6-methoxy-VV-alkyl isatin acylhydrazone derivatives as a novel series of potent selective cannabinoid receptor 2 inverse agonists: Design, synthesis, and binding mode prediction

Article abstract of DOI:10.1021/jm801353p

Recently, we discovered and reported a series of VV-alkyl isatin acylhydrazone derivatives that are potent CB2 agonists. Here, we describe a novel series of selective CB2 inverse agonists resulting from introduction of a methoxy moiety in position 6 of th

Full text of DOI:10.1021/jm801353p

J. Med. Chem. 2009, 52, 433–444
433
6-Methoxy-N-alkyl Isatin Acylhydrazone Derivatives as a Novel Series of Potent Selective
Cannabinoid Receptor 2 Inverse Agonists: Design, Synthesis, and Binding Mode Prediction
Philippe Diaz,† Sharangdhar S. Phatak,‡ Jijun Xu,† Fanny Astruc-Diaz,† Claudio N. Cavasotto,‡ and Mohamed Naguib*,†
Department of Anesthesiology and Pain Medicine, Unit 409, The UniVersity of Texas M. D. Anderson Cancer Center, 1400 Holcombe
BouleVard, Houston, Texas 77030, School of Health Information Sciences, The UniVersity of Texas Health Science Center at Houston,
7000 Fannin, Suite 860B, Houston, Texas 77030
ReceiVed October 27, 2008
Recently, we discovered and reported a series of N-alkyl isatin acylhydrazone derivatives that are potent
CB2 agonists. Here, we describe a novel series of selective CB2 inverse agonists resulting from introduction
of a methoxy moiety in position 6 of the isatin scaffold. These novel 6-methoxy-N-alkyl isatin acylhydrazone
derivatives exhibited high CB2 functional activity and selectivity at human CB2. Compound 16 (MDA77)
had high activity (EC50 ) 5.82 nM) at CB2 and no activity at CB1. Compound 15 (MDA55) (Ki ) 89.9
nM, EC50 ) 88.2 nM at CB2) inhibited the effect of compound 1 (MDA7), a selective CB2 agonist, in an
animal model of neuropathic pain. The molecular modeling study presented here represents a first study of
CB2 based on the structure of ꢀ2-adrenergic receptor. A ligand-based homology model of the CB2 binding
site was developed, and on the basis of our results, we propose a general binding mode for this class of
inverse agonists with CB2.
Introduction
and multiple sclerosis.19-22 CB2 expression within immune
cells, induction of CB2 expression after injury or inflammation,
and the discovery that immunomodulation by cannabinoids is
absent in CB2 knockout mice23 suggest that CB2 plays a key
role in immunomodulatory activity and have identified CB2 as
an attractive therapeutic target for immunomodulation.24-26
Several CB2-selective inverse agonists have been described
in the literature.24The chemical structures of a number of CB2
modulators are shown in Figure 1. Compound 3 is the first
CB2-selective inverse agonist described.27 As in the design
of CB1 antagonists such as N-(piperidinyl)-5-(4-chlorophe-
nyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxa-
mide (SR141716), compound 3 was designed using a pyrazole
moiety.28 Compounds 2 and 3 have been extensively used as
standards against which to measure the specificity of various
cannabinoid agonists at CB2 in animal models. Half-maximal
effective concentration (EC50) values of compound 2 and
compound 3 for inhibition of guanosine-5′-triphosphate
(GTP)γ[35S] binding to CB2 are 76.6 nM and 10.4 nM,
respectively.29
Compound 6, a CB2-selective inverse agonist, has been
shown to inhibit carrageenin-induced mouse paw edema in a
dose-dependent manner.30 In addition, compound 6 inhibited
immunoglobulin E-mediated cutaneous inflammation,31 had
antipruritic activity in a mouse model of chronic dermatitis,32
and was also able to inhibit dinitrofluorobenzene- or 2-arachi-
donoylglycerol-induced ear swelling but had no effect on
arachidonic acid-induced ear swelling.33 On the other hand, in
an animal model of cutaneous contact hypersensitivity, genetic
deletion or pharmacologic blockade of cannabinoid receptors
enhanced contact allergic inflammation.1 The structure-activity
relationships for some analogues of compound 6 have been
described.34
The endogenous cannabinoid (endocannabinoid) system
consists of cannabinoid receptors (cannabinoid receptor 1
[CB1]a and cannabinoid receptor 2 [CB2]), endogenous ligands,
and several proteins responsible for their synthesis, transport,
and hydrolysis. In animal models of allergic contact dermatitis1
and peripheral neuropathy,2 increases in endocannabinoid levels
seem to represent a protective mechanism aimed at counteracting
pain and inflammation.3 The two cannabinoid receptors, CB1
and CB2, have been characterized and cloned.4,5
CB1 is found predominantly in the brain, where the highest
densities are in the hippocampus, cerebellum, and striatum.6
Cognitive impairment (and psychoactivity) induced by ∆9-
tetrahydrocannabinol is mediated by CB1 in the isocortex and
allocortex (i.e., hippocampus).7
CB2 is expressed mainly on immune tissuessthe spleen,
tonsils, monocytes, and B and T lymphocytes.5,8 Selective CB2
agonists appear to be devoid of central effects attributable to
CB1 activation.9,10 CB2 mRNA and/or proteins are increased
during different inflammatory conditions. For example, it has
been shown that CB2 expression is increased in the dorsal root
ganglia and spinal cord of animals after spinal nerve ligation,11,12
sciatic nerve injury,13 or saphenous nerve ligation14,15 in the
bladder of rats after acute or chronic inflammation,16 in women
after endometrial inflammation,17 and in humans and rats with
acute pancreatitis.18 CB2 is up-regulated in reactive microglial
cells in Alzheimer’s disease, Huntington’s disease, simian
immunodeficiency virus-induced encephalitis, HIV encephalitis,
* To whom correspondence should be addressed. Phone: 713-745-4948,
Fax: 713-792-7591, E-mail: Naguib@mdanderson.org.
†
Department of Anesthesiology and Pain Medicine, Unit 409, The
University of Texas M. D. Anderson Cancer Center.
Like compound 6, compound 4 has been shown to be an
immunomodulatory agent against inflammatory disorders in
which leukocyte recruitment is involved.25 The structure-activity
relationships for this class of compounds have been described.35
Compound 4 inhibited leukocyte trafficking induced by chemok-
ines or by antigen challenge in several rodent in vivo models
‡
School of Health Information Sciences, The University of Texas Health
Science Center at Houston.
a Abbreviations: CB1, cannabinoid receptor 1; CB2, cannabinoid receptor
2; DMSO, dimethyl sulfoxide; EC50, half-maximal effective concentration;
GTP, guanosine-5′-triphosphate; hCB1, human CB1; hCB2, human CB2;
IC50, median inhibition concentration; Ki, inhibition constant; NMR, nuclear
magnetic resonance.
10.1021/jm801353p CCC: $40.75
2009 American Chemical Society
Published on Web 12/30/2008

434 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Diaz et al.
Figure 1. Chemical structures of CB2-selective modulators (1-6) and a nonselective CB agonist (7). (1) MDA7;10 (2) AM630;29 (3) SR144528;27
(4) Sch.336;36 (5) N-[1-cyclohexylmethyl-2-(2,3-dihydro-1-benzofuran-5-ylmethyl)-1H-benzimidazol-5-yl]-N-methylbenzenesulfonamide; (6) JTE-
907;30 (7) CP55,940.77
and blocked ovalbumin-induced lung eosinophilia in a mouse
model of allergic asthma;36 these effects have been attributed
to down-regulation of phosphorylation of the monocyte-specific
actin-binding protein L-plastin by compound 4.25 This mecha-
nism may explain the ability of compound 4 to modulate
immune cell mobility and explains its effects on antigen-induced
monoarticular arthritis and autoimmune encephalomyelitis in
the rat.
Figure 2. Modification of isatin acylhydrazone that changed CB2
agonists to CB2 inverse agonists.
In a high-throughput screening program focused on CB2, a
novel series of benzimidazole derivatives exhibiting CB2 affinity
was recently identified.37 After chemical optimization, com-
binding mode for this novel class of inverse agonists and to
pound 5 was described as a CB2 inverse agonist that showed a
identify residues involved in ligand recognition.
binding affinity for CB2 of 0.7 nM and an EC50 of 2.2 nM.37
Compound 5 reversed the effect of a cannabinoid agonist in a
competition assay with an inhibition constant (Ki) of 1.2 nM.
Methods
Chemistry. The synthesis outlined in Scheme 1 proceeded
in two chemical steps from 6-methoxyisatin.39 N-Alkylation of
the 6-methoxyisatin with commercially available alkyl halide
using cesium carbonate with microwave irradiation in N,N-
dimethylformamide afforded the desired products in good yields.
Condensation of the resulting N-substituted isatin with hydrazine
derivatives afforded the desired hydrazone in good to moderate
yields. For hydrazone derivatives 22, 25, 26, and 29, described
below, the 1H nuclear magnetic resonance (NMR) spectra
indicatedthepresenceoftworotamersaspreviouslydescribed.38,40
Crystallographic Analyses. The structure of compound 15
was examined by X-ray diffraction to confirm the Z configu-
ration. Compound 15 yielded crystals of suitable quality for
X-ray diffraction by slow evaporation of an ethyl acetate
solution. As expected, the structure obtained (Figure 3) was the
one in which the hydrogen borne by nitrogen atom N12 might
form a hydrogen bond with oxygen atom O10.
Molecular Modeling Studies. Molecular modeling studies
were performed on the 6-methoxyisatin acylhydrazone deriva-
tives to identify a putative binding site of this class of CB2
inverse agonists. There is uncertainty and limited experimental
evidence about structure-activity relationships and ligand
interactions for CB2-specific inverse agonist compounds.34,41-45
Depending on ligand chemotypes, the ligand:CB2 interaction
In this series, the replacement of an ethoxyphenyl group with a
benzofuran resulted in a change in biological activity from
agonist to inverse agonist in functional assays.37
Recently, we described a novel series of CB2 agonists based
on N-alkyl isatin acylhydrazone derivatives.38 Similar to what
was done by others for compound 5, we sought to design and
synthesize a novel series of CB2 inverse agonists by using our
agonist scaffold and studying the impact of substitutions in the
isatin ring on functional activity. Our first attempt, introducing
electron-withdrawing or electron-donating groups in position 5
or 7 of the isatin ring, resulted in a decrease in functional
activity. However, substitution in position 6 resulted in selective
and potent ligands. A methoxy moiety was chosen to mimic
the oxygen of the compound-5 benzofuran ring. A series of
6-methoxy-N-alkyl isatin acylhydrazone derivatives, based on
our previously reported potent CB2 agonists,38 was prepared
and evaluated (Figure 2). The 6-methoxy-N-alkyl isatin core
was subsequently optimized to find potent selective CB2 inverse
agonists. In this report, we describe the design, synthesis,
structure-activity relationships, and in vivo and in vitro
evaluations of this novel series of potent and selective CB2
inverse agonists. Furthermore, we present the results of molec-
ular modeling studies that we performed in order to suggest a

Isatin Acylhydrazones as SelectiVe CB2 InVerse Agonists
Scheme 1. Synthetic Routea
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 435
a Reagents and conditions: (a) (i) chloral hydrate, hydroxylamine hydrochloride, sodium sulfate, hydrochloric acid, 55 °C, 12 h; (ii) polyphosphoric acid,
55-60 °C, 6 h.39 (b) CsCO3, N,N-dimethylformamide, R1Br, microwave irradiation, 140 °C, 10 min. (c) H2N-HN-CO-R2, AcOH, EtOH, tetrahydrofuran,
room temperature, 12 h.
determined in the presence of 10 µM compound 7 (Tocris
Bioscience). The sample was incubated in a final volume of
0.1 mL for 60 min at 30 °C and then filtered on a GF/B UniFilter
microplate (Perkin-Elmer, catalogue no. 6005177) presoaked
in 0.5% polyethyleneimine for 2 h at room temperature. Filters
were washed six times with 4 mL of cold binding buffer (50
mM Tris, pH 7.4, 2.5 mM EDTA, 0.5% protease-free bovine
serum albumin), and the amount of bound [3H]7 was determined
by liquid scintillation counting. Median inhibition concentration
(IC50) values were determined by nonlinear regression using
the one-site competition equation. The Ki values were calculated
using the Cheng-Prusoff equation (Ki ) IC50/(1 + (L/KD)),
where L ) concentration of radioligand in the assay and KD )
affinity of the radioligand for the receptor.
In Vivo Evaluation: Animals. Adult male Sprague-Dawley
rats (Harlan Sprague-Dawley) weighing 120-150 g were used
in experimental procedures approved by the Animal Care and
Use Committee of The University of Texas M. D. Anderson
Cancer Center. Animals were housed three per cage on a 12 h
light/12 h dark cycle with water and food pellets available ad
libitum.
Figure 3. View of compound 15 showing the atom labeling scheme.
Displacement ellipsoids are scaled to the 50% probability level.
is considered to be mediated by hydrogen bonds and/or π-π
stacking interactions within transmembrane regions 3-7.46-48
Thus, the aim of this molecular modeling study was to suggest
a binding mode for the isatin acylhydrazone class of CB2-
selective inverse agonists and to identify residues involved in
ligand recognition.
Lumbar 5/6 Spinal Nerve Ligation Pain Model. All
surgical procedures were performed while rats were under deep
isoflurane-induced anesthesia in 100% O2. The spinal nerve
ligation was performed as described previously.50 Briefly, a
midline incision was made above the lumbar spine to expose
the left L6 transverse process. The process was then removed,
the left L5 and L6 spinal nerves were isolated, and both nerves
were tightly ligated with 6-0 silk. A prophylactic antibiotic
(norfloxacin 5 mg/kg subcutaneously) and a prophylactic
analgesic (buprenorphine 0.2-0.5 mg/kg subcutaneously or
morphine 2.5 mg/kg subcutaneously) were administered once
daily for 3 days. The rats were allowed to recover for 5-6 days
before being used for behavioral testings. All the experiments
were conducted 10-14 days after spinal nerve ligation.
Assessment of Mechanical Withdrawal Thresholds. For
assessment of antiallodynic effect, rats were placed in a
compartment with a wire mesh bottom and allowed to acclimate
for a minimum of 30 min before testing. Mechanical sensitivity
was assessed using a series of Von Frey filaments with
logarithmic incremental stiffness (0.41, 0.70, 1.20, 2.00, 3.63,
5.50, 8.50, and 15.1 g) (Stoelting, Wood Dale, IL), as previously
described,51 and 50% probability withdrawal thresholds were
calculated with the up-down method.52 In brief, beginning with
the 2.00 g probe, filaments were applied one by one to the
plantar surface of a hind paw for 6-8 s. If no withdrawal
response was observed, the next stiffer filament was applied; if
there was a withdrawal response, the next less stiff filament
was applied. Six consecutive responses after the first change in
the response were used to calculate the withdrawal threshold
(in grams). When response thresholds fell outside the range of
detection, 15.00 g was assigned for absence of response to all
Pharmacology: Cannabinoid Receptor-Mediated Func-
tional Activity. Functional activity for acyl hydrazone was
evaluated using GTPγ[35S] assay in Chinese hamster ovarian
cell membrane extracts expressing recombinant human CB1
(hCB1) or human CB2 (hCB2). The assay relies on the binding
of GTPγ[35S], a radiolabeled nonhydrolyzable GTP analogue,
to the G protein upon binding of an agonist of the G-protein-
coupled receptor. In this system, agonists stimulate GTPγ[35S]
binding, whereas antagonists have no effect and inverse agonists
decrease GTPγ[35S] basal binding. CB1 and CB2 assay data
are presented as the mean of two determinations. Assay
reproducibility was monitored by the use of a reference
compound, compound 7. For replicate determinations, the
maximum variability tolerated in the test was of (20% around
the average of the replicates. Efficacies (Emax) for CB1 and CB2
are expressed as a percentage of the efficacy of compound 7.
Pharmacology: Binding Assays. Compound 15 was screened
in a competitive binding experiment using membranes of CHO-
K1 cells selectively expressing hCB2 at different concentrations
in duplicate.49 The competitive binding experiment was per-
formed in 96-well plates (Masterblock) containing binding buffer
(50 mM Tris, pH 7.4, 2.5 mM EDTA, 0.5% protease-free bovine
serum albumin), recombinant membrane extracts (0.25 µg
protein/well), and 1 nM [3H]7 (Perkin-Elmer, NEX-1051, 161
Ci/mmol, diluted in binding buffer). Nonspecific binding was

436 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Diaz et al.
tested fibers, and 0.25 g was assigned for withdrawal response
to all tested fibers. The percentage maximal possible effect was
calculated as ([postdrug threshold - baseline threshold]/[cutoff
threshold (15 g) - baseline threshold]) × 100.
bonding interactions with the serine residues in transmembrane
region 4 or with lysine or threonine in transmembrane region
3.46,47,60 Hence, the R2 group was placed such that it could
occupy pockets facing toward transmembrane regions 2 and 3
or transmembrane regions 3, 4, and 5. These two conformations
were seeded into the pocket, and an ensemble of 200 structures
for each conformation was generated by randomizing the
position and orientation of the ligand and then performing a
multistep energy minimization in which the van der Waals
interaction was gradually switched from soft to full interaction,
as performed in other cases.61-63 The ligand and receptor were
held flexible in this stage without any restraints. The structures
in the ensemble were then ranked using a crude binding energy
estimation (see Experimental Section). Ten structures were
subjected to a full flexible-ligand:flexible side chain Monte
Carlo-based global energy optimization as has been performed
before in G protein-coupled receptors and other proteins.61-66
From these complexes, two representative structures were
chosen on the basis of binding affinity estimation. The binding
sites were visually inspected, and the final complex was retained
in which the R1 group was oriented toward the lipophilic pocket
as described earlier and the carbonyl was positioned close
enough to any charged-residue side chain to form a hydrogen
bond. As the absence of carbonyl group resulted in lack of
affinity for isatin acylhydrazone compounds, we assumed that
absence of hydrogen bond interaction with the carbonyl possibly
was related to a wrong pose (Figure 4A).
Data Analysis. Statistical analyses were carried out using
BMDP 2007 (Statistical Solutions, Saugus, MA). Data were
analyzed using one-way analysis of variance or t test, where
appropriate. If findings on analysis of variance were significant,
Tukey-Kramer post hoc analysis was used for multiple group
comparison. Area under the curve (AUC) was calculated using
the trapezoidal rule. The results are presented as mean ( SEM
and were considered significant at P < 0.05.
Results and Discussion
In our previously published work, we observed that the
carbonyl group is essential for CB2 binding of N-alkyl isatin
acylhydrazone derivatives.38 Therefore, our first objective in the
present study was to find the optimal building blocks for R1
and R2 (Figure 2) needed for CB2 inverse agonist activity. In
our previously published agonist series,38 we noted that the use
of benzohydrazide moiety (R2 is a phenyl ring) resulted in
compounds with the highest affinities for CB2. Thus, we decided
to study first the substituent effect for R1 and in a second step
R2.
Recently, Cavasotto et al.53 developed and validated a method
of ligand-steered homology modeling of the binding site in
which existing ligands are explicitly used to shape and optimize
the binding site through a docking-based stochastic global
energy minimization procedure. The ligand and receptor are
considered flexible throughout the modeling process, which
ensures a better coverage of the energy landscape. This is
particularly important in the case of CB2,as there is little
structural information available about protein-ligand inter-
actions.41,46-48,54 To accomplish our aim, we constructed a
ligand-steered homology model of CB2 based on the crystal
structure of ꢀ2-adrenergic receptor (the approach is summarized
in Experimental Section).55-57 The template structure of ꢀ2 used
in our study was crystallized in its inactive state55-58 hence,
the CB2 model obtained may have been in an inactive state
suitable for studying the binding of inverse agonist compounds,
as suggested by other studies.48,59
Generation of a CB2 Model in Agreement with Experi-
mental Evidence. Aromatic and hydrogen bond interactions
play an important role in binding of CB2 inverse agonist
compounds. Though CB2 models are not as extensively studied
as CB1 models, modeling and structure-activity relationship
studies have suggested the presence of a hydrophobic pocket
within transmembrane regions 3, 5, 6, and 7 surrounded by
residues F3.36, W6.48, and W5.43.41,45,60 Hydrogen bond
interactions have also been implicated in binding of antagonists
and inverse agonists of CB2.46,47 However, there is not total
agreement on the definition of the binding site and the
interactions between different classes of compounds and the
receptor.
Correlation between Ligand Binding to the CB2 Struc-
tural Model and Structure-Activity Relationship Data.
Because of the limited experimental evidence available with
which to validate our model,41,46-48,54 we evaluated the accuracy
of the model by its ability to explain structure-activity
relationship data on our compounds. In our representative model
with compound 18 (Figure 4B), the carbonyl group is located
at 2 Å from the side chain hydrogen of K3.28, suggesting a
possible ligand-receptor interaction via hydrogen bond. Simi-
larly, the location of the methoxy group seems to indicate a
hydrogen bond interaction with Y5.39. These observations
suggest the possible ligand-receptor interactions and also
explain the orientation of the compound. The cyclohexyl moiety
is properly located in the hydrophobic pocket to form van der
Waals interactions with residues W6.48, F3.36, and W5.43.
Specifically, it is important to note the change in affinities with
different groups at the R1 position facing toward the aromatic
domain. The optimal chain is a n-pentyl group because
compound 16 was 15 times more potent than compound 15 or
compound 17 in terms of efficacy. For compound 16, the R2
group is oriented toward the lipophilic pocket formed by residues
F2.57, F2.61, F2.64, and F7.35, and its overall position suggests
favorable aromatic stacking interaction with F2.61 and F2.64.
It should be mentioned that as in the agonist series, use of the
methylcyclohexyl moiety resulted in loss of selectivity for CB2
compared to CB1. On the other hand, replacing the methylcy-
clohexyl moiety with a benzyl moiety resulted in restoration of
the CB2 selectivity but a decrease in terms of potency for CB2
functional activity. Introducing an oxygen atom in the alkyl
chain was poorly tolerated in compound 21 compared to
compound 17. Increasing the length between the cyclohexyl ring
and the nitrogen atom, such as in compound 20, resulted in a
dramatic decrease in activity but also restored CB2 selectivity
compared to the methylcyclohexyl analogue, compound 18. This
loss of activity might be explained by steric clashes with residues
W6.48 and F3.36, which might result in the loss of hydrogen
bond interaction with K3.28 or Y5.39.
From the set of compounds displaying inverse agonist activity
(Table 1), we selected compound 18 to model the binding site,
in accordance with the ligand-steered homology modeling
method and similar to what has been done with G protein-
coupled receptors and protein kinases.53,61 Because of the
sensitivity of EC50 values to substitutions of the R1 group, we
hypothesized that the R1 group was facing the interior of the
pocket and was not exposed to the free solvent. Thus, in our
initial model, the R1 group was oriented toward the hydrophobic
pocket. Prior modeling studies for antagonists indicate hydrogen

Isatin Acylhydrazones as SelectiVe CB2 InVerse Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 437
Table 1. Determination of Potency (EC50) and Maximal Stimulation (Emax) on hCB1 and hCB2 Receptors of Compounds 15-29a
a CB1 and CB2 assay data are presented as the mean of two determinations. Assay reproducibility was monitored by the use of a reference compound
7. For replicate determinations, the maximum variability tolerated in the test was of (20% around the average of the replicates). Efficacies (Emax) for CB1
or CB2 are expressed as a percentage relative to the efficacy of compound 7. ND ) not determined (plateau was not reached at 10 µM dose).
As the lipophilic pocket formed by residues F2.57, F2.61,
F2.64, and F7.35 seemed to be large, we decided to explore
the substituent effect for R2, keeping a n-pentyl group for R1
as it was the optimal chain length. Replacing the phenyl ring
(compound 16) with a cyclohexyl (compound 22) or a meth-
ylcyclohexyl (compound 26) resulted in a slight decrease in CB2
functional activity, which might be attributed to a loss of the
favorable aromatic stacking interaction with F2.61 and F2.64
and compound 16. Branched alkyl moieties were slightly better
tolerated than the cylohexyl ring, as compounds 23, 24, 25, and
28 had twice the CB2 activity of compound 22. Introduction of
an oxygen atom did not affect the activity (23, 24). Surprisingly,

438 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Diaz et al.
The loss of potency for compounds 15 and 17 can be
explained as follows. We suggest three possible ligand-receptor
interactions: the H-bond with K3.28, the hydrophobic pocket
surrounded by W6.48, F3.36, and W5.43, and the π-π
interactions with F2.61 and F2.64. With a shorter carbon chain,
like that in compound 17, the compound may not satisfy all
three ligand-receptor interactions, resulting in loss of interac-
tions, whereas with a longer carbon chain, like that in compound
15, the affinity loss might be explained by steric clashes in the
hydrophobic pocket after ligand binding. Compound 16 opti-
mally satisfies all three interactions and thus shows higher
affinity. In compound 21, an unfavorable interaction occurs as
a polar atom (oxygen) is introduced in the hydrophobic pocket,
resulting in loss of affinity.
A competitive binding assay was also performed in mem-
branes of CHO-K1 cells selectively expressing the hCB2 for
compound 15. As expected, compound 15 displaced [3H]7 from
hCB2 receptors with a Ki value of 89.9 ( 5.3 nM. In rats, spinal
nerve ligation produced tactile allodynia 1 week after surgery,
as demonstrated by a reduction in paw withdrawal threshold to
mechanical stimulation to 2.3 ( 0.2 g using Von Frey filaments.
Intraperitoneal administration of 10 mg/kg or 15 mg/kg of
compound 15 did not attenuate tactile allodynia, and the
response seen with compound 15 was not different from that
seen with its vehicle (N-methylpyrrolidone [30%], propylene
glycol [30%], ethyl alcohol [10%], Cremophor (10%), and
water) (Figure 5A). Nevertheless, intraperitoneal administration
of 15 mg/kg of compound 1, a selective CB2 agonist,10 resulted
in significant antiallodynic effects (Figure 5B) compared with
its vehicle (hydroxypropyl-ꢀ-cyclodextrin). Intraperitoneal ad-
ministration of 10 mg/kg of compound 15 15 min before
intraperitoneal administration of compound 1 antagonized the
effects of compound 1 (Figure 5B), indicating that compound
15 acts as a CB2 antagonist.
Figure 4. (A) CB2 complexed with compound 18 (yellow carbon
atoms). (B) Possible hydrogen bond with K3.28 is represented as orange
dashes. The π-π stacking can be seen with F2.61 and F2.64. Helices
are colored in red. Helices 1 and 4 are cut away for clarity. The figure
was prepared using PyMol (www.pymol.org).
Conclusions
In summary, we have discovered a novel series of CB2
inverse agonists that are potent and selective. A major focus of
the optimization effort was to increase the functional activity
of this novel series and to avoid potential CB1 central nervous
system adverse effects. Compound 15 inhibited the effect of a
selective CB2 agonist (compound 1)10 in an animal model of
neuropathic pain. More potent CB2-selective compounds, such
as compound 16, and compounds with better druglike profiles,
such as compound 27, were also discovered and will be
evaluated for their in vivo activities in an in vivo model of
inflammation. It should be mentioned that compound 18, a CB1
agonist and CB2 inverse agonist, exhibited an interesting profile,
which we are currently evaluating.
replacing the methylcyclohexyl moiety of compound 26 with a
methylmorpholine (compound 27) resulted in a 4-fold increase
in CB2 functional activity. It is well established that the
morpholine ring usually adopts a chair conformation and
the four carbon atoms deviate only slightly from coplanarity.67
The nitrogen of the compound-27 morpholine ring might form
an intramolecular hydrogen bond with the hydrogen atom borne
by the hydrazoic moiety.68 For compound 27, an additional CH2
group provides flexibility to the morpholine ring, possibly
exposing it to the solvent, and hence is well tolerated. The
resulting restricted conformation adopted by compound 27 might
be greatly different from the conformation adopted by the
methylcyclohexyl moiety (26) and explain the increase in CB2
activity. Replacing the phenyl ring of compound 18 with a
cyclohexyl ring (compound 29) resulted in a decrease in CB2
activity, probably because of loss of aromatic stacking interac-
tion, as previously mentioned for compound 22, as well as loss
of CB1 functional activity. Aromatic stacking interaction of
compound 18 in hCB1 seems to be necessary for CB1 functional
activity. Compared to compound 22, compound 29 exhibited a
lower CB2 activity, probably because of the rigidity induced
by the introduction of two cyclohexyl rings compared to one
cyclohexyl and a n-pentyl moiety.
The molecular modeling study presented in this work provides
a first study of CB2 based on the structure of ꢀ2-adrenergic
receptor. A ligand-based homology model of the CB2 binding
site was developed, and on the basis of our results we propose
a general binding mode for this class of inverse agonists with
CB2. The lipophilic pocket with W6.48, F3.36, V6.51, W5.43,
and W5.46 could be involved in van der Waals interactions with
the R1 group of the ligands. The R2 group interacts with the
hydrophobic pocket surrounded by residues F2.57, F2.61, and
F2.64 by π-π stacking or van der Waals interactions. The
model also suggests that residue K3.28 could be involved in
hydrogen bonding with the carbonyl group of the ligands or

Isatin Acylhydrazones as SelectiVe CB2 InVerse Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 439
Figure 5. Effects of compound 15 administered by intraperitoneal (ip) injection on tactile allodynia in a spinal nerve ligation neuropathic pain
model in rats (n ) 5 per group). Compound 15 did not have any effect on the withdrawal threshold of the nerve-injured paw (ipsilateral paw) or
the normal paw (contralateral paw). (A) The time course of effects of 10 and 15 mg/kg of compound 15. (B) Area under the curve of the %MPE
effects of 10 mg/kg of compound 15 and 15 mg/kg of compound 1, a selective CB2 agonist. All drugs were administered by ip injection. Administration
of 10 mg/kg of compound 15 15 min before administration of compound 1 antagonized the effects of compound 1 (Figure 5B), indicating that
compound 15 acts as a CB2 antagonist.
of fit, S, ) 1.06. Definitions used for calculating R(F), Rw(F2), and
that Y5.39 could be involved in hydrogen bonding with the
methoxy group.
goodness of fit, S, were as follows:
R(F) ) ∑(|Fo| - |Fc|)/∑|Fo|} for reflections with Fo > 4(σ(Fo)).
Rw(F2) ) {∑w(|Fo|2 - |Fc|2)2/∑w(|Fo|)4}1/2, where w is the weight
given each reflection.
Experimental Section
All chemicals were purchased from Sigma-Aldrich or Acros.
Microwave reactions were conducted using an Initiator EXP
Microwave System (Biotage, Charlottesville, VA). Thin-layer
chromatography analyses were performed on Sigma-Aldrich 60
F254 thin-layer chromatography plates. Column chromatography
S ) [∑w(|Fo|2 - |Fc|2)2/(n - p)]1/2, where n is the number of
reflections and p is the number of refined parameters.
The data were corrected for secondary extinction effects. The
correction takes the form: Fcorr ) kFc/[1 + (4.2(4) × 10-5)Fc2 λ3/
(sin 2θ)]0.25, where k is the overall scale factor. Neutral atom
scattering factors and values used to calculate the linear absorption
coefficient were from the International Tables for X-ray Crystal-
lography (1992).72 All figures were generated using SHELXTL/
PC.71
1
was performed with silica gel 230-400 mesh. H NMR spectra
were recorded on a Bruker 300-MHz DPX NMR spectrometer. 13
C
NMR spectra were recorded on a Bruker 500-MHz DRX NMR
spectrometer. Chemical shifts in ppm are reported relative to either
residual dimethyl sulfoxide (3.35 ppm) or CHCl3 (7.24 ppm) as
internal standards. Signals were abbreviated as follows: s ) singlet,
br s ) broad singlet, d ) doublet, t ) triplet, q ) quadruplet, m )
multiplet. Coupling constants (J) are expressed in hertz.
X-ray data for compound 15. Empirical formula: C22H25N3O3.
Formula weight: 379.45. Temperature: 153(2) K. Wavelength:
j
0.71070 Å. Crystal system: Triclinic. Space group: P1. Unit cell
dimensions: a ) 8.3877(2) Å, R ) 100.3570(10)°, b ) 8.4043(2)
Å, ꢀ ) 103.5370(10)°, c ) 15.1821(4) Å, γ ) 101.3700(10)°.
Volume: 991.00(4) Å3. Z ) 2. Density (calculated): 1.272 mg/m3.
Absorption coefficient: 0.086 mm-1. F(000) 404. Crystal size: 0.24
mm × 0.20 mm × 0.20 mm. θ range for data collection: 2.58 to
27.47°. Index ranges: -10 e h e 9, -10 e k e 10, -15 e l e
19. Reflections collected: 7232. Independent reflections: 4516
[R(int) ) 0.0167]. Completeness to θ ) 27.47°, 99.6%. Absorption
correction: None. Refinement method: Full-matrix least-squares on
F2. Data/restraints/parameters: 4516/0/259. Goodness-of-fit on F2:
1.055. Final R indices [I > 2σ(I)]: R1 ) 0.0485, wR2 ) 0.1088.
R indices (all data): R1 ) 0.0688, wR2 ) 0.1192. Largest diff
X-ray Diffraction of Compound 15. X-ray experimental data
for C21H23N3O2: Crystals grew as large, yellow plates by slow
evaporation of an ethyl acetate solution. The data crystal was cut
from a larger crystal and had approximate dimensions of 0.24 mm
× 0.20 mm × 0.20 mm. The data were collected at room
temperature on a Nonius Kappa charge-coupled device diffracto-
meter using a graphite monochromator with Mo KR radiation (λ
) 0.71073Å). A total of 279 frames of data were collected using
ω scans with a scan range of 2° and a counting time of 144 s per
frame. The data were collected at 153 K using an Oxford
Cryostream low-temperature device. Details of crystal data, data
collection, and structure refinement are listed in Table 1. Data
reduction was performed using DENZO-SMN.69 The structure was
solved by direct methods using SIR9770 and refined by full-matrix
least-squares on F2 with anisotropic displacement parameters for
the non-H atoms using SHELXL-97.71 The hydrogen atoms on
carbon were calculated in ideal positions with isotropic displacement
parameters set to 1.2 × Ueq of the attached atom (1.5 × Ueq for
methyl hydrogen atoms). The hydrogen atoms bound to nitrogen
were observed in a ∆F map and refined with isotropic displacement
parameters. The function ∑w(|Fo|2 - |Fc|2)2 was minimized, where
w ) 1/[(σ(Fo))2 + (0.0567P)2 + (0.2659P)] and P ) (|Fo|2 + 2|Fc|2)/
3. Rw(F2) refined to 0.119, with R(F) equal to 0.0485 and a goodness
peak and hole: 0.390 and -0.227 e ·Å-3
.
Sequence Alignment and Homology Model of CB2. The CB2
and ꢀ2 sequences were aligned based on existing information on
conserved residues within class-A G protein-coupled receptors.73
CB2 lacks the conserved proline in helix 5, so the next highly
conserved residue, tyrosine, was used for the alignment as described
by Xie and colleagues.54 The nomenclature of Ballesteros and
colleagues is used, whereby the most conserved residue in helix X
is labeled as X.50.74 The homology model of CB2 was generated
using the recently crystallized structure of ꢀ2-adrenergic receptor
(PDB code 2RH1) as a template.55-57 The N- and C-terminus
residues (amino acids 1-26 and 316-360) of CB2 and the T4L

440 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Diaz et al.
residues connecting helixes V and VI of ꢀ2-adrenergic receptor were
omitted. The resulting alignment was used as an input to MOD-
ELER 9v475 to develop a CB2 model. Mutagenesis and other CB2
modeling studies suggest the possibility of disulfide bond between
residues Cys 174 and Cys 179 in the E2-loop, which is included in
our homology model.46,47,76 This was followed by a restraint-
minimization to relieve the structural strain stemming from the
replacement of nonconserved residues in the homology modeling
process while keeping the pocket intact. A ligand-steered homology
model for CB2 was developed in a way similar to that used to
develop a ligand-steerred homology model for the melanin-
concentrating hormone receptor, in which existing ligands were
explicitly used to shape and optimize the binding site through a
docking-based stochastic global energy minimization procedure.53
Side chains within 6 Å of the ligand were considered free while
the backbone was kept fixed and the ligand-receptor binding energy
was estimated as the ligand-receptor interaction energy, where the
van der Waals, electrostatic, hydrogen bonding, and torsional energy
terms were considered.
1-Butyl-6-methoxy-isatin (12). The title compound was prepared
as a red solid, using 6-methoxy-isatin and 1-bromobutane according
to general procedure A. Column chromatography (silica gel,
heptane/EtOAc: 6/4) afforded the title compound as an orange solid.
1
Yield: 60%; mp: 70.1 °C. H NMR (CDCl3): δ 0.97 (t, J ) 7.5
Hz, 3H), 1.38-1.43 (m, 2H), 1.64-1.68 (m, 2H), 3.68 (t, J ) 7.0
Hz, 2H), 3.93 (s, 3H), 6.37 (d, J ) 2 Hz, 1H), 6.55 (dd, J ) 2 Hz,
J ) 8.5 Hz, 1H), 7.57 (d, J ) 8.5 Hz, 1H). 13C NMR (CDCl3): δ
13.69 (CH3), 20.12 (CH2), 29.48 (CH2), 39.94 (CH2), 56.13 (CH3),
97.59 (CH), 107.41 (CH), 111.36 (C), 128.04 (CH), 153.52 (C),
159.58 (C), 168.26 (CdO). 181.03 (CdO).
1-(2-Cyclohexylethyl)-6-methoxy-isatin (13). The title com-
pound was prepared as a red solid, using 6-methoxy-isatin and
1-bromo-2-cyclohexylethane according to general procedure A.
Column chromatography (silica gel, heptane/EtOAc: 7/3) afforded
1
the title compound as an red solid. Yield: 65%; mp: 94.8 °C. H
NMR (CDCl3): δ 0.91-1.01 (m, 2H), 1.15-1.31 (m, 4H), 1.55 (q,
J ) 8.5 Hz, 2H), 1.64-1.73 (m, 3H), 1.79 (d, J ) 13 Hz, 2H),
3.69 (t, J ) 7.5 Hz, 2H), 3.93 (s, 3H), 6.35 (d, J ) 2 Hz, 1H), 6.54
(dd, J ) 2 Hz, J ) 8.5 Hz, 1H), 7.58 (d, J ) 8.5 Hz, 1H). 13C
NMR (CDCl3): δ 26.10 (CH2), 26.40 (CH2), 33.09 (CH2), 34.63
(CH2), 35.38 (CH), 38.12 (CH2), 56.10 (CH3), 97.55 (CH), 107.32
(CH), 111.44 (C), 128.06 (CH), 153.44 (C), 159.46 (C), 168.22
(CdO). 181.09 (CdO).
6-Methoxy-1-(2-methoxyethyl)-isatin (14). The title compound
was prepared as a red solid, using 6-methoxy-isatin and 1-bromo-
2-cyclohexylethane according to general procedure A. Column
chromatography (silica gel, heptane/EtOAc: 6/4) afforded the title
compound as an orange solid. Yield: 63%; mp: 110.4-11.4 °C.
1H NMR (CDCl3): δ 3.35 (s, 3H), 3.63 (t, J ) 5.4 Hz, 2H), 3.87
(t, J ) 5.4 Hz, 2H), 3.92 (s, 3H), 6.52-6.56 (m, 2H), 7.56 (dd, J
) 1.5, J ) 7.5 Hz, 1H).). 13C NMR (CDCl3): δ 40.48 (CH2), 56.12
(CH3), 59.04 (CH3), 70.26 (CH2), 98.13 (CH), 108.25 (CH), 111.26
(C), 127.72 (CH), 154.14 (C), 159.90 (C), 168.23 (CdO). 180.71
(CdO).
General Procedure for the Synthesis of the N-Alkyl Isatin
(General Procedure A). A mixture of cesium carbonate (1.46 g,
4.48 mmol), 6-methoxyisatin39 (397 mg, 2.24 mmol), and 1-(bro-
momethyl)cyclohexane (476 mg, 2.69 mmol) in dimethylformamide
(20 mL) in sealed vessels was irradiated at 140 °C for 10 min.
After extraction with ethyl acetate, the organic layer was washed
with hydrochloric acid (0.4N) and water. The organic fraction was
dried over MgSO4 and concentrated under vacuum. Compounds
8-14 were synthesized using the same quantity as described in
this general procedure.
1-Hexyl-6-methoxy-isatin (8). The title compound was prepared
as an orange oil, using 6-methoxy-isatin and 1-bromohexane
according to general procedure A. Column chromatography (silica
gel, heptane/EtOAc: 6/4) afforded the title compound as an orange
1
solid. Yield: 63%. H NMR (CDCl3): δ 0.88 (t, J ) 6.9 Hz, 3H),
1.33-1.41 (m, 6H), 1.65-1.72 (m, 2H), 3.67 (t, J ) 7.5 Hz, 2H),
3.93 (s, 3H), 6.36 (d, J ) 1.8 Hz, 1H), 6.54 (dd, J ) 1.8 Hz, J )
8.4 Hz, 1H), 7.58 (d, J ) 8.4 Hz, 1H).
General Procedure for the Synthesis of Hydrazone Deriva-
tives from the Corresponding Isatins (General Procedure B).
A solution of isatin derivative (0.5 mmol) and benzhydrazide (68
mg, 0.5 mmol) in a solution of acetic acid 10%, ethanol 45%, and
tetrahydrofuran 45% (6.5 mL) was stirred at room temperature for
24 h. The mixture was then concentrated under vacuum to afford
the desired product. Compounds 15-29 were synthesized using
the same quantity as described in this general procedure.
1-Cyclohexylmethyl-6-methoxy-isatin (9). The title compound
was prepared as an orange solid, using 6-methoxy-isatin and
1-bromomethylcyclohexane according to general procedure A. The
resulting solid was washed with a mixture of heptane and AcOEt.
1
Yield: 64%; mp: 162-163 °C. H NMR (CDCl3): δ 1.01-1.27
(m, 5H), 1.68-1.79 (m, 6H), 3.51 (d, J ) 7.5 Hz, 2H), 3.94 (s,
3H), 6.37 (d, J ) 2 Hz, 1H), 6.55 (dd, J ) 2 Hz, J ) 8 Hz, 1H),
7.59 (d, J ) 8 Hz, 1H). 13C NMR (CDCl3): δ 25.65 (CH2), 26.12
(CH2), 30.90 (CH2), 36.35 (CH), 46.51 (CH2), 56.14 (CH3), 97.99
(CH), 107.24 (CH), 111.34 (C), 128.00 (CH), 154.01 (C), 159.88
(C), 168.19 (CdO), 180.99 (CdO).
N′-[(3Z)-1-Hexyl-6-methoxy-2-oxo-1,2-dihydro-3H-indol-3-
ylidene]benzohydrazide (15). The title compound was prepared
as a yellow solid, using 1-hexyl-6-methoxy-isatin and benzhydrazide
according to general procedure B. Column chromatography (silica
gel, heptane/EtOAc: 5/5) afforded the title compound as a yellow
solid. Yield: 74%; mp: 111.4 °C. 1H NMR (CDCl3): δ 0.88 (t, J )
7 Hz, 3H), 1.28 to 1.39 (m, 6H), 1.68-1.74 (m, 2H), 3.73 (t, J )
7.5 Hz, 2H), 3.87 (s, 3H), 6.44 (d, J ) 2 Hz, 1H), 6.64 (dd, J ) 2
Hz, J ) 8 Hz, 1H), 7.51 (t, J ) 7.5 Hz, 2H), 7.59 (t, J ) 7.5 Hz,
1H), 7.80 (d, J ) 8 Hz, 1H), 8.00 (d, J ) 7.5 Hz, 1H), 13.98 (br
s, 1H).). 13C NMR (CDCl3): δ 13.99 (CH3), 22.50 (CH2), 26.63
(CH2), 27.58 (CH2), 31.40 (CH2), 39.97 (CH2), 55.75 (CH3), 97.43
(CH), 107.28 (CH), 112.51 (C), 123.67 (CH), 127.74 (CH), 128.91
(CH), 132.38 (C), 132.60 (CH), 137.29 (C), 144.67 (C), 162.56
(C), 163.05 (C), 163.92 (C). Anal. (C22H25N3O3): C, H, N.
1-Benzyl-6-methoxy-isatin (10). The title compound was pre-
pared as an orange solid, using 6-methoxy-isatin and benzyl bromide
according to general procedure A. The resulting solid was washed
with a mixture of heptane and AcOEt. Yield: 67%; mp: 115.8-116.9
°C. 1H NMR (CDCl3): δ 3.82 (s, 3H), 4.89 (s, 2H), 6.25 (d, J ) 2
Hz, 1H), 6.52 (dd, J ) 2 Hz, J ) 8.5 Hz, 1H), 7.29-7.36 (m, 5H),
7.58 (d, J ) 8.5 Hz, 1H). 13C NMR (CDCl3): δ 43.99 (CH2), 56.04
(CH3), 98.31 (CH), 107.93 (CH), 111.41 (C), 127.41 (CH), 128.04
(CH), 128.14 (CH), 129.06 (CH), 134.73 (C), 153.22 (C), 159.71
(C), 168.16 (CdO), 180.59 (CdO).
1-Pentyl-6-methoxy-isatin (11). The title compound was pre-
pared as a red solid, using 6-methoxy-isatin and 1-bromopentane
according to general procedure A. Column chromatography (silica
gel, heptane/EtOAc: 7/3) afforded the title compound as an orange
N′-[(3Z)-6-Methoxy-1-pentyl-2-oxo-1,2-dihydro-3H-indol-3-
ylidene]benzohydrazide (16). The title compound was prepared
as a yellow solid, using 1-pentyl-6-methoxy-isatin and benzhy-
drazide according to general procedure B. The resulting solid was
1
1
solid. Yield: 57%; mp: 77.6-78.4 °C. H NMR (CDCl3): δ 0.91
washed with ethanol. Yellow solid. Yield: 78%; mp:144.8 °C. H
(t, J ) 6.5 Hz, 3H), 1.34-1.39 (m, 4H), 1.67-1.71 (m, 2H), 3.67
(t, J ) 7.0 Hz, 2H), 3.93 (s, 3H), 6.37 (d, J ) 2 Hz, 1H), 6.55 (dd,
J ) 2 Hz, J ) 8 Hz, 1H), 7.58 (d, J ) 8 Hz, 1H). 13C NMR
(CDCl3): δ 13.91 (CH3), 22.29 (CH2), 27.12 (CH2), 28.96 (CH2),
40.17 (CH2), 56.13 (CH3), 97.58 (CH), 107.41 (CH), 111.36 (C),
128.04 (CH), 153.52 (C), 159.57 (C), 168.26 (CdO), 181.04
(CdO).
NMR (CDCl3): δ 0.91 (t, J ) 7 Hz, 3H), 1.35 to 1.39 (m, 4H),
1.69-1.75 (m, 2H), 3.73 (t, J ) 7.5 Hz, 2H), 3.87 (s, 3H), 6.44 (d,
J ) 2 Hz, 1H), 6.64 (dd, J ) 2 Hz, J ) 8.5 Hz 1H), 7.51 (t, J )
7.5 Hz, 2H), 7.59 (t, J ) 7.5 Hz, 1H), 7.80 (d, J ) 8 Hz 1H), 8.00
(d, J ) 7.5 Hz 1H), 13.98 (br s, 1H). 13C NMR (CDCl3): δ 13.92
(CH3), 22.32 (CH2), 27.31 (CH2), 29.05 (CH2), 39.94 (CH2), 55.76
(CH3), 97.43 (CH), 107.28 (CH), 112.51 (C), 123.67 (CH), 127.74

Isatin Acylhydrazones as SelectiVe CB2 InVerse Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 441
(CH), 132.38 (C), 132.60 (CH), 137.29 (C), 144.67 (C), 162.57
(C), 163.05 (C), 163.92 (C). Anal. (C21H23N3O3): C, H, N.
N′-[(3Z)-1-Butyl-6-methoxy-2-oxo-1,2-dihydro-3H-indol-3-
ylidene]benzohydrazide (17). The title compound was prepared
as a yellow solid, using 1-butyl-6-methoxy-isatin and benzhydrazide
according to general procedure B. The resulting solid was washed
dure B. The resulting solid was washed with ethanol. Yellow solid.
Yield: 32%; mp: 99.6 °C. H NMR (CDCl3): δ 0.91 (t, J ) 6 Hz,
1
3H), 1.25 to 1.37 (m, 7H), 1.54-1.60 (m, 3H), 1.70-1.72 (m, 2H),
1.82-1.85 (m, 2H), 1.89 (d, J ) 12 Hz, 0.6H, minor isomer) and
2.00 (d, J ) 12 Hz, 1.4H, major isomer), 2.34 (t, J ) 12 Hz, 0.6H,
major isomer) and 3.33 (t, J ) 12 Hz, 0.4H, minor isomer), 3.70
(t, J ) 7.5 Hz, 2H), 3.87 (s, 3H), 6.43 (d, J ) 2 Hz, 1H), 6.61 (d,
J ) 8.5 Hz, 1H), 7.51 (d, J ) 8.5 Hz, 0.4H, minor isomer), and
7.73 (d, J ) 8.5 Hz, 0.6H, major isomer), 12.28 (br s 0.4H, minor
isomer) and 13.078 (br s 0.6H, major isomer). 13C NMR (CDCl3):
δ 13.91 (CH3), 22.31 (CH2), 25.64 (CH2), 25.75 (CH2), 25.95 (CH2),
27.27 (CH2), 28.80 (CH2) and 29.06 (CH2) for 2 isomers, 29.41
(CH2), 39.22 (CH2) and 39.63 (CH2) for 2 isomers, 39.88 (CH2),
44.72 (CH), 55.73 (CH3), 96.96 (CH) and 97.36 (CH) for 2 isomers,
107.16 (CH), 112.61 (C) and 112.98 (C) for 2 isomers, 121.91 (CH)
and 123.52 (CH) for 2 isomers, 132.25 (C) and 136.42 (C) for 2
isomers, 144.54 (C), 161.49 (C) and 162.36 (C) for 2 isomers,
162.50 (C) and 162.90 (C) for 2 isomers, 173.30 (C) and 178.86
(C) for 2 isomers. Anal. (C21H29N3O3): C, H, N.
1
with ethanol. Yellow solid. Yield: 70%; mp: 183.2 °C. H NMR
(CDCl3): δ 0.98 (t, J ) 5 Hz, 1H), 1.38 to 1.45 (m, 4H), 1.67-1.73
(m, 2H), 3.73 (t, J ) 7 Hz, 2H), 3.87 (s, 3H), 6.44 (d, J ) 2 Hz,
1H), 6.64 (dd, J ) 2, Hz, J ) 8.5 Hz, 1H), 7.51 (t, J ) 7.5 Hz,
2H), 7.59 (t, J ) 7.5 Hz, 1H), 7.80 (d, J ) 8 Hz, 1H), 8.00 (d, J
) 7.5 Hz, 2H), 13.97 (br s, 1H). 13C NMR (CDCl3): δ 13.71 (CH3),
20.23 (CH2), 29.67 (CH2), 39.70 (CH2), 55.76 (CH3), 97.44 (CH),
107.28 (CH), 112.50 (C), 123.66 (CH), 127.73 (CH), 128.90 (CH),
132.38 (C), 132.60 (CH), 137.27 (C), 144.67 (C), 162.59 (C),
163.05 (C). 163.93 (C). Anal. (C20H21N3O3): C, H, N.
N′-[(3Z)-1-(Cyclohexylmethyl)-6-methoxy-2-oxo-1,2-dihydro-
3H-indol-3-ylidene]benzohydrazide (18). The title compound was
prepared as a yellow solid, using 1-cyclohexylmethyl-6-methoxy-
isatin and benzhydrazide according to general procedure B. The
resulting solid was washed with ethyl acetate. Yellow solid. Yield:
2-Hydroxy-N′-[(3Z)-6-methoxy-1-pentyl-2-oxo-1,2-dihydro-
3H-indol-3-ylidene]-2-methylpropanohydrazide (23). The title
compound was prepared as a yellow solid, using 1-pentyl-6-
methoxy-isatin and 2-hydroxy-2-methylpropanohydrazide according
to general procedure B. Column chromatography (silica gel,
heptane/EtOAc: 4/6) afforded the title compound as a yellow solid.
Yield: 86%; mp: 133.2 °C. NMR (CDCl3): δ 0.91 (t, J ) 6.5 Hz,
3H), 1.34 to 1.36 (m, 4H), 1.59 (s, 6H), 1.66-1.75 (m, 2H), 2.63
(s, 1H), 3.68 (t, J ) 7 Hz, 2H), 3.87 (s, 3H), 6.41 (d, J ) 2 Hz,
1H), 6.61 (dd, J ) 2 Hz, J ) 8.5 Hz, 1H), 7.73 (d, J ) 8.5 Hz
1H), 13.71 (br s, 1H). 13C NMR (CDCl3): δ 13.90 (CH3), 22.30
(CH2), 27.25 (CH2), 28.06 (CH3), 29.04 (CH2), 39.89 (CH2), 55.74
(CH3), 73.75 (C), 97.33 (CH), 107.14 (CH), 112.47 (C), 123.70
(CH), 137.84 (C), 144.85 (C), 162.01 (C), 163.12 (C), 174.11 (C).
Anal. (C18H25N3O4 ·0.015heptane): C, H, N.
tert-Butyl-(2Z)-2-(6-methoxy-1-pentyl-2-oxo-1,2-dihydro-3H-
indol-3-ylidene)hydrazinecarboxylate (24). The title compound
was prepared as a yellow solid, using 1-pentyl-6-methoxy-isatin
and tert-butyl carbazate according to general procedure B. Column
chromatography (silica gel, heptane/EtOAc: 5/5) afforded the title
compound as a yellow solid. Yield: 54%; mp: 105.3 °C. 1H NMR
(CDCl3): δ 0.91 (t, J ) 6.5 Hz, 3H), 1.36 to 1.39 (m, 4H),
1.67-1.70 (m, 2H), 3.70 (t, J ) 7 Hz, 2H), 3.86 (s, 3H), 6.41 (d,
J ) 2 Hz, 1H), 6.60 (dd, J ) 2 Hz, J ) 8 Hz, 1H), 7.66 (d, J )
8 Hz, 1H), 12.201 (br s, 1H). 13C NMR (CDCl3): δ 13.91 (CH3),
22.30 (CH2), 27.28 (CH2), 28.17 (CH3), 29.03 (CH2), 39.74 (CH2),
55.69 (CH3), 82.15 (C), 97.08 (CH), 106.96 (CH), 112.97 (C),
122.68 (CH), 144.07 (C), 152.49 (C), 162.16 (C), 162.33 (C). Anal.
(C19H27N3O4): C, H, N.
1
69%; mp: 220 °C. H NMR (CDCl3-d6): δ 1.01-1.09 (m, 2H),
1.16 to 1.26 (m, 3H), 1.68-1.86 (m, 6H), 3.56 (d, 2H), 6.44 (d,
1H), 6.64 (dd, 1H), 7.51 (t, 2H), 7.58 (t, 1H), 7.80 (d, 1H), 8.01
(d, 2H), 13.98 (br s, 1H). Anal. (C23H25N3O3): C, H, N.
N′-[(3Z)-1-Benzyl-6-methoxy-2-oxo-1,2-dihydro-3H-indol-3-
ylidene]benzohydrazide (19). The title compound was prepared
as a yellow solid, using 1-benzyl-6-methoxy-isatin and benzhy-
drazide according to general procedure B. The resulting solid was
washed with ethyl alcohol. Yellow solid. Yield: 62%; mp:
1
186.2-186.7 °C. H NMR (CDCl3): δ 3.77 (s, 3H), 4.94 (s, 2H),
6.35 (d, J ) 2 Hz, 1H), 6.62 (dd, J ) 2 Hz, J ) 8 Hz, 1H),
7.29-7.36 (m, 4H), 7.51 (t, J ) 7.5 Hz, 2H), 7.59 (t, J ) 7.5 Hz,
1H), 7.80 (d, J ) 8 Hz, 1H), 8.01 (d, J ) 7.5 Hz, 2H), 13.95 (br
s, 1H). 13C NMR (CDCl3): δ 43.55 (CH2), 55.67 (CH3), 97.99 (CH),
107.71 (CH), 112.46 (C), 123.63 (CH), 127.30 (CH), 127.76 (CH),
128.11 (CH), 128.92 (CH), 129.06 (CH), 132.33 (C), 132.67
(CH), 137.01 (C), 144.43 (C), 162.63 (C), 162.96 (C), 163.94 (C).
Anal. (C23H19N3O3): C, H, N.
N′-[(3Z)-1-(2-Cyclohexylethyl)-6-methoxy-2-oxo-1,2-dihydro-
3H-indol-3-ylidene]benzohydrazide (20). The title compound was
prepared as a yellow solid, using 1-(2-cyclohexylethyl)-6-methoxy-
isatin and benzhydrazide according to general procedure B. The
resulting solid was washed with ethanol. Yellow solid. Yield: 69%;
mp: 179-180.8 °C. 1H NMR (CDCl3): δ 0.96-1.04 (m, 2H),
1.15-1.38 (m, 4H), 1.59 (q, J ) 7.5 Hz 2H), 1.65-1.75 (m, 3H),
1.81 (d, J ) 12.5 Hz 2H), 3.74 (t, J ) 7.5 Hz, 2H), 3.87 (s, 3H),
6.43 (d, J ) 2 Hz, 1H), 6.64 (dd, J ) 2 Hz, J ) 8 Hz, 1H), 7.51
(t, J ) 7.5 Hz, 2H), 7.59 (t, J ) 7.5 Hz, 1H), 7.80 (d, J ) 8 Hz,
1H), 8.00 (d, J ) 7.5 Hz, 1H), 13.96 (br s, 1H). 13C NMR (CDCl3):
δ 26.10 (CH2), 26.40 (CH2), 33.10 (CH2), 34.82 (CH2), 35.51 (CH),
37.89 (CH2), 55.74 (CH3), 97.39 (CH), 107.23 (CH), 112.58 (C),
123.67 (CH), 127.75 (CH), 128.91 (CH), 132.40 (C), 132.59 (CH),
137.31 (C), 144.6 (C), 162.45 (C), 163.05 (C), 163.92 (C). Anal.
(C24H27N3O3): C, H, N.
N′-[(3Z)-6-Methoxy-1-pentyl-2-oxo-1,2-dihydro-3H-indol-3-
ylidene]-3-methylbutanohydrazide (25). The title compound was
prepared as a yellow solid, using 1-pentyl-6-methoxy-isatin and
3-methylbutanohydrazide according to general procedure B. Column
chromatography (silica gel, heptane/EtOAc: 6/4) afforded the title
1
compound as a yellow solid. Yield: 64%; mp: 47.8-49.4 °C. H
NMR (CDCl3): δ 0.92 (t, J ) 2 Hz, 3H), 1.05 (d, J ) 6.5 Hz, 6H),
1.36 to 1.39 (m, 4H), 1.69-1.75 (m, 2H), 2.30 (m, 2H), 2.72 (d, J
) 6.5 Hz, 1H), 3.72 (t, J ) 7 Hz, 2H), 3.89 (s, 3H), 6.44 (d, J )
2 Hz, 1H), 6.64 (m, 1H), 7.54 (d, J ) 8 Hz, 1/2H) and 7.77 (d, J
) 8 Hz, 1/2H) for 2 isomers, 12.41 and 13.03 (br s for isomers,
1H). 13C NMR (CDCl3): δ 13.91 (CH3), 22.29 (CH2), 22.51 (CH3)
and 22.71 (CH3) for 2 isomers, 25.35 (CH) and 26.25 (CH) for 2
isomers, 27.27 (CH2), 28.98 (CH2) and 29.04 (CH2) for 2 isomers,
39.69 (CH2) and 39.84 (CH2) for 2 isomers, 40.45 (CH2) and 44.85
(CH2) for 2 isomers, 55.72 (CH3), 96.93 (CH) and 97.36 (CH) for
2 isomers, 107.07 (CH) and 107.15 (CH) for 2 isomers, 112.51
(C) and 112.92 (C) for 2 isomers, 121.94 (CH) and 123.54 (CH)
for 2 isomers, 132.36 (C) and 136.24 (C) for 2 isomers, 144.60
(C), 161.49 (C) and 162.33 (C) for 2 isomers, 162.53 (C) and 162.95
(C) for 2 isomers, 169.76 (C) and 175.51 (C) for 2 isomers. Anal.
(C19H27N3O3): C, H, N.
N′-[(3Z)-6-Methoxy-1-(2-methoxyethyl)-2-oxo-1,2-dihydro-
3H-indol-3-ylidene] benzohydrazide (21). The title compound was
prepared as a yellow solid, using 6-methoxy-1-(2-methoxyethyl)-
isatin and benzhydrazide according to general procedure B. The
resulting solid was washed with ethanol. Yellow solid. Yield: 66%;
mp: 168-169 °C. 1H NMR (CDCl3): δ 3.35 (s, 3H), 3.65 (t, 2H),
3.87 (s, 3H), 3.94 (t, 2H), 6.64 (d, 1H), 7.58 (t, 1H), 7.79 (d, 1H),
13.93 (br s, 1H). 13C NMR (CDCl3): δ 40.03 (CH2), 56.73 (CH3),
59.14 (CH3), 69.76 (CH2), 97.72 (CH), 107.84 (CH), 112.33 (C),
123.49 (CH), 127.73 (CH), 128.89 (CH), 132.32 (C), 132.63 (CH),
137.17 (C), 144.93 (C), 162.81 (C), 163.04 (C), 163.91 (C). Anal.
(C19H19N3O4): C, H, N.
N′-[(3Z)-6-Methoxy-1-pentyl-2-oxo-1,2-dihydro-3H-indol-3-
ylidene]cyclohexanecarbohydrazide (22). The title compound was
prepared as a yellow solid, using 1-pentyl-6-methoxy-isatin and
cyclohexanecarbohydrazide according according to general proce-

442 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Diaz et al.
2-Cyclohexyl-N′-[(3Z)-6-methoxy-1-pentyl-2-oxo-1,2-dihydro-
3H-indol-3-ylidene]acetohydrazide (26). The title compound was
prepared as a yellow solid, using 1-pentyl-6-methoxy-isatin and
2-cyclohexylacetohydrazide according to general procedure B.
Column chromatography (silica gel, heptane/EtOAc: 5/5) afforded
the title compound as a yellow solid. Yield: 84%; mp: 63.5 °C. 1H
NMR (CDCl3): δ 0.91 (t, J ) 5.5 Hz, 3H), 1.01-1.07 (m, 2H),
1.18 to 1.39 (m, 7H), 1.60-1.72 (m, 5H), 1.81 (d, J ) 12 Hz, 2
H), 1.92-1.94 (m, 1H), 2.27 (d, J ) 7 Hz, 1H), 2.69 (d, J ) 7 Hz,
1H), 3.70 (t, J ) 5.5 Hz, 2H), 3.87 (s, 3H), 6.42 (s, 1H), 6.62 (d,
J ) 8 Hz, 1H), 7.52 (d, J ) 8 Hz, 1/2H) and 7.74 (d, J ) 8 Hz,
1/2H) for 2 isomers, 12.39 (br s, 1/2H) and 13.99 (br s, 1/2H) for
2 isomers. 13C NMR (CDCl3): δ 13.91 (CH3), 22.29 (CH2), 25.97
(CH2) and 26.15 (CH2) for 2 isomers, 26.29 (CH2), 27.27 (CH2),
28.99 (CH2) and 29.04 (CH2) for 2 isomers, 33.13 (CH2) and 33.32
(CH2) for 2 isomers, 34.66 (CH) and 35.44 (CH) for 2 isomers,
39.05 (CH2), 39.69 (CH2) and 39.84 (CH2) for 2 isomers, 43.66
(CH2), 55.72 (CH3), 96.99 (CH) and 97.34 (CH) for 2 isomers,
107.07 (CH) and 107.14 (CH) for 2 isomers, 112.51 (C) and 112.95
(C) for 2 isomers, 121.95 (CH) and 123.54 (CH) for 2 isomers,
132.36 (C) and 136.20 (C) for 2 isomers, 144.59 (C), 161.48 (C)
and 162.32 (C) for 2 isomers, 162.52 (C) and 162.94 (C) for 2
isomers, 169.75 (C) and 175.52 (C) for 2 isomers. Anal.
(C22H31N3O3): C, H, N.
(CH2) for 2 isomers, 36.50 (CH2) and 39.21 (CH2) for 2 isomers,
44.71 (CH), 46.04 (CH2) and 46.25 for 2 isomers, 55.74 (CH3),
97.38 (CH) and 97.77 (CH) for 2 isomers, 106.89 (CH) and 106.99
(CH) for 2 isomers, 112.55 (C) and 112.93 (C) for 2 isomers, 121.81
(CH) and 123.42 (CH) for 2 isomers, 132.16 (C) and 136.32 (C)
for 2 isomers, 145.02 (C), 161.79 (C) and 162.43 (C) for 2 isomers,
162.66 (C) and 162.82 (C) for 2 isomers, 173.29 (C) and 178.81
(C) for 2 isomers. Anal. (C23H31N3O3 ·0.015heptane): C, H, N.
In Vitro Receptor Radioligand Binding Studies. Compound
15 was also screened in a competitive binding experiment by using
membranes of CHO-K1 cells selectively expressing hCB2 at
different compound-15 concentrations in duplicate.49 The competi-
tive binding experiment was performed in 96-well plates (Master-
block) containing binding buffer (50 mM Tris, pH 7.4, 2.5 mM
EDTA, 0.5% protease-free bovine serum albumin), recombinant
membrane extracts (0.25 µg protein/well), and 1 nM [3H]7 (Perkin-
Elmer, NEX-1051, 161 Ci/mmol, diluted in binding buffer).
Nonspecific binding was determined in the presence of 10 µM
compound 7 (Tocris Bioscience). The sample was incubated in a
final volume of 0.1 mL for 60 min at 30 °C and then filtered on a
GF/B UniFilter microplate (Perkin-Elmer, catalogue no. 6005177)
presoaked in 0.5% polyethyleneimine for 2 h at room temperature.
Filters were washed six times with 4 mL of cold binding buffer
(50 mM Tris, pH 7.4, 2.5 mM EDTA, 0.5% protease-free bovine
serum albumin), and bound [3H]7 was determined by liquid
scintillation counting. The median inhibition concentration (IC50)
was determined by nonlinear regression by using the one-site
competition equation. The inhibition constant (Ki) values were
calculated by using the Cheng-Prusoff equation [Ki ) IC50/(1 +
(L/KD)], where L ) concentration of radioligand in the assay and
KD ) affinity of the radioligand for the receptor.
N′-[(3Z)-6-Methoxy-1-pentyl-2-oxo-1,2-dihydro-3H-indol-3-
ylidene]-2-morpholin-4-ylacetohydrazide (27). The title com-
pound was prepared as a yellow solid, using 1-pentyl-6-methoxy-
isatin and 2-morpholin-4-ylacetohydrazide according to general
procedure B. Column chromatography (silica gel, heptane/EtOAc:
3/7) afforded the title compound as a yellow solid. Yield: 85%;
1
mp: 97.1 °C. H NMR (CDCl3): δ 0.92 (t, J ) 6.5 Hz, 3H), 1.36
to 1.39 (m, 4H), 1.67-1.72 (m, 2H), 2.61 (t, J ) 4 Hz, 4H), 3.29
(s, 2H), 3.70 (t, J ) 6.5 Hz, 2H), 3.85 (t, J ) 4 Hz, 4H), 3.87 (s,
3H), 6.41 (d, J ) 1.5, 1H), 6.60 (dd, J ) 1.5, Hz, J ) 8 Hz, 1H),
7.75 (d, J ) 8 Hz, 2H), 13.86 (br s, 1H). 13C NMR (CDCl3): δ
13.93 (CH3), 22.27 (CH2), 27.23 (CH2), 28.93 (CH2), 29.72 (CH2),
53.91 (CH2), 55.73 (CH3), 61.59 (CH2), 66.80 (CH2), 97.24 (CH),
106.96 (CH), 112.53 (C), 123.64 (CH), 137.68 (C), 145.00 (C),
161.96 (C), 163.11 (C), 168.19 (C). Anal. (C20H28N4O4): C, H, N.
N′-[(3Z)-6-Methoxy-1-pentyl-2-oxo-1,2-dihydro-3H-indol-3-
ylidene]-2,2-dimethylpropanohydrazide (28). The title compound
was prepared as a yellow solid, using 1-pentyl-6-methoxy-isatin
and 2,2-dimethylpropionic acid hydrazide according to general
procedure B. Column chromatography (silica gel, heptane/EtOAc:
5/5) afforded the title compound as a yellow solid. Yield: 98%;
[35S]GTP-γ-S Functional Assays. Functional activity was evalu-
ated using GTPγ[35S] assay in Chinese hamster ovarian cell
membrane extracts expressing recombinant hCB1 or hCB2. The
assay relies on the binding of GTPγ[35S], a radiolabeled nonhy-
drolyzable GTP analogue, to the G protein upon binding of an
agonist of the G-protein-coupled receptor. In this system, agonists
stimulate GTPγ[35S] binding, whereas antagonists have no effect
and inverse agonists decrease GTPγ[35S] basal binding.
Compounds were solubilized in 100% dimethyl sulfoxide at a
concentration of 10 mM within 4 h of the first testing session
(master solution). A predilution for the dose-response curve was
performed in 100% dimethyl sulfoxide and then diluted 100-fold
in assay buffer at a concentration 4-fold higher than the concentra-
tion to be tested. Compounds were tested for agonist activity at
eight concentrations in duplicate: 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and
0.001 µM, with compound 7 (Tocris, 0949) as the reference agonist.
For GTPγ[35S], membranes (Euroscreen SA, Gosselies, Belgium)
were mixed with GDP diluted in assay buffer to give 30 µM solution
(volume:volume) and incubated for at least 15 min on ice. In
parallel, GTP-γ-[35S] (Amersham, SJ1308) was mixed with PVT-
WGA beads (Amersham, RPNQ001) diluted in assay buffer at 50
mg/mL (0.5 mg/10 µL) (volume:volume) just before the reaction
was started. The following reagents were successively added in
the wells of an Optiplate (Perkin-Elmer): 50 µL of ligand, 20 µL
of the membranes:GDP mix, 10 µL of assay buffer for agonist
testing, and 20 µL of the GTP-γ-[35S]:beads mix. The plates were
covered with a topseal, shaken on an orbital shaker for 2 min, and
then incubated for 1 h at room temperature. Then the plates were
centrifuged for 10 min at 2000 rpm, incubated at room temperature
for 1 h, and counted for 1 min/well with a PerkinElmer TopCount
reader. Assay reproducibility was monitored by the use of a
reference compound 7. For replicate determinations, the maximum
variability tolerated in the test was of (20% around the average
of the replicates. Efficacies (Emax) for CB1 and CB2 are expressed
as a percentage relative to the efficacy of compound 7.
1
mp: 56.3 °C. H NMR (CDCl3): δ 0.91 (t, J ) 6.5 Hz, 3H), 1.36
to 1.39 (m, 13H), 1.69-1.75 (m, 2H), 3.70 (t, J ) 7.5 Hz, 2H),
3.87 (s, 3H), 6.42 (d, J ) 2 Hz, 1H), 6.62 (dd, J ) 2 Hz, J ) 8 Hz,
1H), 7.74 (d, J ) 8 Hz, 2H), 13.34 (br s, 1H). 13C NMR (CDCl3):
δ 13.91 (CH3), 22.31 (CH2), 27.29 (CH2), 27.39 (CH3), 29.04 (CH2),
39.00 (C), 39.88 (CH2), 55.72 (CH3), 97.33 (CH), 107.15 (CH),
112.62 (C), 123.90 (CH), 136.80 (C), 144.58 (C), 162.35 (C),
162.87 (C), 175.79 (C). Anal. (C19H27N3O3 · 0.04heptane): C,
H, N.
N′-[(3Z)-1-(Cyclohexylmethyl)-6-methoxy-2-oxo-1,2-dihydro-
3H-indol-3-ylidene]cyclohexanecarbohydrazide (29). The title
compound was prepared as a yellow solid, using 1-pentyl-6-
methoxy-isatin and cyclohexanecarbohydrazide according to general
procedure B. The resulting solid was washed with ethanol. Column
chromatography (silica gel, heptane/EtOAc: 5/5) afforded the title
compound as a yellow solid. Yield: 70%; mp: 147.5 °C. 1H NMR
(CDCl3): δ 1.02-1.07 (m, 2H), 1.19-1.33 (m, 6H), 1.54-1.61 (m,
3H), 1.70-1.88 (m, 9H), 1.89 (d, J ) 12 Hz, 0.66H, minor isomer)
and 2.00 (d, J ) 12 Hz, 1.33H, major isomer), 2.33 (t, J ) 12 Hz,
0.6H, major isomer) and 3.33 (t, J ) 12 Hz, 0.4H, minor isomer),
3.53 (d, J ) 7 Hz, 2H), 3.87 (s, 3H), 6.42 (d, J ) 1.5 Hz, 1H),
6.62 (d, J ) 8.5 Hz, 1H), 7.52 (d, J ) 8.5 Hz, 0.4H, minor isomer),
and 7.73 (d, J ) 8.5 Hz, 0.6H, major isomer), 12.29 (br s 0.4H,
minor isomer) and 13.07 (br s 0.6H, major isomer). 13C NMR
(CDCl3): δ 25.66 (CH2), 25.74 (CH2), 25.95 (CH2), 26.14 (CH2),
28.79 (CH2) and 29.39 (CH2) for 2 isomers, 30.94 (CH2) and 31.02
Acknowledgment. We thank Dr. Kumar Kaluarachchi for
performing the two-dimensional NMR studies. The NMR
Facility at M. D. Anderson Cancer Center is supported by

Isatin Acylhydrazones as SelectiVe CB2 InVerse Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 443
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Cancer Center Support Grant CA16672 awarded by the National
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Supporting Information Available: Crystallographic data for
compound 15, elemental analyses, and 1H NMR spectra for
compounds 15-29. This material is available free of charge via
the Internet at http://pubs.acs.org.
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