Synthesis and structure-activity relationships of amide and hydrazide analogues of the cannabinoid CB1 receptor antagonist N-(piperidinyl)-5-(4-chlorophenyl)-1- (2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716)

Article abstract of DOI:10.1021/jm010498v

Analogues of the biaryl pyrazole N-(piperidinyl)-5-(4-chlorophenyl)-1- (2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716;5) were synthesized to investigate the structure-activity relationship (SAR) of the aminopiperidine region. The structural modifications include the substitution of alkyl hydrazines, amines, and hydroxyalkylamines of varying lengths for the aminopiperidinyl moiety. Proximity and steric requirements at the aminopiperidine region were probed by the synthesis of analogues that substitute alkyl hydrazines of increasing chain length and branching. The corresponding amide analogues were compared to the hydrazides to determine the effect of the second nitrogen on receptor binding affinity. The N-cyclohexyl amide 14 represents a direct methine for nitrogen substitution for 5, reducing the potential for heteroatom interaction, while the morpholino analogue 15 adds the potential for an additional heteroatom interaction. The series of hydroxyalkyl amides of increasing chain length was synthesized to investigate the existence of additional receptor hydrogen binding sites. In displacement assays using the cannabinoid agonist [³H](1R,3R,4R)-3-[2-hydroxy-4- (1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl) cyclohexan-1-ol (CP 55 940; 2) or the antagonist [³H]5, 14 exhibited the highest CB₁ affinity. In general, increasing the length and bulk of the substituent was associated with increased receptor affinity and efficacy (as measured in a guanosine 5′-triphosphate-γ-[³⁵S] assay). However, in most instances, receptor affinity and efficacy increases were no longer observed after a certain chain length was reached. A quantitative SAR study was carried out to characterize the pharmacophoric requirements of the aminopiperidine region. This model indicates that ligands that exceed 3 A? in length would have reduced potency and affinity with respect to 5 and that substituents with a positive charge density in the aminopiperidine region would be predicted to possess increased pharmacological activity.

Full text of DOI:10.1021/jm010498v

2708
J . Med. Chem. 2002, 45, 2708-2719
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s of Am id e a n d Hyd r a zid e
An a logu es of th e Ca n n a bin oid CB1 Recep tor An ta gon ist N-(P ip er id in yl)-
5-(4-ch lor op h en yl)-1-(2,4-d ich lor op h en yl)-4-m eth yl-1H-p yr a zole-3-ca r boxa m id e
(SR141716)
Ma. Elena Y. Francisco,† Herbert H. Seltzman,† Anne F. Gilliam,† Rene´ A. Mitchell,† Sharyl L. Rider,†
Roger G. Pertwee,‡ Lesley A. Stevenson,‡ and Brian F. Thomas*,†
Chemistry and Life Sciences, Research Triangle Institute, Research Triangle Park, North Carolina 27709, and
Department of Biomedical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill,
Aberdeen AB25 2ZD, Scotland, United Kingdom
Received October 25, 2001
Analogues of the biaryl pyrazole N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-carboxamide (SR141716; 5) were synthesized to investigate the structure-
activity relationship (SAR) of the aminopiperidine region. The structural modifications include
the substitution of alkyl hydrazines, amines, and hydroxyalkylamines of varying lengths for
the aminopiperidinyl moiety. Proximity and steric requirements at the aminopiperidine region
were probed by the synthesis of analogues that substitute alkyl hydrazines of increasing chain
length and branching. The corresponding amide analogues were compared to the hydrazides
to determine the effect of the second nitrogen on receptor binding affinity. The N-cyclohexyl
amide 14 represents a direct methine for nitrogen substitution for 5, reducing the potential
for heteroatom interaction, while the morpholino analogue 15 adds the potential for an
additional heteroatom interaction. The series of hydroxyalkyl amides of increasing chain length
was synthesized to investigate the existence of additional receptor hydrogen binding sites.
In displacement assays using the cannabinoid agonist [3H](1R,3R,4R)-3-[2-hydroxy-4-(1,1-
dimethylheptyl)phenyl]-4-(3-hydroxypropyl) cyclohexan-1-ol (CP 55 940; 2) or the antagonist
[3H]5, 14 exhibited the highest CB1 affinity. In general, increasing the length and bulk of the
substituent was associated with increased receptor affinity and efficacy (as measured in a
guanosine 5′-triphosphate-γ-[35S] assay). However, in most instances, receptor affinity and
efficacy increases were no longer observed after a certain chain length was reached. A
quantitative SAR study was carried out to characterize the pharmacophoric requirements of
the aminopiperidine region. This model indicates that ligands that exceed 3 Å in length would
have reduced potency and affinity with respect to 5 and that substituents with a positive charge
density in the aminopiperidine region would be predicted to possess increased pharmacological
activity.
In tr od u ction
ethanolamide (anandamide; 4)5 (Figure 1). These com-
pounds led to the identification and characterization of
cannabinoid receptors in the central nervous system
(CB1)6-8 and the periphery (CB2).9
The use of marijuana and cannabinoid preparations
for medicinal purposes has been known for thousands
of years; yet, its utility in recent years has been limited
because of its psychoactive properties. The isolation and
characterization of the primary psychoactive constituent
of marijuana, ∆9-tetrahydrocannabinol (∆9-THC; 1), led
to the study of the structure-activity relationships
(SAR) of the cannabinoids.1,2 Ultimately, other canna-
bimimetic compounds were discovered, including com-
pounds with structures differing quite dramatically from
that of “classical” cannabinoid compounds, such as
(1R,3R,4R)-3-[2-hydroxy-4-(1,1,-dimethylheptyl)phenyl]-
4-(3-hydroxypropyl) cyclohexan-1-ol (CP 55 940; 2),3
the aminoalkylindole (R)-(+)-[2,3-dihydro-5-methyl-3-
[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-
(1-naphthalenyl)methanone mesylate (WIN55 212-2;
3),4 and the endogenous cannabinoid arachidonyl-
The discovery of N-(piperidinyl)-5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxa-
mide (SR141716; 5), a potent CB1 receptor antagonist
with nanomolar affinity,10,11 provides a unique chemical
tool for further characterization of the cannabinoid
pharmacophore in its relationship to the binding domain
of cannabinoid antagonists. Alkyl hydrazides of increas-
ing steric demand were synthesized to probe the volume
of the aminopiperidine binding site. The amide ana-
logues were compared to the hydrazides to assess the
role of the second nitrogen. The N-cyclohexylamide
analogue represented a direct methine for nitrogen
substitution for 5. The morpholino analogue added a
further heteroatom interaction while the hydroxyalkyl
amides of increasing chain length sought out a receptor
hydrogen-bonding site. Finally, the (R)- and (S)-2-
hydroxymethylethyl amides had an analogy to the (R)-
* To whom correspondence should be addressed. Tel.: (919)541-
6552. Fax: (919)541-6499. E-mail: bft@rti.org.
† Research Triangle Institute.
‡ University of Aberdeen.
10.1021/jm010498v CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/18/2002

Analogues of the Cannabinoid Receptor Antagonist
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13 2709
F igu r e 2. Postulated H bond-stabilized resonance isomers of
27.
cophore models could fit the affinities and efficacies at
the CB1 receptor (with correlation coefficient greater
than that derived for random target variables) but could
not fit the affinities of these compounds at the CB2
receptor. Thus, the results suggest that the SAR derived
from these analogues can aid in the further design
and synthesis of analogues and facilitate the elucidation
of the cannabinoid pharmacophore for CB1 selective
antagonists.
F igu r e 1. Examples of cannabinoid agonists and antagonists.
and (S)-methanandamides12 and probed potential chiral-
ity requirements.
Ch em istr y
The syntheses of the target compounds 7-21 were
carried out in a manner similar to a previously pub-
lished synthesis of 510,26-28 by condensation of the
respective hydrazines and amines with the pyrazole acid
chloride 6 and are shown in Scheme 1.
There is no direct knowledge of the atom-to-atom
interactions between any cannabinoid ligand and the
CB1 or CB2 cannabinoid receptors. Instead, pharma-
cophoric elements of each structural class’s interaction
with these receptors have been inferred using indirect
approaches, such as receptor binding analyses of a
variety of cannabinoid analogues using wild-type and
mutated receptor systems13,14 and through the use of
computer-aided molecular modeling techniques.15-23
In the present study, quenched molecular dynamics
were used to characterize the conformational space
available to each analogue. These conformations were
then included in quantitative SAR (QSAR) analyses and
used to generate three-dimensional pharmacophore
models. The models were derived using receptor binding
affinity and efficacy measurements (guanosine 5′-tri-
phosphate (GTP)-γ-[35S]) as dependent variables. This
approach builds on previous studies, which have utilized
the multiplicity of conformers generated by molecular
dynamics to determine and compare conformationally
accessible regions in structure-activity analyses.21,24,25
The results of the study indicated that the pharma-
The various alkyl hydrazines (R ) Et, Pr, Bu, i-Bu)
were prepared by condensation of tert-butyl carbazate
with the appropriate aldehydes,29 followed by reduction
with lithium aluminum hydride and subsequent acidic
hydrolysis.30 The syntheses of the alkyl hydrazide
analogues 22-27 were then carried out as above
(Scheme 1). The products were characterized by 1H
nuclear magnetic resonance (NMR), high resolution
(electron impact (EI) and fast atom bombardment
(FAB)) mass spectroscopy, high-performance liquid
chromatography (HPLC), and analytical thin-layer chro-
matography (TLC).
1
The N-monoalkylhydrazides exhibited H NMR evi-
dence of a mixture of the H bond-stabilized Z and E
tautomers shown in Figure 2. Using 27 as a representa-
tive example, two sets of peaks were observed for each
of the hydrazide nitrogen, methylene, and methyl
protons of the alkyl group at ambient temperature (295
Sch em e 1

2710 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13
Francisco et al.
1
K). Variable temperature H NMR showed broadening
mean square distance minimization. This alignment
positions all of the molecules in the same three-
dimensional space and superimposes the ring systems
to as great an extent as possible. Because the alkyl
amide and alkyl hydrazide side chains are not part of
the alignment rule, this feature of the molecule could
be compared between conformers of the same compound
as well as between different compounds using the QSAR
techniques described below.
of the above resonances with increasing temperature
that led to coalescence into one set of peaks (δ 4.96, 3.51,
and 0.85) at 329 K. After it was cooled to 295 K, the
original spectrum was regenerated. This trend was
noticeably absent from the amide, N,N-dialkylhydrazide
(i.e., 5 and 15), and hydroxyalkyl amide series, likely
due to the lack of the H-bonding interactions postulated
as a stabilizing influence. The tautomer distribution
ratio was solvent-dependent, increasingly favoring
one tautomer as solvent polarity decreased, MeOH-d4
(1.9:1), DMSO-d6 (2.0:1), pyridine-d5 (2.4:1), and CDCl3
(3.6:1).
Qu a n tita tive Str u ctu r e-Activity An a lyses. The
biological data used as the target values for the struc-
ture-activity analyses included CB1 and CB2 receptor
binding affinity and efficacy in a GTP-γ-[35S] assay.
Comparative molecular field analysis (CoMFA) was
used for the QSAR and involved all of the compounds
shown in Table 1. In this approach, the descriptive
variables are steric and electrostatic descriptions of the
three-dimensional structures of the entire set of com-
pounds. This technique has been previously used suc-
cessfully in QSAR studies of cannabinoids.20,22,25,31,32 The
CoMFA analysis was performed using a proton (H+)
probe atom positioned at lattice points spaced around
the molecules at 2 Å increments. With cross-validation
groups set to 5, a cross-validation study was carried out
by randomly selecting 80% of the compounds to form a
training set, developing a QSAR model based on their
three-dimensional steric and electrostatic properties,
and using this model to predict the dependent variables
of the remaining 20% of the compounds that were not
included in the training set. The predicted dependent
variable of the compounds that was omitted from the
training set was then compared against the actual
dependent variable (e.g., receptor affinity), and a cor-
relation coefficient was obtained. This process was
repeated randomly until every compound had been
omitted from the training set and had its dependent
variables predicted at least once. The correlation coef-
ficients of the entire process were tracked throughout
this process, and the average r2 value was calculated
as a measure of the press or the goodness/robustness of
the model. Cross-validation studies were also conducted
using a “leave-one-out” strategy where the model was
derived repetitively, each new run excluding one par-
ticular compound from the training set. This was
repeated so that each compound was excluded from the
training set and its affinity/potency predicted by a model
derived on the remaining compounds. For each com-
pound, 50 random conformers were selected for each
QSAR analysis. Control studies were also run where
randomized pharmacological data were used in place of
real pharmacological data. These artificial points were
random numbers generated to fall within the range of
the real data. The same cross-validated and final
analyses were performed in the control studies to check
that the r2 values were higher when pharmacological
data were used than when artificial data were used.
The presence of an isomeric mixture with significant
stability of two forms has potential ramifications for the
structure-activity interpretation of SR analogues since
the two isomers are likely to have different affinity and
efficacy profiles at the receptor. The rotamer of the
potent archetype compound 5 that is observed in the
crystal form is the Z isomer. Because this is generally
regarded as the ground state conformation of the
molecule, the corresponding Z tautomers of the N-
monoalkylhydrazides (23-27) can be viewed as the
active conformation corresponding to 5. The design of
future analogues that reside preferentially in the puta-
tive active Z conformation would be anticipated to be
the more active structures. Designing for conformational
preference would, at least, test this concept.
Molecu la r Mod elin g. Molecules and QSAR analyses
were performed using SYBYL (Tripos Inc., St. Louis,
MO). Electrostatic charges of each compound were
calculated with the Gasteiger-Hu¨ckel method. Each
compound was energy-minimized using the SYBYL
force field with a conjugate gradient of 0.001 kcal/mol
or a maximum of 100 000 iterations as termination
criteria.
Qu en ch ed Molecu la r Dyn a m ics. Molecular dy-
namics were computed on each energy-minimized struc-
ture at temperatures from 100 to 1000 K. At each 100
K step, the molecule was allowed to remain at the
specified temperature for 1 ps while snapshots of the
conformation were acquired every 1 ps. After the tem-
perature reached 1000 K, the molecule was held at this
temperature for 1 ps while snapshots were acquired at
1 ps intervals. This procedure yielded a total of 100
snapshots of different conformers for each molecule
investigated. Gasteiger-Hu¨ckel charges were taken into
consideration throughout this molecular dynamics pro-
cedure. Each conformation obtained for a particular
molecule was then energy-minimized again using a
conjugate gradient of 0.01 kcal/mol or a maximum of
100 000 iterations as termination criteria, yielding a
group of 100 energy-minimized conformers per com-
pound.
Molecu la r a n d Con for m a tion a l Align m en t. One
conformation of one compound was used as a template
molecule; it was unimportant which molecule was used
for this template so long as it was applied consistently,
because the pyrazole ring systems of all analogues were
identical. All conformations of all compounds were
aligned in space so as to overlay as closely as possible
the five atoms of their pyrazole ring system with the
corresponding atoms in the template molecule. The
alignment was performed using atom-by-atom root
Resu lts a n d Discu ssion
Recep tor Bin d in g Stu d ies. CB1 Recep tor Affin i-
ties. The binding affinities of each compound as mea-
sured in competition assays (Ki values) with [3H]2 and
[3H]5 in rat whole brain membrane preparations are
provided in Table 1. The Ki of 5 is reported as a
reference. These data demonstrate that as the size of

Analogues of the Cannabinoid Receptor Antagonist
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13 2711
Ta ble 1. Radioligand Binding Data for Amide and Hydrazide Analogues of 5
a
b
d
n ) 2 unless otherwise noted. n ) 1. c n ) 6. Value greater than highest point on displacement curve.

2712 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13
Ta ble 2. Receptor Antagonism in Mouse Vas Deferens
Francisco et al.
a
Concentration of compound used to determine the dissociation constant, KB, which was determined in the mouse-isolated vas deferens
using the cannabinoid receptor agonist 3.
the carbon chain is increased, modest increases in the
binding affinity are observed, up to the C-5 chain. With
the C-6 chain, increases in binding affinity are no longer
evident. This trend is apparent in the alkyl amides
7-13, the hydroxyalkyl amides 16-21, and the alkyl
hydrazides 22-27. The presence of a chiral center in
the compound appeared not to be a factor as there was
not a significant difference in the binding affinity to the
receptor between the (R)- and (S)-hydroxymethylethyl
amides (20 and 21). They both exhibited a 20-fold
decrease in binding affinity over 5, as did the rest of
the hydroxyalkyl amide series. This implies that the
presence of the second electronegative heteroatom de-
creases the receptor binding affinity.
When a methylene unit in the piperidinyl ring is
replaced with an oxygen, as in the morpholino derivative
15, only moderate binding affinity is observed, again
implying that the electronegativity of the oxygen de-
creases receptor binding affinity. Similarly, the replace-
ment of the nitrogen atom in the piperidinyl ring of 5
with a methylene group (14) results in modest increases
in CB1 affinity (when measured with [3H]2) and efficacy
(discussed later). This suggests that the piperidinyl ring
nitrogen is not likely involved in electrostatic interac-
tions or hydrogen bond formation with the receptor that
promotes higher affinity/efficacy.
In general, EC50 values in this test system paralleled
observed trends in CB1 receptor affinity. For example,
the EC50 values of the alkyl amides decreased as the
carbon chain increased from ethyl to pentyl but then
increased again with the hexyl analogue. In all in-
stances, these compounds behaved as inverse agonists.
The Emax values in the alkyl amides were similar to that
observed with 5 up until a straight chain length of four
was reached, after which the Emax values were lower.
In one compound (12), the inverse agonist activity was
only -7.4% of control, indicating that this compound
was a very weak inverse agonist in this system.
In Vit r o P h a r m a cology: Mou se Va s Defer en s
Assa y. It has been established that cannabinoid ago-
nists, such as 3, can inhibit electrically evoked contrac-
tions of isolated tissue preparations such as the mouse
vas deferens.34 Five compounds with reasonably high
affinity for the CB1 receptor were tested for their ability
to antagonize this effect (Table 2). In all of these
experiments, a dose-response curve to 3 was con-
structed 30 min after administration of either the test
compound, compound 5, or vehicle (n ) 5 or 6). All five
of the compounds produced parallel dextral shifts in the
dose-response curve of 3, with 14 being the most potent
of the compounds tested. Compounds 12, 14, and 15 also
exhibited a significant inverse effect (enhancement of
twitch amplitude immediately before the first addition
of 3). Compounds 11 and 13 did not exhibit this inverse
effect at the concentrations tested.
CB2 Recep tor Affin ities. Most of the structural
modifications did not appear to have much effect on CB1/
CB2 selectivity, primarily because none of the com-
pounds tested were found to possess high affinity for
the CB2 receptor. The majority of those compounds
showing a modest increase in selectivity for the CB1
receptor over that of 5 were in the alkyl amide and cyclic
analogues. Typically, the affinities of the hydrazide
analogues for the CB2 receptors were even lower than
that of 5, but their CB1 affinity was even more dramati-
cally reduced. Thus, in this set of compounds, both
affinity and selectivity for the CB1 receptor were de-
creased.
Molecu la r Mod elin g a n d QSAR An a lyses.
Qu en ch ed Molecu la r Dyn a m ics. The quenched
molecular dynamics approach used for conformational
sampling generated 100 low-energy conformations for
each molecule. The conformations were quite diverse for
certain molecules, while other molecules repeatedly
yielded a small number of similar conformations. These
differences in conformational mobility can be visualized
graphically by overlaying the conformations for a par-
ticular molecule (Figure 3). Visual inspection of the
conformational ensembles for this series of analogues
GTP -γ-[35S] Bin d in g Assa y. The GTP-γ-[35S] assays
were conducted according to a variation of Sim et al.33

Analogues of the Cannabinoid Receptor Antagonist
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13 2713
F igu r e 3. Graphical representation of the conformational
ensemble obtained by quenched molecular dynamics for 14.
F igu r e 4. Potency of analogues of 5 as predicted by QSAR
model.
of 5 suggests that a full and representative sample of
conformations was afforded by this technique.
Ta ble 3. QSAR Model Derived with Real or Random Data
r2 values
CoMF A. The CoMFA approach is typically applied
to single conformations selected for each analogue and
usually involves the identification of a bioactive con-
former or the selection of a putative bioactive conforma-
tion. The data and results achieved during these QSAR
studies are dependent on the molecular alignment
system used to compare analogues. We chose to align
molecules by overlaying their pyrazole rings using a root
mean square minimization procedure, thereby maximiz-
ing the structural differences of the training set in the
aminopiperidinyl region. This alignment strategy is
arguably the most appropriate, because the two-
dimensional structure of the compounds differed only
in the aminopiperidinyl region. Furthermore, the three-
dimensional conformational mobility of these analogues
also differed primarily in the aminopiperidinyl region.
Regardless of the number of conformations used for
each analogue or sampling strategy, the cross-validated
analysis of the relationship between the CoMFA mo-
lecular fields and the pharmacological affinity and
potency measurements generally indicated that a model
derived with (the maximum of) five components was
optimal. Components are the variables used by SYBYL
in developing the QSAR model; five components indicate
that five parameters were varied to achieve the pre-
dicted data used in the “predicted vs actual” linear
regression fit. Note that components do not have a
specific physical analogue: they are neither regions of
the molecule nor pharmacological assays but simply a
facet of the numerical techniques used to develop a
QSAR model. The larger number of components in an
equation created to explain activity indicates a more
complex and less robust model.
real data
random data
dependent variable
cross
final
cross
final
pKi, CB1 ([3H]2)
pKi, CB1 ([3H]5)
pKi, CB2 ([3H]2)
pKEC50 (GTP-γ-[35S])
0.675
0.691
0.402
0.168
0.701
0.717
0.464
0.276
0.145
0.128
0.054
0.310
0.263
0.224
0.213
0.365
0.168 were obtained for the CB1 receptor affinities
utilizing [3H]2 and [3H]5, CB2 receptor affinity, and
efficacy in a GTP-γ-[35S] assay, respectively. When
random pharmacological data were used, values of
0.145, 0.128, 0.054, and 0.310 were obtained for the CB1
receptor affinities utilizing [3H]2 and [3H]5, CB2 receptor
affinity, and efficacy in a GTP-γ-[35S] assay, respectively.
The final analysis (based on all 1050 conformers being
included in the training set) resulted in r2 values of
0.701, 0.717, 0.464, and 0.276 with real data and 0.263,
0.224, 0.213, and 0.365 for random data (in models
derived for CB1 receptor affinities utilizing [3H]2 and
[3H]5, CB2 receptor affinity, and efficacy in a GTP-γ-
[35S] assay, respectively). The results, which are listed
in Table 3, indicated that pharmacophore models could
fit the affinities of these compounds at the CB1 receptor
but could not fit the affinities of these compounds at
the CB2 receptor nor their efficacies in a GTP-γ-[35S]
assay.
Visu a liza tion of CoMF A F ield s. Three-dimensional
contour plots of the CoMFA model allow the visualiza-
tion of regions where changes in steric or electrostatic
properties are correlated with experimentally deter-
mined differences in biological properties. The contour
plots in Figures 5 and 6 display the QSAR model for
receptor affinity in the CB1 receptor assay utilizing [3H]-
2 as radioligand when derived from a training set of
1050 conformers (50 random conformations from each
of 21 molecules). Some of the most heavily weighted
regions in the model are depicted in the contour plot at
the 80/20 level.
The strength of the cross-validated and final models
was demonstrated by comparison with relationships
derived with random pharmacological data that spanned
the same range as the real pharmacological data, and
this is illustrated in Figure 4. This CoMFA analysis
utilized 50 conformations of each of the 21 molecules
listed in Table 1 (1050 conformers total) in the training
set. Cross-validated r2 values of 0.675, 0.691, 0.402, and
Inspection of the steric contour plot (Figure 5) reveals
that the aminopiperidinyl region of 5 was surrounded

2714 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13
Francisco et al.
F igu r e 5. Stereoview of the QSAR derived for the steric fields (yellow and green contours) for CB1 binding affinity utilizing
[3H]2 as radioligand. The steric plot is depicted so that steric bulk should be moved closer to areas contoured in green and farther
from regions contoured in yellow in order to increase the predicted target property (i.e., affinity). For this model, an 80/20 level
of contribution is depicted; 80 indicates that the region displayed is that which contributes within the top 20% of the (green)
interaction, and 20 indicates that the region displayed is that which contributes within the top 20% of negative (yellow) interactions.
F igu r e 6. Stereoview of the QSAR derived for the electrostatic field (blue and red contours) for CB1 binding affinity utilizing
[3H]2 as radioligand. The electrostatic plot is contoured such that the positive charge should be moved closer to regions contoured
in blue and farther from regions contoured in red in order to increase the target property being contoured.
by a green contour, indicating a region where steric bulk
is associated with increased predicted pharmacological
potency. Ligands substituted at the aminopiperidinyl
region of 5 that exceed 3 Å in length are predicted to
have reduced potency and affinity with respect to 5. This
predicted decrease in potency and affinity corresponds
to the amide and hydrazide analogues exceeding five
carbons in length.
Examination of the electrostatic plot in Figure 6
reveals a large contour in blue that completely sur-
rounds the aminopiperidinyl region and a smaller red
contour that covers only the amide linkage. The blue
contour indicates that compounds with positive charge
densities in this region would be predicted to possess
increased pharmacological activity while the regions
contoured in red are indicative of where negative charge
is associated with increased pharmacological activity.
Therefore, one could suggest that in the hydroxyalkyl
amides, the presence of the electronegative oxygen atom
in the blue-contoured region leads to their decreased
affinity, resulting in compounds with Ki values ranging
from 117 to 1686 nM.
that the CB2 selective antagonist N-[(1S)-endo-1,3,3-tri-
methylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methyl-
phenyl)-1-(4-methylbenzyl)-1H-pyrazole-3-carboxamide
(SR144528; 28) has structural modifications in addition
to the change at the aminopiperidine moiety (replaced
by the (1S)-endo-fenchylamine group in 28) that was
explored. These changes, which include replacement of
the phenyl group on nitrogen 1 with a substituted
benzyl group and the elimination of the methyl group
on the pyrazole ring, could be more responsible for the
complete inversion of the CB1/CB2 selectivity of 28 as
compared to 5. Indeed, one might suggest that these
regions need to be included in QSAR studies involving
CB2 affinity, thereby illustrating how the SAR derived
from these analogues can aid in the further design and
synthesis of analogues of 5.
The results of the study demonstrate that three-
dimensional pharmacophore models derived for a vari-
ety of analogues of 5 can fit their affinities and efficacies
at the CB1 receptor (with a correlation coefficient
greater than that derived for random target variables)
but cannot fit the affinities of these compounds at the
CB2 receptor. This is consistent with the observation
Con clu sion s
On the basis of the results reported here, as the length
of the carbon chain increases, the affinity of the ana-

Analogues of the Cannabinoid Receptor Antagonist
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13 2715
N-E t h yl-5-(4-ch lor op h en yl)-1-(2,4-d ich lor op h en yl)-4-
m eth yl-1H-p yr a zole-3-ca r boxa m id e (7). Ethylamine (2.0 M
solution in THF, 0.5 mL, 1.00 mmol), triethylamine (280 µL,
2.00 mmol), and CH2Cl2 (5 mL) were cooled to 0 °C under argon
and treated dropwise over 5 min with a solution of 627,28 (218.6
mg, 0.55 mmol) in CH2Cl2 (5 mL). The solution was allowed
to warm slowly to 25 °C under Ar and stirred for an additional
2 h. The reaction was quenched by addition of 10 mL of H2O
and partitioned. The organic layer was washed with 2 × 20
mL of 1 N HCl. The combined aqueous layers were back-
extracted with CH2Cl2, and the combined CH2Cl2 layers were
washed with saturated aqueous NaHCO3. The aqueous layer
was back-extracted with CH2Cl2, and the combined CH2Cl2
layers were dried over Na2SO4. The CH2Cl2 was evaporated
in vacuo, yielding a brown foam. Flash chromatography (1:1
EtOAc-hexanes) yielded the product as a white foam (200 mg,
logues of 5 increases but only up to the C-5 chain. When
the chain is six carbons long, gains in affinity toward
the CB1 receptor are no longer observed. This trend is
consistent over the alkyl hydrazide, the alkyl amide, and
the hydroxyalkyl amide series. When comparing the
receptor binding affinities of 5 and 14, it is apparent
that the nitrogen atom in the piperidinyl moiety is not
contributing to the affinity of 5. In addition, the binding
affinity for the alkyl hydrazide series decreased relative
to 5, which lends support to the theory that the second
nitrogen atom as a 2° nitrogen is not contributing to
the overall binding affinity. The presence of the second
heteroatom in the case of the morpholino amide 15 and
the hydroxyalkyl amide series 16-21 also decreased the
receptor binding affinity.
1
90% yield); mp 119.2-119.8 °C. H NMR: δ 7.57 (d, J ) 2.2
Hz, 1H, Ar 3-H), 7.52 (d, J ) 8.5 Hz, 1H, Ar 6-H), 7.44 (dd, J
) 2.2, 8.5 Hz, 1H, Ar 5-H), 7.36 (d, J ) 8.5 Hz, 2H, Ar′ 3,5-H),
7.19 (d, J ) 8.5 Hz, 2H, Ar′ 2,6-H), 3.39 (q, J ) 7.2 Hz, 2H,
N-CH2-CH3), 2.30 (s, 3H, CH3), 1.21 (t, J ) 7.2 Hz, 3H,
N-CH2-CH3). HPLC: 65% CH3CN-H2O, Rt 10.4 min (100%);
85% CH3OH-H2O, Rt 4.3 min (100%). TLC: 4:1 EtOAc-Hex,
Rf 0.68; 10% MeOH-CHCl3, Rf 0.70. HREIMS: m/z 407.0360
(calcd for C19H1635Cl3N3O, 407.0359).
From the SAR studies, consistent data have been
acquired, which indicates that the pharmacophoric
requirements of the aminopiperidine region involve
having a side chain no greater than 3 Å in length. In
addition, having a substituent with a positive charge
density in the aminopiperidine region is predicted to
result in compounds with increased affinity and potency
toward the CB1 receptor. The consistent applications of
the methods and the comparison of these results with
the randomized data lend support for these pharma-
cophoric requirements of the aminopiperidine region.
N-(1-P r opyl)-5-(4-ch lor oph en yl)-1-(2,4-dich lor oph en yl)-
4-m eth yl-1H-p yr a zole-3-ca r boxa m id e (8). Compound 8
was obtained from 6 and 1-propylamine according to the
procedure described for 7 and was isolated as a white semisolid
1
(208 mg, 88% yield). H NMR: δ 7.57 (d, J ) 2.2 Hz, 1H, Ar
3-H), 7.53 (d, J ) 8.5 Hz, 1H, Ar 6-H), 7.44 (dd, J ) 2.2, 8.5
Hz, 1H, Ar 5-H), 7.36 (d, J ) 8.5 Hz, 2H, Ar′ 3,5-H), 7.19 (d,
J ) 8.6 Hz, 2H, Ar′ 2,6-H), 3.35 (t, J ) 7.7 Hz, 2H, N-CH2-
CH2-CH3), 2.30 (s, 3H, CH3), 1.64 (m, 2H, N-CH2-CH2-
CH3), 0.97 (t, J ) 7.4 Hz, 3H, N- CH2-CH2-CH3). HPLC:
65% CH3CN-H2O, Rt 14.9 min (100%); 85% CH3OH-H2O,
Rt 4.8 min (100%). TLC: 4:1 EtOAc-Hex, Rf 0.72; 10%
MeOH-CHCl3, Rf 0.73. HREIMS: m/z 421.0519 (calcd for
Exp er im en ta l Section
Gen er a l Meth od s. Reactions were conducted under N2 or
Ar atmospheres using oven-dried glassware. All solvents and
chemicals used were reagent grade. Tetrahydrofuran (THF)
and Et2O were distilled from sodium benzophenone ketyl
under N2. CH2Cl2, hexanes, and toluene were passed through
basic alumina and stored over 4 Å molecular sieves under Ar.
Et3N was distilled from CaH2 and stored over NaOH pellets
under Ar. Unless otherwise mentioned, reagents were obtained
from commercial sources and used without further purification.
Melting points were determined on a Mel-Temp apparatus and
are uncorrected. The optical rotations were determined on a
Rudolph Autopol III spectropolarimeter. Flash column chro-
matography was carried out with Whatman silica gel 60 (230-
400 mesh). Purity and characterization of compounds were
established by a combination of HPLC, TLC, gas chromatog-
raphy mass spectrometry (GC-MS), high-resolution mass
spectrometry (HRMS), and NMR analytical techniques de-
scribed below. Compounds were shown to be homogeneous by
HPLC employing two diverse solvent mixtures on a Waters
dual pump chromatograph operating at 2.0 mL/min with a
model 484 tunable absorbance detector, Waters Nova-Pak
reversed phase C-18 (4 µm) RCM 8 mm × 100 mm column,
UV detection at 280 nm; eluants utilized were either CH3CN-
H2O or CH3OH-H2O mixtures as indicated in each experi-
mental procedure. TLC on Whatman Si254F silica gel glass
plates eluting with the solvents indicated in each experimental
procedure, and employing UV and phosphomolybdic acid-ceric
sulfate spray and/or iodine detection, similarly showed the
homogeneity of the compounds. GC-MS was measured on a
Hewlett-Packard 6890 GC System with a model 5973 Mass
Selective Detector using EI ionization. HRMS were determined
on a VG-70S Mass Spectrometer (Micromass; Beverly, MA)
and were performed by the mass spectrometry laboratory at
the University of South Carolina. 1H NMR spectra were
recorded on a Bruker Avance DPX-300 (300 MHz) spectrom-
eter and were determined in MeOH-d4 with tetramethylsilane
(TMS) (0.00 ppm) or MeOH (3.30 ppm) as the internal
reference unless otherwise noted. Variable temperature 1H
NMR spectra were recorded on a Bruker AMX-500 (500 MHz)
and were determined in DMSO-d6 with TMS (0.00 ppm) as
the internal reference. Temperatures were uncorrected. For
tautomeric mixtures (25-27), fractional proton resonances
were reported as observed.
C
20H1835Cl3N3O, 421.0515).
N-(2-P r opyl)-5-(4-ch lor oph en yl)-1-(2,4-dich lor oph en yl)-
4-m eth yl-1H-p yr a zole-3-ca r boxa m id e (9). Compound 9
was obtained from 6 and 2-propylamine according to the
procedure described for 7 and was isolated as a white semisolid
1
(207 mg, 95% yield). H NMR: δ 7.57 (d, J ) 2.2 Hz, 1H, Ar
3-H), 7.54 (d, J ) 8.5 Hz, 1H, Ar 6-H), 7.45 (dd, J ) 2.2, 8.4
Hz, 1H, Ar 5-H), 7.37 (d, J ) 8.5 Hz, 2H, Ar′ 3,5-H), 7.19 (d,
J ) 8.5 Hz, 2H, Ar′ 2,6-H), 4.20 (hept, J ) 6.6 Hz, 1H, N-CH-
(CH3)2), 2.30 (s, 3H, CH3), 1.24 (d, J ) 6.6 Hz, 6H, N-CH-
(CH3)2). HPLC: 65% CH3CN-H2O, Rt 14.5 min (100%); 85%
CH3OH-H2O, Rt 5.1 min (100%). TLC: 4:1 EtOAc-Hex, Rf
0.73; 10% MeOH-CHCl3, Rf 0.73. HREIMS: m/z 421.0502
(calcd for C20H1835Cl3N3O, 421.0515).
N-(1-Bu tyl)-5-(4-ch lor op h en yl)-1-(2,4-d ich lor op h en yl)-
4-m eth yl-1H-p yr a zole-3-ca r boxa m id e (10). Compound 10
was obtained from 6 and 1-butylamine according to the
procedure described for 7 and was isolated as a white semisolid
1
(185 mg, 84% yield). H NMR: δ 7.57 (d, J ) 2.2 Hz, 1H, Ar
3-H), 7.53 (d, J ) 8.5 Hz, 1H, Ar 6-H), 7.45 (dd, J ) 2.2, 8.5
Hz, 1H, Ar 5-H), 7.37 (d, J ) 8.5 Hz, 2H, Ar′ 3,5-H), 7.19 (d,
J ) 8.5 Hz, 2H, Ar′ 2,6-H), 3.36 (t, J ) 7.1 Hz, 2H, N-CH2-
CH2-CH2-CH3), 2.30 (s, 3H, CH3), 1.59 (m, 2H, N-CH2-
CH2-CH2-CH3), 1.41 (m, 2H, N-CH2-CH2-CH2-CH3),
0.96 (t, J ) 7.3 Hz, 3H, N-CH2-CH2-CH2-CH3). HPLC:
65% CH3CN-H2O, Rt 20.5 min (100%); 85% CH3OH-H2O,
Rt 5.7 min (100%). TLC: 4:1 EtOAc-Hex, Rf 0.75; 10%
MeOH-CHCl3, Rf 0.77. HREIMS: m/z 435.0679 (calcd for
C
21H2035Cl3N3O, 435.0672).
N-(2-Met h ylp r op yl)-5-(4-ch lor op h en yl)-1-(2,4-d ich lo-
r oph en yl)-4-m eth yl-1H-pyr azole-3-car boxam ide (11). Com-
pound 11 was obtained from 6 and 2-methylpropylamine
according to the procedure described for 7 and was isolated
1
as a white semisolid (196 mg, 89% yield). H NMR: δ 7.57 (d,
J ) 2.2 Hz, 1H, Ar 3-H), 7.53 (d, J ) 8.5 Hz, 1H, Ar 6-H), 7.44
(dd, J ) 2.2, 8.5 Hz, 1H, Ar 5-H), 7.36 (d, J ) 8.5 Hz, 2H, Ar′

2716 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13
Francisco et al.
3,5-H), 7.19 (d, J ) 8.5 Hz, 2H, Ar′ 2,6-H), 3.18 (d, J ) 7.0 Hz,
2H, N-CH2-CH-(CH3)2), 2.30 (s, 3H, CH3), 1.89 (m, 1H,
N-CH2-CH-(CH3)2), 0.96 (d, J ) 6.7 Hz, 6H, N-CH2-CH-
(CH3)2). HPLC: 65% CH3CN-H2O, Rt 13.4 min (100%); 85%
CH3OH-H2O, Rt 6.2 min (100%). TLC: 4:1 EtOAc-Hex, Rf
0.77; 10% MeOH-CHCl3, Rf 0.72. HREIMS: m/z 435.0675
(calcd for C21H2035Cl3N3O, 435.0672).
with saturated aqueous NaHCO3. The aqueous layer was back-
extracted with CH2Cl2, and the combined CH2Cl2 layers were
dried over Na2SO4. The CH2Cl2 was evaporated in vacuo,
yielding a brown foam. Flash chromatography (2:3 EtOAc-
hexanes) yielded the product as a white foam (144 mg, 73%
yield); mp 187.6-188.7 °C. 1H NMR: δ 7.55 (d, J ) 2.2 Hz,
1H, Ar 3-H), 7.51 (d, J ) 8.5 Hz, 1H, Ar 6-H), 7.43 (dd, J )
2.2, 8.5 Hz, 1H, Ar 5-H), 7.36 (d, J ) 8.5 Hz, 2H, Ar′ 3,5-H),
7.18 (d, J ) 8.5 Hz, 2H, Ar′ 2,6-H), 2.29 (s, 3H, CH3). HPLC:
65% CH3CN-H2O, Rt 3.6 min (100%); 80% CH3OH-H2O, Rt
5.1 min (100%). TLC: 7:1 EtOAc-Hex, Rf 0.51; 20% MeOH-
CHCl3, Rf 0.67. HRFABMS: m/z 396.0082 (calcd for [M + H]
N-(1-P en tyl)-5-(4-ch lor oph en yl)-1-(2,4-dich lor oph en yl)-
4-m eth yl-1H-p yr a zole-3-ca r boxa m id e (12). Compound 12
was obtained from 6 and 1-pentylamine according to the
procedure described for 7 and was isolated as a colorless oil
1
(206 mg, 88% yield). H NMR: δ 7.61 (d, J ) 2.2 Hz, 1H, Ar
C
17H1335Cl3N3O2, 396.0073).
3-H), 7.56 (d, J ) 8.4 Hz, 1H, Ar 6-H), 7.48 (dd, J ) 2.2, 8.4
Hz, 1H, Ar 5-H), 7.40 (d, J ) 8.5 Hz, 2H, Ar′ 3,5-H), 7.23 (d,
J ) 8.5 Hz, 2H, Ar′ 2,6-H), 3.39 (t, J ) 7.2 Hz, 2H, N-CH2-
(CH2)3-CH3), 2.34 (s, 3H, CH3), 1.65 (m, 2H, N-CH2-CH2-
(CH2)2-CH3), 1.40 (m, 4H, N-CH2-CH2-(CH2)2-CH3), 0.96 (t,
J ) 6.8 Hz, 3H, N-(CH2)4-CH3). HPLC: 65% CH3CN-H2O,
Rt 19.3 min (100%); 85% CH3OH-H2O, Rt 6.4 min (100%).
TLC: 4:1 EtOAc-Hex, Rf 0.81; 10% MeOH-CHCl3, Rf 0.81.
HREIMS: m/z 449.0838 (calcd for C22H2235Cl3N3O, 449.0828).
N-(2-H yd r oxyet h yl)-5-(4-ch lor op h en yl)-1-(2,4-d ich lo-
r oph en yl)-4-m eth yl-1H-pyr azole-3-car boxam ide (17). Com-
pound 17 was obtained from 6 and 2-aminoethanol according
to the procedure described for 7 and was isolated as a colorless
1
oil (196 mg, 88% yield). H NMR: δ 7.57 (d, J ) 2.2 Hz, 1H,
Ar 3-H), 7.52 (d, J ) 8.5 Hz, 1H, Ar 6-H), 7.44 (dd, J ) 2.2,
8.5 Hz, 1H, Ar 5-H), 7.36 (d, J ) 8.5 Hz, 2H, Ar′ 3,5-H), 7.19
(d, J ) 8.5 Hz, 2H, Ar′ 2,6-H), 3.69 (t, J ) 5.8 Hz, 2H, N-CH2-
CH2-OH), 3.49 (t, J ) 5.8 Hz, 2H, N-CH2-CH2-OH) 2.30
(s, 3H, CH3). HPLC: 70% CH3CN-H2O, Rt 3.9 min (100%);
80% CH3OH-H2O, Rt 6.3 min (100%). TLC: 10:1 EtOAc-Hex,
Rf 0.43; 20% MeOH-CHCl3, Rf 0.66. HREIMS: m/z 405.0200
(calcd for [M - H2O] C19H1435Cl3N3O, 405.0202).
N-(1-Hexyl)-5-(4-ch lor op h en yl)-1-(2,4-d ich lor op h en yl)-
4-m eth yl-1H-p yr a zole-3-ca r boxa m id e (13). Compound 13
was obtained from 6 and 1-hexylamine according to the
procedure described for 7 and was isolated as a colorless oil
1
(111 mg, 60% yield). H NMR: δ 7.59 (d, J ) 2.2 Hz, 1H, Ar
3-H), 7.54 (d, J ) 8.5 Hz, 1H, Ar 6-H), 7.46 (dd, J ) 2.2, 8.5
Hz, 1H, Ar 5-H), 7.38 (d, J ) 8.6 Hz, 2H, Ar′ 3,5-H), 7.21 (d,
J ) 8.6 Hz, 2H, Ar′ 2,6-H), 3.39 (t, J ) 7.1 Hz, 2H, N-CH2-
(CH2)4-CH3), 2.32 (s, 3H, CH3), 1.62 (m, 2H, N-CH2-CH2-
(CH2)3-CH3), 1.37 (m, 6H, N-CH2-CH2-(CH2)3-CH3), 0.93 (t,
J ) 6.8 Hz, 3H, N-(CH2)5-CH3). HPLC: 75% CH3CN-H2O,
Rt 17.3 min (100%); 82% CH3OH-H2O, Rt 15.8 min (100%).
TLC: 4:1 EtOAc-Hex, Rf 0.79; 10% MeOH-CHCl3, Rf 0.82.
HREIMS: m/z 463.0984 (calcd for C23H2435Cl3N3O, 463.0985).
N-(3-Hyd r oxyp r op yl)-5-(4-ch lor op h en yl)-1-(2,4-d ich lo-
r oph en yl)-4-m eth yl-1H-pyr azole-3-car boxam ide (18). Com-
pound 18 was obtained from 6 and 3-amino-1-propanol ac-
cording to the procedure described for 7 and was isolated as a
1
colorless oil (208 mg, 92% yield). H NMR: δ 7.56 (d, J ) 2.2
Hz, 1H, Ar 3-H), 7.52 (d, J ) 8.5 Hz, 1H, Ar 6-H), 7.44 (dd, J
) 2.2, 8.5 Hz, 1H, Ar 5-H), 7.36 (d, J ) 8.5 Hz, 2H, Ar′ 3,5-H),
7.19 (d, J ) 8.5 Hz, 2H, Ar′ 2,6-H), 3.64 (t, J ) 6.2 Hz, 2H,
N-CH2-CH2-CH2-OH), 3.46 (t, J ) 6.8 Hz, 2H, N-CH2-
CH2-CH2-OH) 2.30 (s, 3H, CH3), 1.81 (p, J ) 6.5 Hz, 2H,
N-CH2-CH2-CH2-OH). HPLC: 70% CH3CN-H2O, Rt 4.4
min (100%); 80% CH3OH-H2O, Rt 6.9 min (100%). TLC: 10:1
EtOAc-Hex, Rf 0.42; 20% MeOH-CHCl3, Rf 0.66. HREIMS:
m/z 437.0469 (calcd for C20H1835Cl3N3O2, 437.0465).
N-Cycloh exyl-5-(4-ch lor oph en yl)-1-(2,4-dich lor oph en yl)-
4-m eth yl-1H-p yr a zole-3-ca r boxa m id e (14). Compound 14
was obtained from 6 and cyclohexylamine according to the
procedure described for 7 and was isolated as a white semisolid
1
(214 mg, 92% yield). H NMR: δ 7.57 (d, J ) 2.2 Hz, 1H, Ar
3-H), 7.53 (d, J ) 8.5 Hz, 1H, Ar 6-H), 7.45 (dd, J ) 2.2, 8.5
Hz, 1H, Ar 5-H), 7.36 (d, J ) 8.5 Hz, 2H, Ar′ 3,5-H), 7.19 (d,
J ) 8.5 Hz, 2H, Ar′ 2,6-H), 3.83 (m, 1H, N-CH), 2.29 (s, 3H,
CH3), 1.94 (m, 2H, cyclohex), 1.78 (m, 2H, cyclohex), 1.66 (m,
1H, cyclohex), 1.36 (m, 5H, cyclohex). HPLC: 65% CH3CN-
H2O, Rt 31.8 min (100%); 85% CH3OH-H2O, Rt 8.8 min
(100%). TLC: 4:1 EtOAc-Hex, Rf 0.71; 10% MeOH-CHCl3,
Rf 0.72. HREIMS: m/z 461.0834 (calcd for C23H2235Cl3N3O,
461.0828).
N-(4-H yd r oxyb u t yl)-5-(4-ch lor op h en yl)-1-(2,4-d ich lo-
r oph en yl)-4-m eth yl-1H-pyr azole-3-car boxam ide (19). Com-
pound 19 was obtained from 6 and 4-amino-1-butanol accord-
ing to the procedure described for 7 and was isolated as a white
semisolid (202 mg, 86% yield). 1H NMR: δ 7.56 (d, J ) 2.2
Hz, 1H, Ar 3-H), 7.52 (d, J ) 8.5 Hz, 1H, Ar 6-H), 7.44 (dd, J
) 2.2, 8.5 Hz, 1H, Ar 5-H), 7.36 (d, J ) 8.5 Hz, 2H, Ar′ 3,5-H),
7.19 (d, J ) 8.5 Hz, 2H, Ar′ 2,6-H), 3.58 (t, J ) 6.2 Hz, 2H,
N-CH2-CH2-CH2-CH2-OH), 3.38 (t, J ) 6.8 Hz, 2H,
N-CH2-CH2-CH2-CH2-OH) 2.30 (s, 3H, CH3), 1.62 (m, 4H,
N-CH2-(CH2)2-CH2-OH). HPLC: 65% CH3CN-H2O, Rt 4.2
min (100%); 80% CH3OH-H2O, Rt 7.3 min (100%). TLC: 30:1
EtOAc-Hex, Rf 0.41; 20% MeOH-CHCl3, Rf 0.65. HREIMS:
m/z 451.0626 (calcd for C21H2035Cl3N3O2, 451.0621).
N-(Mor p h olin -4-yl)-5-(4-ch lor op h en yl)-1-(2,4-d ich lo-
r oph en yl)-4-m eth yl-1H-pyr azole-3-car boxam ide (15). Com-
pound 15 was obtained from 6 and N-aminomorpholine
according to the procedure described for 7 and was isolated
as a white foam (186 mg, 78% yield); mp 235.3-236.2 °C
(literature35 mp 247-249 °C). 1H NMR: δ 7.57 (d, J ) 2.2 Hz,
1H, Ar 3-H), 7.54 (d, J ) 8.5 Hz, 1H, Ar 6-H), 7.46 (dd, J )
2.2, 8.5 Hz, 1H, Ar 5-H), 7.37 (d, J ) 8.5 Hz, 2H, Ar′ 3,5-H),
7.20 (d, J ) 8.5, 2H, Ar′ 2,6-H), 3.80 (br t, J ) 4.7 Hz, 4H,
N-CH2), 2.90 (br t, J ) 4.6 Hz, 4H, O-CH2), 2.31 (s, 3H, CH3).
HPLC: 70% CH3CN-H2O, Rt 4.5 min (100%); 85% CH3OH-
H2O, Rt 3.6 min (100%). TLC: 30:1 EtOAc-Hex, Rf 0.39; 20%
MeOH-CHCl3, Rf 0.78. HREIMS: m/z 464.0581 (calcd for
(S)-(+)-N-(2-Hydr oxy-1-m eth yleth yl)-5-(4-ch lor oph en yl)-
1-(2,4-d ich lor op h en yl)-4-m eth yl-1H-p yr a zole-3-ca r boxa -
m id e (20). Compound 20 was obtained from 6 and (S)-(+)-2-
amino-1-propanol according to the procedure described for 7
and was isolated as a white semisolid (200 mg, 90% yield).
1
[R]20 +8.8° (c 0.08, CHCl3). H NMR: δ 7.57 (d, J ) 2.2 Hz,
D
1H, Ar 3-H), 7.53 (d, J ) 8.5 Hz, 1H, Ar 6-H), 7.45 (dd, J )
2.2, 8.5 Hz, 1H, Ar 5-H), 7.36 (d, J ) 8.5 Hz, 2H, Ar′ 3,5-H),
7.19 (d, J ) 8.5 Hz, 2H, Ar′ 2,6-H), 4.16 (m, 1H, N-CH-
(CH3)-CH2-OH), 3.58 (d, J ) 5.2 Hz, 2H, N-CH-(CH3)-
CH2-OH), 2.30 (s, 3H, CH3), 1.23 (d, J ) 6.8 Hz, 3H, N-CH-
(CH3)-CH2-OH). HPLC: 75% CH3CN-H2O, Rt 3.8 min
(100%); 80% CH3OH-H2O, Rt 7.5 min (100%). TLC: 10:1
EtOAc-Hex, Rf 0.49; 20% MeOH-CHCl3, Rf 0.72. HREIMS:
m/z 437.0481 (calcd for C20H1835Cl3N3O2, 437.0465).
C
21H1935Cl3N4O2, 464.0574).
N-Hyd r oxy-5-(4-ch lor op h en yl)-1-(2,4-d ich lor op h en yl)-
4-m eth yl-1H-p yr a zole-3-ca r boxa m id e (16). The di-HCl salt
of hydroxamic acid (76 mg, 1.09 mmol), triethylamine (500 µL,
3.59 mmol), and 95% EtOH (5 mL) was cooled to 0 °C under
argon and treated dropwise over 5 min with a solution of 6
(218.6 mg, 0.55 mmol) in CH2Cl2 (5 mL). The solution was
allowed to warm slowly to 25 °C under Ar and stirred for an
additional 2 h. The reaction was quenched by addition of 10
mL of H2O. The organic layer was washed with 2 × 20 mL of
1 N HCl. The combined aqueous layers were back-extracted
with CH2Cl2, and the combined CH2Cl2 layers were washed
(R)-(-)-N-(2-Hydr oxy-1-m eth yleth yl)-5-(4-ch lor oph en yl)-
1-(2,4-d ich lor op h en yl)-4-m eth yl-1H-p yr a zole-3-ca r boxa -
m id e (21). Compound 21 was obtained from 6 and (R)-(-)-2-
amino-1-propanol according to the procedure described for 7
and was isolated as a white semisolid (196 mg, 88% yield).

Analogues of the Cannabinoid Receptor Antagonist
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13 2717
1
[R]20 -3.4° (c 0.09, CHCl3). H NMR: δ 7.57 (d, J ) 2.2 Hz,
H2O, Rt 8.3 min (100%); 82% CH3OH-H2O, Rt 6.3 min (100%).
TLC: 10:1 EtOAc-Hex, Rf 0.47; 10% MeOH-CHCl3, Rf 0.42.
HREIMS: m/z 450.0770 (calcd for C21H2135Cl3N4O, 450.0781).
N-(2-Met h ylp r op yl)-5-(4-ch lor op h en yl)-1-(2,4-d ich lo-
r op h en yl)-4-m eth yl-1H-p yr a zole-3-ca r boh yd r a zid e (27).
Compound 27 was obtained from 6 and the di-HCl salt of
2-methylpropylhydrazine according to the procedure described
for 16 and was isolated as a white foam (132 mg, 60% yield).
1H NMR (DMSO-d6, 300 MHz): δ 7.81 (d, J ) 2.2 Hz, 1H, Ar
3-H), 7.61 (d, J ) 8.5 Hz, 1H, Ar 6-H), 7.54 (dd, J ) 2.2, 8.5
Hz, 1H, Ar 5-H), 7.45 (d, J ) 8.5 Hz, 2H, Ar′ 3,5-H), 7.22 (d,
J ) 8.5 Hz, 2H, Ar′ 2,6-H), 5.06 (br s, 0.67H, NH-CH2-CH-
(CH3)2), 4.94 (br s, 0.33H, NH′-CH2-CH-(CH3)2), 3.49 (br d,
J ) 5.3 Hz, 1.5H, N-CH2-CH-(CH3)2), 3.40 (d, J ) 7.4 Hz,
0.5H, N-CH2′-CH-(CH3)2), 2.08 (m, 1H, N-CH2-CH-
(CH3)2), 2.05 (s, 3H, CH3), 0.89 (d, J ) 6.6 Hz, 2H, N-CH2-
CH-(CH3′)2), 0.75 (d, J ) 6.7 Hz, 4H, N-CH2-CH-(CH3)2).
HPLC: 75% CH3CN-H2O, Rt 6.5 min (100%); 82% CH3OH-
H2O, Rt 6.0 min (100%). TLC: 10:1 EtOAc-Hex, Rf 0.47; 20%
MeOH-CHCl3, Rf 0.78. HREIMS: m/z 450.0779 (calcd for
D
1H, Ar 3-H), 7.53 (d, J ) 8.5 Hz, 1H, Ar 6-H), 7.45 (dd, J )
2.2, 8.5 Hz, 1H, Ar 5-H), 7.36 (d, J ) 8.5 Hz, 2H, Ar′ 3,5-H),
7.19 (d, J ) 8.5 Hz, 2H, Ar′ 2,6-H), 4.16 (m, 1H, N-CH-
(CH3)-CH2-OH), 3.58 (d, J ) 5.3 Hz, 2H, N-CH-(CH3)-
CH2-OH), 2.30 (s, 3H, CH3), 1.23 (d, J ) 6.8 Hz, 3H, N-CH-
(CH3)-CH2-OH). HPLC: 75% CH3CN-H2O, Rt 3.8 min
(100%); 80% CH3OH-H2O, Rt 7.6 min (100%). TLC: 10:1
EtOAc-Hex, Rf 0.53; 20% MeOH-CHCl3, Rf 0.72. HREIMS:
m/z 437.0458 (calcd for C20H1835Cl3N3O2, 437.0465).
5-(4-Ch lor op h en yl)-1-(2,4-d ich lor op h en yl)-4-m et h yl-
1H-p yr a zole-3-ca r boh yd r a zid e (22). Compound 22 was
obtained from 6 and the di-HCl salt of hydrazine according to
the procedure described for 16 and was isolated as a white
1
semisolid (97 mg, 51% yield). H NMR: δ 7.51 (d, J ) 2.2 Hz,
1H, Ar 3-H), 7.50 (d, J ) 8.5 Hz, 1H, Ar 6-H), 7.41 (dd, J )
2.2, 8.5 Hz, 1H, Ar 5-H), 7.33 (d, J ) 8.5 Hz, 2H, Ar′ 3,5-H),
7.16 (d, J ) 8.5 Hz, 2H, Ar′ 2,6-H), 2.28 (s, 3H, CH3). HPLC:
60% CH3CN-H2O, Rt 6.0 min (98%); 80% CH3OH-H2O,
Rt 6.5 min (96%). TLC: 30:1 EtOAc-Hex, Rf 0.49; 20%
MeOH-CHCl3, Rf 0.70. HREIMS: m/z 394.0150 (calcd for
C
21H2135Cl3N4O, 450.0781).
Solven t-Dep en d en t NMR
C
17H1335Cl3N4O, 394.0155).
N-Meth yl-5-(4-ch lor op h en yl)-1-(2,4-d ich lor op h en yl)-4-
solvent
CDCl3
isomer (ratio)a
CH2 (ppm)
(CH3)2 (ppm)
m eth yl-1H-p yr a zole-3-ca r boh yd r a zid e (23). Compound 23
was obtained from 6 and methylhydrazine according to the
procedure described for 7 and was isolated as a white solid
major (3.6)
minor (1.0)
major (2.4)
minor (1.0)
major (2.0)
minor (1.0)
major (1.9)
minor (1.0)
3.69
3.54
3.93
3.79
3.49
3.40
3.62
3.54
0.85
0.99
0.90
1.00
0.75
0.89
0.83
0.98
1
pyridine-d5
DMSO-d6
MeOH-d4
(184 mg, 90% yield); mp 163.9-166.1 °C. H NMR: δ 7.57 (br
s, 1H, Ar 3-H), 7.45 (br d, J ) 8.5 Hz, 1H, Ar 6-H), 7.40 (dd,
J ) 2.1, 8.5 Hz, 1H, Ar 5-H), 7.35 (br d, J ) 8.2 Hz, 2H, Ar′
3,5-H), 7.19 (d, J ) 8.2 Hz, 2H, Ar′ 2,6-H), 3.39 (s, 3H, N-CH3),
2.14 (s, 3H, CH3). HPLC: 60% CH3CN-H2O, Rt 5.6 min (99%);
80% CH3OH-H2O, Rt 5.5 min (100%). TLC: 30:1 EtOAc-Hex,
Rf 0.33; 20% MeOH-CHCl3, Rf 0.71. HREIMS: m/z 408.0307
(calcd for C18H1535Cl3N4O, 408.0311).
a
Ratios were taken of the geminal dimethyl protons, calculated
from their integration areas.
N-E t h yl-5-(4-ch lor op h en yl)-1-(2,4-d ich lor op h en yl)-4-
m eth yl-1H-p yr a zole-3-ca r boh yd r a zid e (24). Compound 24
was obtained from 6 and the di-HCl salt of ethylhydrazine
according to the procedure described for 16 and was isolated
as a white semisolid (115 mg, 56% yield). 1H NMR: δ 7.57 (br
s, 1H, Ar 3-H), 7.41 (br s, 2H, Ar 6-H, Ar 5-H), 7.35 (br d, J )
8.4 Hz, 2H, Ar′ 3,5-H), 7.19 (d, J ) 8.4 Hz, 2H, Ar′ 2,6-H),
3.74 (q, J ) 6.8 Hz, 2H, N-CH2-CH3), 2.15 (s, 3H, CH3), 1.26
(t, J ) 6.8 Hz, 3H, N-CH2-CH3). HPLC: 60% CH3CN-H2O,
Rt 7.8 min (98%); 80% CH3OH-H2O, Rt 7.2 min (100%).
TLC: 30:1 EtOAc-Hex, Rf 0.46; 10% MeOH-CHCl3, Rf 0.44.
HREIMS: m/z 422.0473 (calcd for C19H1735Cl3N4O, 422.0468).
N-(1-P r opyl)-5-(4-ch lor oph en yl)-1-(2,4-dich lor oph en yl)-
4-m eth yl-1H-p yr a zole-3-ca r boh yd r a zid e (25). Compound
25 was obtained from 6 and the di-HCl salt of 1-propylhydra-
zine according to the procedure described for 16 and was
isolated as a white foam (137 mg, 62% yield); mp 126.7-128.5
°C. 1H NMR: δ 7.61 (br s, 1H, Ar 3-H), 7.43 (br s, 2H, Ar 6-H,
Ar 5-H), 7.37 (br d, J ) 8.4 Hz, 2H, Ar′ 3,5-H), 7.20 (d, J ) 8.4
Hz, 2H, Ar′ 2,6-H), 3.68 (t, J ) 7.1 Hz, 2H, N-CH2-CH2-
CH3), 2.14 (s, 3H, CH3), 1.76 (m, 2H, N-CH2-CH2-CH3), 0.99
(t, J ) 7.3 Hz, 1H, N-CH2-CH2-CH3′), 0.83 (t, J ) 7.4 Hz,
2H, N-CH2-CH2-CH3). HPLC: 75% CH3CN-H2O, Rt 5.5
min (100%); 82% CH3OH-H2O, Rt 5.1 min (100%). TLC: 10:1
EtOAc-Hex, Rf 0.42; 10% MeOH-CHCl3, Rf 0.41. HREIMS:
m/z 436.0625 (calcd for C20H1935Cl3N4O, 436.0624).
N-(1-Bu tyl)-5-(4-ch lor op h en yl)-1-(2,4-d ich lor op h en yl)-
4-m eth yl-1H-p yr a zole-3-ca r boh yd r a zid e (26). Compound
26 was obtained from 6 and the di-HCl salt of 1-butylhydrazine
according to the procedure described for 16 and was isolated
as a white semisolid (136 mg, 62% yield). 1H NMR: δ 7.58 (br
s, 1H, Ar 3-H), 7.41 (br s, 2H, Ar 6-H, Ar 5-H), 7.36 (br d, J )
8.4 Hz, 2H, Ar′ 3,5-H), 7.19 (d, J ) 8.4 Hz, 2H, Ar′ 2,6-H),
3.72 (m, 1.33H, N-CH2-CH2-CH2-CH3), 3.35 (m, 0.67H,
N-CH2′-CH2-CH2-CH3), 2.29 (s, 1H, CH3′), 2.14 (s, 2H,
CH3), 1.71 (m, 1.33H, N-CH2-CH2-CH2-CH3), 1.57 (m,
0.67H, N-CH2-CH2′-CH2-CH3), 1.40 (m, 0.67H, N-CH2-
CH2-CH2′-CH3), 1.27 (m, 1.33H, N-CH2-CH2-CH2-CH3),
0.95 (t, J ) 7.3 Hz, 2H, N-CH2-CH2-CH2-CH3), 0.84 (t, J
) 7.4 Hz, 1H, N-CH2-CH2-CH2-CH3′). HPLC: 75% CH3CN-
Recep tor Bin d in g Assa ys. 1. Ra t Mem br a n e Assa y. For
the competition assays utilizing rat brain membrane prepara-
tions, male CD rats (Charles River Laboratories, Raleigh, NC)
weighing 220-225 g were sacrificed. The whole brains were
quickly removed and placed into a 55 mL Potter-Elvehjem
glass homogenizer tube maintained on ice. Alternatively, the
brains were placed on dry ice and subsequently frozen at -70
°C until processing. The tissue was subjected to a homogeniza-
tion and centrifugation procedure described previously6 to yield
the final membrane preparation used in the binding assay.
Total protein concentration of the resuspended membrane
pellet was determined by a dye-binding assay commercially
available from Biorad Laboratories (Hercules, CA). Aliquots
of the membrane preparation were stored at -70 °C until use.
Compounds 7-27 were evaluated for their ability to compete
with the binding of [3H]2 or [3H]5. Competing compounds were
prepared in buffer consisting of 50 mM Tris-HCl, pH 7.4, 1
mM EDTA, 3 mM MgCl2, and 0.5% (w/v) bovine serum
albumin (BSA) (buffer A). Tritiated compounds were diluted
in buffer A to yield concentrations of 7.2 nM for [3H]2 and 20
nM for [3H]5 so that addition to the incubation mixture yielded
a
final concentration in the assay of 0.72 and 2.0 nM,
respectively. Unlabeled drug for determination of nonspecific
binding (unlabeled 2 in assays using [3H]2 and unlabeled 5 in
assays using [3H]5) was at a final concentration of 10 µM.
The competition assays were conducted in a total volume
of 0.5 mL in 1.2 mL polypropylene test tubes. The reaction
mixtures (in duplicate) consisted of 50 µL of tritiated drug, 50
µL of unlabeled drug dilution, and sufficient buffer A such that
a total volume of 0.5 mL was achieved with the addition of
brain membrane extract. Duplicate tubes for nonspecific
binding and total binding were prepared by adding 50 µL
aliquots of the unlabeled compound to be displaced and of
buffer A, respectively. An aliquot of brain membrane extract
equivalent to 20-25 µg of protein was added to each tube. The
final volume of the reaction mixture was brought to a total of
0.5 mL by the addition of buffer A. After they were mixed by
vortex, the reaction tubes were incubated at 30 °C for 1 h.
A 96 manifold Brandel (Gaithersburg, MD) cell harvester
was prepared by priming approximately 1 L of cold 50 mM

2718 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13
Francisco et al.
Tris-HCl, pH 7.4, containing 0.1% (w/v) BSA buffer (buffer B)
through the harvester. A 96 well shallow well filter plate (GF/
C) pretreated for approximately 1 h in buffer B was placed
into the cell harvester. After the incubation period was
complete, the reaction was terminated by vacuum filtration
of the reaction mixture. The reaction tubes were then rinsed
with approximately 4 mL of buffer B. After the tubes were
rinsed, the filter plate was removed and allowed to dry
thoroughly. Approximately 50 µL of MicroScint 20 liquid
scintillation cocktail (Packard; Meriden, CT) was added to each
plate well with an automated cocktail dispenser. The plates
were sealed and allowed to sit overnight and then counted in
a liquid scintillation counter for a statistically appropriate
amount of time.
The amount of radiolabel specifically bound in the absence
of competing compounds was calculated by subtracting non-
specific binding from total binding. The percentage of this
specific binding was then calculated for the amount of radio-
label bound in the presence of various concentrations of each
competing compound.
switched off and an antagonist or its vehicle added. Thirty
minutes later, the first addition of R-(+)-3 was made. Additions
of R-(+)-3 were made cumulatively at 5 min intervals without
washout, the tissues being stimulated for the final 2 min of
exposure to each concentration of this agonist. Compounds 11-
15 were dissolved in DMSO and R-(+)-3 in a solution consist-
ing of 50% DMSO and 50% saline. By themselves, these
vehicles did not inhibit the twitch response. Drug additions
were made in a volume of 10 µL. Values have been expressed
as means and variability as 95% confidence limits. The degree
of inhibition of evoked contractions induced by R-(+)-3 was
calculated in percentage terms by comparing the amplitude
of the twitch response after each addition of R-(+)-3 with its
amplitude immediately before the first addition of this agonist.
KB values for antagonism of R-(+)-3 were calculated by
substituting a single concentration ratio value into the equa-
tion (x - 1) ) B/KB, where x (the concentration ratio) is the
concentration of R-(+)-3 that produced a particular degree of
inhibition in the presence of antagonist at a concentration, B,
divided by the concentration of R-(+)-3 that produced an
identical degree of inhibition in the absence of antagonist.36
Values of the concentration ratio and its 95% confidence limits
were determined by symmetrical (2 + 2) dose parallel line
assays.37 This method was also used to establish whether two
point log concentration-response plots deviated significantly
from parallelism.
The data were then analyzed using GraphPad Prism
(GraphPad Software, Inc.; San Diego, CA), which fit the
displacement data to a one-binding site model using a good-
ness-of-fit quantification based on sum of squares and calcu-
lated the Ki for the competing compound. The Ki values are
presented as means ( SEM (n ) 2) in Table 1.
2. CB2 Assa y. The test compounds 7-27 were further
evaluated for their ability to bind to the CB2 cannabinoid
receptor (human, CHO-K1). These assays were conducted as
described for rat membrane binding assays above using [3H]2
as radioligand. The protein sources were membranes from
CHO-K1 cells transfected with the human recombinant CB2
cannabinoid receptor (Sigma Chemical Co.; St. Louis, MO) at
a final assay concentration of 80 pM. The data were analyzed
as described above.
3. GTP -γ-[35S] Bin d in g Assa y. GTP-γ-[35S] assays of test
compounds 7-27 were conducted according to a variation of
Sim et al.33 The reaction mixture consisted of test compound
(105 nM-400 µM), GDP (200 µM), GTP-γ-[35S] (100 pM), and
rat membrane preparation (45 µg/mL) in a total volume of 0.5
mL of buffer A above. Nonspecific binding was determined in
the presence of 100 µM unlabeled GTP-γ-S, and basal binding
was determined in the absence of drug. Duplicate samples
were incubated for 1 h at 30 °C, and the bound complex was
filtered from the reaction mixture as described previously and
counted in a liquid scintillation counter. Specific binding was
calculated by subtracting nonspecific binding from total bind-
ing and dividing by the total basal binding minus nonspecific
binding. Data were analyzed as described above.
In Vitr o P h a r m a cology. Mou se Va s Defer en s Assa y.
Vasa deferentia were obtained from albino MF1 mice weighing
26-44 g. Each tissue was mounted in a 4 mL organ bath at
an initial tension of 0.5 g. The baths contained Mg2+-free Krebs
solution, which was kept at 35 °C and bubbled with 95% O2
and 5% CO2. The composition of the Krebs solution was (mM)
as follows: NaCl 118.2, KCl 4.75, KH2PO4 1.19, NaHCO3 25.0,
glucose 11.0, and CaCl2‚6H2O 2.54. Isometric contractions were
evoked by stimulation with 0.5 s trains of three pulses of 110%
maximal voltage (train frequency 0.1 Hz; pulse duration 0.5
ms) through a platinum electrode attached to the upper end
and a stainless steel electrode attached to the lower end of
each bath. Stimuli were generated by a Grass S48 stimulator
and then amplified (Med-Lab channel attenuator) and divided
to yield separate outputs to eight organ baths (Med-Lab
StimuSplitter). Contractions were monitored by computer
using a data recording and analysis system (MacLab) that was
linked via preamplifiers (Macbridge) to UF1 transducers. After
placement in an organ bath, each tissue was subjected to a
stimulation-free period of 15 min and then stimulated for 10
min. Tissues were then subjected to alternate periods of
stimulation (5 min) and rest (10 min) until consistent twitch
amplitudes were obtained. This equilibration procedure was
followed by a stimulation-free period of 10 min. Tissues were
then stimulated for 10 min after which the stimulator was
Ack n ow led gm en t. The authors acknowledge and
appreciate the editorial assistance of Ms. Kathleen G.
Ancheta and the NMR expertise of Mr. Gregory S.
Bailey and Dr. J ason P. Burgess. The authors also thank
Dr. Michael D. Walla, University of South Carolina, for
the high-resolution mass spectra and Dr. Clifford George,
Laboratory for the Structure of Matter, Naval Research
Laboratory, Washington, DC, for the X-ray crystallo-
graphic data. This work was supported by the National
Institute on Drug Abuse (NIDA) Grant DA-11638
(B.F.T.) and NIDA Grant DA-09789 (R.G.P.).
Su p p or tin g In for m a tion Ava ila ble: Full spectrum fig-
ures of temperature-dependent NMR of 27. This material is
available free of charge via the Internet at http://pubs.acs.org.
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