Conformationally constrained analogues of N-(piperidinyl)-5-(4- chlorophenyl)-1-(2,4- dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716): Design, synthesis, computational analysis, and biological evaluations

Article abstract of DOI:10.1021/jm8000778

Structure-activity relationships (SARs) of 1 (SR141716) have been extensively documented, however, the conformational properties of this class have received less attention. In an attempt to better understand ligand conformations optimal for receptor recognition, we have designed and synthesized a number of derivatives of 1, including a four-carbon-bridged molecule (11), to constrain rotation of the diaryl rings. Computational analysis of 11 indicates a ～20 kcal/mol energy barrier for rotation of the two aryl rings. NMR studies have determined the energy barrier to be ～18 kcal/mol and suggested atropisomers could exist. Receptor binding and functional studies with these compounds displayed reduced affinity and potency when compared to 1. This indicates that our structural modifications either constrain the ring systems in a suboptimal orientation for receptor interaction or the introduction of steric bulk leads to disfavored steric interactions with the receptor, and/or the relatively modest alterations in the molecular electrostatic potentials results in disfavored Coulombic interactions.

Full text of DOI:10.1021/jm8000778

3526
J. Med. Chem. 2008, 51, 3526–3539
Conformationally Constrained Analogues of N-(Piperidinyl)-5-(4-Chlorophenyl)-1-
(2,4- Dichlorophenyl)-4-Methyl-1H-Pyrazole-3-Carboxamide (SR141716): Design, Synthesis,
Computational Analysis, And Biological Evaluations
Yanan Zhang,* Jason P. Burgess, Marcus Brackeen, Anne Gilliam, S. Wayne Mascarella, Kevin Page, Herbert H. Seltzman, and
Brian F. Thomas*
Chemistry and Life Sciences, Research Triangle Institute, Research Triangle Park, North Carolina 27709
ReceiVed January 25, 2008
Structure-activity relationships (SARs) of 1 (SR141716) have been extensively documented, however, the
conformational properties of this class have received less attention. In an attempt to better understand ligand
conformations optimal for receptor recognition, we have designed and synthesized a number of derivatives
of 1, including a four-carbon-bridged molecule (11), to constrain rotation of the diaryl rings. Computational
analysis of 11 indicates a ∼20 kcal/mol energy barrier for rotation of the two aryl rings. NMR studies have
determined the energy barrier to be ∼18 kcal/mol and suggested atropisomers could exist. Receptor binding
and functional studies with these compounds displayed reduced affinity and potency when compared to 1.
This indicates that our structural modifications either constrain the ring systems in a suboptimal orientation
for receptor interaction or the introduction of steric bulk leads to disfavored steric interactions with the
receptor, and/or the relatively modest alterations in the molecular electrostatic potentials results in disfavored
Coulombic interactions.
Introduction
stituent, the conformational properties of 1 and its analogues
have only recently received attention.24,26 Thus, further studies
are needed to determine with certainty what the optimal ligand
conformations of 1 are for maximal receptor recognition and
inverse agonist activity or what influence the energy manifold
of conformation has on a particular ligand’s affinity and efficacy.
Cannabinoid research has witnessed dramatic progress since
the discovery and cloning of the cannabinoid CB11 and CB2
receptors2 and subsequent identification of the endogenous
ligands.3–5 The cannabinoid CB1 receptor, in particular, has
attracted significant interest due to its high abundance in the
central nervous system (CNS)a and its involvement in a variety
of physiological processes, including feeding, energy homeo-
stasis, analgesia, learning, and memory.6–11 The discovery of 1
(SR141716, or Rimonabant, Figure 1),12 the first drug that
selectively blocks both the in vitro and in vivo effects of
cannabinoids mediated by the CB1 receptor, marked a turning
point in cannabinoid research. Not only has it provided an
important tool for the investigation of the endocannabinoid
system, it offered a completely different structural scaffold than
the previous cannabinoids with which researchers can develop
new cannabinoid ligands. Indeed, several hundred compounds
based on the structure of 1 have been developed.13–16 However,
1 still remains one of the most potent cannabinoid antagonists.
The structure-activity relationships (SARs) of cannabinoid
antagonists have primarily been defined by substitutions made
at the 1, 3, 4, and 5 positions of the pyrazole of 117 as well as
complementary receptor mutation studies.18–22 It is generally
accepted that: (1) the carboxamide oxygen of the 3-position
substituent in 1 is involved in receptor recognition and inverse
agonist activity through the formation of a hydrogen bond,23,24
(2) the 1- and 5-position aryl ring systems interact with the
receptor through aromatic stacking,24 and (3) the methyl group
at the 4-position interacts with a hydrophobic pocket.25 While
these studies have defined putative interactions of each sub-
In this study, we will further examine the relationships between
conformation, conformational mobility, and conformational prefer-
ence for analogues of 1 as they relate to CB1 receptor affinity and
efficacy. In doing so, this work expands upon our previous studies
with rigid analogues of 1, such as with compound 2, in which the
three aryl rings are locked into an almost planar conformation.27
While this analogue showed slightly decreased affinity (∼40 nM)
for the CB1 receptor, it retained excellent selectivity (CB1/CB2
) ∼80). The work described herein also addresses an interesting
paradox observed in the tricyclic pyrazole series previously
described by Pinna et al.28–30 The analogue of 1 with a one carbon
bridge (3) was reported to be a high affinity, selective CB2 ligand.
When the bridge was extended to two carbons in compound 4, a
less selective ligand was obtained (Ki(CB1) ) 15 nM, Ki(CB2) )
230 nM). Finally, they reported that compound 5 (NESS 0327)
with a three-carbon bridge was a CB1 receptor antagonist with
remarkable affinity (Ki(CB1) ) 0.00035 nM, Ki(CB2) ) ∼20
nM).28 Interestingly, this compound had been previously prepared
in a different laboratory and only modest nanomolar affinity was
observed.31 Initial analysis by molecular modeling suggested certain
conformations of compound 5 may be preferable and the energy
differences between these conformations are significant. Therefore,
we hypothesized that differing conformations (atropisomers) of 5
may have contributed to the discrepancy in reported affinities. To
this end, we have resynthesized compound 5 and have prepared
the four-carbon-bridged analogue 11, in which a higher energy
barrier to conformational interconversion is present. We have also
created a number of additional analogues 6-10 with conforma-
tionally constrained aromatic rings (Figure 2). It was anticipated
that our structural modifications of 1 would create energy barriers
that restrain the rotation of the aryl rings so that certain low energy
* To whom correspondence should be addressed. Phone: 919-541-1235
(Y.Z.); 919-541-6552 (B.F.T.). Fax: 919-541-6499 (Y.Z.); 919-541-6499
(B.F.T.). E-mail: yzhang@rti.org. (Y.Z.); bft@rti.org.
a Abbreviations: SAR, structure-activity relationship; CB1, cannabinoid
receptor 1; CB2, cannabinoid receptor 2; CNS, central nervous system;
CoMFA, comparative molecular field analysis; NMR, nuclear magnetic
resonance; VT NMR, variable temperature NMR.
10.1021/jm8000778 CCC: $40.75
2008 American Chemical Society
Published on Web 05/31/2008

Conformationally Constrained SR141716 Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3527
Figure 1. 1 and selected conformationally constrained analogues. Four dihedral angles (τ1-4) are defined in 1.
29 using procedures described for 21. Target compound 10 was
then prepared in a manner analogous to that described for 6
and 7.
Key intermediate 36 to compound 11 was synthesized from
3-chlorobenzaldehyde (33) using the route described in Scheme
4.31 Wittig olefination of 33 with (4-carboxybutyl)triphenylphos-
phonium bromide afforded 34 as a mixture of E and Z isomers.
Catalytic hydrogenation of 34 using PtO2 provided saturated
derivative 35. Conversion of acid 35 with oxalyl chloride and
catalytic dimethylformamide gave the intermediate acid chloride,
which was immediately cyclized in an intramolecular Friedel-
Craft acylation reaction to give compound 36. Compound 36
was transformed to target compound 11 using the procedures
described for compounds 6 and 7.
Figure 2. Conformationally constrained analogues of 1 developed in
Conformational Analysis. The conformation of 1 can rea-
sonably be defined by four dihedral angles (τ1-4) as shown in
Figure 1. Quenched molecular dynamics simulations indicate
this molecule has clear conformational preferences for its ring
substituents (Figure 3). For example, the trans conformation
(carbonyl oxygen and pyrazole nitrogen on opposite sides),
where τ1 is ∼180°, is adopted in low energy conformations.
Torsion angle τ2 describes the movement of the piperidine ring,
where some flexibility is generally allowed, albeit symmetry
of the piperidine ring simplifies the conformational freedom at
this position. The dihedral angles between the pyrazole and
para-chlorophenyl and 2,4-dichlorophenyl rings are defined as
torsion angles τ3 and τ4, respectively. The two torsion angles
of the aromatic rings, τ3 and τ4, are energetically “cogged,”
such that if τ4 is at 60°/-120°, τ3 falls into energy minima at
40° or -140°, but if τ4 is at 120°/-60°, τ3 has its energy
minima at 140° and -40°. Because of the symmetry of the para-
chloro ring system, the τ4 torsion angle at 60° is equivalent to
-120°, and 120° is the equivalent of -60°. These interdepen-
dent conformational characteristics are quite apparent in the
energy plot obtained through a grid search of the τ3 and τ4
torsion angles in Spartan (version 4.0, Wave Function, Irvine,
CA), as shown in Figure 4. It is interesting to note that there is
a considerable energy barrier (>10 kcal/mmol), for all of these
compounds, to rotation of the monochlorinated ring (τ4) through
coplanarity with the pyrazole ring due to the presence of the methyl
group at the 4-position and the dichlorinated ring system at the
1-position. In comparison, the dichlorinated ring system (τ3) has
a relatively lower barrier (∼6 kcal/mol) rotating across its full 360°
range.
our group.
conformations would be favored. It was also predicted that in
certain instances when the energy barrier is high enough, atropi-
somers could be formed or even separated and perhaps display
different biological characteristics. By characterizing their structural
properties using computational chemistry and NMR and determin-
ing their receptor binding affinity and ability to modulate signal
transduction, we further define the relationship between ring
orientation and interaction/activation at the CB1 receptor.
Results and Discussions
Chemistry. Compounds 6 and 7 were prepared following
procedures reported for the synthesis of 1 and its derivatives32,33
(Scheme 1). Treatment of commercially available 2,4-dichlo-
ropropiophenone (12) with diethyl oxalate and sodium ethoxide
afforded 13, which existed in equilibrium with the tautomeric
1
enone 14 as shown in the H NMR. Condensation of 13 with
2,4-dichlorophenylhydrazine or 2,4,6-trichlorophenylhydrazine
in refluxing ethanol provided pyrazole esters 15 and 16,
respectively. Corresponding acids 17 and 18 were obtained by
hydrolysis in methanolic sodium hydroxide and were then
coupled to 1-aminopiperidine using isobutylchloroformate to
afford hydrazides 6 and 7.
Compounds 8 and 9 were prepared in a manner analogous
to that described for 6 and 7 (Scheme 2). Starting propiophenone
21 was obtained in two steps from bromide 19. Lithiation of
19 provided the lithium species, which was quenched with
propionaldehyde to yield alcohol 20. Subsequent oxidation with
pyridinium chlorochromate of 20 afforded ketone 21 in excellent
yield. Compound 21 was advanced to target compounds 8 and
9 using procedures described for 6 and 7.
Figure 4 also reveals that when the para-chlorinated ring
system of 1 is modified by addition of an ortho substituent (6),
neither aryl ring now has symmetry. The added steric bulk does
not significantly change the preferred torsion angles or the
energy barrier for rotation of τ3, but the energy barrier for
complete rotation of τ4 doubles to approximately 20 kcal/mol.
Propiophenone 29 was synthesized from aniline 26 (Scheme
3). Diazotization of 26 using sodium nitrite and concentrated
hydrochloric acid followed by potassium iodide afforded 27.34
Iodide 27 was then converted to the requisite propiophenone

3528 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Scheme 1. Synthesis of Compounds 6 and 7a
Zhang et al.
a Reagents and conditions: (a) NaOEt, EtOH, diethyloxalate. (b) 2,4-Dichlorophenylhydrazine hydrochloride, EtOH. (c) 2,4,6-Trichlorophenylhydrazine
hydrochloride, EtOH. (d) NaOH, MeOH, H2O. (e) Isobutylchloroformate, Et3N, THF, then 1-aminopiperidine.
Scheme 2. Synthesis of Compounds 8 and 9a
a Reagents and conditions: (a) n-BuLi, THF, then propionaldehyde. (b) PCC, CH2Cl2. (c) NaOEt, EtOH, diethyloxalate. (d) 2,4-Dichlorophenylhydrazine
hydrochloride, EtOH. (e) 2,4,6-Trichlorophenylhydrazine hydrochloride, EtOH. (f) NaOH, MeOH, H2O. (g) Isobutylchloroformate, Et3N, THF, then
1-aminopiperidine.
Scheme 3. Synthesis of Compound 10a
a Reagents and conditions: (a) NaNO2, HCl, KI, H2O. (b) n-BuLi, THF, then propionaldehyde. (c) PCC, CH2Cl2. (d) NaOEt, EtOH, diethyloxalate. (e)
2,4-Dichlorophenylhydrazine hydrochloride, EtOH. (f) NaOH, MeOH, H2O. (g) Isobutylchloroformate, Et3N, THF, then 1-aminopiperidine.
Therefore, one might predict that if the “active” conformation
requires τ4 angles near 0°, one would expect the activity of 6
to be decreased. Compound 7, resulting from one o-chlorine
addition on each ring, results in an increased rotational barrier
at τ3 and τ4 up to approximately 20 kcal/mol. Thus, the ring
systems have difficulty crossing the plane of the pyrazole ring.
Instead of the relatively free range of motion across the entire
range of τ3 and τ4 as seen with 1, the range of motion of the
aryl ring systems in compound 7 is limited to approximately
60° on either side of the pyrazole plane. As might be expected,

Conformationally Constrained SR141716 Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3529
Scheme 4. Synthesis of Compound 11a
a Reagents and conditions: (a) (4-Carboxybutyl)triphenylphosphonium bromide, NaHMDS, DMF. (b) H2/PtO2, EtOH. (c) Oxalyl chloride, DMF (cat.),
CH2Cl2. (d) AlCl3, CH2Cl2. (e) Diethyloxalate, NaOEt, EtOH. (f) 2,4-Dichlorophenylhydrazine hydrochloride, EtOH. (g) NaOH, MeOH, H2O. (h)
Isobutylchloroformate, Et3N, THF, then 1-aminopiperidine.
Figure 3. Conformational distributions of 1 from quenched molecular dynamics performed in SYBYL (Tripos, St Louis, MO).
the energy manifold for compound 8 is similar to that calculated
for compound 6, and the energy manifold calculated for compound
9 is similar to that of compound 7. Finally, dimethylation of the
ortho positions on the para-chlorophenyl ring (10) also produces
an energy manifold similar to compound 6.
A torsional grid search was also performed on the tricyclic
pyrazole series (3-5, Figure 5). The one-carbon-bridged com-
pound (3) exists in an almost planar conformation with τ4 less
than 10°. The two-carbon-bridged compound (4) has slightly
more flexibility than 3, and τ4 are at around 20°. The three-
carbon-bridged compound (5) is further off the pyrazole plane,
with a dihedral angle τ4 of around 45°. This dihedral angle is
about 60° in the four-carbon-bridged compound (11), which is
close to that of 1. From the calculations obtained in Spartan, it
appears that the energy barrier for ring rotation around τ4 is
approximately 10 kcal/mol for the three-carbon-bridged mol-
ecule (5), whereas the energy barrier increases to approximately
20 kcal/mol in the four-carbon-bridged molecule (11). The
higher energy barrier in compounds bridged with the longer
four-carbon bridge as compared to the three-carbon bridge is
consistent with calculations and observations on bridged biaryls
reported by Jaime and Font.35 It appears that the energy barrier
to inversion is sufficiently large in the four carbon-bridged
compound (11) such that a stereogenic axis is formedsthe single
bond between the para-chlorophenyl and pyrazole, therefore
atropisomers could potentially exist. Furthermore, atropisomers
should be observed by NMR and could possibly even be
isolated.

3530 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Zhang et al.
Figure 4. Conformational mobility of the diaryl ring systems in analogues of 1 with substitution on diaryls. Calculation was performed in Spartan
(version 4.0, Wave Function, Irvine, CA). Results were plotted in JMP (version 7.0.1, SAS Institute, Cary, NC) as graphs of energy and dihedral
angles τ3 and τ4 across a 30 kcal/mol range, with the minimum energy in red and increasing energy contoured towards green. Numbering denotes
the energy minimum. Because of the symmetry of the molecules, one conformation may have more than one energy minimum.
Computation of Electrostatic Potential. Spartan was used
to calculate and map the electrostatic potential of all molecules.
In general, the ranges of the molecular electrostatic potentials
were quite similar across all molecules, and only in those regions
where substitutions were made were the electrostatic potentials
modestly affected (data not shown). Thus, it appeared that steric
and conformational factors would play a larger role in deter-
mination of receptor affinity and biological activity than elec-

Conformationally Constrained SR141716 Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3531
Figure 5. Conformational mobility of the diaryl ring systems in tricyclic analogues of 1. Calculation results from Spartan were plotted in JMP as
graphs of energy and the dihedral angles τ3 and τ4 across a 30 kcal/mol range, with the minimum energy in red and increasing energy contoured
towards green.
Figure 6. (a) Possible atropisomers of compound 11. Atropisomers are also present in the corresponding ester (39) and acid (40). (b) 2, 2′-
Binaphthyridine.
trostatic differences, although further studies using comparative
molecular field analysis (CoMFA) and/or other approaches are
required to adequately characterize the weighting of these
various structural features.
NMR Study. Conformational analysis described above sug-
gested a ∼20 kcal/mol energy barrier between the conformations
of 11, which is sufficiently high that enantiomers may exist.
Indeed, these bridged biaryl structures have been previously
studied by Thummel and co-workers in their research on polyaza
cavity-shaped molecules,36–40 such as 2,2′-binaphthyridine
shown in Figure 6.37 They reported that splitting of geminal
protons, or presence of an AB pattern, on the carbon bridge
was observed in the 1H NMR of these molecules. Because
geminal protons on bridge carbons are magnetically equivalent
if rapid interconversion is present, this observation clearly
demonstrates that at room temperature these molecules are either
conformationally rigid or the interconversion between conform-
ers is slow enough to be detected on the NMR time scale.
Indeed, the room-temperature 1H NMR spectra of compounds
11, 39, and 40 in CDCl3 all showed in the aliphatic regions a
multiplet at around 3.3 ppm, which was integrated for one
proton. COSY, HSQC, and HMBC 2D NMR study determined
that this signal was one of the benzylic protons on the carbon
bridge, while the other methylene proton was at around 2.5 ppm.

3532 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Zhang et al.
Figure 7. Derivatives of compounds 11, 1, and 5 with chiral auxiliary.
Figure 8. 1H NMR temperature study on compound 41.
Therefore, the presence of this well-resolved diastereotopic AB
system of geminal protons clearly demonstrates that at this
temperature the interconversion of the system is either not
allowed or the inversion rate is slow enough to be detected on
the NMR time scale. In comparison, the three-carbon-bridge
derivative (5) was prepared following a similar route as that of
11 (data not shown). In the 1H NMR spectrum of 5, separation
of geminal protons at the benzylic positions on the bridge was
not observed and only broad peaks are present. This evidence
suggests that a rigid conformation is not adopted by this com-
pound at room temperature. This observation is in accordance
with our calculation results.
In an effort to further study the conformational properties of
the structure and possibly isolate diastereomers, a chiral oxazol-
one, (3aR,8aS)-3,3a,8,8a-tetrahydro-indeno[1,2-d]oxazol-2-one,
was introduced as chiral auxiliary and compound 41 was
prepared (Figure 7).41 In addition to the expected isolation of
the carbon bridge protons at around 3.3 ppm in CDCl3 in the
1H NMR of compound 41, several interesting and encouraging
observations were made, suggesting the presence of atropiso-
mers. For example, the 28-H displayed two distinct sets of
signals at 6.5 ppm, which coupled to two C-13 signals separated
by 1.5 ppm in HSQC, suggesting the presence of two isomers.
The 27-H, which showed a doublet-doublet signal, also coupled
to two C13 signals that are 1.0 ppm apart. However, the
possibility of the existence of rotamers, as is commonly observed
in tertiary amides,42,43 has to be considered. To this end, the
same auxiliary was installed to 1 and the three-carbon-bridged
pharmacophore to afford 42 and 43, respectively. In these
compounds, the splitting of the hydrogen on the auxiliary was
not observed; instead, a clean doublet was assigned. Therefore,
the two sets of signals observed in 11 and 41 reflected the
rigidity of this four-carbon-bridged structure, rather than the
rotation around the amide bond. This is in agreement with our
findings that a 5-6 kcal/mol energy barrier for rotation around
the CO-NCO bond in 41 was obtained in calculations per-
formed in Spartan (data not shown). Taken together, these
observations strongly support the presence of two atropisomers.
Variable Temperature NMR Study. It is anticipated that
the interconversion between the conformations will occur more
readily with the elevation of temperature and coalescence will
take place when the temperature is sufficiently high. Therefore,
variable temperature NMR (VT NMR) study was carried out
on compound 41, as shown in Figure 8. While it was expected
that similar results would be obtained on other four-carbon-
bridged compounds, such as 11, compound 41 was chosen in
hope of more resolved signals due to the chiral effects of the
auxiliary. Therefore, a sample of 41 dissolved in DMF-d6 was
first cooled to -30 °C and then slowly heated to 100 °C. The
1H NMR spectra as a function of temperature were recorded.
While the two sets of signals for the protons at the 28-position
stay resolved at relatively low temperature, they coalesced to
one broad single peak when the temperature reaches 100 °C.
Similarly, signals at 7.6-7.8 ppm merged into broad peaks at that

Conformationally Constrained SR141716 Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3533
Figure 9. 1H NMR temperature study on compound 43.
temperature. This transition in pattern is also present for peaks at
3.3 and 3.5 ppm in the aliphatic region. These observations suggest
that at higher temperature, such as 100 °C, rapid interconversion
between conformations is present.
CH2) in compound 41, which all demonstrated coalescence at
100 °C, were used in the calculations and an energy barrier of
17.8 ( 0.2 kcal/mol was obtained. These data are consistent
with our computation results that showed an energy barrier of
∼20 kcal/mol for 11 with the four-carbon bridge. Similarly, an
energy barrier of 12.6 ( 0.3 kcal/mol for compound 43 was
obtained, following calculations that used signals at 7.8 ppm
(17-H), 6.8 ppm (19-H), 6.3 ppm (27-H), 5.6 ppm (26-H), and
3.3 ppm (30-CH2), which coalesced at between -10 and 0 °C.
Again, these energies are in agreement with our calculations
that demonstrate activation energy of ∼10 kcal/mol for 5 with
the three-carbon bridge.
Separation of Atropisomers. Attempt to recrystalize acid
40 with a chiral amine to form chiral salts were unsuccessful.
Efforts to separate the two possible atropisomers of 11 via HPLC
under various conditions, including using chiral stationary
phases, did not lead to separation of the possible atropisomers.
Taken together, inseparable atropisomers at room temperature
and an energy barrier of ∼18 kcal/mol for inversion of the bridge
suggest a half-life that is in the magnitude of seconds.47 It
appears that introduction of more ortho-substituents that further
increase the rotational barrier might be necessary to further
differentiate between the two isomers, thus making the separa-
tion possible.
Pharmacology. Most compounds showed nM affinity for the
CB1 receptor in competition assays against [3H]-(1R,3R,4R)-
3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypro-
pyl) cyclohexan-1-ol (44, CP-55940)48 and [3H]-1 (Table 1).
The observation that 1 remained the compound with the highest
affinity for the CB1 receptor suggests that the conformational
mobility and low energy conformations of the aryl ring systems
in this compound are optimal for CB1 receptor interaction. In
support of this conclusion, one can see that the ring-substituted
analogues with the highest affinity (6 and 8) retained confor-
mational preferences most similar to 1. Ring-substituted ana-
In comparison, the three carbon bridge derivative (43) was
1
also submitted to VT NMR study. H NMR spectra of 43 in
DMF-d6 were obtained in the range between -40 and 40 °C
(Figure 9). As the temperature decreased, splitting was observed
for most of the aromatic protons, such as the doublet at around
6.8ppmatroomtemperature,whichturnedintoadoublet-doublet
at -40 °C. More dramatic pattern changes were observed
between 7.65-7.95 ppm, with additional signals appearing, as
shown in Figure 9. In the aliphatic region, as the temperature
lowers, the broad peaks for the bridge protons split and signals
appeared at 2.4, 2.7, and 2.8 ppm, respectively. Furthermore,
close similarity in patterns between 43 at -40 °C and 41 at
room temperature was observed. All these findings indicate that
at lower temperature, the inversion of conformation for 43 was
slow enough that it can be detected on the NMR time scale.
To validate our computational study, energy barriers of
rotation, or the free energies of activation for interconversion
of conformers, were calculated by the coalescence method in
the VT NMR study.44 We identified four sets of signals for 41
and five sets for 43 that demonstrated coalescence over the
temperature ranges investigated. The coalescence temperatures
were measured and the free energies were calculated using the
Eyring equation [∆G ) 4.569 × 10-3 × Tc × (10.319 + log
Tc/kc)] and the rate constant (kc ) 2.22 ∆ν).45,46 While the bridge
protons at 1.5-1.6 ppm in 41 demonstrated coalescence at 100
°C and would serve as an ideal probe for interconversion, they
were complicated due to overlapping resonances and were,
therefore, not deployed in our calculation. Similarly, bridge
signals at 2.2 and 2.4 ppm in 43, which coalesced at around 0
°C, were not included in the calculation. Signals at 7.5 ppm
(18-H), 6.3 ppm (28-H), 3.5 ppm (27-H), and 3.3 ppm (31-

3534 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Zhang et al.
Table 1. Cannabinoid Receptor Binding Affinities of the analogues
Ki (nM) in hCB1 (n ) 2)
Table 2. Functionality of the Analogues at the CB1 Receptor Using
[
35S]-GTP-γ-S Assay
competition vs
compound
pA2 (nM)
SEM
Ki (nM) in
tritiated ligands
hCB2 competition
6
7
8
9
10
11
1
7.72 (19.1 nM)
6.57 (269 nM)
7.92 (12.0 nM)
7.11 (77.6 nM)
7.03 (93.3 nM)
7.39 (40.7 nM)
8.59 (2.6 nM)a
0.54
0.25
0.35
0.19
0.63
0.59
0.08a
compound
3H-44 SEM
34.2 2.85
261 25.0
16.1 3.55
251 19.0
194
40.9
6.18
3H-1
SEM
vs3H-44
6
7
8
9
17.9
0.40
23.5
0.14
5.50
19.2
3.65
0.1
1044
1109
1124
1781
758
148
5.83
152
116
14.7
1.18
10
11
20.0
10.4
1.2
a Mean and SEM based on 12 individual pA2 experiments.
492
313
1 (Thomas
et al., 2005)
provided in Table 2. The pA2 values for each compound were
in close correspondence with their affinity. Similar to 1, these
compounds typically required µM concentrations to show
inverse agonist activity in hCB1 transfects, and the change from
basal was relatively modest (<25% decrease in basal binding
under the conditions used, data not shown).
logues with the lowest affinity were those that had three
substituents on one of the rings, suggesting that the ability to
rotate steric bulk out of a region occupied by the receptor is
compromised when both ortho positions are occupied on a
particular aryl ring. One might infer from the data that the steric
region of conflict is located between the ring systems because
full ortho-substitution of either ring results in decreased affinity,
while mono-ortho-substitution of either ring is tolerated. How-
ever, this remains to be unequivocally determined.
In our laboratory, the CB1 receptor affinity of the three-
carbon-bridged compound (5) was determined to be lower than
the prototype 1, specifically 18.4 ( 1.62 nM against [3H]-44
and 5.55 ( 0.25 nM against [3H]-1. These values are closer to
the Ki value reported by Stoit et al.31 with [3H]-44 in hCB1
transfected Chinese hamster ovary (CHO) cells (126 nM) than
to the 350 fM affinity reported by Ruiu et al.49 using mouse
brain membrane (minus cerebellum) and [3H]-44. Thus, we were
unable to confirm the remarkable high affinity reported by Ruiu.
Furthermore, the four carbon-bridged molecule (11), which
appeared to further constrain the aryl ring systems when assessed
by molecular modeling and NMR studies, had a slightly lower
affinity than 5 (40.9 ( 10.4 nM versus [3H]-44 and 14.7 (
3.65 against [3H]-1) when tested in our laboratory against hCB1
transfected CHO cells. The order of decreasing affinity seen in
our laboratory, from 1, to the three-carbon bridge, and then to
the four-carbon bridge, is then consistent with the increasing
constraint of the ring system from 1, to the three-, and then to
the four-carbon-bridged system.
All of the compounds synthesized and tested had significantly
lower affinity for CB2 than CB1. However, all of the analogues
showed CB1 to CB2 receptor selectivity lower than 1 and, in
some analogues, the selectivity was only approximately 10-fold.
For example, compounds 7 and 9 had a greater decrease in CB1
than in CB2 affinity, such that their CB2/CB1 affinity ratios
were considerably less than 1. In these experiments, the three-
carbon-bridged compound (5) had a CB2 Ki value of 758 nM,
which differs from the 21 nM value reported by Ruiu et al.
(2003) as well as in subsequent publications from this group
(Murineddu et al. 2005). Thus, the CB1 to CB2 selectivity in
these experiments is approximately 10-fold, whereas Ruiu et
al. (2003) reported a selectivity over 60000 fold. Finally, 11
had reasonable affinity for CB1 (Ki approximately 15 nM when
measured with the antagonist [3H]-1) and a Ki at the CB2
receptor of 492 nM when measured with [3H]-44. Therefore,
in these experiments, it appears that ring constraint decreases
CB1 receptor affinity more dramatically than CB2 receptor
affinity when compared against the prototype 1.
Conclusion
We had initially hypothesized conformational restriction of
the aryl rings afforded by the three-carbon bridge in 5 increased
the population of conformers that could bind to the CB1 receptor
with high affinity and that this was responsible for the sub-pM
affinity reported by Pinna and collegues research group.7–9
Molecular modeling calculations indicated that this was the case,
and that a four-carbon-bridged analogue would further restrict
the conformational mobility of the aryl rings and possibly
produce an even greater increase in affinity and activity.
Similarly, substitution of the aryl ring protons to restrict or alter
the conformational preferences of the aryl rings could also have
dramatic impact on affinity and activity. We had also noted that
Stoit et al.31 had reported the three-carbon-bridged compound
earlier and found that it had lower affinity for the CB1 receptor
than 1. It is interesting to note that the research reported by
Stoit et al. included in vivo administration of the three-carbon-
bridged compound by ip and po routes, and the authors reported
that no activity was detected, which is also consistent with a
compound that had decreased affinity as compared to 1. To
clarify this discrepancy and further characterize the structure-acti-
vity relationships of CB1 receptor antagonists, we designed and
synthesized a series of derivatives of 1 that further modified
and constrained the conformational mobility of the diaryl ring
systems. Specifically, we synthesized and tested the three-
carbon-bridged molecule (5) as well as a four-carbon-bridged
molecule (11) and introduced ortho substituents into the two
phenyl ring systems (as shown in Figure 2). These structural
modifications create energy barriers that restrain the rotation of
the aryl rings to distinct low energy conformations. Indeed, in
the bridged compounds, eclipsing interactions or torsional strain
in the annelated bridge can resist rotation around the single bond
between the pyrazole and the 4-chlorophenyl group, which will
in turn restrict the rotation of the 2,4-dichlorophenyl ring. As
the system can no longer interconvert between the two minimum
energy conformations of the annelated ring, conformational
enantiomerism occurs. That is, in the four-carbon-bridged
system, a sufficiently high energy barrier to inversion could
allow for the isolation of atropisomers. Indeed, an energy barrier
of ∼20 kcal/mol was obtained through our calculation in
Spartan. VT NMR study on 41 suggested that a barrier of ∼18
kcal/mol for interconversion was present in this four-carbon-
bridged structure and atropisomers could be formed. Unsuc-
cessful attempts to isolate the atropisomers indicated that further
introduction of steric hindrance is necessary. The eventual
isolation of atropisomers would certainly allow for an interesting
The conformationally constrained compounds were able to
shift the dose response curve for 44 in [35S]-GTP-γ-S functional
assays, indicating that they were antagonists. Their pA2 values
against 44 stimulated [35S]-GTP-γ-S binding in hCB1 cells are

Conformationally Constrained SR141716 Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3535
δ: 1.43 (t, J ) 6.0, 3H), 2.25 (s, 3H), 4.46 (q, J ) 6.0, 2H), 7.15
(m, 2H), 7.29 (s, 1H), 7.40 (s, 1H), 7.48 (s, 1H).
study of the orientation of aryl rings when interacting with the
CB1 receptor. Because the conformationally constrained com-
pounds show decreased affinity as compared to 1, we conclude
that the approaches we have used either (1) constrain the ring
systems in an orientation less optimal for receptor interaction
than afforded by the conformational mobility of the rings in 1,
(2) that the addition of steric bulk to constrain the conformations
leads to disfavored steric interactions with the receptor, and/or
(3) that the relatively modest alterations in the molecular
electrostatic potentials of the molecules seen in the analogues
results in disfavored Coulombic interactions.
1,5-bis(2,4-Dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxy-
lic acid (17). To a solution of 15 (1.77 g) in 30 mL of methanol
was added 30 mL of 2 N NaOH. The mixture was stirred at room
temperature for 14 h. The reaction was concentrated to about half
the volume and then extracted with ether (2 × 20 mL). The aqueous
layer was acidified with 37% HCl and extracted with ethyl acetate
(3 × 50 mL). The combined organic layers are washed with water
and brine and then dried with Na2SO4. The solvent was removed
in vacuo to give 17 (1.35 g, 95% over two steps from 13) as an
1
off-white solid. H NMR (CDCl3) δ: 2.22 (s, 3H), 7.15 (d, J )
9.0, 1H), 7.24 (m, 2H), 7.33 (d, J ) 9.0, 1H), 7.40 (s, 1H), 7.44 (s,
1H).
Experimental Section
This acid was analytically pure and was used in the following
step without further purification.
Chemistry. General Methods. Reactions were conducted under
N2 or Ar atmospheres using oven-dried glassware. All solvents and
chemicals used were reagent grade. Anhydrous tetrahydrofuran
(THF), dichloromethane, and N,N-dimethylformamide (DMF) were
purchased from Aldrich and used as such. Unless otherwise
mentioned, all reagents and chemicals were purchased from
commercial vendors and used as received. Flash column chroma-
tography 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 chromatography-mass
spectrometry (GC-MS), and NMR analytical techniques described
below. 1H and 13C NMR spectra were recorded on a Bruker Avance
DPX-300 (300 MHz) spectrometer and were determined in CHCl3-d
or MeOH-d4 with tetramethylsilane (TMS) (0.00 ppm) or solvent
peaks as the internal reference unless otherwise noted. Chemical
shifts are reported in ppm relative to the solvent signal, and coupling
constant (J) values are reported in Hertz (Hz). Variable temperature
NMR spectra were recorded on a Bruker AMX-500 (500 MHz)
and were determined in DMF-d7 with TMS (0.00 ppm) as the
internal reference. Temperatures were uncorrected. Thin-layer chro-
matography (TLC) was performed on EMD precoated silica gel
60 F254 plates, and spots were visualized with UV light or I2
detection. Low-resolution mass spectra were obtained using a
Waters Alliance HT/Micromass ZQ system (ESI).
Ethyl 4-(2,4-Dichlorophenyl)-3-methyl-2,4-dioxobutanoate
(13). To a solution of sodium (2.3 g, 98 mmol) dissolved in 40 mL
of ethanol was added diethyl oxalate (7.4 mL, 54 mmol). The
mixture was stirred for 30 min before a solution of 12 (10 g, 49.2
mmol) in 200 mL of ethanol was added dropwise. The resulting
reaction mixture was stirred at room temperature overnight. The
reaction was concentrated in vacuo, and the resulting slurry was
dissolved in 200 mL of water and then extracted with methylene
chloride (3 × 150 mL). The combined organic layers were washed
with water, brine, and then dried with Na2SO4 and concentrated. The
resulting slurry was purified by chromatography on silica gel to give
13 and 14 (6.9 g, 46%) as a mixture of two isomers (13:14 ) 2:3).
13: 1H NMR (CDCl3) δ: 1.40 (d, J ) 3.0, 3H), 1.41 (t, J ) 7.5, 3H),
4.39 (q, J ) 6.0, 2H), 5.03 (q, J ) 6.0, 1H), 7.38 (d, J ) 7.5, 1H),
7.48 (s, 1H), 7.65 (d, J ) 9.0, 1H). 14: 1H NMR (CDCl3) δ: 1.35 (t,
J ) 7.5, 3H), 1.82 (s, 3H), 4.32 (q, J ) 7.5, 2H), 7.24 (d, J ) 9.0,
1H), 7.37 (d, J ) 7.5, 1H), 7.48 (s, 1H).
5-(2,4-Dichlorophenyl)-4-methyl-1-(2,4,6-trichlorophenyl)-1H-
pyrazole-3-carboxylic Acid (18). Following the procedure in the
preparation of 17, 18 was synthesized (1.37 g, 92% from 13) as an
off white solid. H NMR (CDCl3) δ: 2.28 (s, 3H), 7.17 (m, 2H),
7.32 (s, 1H), 7.43 (s, 1H), 7.50 (s, 1H).
1
1,5-bis(2,4-Dichlorophenyl)-4-methyl-N-piperidin-1-yl-1H-
pyrazole-3-carboxamide (6). To a solution of 200 mg (0.46 mmol)
of acid 17 in 30 mL of THF at 0 °C was added sequentially
isobutylchloroformate (66.2 µL, 0.51 mmol) and triethylamine (77.0
µL, 0.55 mmol). The resulting cloudy mixture was stirred for 30
min before 106 µL of 1-aminopiperidine (0.92 mmol) was added.
The mixture was allowed to warm to room temperature in 2 h.
The reaction was diluted with 100 mL of ethyl acetate and washed
with 1 N NaOH, H2O, brine, and then dried with Na2SO4. The
solvent was removed in vacuo, and the resulting slurry was purified
by chromatography on silica gel to give 6 (186 mg, 80.5%) as a
white solid. 1H NMR (CDCl3) δ: 1.44 (m, 2H), 1.74 (m, 4H), 2.24
(s, 3H), 2.87 (m, 4H), 7.11 (d, J ) 9.0, 1H), 7.22 (m, 3H), 7.42
(m, 2H), 7.64 (br s, 1H). 13C NMR (CDCl3) δ: 9.28, 23.38, 25.46,
57.08, 120.13, 126.57, 127.29, 129.87, 130.27, 130.35, 132.90,
133.06, 135.56, 136.05, 136.35, 140.41, 144.21, 159.89. MS m/z
499 [M + H]+. Anal. (C22H20Cl4N4O), C, H, N.
5-(2,4-Dichlorophenyl)-4-methyl-N-piperidin-1-yl-1-(2,4,6-
trichlorophenyl)-1H-pyrazole-3-carboxamide (7). Following the
procedure in the preparation of 6, 7 (77.0%) was obtained from 18
as a white solid. 1H NMR (CDCl3) δ: 1.43 (m, 2H), 1.75 (m, 4H),
2.28 (s, 3H), 2.88 (m, 4H), 7.10 (d, J ) 6.0, 1H), 7.14 (d, J ) 6.0,
1H), 7.33 (s, 1H), 7.43 (s, 1H), 7.47 (s, 1H), 7.62 (br s, 1H). 13C
NMR (CDCl3) δ: 9.70, 23.39, 25.46, 57.04, 120.49, 125.95, 127.14,
128.61, 128.95, 130.26, 132.26, 133.79, 135.76, 135.79, 136.33,
136.43, 140.46, 144.89, 159.84. MS m/z 533 [M + H]+. Anal.
(C22H19Cl5N4O), C, H, N.
1-(4-Chloro-2-methylphenyl)propan-1-ol (20). To a solution
of 19 (10 g, 49 mmol) in 80 mL of anhydrous diethyl ether cooled
to -78 °C was added n-butyl lithium (36.5 mL, 1.6 M solution in
hexanes) dropwise. The reaction was stirred at that temperature
for 10 min and then warmed to room temperature. After 30 min,
the reaction was cooled to -78 °C, and propioaldehyde was added.
The reaction was allowed to warm to room temperature over 4 h.
The reaction was poured into 50 mL of 2 N HCl and extracted
with ethyl acetate (3 × 60 mL). The combined organic layers were
washed with water, brined, and dried with Na2SO4. The solvent
was removed, and the resulting slurry was purified on silica gel to
give 20 as an oil. 1H NMR (CDCl3) δ: 0.93 (t, J ) 7.5, 3H), 1.68
(q, J ) 7.0, 2H), 2.27 (s, 3H), 4.77 (t, J ) 6.3, 1H), 7.10 (s, 1H),
7.17 (d, J ) 8.1, 1H), 7.35 (d, J ) 8.1, 1H).
Ethyl 1,5-bis(2,4-Dichlorophenyl)-1H-pyrazole-3-carboxylate
(15). A mixture of 13 (1g, 3.3mmol) with 2,4-dichlorophenylhy-
drazine (0.78 g, 3.6 mmol) in 20 mL of ethanol was stirred at room
temperature for 2 h, then heated to reflux for 12 h. The reaction
was cooled to room temperature. To the reaction was added 100
mL of water and extracted with ethyl acetate (3 × 80 mL). The
combined organic layers were washed with water and brine and
dried with Na2SO4. The solvent was removed in vacuo, and the
resulting slurry was purified on silica gel to give 15 as an oil (1.77
The compound was submitted to the following step without
further purification.
1-(4-Chloro-2-methylphenyl)propan-1-one (21). To a solution
of the above alcohol in 100 mL of methylene chloride was added
21 g of celite, followed by the addition of PCC. The reaction
mixture was stirred for 1 h before filtering through a pad of silica.
The filtrate was concentrated to give 21 (8.5 g, 96% from 19) as a
yellow oil. 1H NMR (CDCl3) δ: 1.18 (t, J ) 7.2, 3H), 2.48 (s,
3H), 2.89 (q, J ) 7.2, 2H), 7.22 (m, 2H), 7.58 (d, J ) 9, 1H).
1
g). H NMR (CDCl3) δ: 1.43 (t, J ) 6.9, 3H), 2.21 (s, 3H), 4.45
(q, J ) 7.2, 2H), 7.15 (m, 1H), 7.23 (m, 2H), 7.33 (d, J ) 8.4,
1H), 7.38 (s, 1H), 7.43 (s, 1H).
Ethyl 5-(2,4-Dichlorophenyl)-1-(2,4,6-trichlorophenyl)-1H-
pyrazole-3-carboxylate (16). Following the procedure in the
preparation of 15, 16 was synthesized from 13. 1H NMR (CDCl3)

3536 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Zhang et al.
Ethyl 4-(4-Chloro-2-methylphenyl)-3-methyl-2,4-dioxobu-
tanoate (22). Following the procedure in the preparation of 13, 22
and 23 (5.6 g, 45%) was prepared from of 21 (8 g, 44 mmol) as a
1-(4-Chloro-2,6-dimethylphenyl)propan-1-one (29). A solution
of the above alcohol 28 in 100 mL of CH2Cl2 was added to celite.
To the resulting suspension was added PCC (4.87 g, 22.6 mmol).
The reaction was stirred at room temperature for 1 h and then
filtered through a pad of silica. The filtrate was concentrated to
give 29 (2.1 g, 95% from 27) as an orange oil. H NMR (CDCl3)
δ: 1.18 (t, J ) 7.2, 3H), 2.17 (s, 6H), 2.68 (q, J ) 7.2, 2H), 7.00
(s, 2H).
1
mixture of two isomers. 22: H NMR (CDCl3) δ: 1.38 (m, 6H),
2.46 (s, 3H), 4.28 (q, J ) 7.2, 2H), 4.91 (q, J ) 7.2, 1H), 7.28 (m,
1
1
2H), 7.74 (d, J ) 9.0, 1H). 23: H NMR (CDCl3) δ: 1.32 (t, J )
7.2), 1.78 (s, 3H), 2.30 (s, 3H), 4.39 (q, J ) 7.2, 2H), 7.14 (d, J )
9.0, 1H), 7.28 (m, 2H).
Ethyl 4-(4-Chloro-2,6-dimethylphenyl)-3-methyl-2,4-dioxobu-
tanoate (30). Following the procedure for 13, 30 (63.9%) was
prepared from 30. 1H NMR (CDCl3) δ: 1.38 (t, J ) 3.0, 3H), 1.70
(s, 3H), 2.20 (s, 6H), 4.39 (q, J ) 6.0, 2H), 7.08 (s, 2H), 15.39 (br
s, 1H).
Ethyl 5-(4-Chloro-2-methylphenyl)-1-(2,4-dichlorophenyl)-
4-methyl-1H-pyrazole-3-carboxylate (24). 24 was obtained from
1
22 as an off-white solid following the procedure for 15. H NMR
(CDCl3) δ: 1.43 (t, J ) 7.2, 3H), 2.13 (s, 3H), 2.17 (s, 3H), 4.46
(q, J ) 7.2, 2H), 6.98 (d, J ) 8.1, 1H), 7.10 (d, J ) 8.1, 1H), 7.22
(m, 3H), 7.38 (s, 1H).
Ethyl 5-(4-Chloro-2,6-dimethylphenyl)-1-(2,4-dichlorophenyl)-
4-methyl-1H-pyrazole-3-carboxylate (31). 31 (25.5%) was ob-
tained from 30 following the procedure for 15. H NMR (CDCl3)
δ: 1.44 (t, J ) 7.2, 3H), 2.04 (s, 6H), 2.11 (s, 3H), 4.46 (q, J )
7.2, 2H), 6.98 (d, J ) 8.4, 1H), 7.04 (s, 2H), 7.12 (d, J ) 8.4, 1H),
7.43 (s, 1H).
Ethyl 5-(4-Chloro-2-methylphenyl)-4-methyl-1-(2,4,6-trichlo-
1
rophenyl)-1H-pyrazole-3-carboxylate (25). 25 was obtained from
1
22 as an off-white solid. H NMR (CDCl3) δ: 1.43 (t, J ) 6.0,
3H), 2.19 (s, 3H), 2.26 (s, 3H), 4.46 (q, J ) 6.0, 2H), 7.06 (s, 2H),
7.28 (m, 2H), 7.41 (s, 1H).
5-(4-Chloro-2,6-dimethylphenyl)-1-(2,4-dichlorophenyl)-4-
5-(4-Chloro-2-methylphenyl)-1-(2,4-dichlorophenyl)-4-meth-
yl-N-piperidin-1-yl-1H-pyrazole-3-carboxamide (8). Following
the procedure for the preparation of 17, the ester 24 was hydrolyzed
methyl-1H-pyrazole-3-carboxylic Acid (32). 31 was hydrolyzed
1
to give 32 (90.0%) as a white solid. H NMR (CDCl3) δ: 2.04 (s,
1
6H), 2.12 (s, 3H), 6.96 (d, J ) 8.4, 1H), 7.05 (s, 2H), 7.12 (d, J )
to the acid (83.5% over 2 steps from 22). H NMR (CDCl3) δ:
8.4, 1H), 7.46 (s, 1H).
2.14 (s, 3H), 2.19 (s, 3H), 6.99 (d, J ) 8.1, 1H), 7.10 (d, J ) 6.0,
1H), 7.21 (m, 3H), 7.41 (s, 1H).
5-(4-Chloro-2,6-dimethylphenyl)-1-(2,4-dichlorophenyl)-4-
methyl-N-piperidin-1-yl-1H-pyrazole-3-carboxamide (10). 10
(65.8%) was obtained as a white solid from 32 following procedure
The above acid was converted to 8 (75.5%) following the pro-
1
cedure for 6. H NMR (CDCl3) δ: 1.45 (m, 2H), 1.75 (m, 4H),
1
of 6. H NMR (CDCl3) δ: 1.45 (m, 2H), 1.77 (m, 4H), 2.02 (s,
2.13 (s, 3H), 2.20 (s, 3H), 2.88 (m, 4H), 6.96 (d, J ) 8.1, 1H),
7.10 (d, J ) 8.1, 1H), 7.20 (m, 3H), 7.41 (s, 1H), 7.82 (br s, 1H).
13C NMR δ: 9.62, 19.91, 22.37, 24.66, 56.16, 119.72, 126.44,
126.52, 128.09, 130.60, 130.73, 130.81, 132.47, 132.96, 135.84,
136.37, 140.28, 143.05, 143.37, 161.40. MS m/z 477 [M + H]+.
Anal. (C23H23Cl3N4O·0.5CH3OH) C, H, N.
6H), 2.14 (s, 3H), 2.89 (m, 4H), 6.90 (d, J ) 8.4, 1H), 7.03 (s,
2H), 7.14 (d, J ) 8.4, 1H), 7.48 (s, 1H), 7.71 (br s, 1H). 13C NMR
δ: 9.37, 20.46, 23.73, 25.80, 57.42, 119.65, 126.51, 127.75, 129.26,
129.34, 131.31, 132.45, 135.50, 135.69, 135.78, 140.76, 141.84,
144.40, 160.56. MS m/z 491 [M + H]+. Anal. (C24H25Cl3N4O·
0.5CH3OH) C, H, N.
5-(4-Chloro-2-methylphenyl)-4-methyl-N-piperidin-1-yl-1-
(2,4,6-trichlorophenyl)-1H-pyrazole-3-carboxamide (9). Follow-
ing the procedure for the preparation of 17, ester 25 was hydrolyzed
to the acid (79.8% over 2 steps from 22). 1H NMR δ: 2.20 (s, 3H),
2.26 (s, 3H), 7.07 (s, 2H), 7.28 (m, 2H), 7.43 (s, 1H).
(5E,Z)-6-(3-Chlorophenyl)hex-5-enoic acid (34). To a suspen-
sion of (4-carboxybutyl)triphenylphosphonium bromide (5 g, 141
mmol) in 50 mL of DMF was added 71.1 mL of sodium
hexamethyldisilazane (NaHMDS, 1 M in THF) at room temperature.
To the resulting red solution was added 3-chlorobenzaldehyde. The
reaction was heated to 100 °C for 0.5 h and then cooled to room
temperature. The reaction was poured into 200 mL of water and
extracted with ether (2 × 100 mL). The aqueous layer was then
acidified with concentrated HCl and extracted with ethyl acetate
(3 × 150 mL). The combined organic layers were washed
sequentially with water, brine, and then dried with Na2SO4 and
The above acid was converted to 9 (77.8%) following the
procedure for 6. 1H NMR (CDCl3) δ: 1.42 (m, 2H), 1.76 (m, 4H),
2.21 (s, 3H), 2.25 (s, 3H), 2.88 (m, 4H), 7.03 (s, 2H), 7.28 (m,
2H), 7.43 (s, 1H), 7.63 (br s, 1H). 13C NMR δ: 10.78, 20.76, 24.59,
26.66, 58.24, 120.47, 127.16, 129.94, 129.96, 131.82, 132.27,
135.28, 136.43, 136.57, 137.36, 137.51, 141.49, 143.90, 146.02,
161.22. MS m/z 513 [M + H]+. Anal. (C23H22Cl4N4O) C, H, N.
5-Chloro-2-iodo-1,3-dimethylbenzene (27). A suspension of
4-chloro-2,6-xylidine (26) in 40 mL of water and conc HCl (35
mL) was cooled to 0 °C. To the above suspension was added an
aqueous solution of NaNO2 dropwise until excess HNO2 was
detected by starch-iodide paper. A solution of KI in 30 mL of water
was added. The mixture was stirred overnight before it was heated
to reflux until no gas evolution was observed. The mixture was
cooled to room temperature and extracted with CH2Cl2 (3 × 120
mL). The combined organic layers were washed with 5% aqueous
NaOH, water, and brine and dried over Na2SO4. The solvent was
1
concentrated to give 34 (6g) as a brown oil. H NMR (CDCl3) δ:
1.78-1.84 (m, 2H), 2.22-2.43 (m, 4H), 6.14-6.22 (m, 1H),
6.31-6.40 (m, 1H), 7.13-7.24 (m, 4H), 7.31 (s, 1H), 11.29 (br s,
1H).
The material was of suitable purity and submitted to the next
step without further purification.
6-(3-Chlorophenyl)hexanoic acid (35). A mixture of acid 34
and PtO2 was stirred at room temperature under hydrogen at 45
psi for 1 h. The reaction was filtered through celite and concentrated.
TLC and 1H NMR showed the presence of the corresponding ethyl
ester.
1
removed in vacuo to give 27 (8.9 g, 52%) as a brown solid. H
The mixture was then dissolved in 40 mL of methanol and heated
to reflux for 30 min. Methanol was removed in vacuo, and the
resulting slurry was diluted with 40 mL 1 N HCl. The aqueous
layer was extracted with ethyl acetate (3 × 100 mL). The combined
organic layers were washed with water and brine and concentrated
to give the targeted acid 35 as a brown oil. H NMR (CDCl3) δ:
1.41 (m, 2H), 1.65 (m, 4H), 2.36 (t, 2H, J ) 7.2), 2.59 (t, 2H, J )
7.5), 7.05 (d, 1H, J ) 6.9), 7.17 (m, 2H), 7.26 (s, 1H).
2-Chloro-7,8,9,10-tetrahydrobenzo[8]annulen-5(6H)-one (36).
To a solution of acid 35 in CH2Cl2 was added oxalyl chloride, and
the mixture was cooled to 0 °C. To the above mixture was added
2 drops of DMF, and the resulting mixture was allowed to warm
to room temperature. After 2 h, the solvent and excess oxalyl
chloride were removed in vacuo to give 36 as a brown oil.
NMR and mass spectral data are in accordance with published
data.34
1-(4-Chloro-2,6-dimethylphenyl)propan-1-ol (28). To a solu-
tion of 27 (3 g, 11.3 mmol) in 50 mL of anhydrous ether cooled to
-78 °C was added n-BuLi (2.5 M in hexane, 5.0 mL). The mixture
was stirred at that temperature for 3 h before propioaldehyde was
added. The reaction was allowed to warm to room temperature
overnight. To the reaction was added 1 N HCl and extracted with
ether (3 × 40 mL). The combined organic layers were washed with
water, brined, and dried with Na2SO4. The solvent was removed
in vacuo, and the resulting slurry was purified by chromatography
1
1
on silica gel to give 28 as a yellow oil. H NMR (CDCl3) δ: 0.94
(t, J ) 7.2, 3H), 1.70-1.94 (m, 2H), 2.37 (s, 6H), 4.98 (t, J ) 7.2,
1H), 6.95 (s, 2H).

Conformationally Constrained SR141716 Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3537
The crude acid chloride in 30 mL of CH2Cl2 was added dropwise
to a suspension of 3.36 g (25.2 mmol) of anhydrous AlCl3 in 30
mL of CH2Cl2. The resulting dark solution was stirred overnight.
The reaction was poured into ice-water and then extracted with
CH2Cl2 (3 × 100 mL). The combined organic layers were washed
with 5% NaHCO3, water, and dried with Na2SO4. The solvent was
removed in vacuo, and the resulting slurry was purified on silica
To a solution of (3aR-cis)-(+)-3,3a,8,8a-tetrahydro-2H-indeno[1,2-
d]oxazol-2-one (164.5 mg, 0.94 mmol) in 10 mL of THF at -78
°C was added n-butyllithium (1.6 M in hexane, 0.64 mL, 1.02
mmol). The resulting mixture was stirred for 20 min at -78 °C
and a solution of the above acyl chloride in 5 mL of THF was
added. The reaction mixture was stirred for an additional 30 min
at -78 °C and then warmed to room temperature. Saturated aqueous
ammonium chloride (3 mL) was added to the mixture and then
diluted with water (30 mL). The reaction was extracted with ethyl
acetate (3 × 40 mL), and the combined organics were dried over
Na2SO4, filtered, and evaporated. The crude product was subse-
quently purified using chromatography on silica gel to give 41 (260
mg, 53.8%). 1H NMR (DMF-d7) δ: 1.50-1.90 (m, 3H), 2.05-2.25
(m, 1H), 2.42-2.62 (m, 1H), 2.83 (m, 1H), 3.05 (m, 1H), 3.43 (d,
1H), 3.55-4.0 (m, 2H), 5.70 (t, J ) 6.3, 1H), 6.38 (m, 1H), 7.10
(m, 1H), 7.25 (dd, J1 ) 8.1, J2 ) 2.1, 1H), 7.50 (m, 3H), 7.57 (m,
1H), 7.60-7.95 (m, 4H).
(3aR,8aS)-3-{[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazol-3-yl]carbonyl}-3,3a,8,8a-tetrahydro-2H-in-
deno[1,2-d][1,3]oxazol-2-one (42). 42 (75.5%) was obtained
following the above procedure from the corresponding acid. 1H
NMR (CDCl3) δ: 2.15 (s, 3H), 3.35 (d, J ) 3.0, 2H), 5.30 (dd, J1
) J2 ) 3.0), 6.08 (d, J ) 6.0, 1H), 7.01 (m, 2H), 7.19-7.32 (m,
8H), 7.72 (d, J ) 8.1, 1H).
(3aR,8aS)-3-{[8-chloro-1-(2,4-dichlorophenyl)-1,4,5,6-
tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazol-3-yl]carbonyl}-
3,3a,8,8a-tetrahydro-2H-indeno[1,2-d][1,3]oxazol-2-one (43). 43
(68.0%) was obtained from the corresponding acid. 1H NMR (DMF-
d7) δ: 2.24 (m, 2H), 2.55 (m, 1H), 2.77 (m, 3H), 5.66 (t, J ) 6.3
Hz, 1H), 6.32 (d, J ) 6.6 Hz, 1H), 6.87 (d, J ) 8.4, 1H), 7.25 (dd,
J1 ) 8.4 Hz, J2 ) 2.1, 1H), 7.40 (m, 3H), 7.56 (d, J ) 1.8, 1H),
7.73 (dd, J1 ) 8.1, J2 ) 1.5, 2H), 7.81 (d, J ) 2.1, 1H), 7.87 (d,
J ) 8.7, 1H).
Molecular Modeling. Molecular dynamics were performed using
the MMFF94 force field within SYBYL (Tripos, St Louis, MO).
This approach involves molecular dynamics simulations on the
energy-minimized molecules. During the molecular dynamics
simulation, the molecule was heated from 0 to 2000 K at 100 K
steps lasting 10 ps each, with snapshot conformations taken at 10
ps intervals. Upon reaching 2000 K, the molecule was held at this
temperature for 1000 ps, while additional snapshots were acquired
at 10 ps intervals. Each of the snapshot conformations obtained
was energy minimized again using a conjugate gradient of 0.01
kcal/mol or a maximum of 100000 iterations as termination criteria,
yielding a group of energy-minimized conformers. All conforma-
tions were overlaid using a single template molecule, and the
pyrazole ring atoms were used for atom-by-atom root-mean-square
distance minimization. This alignment positioned all of the
molecules in the same 3-dimensional space and superposed the
central ring systems to as great an extent as possible.
Conformational profiles (Figures 4 and 5) for rotations about τ3
and τ4 were calculated using Spartan version 4.0 from Wave
Function, Inc. (Irvine, CA). The Merck molecular force field
(MMFF94) was used to investigate the conformational dynamics
of the two aromatic substituents of 1 and its derivatives. Starting
from the lowest energy conformation, two dihedral angles τ3 and
τ4 were defined and dynamically constrained to rotate 360° in a
succession of 72 steps, or in 5° increments. A systematic grid scan
of the two dihedral angles was then executed using an energy profile
calculation.
Electrostatic potentials (ESP) were calculated using semi-empir-
ical AM1 force field in Spartan version 4.0. All analogues were
overlaid using the pyrazole atoms and their electrostatic potential
charges were mapped and examined.
Receptor Binding Assays. CB1 and CB2 Receptor Binding
Assays. The CB1 receptor binding assay involves membranes
isolated from a HEK-293 expression system, whereas the CB2
receptor is expressed in CHO-K1 cells (Sigma-Aldrich Chemical
Co., St. Louis, Mo). The methods used for performing binding
assays in transfected cells expressing human CB1 or CB2 receptors
are similar to those previously described for rat brain membrane
1
gel to yield the ketone (36) as a yellow oil. H NMR (CDCl3) δ:
1.47-1.55 (m, 2H), 1.76-1.88 (m, 4H), 1.93 (t, J ) 6.9, 2H), 3.04
(t, J ) 6.6, 2H), 7.19 (s, 1H), 7.26 (d, J ) 8.1, 1H), 7.67 (d, J )
8.1, 1H).
Ethyl (2-Chloro-5-oxo-5,6,7,8,9,10-hexahydrobenzo[8]annulen-
6-yl)(oxo)acetate (37). First, 242 mg (10.5 mmol) of sodium was
dissolved in 6 mL of absolute ethanol. Then 0.72 mL (5.27 mmol)
of diethyl oxalate was added. To the above mixture was added
dropwise a solution of 1.1 g (5.27 mmol) of ketone 36 in 50 mL of
absolute ethanol. The resulting mixture was stirred overnight. The
reaction was concentrated and then diluted with water and extracted
with ethyl acetate. The aqueous layer was acidified with conc HCl
and extracted with ethyl acetate (3 × 60 mL). The combined organic
layers were washed with water and brine and then concentrated.
The resulting slurry was purified by chromatography on silica gel
to give 37 (1.5 g, 20% over 4 steps from 33) as a colorless oil. 1H
NMR (CDCl3) δ: 1.37 (t, J ) 7.2, 3H), 1.60-2.00 (m, 5H),
2.60-2.80 (m, 3H), 2.70 (t, J ) 7.2, 1H), 4.37 (q, J ) 7.2, 2H),
7.27 (m, 2H), 7.35 (d, J ) 9.0, 1H).
Ethyl 9-Chloro-1-(2,4-dichlorophenyl)-4,5,6,7-tetrahydro-1H-
benzo[7,8]cycloocta[1,2-c]pyrazole-3-carboxylate (39). A mixture
of the ester 37 and 2,4-dichlorophenylhydrazine hydrochloride salt
in 30 mL of absolute ethanol was stirred at room temperature for
2 h, then was heated to reflux for 12 h. The reaction was cooled to
room temperature, and most of the ethanol was removed in vacuo.
The resulting slurry was poured into 60 mL of water and extracted
with ethyl acetate (3 × 50 mL). The combined organic layers were
washed with aqueous NaHCO3, water, dried with Na2SO4, and
concentrated. The residue was purified by column chromatography
on silica gel to yield 39 (0.94 g, 60.0%) as a yellow solid. 1H NMR
(CDCl3) δ: 1.42 (t, J ) 6.0, 3H), 1.58 (m, 2H), 1.80 (m, 1H), 2.07
(m, 1H), 2.45-2.90 (m, 3H), 3.28 (m, 1H), 4.45 (q, J ) 6.0, 2H),
6.73 (d, J ) 9.0, 1H), 6.97 (d, J ) 9.0, 1H), 7.20-7.45 (m, 4H).
9-Chloro-1-(2,4-dichlorophenyl)-4,5,6,7-tetrahydro-1H-
benzo[7,8]cycloocta[1,2-c]pyrazole-3-carboxylic acid (40). Ethyl
ester 39 (210 mg, 0.47mmol) was dissolved in 30 mL of MeOH.
To the above solution was added 30 mL of 1 N NaOH. The mixture
was stirred at room temperature overnight. The methanol was
removed in vacuo. The resulting solution was acidified with HCl
and extracted with ethyl acetate (3 × 50 mL). The combined organic
phases were washed with H2O and brine and dried with Na2SO4.
The solvent was removed to give 40 as a white solid (190 mg,
96%). 1H NMR (CDCl3) δ: 1.53-1.62 (m, 2H), 1.74-1.86 (m,
2H), 2.39-2.88 (m, 3H), 3.23-3.31 (m, 1H), 6.71 (d, J ) 6.3,
1H), 6.98 (d, J ) 8.1, 1H), 7.26-7.39 (m, 4H).
9-Chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-4,5,6,7-tet-
rahydro-1H-benzo[7,8]cycloocta[1,2-c]pyrazole-3-carboxam-
ide (11). Following the procedure in the preparation of 6, 11 (67%)
was obtained from 40 as a white solid. 1H NMR (CDCl3) δ:
1.35-1.85 (m, 10H), 2.50 (m, 1H), 2.60-2.85 (m, 2H), 2.88 (m, 4H),
3.40 (m, 1H), 6.68 (d, J ) 8.4 Hz, 1H), 6.95 (dd, J ) 8.1), 7.28 (m,
3H), 7.38 (s, 1H), 7.63 (s, 1H). 13C NMR δ: 23.75, 23.93, 24.45, 25.85,
30.61, 32.87, 124.91, 126.09, 127.08, 128.20, 130.18, 130.54, 130.62,
131.11, 133.25, 135.50, 136.11, 136.31, 143.75, 145.08, 160.36. MS
m/z 503 [M + H]+. Anal. (C25H25Cl3N4O), C, H, N.
(3aR,8aS)-3-{[9-chloro-1-(2,4-dichlorophenyl)-4,5,6,7-tetrahy-
dro-1H-benzo[7,8]cycloocta[1,2-c]pyrazol-3-yl]carbonyl}-3,3a,
8,8a-tetrahydro-2H-indeno[1,2-d][1,3]oxazol-2-one (41). To a
solution of 40 (360 mg, 0.85 mmol) in 15 mL of dichloromethane
at 0 °C was added oxalyl chloride (0.15 mL, 1.71 mmol). Ten min
later, 2 drops of DMF was added and the reaction was allowed to
warm to room temperature. After stirring for 2 h, the solvent was
removed in vacuo. Toluene was added to the mixture and then
removed in vacuo to give the acyl chloride.

3538 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Zhang et al.
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preparations. Binding is initiated with the addition of 40 pM of
cell membrane protein to assay tubes containing [3H]-44 (ca. 130
Ci/mmol) or [3H]-1 (ca. 22.4 Ci/mmol), a cannabinoid analogue
(for displacement studies), and a sufficient quantity of buffer (50
mM Tris ·HCl, 1 mM EDTA, 3 mM MgCl2, 5 mg/mL BSA, pH
7.4) to bring the total incubation volume to 0.5 mL. All assays are
performed in polypropylene test tubes. In the displacement assays,
the concentrations of [3H]-44 and [3H]-1 are 7.2 nM and 20 nM,
respectively. Nonspecific binding is determined by the inclusion
of 10 µM unlabeled 44 or 1. All cannabinoid analogues are prepared
by suspension in buffer A from a 1 mg/mL ethanol stock. Following
incubation at 30 °C for 1 h, binding is terminated by vacuum
filtration through GF/C glass fiber filter plates (Packard, Meriden,
CT, pretreated in buffer B for at least 1 h) in a 96-well sampling
manifold (Millipore, Bedford, MA). Reaction vessels are washed
twice with 4 mL of ice cold buffer (50 mM Tris·HCl, 1 mg/mL
BSA). The filter plates are air-dried and sealed on the bottom.
Liquid scintillate is added to the wells and the top sealed. After
incubating the plates in cocktail for at least 2 h, the radioactivity
present is determined by liquid scintillation spectrometry. Assays
are done in duplicate, and results represent the combined data of
3-6 independent experiments. Saturation and displacement data
are analyzed by unweighted nonlinear regression of receptor binding
data For displacement studies, curve-fitting and IC50 calculation
are done with GraphPad Prism (GraphPad Software, Inc., San
Diego, CA), which fits the data to one- and two-site models and
compares the two fits statistically.
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N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazole-3-carboxamide (SR141716A) interaction with LYS
3.28(192) is crucial for its inverse agonism at the cannabinoid CB1
receptor. Mol. Pharmacol. 2002, 62 (6), 1274–1287.
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N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazole-3-carboxamide (SR141716A) interaction with LYS
3.28(192) is crucial for its inverse agonism at the cannabinoid CB1
receptor. Mol. Pharmacol. 2002, 62 (6), 1274–1287.
(24) Hurst, D.; Umejiego, U.; Lynch, D.; Seltzman, H.; Hyatt, S.; Roche,
M.; McAllister, S.; Fleischer, D.; Kapur, A.; Abood, M.; Shi, S.; Jones,
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Cannabinoid Receptor Ligands with Lower Lipophilicity. J. Med.
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of SR141716A with the CB1 cannabinoid receptor as determined
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GTP-γ-[35S] Assay. GTP-γ-[35S] assays were also performed
to determine the ability of 6-11 to shift the binding curves of the
agonists 44 or 1. Reaction mixtures consisted of either 44 (2.5 pM
to 25 µM) or 1 (10 pM to 100 µM), 20 µM GDP, and 100 pM
GTP-γ-[35S] in 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 5 mM
MgCl2, 100 mM NaCl, and 1 mg/mL BSA. The effects of these
compounds on agonist binding were compared at concentrations
of 1, 10, and 100 nM vs reactions with no antagonist in a final
reaction mixture volume of 0.5 mL. Binding was determined using
membrane preparations as previously described.17 Data analysis
was performed using global nonlinear regression analysis of the
dose-response curves (Prism, GraphPad), and pA2 values were
calculated. The calculations were performed with the slope of the
Schild line constrained to 1, as well as unconstrained, and an F-test
(P < 0.05) was used to determine the best model.
Acknowledgment. This work was supported by the National
Institute of Drug Abuse, National Institutes of Health, Bethesda,
MD (grant no. DA019217).
Supporting Information Available: Elemental analysis data of
compounds 6-11. This material is available free of charge via the
Internet at http://pubs.acs.org.
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