)-CP-55940, the
endogenous cannabinoid ligand anandamide and its analogs,
the aminoalkylindoles (WIN 55212-2), and cannabinoid
antagonists. The most representative member of the latter class
is Rimonabant (AcompliaÔ or SR141716) 1 (Fig. 1) which is
a first highly selective CB1 antagonist developed by Sanofi-
Aventis [4]. Rimonabant 1 was granted authorization for mar-
keting in European Union states in 2006 as an anti-obesity drug
[5,6]. Cannabinoid ligands and, particularly, CB1 antagonists
are an emerging class of drugs for control of appetite [5e8],
treatment of neuropsychiatric disorders [9e13], and drug
dependence [14e16]. In addition, there is a growing number
of structurally diverse CB1 antagonists/inverse agonists [17]
that possess a pharmacophoric arrangement similar to that of 1.
To date, at least two subtypes of the cannabinoid receptor,
CB1 and CB2, have been cloned [1]. CB1 receptors are located
predominantly in the brain and to a lesser extent in ganglionic
system whereas CB2 receptors are found mainly on immune
cells [1] and, in low density, in the brain [2]. The currently
known classes of cannabinoid receptor ligands (see for review
[3]) include tetrahydrocannabinol (D9-THC), the principal
* Corresponding author. PET Center, Division of Nuclear Medicine,
Radiology, Johns Hopkins Medicine, 600 North Wolfe Street, Nelson
B1-122, Baltimore, MD 21287-0816, USA. Tel.: þ1 410 614 5130.
E-mail address: ahorti1@jhmi.edu (A.G. Horti).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.03.040

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H. Fan et al. / European Journal of Medicinal Chemistry 44 (2009) 593e608
O
N
NH
O
O
O
N
N
NH
N
N
H
H3C
H3C
H3C
N
N
N
N
N
Cl
Cl
Cl
Cl
123/124I
H311CO
Cl
Cl
Cl
123/124I]2, [123/124I]AM281
1, Rimonabant
(SR141716, AcompliaTM)
[
[
11C]3, [11C]NIDA41020 or
[
11C]SR144380
Fig. 1. Representative cannabinoid ligands and radioligands.
The ability to image CB1 receptors in the human brain using
Recently we reported development of JHU75528 4 (Fig. 2,
Table 1), an analog of Rimonabant having a combination of
greater CB1 in vitro binding affinity and lower lipophilicity
than those of the previously studied CB1 in vivo radioligands
[30]. Compound [11C]4 is the first CB1 PET radioligand
manifesting reasonable imaging properties in animals [31]
and human PET studies with [11C]4 is currently in progress.
New reports of other groups described the development of
[11C]Me-PPEP ([11C]5) [32] and [18F]MK-9470 ([18F]6) [33]
(Fig. 2) that are also suitable for quantitative PET imaging
of CB1 radioligands. Yet, PET imaging properties of all three
compounds [31e33] are not ideal and exhibit certain draw-
backs including modest binding potential and/or moderate
brain uptake and/or slow brain kinetics. Thus, the binding po-
tentials of all three compounds [11C]4, [11C]5 and [18F]6 are
moderate [31e33]. However, unlike the case with [11C]5
and [18F]6 [32,33] having very slow brain kinetics the radioli-
gand [11C]4 manifests ideal brain kinetics in non-human pri-
mate studies for PET quantification [31]. We suggested that
improvement of imaging properties of [11C]4 including bind-
ing potential and total brain uptake could be achieved by de-
velopment of analogs of [11C]4 with better in vitro
properties: higher binding affinity and reduced lipophilicity
within the optimal range for PET radioligands (log D ¼ 1e3).
Here we are presenting a series of analogs of 4 with improved
binding affinity and lipophilicity.
positron emission tomography (PET) or single photon emission
computed tomography (SPECT) would provide the possibility
to conduct non-invasive receptor studies under normal physio-
logical conditions and in disease states. The development of
useful PET radiotracer would assist in the design and testing
of promising pharmaceuticals targeting the CB1 receptor.
Initial attempts by several groups to image CB1 in animal
brains examined the feasibility of specific labeling of CB1
in vivo [18e28]. Unfortunately, all of the radioligands initially
studied (more than 10; mostly analogs of D9-tetrahydrocan-
nabinol, the principal psychoactive component of marijuana,
cannabinoid agonist WIN 55212-2 and 1) exhibited insuffi-
cient properties for quantification of CB1 receptor by PET
(low specific binding, high non-specific binding and/or low
brain uptake). The attempts to quantify CB1 in the living hu-
man brain by SPECT and PET demonstrated low binding po-
tential (BP ¼ 0.21 and 0.37) for the radioligands [123I]2 [27]
and [124I]2 [28], correspondingly (Fig. 1). High lipophilicity
and tight association to proteins were likely causes of high
non-specific binding that resulted in low contrast of the in
vivo images with labeled 2 [27,28]. Compound [11C]3
(Fig. 1) synthesized by our group in the past [21,29] also dis-
played a low BP (0.6) and relatively low brain uptake in the
Rhesus monkey brain due to its high lipophilicity and moder-
ate binding affinity [21,29].
CN
O
N
NH
O
NC
O
O
N
N
F3C
N
N
N
Me
H
Cl
H311CO
CH3
18F
O
O11CH3
11C]MePPEP, [11C]5
Cl
[
18F]MK9470, [18F]6
11C]JHU75528, [11C]4
[
[
Fig. 2. Latest radioligands for emission tomography imaging of CB1 receptors.

H. Fan et al. / European Journal of Medicinal Chemistry 44 (2009) 593e608
595
Table 1
Comparison of inhibition binding affinity ([3H]2, recombinant hCB1) and lipophilicity of compounds 1, 2, 4, 15, 19 and 7ae7j
H
O
N
R4
R5
N
N
R2
R1
R3
a
c
Compound
R1
R2
R3
R4
R5
Binding affinity,b Ki, nM
Experimental, log D7.4
HU210
1
e
Cl
e
e
e
e
1.5 ꢁ 0.8 (18)
40 ꢁ 8 (3)
422, 525 [30]
11 ꢁ 7 [30]
4.7 [30]
13, 18
e
Cl
Cl
Cl
Br
Cl
Br
Cl
Cl
Br
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Pip
Mor
Pip
Pip
Pip
Pip
Pip
Pip
Pip
Mor
Mor
Pyr
Pip
Pip
CH3
CH3
CN
CN
CN
CN
CN
CN
4.6 [30]
e
2
I
4
OMe
OMe
OMe
OMe
I
3.3e3.6 [30]
3.4 [30]
2.8 ꢁ 0.2 (3)
2.9 ꢁ 0.2 (3)
3.9
15
7a
7b
7c
7d
7e
7f
F
F
32, 37
33
14, 20
Cl
Cl
H
Br
3.7
2.5
OMe
I
NH2CO
CN
CN
>10,000
131
>1000
Cl
Cl
Cl
Cl
Cl
3.2
3.1
7g
7h
7i
Br
OMe
OCH2F
Cl
CN
CN
2.0, 3.7
10.3 ꢁ 3.2 (3)
24
2.8 ꢁ 0.1 (3)
2.9 ꢁ 0.3(3)
3.5 ꢁ 0.2 (3)
7jd
a
CN
Pip ¼ piperidinyl, Mor ¼ morpholinyl, Pyr ¼ pyrrolidinyl.
b
Determination of inhibition binding affinity of all compounds in the table has been done commercially by Novascreen under the same conditions as described
[30], Ki ¼ mean ꢁ SD (number of independent assays), high affinity synthetic cannabinoid HU210 [1,38] was used here as a reference standard.
c
The octanol/phosphate buffer (pH ¼ 7.4) partition coefficients (log D7.4) of compounds 7ae7j were measured using conventional flask-shake technique,
mean ꢁ SD (number of independent assays).
d
Synthesized for this study as described elsewhere [34].
2. Results and discussion
obtained as a major by-product of ethyl 1-(2-bromophenyl)-4-
cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxylate 12e
that is described elsewhere [30].
2.1. Chemistry
The ethyl esters 12ae12d, 12e0 were saponified with lithium
hydroxide (Scheme 2) to give the carboxylic acids 13ae13e
that were further coupled with either 1-aminopiperidine 10a,
1-aminopyrrolidine 10b or 1-aminomorpholine 10c in the
presence of benzotriazol-1-yloxytris(dimethylamino)-
phosphonium hexafluorophosphate (BOP) to produce 7ae
7h in reasonable yields.
Novel analogs of 4 (compounds 7ae7i) were synthesized
in this study via the general methods described in the indus-
trial patent [34] and our paper [30] using three building
blocks: derivatives of benzoylacetonitrile 8, derivatives of
ethyl 2-chloro-2-(2-benzylhydrazono)acetate 9 and cyclic
1,1-dialkylhydrazines 10 (Schemes 1 and 2).
Ethyl 2-chloro-2-(2-benzylhydrazono)acetates 9a and 9b
(Scheme 1) were obtained in two steps by conversion of cor-
responding commercially available anilines 11a and 11b into
arenediazonium salts followed by reaction with ethyl 2-
chloro-acetoacetate using the previously published method
[35]. The intermediate benzoylacetonitrile 8a was prepared
by reaction of ethyl 4-iodobenzoate with acetonitrile in the
presence of sodium ethoxide [36] (Scheme 1).
The cycloaddition (Scheme 1) of benzoylacetonitriles 8ae
8c and ethyl 2-chloro-2-(2-benzylhydrazono)acetate deriva-
tives 9ae9d in the presence of sodium ethoxide yielded ethyl
4-cyano-1,5-diaryl-1H-pyrazole-3-carboxylates 12ae12e. Un-
der the conditions of this reaction, the cyano group can undergo
a side reaction yielding the corresponding 4-carbamoylpyrazole
as a by-product. Thus, ethyl 1-(2-bromophenyl)-4-carbamoyl-
5-(4-methoxyphenyl)-1H-pyrazole-3-carboxylate 12e0 was
The fluoromethoxy derivative 7i was prepared by fluoro-
methylation of the corresponding phenol 14 [30] with fluoro-
methyl tosylate [37] (Scheme 3).
2.2. Structureeactivity relationships
The overall objective of this study is in development of an-
alogs of 4 with reduced lipophilicity and improved binding
affinity as potential PET ligands for imaging CB1 receptor. In
our previous structureeactivity relationship study with
derivatives of Rimonabant 1 replacement of a lipophilic substit-
uent attached to the C5 benzene ring of 1 with a substituent with
reduced hydrophobic constant p led to a reduction in binding
affinity whereas there was no clear correlation of the binding af-
finity vs. hydrophobic properties of the substituents of the N1
benzene ring [21,29]. Therefore, here we have initially chosen

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H. Fan et al. / European Journal of Medicinal Chemistry 44 (2009) 593e608
O
O
NH2
NC
R1
R1
OEt
N
a,b
c
N
N
R2
R3
O
R2
N
H
Cl
R2
O
11a R1 = Cl, R2 = F
11b R1 = Br, R2 = F
9a R1 = Cl, R2 = F
9b R1 = Br, R2 = F
9c R1 = Br, R2 = H
12a R2 = Cl, R3 = F
12b R2 = Br, R3 = F
O
O
NH2
O
O
N
NC
O
N
N
N
Br
Br
+
c
O
O
12e'
12e
O
O
O
NC
EtOOC
N
d
CN
N
e
Cl
Cl
X
I
R1
12c R1 = I
12d R1 = Br
8a X = I
8b X = Br
Scheme 1. Reagents: (a) NaNO2, HCl; (b) ethyl 2-chloro-acetoacetate, CH3COONa, ethanol; (c) 4-methoxybenzoylacetonitrile 8c, sodium ethanolate; (d) sodium
ethanolate, CH3CN; (e) ethyl 2-chloro-2-(2-(2,4-dichlorophenyl)hydrazono)acetate 9d, sodium ethanolate.
to replace the chloro substituents of N1 benzene ring of 4. Re-
placement of the 4-chloro substituent with fluorine gave the
chloroefluoro derivative 7a with comparable binding affinity
and reduced lipophilicity (Table 1). When both chlorine atoms
of 4 were replaced with fluorine and bromine, derivative 7b
with lower binding affinity (Table 1) was obtained.
Reduction of the number of C-atoms in the molecule usually
reduces a compound lipophilicity. Shrinkage of the piperidine
ring of 4 to a pyrrolidine ring revealed analog 7h with lower
lipophilicity and slightly higher binding affinity. The previous
studies did not demonstrate an improvement of the binding af-
finity of pyrrolidinyl vs. piperidinyl analogs in the 5 series [39].
O
H
N
O
OH
R4
R5
R5
N
N
N
N
R2
R2
a
LiOH
12a-12d, 12e'
BOP
H2O/THF
R1
R1
R3
R3
7a R1=OMe, R2=Cl, R3=F, R4=piperidinyl, R5=CN
7b R1=OMe, R2=Br, R3=F, R4=piperidinyl, R5=CN
7c R1=I, R2=Cl, R3=Cl, R4=piperidinyl, R5=CN
7d R1=Br, R2=Cl, R3=Cl, R4=piperidinyl, R5=CN
7e R1=OMe, R2=Br, R3=H, R4=piperidinyl, R5=CO-NH2
7f R1=I, R2=Cl, R3=Cl, R4=morpholinyl, R5=CN
7g R1=Br, R2=Cl, R3=Cl, R4=morpholinyl, R5=CN
7h R1=OMe, R2=Cl, R3=Cl, R4=pyrrolidinyl, R5=CN*
13a R1=OMe, R2=Cl, R3=F, R5=CN
13b R1=OMe, R2=Br, R3=F, R5=CN
13c R1=I, R2=Cl, R3=Cl, R5=CN
13d R1=Br, R2=Cl, R3=Cl, R5=CN
13e R1=OMe, R2=Br, R3=H, R5=CO-NH2
Scheme 2. Reagents: BOP ¼ benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate, (a) N-aminopiperidine or N-aminomorpholine or
N-aminopyrrolidine; * 7h was prepared with 1-(2,4-dichlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxylic acid 13f [30].

H. Fan et al. / European Journal of Medicinal Chemistry 44 (2009) 593e608
597
studies suggested that the binding site of the CB1 inverse ag-
onist/antagonist, 1, is within the transmembrane helix
(TMH)3-4-5-6 aromatic microdomain and involves direct aro-
matic stacking interactions with F3.36(200), Y5.39(275), and
W5.43(279) [40]. Our modeling studies also suggested that al-
though 1 can engage in aromatic stacking interactions in the
inactive (R) and active (R*) states of CB1, the C3 substituent
carboxamide oxygen of 1 can hydrogen bond with K3.28(192)
only in the CB1 inactive (R) state [41]. Through mutant cycle
[41] and SAR studies [42], we have demonstrated that the in-
verse agonism produced by 1 is likely due to this hydrogen-
bonding interaction with K3.28(192).
N
NH
N
NH
O
O
NC
NC
N
N
a
N
N
Cl
Cl
F
O
HO
14
7i
Cl
Cl
Scheme 3. Reagents: (a) FCH2OTs, K2CO3, acetone.
Conversion of the methoxy group of 4 to the fluorome-
thoxy-group gave an equally potent CB1 ligand 7i with re-
duced lipophilicity.
2.4. Conformational analysis
The global minimum energy conformer of 4 (see Fig. 3) has
the carboxamide oxygen of the C3 substituent nearly in plane
with the pyrazole ring and pointing in the direction of the C4
cyano group (OeC10eC3eC4 ¼ 2.3ꢂ). The piperidine ring is
in a chair conformation with the lone pair of the nitrogen
electrons pointing in the same direction as the carboxamide
hydrogen (LPeN30eN20eH ¼ ꢀ1.0ꢂ). The C5 substituent
para-methoxy ring is out of plane with the pyrazole ring
(C4eC5eC1000eC2000 ¼ ꢀ47.3ꢂ) and the methoxy group is
nearly in the plane of the aromatic ring (C3000eC4000eOe
C ¼ 3.3ꢂ). The N1 substituent dichlorophenyl ring is also out
It is noteworthy that replacement of the 4-cyano group of
the high affinity ligand 15 [30] (Table 1) with 4-carbamoyl
group yielded derivative 7e that did not bind with the CB1
receptor. This finding is disappointing because compound 7e
manifests a lipophilicity value in the target range (Table 1).
The binding affinity matter of 7e is discussed in greater details
below in Section 2.3.
Radioligands [123I]2 and [124I]2 (Fig. 1), radioiodinated
analogs of 1, were developed as SPECT and PET ligands for
studying CB1 receptors in human subjects [27,28]. We hy-
pothesized that replacement of the 4-methyl group in the
molecule 2 with a cyano substituent might give a better ligand.
As we expected, the binding affinity of the 4-cyano compound
7f was greater than that of 2 (Table 1). Compound 7c, a piper-
idinyl analog of the morpholinyl derivative 7f, manifested
even better binding affinity but, also, substantially higher
lipophilicity than that of 7f. Surprisingly, brominated ligand
7d shows an improved binding affinity as compared with its
iodinated congener 7c. Previous SAR studies with 4-Br and
4-I analogs of 1 demonstrated a higher binding affinity of
the iodo derivative [39].
Replacement of the 4-methyl group in 1 with a 4-cyano
group yielded compound 7j previously described in an indus-
trial patent [34]. The binding affinity of 7j was not reported in
the patent. Experimental determination of properties of 7j
demonstrated that this CN-analog of 1 displays higher binding
affinity and lower lipophilicity than those of 1 (Table 1). The
role of the cyano group in the binding with CB1 receptor is
discussed below.
Good binding affinity and reduced lipophilicity of 7a, 7h
and 7i suggest that if radiolabeled with positron-emitting
isotopes 11C and 18F these ligands might be better than
[11C]4 as PET radioligands.
2.3. Molecular modeling studies
The goal of modeling studies was to assess if the cyano com-
pound 4 could occupy the same binding site at CB1 receptor as
the methylated lead compound 1 and to probe the CB1 receptor
state model for the molecular origins of the low binding affinity
of compound 7e. Our previous combined mutation/modeling
Fig. 3. The global minimum energy conformers of compounds 1, 4 and 7e are
illustrated here. Compound 7e was found to have an intramolecular hydrogen
bond between the C4 carboxamide nitrogen and the C3 carboxamide oxygen.

598
H. Fan et al. / European Journal of Medicinal Chemistry 44 (2009) 593e608
of plane with the pyrazole ring (N2eN1eC100e
C200 ¼ ꢀ74.7ꢂ). In this position the ortho-chloro is in the
bottom face of the molecule. This conformation is very similar
to the previously calculated global minimum energy conformer
of SR141716 (1) [41,42] (see Fig. 3).
the CB1 inactive state TMH3-4-5-6 aromatic microdomain re-
gion. In Fig. 4(B), SR141716 (1) is docked in the same
TMH3-4-5-6 aromatic microdomain in a minimum energy
conformation (DE ¼ 0.92 kcal/mol above its global minimum)
[41,42]. The view in Fig. 4 is from lipid, looking towards
TMHs 3 and 4. TMHs 1, 2 and 7 are not shown in order to sim-
plify the view. Aromatic residues for which 4 and 1 have
a direct aromatic stacking interaction are colored orange
here. Aromatic residues that are part of the extended aromatic
cluster are colored yellow, while those that are not part of the
aromatic cluster are colored cyan. In Fig. 4(A), boꢂth the
The global minimum energy conformer of 7e (see Fig. 3) has
the carboxamide oxygen of the C3 substituent slightly out of
plane with the pyrazole ring and pointing in the direction of
the C4 amide group (OeC10eC3eC4 ¼ ꢀ5.5ꢂ). The piperidine
ring is in a chair conformation with the lone pair of the nitrogen
electrons pointing in the same direction as the carboxamide
hydrogen (LPeN30eN20eH ¼ ꢀ1.7ꢂ). The C5 substituent
para-methoxy ring is out of plane with the pyrazole ring
(C4eC5eC1000eC2000 ¼ ꢀ55.6ꢂ) and the methoxy group is
nearly in the plane of the aromatic ring (C3000eC4000eOe
C ¼ 3.2ꢂ). The N1 substituent bromophenyl ring is also out of
plane with the pyrazole ring (N2eN1eC100eC200 ¼ ꢀ71.9ꢂ).
In this position, the ortho-bromo is in the bottom face of the
molecule. Compound 7e also has an intramolecular hydrogen
bond that locks the position of the carboxamide oxygen of the
C3 substituent and the amide nitrogen of the C4 substituent in
place. The hydrogen bond distance (N/O) and angle (NeHeO)
˚
carboxamide oxygen (N/O ¼ 2.65 A, NeHeO ¼ 157 ) and
ꢂ
˚
the cyano group of 4 (N/N ¼ 2.81 A, NeHeN ¼ 122 )
have hydrogen-bonding interactions with K3.28(192), a residue
that is part of a K3.28(192)/D6.58(366) salt bridge that is
a characteristic of the R state model [42]. Compound 4
˚
directly stacks with W5.43(279) (methoxy ring d ¼ 4.9 A
ꢂ
ꢂ
˚
and a ¼ 85 , dichloro (DC) ring d ¼ 4.6 A and a ¼ 78 ) and
ꢂ
˚
F3.36(200) (DC ring d ¼ 5.1 A and a ¼ 83 ). While the C5
substituent para-methoxy ring cannot have a direct aromatic
stacking interaction with Y5.39(275) due to the intervening
methoxy group, this substituent does have a strong interaction
with Y5.39(275) (see Table A3). In this way, the ligand acts as
a bridge between the F3.36(200)/W5.43(279)/W6.48(356) and
Y5.39(275)/W4.64(255)/F5.42(278) aromatic clusters present
in the TMH3-4-5-6 aromatic microdomain in the minimized
complex. In Fig. 4(B), the cꢂarboxamide oxygen of 1
ꢂ
˚
are 2.70 A and 156.0 , respectively.
2.5. CB1 receptor state model docking studies
Based on our recent paper [42], we docked in our model of
the CB1 inactive (R) state, a minimum energy conformer of 4
in which the lone pair of electrons of the piperidine nitrogen
points in the direction opposite to the carboxamide hydrogen
(see Fig. 4 and also Tables A1 and A2 in appendix for further
information). Modeling studies revealed that compound 4 can
occupy a similar orientation within the CB1 R binding pocket
as SR141716 (1). The energy minimized ligand/CB1 inactive
(R) state complexes for 4 and 1 [42] are illustrated in Fig. 4. In
Fig. 4(A), Compound 4 is docked in a minimum energy con-
formation (DE ¼ 1.25 kcal/mol above its global minimum) in
˚
(N/O ¼ 2.65 A, NeHeO ¼ 156 ) has a hydrogen-bonding
interaction with K3.28(192) in the K3.28(192)/D6.58(366)
salt bridge. Compound 1 has direct aromatic stacking interac-
˚
tions with F3.36(200) (monochloro (MC) ring d ¼ 6.9 A and
ꢂ
ꢂ
˚
a ¼ 56 , DC ring d ¼ 5.3 A and a ¼ 65 ) and W5.43(279)
ꢂ
˚
˚
(MC ring d ¼ 4.7 A and a ¼ 44 , DC ring d ¼ 5.2 A and
ꢂ
˚
a ¼ 86 ) and with Y5.39(275) (MC ring d ¼ 6.0 A and
a ¼ 60ꢂ) bridging the F3.36(200)/W5.43(279)/W6.48(356)
and Y5.39(275)/W4.64(255)/F5.42(278) aromatic clusters and
forming one large extended cluster in the minimized complex.
Fig. 4. (A) Compound 4 in a minimum energy conformation (DE ¼ 1.25 kcal/mol) and (B) compound 1 in a minimum energy conformation (DE ¼ 0.92 kcal/mol)
are shown here docked in the TMH3-4-5-6 aromatic microdomain within a CB1 TMH bundle model of the R state. The view here is from lipid, looking towards
TMHs 3 and 4. TMHs 1, 2 and 7 are not shown in order to simplify the view. Aromatic residues for which each ligand has a direct aromatic stacking interaction are
colored orange. Aromatic residues that are part of the extended aromatic cluster, but that do not stack directly with the ligand are colored yellow, while those
aromatic residues that have no direct or indirect interaction with the ligand are colored cyan.

H. Fan et al. / European Journal of Medicinal Chemistry 44 (2009) 593e608
599
The pairwise interaction energy analysis of the 4/CB1 R
and 1/CB1 R complexes revealed that the greatest coulombic
interactions for 4 (ꢀ92.84 kJ/mol) and 1 (ꢀ72.02 kJ/mol)
were with K3.28. The greater coloumbic interactions for 4
reflect the fact that this compound forms two hydrogen bonds
with K3.28, while 1 forms one hydrogen bond. High van der
Waals’ interactions were found for both ligands with W5.43
(4, ꢀ29.34 kJ/mol; 1, ꢀ25.10 kJ/mol) and with M6.55 (4,
ꢀ32.22 kJ/mol; 1, ꢀ28.36 kJ/mol). In addition, 4 was found
to have a strong van der Waals interaction with D6.58(366)
(ꢀ15.72 kJ/mol) and 1 with V3.32(196) (ꢀ20.29 kJ/mol). A
table that includes all of the calculated pairwise interaction en-
ergies for 4 and 1 at CB1 R is included in the Appendix here
(Table A3). The fact that the combined total energy of pairwise
interaction is lower for 4 (ꢀ261.90 kJ/mol or ꢀ62.55 kcal/
mol) than for 1 (ꢀ218.63 kJ/mol or ꢀ52.22 kcal/mol), may
be the reason why 4 has higher CB1 affinity than 1. It is impor-
tant to note, however, that pairwise interaction energies may
not be directly comparable with changes in affinities. The
experimentally measured change in affinity includes not only
the strength of ligandereceptor interactions in the newly
formed complex, but also the possible loss of intrareceptor
interactions in the unoccupied receptor resulting from ligand
binding. While Table A3 should reflect the former, it does
not take into consideration the latter.
same CB1 R TMH3-4-5-6 aromatic microdomain binding
site as 1 and 4, it exceeds the steric limitations of this site in
nearly every direction. The most severe steric clash results
from the substitution of the amide group for the cyano group
at C4 in 4. This produces a severe steric clash between the
C4 substituent amide nitrogen of 7e and residues K3.28(192)
and D6.58(366), as well as a clash between the C4 carboxa-
mide oxygen and residue L3.29(193). A severe steric clash
also exists between the 7e piperidine ring and residues
C7.42(386) and S7.39(383), as well as between the methoxy
group of the C5 substituent para-methoxyphenyl ring and
residues Y5.39(275) and W5.43(279). Finally, there is a steric
clash between the bromine of the 7e N1 substituent ortho-
bromophenyl ring and residue V3.32(196) (not shown in
Fig. 5) and the phenyl ring of the 7e N1 substituent ortho-
bromophenyl ring and residue M6.55(363).
2.6. Functional assay
Given the structural similarity of the series that include 4
and its analogs (Table 1) to the antagonist 1, we wondered if
Compound 7e has a Ki > 10,000 nM at CB1 (see Table 1).
Fig. 5 illustrates that when this compound is docked in the
Fig. 5. Compound 7e has a Ki > 10,000 nM at CB1 (see Table 1). This figure
illustrates that compound 7e exceeds the steric limitations of the CB1 R
TMH3-4-5-6 aromatic microdomain binding site in nearly every direction.
The most severe steric clash results from the substitution of the amide group
for the cyano group in 4. This produces a severe steric clash between the C4
substituent amide nitrogen of 7e and residues K3.28(192) and D6.58(366), as
well as a clash between the C4 carboxamide oxygen and residue L3.29(193).
A severe steric clash also exists between the 7e piperidine ring and residues
C7.42(386) and S7.39(383), as well as between the methoxy group of the
C5 substituent para-methoxyphenyl ring and residues Y5.39(275) and
W5.43(279). Finally, there is a steric clash between the bromine of the 7e
N1 substituent ortho-bromophenyl ring and residue V3.32(196) (not shown
here) and the phenyl ring of the 7e N1 substituent ortho-bromophenyl ring
and residue M6.55(363).
Fig. 6. Compound 4 (JHU75528) reverses the effects of the CB agonist WIN
55212-2 on glutamate release in striatal brain slices. (A) Extracellular record-
ing of locally evoked, glutamatergic striatal population spikes (PS) was per-
formed in mouse brain slices. Bath superfusion of 1 mM WIN 55212-2
produced a significant decrease in the PS amplitude, and this recovered to con-
trol (pre-drug) levels following superfusion with 4 (3 mM). (B) Summary of
the reversal of WIN 55212-2 effects on glutamatergic responses by 4 in mouse
brain slices (*P < 0.05, paired t-test, n ¼ 5 slices from three animals). This
confirms the CB antagonist properties of 4.

600
H. Fan et al. / European Journal of Medicinal Chemistry 44 (2009) 593e608
purity was 98%. The precursor 16 was synthesized via the
N
N
carboxylic acid 17 [30] and the intermediate 18 (Scheme 5).
The preliminary in vivo evaluation of [11C]7h was per-
formed in CD-1 mice. We also performed regional brain
distribution studies in mice with 1-(2-bromophenyl)-4-cyano-
5-(4-[11C]methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-
carboxamide ([11C]JHU75575) [11C]15, a monobromo analog
of 4 that we synthesized in the past [30] (Table 1).
O
O
NH
Cl
NH
Cl
NC
NC
[
11C]CH3Tf
acetone/NaOH,
N
N
N
N
H311CO
HO
Cl
Cl
16
The mouse brain uptake of [11C]7h and [11C]15 was quite
high (Fig. 7) and comparable with previously studied [11C]4
([11C]JHU75528 [31]). The highest accumulation of 11C radio-
activity occurred in the hippocampus, frontal cortex and cere-
bellum and lowest radioactivity was seen in the brainstem.
This distribution of radioactivity of [11C]7h and [11C]15 in
the mouse brain matches the data obtained in the previous au-
toradiographic studies [46] and distribution of [11C]4 [31]. The
clearance rates of [11C]7h and [11C]15 from the brainstem
were higher than from any other region studied. The hippo-
campus-to-brainstem ratio of [11C]7h was greater than that
of [11C]15 and reached values of 2.3, 3.0 and 3.5 at 30, 45
and 90 min post-injection, correspondingly. These target-to-
non-target ratios are the highest reported to date and are
10e30% greater than those previously obtained for [11C]4
[31]. The superior ratio of [11C]7h vs. [11C]4 [31] or [11C]15
is in agreement with better combination of binding affinity
and lower lipophilicity of [11C]7h (Table 1).
A blocking dose of the selective CB1 antagonist 1 signifi-
cantly inhibited [11C]7h binding at 60 min after administration
of the radiotracer in the hippocampus, a region with highest
density of CB1 receptors, but it failed to significantly block ac-
cumulation of radioactivity in the brainstem, a region with
a low density of CB1 (Fig. 8). This study demonstrated that
uptake of [11C]7h in the receptor-rich hippocampus is CB1-
mediated.
[
11C]7h ([11C]JHU76609)
Scheme 4. Radiosynthesis of [11C]7h ([11C]JHU76609).
any of these compounds might functionally reverse the actions
of the known CB agonist WIN 55212-2. Extracellular electro-
physiological recordings were performed in rodent brain slices
containing the striatum and nucleus accumbens. In this prepa-
ration, glutamatergic field potentials are known to be inhibited
by CB agonists via presynaptic activation of CB1 receptors
[43,44]. Bath application of 1 mM WIN 55212-2 inhibited glu-
tamate release by w30% (Fig. 6). When 3 mM 4 was applied
subsequent to WIN 55212-2, this inhibitory effect was rapidly
reversed. These data demonstrate that 4 exhibits functional CB
antagonist properties, consistent with its structural relationship
to 1.
Cannabinoid antagonists have an advantage over agonists
for in vivo imaging of CB1 receptors because antagonists
bind to a greater population of the receptor than agonists do
[24,45]. Therefore, if all other properties are the same, the
BP value of a radiolabeled CB1 antagonist will be greater
than that of a radiolabeled agonist. In addition, the side effects
of CB1 antagonists in vivo are lower. Perhaps the antagonistic
properties of [11C]4 is one of the reasons for its success in the
animal imaging experiments [31].
2.7. Radiochemistry and in vivo studies
3. Conclusion
Having the best combination of high in vitro binding
affinity and lowest lipophilicity (Table 1), compound 7h
was labeled with [11C]methyl triflate by radiomethylation
of corresponding phenol precursor 16 (Scheme 4). After
purification and formulation the radiochemical yield of
[11C]7h was 15e24%, specific radioactivity was 482 ꢁ
288 GBq/mmol (13,019 ꢁ 7800 mCi/mmol) and radiochemical
A novel series of analogs of JHU75528 4 with high affinity
for CB1 receptor and reduced lipophilicity have been synthe-
sized. Given the excellent in vitro properties of the best com-
pounds of the series (7a, 7h and 7i), these are the targets for
the future evaluation as potential radioligands with enhanced
properties for PET imaging of CB1 receptors in human
N
N
O
O
O
NH
NH
OH
NC
NC
Pd(PPh3)4,
PhSiH3
NC
N
a
N
N
N
N
Cl
N
Cl
Cl
Allyl
Allyl
HO
O
O
Cl
Cl
Cl
17
18
16
Scheme 5. Synthesis of precursor 16 for radiolabeling of [11C]7h. Reagents: (a) benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate
(BOP), N-aminopyrrolidine.

H. Fan et al. / European Journal of Medicinal Chemistry 44 (2009) 593e608
601
Hip/BS ratio
CB
Hip
FrCtx
BS
6
6
5
4
3
2
1
0
* * *
******
**** **
[11C]15
* * *
[11C]7h
5
4
* * *
**
** **
3
2
1
0
30 min
45 min
90 min
30 min
45 min
90 min
Fig. 7. Regional brain distribution of [11C]7h (left) and [11C]15 (right) in CD-1 mice. CB ¼ cerebellum; Hip ¼ hippocampus; FrCtx ¼ frontal cortex; BS ¼ brain-
stem. Data are mean %ID/g tissue ꢁ SD. Black bars are hippocampus/brainstem ratio. Data are mean ꢁ SD. There was a significant difference between the uptake
in brainstem and all other regions ([11C]7h, *P < 0.03; [11C]15, **P < 0.05), data analysis is ANOVA single-factor.
subjects. Preliminary evaluation of [11C]7h in the mouse brain
demonstrated a better target-to-non-target ratio of [11C]7h
than that of [11C]4 and [11C]15.
Extracellular electrophysiological recordings in rodent
brain slices demonstrated that 4, the lead compound of the
new series and potential PET radioligand for imaging of
CB1 receptors in humans, has functional CB antagonist prop-
erties, consistent with its structural relationship to Rimona-
bant 1.
Compound 4, the lead of the series described here, displays
a higher binding affinity than that of its congener Rimonabant
1. This finding is in agreement with the conformational anal-
ysis and CB1 receptor docking studies that demonstrated
that even though 4 has a similar binding arrangement as 1,
the former forms an additional hydrogen bond between the
cyano group with the K3.28(192)/D6.58(366) salt bridge.
Replacement of 4-cyano group with 4-carbamoyl group in
the molecule 7e led to severe steric clash between amide nitro-
gen and piperidine ring and the receptor binding site. This
finding is in agreement with very low CB1 binding affinity
of 7e.
4. Experimental protocols
4.1. General
All chemicals and solvents were of reagent grade, and were
used as received from Aldrich. 1H NMR spectra were obtained
with a Varian 400 MHz spectrometer. Chemical shifts are re-
ported in ppm (d) relative to internal tetramethylsilane in
CDCl3. High resolution mass spectrometry was performed at
the University of Notre Dame Mass Spectrometry Facility.
Galbraith Laboratories Inc. (Knoxville, TN) did elemental
analysis. Flash chromatography purification was performed
using E. Merck 7729 (<230 mesh) silica gel. All HPLC chro-
matograms were recorded by a Varian Galaxie system. Semi-
preparative (10 mm ꢃ 300 mm) and analytical (4.6 mm ꢃ
100 mm) 10 mM C18 Luna columns (Phenomenex Torrance,
CA) were used for purification and quality control, respectively.
5
Hip
BS
4
3
2
1
0
4.2. 3-(4-Iodophenyl)-3-oxopropanenitrile (8a)
*
**
A mixture of ethyl 4-iodobenzoate (2.76 g, 10 mmol),
NaOEt (0.748 g, 11 mmol), and acetonitrile (0.65 mL,
12 mmol) in anhydrous toluene (5 mL) was stirred at 110 ꢂC
for 20 h. The reaction mixture was cooled and diluted with
30 mL water to dissolve solids. The mixture was washed
with Et2O (2 ꢃ 30 mL). The aqueous layer was acidified
with 1 N HCl to pH 7, and then was extracted with CH2Cl2
(3 ꢃ 30 mL), washed with brine and dried over Na2SO4. The
organic solvent was removed and the desired product 8a was
control
block
Fig. 8. Comparison of regional brain uptake (%ID/g tissue) of [11C]7h in three
CD-1 mice at 60 min time point in control and blocking experiments with Ri-
monabant 1 (1 mg/kg, i.v.). Data are mean ꢁ SD. There was significant block-
ing in the hippocampus (Hip) (*P ¼ 0.03). The blocking in the brainstem (BS)
was insignificant (**P ¼ 0.34). Data analysis is ANOVA single-factor.
1
obtained (980 mg, 36%). H NMR (CDCl3, d) 4.04 (s, 2H,
CH2), 7.63 (d, J ¼ 8.0 Hz, 2H, ArH), 7.91 (d, J ¼ 8.0 Hz,
2H, ArH). FAB-HRMS: calcd. for C9H6INO: 270.9494; found:
271.9590 (M þ H)þ.

602
H. Fan et al. / European Journal of Medicinal Chemistry 44 (2009) 593e608
4.3. Chloro[(2-chloro-4-fluorophenyl)hydrazono]ethyl
acetate (9a)
(m, 1H, ArH), 7.26 (d, J ¼ 8.8 Hz, 2H, ArH), 7.36 (m, 1H,
ArH), 7.46 (m, 1H, ArH). FAB-HRMS: calcd. for
C20H15BrFN3O3: 443.0281; found: 443.0287.
A
mixture of 2-chloro-4-fluoroaniline 11a (3.22 g,
22.5 mmol) in 37.5 mL 24% HCl, and 100 mL water was
stirred for 2 h at room temperature. The reaction mixture
was cooled with ice and a solution of sodium nitrite
(1.59 g, 23 mmol) in 11 mL water was added dropwise for
30 min. The mixture was then added to a solution of sodium
acetate (1.76 g, 21.5 mmol) and ethyl 2-chloro-acetoacetate
(3.11 mL, 22.5 mmol) in 225 mL EtOH, and cooled with
ice. The temperature was allowed to increase slowly for
2 h. The precipitate was filtered and washed with water and
dried to give the desired product 4.79 g (76%). 1H NMR
(CDCl3, d) 1.41 (t, J ¼ 6.8 Hz, 3H, CH3), 4.40 (q,
J ¼ 6.8 Hz, 2H, CH2), 7.03 (m, 1H, ArH), 7.12 (dd,
J1 ¼ 7.6 Hz, J2 ¼ 2.4 Hz, 1H, ArH), 7.59 (m, 1H, ArH),
8.69 (br, 1H, NH).
4.7. Ethyl 1-(2,4-dichlorophenyl)-4-cyano-5-(4-
iodophenyl)-1H-pyrazole-3-carboxylate (12c)
Compound 12c was prepared from ethyl chloro[(2,4-dichlor-
ophenyl)hydrazono]acetate 9d [34] and 3-(4-iodophenyl)-3-
oxopropanenitrile 8a with the same procedure described for
1
12a. Yield was 25%. H NMR (CDCl3, d) 1.47 (t, J ¼ 6.8 Hz,
3H, CH3), 4.53 (q, J ¼ 1.2 Hz, 2H, CH2), 7.04 (d, J ¼ 8.8 Hz,
2H, ArH), 7.39e7.47 (m, 3H, ArH), 7.75 (d, J ¼ 7.6 Hz, 2H,
ArH). FAB-HRMS: calcd. for C19H12Cl2IN3O2: 510.9351;
found: 511.9435 (M þ H)þ.
4.8. Ethyl 1-(2,4-dichlorophenyl)-4-cyano-5-(4-
bromophenyl)-1H-pyrazole-3-carboxylate (12d)
4.4. Chloro[(2-bromo-4-fluorophenyl)hydrazono]ethyl
acetate (9b)
Compound 12d was prepared from ethyl chloro[(2,4-
dichlorophenyl)hydrazono]acetate 9d [34] and commercially
available 3-(4-bromophenyl)-3-oxopropanenitrile 8b with the
1
Compound 9b was prepared from 2-bromo-4-fluoroaniline
11b with the same procedure described for 9a. Yield was
66%. 1H NMR (CDCl3, d) 1.41 (t, J ¼ 6.8 Hz, 3H, CH3),
4.40 (q, J ¼ 6.8 Hz, 2H, CH2), 7.08 (m, 1H, ArH), 7.28 (dd,
J1 ¼ 8.0 Hz, J2 ¼ 2.8 Hz, 1H, ArH), 7.58 (m, 1H, ArH), 8.73
(br, 1H, NH).
same procedure described for 12a. Yield was 41%. H NMR
(CDCl3, d) 1.47 (t, J ¼ 6.8 Hz, 3H, CH3), 4.52 (q, J ¼ 6.8 Hz,
2H, CH2), 7.19 (d, J ¼ 8.4 Hz, 2H, ArH), 7.39e7.47 (m, 3H,
ArH), 7.54 (d, J ¼ 8.4 Hz, 2H, ArH). FAB-HRMS: calcd. for
C19H12BrCl2N3O2: 462.9490; found: 463.9588 (M þ H)þ.
4.9. Ethyl 1-(2-bromophenyl)-4-carbamoyl-5-(4-
4.5. Ethyl 1-(2-chloro-4-fluorophenyl)-4-cyano-5-(4-
methoxyphenyl)-1H-pyrazole-3-carboxylate (12e0)
methoxyphenyl)-1H-pyrazole-3-carboxylate (12a)
A mixture of chloro[(2-bromophenyl)hydrazono]ethyl ace-
tate 9c [30] (1.63 g, 5.5 mmol), 4-methoxybenzoylacetonitrile
8c (0.97 g, 5.5 mol), 60 mL EtOH, and sodium ethoxide,
which was prepared by dissolving 0.14 g sodium in 12.5 mL
EtOH, was heated to reflux for 18 h. After cooling to room
temperature, the solvent was removed by reduced pressure.
EtOAc (75 mL) was added and the precipitate was filtered.
The organic layer was washed with water and brine, dried
over Na2SO4, and evaporated with a rotary evaporator. The
crude product was purified by flash chromatography 10:90
EtOAc/CH2Cl2 to afford two compounds: ethyl 1-(2-bromo-
phenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carbox-
ylate 12e [30] with yield 19% (first major peak) and the
desired product 12e0 with yield 12% (second major peak).
1H NMR (CDCl3, d) 0.95 (t, J ¼ 6.8 Hz, 3H, CH3), 3.85 (s,
3H, OCH3), 3.92 (q, J ¼ 6.8 Hz, 2H, CH2), 5.65 (br,
NH2CO), 6.92 (d, J ¼ 8.8 Hz, 2H, ArH), 7.38 (m, 1H, ArH),
7.44e7.52 (m, 2H, ArH), 7.68 (d, J ¼ 8.8 Hz, 2H, ArH),
7.73 (dd, J1 ¼ 8.0 Hz, J2 ¼ 1.2 Hz, 1H, ArH). FAB-HRMS:
calcd. for C20H19BrN3O4: 443.0481; found: 444.0538.
A mixture of chloro[(2-chloro-4-fluorophenyl)hydrazono]
ethyl acetate 9a (1.53 g, 5.5 mmol), 4-methoxybenzoylaceto-
nitrile 8c (0.97 g, 5.5 mmol), 60 mL EtOH, and sodium ethox-
ide, that was prepared by dissolving 0.14 g sodium in 12.5 mL
EtOH, was heated to reflux for 18 h and solvent was evapo-
rated under reduced pressure. Ethyl acetate (75 mL) was
added and the precipitate was filtered. The organic layer was
washed with water and saturated NaCl. The residue was puri-
fied by flash chromatography (silica gel, 10:90 EtOAc/hexane
to 50:50 EtOAc/CH2Cl2) to afford the desired product. Yield:
1
155 mg (7%). H NMR (CDCl3, d) 1.45 (t, J ¼ 7.2 Hz, 3H,
CH3), 3.81 (s, 3H, OCH3), 4.53 (q, J ¼ 6.8 Hz, 2H, CH2),
6.86 (d, J ¼ 8.8 Hz, 2H, ArH), 7.11 (m, 1H, ArH), 7.19 (m,
1H, ArH), 7.28 (d, J ¼ 8.8 Hz, 2H, ArH), 7.48 (m, 1H,
ArH). FAB-HRMS: calcd. for C20H15ClFN3O3: 399.0786;
found: 399.0777.
4.6. Ethyl 1-(2-bromo-4-fluorophenyl)-4-cyano-5-(4-
methoxyphenyl)-1H-pyrazole-3-carboxylate (12b)
Compound 12b was prepared from chloro[(2-bromo-4-fluo-
rophenyl)hydrazono]ethyl acetate (9b) with the same proce-
1
dure described for 12a. Yield was 10%. H NMR (CDCl3, d)
4.10. 1-(2-Chloro-4-fluorophenyl)-4-cyano-5-(4-
methoxyphenyl)-1H-pyrazole-3-carboxylic acid (13a)
1.47 (t, J ¼ 7.2 Hz, 3H, CH3), 3.81 (s, 3H, OCH3), 4.52 (q,
A mixture of ethyl 1-(2-chloro-4-fluorophenyl)-4-cyano-5-
(4-methoxyphenyl)-1H-pyrazole-3-carboxylate 12a (0.155 g,
J ¼ 7.2 Hz, 2H, CH2), 6.88 (d, J ¼ 8.4 Hz, 2H, ArH), 7.15

H. Fan et al. / European Journal of Medicinal Chemistry 44 (2009) 593e608
603
0.39 mmol) in 10 mL THF and LiOH (12 mg, 0.49 mmol) in
1 mL water was heated overnight at 65 ꢂC. The reaction mix-
ture was cooled to room temperature, water (25 mL) and 5%
HCl (2.5 mL) were added and the mixture was extracted with
EtOAc (3 ꢃ 20 mL). The organic layer was washed with
brine and dried over Na2SO4. The solvent was removed to
afford the desired product 13a with yield 137 mg (95%).
1H NMR (CDCl3, d) 3.81 (s, 3H, CH3), 6.89 (d,
J ¼ 6.8 Hz, 2H, ArH), 7.11 (m, 1H, ArH), 7.19 (m, 1H,
ArH), 7.26 (d, J ¼ 6.8 Hz, 2H, ArH), 7.49 (m, 1H, ArH).
FAB-HRMS: calcd. for C18H11ClFN3O3: 371.0473; found:
372.0549 (M þ H)þ.
(m, 1H, ArH), 7.49 (m, 2H, ArH), 7.71 (d, J ¼ 8.8 Hz, 2H,
ArH). FAB-HRMS: calcd. for C18H14BrN3O4: 415.0168;
found: 416.0265 (M þ H)þ.
4.15. 1-(2-Chloro-4-fluorophenyl)-4-cyano-5-(4-
methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-
carboxamide (7a)
Compound 13a (0.13 g, 0.35 mmol) was added to a solution
containing 1-aminopiperidine (0.07 mL, 0.63 mmol) and trie-
thylamine (0.18 mL) in dichloromethane (10 mL). Then
BOP (0.28 g, 0.62 mmol) was added. The mixture was stirred
at room temperature for 20 h. The reaction mixture was
quenched with 10 mL of cold water and the organic phase
was washed with 2% HCl (7 mL), 5% sodium carbonate and
brine and dried over anhydrous Na2SO4. The solvent was re-
moved and the residue was purified by flash chromatography
(EtOAc/CH2Cl2 1:1). The final product was obtained with yield
46 mg (29%). 1H NMR (CDCl3, d) 1.44 (br, 2H, CH2-py), 1.76
(m, 4H, CH2-py), 2.91 (br, 4H, CH2-py), 3.81 (s, 3H, CH3),
6.87 (d, J ¼ 8.8 Hz, 2H, ArH), 7.13 (m, 1H, ArH), 7.21e
7.25 (m, 3H, ArH), 7.45 (m, 1H, ArH), 7.56 (br, 1H, NH).
FAB-HRMS: calcd. for C23H21ClFN5O2: 453.1368; found:
454.1423 (M þ H)þ.
4.11. 1-(2-Bromo-4-fluorophenyl)-4-cyano-5-(4-
methoxyphenyl)-1H-pyrazole-3-carboxylic acid (13b)
Compound 13b was prepared from ethyl 1-(2-bromo-4-fluoro-
phenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxylate
12b with the same procedure described for 13a. Yield was
98%. 1H NMR (CDCl3, d) 3.81 (s, 3H, CH3), 6.88 (d,
J ¼ 8.8 Hz, 2H, ArH), 7.16 (m, 1H, ArH), 7.27 (d,
J ¼ 8.8 Hz, 2H, ArH), 7.36 (dd, J1 ¼ 7.6 Hz, J2 ¼ 2.8 Hz,
2H, ArH), 7.48 (dd, J1 ¼ 8.8 Hz, J2 ¼ 5.2 Hz, 2H, ArH).
FAB-HRMS: calcd. for C20H11BrlFN3O3: 414.9968; found:
416.0048 (M þ H)þ.
4.16. 1-(2-Bromo-4-fluorophenyl)-4-cyano-5-(4-
methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-
carboxamide (7b)
4.12. 1-(2,4-Dichlorophenyl)-4-cyano-5-(4-iodophenyl)-
1H-pyrazole-3-carboxylic acid (13c)
Compound 13c was prepared from ethyl 1-(2,4-dichloro-
phenyl)-4-cyano-5-(4-iodophenyl)-1H-pyrazole-3-carboxylate
12c with the same procedure described for 13a. Yield was
Compound 7b was prepared from 1-(2-bromo-4-fluoro-
phenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxylic
acid 13b with the same procedure described for 7a. Yield was
1
1
99%. H NMR (CDCl3, d) 7.05 (d, J ¼ 8.8 Hz, 2H, ArH),
32%. H NMR (CDCl3, d) 1.43 (br, 2H, CH2-py), 1.75 (m, 4H,
7.42 (m, 2H, ArH), 7.49 (d, J ¼ 2.0 Hz, 1H, ArH), 7.76 (d,
CH2-py), 2.89 (br, 4H, CH2-py), 3.80 (s, 3H, CH3), 6.87 (d,
J ¼ 8.8 Hz, 2H, ArH), 7.25 (d, J ¼ 8.8 Hz, 2H, ArH), 7.39e7.47
(m, 2H, ArH), 7.57 (br, 1H, NH). FAB-HRMS: calcd. for
C23H21BrFN5O2: 497.0863; found: 498.0921 (M þ H)þ.
J ¼ 8.4 Hz,
2H,
ArH).
FAB-HRMS:
calcd.
for
C17H8Cl2IN3O2: 482.9038; found: 483.9122 (M þ H)þ.
4.13. 1-(2,4-Dichlorophenyl)-4-cyano-5-(4-
bromophenyl)-1H-pyrazole-3-carboxylic acid (13d)
4.17. 1-(2,4-Dichlorophenyl)-4-cyano-5-(4-iodophenyl)-
N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (7c)
Compound 13d was prepared from ethyl 1-(2,4-di-
chlorophenyl)-4-cyano-5-(4-bromophenyl)-1H-pyrazole-
3-carboxylate 12d with the same procedure described
for 13a. Yield was 99%. 1H NMR (CDCl3, d) 7.20
(d, J ¼ 8.4 Hz, 2H, ArH), 7.42 (m, 2H, ArH), 7.49 (d,
J ¼ 2.0 Hz, 1H, ArH), 7.55 (d, J ¼ 8.4 Hz, 2H, ArH).
FAB-HRMS: calcd. for C17H8BrCl2N3O2: 434.9177;
found: 435.9245 (M þ H)þ.
Compound 7c was prepared from 1-(2,4-dichlorophenyl)-4-
cyano-5-(4-iodophenyl)-1H-pyrazole-3-carboxylic acid 13c
with the same procedure described for 7a. Yield was 32%.
1H NMR (CDCl3, d) 1.44 (br, 2H, CH2-py), 1.75 (m, 4H,
CH2-py), 2.91 (br, 4H, CH2-py), 7.04 (d, J ¼ 8.8 Hz, 2H,
ArH), 7.42 (dd, J1 ¼ 8.4 Hz, J2 ¼ 2.0 Hz, 1H, ArH), 7.47e
7.95 (m, 2H, ArH), 7.57 (br, 1H, NH), 7.73 (d, J ¼ 8.8 Hz,
2H, ArH). FAB-HRMS: calcd. for C22H18Cl2IN5O:
564.9933; found: 565.9987 (M þ H)þ.
4.14. 1-(2-Chloro-4-fluorophenyl)-4-carbamoyl-5-(4-
methoxyphenyl)-1H-pyrazole-3-carboxylic acid (13e)
4.18. 1-(2,4-Dichlorophenyl)-4-cyano-5-(4-
bromophenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-
carboxamide (7d)
Compound 13e was prepared from ethyl 1-(2-bromo-
phenyl)-4-carbamoyl-5-(4-methoxyphenyl)-1H-pyrazole-3-
carboxylate 12e0 with the same procedure described for 13a.
Yield was 95%. 1H NMR (CDCl3, d) 3.84 (s, 3H, CH3),
5.04 (br, NH2CO), 6.95 (d, J ¼ 8.8 Hz, 2H, ArH), 7.40
Compound 7d was prepared from 1-(2,4-dichlorophenyl)-4-
cyano-5-(4-bromophenyl)-1H-pyrazole-3-carboxylic acid 13d

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H. Fan et al. / European Journal of Medicinal Chemistry 44 (2009) 593e608
1
with the same procedure described for 7a. Yield was 17%. H
NMR (CDCl3, d) 1.44 (br, 2H, CH2-py), 1.75 (m, 4H, CH2-
py), 2.89 (br, 4H, CH2-py), 7.18 (d, J ¼ 9.6 Hz, 2H, ArH),
7.42e7.43 (m, 2H, ArH), 7.50 (d, J ¼ 1.2 Hz, 1H, ArH), 7.53
(d, J ¼ 8.8 Hz, 2H, ArH), 7.55 (br, 1H, NH). FAB-HRMS: calcd.
for C22H18BrCl2N5O: 517.0072; found: 518.0172 (M þ H)þ.
(d, J ¼ 8.4 Hz, 2H, ArH), 7.37e7.39 (m, 2H, ArH), 7.49 (d,
J ¼ 1.6 Hz, 1H, ArH), 7.54 (br, 1H, NH). FAB-HRMS: calcd.
for C22H19Cl2N5O2: 455.0916; found: 456.0977 (M þ H)þ.
4.23. 1-(2,4-Dichlorophenyl)-4-cyano-5-(4-
(fluoromethoxy)phenyl)-N-(piperidin-1-yl)-1H-pyrazole-
3-carboxamide (7i)
4.19. 1-(2-Bromophenyl)-5-(4-methoxyphenyl)-N3-
(piperidin-1-yl)-1H-pyrazole-3,4-dicarboxamide (7e)
Fluoromethyltosylate [37] (41 mg, 0.24 mmol) was added
to a solution of 1-(2,4-dichlorophenyl)-4-cyano-5-(4-hydroxy-
phenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide [30]
(91 mg, 0.2 mmol) and potassium carbonate (55 mg,
0.4 mmol) in acetone (10 mL) and the mixture was refluxed
for 2 days. The reaction mixture was cooled and solvent was
removed. The residue was diluted with saturated ammonium
chloride solution, extracted with ethyl acetate and washed
with water. The organic layer was dried over Na2SO4 and sol-
vent was removed at reduced pressure. The residue was sepa-
rated by flash chromatography (hexanes/EtOAc (3:1)). Yield
was 35 mg (36%). 1H NMR (CDCl3, d) 1.44 (br, 2H, CH2-py),
1.75 (m, 4H, CH2-py), 2.90 (br, 4H, CH2-py), 5.67 (d,
J ¼ 54.4 Hz, 2H, OCH2F), 7.07 (d, J ¼ 8.4 Hz, 2H, ArH), 7.29
(d, J ¼ 8.8 Hz, 2H, ArH), 7.40 (d, J ¼ 1.6 Hz, 2H, ArH), 7.48
(t, J ¼ 1.6 Hz, 1H, ArH), 7.54 (br, 1H, NH). FAB-HRMS: calcd.
for C23H20Cl2FN5O2: 487.0978; found: 488.1068 (M þ H)þ.
Compound 7e was prepared from 1-(2-chloro-4-fluoro-
phenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carbox-
ylic acid 13e with the same procedure described for 7a. Yield
was 22%. 1H NMR (CDCl3, d) 1.36 (br, 2H, CH2-py), 1.64 (m,
4H, CH2-py), 2.69 (br, 4H, CH2-py), 3.85 (s, 3H, CH3), 5.45
(br, 2H, NH2CO), 6.88 (d, J ¼ 8.8 Hz, 2H, ArH), 7.43 (m,
1H, ArH), 7.50 (m, 2H, ArH), 7.71 (d, J ¼ 8.8 Hz, 2H,
ArH), 7.77 (br, 1H, NH). FAB-HRMS: calcd. for
C23H24BrN5O3: 497.1063; found: 498.1141 (M þ H)þ.
4.20. 1-(2,4-Dichlorophenyl)-4-cyano-5-(4-iodophenyl)-
N-morpholino-1H-pyrazole-3-carboxamide (7f)
Compound 7f was prepared from 1-(2,4-dichlorophenyl)-4-
cyano-5-(4-iodophenyl)-1H-pyrazole-3-carboxylic acid 13c
and N-aminomorpholine with the same procedure described
for 7a. Yield was 35%. 1H NMR (CDCl3, d) 2.98 (t,
J ¼ 4.4 Hz, 4H, CH2-py), 3.86 (t, J ¼ 4.4 Hz, 4H, CH2-py),
7.03 (d, J ¼ 8.0 Hz, 2H, ArH), 7.42 (m, 2H, ArH), 7.51 (m,
1H, ArH), 7.60 (br, 1H, NH), 7.74 (d, J ¼ 8.0 Hz, 2H, ArH).
FAB-HRMS: calcd. for C21H16Cl2IN5O2: 566.9726; found:
567.9792 (M þ H)þ.
4.24. 1-(2,4-Dichlorophenyl)-4-cyano-5-(4-
hydroxyphenyl)-N-(pyrrolidin-1-yl)-1H-pyrazole-3-
carboxamide (16)
5-(4-(Allyloxy)phenyl)-1-(2,4-dichlorophenyl)-4-cyano-N-
(pyrrolidin-1-yl)-1H-pyrazole-3-carboxamide 18 was prepared
from 5-(4-(allyloxy)phenyl)-1-(2,4-dichlorophenyl)-4-cyano-
1H-pyrazole-3-carboxylic acid 17 [30] and 1-aminopyrrolidine
hydrochloride with the same procedure described for 7a. Yield
of 18 was 72%. A mixture of 18 (172 mg, 0.36 mmol),
Pd(PPh3)4 (8.2 mg, 7.1 mmol), and PhSiH3 (78 mg,
0.72 mmol) in 150 mL CH2Cl2 was stirred at room temperature
for 1 h. Then the solvent was removed and EtOAc (20 mL) was
added to the residue. The organic layer was washed with satu-
rated NaHCO3, brine, and dried over Na2SO4. The crude product
was purified by flash chromatography, EtOAc/CH2Cl2 (1:1).
The final product 16 was obtained with yield 95 mg (60%). 1H
NMR (CDCl3, d) 1.91 (m, 4H, CH2-py), 3.05 (br, 4H, CH2-
py), 6.87 (dd, J1 ¼ 6.4 Hz, J2 ¼ 1.6 Hz, 2H), 7.19 (dd,
J1 ¼ 6.2 Hz, J2 ¼ 1.5 Hz, 2H), 7.40 (m, 2H), 7.54 (d,
J ¼ 2.0 Hz, 1H), 7.64 (br, 1H). FAB-HRMS: calcd. for
C21H17Cl2N5O2: 441.0759; found: 442.0838 (M þ H)þ.
4.21. 1-(2,4-Dichlorophenyl)-4-cyano-5-(4-
bromophenyl)-N-morpholino-1H-pyrazole-3-
carboxamide (7g)
Compound 7g was prepared from 1-(2,4-dichlorophenyl)-4-
cyano-5-(4-bromophenyl)-1H-pyrazole-3-carboxylic acid 13d
and N-aminomorpholine with the same procedure described
for 7a. Yield was 35%. 1H NMR (CDCl3, d) 2.98 (t,
J ¼ 4.8 Hz, 4H, CH2-py), 3.85 (t, J ¼ 4.8 Hz, 4H, CH2-py),
7.18 (d, J ¼ 8.4 Hz, 2H, ArH), 7.44 (m, 2H, ArH), 7.50 (m,
1H, ArH), 7.53 (d, J ¼ 8.8 Hz, 2H, ArH), 7.62 (br, 1H, NH).
FAB-HRMS: calcd. for C21H16BrCl2N5O2: 518.9864; found:
519.9921 (M þ H)þ.
4.22. 1-(2,4-Dichlorophenyl)-4-cyano-5-(4-
methoxyphenyl)-N-(pyrrolidin-1-yl)-1H-pyrazole-3-
carboxamide (7h)
4.25. In vitro inhibition binding assay
Compound 7h was prepared from 1-(2,4-dichlorophenyl)-
4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxylic acid
13f [30] and 1-aminopyrrolidine hydrochloride with the
same procedure described for 7a. Yield was 15%. H NMR
(CDCl3, d) 1.91 (m, 4H, CH2-py), 3.05 (br, 4H, CH2-py),
3.81 (s, 3H, CH3), 6.88 (d, J ¼ 8.4 Hz, 2H, ArH), 7.23
The in vitro inhibition binding assays of all CB1 ligands
(Table 1) were performed commercially by NovaScreen (Han-
over, MD) under the experimental conditions similar to those pre-
viously published [30,47]. Briefly, membranes from HEK-293
cells expressing the human recombinant cannabinoid receptor
CB1 were incubated with [3H]CP-55940 (Kd ¼ 0.6 nM) at
1

H. Fan et al. / European Journal of Medicinal Chemistry 44 (2009) 593e608
605
a concentration of 0.5 nM in 50 mM TriseHCl buffer with
5 mM MgCl2, 5 mg/mL BSA and 2.5 mM EDTA at pH 7.4
for 90 min at 30 ꢂC. The binding reaction was terminated by
rapid vacuum filtration of the assay contents onto presoaked
(0.5% PEI) Whatman GF/C filters. Radioactivity trapped
onto the filters was assessed using liquid scintillation counting.
Non-specific binding was defined as that remaining in the
presence of 1 mM HU210. The assays were done in duplicate
at multiple concentrations of the test compounds. Binding as-
say results were analyzed using one site competition models
and IC50 curves were generated based on a sigmoidal dose re-
sponse with variable slope. Values of Ki were calculated using
ChengePrusoff equation [48].
3-21G* 6-fold conformer searches were performed for the
rotatable bonds (C3 substituent: C3eC10and N20eN30; C5
substituent: C5eC1000; N1 substituent: N1eC100). In each
conformer search, local energy minima were identified by
rotation of a subject torsion angle through 360ꢂ in 60ꢂ in-
crements (6-fold search), followed by HF 3-21G* energy
minimization of each rotamer generated.
In order to compare the energy of the conformers of 4 to that of
1 [42], an ab initio geometry optimization at the HF 6-31G* level
was performed for the global minimum energy conformer of 4
identified by the HF 3-21G* conformational search and for the
second to global minimum energy conformer of 4. The energy
separation between conformers reported in the text was calcu-
lated using results from these ab initio HF calculations at the 6-
31G* level as encoded in Jaguar (version 6.0, Schrodinger,
LLC, New York, NY). To calculate the energydifferencebetween
the global minimum energy conformer of the compound and its
final docked conformation, rotatable bonds in the global mini-
mum energy conformer were driven to their corresponding value
inthefinaldockedconformationandthesinglepointenergyofthe
resultant structure was calculated at the HF 6-31G* level.
4.26. Functional assay
Coronal brain slices (250e300 mm) containing the striatum
were prepared from 2e4 month old C57/BL6 mice based on pre-
viously publishedprotocols [49,50]. Extracellular recordings were
performed using a differential AC amplifier (A-M Systems, Model
1700) and electrodes pulled from thick-walled borosilicate capil-
lary tubing (0.75 mm ID, 1.5 mm OD, Sutter Instruments, Novato,
CA) filled with 3 M NaCl solution. During the recording, slices
were maintained at 32e33 ꢂC and were continuously superfused
at a rate of 2 mL/min with a modified artificial cerebrospinal fluid
(aCSF) consisting of (in mM) NaCl, 126; KCl, 3.0; MgCl2, 1.5;
CaCl2, 2.4; NaH2PO4, 1.2; glucose, 11.0; NaHCO3, 26, and
saturated with 95% O2 and 5% CO2. In order to isolate gluta-
mate-driven synaptic potentials [51] the aCSF also contained the
GABAA-receptor antagonist picrotoxin (100 mM) and the N-
methyl-D-aspartate (NMDA)-type receptor antagonist APV
(40 mM). Electrical stimulation was performed using a bipolar
tungsten stimulating electrode placed near (<100 mm) the record-
ing electrode, in the dorsal striatum. Single, 0.1 ms pulses (10e
30 V) were delivered to the stimulating electrode at a frequency
of 0.033 Hz. Followingestablishment of a stable baselineresponse
(ꢄ10 min), drugs were applied via bath superfusion. Data were ac-
quired and stored on a Pentium-based PC computer via an A/D
board(NationalInstrumentsPCI6024E, Austin, TX), usingaWin-
dows-based software package (courtesy of Dr. John Dempster,
University of Strathclyde, Glasgow, UK). Analyses of peak synap-
tic potential amplitudes were performed off-line using the same
software. Responses were normalized by averaging the control
(baseline, pre-drug) responses, and post-drug effects were deter-
mined by averaging 5e10 sweeps during the peak of the response.
A paired, two-tailed t-test (GraphPad Prism v 5.0, GraphPad Sci-
entific, San Diego CA) was then performed between the pre-drug
and post-drug responses in order to determine statistical signifi-
cance at an a value of 0.05 (e.g., p ꢅ 0.05 was considered statis-
tically significant).
4.28. Ligandereceptor complex modeling
Ligand docking and energy minimization of ligand/CB1 R
complex. Receptor residues are numbered here using the amino
acid numbering scheme proposed by Ballesteros and Weinstein
[52]. Compound 4 was docked using interactive computer
graphics in a recently described TMH model of the CB1 receptor
inactive state (R) [42]. The ligand was docked within the aro-
matic residue rich TMH3-4-5-6 region of the bundle in the
same orientation as its structural analog 1. The complex was
then energyminimized usingthe Amber* united atom force field
in Macromodel (version 8.6, Schrodinger, LLC, New York, NY)
¨
and our previously published protocol [42]. Interactive docking
methods were used here because the binding region in CB1 for 1
(TMH3-4-5-6) and the nature of specific ligand functional
group/specific amino acid interactions are known from muta-
tion/chimera studies. The combined information limits the li-
gand to one particular region of CB1 and to one particular
orientation in this region. Given the following experimental ev-
idence, we consider the approach here to be appropriate: Shire
and co-workers have shown in CB1/CB2 chimera studies that
the TMH4-EC2-TMH5 region of CB1 contains residues critical
for the binding of 1 [53]. Subsequent CB1 F3.36(200)A,
W5.43(279)A, and W6.48(356)A mutation studies published
by our group indicated that the binding of 1 is affected by
each of these mutations, suggesting that these residues are part
of the binding site for 1 [40]. Our previous mutant cycle study
indicated that K3.28(192) is involved in a direct interaction
with the C3 substituent of 1 in wild-type (WT) CB1 [41]. Re-
cent analog synthesis/CB1 binding results suggest that this di-
rect interaction is between K3.28(192) and the carboxamide
oxygen of 1 [42]. This result fixes the orientation of 1 in the
CB1 bundle to be that pictured in Fig. 4B.
4.27. Molecular modeling
Conformational analysis. Complete conformational analy-
ses of 4 and 7e were performed using ab initio HartreeeFock
calculations at the 3-21G* level, within the Spartan molec-
ular modeling program (Wavefunction, Inc., Irvine, CA). HF
The energy of each ligand/CB1 R TMH bundle complex was
minimized using the AMBER* united atom force field in

606
H. Fan et al. / European Journal of Medicinal Chemistry 44 (2009) 593e608
Macromodel (version 8.6, Schrodinger, LLC, New York, NY).
˚
˚
cutoff (updated every 10 steps), 20.0 A electrostatic cutoff,
solvent was removed under reduced pressure. The product was
reconstituted in ethanol (1 mL) and sterile 0.9% saline (9.0 mL)
and passed through a 0.2 mM sterile filter (Millex-FG, Millipore)
into a sterile, pyrogen-free multi-dose vial. The non-decay cor-
rected radiochemical yield for [11C]7h was 15e24%.
An aliquot of the final solution of knownvolume and radioactiv-
ity was applied to an analytical reverse-phase HPLC column (Luna
C18 column, 250 mmꢃ4.6 mm; a mobile phase of CH3CN:0.1 M
aqueous ammonium formate buffer 60:40; flow rate of 5 mL/min).
The desired product was eluted with a retention time of 2.5 min.
The area of the UV absorbance peak at 254 nm corresponding to
the product was measured and compared to a standard curve re-
lating mass to UV absorbance. The average specific radioactivity
of the final product ([11C]7h) was 482 ꢁ 288 GBq/mmol
(13,019 ꢁ 7800 mCi/mmol) (n ¼ 5). The radiochemical product
was co-eluted with a standard of ‘‘cold’’ 7h.
¨
A distance dependent dielectric, 8.0 A extended non-bonded
˚
and 4.0 A hydrogen bond cutoff were used. During the minimi-
zation a 10,000 kcal/mol force was used to restrain the rotation
of the N1 substituent dichlorophenyl, the C5 substituent para-
methoxyphenyl and the piperidine rings. The first stage of the
calculation consisted of 2000 steps of PolakeRibier conjugate
gradient (CG) minimization in which a force constant of
225 kJ/mol was used on the helix backbone atoms in order to
hold the TMH backbones fixed, while permitting the side chains
to relax. The second stage of the calculation consisted of 100
steps of CG in which the force constant on the helix backbone
atoms was reduced to 50 kJ/mol in order to allow the helix back-
bones to adjust. Stages one and two were repeated with the num-
ber of CG steps in stage two incremented from 100 to 500 steps
2
until a gradient of 0.04 kJ/(mol A ) was reached.
˚
Assessment of aromatic stacking interactions. Aromatice
aromatic (pep) stacking interactions were identified in the
minimized ligand/receptor complexes based upon criteria de-
fined by Burley and Petsko for the ring centroid to centroid
distance (d ) and the angle between normal vectors of interact-
ing aromatic rings (a) [54]. These interactions were further
classified as tilted-T arrangements if 30ꢂ<q<90ꢂ and as paral-
lel arrangements for q < 30ꢂ. Parallel arrangements were con-
sidered favorable only if the interacting rings were offset from
each other [55]. All measurements were made using Maestro
(version 7.0, Schrodinger, LLC, New York, NY).
4.30. Mice studies
Baseline study. Male CD-1 mice weighing 25e30 g from
Charles River Laboratories (Wilmington, MA) were used for
biodistribution studies. The animals were sacrificed by cervi-
cal dislocation at various times (three animals per time point)
following injection of [11C]7h or [11C]15 (w7.4 MBq;
w200 mCi, specific radioactivity was about 200e240 GBq/
mmol (5400e6400 mCi/mmol) in 0.2 mL saline) into a lateral
tail vein. The brains were rapidly removed and dissected on
ice. The brain regions of interest were weighed and their radio-
activity content was determined in an automated g-counter
with a counting error below 3%. Aliquots of the injectate
were prepared as standards and their radioactivity content
was counted along with the tissue samples. The percent of in-
jected dose per gram of tissue (%ID/g tissue) was calculated.
All experimental protocols were approved by the Animal Care
and Use Committee of the Johns Hopkins Medical Institutions.
Blocking with Rimonabant 1. In vivo CB1 receptor blocking
studies were performed by intravenous (i.v.) administration of
1 mg/kg of Rimonabant 1 followed by i.v. injection in three
animals of [11C]7h (w7.4 MBq; w200 mCi) with specific ra-
dioactivity w300 GBq/mmol (8100 mCi/mmol) 15 min there-
after. Rimonabant 1 was dissolved in a vehicle solution
(saline:alcohol:Cremophore-EL (9:1:0.06)) and administered
in a volume of 0.1 mL. Control animals (three animals) were
injected with 0.1 mL of the vehicle solution. Sixty minutes af-
ter administration of the tracer the brain tissues were harvested
and their radioactivity content was determined.
Assessment of pairwise interaction energies. After defining
the atoms of each ligand as one group (Group 1) and the atoms
corresponding to a residue that lines the binding site in the fi-
nal ligand/CB1 R complex as another group (Group 2), Macro-
model (version 8.6, Schrodinger, LLC, New York, NY) was
¨
used to output the pair interaction energy (coulombic and
van der Waals) for a given pair of atoms. The pairs corre-
sponding to Group 1 (ligand) and Group 2 (residue of interest)
were then summed to yield the interaction energy between the
ligand and that residue.
4.29. Radiochemistry
4.29.1. 1-(2,4-Dichlorophenyl)-4-cyano-[11C]
5-(4-methoxyphenyl)-N-(pyrrolidin-1-yl)-1H-pyrazole-3-
carboxamide ([11C]7h)
Precursor, 1-(2,4-dichlorophenyl)-4-cyano-5-(4-hydroxy-
phenyl)-N-(pyrrolidin-1-yl)-1H-pyrazole-3-carboxamide 16
(1 mg), was added to a 1 mL reaction vial. The precursor
was dissolved in 0.2 mL of acetone. Five microliters of 2 M so-
dium hydroxide were added, and the vial was capped with a sep-
tum sealand shaken. No-carrier-added11C-labeled methyltriflate
prepared as described previously [56] was swept by nitrogen flow
into the vial. The reaction mixture was heated for 3 min at 45 ꢂC
and diluted with 200 mL of water. The mixture was injected into
a Phenomenex Luna C18 column (10ꢃ250 mm) and eluted with
CH3CN:0.1 Maqueousammoniumformatebuffer60:40ataflow
rate of 10 mL/min. The radioactive [11C]7h (peak retention time
of 7e8 min) was collected in a rotary evaporator flask and the
The animal statistical data analysis was performed with Mi-
crosoft Excel software with ANOVA single-factor analysis
tool kit, p ꢅ 0.05 was considered statistically significant.
Acknowledgements
The authors are grateful to Ms. Paige Finley for the rodent
experiments and Ms. Judy W. Buchanan for editorial work.
This work was supported in part by NIH/NIMH grant MH-
079017 (AGH) and NIH/NIDA Grants DA-03934 and DA-
021358 (PHR).

H. Fan et al. / European Journal of Medicinal Chemistry 44 (2009) 593e608
607
References
Appendix.
[1] A.C. Howlett, F. Barth, T.I. Bonner, G. Cabral, P. Casellas, W.A. Devane,
C.C. Felder, M. Herkenham, K. Mackie, B.R. Martin, R. Mechoulam,
R.G. Pertwee, Pharmacol. Rev. 54 (2002) 161e202.
Table A1
Compounds 4 and 7e global minimum energy conformer measurements
Substituent
Definition of torsion/
angle/distance
Measurements
[2] J.P. Gong, E.S. Onaivi, H. Ishiguro, Q.R. Liu, P.A. Tagliaferro,
A. Brusco, G.R. Uhl, Brain Res. 1071 (2006) 10e23.
[3] R.G. Pertwee, Int. J. Obes. 30 (2006) S13eS18.
[4] M. Rinaldi-Carmona, F. Barth, M. Heaulme, D. Shire, B. Calandra,
C. Congy, S. Martinez, J. Maruani, G. Neliat, D. Caput, et al., FEBS
Lett. 350 (1994) 240e244.
Compound 4
Compound 7e
C4 substituent
Piperidine
OeC10eC3eC4
LPeN30eN20eH
C4eC5eC1000eC2000
C3000eC4000eOeC
N2eN1eC100eC200
N/O
2.3ꢂ
ꢀ1.0ꢂ
ꢀ47.3ꢂ
3.3ꢂ
ꢀ5.5ꢂ
ꢀ1.7ꢂ
ꢀ55.6ꢂ
3.2ꢂ
C5 substituent
Methoxy group
N1 substituent
HB distance
HB angle
[5] L.F. Van Gaal, A.M. Rissanen, A.J. Scheen, O. Ziegler, S. Rossner,
Lancet 365 (2005) 1389e1397.
ꢀ74.7ꢂ
ꢀ71.9ꢂ
˚
e
2.70 A
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NeHeO
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156.0ꢂ
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Table A2
Torsion changes of the docked compounds 4 and 7e
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Substituent
Definition of torsion/
angle/distance
Measurements
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Compound 4
Compound 7e
Piperidine
Methoxy group
LPeN30eN20eH
C3000eC4000eOeC
178.8ꢂ
ꢀ165.1ꢂ
179.5ꢂ
177.1ꢂ
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Compare to Table A1 above. The minimum energy conformer of 4 (lone pair
pointing in the direction opposite to the carboxamide hydrogen; methoxy
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Docking studies of 7e also necessitated using the equivalent minimum energy
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Table A3
Pairwise interaction energies for 4 and 1 with CB1 R binding pocket residues,
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Residues
Compound 4
Compound 1
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Coulomb
(kJ/mol)
vdW Coulomb vdW
(kJ/mol) (kJ/mol) (kJ/mol)
K3.28
L3.29
V3.32
T3.33
F3.36
T3.37
I4.56
ꢀ92.84
ꢀ2.26
0.71
ꢀ3.34 ꢀ72.02
ꢀ8.81
ꢀ9.99
ꢀ20.29
ꢀ11.01
ꢀ14.39
ꢀ2.86
ꢀ3.61
ꢀ6.59
ꢀ25.10
ꢀ0.84
ꢀ3.13
ꢀ28.36
ꢀ6.75
ꢀ1.03
ꢀ0.61
ꢀ5.75
ꢀ6.03
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ꢀ13.49
ꢀ14.14
ꢀ4.78
ꢀ14.47
ꢀ0.28
ꢀ0.46
ꢀ6.69
ꢀ29.34
ꢀ0.76
ꢀ5.21
ꢀ32.22
ꢀ15.72
ꢀ9.98
ꢀ5.28
ꢀ4.81
ꢀ5.06
0.53
ꢀ1.04
ꢀ1.29
ꢀ1.11
0.02
ꢀ0.23
ꢀ4.32
0.05
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0.07
ꢀ0.74
ꢀ1.02
ꢀ0.70
ꢀ0.31
0.25
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Y5.39
W5.43
W6.48
I6.54
ꢀ3.32
ꢀ1.41
ꢀ0.22
0.26
M6.55
D6.58
F7.35
C7.38
S7.39
C7.42
1.48
3.31
2.49
5.67
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ꢀ2.20
0.23
0.08
ꢀ0.12
ꢀ0.24
ꢀ0.85
1.71
ꢀ0.65
Subtotal (kJ/mol)
Subtotal (kcal/mol)
Total (kJ/mol)
ꢀ101.27 ꢀ166.03 ꢀ68.77
ꢀ155.14
ꢀ24.19 ꢀ39.66 ꢀ16.43
ꢀ37.05
ꢀ267.30
ꢀ223.91
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Total (kcal/mol)
ꢀ63.84
1.29
ꢀ53.48
1.26
Ligand Conf. Costa (kcal/mol)
Combined total (kcal/mol)
a
ꢀ62.55
ꢀ52.22
Energy calculated at the HF-6-31G* level (see Section 4).

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