Tricyclic pyrazoles. 4. Synthesis and biological evaluation of analogues of the robust and selective CB2 cannabinoid ligand 1-(2′, 4′-dichlorophenyl)-6-methyl-N-piperidin-1-yl-1,4-dihydroindeno[1,2-c] pyrazole-3-carboxamide

Article abstract of DOI:10.1021/jm060920d

New analogues (2a-p) of the previously reported CB₂ ligands 6-methyl- and 6-chloro-1-(2′,4′-dichlorophenyl)-N-piperidin-1-yl-1, 4-dihydroindeno[1,2-c]pyrazole-3-carboxamides (1a,b) have been synthesized and evaluated for cannabinoid receptor af

Full text of DOI:10.1021/jm060920d

7502
J. Med. Chem. 2006, 49, 7502-7512
Tricyclic Pyrazoles. 4. Synthesis and Biological Evaluation of Analogues of the Robust and
Selective CB2 Cannabinoid Ligand 1-(2′,4′-Dichlorophenyl)-6-methyl-N-piperidin-1-yl-
1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide
Gabriele Murineddu,† Paolo Lazzari,‡ Stefania Ruiu,‡ Angela Sanna,‡ Giovanni Loriga,† Ilaria Manca,† Matteo Falzoi,‡
Christian Dess`ı,‡ Maria M. Curzu,† Giorgio Chelucci,§ Luca Pani,‡,| and Ge´rard A. Pinna*,†
Dipartimento Farmaco Chimico Tossicologico, UniVersita` di Sassari, Via F. Muroni 23/A, 07100 Sassari, Italy, Neuroscienze PharmaNess
S.c.a r.l., c/o POLARIS, Edificio 5, Loc.Piscinamanna, 09010 Pula (CA), Italy, Dipartimento di Chimica, UniVersita` di Sassari, Via Vienna 2,
07100 Sassari, Italy, and C.N.R. Istituto di Tecnologie Biomediche, Sezione di Cagliari, c/o Neuroscienze PharmaNess S.c.a r.l.
ReceiVed August 1, 2006
New analogues (2a-p) of the previously reported CB2 ligands 6-methyl- and 6-chloro-1-(2′,4′-dichlorophe-
nyl)-N-piperidin-1-yl-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamides (1a,b) have been synthesized and
evaluated for cannabinoid receptor affinity. One example, 1-(2′,4′-dichlorophenyl)-6-methyl-N-cyclohexy-
ilamine-1,4-dihydroindeno[1,2-c] pyrazole-3-carboxamide (2a) was shown to have single digit nanomolar
affinity for cannabinoid CB2 receptors. Furthermore, compounds 2a and 2b, as well as lead structures 1a,b,
were also shown to be agonist in an in vitro model based on human promyelocytic leukemia HL-60 cells.
Introduction
maturation process. A role of the CB2 receptor in inhibiting the
growth of murine lymphoma and leukemia cells has also been
described.20
The CB2 receptor, first described in 1993, is a member of
the cannabinoid receptor family and possesses, in humans,
∼44% overall homology to the CB1 receptor,1-3 with 68%
amino acid sequence identity within the transmembrane do-
mains.2 Unlike the CB1 receptor, this cannabinoid receptor
subtype is rather divergent across human, rat, and mouse, as
indicated by the following amino acid identity percentage: 93%
between rat and mouse; 81% between rat and human.3
Despite the general opinion that CB2 receptors are almost
exclusively expressed in peripheral tissue, the presence of this
cannabinoid receptor subtype in the central nervous system has
also directly or indirectly been evidenced, as demonstrated by
CB2 receptor expression in rat microglial cells,18 adult rat
retina,21 human perivascular microglial cells,22 and neuritic
plaques-associated microglial cells in Alzheimer’disease brain.23
The correlation between CB2 receptor and central nervous
system has been recently supported by a work based on
immunohistochemical and gene expression analysis in rat
brain.24 In fact, a widespread expression of CB2 receptors in
the brain and in hippocampal neuron-specific-enolase-positive
neuronal cells has been evidenced in this study.24 Moreover, a
possible direct involvement of neural mechanisms on CB2
receptor-mediated antihyperalgesia has been proposed by Bel-
tramo et al.25
CB2 receptors, as well as the CB1, belong to the large family
of rhodospin-like family of G-protein-coupled receptors (GPCRs),
which control different multiple intracellular signal transduction
pathways; that is, CB1 and CB2 receptor agonists inhibit
forskolin-stimulated adenylyl cyclase by activation of a pertussis
toxin-sensitive G-protein; CB2 receptor agonists can produce
stimulation of cAMP formation; Gi/o MAP kinase is activated
both in cultured human promyelocytic HL-60 and in CHO cells
possessing endogenous or expressing recombinant CB2 recep-
tors, respectively.4
The multifocal expression of CB2 immunoreactivity in glial
and neuronal patterns in different brain regions suggests re-
evaluation of the possible roles played by this receptor subtype
in CNS. Consequently, the relevance of ligands able to
selectively interact with CB2 receptors could be higher than that
related only to the known pathophysiological processes involv-
ing this cannabinoid receptor subtype (i.e., peripheral antinoci-
ception, immunomodulation, inflammation, neurodegeneration,
uncontrolled cell proliferation).26-28 Therefore, in addition to
the discovery of new CB1 selective compounds, with significant
applications in different therapeutic areas (i.e., smoking cessa-
tion, obesity, sexual disorders, pain, gastrointestinal disorders,
and glaucoma),4 the individuation of new classes of CB2 active
and selective ligands is of great interest, too.
CB2 receptors are expressed in peripheral tissue (tonsils,
thymus, spleen, pancreas), peripheral nerve terminals, and
skin tumor cells,4-6 while CB1 receptors are localized
both in brain (cerebellum, hippocampus, cortex, basal ganglia)
and in peripheral tissues and organs (testis, eye, urinary bladder,
gastrointestinal tract).4,5,7-10 Moreover, CB2 is expressed
abundantly in various types of inflammatory cells and immune
competent cells altering the maturation of macrophages, eosi-
nophils and natural killer and B lymphocytes.11-16 According
to its predominant expression in immune tissues and lympho-
cytes, CB2 receptors appear to specifically mediate both
immunosuppressive and immunostimulatory effects.15-19
Gene expression studies showed that activation of the CB2
receptor is involved in initialization of the immune cell
Among the different classes of synthetic compounds assuring
CB2 affinity and selectivity, the relevance of pyrazole derivatives
have been consolidated by various studies.29,30 In this context,
we have recently reported the synthesis and the CB2 affinity of
several new 1,4-dihydroindeno[1,2-c]pyrazol-based ligands 1,
among which compound 1a, and to minor extend 1b, exerted
robust affinity and selectivity for CB2 receptor (Figure 1).31
* To whom correspondence should be addressed. Phone: +39079228721.
Fax: +39079228720. E-mail: pinger@uniss.it.
† Dipartimento Farmaco Chimico Tossicologico.
‡ Neuroscienze PharmaNess S.c.a r.l.
§ Dipartimento di Chimica.
| C.N.R. Instituto di Tecnologie Biomediche.
10.1021/jm060920d CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/15/2006

Analogues of the Cannabinoid Ligand
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7503
Table 1. Structures and Binding Data of Compounds 2a-p
a Affinity of compounds for the CB2 receptor was assayed using mouse spleen homogenate and [3H]-CP-55 940. b Affinity of compounds for the CB1
receptor was evaluated using mouse brain (minus cerebellum) homogenate and [3H]-CP 55 940. Ki values were obtained from five independent experiments
carried out in triplicate and are expressed as mean ( standard error.
This finding prompted us to investigate new 1,4-dihydroin-
deno[1,2-c]pyrazoles 2a-p (Table 1), which were obtained by
modifying the carbamoyl moiety and the aryl substitution of
the lead compounds 1a,b.
This study also sought to establish how these compounds
modulate CB2 receptors (intrinsic activity) using an in
vitro model based on human promyelocytic leukemia HL-60
cells.

7504 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Murineddu et al.
mouse spleen were used as starting material. Mouse brain (minus
cerebellum) homogenates were instead employed for CB1
3
affinity evaluation. H-CP 55 940 was used as radioligand in
both cases. The results from the in vitro binding assay were
compared with the Ki values of the prototypical cannabinoid
ligands 1a and 1b and of the reference compounds SR144528
and CP55 940.
The capability of these new derivatives and of the lead
compounds to modulate CB2 receptors has been further inves-
tigated through an in vitro model based on human promyelocytic
leukemia HL-60 cells. This cell line, selectively expressing the
CB2 but not the CB1 receptor, represents a reliable platform for
the study of the mechanism of action of cannabinoids.11,15,17,34
In fact, it has been shown that the exposure of HL-60 cells to
the cannabinoid nonselective synthetic agonist CP55 940 and
the endogenous ligand 2-AG induces a rapid phosphorylation
and activation of the extracellular signal-related kinases (ERK
1/2 or p44/p42-MAPK) and that the addition of SR144528, a
CB2 receptor-specific antagonist, abolished the response induced
by the agonists, thus indicating that the P-ERK 1/2 activation
is CB2 specific.12,17,35 Accordingly, the activity of both the new
derivatives with the best CB2 affinity (2a and 2b) and the
reference compounds (1a, 1b, and CP55 940) was assayed by
the exposure of human promyelocytic leukemia HL-60 cells to
the potential CB2 ligands and the consequent western blot
analysis of the cell extract proteins. In the case of P-ERK 1/2
activation, to establish CB2-specific activity of the compounds,
experiments involving a preliminary step characterized by the
adding of the CB2 receptor antagonist SR144528, with the
subsequent exposure with the new derivatives, were also carried
out.
Figure 1.
Scheme 1. Synthesis of Compounds 2a-p
Chemistry
The compounds described in this report were prepared as
shown in Scheme 1.
Target compounds 2a-p were obtained by amidation of the
appropriate amine or hydrazine 5 and the necessary 1-(2′,4′-
dichlorophenyl)-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxyl-
ic acid (4a,31 4b, or 4c), using thionyl chloride (method A) or
1-hydroxybenzotriazole (BtOH) and 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide (EDC; method B).
Results and Discussion
Radioligand Binding Assays. The cannabinoid receptor
affinities of the new 1,4-dihydroindeno[1,2-c]pyrazole deriva-
tives are shown in Table 1. For comparison, the Ki values of
the lead compounds 6-methyl- and 6-chloro-1-(2′,4′-dichlo-
rophenyl)-N-piperidin-1-yl-1,4-dihydroindeno[1,2-c]pyrazole-3-
carboxamides (1a,b) and reference CB2 ligands SR144528 and
CP55 940 have been reported. Results are the average of five
independent experiments with three replicates at each concentra-
tion.
Our initial result showed that isosteric replacement of the
carboxamide N-piperidinyl moiety of 1a with the cyclohexyl
ring gave the analogue 2a with nanomolar CB2 affinity and
acceptable subtype receptor selectivity (KiCB2 ) 7.6 nM; Ki-
CB1/KiCB2 ) 118). Because the bioisosteric analogue 2a
displayed reasonable CB2 affinity and selectivity, we decided
to do a detailed structure-activity relationship (SAR) investiga-
tion of 2a to provide an understanding of the structural features
that influence the affinity of this novel compound.
Our initial strategy was to prepare analogues of 2a by
stepwise introduction of various carbamoyl moieties at the
pyrazole ring of the N-cyclohexyl 1,4-dihydroindeno[1,2-c]-
pyrazole-3-carboxamide template of 2a. In addition, analogues
of 2a having a modified aromatic substitution of the tricyclic
ring system and carbamoyl unit were also prepared. As
exemplified by compound 2b, replacement of the cyclohexyl
ring of the carbamoyl moiety of 2a with a phenyl group led to
a 4-fold affinity decrease (KiCB2 ) 32.8 nM).
The required unknown tricyclic acids (4b,c) were synthesized
as reported in the Scheme 2.
The 1,3-diketoesters 7b and 7c, as a tautomeric equilibrium
shifted toward the alkenylidene structure (7′), were prepared
from the disubstituted indanones 6b and 6c,32 respectively, and
diethyl oxalate in the presence of sodium ethylate.
Compounds 7b,c and the 2,4-dichlorophenylhydrazine hy-
drochloride were heated in acetic acid to afford the desired
dihydroindenopyrazoles 8b,c.
Saponification of the esters 8b,c furnished acids 4b,c.
The 6-chloro-5-methylindanone (6b), so far undescribed in
literature, was prepared starting from the 5-methylindanone (9),31
as depicted in Scheme 3.
Briefly, indanone 9 was nitrated with potassium nitrate in
concentrated sulfuric acid, to give a mixture of isomers 10a
and 10b, which could be chromatographically separated and
1
characterized via H NMR.
Subsequent reduction of the nitro group of 10a by palladium-
catalyzed hydrogenation afforded 6-amino-5-methylindanone 11,
which then underwent diazoniation and final reaction with
copper(I) chloride to form indanone 6b.
Biology
Cannabinoid receptor affinities of the 1,4-dihydroindeno[1,2-
c]pyrazoles were determined by radioligand binding experiments
according to the previously reported procedures by Ruiu33 and
expressed as Ki values. In the CB2 assays, homogenates of
Benzamides with a p-substituent as chlorine (2c), fluoro (2d),
methyl (2e), and methoxy (2g), resulted in a somewhat lower
CB2 affinity as compared to the phenyl derivative 2b. Moreover,
the presence of the strongly electron-withdrawing trifluorom-

Analogues of the Cannabinoid Ligand
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7505
Scheme 2
Reagents and conditions: (i) Na, dry EtOH, (COOEt)2; (ii) 2,4-Cl2C6H3NHNH2·HCl, CH3CO2H; (iii) KOH, EtOH/H2O.
Scheme 3
60 cells (Figure 2). In particular, through the adopted procedure,
CP55 940 induced a time- (data not shown) and dose-related
induction of P-ERK 1/2 expression in HL-60 cells (Figure 2A),
peaking at the concentration of 100 nM, 10 min after exposure
(+103.0 ( 8.2% vs vehicle). Moreover, the CB2 receptor
antagonist SR144528, which does not alter the P-ERK 1/2
expression, completely blocked this effect if used at a concen-
tration of 50 nM and added 5 min before the exposure with the
CB2 agonist (+14.5 ( 12.4% vs vehicle and -44.0 ( 8.5% vs
CP55 940; Figure 2B).
As shown in Figure 3, the tested compounds 1a, 1b, 2a, and
2b significantly increased P-ERK 1/2 expression in HL-60 cells.
Time course studies showed that the tested compound reached
the maximum effect after 10 min of treatment (data not shown)
and, as indicated in Figure 3, the plateau was reached at the
concentration of 10 nM (+83.0 ( 4.2%, +65.0 ( 18.5%, +61.3
( 12.4%, and +125.0 ( 35.8% vs vehicle for 1a, 1b, 2a, and
2b, respectively). The increase of P-ERK 1/2 was comparable
with that elicited by CP55 940. The exposure to the CB2 receptor
antagonist SR144528 (50 nM) 5 min before the adding of the
tested compounds was able to reverse this effect, as shown in
Figure 4. Altogether, our data showed that the tested compounds
act as CB2 receptor agonists and that their effect is specific as
it was blocked by a CB2 receptor antagonist.
Reagents and conditions: (i) H2SO4, KNO3; (ii) H2, Pd/C 10%, EtOH;
(iii) NaNO2, HCl, CuCl.
ethyl group at the phenyl para-position (2f), as well as the
disubstitution of the same phenyl ring (2h and 2i), had marked
unfavorable effect on CB2 affinity. In fact, in these last
mentioned cases, Ki values higher than 5000 were determined
for the CB2 receptor, suggesting a negative influence of the steric
effect of these substituents on the carboxamide phenyl group.
Compounds 2j-m were prepared to investigate the influence
of several arylhydrazide derivatives on the affinity toward CB2
receptors. In particular, the phenyl (2j), p-chlorophenyl (2k),
p-nitrophenyl (2l), and p-methoxyphenyl (2m) hydrazides were
assayed. All these compounds showed no significant affinity
for CB2 receptors except 2j, the unsubstituted phenyl hydrazide,
which displayed a moderate CB2 affinity (KiCB2 ) 63 nM).
Three analogues were prepared to explore alternative sub-
stitution on the aryl moiety of the core 1,4-dihydroindeno[1,2-
c]pyrazole ring system. The 6-methyl-7-chloro-disubstituted
analogue 2n bound the CB2 receptors with a Ki of 25 nM, 3-fold
less potent than 2a. A more marked reduction of affinity was
observed for 6-methyl-7-chloro- and the reversed 6-chloro-7-
methyl-disubstituted derivatives 2o and 2p, respectively.
Generally, the CB1 receptor affinities of all the investigated
compounds were lower than their CB2 receptor affinities.
In Vitro CB2 Activity Evaluation. To assess in vitro
function, 2a,b and leads 1a,b were tested for intrinsic activity
in CB2 receptors. Our data confirmed previously reported
results12,17,35 on the effect of the CB2 receptor agonist CP55 940
toward phosphorylated ERK 1/2 (P-ERK 1/2) expression in HL-
Conclusions
In the present study, the preparation of a series of new ligands
with affinity for the CB2 subtype of the cannabinoid receptors
is reported.
The results obtained from radioligand binding experiments
on compounds 2 suggest that substitution of the cyclohexyl
carbamoyl groups of 2a with a phenyl carbamoyl moiety (2b)
led to a decrease of affinity. Moreover, substitution of the
carbamoyl phenyl ring was less tolerated, however, fluoro (2d),
methyl (2e), and methoxyl (2g) derivatives maintained a
moderate CB2 affinity. On the other hand, the introduction of
an additional substituent at position 3′ or 2′ of the phenyl
carbamoyl group led to a substantial decrease of affinity.
We found that substitution of the carbamoyl moiety with the
phenylhydrazide unit at position 3 of the indenopyrazole ring
system resulted in a first-order decrease of activity. The presence
of substituents on the phenyl ring of phenylhydrazide 2j was
detrimental for the CB2 affinity.

7506 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Murineddu et al.
Figure 2. Panel A shows a dose response study of P-ERK 1/2 expression following a 10 min exposure to CB2 receptor agonist CP55 940 (CP).
Doses are expressed in nM. The effect of a 5 min pretreatment with the CB2 receptor antagonist SR144528 (50 nM; SR) on the agonist-induced
P-ERK 1/2 is shown in panel B. On the right are shown representative western blots of the P-ERK expression. Data are expressed as a mean
percentage of vehicle ( SEM and are the results of five separate experiments. *p < 0.05 vs vehicle; #p < 0.05 vs CP55 940.
sensitive compounds were performed under an argon atmosphere.
Flash chromatography (FC) was performed using Merck silica gel
60 (230-400 mesh ASTM). Thin layer chromatography was
performed with Polygram SIL N-HR/HV254 precoated plastic sheets
(0.2 mm). The pyrazole acid 4a31 and the indanone 6c32 were
prepared according to the previously described literature.
Finally, the simultaneous introduction of two substituents
(CH3 and Cl) at positions 6 and 7 of the indenopyrazole core
gave rise to compounds that retained good to moderate activity.
For the first time, in vitro CB2 intrinsic activity evaluation
assays, based on the determination of P-ERK 1/2 expression
increasing in HL-60 cells exposed to the compounds to be
assayed, highlighted agonist activity toward CB2 receptors for
two representative terms 2a and 2b and for prototype 1a and
1b. These results could open interesting pharmacological
developments for the potent selective CB2 agonists 1a and 1b,
or other analogues, especially for the treatment of immune
disorders or, as recently emerged from literature,36,37 as anti-
nociceptive agents. However, further in vitro and in vivo studies
of the more active compounds in this series are needed, in order
to define the full range of their pharmacological actions and to
demonstrate the therapeutic potential of these molecules.
General Procedure I: Synthesis of Carboxamides (2a-i,n)
and Carbohydrazides (2j-m,o,p). Method A. A mixture of
1-(2′,4′-dichlorophenyl)-6-methyl-1,4-dihydroindeno[1,2-c]pyrazole-
3-carboxilic acid 4a31 (1 equiv, 0.69 mmol) and thionyl chloride
(3.0 equiv) in toluene (5.22 mL) was refluxed for 3 h. The solvent
and the excess of SOCl2 were removed under reduced pressure,
and the resulting dark solid in CH2Cl2 (2.6 mL) was dropwise added
to a solution of requisite amine (1.5 equiv) in CH2Cl2 (2.6 mL) at
0 °C. The mixture was warmed to room temperature for 0.5-3 h.
The mixture was then poured into a separatory funnel, and brine
was added. The aqueous layer was separated and extracted with
CH2Cl2. The combined organic layers were washed with water,
dried over Na2SO4, and concentrated under reduced pressure. The
analytically pure product was isolated by an appropriate method
of purification as indicated below.
Method B. A mixture of the appropriate carboxylic acid 4 (1
equiv, 0.69 mmol), EDC (1.2 equiv), and BtOH (1.2 equiv) in
acetonitrile (6.9 mL) was stirred at room temperature for 15 min.
A solution of the requisite amine or hydrazine 5 (4 equiv) in
acetonitrile (0.7 mL) was dropwise added, and the whole was stirred
at room temperature for 2-20 h. The solution was taken up with
water, and the precipitate was filtered off and air-dried. The
analytically pure product was isolated by trituration with an
appropriate solvent, as indicated below.
Experimental Section
General Procedures. Melting points were obtained on a Ko¨fler
melting point apparatus and are uncorrected. IR spectra were
recorded as thin films (for oils) or nujol mulls (for solids) on NaCl
plates with a Perkin-Elmer 781 IR spectrophotometer and are
expressed in ν (cm-1). All NMR spectra were taken on a Varian
XL-200 NMR spectrometer with 1H and 13C being observed at 200
1
and 50 MHz, respectively. Chemical shifts for H and 13C NMR
spectra were reported in δ or ppm downfield from TMS [(CH3)4-
Si]. Multiplicities are recorded as s (singlet), br s (broad singlet),
d (doublet), t (triplet), q (quartet), qu (quintuplet), dd (doublet of
doublets), and m (multiplet). Atmospheric pressure ionization
electrospray (API-ES) mass spectra, when reported, were obtained
on an Agilent 1100 series LC/MSD spectrometer. Elemental
analyses were performed by Laboratorio di Microanalisi, Diparti-
mento di Chimica, Universita` di Sassari, Italy, and are within (0.4%
of the calculated values. All reactions involving air- or moisture-
1-(2′,4′-Dichlorophenyl)-6-methyl-N-cyclohexylamine-1,4-di-
hydroindeno[1,2-c]pyrazole-3-carboxamide (2a). Method A was
used to convert 4a and cyclohexylamine into the title product. The
mixture was stirred at room temperature for 3 h and purified by
trituration with petroleum ether to afford 2a (0.17 g, 56.7%) as a

Analogues of the Cannabinoid Ligand
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7507
Figure 3. Panels A-D show the effect of different doses (nM) of CB2 receptor ligands on P-ERK 1/2 expression in HL-60 cells after 10 min of
treatment (1a, 1b, 2a, and 2b, respectively). On the right side are shown representative western blots of P-ERK expression. Data are expressed as
a mean percentage of vehicle ( SEM and are the results of five separate experiments. *p < 0.05 vs vehicle; #p < 0.05 vs CB2 ligands (1a, 1b, 2a,
and 2b, respectively).
cream solid. Rf ) 0.136 (petroleum ether/EtOAc 9.5:0.5); mp 190.5
°C; IR 1658, 3413; 1H NMR (CDCl3) δ 1.10-1.55 (m, 4H), 1.58-
1.85 (m, 4H), 1.97-2.10 (m, 2H), 2.40 (s, 3H), 3.85 (s, 2H), 3.90-
4.07 (m, 1H), 6.80 (d, 1H, J ) 7.8 Hz, NH, exch. with D2O), 6.88
(d, 1H, J ) 7.8 Hz), 7.04 (d, 1H, J ) 7.8 Hz), 7.39 (s, 1H), 7.45
(dd, 1H, J ) 8.4 Hz, J ) 2.2 Hz), 7.55 (d, 1H, J ) 8.4 Hz), 7.66
(d, 1H, J ) 2.2 Hz); 13C NMR (CDCl3) δ 21.60 (CH3), 25.00 (CH2
× 2), 25.60 (CH2), 29.65 (CH2), 33.20 (CH2 × 2), 48.10 (CH),
118.67 (CH), 127.14 (CH), 127.28 (CH), 127.79 (C), 128.14 (CH),
128.67 (C), 129.75 (CH), 130.46 (CH), 131.94 (C), 135.91 (C),
136.09 (C), 137.13 (C), 142.28 (C), 150.05 (C), 152.04 (C), 161.00
(CO). API-ES calcd, 440.36; found, 440.20.
1-(2′,4′-Dichlorophenyl)-6-methyl-N-phenyl-1,4-dihydroindeno-
[1,2-c]pyrazole-3-carboxamide (2b). Method A was used to
convert 4a and aniline into the title product. The mixture was stirred
at room temperature for 2 h and purified by FC (petroleum ether/
EtOAc 7:3) to afford 2b (0.18 g, 60.0%) as a cream solid. Rf )
0.204 (petroleum ether/EtOAc 9.5:0.5); mp 197.8 °C; IR 1687,
3389; 1H NMR (CDCl3) δ 2.41 (s, 3H), 3.91 (s, 2H), 6.91 (d, 1H,
J ) 7.8 Hz), 7.04-7.20 (m, 2H), 7.30-7.45 (m, 3H), 7.49 (dd,
1H, J ) 8.0 Hz, J ) 2.2 Hz), 7.59 (d, 1H, J ) 8.4 Hz), 7.69-7.34
(m, 3H), 8.73 (br s, 1H, NH, exch. with D2O); 13C NMR (CDCl3)
δ 21.63 (CH3), 29.69 (CH2), 118.76 (CH), 119.62 (CH × 2), 124.11
(CH), 127.42 (CH), 128.00 (C), 128.21 (CH), 128.22 (CH), 128.50
(C), 129.01 (CH × 2), 129.68 (CH), 130.56 (CH), 130.90 (C),
132.00 (C), 135.90 (C), 136.20 (C), 137.65 (C), 137.85 (C), 142.00
(C), 149.80 (C), 159.80 (CO); API-ES calcd, 434.32; found, 434.20.
1-(2′,4′-Dichlorophenyl)-6-methyl-N-p-chlorophenyl-1,4-dihy-
droindeno[1,2-c]pyrazole-3-carboxamide (2c). Method A was
used to convert 4a and p-chloroaniline into the title product. The
mixture was stirred at room temperature for 2 h and purified by
trituration with petroleum ether to afford 2c (0.18 g, 57.6%) as a
cream solid. Rf ) 0.227 (petroleum ether/EtOAc 9.5:0.5); mp 211.5
°C; IR 1678, 3374; 1H NMR (CDCl3) δ 2.41 (s, 3H), 3.90 (s, 2H),
6.90 (d, 1H, J ) 7.8 Hz), 7.06 (d, 1H, J ) 7.8 Hz), 7.32 (d, 2H,
J ) 8.8 Hz), 7.41 (s, 1H), 7.49 (dd, 1H, J ) 6.8 Hz, J ) 1.8 Hz),
7.58 (d, 1H, J ) 8.2 Hz), 7.67 (d, 2H, J ) 8.8 Hz), 7.69 (d, 2H,
J ) 2.2 Hz), 8.74 (s, 1H, NH, exch. with D2O); 13C NMR (CDCl3)
δ 21.64 (CH3), 29.62 (CH2), 118.79 (CH), 120.78 (CH × 2), 127.22
(CH), 127.47 (CH), 127.95 (C), 128.24 (CH), 128.43 (C), 129.04
(CH × 2), 129.65 (CH), 130.58 (CH), 131.97 (C), 135.84 (C),
136.25(C), 136.40 (C), 137.53 (C), 141.68 (C), 149.91 (C × 2),
152.57 (C), 159.72 (CO); API-ES calcd, 468.76; found, 469.20.
1-(2′,4′-Dichlorophenyl)-6-methyl-N-p-fluorophenyl-1,4-dihy-
droindeno[1,2-c]pyrazole-3-carboxamide (2d). Method A was
used to convert 4a and p-fluoroaniline into the title product. The
mixture was stirred at room temperature for 1 h and purified by
trituration with petroleum ether to afford 2d (0.20 g, 65.8%) as a

7508 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Murineddu et al.
Figure 4. Panels A-D show the CB2 receptor antagonist SR144528 (SR) inhibition of P-ERK 1/2 activation. CB2 receptor ligands were used at
a concentration of 50 nM. On the right side are shown representative western blots of P-ERK expression. Data are expressed as a mean percentage
of vehicle ( SEM and are the results of five separate experiments. *p < 0.05 vs vehicle; #p < 0.05 vs CB2 ligands (1a, 1b, 2a, and 2b, respectively).
cream solid. Rf ) 0.189 (petroleum ether/EtOAc 9.5:0.5); mp 206-
208 °C; IR 1689, 3373; 1H NMR (CDCl3) δ 2.41 (s, 3H), 3.89 (s,
2H), 6.89 (d, 1H, J ) 7.8 Hz), 7.00-7.12 (m, 3H), 7.40 (s, 1H),
7.48 (dd, 1H, J ) 8.4 Hz, J ) 1.8 Hz), 7.58 (d, 1H, J ) 8.6 Hz),
7.62-7.75 (m, 3H), 8.73 (s, 1H, NH, exch. with D2O); 13C NMR
(CDCl3) δ 21.60 (CH3), 29.59 (CH2), 115.39 (CH), 115.83 (CH),
118.75 (CH), 121.19 (CH), 121.35 (CH), 127.18 (CH), 127.43 (CH),
127.91 (C), 128.20 (CH), 128.42 (CH), 129.62 (C), 130.54 (CH),
131.94 (C), 133.79 (C), 135.83 (C), 136.17 (C), 137.45 (C), 141.75
(C), 149.90 (C), 152.47 (C), 156.79 (C), 159.65 (CO); API-ES
calcd, 452.31; found, 452.20.
1-(2′,4′-Dichlorophenyl)-6-methyl-N-p-methylphenyl-1,4-dihy-
droindeno[1,2-c]pyrazole-3-carboxamide (2e). Method A was
used to convert 4a and p-toluidine into the title product. The mixture
was stirred at room temperature for 2 h and purified by trituration
with petroleum ether to afford 2e (0.22 g, 73.0%) as a pink solid.
Rf ) 0.212 (petroleum ether/EtOAc 9.5:0.5); mp 119 °C; IR 1684,
3389; 1H NMR (CDCl3) δ 2.34 (s, 3H), 2.41 (s, 3H), 3.90 (s, 2H),
6.90 (d, 1H, J ) 7.6 Hz), 7.06 (d, 1H, J ) 7.6 Hz), 7.16 (d, 2H,
J ) 8.4 Hz), 7.41 (s, 1H), 7.49 (dd, 1H, J ) 8.4 Hz, J ) 2.2 Hz),
7.57-7.62 (m, 3H), 7.68 (d, 1H, J ) 2.2 Hz), 8.69 (br s, 1H, NH,
exch. with D2O); 13C NMR (CDCl3) δ 20.88 (CH3), 21.61 (CH3),
29.65 (CH2), 118.75 (CH), 119.63 (CH × 2), 127.19 (CH), 127.39
(CH), 127.96 (C), 128.20 (CH), 128.53 (C), 129.50 (CH × 2),
129.69 (CH), 130.54 (CH), 131.97 (C), 133.68 (C), 135.25 (C),
135.95 (C), 136.11 (C), 137.37 (C), 142.09 (C), 149.98 (C), 152.40
(C), 159.62 (CO); API-ES calcd, 448.34; found, 448.20.
1-(2′,4′-Dichlorophenyl)-6-methyl-N-p-trifluoromethylphenyl-
1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide (2f). Method A
was used to convert 4a and p-trifluoroaniline into the title product.
The mixture was stirred at room temperature for 2.5 h and purified
by FC (petroleum ether/EtOAc 9.5:0.5) to afford 2f (0.06 g, 19.0%)
as an orange solid. Rf ) 0.303 (petroleum ether/EtOAc 9.5:0.5);
mp 223 °C; IR 1696, 3373; 1H NMR (CDCl3) δ 2.42 (s, 3H), 3.91
(s, 2H), 6.90 (d, 1H, J ) 7.8 Hz), 7.07 (d, 1H, J ) 8.0 Hz), 7.42
(s, 1H), 7.50 (dd, 1H, J ) 8.0 Hz, J ) 1.8 Hz), 7.56-7.62 (m,
3H), 7.69 (d, 1H, J ) 2.2 Hz), 7.84 (d, 2H, J ) 8.6 Hz), 8.89 (br
s, 1H, NH, exch. with D2O); 13C NMR (CDCl3) δ 21.63 (CH3),
29.63 (CH2), 118.82 (CH), 119.16 (CH), 126.19 (CH), 126.26 (CH),
126.34 (CH), 127.23 (CH), 127.52 (CH), 128.02 (C), 128.26 (CH),
128.38 (C), 129.65 (CH), 130.61 (CH), 132.01 (C), 135.80 (C),
136.34 (C), 137.62 (C × 2), 140.88 (C), 141.49 (C), 149.88 (C ×
2), 152.72 (C), 159.93 (CO); API-ES calcd, 502.31; found, 502.20.
1-(2′,4′-Dichlorophenyl)-6-methyl-N-p-methoxyphenyl-1,4-di-
hydroindeno[1,2-c]pyrazole-3-carboxamide (2g). Method A was
used to convert 4a and p-anisidine into the title product. The mixture
was stirred at room temperature for 0.5 h and purified by trituration
with petroleum ether to afford 2e (0.12 g, 38.5%) as a gray solid.
Rf ) 0.341 (petroleum ether/EtOAc 8:2); mp 148 °C; IR 1675,
3358; 1H NMR (CDCl3) δ 2.41 (s, 3H), 3.82 (s, 3H), 3.90 (s, 2H),
6.90 (d, 3H), 7.06 (d, 1H, J ) 7.6 Hz), 7.41 (s, 1H), 7.49 (dd, 1H,
J ) 6.2 Hz, J ) 1.8 Hz), 7.57-7.65 (m, 3H), 7.69 (d, 1H, J ) 2.2
Hz), 8.64 (br s, 1H, NH, exch. with D2O); 13C NMR (CDCl3) δ
21.62 (CH3), 29.65 (CH2), 55.48 (CH3), 114.16 (CH), 118.75 (CH),

Analogues of the Cannabinoid Ligand
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7509
121.29 (CH × 2), 127.20 (CH), 127.40 (CH), 127.92 (C), 128.21
(CH), 128.43 (C), 129.68 (CH × 2), 130.55 (CH), 130.97 (C),
131.97 (C), 136.24 (C), 136.40 (C), 137.37 (C), 141.68 (C), 149.99
(C), 152.57 (C), 156.25 (C), 159.44 (CO); API-ES calcd, 464.34;
found, 467.20.
a yellowish solid. Rf ) 0.10 (petroleum ether/EtOAc 8:2); mp
1
121.7-122 °C; IR 1682, 3347; H NMR (CDCl3) δ 2.41 (s, 3H),
3.84 (s, 2H), 6.52 (br s, 1H, NH, exch. with D2O), 6.91 (d, 1H, J
) 7.8 Hz), 6.97 (d, 1H, J ) 9.0 Hz), 7.07 (d, 1H, J ) 8.2 Hz),
7.09 (d, 1H, J ) 8.2 Hz), 7.39 (s, 1H), 7.50 (dd, 1H, J ) 8.2 Hz;
J ) 2 Hz), 7.58 (d, 1H, J ) 8.4 Hz), 7.70 (d, 1H, J ) 1.8 Hz),
1-(2′,4′-Dichlorophenyl)-6-methyl-N-m, p-dichlorophenyl-1,4-
dihydroindeno[1,2-c]pyrazole-3-carboxamide (2h). Method B
was used to convert 4a and 2,4-dichloroaniline into the title product.
The mixture was stirred at room temperature for 2 h and purified
by trituration with petroleum ether to afford 2h (0.22 g, 62.8%) as
a white solid. Rf ) 0.204 (petroleum ether/EtOAc 9.5:0.5); mp 186-
187 °C; IR 1710, 3347; 1H NMR (CDCl3) δ 2.43 (s, 3H), 3.84 (s,
2H), 6.95-7.14 (m, 3H), 7.40-7.70 (m, 6H), 8.12 (br s, 1H, NH,
exch. with D2O); 13C NMR (CDCl3) δ 20.68 (CH3), 29.62 (CH2),
121.71 (CH), 121.98 (CH), 124.68 (CH), 126.21 (CH), 127.71 (CH),
129.45 (CH), 129.86 (CH × 2), 130.10 (CH), 132.40 (C), 132.00
(C), 132.72 (C), 133.14 (C), 133.87 (C), 134.21 (C), 135.61 (C),
142.05 (C), 146.01 (C), 151.27 (C), 152.91 (C), 156.63 (C), 158.03
(CO); API-ES calcd, 503.21; found, 503.10.
1-(2′,4′-Dichlorophenyl)-6-methyl-N-o, p-dichlorophenyl-1,4-
dihydroindeno[1,2-c]pyrazole-3-carboxamide (2i). Method A was
used to convert 4a and 3,4-dichloroaniline into the title product.
The mixture was stirred at room temperature for 3 h and purified
by trituration with petroleum ether to afford 2i (0.20 g, 58.8%) as
a white solid. Rf ) 0.166 (petroleum ether/EtOAc 9.5:0.5); mp 259-
269 °C; IR 1693, 3390; 1H NMR (CDCl3) δ 2.42 (s, 3H), 3.90 (s,
2H), 6.90 (d, 1H, J ) 8.0 Hz), 7.06 (d, 1H, J ) 7.8 Hz), 7.35-
7.61 (m, 5H), 7.69 (s, 1H), 8.02 (s, 1H), 8.77 (br s, 1H, NH, exch.
with D2O); 13C NMR (CDCl3) δ 20.68 (CH3), 29.44 (CH2), 121.71
(CH), 121.98 (CH), 124.68 (CH), 126.21 (CH), 127.71 (CH), 129.45
(CH), 129.86 (CH × 2), 130.10 (CH), 131.40 (C), 132.00 (C),
132.72 (C), 133.14 (C), 133.87 (C), 134.21 (C), 135.61 (C), 142.05
(C), 146.01 (C), 151.27 (C), 151.91 (C), 156.63 (C), 158.03 (CO);
API-ES calcd, 503.21; found, 503.10.
8.16 (d, 2H, J ) 9.0 Hz), 8.65 (br s, 1H, NH, exch. with D2O); 13
C
NMR (CDCl3) δ 21.64 (CH3), 29.49 (CH2), 112.19 (CH), 118.87
(CH × 2), 125.86 (CH × 2), 127.21 (CH), 127.56 (CH), 128.20
(CH), 128.29 (C × 2), 129.50 (CH), 130.66 (CH), 131.86 (C),
135.70 (C), 136.38 (C), 137.73 (C), 139.52 (C), 141.26 (C), 149.73
(C), 152.35 (C), 153.56 (C), 161.94 (CO); API-ES calcd, 494.33;
found, 434.23.
1-(2′,4′-Dichlorophenyl)-6-methyl-N-p-methoxyphenyl-1,4-di-
hydroindeno[1,2-c]pyrazole-3-carbohydrazide (2m). Method B
was used to convert 4a and p-methoxyphenylhydrazine into the
title product. The mixture was stirred at room temperature for 9 h
and purified by FC (petroleum ether/EtOAc 6:4) to afford 2m (0.07
g, 20.9%) as an orange solid. Rf ) 0.151 (petroleum ether/EtOAc
1
9.5:0.5); mp 62-63 °C; IR 1670, 3347; H NMR (CDCl3) δ 2.40
(s, 3H), 3.75 (s, 3H), 3.84 (s, 2H), 6.10 (br s, 1H, NH, exch. with
D2O), 6.82 (d, 2H, J ) 8.6 Hz), 6.89-6.98 (m, 3H), 7.07 (d, 1H,
J ) 8.2 Hz), 7.39 (s, 1H), 7.49 (dd, 1H, J ) 8.2 Hz; J ) 2 Hz),
7.56 (d, 1H, J ) 8.0 Hz), 7.68 (d, 1H, J ) 2.0 Hz), 8.65 (br s, 1H,
NH, exch. with D2O); 13C NMR (CDCl3) δ 21.62 (CH3), 29.54
(CH2), 55.63 (CH3), 114.60 (CH), 115.72 (CH), 118.79 (CH),
127.18 (CH), 127.42 (CH), 128.14 (C), 128.21 (CH), 128.42 (C),
128.82 (CH), 129.59 (CH), 130.57 (CH), 130.90 (CH), 131.86 (C),
135.89 (C), 135.12 (C), 137.43 (C), 140.24 (C), 141.77 (C), 149.85
(C), 152.04 (C), 154.74 (C), 161.97 (CO); API-ES calcd, 479.36;
found, 479.26.
7-Chloro-1-(2′,4′-dichlorophenyl)-6-methyl-N-cyclohexylamine-
1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide (2n). Method
B was used to convert 4b and cyclohexylamine into the title product
using CH2Cl2 instead of acetonitrile. The mixture was stirred at
room temperature for 20 h and purified by FC (petroleum ether/
EtOAc 9:1) to afford 2n (0.20 g, 63%) as a pale yellow solid. Rf )
0.24 (petroleum ether/EtOAc 9:1); mp 241-243 °C; IR 1658, 3413;
1H NMR (CDCl3) δ 1.07-2.10 (m, 10H), 2.41 (s, 3H), 3.84 (s,
2H), 3.76-4.10 (m, 1H), 6.79 (d, 1H, J ) 8.4 Hz, NH, exch. with
D2O), 6.94 (s, 1H), 7.43 (s, 1H), 7.43-7.75 (m, 3H); 13C NMR
(CDCl3) δ 20.42 (CH3), 24.96 (CH2 × 2), 25.55 (CH2), 29.34 (CH2),
33.15 (CH2 × 2), 48.10 (CH), 119.43 (CH), 128.31 (CH), 128.56
(CH), 128.69 (C), 129.62 (CH), 130.33 (C), 130.61 (CH), 131.76
(C), 132.70 (C), 134.67 (C), 135.73 (C), 136.20 (C), 142.29 (C),
148.09 (C), 150.73 (C), 160.76 (CO); API-ES calcd, 474.81; found,
474.25.
7-Chloro-1-(2′,4′-dichlorophenyl)-6-methyl-N-piperidin-1-yl-
1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide (2o). Method
B was used to convert 4b and N-aminopiperidine into the title
product using CH2Cl2 instead of acetonitrile. The mixture was stirred
at room temperature for 7 h and purified by FC (petroleum ether/
EtOAc 8:2) to afford 2o (0.21 g, 64%) as a yellow solid. Rf ) 0.14
(petroleum ether/EtOAc 8:2); mp 230-232 °C; IR 1685, 3395; 1H
NMR (CDCl3) δ 1.33-1.90 (m, 6H), 2.41 (s, 3H), 2.73-3.09 (m,
4H), 3.84 (s, 2H), 6.94 (s, 1H), 7.42 (s, 1H), 7.43-7.76 (m, 4H);
13C NMR (CDCl3) δ 20.42 (CH3), 23.29 (CH2), 25.36 (CH2 × 2),
29.32 (CH2), 57.07 (CH2 × 2), 119.45 (CH), 128.32 (CH), 128.56
(CH), 129.17 (C), 129.58 (CH), 130.21 (C), 130.64 (CH), 131.74
(C), 132.71 (C), 134.78 (C), 135.64 (C), 136.26 (C), 141.43 (C),
148.09 (C), 150.64 (C), 158.95 (CO). API-ES calcd, 475.80; found,
476.05.
1-(2′,4′-Dichlorophenyl)-6-methyl-N-phenyl-1,4-dihydroindeno-
[1,2-c]pyrazole-3-carbohydrazide (2j). Method B was used to
convert 4a and phenylhydrazine into the title product. The mixture
was stirred at room temperature for 9 h and purified by trituration
with petroleum ether to afford 2j (0.26 g, 83.1%) as an orange
solid. Rf ) 0.194 (petroleum ether/EtOAc 8:2); mp 195-196 °C;
1
IR 1685, 3347; H NMR (CDCl3) δ 2.40 (s, 3H), 3.84 (s, 2H),
6.21 (d, 1H, J ) 4.2 Hz, NH, exch. with D2O), 6.85-7.10 (m,
5H), 7.20-7.30 (m, 2H), 7.38 (s, 1H), 7.48 (dd, 1H, J ) 8.6 Hz,
J ) 2.2 Hz), 7.58 (d, 1H, J ) 8.6 Hz), 7.69 (d, 1H, J ) 2.2 Hz),
8.59 (d, 1H, J ) 4.2 Hz, NH, exch. with D2O); 13C NMR (CDCl3)
δ 21.61 (CH3), 29.52 (CH2), 113.72 (CH × 2), 118.80 (CH), 121.24
(CH), 127.17 (CH), 127.43 (CH), 128.20 (CH), 128.41 (C), 129.17
(CH × 2), 129.59 (CH), 130.58 (CH), 131.86 (C), 135.88 (C),
136.14 (C), 137.44 (C × 2), 140.19 (C), 148.10 (C), 149.86 (C),
152.07 (C), 161.94 (CO); API-ES calcd, 449.33; found, 449.23.
1-(2′,4′-Dichlorophenyl)-6-methyl-N-p-chlorophenyl-1,4-dihy-
droindeno[1,2-c]pyrazole-3-carbohydrazide (2k). Method B was
used to convert 4a and p-chlorophenylhydrazine into the title
product. The mixture was stirred at room temperature for 3 h and
purified by trituration with petroleum ether to afford 2k (0.30 g,
90.8%) as a cream solid. Rf ) 0.070 (petroleum ether/EtOAc 8:2);
1
mp 245.5 °C; IR 1658, 3370; H NMR (CDCl3) δ 2.41 (s, 3H),
3.84 (s, 2H), 6.91 (d, 1H, J ) 7.8 Hz), 7.06 (d, 1H, J ) 7.8 Hz),
7.35-7.52 (m, 4H), 7.60 (d, 1H, J ) 8.2 Hz), 7.68 (d, 1H, J ) 2.2
Hz), 7.94 (d, 2H, J ) 8.4 Hz), 9.21 (d, 1H, NH, exch. with D2O),
10.26 (br s, 1H, NH, exch. with D2O); 13C NMR (CDCl3) δ 21.40
(CH3), 29.22 (CH2), 118.56 (CH), 126.81 (CH), 127.18 (CH),
127.71 (C), 127.65 (CH), 128.12 (C), 128.35 (CH × 2), 129.07
(CH × 2), 129.50 (CH), 130.12 (CH), 131.30 (C), 135.64 (C),
135.75 (C), 137.08 (C × 2), 137.84 (C), 139.74 (C), 149.38 (C ×
2), 160.02 (CO); API-ES calcd, 483.78; found, 483.68.
6-Chloro-1-(2′,4′-dichlorophenyl)-7-methyl-N-piperidin-1-yl-
1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide (2p). Method
B was used to convert 4c and N-aminopiperidine into the title
product using CH2Cl2 instead of acetonitrile. The mixture was stirred
at room temperature for 20 h and purified by FC (petroleum ether/
EtOAc 6:4) to afford 2p (0.18 g, 54%) as a yellow solid. Rf )
0.25 (petroleum ether/EtOAc 6:4); mp 235-237 °C; IR 1684, 3346;
1H NMR (CDCl3) δ 1.05-1.93 (m, 6H), 2.34 (s, 3H), 2.78-3.05
(m, 4H), 3.85 (s, 2H), 6.80 (s, 1H), 7.40-7.60 (m, 2H), 7.53 (s,
1-(2′,4′-Dichlorophenyl)-6-methyl-N-p-nitrophenyl-1,4-dihy-
droindeno[1,2-c]pyrazole-3-carbohydrazide (2l). Method B was
used to convert 4a and p-nitrophenylhydrazine into the title product.
The mixture was stirred at room temperature for 9 h and purified
by FC (petroleum ether/EtOAc 8:2) to afford 2l (0.17 g, 49.4%) as

7510 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Murineddu et al.
1H), 7.60-7.79 (m, 2H); 13C NMR (CDCl3) δ 20.36 (CH3), 23.29
(CH2), 25.36 (CH2 × 2), 29.31 (CH2), 57.06 (CH2 × 2), 120.82
(CH), 126.91 (CH), 128.29 (CH), 129.02 (C), 129.69 (CH), 129.81
(C), 130.55 (CH), 131.85 (C), 133.23 (C), 134.48 (C), 135.77 (C),
136.19 (C), 141.43 (C), 148.48 (C), 150.87 (C), 158.97 (CO); API-
ES calcd, 475.80; found, 475.25.
6b into the title product. Compound 7a (0.51 g, 96.0%) was isolated
as a white solid. Rf ) 0.21 (petroleum ether/EtOAc 9:1); IR 1680,
1730, 3440; 1H NMR (CDCl3) δ 1.43 (t, 3H, J ) 7.2 Hz), 2.49 (s,
3H), 3.92 (s, 2H), 4.42 (q, 2H, J ) 7.2 Hz), 7.42 (s, 1H), 7.82 (s,
1H), 13.20 (br s, 1H, OH exchange with D2O); 13C NMR (CDCl3)
δ 14.15 (CH3), 21.27 (CH3), 31.01 (CH2), 62.31 (CH2), 116.58 (C),
124.08 (CH), 128.22 (CH), 134.58 (C), 136.38 (C), 144.00 (C),
148.71 (C), 153.91 (C), 162.59 (CO), 197.30 (CO).
Synthesis of 5-Methyl-6-nitroindanone (10a) and 5-Methyl-
4-nitroindanone (10b). To a mixture of 5-methylindanone 931
(20.52 mmol, 1 equiv) in concentrated H2SO4 (26 mL) cooled at
-5 °C, a solution of KNO3 (0.9 equiv) in concentrated H2SO4 (6.7
mL) was dropwise added in 2 h. The mixture was stirred for 2 h at
the same temperature and then poured in ice. The resulting mixture
was stirred for 13 h at room temperature, then extracted with Et2O
and washed (H2O). The organic layers, dried (Na2SO4) and
concentrated, afforded a crude product that was purified by FC
(petroleum ether/EtOAc, 9:1) to afford the analytically pure
products, which showed two bands at Rf 0.20 and 0.41. The
Ethyl R-(5-Chloro-6-methyl-1-oxo-2,3-dihydro-1H-inden-2-
yl)-R-oxo-acetate (7c). General procedure IV was used to convert
6c into the title product. Compound 7c (0.46 g, 87.0%) was isolated
as a white solid. Rf ) 0.50 (petroleum ether/EtOAc 7:3); IR 1685,
1725, 3445; 1H NMR (CDCl3/DMSO) δ 1.44 (t, 3H, J ) 7.2 Hz),
2.45 (s, 3H), 3.93 (s, 2H), 4.45 (q, 2H, J ) 7.2 Hz), 7.54 (s, 1H),
7.71 (s, 1H), 14.50 (br s, 1H, OH exchange with D2O); 13C NMR
(CDCl3) δ 14.15 (CH3), 21.27 (CH3), 31.01 (CH2), 62.31 (CH2),
116.38 (C), 124.08 (CH), 128.22 (CH), 134.58 (C), 136.38 (C),
144.00 (C), 148.71 (C), 153.75 (C), 162.59 (CO), 197.30 (CO).
General Procedure III: Synthesis of Tricyclic Esters (8b,c).
A stirred mixture of diketoester 7b,c (1.0 equiv, 7.20 mmol) and
2,4-dichlorophenylhydrazine hydrochloride (1.1 equiv) in CH3-
COOH (54 mL) was heated under reflux for 8 h. The reaction was
allowed to cool to room temperature, and the precipitate was filtered,
washed with water, and air-dried to yield the analytically pure ester.
Ethyl 7-Chloro-1-(2′,4′-dichlorophenyl)-6-methyl-1,4-dihy-
droindeno[1,2-c]pyrazole-3-carboxylate (8b). General procedure
III was used to convert 7b and 2,4-dichlorophenylhydrazine
hydrochloride into the title product 8b (3.03 g, quantitative yield)
as a pale yellow solid. Rf ) 0.24 (petroleum ether/EtOAc 9:1); mp
1
component at Rf 0.20 (2.31 g, 66%) is a pale yellow solid. IR, H
NMR spectra, and elemental analysis showed it to be 5-methyl-6-
1
nitroindanone (10a); IR 1690; H NMR (CDCl3) δ 2.68 (s, 3H),
2.73-2.91 (m, 2H), 3.14-3.31 (m, 2H), 7.47 (s, 1H), 8.31 (s, 1H);
13C NMR (CDCl3) δ 21.13 (CH3), 25.76 (CH2), 36.52 (CH2), 120.08
(CH), 130.74 (CH), 135.94 (C), 139.88 (C), 149.16 (C), 158.55
(C), 204.40 (CO). The component at Rf 0.41 (0.93 g, 26%) is a
yellow solid. IR, 1H NMR spectra, and elemental analysis showed
it to be 5-methyl-4-nitroindanone (10b); IR 1690; 1H NMR (CDCl3)
δ 2.59 (s, 3H), 2.70-2.87 (m, 2H), 3.25-3.42 (m, 2H), 7.40 (d,
1H, J ) 7.8 Hz), 7.83 (d, 1H, J ) 7.8 Hz); 13C NMR (CDCl3) δ
19.56 (CH3), 24.69 (CH2), 35.99 (CH2), 126.59 (CH), 132.02 (CH),
137.45 (C), 138.97 (C), 148.32 (C), 148.33 (C), 204.14 (CO).
Synthesis of 6-Amino-5-methylindan-1-one (11). A solution
of 5-methyl-6-nitroindanone 10a (2.51 g, 13.13 mmol) in 38 mL
of EtOH was hydrogenated in a Parr shaker over 0.28 g (0.26 mmol)
of 10% Pd/C under a hydrogen pressure of 2 atm at room
temperature for 1 h. The mixture was filtered through Celite, and
the catalyst was washed with several portions of hot EtOH. The
solution was evaporated, and the residue was purified by FC
(CHCl3/MeOH 9.8:0.2) to afford a pale yellow solid (1.58 g, 75%).
Rf ) 0.41 (CHCl3/MeOH 9.8:0.2); mp 186-186.5 °C; IR 1700;
1H NMR (CDCl3) δ 2.26 (s, 3H), 2.60-2.80 (m, 2H), 2.93-3.14
(m, 2H), 3.72 (br s, 2H), 7.00 (s, 1H), 7.17 (s, 1H); 13C NMR
(CDCl3) δ 18.43 (CH3), 24.95 (CH2), 36.73 (CH2), 107.53 (CH),
127.95 (CH), 131.42 (C), 141.52 (C), 144.27 (C), 146.03 (C),
207.14 (CO).
1
216-218 °C; IR 1725; H NMR (CDCl3/DMSO) δ 1.44 (t, 3H, J
) 7.0 Hz), 2.41 (s, 3H), 3.80 (s, 2H), 4.44 (q, 2H, J ) 7.2 Hz),
6.93 (s, 1H), 7.43-7.75 (m, 4H); 13C NMR (CDCl3) δ 14.46 (CH3),
20.52 (CH3), 29.41 (CH2), 61.25 (CH2), 120.95 (CH), 126.85 (CH),
128.21 (CH), 129.94 (C), 129.96 (CH), 130.33 (CH), 131.86 (C),
133.26 (C), 134.69 (C), 135.78 (C), 136.34 (C), 139.35 (C), 147.88
(C × 2), 150.76 (C), 162.09 (CO).
Ethyl 6-Chloro-1-(2′,4′-dichlorophenyl)-7-methyl-1,4-dihy-
droindeno[1,2-c]pyrazole-3-carboxylate (8c). General procedure
III was used to convert 7c and 2,4-dichlorophenylhydrazine
hydrochloride into the title product 8c (1.24 g, 89.0%) as a pale
yellow solid. Rf ) 0.31 (petroleum ether/EtOAc 9:1); mp 245-
1
246 °C; IR 1720; H NMR (CDCl3) δ 1.44 (t, 3H, J ) 7.2 Hz),
2.34 (s, 3H), 3.81 (s, 2H), 4.47 (q, 2H, J ) 7.2 Hz), 6.93 (s, 1H),
7.43-7.75 (m, 4H); 13C NMR (CDCl3) δ 14.44 (CH3), 20.37 (CH3),
29.40 (CH2), 61.27 (CH2), 120.95 (CH), 126.85 (CH), 128.21 (CH),
129.94 (C), 129.96 (CH), 130.33 (CH), 131.86 (C), 133.27 (C),
134.69 (C), 135.80 (C), 136.28 (C), 139.35 (C), 147.88 (C × 2),
150.76 (C), 162.09 (CO).
Synthesis of 6-Chloro-5-methylindan-1-one (6b). To a mixture
of aminoketone 11 (1.86 mmol, 1 equiv) in a 15% solution of HCl
(2.50 mL) was cautiously added an aqueous solution of NaNO2
(1.2 equiv, 1 mL), and the whole was stirred at 0 °C. The resulting
solution was dropwise added to a mixture of CuCl (3.6 equiv) in
concentrated HCl (5.30 mL) at the same temperature. The resulting
mixture was stirred for 24 h at room temperature, then poured in
water, and extracted with EtOAc. The organic layers, dried (Na2-
SO4) and concentrated, afforded a crude product that was purified
by FC (petroleum ether/EtOAc, 8:2), furnishing the analytically
pure products as pale yellow solid (0.34 g, quantitative yield). Rf
General Procedure IV: Synthesis of Carboxylic Acids (4b,c).
A mixture of ester 8b,c (1.0 equiv, 0.5 mmol) and potassium
hydroxide in pellets (13 equiv) in EtOH/H2O 1:1 (6 mL) was heated
under reflux for 4 h. The mixture was concentrated and then
acidified with 37% HCl. The precipitate was filtered, washed with
water, and air-dried to yield the analytically pure acid.
7-Chloro-1-(2′,4′-dichlorophenyl)-6-methyl-1,4-dihydroindeno-
[1,2-c]pyrazole-3-carboxylic Acid (4b). General procedure II was
used to convert 8b into the title product 4b (0.20 g, quantitative
yield) as a yellow solid. Rf ) 0.47 (CHCl3/MeOH 9:1); mp 239-
239.5 °C; IR 1690, 3410; 1H NMR (CDCl3/DMSO) δ 2.41 (s, 3H),
3.79 (s, 2H), 6.94 (s, 1H), 7.35-7.75 (m, 4H); 13C NMR (CDCl3/
DMSO) δ 19.78 (CH3), 28.79 (CH2), 118.79 (CH), 127.70 (CH),
127.93 (CH), 129.31 (CH), 129.68 (CH), 130.81 (C), 132.06 (C),
133.99 (C × 2), 135.12 (C), 135.49 (C), 139.30 (C), 146.97 (C ×
2), 149.75 (C), 163.06 (CO).
6-Chloro-1-(2′,4′-dichlorophenyl)-7-methyl-1,4-dihydroindeno-
[1,2-c]pyrazole-3-carboxylic Acid (4c). General procedure II was
used to convert 8c into the title product 4c (0.18 g, 93%) as a yellow
solid. Rf ) 0.28 (CHCl3/MeOH 9:1); mp 229-231 °C; IR 1690,
3410; 1H NMR (CDCl3/DMSO) δ 2.33 (s, 3H), 3.79 (s, 2H), 6.84
(s, 1H), 7.48-7.93 (m, 4H); 13C NMR (CDCl3/DMSO) δ 19.91
(CH3), 29.00 (CH2), 120.52 (CH), 126.47 (CH), 128.25 (CH),
1
) 0.48 (petroleum ether/EtOAc 8:2); mp 96-98 °C; IR 1690; H
NMR (CDCl3) δ 2.46 (s, 3H), 2.67-2.73 (m, 2H), 3.05-3.11 (m,
2H), 7.36 (s, 1H), 7.72 (s, 1H); 13C NMR (CDCl3) δ 21.08 (CH3),
25.27 (CH2), 36.60 (CH2), 123.90 (CH), 128.71 (CH), 136.45 (C),
141.50 (C), 143.29 (C), 153.34 (C), 205.18 (CO).
General Procedure II: Synthesis of R,γ-Diketoesters (7b,c).
Sodium metal (2.0 equiv) was added in small portion to dry ethanol
(1.8 mL) and stirred until all the sodium had reacted. Ethyl oxalate
(2.0 eq) was added, followed by dropwise addition of a solution of
appropriate ketone 6b,c starting material (1.0 equiv, 1.88 mmol)
in dry ethanol (18 mL). The mixture was stirred at room temperature
for 24 h and than it was slowly poured into ice and 2 N HCl was
added. The precipitate was filtered, washed with cool ethanol, and
air-dried to yield the analytically pure diketoester.
Ethyl R-(6-Chloro-5-methyl-1-oxo-2,3-dihydro-1H-inden-2-
yl)-R-oxo-acetate (7b). General procedure IV was used to convert

Analogues of the Cannabinoid Ligand
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7511
129.32 (C), 129.61 (C), 129.84 (CH × 2), 130.90 (C), 132.25 (C),
133.96 (C), 135.35 (C), 135.53 (C), 139.64 (C), 147.66 (C),149.96
(C), 162.98 (CO).
shown by western blot analysis). The dose of SR144528 was chosen
among the doses lacking an intrinsic activity and providing an
appropriate block of the CB2 receptor.12,30,34,40
Animals. Male CD-1 mice (Charles River S.p.A., Calco, LC,
Italy), weighing from 20 to 35 g were used. Mice were housed in
plastic cages under a 12 h artificial light-dark cycle (lights off at
8.00 p. m.), at a constant temperature (22 ( 2 °C). Water and
laboratory rodent chow (MIL Morini, San Polo D’Enza, RE, Italy)
were provided ad libitum. All experimental procedures were
performed in strict accordance with the E.C. regulation for care
and use of experimental animals (EEC No 86/609).
For western blot analysis after appropriate time of exposure, cells
were collected by centrifugation at 1000 × g and the resulting
pellets were washed in ice-cold PBS buffer by centrifugation at
1000 × g. The cells were then lysed at 4 °C in 50 µL of 20 mM
HEPES buffer (pH 7.9) containing NaCl 125 mM; MgCl2 5 mM;
glycerol 12%; ethylenediaminetetracetic acid (EDTA) 0.2 mM;
Nonidet P-40 0.1%; dithithreitol (DTT) 5 mM; phenilmethylsul-
phonil fluoride (PMSF) 0.5 mM; leupeptin 0.5 µg/mL; and pepstatin
A 0.7 µg/mL. The extracts were then centrifuged at 10 000 × g (at
4 °C) for 15 min, and the resulting supernatant was collected as
total cell extracts. An aliquot was analyzed for protein concentration
determination by using protein assay kit II (Bio-Rad Laboratories,
Hercules, CA), and the rest was frozen at -80 °C until assayed.
Western blot studies were performed as previously described.41
Briefly, aliquots of cell extracts containing 50 µg of total protein
were separated by 10% sodium dodecyl sulfate-polyacrilamide gels
(SDS PAGE, Bio-Rad) and transferred to nitrocellulose membranes
(Bio-Rad). Blots were blocked with 5% nonfat dry milk in TBST
(0.1% Tween 20 inTris borate saline; Bio-Rad) and probed with a
specific antibody against Phospho MAPK 1/2 (Cell Signaling
Technology, Beverly, MA) with a 1:1000 dilution in 1% BSA TBST
overnight. After washing in TBST, blots were probed with
horseradish peroxidase conjugated antibodies (Cell Signaling
Technology) with a 1:2000 dilution in TBST plus 5% milk, and
after washing in TBST, chemiluminescence was detected by West
Pico chemiluminescent substrate (Pierce, Rockford, IL). Immu-
noreactive bands were visualized with a Fuji Las 1000 image
analyzer (Raytest Isotopenmessgera¨te GmbH, Straubenhartd, Ger-
many), and the optical density of immunoreactive bands was
measured using a specific software (AIDA 2.11, Raytest Isotopen-
messgera¨te GmbH, Straubenhartd, Germany). One-way ANOVA
was performed as a statistical analysis using Graph Pad Prism
program (San Diego, CA).
Chemicals and Drugs. [3H]-CP-55 940 (specific activity 180
Ci/mmol) was purchased from New England Nuclear (Boston, MA).
CP-55 940 was obtained from Tocris Cookson Ltd (Bristol, U.K.).
SR144528 was kindly provided by Sanofi-Synthe´labo (now
Sanofi-Aventis). For binding experiments, drugs were dissolved
in dimethyl sulfoxide (DMSO). DMSO concentration in the
different assays never exceeded 0.1% (v/v) and was without effect
on radioligand binding.
For radioligand binding assays and in vitro CB2 activity
evaluation, if not otherwise specified, chemicals were purchased
from Sigma Aldrich (Milano, Italy).
Radioligand Binding Methods. Mice were killed by cervical
dislocation, and the brain (minus cerebellum) and spleen were
rapidly removed and placed on an ice-cold plate. After thawing,
tissues were homogenated in 20 vol (wt/v) of ice-cold TME buffer
(50 mM Tris-HCl, 1 mM EDTA and 3.0 mM MgCl2, pH 7.4). The
homogenates were centrifuged at 1086 × g for 10 min at 4 °C,
and the resulting supernatants were centrifuged at 45 000 × g for
30 min.
[3H]-CP-55 940 binding was performed by the method previously
described by Ruiu et al.33 Briefly, the membranes (30-80 µg of
protein) were incubated with 0.5-1 nM of [3H]-CP-55 940 for 1 h
at 30 °C in a final volume of 0.5 mL of TME buffer containing 5
mg/mL of fatty acid-free bovine serum albumin (BSA). Nonspecific
binding was estimated in the presence of 1 µM of CP-55 940. All
binding studies were performed in disposable glass tubes pretreated
with Sigma-Cote (Sigma Chemical Co., Ltd., Poole, U.K.) to reduce
nonspecific binding. The reaction was terminated by rapid filtration
through Whatman GF/C filters presoaked in 0.5% polyethylene-
imine (PEI) using a Brandell 36-sample harvester (Gaithersburg,
MD). Filters were washed five times with 4 mL aliquots of ice-
cold Tris HCl buffer (pH 7.4) containing 1 mg/mL BSA The filter
bound radioactivity was measured in a liquid scintillation counter
(Tricarb 2900, Packard, Meridien, U.S.A.) with 4 mL of scintillation
fluid (Ultima Gold MV, Packard).
Protein determination was performed by means of Bradford
protein assay38 using BSA as a standard according to the protocol
of the supplier (Bio-Rad, Milan, Italy).
All experiments were performed in triplicate and results were
confirmed in at least five independent experiments. Data from
radioligand inhibition experiments were analyzed by nonlinear
regression analysis of a sigmoid curve using Graph Pad Prism
program. IC50 values were derived from the calculated curves and
converted to Ki values as previously described.39
In Vitro CB2 Activity Evaluation. Human promyelocytic
leukemia HL-60 cells from the European Collection of Cell Cultures
(ECACC, Salisbury, U.K.) were purchased from Sigma-Aldrich
(Milano, Italy). Cell lines were grown at 37 °C in humidified 5%
CO2 in RPMI 1640 medium (Gibco-BRL, Gaithersburg, MA)
supplemented with 10% heat-inactivated fetal bovine serum FBS
(Gibco-BRL), 25 mM HEPES, 2.5 mM sodium pyruvate, and 20
µg/mL gentamicin. Culture medium was added every 2 days and
experiments were made at 80% cell confluency. Tested and
reference compounds were dissolved in culture medium with 1%
DMSO and treatments were made for time course, dose response,
and competition studies in a volume of 10 µL/mL of cell suspension.
To verify whether the observed effects were CB2 receptor
specific, a 5 min pretreatment with the CB2 receptor antagonist
SR144528 (50 nM) was performed before the exposure to the
compounds that displayed a significant induction of P-ERK (as
Supporting Information Available: Table of elemental analy-
ses of compounds 2a-p are included. This material is available
free of charge via the Internet at http://pubs.acs.org.
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