Rational design, synthesis and anti-proliferative properties of new CB2 selective cannabinoid receptor ligands: An investigation of the 1,8-naphthyridin-2(1H)-one scaffold

Article abstract of DOI:10.1016/j.ejmech.2012.03.031

CB2 receptor ligands are becoming increasingly attractive drugs due to the potential role of this receptor in several physiopathological processes. Using our previously described series of 1,8-naphthyridin-2(1H)-on-3-carboxamides as a lead class, several nitrogen heterocyclic derivatives, characterized by different central cores, were synthesized and tested for their affinity toward the human CB1 and CB2 cannabinoid receptors. The obtained results suggest that the new series of quinolin-2(1H)-on-3-carboxamides, 4-hydroxy-2-oxo-1,2-dihydro- 1,8-naphthyridine-3-carboxamides and 1,2-dihydro-2-oxopyridine-3-carboxamides represent novel scaffolds very suitable for the development of promising CB2 ligands. Furthermore, the newly synthesized CB2 ligands inhibit proliferation of several cancer cell lines. In particular, it was demonstrated that in DU-145 cell line these ligands exert a CB2-mediated anti-proliferative action and decrease the CB2 receptor expression levels.

Full text of DOI:10.1016/j.ejmech.2012.03.031

European Journal of Medicinal Chemistry 52 (2012) 284e294
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Rational design, synthesis and anti-proliferative properties of new CB2 selective
cannabinoid receptor ligands: An investigation of the 1,8-naphthyridin-2(1H)-one
scaffold
,
a
b
a
Clementina Manera a , Giuseppe Saccomanni , Anna Maria Malfitano , Simone Bertini ,
*
Francesca Castelli a, Chiara Laezza c, Alessia Ligresti d, Valentina Lucchesi a, Tiziano Tuccinardi a
,
,
e
Flavio Rizzolio e g, Maurizio Bifulco b, Vincenzo Di Marzo b, Antonio Giordano e f, Marco Macchia a,
,
,
Adriano Martinelli a
a Dipartimento di Scienze Farmaceutiche, Università di Pisa, via Bonanno 6, 56126 Pisa, Italy
b Dipartimento di Scienze Farmaceutiche e biomediche, Università di Salerno, 84084 Fisciano (SA), Italy
c Institute of Endocrinology and Experimental Oncology, IEOS, CNR, Naples, Italy
d Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy
e Department of Biology, Sbarro Institute for Cancer Research and Molecular Medicines, Centre for Biotechnology, College of Science and Technology, Temple University, Philadelphia,
Pennsylvania 19122, USA
f Program in Genetic Oncology, Department of Human Pathology and Oncology, University of Siena, Siena, Italy
g Experimental and Clinical Pharmacology, Centro di Riferimento Oncologico, IRCCS, Aviano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
CB2 receptor ligands are becoming increasingly attractive drugs due to the potential role of this receptor
in several physiopathological processes. Using our previously described series of 1,8-naphthyridin-2(1H)-
on-3-carboxamides as a lead class, several nitrogen heterocyclic derivatives, characterized by different
central cores, were synthesized and tested for their affinity toward the human CB1 and CB2
cannabinoid receptors. The obtained results suggest that the new series of quinolin-2(1H)-on-3-
carboxamides, 4-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides and 1,2-dihydro-2-
oxopyridine-3-carboxamides represent novel scaffolds very suitable for the development of promising
CB2 ligands. Furthermore, the newly synthesized CB2 ligands inhibit proliferation of several cancer cell
lines. In particular, it was demonstrated that in DU-145 cell line these ligands exert a CB2-mediated anti-
proliferative action and decrease the CB2 receptor expression levels.
Received 11 November 2011
Received in revised form
13 March 2012
Accepted 15 March 2012
Available online 24 March 2012
Keywords:
Cannabinoid
CB1 receptor
CB2 receptor
Anti-proliferative
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
mammalian tissues and cloned. CB1R mediates the psychotropic
actions of cannabis. It is mainly expressed in neurons of the CNS
Over the past 15 years a considerable number of studies have
been conducted in order to understand the biological role of the
endocannabinoid system (ECS) and its regulatory functions in
health and disease. The ECS consists of the G-protein coupled
cannabinoid receptors; the endogenous ligands anandamide (AEA)
and 2-arachidonoylglycerol (2-AG); and the enzymes responsible
for endocannabinoid biosynthesis, cellular uptake and metabolism
[1e3].
[4], and in dorsal root ganglion neurons [5], but there is also
evidence for the expression of CB1R in non-neural tissues [6].
CB1R inhibits ongoing release of various neurotransmitters such
as acetylcholine, noradrenaline, dopamine, 5-hydroxytryptamine,
g-aminobutyric acid and glutamate [7]. CB2R was originally
described as being restricted to cells of immune origin [3,8].
However, recent evidence points to a neuronal localization in
some regions of the brain [9], and CB2R immunoreactivity has
also been shown in activated microglia in affected regions of
multiple sclerosis and amyotrophic lateral sclerosis post mortem
human spinal cord [10]. Furthermore, various studies have shown
that the activation of CB2R could block activation of microglia
cells, but has little effect on the normal functioning of neurons
within the CNS. Therefore, CB2R agonists may be potential
To date, two distinct cannabinoid receptors, CB1 receptor
(CB1R) and CB2 receptor (CB2R), have been identified in
* Corresponding author. Tel.: þ39 0 502219548; fax: þ39 0 502219605.
E-mail address: manera@farm.unipi.it (C. Manera).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.031

C. Manera et al. / European Journal of Medicinal Chemistry 52 (2012) 284e294
285
alternatives to immunosuppressants currently used during neu-
roinflammation [11].
central core would also need to be connected with a lipophilic
substituent in position 1 with an H-bond acceptor atom, capable of
interacting in the CB2R with the nonconserved S3.31(112). Some of
the new tested compounds exhibited a subnanomolar CB2R affinity
and a high selectivity over CB1R. Furthermore, the concentration-
dependent inhibitory action on human basophils activation and
the concentration-dependent decrease of cell viability in Jurkat
cells shown by one of these derivatives strongly suggest that these
compounds possess agonist properties at CB2R.
In the present study we evaluated the effects on the CBRs’
affinity of the lipophilic central core properties. For this reason,
using compounds A as a lead class, the derivatives of general
structure BeF, characterized by different central cores, were
synthesized and tested for their affinity toward the human CB1R
and CB2R (see Fig. 1).
On the basis of our previous modeling studies [33,34], these new
scaffolds should correctly orient the nonaromatic carboxamide
group in position 3 capable of interacting in the CB2R with the
nonconserved residue F5.46(197) and the lipophilic substituent in
position 1 with the H-bond acceptor atom capable of interacting
with S3.31(112). As shown in Fig. 2, all the derivatives possesses an
aromatic feature and are able to orient the two substituents in the
hypothesized way.
Finally, the newly synthesized CB2R ligands, showing the best
binding profiles in the series, were also evaluated in vitro for their
anti-proliferative activity against a large panel of human tumor-
derived cell lines selected on the basis of previously published
observation [35,36].
CB1R/CB2R agonists can of course produce adverse effects in
patients, and many of these are probably caused by the activation of
central CB1R, rather than of CB2R or peripheral CB1R. Several
experimental data suggest that an interesting strategy for circum-
venting the unwanted consequences of cannabinoid CB1R activa-
tion, may be to target CB2R [12] The potential therapeutic targets
for CB2R-selective agonists include neuropathic and inflammatory
pain [13,14] multiple sclerosis [15,16], amyotrophic lateral sclerosis
[17,18], Huntington’s disease [19], stroke [20], atherosclerosis [21],
gastrointestinal inflammatory states [22,23], chronic liver diseases
[23,24], and cancer [25]. Numerous pharmacological studies have
shown that CB2R agonists might directly inhibit tumor growth
in vitro and in animal models such as xenograft tumors chemically
or genetically induced in mice. The responsible mechanisms may
involve cytotoxic or cytostatic effects, apoptosis induction, anti-
metastatic effect accompanied by inhibition of neo-angiogenesis,
and tumor cell migration [26]. Therefore, the development of
CB2R-selective agonists for the treatment of tumors represent an
efficacious e and increasingly intriguing e alternative. These
agents seem to discern between tumor cells and their non-
transformed counterparts, displaying a tumor-selectivity not seen
in common cytotoxic agents. The potential adverse effects are
acceptable and lie well within the range of those induced by
common anti-tumor drugs. Moreover, CB2R-selective agonists
could be used in combination with other chemotherapeutic drugs
to reduce dosages and exert a more potent clinical impact [27,28].
Finally, there is evidence that CB2R expression is higher in some
human cancer cells than in normal cells [29], and this would justify
the development of a diagnostic test based on CB2R levels alone or
in association with other recognized prognostics.
2. Chemistry
The synthesis of the new compounds BeF was accomplished as
depicted in Schemes 1e4. As shown in Scheme 1, the reaction of
2-nitrobenzaldehyde and diethylmalonate in acetic anhydride with
NaHCO3 at 110 ꢀC afforded the 2-nitrobenzylidene derivative 1 [37].
Reduction of 1 by iron powder in acetic acid yielded the ethyl
quinolin-2(1H)-on-3-carboxylate 2. Heating of 2 with cyclohexyl-
amine or cycloheptylamine at 120 ꢀC in a sealed tube provided the
corresponding carboxamide derivatives 3 and 4. N1-alkylation of
these carboxamides in anhydrous DMF with benzylchloride or
p-fluorobenzylchloride or 4-(2-chloroethyl)morpholine in the
presence of NaH at room temperature afforded the desired quino-
lin-2-one derivatives B1eB5. When the reaction to obtain the
compounds B2 was carried out at 70 ꢀC, the 2-(p-fluorobenzyloxy)-
quinoline derivative C1 was also isolated.
In a research program designed with the goal of obtaining CB2R-
selective ligands [30e33], and on the basis of docking studies
developed using three-dimensional models of the two CBRs [34],
we recently described the synthesis, pharmacological character-
ization and molecular modeling studies of
a series of 1,8-
naphthyridin-2(1H)-on-3-carboxamides of general structure
A
(see Fig. 1) [33]. The docking analysis suggested that the preser-
vation of a good CB2/CB1 selectivity and the improvement of the
CB2R affinity required a lipophilic central core connected with
a nonaromatic carboxamide group in position 3 capable of inter-
acting in the CB2R with the nonconserved residue F5.46(197). The
O
The
synthesis
of
4-hydroxy-2-oxo-1,2-dihydro-1,8-
NHR2
naphthyridine-3-carboxamide derivatives D1eD5 is outlined in
Scheme 2. Esterification of 2-aminonicotinic acid with concentrated
H2SO4 in MeOH afforded the methyl 2-amino-3-pyridinecarboxylate
(5) [38]. Heating of 5 with EtONa and diethyl malonate, in anhydrous
ethanol at reflux yielded the 4-hydroxy-2-oxo-1,8-naphthyridine
derivative 6 [39]. Reaction of 6 with cyclohexylamine or cyclo-
heptylamine provided the corresponding carboxamide derivatives 7
and 8, respectively, which, as above reported for the preparation of
compounds B, by treatment with NaH and then with the benzyl-
chloride or p-fluorobenzylchloride or 4-(2-chloroethyl)-morpho-
line, gave the desired compounds D1eD5.
N
R1
O
O
O
B
O
O
NHR2
NHR2
NHR2
N
O
N
N
N
O
R1
R1
R1
F
A
C
Synthetic routes of 2-oxopyridine derivatives E1eE5 and
2-substituted pyridines F1eF3 are outlined in Schemes 3 and 4.
Reaction of 2-hydroxynicotinic acid with concentrated H2SO4 and
MeOH at 90 ꢀC for 60 min in microwave gave the methyl ester 9 [40],
which was heated with the appropriate amine at 150 ꢀC to give the
carboxamide derivatives 10 and 11 (Scheme 3). Treatment of car-
boxamide derivatives 10 and 11 in anhydrous DMF with NaH
and then with the benzylchloride or p-fluorobenzylchloride or
O
O
O
O
OH
NHR2
NHR2
N
N
R1
N
R1
D
E
Fig. 1. Schematic representation of the analyzed scaffolds.

286
C. Manera et al. / European Journal of Medicinal Chemistry 52 (2012) 284e294
Fig. 2. Pharmacophoric superimposition of the analyzed scaffolds. The cyclohexyl (R1) and ethylmorpholino (R2) groups were used as reference substituents.
4-(2-chloroethyl)-morpholine afforded the 2-oxopyridine deriva-
tives E1eE5. In the case of the reaction with benzylchloride or
O
CHO
NO2
COOEt
COOEt
OEt
ii
i
p-fluorobenzylchloride the 2-benzyloxypyridine derivative F1 or
2-(p-fluorobenzyloxy)pyridine derivatives F2 and F3 were also iso-
lated (Scheme 3). However the compounds F1eF3 were obtained
in very low yield, so these compounds were obtained by
the alternative synthetic method as outlined in Scheme 4.
Reaction of 2-chloronicotinic acid with SOCl2 at 90 ꢀC gave the
2-chloronicotinoyl chloride which, without purification, was treated
with triethylamine and the cyclohexylamine or cycloheptylamine
in toluene to afford the corresponding 2-chloropyridine-3-
carboxamide derivatives 12 and 13, respectively. These compounds
were treated with benzyl alcohol or p-fluorobenzyl alcohol, potas-
sium t-butoxide and tricaprylylmethylammonium chloride (Aliquat
336) at 120 ꢀC for 2 h to give the desired pyridine derivatives F1eF3.
N
H
O
NO2
2
1
iii
O
O
NHR1
N
H
3, 4
iv
O
O
O
NHR1
N
H
N
O
N
R2
B1-B5
C1
3. Results and discussion
F
3.1. CB1 and CB2 receptor affinity
Scheme 1. Synthesis of N1-substituted quinolin-2(1H)-on-3-carboxamide derivatives
B1eB5.
The binding affinities (Ki values) of compounds BeF was eval-
uated in competitive binding assays against [3H]CP-55,940 toward
OH
O
O
O
O
COOCH3
COOH
NH2
ii
OCH2CH3
i
N
NH2
N
N
N
H
5
6
iii
OH
NHR1
N
7, 8
N
H
iv
OH
O
O
NHR1
N
N
R2
D1-D5
Scheme 2. Synthesis of N1-substituted 4-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide derivatives D1eD5.

C. Manera et al. / European Journal of Medicinal Chemistry 52 (2012) 284e294
287
Table 1
O
O
Radioligand binding data of compounds BeF.a
OH
OH
OCH3
OH
i
Ki (nM)
N
N
ii
9
Compd
R2
R1
CB1b
CB2c
SId
A133
A233
A333
A433
A533
B1
B2
B3
B4
B5
D1
D2
D3
D4
D5
E1
E2
E3
E4
E5
C1
F1
F2
F3
SR144528
JWH133
Benzyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cycloheptyl
Cycloheptyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cycloheptyl
Cycloheptyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cycloheptyl
Cycloheptyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cycloheptyl
Cycloheptyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cycloheptyl
560
56
560
9.6
18
67
16.5
300
11.6
206
560
NT
64.6
NT
234
800
70
31
18
25
71
14
6
4.2
5.7
33
48
103
7.1
e
O
p-Fluorobenzyl
Ethylmorpholino
p-Fluorobenzyl
Ethylmorpholino
Benzyl
p-Fluorobenzyl
Ethylmorpholino
p-Fluorobenzyl
Ethylmorpholino
Benzyl
p-Fluorobenzyl
Ethylmorpholino
p-Fluorobenzyl
Ethylmorpholino
Benzyl
p-Fluorobenzyl
Ethylmorpholino
p-Fluorobenzyl
Ethylmorpholino
p-Fluorobenzyl
Benzyl
2.2
7.9
0.7
3
NHR1
OH
N
iii
10, 11
16
2.9
9.0
0.25
2.0
79
NT
2.4
NT
1.8
200
20
7900
7.8
790
130
916
250
25
O
O
O
NHR1
NHR1
N
N
O
R2
R2
F1-F3
E1-E5
26.6
e
Scheme 3. Synthesis of N1-substituted 1,2-dihydro-2-oxo-pyridine-3-carboxamide
derivatives E1eE5.
130
4
3.5
e
>10,000
43
both human recombinant CB1R and CB2R expressed in CHO cells as
previously described [33]. The results are reported in Table 1 with
the Ki values of the previously reported 1,8-naphthyridin-2(1H)-on-
3-carboxamide derivatives A1eA5 [33]. The Ki values of SR144528
[41] and JWH133 [42] as reference compounds are also included in
Table 1.
The low solubility of D2 and D4 hindered their biological testing
in DMSO/water solution during the cannabinoid receptor binding
assay.
5.5
1.2
9.2
e
950
1200
>10,000
1811
100
121.2
116.8
p-Fluorobenzyl
p-Fluorobenzyl
7.3
4
4.35
1.82
a
Data represent mean values for at least three separate experiments performed
in duplicate and are expressed as Ki (nM), for CB1 and CB2 binding assays. Standard
error of means (SEM) are not shown for the sake of clarity and were never higher
than 5% of the means.
Our previous studies showed that 1,8-naphthyridin-2(1H)-on-
3-carboxamide derivatives A constitute a suitable template in the
design of potent and selective CB2R ligands [33]. In order to eval-
uate the effects on the CBRs’ affinity determined by the presence of
different central scaffolds, the 1,8-naphthyridin-2(1H)-one ring
system of derivatives A was replaced. The first step of our plan was
to investigate the influence for binding to the CBRs of the N atom in
position 8 of the 1,8-naphthyridine nucleus of compounds A
through the synthesis of quinolin-2(1H)-on-3-carboxamide deriv-
atives B1eB5. The aliphatic carboxamide groups in position 3 and
the substituents in position 1 have been selected on the basis of the
binding results obtained to CB2R for the derivatives A [33]. With
regard to the CB1R affinity, the results reported in Table 1 showed
that the quinolin-2(1H)-on-3-carboxamide derivatives B1eB5 have
a variable affinity with Ki values in the range of 300 nM (B3) to
11.6 nM (B4). Concerning the CB2R, the new compounds B1eB5
proved to be high-affinity CB2R ligands with Ki values ranging
from 16 nM (B2) to 0.25 nM (B4). Furthermore, in an agreement
with a previous report concerning derivatives A [33], the binding
data revealed that the compounds with a p-fluorobenzyl group in
position 1 (B2 and B4) exhibited CB2R affinity higher than that of
the corresponding ethylmorpholino derivatives (B3 and B5). In
addition, the presence of a fluorine atom para to the benzyl group in
position 1 of the quinoline nucleus (B2) increased affinity with
b
NT ¼ not tested, as it is insoluble in the solvent normally used in binding assays.
Affinity of compounds for CB1R was evaluated using membranes from HEK-293
cells transfected and [3H]CP55,940.
c
Affinity of compounds for CB2R was evaluated using membranes from HEK-293
cells transfected and [3H]CP-55,940.
d
SI: selectivity index for CB2R calculated as Ki(CB1)/Ki(CB2) ratio.
respect to the benzyl group (B1). Finally, the p-fluorobenzyl quin-
oline derivative B4 proved to be the compound with the highest
affinity in this series, with CB2R Ki values of 0.25 nM. These results
suggest that the N atom in position 8 of the 1,8-naphthyridin-
2(1H)-on-3-carboxamide derivatives (A) played a secondary role in
the binding to CB2R. In fact, the comparison of the Ki values of
compounds B with those of the corresponding compounds A shows
that the CB2R affinity of the two series are very similar (see Table 1).
This conclusion is in agreement with the results recently reported
by Pasquini et al. [43]. In fact these authors described the phar-
macological properties of several quinolone derivatives including
a quinolin-2-one derivative which possess CB2R affinity and
selectivity comparable to that of compound D5.
The second step in our strategy was to study the effect of
the introduction of
a hydroxy group in position 4 of the
1,8-naphthyridine nucleus of compounds A. For this reason,
4-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide
derivatives of general structure D were synthesized. The aliphatic
carboxamide groups in position 3 and the substituents in position 1
are the same as those of compounds A and B. Unfortunately, these
compounds have low solubility, so the analysis of the results was
limited. However, for CB1R, compound D3 showed the highest
affinity, with Ki value of 64.6 nM. The other compounds showed
lower affinity, with Ki value > 200 nM. With regard to CB2R, the
derivatives D5 and D3 were found to possess a remarkable affinity,
with Ki values of 1.8 nM and 2.4 nM, respectively.
O
O
Cl
O
NHR1
NHR1
OH
i, ii
N
Cl
N
12, 13
iii
N
O
We next decided to verify the importance of the lipophilic
interactions of the 1,8-naphthyridine nucleus of compounds A on
affinity to the CB2R, replacing it with a monocyclic nucleus. For this
R2
F1-F3
Scheme 4. Synthesis of 2-substituted pyridine-3-carboxamide derivatives F1eF3.

288
C. Manera et al. / European Journal of Medicinal Chemistry 52 (2012) 284e294
reason, the synthesis of 1,2-dihydro-2-oxopyridine-3-carboxamide
derivatives of general structure was performed. These
3.2. Analysis of compounds D
E
compounds were characterized in positions 1 and 3 of the pyridine
nucleus by the same substituents of the compounds A, B and D. The
binding data reported in Table 1 indicated that with regard to CB1R,
compounds E possess a high variability of affinity with Ki values in
the range of >10000 nM (E3) to 43 nM (E4). Also, in the case of
CB2R, these compounds showed a high variability of affinity, with
Ki values in the range of 7900 nM (E3) to 7.8 nM (E4). Furthermore,
the binding data revealed that for this series the presence of a p-
fluorine atom on the benzyl group in position 1 increased affinity,
as shown by the comparison between Ki values of the p-fluo-
robenzyl derivative E2 (Ki ¼ 20 nM) and Ki values of the benzyl
derivative E1 (Ki ¼ 200 nM). From these data it emerged that the
reduction of 1,8-naphthyridine nucleus to pyridine nucleus deter-
mined an overall reduction of the CB2R affinity, supporting the
importance of the lipophilic interaction of the second aromatic
ring.
This class of compounds can form different intramolecular H
bonds. In order to define the preferred tautomer, the three main
forms were submitted to quantum mechanics calculations. These
studies suggested that tautomer 1 is preferred, as it proved to be
about 5 and 15 kcal/mol more stable than 2 and 3, respectively (see
Table 2).
3.3. Cell proliferation assay
The newly synthesized CB2R ligands showing the best binding
profiles in the series were evaluated in vitro for their ability to
reduce cell proliferation on a collection of tumoral cell lines (human
breast carcinoma cells MCF-7, human prostate carcinoma cells DU-
145, human gastric adenocarcinoma cells AGS, glioblastoma cells
T98G). A 1 mM fixed concentration dose of compounds B5, D5 and
Finally, we examined the effect on the CBRs’ affinity of the
shifting of the substituent from the N1 to the oxygen in position 2 of
the heterocyclic nucleus for compounds of general structure B and
E. For this aim, the N-cyclohexyl-2-(p-fluorobenzyloxy)-quinoline-
3-carboxamide C1 and the N-cyclohexyl and N-cyclohepthyl-2-
substituted-pyridine-3-carboxamide derivatives F1eF3 were
studied. The binding data obtained showed that all these
compounds possess moderate affinity for the two subtypes of CBRs,
although the affinity is slightly higher for CB2R.
Overall, the reported data suggest that, with regard to CB2R
selectivity, the 2-oxopyridine derivatives E and O-substituted
compounds C1 and F1eF3 showed a low selectivity. Derivatives B
and D, on the other hand, showed a good selectivity, in particular
compounds B5 and D5, which exhibited significant selectivity, with
Ki(CB1)/Ki(CB2) ratios of 103 and 130, respectively. These values are
greater than that of the corresponding compound A5, which
possesses a Ki(CB1)/Ki(CB2) selectivity ratio of 6. Furthermore, no
compound exhibited a reversed selectivity, as in no case was the
Ki(CB1)/Ki(CB2) ratio lower than 1.
With regards to the substituents in position 1 of the heterocyclic
nucleus, the ethylmorpholino group generally determined a high
degree of CB2/CB1 selectivity, in an agreement with our previously
studies that suggested that this group interacted in the CB2R with
the nonconserved S3.31(112) [33]. However, the presence of
a p-fluorobenzyl seemed to preserve a certain degree of CB2/CB1
selectivity, even if generally lower than that observed for the eth-
ylmorpholino derivatives. Even if such interactions cannot be
classified as strong hydrogen bonds, interactions between CF and
polar hydrogen atoms HX (where X ¼ O, N) frequently occur in the
Protein Data Bank and Cambridge Structural Database [44] there-
fore the weak interaction in the CB2 of the fluorine atom of this
derivatives with S3.31(112) could contribute to the maintenance of
a certain degree of CB2/CB1 selectivity.
E4 was utilized. These compounds were evaluated in parallel with
derivative A5 [33] structurally analogous of B5 and D5. Overall, all
the new tested compounds showed better activity profiles than the
starting lead derivative A5 (Fig. 3). Compound D5 always exhibited
the highest potency, with a reduction of proliferation values
ranging between 15% and 62% (26% MCF7, 33% DU-145, 15% T98G,
62% AGS) (Fig. 3). Compound E4 inhibits significantly the prolifer-
ation of MCF7 and DU145 cells of 12% and 15%, respectively.
Compound B5 is the least active and is effective only in DU145 cells
(11%). Finally, to investigate the selectivity of the ligands’ effect in
tumoral versus non-tumoral cells, normal fibroblasts (WS-1) were
treated with the same dose of ligands as tumor cells. Compound D5
inhibits the proliferation rate of 21%, whereas compounds B5 and
E4 are not effective on normal cells. These results suggest that at
the same dose, compounds B5 and E4 are more effective on tumor
cells than compound D5. Furthermore the prostate and breast
cancer cells are more sensitive to the action of B5 and E4, compared
to glioblastoma and gastric adenocarcinoma cell lines.
In summary, all the CB2 ligands exert anti-proliferative effects
on DU-145 cell line with respect to the other cell lines, thus we
further explored their cytotoxic effects on DU-145 cell proliferation
and the potential involvement of the CB2 receptor. We observed
inhibition of cell proliferation at concentrations ranging from 1 to
0.1
assess the agonist activity of these compounds, we used B5, D5 and
E4 at the concentration of 0.3 M in the presence and in the
absence of the CB2 antagonist SR144528 used at 3 M. We observed
mM after 24 h of treatment (data not shown). Indeed, in order to
m
m
that the CB2 antagonist reverted the effects of the agonists, thus
suggesting a CB2emediated effect (see Fig. 4). The concentration of
0.3 mM was selected, since we showed that this dose did not inhibit
the proliferation of normal myelin basic protein-activated human
lymphocytes isolated from buffy coat of healthy donors (data not
shown).
Table 2
Calculated energy of the three different tautomers.
Tautomer 1
Tautomer 2
Tautomer 3
Energy (kcal/mol)
ꢁ404308.92
Energy (kcal/mol)
ꢁ404304.15
Energy (kcal/mol)
ꢁ404293.58

C. Manera et al. / European Journal of Medicinal Chemistry 52 (2012) 284e294
289
Fig. 5. CB2 agonist down-regulation of the CB2 receptor expression levels is reversed
by CB2 antagonist. Cell extracts were prepared following 24 h incubation of DU-145
Fig. 3. Effect of CB2 ligands on cancer cell proliferation. Various tumor cell lines of
different origin were treated with a ligand concentration of 1 M, and after four days
m
the cell number was measured with XTT staining (see experimental section). Statistical
analysis was performed by two-tailed t-test comparing vehicle (NT) and drug treated
samples *p value < 0.05, **p value < 0.001. y axis: treated/untreated O.D. ratio.
cells with the CB2 agonists at 0.3
SR144528 at the concentration of 3
assessed and normalized by actin.
m
M
in the presence and in the absence of
m
M. The expression of the CB2 receptor was
Furthermore, we analyzed the effects of the CB2 agonists in the
presence and in the absence of SR144528 on the expression of the
CB2 receptor in DU-145 cells. We observed a down-regulation of
our compounds and no difference with respect to the control was
observed (data not showed). Indeed to further confirm this finding,
we performed a similar experiment as in Fig. 5, but using the CB1
the CB2 levels by B5, D5 and E4 used at 0.3
mM, the effect was
antagonist SR141716, we found that used at the dose of 3
not revert the CB2 agonist effects (data not showed).
mM it did
reverted by SR144528 used at 3 M (Fig. 5) after 24 h treatment,
m
thus confirming the results observed on cell proliferation. We
found that SR144528 has an effect by itself on DU-145 cells as
showed in Fig. 4, this result might be due to its inverse agonist
properties as reported by other studies [45], however its effect on
the CB2 receptor expression is not significantly different with
respect to the control. This finding might exclude an antagonist or
inverse agonist like effect of our compounds since although
SR144528 and D5, B5 and E4 exert a similar effect on DU-145 cell
growth (Fig. 4), their effects on CB2 levels are different, thus sug-
gesting different mechanism of actions of the CB2 antagonist and
our compounds. Clearly appropriate in vitro functional assays will
have to be carried out to confirm the agonist activity of these
compounds. These findings suggest the efficacy of CB2 agonists to
reduce cancer cell proliferation and the involvement of the CB2
receptors in the effects observed in prostate cancer cells. Indeed,
the CB2-mediated effect supports the CB2 activity of these novel
compounds. In order to exclude a residual CB1 agonism, we
analyzed the expression of CB1 receptor following treatment with
Studies on further applications of these compounds are ongoing
to assess their effect on autoimmune diseases like multiple scle-
rosis and some of our data suggest anti-proliferative and anti-
inflammatory properties of these compounds on activated
lymphocytes.
4. Conclusions
Starting from compounds 1,8-naphthyridin-2(1H)-on-3-
carboxamides A, in order to evaluate the effects of the modification
of the 1,8-naphthyridin-2(1H)-one ring system, new derivatives
characterized by a different central scaffold were synthesized and
tested on CB1R and CB2R.
The obtained results clearly suggest that the modification of the
central scaffold have interesting effects on the CB2R affinity.
Compounds B and D emerged as promising CB2R ligands; in
particular compounds B5 and D5 are characterized by high affinity,
accompanied by very important Ki(CB1)/Ki(CB2) selectivity ratio.
Furthermore the new scaffold E, even if it is associated with an
overall decrease of CB2R affinity, has better solubility characteris-
tics than compounds B and D. Therefore, the 1,2-dihydro-2-
oxopyridine-3-carboxamide derivatives
E can be useful for
designing CB2R ligands with improved bioavailability. Finally
these newly synthesized CB2R ligands are effective on different
tumor cell lines. In particular, they inhibit proliferation of several
cancer cell lines and show CB2 activity, as observed in DU-145 cells.
In this cell line the anti-proliferative effect of compounds B5, D5
and E4 is mediated by the CB2 receptor, indeed these compounds
decrease the CB2 receptor expression levels and their effect is
reverted by the CB2 antagonist. However, further studies are
needed to identify the mechanism linked to the anti-proliferative
effect of these compounds and their potential application in cancer.
Fig. 4. Cytotoxic effect of CB2 ligands on DU-145 prostate cancer cell proliferation is
mediated by the CB2 receptor. DU-145 cells were treated with the CB2 agonists C5, B5
5. Experimental section
and D4 at the concentration of 0.3
mM in the presence and in the absence of SR144528
at the concentration of 3 M for 24 h, the proliferative response was measured by
m
5.1. Chemistry
sulforhodamine B assay. The histogram showed is representative of three independent
experiments. Statistical analysis was performed by two-tailed t-test comparing vehicle
(CTR) and CB2 agonist treated samples (*p < 0.05) or CB2 treated samples and CB2
treated samples in combination with SR144528 (**p < 0.05).
Melting points were determined on a Kofler hot stage apparatus
and are uncorrected. IR spectra in Nujol mulls were recorded on an

290
C. Manera et al. / European Journal of Medicinal Chemistry 52 (2012) 284e294
ATI Mattson Genesis Series FTIR spectrometer. 1H NMR and 13C
NMR spectra were recorded with a Bruker ACe200 spectrometer in
(s, 1H, H4), 8.04 ( d, J ¼ 7.8 Hz, 1H, Ar), 7.52e7.12 (m, 8H, Ar), 5.63
(s, 2H, CH2), 3.79 (m, 1H, CH), 1.88e1.23 (m, 10H, cyclohexyl).
Anal. C23H24N2O2 (C, H, N, O).
d
units from TMS as an internal standard. Microwave-assisted
reactions were run in a CEM microwave synthesizer. Mass spectra
were performed with Thermo Qest Finningan GCQ plus.
a
5.1.5.2. N-Cyclohexyl-1-(p-fluorobenzyl)-quinolin-2(1H)-on-3-carb-
oxamide (B2). Yield 52%; mp 162e164 ꢀC (crystallized from ethyl
Elemental analyses (C, H, N) were within ꢂ0.4% of theoretical
values, and were performed on a Carlo Erba elemental analyzer
model 1106 apparatus.
acetate); MS m/z 378 (Mþ); 1H NMR (DMSO)
d
9.77 (d, J ¼ 7.9 Hz,
1H, NH), 8.94 (s, 1H, H4), 8.06 (d, J ¼ 7.8 Hz 1H, Ar), 7.73e7.12 (m,
7H, Ar), 5.62 (s, 2H, CH2), 3.86 (m, 1H, CH); 1.78e1.39 (m, 10H,
5.1.1. Diethyl (2-nitrobenzylidene)malonate (1)
cyclohexyl). 13C NMR (DMSO)
d 163,20, 161.45, 161.25, 143.56,
Diethyl malonate (6.57 mmol; 1 ml) and 0.834 g of NaHCO3
(9.70 mmol) were added to a solution of 1.0 g (6.57 mmol) of
2-nitrobenzaldehyde in 2.3 ml of acetic anhydride. The reaction
mixture was stirred at 110 ꢀC for 24 h. After cooling, the mixture
was portioned between ethyl acetate and water. The organic layer
was washed successively with water, 5% aq Na2CO3 and brine. The
solvent was removed in vacuo to give a brown residue which was
used for the next reaction without purification.
139.40, 132.98, 132.36, 130.94, 128.61, 128.44, 123.09, 121.10, 119.47,
115,64, 115.33, 115.21, 47.42, 44.83, 32.25, 25.17, 24.18. Anal.
C23H23FN2O2 (C, H, N, O).
5.1.5.3. N-Cyclohexyl-1-(2-morpholin-4-ylethyl)-quinolin-2(1H)-on-
3-carboxamide (B3). Yield 47%; mp 195e197 ꢀC (crystallized from
ethyl acetate); MS m/z 383 (Mþ); 1H NMR (DMSO)
d 9.81
(d, J ¼ 7.8 Hz, 1H, NH), 8.85 (s, 1H, H4), 8.01 ( d, J ¼ 7.4 Hz, 1H, Ar),
7.65e7.38 (m, 3H, Ar), 4.45 (m, 2H, CH2), 3,85 (m, 1H, CH), 3.59
(m, 4H, morpholine), 2.62 (m, 6H, morpholine þ CH2), 1.98e1.33
(m, 10H, cyclohexyl). Anal. C22H29N3O3 (C, H, N, O).
5.1.2. Ethyl quinolin-2(1H)-on-3-carboxylate (2)
Iron powder (11.54 g; 206.7 mmol) was added to a pre-heated
100 ꢀC solution of compound 1 (11.76 g; 51.7 mmol) in 30. 0 mL
of acetic acid. The reaction mixture was maintained at 100 ꢀC for
24 h, cooled and filtered through Celite pad. The filtrate was
evaporated in vacuo to leave a brown residue which was purified by
flash chromatography using ethyl acetate/hexane (8:2) to obtain 2
5.1.5.4. N-Cyclohepthyl-1-(p-fluorobenzyl)-quinolin-2(1H)-on-3-carb-
oxamide (B4). Yield: 60%; mp 185e187 ꢀC (crystallized from ethyl
acetate); MS m/z 392 (Mþ); 1H NMR (DMSO)
d
9.82 (d, J ¼ 7.8 Hz,
1H, NH), 8.94 (s, 1H, H4), 8.05 ( d, J ¼ 7.2 Hz, 1H, Ar), 7.76e7.09 (m,
7H, Ar), 5.63 (s, 2H, CH2), 4.01 (m, 1H, CH), 1.91e1.24 (m, 12H,
(6.45 g, 51%): mp 177ꢀe179 ꢀC; 1H NMR (CDCl3)
d 10.87 (br, 1H, NH),
8.59 (s, 1H, H4), 7.70 (m, 2H, Ar), 7.52 (m, 1H, Ar), 7.39 (m, 1H, Ar),
cycloheptyl). 13C NMR (DMSO)
d 163.01, 160.90, 160.48, 142.93,
4.53 (q, J ¼ 7.1 Hz, 2H, CH2), 1.51 (t, J ¼ 7.1 Hz, 3H, CH3).
138.95, 132.39, 131.79, 131.73, 130.37, 128.06, 127.89, 122.52, 120.63,
118.91, 115.07, 114.76, 49.09, 40.24, 33.68, 27.02, 23.01. Anal.
C24H25FN2O2 (C, H, N, O).
5.1.3. N-Cyclohexyl-quinolin-2(1H)-on-3-carboxamide (3)
A mixture of quinolone 2 (0.200 g, 0.92 mmol) and cyclohex-
ylamine (0.91 g, 9.2 mmol) was heated in a sealed tube at 120 ꢀC for
24 h. After cooling, the reaction mixture was treated with ethyl
ether to give a solid residue which was collected by filtration and
crystallized from ethyl acetate to obtain 3. Yield 87%; mp
5.1.5.5. N-Cyclohepthyl-1-(2-morpholin-4-ylethyl)-quinolin-2(1H)-on-
3-carboxamide (B5). Yield: 47%; mp 198e200 ꢀC (crystallized from
ethyl acetate); MS m/z 397 (Mþ); 1H NMR (DMSO)
d 9.83
(d, J ¼ 7.9 Hz, 1H, NH), 8.85 (s, 1H, H4), 8.02 (d, J ¼ 7.4 Hz, 1H, Ar),
7.85e7.50 (m, 2H, Ar), 7.35 (m,1H, Ar), 4.52 (m, 2H, CH2), 4.00 (m,1H,
CH), 3.58 (m, 4H, morpholine), 2.58 (m, 6H, morpholine þ CH2),
1.98e1.18 (m, 12H, cycloheptyl). Anal. C23H31N3O3 (C, H, N, O).
248e250 ꢀC; 1H NMR (CDCl3)
d
12.05 (br,1H, NH), 9.85 (d, J ¼ 7.9 Hz,
1H, NH), 9.07 (s, 1H, H4), 7.83 (d, J ¼ 7.4 Hz, 1H, Ar), 7.64 (m, 1H, Ar),
7.34 (m, 2H, Ar), 4.14 (m, 1H, CH), 2.07e1.30 (m, 10H, cyclohexyl).
Anal. C16H18N2O2 (C, H, N,O).
5.1.6. N-Cyclohexyl-2-(p-fluorobenzyloxy)-quinoline-3-
carboxamide (C1)
5.1.4. N-Cycloheptyl-quinolin-2(1H)-on-3-carboxamide (4)
A mixture of quinolone 2 (0.430 g, 1.98 mmol) and cyclo-
heptylamine (2.24 g, 19.8 mmol) was heated in a sealed tube at
120 ꢀC for 24 h. After cooling, the reaction mixture was treated as
reported for compound 3. Yield 73%; mp > 300 ꢀC (crystallized
An amount 0.723 g of NaH (15.0 mmol) was added to a solution
of 1.35
g (5.02 mmol) of N-cyclohexyl-quinolin-2(1H)-on-3-
carboxamide (3) in 50 ml of dry DMF. After 1 h, 0.90 ml of p-fluo-
robenzylchloride (1.09 g; 7.53 mmol) was added and the reaction
was heated at 70 ꢀC for 24 h. The mixture was concentrated under
reduced pressure and the residue was treated with water to give
a solid residue which was collected by filtration and purified by
flash chromatography on a silica gel using ethyl acetate/hexane 1:2
as eluent to give B2 (0.417 g, 22%) and compound C1 (0.284 g, 15%);
from ethyl acetate); 1H NMR (CDCl3)
d 11.10 (br, 1H, NH), 9.73
(d, J ¼ 7.9 Hz, 1H, NH), 9.00 (s, 1H, H4), 7.83 (d, J ¼ 7.4 Hz, 1H, Ar),
7.64 (m, 1H, Ar), 7.24 (m, 2H, Ar), 4.18 (m, 1H, CH), 2.12e1.60
(m, 12H, cycloheptyl). Anal. C17H20N2O2 (C, H, N, O).
5.1.5. General procedure for the preparation of N1-substituted
quinolin-2(1H)-on-3-carboxamide derivatives B1eB5
mp 202e204 ꢀC (crystallized from hexane); 1H NMR (DMSO)
d 9.77
(d, J ¼ 7.9 Hz, 1H, NH), 8.62 (s, 1H, H4), 8.21 (d, J ¼ 7.6 Hz, 1H, Ar),
An amount of 4.50 mmol of NaH was added to a solution of
1.50 mmol of carboxamide derivative 3 or 4 in 20 ml of dry DMF.
After 1 h 2.20 mmol of benzylchloride or p-fluorobenzylchloride or
4-(2-cloroethyl)morpholine was added and the reaction mixture
was stirred for 24 h at room temperature. The mixture was then
concentrated under reduced pressure and treated with water to
give a solid residue which was collected by filtration and purified by
crystallization to obtain the title compounds.
8.10 (m,1H, Ar), 7.87e7.27 (m, 6H, Ar), 5.54 (s, 2H, CH2), 3.79 (m,1H,
CH), 1.78e1.24 (m, 10H, cyclohexyl). 13C NMR (DMSO)
d 164.28,
162.57, 157.54, 144.86, 140.12, 132.78, 131.07, 130.69, 130.52, 128.58,
126.32, 124.81, 120.31, 115.32, 114.90, 114.54, 67.25, 47.62, 31.96,
30.67, 23.99. Anal. C23H23FN2O2 (C, H, N, O).
5.1.7. Methyl 2-amino-3-pyridinecarboxylate (5)
A mixture of 2-aminonicotinic acid (0.25 g, 1.80 mmol) and
1.80 mL of concentrated H2SO4 in 3.6 ml of methanol was heated at
80 ꢀC for 7 h. After cooling, the methanol was removed by evapo-
ration under reduced pressure and the obtained mixture was
treated with the solution of NaHCO3 (pH 7e8). The precipitate thus
5.1.5.1. N-Cyclohexyl-1-benzyl-quinolin-2(1H)-on-3-carboxamide
(B1). Yield 45%; mp 149e151 ꢀC (crystallized from hexane); MS
m/z 360 (Mþ); 1H NMR (DMSO)
d
9.77 (d, J ¼ 7.9 Hz, 1H, NH), 9.00

C. Manera et al. / European Journal of Medicinal Chemistry 52 (2012) 284e294
291
formed was collected by filtration to give the title compound 5
5.1.11.2. N-Cyclohexyl-1-(p-fluorobenzyl)-4-hydroxy-2-oxo-1,2-dihy-
dro-1,8-naphthyridine-3-carboxamide (D2). Yield 53%þ; m.p. 138ꢀ-
140 ꢀC (crystallized from CHCl3); MS m/z 395 (M ); 1H NMR
(0.17 g, yield: 63%) which was used for the next reaction without
purification. 1H NMR (DMSO)
d
8.20 (dd, J ¼ 4.9 and 2.0 Hz, 1H, Ar),
8.05 (dd, J ¼ 7.7 and 2.0 Hz, 1H, Ar), 7.12 (br, 2H, NH2), 6.63 (m, 1H,
(DMSO)
d
17.68 (brs, 1H, OH), 11.02 (d, J ¼ 7.7 Hz, 1H, NH), 8.43
Ar), 3.80 (s, 3H, OCH3).
(m, 2H, Ar), 7.65 (m, 1H, Ar), 7.37 (m, 2H, Ar), 7.16 (m, 2H, Ar), 5.50
(s, 2H, CH2), 3.95 (m, 1H, CH), 1.85e1.26 (m, 10H, cyclohexyl). Anal.
C22H22FN3O3 (C, H, N, O).
5.1.8. Ethyl 4-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-
carboxylate (6)
To a solution of 0.22 g of Na (9.77 mmol) in 8.20 mL anhydrous
ethanol was added 1.58 g of diethylmalonate (9.87 mmol) and the
mixture was stirred at room temperature for 5 min. Then 0.50 g of
pyridinecarboxilate 5 (3.29 mmol) was added and the mixture was
heated under reflux for 6 h. The cooled mixture was diluted with
9.0 mL of water and then acidified (pH 3e4) with concentrated HCl
to give a precipitate which was collected by filtration and crystal-
lized from ethanol to obtain derivative 6 (0.45 g, 58%); m.p.
5.1.11.3. N-Cyclohexyl-4-hydroxy-1-(2-morpholin-4-ylethyl)-2-oxo-
1,2-dihydro-1,8-naphthyridine-3-carboxamide (D3). Yield 17%þ; m.p.
248e250 ꢀC (crystallized from ethyl acetate); MS m/z 400 (M ); 1H
NMR (DMSO)
d
17.60 (br, 1H, OH), 11.20 (d, J ¼ 7.8 Hz, 1H, NH), 8.80
(dd, J ¼ 4.8 and 1.9 Hz, 1H, Ar), 8.40 (dd, J ¼ 8.0 and 1.9 Hz, 1H, Ar),
7.40 (m, 1H, Ar), 4.52 (m, 2H, CH2), 3.90 (m, 1H, CH), 3.52 (m, 4H,
morpholine), 2.50 (m, 6H, morpholine þ CH2), 1.88e1.22 (m, 10H,
cyclohexyl). Anal. C21H28N4O4 (C, H, N, O).
146e148 ꢀC; 1H NMR (DMSO)
d 17.56 (br,1H, OH),11.30 (br, 1H, NH),
8.63 (dd, J ¼ 4.5 and 1.5 Hz, 1H, Ar), 8.33 (dd, J ¼ 7.8 and 1.5 Hz, 1H,
Ar), 7.27 (m, 1H, Ar), 4.31 (q, J ¼ 7.1 Hz, 2H, CH2), 1.18 (t, J ¼ 7.1 Hz,
3H, CH3).
5.1.11.4. N-Cycloheptyl-1-(p-fluorobenzyl)-4-hydroxy-2-oxo-1,2-dihy-
dro-1,8-naphthyridine-3-carboxamide
(D4). Yield
59%; þ m.p.
164e166 ꢀC (crystallized from ethyl acetate); MS m/z 409 (M ); 1H
NMR: (DMSO)
d
17.62 (br, 1H, OH), 11.05 (d, J ¼ 7.8 Hz, 1H, NH), 8.38
5.1.9. N-Cyclohexyl-4-hydroxy-2-oxo-1,2-dihydro-1,8-
naphthyridine-3-carboxamide (7)
(m, 2H, Ar), 7.67 (m,1H, Ar), 7.32 (m, 2H, Ar), 7.09 (m, 2H, Ar), 5.52 (s,
2H, CH2), 4.03 (m, 1H, CH), 1.91e1.24 (m, 12H, cycloheptyl). Anal.
C23H24FN3O3 (C, H, N, O).
A mixture of compound 6 (0.24 g, 0.85 mmol) and cyclohexyl-
amine (0.84 g, 8.50 mmol) in a sealed tube was placed in the oil
bath. The reaction was heated at 120 ꢀC for 24 h. After cooling, the
reaction mixture was treated with ethyl ether to give a solid residue
which was collected by filtration and crystallized from ethyl acetate
5.1.11.5. N-Cycloheptyl-4-hydroxy-1-(2-morpholin-4-ylethyl)-2-oxo-
1,2-dihydro-1,8-naphthyridine-3-carboxamide (D5). Yieþld 22%; m.p.
181e184 ꢀC (crystallized from CHCl3); MS m/z 414 (M ); 1H NMR:
to obtain 7. (0.220 g, 90%); mp 253e255 ꢀC; 1H NMR (DMSO)
d
17.78
(DMSO)
d
17.68 (br, 1H, OH), 10.82 (d, J ¼ 7.8 Hz 1H, NH), 8.80
(br, 1H, OH), 12.05 (br, 1H, NH), 10.04 (br, 1H, NH), 8.79 (dd, J ¼ 4.6
and 1.9 Hz, 1H, Ar), 8.46 (dd, J ¼ 7.7 and 1.9 Hz, 1H, Ar), 7.31 (m, 1H,
Ar), 3.97 (m, 1H, CH), 1.98e1.12 (m, 10H, cyclohexyl). Anal.
C15H17N3O3 (C, H, N, O).
(dd, 1H, Ar), 8.45 (dd, 1H, Ar), 7.44 (m, 1H, Ar), 4.51 (m, 2H, CH2),
4.04 (m, 1H, CH), 3.60 (m, 4H, morpholine), 2.60 (m, 6H,
morpholine þ CH2), 1.87e1.22 (m, 12H, cycloheptyl). 13C NMR
(DMSO)
d 171.78, 169.702, 162.25, 152.98, 149.40, 134.32, 118.47,
112.64, 97.33, 67.30, 56.41, 54.21, 50.67, 38.33, 35.05, 28.27, 24.43.
Anal. C22H30N4O4 (C, H, N, O).
5.1.10. N-Cycloheptyl-4-hydroxy-2-oxo-1,2-dihydro-1,8-
naphthyridine-3-carboxamide (8)
A mixture of compound 6 (0.50 g, 2.14 mmol) and cyclo-
heptylamine (2.42 g; 21.4 mmol) in a sealed tube was placed in the
oil bath and heated at 120 ꢀC for 24 h. After cooling, the reaction
mixture was treated as reported for compound 7. Yield 98%; mp >
5.1.12. Methyl 2-hydroxypyridine-3-carboxylate (9)
A mixture of 1.00 g (7.20 mmol) of 2-hydroxypyridine-3-
carboxylic acid and 7.20 mL of concentrated H2SO4 in 15.0 mL of
CH3OH was heated in microwave at 90 ꢀC for 60 min (power 200 W,
pressure 100 psi, stirring off). After cooling the CH3OH was
removed by evaporation under reduced pressure. The obtained
mixture was treated with a solution of NaHCO3 (pH 7e8) and
extracted with CH2Cl2. The organic layers were washed with brine,
dried over anhydrous Na2SO4, and evaporated to dryness to give the
desired compound as a white solid. (0.92 g, yield 83%) mp:
300 ꢀC (crystallized from ethyl acetate); 1H NMR (CDCl3)
d 17.73
(br, 1H, OH), 12.02 (br, 1H, NH), 10.11 (d, J ¼ 7.8 Hz, 1H, NH), 8.82
(dd, J ¼ 4.6 and 1.7 Hz, 1H, Ar), 8.49 (dd, J ¼ 7.9 and 1.9 Hz, 1H, Ar),
7.33 (m, 1H, Ar), 4.15 (m, 1H, CH), 2.19e1.74 (m, 12H, cycloheptyl).
Anal. C16H19N3O3 (C, H, N, O).
5.1.11. General procedure for the preparation of N1-substituted 4-
hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide
derivatives D1eD5
152e154 ꢀC; 1H NMR (DMSO)
d
12.09 (br, 1H, OH), 8.07 (dd, J ¼ 7.1
and 2.2 Hz, 1H, Ar), 7.68 (dd, J ¼ 6.4 and 2.2 Hz, 1H, Ar), 6.29 (m, 1H,
Ar), 3.72 (s, 3H, CH3).
An amount of 4.68 mmol of NaH was added to a solution of
1.56 mmol of carboxamide derivative 7 or 8 in 17.0 mL of dry
DMF. After 1 h 3.12 mmol of the suitable chloride was added and
the reaction was stirred for 24 h at room temperature. The
mixture was concentrated under reduced pressure and the
residue was treated with water to give a solid residue which was
collected by filtration and purified by crystallization to obtain the
title compounds.
5.1.13. N-Cyclohexyl-2-hydroxypyridine-3-carboxamide (10)
A mixture of methyl 2-hydroxypyridine-3-carboxylate (0.550 g,
3.60 mmol) and cyclohexylamine (1.06 g, 10.7 mmol) was heated at
150 ꢀC for 48 h. After cooling the reaction mixture was poured into
ice and treated with HCl 5% until pH ¼ 4. The precipitated solid was
collected by filtration and purified by crystallization from ethyl
acetate to give the pure amide 10 (0.50 g, yield 63.2%); m.p.
196e198 ꢀC; 1H NMR (DMSO)
1H, NH), 8.35 (dd, J ¼ 6.7 and 2.2 Hz, 1H, Ar), 7.72 (dd, J ¼ 6.0 and
2.2 Hz, 1H, Ar), 6.50 (m, 1H, Ar), 3.80 (m, 1H, CH), 1.85e1.29 (m, 10H,
cyclohexyl). Anal. C12H16N2O2 (C, H, N, O).
d
10.09 (br,1H, OH), 9.87 (d, J ¼ 7.1 Hz,
5.1.11.1. N-Cyclohexyl-1-benzyl-4-hydroxy-2-oxo-1,2-dihydro-1,8-nap-
hthyridine-3-carboxamide (D1). Yield: 22%; m.p. 143e145 ꢀC (crys-
tallized from CHCl3); MS m/z 377 (Mþ); 1H NMR (DMSO)
d 17.72 (br,
1H, OH), 10.20 (d, J ¼ 7.7 Hz, 1H, NH), 8.76 (dd, J ¼ 4.6 and 1.8 Hz, 1H,
Ar), 8.48 (dd, J ¼ 7.7 and 1.8 Hz, 1H, Ar), 7.44 (m, 1H, Ar), 7.26 (m, 5H,
Ar), 5.61 (s, 2H, CH2), 3.85 (m,1H, CH),1.87e1.22 (m,10H, cyclohexyl).
Anal. C22H23N3O3 (C, H, N, O).
5.1.14. N-Cyclohepthyl-2-hydroxypyridine-3-carboxamide (11)
A mixture of methyl 2-hydroxypyridine-3-carboxylate (0.780 g,
5.10 mmol) of and cyclohepthylamine (1.72 g, 15.2 mmol) was

292
C. Manera et al. / European Journal of Medicinal Chemistry 52 (2012) 284e294
heated at 150 ꢀC in oil bath for 48 h. After cooling, the reaction
mixture was treated as reported for compound 10 (1.10 g, yield
92%.); m.p. 143e145 ꢀC (crystallized from ethyl acetate); 1H NMR
5.1.16. N-Cyclohexyl-2-chloropyridine-3-carboxamide (12)
A suspension of 2-chloronicotinic acid (0.50 g, 3.20 mmol) and
6.40 mL of SOCl2 was heated on an oil bath at 90 ꢀC for 3 h. After
cooling the excess of SOCl2 was distilled off in vacuum and then the
residue obtained was dissolved in 7.0 mL of toluene and treated
with 1.0 mL of triethylamine and cyclohexylamine (0.32 g,
3.20 mmol). The reaction mixture was heated at 50 ꢀC for 1 h, then
diluted with water and extracted with CH2Cl2. The organic layers
were washed with brine, dried over anhydrous Na2SO4 and evap-
orated to dryness. The crude residue was crystallized from petro-
leum ether to give 0.65 g (yield 85%) of the title compound; m.p.
(DMSO)
d
10.07 (br, 1H, OH), 9.88 (d, J ¼ 7.3 Hz, 1H, NH), 8.32 (dd,
J ¼ 6.8 and 2.2 Hz, 1H, Ar), 7.71 (dd, J ¼ 6.2 and 2.2 Hz, 1H, Ar), 6.48
(m, 1H, Ar), 3.99 (m, 1H, CH), 2.10e1.48 (m, 12H, cycloheptyl). Anal.
C13H18N2O2 (C, H, N, O).
5.1.15. General procedure for the preparation of N1-substituted 1,2-
dihydro-2-oxo-pyridine-3-carboxamide derivatives E1eE5
An amount of 13.0 mmol of NaH was added to a solution of
3.20 mmol of 2-hydroxypyridine-3-carboxamide derivatives 10 or
11 in 19.0 ml of dry DMF and the mixture was heated at 70 ꢀC under
stirred. After 2 h, 3.84 mmol of suitable chloride was added and the
mixture was stirred at 70 ꢀC for 2 h. After the mixture was
concentrated under reduced pressure and the residue was treated
with water to give a solid which was collected by filtration and
purified by flash chromatography (exane/ethyl acetate 2:1) to
obtain the title compounds. In the case of the reaction with ben-
zylchloride or with p-fluorobenzylchloride the 2-benzyloxy or
2-(p-fluorobenzyloxy)-pyridine derivatives F1eF3 were also
isolated.
128e130 ꢀC; 1H NMR (DMSO)
and 2.9 Hz, 1H, Ar), 7.48 (m, 1H, Ar), 3.72 (m, 1H, NCH), 1.87e1.16
d
8.45 (m, 2H, NH, Ar), 7.85 (dd, J ¼ 7.6
(m, 10H, cyclohexyl). Anal. C12H15ClN2O (C, H, N, O).
5.1.17. N-Cyclohepthyl-2-chloropyridine-3-carboxamide (13)
This compound was prepared as above reported for the
synthesis of 12; yield: 92.6%; m.p. 148e150 ꢀC (crystallized from
petroleum ether); 1H NMR (DMSO)
d 8.90 (m, 2H, NH, Ar), 8.25
(dd, J ¼ 7.6 and 2.0 Hz, 1H, Ar), 7.88 (m, 1H, Ar), 4.32 (m, 1H, CH),
2.34e1.92 (m, 12H, cycloheptyl). Anal. C13H17ClN2O (C, H, N, O).
5.1.18. General procedure for the preparation of 2-substituted
pyridine-3-carboxamide derivatives F1eF3
1.10 mmol of suitable alcohol were added to 2.20 mmol of
potassium t-butoxide and 0.10 mmol of the tetraalkylammonium
salt (Aliquat 336). The mixture was shaken for 5 min with
a mechanical stirrer and then 3.00 mmol of N-cyclohexyl-2-
5.1.15.1. N-Cyclohexyl-1-benzyl-1,2-dihydro-2-oxo-pyridine-3-carbo-
xamide (E1). yield: 43%; m.p. 76e78 ꢀC (crystallized from hexane);
MS m/z 310 (Mþ); 1H NMR (CDCl3)
d
9.75 (d, J ¼ 6.3 Hz,1H, NH), 8.56
(dd, J ¼ 7.1 and 2.3 Hz, 1H, Ar), 7.50 (dd, J ¼ 6.8 and 2.3 Hz, 1H, Ar),
7.34 (m, 5H, Ar), 6.42 (m, 1H, Ar), 5.25 (s, 2H, CH2), 3.98 (m, 1H, CH),
1.84e1.28 (m, 10H, cyclohexyl). Anal. C19H22N2O2 (C, H, N, O).
chloropyridine-3-carboxamide
(12)
or
N-cyclohepthyl-2-
chloropyridine-3-carboxamide (13) were added. The mixture was
stirred 5 more min and then heated at 120 ꢀC for 2 h. After cooling
the reaction mixture was treated with 20 ml of CH2Cl2 and filtered.
The filtrate was washed with H2O, brine, dried over anhydrous
Na2SO4 and evaporated to dryness. The solid residue was purified
by flash chromatography (hexane/ethyl acetate 3:1).
5.1.15.2. N-Cyclohexyl-1-(p-fluorobenzyl)-1,2-dihydro-2-oxo-pyridine-
3-carboxamide (E2). Yield 19%; m.p. 121e124 ꢀC (crystallized from
hexane); MS m/z 328 (Mþ); 1H NMR (CDCl3)
d
9.71 (d, J ¼ 6.2 Hz, 1H,
NH), 8.56 (dd, J ¼ 7.1 and 2.2 Hz 1H, Ar), 7.50 (dd, J ¼ 6.6 and 2.2 Hz,
1H, Ar), 7.33 (m, 2H, Ar), 7.08 (m, 2H, Ar), 6.43 (m, 1H, Ar), 5.21 (s, 2H,
CH2), 3.99 (m, 1H, CH), 2.03e1.23 (m, 10H, cyclohexyl). Anal.
C19H21FN2O2 (C, H, N, O).
5.1.18.1. N-Cyclohexyl-2-(benzyloxy)pyridine-3-carboxamide (F1).
Yield 56%; m.p.: 68e70 ꢀC (crystallized from hexane); MS m/z 310
5.1.15.3. N-Cyclohexyl-1-(2-morpholin-4-ylethyl)-1,2-dihydro-2-oxo-
(Mþ); 1H NMR (DMSO)
d
8.34 (dd, J ¼ 4.9 and 2.2 Hz, 1H, Ar), 8.15
pyridine-3-carboxamide (E3). Yield: 51%; m.p. 96e98 ꢀC (crystal-
(dd, J ¼ 7.4 and 2.2 Hz, 1H, Ar), 8.05 (d, J ¼ 8.0 Hz, 1H, NH), 7.53 (m,
2H, Ar), 7.42 (m, 3H, Ar), 7.16 (m, 1H, Ar), 5.45 (s, 2H, CH2), 3.75 (m,
1H, CH), 1.76e1.11 (m, 10H, cyclohexyl). Anal. C19H22N2O2 (C, H, N,
O).
lized from hexane); MS m/z 333 (Mþ); 1H NMR (CDCl3)
d 9.74 (d,
J ¼ 7.1 Hz, 1H, NH), 8.55 (dd, J ¼ 7.3 and 2.2 Hz, 1H, Ar), 7.53 (dd,
J ¼ 6.6 and 2.2 Hz, 1H, Ar), 6.42 (m, 1H, Ar), 4.13 (t, J ¼ 6.0 Hz, 2H,
CH2), 4.00 (m, 1H, CH), 3.70 (m, 4H, morpholine), 2.73 (t, J ¼ 6.0 Hz,
2H, CH2), 2.52 (m, 4H, morpholine), 2.02e1.27 (m, 10H, cyclohexyl).
Anal. C18H27N3O3 (C, H, N, O).
5.1.18.2. N-Cyclohexyl-2-(4-fluorobenzyloxy)pyridine-3-carboxamide
(F2). Yield 69%. m.p. 94e96 ꢀC (crystallized from hexane); MS m/z
328 (Mþ); 1H NMR (DMSO)
8.28 (dd, J ¼ 7.2 and 2.0 Hz, 1H, Ar), 7.98 (d, J ¼ 7.9 Hz, 1H, NH), 7.37
(m, 2H, Ar), 7.21 (m, 3H, Ar), 5.47 (s, 2H, CH2), 4.00 (m, 1H, CH),
1.86e1.07 (m, 10H, cyclohexyl). Anal. C19H21FN2O2 (C, H, N, O).
d
8.55 (dd, J ¼ 5.6 and 2.0 Hz, 1H, Ar),
5.1.15.4. N-Cyclohepthyl-1-(p-fluorobenzyl)-1,2-dihydro-2-oxo-pyri-
dine-3-carboxamide(E4). Yield: 63%; m.p.: 118e120 ꢀC (crystallized
from hexane); MS m/z 342 (Mþ); 1H NMR (DMSO)
d 9.76 (d, 1H,
J ¼ 7.1 Hz, NH), 8.35 (dd, J ¼ 7.0 and 2.2 Hz, 1H, Ar), 8.19 (dd, J ¼ 6.6
and 2.2 Hz, 1H, Ar), 7.40 (m, 2H, Ar), 7.33 (m, 2H, Ar), 6.55 (m, 1H,
Ar), 5.22 (s, 2H, CH2), 3.95 (m, 1H, CH), 2.01e1.35 (m, 12H, cyclo-
5.1.18.3. N-Cyclohepthyl-2-(4-fluorobenzyloxy)pyridine-3-carboxamide
(F3). Yield 48%; mp 60e62 ꢀC (crystallized from hexane); MS m/z 342
heptyl). 13C NMR (CDCl3)
d
162.36, 162.10, 160.21, 143.58, 143.55,
(Mþ); 1H NMR (DMSO)
d
8.34 (dd, J ¼ 4.9 and 2.0 Hz, 1H, Ar), 8.15 (dd,
140.03, 130.02, 129.86, 122.41, 116.34, 115.91, 107.00, 52.05, 50.58,
35.08, 28.27, 24.37. Anal. C20H23FN2O2 (C, H, N, O).
J ¼ 7.3 and 2.0 Hz,1H, Ar), 8.03 (d, J ¼ 7.1 Hz, 1H, NH), 7.61 (m, 2H, Ar),
7.25 (m, 3H, Ar), 5.42 (s, 2H, CH2), 4.00 (m, 1H, CH), 1.75e1.05 (m, 12H,
cyclohepthyl). Anal. C20H23FN2O2 (C, H, N, O).
5.1.15.5. N-Cyclohepthyl-1-(2-morpholin-4-ylethyl)-1,2-dihydro-2-oxo-
pyridine-3-carboxamide (E5). Yield: 43,%; m.p: 68e70 ꢀC (crystallized
5.2. CB1 and CB2 receptor binding assays
from hexane); MS m/z 347 (Mþ); 1H NMR (CDCl3)
d
9.90 (d, J ¼ 7.1 Hz,
1H, NH), 8.53 (dd, J ¼ 7.3 and 2.2 Hz, 1H, Ar), 7.52 (dd, J ¼ 6.6 and
2.2 Hz, 1H, Ar), 6.40 (m, 1H, Ar), 4.12 (t, J ¼ 6.0 Hz, 2H, CH2), 3.99 (m,
1H, CH), 3.68 (m, 4H, morpholine), 2.73 (t, J ¼ 6.0 Hz, 2H, CH2), 2.53
(m, 4H, morpholine), 2.06e1.37 (m, 12H, cycloheptyl). Anal.
C19H29N3O3 (C, H, N, O).
The new compounds were evaluated in CB1R and CB2R binding
assays using membranes from HEK-293 cells transfected with
cDNAs encoding the human recombinant CB1R (Bmax) 2.5 pmol/
mg protein and human recombinant CB2R (Bmax) 4.7 pmol/mg
protein (PerkineElmer, Italy). These membranes were incubated

C. Manera et al. / European Journal of Medicinal Chemistry 52 (2012) 284e294
293
with [3H]CP55,940) [46] (0.14 nM/kd ¼ 0.18 nM and 0.084 nM/
5.6. Analysis of the C derivative geometry
kd ¼ 0.31 nM for CB1 and CB2 receptors, respectively) as the high
affinity ligand and displaced with 100 nM of WIN55212-2 [47] as
the heterologous competitor for nonspecific binding (Ki values 9.2
and 2.1 nM, respectively, for CB1R and CB2R). All compounds were
tested following the procedure described by the cell membrane
manufacturer. Displacement curves were generated by incubating
drugs with [3H]CP55,940 for 90 min at 30 ꢀC. Ki values were
calculated by applying the ChengePrusoff equation [48] to the IC50
values (obtained by GraphPad) for the displacement of the bound
radioligand by increasing concentrations of the test compound.
Data are the mean (SEM of at least n ¼ 3 experiments).
All the calculations were carried out using Gaussian09 [50]. To
compare the energy of the three different tautomers, they were
optimized using the MP2 chemical model with a 6-31Gþþ** basis
set. The solvent effects were estimated using the Polarizable
Continuum Model using the integral equation formalism variant as
implemented in Gaussian09 [50].
Acknowledgment
This work was partially supported by National Interest Research
Projects (PRIN 2008, Grant “Pharmacological characterization of
novel direct and indirect agonists of endocannabinoid receptors and
their potential use for the treatment of colorectal carcinoma”) and by
FISM e Fondazione Italiana Sclerosi Multipla e Cod. 2009/R/3/C1.
Authors from the CNR of Pozzuoli thank Marco Allarà for technical
5.3. Cell cultures
Different tumor cell lines (MCF-7 human breast carcinoma cells,
DU-145 human prostate carcinoma cells, AGS human gastric
adenocarcinoma cells, T98G glioblastoma cells) and normal cells
(WS-1) were purchased from ATCC and maintained at 37 ꢀC in
a humidified atmosphere containing 5% CO2 accordingly to the
supplier.
assistance. Authors thank Associazione Educazione
Medica Salernitana (ERMES).
e Ricerca
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