Novel 4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives as new CB 2 cannabinoid receptors agonists: Synthesis, pharmacological properties and molecular modeling

Article abstract of DOI:10.1021/jm050467q

Recent data indicated that the CB₂ cannabinoid receptor constitutes an attractive drug target due to its potential functional role in several physiological and pathological processes. A set of 4-oxo-1,4- dihydroquinoline-3-carboxamide derivatives, characterized by the presence of some important structural requirements exhibited by other classes of cannabinoid ligands, such as an aliphatic or aromatic carboxamide group in position 3, and an alkyl or benzyl group in position 1, was synthesized and assayed to measure their respective affinity for both human CB₁ and CB₂ cannabinoid receptors. The results indicate that these 3-carboxamido-quinolones derivatives exhibited a CB₂ receptor selectivity, particularly derivatives 28-30, and 32R. Moreover, in the [₃₅S]-GTPγS binding assay, all the compounds behaved as CB₂ receptor agonists. Molecular modeling studies showed that compound 30 interacts with the CB ₂ receptor through a combination of hydrogen bond and aromatic/hydrophobic interactions. In conclusion, 4-oxo-1,4-dihydroquinoline-3- carboxamide derivatives constitute a new class of potent and selective CB ₂ cannabinoid receptors agonists.

Full text of DOI:10.1021/jm050467q

70
J. Med. Chem. 2006, 49, 70-79
Novel 4-Oxo-1,4-dihydroquinoline-3-carboxamide Derivatives as New CB2 Cannabinoid
Receptors Agonists: Synthesis, Pharmacological Properties and Molecular Modeling
Eric Stern,†,‡ Giulio G. Muccioli,†,§ Re´gis Millet,‡ Jean-Franc¸ois Goossens,‡ Amaury Farce,| Philippe Chavatte,|
Jacques H. Poupaert,§ Didier M. Lambert,§ Patrick Depreux,*,‡ and Jean-Pierre He´nichart‡
Institut de Chimie Pharmaceutique Albert Lespagnol, UniVersite´ de Lille 2, EA 2692, 3 rue du Pr. Laguesse, B.P. 83, F-59006 Lille, France,
Unite´ de Chimie pharmaceutique et de Radiopharmacie, Ecole de Pharmacie, Faculte´ de Me´decine, UniVersite´ catholique de LouVain, 73
aVenue E. Mounier UCL-CMFA (7340), B-1200 Bruxelles, Belgium, and Faculte´ de Pharmacie, Laboratoire de Chimie The´rapeutique,
UniVersite´ de Lille 2, EA 1043, 3 rue du Professeur Laguesse, B.P. 83, F-59006 Lille, France
ReceiVed May 18, 2005
Recent data indicated that the CB2 cannabinoid receptor constitutes an attractive drug target due to its potential
functional role in several physiological and pathological processes. A set of 4-oxo-1,4-dihydroquinoline-
3-carboxamide derivatives, characterized by the presence of some important structural requirements exhibited
by other classes of cannabinoid ligands, such as an aliphatic or aromatic carboxamide group in position 3,
and an alkyl or benzyl group in position 1, was synthesized and assayed to measure their respective affinity
for both human CB1 and CB2 cannabinoid receptors. The results indicate that these 3-carboxamido-quinolones
derivatives exhibited a CB2 receptor selectivity, particularly derivatives 28-30, and 32R. Moreover, in the
[35S]-GTPγS binding assay, all the compounds behaved as CB2 receptor agonists. Molecular modeling studies
showed that compound 30 interacts with the CB2 receptor through a combination of hydrogen bond and
aromatic/hydrophobic interactions. In conclusion, 4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives
constitute a new class of potent and selective CB2 cannabinoid receptors agonists.
Introduction
associated metabolic syndrome.11 On the contrary, ∆9-THC and
nabilone are currently marketed to reduce emesis and/or prevent
cachexia in AIDS or cancer patients.12 Other therapeutic
applications currently studied include multiple sclerosis, neu-
ropathic, and cancer pain.
Despite the discovery by sequence homology of the CB2
cannabinoid receptor in the same years, the physiological as
well as putative therapeutic potential of this receptor largely
remains unexplored. However, recent data indicate that CB2
cannabinoid receptors participate in the control of peripheral
pain,13,14 inflammation,15 and cancer proliferation.16 It also has
an antifibrogenic role in the liver.17 Moreover, the recent
discovery of the presence of the CB2 cannabinoid receptors in
the brain microglial cells18,19 gave a rationale for prevention of
Alzheimer’s disease pathology by cannabinoid agents. Indeed,
it was recently shown that CB2 cannabinoid receptor agonists
might provide neuroprotection by blockade of microglial
activation.20
Hashish and marijuana, two preparations derived from the
Indian hemp Cannabis SatiVa L., have been used since im-
memorial time for their medicinal and psychoactive properties.
Despite the isolation of ∆9-tetrahydrocannabinol (∆9-THC, Chart
1) as the major psychoactive component of Cannabis SatiVa
L.1 in the 1960s, the molecular targets of ∆9-THC were only
discovered in the late 1980s, with on one hand the help of a
synthetic radioligand [3H]-CP-55,940
(Chart 1) allowing
2
suitable cannabinoid receptor binding assays and, on the other
hand, the characterization of the biochemistry and the molecular
biology of cannabinoid receptors, which led to two subtypes of
G-protein coupled receptors named CB13 and CB24 cannabinoid
receptors. They differ in sequences, tissue localization, and, to
some extent, signal transduction mechanisms. In addition,
generation of knock-out mice models where either one subtype
or two subtypes genes have been deleted suggest the putative
existence of additional cannabinoid receptors.5
Both types of cannabinoid receptors are sensitive to pertussis
toxin, suggesting their predominant coupling to Gi-type proteins
allowing the use of [35S]-GTPγS assay21 to characterize the
functionality of cannabinoid ligands. Cannabinoid receptors are
activated by endocannabinoids, long-chain polyunsaturated fatty
acids where the carboxylate group is either amidated by
ethanolamine or esterified by glycerol.22 The two major
representatives are anandamide (N-arachidonylethanolamine)
and 2-AG (2-arachidonoylglycerol) (Chart 1), respectively.
On a pharmacological point of view, up to now, the
cannabinoid ligands might be divided into three major catego-
ries: (a) compounds which are not, or poorly, selective for one
cannabinoid receptor subtype such as classical cannabinoids (∆9-
THC and related compounds), nonclassical cannabinoids (e.g.,
CP-55,940 and derivatives), and aminoalkylindoles (e.g., WIN-
55,212-2); (b) compounds which are selective for the canna-
binoid CB1 receptor subtype such as some biarylpyrazoles (e.g.,
SR-141716A) and many other diverse heterocyclic structures
The cannabinoid CB1 receptor is nowadays extensively
studied due to its implication both in the therapeutic and
psychoactive effects of cannabinoids in the central nervous
system. Transduction mechanisms of cannabinoid CB1 receptors
involve inhibition of cAMP production through inhibition of
adenylate cyclase,6 inhibition of calcium influx,7,8 activation of
potassium channels,9 and activation of the MAP kinase path-
way.10
Promising selective cannabinoid CB1 receptor antagonists
such as rimonabant or SLV-319 are currently under investigation
in clinical human studies for the treatment of obesity and the
† Both authors contributed equally to this work.
‡ Institut de Chimie Pharmaceutique Albert Lespagnol, Universite´ de Lille
2.
§ Universite´ Catholique de Louvain.
| Faculte´ de Pharmacie, Universite´ de Lille 2.
* To whom correspondence should be addressed. Phone: +33-3-2096-
4379. Fax: +33-3-2096-4906. E-mail: pdepreux@pharma.univ-lille2.fr.
10.1021/jm050467q CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/13/2005

New CB2 Cannabinoid Receptors Agonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 71
Chart 1. Representative Ligands of the Cannabinoid Receptors
such as triazoles,23 thiazoles,24 imidazoles,24,25 imidazolidinedi-
ones,26 2-thioxoimidazolidinones,27 and pyridines28 that have
been recently reported; and (c) a few compounds that are
selective for the CB2 cannabinoid receptor subtype such as some
biarylpyrazoles (e.g., SR-144528), 1,2-dihydroquinoline-3-car-
boxamide (JTE-907, Chart 1),29 1,8-naphthyridines,30 and triaryl
bis-sulfones.31
Chart 2. General Structure of
4-Oxo-1,4-dihydroquinoline-3-carboxamide Derivatives 11-36
At present, new potent CB2-selective cannabinoid receptor
ligands are important to understand some of the physiological
effects of cannabinoids, such as their immunosuppressive, anti-
inflammatory, and antinociceptive activities.32-37 For instance,
it was very recently shown that low doses of ∆9-THC could
reduce atherosclerosis in mice by acting at the CB2 cannabinoid
receptor.38 Finally but not least, cannabinoid agonists that
selectively target CB2 cannabinoid receptors should be devoid
of psychoactive effects.
Along this line, the present paper describes the synthesis and
the pharmacological properties of a set of 27 4-oxo-1,4-
dihydroquinoline-3-carboxamide derivatives, with various sub-
stitutions on the heterocyclic nucleus, as illustrated in Chart 2.
Aliphatic or aromatic carboxamide groups in position 3 have
been selected on the basis of other cannabinoid pharmacophoress
such as those present both in the quinolone derivative JTE-907
and in the biarylpyrazole SR-144528sand a hydrophobic
substituent in position 1 as in the aminolakylindole derivatives.39
They were tested in competitive binding assays toward both
human CB1 (hCB1) and CB2 (hCB2) cannabinoid receptors
expressed in CHO cells40 and were found selective for the CB2
cannabinoid receptor subtype. For compounds exhibiting a Ki
value lower than 1 µM, [35S]-GTPγS binding assays were
performed to determine their functionality.32 Additionally,
overlay of WIN-55,212-2, CP-55,940, and 30 allowed us to
study their structural similarities. Docking was finally performed
to elucidate the mode of interaction of compound 30 with a
CB2 cannabinoid receptor model.
Results
Chemistry. The synthetic routes to obtain the target 4-oxo-
1,4-dihydroquinoline-3-carboxamide derivatives 11-36 are
outlined in Scheme 1. All substituents are summarized in Table

72 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Stern et al.
Scheme 1. Synthesis of the 4-Oxo-1,4-dihydroquinoline-3-carboxamide Derivatives 11-36a
a Reagents and conditions: (i), 100 °C, 91%; (ii) Ph-O-Ph, reflux, 77%; (iii) R-X, NaH, DMF, 90 °C, 50-93%; (iV) NaOH, EtOH, 100 °C, 70-76%;
(V) R′-NH2 or morpholine, PyBRoP, PS-HOBt (HL), DIEA, DMF, rt, 14-97%.
1. Compounds 11-36 were obtained by a coupling reaction
between selected amines and 4-oxo-1,4-dihydroquinoline-3-
carboxylic acids 7-10 obtained in three steps following Gould-
Jacobs’ procedure.41,42
Table 1. Structures and Percentages of Displacement of
[3H]-SR-141716A and [3H]-CP-55,940, Respectively, by Compounds
8-36 (10 µM) on hCB1 and hCB2 Cannabinoid Receptorsa
To afford the 4-oxo-1,4-dihydroquinoline-3-carboxylic acid
ethyl ester 2, extreme conditions (i.e., reflux of 1 at ∼ 255°C
in diphenyl ether43) were required to induce cyclization. In this
process, as observed by 1H NMR, the 4-quinolone form 2, and
not its 4-hydroxy-quinoline tautomeric form 2′, was obtained.
N-alkylation of 2 in anhydrous DMF with appropriate alkyl
bromides or benzylbromide in the presence of sodium hydride
gave the 1-alkyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
ethyl esters 3-6.44 Hydrolysis in 10% aqueous NaOH yielded
the corresponding 1-alkyl-4-oxo-1,4-dihydroquinoline-3-car-
boxylic acids 7-10.45
% of displacement
compd
R′
R
CB1-R
CB2-R
<15
50.5 ( 6.9
60.1 ( 7.6
28.2 ( 8.0
8
n-pentyl <20
n-butyl <20
n-pentyl <20
n-hexyl <10
11 4-methoxyphenyl
12 4-methoxyphenyl
13 4-methoxyphenyl
14 1-naphthyl
15 1-naphthyl
16 1-naphthyl
17 benzyl
18 benzyl
19 benzyl
20 4-methoxybenzyl
21 2-naphthyl
n-butyl 45.3 (.7.4 85.4 ( 6.3
n-pentyl 65.9 ( 6.8 90.6 ( 1.3
n-hexyl 53.3 ( 7.4 80.0 ( 6.6
n-butyl 33.6 ( 6.5 44.9 ( 6.7
n-pentyl 50.5 ( 5.5 66.7 ( 8.1
n-hexyl 37.4 ( 4.0 54.6 ( 6.5
To obtain some chemical diversity among 1-alkyl-4-oxo-1,4-
dihydroquinoline-3-carboxamides, a solid phase procedure was
set up using a previously reported polystyrene-supported HOBt
as coupling reagent.46,47 The reaction performed on a Quest 205
synthesizer according to the general reaction pathway outlined
in Scheme 2 generated compounds 11-36. This is a two-step
procedure: (i) formation of the polymer bound activated ester
of the carboxylic acid (7-10) using bromotrispyrrolidinophos-
phonium hexafluorophosphate (PyBrop); (ii) release of the target
amide in solution by addition of the amine. Optimization of
both steps was performed to obtain a solution of amide free of
byproducts. The best conditions for activation were found to
be 2 × 3 h activating time step in DMF. The coupling step was
achieved in 24 h with 0.9 equiv of the amine in a DMF solution.
The target compounds 11-36 were purified either by recrys-
tallization or by preparative thick-layer chromatography. The
n-pentyl <10
n-pentyl <10
51.7 ( 4.3
63.9 ( 4.1
22 2-phenylethyl
n-pentyl 64.8 ( 5.1 98.2 ( 2.9
n-pentyl 32.8 ( 4.8 85.5 ( 6.0
23 3-phenylpropyl
24 3,4-dichlorophenyl
25 4-cyanophenyl
26 4-biphenyl
27 2-(benzo[1,3]dioxol-5-yl)ethyl
28 1-adamantyl
n-pentyl <10
n-pentyl 49.6 ( 3.3 79.4 ( 6.0
n-pentyl <20 <20
68.1 ( 3.0
n-pentyl 43.6 ( 2.0 88.7 ( 1.7
n-pentyl 59.0 ( 3.9 104.0 ( 3.9
n-pentyl 37.8 ( 3.3 103.8 ( 1.8
n-pentyl 54.6 ( 2.4 95.6 ( 5.4
benzyl 32.5 ( 1.4 86.3 ( 2.1
n-pentyl 68.3 ( 3.1 101.1 ( 3.2
29 2-adamantyl
30 1-(3,5-dimethyl)adamantyl
31 1-(3,5-dimethyl)adamantyl
32 (RS)-1-phenylethyl
33 (RS)-1-(2-naphthyl)ethyl
34 (RS)-1-(1-naphthyl)ethyl
n-pentyl <10
82.9 ( 3.7
n-pentyl 35.5 ( 3.7 98.9 ( 4.1
1
35 (RS)-1-(1,2,3,4-tetrahydro-naphthyl ) n-pentyl 79.7 ( 4.1 96.5 ( 4.0
36 n-pentyl <10 36.5 ( 3.4
N(1) alkylation was confirmed by 2D H NMR (ROESY) as
evidenced by the presence of cross-peaks between H(2) and
N-CH2 protons.
a Mean ( SEM of at least four experiments performed in duplicate.

New CB2 Cannabinoid Receptors Agonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 73
Scheme 2. Solid-Phases Procedure of the Synthesis of the 4-Oxo-1,4-dihydroquinoline-3-carboxamide Derivatives 11-36a
a Reagents and conditions: (i) PyBRoP, PS-HOBt (HL), DIEA, DMF, rt; (ii) R′-NH2 or morpholine, DMF, rt.
Table 2. Affinities of Selected Derivatives and of Reference
Compounds (SR-144528, WIN-55,212-2, CP-55,940, JWH-133, and
HU-210) at the hCB2 Cannabinoid Receptora
Table 3. Affinities of Selected Derivatives (Compounds 15, 29, 32R,
and 35) and of Reference Cannabinoid (SR-141716A) at the hCB1
Cannabinoid Receptora
hCB2
hCB1
cannabinoid receptor
cannabinoid receptor
selectivity ratio
CB2 versus CB1
compd
Ki (nM)
compd
Ki (nM)
14
15
16
18
22
23
24
25
27
28
29
30
31
32
455 ( 63
371 ( 34
844 ( 78
>1000
15
29
32R
4083 ( 375
1925 ( 179
1154 ( 108
1045 ( 96
5.37 ( 0.6
11
143
31
35
17
201 ( 28
>1000
SR-141716A
a The Ki values were obtained from nonlinear analysis of competition
curves using [3H]-SR-141716A as radioligand. Data are the mean ( SEM
of at least four experiments performed in duplicate.
>1000
772 ( 72
426 ( 39
16.4 ( 1.5
13.4 ( 1.2
15.8 ( 1.4
664 ( 62
70.8 ( 9.1
37.1 ( 3.4
784 ( 71
>1000
1,4-dihydroquinoline-3-carboxamide derivatives (11-36) are
selective for the CB2 cannabinoid receptors.
The question of the 4-oxo-1,4-dihydroquinoline-3-carboxa-
mide derivatives functionality was investigated by using a [35S]-
GTPγS binding assay.49 This assay constitutes a functional
measure of the interaction of the receptor and the G-protein,
the first step in activation of the G-protein coupled receptors.
It is a useful tool to distinguish between agonists (increasing
the nucleotide binding), inverse agonists (decreasing the nucle-
otide binding), and neutral antagonists (not affecting the
nucleotide binding). The reference cannabinoid agonists JWH-
133, HU-210, WIN-55,212-2, and CP-55,940 as well as the
inverse agonist SR-144528 were assayed in a [35S]-GTPγS
binding stimulation assay at the hCB2 cannabinoid receptors.
Results expressed as percentages of the basal stimulation (set
at 100%) are summarized in Table 4. In this model, agonists at
10 µM give a [35S]-GTPγS binding stimulation from 128 to
149% of basal stimulation, while the inverse agonist SR-144528
abolishes it to 22%. 4-Oxo-1,4-dihydroquinoline-3-carboxamide
derivatives 14-16, 18, 22-25, and 27-35 were challenged in
this assay and gave values from 117 up to 137% of basal
stimulation indicating that these compounds act as agonists at
the hCB2 cannabinoid receptors. To further investigate the
agonistic properties, the potency and efficacy of compounds
29, 30, 32R, and 35 were determined (Table 5). These
compounds dose-dependently increased the [35S]-GTPγS bind-
ing, exhibiting EC50 values of the same magnitude as the
respective Ki values. For instance, compounds 30 and 32R
exhibited EC50 values of 14.1 and 16.8 nM, respectively.
Structure-Affinity Relationships. Preliminary screening
results showed that the first 3-carboxamidoquinolone derivatives
studied displayed affinity for the cannabinoid receptors and
therefore could constitute a suitable template in the design of
new cannabinoid derivatives. In addition, a selectivity profile
for the hCB2 subtype was observed. To define the correct profile
for binding to the hCB2 cannabinoid receptor, we decided to
32R
32S
33
33R
33S
34
34R
34S
35
SR-144528
WIN-55,212-2
CP-55,940
JWH-133
584 ( 54
>5000
174 ( 16
125 ( 12
>1000
60.2 ( 5.5
51.7 ( 4.8
9.1 ( 0.8
15.4 ( 1.4
20.3 ( 2.6
7.3 ( 0.9
HU-210
a The Ki values were obtained from nonlinear analysis of competition
curves using [3H]-CP-55,940 as radioligand. Data are the mean ( SEM of
at least four experiments performed in duplicate.
Pharmacology. The 4-oxo-1,4-dihydroquinoline-3-carboxa-
mide derivatives (11-36) were screened at 10 µM concentra-
tions for their affinity and selectivity toward the hCB1 and hCB2
cannabinoid receptors in a competitive binding experiment as
previously described.48 Membranes from Chinese hamster
ovarian (CHO) cells expressing either the hCB1 or the hCB2
cannabinoid receptors were used in these experiments. [3H]-
SR-141716A and [3H]-CP-55,940 at concentrations of 1 nM
were used as radioligands for the hCB1 and the hCB2 canna-
binoid receptor, respectively. The results expressed as the
displacement percentages of the radioligand from its binding
site are summarized in Table 1. The Ki values were then
determined for compounds exhibiting a specific displacement
superior to 60% either for the cannabinoid hCB2 (Table 2) or
the hCB1 receptors (Table 3). The selectivity ratio is presented
in the Table 3 when Ki values have been obtained for both
receptors. All together these results indicated that the 4-oxo-

74 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Stern et al.
Table 4. [35S]-GTPγS Binding Stimulation Assays (10 µM) of Selected
Compounds and Reference Cannabinoid Ligands (SR-144528,
WIN-55,212-2, CP-55,940, JWH-133, and HU-210) for the hCB2
Cannabinoid Receptorsa
tions. Indeed, replacement of 1-naphthyl (15) by the 2-naphthyl
isomer (21) resulted in a decreased affinity. However, replace-
ment of the benzyl group (18) by 4-methoxybenzyl (20) and
replacement of the 4-methoxyphenyl group (12) by 4-cyanophe-
nyl (25) or 3,4-dichlorophenyl (24) did not elicit a clear effect
on affinity. Therefore, electronic effects on the aromatic moiety
did not influence the affinity for the cannabinoid CB2 receptor.
hCB2 cannabinoid receptor
[35S]-GTPγS specific binding
compd
(% of basal)
14
15
16
18
22
23
24
25
27
28
29
30
31
32
32R
33
33R
34
34R
35
131.8 ( 3.5**
123.7 ( 3.8**
123.9 ( 2.4**
128.6 ( 2.3**
135.7 ( 2.5**
133.7 ( 2.2**
116.5 ( 0.8**
133.1 ( 1.1**
136.5 ( 1.3**
125.6 ( 2.5**
129.6 ( 1.1**
130.7 ( 1.8**
121.0 ( 2.0**
130.5 ( 1.0**
123.6 ( 1.4**
122.5 ( 1.7**
122.3 ( 2.1**
116.8 ( 1.0**
119.2 ( 0.8**
123.1 ( 1.1**
21.6 ( 2.7**
127.8 ( 2.3**
145.6 ( 1.8**
148.0 ( 2.5**
135.0 ( 1.4**
We also synthesized 2-phenylethyl (22), 3-phenylpropyl (23),
4-biphenyl (26), and 2-(benzo[1,3]dioxol-5yl)ethyl (27) deriva-
tives in order to evaluate the importance of hydrophobic
character in the 3-position, since 1-naphthyl (15) revealed a good
affinity for CB2 receptor (Ki ) 371 nM). The best result was
found with 22 (Ki ) 204 nM) when the phenyl group was spaced
from the 3-carboxamidoquinolone ring by an ethyl link.
Replacement of this ethyl link by methyl or propyl homologues
led to a decreased affinity (Ki values > 1000 nM) that was even
more markedly observed in the 4-biphenyl derivative (26, <20%
displacement at 10 µM). Compound 27, in which the phenethyl
substituent of 22 is replaced by a 2-(benzo[1,3]dioxol-5yl)ethyl,
showed about 2-fold less affinity (Ki ) 426 nM).
Nonaromatic compounds with different adamantyl substitu-
ents (28, 29, 30, 31) were also investigated. These moieties are
widely found in the structure of cannabinoid ligands11,50 with
antagonistic properties. They can also generate hydrophobic
interactions. These modifications induced a marked improve-
ment in affinity (Ki values of 16.4 nM and 13.4 nM for 28 and
29, respectively). Interestingly, in the [35S]-GTPγS binding
assay, these compounds kept their agonistic properties. En-
hancement of adamantyl hydrophobicity by addition of two
methyl groups (in position 3 and 5) did not elicit a marked effect
on affinity as shown by compound 30 (Ki ) 15.8 nM).
Compound 31 in which the n-pentyl side chain is replaced by
a benzyl showed about 44-fold less affinity (Ki ) 664 nM).
Replacement of the adamantyl substituent by a morpholino
group, yielding compound 36, resulted in a decreased affinity.
SR-144528
WIN-55,212-2
CP-55,940
JWH-133
HU-210
a Results are expressed as the percentages of stimulation of [35S]-GTPγS
binding (basal value set at 100%). Data are the mean ( SEM of three
experiments performed in duplicate. Statistical significance was assessed
by one-way ANOVA followed by a Dunett post-test (*P < 0.05 and **P
< 0.01).
Table 5. Determination of the Potency and Efficacy of Compounds 29,
30, 32R, 35, SR-144528, WIN-55,212-2, CP-55,940, JWH-133, and
HU-210 at the hCB2 Cannabinoid Receptors
compd
EC50 (nM)
Emax (%)
The cannabinoid hCB2 receptor binding data reported herein
suggested that the hydrophobicity of the carboxamido substituent
appears to be as important as the aromatic character. In addition,
the poor affinity of the 3-carboxylic acid intermediate 8 (<15%
displacement at 10 µM) underlines the importance of the amide
substitution. The carboxamido group is most likely located in
a large hydrophobic cluster. To study the pocket, for the
aromatic carboxamide compounds, a steric constraint by modu-
lation of 22 using its 1-phenylethyl isomer (32) was introduced.
The steric constraint imposed by a methyl group on the
methylene spacer induced a particular orientation of the aromatic
group. In this series, we also replaced the phenyl moiety by
other aromatic nuclei: 1- and 2-naphthyl (33 and 34). Com-
pounds 32-34 were first tested as racemates, as shown in Table
2. Introduction of this pseudo-rigidification led to an increase
of the hCB2 cannabinoid receptor affinity as illustrated by
compounds 32 and 18 (Ki values of 70.8 and >1000 nM,
respectively). Modification of the aromatic group, in compounds
33 (Ki > 1000 nM) and 34 (Ki ) 174 nM), resulted in a
decreased affinity. Compound 35, corresponding to a constrained
analogue of 32, showed an affinity of the same magnitude (Ki
values of 60.2 nM and 70.8 nM for 35 and 32, respectively).
29
30
24.8 ( 3.4
14.1 ( 5.6
16.8 ( 4.1
157.4 ( 54
2.1 ( 1.1
126.0 ( 1.0
124.1 ( 0.8
128.7 ( 1.2
123.5 ( 1.5
21.6 ( 2.7
126.2 ( 1.5
145.6 ( 2.98
149.0 ( 3.2
135.1 ( 3.4
32R
35
SR-144528
WIN-55,212-2
CP-55,940
JWH-133
24.6 ( 1.7
6.1 ( 2.1
145.6 ( 3.0
4.1 ( 1.3
HU-210
a Emax results are expressed as the percentages of stimulation of [35S]-
GTPγS binding (basal value set at 100%). Data are the mean ( SEM of
three experiments performed in duplicate. The EC50 values were obtained
from nonlinear analysis of [35S]-GTPγS binding curves. Data are the mean
( SEM of at least three experiments performed in duplicate.
investigate structure modifications on quinolone nucleus either
by introducing various alkyl side chains on the N1 nitrogen or
by varying the substitution of the amide nitrogen.
A first set of nine compounds was synthesized (11-19).
These compounds encompassed an N1-alkyl side chain (butyl,
pentyl or hexyl) and a 4-methoxyphenyl, 1-naphthyl, or benzyl
carboxamido substituent. In these series, the 1-naphthyl deriva-
tives showed the highest affinity and, regardless of the nature
of the carboxamido substituent, the n-pentyl group proved to
be more potent than the n-butyl or n-hexyl. The highest hCB2
cannabinoid receptor affinity was found for compound 15 (Ki
) 371 nM). Therefore, to continue our investigation, we decided
to keep the n-pentyl residue constant and develop a library of
compounds with various 3-carboxamido substituents.
The enantiopure forms of 32-34, respectively noted 32-
34R and 32-34S, synthesized starting from the corresponding
enantiopure amine were also tested, as summarized in Table 2.
In all cases, the R enantiomers exhibited about 10-fold higher
affinity than the S enantiomers. The highest affinity was found
in the eutomer 32R (Ki ) 37.1 nM). The distomer 32S with an
affinity of 776 nM highlighted that this chiral ligand binds
The hCB2 receptor binding data (Table 2) revealed that the
affinity is quite sensitive to the 3-carboxamido group modifica-

New CB2 Cannabinoid Receptors Agonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 75
Using this receptor model, docking of the 3-carboxamido-
4-quinolone derivatives was performed, followed by energy
minimization. Nonselective CB2 ligands, WIN-55,212-2 and
CP-55,940, were used as template molecules, using the informa-
tion obtained in mutants receptors to delineate the binding site.
The resulting receptor-based alignments of WIN-55,212-2,
CP-55,940, and 30 are illustrated in Figure 1. These results
suggested that the 4-oxo-1,4-dihydroquinoline derivatives in-
teract in the same region of the CB2 receptor as WIN-55,212-2
and CP-55,940. However, the CB2 cannabinoid receptor residues
involved in the interactions with the ligands slightly differ
between WIN-55,212-2 and CP-55,940 and derivative 30.
Compound 30 was also docked in the cannabinoid CB2
cannabinoid receptor active site, as shown in Figure 2. Aromatic
residues are abundant in the region encompassed by transmem-
brane regions 3 to 5 (TM 3-TM 5), particularly near the
extracellular side of the cannabinoid receptor. The hydrophobic
N(1) alkyl side chain is perfectly positioned to interact with
hydrophobic residues Ile110, Cys175, Pro178, and Leu182. The
Figure 1. Receptor-based alignment results of WIN-55,212-2 (red),
CP-55,940 (cyan), and 30 (green).
stereoselectively to the hCB2 receptor. Introduction of this
pseudo-rigidification induced an improvement of affinity for
both of the two receptor subtypes.
amide oxygen atom in 30 forms a hydrogen bond with Ser193
The quinolone nucleus is in interaction with aromatic or
hydrophobic residues Pro168, Thr173, Leu182, Leu196, and Phe200
.
Superposition and Docking Studies. Bovine rhodopsin was
used for homology modeling of the CB2 cannabinoid receptor
as it is the only currently solved G-protein coupled receptor
tridimensional structure. However, its crystallographic structure
has been determined in a dark-adapted conformation which is
probably an inactive form. Therefore, besides pure homology
modeling, a more elaborated construction of the CB2 canna-
binoid receptor was required to approach its putative active
conformation. Previous works performed on the CB1 cannab-
inoid receptor have shown that transmembrane domains 3 and
6 (TM 3 and TM 6) should undergo a rotation around their
axis to render the effect of an agonist binding.51 We followed
these conclusions to build a model of CB2 cannabinoid receptor
able to bind the 3-carboxamido-4-quinolone agonists. TM3 was
rotated by 20° anticlockwise when seen from the extracellular
side. TM6 was also rotated until Cys257 pointed toward the
pocket, resulting in a 30° anticlockwise rotation. The geometry
of this final model was optimized until convergence to a 0.01
kcal/mol‚Å gradient with the backbone hydrogen bonds con-
strained and subsequently checked to verify its structural
validity. The ligands were then docked in a pocket defined as
a sphere of 15 Å around Lys98 using the Gold software.
.
The 1-(3,5-dimethyl)adamantyl carboxamido substituent fits well
in a pocket formed by various lipophilic or aromatic residues
Phe183, Pro184, Pro187, Phe197, and Leu269
.
Conclusion
In light of these results, this series of 4-oxo-1,4-dihydro-
quinoline-3-carboxamide derivatives represents a new class of
heterocyclic derivatives, acting as potent CB2-selective receptor
ligands. The [35S]-GTPγS binding data revealed the CB2
cannabinoid receptor agonistic properties of these derivatives.
Best affinity values were obtained with compounds 28-30,
bearing an adamantyl-carboxamido substituent. Compounds con-
taining aromatic moieties on the amide resulted in a decreased,
albeit notable, affinity for the hCB2 cannabinoid receptors, as
shown by 15, 22, and 27. The introduction of a steric constraint
induced a marked improvement in affinity. Moreover, interac-
tions of 32R, 33R, and 34R with the CB2 cannabinoid receptor
are highly stereoselective. Whatever the carboxamido sub-
stitutent, the preferred N1 side chain was the n-pentyl one.
Finally, molecular modeling of 30 obtained either by superim-
Figure 2. Compound 30 docked in the putative active site of the CB2 receptor. Hydrogen bond is colored in yellow.

76 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Stern et al.
adjusted to pH 4 with aqueous 10% hydrochloric acid. The resulting
precipitate was collected by filtration, washed with H2O, and
recrystallized from diisopropyl ether.
position or by docking indicated that these derivatives may share
to some extent the binding mode of WIN-55,212-2.
1-Butyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic Acid (7).
White solid (0.71 g, 70%); mp 165-166 °C; IR; 1H NMR (DMSO-
d6).
4-Oxo-1-pentyl-1,4-dihydro-quinoline-3-carboxylic Acid (8).
White solid (0.81 g, 76%); mp 135-137 °C; IR; 1H NMR (DMSO-
d6).
1-Hexyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic Acid (9).
White solid (0.67 g, 75%); mp 150-151 °C; IR; 1H NMR (DMSO-
d6).
1-Benzyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic Acid (10).
White solid (0.86 g, 75%); mp 175-176 °C; IR; 1H NMR (DMSO-
d6).
Experimental Section
Chemistry. All commercial reagents and solvents were used
without further purification. Analytical thin-layer chromatography
was performed on precoated Kieselgel 60F254 plates (Merck); the
spots were located by UV (254 and 366 nm) and/or with iodine;
Rf values are given for guidance. Silica gel 60 230-400 mesh
purchased from Merck was used for column chromatography.
Preparative thick-layer chromatography was performed using silica
gel from Merck, and the compounds were extracted from silica
gel by the following solvent system: CH2Cl2/MeOH 70:30. All
melting points were determinated with a Bu¨chi 535 capillary
1
appartus and remain uncorrected. H NMR spectra were obtained
General Procedure for the Preparation of N3-aryl-1-alkyl-
4-oxo-1,4-dihydroquinoline-3-carboxamide (11-36). To a solu-
tion of PybrOP (1.5 mmol) in 3 mL of dry DMF were added at
room temperature compounds 7-10 and diisopropylethylamine (3.0
mmol). The preswollen resin (0.75 g) in dry DMF was treated with
the above mixture at room temperature for 3 h, after which time
the resin was washed three times with dry DMF and three times
with dichloromethane. The same activation procedure was repeated
a second time. The appropriate amine (0.67 mmol) dissolved in
dry DMF was reacted with the polymer-bound activated ester for
24 h at room temperature. The supernatant was then separated from
the resin by filtration and the polymer beads washed three times
with dry DMF and three times with dichloromethane. The combined
solutions were concentrated and, the residue was purified either
by crystallization or preparative TLC.
N3-(4-Methoxyphenyl)-1-butyl-4-oxo-1,4-dihydroquinoline-3-
carboxamide (11). Recrystallization from diisopropyl ether, yellow
solid (57 mg, 24%); mp 129-130 °C; IR; 1H NMR (CDCl3); LC-
MS (APCI+) m/z 351 (MH+). Anal. (C21H22N2O3) C, H, N.
N3-(4-Methoxyphenyl)-4-oxo-1-pentyl-1,4-dihydroquinoline-
3-carboxamide (12). Recrystallization from diisopropyl ether,
yellow solid (34 mg, 14%); mp 134-135 °C; IR; 1H NMR (CDCl3);
LC-MS (APCI+) m/z 365 (MH+). Anal. (C22H24N2O3) C, H, N.
N3-(4-Methoxyphenyl)-1-hexyl-4-oxo-1,4-dihydroquinoline-
3-carboxamide (13). Recrystallization from diisopropyl ether,
yellow solid (130 mg, 51%); mp 119-120 °C; IR; 1H NMR
(CDCl3); LC-MS (APCI+) m/z 379 (MH+). Anal. (C23H26N2O3) C,
H, N.
using a Bru¨cker 300 MHz spectrometer, chemical shifts (δ) were
expressed in ppm relative to tetramethylsilane used as an internal
standard, J values are in hertz, and the splitting patterns were
designated as follows: s, singlet; d, doublet; t, triplet; m, multiplet.
IR spectra were determined with a Bru¨cker Vector 22 spectrometer
on a germanium crystal. APCI+ (atmospheric pressure chemical
ionization) mass spectra were obtained on an LC-MS system
Thermo Electron Surveyor MSQ. Optical rotations ([R]D) were
measured on a Perkin-Elmer 343 polarimeter. Specific rotations
are given as deg/dm; the concentration values are reported as g/mL
of the specified solvent and were recorded at 25 °C. Elemental
analyses were performed by the “Service Central d′Analyses” at
the CNRS, Vernaison (France).
2-Phenylaminomethylene-malonic Acid Diethyl Ester (1). A
mixture of 2-ethoxymethylene-malonic acid diethyl ester (9.30 mL,
46 mmol) and aniline (4.20 mL, 46 mmol) was heated at 100 °C
for 4 h. Petroleum ether (100 mL) was added, and the resulting
solution was cooled in an ice bath. The resulting precipitate was
collected by filtration, washed with petroleum ether, and recrystal-
lized from petroleum ether to provide 11.02 g (91%) of compound
1
1 as a white solid: mp 54-55 °C; IR; H NMR (CDCl3).
4-Oxo-1,4-dihydro-quinoline-3-carboxylic Acid Ethyl Ester
(2). Phenyl ether (40 mL) was heated under stirring at 240 °C. The
malonic acid diethyl ester (1) (9.50 g, 36 mmol) was slowly added,
and the resulting mixture was refluxed for 4 h. After the mixture
was cooled at room temperature, the resulting precipitate was
collected by filtration, washed with petroleum ether, and recrystal-
lized from DMF to provide 6.02 g (77%) of compound 2 as a white
N3-(1-Naphthyl)-1-butyl-4-oxo-1,4-dihydroquinoline-3-car-
boxamide (14). Recrystallization from ethyl acetate, white solid
1
solid: mp >250 °C; IR; H NMR (DMSO-d6).
1
General Procedure for the Preparation of 1-Alkyl-4-oxo-1,4-
dihydro-quinoline-3-carboxylic Acid Ethyl Ester (3-6). NaH
60% (0.22 g, 5.52 mmol) was added to a mixture of quinoline
carboxilic acid ethyl ester 2 (1.00 g, 4.60 mmol) and anhydrous
DMF (30 mL) under stirring at room temperature. The appropriate
alkyl bromide (5.52 mmol) was added, and the mixture was stirred
at 90 °C for 3 h (3-4) and 6 h (5-6). The solvent was removed
under reduced pressure, and the residue subsequently dissolved in
ethyl acetate. The precipitate was eliminated by filtration, and the
organic layer was concentrated under reduced pressure and finally
purified by flash chromatography, using dichloromethane/ethyl
acetate 5:5 as eluent.
(90 mg, 36%); mp 174-175 °C; IR; H NMR (CDCl3); LC-MS
(APCI+) m/z 371 (MH+). Anal. (C24H22N2O2) C, H, N.
N3-(1-Naphthyl)-4-oxo-1-pentyl-1,4-dihydroquinoline-3-car-
boxamide (15). Recrystallization from ethyl acetate, white solid
1
(184 mg, 71%); mp 184-185 °C; IR; H NMR (CDCl3); LC-MS
(APCI+) m/z 385 (MH+). Anal. (C25H24N2O2) C, H, N.
N3-(1-Naphthyl)-1-hexyl-4-oxo-1,4-dihydroquinoline-3-car-
boxamide (16). Recrystallization from ethyl acetate, white solid
1
(81 mg, 30%); mp 158-159 °C; IR; H NMR (CDCl3); LC-MS
(APCI+) m/z 399 (MH+). Anal. (C26H26N2O2) C, H, N.
N3-(Benzyl)-1-butyl-4-oxo-1,4-dihydroquinoline-3-carboxam-
ide (17). Recrystallization from n-heptane, white solid (70 mg,
1
31%); mp 165-166 °C; IR; H NMR (CDCl3); LC-MS (APCI+)
1-Butyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic Acid Ethyl
1
m/z 335 (MH+). Anal. (C21H22N2O2) C, H, N.
Ester (3). Orange oil (0.72 g, 57%); IR; H NMR (CDCl3).
4-Oxo-1-pentyl-1,4-dihydro-quinoline-3-carboxylic Acid Ethyl
N3-(Benzyl)-4-oxo-1-pentyl-1,4-dihydroquinoline-3-carboxa-
mide (18). Recrystallization from n-heptane, white solid (143 mg,
1
Ester (4). Orange oil (1.23 g, 93%); IR; H NMR (CDCl3).
1
61%); mp 170-171 °C; IR; H NMR (CDCl3); LC-MS (APCI+)
1-Hexyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic Acid Ethyl
1
m/z 349 (MH+). Anal. (C22H24N2O2) C, H, N.
Ester (5). Orange oil (0.69 g, 50%); IR; H NMR (CDCl3).
1-Benzyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic Acid Ethyl
Ester (6). Beige solid (1.06 g, 75%); mp 120-121 °C; IR; 1H NMR
(DMSO-d6).
General Procedure for the Preparation of 1-Alkyl-4-oxo-1,4-
dihydro-quinoline-3-carboxylic Acid (7-10). The appropriate
quinoline-3-carboxylic acid ethyl ester 3-6 (4.13 mmol) was
refluxed for 3 h in a mixture of aqueous 10% sodium hydroxyde
(5 mL) and ethyl alcohol (5 mL). After cooling, the solution was
N3-(Benzyl)-1-hexyl-4-oxo-1,4-dihydroquinoline-3-carboxa-
mide (19). Recrystallization from n-heptane, white solid (102 mg,
1
42%); mp 176-177 °C; IR; H NMR (CDCl3); LC-MS (APCI+)
m/z 363 (MH+). Anal. (C23H26N2O2) C, H, N.
N3-(4-Methoxybenzyl)-4-oxo-1-pentyl-1,4-dihydroquinoline-
3-carboxamide (20). Purified by TLC eluting from cyclohexane/
ethyle acetate 6:4, yellow oil (140 mg, 55%); IR; LC-MS (APCI+)
m/z 379 (MH+). Anal. (C23H26N2O3) C, H, N.

New CB2 Cannabinoid Receptors Agonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 77
N3-(2-Naphthyl)-4-oxo-1-pentyl-1,4-dihydroquinoline-3-car-
boxamide (21). Recrystallization from ethyl acetate, white solid
hexane/ethyl acetate 6:4, white oil (203 mg, 73%); [R]D25 ) - 151°,
1
c ) 0.01, CH2Cl2; IR; H NMR (CDCl3); LC-MS (APCI+) m/z
1
(129 mg, 50%); mp 162-163 °C; IR; H NMR (CDCl3); LC-MS
413 (MH+). Anal. (C27H28N2O2) C, H, N.
(APCI+) m/z 385 (MH+). Anal. (C25H24N2O2) C, H, N.
(S)-N3-(1-(2-Naphthyl)ethyl)-4-oxo-1-pentyl-1,4-dihydroquin-
oline-3-carboxamide (33S). Purified by TLC eluting from cyclo-
hexane/ethyl acetate 6:4, white oil (239 mg, 86%); [R]D25 ) + 151°,
N3-(Phenethyl)-4-oxo-1-pentyl-1,4-dihydroquinoline-3-car-
boxamide (22). Purified by TLC eluting from cyclohexane/ethyle
acetate 6:4, yellow oil (188 mg, 77%); IR; 1H NMR (CDCl3); LC-
MS (APCI+) m/z 363 (MH+). Anal. (C23H26N2O2) C, H, N.
N3-(3-Phenylpropyl)-4-oxo-1-pentyl-1,4-dihydroquinoline-3-
carboxamide (23). Purified by TLC eluting from dichloromethane/
methyl alcohol 95:5, white solid (162 mg, 64%); mp 78-79 °C;
1
c ) 0.01, CH2Cl2; IR; H NMR (CDCl3); LC-MS (APCI+) m/z
413 (MH+). Anal. (C27H28N2O2) C, H, N.
(RS)-N3-(1-(1-Naphthyl)ethyl)-4-oxo-1-pentyl-1,4-dihydro-
quinoline-3-carboxamide (34). Purified by TLC eluting from
25
cyclohexane/ethyl acetate 6:4, white oil (150 mg, 54%); [R]D
)
1
IR; H NMR (CDCl3); LC-MS (APCI+) m/z 377 (MH+). Anal.
0°, c ) 0.01, CH2Cl2; IR; 1H NMR (CDCl3); LC-MS (APCI+) m/z
(C24H28N2O2) C, H, N.
413 (MH+). Anal. (C27H28N2O2) C, H, N.
N3-(3,4-Dichlorophenyl)-4-oxo-1-pentyl-1,4-dihydroquinoline-
3-carboxamide (24). Purified by TLC eluting from cyclohexane/
ethyl acetate 6:4, white solid (201 mg, 74%); mp 175-176 °C;
(R)-N3-(1-(1-Naphthyl)ethyl)-4-oxo-1-pentyl-1,4-dihydroquin-
oline-3-carboxamide (34R). Purified by TLC eluting from cyclo-
hexane/ethyl acetate 6:4, white oil (228 mg, 82%); [R]D25 ) - 200°,
1
1
IR; H NMR (CDCl3); LC-MS (APCI+) m/z 404 (MH+). Anal.
c ) 0.01, CH2Cl2; IR; H NMR (CDCl3); LC-MS (APCI+) m/z
(C21H20N2O2Cl2) C, H, N.
413 (MH+). Anal. (C27H28N2O2) C, H, N.
N3-(4-Cyanophenyl)-4-oxo-1-pentyl-1,4-dihydroquinoline-3-
carboxamide (25). Purified by TLC eluting from cyclohexane/ethyl
(S)-N3-(1-(1-Naphthyl)ethyl)-4-oxo-1-pentyl-1,4-dihydroquin-
oline-3-carboxamide (34S). Purified by TLC eluting from cyclo-
hexane/ethyl acetate 6:4, white oil (217 mg, 78%); [R]D25 ) + 200°,
1
acetate 6:4, white solid (111 mg, 46%); mp 119-120°C; IR; H
NMR (CDCl3); LC-MS (APCI+) m/z 360 (MH+). Anal. (C22H21N3O2)
C, H, N.
1
c ) 0.01, CH2Cl2; IR; H NMR (CDCl3); LC-MS (APCI+) m/z
413 (MH+). Anal. (C27H28N2O2) C, H, N.
N3-(4-Biphenyl)-4-oxo-1-pentyl-1,4-dihydroquinoline-3-car-
boxamide (26). Purified by TLC eluting from cyclohexane/ethyl
(RS)-N3-(1-(1,2,3,4-Tetrahydronaphthyl))-4-oxo-1-pentyl-1,4-
dihydroquinoline-3-carboxamide (35). Purified by TLC eluting
1
25
acetate 6:4, white solid (108 mg, 39%); mp 153-154°C; IR; H
from cyclohexane/ethyl acetate 7:3, white oil (143 mg, 55%); [R]D
NMR (CDCl3); LC-MS (APCI+) m/z 411 (MH+). Anal. (C27H26N2O2)
C, H, N.
1
) 0°, c ) 0.01, CH2Cl2; IR; H NMR (CDCl3); LC-MS (APCI+)
m/z 389 (MH+). Anal. (C25H28N2O2) C, H, N.
N3-(2-(Benzo[1,3]dioxol-5-yl)ethyl)-4-oxo-1-pentyl-1,4-dihy-
droquinoline-3-carboxamide (27). Purified by TLC eluting from
dichloromethane/methyl alcohol 95:5, white solid (121 mg, 44%);
3-(Morpholino-4-carbonyl)-1-pentyl-1,4-dihydroquinolin-4-
one (36). Purified by TLC eluting from n-heptane/ethyl acetate 6:4,
white oil (177 mg, 80%); IR; 1H NMR (DMSO-d6); LC-MS
(APCI+) m/z 329 (MH+). Anal. (C25H32N2O2) C, H, N.
Pharmacology. Fatty acid free bovine serum albumin (BSA)
was purchased from Sigma Chemical Co (St. Louis, MO). WIN-
55,21-2 was purchased from RBI (Natick, MA), HU-210 and CP-
55,940 were acquired from Tocris (Bristol, U.K.). SR-141716A and
SR-144528 were kindly donated by Sanofi Recherche (Montpellier,
France).
Cell Culture and Preparation of hCB1- or hCB2-Transfected
CHO Cell Membranes. CHO cells stably transfected with the
cDNA sequences encoding either the human CB1 or the human
CB2 cannabinoid receptors were kindly donated by Dr. M. Detheux
and Dr. P. Nokin, respectively (Euroscreen s.a., Gosselies, Belgium).
Cells were grown in Ham’s F12 nutrient mixture supplemented with
10% FBS, 2.5 µL/mL fungizone, 100 U/mL penicillin, 100 µg/mL
streptomycin, and 400 µg/mL G418. Once at confluence, the cells
were trypsinized and collected by centrifugation at 100g for 10
min. The following steps were performed on ice. The pellet was
lysed in ice-cold 50 mM Tris-HCl, pH 7.4, and the homogenate
was centrifuged at 15 000g for 10 min. The resulting pellet
(membranes) was washed twice with the same solution under
identical conditions. The protein content was determined as
described by Bradford52 using Coomasie Blue (Biorad, Belgium)
with bovine serum albumin as standard.
1
mp 85-86°C; IR; H NMR (CDCl3); LC-MS (APCI+) m/z 407
(MH+). Anal. (C24H26N2O4) C, H, N.
N3-(1-Adamantyl)-4-oxo-1-pentyl-1,4-dihydroquinoline-3-car-
boxamide (28). Recrystallization from n-heptane, white solid (144
mg, 55%); mp 184-185 °C; IR; 1H NMR (CDCl3); LC-MS
(APCI+) m/z 393 (MH+). Anal. (C25H32N2O2) C, H, N.
N3-(2-Adamantyl)-4-oxo-1-pentyl-1,4-dihydroquinoline-3-car-
boxamide (29). Recrystallization from n-heptane, white solid (118
mg, 45%); mp 180-181 °C; IR; 1H NMR (CDCl3); LC-MS
(APCI+) m/z 393 (MH+). Anal. (C25H32N2O2) C, H, N.
N3-(1-(3,5-Dimethyl)adamantyl)-4-oxo-1-pentyl-1,4-dihydro-
quinoline-3-carboxamide (30). Recrystallization from n-heptane,
white solid (190 mg, 67%); mp 164-165 °C; IR; 1H NMR (CDCl3);
LC-MS (APCI+) m/z 421 (MH+). Anal. (C27H36N2O2) C, H, N.
N3-(1-(3,5-Dimethyl)adamantyl)-4-oxo-1-benzyl-1,4-dihydro-
quinoline-3-carboxamide (31). Recrystallization from n-heptane,
white solid (221 mg, 70%); mp 174-175 °C; IR; 1H NMR (CDCl3);
LC-MS (APCI+) m/z 441 (MH+). Anal. (C29H32N2O2) C, H, N.
(RS)-N3-(1-Phenylethyl)-4-oxo-1-pentyl-1,4-dihydroquinoline-
3-carboxamide (32). Purified by TLC eluting from cyclohexane/
ethyl acetate 6:4, white oil (188 mg, 77%); [R]D25 ) 0°, c ) 0.01,
1
CH2Cl2; IR; H NMR (CDCl3); LC-MS (APCI+) m/z 363 (MH+).
Anal. (C23H26N2O2) C, H, N.
(R)-N3-(1-Phenylethyl)-4-oxo-1-pentyl-1,4-dihydroquinoline-
3-carboxamide (32R). Purified by TLC eluting from cyclohexane/
Competition Binding Assay. [3H]-SR-141716A (52 Ci/mol) was
purchased from Amersham (Roosendaal, The Netherlands) and
[3H]-CP-55,940 (101 Ci/mol) from NEN Life Science (Zaventem,
Belgium). Glass fiber filters were purchased from Whatman
(Maidstone, U.K.), while Aqualuma was from PerkinElmer (Schaes-
berg, The Netherlands). Stock solutions of the compounds were
prepared in DMSO and further diluted (100×) with the binding
buffer to the desired concentration. Final DMSO concentrations in
the assay were less than 0.1%.
25
ethyl acetate 6:4, white oil (237 mg, 97%); [R]D ) - 95°, c )
1
0.01, CH2Cl2; IR; H NMR (CDCl3); LC-MS (APCI+) m/z 363
(MH+). Anal. (C23H26N2O2) C, H, N.
(S)-N3-(1-Phenylethyl)-4-oxo-1-pentyl-1,4-dihydroquinoline-
3-carboxamide (32S). Purified by TLC eluting from cyclohexane/
25
ethyl acetate 6:4, white oil (234 mg, 96%); [R]D ) + 95°, c )
1
0.01, CH2Cl2; IR; H NMR (CDCl3); LC-MS (APCI+) m/z 363
(MH+). Anal. (C23H26N2O2) C, H, N.
Under these conditions, using [3H]-SR-141716A, the Bmax value
was 57 pmol/mg protein and the Kd value was 1.13 (0.13 nM) for
the hCB1 cannabinoid receptor. Using [3H]-CP-55,940, the Bmax
value was 57 pmol/mg protein and the Kd value was 4.3 (0.13 nM)
for the hCB2 cannabinoid receptor.
(RS)-N3-(1-(2-Naphthyl)ethyl)-4-oxo-1-pentyl-1,4-dihydro-
quinoline-3-carboxamide (33). Purified by TLC eluting from
25
cyclohexane/ethyl acetate 6:4, white oil (220 mg, 79%); [R]D
)
0°, c ) 0.01, CH2Cl2; IR; 1H NMR (CDCl3); LC-MS (APCI+) m/z
413 (MH+). Anal. (C27H28N2O2) C, H, N.
The competitive binding experiments were performed using [3H]-
SR-141716A (1 nM) or [3H]-CP-55,940 (1 nM) as radioligands
for the hCB1 and the hCB2 cannabinoid receptor, respectively, at
(R)-N3-(1-(2-Naphthyl)ethyl)-4-oxo-1-pentyl-1,4-dihydroquin-
oline-3-carboxamide (33R). Purified by TLC eluting from cyclo-

78 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Stern et al.
30 °C in plastic tubes, and 40 µg of membranes per tube
resuspended in 0.5 mL (final volume) of binding buffer (50 mM
Tris-HCl, 3 mM MgCl2, 1 mM EDTA, 0.5% bovine serum
albumine, pH 7.4). The test compounds were present at varying
concentrations, and the nonspecific binding was determined in the
presence of 10 µM HU-210. After 1 h the incubation was stopped,
and the solutions were rapidly filtered through 0.5% PEI pretreated
GF/B glass fiber filters on a M-48T Brandell cell harvester and
washed twice with 5 mL ice-cold binding buffer without serum
albumin. The radioactivity on the filters was measured in a
Pharmacia Wallac 1410 â-counter using 10 mL of Aqualuma, after
10 s shaking and 3 h resting. Assays were performed at least in
triplicate.
[35S]-GTPγS Assays. [35S]-GTPγS (1173 Ci/mmol) was pur-
chased from Amersham (Roosendaal, The Netherlands). The
binding experiments were performed at 30 °C in plastic tubes
containing 40 µg of protein in 0.5 mL (final volume) of binding
buffer (50 mM Tris-HCl, 3 mM MgCl2, 1 mM EDTA, 100 mM
NaCl, 0.1% bovine serum albumin, pH 7.4) supplemented with 20
µM GDP. The test compounds were present at varying concentra-
tions, and the nonspecific binding was measured in the presence
of 100 µM Gpp(NH)p. The assay was initiated by the addition of
[35S]-GTPγS (0.05 nM, final concentration). The tubes were
incubated for 1 h. The incubations were terminated by the addition
of 5 mL ice-cold washing buffer (50 mM Tris-HCl, 3 mM MgCl2,
1 mM EDTA, 100mM NaCl). The suspension was immediately
filtered through GF/B filters using a 48-well Brandell cell harvester
and washed twice with the same ice-cold buffer. The radioactivity
on the filters was counted as mentioned above. Assays were
performed in triplicate.
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1
Supporting Information Available: 2D H NMR (ROESY)
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