Synthesis and Characterization of NESS 0327: A Novel Putative Antagonist of the CB1 Cannabinoid Receptor

Abstract

The compound N-piperidinyl-[8-chloro-1-(2,4-dichlorophenyl)-1,4,5,6-tetrahydrobenzo [6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide] (NESS 0327) was synthesized and evaluated for binding affinity toward cannabinoid CB1 and CB2 receptor. NESS 0327 exhibited a stronger selectivity for CB1 receptor compared with N-piperidinyl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR 141716A), showing a much higher affinity for CB1 receptor (Ki = 350 +/- 5 fM and 1.8 +/- 0.075 nM, respectively) and a higher affinity for the CB2 receptor (Ki = 21 +/- 0.5 nM and 514 +/- 30 nM, respectively). Affinity ratios demonstrated that NESS 0327 was more than 60,000-fold selective for the CB1 receptor, whereas SR 141716A only 285-fold. NESS 0327 alone did not produce concentration-dependent stimulation of guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding in rat cerebella membranes. Conversely, NESS 0327 antagonized [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol [1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate] (WIN 55,212-2)-stimulated [35S]GTPgammaS binding. In functional assay, NESS 0327 antagonized the inhibitory effects of WIN 55,212-2 on electrically evoked contractions in mouse isolated vas deferens preparations with pA2 value of 12.46 +/- 0.23. In vivo studies indicated that NESS 0327 antagonized the antinociceptive effect produced by WIN 55,212-2 (2 mg/kg s.c.) in both tail-flick (ID50 = 0.042 +/- 0.01 mg/kg i.p.) and hot-plate test (ID50 = 0.018 +/- 0.006 mg/kg i.p.). These results indicated that NESS 0327 is a novel cannabinoid antagonist with high selectivity for the cannabinoid CB1 receptor.

Full text

JPET #49924
1
Journal of Pharmacology and Experimental Therapeutics
Synthesis and characterization of NESS 0327: a novel putative antagonist
of the CB
1
cannabinoid receptor
Stefania Ruiu, Gèrard A. Pinna
2
, Giorgio Marchese, Jean-Mario Mussinu
2
, Pierluigi Saba
3
,
Simone Tambaro, Paola Casti, Romina Vargiu
4
and Luca Pani
5
Neuroscienze S.c.a r.l., 09123 Cagliari, Italy;
Copyright 2003 by the American Society for Pharmacology and Experimental Therapeutics.
JPET Fast Forward. Published on March 27, 2003 as DOI:10.1124/jpet.103.049924

JPET #49924
2
NESS 0327: a novel putative CB1 cannabinoid receptor antagonist.
Correspondence to:
Luca Pani
Institute of Neurogenetic and Neuropharmacology
Via Boccaccio 8, 09047 Selargius (Ca), Italy.
Tel: +39 070 2548079 fax: +39 070 254275
e.mail: L.Pani@inn.cnr.it
Abbreviations: NESS 0327, N-piperidinyl-[8-chloro-1-(2,4-dichlorophenyl)-1,4,5,6-tetrahydrobenzo
[6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide], SR 141716A, N-piperidinyl-5-(4-chlorophenyl)-1-
(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, [
35
S]GTPγS, guanosine 5’-O-(3-
[
35
S]thio)-triphosphate, WIN 55,212-2, [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl) methyl]
pyrolol [1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl) methanone mesylate]
Text pages: 28
Tables:1
Figures: 4
References:36
Words in the Abstract: 214
Words in the Introduction : 369
Words in the Discussion: 749

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Abstract
The compound N-piperidinyl-[8-chloro-1-(2,4-dichlorophenyl)-1,4,5,6-tetrahydrobenzo
[6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide] (NESS 0327) was synthesized and evaluated
for binding affinity towards cannabinoid CB
1
and CB
2
receptor. NESS 0327 exhibited a
stronger selectivity for CB
1
receptor when compared with N-piperidinyl-5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR 141716A) showing a
much higher affinity for CB
1
receptor (K
i
= 350 ± 5 fM and 1.8 ± 0.075 nM, respectively)
and a higher affinity for the CB
2
receptor (K
i
= 21 ± 0.5 nM and 514 ± 30 nM, respectively).
Affinity ratios demonstrated that NESS 0327 was more than 60,000-fold selective for the
CB
1
receptor, while SR 141716A only 285 fold. NESS 0327 alone did not produce
concentration-dependent stimulation of guanosine 5’-O-(3-[
35
S]thio)-triphosphate
([
35
S]GTPγS) binding in rat cerebella membranes. Conversely, NESS 0327 antagonized
[R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl) methyl] pyrolol [1,2,3-de]-1,4-benzoxazin-
yl]-(1-naphthalenyl) methanone mesylate] (WIN 55,212-2)-stimulated [
35
S]GTPγS binding.
In functional assay, NESS 0327 antagonized the inhibitory effects of WIN 55,212-2 on
electrically evoked contractions in mouse isolated vas deferens preparations with pA
2
values
of 12.46 ± 0.23. In vivo studies indicated that NESS 0327 antagonized the antinociceptive
effect produced by WIN 55,212-2 (2 mg/kg, s.c.) in both tail flick (ID
50
= 0.042 ± 0.01
mg/kg i.p.) and hot plate test (ID
50
= 0.018 ± 0.006 mg/kg i.p.). These results indicated that
NESS 0327 is a novel cannabinoid antagonist with high selectivity for the cannabinoid CB1
receptor.

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Interest in the pharmacology of cannabinoids has rapidly increased after the cloning of
cannabinoid receptors and the discovery of their endogenous ligand:
arachidonylethanolamide (anandamide) (Devane et al., 1988, 1992; Munro et al., 1993). Two
types of cannabinoid receptors, CB
1
and CB
2,
have been characterized, both of which have
distinct anatomical distributions and ligand binding profiles. Cannabinoid CB
1
receptors are
present in the central nervous system (CNS) with the highest densities in the hippocampus,
cerebellum and striatum (Herkenham et al., 1990; Howlett, 1998) and, to a lesser extent, in
several peripheral tissues. Cannabinoid CB
2
receptors appear to be predominantly located in
peripheral tissues (Pertwee, 1997, 1999; Galiègue et al., 1995). Both receptors belong to the
G protein-coupled family of receptors that negatively regulate adenylate cyclase and control
the release of arachidonic acid (Howlett, 1995). Naturally occurring [
∆
9
-
tetrahydrocannabinol (
∆
9
-THC) and
∆
8
-THC] and synthetic cannabinoid agonists (HU-210,
CP 55,940 and WIN 55,212-2) produce a number of effects in mice (hypoactivity, catalepsy,
hypothermia and antinociception) that are collectively known as the tetrad of cannabinoid-
induced behaviours (Abood and Martin, 1992; Compton et al., 1992, 1993). These behaviors
are of a central origin and are thought to be mediated via the cannabinoid CB
1
receptor
(Compton et al., 1996; Lichtman and Martin, 1997; Rinaldi Carmona et al., 1994), while the
CB
2
receptor may mediate some of the peripheral effects of ∆
9
-THC, such as
immunosuppression (Martin, 1986).
The cloning of CB
1
and CB
2
receptors and the subsequent development of selective tools has
advanced the concept of therapeutically targeting cannabinoid receptors. Besides their
established clinical antiemetic action (Gralla 1999; Voth and Schwartz, 1997), cannabinoid
receptor agonists also possess appetite stimulant, anticonvulsant, antinociceptive,
hypothermic and antiglaucoma properties (Formukong et al., 1989; Mattes et al., 1994;
Pertwee, 1999; Porcella et al., 2001).

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Recently, several groups have become interested in the development of cannabinoid
antagonists, hoping to develop new drugs to cure diseases connected with possible
malfunctions of “cannabinoid/anandamide” system.
We report the synthesis of a putative cannabinoid ligand, code named NESS 0327, its
differential binding to CB
1
and CB
2
cannabinoid receptors, its ability to stimulate [
35
S]GTPγS
binding in rat brain, its effect on mouse vas deferens and its action on an in vivo assay known
to be affected by cannabinoids.

JPET #49924
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Methods
(
Z
,
E
)-5-(3-Chlorophenyl)-pent-4-enoic acid (3)
: A solution of (3-carboxypropyl)
triphenylphosphonium bromide (2) (14 g, 32.61 mM) in anhydrous dimethylsulfoxide
(DMSO) (40 ml), with 2.6 M of the sodium salt of DMSO in anhydrous DMSO (24 ml,
62.25 mM), below 10°C, was added to a solution of 3-chlorobenzaldehyde (3.06 g, 21.74
mM) in anhydrous tetrahydrofuran (8 ml). The resulting solution was heated at 50°C for 18
h; subsequently, it was allowed to return to room temperature and poured into water. The
mixture was acidified with 6 N hydrochloric acid and extracted with ethyl acetate (3x25 ml).
The combined extracts were washed with brine, water and then dried over anhydrous sodium
sulphate, to provide a browning compound after evaporation. The crude compound was
purified by flash column chromatography on silica gel eluting with dichloromethane
/acetone
9/1 to afford the desired diastereomeric mixture 3 (42% yield); Rf 0.51
(dichloromethane/acetone 9/1); IR (nujol): 3200-2500 (OH), 1720 (C=O), 1590 (Ar);
1
H-
NMR: 2.40-2.75 (m, 8H), 5.60-5.75 (m, 2H), 6.15-6.45 (m, 2H), 7.10-7.28 (m, 6H), 7.32 (s,
2H), 9.50 (br s, 2H, exch. with D
2
O). Anal. C
11
H
11
ClO
2
(C, H, Cl).
5-(3-Chlorophenyl)-pentanoic acid (4): A suspension of the diastereomeric mixture of
pentenoic acid derivate 3 (1 g, 4.75 mM) was subjected to catalytic hydrogenation over PtO
2
(Adams‘ catalyst, 0.1 g, 10% w/w) in ethanol (EtOH) (50 ml) for 2.5 h at room temperature
and 45 psi of hydrogen pressure. The mixture was filtered through a paper filter and the
filtrate concentrated under reduced pressure to yield the desired acid 4 (100% yield) as a
yellow solid, m.p.56-58°C. Rf 0.84 (petroleum ether/ethyl acetate 1/1); IR(nujol): 3300
(OH), 1710 (C=O), 1600 (Ar);
1
H-NMR: 1.60-1.80 (m, 4H), 2.30-2.45 (m, 2H), 2.55-2.71
(m, 2H), 7.04 (d, 1H, J = 6.4 Hz), 7.12-7.35 (m, 3H), 9.65 (br s, 1H, exch. with D
2
O). Anal.
C
11
H
13
ClO
2
(C, H, Cl).
2-Chloro-6,7,8,9-tetrahydro-benzocyclohepten-5-one (5): A suspension of pentanoic acid
4 (0.5 g, 2.36 mM) and thionyl chloride (0.63 ml, 8.5 mM) was heated for 30 min at 50°C.

JPET #49924
7
Thionyl chloride in excess was subsequently removed under reduced pressure and the residue
was added for 3 times to dichloromethane (3 ml) which was evaporated under reduced
pressure. A solution of the crude acyl chloride in dichloromethane (3 ml) was added drop
wise to a magnetically stirred suspension of AlCl
3
(0.32 g, 2.36 mM) in dichloromethane (3
ml). The resulting mixture was stirred at room temperature overnight then poured into ice
and the whole extracted with dichloromethane (3x5 ml). The combined extracts were washed
with (5%) aqueous sodium bicarbonate solution, water and, after drying over anhydrous
sodium sulfate, filtered and evaporated to provide a brownish compound. The crude
compound was purified by flash chromatography on silica gel eluting with petroleum ether/
ethyl acetate 9/1 to afford the attempt compound 5 (77% yield) as a yellow orange oil, b.p.
94-97°C/0.05 mm Hg (lit
1
128-131/0.35 mmHg); Rf 0.65 (petroleum ether/ethyl acetate 9/1);
IR (film): 3350 (OH), 1680 (C=O), 1590 (Ar);
1
H-NMR: 1.72-1.98 (m, 4H), 2.73 (t, 2H, J =
6.2 Hz), 2.91 (t, 2H, J = 6.0 Hz), 7.21 (s, 1H), 7.28 (d, 1H, J = 8.8 Hz), 7.68 (d, 1H, J = 8.6
Hz). Anal. C
11
H
11
ClO (C, H, Cl).
(2-Chloro-5-oxo-6,7,8,9-tetrahydro-5H-benzocyclohepten-6-yl)-oxo-acetic acid ethyl
ester (6): A mixture of EtONa (7.5 mM) in absolute EtOH, (3.5 ml) and diethyl oxalate (0.51
ml, 3.75 mM) was stirred for 30 min at room temperature, and a solution of compound 5
(0.73 g, 3.75 mM) in absolute ethanol (27 ml) was added over 30 min. The resulting mixture
was reacted at room temperature for 9 h then poured onto crushed ice and the whole acidified
with 2 N hydrochloric acid and extracted with chloroform (3x15 ml). The combined extracts
were washed with water, dried over anhydrous sodium sulfate, filtered and evaporated to
afford the β-dichetoester 6 as an orange oil, which was used in the next step without further
purification, (84% yield); b.p. 95-98°C/0.05 mm Hg; Rf 0.78 (petroleum ether/ethyl acetate
9/1); IR (film): 3440 (OH), 1730 (C=O), 1680 (C=O), 1600 (Ar);
1
H-NMR: 1.41 (t, 3H, J = 7
Hz), 2.08 (quint, 2H), 2.32 (t, 2H, J = 7.2Hz), 2.72 (t, 2H, J = 7 Hz), 3.88 (q, 2H, J = 7 Hz),
7.23 (d, 1H, J = 1.8 Hz), 7.34 (dd, 1H), 7.58 (d, 1H, J = 8.2 Hz), 15.37 (br s, 1H, exch. with
D
2
O). Anal. C
15
H
15
ClO
4
(C, H, Cl).

JPET #49924
8
8-Chloro-1-(2,4-dichlorophenyl)-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-
3-carboxylic acid ethyl ester (8): 2,4-dichlorophenylhydrazine hydrochloride (7) (0.72 g,
3.38 mM) was added to a magnetically stirred solution of ester 6 (0.9 g, 3.05 mM) in EtOH
(21 ml) and the resulting mixture heated under reflux for 3 h; subsequently the solvent was
removed under reduced pressure to yield the crude ester. Purification by flash
chromatography on silica gel eluting with petroleum ether/ethyl acetate 8.5/1.5 gave the
attempt compound 8 as a yellow solid (58% yield); mp 160-161°C (crumbled with petroleum
ether); Rf 0.47 (petroleum ether/ethyl acetate 9/1); IR (nujol): 1725 (C=O), 1605 (Ar);
1
H-
NMR: 1.43 (t, 3H, J = 7 Hz), 2.13-2.40 (m, 2H), 2.67 (t, 2H, J = 6.4 Hz), 3.09-3.40 (m, 2H),
4.46 (q, 2H, J = 7 Hz), 6.60 (d, 1H, J = 8.2 Hz), 7.02 (dd, 1H), 7.31 (d, 1H, J = 2.2 Hz), 7.36-
7.49 (m, 2H), 7.54 (d, 1H, J = 9.2 Hz). Anal. C
21
H
17
Cl
3
N
2
O
2
(C, H, Cl, N).
8-Chloro-1-(2,4-dichlorophenyl)-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-
3-carboxylic acid (9) :A solution of potassium hydroxide (0.17 g, 2.94 mM) in methanol (5
ml) was added to a magnetically stirred solution of ester 8 (0.64 g, 1.47 mM) in methanol (7
ml), the mixture was refluxed for 9 h and the cooling reaction mixture poured onto crushed
ice and acidified with 1 M hydrochloric acid. The precipitate was filtered, washed with water
and dried under vacuum to yield the corresponding acid as a white solid. (97% yield); mp
270°C (EtOH); Rf 0.51 (chloroform/methanol 9/1); IR (nujol): 3410 (OH), 1690 (C=O);
1
H-
NMR: 2.20-2.39 (m, 2H), 2.50-3.35 (m, 4H), 6.61 (d, 1H, J = 8.2 Hz), 7.03 (dd, 1H), 7.32 (d,
1H, J = 1.8 Hz), 7.39-7.49 (m, 2H), 7.53 (d, 1H, J = 8.2 Hz), 13.25 (br s, 1H, exch. with
D
2
O). Anal. C
19
H
13
Cl
3
N
2
O
2
(C, H, Cl, N).
N-Piperidinyl-[8-chloro-1-(2,4-dichlorophenyl)-1,4,5,6 tetrahydrobenzo [6,7] cyclohepta
[1,2-c] pyrazole-3-carboxamide] (NESS 0327)
:
A solution of the acid 9 (0.50 g, 1.23 mM)
and thionyl chloride (0.24 ml, 3.69 mM) in toluene (10 ml) was refluxed for 3 h. Solvent was
evaporated under reduced pressure and the residue redissolved in toluene (3x5 ml) and
evaporated to yield the crude carboxylic chloride. A solution of the above carboxylic chloride
in dichloromethane (6 ml) was added dropwise to a solution of 1-aminopiperidine (10) (0.19

JPET #49924
9
ml, 1.65 mM) and triethylamine (0.23 ml, 1.65 mM) in dichloromethane (6.2 ml). After
stirring at room temperature for 1 h, the reaction mixture was added with brine and extracted
with dichloromethane (3x15 ml). The combined extracts were washed with brine, dried over
anhydrous sodium sulfate, filtered and evaporated to give a yellowish compound. The crude
compound was purified by flash chromatography on silica gel eluting with petroleum
ether/ethyl acetate 1/1 to afford the desired carboxamide NESS 0327 as a white solid, (93%
yield); mp 205-206°C (acetone), (lit
2
202°C); Rf 0.68 (petroleum ether/ethyl acetate 1/1);
IR(nujol): 3200 (NH), 1650 (C=O), 1600 (Ar);
1
H-NMR: 1.35-1.53 (m, 2H), 1.58-1.89 (m,
6H), 2.15-2.36 (m, 2H), 2.66 (t, 2H, J = 6.4 Hz), 2.87(t, 4H, J = 5.0 Hz), 6.56 (d, 1H, J = 8.2
Hz), 7.01 (dd, 1H), 7.31 (d, 1H, J = 1.8 Hz), 7.37-7.54 (m, 3H), 7.66 (br s, 1H, exch. with
D
2
O). Anal. C
24
H
23
Cl
3
N
4
O (C, H, Cl, N).
Radioligand Binding Methods. Male CD1 mice weighing 20-25 g (Charles River, Calco,
LC, Italy) were housed in the animal care quarters; temperatures were maintained at 22 ± 2°C
on a 12 h light/dark cycle and food and water were available ad libitum. All experimental
protocols were authorized by the Ethical Committee at the University of Cagliari and
performed in strict accordance with the E.C. regulations for care and use of experimental
animals (EEC N°86/609).
Mice were killed by cervical dislocation, brains (minus cerebellum) and spleens were rapidly
removed and placed on an ice-cold plate. After thawing, tissues were homogenated in 20 vol.
(w/v) of ice-cold TME buffer (50 mM Tris-HCl, 1 mM EDTA and 3.0 mM MgCl
2
, pH 7.4).
The homogenates were centrifuged at 1,086 x g for 10 min at 4°C, and the resulting
supernatants were centrifuged at 45,000 x g for 30 min at 4°C.
[
3
H]-CP 55,940 binding was performed by a modification of the method previously described
(Rinaldi-Carmona et al., 1994). Briefly, the membranes (30-80 µg of protein) were incubated
with 0.5 nM of [
3
H]-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). Non-specific binding
was estimated in the presence of 1 µM of CP 55,940. All binding studies were performed in

JPET #49924
10
disposable glass tubes pre-treated with Sigma-Cote (Sigma Chemical Co. Ltd., Poole, UK),
in order to reduce non-specific binding. The reaction was terminated by rapid filtration
through Whatman GF/C filters pre-soaked in 0.5% polyethyleneimine using a Brandell 96-
sample harvester (Gaithersburg, MD, USA). 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,
USA) with 4 ml of scintillation fluid (Ultima Gold MV, Packard). Protein determination was
performed by means of Bradford (1976) protein assay using BSA as a standard, according to
the protocol of the supplier (Bio-Rad, Milan, Italy). Drugs were dissolved in DMSO. To
avoid possible undesired effects on radioligand binding, DMSO concentration in the different
assays never exceeded 0.1% (v/v). All experiments were performed in triplicate and results
were confirmed in at least four independent experiments. Data from radioligand inhibition
experiments were analyzed by non-linear regression analysis of a Sigmoid Curve using
Graph Pad Prism program (Graph Pad Software, Inc. San Diego, CA, USA). IC
50
values
were derived from the calculated curves and converted to K
i
values as described previously
(Cheng and Prusoff, 1973).
Mouse Vas Deferens Experiments. Vasa deferentia were obtained from albino CD1 mice
weighing 25-40 g. Tissue was mounted in 10 ml organ bath at an initial tension of 0.5 g using
the method described by Pertwee et al. (1993). The bath contained Krebs-Henseleit solution
(118.2 mM NaCl, 4.75 mM KCl, 1.19 mM KH
2
PO
4
,
25.0 mM NaHCO
3
, 11.0 mM glucose
and 2.54 mM CaCl
2
) which was kept at 37°C and bubbled with 95% O
2
and 5% CO
2
.
Isometric contractions were evoked by stimulation with 0.5 s trains of three pulses of 110%
maximal voltage (train frequency, 0.1 Hz; pulse duration, 0.5 ms) through platinum
electrodes attached to the upper end of each bath and a stainless steel electrode attached to
the lower end. Stimuli were generated by Grass S88K stimulator then amplified
(Multiplexing pulse booster 316S, Ugo Basile Comerio, Va, Italy) and divided to yield
separate outputs to four organ baths. Contractions were monitored by computer using a data

JPET #49924
11
recording and analysis system (PowerLab 400) linked via preamplifiers (QuadBridge) to an
F10 transducer (2Biological Instruments, Besozzo, Va, Italy).
Each tissue was subject to several periods of stimulation. The first of these began after the
tissue had equilibrated in the buffering medium but before drug administration, and
continued for 10 min. The stimulator was then switched off for 15 min, after which the
tissues were subjected to further periods of stimulation each lasting 5 min and separated by a
stimulation-free period. The drugs were added once the contractile responses to electrical
stimulation were reproducible. Preparations were exposed to cumulative increasing
concentrations of WIN 55,212-2 to obtain concentration-response curves either in the
absence (control) or in the presence of NESS 0327 (1 pM, 10 pM or 100 pM) added at a
fixed concentration 20 min before the first concentration of WIN 55,212-2. It was not
possible to reverse the inhibitory effect of cannabinoid on the twitch response by washing
them out of the organ bath. Consequently only one concentration-response curve was
constructed per tissue. DMSO was added instead of the drug. The control dose of DMSO was
the same as the dose added in combination with the highest dose of drug used. DMSO alone
did not inhibit the twitch response (n=6) at the maximum concentration used in the bath (4
µl/ml).
Drug additions were performed in volumes of 10 µl. The effects of the antagonists or
agonists were calculated as percentage of decrease in the pre-drug twitch force. Inhibition of
the electrically evoked twitch response is expressed in percentage terms and has been
calculated by comparing the amplitude of the twitch response after each addition of an
agonist with the amplitude immediately prior to the first addition of the agonist. The pA
2
values for competitive antagonists were calculated by Schild regression analysis
(Arunlakshana and Schild, 1959). Data were plotted as log antagonist concentrations (M) vs.
log (concentration-ratios,-1). It is assumed that when the slope value of the regression line in
the Schild plot does not differ statistically from unity, the pA
2
value represents the
dissociation constant of the antagonist (pK
B
). In each estimate eight isolated tissue

JPET #49924
12
preparations were used. Statistical significance was determined by use of Student’s test and
P<0.05 was considered significant.
[
35
S]GTP
γ
S Binding Assay. Male Sprague-Dawley rats (Charles River, Como, Italy),
weighing 200-250 g, were used in all experiments. Rats were killed by decapitation, their
brains rapidly removed and cerebella were dissected on ice. Cerebella tissue was suspended
in 20 volumes of cold centrifugation buffer (50 mM Tris-HCl, 3 mM MgCl
2
, 1 mM EDTA,
pH 7.4) and homogenated using a homogenizer system (Glas-Col, Terre Haute, IN, USA).
The homogenate was centrifuged at 48,000 x g for 10 min at 4°C. The pellet was then
resuspended in the same buffer, homogenized, and centrifuged as previously. The final P2
pellet was subsequently resuspended in assay buffer (50 mM Tris-HCl, 3 mM MgCl
2
, 0.2
mM EGTA, 100 mM NaCl, pH 7.4), homogenized and diluted to a concentration of
∼
2
µg/µl with assay buffer. The protein concentration was determined by the method of
Bradford (1976) using bovine serum albumin as a standard according to the protocol of the
supplier (Bio-Rad, Milan, Italy). Membrane aliquots were then stored at –80°C until use.
[
35
S]GTPγS binding was measured as described by Selley et al. (1996). Briefly, rat cerebella
membranes (15 µg of protein) were incubated with drugs for 60 min at 30°C in assay buffer
containing 0.1% fatty acid free bovine serum albumin in the presence of 0.05 nM
[
35
S]GTPγS and 30 µM guanosine-5’-diphosphate (GDP), in a final volume of 1 ml. The
reaction was terminated by rapid filtration using a Packard Unifilter-GF/B, washed 2 times
with 1 ml of ice-cold 50 mM Tris-HCl, pH 7.4 buffer and dried 1 h at 30°C. The radioactivity
on the filters was counted in a liquid microplate scintillation counter (TopCount NXT,
Packard, Meridien, USA) using 50 µl of scintillation fluid (Microscint
TM
20, Packard,
Meridien, USA).
Stock solution of WIN 55,212-2 and NESS 0327 were prepared in DMSO and then diluted in
assay buffer. The final concentration of DMSO was < 0.01%, which had no effect either on
basal or stimulated [
35
S]GTP
γ
binding. WIN 55,212-2 concentration effect curves were

JPET #49924
13
determined by incubating membranes with various concentrations of WIN 55,212-2 (10-
10,000 nM) in the presence of 0.05 nM of [
35
S]GTPγS and 30 µM GDP.
Non specific binding was measured in the presence of 10 µM unlabeled GTPγS. Basal
binding was assayed in the absence of agonist and in the presence of GDP. The stimulation
by agonist was defined as a percentage increase above basal levels (i.e., {[dpm(agonist)-dpm
(no agonist)]/dpm (no agonist)}x 100).
Data are reported as mean
±
S.E.M. of three to six experiments, performed in triplicate.
Nonlinear regression analysis of concentration-response data was performed using Prism 2.0
software (GraphPad Prism Program, San Diego, CA, USA) to calculate E
max
and EC
50
values.
The resulting ED
50
values were used to determine K
e
values for antagonism of the agonist-
stimulated response by antagonist, using the relationship K
e
= [Ant]/(Dr-1), where [Ant] is
the concentration of antagonist, and DR is the ratio of ED
50
values in the presence and
absence of antagonist (Sim et al., 1995). Statistical analyses were carried out using one way
ANOVA followed by Newman-Keuls post-hoc test.
Determination of mouse antinociception. Male CD1 mice weighing 20-25 g (Charles
River, Calco, LC, Italy) were used to assess antinociception by means of the tail flick and hot
plate test. A tail flick meter (Ugo Basile Instruments, Italy) equipped with an irradiant heat
source that focused 2.5 cm of the distal tip of the tail was used. A 15 s cut-off time for heat
exposure was used to avoid cutaneous damage and the intensity of the thermal source was
adjusted to produce a 3-5 s latency in vehicle treated rats.
The effect of the compounds on the reaction time of mice placed on the hot plate (Ugo Basile
Instruments, Italy) (55 ± 0.8°C) was assessed determining the time at which animals first
displayed a nociceptive response (licking the front paws, fanning the hind paws or jumping).
To avoid skin damage, after 40 s (cut-off) the animal was removed from the hot plate. In both
tests each animal was tested prior to drug administration to determine control latency and the

JPET #49924
14
animals were used only in the determination of one time point. Data were transformed to the
% MPE by the following equation (Harris and Pierson, 1964); %MPE = [(test latency –
control latency) / (cut-off - basal latency)] X 100; where the latencies were expressed in
seconds and the cut-off varied depending on the test (tail flick = 15 s; hot plate = 40 s). To
establish the dose-dependent curves, at least four drug doses were used on 10 mice per each
dose and each animal group was used only in the determination of one time point. Mice were
tested 30 minutes after WIN 55,212-2 (2 mg/kg s.c.) or vehicle and up to 120 minutes. NESS
0327 (0.01-1 mg/kg, i.p.) or vehicle were given 20 min before WIN 55,212-2 administration.
WIN 55,212-2 was dissolved (5 ml/kg) in an emulsion of ethanol-cremophor-saline (1:1:18),
NESS 0327 was dissolved in two drops of Tween-80 diluted in distilled water to a volume of
5 ml/kg. Three independent experiments were carried out for ID
50
± S.E.M. determination.
Statistical analyses were carried out using two ways ANOVA followed by Newman-Keuls
post-hoc test.
Materials
. Unless otherwise stated, all materials were obtained from commercial suppliers
and used without purification. Anhydrous solvents such as ethanol, tetrahydrofuran and
DMSO were obtained from Sigma-Aldrich in sure-seal bottles. All reactions involving air- or
moisture-sensitive compounds were performed under a nitrogen atmosphere. Flash column
chromatography was carried out using Merck Silica gel 60 (230-400 mesh ASTM). Thin
layer chromatography was performed with Polygram
SIL N-HR-/HV
254
precoated plastic
sheet (0.2 mm).
1
H-NMR spectra were determined in CDCl
3
with super conducting FT-NMR
using a XL-200 Varian apparatus at 200 MHz. Chemical shifts are reported in
δ
(ppm)
relative to TMS as the internal standard and coupling constants in Hz. Significant
1
H-NMR
data are reported in the following order: multiplicity (s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; dd, double doublet; br s, broad singlet), number of protons, coupling
constants (J) in Hz. IR spectra were recorded as thin films or nujol mulls on NaCl plates with
a Perkin-Elmer 781 IR spectrophotometer and are expressed in
ν
(cm
-1
). Melting points were

JPET #49924
15
determined on a K fler melting point apparatus and are uncorrected. Compounds are
indicated by the molecular formula followed by the symbols for the elements (C, H, N) and
were found to be within
±
0.4% of their theoretical values. [
3
H]-CP 55, 940 (180 Ci/mmol)
and [
35
S]GTPγS (1200-1350 Ci/mmole) were purchased from New England Nuclear (Boston,
MA, USA). CP 55,940 and WIN 55,212-2 were obtained from Tocris Cookson Ltd (Bristol,
UK). GDP and GTPγS were obtained from Sigma/RBI (St. Louis, MO, USA). SR 141716A
and SR 144528 were kindly provided by Sanofi-Synthélabo (Bagneux, France).

JPET #49924
16
Results
Chemistry
Target compound NESS 0327 was prepared as shown in fig 1. Acid 9, prepared via the ester
8 by saponification, was activated with thionyl chloride and, without isolation of the
intermediate acyl chloride, reacted with a stoichiometric amount of N-amino-piperidine, in
presence of triethylamine. Ester 8 was prepared starting from the aldehyde 1, which
submitted to a Wittig condensation with the phosphonium bromide 2 yielded the pentenoic
acid derivate 3. Reduction of the double bond of 3 to give 4 with H
2
over PtO
2
in EtOH
(Adams’ catalyst), followed by transformation into the corresponding acyl chloride with
thionyl chloride, and cyclization, with AlCl
3
in dichloromethane, afforded the
benzocycloheptanone 5. This benzocyclanone reacted with diethyl oxalate by means of
NaOEt in EtOH to provide the
α,γ
-diketoester 6, that was allowed to react with 2,4-
dichlorophenylhydrazine hydrochloride 7 to yield the educt 1H-pyrazole-3-carboxylic acid
ethyl ester 8. (fig 1)
Biology
The affinity of NESS 0327 for the cannabinoid CB
1
receptor in mouse forebrain membranes
was evaluated using competitive binding assay. As shown in fig. 2A the specific binding of
[
3
H]-CP 55,940 to its high affinity receptor in mouse brain synaptosomal membranes was
totally displaced by NESS 0327 in a concentration dependent manner with K
i
values of 350 ±
5 fM (n=4). Both SR 141716A and SR 144528 compete for CB
1
receptor with K
i
values of
1.8 ± 0.075 nM and 70 ± 7 nM (n=4) respectively, in close agreement with published values
(Rinaldi-Carmona et al., 1994, 1998). The affinities of NESS 0327, SR 141716A and SR
144528 for CB
2
receptor were determined in mouse spleen (fig. 2B). The concentration-
response gave K
i
values of 21 ± 0.5 nM, 514 ± 30 nM and 0.28 ± 0.04 nM (n=4) for NESS
0327, SR 141716A and SR 144528, respectively. These results show that NESS 0327 is over
60,000 fold selective for the CB
1
receptor versus CB
2
receptor. NESS 0327 was screened for

JPET #49924
17
cannabinoid agonist activity using mouse vas deferens model. Cannabinoid agonists inhibit
the electrically induced contractions of the mouse vas deferens via activation of inhibitory
CB
1
receptors present on the sympathetic nerve terminals (Pertwee, 1997). As shown in fig.
3, WIN 55,212-2 induced a concentration-dependent inhibition of the twitch contractions in
the mouse isolated vas deferens preparations, with pD
2
values of 8.45 ± 0.05. NESS 0327,
which alone had no effect up to 1 µM, produced a concentration-dependent rightward and
almost parallel shift of the concentration response-curve for WIN 55,212-2 showing that it
behaved as a competitive antagonist versus the synthetic cannabinoid agonist with pA
2
value
of 12.46 ± 0.23 and with a slope in the Schild plot not significantly different from unity (1.03
± 0.05, P>0.05).
Efficacy of the compound at the CB
1
receptor was measured using ligand stimulation of
[
35
S]GTPγ binding to cerebellar membranes. [
35
S]GTPγ binding was stimulated in a
concentration-dependent and saturable manner by WIN 55,212-2 with ED
50
and E
max
values
of 0.16 ± 0.01 µM and 286 ± 24% (stimulation above basal binding), respectively (table 1).
In order to determine the ability of NESS 0327 to antagonize CB
1
agonist-stimulated
activation of G-protein the effect of three concentrations of NESS 0327 (0.1, 1, 10 nM) on
the log concentration-response curve of WIN 55,212-2 was investigated. NESS 0327
produced concentration-dependent rightward shift of the WIN 55,212-2 concentration
response-curve (one way ANOVA: F(3,14) = 43.35, P<0.01) without affecting the E
max
of
the agonist (table 1). NESS 0327 at concentrations of 0.1, 1 and 10 nM shifted the dose–
response curve for WIN 55,212-2 to the right with calculated K
e
values of 80.3 ± 20, 283 ±
11 and 2016 ± 226 pM, respectively. NESS 0327, at concentrations from 0.1 through 1 µM,
had no effect on [
35
S]GTPγS binding, while, in the same conditions, SR 141716A at
concentration of 1 µM produced an inhibition of 21 ± 2 % of basal [
35
S] GTPγS binding
(data not shown). The lack of effect on basal [
35
S]GTP
γ
S binding suggests that NESS 0327
had no appreciable negative intrinsic activity in brain under the conditions used in this study.

JPET #49924
18
The in vivo antagonism of NESS 0327 for the cannabinoid receptor was investigated in an
animal model classically used to study cannabinoid drug effects. As shown in fig. 4A and fig.
4B, NESS 0327 dose dependently reduced the analgesia induced by the cannabinoid agonist
WIN 55,212-2 (2 mg/kg s.c.) on both tail flick (Two ways ANOVA: F
dose
(6, 189) = 10.26,
P<0.01; F
time
(2, 189) = 7.22, P<0.01; F
interact
(12, 189) = 3.7, P<0.01 and hot plate (Two
ways ANOVA: F
dose
(6, 189) = 42.37, P<0.01; F
time
(2, 189) = 14.20, P<0.01; F
interact
(12,
189) = 5.4, P<0.01; a complete antagonism was reached at the dose of 0.1 mg/kg in the tail
flick test (P>0.05 vs. vehicle treated rats) and of 0.05 mg/kg in the hot plate test (P>0.05 vs.
vehicle treated rats). The ability of NESS 0327 to inhibit the antinociceptive effect induced
by WIN 55,212-2 was maintained during the observation period. 30 min after WIN 55,212-2
injection, NESS 0327 showed a ID
50
= 0.042 ± 0.01 mg/kg i.p. in the tail flick and ID
50
=
0.018 ± 0.006 mg/kg i.p.in the hot plate test. Furthermore, NESS 0327 did not show any
antinociception activity per se (data not shown), suggesting that it is devoid of inverse
agonist activity and it should be regarded as a pure antagonist.

JPET #49924
19
Discussion
Given the role of the endogenous cannabinoid system in different physiological responses
and its involvement in numerous pathological processes, the search of new and selective
agonists/antagonists of the CB
1
and CB
2
cannabinoid receptor will constitute an important
line of research in the forthcoming years. In this respect, NESS 0327 showed an high
selectivity for CB
1
vs. CB
2
receptors and the in vitro functional assays (isolated organ and
GTPγS) as well the in vivo antinociceptive studies indicated that the compound behaves as
antagonist of the CB
1
receptor. However since the relative binding affinity of NESS 0327 for
the CB1 receptor is about 5,000 times more than that of SR 141716A, the in vivo experiment
whereas the relative difference in activity is only ten times might suggest a poor central
bioavailability of NESS 0327.
NESS 0327 was selected as a lead compound from a series of potential cannabinoid receptor
antagonists (not shown) because it displayed the highest affinity for the CB
1
subtype of the
cannabinoid receptor. Structure relationship inferential reasoning would suggest that a proper
low-energy constrained conformation of the NESS 0327 semirigid tricyclic unit may relate to
its potent and selective affinity for the CB
1
receptor with respect to the parent compound SR
141716A. On the basis of the remarkable result further synthesis of analogues derived from
manipulation in the tricyclic 1,4,5,6-tetrahydrobenzo [6,7] cyclohepta [1,2-c] pyrazole
backbone and variation of substitution on either N
1
-aromatic ring and the aminopiperidine
carboxamide region, may facilitate the elucidation of the cannabinoid pharmacophore for
CB
1
selective antagonist.
Development of cannabinoid receptor selective antagonists will provide the tools necessary
for a better understanding of the cannabinoid receptor functions both in the central nervous
system and in the peripheral immune system. In this respect, considering the higher
selectivity for the CB1 receptor, NESS 0327 may prove to be more advantageous when
compared to the classical CB
1
receptor antagonist SR 141716A.

JPET #49924
20
Current views of the interaction between CB
1
/CB
2
receptors and signal transducting G-
proteins interaction are described in the general framework of allosteric modulation, in which
the receptor isomerizes between an active or inactive form (Samama et al., 1993; Nakamura-
Palacios et al., 1999). Therefore, more detailed studies will be needed to address whether
NESS 0327 may affect the distribution between the active or inactive states of the
cannabinoid receptor (as for a neutral-competitive antagonist) or, on the contrary, may
enhance the accumulation of the receptor in the inactive state (as for an inverse agonist). SR
141716A for instance, has been shown to stimulate cAMP production, providing evidence for
an inverse agonist effect (Mato et al., 2002). It has been further demonstrated that SR
141716A has a peculiar inverse-antagonist activity that is consistent with the stabilization of
an inactive receptor/Gi protein complex. Accordingly, SR 141716A could cause a depletion
of Gi and thus render the protein unavailable for the inhibitory action of other ligands
(Bouaboula et al., 1997). The availability of new and selective ligands, such as NESS 0327,
for the cannabinoid receptor CB
1
would allow a better conceptualisation of the rather
complex mode of cannabinoid receptor/ligand interaction, since ∆
9
-THC itself has been
shown to be a weak but very selective antagonist for the cannabinoid receptor CB
2
(Bayewitch et al., 1996; Barth and Rinaldi-Carmona, 1999). Since recent data using SR
141716A seem to suggest a ligand-independent activity for cannabinoid receptor signalling
(Mato et al., 2002), NESS 0327 could be employed as a more selective antagonist for the
CB
1
receptors, to study the recent proposed ability of the CB1 receptor to sequester G-
proteins from a common pool and prevent other G-protein-coupled receptors from signalling
(Vasquez and Lewis, 1999).
The use of antagonists in studies investigating the biology of cannabinoid receptors may help
to distinguish between receptor-dependent and receptor-independent effects elicited by
cannabinoid agonists. A large arsenal of cannabinoid receptor antagonists will be
instrumental in characterizing both the well-known and eventually, newly discovered,
cannabinoid receptor subtypes. The availability of a compound such as NESS 0327

JPET #49924
21
displaying femtomolar affinity for the CB1 receptor would consequently allow radioactive
labelling of the latter, thus enabling the study of CB1 cellular and tissutal distribution in
further detail. Stringent screening techniques might also be of use in the characterization of
new cannabinoid receptors.
Additional in vivo experiments should provide further evidence for the clinical potential of
this powerful CB
1
antagonist. It should be determined whether NESS 0327 would show
better efficacy as a CB
1
antagonist in animal models of excessive food-intake, psychosis and
cognitive impairment, three area of possible interest for a novel CB
1
selective antagonist.

JPET #49924
22

JPET #49924
23
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27
Footnotes
2
Dpt. Farmaco Chimico Tossicologico, University of Sassari, 07100 Sassari, Italy;
3
“B.B.
Brodie” Dpt of Neuroscience, University of Cagliari, 09124 Cagliari, Italy,
4
Section of
Human Physiology and Nutrition, Dpt. of Applied Sciences to Biosystems, University of
Cagliari, 09124 Cagliari, Italy,
5
C.N.R. Institute of Neurogenetics and Neuropharmacology
and Neuroscienze S.c.a r.l.

JPET #49924
28
Legends
Fig.1. Schematic synthesis of NESS 0327
Fig.2. Competitive inhibition of [
3
H]-CP 55,940 binding in mouse brain (A) and mouse
spleen (B) by NESS 0327, SR 141716A and SR 144528. Binding assays were carried out at
30°C using 0.5 nM [
3
H]-CP 55,940 and increasing concentrations of drugs. Data are mean ±
S.E.M. of at least four different experiments, each performed in triplicate.
Fig. 3. Cumulative concentration –response curves for WIN 55,212-2 on the amplitude of
twitch contractions elicited by electrical field stimulation of the mouse vas deferens obtained
in the presence of its vehicle, DMSO, (
) (control) and in the presence of NESS 0327 at 1
pM (
), 10 pM ( ) or 100 pM ( ). Assays were performed as described under “Methods”.
Each symbol represents the mean value ± S.E.M of inhibition of electrically evoked
contractions of vasa deferentia expressed as a percentage of the amplitude of the twitch
response measured before the first addition of WIN 55,212-2 to the organ bath (n= 6-8
different preparations). NESS 0327 was added 20 min before the first addition of WIN
55,212-2.
Fig. 4. Inhibition by NESS 0327 of WIN 55,212-2 induced antinociception in the tail flick
(A) and in the hot plate test (B). Mice were tested after 30, 60 and 120 min after
administration of WIN 55,212-2 (2 mg/kg s.c.) or vehicle. NESS 0327 (0.01-1 mg/kg) or
vehicle were administered i.p. 20 minutes before WIN 55,212-2 injection. Each column
represents the mean
±
S.E.M of the %MPE obtained from ten animals. Statistical analysis
was carried out using two ways ANOVA followed by Newman Keuls post-hoc test (* P<0.05
and ** P<0.01).

JPET #49924
29
Table 1
ED
50
and E
max
values
of WIN 55,212-2 in stimulating [
35
S]GTP
γ
S binding in the presence or
absence of NESS 0327
Compounds ED
50
(µM) E
max
(% Basal Binding)
WIN 55,212-2
0.16
±
0.01
**
286
±
24
WIN 55,212-2 + NESS 0327 (0.1 nM)
0.36
±
0.07
**
269
±
42
WIN 55,212-2 + NESS 0327 (1 nM)
0.79
±
0.13
**
362
±
42
WIN 55,212-2+ NESS 0327 (10 nM)
1.11
±
0.03
**
200
±
21
[
35
S]GTP
γ
S binding was performed in rat cerebella membranes with 30 µM of GDP and 8-10
concentrations of WIN 55,212-2 in the absence and presence of 0.1, 1, and 10nM of NESS
0327. Data are expressed as percent of basal [
35
S]GTP
γ
S binding. The values represent mean
± S.E.M. of 4-6 separate experiments, each performed in triplicate. ED
50
and E
max
values
were calculated from non-linear
regression curve fitting using Graphpad prism program.
The statistical significance of differences between the groups was assessed by one-way
analysis of variance (ANOVA) following by the Neuman-Keuls test (*P<0.05 and
**
P<0.001
versus WIN 55,212-2)