Tricyclic pyrazoles. Part 6. Benzofuro[3,2-c]pyrazole: A versatile architecture for CB2 selective ligands

Article abstract of DOI:10.1016/j.ejmech.2014.05.055

A new series of 1H-benzofuro[3,2-c]pyrazole-3-carboxamides was synthesized. The novel compounds (15-24) were evaluated for their affinity to CB₂ and CB₁ cannabinoid receptors. The synthesis of the title compounds takes advantage of the acid-catalysed thermal cyclization of bicyclic hydrazone ethyl 2-(2-(2,4-dichlorophenyl)hydrazono)-2-(6-methyl-3-oxo-2,3- dihydrobenzofuran-2-yl)acetate to tricyclic ethyl 1-(2,4-dichlorophenyl)-6- methyl-1H-benzofuro[3,2-c]pyrazol-3-carboxylate. All the obtained derivatives showed high affinity to CB₂ receptors. Moreover, significant selectivity for CB₂ over CB₁ receptors was highlighted for lead derivatives amongst the novel series. The best binding profiles were determined for homologues bearing monocyclic and bicyclic monoterpenic substituents at the carbamoyl group at 3 position of the pyrazole ring (K _iCB₂ < 4 nM). In particular, the isopinocampheyl- substituted derivative 22 exhibited the highest selectivity for CB₂ receptors with K_i values of 3.7 and 2398 nM for CB₂ and CB₁ receptors, respectively. Preliminary functional assays evidenced CB₂ agonism behaviour for all the assayed novel derivatives.

Full text of DOI:10.1016/j.ejmech.2014.05.055

European Journal of Medicinal Chemistry 82 (2014) 281e292
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Tricyclic pyrazoles. Part 6. Benzofuro[3,2-c]pyrazole: A versatile
architecture for CB2 selective ligands
,
,
c
,
, f
Giovanni Pinna a *, Giovanni Loriga b, Paolo Lazzari a d, Stefania Ruiu b, Matteo Falzoi e
,
Simona Frau a, Amedeo Pau a, Gabriele Murineddu a, Battistina Asproni a,
Gerard A. Pinna a
a
ꢀ
Dipartimento di Chimica e Farmacia, Universita di Sassari, Via F. Muroni 23/A, 07100 Sassari, SS, Italy
b CNR, Istituto di Farmacologia Traslazionale, U.O.S. Cagliari, Edificio 5, Loc. Piscinamanna, 09010 Pula, CA, Italy
c KemoTech Srl, Edificio 3, Loc. Piscinamanna, 09010 Pula, CA, Italy
d PharmaNess Scarl, Edificio 5, Loc. Piscinamanna, 09010 Pula, CA, Italy
e Consorzio ELPRO, Edificio 5, Loc. Piscinamanna, 09010 Pula, CA, Italy
f
ꢀ
Dipartimento di Scienze della Vita e dell'Ambiente, Lab. Genetica, Universita di Cagliari, Via T. Fiorelli 1, 09126 Cagliari, CA, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 1H-benzofuro[3,2-c]pyrazole-3-carboxamides was synthesized. The novel compounds
(15e24) were evaluated for their affinity to CB2 and CB1 cannabinoid receptors. The synthesis of the title
compounds takes advantage of the acid-catalysed thermal cyclization of bicyclic hydrazone ethyl 2-(2-
(2,4-dichlorophenyl)hydrazono)-2-(6-methyl-3-oxo-2,3-dihydrobenzofuran-2-yl)acetate to tricyclic
ethyl 1-(2,4-dichlorophenyl)-6-methyl-1H-benzofuro[3,2-c]pyrazol-3-carboxylate.
Received 26 February 2014
Received in revised form
21 May 2014
Accepted 23 May 2014
Available online 24 May 2014
All the obtained derivatives showed high affinity to CB2 receptors. Moreover, significant selectivity for
CB2 over CB1 receptors was highlighted for lead derivatives amongst the novel series.
The best binding profiles were determined for homologues bearing monocyclic and bicyclic mono-
terpenic substituents at the carbamoyl group at 3 position of the pyrazole ring (KiCB2 < 4 nM). In
particular, the isopinocampheyl-substituted derivative 22 exhibited the highest selectivity for CB2 re-
ceptors with Ki values of 3.7 and 2398 nM for CB2 and CB1 receptors, respectively.
Keywords:
Cannabinoid receptors
CB2 ligands
benzofuro[3,2-c]pyrazole derivatives
Monoterpenes
CB2 agonists
Preliminary functional assays evidenced CB2 agonism behaviour for all the assayed novel derivatives.
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
lymph nodes) and haematopoietic cells, with high levels of
expression in B-cells, natural killer cells, T4 and T8 cells and
In the past few years the CB2 cannabinoid (CB2) receptor has
emerged as a critical player in the regulation of inflammation and
pain [1] as well as in atherosclerosis [2], osteoporosis [3] and
demyelinating diseases [4].
The CB2 receptor, isolated from HL-60 cells (the human mono-
cytic cell line [5]), belongs to the large superfamily of G protein-
coupled receptors (GPCRs).
The activation of CB2 receptor leads to inhibition of adenyl
cyclase and to stimulation of mutagen-activated protein kinase
pathway [6].
Although the function of this cannabinoid receptor subtype has
not been fully elucidated, CB2 is known to be expressed almost
exclusively in tissues of the immune system (spleen, tonsils and
microglial cells [7].
However, recent studies have also intriguingly demonstrated
the presence of CB2 receptors in brain [8], myocardium, car-
diomyoblasts and endothelial cells of various origins [9].
The pharmacology of the CB2 receptor has revealed significant
differences compared with that of CB1 cannabinoid (CB1) receptor
subtype. In particular, a relative absence of the unfavourable central
nervous system (CNS) side effects has been observed following the
administration of CB2 cannabinoidergic compounds. In contrast,
CB1 agents acting at CNS level determined psychoactive effects, as
in the case of the major component of Cannabis sativa L.
D
9-tetra-
hydrocannabinol (
D
9-THC) [10].
These considerations suggest that novel pharmacotherapies
targeting CB2 receptors may have considerable therapeutic poten-
tial, with particular emphasis to the treatment of inflammatory and
neuropathic pain [1f,11,12], immune disorders [13], and neurode-
generative diseases [14,15].
* Corresponding author.
E-mail addresses: giopinna@uniss.it (G. Pinna), paolo.lazzari@kemotech.it
(P. Lazzari), pinger@uniss.it (G.A. Pinna).
http://dx.doi.org/10.1016/j.ejmech.2014.05.055
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

282
G. Pinna et al. / European Journal of Medicinal Chemistry 82 (2014) 281e292
Various medicinal chemistry strategies have been adopted to
into the cyclic derivative 28 upon treatment with p-toluenesulfonic
acid at reflux temperature. The hydrolysis of ester 28 with KOH in
H2O/EtOH afforded the acid precursor 29 in quantitative yield.
The final coupling reaction of 29 with the required amines in
dichloromethane in the presence of BtOH/EDC gave the desired
carboxamides and carbohydrazides 15e24 (Fig. 3).
develop selective CB2 compounds. The identified CB2 ligands may
be classified into at least six different chemical families:(1) tradi-
tional cannabinoid ligands with a partially reduced dibenzopyran
template (e.g. JWH-133 (1) [16] and JWH-361 (2) [17]), (2) indoles
(e.g. AM-1241 (3) [18], GW405833/L768242 (4) [19,20], A-796260
(5) [21], and AM630 (6) [22]), (3) resorcinol compounds (e.g. HU308
(7) [23] and AM-1703 (8) [24]), (4) pyrazoles (e.g. SR144528 (9) [25]
and NESS (10) [26]), (5) quinolones (e.g. JTE-907 (11) [27] and 12
[28]) and (6) triarylbis-sulfones (e.g. Sch. 336 (13) [29]) (Fig. 1).
With regard to the pyrazole-based cannabinoids, interesting
pharmacological profiles have been evidenced by compounds
characterized by 1,4-dihydroindeno[1,2-c]pyrazole planar scaffold.
N-piperidin-1-yl-1-(2,4-dichlorophenyl)-6-methyl-1,4-
dihydroindeno[1,2-c]pyrazole-3-carboxamide (10) demonstrated
both marked affinity and remarkable selectivity for CB2 receptors
(KiCB2 ¼ 0.037 nM; KiCB1/KiCB2 ¼ 9810:1) [26]. Moreover, the lead
compound as well as a series of corresponding homologues showed
CB2 agonist activity in an in vitro model based on the evaluation of
the extracellular signal-related kinases (ERK 1/2 or p44/p42-MAPK)
expression [31,32].
3. Biology
3.1. Radioreceptor binding assays
Previously reported procedures based on radioreceptor binding
assays were adopted to determine the affinity of the novel com-
pounds to both CB2 and CB1 receptors [31]. Mouse spleen and
mouse brain (minus cerebellum) were employed as starting ma-
trixes for CB2 and CB1 affinity tests, respectively. [3H]CP-55,940 was
employed as radio-labelled ligand in both the procedures. The
experimental IC50 data were converted into the corresponding Ki
values through Cheng-Prusoff's equation [35]. The CB2 and CB1 af-
finity values of the novel derivatives, expressed as KiCB2 and KiCB1,
respectively, were compared with those of 1,4-dihydroindeno[1,2-
c]pyrazole lead compounds 10 and 14, as well as with that of the
reference CB2 antagonist SR144528 (9).
Structural modification of compound 10 by replacement of the
carbamoyl piperidine ring with several cyclic moieties resulted in
analogues expressing lower CB2 receptor affinity, with the excep-
tion of the compound N-cyclohexyl-1-(2,4-dichlorophenyl)-6-
3.2. Intrinsic activity by in vitro assays
methyl-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide
(14)
[31]. In fact, the homologue bearing a cyclohexyl carbamoyl group
at 3 position of the pyrazole ring showed a Ki value of 7.6 nM for CB2
receptor and a selectivity expressed as KiCB1/KiCB2 of 118 (Fig. 2).
Moreover, compound 14, namely NESS400, evidenced significant
antinociceptive activity in Spared Nerve Injury (SNI) neuropathic
mice by alleviating both mechanical allodynia and thermal hyper-
algesia [33].
CB2 intrinsic activity of the novel derivatives was evaluated
through in vitro tests based on the determination of phosphory-
lated ERK 1/2 (P-ERK 1/2) expression in human promyelocytic
leukaemia HL-60 (HL-60) cell line, following the cell treatment
with the cannabinoid compounds. Accordingly to previously re-
ported results, the exposure of HL-60 cells to cannabinoid agonists
induced rapid phosphorylation and activation of the ERK 1/2 which
were reversed by a pre-treatment of the cells with the reference
CB2 selective antagonist SR144528 [31,32]. Moreover, no effect was
highlighted by the treatment of the same cell line with CB1 selective
agonists as consequence of the expression of CB2 but not of CB1
receptors by HL-60 cells.
Analogue procedure was adopted to investigate CB1 intrinsic
activity of novel compounds characterized by good affinity to this
cannabinoid receptor subtype. In this case, mouse neuroblastoma
N1E-115 cell line was employed instead of HL-60 cell line. In fact,
N1E-115 cells express CB1 but not CB2 receptors, and their exposure
to the cannabinoid agonists ACEA and WIN55-212,2 induces a rapid
phosphorylation and activation of the ERK 1/2 [36]. This effect is
counteracted by a pre-treatment of the cells with CB1 antagonists
[37].
To further investigate the potentiality of the planar pyrazole
tricyclic scaffold to determine CB2 affinity and selectivity and with
the purpose to possibly improve CB2 binding profile relative to 10,
in a continuing effort to identify the pharmacophoric groups in this
class of potent CB2 ligands, we designed a new series of 1,4-
dihydroindeno[1,2-c]pyrazole analogues, namely benzofuro[3,2-c]
pyrazoles, containing an oxygen atom at 4 position in place of a
methylene unit (Fig. 3). To ascertain bioisosterism of the novel
compounds relative to 10 and homologues (series I) we have
synthesized compounds 15e24 belonging to the novel benzofuro
[3,2-c]pyrazole series (Fig. 3) and we have evaluated their biological
profiles (affinity to cannabinoid receptors and intrinsic activity).
Interesting profiles were determined for the new compounds,
particularly in the case of derivatives containing monocyclic or
bicyclic monoterpene units at the carbamoyl function at 3 position
of the pyrazole ring. According to the determined cannabinoid
binding affinity and CB2 agonism behaviour, as well as with refer-
ence to previously reported data evidencing pharmaceutical
properties of CB2 agonists, selected compounds of the novel series
could represent useful candidates for the development of innova-
tive strategies for the treatment of various pathologies and disturbs
involving CB2 receptors, i.e. inflammatory and neuropathic pain,
immune disorders, and neurodegenerative diseases.
4. Results and discussion
4.1. Cannabinoid receptor affinities
The cannabinoid receptor affinities of the novel benzofuro[3,2-
c]pyrazole derivatives are shown in Table 1. By comparison, the Ki
values of the lead compounds N-piperidin-1-yl-6-methyl-1-(20,40-
dichlorophenyl)-1,4-dihydroindeno[1,2-c]pyrazole
(10),
carboxamide
N-cyclohexyl-6-methyl-1-(20,40-dichlorophenyl)-1,4-
2. Chemistry
dihydroindeno[1,2-c]pyrazole-3-carboxamide (14), and reference
CB2 ligand SR144528 (9) are reported.
Benzofuro[3,2-c]pyrazole analogues 15e24 were prepared
starting from the synthesis of 6-methylbenzofuran-3(2H)-one 25 as
obtained according to the previously reported procedure [34]
Our initial results showed that the replacement of the 1,4-
dihydroindeno[1,2-c]pyrazole scaffold of 10 and 14 with the ben-
zofuro[3,2-c]pyrazole ring system gave the analogue 15 with a
nanomolar CB2 affinity and an acceptable subtype receptor selec-
tivity (KiCB2 ¼ 12.9 nM; KiCB1/KiCB2 ¼ 138.6). Because the analogue
15 displayed reasonable CB2 affinity and selectivity, we conducted a
(Scheme 1). The
the -diketoester 26 as a tautomeric mixture. Reaction of 26 with
2,4-dichlorophenyl hydrazine gave hydrazone 27, then converted
a-acylation of 25 with diethyl oxalate furnished
a,g

G. Pinna et al. / European Journal of Medicinal Chemistry 82 (2014) 281e292
283
Fig. 1. Chemical structures of CB2 selective ligands [30].

284
G. Pinna et al. / European Journal of Medicinal Chemistry 82 (2014) 281e292
comparison of 18 with 14, the 1H-benzofuro[3,2-c]pyrazole skel-
eton did not influence the cannabinoid binding profile of the
cyclohexyl substituted carbamoyl compounds. In contrast,
remarkable differences were observed by replacing 1,4-
dihydroindeno[1,2-c]pyrazole scaffold with the novel 1H-benzo-
furo[3,2-c]pyrazole in the case of N-piperidinyl carboxamide de-
rivatives, as suggested by KiCB2 and KiCB1 values of 15 relative to
those of 10.
Changes of the size and shape of the cyclohexyl unit of 18 (see
compounds 19e24) had interesting but unpredictable effects on
cannabinoid binding affinity and subtype selectivity.
Indeed the adamantyl analogues 19 (KiCB2 ¼ 17.5 nM) and 20
(KiCB2 ¼ 28.0 nM) showed CB2 affinity 1.1 and 1.8-fold lower than
that of compound 18 and significant higher affinity to CB1 subtype
compared to the same derivative. Consequently, a loss of CB2
selectivity was recorded for both 19 and 20 relative to 18.
The introduction of various monoterpenic moieties in place of
the cyclohexyl group of 18 to afford compounds 21e24 elicited
KiCB2 values lower than 4.0 nM 4.2e6.8-fold enhancements of CB2
receptor affinity compared to 18 were in particular determined for
this sub-class of the novel 1H-benzofuro[3,2-c]pyrazole derivatives.
Menthyl homologue 21 evidenced a high affinity to CB2 re-
ceptors (KiCB2 ¼ 2.3 nM) as well as an interesting CB1 affinity
(KiCB1 ¼ 38.2 nM), with a very low CB1 to CB2 affinity ratio (KiCB1/
KiCB2 ¼ 16.6).
Fig. 2. Structureandcannabinoidreceptor profileofN-cyclohexyl-1-(2,4-dichlorophenyl)-
6-methyl-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide (14), namely NESS400.
detailed structureeactivity relationship (SAR) investigation in or-
der to determine the structural features influencing the affinity to
cannabinoid receptors of these novel compounds.
Our strategy was to prepare analogues of 15 by stepwise intro-
duction of various carbamoyl moieties at the pyrazole ring of the
1H-benzofuro[3,2-c]pyrazole-3-carboxamide architecture of 15.
Compounds 16 and 17 were obtained by replacing the piperi-
dine ring of 15 with a pyrrolidine and a morpholine ring, respec-
tively. Both derivatives displayed acceptable affinities to CB2
receptor (KiCB2 < 40.0 nM), although 2e3-fold lower than that of
15. A comparable loss of affinity was detected for 16 and 17 also to
CB1 receptors, with consequent values of CB2 selectivity for both
derivatives in the same order of that of 15: KiCB1/KiCB2 ¼ 92.3 and
107.8 nM for 16 and 17, respectively. The CB2 affinity decreasing of
16 relative to 15 was in line with previously reported data con-
cerning 6-chloro-1-(20,40-dichlorophenyl)-1,4-dihydroindeno[1,2-
c]pyrazole carboxamide homologues [26].
Amongst the tested compounds, the isopinocampheyl deriva-
tive 22 exhibited the most suitable cannabinoid profile relative to
the definition of novel selective CB2 ligands. The compound showed
in fact KiCB2 ¼ 3.7 nM, KiCB1 ¼ 2398 nM, and KiCB1/KiCB2 ¼ 648.
However, interesting behaviours were also determined for the
bornyl (23) and fenchyl (24) compounds, which showed KiCB2
values of 3.5 and 2.5, respectively, and KiCB1/KiCB2 values higher
than 100.
4.2. Intrinsic activity by in vitro assays
Removal of the carbamoyl piperidine-nitrogen atom to give the
corresponding cyclohexyl derivative 18 induced no significant
variation of KiCB2 value and a slight increase of binding affinity to
CB1 receptors. Therefore, a lower CB2 selectivity was determined for
18 relative to 15 (KiCB1/KiCB2 ¼ 79.9 for 18). These marginal
changes in cannabinoid receptor affinity suggested that the nitro-
gen atom in the piperidine ring of the carbamoyl function at 3
position of the pyrazole ring of the novel series did not play a
relevant role in binding to CB2 receptors. As evidenced by the
CB2 intrinsic activity of all the synthesized compounds was
ascertained by in vitro test based on HL-60 cells through the eval-
uation of P-ERK 1/2 expression induced by the novel cannabinoid
ligands. CB2 agonism behaviour was highlighted for all the novel
derivatives. According to the reference cannabinoid agonist
WIN55,212-2, all the assayed compounds induced in fact significant
up-regulation of P-ERK 1/2. Fig. 4 reports the P-ERK 1/2 expression
profiles elicited by compounds 15 and 22, as examples of the ob-
tained results. Analogue behaviours were recorded with other
Fig. 3. Design of CB2 cannabinoid receptor ligands: from 1,4-dihydroindeno[1,2-c]pyrazole scaffold (Series I) to 1H-benzofuro[3,2-c]pyrazole skeleton (compounds 15e24).

G. Pinna et al. / European Journal of Medicinal Chemistry 82 (2014) 281e292
285
Scheme 1. Reagents and conditions: (i) Na, (CO2Et)2, dry EtOH, rt, 20 h; (ii) C6H3Cl2NHNH2∙HCl, dry EtOH, 78 ꢁC, 1.5 h; (iii) p-TsOH∙H2O, C6H5Me, 110 ꢁC, 30 h; (iv) KOH, EtOH/H2O
80 ꢁC, 1 h; (v) HOBt/EDC, CH2Cl2, rt, 1 h, then Z-NH2, (Et3N for 16 and 19), CH2Cl2, rt, 22 h.
derivatives, which determined statistically significant increasing of
P-ERK 1/2 at the concentrations reported in Table 2. As evidenced in
Fig. 5, induced P-ERK 1/2 up-regulation was reversed by the pre-
treatment of HL-60 cells with the reference selective CB2 antago-
nist AM630, by confirming the effect elicited by the novel com-
pounds as mediated by CB2 receptors. The same profiles reported in
Fig. 5 for 15 and 22 were determined for all other novel derivatives
as well as for WIN55,212-2 (data not shown).
Considering the cannabinoid binding profile (Table 1), com-
pound 21 characterized by KiCB1 ¼ 38.2 nM was also assayed to-
wards CB1 activity. According to previously reported procedure
[36], CB1 expressing N1E-115 cell line was adopted for the test and
the intrinsic activity of the novel compound was determined by
evaluating the P-ERK 1/2 expression profile. As in the case of
reference cannabinoid agonist WIN55,212-2, compound 21
enhanced P-ERK 1/2 expression in N1E-115 cells (Fig. 6a). CB1
agonism behaviour of the novel cannabinoid ligand was further
supported by the inhibition of its effect on P-ERK 1/2 expression by
a pre-treatment step of N1E-115 cells with the reference selective
CB1 antagonist/inverse agonist rimonabant (Fig. 6b).
characterized by 1,4-dihydroindeno[1,2-c]pyrazole skeleton (Series
I) [26,31]. Differences of two orders of magnitude were instead
observed by the comparison of KiCB2 of lead 10 and that of the
closest homologue of the novel series (15). Further approaches will
be adopted in the future to investigate the reasons of the deter-
mined differences concerning these two specific homologues.
Amongst the novel series, the compounds containing mono-
cyclic or bicyclic monoterpene units at the carbamoyl function
displayed the highest CB2 affinities. Derivatives 22 and 23, bearing
as terpene motif the isopinocampheyl and the bornyl moiety,
respectively, evidenced the best cannabinoid binding profiles.
Compound 21 showed also a good CB1 affinity.
Bioisosterism of the novel 1H-benzofuro[3,2-c]pyrazole de-
rivatives with the previously reported homologues (Series I) was
also confirmed by in vitro tests aimed to evaluate CB2 intrinsic ac-
tivity. As in the case of the reference 1,4-dihydroindeno[1,2-c]pyr-
azole compounds [31,32]. All the novel synthesized derivatives
evidenced CB2 agonism profiles. CB1 agonism behaviour was also
ascertained for compound 21 by in vitro test.
6. Experimental protocols
5. Conclusion
6.1. Chemistry. General methods
We have synthesized novel cannabinoid receptor ligands
(15e24) that combine a new benzofuropyrazole tricyclic scaffold
with various carbamoyl moieties to probe the size and shape of the
ligand binding pockets of CB receptors. The novel cannabinoid li-
gands show good to high affinity to CB2 receptors. The obtained
results are qualitatively comparable and in agreement with those of
our lead homologue 14, as well as of corresponding analogues,
€
Melting points were determined using a Kofler melting point
apparatus and are uncorrected. Infrared spectra (IR) were measured
as Nujol mulls on NaCl plates with a Jasco FT/IR 460 plus spectro-
photometer and are expressed in cmꢀ1 1H NMR spectra were
.
recorded on a 400 MHz instrument, while 13C NMR spectra were
registered at 100 MHz, using deuterated dimethyl sulfoxide

286
G. Pinna et al. / European Journal of Medicinal Chemistry 82 (2014) 281e292
Table 1 (continued)
Table 1
Structures and binding data of compounds 15e24.
Compd
Z
Receptor affinitya,b (nM) CB2 selectivity
a,c
b,c
KiCB2
KiCB1
KiCB1/KiCB2
23
3.5 0.6
469 64
134:1
24
2.5 0.3
257
7
102.8:1
Compd
Z
Receptor affinitya,b (nM) CB2 selectivity
a,c
b,c
KiCB2
KiCB1
KiCB1/KiCB2
15
12.9 1.1 1788 330 138.6:1
10 (comp)26
0.037 0.03 363 30 9810:1
14 (comp)31
7.6 0.7
900 45
70 10
118:1
250:1
SR144528 (comp)25
0.28 0.04
a
Affinity of compounds to CB2 receptor was evaluated using mouse spleen ho-
mogenate and [3H]-CP-55,940.
16
17
29.5 8.7 2722 652 92.3:1
b
Affinity of compounds to CB1 receptors was assayed using mouse brain (minus
cerebellum) homogenate and [3H]-CP-55,940.
c
Ki values were obtained from five independent experiments run in triplicate and
are expressed as the mean of the five values standard error.
37.7 6.3 4064 64 107.8:1
(DMSO-d6) or chloroform (CDCl3) as solvents, and tetramethylsi-
lane (TMS) as internal standard at room temperature. Chemical
shifts are reported as d values in parts per million (ppm) downfield
18
19
15.6
4
1247 129 79.9:1
from TMS. Multiplicities are described as broad singlet (br s),
singlet (s), doublet (d), doublet of doublet (dd), double double
doublet (ddd), triplet (t), quartet (q), multiplet (m). The coupling
constants (J) are expressed in hertz (Hz). Gas chromatogra-
phyemass spectrometry measurements were performed either on
a Hewlett Packard 5790A/5970A or an Agilent 6850/5973 GC/MS
system by electron impact.
17.5 0.9
537 132 30.7:1
Thin-layer chromatography was performed on TLC silica gel 60
F254 aluminium and glass-plates. Visualization was carried out by
UV light (254 nm), KMnO4 and/or Hanessian stain. Flash chroma-
tography was performed with Merck silica gel 60 (230e400 mesh).
Specific rotation was recorded at 25 ꢁC with a PerkineElmer 241
apparatus, using the sodium D line (589 nm), and chloroform as
solvent.
All the solvents and reagents were obtained from Sigma Aldrich
and used without further purification. Air-sensitive reactions were
conducted in anhydrous solvents under dry argon atmosphere and
in oven-dried glassware. 2-Bromo-20-hydroxy-40-methyl-aceto-
phenone and fenchylamine were prepared according to the re-
ported procedures [38,39]. 6-Methylbenzofuran-3(2H)-one was
obtained by refinement of the procedure described on literature
[34,40].
20
21
28 6.6
363 92
13:1
2.3 0.3 38.2
6
16.6:1
6.2. Preparation of compounds 26e29
22
3.7 0.3 2398 629 648:1
6.2.1. Ethyl 2-(3-hydroxy-6-methylbenzofuran-2-yl)-2-oxoacetate
(26)
Sodium (0.136 g, 5.9 mmol) was dissolved in absolute ethanol
(3.0 mL) under argon atmosphere. To the solution were added
diethyl oxalate (0.80 ml, 5.87 mmol) and a solution of the ketone 25
(0.39 g, 2.63 mmol) in absolute ethanol (30 mL). The resulting
mixture was left stirring at room temperature for 20 h, poured into
a mixture of ice and 2 N HCl, and extracted with chloroform. The

G. Pinna et al. / European Journal of Medicinal Chemistry 82 (2014) 281e292
287
Fig. 4. Effect of different doses of the novel CB2 receptor ligands on P-ERK 1/2 expression in HL-60 cells after 15 min of treatment: a) 15; b) 22. Data are expressed as mean
percentage vs vehicle (100%) SEM and are the results of five independent experiments. *p < 0.05; **p < 0.01.
organic phases were dried over Na2SO4, and evaporated under
reduced pressure. The crude residue was triturated with petroleum
ether/Et2O to give 0.47 g (73%) of the title compound as a fluores-
cent yellow solid. Rf 0.48 (CH2Cl2/acetone 7:3); mp 113.0e115.0 ꢁC;
IR (cmꢀ1): 3406, 1746, 1691, 1651; 1H NMR (400 MHz, CDCl3):
6.2.2. Ethyl 2-(2-(2,4-dichlorophenyl)hydrazono)-2-(6-methyl-3-
oxo-2,3-dihydrobenzofuran-2-yl)acetate (27)
2,4-Dichlorophenyl
hydrazine
hydrochloride
(1.20
g,
5.60 mmol) was added to a solution of 26 (1.07 g, 4.31 mmol) in
absolute ethanol (1.15 mL). After stirring at reflux temperature for
1.5 h, the mixture was cooled to room temperature and then poured
into an ice bath. The resulting precipitate was filtered under vac-
uum, washed with cold ethanol and dried to give 1.37 g (78%) of the
hydrazone as a yellow solid. Rf 0.42 (petroleum ether/EtOAc 95:5);
mp 190.0e192.0 ꢁC; IR (cmꢀ1): 3423, 1718, 1619, 1502, 1166; 1H
d
11.80 (br s, 1H), 7.66 (d, J ¼ 8.0 Hz, 1H), 7.23 (s, 1H), 7.10 (d,
J ¼ 8.0 Hz,1H), 4.55 (q, J ¼ 6.8 Hz, 2H), 2.48 (s, 3H),1.49 (t, J ¼ 6.8 Hz,
3H); 13C NMR (100 MHz, CDCl3):
166.9, 165.3, 157.1, 153.4, 143.7,
d
136.6, 125.3, 122.2, 118.4, 112.6, 64.7, 22.4, 13.9; GCeMS m/z (%
relative intensity, ion): 248 (25, Mþ), 249 (3, M þ 1).
NMR (400 MHz, CDCl3):
d 12.78 (s, 1H), 7.61e7.57 (m, 2H), 7.34 (s,
1H), 7.23 (d, J ¼ 8.8 Hz, 1H), 6.93 (d, J ¼ 8.0 Hz, 2H), 5.28 (s, 1H), 4.07
(q, J ¼ 7.2 Hz, 2H), 2.45 (s, 3H), 0.84 (t, J ¼ 7.2 Hz, 3H); 13C NMR
Table 2
Effect of novel CB2 compounds 15e24 on P-ERK 1/2 expression in HL-60 cells after
15 min of treatment.
(100 MHz, CDCl3):
d 198.1, 172.8, 161.9, 149.9, 137.7, 129.1, 128.2,
127.9, 124.9, 123.9, 123.5, 120.1, 119.2, 116.0, 113.3, 116.0, 86.0, 61.4,
Compd
Dosea,* (nM)
Statistically significant %
22.5, 13.1; GCeMS m/z (% relative intensity, ion): 134 (100,
of P-ERK 1/2 expression
M
þ ꢀ 273).
vs vehicleb,
*
15
16
17
18
19
20
21
50
75
75
50
50
75
5
205 12
185 18
178 13
196 20
187 20
175 22
193 11
6.2.3. Ethyl 1-(2,4-dichlorophenyl)-6-methyl-1H-benzofuro[3,2-c]
pyrazole-3-carboxylate (28)
p-Toluenesulfonic acid (0.023 g, 0.123 mmol) was added to a
solution of the hydrazone 27 (0.50 g, 1.23 mmol) in toluene
(6.0 mL). The resulting mixture was heated at reflux temperature
for 30 h and cooled to room temperature. The mixture was
concentrated under reduced pressure and the crude was purified
by flash-chromatography (petroleum ether/Et2O 95:5 / 8:2) to
give 0.25 g (52%) of the title compound as a white solid. Rf 0.30
(petroleum ether/Et2O 8:2); mp 138.0e140.0 ꢁC; IR (cmꢀ1) 3430,
22
23
24
5
205
6
10
10
75
189 11
200 15
190 17
WIN55,212-2 (comp)
a
Lower dose of the compounds determining statistically significant increase of P-
ERK 1/2 expression vs vehicle assumed as 100% (*p < 0.005). Doses from 0.5 to
125 nM were assayed for each novel derivative. The reference CB2 agonist
WIN55,212-2 was tested at the dose of 75 nM according to previously reported
results [32].
2098,1635,1521,1371,1016; 1H NMR (400 MHz, CDCl3):
d 7.59e7.57
(m, 2H), 7.39e7.37 (m, 2H), 7.24 (d, J ¼ 8.0 Hz, 1H), 7.04 (d,
J ¼ 8.0 Hz, 1H), 4.48 (q, J ¼ 7.2 Hz, 2H), 2.43 (s, 3H), 1.41 (t, J ¼ 7.2 Hz,
b
Data obtained from western blots of P-ERK 1/2 expression in the case of the
3H); 13C NMR (100 MHz, CDCl3):
d 162.8, 160.8, 147.4, 138.0, 136.2,
Lower Dose. Data are expressed as a mean percentage of vehicle SEM and are the
results of five separate experiments.
136.1, 135.9,130.5,130.4, 129.6, 128.3, 126.8, 124.7, 119.3, 114.1, 113.9,

288
G. Pinna et al. / European Journal of Medicinal Chemistry 82 (2014) 281e292
Fig. 5. Effect of the CB2 receptor antagonist AM630 (75 nM) on P-ERK 1/2 expression induced on HL-60 cells by: a) the reference cannabinoidergic compound WIN55,212-2 (75 nM)
or 15 (50 nM); b) WIN55,212-2 (75 nM) or 22 (5.0 nM). Cells were pre-treated with AM630 for 5 min, then a 15 min of exposure with WIN55,212-2 (25 nM), 15, or 22 was adopted.
Reference assays were also carried out with AM630 alone or with the cannabinoidergic compounds to be tested without AM630 pre-treatment. Data are expressed as a mean
percentage of vehicle (100%) SEM. *p < 0.05 vs vehicle; #p < 0.05 vs WIN55,212-2; xp < 0.05 vs 15 or 22.
61.6, 22.0, 14.5; GCeMS m/z (% relative intensity, ion): 389 (21, Mþ),
388 (100, M ꢀ 1), 390 (65, M þ 1), 391 (13, M þ 2), 393 (2, M þ 4).
vacuum and the residue was purified by flash-chromatography to
afford the desired compound.
When the starting amine/hydrazide was used as hydrochloride
salt, triethylamine (Et3N) (2.0 eq, 0.50 mmol) was added to the
mixture.
6.2.4. 1-(2,4-Dichlorophenyl)-6-methyl-1H-benzofuro[3,2-c]
pyrazole-3-carboxylic acid (29)
A mixture of ester 28 (0.17 g, 0.44 mmol) and KOH (0.32 g,
5.70 mmol) in EtOH/H2O 1:1 (5.6 mL) was heated at 80 ꢁC for 1 h.
The mixture was cooled to 0 ꢁC and acidified with concentrated
HCl. The resulting precipitate was filtered under vacuum, washed
with cold water and dried to afford the desired acid in quantitative
6.3.1. N-(Piperidin-1-yl)-1-(2,4-dichlorophenyl)-6-methyl-1H-
benzofuro[3,2-c]pyrazole-3-carbohydrazide (15)
General procedure I was used to convert 29 into the title
product. White solid; yield: 81%; Rf 0.10 (petroleum ether/Et2O
6:4); mp 155.0e157.0 ꢁC; IR (cmꢀ1) 3434, 2938, 2856, 1648; 1H
yield as
228.0e230.0 ꢁC; IR (cmꢀ1) 3417, 2096, 1637, 1484, 1434; 1H NMR
(400 MHz, DMSO-d6):
a white solid. Rf 0.30 (CHCl3/MeOH 8:2); mp
NMR (400 MHz, CDCl3):
d
7.66 (d, J ¼ 4.0 Hz, 1H), 7.61e7.58 (m, 2H),
d
8.06 (d, J ¼ 4.0 Hz, 1H), 7.85 (d, J ¼ 8.0 Hz,
7.49e7.45 (m, 2H) 7.29 (d, J ¼ 8.0 Hz, 1H), 7.09 (d, J ¼ 8.0 Hz, 1H),
2.92 (t, J ¼ 4.8 Hz, 4H), 2.49 (s, 3H), 1.81e1.75 (m, 4H), 1.49e1.43 (m,
1H), 7.74 (dd, J ¼ 4.0, 12.0 Hz, 1H), 7.64 (s, 1H), 7.37 (d, J ¼ 8.0 Hz,
1H), 7.20 (d, J ¼ 12.0 Hz, 1H), 2.47 (s, 3H); 13C NMR (100 MHz,
2H); 13C NMR (100 MHz, CDCl3):
d 163.0, 157.6, 146.9, 137.9, 136.3,
DMSO-d6):
d 162.4, 161.8, 147.2, 138.4, 136.0, 135.9, 135.5, 130.7,
136.1, 135.7, 130.6, 130.5, 129.2, 129.0, 128.3, 124.5, 119.1, 114.1, 114.0,
57.1, 25.3, 23.3, 21.9; GCeMS m/z (% relative intensity, ion): 443 (5,
Mþ), 445 (1, M þ 2), 98 (100, M ꢀ 345).
130.3, 130.1, 129.4, 127.3, 125.5, 119.6, 114.0, 113.8, 21.8; GCeMS m/z
(% relative intensity, ion): 360 (1, M ꢀ 1), 83 (100, M ꢀ 278).
6.3. General procedure I: preparation of carbohydrazides 15e17
and carboxamides 18e24
6.3.2. N-(Pyrrolidin-1-yl)-1-(2,4-dichlorophenyl)-6-methyl-1H-
benzofuro[3,2-c]pyrazole-3-carbohydrazide (16)
A mixture of the benzofuro[3,2-c]pyrazole-3-carboxylic acid 29
(1.0 eq, 0.25 mmol), 1-hydroxybenzotriazole (HOBt) (1.2 eq,
0.30 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(EDC) (1.2 eq, 0.30 mmol) in dichloromethane (2.0 mL) was stir-
red at room temperature for 1 h. A solution of the appropriate
amine or hydrazine (2.0 eq, 0.50 mmol) in dichloromethane
(3.0 mL) was added drop-wise. The resulting mixture was stirred at
room temperature for 22 h. The solvent was removed under
General procedure I was used to convert 29 into the title
product. White solid; yield: 94%; Rf 0.30 (petroleum ether/EtOAc
1:1); mp 90.0e92.0 ꢁC; IR (cmꢀ1): 3405, 2977, 1677; 1H NMR
(400 MHz, CDCl3):
d
7.66 (d, J ¼ 4.0 Hz, 1H), 7.59 (d, J ¼ 8.0 Hz, 1H),
7.47 (dd, J ¼ 4.0, 8.0 Hz, 1H), 7.45 (s, 1H), 7.29 (d, J ¼ 8.0 Hz, 1H), 7.09
(d, J ¼ 8.0 Hz, 1H), 3.08e3.05 (m, 4H), 2.49 (s, 3H), 1.95e1.92 (m,
4H); 13C NMR (100 MHz, CDCl3):
d 163.0, 158.5, 146.9, 137.9, 136.3,
136.1, 135.7, 130.6,130.5,129.2,128.8,128.3,124.5, 119.1, 114.0, 113.9,

G. Pinna et al. / European Journal of Medicinal Chemistry 82 (2014) 281e292
289
Fig. 6. a) Effect of different doses of the novel CB1 receptor ligand 21 on P-ERK 1/2 expression in N1E-115 cells after 10 min of treatment. Data are expressed as mean percentage vs
vehicle (100%) SEM and are the results of five independent experiments. *p < 0.05. b) Effect of the CB1 receptor antagonist/inverse agonist rimonabant (SR) at the dose of 1.0 M on
m
P-ERK 1/2 expression induced by the reference cannabinoidergic compound WIN55,212-2 (25 nM) or 21 (125 nM). Cells were pre-treated with SR for 5 min, then a 10 min of
exposure with WIN55,212-2 (25 nM) or 21 was adopted. Reference assays were also carried out with SR alone or with the cannabinoidergic compounds to be tested without SR pre-
treatment. Data are expressed as a mean percentage of vehicle (100%) SEM. *p < 0.05 vs vehicle; #p < 0.05 vs WIN55,212-2; xp < 0.05 vs 21.
55.6, 22.3, 21.9; GCeMS m/z (% relative intensity, ion): 429 (1, Mþ),
430 (1, M þ 1), 85 (100, M ꢀ 344).
119.1, 114.1, 113.9, 48.0, 33.1, 25.6, 24.9, 21.9; GCeMS m/z (% relative
intensity, ion): 442 (3, Mþ), 443 (6, M þ 1), 444 (1, M þ 2), 98 (100,
M ꢀ 344).
6.3.3. N-(Morpholin-1-yl)-1-(2,4-dichlorophenyl)-6-methyl-1H-
benzofuro[3,2-c]pyrazole-3-carbohydrazide (17)
General procedure I was used to convert 29 into the title
product. White solid; yield: quant.; Rf 0.14 (petroleum ether/EtOAc
1:1); mp 178.0e180.0 ꢁC; IR (cmꢀ1) 3430, 2925, 2857,1673; 1H NMR
6.3.5. N-Adamant-2-yl-1-(2,4-dichlorophenyl)-6-methyl-1H-
benzofuro[3,2-c]pyrazole-3-carboxamide (19)
General procedure I was used to convert 29 into the title
product. White solid; yield: 76%; Rf 0.20 (petroleum ether/Et2O
8:2); mp 209.0e211.0 ꢁC; IR (cmꢀ1) 3417, 1664; 1H NMR (400 MHz,
(400 MHz, CDCl3):
d
7.66 (d, J ¼ 4.0 Hz, 1H), 7.65 (s, 1H), 7.60 (d,
J ¼ 8.0 Hz, 1H), 7.47 (dd, J ¼ 4.0, 8.0 Hz, 1H), 7.45 (s, 1H), 7.30 (d,
J ¼ 8.0 Hz, 1H), 7.10 (d, J ¼ 8.0 Hz, 1H), 3.89 (t, J ¼ 4.4 Hz, 4H), 3.02 (t,
CDCl3):
d
7.66 (d, J ¼ 4.0 Hz, 1H), 7.62 (d, J ¼ 8.0 Hz, 1H), 7.49e7.45
(m, 2H), 7.22 (d, J ¼ 8.0 Hz, 1H), 7.09 (d, J ¼ 8.0 Hz, 1H), 4.33 (d,
J ¼ 4.4 Hz, 4H), 2.49 (s, 3H); 13C NMR (100 MHz, CDCl3):
d 163.0,
J ¼ 8.0 Hz, 1H), 2.49 (s, 3H), 2.10 (s, 2H), 1.95e1.91 (m, 8H), 1.78 (s,
157.9, 146.9, 138.1, 136.4, 136.0, 135.8, 130.7, 130.5, 129.2, 128.6,
128.4, 124.6, 119.2, 114.1, 113.9, 66.5, 56.0, 22.0; GCeMS m/z (%
relative intensity, ion): 445 (2, Mþ), 446 (3, M þ 1), 447 (1, M þ 2),
83 (100, M ꢀ 362).
2H), 1.69 (d, J ¼ 12.0 Hz, 2H); 13C NMR (100 MHz, CDCl3):
d 163.0,
159.4, 146.8, 137.8, 136.3, 136.2, 135.5, 130.6, 130.4, 130.0, 129.3,
128.3, 124.5, 119.1, 114.1, 114.0, 53.1, 37.6, 37.1, 32.0, 27.3, 27.2, 21.9;
GCeMS m/z (% relative intensity, ion):495 (15, M þ 1), 496 (4,
M þ 2), 498 (3, M þ 4), 83 (100, M ꢀ 411).
6.3.4. N-Cyclohexyl-1-(2,4-dichlorophenyl)-6-methyl-1H-
benzofuro[3,2-c]pyrazole-3-carboxamide (18)
6.3.6. N-Adamant-1-yl-1-(2,4-dichlorophenyl)-6-methyl-1H-
benzofuro[3,2-c]pyrazole-3-carboxamide (20)
General procedure I was used to convert 29 into the title
product. White solid; yield: 81%; Rf 0.36 (petroleum ether/Et2O
8:2); mp 210.0e212.0 ꢁC; IR (cmꢀ1) 3399, 1670; 1H NMR (400 MHz,
General procedure I was used to convert 29 into the title
product. Off-white solid; yield: quant.; Rf 0.16 (petroleum ether/
Et2O 8:2); mp 140.0e142.0 ꢁC; IR (cmꢀ1) 3409, 3092, 3009, 2932,
2855, 1665; 1H NMR (400 MHz, CDCl3):
d 7.65e7.59 (m, 2H),
7.47e7.45 (m, 2H), 7.29 (d, J ¼ 8.0 Hz, 1H) 7.08 (d, J ¼ 8.0 Hz, 1H),
6.73 (d, J ¼ 8.0 Hz, 1H), 4.09e3.99 (m, 1H), 2.49 (s, 3H), 2.08e2.05
(m, 2H), 1.78e1.75 (m, 2H), 1.69e1.59 (m, 1H), 1.49e1.39 (m, 2H),
CDCl3):
d
7.65 (d, J ¼ 4.0 Hz, 1H), 7.60 (d, J ¼ 8.0 Hz, 1H), 7.46 (dd,
J ¼ 4.0 Hz, 8.0 Hz, 1H), 7.42 (s, 1H) 7.29 (d, J ¼ 8.0 Hz, 1H), 7.08 (d,
J ¼ 8.0 Hz, 1H), 6.58 (s, 1H), 2.49 (s, 3H), 2.18 (s, 6H), 2.13 (s, 3H),
1.34e1.16 (m, 3H); 13C NMR (100 MHz, CDCl3):
d
162.9, 159.3, 146.8,
1.77e1.70 (m, 6H); 13C NMR (100 MHz, CDCl3):
d 163.0, 159.4, 147.0,
137.8, 136.4, 136.2, 135.6, 130.6, 130.5, 129.9, 129.3, 128.3, 124.5,
137.8, 136.4, 136.2, 135.5, 130.7, 130.6, 130.4, 129.3, 128.3, 124.4,

290
G. Pinna et al. / European Journal of Medicinal Chemistry 82 (2014) 281e292
119.1, 114.1, 114.0, 52.3, 41.7, 36.4, 29.5, 21.9; GCeMS m/z (% relative
intensity, ion): 494 (3, Mþ), 495 (6, M þ 1), 496 (2, M þ 2), 83 (100,
M ꢀ 411).
M þ 1), 498 (4, M þ 2), 343 (100, M ꢀ 153); ½aꢂ2D0 ꢀ11.0 (c 10ꢀ3
,
CHCl3).
6.4. Pharmacology
6.3.7. N-Menthyl-1-(2,4-dichlorophenyl)-6-methyl-1H-benzofuro
[3,2-c]pyrazole-3-carboxamide (21)
General procedure I was used to convert 29 into the title
product. Off-white solid; yield: 96%; Rf 0.42 (petroleum ether/Et2O
8:2); mp 70.0e72.0 ꢁC; IR (cmꢀ1) 3403, 3010, 2954, 2867, 1666; 1H
6.4.1. Chemicals, drugs and cells for in vitro assays
[3H]-CP-55,940 (180 Ci/mmol) was obtained from PerkinElmer
Italia. N-piperidinyl-5-(4-chlorophenyl)-1-(2,4-dicholorophenyl)-
4-methyl-1H-pyrazole-3-carboxamide (rimonabant) was pur-
chased from Kemprotech Limited (Middlesbrough, UK) while
WIN55,212-2 and AM630 from Tocris Bioscience.
NMR (400 MHz, CDCl3):
d
7.67 (d, J ¼ 4.0 Hz, 1H), 7.62 (d, J ¼ 8.0 Hz,
1H), 7.47 (dd, J ¼ 4.0, 8.0 Hz, 1H), 7.44 (s, 1H), 7.30 (d, J ¼ 8.0 Hz, 1H),
7.09 (d, J ¼ 8.0 Hz, 1H), 6.55 (d, J ¼ 8.0 Hz, 1H), 4.10e4.01 (m, 1H),
2.49 (s, 3H), 2.14e1.99 (m, 2H),1.77e1.70 (m, 2H),1.27e1.12 (m, 2H),
HL-60 and N1E-115 cell lines from the European Collection of
Cell Cultures (ECACC), Foetal Bovine Serum (FBS), Penicillin-
Streptomycin, L-Glutamine, Amphotericin B, Gentamicin, and
1.01e0.87 (m, 12H); 13C NMR (100 MHz, CDCl3):
d
163.0, 159.5,
146.8, 137.8, 136.4, 136.2, 135.6, 130.6, 130.5, 129.9, 129.3, 128.3,
124.5, 119.1, 114.1, 114.0, 49.7, 48.2, 43.1, 34.6, 31.9, 26.9, 23.9, 22.2,
21.9, 21.2, 16.3; GCeMS m/z (% relative intensity, ion): 498 (2, Mþ),
499 (4, M þ 1), 83 (100, M ꢀ 415); ½aꢂ2D0 ꢀ36.0 (c 10ꢀ3, CHCl3).
dimethyl-sulfoxide (DMSO) were purchased from SigmaeAldrich
(Milan, Italy). RPMI 1640 Medium and Phosphate Buffered Saline
(PBS) were obtained from Gibco-BRL (Gaithensburg, MA).
For radioreceptor binding experiments, compounds to be tested
were dissolved in DMSO. The solvent concentration in the different
assays never exceeded 0.1% (v/v) and was without effects.
Regarding cannabinoid intrinsic activity evaluation, tested and
reference compounds were dissolved in culture medium with 1%
DMSO.
6.3.8. N-Isopinocampheyl-1-(2,4-Dichlorophenyl)-6-methyl-1H-
benzofuro[3,2-c]pyrazole-3-carboxamide (22)
General procedure I was used to convert 29 into the title
product. Off-white solid; yield: 89%; Rf 0.32 (petroleum ether/Et2O
8:2); mp 85.0e87.0 ꢁC; IR (cmꢀ1) 3407, 2935, 1666; 1H NMR
(400 MHz, CDCl3):
d
7.66 (d, J ¼ 4.0 Hz, 1H), 7.62 (d, J ¼ 8.0 Hz, 1H),
6.4.2. Animals
7.47 (dd, J ¼ 4.0, 8.0 Hz, 1H), 7.45 (s, 1H), 7.30 (d, J ¼ 8.0 Hz, 1H), 7.09
(d, J ¼ 8.0 Hz, 1H), 6.75 (d, J ¼ 8.0 Hz, 1H), 4.59e4.51 (m, 1H),
2.75e2.68 (m, 1H), 2.49 (s, 3H), 2.47e2.41 (m, 1H), 2.02e1.94 (m,
2H), 1.89e1.86 (m, 1H), 1.72 (ddd, J ¼ 4.0 Hz, 8.0 Hz, 16.0 Hz, 1H),
1.25 (s, 3H), 1.20 (d, J ¼ 7.2 Hz, 3H), 1.11 (s, 3H), 0.97 (d, J ¼ 10.0 Hz,
Male CD1 mice (Charles River, Calco, LC, Italy) were housed in
the animal care quarters at the following conditions: temperature
22 2 ꢁC, humidity 55 5%, 12 h light/dark cycles, food and water
available ad libitum. Animal management was performed accord-
ing to the UE guidelines for the care and use of experimental ani-
mals (CEE Nꢁ 86/609) and the protocol authorized by Italian Health
Ministry. Experiments were carried out with animals weighing
30e35 g.
1H); 13C NMR (100 MHz, CDCl3):
d 163.0, 159.7, 146.9, 137.8, 136.4,
136.2,135.6,130.6,130.5,129.9,129.3,128.3,124.5,119.1,114.1,114.0,
47.9, 47.6, 46.2, 41.6, 38.5, 37.1, 35.1, 28.1, 23.5, 21.9, 20.9; GCeMS m/
z (% relative intensity, ion): 496 (2, Mþ), 497 (4, M þ 1), 498 (1,
M þ 2), 343 (100, M ꢀ 153); ½aꢂ2D0 ꢀ12.0 (c 10ꢀ3, CHCl3).
6.4.3. Radioreceptor binding assays
Assays were performed according to previously reported pro-
cedure [31]. Spleen and brain (minus cerebellum) tissues were
employed as starting matrixes for CB2 and CB1 binding assays,
respectively. Both the tissues were removed from male CD1 mice
killed by cervical dislocation. Tissues were homogenated in 20 vol.
(wt/v) of ice-cold TME buffer (50 mM TriseHCl, 1 mM EDTA and
3.0 mM MgCl2, pH 7.4). The homogenates were centrifuged at
1086 ꢃ g for 10 min at 4 ꢁC, and the resulting supernatants were
centrifuged at 45,000 ꢃ g for 30 min.
6.3.9. N-Bornyl-1-(2,4-dichlorophenyl)-6-methyl-1H-benzofuro
[3,2-c]pyrazole-3-carboxamide (23)
General procedure I was used to convert 29 into the title prod-
uct. Off-white solid; yield: 89%; Rf 0.26 (petroleum ether/Et2O 8:2);
mp 78.0e80.0 ꢁC; IR (cmꢀ1) 3414, 2956, 1669; 1H NMR (400 MHz,
CDCl3):
d
7.66e7.62 (m, 2H), 7.49e7.44 (m, 2H), 7.30 (d, J ¼ 8.0 Hz,
1H), 7.08 (d, J ¼ 8.0 Hz, 1H), 6.90 (d, J ¼ 8.8 Hz, 1H), 4.55e4.49 (m,
1H), 2.49 (s, 3H),1.86e1.77 (m,1H),1.72e1.63 (m, 2H),1.46e1.40 (m,
1H), 1.30e1.13 (m, 3H), 1.02 (s, 3H), 0.92 (d, J ¼ 8.0 Hz, 6H); 13C NMR
To reduce non-specific binding, all binding studies were per-
formed in Sigma-Cote pre-treated glass tubes (Sigma Chemical Co.
(100 MHz, CDCl3):
d 163.0, 160.3, 146.8, 137.8, 136.3, 136.2, 135.6,
130.6, 130.4, 130.0, 129.3, 128.3, 124.4, 119.1, 114.1, 114.0, 53.6, 49.9,
48.3, 45.0, 37.5, 28.4, 28.1, 21.9, 19.9, 18.8, 13.8; GCeMS m/z (%
relative intensity, ion): 496 (7,M.þ), 497 (16, M þ 1), 498 (4, M þ 2),
83 (100, M ꢀ 414); ½aꢂ2D0 þ3.0 (c 10ꢀ3, CHCl3).
Ltd., Poole, UK). The obtained membranes (30e80 mg of protein as
determined by Bradford protein assay according to the supplier
protocol by Bio-Rad, Milan, Italy) were incubated with 0.5e1.0 nM
of [3H]-CP-55,940 for 1 h at 30 ꢁC. A final volume of 0.5 mL of TME
buffer containing 5 mg/mL of fatty acid-free bovine serum albumin
was adopted. Non-specific binding was estimated in the presence
6.3.10. N-Fenchyl-1-(2,4-dichlorophenyl)-6-methyl-1H-benzofuro
[3,2-c]pyrazole-3-carboxamide (24)
of 1.0 mM of CP-55,940. The reaction was blocked by rapid filtration
General procedure I was used to convert 29 into the title
product. White solid; yield: 96%; Rf 0.30 (petroleum ether/Et2O
8:2); mp 84.0e86.0 ꢁC; IR (cmꢀ1) 3416, 2853, 2360, 1671, 1458,
through Whatman GF/C filters presoaked in 0.5% poly-
ethyleneimine (PEI). A Brandell 36-sample harvester (Gaithersburg,
MD, USA) was used. Filters were washed five times with 4 mL ali-
quots of ice cold Tris HCl buffer (pH 7.4) containing 1.0 mg/mL BSA.
The filter bound radioactivity was measured in a liquid scintillation
counter (Trisarb 2900, Packard, Meridien, USA) with 4 mL of scin-
tillation fluid (Ultima Gold MV, Packard).
All experiments were performed in triplicate and the results
were confirmed in five independent assays. Non-linear regression
analysis of a Sigmoid Curve using Graph Pad Prism program was
adopted to analyse radioligand inhibition experiments and to
determine IC50 values. Affinities to CB1 and CB2 receptors were
1376; 1H NMR (400 MHz, CDCl3):
d
7.67 (d, J ¼ 2.4 Hz, 1H), 7.63 (d,
J ¼ 8.4 Hz, 1H), 7.47 (dd, J ¼ 8.4, 2.4 Hz, 1H), 7.44 (s, 1H), 7.31 (d,
J ¼ 8.0 Hz, 1H), 7.09 (d, J ¼ 8.0 Hz, 1H), 6.92 (d, J ¼ 9.6 Hz, 1H), 3.89
(d, J ¼ 9.6 Hz, 1H), 2.50 (s, 3H) 1.81e1.80 (m, 1H), 1.75e1.70 (m, 2H),
1.54e1.39 (m, 2H), 1.28e1.21 (m, 5H), 1.14 (s, 3H), 0.89 (s, 3H); 13C
NMR (100 MHz, CDCl3):
d 163.0, 160.8, 146.8, 137.8, 136.3, 136.2,
135.5, 130.6, 130.4, 129.8, 129.2, 128.3, 124.5, 119.1, 114.2, 114.0, 63.1,
48.6, 48.2, 42.7, 39.6, 30.9, 27.4, 26.0, 21.9, 21.3, 19.7; GCeMS m/z (%
relative intensity, ion): 496 (8, Mþ), 495 (30, M ꢀ 1), 497 (19,

G. Pinna et al. / European Journal of Medicinal Chemistry 82 (2014) 281e292
291
expressed as Ki (mean standard error) obtained from IC50 values
References
according to previously described procedures [35].
[1] (a) Y. Cheng, S.A. Hitchcock, Targeting cannabinoid agonists for inflammatory
and neuropathic pain, Expert Opin. Invest. Drugs 16 (2007) 951e965;
(b) K. Worm, Q. Jean Zhou, C.T. Saeui, R.C. Green, J.A. Cassel, G.J. Stabley,
R.N. DeHaven, N. Conway-James, C.T. LaBuda, M. Koblish, T.P. Little, R.E. Dolle,
Sulfamoyl benzamides as novel CB2 cannabinoid receptor ligands, Bioorg.
Med. Chem. Lett. 18 (2008) 2830e2835;
6.4.4. Intrinsic activity by in vitro assays
CB2 and CB1 intrinsic activity was evaluated by analysing P-
ERK 1/2 expression induced by the compounds on HL-60 and
N1E-115 cell lines, respectively [31,32,37]. In first experiment sets,
P-ERK 1/2 was determined after 15 min treatment of HL-60 cells,
or 10 min treatment of N1E-115 cells, with the compounds to be
assayed. Successively, to confirm the capability of the novel de-
rivatives to act as CB2 agonists, a 5 min pre-treatment with the
selective CB2 receptor antagonist AM630 (75 nM) was carried out
before the exposure of HL-60 cells to the cannabinoid com-
pounds. Analogous assays were performed to ascertain CB1
intrinsic activity. In these cases, a 5 min pre-treatment with the
(c) K.L. Wright, H. Duncan, K.A. Sharkey, Cannabinoid CB2 receptors in the
gastrointestinal tract: a regulatory system in states of inflammation, Br. J.
Pharmacol. 153 (2008) 263e270;
ꢁ
~
(d) C. Benito, R.M. Tolon, M.R. Pazos, E. Núnez, A.I. Castillo, J. Romero,
Cannabinoid CB2 receptors in human brain inflammation, Br. J. Pharmacol. 153
(2008) 277e285;
(e) G.T. Whiteside, G.P. Lee, K.J. Valenzano, The role of the cannabinoid CB2
receptor in pain transmission and therapeutic potential of small molecule CB2
receptor agonists, Curr. Med. Chem. 15 (2007) 917e936;
(f) J. Guindon, A.G. Hohmann, Cannabinoid CB2 receptors: a therapeutic target
for the treatment of inflammation and neuropathic pain, Br. J. Pharmacol. 153
(2008) 319e334.
selective CB1 antagonist/inverse agonist rimonabant (1.0 mM) was
[2] F. Mach, F. Montecucco, S. Steffens, Cannabinoid receptors in acute and
chronic complications of atherosclerosis, Br. J. Pharmacol. 153 (2008)
290e298.
adopted before the administration of the novel compounds to
N1E-115 cells.
[3] I. Bab, A. Zimmer, Cannabinoid receptors and the regulation of bone mass, Br.
J. Pharmacol. 153 (2008) 182e188.
Cells were grown at 37 ꢁC in humidified 5% CO2 in RPMI 1640
ꢁ
ꢁ
[4] A. Arevalo-Martin, D. García-Ovejero, O. Gomez, A. Rubio-Araiz, B. Navarro-
Galve, E. Guaza Molina-Holgado, F. Molina-Holgado, CB2 cannabinoid receptors
as an emerging target for demyelinating diseases: from neuroimmune in-
teractions to cell replacement strategies, Br. J. Pharmacol. 153 (2008) 216e225.
[5] S. Munro, K.L. Thomas, M. Abu-Shaar, Molecular characterization of a pe-
ripheral receptor for cannabinoids, Nature 365 (1993) 61e65.
[6] V. Di Marzo, L. De Petrocellis, Plant, synthetic and endogenous cannabinoids in
medicine, Annu. Rev. Med. 57 (2006) 553e574.
Medium supplemented with 10% FBS, 1.0
Streptomycin, 2.0 mM -Glutamine, 2.5 g/mL Amphotericin B,
and 50 g/mL Gentamicin. Treatments with cannabinoid agents
were performed in a volume of 10 l/ml of cell suspension. After
mg/ml Penicillin-
L
m
m
m
appropriate time of exposure, cells were collected by centrifugation
at 1000 ꢃ g. The resulting pellets were washed in ice-cold PBS
buffer by centrifugation at 1000 ꢃ g. The pellet was lysed at 4 ꢁC in
HEPES based buffer containing other components (for details see
Ref. [31]). The extracts were centrifuged at 10,000 ꢃ g for 15 min at
4 ꢁC. The resulting supernatant was collected as total cell extracts.
Extracted total proteins were quantified using a Quant-iT™Protein
Assay Kit (Invitrogen™) by a Qubit Quantitation platform system
(Invitrogen™). Each western blot assay was carried out at fixed
[7] B.S. Basavarajappa, Neuropharmacology of the endocannabinoid signaling
system-molecular mechanisms, biological actions and synaptic plasticity,
Curr. Neuropharmacol. 5 (2007) 81e97.
[8] (a) A. Ellert-Miklaszlewska, W. Grajkowska, K. Gabrusiewicz, B. Kaminska,
L. Konarska, Distinctive pattern of cannabinoid receptor subtype II (CB2)
expression in adult and pediatric brain tumors, Brain Res. 1137 (2006)
161e169;
(b) J.P. Gong, E.S. Onaivi, H. Ishiguro, Q.R. Liu, P.A. Tagliaferro, A. Brusco,
G.R. Uhl, Cannabinoid CB2 receptors: immunohistochemical localization in rat
brain, Brain Res. 1071 (2006) 10e23;
(c) E.S. Onaivi, H. Ishiguro, J.P. Gong, S. Patel, A. Perchuk, P.A. Meozzi, L. Myers,
Z. Mora, P. Tagliaferro, E. Gardner, A. Brusco, B.E. Akinshola, Q.R. Liu, B. Hope,
S. Iwasaki, T. Arinami, L. Teasenfitz, G.R. Uhl, Discovery of the presence and
functional expression of cannabinoid CB2 receptors in brain, Ann. N. Y. Acc. Sci.
1074 (2006) 514e536;
(d) M.D. Van Sickle, M. Duncan, P.J. Kingsley, A. Mouihate, P. Urbani,
K. Mackie, N. Stella, A. Makriyannis, D. Piomelli, J.S. Davison, L.J. Marnett, V. Di
Marzo, Q.J. Pittman, K.D. Patel, K.A. Sharkey, Identification and functional
characterization of brainstem cannabinoid CB2 receptors, Science 310 (2005)
329e332.
total protein amount of 40 mg. Separation was performed by 10%
Bis-Tris Gel NuPAGE® Novex (Invitrogen) and nitrocellulose mem-
branes (Bio-Rad). Blots were blocked through Chemi BLOCKER™
(Millipore) in TBST (0.1% Tween 20 in Tris borate saline, Bio-Rad)
and probed with Anti-phospho-ERK 1/2 recombinant clone
AW39R (Millipore) with a 1:1000 dilution in 1% BSA (Bovine Serum
Albumin) in TBST (10 min). After three cycles of washing in TBST,
membranes were probed with the secondary antibody Goat anti-
Rabbit IgC e HRP (Invitrogen) with a 1:2000 dilution in Chemi
BLOCKER™-TBST 1:4. After three cycles of washing in TBST, blots
were treated for 5 min with Immobilon Western Chemiluminescent
HRP substrate (Millipore). Immunoreactive bands were visualized
[9] M. Rajesh, P. Mukhopadhyay, G. Hasko, T.W. Huffman, K. Mackie, P. Pacher,
CB2 cannabinoid receptor agonists attenuate TNF-a-induced human vascular
smooth muscle cell proliferation and migration, Br. J. Pharmacol. 153 (2008)
347e357.
[10] (a) Y. Gaoni, R. Mechoulam, Isolation, structure, and partial synthesis of an
active constituent of hashish, J. Am. Chem. Soc. 86 (1964) 1646e1647;
(b) R.G. Pertwee, The diverse CB1 and CB2 receptor pharmacology of three
€
by a Fujifilm Las-1000 analyzer (Raytest Isotopenmessgerate
GmbH) and immunoreactive band optical density was measured
using AIDA 2.11 software (Raytest Isotopenmessgerate GmbH).
plant cannabinoids: D9 tetrahydrocannabinol, cannabidiol and
drocannabivarin, Br. J. Pharmacol. 153 (2008) 199e215.
D
9tetrahy-
€
Data were expressed as mean percentage of vehicle
SEM
[11] G.C. Hsieh, M. Pai, P. Chandran, B.A. Hooker, C.Z. Zhu, A.K. Salyers,
E.J. Wensink, C.C. Zhan, W.A. Carroll, M.J. Dart, B.B. Yao, P. Honore, M.D. Meyer,
Central and peripheral sites of action for CB2 receptor mediated analgesic
activity in chronic inflammatory and neuropathic pain models in rats, Br. J.
Pharmacol. 162 (2011) 428e440.
(results of five independent experiments). Statistical analysis was
performed by One-way ANOVA followed by Dunnet's multiple
comparison using Graph Pad Prism 5 program (San Diego).
[12] A.G. Nackley, A. Makriyannis, A.G. Hohmann, Selective activation of canna-
binoid CB(2) receptors suppresses spinal fos protein expression and pain
behavior in a rat model of inflammation, Neuroscience 119 (2003) 747e757.
[13] (a) A.J. Sanchez, P. Gonzalez-Perez, I. Galve-Roperh, A. García-Merino, R-
(þ)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo-[1,2,3-de]-1,4-
benzoxazin-6-yl]-1-naphtalenylmethanone (WIN-2) ameliorates experi-
mental autoimmune encephalomyelites and induces encephalitogenic T cella
poptosis: partial involvement of the CB2 receptor, Biochem. Pharmacol. 72
(2006) 1697e1706;
Acknowledgements
ꢁ
ꢁ
ꢁ
This study was supported in part by grant from Regione
Autonoma della Sardegna (project: “CB2 CANNABINOIDI: Sintesi e
valutazione farmacologica di nuovi ligandi dei recettori dei can-
nabinoidi CB2 con implicazioni nel trattamento del dolore, di stati
infiammatori e di malattie neurodegenerative”; L.R.7, Bando 2008).
(b) H. Xu, C.L. Cheng, M. Chen, A. Manivannan, L. Cabay, R.G. Pertwee,
A. Coutts, J.V. Forrester, Anti-inflammatory property of the cannabinoid re-
ceptor-2-selective agonist JWH-133 in
a rodent model of autoimmune
uveoretinitis, J. Leukoc. Biol. 82 (2007) 532e541.
Appendix A. Supplementary data
ꢁ
[14] J. Fernandez-Ruiz, M. Moreno-Martet, C. Rodríguez-Cueto, C. Palomo-Garo,
ꢁ
~
ꢁ
M. Gomez-Canas, S. Valdeolivas, C. Guaza, J. Romero, M. Guzman,
R. Mechoulam, J.A. Ramos, Prospects for cannabinoid therapies in basal
ganglia disorders, Br. J. Pharmacol. 63 (2011) 1365e1378.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.05.055.

292
G. Pinna et al. / European Journal of Medicinal Chemistry 82 (2014) 281e292
ꢁ
[15] (a) M. García-Arencibia, S. Gonzalez, E. de Lago, J.A. Ramos, R. Mechoulam,
[27] H. Iwamura, H. Suzuki, Y. Ueda, T. Kaya, T. Inaba, In vitro and in vivo charac-
terization of JTE-907, a novel selective ligand for the cannabinoid CB2 re-
ceptor, J. Pharmacol. Exp. Ther. 296 (2001) 420e425.
ꢁ
J. Fernandez-Ruiz, Evaluation of the neuroprotective effect of cannabinoids in
rat model of Parkinson's diseases: importance of antioxidant and cannabinoid
receptor-independent properties, Brain Res. 1134 (2007) 162e170;
(b) J.L. Shoemaker, K.A. Seely, R.L. Reed, J.P. Crow, P.L. Prather, The CB2
cannabinoid agonist AM-1241 prolongs survival in a transgenic mouse model
of amyotrophicl ateral sclerosis when initiated at sympton onset,
J. Neurochem. 101 (2007) 87e98.
[28] E. Stern, G.G. Muccioli, R. Millet, J.-F. Goossens, A. Farce, P. Chavatte,
ꢁ
J.H. Poupaert, D.M. Lambert, P. Depreux, J.-P. Henichart, Novel 4-oxo-1,4-
dihydroquinoline-3-carboxamide derivatives as new CB2 cannabinoid recep-
tor agonists: synthesis, pharmacological properties and molecular modelling,
J. Med. Chem. 49 (2006) 70e79.
[16] J.W. Huffman, J. Liddle, S. Yu, M.M. Aung, M.E. Abood, J.L. Wiley, B.R. Martin, 3-
[29] C.A. Lunn, J.S. Fine, A. Rojas-Triana, J.V. Jackson, X. Fan, T.T. Kung,
W. Gonsiorek, M.A. Shwarz, B. Lavey, J.A. Kozlowski, S.K. Narula, D.J. Lundell,
R.W. Hipkin, L.A. Bober, A novel cannabinoid peripheral cannabinoid receptor-
selective inverse agonist blocks leukocyte recruitment in vivo, J. Pharmacol.
Exp. Ther. 316 (2006) 780e788.
[30] K.S. Marriott, J.W. Huffman, Recent advances in the development of selective
ligands for the cannabinoid CB2 receptor, Curr. Top. Med. Chem. 8 (2008)
187e204.
[31] G. Murineddu, P. Lazzari, S. Ruiu, A. Sanna, G. Loriga, I. Manca, M. Falzoi,
C. Dessì, M.M. Curzu, G. Chelucci, L. Pani, G.A. Pinna, 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, J. Med. Chem. 49 (2006)
7502e7512.
[32] G. Murineddu, B. Asproni, S. Ruiu, F. Deligia, M. Falzoi, A. Pau, B.F. Thomas,
Y. Zhang, G.A. Pinna, L. Pani, P. Lazzari, Tricyclic pyrazoles. Part 5. Novel 1,4-
Dihydroindeno[1,2-c]pyrazole CB2 ligands using molecular hybridization
based on scaffold hopping, Open. Med. Chem. J. 6 (2012) 1e14.
[33] L. Luongo, E. Palazzo, S. Tambaro, C. Giordano, L. Gatta, M.A. Scafuro, F. sca
Rossi, P. Lazzari, L. Pani, V. de Novellis, M. Malcangio, S. Maione, 1-(20,40-
dichlorophenyl)-6-methyl-N-cyclohexylamine-1,4-dihydroindeno[1,2-c]pyr-
(10,10-Dimethylbutyl)-1-deoxy- 8-THC and related compounds: synthesis of
D
selective ligands for the CB2 receptor, Bioorg. Med. Chem.
7 (1999)
2905e2914.
[17] K.C. Marriott, J.W. Huffman, J.L. Wiley, B.R. Martin, Synthesis and pharma-
cology of 11-nor-1-methoxy-9-hydroxy-hexahydrocannabinols and 11-nor-
1-deoxy-9-hydroxyhexahydrocannabinols: new selective ligands for the
cannabinoid CB2 receptor, Bioorg. Med. Chem. 14 (2006) 2386e2397.
[18] M.M. Ibrahim, H. Deng, A. Zvonok, D.A. Cockayne, J. Kwan, H.P. Mata,
T.W. Vanderah, J. Lai, F. Porreca, A. Makriyannis, T.P. Malan Jr., Activation of
CB2 receptors by AM1241 inhibits experimental neuropathic pain: pain in-
hibition by receptors not present in the CNS, Proc. Natl. Acad. Sci. U. S. A. 100
(2003) 10529e10533.
[19] K.J. Valenzano, L. Tafesse, G. Lee, J.E. Harrison, J.M. Boulet, S.L. Gootshall,
L. Mark, M.S. Pearson, W. Miller, S. Shan, L. Rabadi, Y. Rotshteyn, S.M. Chaffer,
P.I. Turchin, D.A. Elsemore, M. Toth, L. Koetzner, G.T. Whiteside, Pharmaco-
logical and pharmacokinetic characterization of the cannabinoid receptor 2
agonist, GW405833, utilizing rodent models of acute and chronic pain, anx-
iety, atoxia and catalepsy, Neuropharmacology 48 (2005) 658e672.
[20] J.F. Bouchard, D. Lamontagne, Cannabinoids as Vasodilators and Car-
dioprotectors Against Ischemia, W.O. Patent 028588, 2001.
[21] B.B. Yao, G.C. Hsieh, J.M. Frost, Y. Fan, T.R. Garrison, A.V. Daza, G.K. Grayson,
C.Z. Zhu, M. Pai, P. Chandran, A.K. Salyers, E.J. Wensink, P. Honore, J.P. Sullivan,
M.J. Dart, M.D. Meyer, In vitro and in vivo characterization of A-796260: a
selective cannabinoid CB2 receptor agonist exhibiting analgesic activity in
rodent pain models, Br. J. Pharmacol. 153 (2008) 390e401.
azole-3-carboxamide, a novel CB2 agonist, alleviates neuropathic pain
through functional microglial changes in mice, Neurobiol. Dis. 37 (2010)
177e185.
[34] A.G. Gonzales, T.B. Barrera, C.Y. Hernandez, A synthesis of 3-hydroxymethyl-
6-methylbenzofuran, Heterocycles 34 (1992) 1311e1315.
[22] R.A. Ross, H.C. Brockie, L.A. Stevenson, V.L. Murphy, F. Templeton,
A. Makriyannis, R.G. Pertwee, Agonist-inverse agonist characterization at CB1
and CB2 cannabinoid receptors of L759633, L759656 and AM630, Br. J. Phar-
macol. 126 (1999) 665e672.
[35] Y. Cheng, W.H. Prusoff, Relationship between the inhibition constant (Ki) and
the concentration of inhibitor which causes 50 per cent inhibition (I50) of an
enzymatic reaction, Biochem. Pharmacol. 22 (1973) 3099e3108.
[36] M.I. Davis, J. Ronesi, D.M. Lovinger, A predominant role for inhibition of the
adenylate cyclase/protein kinase A pathway in ERK activation by cannabinoid
ꢂ
[23] L. Hanus, A. Breuer, S. Tchilibon, S. Shiloah, D. Goldenberg, M. Horowitz,
R.G. Pertwee, R.A. Ross, R. Mechoulam, E. Fride, HU-308: a specific agonist for
CB2, a peripheral cannabinoid receptor, Proc. Natl. Acad. Sci. U. S. A. 96 (1999)
14228e14233.
receptor 1 in N1E-115 neuroblastoma cells, J. Biol. Chem. 278 (2003)
48973e48980.
[37] I. Manca, A. Mastinu, F. Olimpieri, M. Falzoi, M. Sani, S. Ruiu, G. Loriga,
A. Volonterio, S. Tambaro, M.E.H. Bottazzi, M. Zanda, G.A. Pinna, P. Lazzari,
Novel pyrazole derivatives as neutral CB1 antagonists with significant activity
towards food intake, Eur. J. Med. Chem. 62 (2013) 256e269.
[38] K.L. Carrol, O.G. Kenneth, Selective bromination with copper(II) bromide,
J. Org. Chem. 29 (1964) 3459e3461.
[24] A. Makriyannis, A. Khanolkar, Novel Bicyclic Cannabinoid Agonists for the
Cannabinoid Receptor, W.O. Patent 028497, 2001.
[25] M. Rinaldi-Carmona, F. Barth, J. Millan, J.-M. Derocq, P. Casellas, C. Congy,
D. Oustric, M. Sarran, M. Bouaboula, B. Calandra, M. Portier, D. Shire, J.-
ꢀ
C. Breliere, G. Le Fur, SR144528, the first potent and selective antagonist
of the CB2 cannabinoid receptor, J. Pharmacol. Exp. Ther. 284 (1998)
[39] J.A. Suchocki, E.L. May, T.J. Martin, C. George, B.R. Martin, Synthesis of 2-exo-
and 2-endo-mecamylamine analogs. Structure-activity relationships for
nicotinic antagonism in the central nervous system, J. Med. Chem. 34 (1991)
1003e1010.
[40] A.R. Deshpande, M.V. Paradkar, Synthesis of 3-(3-benzofuranyl)coumarins,
Synth. Commun. 20 (1990) 809e816.
644e650.
ꢀ
[26] J.-M. Mussinu, S. Ruiu, A.C. Mule, A. Pau, M.A. Carai, G. Loriga, G. Murineddu,
G.A. Pinna, Tricyclic pyrazoles. Part 1: synthesis and biological evaluation of
novel 1,4-dihydroindeno[1,2-c]pyrazol-based ligands for CB1 and CB2 canna-
binoid receptors, Bioorg. Med. Chem. 11 (2003) 251e263.