Synthesis, biological evaluation, and structural studies on N1 and C5 substituted cycloalkyl analogues of the pyrazole class of CB1 and CB2 ligands

Article abstract of DOI:10.1016/j.bmc.2003.10.045

A series of N1 and C5 substituted cycloalkyl and C5 4-methylphenyl analogues of the N-(piperidin-1-yl)-4-methyl-1H-pyrazole-3-carboxamide class of cannabinoid ligands were synthesized. The analogues were evaluated for CB1 and CB2 receptor binding affinities and receptor subtype selectivity. The effects of pyrazole substitution on ligand conformation and as such receptor affinities was not readily apparent; therefore, the geometries of the N1 and C5 substituents relative to the pyrazole ring were studied using high field NMR spectroscopy and systematic molecular mechanics geometry searches. An analysis of the relative ring geometries and functional group orientations provides new insight into the structural requirements of the CB1 and CB2 ligand binding pocket.

Full text of DOI:10.1016/j.bmc.2003.10.045

Bioorganic & Medicinal Chemistry 12 (2004) 393–404
Synthesis, biological evaluation, and structural studies on N1 and
C5 substituted cycloalkyl analogues of the pyrazole class of CB1
and CB2 ligands
Mathangi Krishnamurthy, Wei Li and Bob M. Moore, II*
Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee-Memphis, Memphis, TN 38103, USA
Received 9 September 2003; revised 21 October 2003; accepted 25 October 2003
Abstract—A series of N1 and C5 substituted cycloalkyl and C5 4-methylphenyl analogues of the N-(piperidin-1-yl)-4-methyl-1H-
pyrazole-3-carboxamide class of cannabinoid ligands were synthesized. The analogues were evaluated for CB1 and CB2 receptor
binding affinities and receptor subtype selectivity. The effects of pyrazole substitution on ligand conformation and as such receptor
affinities was not readily apparent; therefore, the geometries of the N1 and C5 substituents relative to the pyrazole ring were studied
using high field NMR spectroscopy and systematic molecular mechanics geometry searches. An analysis of the relative ring geo-
metries and functional group orientations provides new insight into the structural requirements of the CB1 and CB2 ligand binding
pocket.
# 2003 Elsevier Ltd. All rights reserved.
there are no clinically relevant drugs.7ꢀ9 Similarly, dis-
covery of leptin10 and the leptin receptor11 opened the
1.Introduction
Obesity is rapidly becoming one of the major health
threats facing industrialized nations. In the United
States it is estimated that over one third of the popul-
ation is overweight with a statewide average of 20per-
cent being obese.1,2 This condition is a complicating
factor in diabetes and cardiovascular disease as well as
causing an increased risk of certain types of cancer.3,4
Overall, obesity ranks second to smoking in preventable
deaths contributing to an estimated 300,000 deaths
annually.5 Diet and exercise is the first line therapy for
this disease but this often fails due to non-compliance or
poor education. Ultimately, many individuals afflicted
with obesity seek chemotherapeutic intervention as a
means of weight reduction. Stimulant drugs such as
amphetamine, fenfluramine, and ephedrine have been
used to treat obesity, however adverse effects and
addiction can severely reduce the efficacy of these treat-
ments.6 Agonists of the b3-adrenergic receptor have
been shown to regulate fat cell thermogenesis and are
under investigation for obesity therapy, however to date
possibility of a new therapeutic target for the treatment
of obesity but molecules designed to target this pathway
have yet to emerge. One aspect of the leptin pathway
that provides another target for treating obesity is the
receptor mediated regulation of the release of endo-
cannabinoids thus regulating food intake.12 This com-
bined with the demonstrated appetite stimulation
associated with other cannabinoid receptor 1 (CB1)
agonists,13,14 e.g., Á9-THC; 1, indicated that antagonists
of the receptor provided a novel target for treating obe-
sity. In fact, this hypothesis is being tested by Sanofi-
Synthelabo in Phase III clinical trails utilizing the diaryl
pyrazole CB1 antagonist SR141716 (2; rimonabant).15
The apparent success of SR141716 suggests that the
development of new selective CB1 antagonists holds
promise for the identification of novel anti-obesity drugs.
Keywords: Cannabinoid; Pyrazole; Cycloalkyl; NMR; Molecular
modelling.
* Corresponding author at current address: Room 327D, 847 Monroe
Ave. Memphis, TN 38103, USA. Tel.: +1-901-448-6085; fax: +1-
901-448-6828; e-mail: bmoore@utmem.edu
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.10.045

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M. Krishnamurthy et al. / Bioorg. Med. Chem. 12 (2004) 393–404
The CB1 antagonist SR14171616 is one of a large num-
ber of analogues that have been developed around the
pyrazole scaffold.17,16,18ꢀ21 Efforts to develop the SAR of
this class of CB1 ligands have focused on the substitution
and orientation of the N1 and C5 aryl functionalities and
the substituent at the 3-carboxamide position. Structure
activity studies on 3-carboxamide derivatives indicate
that one or more hydrogen-bond donor-acceptor pairs
between the ligand and the ligand binding pocket (LBP)
exist;22,23 furthermore, functional groups that occupy
the aminopiperidine region of the LBP have an opti-
mum length of 3 A.21 Synthetic analogues developed to
probe the C5 and N1 positions have generally retained
the aminopiperidine of SR141716 and one aromatic ring
at either the C5 or N1 positions. The overall trend
observed for CB1 activity shows that a 4-substituted
phenyl ring at the C5 position is associated with high
affinity receptor binding. In the N1 position 2,4-
dichlorophenyl is the optimum substituent for both high
CB1 affinity and receptor subtype selectivity.24,20 The
inclusion of an N1 4-butylphenyl, 4-pentylphenyl or
phenyl group significantly reduces affinity (Kis=150–433
nM)18 although the introduction of n-pentyl, n-hexyl,
and n-heptyl retains affinity (Kis=23–47 nM)23
comparable to the N1 4-chlorophenyl analogue. Unfor-
tunately, it is not possible to make a comparison relative
to the CB1 versus CB2 selectivity since CB2 receptor
binding affinity is not reported for many of the analogues.
hydrazines.20 The intermediate cycloalkyl hydrazines
were prepared by reacting ketones 3-7 with t-butyl car-
bazate followed by the reduction of the N0-cycloalkyl-
idene-hydrazinecarboxylic acid ethyl ester with diborane
in 6N HCl yielding compounds 8–12 as the hydrochlo-
ride salts (Scheme 1).27 The 5-aryl-3-methyl-2,4-dioxo-
pentanoic acid ethyl esters were prepared as previously
described by reacting the 4-chloro-1-phenyl-propan-1-
one 13 with diethyl oxalate in the presence of lithium
bis(trimethylsilyl)amide to provide 14 as the lithium salt
(Scheme 2).28 Compound 14 was then allowed to react
with the hydrazine salts of 8–12 yielding the substituted
4-methyl-1H-pyrazole-3-carboxylic acid ethyl esters 15–
19 (Scheme 2).29 The resulting esters were hydrolyzed,
converted to their acid chlorides utilizing SOCl2 then
reacted with 1-amino-piperidine in the presence of trie-
thylamine to yield the corresponding carboxamides 20–
24 of which 23 was isolated as a diastereomeric mixture.20
In a similar fashion carboxamide 27 was synthesized from
4-methyl-1-phenyl-propan-1-one and 2,4-dichlorophenyl
hydrazine (Scheme 3). The requisite 4-cycloalkyl-3-
methyl-2,4-dioxo-butyric acid ethyl esters for the C5
cycloalkyl pyrazoles were prepared from either aldehyde
28 via reaction with ethyl magnesium bromide, oxidation
of the resulting alcohol with PCC30 or by the reaction of
the Grignard of 29 with propionaldehyde and then oxi-
dation with PCC (Scheme 4). The ketones 30 and 31 were
converted to 32 and 33 as previously described then
allowed to react with 4-chloro-phenyl hydrazine each
yielding the C5 and N1 substituted cycloalkyl pyrazoles
which were resolved via flash chromatography. Utilizing
the aforementioned chemistry compounds 34 and 35
were converted to the aminopiperidine carboxamides 36
and 37. The dicyclohexyl pyrazole analogue 39 was
prepared from 32 and 9 (Scheme 5).
It is interesting to note that linear chain alkyl sub-
stituents in the N1 position cause only a modest reduc-
tion in binding.23 This prompted us to question the
effects of substituting cycloalkyl groups in either or both
the C5 and N1 positions based on the following: 1) we
have previously demonstrated that there is a pocket in
both the CB1 and CB2 receptors capable of accom-
modating a cycloalkyl substituent at the C3 position of
classical cannabinoids (CCBs);25 2) Shim et al. has sug-
gested that the N1 substituent on the diaryl pyrazoles
may occupy the same receptor subsite as the C3 side chain
of CCBs;23 and 3) it has been proposed that a cyclohexyl
ring can serve as an isosteric replacement for a phenyl
ring.26 Based on these points a series of N1 and C5
substituted cycloalkyl 4-chlorophenylpryazoles and a
dicycloalkylpyrazole were designed and synthesized.
Additionally, the C5 4-methylphenyl N1 2,4-dichloro-
phenyl was synthesized to study if a halogen is required for
the high affinity and selectivity reported for SR141716
and AM-251.20 In this study we report the binding
affinities of these compounds with the CB1 and CB2
receptors as well as 1D and 2D NMR studies directed at
determining the solution conformation of these compounds
relative to the diarylpryazoles analogues.
2.2. Receptor binding assays
Cell membranes from HEK293 cells transfected with the
human CB1 receptor and membranes from CHO-K1
cells transfected with the human CB2 receptor were
used in the receptor binding assays.25 Binding affinities
of compounds 20–24, 27, 36, 37, and 39 were deter-
mined by assaying the displacement of [3H]-CP 55,940
from the CB1 and CB2 receptor preparations by
increasing concentrations of the pyrazole analogues
(Table 1). The receptor affinities of our analogues dis-
played unique affinities for the CB1 and CB2 receptors;
2.Results and discussion
2.1. Chemistry
The synthesis of the cycloalkyl substituted pyrazoles
was carried out via the condensation of the appro-
priately 5 position substituted 3-methyl-2,4-dioxo-pen-
tanoic acid ethyl ester with the required alkyl or aryl
Scheme 1.

M. Krishnamurthy et al. / Bioorg. Med. Chem. 12 (2004) 393–404
395
Scheme 2.
Table 1. Binding affinities of the pyrazole analogues 20–24, 27, 36,
37, and 39 for the CB1 and CB2 receptors
Compd
CB1 Ki (nM)a
CB2 Ki (nM)
Ratio CB1/CB2
20
21
22
23
24
27
36
37
39
1560( Æ77)
351 (Æ1.5)
275 (Æ67)
494 (Æ57)
264 (Æ26)
39 (Æ2.0)
1020 (Æ22)
3210( Æ45)
2197 (Æ21)
281 (Æ11)
479 (Æ50)
2490 (Æ102)
133 (Æ30)
410( Æ10)
1 : 0.65
1 : 9.1
1 : 8.0
1 : 0.56
1 : 1.8
1 : 64
1 : 0.42
1 : 1.5
1 : 49
318 (Æ8.5)
273 (Æ19)
5110( Æ110)
>2.5Â105
a The Ki values for the analogues were obtained from nꢁ3 indepen-
dent experiments run in triplicate showing the standard error of the
mean in parentheses.
410nM affinities that were relatively non-selective for
the receptors subtypes. With respect to the equivalent
C5 substituted analogues 21 and 22, the CB1 affinities
are essentially the same; however, analogue 36 has a 24-
fold higher affinity for the CB2 receptor relative to 21
while the affinity of 37 for the same receptor shows a 5-
fold increase. These data suggest that CB2 selective
pyrazole analogues can be developed utilizing 36 as the
lead structure. Substitution of the pyrazole scaffold with
two cyclohexyl groups, compound 39, drastically
reduced the CB1 affinity and totally abolished binding
to the CB2 receptor. In combination these data sug-
gested that interesting information regarding the
ligand–receptor interactions could be derived; therefore,
these compounds were studies by high field NMR spec-
troscopy and molecular modeling studies.
Scheme 3.
however, only compound 27 had an affinity and selec-
tivity (CB1 Ki=39 nM, CB1–CB2=1:64) comparable to
the benchmark diarylpryazole SR141716 (CB1 Ki
range=1.98–12.3 nM, CB1–CB2 ratio range=1:57–
1:505)31ꢀ33,16,20 and AM-251 (CB1 Ki=7.49 nM, CB1–
CB2=1:306).20 Within the C5 substituted cycloalkyl
series, the cyclohexyl, 21, and cycloheptyl, 22,
derivatives had a modest 9.1 and 8.0CB1–CB2 ratio,
respectively, with CB1 affinities of 351 and 275 nM.
Interestingly, substitution of the C5 cyclohexyl group
for 3-methyl- and 4-methyl cyclohexyl had little effect
on the CB1 affinity but did result in an 11.4- and 4.6-
fold increases in CB2 affinity for the 3-methyl, 23, and
4-methyl, 24, compounds, respectively. Despite the
modest affinities of the C5 substituted analogues for
both receptor subtypes it is interesting to note the that
the C5 phenyl N1 2,4-dichlorophenyl pyrazole (CB1
Ki=123 nM, CB1–CB2 ratio=1:1.8) reported by Lan et
al. had modestly higher affinities, i.e., 1.3–4-fold,20 com-
pared to those observed for the methylcycloalkyl anao-
logues. This suggests that under certain conditions the
cyclohexyl group can be substituted for a phenyl ring.
2.3. NMR and molecular modeling studies
The affinity spectrum observed for our compounds sug-
gested that conformational studies on the series could
provide insight into the structural elements underlying
the activity. It was predicted that the increased steric
bulk of the cycloalkyl group in either the N1 or C5
position would affect the orientation of the remaining
aromatic ring relative to the pyrazole ring. Due to the
fact that the substitution of the 4-methylphenyl of 27 for
the 4-iodophenyl of AM-251 for cycloalkyl groups has
negligible effects on the calculated logP, it was proposed
that the spatial orientation of the N1 and C5 sub-
Substitution of the N1 aryl group for cyclohexyl, 36,
and cycloheptyl, 37, resulted in compounds with 133–

396
M. Krishnamurthy et al. / Bioorg. Med. Chem. 12 (2004) 393–404
Scheme 4.
Scheme 5.
Figure 1. Partial 1D NMR spectrum of 21 showing the axial and
equatorial coupling constants of the cyclohexyl methyne proton.
stituents played a major role in binding affinity. To
examine this effect 1D and 2D high field NMR experi-
ments were conducted on compounds 21, 24, 27, 36, 39,
and AM-251. The chemical shifts of the protons in the
aforementioned compounds were first assigned using
1D, gHSQC, gHMBC, and gCOSY experiments. An
analysis of the methyne proton signals in the 1D spectra
of 21, 24, 36, and 39 showed a clear triplet of triplets
(Fig. 1) indicating that the methyne proton was in the
axial position for all the cycloalkyl derivatives. To
understand the relative spatial orientation of all the
assigned protons 2D NOESY experiments were con-
ducted on the analogues. In all cases a network of
NOEs was observed interconnecting the C4 methyl, the
C5 substituent and the N1 substituent (Fig. 2). This fig-
ure only shows the NOEs that were used to establish the
relative orientation of the pyrazole substituents and not
intra-functional group signals. Interestingly, in com-
pounds 21, 24, and 39 weak signals between the cyclo-
hexyl protons and the NH of the carboxamide were
observed with a clear absence of a NH to C3 methyl
NOE. This suggests that, in chloroform and for the
compounds showing the NOE, the geometry of the car-
boxamide is the Ts and Tg conformation as previously
defined by Shim et al. (Fig. 3).23 In addition, the
degeneracy of the methylene protons in the piperidine
ring, that is, no resolvable axial equatorial coupling,
suggest that the piperidine ring is rapidly inter-convert-
ing between the s and g form in the NMR time scale.
A qualitative analysis of the NOE intensities as well as
the presence or absence of NOEs suggested that intro-
duction of a cycloalkyl at either or both the N1 and C5

M. Krishnamurthy et al. / Bioorg. Med. Chem. 12 (2004) 393–404
397
positions resulted in geometries unique to each class of
the analogues. In order to more accurately define the
relative orientation of the N1 and C5 substitutents with
respect to the pyrazole ring a quantitative analysis of
the NOESY spectra was conducted. This was accom-
plished by integrating of the non-overlapping unam-
biguously assigned peaks utilizing TRIPOS TRIAD
followed by constraint generation utilizing MARDI-
GRAS.34 Due to the degeneracy of the piperidine ring,
no constraints were generated for NOEs between this
ring and the carboxamide NH. The number of con-
straints generated and their location are shown in Fig-
ure 2. Utilizing these constraints the models were
subjected to 10cycles of simulated annealing to deter-
mine the populations of conformers associated with the
NMR data. Compounds 36 and 39 converged on a sin-
gle conformer while compounds 27, 21, 24, and AM-251
had two conformers that could be found on the poten-
tial energy surface derived from a systematic molecular
mechanics search of t1 and t2 (Table 2). The torsional
values for 27 and AM-251 are consistent with the low
energy conformations of unprotonated SR141716
reported by Shim et al.23 Taken together these data
support the NMR derived structures thus identifying
the predominant conformation of the compounds in
chloroform; furthermore, the conformations may reflect
the aqueous structure when considering the limited
steric space available to cyclic substituents appended
from the N1 and C5 positions of the pyrazole ring.
ligand binding pockets of the CB1 and CB2 receptors
could be derived from a structural analysis. A detailed
analysis of the receptor interactions utilizing CoMFA or
a related method was not deemed appropriate due to a
lack of functional data; however, a qualitative analysis
of the molecular surfaces could identify basic elements
that give rise to the observed binding affinities. To this
end, the NMR derived average structures were aligned
by overlaying the pyrazole atoms of each compound
followed by Connolly molecular surface generation uti-
lizing MOLCAD (Fig. 2).34 In general, 27 and AM-251
are identical with the exception of a 21 A3 increase in
volume arising from the iodo group and slight increase
in clogP from 6.19 to 6.28 for the unprotonated forms of
27 and AM-251, respectively. However, the differences
in the receptor affinities and selectively are significant
suggesting that there are electronic effects that cannot
be studied via molecular mechanics based calculations.
This possible role is presently being investigated using
quantum mechanical calculations to assess potential
electronic effects.
The role of hydrophobic interactions between the
receptor and the pyrazole N1 and C5 substitutents has
been studied by several groups.18ꢀ20,23 Compounds
containing hydrophobic groups, such as aryl, alkylaryl,
and N-alkyl, at the N1 and C5 positions and analogues
containing a 1,4-dihydroindeno35 (Fig. 4) group gen-
erally bind to both CB receptor types; however, the
spectrum of affinities as well as CB1 and CB2 selectivity
appear to be dependent on the spatial orientation of the
groups relative to the pyrazole ring. In the context of
The NMR derived structures combined with the recep-
tor affinity data suggested that information on the
Table 2. Low energy pyrazole analogue conformations of t1 and t2 as determined by NMR and systematic conformational searches
Simulated Annealing
Rotamersa
Systematic search
Unique rotomersb
4
Compd
t1, t2
Local minima
8
t1, t2
27 and
AM-251
2
48, 234
310, 305
40, 230
120, 320
230, 230
310, 310
10, 250
50, 300
60, 240
110, 300
21 and 24
2
10, 249
61, 285
6
4
4
36
39
1
1
98, 2904
80, 325
2
, 120
80
90, 290
60, 110
80, 330
250, 280
270, 270
4
a Average minimized structure of rotomer populations.
b Conformations not arising from the 2-fold symmetry of the cyclohexyl 4-methylphenyl, 4-iodophenyl, and 4-chlorophenyl group are defined as
unique.

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M. Krishnamurthy et al. / Bioorg. Med. Chem. 12 (2004) 393–404
Figure 2. The upper panel shows the inter-proton NOEs (curved lines) that were utilized to calculate the inter-proton distance constraints. The lower
panel shows the average structures calculated utilizing simulated annealing studies that are surrounded by the molecular surfaces of the pyrazole,
N1, and C5 substituents. White areas indicate potential volume excluded regions in the CB2 receptor.
Figure 3. Proposed conformations of the N-piperidinyl carboxamide
moiety in compounds 21, 24, and 39. The NOEs between the carbox-
amide NH and the cyclohexyl protons combined with the degenerate
N-a methylene protons suggests that the N-piperidinyl carboxamide is
interconverting between the Ts and Tg forms.
the present study, the orientation of the cycloalkyl
groups and directing effects of the cycloalkyl groups on
the aromatic ring were of particular interest. In the
report by Shim et al.23 the N1 cyclohexyl C5 phenyl
analogue had comparable affinity to our N1 cyclohexyl
C5 4-chlorophenyl analogue 21 for the CB1 receptor
(391 versus 351 nM); however, the affinity of the N1 n-
hexyl and n-heptyl C5 phenyl analogues reported by
Shim et al. are, on average, 10-fold higher for the CB1
receptor. The aforementioned compounds are the car-
bon equivalents of 21 and 22 with one distinct differ-
ence; the conformational space available is drastically
reduced in the cyclic analogues. In combination these
results support the hypothesis that the N1 linear side-
chains adopt a conformation in the receptor that
approximates the geometry of an N1 aryl group. Addi-
tionally, for compounds 27, AM-251, 21, and 22 that
exhibit high to modest receptor selectivity, the structural
data suggest that a t2 in the range of 230degrees is
optimum for selectivity. This result may suggest that the
high CB2 selectivity observed for the 1,4-dihydroindeno
is in part due to locking t2 into a low energy torsional
well between ꢀ10and 10degrees.
Figure 4. Molecular structures of related pyrazole CB1 and CB2
receptor ligands.
It is clear that the angle of t2 is not the sole determinate
of subtype selectivity when considering that SR144528
is a CB2 selective pyrazole analogue.36 Two important
modifications in SR144528 are the replacement of the
aminopiperidine by (1S)-endo-fenchylamine and the
introduction of a 4-methylbenzyl group in the N1 posi-
tion. Although the (1S)-endo-fenchylamine function-
ality likely contributes significantly to CB2 selectivity,
the introduction of the 4-methylbenzyl is of particular
interest in this study since: (1) introduction of the
methyl groups in compounds 23 and 24 increased
the CB2 affinity relative to 21 and; (2) a comparison of
the molecular surfaces in Figure 2 suggest that there is a
receptor excluded volume in the CB2 receptor. This
excluded volume may be, in part, responsible for the

M. Krishnamurthy et al. / Bioorg. Med. Chem. 12 (2004) 393–404
399
selectivity of SR144528 wherein the methylene spacer
effectively projects the 4-methylphenyl past the sterically
restricted region of the CB2 receptor. Interestingly, the
increased steric bulk of the 4-methylphenyl and analo-
gue 23 does correlate with the reported decreased affi-
nity for the CB1 receptor when hydrophobic groups are
extended beyond the N1 2,4-dichlorophenyl moieties in
SR141716 related compounds.23
Routine NMR spectra were obtained on a Bruker
ARX-300MHz NMR and were consistent with the
assigned structure, high resolution 1D and 2D spectra
were obtained on a Varian Inova 500 MHz NMR. All
NMR were recorded in CDCl3 unless otherwise speci-
fied. Routine mass spectra were determined on a Bruker
ESQUIRE Ion Trap LC/MS(n) system. Elemental ana-
lyses were performed by Atlantic Microlabs. Corrected
melting points were determined on an Electrothermal
IA9000 digital melting point apparatus. IR spectra were
measured on a Perkin–Elmer Model 1605 FT infrared
spectrophotometer. Thin layer chromatography was
performed on silica gel plates (Merck TLC plates, silica
gel 60, F254).
The 5- to 24-fold higher CB2 affinities of the C5
cycloalkyl derivatives 34 and 35 relative to the equiva-
lent N1 congeners 21 and 22 was surprising when con-
sidering that the CB1 affinities were essentially
equivalent. The solution structure models suggest that
the steric bulk of the cyclohexyl ring directs both the N1
and C5 substituents into a geometry that is orthogonal
to the pyrazole ring (Fig. 2). This finding is significant in
that, of the compounds that have affinity for either
receptor, only compound 34 is predicted to be restricted
to two doubly orthogonal rotomers of which only one
satisfies the NMR data. It is difficult to assess the sig-
nificance of this result since ligands do not necessarily
bind to receptors in low energy conformations; how-
ever, this geometry does provide a novel template
molecule from which QSAR studies can be based. To
this end, the relative lack of affinity of 39 may further
aid in defining the steric limitations of the CB1 and CB2
receptor ligand binding pocket.
4.1. Cyclopentylidene carbazate (3)
Cyclopentanone (2.65 mL, d=0.951, 30 mmol) and tert-
butyl carbazate (3.96 g, 30mmol) in 33 mL hexane were
heated to reflux for 20min. The reaction mixture was
then cooled to room temperature and the precipitate
filtered and dried to afford the carbazate as a white solid
1
5.28 g (88%). H NMR (300 MHz, CDCl3): d (ppm)
1.51 (s, 9H), 1.84 (m, 4H), 2.19 (t, J=7.2 Hz, 2H), 2.49
(t, J=7.2 Hz, 2H), 7.23 (brs, 1H); MS: (ESI, Pos) m/z
221.1 [(M+23)+].
Utilizing the appropriate cycloalkyl ketones the follow-
ing cycloalkyl carbazates were similarly prepared.
4.1.1. Cyclohexylidene carbazate (4). Yield 5.92 g
(92.2%) as a white solid. 1H NMR (300MHz, CDCl3): d
(ppm) 1.52 (s, 9H), 1.66 (m, 6H), 2.20(t, J=6 Hz, 2H),
2.36 (t, J=6 Hz, 2H), 7.52 (brs, 1H); MS: (ESI, Pos) m/
z 235.1 [(M+23)+].
3.Conclusions
A series of N1 and C5 cycloalkyl substituted pyrazole
aminopiperizine carboxamides as well as the C5 4-
methylphenyl analogue of SR141716 were synthesized
and evaluated for CB1 and CB2 affinity. Within this
series the 4-methylphenyl derivative 25 had an affinity
and subtype selectivity comparable to the benchmark
compound SR141716 while the N1 cyclohexyl and
cycloheptyl analogues had modest selectivity that is
comparable to several diaryl pyrazoles. The qualitative
analysis of the NMR derived structures has identified
structural features that are consistent with and suppor-
tive of previous QSAR studies. Our studies have also
identified additional elements in the pyrazole structures
that may provide new insights into the requirements of
the ligand binding pockets of the CB1 and CB2 recep-
tors. We believe that the results of this study, in
combination with the recently reported 1,4-dihy-
droindeno pyrazole analogues, will enable researchers
to develop and refine the pyrazole QSAR models of the
ligand binding pockets of the CB1 and CB2 receptors.
4.1.2. Cycloheptylidene carbazate (5). Yield 12.37 g
1
(91.1%) as a white solid. H NMR (300 MHz, CDCl3):
d (ppm) 1.50(s, 9H), 1.59 (m, 6H), 1.73 (m, 2H), 2.28 (t,
J=6 Hz, 2H), 2.53 (t, J=6.3 Hz, 2H), 7.38 (brs, 1H);
MS: (ESI, Pos) m/z 249.1 [(M+23)+].
4.1.3. 3-Methyl-cyclohexylidene carbazate (6). Yield
1
6.12 g (90.0%) as a white solid. H NMR (300 MHz,
CDCl3): d (ppm) 1.00 (m, 3H), 1.18 (m, 1H), 1.51 (s,
9H), 1.64 (m, 2H), 1.82 (m, 3H), 2.10(m, 1H), 2.58 (m,
2H), 7.59 (brs, 1H); MS: (ESI, Pos) m/z 249.1
[(M+23)+].
4.1.4. 4-Methyl-cyclohexylidene carbazate (7). Yield
1
6.42 g (94.7%) as a white solid. H NMR (300 MHz,
CDCl3): d (ppm) 0.95 (d, J=6.6 Hz, 3H), 1.20(m, 2H),
1.50(s, 9H), 1.69 (m, 1H), 1.88 (m, 3H), 2.21 (m, 1H),
2.55 (m, 2H), 7.58 (brs, 1H); MS: (ESI, Pos) m/z 249.1
[(M+23)+].
4.Experimental
All chemicals and reagents were purchased from Sigma-
Aldrich or Fisher Scientific Inc. AM-251 was purchased
form Tocris. Anhydrous solvents were prepared by dis-
tillation over sodium metal or calcium hydride just prior
to use. All reactions were carried out under dry condi-
tions and under an argon atmosphere. Silica Gel 60,
200–425 mesh was used for flash chromatography.
4.1.5. Cyclopentyl hydrazine hydrochloride (8). Dibor-
ane (25 mL of 1M solution in THF, 25 mmol) was
added to compound 3 (5.0g, 25.3 mmol) and the reac-
tion mixture was allowed to stir at room temperature
for 15 min after which 6N HCl (13 mL) was added
dropwise. The reaction mixture was then heated over a
steam bath for 15 min and the solvent removed under

400
M. Krishnamurthy et al. / Bioorg. Med. Chem. 12 (2004) 393–404
reduced pressure. The residue was then dissolved in
THF and boric acid was filtered off. The solvent was
then evaporated under reduced pressure and the residue
recrystallized from THF/ether to afford 2.64 g (76.7%)
of compound 6 as white crystalline solid. Mp 129 ꢂC. 1H
NMR (300 MHz, DMSO): d (ppm) 1.70(m, 8H), 3.51
(m, 1H), 5.02 (brs, 4H); MS: (ESI, Pos) m/z 101.4
[(M+1)+].
perature for 18 h. It was then diluted with ether and stir-
red for 30min, then filtered over a pad of silica gel,
washed with ether and the ether evaporated under
reduced pressure to give the crude product. The crude
product was then purified by column chromatography
using EtOAc–hexane (4:96) to yield the ketone as a yel-
1
low oil (8.08 g, 68.3%). H NMR (300 MHz, CDCl3): d
(ppm) 1.03 (t, J=7.2 Hz, 3H), 1.28 (m, 5H) 1.71 (m, 5H),
2.35 (m, 1H), 2.46 (q, J=7.2 Hz, 2H); IR (KBr, neat): u
1712 cmꢀ1 (C¼O); MS: (ESI, Pos) m/z 163.1 [(M+23)+].
Using the appropriate cycloalkyl carbazates the follow-
ing cycloalkyl hydrazine hydrochlorides were similarly
prepared.
4.1.11. 1-cycloheptyl-propanone (31). Cycloheptyl bro-
mide (29, 10mL, d=1.289, 72.8 mmol) was added to
dried magnesium turnings in 90mL of dry THF and the
reaction mixture was heated to reflux for 30min to form
the Grignard reagent. Propionaldehyde (2.08 g, 35.9
mmol) was added to dry THF (56 mL) and the solution
was cooled to ꢀ20 ꢂC. To this solution, the Grignard
reagent was added dropwise and the reaction mixture
was allowed to stir at the same temperature for 6 h. 1N
HCl was then added to the reaction mixture and it was
extracted with ether. The organic layer was separated,
dried over sodium sulfate and then evaporated under
reduced pressure to afford 1 g of the crude alcohol as
colorless oil. The crude alcohol was then oxidized to the
corresponding ketone using PCC as described above.
Yield 0.84 g (85%) as a yellow oil. 1H NMR (300 MHz,
CDCl3): d (ppm) 1.05 (t, J=6 Hz, 3H), 1.45 (m, 8H),
1.77 (m, 4H), 2.45 (q, J=6 Hz, 2H), 2.53 (m, 1H); IR
(KBr, neat): u 1713 cmꢀ1 (C¼O); MS: (ESI, Pos) m/z
177.3 [(M+23)+].
4.1.6. Cyclohexyl hydrazine hydrochloride (9). Yield 1.64
g (46.5%). Mp 112–113 ꢂC. 1H NMR (300 MHz,
DMSO): d (ppm) 1.12 (m, 5H), 1.59 (m, 1H), 1.74 (m,
2H), 1.96 (s, 2H), 2.83 (m, 1H), 5.00 (brs, 4H); MS:
(ESI, Pos) m/z 115.2 [(M+1)+].
4.1.7. Cycloheptyl hydrazine hydrochloride (10). Yield
2.30g (46.7%) as white crystalline solid. Mp 80 ꢂC H
1
NMR (300 MHz, DMSO): d (ppm) 1.53 (m, 10H), 2.02
(m, 2H), 3.04 (m, 1H), 5.42 (brs, 4H); MS: (ESI, Pos)
m/z 129.1 [(M+1)+].
4.1.8. 3-Methyl-cyclohexyl-hydrazine hydrochloride (11).
Diborane (26.6 mL of 1M solution in THF, 26.6 mmol)
was added to compound 6 (6.00 g, 26.6 mmol) and the
reaction mixture was allowed to stir at room tempera-
ture for 15 min after which 6N HCl (14 mL) was added
to it dropwise. The reaction mixture was then heated
over a steam bath for 15 min and the solvent removed
under reduced pressure. The residue was then dissolved
in THF and boric acid was filtered off. The solvent was
then evaporated under reduced pressure and the residue
was taken to the next step without further purification.
4.1.12. 1-(Cyclopentyl)-5-(4-chlorophenyl)-4-methyl-1H-
pyrazole-3-carboxylic acid, ethyl ester (15). 40-chlor-
opropiophenone (13, 1.55 g, 9.23 mmol) was dissolved
in anhydrous THF (24 mL) and cooled to ꢀ78 ꢂC. To
this LiHMDS (9.3 mL of 1M solution in hexane, 9.3
mmol) was added and the reaction mixture was stirred
at the same temperature for 45 min after which it was
warmed to ꢀ20 ꢂC and then cooled back to ꢀ78 ꢂC.
Then, diethyl oxalate (1.46 mL, 10.4 mmol) in dry THF
(3 mL) was added to it and the reaction mixture was
allowed to stir at room temperature for 20h. It was then
diluted with ether and washed with 1N HCl. The
organic layer was then separated, dried over sodium
sulfate and then evaporated under reduced pressure to
give 2.43 of crude ethyl-2,4-dioxo-3-methyl-4- (4-chlor-
ophenyl)-butanoate as yellow oil. The crude product
(2.43 g, 8.99 mmol) was dissolved in ethanol (24.3 mL)
and to this compound 8 (1.27 g, 8.99 mmol) and triethyl
amine (0.91 g, 8.99 mmol) were added. The reaction
mixture was then heated to reflux for 9 h. It was then
cooled, washed with brine and extracted with tert-butyl
ethyl ether. The organic layer was then separated, dried
over sodium sulfate and evaporated. The crude product
was then purified by column chromatography with pet-
roleum ether–ethyl acetate (95:5) to give compound 15
as a yellow solid (697 mg, 23.4%). Rf=0.3 (ethyl ace-
tate–petroleum ether 5:95). 1H NMR (300 MHz,
CDCl3): d (ppm) 1.40(m, 4H), 1.56 (m, 2H), 1.69 (s,
1H), 1.93 (m, 4H), 2.13 (s, 3H), 4.41 (m, 3H), 7.22 (d,
J=8.1 Hz, 2H), 7.48 (d, J=8.4 Hz, 2H); MS: (ESI, Pos)
m/z 333.4 [(M+1)+] and 355.4 [(M+23)+].
4.1.9. 4-Methyl-cyclohexyl-hydrazine hydrochloride (12).
Diborane (26.6 mL of 1M solution in THF, 26.6 mmol)
was added to compound 7 (6.01 g, 26.6 mmol) and the
reaction mixture was allowed to stir at room tempera-
ture for 15 min after which 6N HCl (14 mL) was added
to it dropwise. The reaction mixture was then heated
over a steam bath for 15 min and the solvent removed
under reduced pressure. The residue was then dissolved
in THF and boric acid was filtered off. The solvent was
then evaporated under reduced pressure and the residue
was taken to the next step without further purification.
4.1.10. 1-cyclohexyl-propanone (30). Cyclohexane car-
boxaldehye (28, 12.1 mL, d=0.926, 100 mmol) was dis-
solved in dry THF (250mL) and cooled to ꢀ20 ꢂC. To
this solution ethyl magnesium bromide (40mL of 3M
solution in ether, 120mmol) was added slowly and the
reaction mixture was then allowed to stir at the same
temperature for 5 h. 1N HCl was then added to the reac-
tion mixture and it was extracted with ether. The organic
layer was separated, dried over sodium sulfate and then
evaporated under reduced pressure to afford the 12 g of
crude alcohol as a colorless oil. The crude alcohol was
dissolved in methylene chloride and to this a mixture of
Celite (43 g) and PCC (43 g, 200 mmol) was added and
the reaction mixture was allowed to stir at room tem-

M. Krishnamurthy et al. / Bioorg. Med. Chem. 12 (2004) 393–404
401
Utilizing the appropriate cycloalkyl or substituted
phenyl hydrazine hydrochlorides and the appropriate
diketo esters the following pyrazole esters were similarly
prepared.
4.1.19.
1,5-Dicyclohexyl-4-methyl-1H-pyrazole-3-car-
boxylic acid, ethyl ester (38). Yield 286 mg (10.2%) as a
yellow oil. Rf=0.29 (ethyl acetate–petroleum ether
1
5:95). H NMR (300 MHz, CDCl3): d (ppm) 1.38 (m,
7H), 1.85 (m, 16H), 2.31 (s, 3H), 2.71 (m, 1H), 4.11 (m,
1H), 4.38 (q, J=7.2 Hz, 2H); MS: (ESI, Pos) m/z 319.4
[(M+1)+] and 341.4 [(M+23)+].
4.1.13. 1-(Cyclohexyl)-5-(4-chlorophenyl)-4-methyl-1H-
pyrazole-3-carboxylic acid, ethyl ester (16). Yield 554
mg (24.9%) as a white solid. Rf=0.39 (ethyl acetate–
1
petroleum ether 5:95) H NMR (300MHz, CDCl3): d
4.1.20. 1-(2,4-Dichloro phenyl)-5-(4-methylphenyl)-4-
methyl-1H-pyrazole-3-carboxylic acid, ethyl ester (27).
Yield 521 mg (20.8%) as cream colored solid. Rf=0.33
(ethyl acetate–hexane 6:94) 1H NMR (300 MHz,
CDCl3): d (ppm) 1.32 (t, J=7.2 Hz, 3H), 2.34 (s, 3H),
2.40(s, 3H), 4.29 (q, J=7.2 Hz, 2H), 7.02 (d, J=8.1 Hz,
2H), 7.13 (d, J=8.4 Hz, 2H), 7.28 (m, 2H), 7.44 (m,
1H); MS: (ESI, Pos) m/z 390.4 [(M+1)+] and 412.3
[(M+23)+].
(ppm) 1.22 (m, 3H), 1.41 (t, J=7.2 Hz, 3H), 1.57 (s,
3H), 1.83 (m, 4H), 2.13 (s, 3H), 3.91 (m, 1H), 4.42 (q,
J=7.2 Hz, 2H), 7.19 (d, J=8.4 Hz, 2H), 7.48 (d, J=8.1
Hz, 2H); MS: (ESI, Pos) m/z 347.4 [(M+1)+] and 369.4
[(M+23)+].
4.1.14. 1-(Cycloheptyl)-5-(4-chlorophenyl)-4-methyl-1H-
pyrazole-3-carboxylic acid, ethyl ester (17). Yield 1.91 g
(39.3%) as a white solid. Rf=0.52 (petroleum ether–
ethyl acetate 95:5) 1H NMR (300 MHz, CDCl3): d
(ppm) 1.41 (t, J=7.2 Hz, 3H), 1.58 (m, 6H), 1.82 (m,
4H), 2.12 (s, 3H), 2.21 (m, 2H), 4.09 (m, 1H), 4.42 (q,
J=7.2 Hz, 2H), 7.19 (d, J=8.4 Hz, 2H), 7.48 (d, J=8.7
Hz, 2H); MS: (ESI, Pos) m/z 361.4 [(M+1)+] and 383.3
[(M+23)+].
4.1.21. N-piperidinyl-1-(cyclopentyl)-5-(4-chlorophenyl)-
4-methyl-1H-pyrazole-3-carboxamide (20). Compound
15 (0.697 g, 2.1 mmol) was dissolved in methanol
(14 mL) and to this a solution of KOH (0.24 g,
4.2 mmol) in methanol (5 mL) was added. The reaction
mixture was heated to reflux for 3 h, cooled and poured
into water. It was acidified with 10% HCl and the
precipitate was filtered, washed with water and dried
under vacuum to give the crude acid (0.581 g, 91%).
The crude acid (0.581 g, 1.91 mmol) was dissolved in
toluene (18 mL) and to this solution thionyl chloride
(0.52 mL, d=1.631, 7.05 mmol) was added and the
reaction mixture was heated to reflux for 3 h. It
was then cooled and the solvent evaporated under
reduced pressure. The residue was then dissolved in
toluene and solvent was again evaporated under
reduced pressure to yield the crude acid chloride
(0.391 g, 63.3%).
4.1.15. 1-(3-Methyl-cyclohexyl)-5-(4-chlorophenyl)-4-
methyl-1H-pyrazole-3-carboxylic acid, ethyl ester (18).
Yield 1.04 g (31.3%) as a yellow oil. Rf=0.49 (hexane/
1
ethyl acetate 9:1) H NMR (300 MHz, CDCl3): d (ppm)
0.90 (m, 3H), 1.38 (t, J=6.9 Hz, 3H), 1.67 (m, 1H),
1.80(m, 2H), 2.02 (m, 2H), 2.13 (s, 3H), 2.24 (m, 2H),
2.36 (m, 1H), 3.97 (m, 1H), 4.21 (m, 1H), 4.36 (q,
J=6.9 Hz, 2H), 7.20(d, J=8.1 Hz, 2H), 7.48 (d,
J=8.1 Hz, 2H); MS: (ESI, Pos) m/z 361.4 [(M+1)+]
and 383.4 [(M+23)+].
4.1.16. 1-(4-Methyl-cyclohexyl)-5-(4-chlorophenyl)-4-
methyl-1H-pyrazole-3-carboxylic acid, ethyl ester (19).
The crude acid chloride (0.391 g, 1.21 mmol) was dis-
solved in dry methylene chloride (5.5 mL) and this
solution was added drop wise to a solution of triethyl
amine (0.19 g, 1.87 mmol) and 1-amino piperidine
(0.2 mL, d=0.928, 1.87 mmol) in methylene chloride
(4.3 mL) maintained at 0 ꢂC. The reaction mixture
was stirred at room temperature for 8 h after which
it was added to brine and extracted with methylene
chloride. The organic layer was separated, dried
over sodium sulfate and purified by column chro-
matography with petroleum ether–ethyl acetate (3:1)
to yield a white solid which was then recrystallised
with methylene chloride/hexane to yield the amide as
a white crystalline solid (110mg, 23.6%). Rf=0.34
(ethyl acetate–petroleum ether 1:3), mp: 185 ꢂC. 1H
NMR (500 MHz, CDCl3): d (ppm) 1.52 (m, 2H), 1.64
(m, 2H), 1.82 (p, J=5.5 Hz, 4H), 1.97 (m, 4H), 2.10
(m, 2H), 2.25 (s, 3H), 2.98 (t, J=5 Hz, 4H), 4.49 (p,
J=7 Hz, 1H), 7.26 (d, J=9 Hz, 2H), 7.52 (d, J=8.5 Hz,
2H), 7.74 (s, 1H); MS: m/z 387.4 (M+1). Anal. (calcd
for C21H27ClN4O) C, H, N (Theoretical: C-65.19%,
H-7.03%, N-14.48% Found: C-65.23%, H-6.96%,
N-14.47).
Yield 1.22 g (36.6%) as a white solid. Rf=0.49 (hex-
1
ane–ethyl acetate 92:8) H NMR (300 MHz, CDCl3): d
(ppm) 1.04 (d, J=7.2 Hz, 3H), 1.41 (t, J=6.9 Hz, 3H),
1.62 (m, 6H), 1.88 (m, 1H), 2.12 (s, 3H), 2.20(m, 2H),
3.93 (m, 1H), 4.41 (q, J=7.2 ppm, 2H), 7.20(d, J=8.7
Hz, 2H), 7.47 (d, J=8.7 Hz, 2H); MS: (ESI, Pos) m/z
361.4 [(M+1)+] and 383.4 [(M+23)+].
4.1.17. 1-(4-Chlorophenyl)-5-(cyclohexyl)-4-methyl-1H-
pyrazole-3-carboxylic acid, ethyl ester (34). Yield 525
mg (12.1%) as a yellow oil. Rf=0.45 (ethyl acetate–
petroleum ether 1:3) 1H NMR (300 MHz, CDCl3): d
(ppm) 1.17 (m, 2H), 1.39 (t, J=7.2 Hz, 3H), 1.75 (m,
8H), 2.43 (s, 3H), 2.59 (m, 1H), 4.40(q, J=7.2 Hz, 2H),
7.30(d, J=8.4 Hz, 2H), 7.45 (d, J=8.4 Hz, 2H); MS:
(ESI, Pos) m/z 347.4 [(M+1)+] and 369.4 [(M+23)+].
4.1.18. 1-(4-Chlorophenyl)-5-(cycloheptyl)-4-methyl-1H-
pyrazole-3-carboxylic acid, ethyl ester (35). Yield 302
mg (15.8%) as yellow oil. Rf=0.24 (ethyl acetate–hex-
1
ane 1:9) H NMR (300 MHz, CDCl3): d (ppm) 1.39 (t,
J=6.9 Hz, 3H), 1.61 (m, 7H), 1.79 (m, 5H), 2.38 (s, 3H),
2.75 (m, 1H), 4.39 (q, J=6.9 Hz, 2H), 7.30(d, J=8.7
Hz, 2H), 7.45 (d, J=8.7 Hz, 2H); MS: (ESI, Pos) m/z
361.5[(M+1)+] and 383.4 [(M+23)+].
Utilizing the appropriate pyrazole esters the following
pyrazole carboxamides were similarly prepared.

402
M. Krishnamurthy et al. / Bioorg. Med. Chem. 12 (2004) 393–404
4.1.22. N-piperidinyl-1-(cyclohexyl)-5-(4-chlorophenyl)-
4-methyl-1H-pyrazole-3-carboxamide (21). Yield 151 mg
(32.4%) as a white crystalline solid. Rf=0.42 (ethyl
acetate/petroleum ether 1:3), mp: 150 ꢂC. 1H NMR
(500 MHz, CDCl3): d (ppm) 1.15 (m, 2H), 1.38 (m, 2H),
1.59 (m, 2H), 1.69 (p, J=5.5 Hz, 4H), 1.76 (m, 4H), 1.86
(m, 2H), 2.11 (s, 3H), 2.84 (brs, 4H), 3.79 (tt, J=3.5,
11.5 Hz, 1H), 7.11 (d, J=8.5 Hz, 2H), 7.40(d, J=8.5
Hz, 2H), 7.61 (s, 1H); MS: m/z 423.9 (M+23) Anal.
(calcd for C22H29ClN4O) C, H, N (Theoretical: C-
65.90%, H-7.29%, N-13.97% Found: C-65.78%, H-
7.35%, N-13.55).
4.1.27. N-piperidinyl-1- (4-chlorophenyl)-5-(cyclohexyl)-
4-methyl-1H-pyrazole-3-carboxamide (36). Yield 104 mg
(20%) as a white crystalline solid. Rf=0.45 (ethyl acetate/
petroleum ether 1:3), mp: 165 ꢂC. H NMR (500 MHz,
1
CDCl3): d (ppm) 1.19 (m, 3H), 1.41 (m, 2H), 1.73 (m,
11H), 2.46 (s, 3H), 2.57 (tt, J=4.5, 11.5 Hz, 1H), 2.82
(brs, 4H), 7.28 (d, J=8.4 Hz, 2H), 7.48 (d, J=8.5 Hz,
2H), 7.59 (s, 1H); MS: m/z 401.5 (M+1) Anal. (calcd for
C22H29ClN4O) C, H, N (Theoretical: C-65.9%, H-7.29%,
N-13.97% Found: C-65.63%, H-7.18%, N-13.81).
4.1.28. N-piperidinyl-1-(4-chlorophenyl)-5-(cycloheptyl)-
4-methyl-1H-pyrazole-3-carboxamide (37). Yield 84 mg
(25%) as a white crystalline solid. Rf=0.3 (ethyl ace-
tate/petroleum ether 1:4), mp: 175–176 ꢂC. 1H NMR
(500 MHz, CDCl3): d (ppm) 1.32 (m, 4H), 1.46 (m, 4H),
1.68 (m, 8H), 1.79 (m, 2H), 2.35 (s, 3H), 2.65 (tt, J=3.5,
11 Hz, 1H), 2.76 (brs, 4H), 7.22 (d, J=8.5 Hz, 2H), 7.41
(d, J=8.5 Hz, 2H), 7.52 (s, 1H); MS: m/z 415.4 (M+1)
Anal. (calcd for C23H31ClN4O. 0.1H2O) C, H, N (The-
oretical: C-66.28%, H-7.55%, N-13.44% Found: C-
65.9%, H-7.55%, N-13.19).
4.1.23. N-piperidinyl-1- (cycloheptyl)-5-(4-chlorophenyl)-
4-methyl-1H-pyrazole-3-carboxamide (22). Yield 614 mg
(30.8%) as a white crystalline solid. Rf=0.42 (ethyl
acetate–petroleum ether 15:85), mp: 154–155 ꢂC. 1H
NMR (500 MHz, CDCl3): d (ppm) 1.42 (m, 2H), 1.51
(m, 2H), 1.63 (m, 4H), 1.82 (m, 6H), 1.92 (m, 2H), 2.14
(m, 2H), 2.24 (s, 3H), 2.98 (t, J=4.5 Hz, 4H), 4.14 (h,
J=4.5 Hz, 1H), 7.24 (d, J=8.5 Hz, 2H), 7.52 (d, J=8.5
Hz, 2H), 7.75 (s, 1H); MS: m/z 415.5 (M+1) Anal.
(calcd for C23H31ClN4O) C, H, N (Theoretical: C-66.57%,
H-7.53%, N-13.5% Found: C-66.53%, H-7.47%, N-
13.52).
4.1.29. N-piperidinyl-1,5-dicyclohexyl-4-methyl-1H-pyra-
zole-3-carboxamide (39). Yield 55 mg (25%) as a white
crystalline solid. Rf=0.54 (ethyl acetate–petroleum
ether 1:3), mp: 197–198 ꢂC. 1H NMR (500 MHz,
CDCl3): d (ppm) 1.34 (m, 8H), 1.74 (m, 10H), 1.89 (m,
8H), 2.36 (s, 3H), 2.68 (p, J=7.5 Hz, 1H), 2.87 (brs,
4H), 4.02 (tt, J=3.5, 11 Hz, 1H), 7.63 (s, 1H); MS: m/z
373.5 (M+1) Anal. (calcd for C22H36N4O. 0.5H2O) C,
H, N (Theoretical: C-69.25%, H-9.77%, N-14.68%
Found: C-69.12%, H-9.53%, N-14.58).
4.1.24.
N-piperidinyl-1-
(3-methyl-cyclohexyl)-5-(4-
chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (23).
Yield 473 mg (42.4%) as a white crystalline solid.
Rf=0.4 (ethyl acetate–hexane 1:3), mp: 151 ꢂC. 1H
NMR (500 MHz, CDCl3): d (ppm) 1.28 (m, 4H), 1.50
(m, 2H), 1.77 (m, 7H), 2.00 (m, 2H), 2.12 (s, 3H), 2.85
(t, J=5.4 Hz, 4H), 4.13 (J=4, 10.5 Hz, 1H), 7.12 (d,
J=8.4 Hz, 2H), 7.39 (d, J=8.7 Hz, 2H), 7.61 (s, 1H);
MS:
m/z
415.4
(M+1)
C23H31ClN4O.0.5H2O) C, H,
Anal.
N
(calcd
for
(Theoretical: C-
4.2. NMR studies and molecular modeling
65.16%, H-7.61%, N-13.21% Found: C-64.97%, H-
7.61%, N-13.16).
All spectra were acquired at 23 ꢂC and 500 MHz on a
Varian Inova-500 spectrometer using a 5-mm HCN tri-
ple resonance probe. Both proton and carbon chemical
shifts were referenced to the residual solvent peak of
DMSO (2.49 ppm for proton and 40ppm for carbon).
For two-dimensional NOESY measurements, a total of
1024 fids were recorded for the indirect dimension, with
a 2 second recycle delay, with a 500 ms mixing time. The
TRIAD NMR package within the Sybyl software was
used for data processing and analysis.34 Peaks in the
NOESY spectra were assigned and integrated using
TRIAD standard functions. MARDIGRAS was then
used to generate distance constraints using these peak
integrals. The resulting constraints were then examined
to ensure that the error in distances conformed to
established errors for NOE constraints wherein; x<2.5
A was Æ0.1 A; xꢃ3.0A was Æ0.2 A; xꢃ3.5 A was
Æ0.3 A; and xꢁ3.5 was Æ0.4 A.
4.1.25. N-piperidinyl-1-(4-methyl-cyclohexyl)-5-(4-chlor-
ophenyl)-4-methyl-1H-pyrazole-3-carboxamide
(24).
Yield 645 mg (47.2%) as a white crystalline solid.
Rf=0.3 (ethyl acetate–hexane 1:4), mp: 171 ꢂC.
1H NMR (500 MHz, CDCl3): d (ppm) 1.55 (m, 4H),
1.76 (m, 9H), 2.17 (m, 2H), 2.18 (s, 3H), 2.92 (t,
J=5.4 Hz, 4H), 3.88 (tt, J=3.0, 11.0 Hz, 1H), 7.18
(d, J=8.4 Hz, 2H), 7.46 (d, J=8.4 Hz, 2H), 7.71
(s, 1H); MS: m/z 415.5 (M+1) Anal. (calcd for
C23H31ClN4O) C, H,
N (Theoretical: C-66.57%,
H-7.53%, N-13.5% Found: C-66.4%, H-7.53%, N-
13.30).
4.1.26. N-piperidinyl-1- (2,4-dichlorophenyl)-5-(4-methyl-
phenyl)-4-methyl-1H-pyrazole-3-carboxamide (27). Yield
613 mg (30.8%) as a white crystalline solid. Rf=0.32
(ethyl acetate–hexane 6:94), mp: 154 ꢂC. 1H NMR
(500 MHz, CDCl3): d (ppm) 1.49 (m, 2H), 1.81 (p, J=6
Hz, 4H), 2.39 (s, 3H), 2.42 (s, 3H), 2.92 (brs, 4H), 7.05
(d, J=8 Hz, 2H), 7.17 (d, J=8 Hz, 2H), 7.32 (m, 2H),
7.48 (dd, J=1, 2 Hz, 1H), 7.69 (s, 1H); MS: m/z 415.5
(M+1) Anal. (calcd for C23H31ClN4O) C, H, N (Theo-
retical: C-62.31%, H-5.46%, N-12.64% Found: C-
62.08%, H-5.51%, N-12.48).
Ten cycles of simulated annealing using the constraints
generated by MARDIGRAS was performed on com-
pounds 21, 24, 27, 36, 39, and AM-251 by heating to
1000 K for 1ps followed by exponential cooling to 200 K
then equilibrating for 5 ps. The experimentally obtained
NOE distance constraints were applied during all steps
of the simulated annealing runs. These averaged con-
formations of unique rotomers were then minimized

M. Krishnamurthy et al. / Bioorg. Med. Chem. 12 (2004) 393–404
403
ꢀ1
ꢀ1 with-
.
A
individual molar IC50 values were determined using
GraphPad Prism. The corresponding Ki values for each
drug were determined utilizing the Cheng and Prusoff
equation37 and final data are presented as KiÆS.E.M. of
nꢁ2 experiments.
.
with a gradient tolerance of 0.005 Kcal mol
out experimental NOE distance constraints to obtain
the final average conformations. Systematic searches
were performed around t1 and t2 from 0to 350 using
10degree increments. Additional parameters included
the Tripos force field with MMFF94 charges, an 8 A
nonbonding cutoff, and distance dependent dielectric
constant function.
ꢂ
Acknowledgements
This research was supported by the College of Phar-
macy, University of Tennessee Health Sciences Center,
Memphis, TN.
4.3. Receptor binding assays
Cell membranes from HEK293 cells transfected with the
human CB1 receptor (Lot #1929, Bmax: 1.7 pmol/mg
protein, Kd for [1H]CP 55,940binding: 186 pM) and
membranes from CHO-K1 cells transfected with the
human CB2 receptor (Lot #1930, Bmax: 3.3 pmol/mg
protein, Kd for [3H]CP 55,940binding: 0.12 nM) were
purchased from Perkin–Elmer Life Sciences, Inc.
[3H]CP 55,940having a specific activity of 120Ci/mmol
was obtained from Perkin–Elmer Life Sciences, Inc. All
other chemicals and reagents were obtained from
Sigma-Aldrich. The assays were carried out in 96-well
plates obtained from Millipore, Inc. fitted with glass
fiber filters (hydrophilic, GFC filters) having a pore size
of 1.2 m. The filters were soaked with 0.05% poly-
ethyleneimine solution and washed 5x with deionized
water prior to carrying out the assays. The filtrations
were carried out on a 96-well vacuum manifold (Milli-
pore Inc.), the filters punched out with a pipette tip
directly into scintillation vials at the end of the experi-
ment and vials filled with 5 mL scintillation cocktail
Ecolite (+) (Fisher Scientific). Counting was carried out
on a Beckmann Scintillation Counter model LS6500.
Drug solutions were prepared in DMSO and the radi-
oligand was dissolved in ethanol.
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