Synthesis and characterization of NESS 0327: A novel putative antagonist of the CB1 cannabinoid receptor

Article abstract of DOI:10.1124/jpet.103.049924

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.

Full text of DOI:10.1124/jpet.103.049924

0022-3565/03/3061-363–370$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics
JPET 306:363–370, 2003
Vol. 306, No. 1
49924/1071944
Printed in U.S.A.
Synthesis and Characterization of NESS 0327: A Novel
Putative Antagonist of the CB1 Cannabinoid Receptor
`
STEFANIA RUIU, GERARD A. PINNA, GIORGIO MARCHESE, JEAN-MARIO MUSSINU, PIERLUIGI SABA,
SIMONE TAMBARO, PAOLA CASTI, ROMINA VARGIU, and LUCA PANI
Neuroscienze S.c.a r.l., Cagliari, Italy (S.R., G.M., S.T., P.C.); Department of Pharmacology, Chemistry, and Toxicology, University of Sassari,
Sassari, Italy (G.A.P., J.-M.M.); “B.B. Brodie” Department of Neuroscience, University of Cagliari, Cagliari, Italy (P.S.); Section of Human
Physiology and Nutrition, Department of Applied Sciences to Biosystems, University of Cagliari, Cagliari, Italy (R.V.); and C.N.R. Institute of
Neurogenetics and Neuropharmacology and Neuroscienze S.c.a r.l, Selargius, Italy (L.P.)
Received February 3, 2003; accepted March 26, 2003
ABSTRACT
The compound N-piperidinyl-[8-chloro-1-(2,4-dichlorophenyl)- binding in rat cerebella membranes. Conversely, NESS 0327 an-
1,4,5,6-tetrahydrobenzo
boxamide] (NESS 0327) was synthesized and evaluated for bind- rolol
[6,7]cyclohepta[1,2-c]pyrazole-3-car- tagonized [R(ϩ)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]py-
[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone
ing affinity toward cannabinoid CB1 and CB2 receptor. NESS 0327 mesylate] (WIN 55,212-2)-stimulated [35S]GTP␥S binding. In func-
exhibited a stronger selectivity for CB1 receptor compared with tional assay, NESS 0327 antagonized the inhibitory effects of WIN
N-piperidinyl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl- 55,212-2 on electrically evoked contractions in mouse isolated
1H-pyrazole-3-carboxamide (SR 141716A), showing a much vas deferens preparations with pA2 value of 12.46 Ϯ 0.23. In vivo
higher affinity for CB1 receptor (Ki ϭ 350 Ϯ 5 fM and 1.8 Ϯ 0.075 studies indicated that NESS 0327 antagonized the antinociceptive
nM, respectively) and a higher affinity for the CB2 receptor (Ki ϭ effect produced by WIN 55,212-2 (2 mg/kg s.c.) in both tail-flick
21 Ϯ 0.5 nM and 514 Ϯ 30 nM, respectively). Affinity ratios (ID50 ϭ 0.042 Ϯ 0.01 mg/kg i.p.) and hot-plate test (ID50 ϭ 0.018 Ϯ
demonstrated that NESS 0327 was more than 60,000-fold selec- 0.006 mg/kg i.p.). These results indicated that NESS 0327 is a
tive for the CB1 receptor, whereas SR 141716A only 285-fold. novel cannabinoid antagonist with high selectivity for the canna-
NESS 0327 alone did not produce concentration-dependent stim- binoid CB1 receptor.
ulation of guanosine 5Ј-O-(3-[35S]thio)-triphosphate ([35S]GTP␥S)
Interest in the pharmacology of cannabinoids (CBs) has 1995). Both receptors belong to the G protein-coupled family
rapidly increased after the cloning of cannabinoid receptors
and the discovery of their endogenous ligand: arachido-
nylethanolamide (anandamide) (Devane et al., 1988, 1992;
Munro et al., 1993). Two types of cannabinoid receptors, CB1
and CB2, have been characterized, both of which have dis-
tinct anatomical distributions and ligand binding profiles.
Cannabinoid CB1 receptors are present in the central ner-
vous system 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 CB2 receptors seem to be predominantly located
in peripheral tissues (Pertwee, 1997, 1999; Galie`gue et al.,
of receptors that negatively regulate adenylate cyclase and
control the release of arachidonic acid (Howlett, 1995). Nat-
urally 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 antinocicep-
tion) that are collectively known as the tetrad of cannabinoid-
induced behaviors (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 CB1 receptor
(Rinaldi-Carmona et al., 1994; Compton et al., 1996; Licht-
man and Martin, 1997), whereas the CB2 receptor may me-
diate some of the peripheral effects of ⌬9-THC, such as im-
munosuppression (Martin, 1986).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.103.049924.
The cloning of CB1 and CB2 receptors and the subsequent
development of selective tools have advanced the concept of
ABBREVIATIONS: CB, cannabinoid; THC, tetrahydrocannabinol; WIN 55,212-2, R(ϩ)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol[1,2,3-
de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate; 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; [35S]GTP␥S, guanosine 5Ј-O-(3-[35S]thio)-triphosphate; DMSO, dimethyl sulfoxide; EtOH, ethanol; mp, melting point; BSA, bovine
serum albumin; ANOVA, analysis of variance; %MPE, percent maximal possible effect; Hu-210, R(Ϫ)-7-hydroxy-⌬-tetrahydrocannabinol-dimeth-
ylheptyl; CP 55,960, (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-3-hydroxy-propyl)cyclohexan-1-ol; IR, infrared spectroscopy.
363

364
Ruiu et al.
therapeutically targeting cannabinoid receptors. Besides yellow orange oil, boiling point 94–97°C/0.05 mm Hg (lit1 128–131/
0.35 mm Hg); Rf 0.65 (petroleum ether/ethyl acetate, 9:1); IR (film):
3350 (OH), 1680 (CϭO), 1590 (Ar); 1H NMR: 1.72 to 1.98 (m, 4H),
2.73 (t, 2H, J ϭ 6.2 Hz), 2.91 (t, 2H, J ϭ 6.0 Hz), 7.21 (s, 1H), 7.28 (d,
1H, J ϭ 8.8 Hz), 7.68 (d, 1H, J ϭ 8.6 Hz). Anal. C11H11ClO (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
and then poured onto crushed ice and the whole acidified with 2 N
hydrochloric acid and extracted with chloroform (3 ϫ 15 ml). The
combined extracts were washed with water, dried over anhydrous
sodium sulfate, filtered, and evaporated to afford the ␤-dichetoester
6 as an orange oil, which was used in the next step without further
purification (84% yield); boiling point 95–98°C/0.05 mm Hg; Rf 0.78
(petroleum ether/ethyl acetate, 9:1); IR (film): 3440 (OH), 1730
(CϭO), 1680 (CϭO), 1600 (Ar); 1H NMR: 1.41 (t, 3H, J ϭ 7 Hz), 2.08
(quint, 2H), 2.32 (t, 2H, J ϭ 7.2Hz), 2.72 (t, 2H, J ϭ 7 Hz), 3.88 (q,
2H, J ϭ 7 Hz), 7.23 (d, 1H, J ϭ 1.8 Hz), 7.34 (dd, 1H), 7.58 (d, 1H, J ϭ
8.2 Hz), 15.37 (br s, 1H, exch. with D2O). Anal. C15H15ClO4 (C, H, Cl).
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 pres-
sure 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 (CϭO), 1605 (Ar); 1H NMR: 1.43 (t, 3H,
J ϭ 7 Hz), 2.13 to 2.40 (m, 2H), 2.67 (t, 2H, J ϭ 6.4 Hz), 3.09 to 3.40
(m, 2H), 4.46 (q, 2H, J ϭ 7 Hz), 6.60 (d, 1H, J ϭ 8.2 Hz), 7.02 (dd, 1H),
7.31 (d, 1H, J ϭ 2.2 Hz), 7.36–7.49 (m, 2H), 7.54 (d, 1H, J ϭ 9.2 Hz).
Anal. C21H17Cl3N2O2 (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 (CϭO); 1H NMR: 2.20 to 2.39 (m,
2H), 2.50 to 3.35 (m, 4H), 6.61 (d, 1H, J ϭ 8.2 Hz), 7.03 (dd, 1H), 7.32
(d, 1H, J ϭ 1.8 Hz), 7.39 to 7.49 (m, 2H), 7.53 (d, 1H, J ϭ 8.2 Hz),
13.25 (br s, 1H, exch. with D2O). Anal. C19H13Cl3N2O2 (C, H, Cl, N).
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 (3 ϫ 5 ml) and evaporated to yield the crude
carboxylic chloride. A solution of the above-mentioned carboxylic
chloride in dichloromethane (6 ml) was added dropwise to a solution
their established clinical antiemetic action (Voth and
Schwartz, 1997; Gralla, 1999), cannabinoid receptor agonists
also possess appetite stimulant, anticonvulsant, antinocicep-
tive, hypothermic, and antiglaucoma properties (Formukong
et al., 1989; Mattes et al., 1994; Pertwee, 1999; Porcella et al.,
2001).
Recently, several groups have become interested in the
development of cannabinoid antagonists, hoping to develop
new drugs to cure diseases connected with possible malfunc-
tions of “cannabinoid/anandamide” system.
We report the synthesis of a putative cannabinoid ligand,
code named NESS 0327, its differential binding to CB1 and
CB2 cannabinoid receptors, its ability to stimulate
[
35S]GTP␥S binding in rat brain, its effect on mouse vas
deferens, and its action on an in vivo assay known to be
affected by cannabinoids.
Materials and 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 dimethyl sulfoxide (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 solu-
tion 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
(3 ϫ 25 ml). The combined extracts were washed with brine, water,
and then dried over anhydrous sodium sulfate, to provide a browning
compound after evaporation. The crude compound was purified by
flash column chromatography on silica gel eluting with dichlo-
romethane/acetone (9:1) to afford the desired diastereomeric mixture
3 (42% yield); Rf 0.51 (dichloromethane/acetone, 9:1); IR (nujol): 3200
to 2500 (OH), 1720 (CϭO), 1590 (Ar); 1H NMR: 2.40 to 2.75 (m, 8H),
5.60 to 5.75 (m, 2H), 6.15 to 6.45 (m, 2H), 7.10 to 7.28 (m, 6H), 7.32
(s, 2H), 9.50 (br s, 2H, exch. with D2O). Anal. C11H11ClO2 (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 PtO2 [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, mp 56–58°C. Rf 0.84 (petroleum ether/ethyl acetate, 1:1); IR
(nujol): 3300 (OH), 1710 (CϭO), 1600 (Ar); 1H NMR: 1.60 to 1.80 (m,
4H), 2.30 to 2.45 (m, 2H), 2.55 to 2.71 (m, 2H), 7.04 (d, 1H, J ϭ 6.4
Hz), 7.12 to 7.35 (m, 3H), 9.65 (br s, 1H, exch. with D2O). Anal.
C
11H13ClO2 (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. Thionyl chloride in
excess was subsequently removed under reduced pressure and the
residue was added for three times to dichloromethane (3 ml), which of 1-aminopiperidine (10) (0.19 ml, 1.65 mM) and triethylamine (0.23
was evaporated under reduced pressure. A solution of the crude acyl ml, 1.65 mM) in dichloromethane (6.2 ml). After stirring at room
chloride in dichloromethane (3 ml) was added drop wise to a mag-
netically stirred suspension of AlCl3 (0.32 g, 2.36 mM) in dichlo- extracted with dichloromethane (3 ϫ 15 ml). The combined extracts
romethane (3 ml). The resulting mixture was stirred at room tem-
were washed with brine, dried over anhydrous sodium sulfate, fil-
temperature for 1 h, the reaction mixture was added with brine and
perature overnight then poured into ice and the whole extracted with tered, and evaporated to give a yellowish compound. The crude
dichloromethane (3 ϫ 5 ml). The combined extracts were washed compound was purified by flash chromatography on silica gel eluting
with (5%) aqueous sodium bicarbonate solution, water, and after with petroleum ether/ethyl acetate, 1:1, to afford the desired carbox-
drying over anhydrous sodium sulfate, filtered and evaporated to amide NESS 0327 as a white solid (93% yield); mp 205–206°C (ace-
provide a brownish compound. The crude compound was purified by tone), (lit2 202°C); Rf 0.68 (petroleum ether/ethyl acetate, 1:1); IR
flash chromatography on silica gel eluting with petroleum ether/ (nujol): 3200 (NH), 1650 (CϭO), 1600 (Ar); 1H NMR: 1.35 to 1.53 (m,
ethyl acetate (9:1) to afford the attempt compound 5 (77% yield) as a 2H), 1.58 to 1.89 (m, 6H), 2.15 to 2.36 (m, 2H), 2.66 (t, 2H, J ϭ 6.4

NESS 0327: A Novel Putative CB1 Cannabinoid Receptor Antagonist
365
Hz), 2.87 (t, 4H, J ϭ 5.0 Hz), 6.56 (d, 1H, J ϭ 8.2 Hz), 7.01 (dd, 1H),
7.31 (d, 1H, J ϭ 1.8 Hz), 7.37 to 7.54 (m, 3H), 7.66 (br s, 1H, exch.
with D2O). Anal. C24H23Cl3N4O (C, H, Cl, N).
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
Radioligand Binding Methods. Male CD1 mice weighing 20 to reproducible. Preparations were exposed to cumulative increasing
25 g (Charles River, Calco, Italy) were housed in the animal care concentrations of WIN 55,212-2 to obtain concentration-response
quarters; temperature was maintained at 22 Ϯ 2°C on a 12-h light/ curves either in the absence (control) or in the presence of NESS
dark cycle and food and water were available ad libitum. All exper- 0327 (1, 10, or 100 pM) added at a fixed concentration 20 min before
imental protocols were authorized by the Ethical Committee at the the first concentration of WIN 55,212-2. It was not possible to reverse
University of Cagliari and performed in strict accordance with the the inhibitory effect of cannabinoid on the twitch response by wash-
EC regulations for care and use of experimental animals (EEC no. ing them out of the organ bath. Consequently, only one concentra-
86/609).
tion-response curve was constructed per tissue. DMSO was added
Mice were killed by cervical dislocation, and brains (minus cere- instead of the drug. The control dose of DMSO was the same as the
bellum) and spleens were rapidly removed and placed on an ice-cold dose added in combination with the highest dose of drug used. DMSO
plate. After thawing, tissues were homogenated in 20 volumes (w/v)
alone did not inhibit the twitch response (n ϭ 6) at the maximum
of ice-cold TME buffer (50 mM Tris-HCl, 1 mM EDTA, and 3.0 mM concentration used in the bath (4 ␮l/ml).
MgCl2, pH 7.4). The homogenates were centrifuged at 1,086g for 10
Drug additions were performed in volumes of 10 ␮l. The effects of
min at 4°C, and the resulting supernatants were centrifuged at the antagonists or agonists were calculated as percentage of decrease
45,000g for 30 min at 4°C.
in the predrug twitch force. Inhibition of the electrically evoked
[3H]CP 55,940 binding was performed by a modification of the twitch response is expressed in percentage terms and has been
method described previously (Rinaldi-Carmona et al., 1994). Briefly, calculated by comparing the amplitude of the twitch response after
the membranes (30–80 ␮g of protein) were incubated with 0.5 nM each addition of an agonist with the amplitude immediately before
[3H]CP 55,940 for 1 h at 30°C in a final volume of 0.5 ml of TME the first addition of the agonist. The pA2 values for competitive
buffer containing 5 mg/ml fatty acid-free bovine serum albumin antagonists were calculated by Schild regression analysis (Arunlak-
(BSA). Nonspecific binding was estimated in the presence of 1 ␮M CP shana and Schild, 1959). Data were plotted as log antagonist con-
55,940. All binding studies were performed in disposable glass tubes centrations (molar) versus log (concentration-ratios, Ϫ1). It is as-
pretreated with Sigma-Cote (Sigma Chemical, Poole, Dorset, UK), to sumed that when the slope value of the regression line in the Schild
reduce nonspecific binding. The reaction was terminated by rapid plot does not differ statistically from unity, the pA2 value represents
filtration through GF/C filters (Whatman, Maidstone, UK) pre- the dissociation constant of the antagonist (pKB). In each estimate,
soaked in 0.5% polyethyleneimine using a 96-sample harvester eight isolated tissue preparations were used. Statistical significance
(Brandel, Inc., Gaithersburg, MD). Filters were washed five times was determined by use of Student’s test and P Ͻ 0.05 was considered
with 4-ml aliquots of ice-cold Tris HCl buffer (pH 7.4) containing 1 significant.
mg/ml BSA The filter bound radioactivity was measured in a liquid
scintillation counter (Tricarb 2900; PerkinElmer Life Sciences, Bos- (Charles River), weighing 200 to 250 g, were used in all experiments.
ton, MA) with ml of scintillation fluid (Ultima Gold MV; Rats were killed by decapitation, their brains rapidly removed, and
[
35S]GTP␥S Binding Assay. Male Sprague-Dawley rats
4
PerkinElmer Life Sciences). Protein determination was performed cerebella dissected on ice. Cerebella tissue was suspended in 20
by means of Bradford (1976) protein assay using BSA as a standard, volumes of cold centrifugation buffer (50 mM Tris-HCl, 3 mM MgCl2,
according to the protocol of the supplier (Bio-Rad, Milan, Italy). and 1 mM EDTA, pH 7.4) and homogenated using a homogenizer
Drugs were dissolved in DMSO. To avoid possible undesired effects system (Glas-Col, Terre Haute, IN). The homogenate was centri-
on radioligand binding, DMSO concentration in the different assays fuged at 48,000g for 10 min at 4°C. The pellet was then resuspended
never exceeded 0.1% (v/v). All experiments were performed in trip- in the same buffer, homogenized, and centrifuged as described pre-
licate, and results were confirmed in at least four independent ex- viously. The final P2 pellet was subsequently resuspended in assay
periments. Data from radioligand inhibition experiments were ana- buffer (50 mM Tris-HCl, 3 mM MgCl2, 0.2 mM EGTA, and 100 mM
lyzed by nonlinear regression analysis of a Sigmoid Curve using NaCl, pH 7.4), homogenized, and diluted to a concentration of ϳ2
GraphPad Prism program (Graph Pad Software, Inc., San Diego,
␮g/␮l with assay buffer. The protein concentration was determined
CA). IC50 values were derived from the calculated curves and con- by the method of Bradford (1976) using bovine serum albumin as a
verted to Ki values as described previously (Cheng and Prusoff, standard according to the protocol of the supplier (Bio-Rad). Mem-
1973).
brane aliquots were then stored at Ϫ80°C until use.
[
Mouse Vas Deferens Experiments. Vasa deferentia were ob-
35S]GTP␥S binding was measured as described by Selley et al.
tained from albino CD1 mice weighing 25 to 40 g. Tissue was (1996). Briefly, rat cerebella membranes (15 ␮g of protein) were
mounted in a 10-ml organ bath at an initial tension of 0.5 g using the incubated with drugs for 60 min at 30°C in assay buffer containing
method described by Pertwee et al. (1993). The bath contained 0.1% fatty acid-free bovine serum albumin in the presence of 0.05 nM
Krebs-Henseleit solution (118.2 mM NaCl, 4.75 mM KCl, 1.19 mM
KH2PO4, 25.0 mM NaHCO3, 11.0 mM glucose, and 2.54 mM CaCl2), was terminated by rapid filtration using
[
35S]GTP␥S and 30 ␮M GDP, in a final volume of 1 ml. The reaction
Unifilter-GF/B
a
which was kept at 37°C and bubbled with 95% O2 and 5% CO2. (PerkinElmer Life Sciences), washed two times with 1 ml of ice-cold
Isometric contractions were evoked by stimulation with 0.5-s trains 50 mM Tris-HCl, pH 7.4, buffer, and dried 1 h at 30°C. The radio-
of three pulses of 110% maximal voltage (train frequency, 0.1 Hz; activity on the filters was counted in a liquid microplate scintillation
pulse duration, 0.5 ms) through platinum electrodes attached to the counter (TopCount NXT’ PerkinElmer Life Sciences) using 50 ␮l of
upper end of each bath and a stainless steel electrode attached to the
lower end. Stimuli were generated by Grass S88K stimulator then
scintillation fluid (Microscint 20; PerkinElmer Life Sciences).
Stock solution of WIN 55,212-2 and NESS 0327 were prepared in
amplified (multiplexing pulse booster 316S; Ugo Basile, Comerio, Italy) DMSO and then diluted in assay buffer. The final concentration of
and divided to yield separate outputs to four organ baths. Contractions DMSO was Ͻ0.01%, which had no effect either on basal or stimu-
were monitored by computer using a data recording and analysis sys- lated [35S]GTP␥ binding. WIN 55,212-2 concentration effect curves
tem (PowerLab 400) linked via preamplifiers (QuadBridge) to an F10 were determined by incubating membranes with various concentra-
transducer (Biological Instruments, Besozzo, Italy).
Each tissue was subject to several periods of stimulation. The first
of these began after the tissue had equilibrated in the buffering
tions of WIN 55,212-2 (10–10,000 nM) in the presence of 0.05 nM
[
Nonspecific binding was measured in the presence of 10 ␮M un-
35S]GTP␥S and 30 ␮M GDP.
medium but before drug administration, and continued for 10 min. labeled GTP␥S. Basal binding was assayed in the absence of agonist
The stimulator was then switched off for 15 min, after which the and in the presence of GDP. The stimulation by agonist was defined

366
Ruiu et al.
as a percentage increase above basal levels (i.e., {[dpm(agonist) Ϫ 55,212-2 were obtained from Tocris Cookson, Inc. (Bristol, UK). GDP
dpm (no agonist)]/dpm (no agonist)} ϫ 100). and GTP␥S were obtained from Sigma-Aldrich. SR 141716A and SR
Data are reported as mean Ϯ S.E.M. of three to six experiments, 144528 were kindly provided by Sanofi-Synthe´labo (Bagneux,
performed in triplicate. Nonlinear regression analysis of concentra- France).
tion-response data was performed using Prism 2.0 software (Graph-
Pad Prism program) to calculate Emax and EC50 values.
Results
The resulting ED50 values were used to determine Ke values for
antagonism of the agonist-stimulated response by antagonist, using
the relationship Ke ϭ [Ant]/(Dr Ϫ 1), where [Ant] is the concentration
of antagonist, and DR is the ratio of ED50 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 to 25 g (Charles River), were used to assess antinoci-
ception by means of the tail-flick and hot-plate test. A tail-flick meter
(Ugo Basile) 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- to 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) (55 Ϯ 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 before drug administra-
tion to determine control latency and the 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 ϭ [(test latency Ϫ control latency)/(cut-off Ϫ basal latency)] ϫ
100; where the latencies were expressed in seconds and the cut-off
varied depending on the test (tail-flick ϭ 15 s; hot-plate ϭ 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 min after
WIN 55,212-2 (2 mg/kg s.c.) or vehicle and up to 120 min. 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
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, with-
out 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 alde-
hyde 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 H2 over
PtO2 in EtOH (Adams’ catalyst), followed by transformation
into the corresponding acyl chloride with thionyl chloride,
and cyclization, with AlCl3 in dichloromethane, afforded the
benzocycloheptanone 5. This benzocyclanone reacted with
diethyl oxalate by means of NaOEt in EtOH to provide the
␣,␥-diketoester 6, which was allowed to react with 2,4-dichlo-
rophenylhydrazine 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
CB1 receptor in mouse forebrain membranes was evaluated
using competitive binding assay. As shown in Fig. 2A, the
specific binding of [3H]CP 55,940 to its high-affinity receptor
in mouse brain synaptosomal membranes was totally dis-
placed by NESS 0327 in a concentration dependent manner
with Ki values of 350 Ϯ 5 fM (n ϭ 4). Both SR 141716A and
SR 144528 compete for CB1 receptor with Ki values of 1.8 Ϯ
0.075 nM and 70 Ϯ 7 nM (n ϭ 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 CB2 receptor were determined in mouse spleen
(Fig. 2B). The concentration-response gave Ki values of 21 Ϯ
0.5, 514 Ϯ 30, and 0.28 Ϯ 0.04 nM (n ϭ 4) for NESS 0327, SR
141716A, and SR 144528, respectively. These results show
that NESS 0327 is over 60,000-fold selective for the CB1
receptor versus CB2 receptor. NESS 0327 was screened for
cannabinoid agonist activity using mouse vas deferens
model. Cannabinoid agonists inhibit the electrically induced
contractions of the mouse vas deferens via activation of in-
hibitory CB1 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 pD2 values of 8.45 Ϯ 0.05. NESS 0327, which alone had
no effect up to 1 ␮M, produced a concentration-dependent
rightward and almost parallel shift of the concentration re-
sponse-curve for WIN 55,212-2, showing that it behaved as a
ml/kg. Three independent experiments were carried out for ID50
S.E.M. determination. Statistical analyses were carried out using
two-way ANOVA followed by Newman-Keuls post hoc test.
Ϯ
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 (St. Louis, MO) in sure-seal bottles. All reactions
involving air- or moisture-sensitive compounds were performed un-
der a nitrogen atmosphere. Flash column chromatography was car-
ried out using Merck Silica gel 60 (230–400 mesh ASTM). Thin-layer
chromatography was performed with Polygram SIL N-HR-/HV254
precoated plastic sheet (0.2 mm). 1H NMR spectra were determined
in CDCl3 with super conducting FT-NMR using a XL-200 Varian
apparatus at 200 MHz. Chemical shifts are reported in ␦ (ppm)
relative to tetramethylsilane as the internal standard and coupling
constants in Hertz. Significant 1H NMR data are reported in the competitive antagonist versus the synthetic cannabinoid ag-
following order: multiplicity (s, singlet; d, doublet; t, triplet; q, quar-
tet; m, multiplet; dd, double doublet; br s, broad singlet), number of
protons, coupling constants (J) in Hertz. IR spectra were recorded as
thin films or nujol mulls on NaCl plates with a PerkinElmer 781 IR
spectrophotometer and are expressed in ␯ (per centimeter). Melting
points were determined on a Ko¨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 Ϯ 0.4% of their theoretical values. [3H]CP 55,940 (180
Ci/mmol) and [35S]GTP␥S (1200–1350 Ci/mmol) were purchased
onist with pA2 value of 12.46 Ϯ 0.23 and with a slope in the
Schild plot not significantly different from unity (1.03 Ϯ 0.05,
P Ͼ 0.05).
Efficacy of the compound at the CB1 receptor was mea-
sured using ligand stimulation of [35S]GTP␥ binding to cer-
ebellar membranes. [35S]GTP␥ binding was stimulated in a
concentration-dependent and saturable manner by WIN
55,212-2 with ED50 and Emax values of 0.16 Ϯ 0.01 ␮M and
286 Ϯ 24% (stimulation above basal binding), respectively
from PerkinElmer Life Sciences (Boston, MA). CP 55,940 and WIN (Table 1).

NESS 0327: A Novel Putative CB1 Cannabinoid Receptor Antagonist
367
Fig. 1. Schematic synthesis of NESS 0327.
To determine the ability of NESS 0327 to antagonize CB1 used to study cannabinoid drug effects. As shown in Fig. 4, A
agonist-stimulated activation of G protein the effect of three and B, NESS 0327 dose dependently reduced the analgesia
concentrations of NESS 0327 (0.1, 1, and 10 nM) on the log induced by the cannabinoid agonist WIN 55,212-2 (2 mg/kg
concentration-response curve of WIN 55,212-2 was investi- s.c.) on both tail-flick [two-way ANOVA: Fdose (6,189) ϭ
gated. NESS 0327 produced concentration-dependent right- 10.26, P Ͻ 0.01; Ftime (2,189) ϭ 7.22, P Ͻ 0.01; Finteract
ward shift of the WIN 55,212-2 concentration response-curve (12,189) ϭ 3.7, P Ͻ 0.01] and hot-plate (two-way ANOVA:
[one-way ANOVA: F(3,14) ϭ 43.35, P Ͻ 0.01) without affect- Fdose (6,189) ϭ 42.37, P Ͻ 0.01; Ftime (2,189) ϭ 14.20, P Ͻ
ing the Emax of the agonist (Table 1). NESS 0327 at concen- 0.01; Finteract (12,189) ϭ 5.4, P Ͻ 0.01]; a complete antago-
trations of 0.1, 1, and 10 nM shifted the dose-response curve nism was reached at the dose of 0.1 mg/kg in the tail-flick test
for WIN 55,212-2 to the right with calculated Ke values of (P Ͼ 0.05 versus vehicle-treated rats) and of 0.05 mg/kg in
80.3 Ϯ 20, 283 Ϯ 11, and 2016 Ϯ 226 pM, respectively. NESS the hot-plate test (P Ͼ 0.05 versus vehicle-treated rats). The
0327, at concentrations from 0.1 through 1 ␮M, had no effect ability of NESS 0327 to inhibit the antinociceptive effect
on [35S]GTP␥S binding, whereas, in the same conditions, SR induced by WIN 55,212-2 was maintained during the obser-
141716A at concentration of 1 ␮M produced an inhibition of vation period. Thirty minutes after WIN 55,212-2 injection,
21 Ϯ 2% of basal [35S]GTP␥S binding (data not shown). The NESS 0327 showed a ID50 ϭ 0.042 Ϯ 0.01 mg/kg i.p. in the
lack of effect on basal [35S]GTP␥S binding suggests that tail-flick and ID50 ϭ 0.018 Ϯ 0.006 mg/kg i.p. in the hot-plate
NESS 0327 had no appreciable negative intrinsic activity in test. Furthermore, NESS 0327 did not show any antinocicep-
brain under the conditions used in this study.
tion activity per se (data not shown), suggesting that it is
The in vivo antagonism of NESS 0327 for the cannabinoid devoid of inverse agonist activity and it should be regarded as
receptor was investigated in an animal model classically a pure antagonist.

368
Ruiu et al.
Fig. 3. Cumulative concentration-response curves for WIN 55,212-2 on
the amplitude of twitch contractions elicited by electrical field stimula-
tion of the mouse vas deferens obtained in the presence of its vehicle,
DMSO (f) (control), and in the presence of NESS 0327 at 1 pM (ƒ), 10 pM
(F), or 100 pM (E). Assays were performed as described under Materials
and Methods. Each symbol represents the mean value Ϯ S.E.M of inhi-
bition 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 ϭ 6–8 different
preparations). NESS 0327 was added 20 min before the first addition of
WIN 55,212-2.
TABLE 1
ED50 and Emax values of WIN 55,212-2 in stimulating [35S]GTP␥S
binding in the presence or absence of NESS 0327
[
35S]GTP␥S binding was performed in rat cerebella membranes with 30 ␮M of GDP
and 8 to 10 concentrations of WIN 55,212-2 in the absence and presence of 0.1, 1, and
10 nM NESS 0327. Data are expressed as percentage of basal [35S]GTP␥S binding.
The values represent mean Ϯ S.E.M. of four to six separate experiments, each
performed in triplicate. ED50 and Emax values were calculated from nonlinear re-
gression curve fitting using GraphPad Prism program. The statistical significance of
differences between the groups was assessed by one-way ANOVA followed by the
Newman-Keuls test (* P Ͻ 0.05 and ** P Ͻ 0.001 versus WIN 55,212-2).
Fig. 2. Competitive inhibition of [3H]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 [3H]CP 55,940 and
increasing concentrations of drugs. Data are mean Ϯ S.E.M. of at least
four different experiments, each performed in triplicate.
Compounds
ED50
Emax
␮M
% basal
binding
WIN 55,212-2
0.16 Ϯ 0.01
286 Ϯ 24
269 Ϯ 42
362 Ϯ 42
200 Ϯ 21
WIN 55,212-2 ϩ NESS 0327 (0.1 nM)
WIN 55,212-2 ϩ NESS 0327 (1 nM)
WIN 55,212-2 ϩ NESS 0327 (10 nM)
0.36 Ϯ 0.07*
0.79 Ϯ 0.13**
1.11 Ϯ 0.03**
Discussion
Given the role of the endogenous cannabinoid system in
different physiological responses and its involvement in nu-
merous pathological processes, the search of new and selec- clic unit may relate to its potent and selective affinity for the
tive agonists/antagonists of the CB1 and CB2 cannabinoid CB1 receptor with respect to the parent compound SR
receptor will constitute an important line of research in the 141716A. On the basis of the remarkable result further syn-
forthcoming years. In this respect, NESS 0327 showed a high thesis of analogs derived from manipulation in the tricyclic
selectivity for CB1 versus CB2 receptors and the in vitro 1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole back-
functional assays (isolated organ and GTP␥S) as well the in bone and variation of substitution on either N1-aromatic ring
vivo antinociceptive studies indicated that the compound and the aminopiperidine carboxamide region, may facilitate
behaves as antagonist of the CB1 receptor. However, because the elucidation of the cannabinoid pharmacophore for CB1-
the relative binding affinity of NESS 0327 for the CB1 recep- selective antagonist.
tor is about 5000 times more than that of SR 141716A, the in
Development of cannabinoid receptor selective antagonists
vivo experiment where the relative difference in activity is will provide the tools necessary for a better understanding of
only 10 times might suggest a poor central bioavailability of the cannabinoid receptor functions both in the central ner-
NESS 0327.
vous system and in the peripheral immune system. In this
NESS 0327 was selected as a lead compound from a series respect, considering the higher selectivity for the CB1 recep-
of potential cannabinoid receptor antagonists (data not tor, NESS 0327 may prove to be more advantageous com-
shown) because it displayed the highest affinity for the CB1 pared with the classical CB1 receptor antagonist SR
subtype of the cannabinoid receptor. Structure relationship 141716A.
inferential reasoning would suggest that a proper low-energy
Current views of the interaction between CB1/CB2 recep-
constrained conformation of the NESS 0327 semirigid tricy- tors and signal transducting G proteins interaction are de-

NESS 0327: A Novel Putative CB1 Cannabinoid Receptor Antagonist
369
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 was ad-
ministered i.p. 20 min before WIN
55,212-2 injection. Each column repre-
sents the mean Ϯ S.E.M of the %MPE
obtained from ten animals. Statistical
analysis was carried out using two-way
ANOVA followed by Newman-Keuls post
hoc test (ء, P Ͻ 0.05 and ءء, P Ͻ 0.01).
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Address correspondence to: Dr. Luca Pani, Institute of Neurogenetic and
Neuropharmacology, Via Boccaccio 8, 09047 Selargius, Italy. E-mail:
l.pani@inn.cnr.it