Design, synthesis, and biological evaluation of new 1,8-naphthyridin-4(1H)- on-3-carboxamide and quinolin-4(1H)-on-3-carboxamide derivatives as CB 2 selective agonists

Article abstract of DOI:10.1021/jm0603466

On the basis of docking studies carried out using the recently published cannabinoid receptor models, new 1,8-naphthyridin-4(1H)-on-3-carboxamide and quinolin-4(1H)-on-3-carboxamide derivatives were designed, synthesized, and tested for their affinities toward the cannabinoid CB₁ and CB ₂ receptors. Compound 10, which presented p-fluorobenzyl and carboxycycloheptylamide substituents bound in the 1 and 3 positions of the 1,8-naphthyiridine-4-one nucleus, showed a high CB₂ affinity with a K_i of 1.0 nM. The substitution of the naphthyridine-4-one nucleus with the quinoline-4-one system determined a general increase in CB₂ affinity. In particular, the N-cyclohexyl-7-chloro-1-(2-morpholin-4-ylethyl) quinolin-4(1H)-on-3-carboxamide (40) possessed a remarkable affinity, with K_i of 3.3 nM, which was also accompanied by a high selectivity for the CB₂ receptor (K_i(CB₁/K_i(CB ₂) ratio greater than 303). Moreover, the [³⁵S]GTPγ binding assay and functional studies on human basophils indicated that the 1,8-naphthyridin-4(1H)-on-3-carboxamide derivatives behaved as CB₁ and CB₂ receptor agonists.

Full text of DOI:10.1021/jm0603466

J. Med. Chem. 2006, 49, 5947-5957
5947
Design, Synthesis, and Biological Evaluation of New 1,8-Naphthyridin-4(1H)-on-3-carboxamide
and Quinolin-4(1H)-on-3-carboxamide Derivatives as CB2 Selective Agonists
Clementina Manera,*,† Veronica Benetti,† M. Paola Castelli,‡ Tiziana Cavallini,† Sara Lazzarotti,† Fabio Pibiri,‡
Giuseppe Saccomanni,† Tiziano Tuccinardi,*,† Alfredo Vannacci,§ Adriano Martinelli,† and Pier Luigi Ferrarini†
Dipartimento di Scienze Farmaceutiche, UniVersita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy, Dipartimento di Neuroscience B.B. Brodie,
UniVersita` di Cagliari, Cittadella UniVersitaria, SS 554 Km 4.5, 09042 Monserrato, Italy, and Dipartimento di Farmacologia Preclinica e
Clinica, UniVersita` di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy
ReceiVed March 24, 2006
On the basis of docking studies carried out using the recently published cannabinoid receptor models,35 new
1,8-naphthyridin-4(1H)-on-3-carboxamide and quinolin-4(1H)-on-3-carboxamide derivatives were designed,
synthesized, and tested for their affinities toward the cannabinoid CB1 and CB2 receptors. Compound 10,
which presented p-fluorobenzyl and carboxycycloheptylamide substituents bound in the 1 and 3 positions
of the 1,8-naphthyiridine-4-one nucleus, showed a high CB2 affinity with a Ki of 1.0 nM. The substitution
of the naphthyridine-4-one nucleus with the quinoline-4-one system determined a general increase in CB2
affinity. In particular, the N-cyclohexyl-7-chloro-1-(2-morpholin-4-ylethyl)quinolin-4(1H)-on-3-carboxamide
(40) possessed a remarkable affinity, with Ki of 3.3 nM, which was also accompanied by a high selectivity
for the CB2 receptor (Ki(CB1)/Ki(CB2) ratio greater than 303). Moreover, the [35S]GTPγ binding assay and
functional studies on human basophils indicated that the 1,8-naphthyridin-4(1H)-on-3-carboxamide derivatives
behaved as CB1 and CB2 receptor agonists.
Introduction
the molecular characterization of these receptors allowed the
development of synthetic compounds with cannabinoid and
noncannabinoid structures, which have found pharmaceutical
application.12,13 Although the physiological role of CB receptors
is not yet completely explained, these receptors seem to be
involved in several pathophysiological diseases.14 In particular,
selective CB1 receptor antagonists such as rimonabant15 are
currently under investigation in clinical human studies for
treating obesity through the control of appetite16,17 and may be
a helpful tool to stop smoking.18 In contrast, ∆9-THC and
nabilone are currently marketed to reduce emesis and/or prevent
cachexia in AIDS or cancer patients.19 Unlike the CB1 receptor,
the physiological and putative therapeutic potential of the CB2
receptor largely remains unexplored. However, selective ligands
could be useful for the treatment of pain,20 inflammation,21
osteoporosis,22 growth of malignant gliomas,23 tumors of
immune origin,24 and immunological disorders such as multiple
sclerosis.25 Furthermore, CB2 agents could be exploited for
prevention of Alzheimer’s disease pathology, given of the
presence of the CB2 receptor in the brain microglial cells.26,27
Finally, it has recently been shown that CB2 receptor agonists
might provide neuroprotection by blockade of microglial
activation.28
Cannabinoid ligands are currently classified into different
structural classes, namely, classic cannabinoids (tricyclic diben-
zopyran derivatives produced by the Cannabis plant and their
synthetic derivatives) such as ∆9-THC, nonclassic cannabinoids
(bicyclic or tricyclic THC derivatives) such as [3H]CP-55,-
940,29,30 endocannabinoids such as arachidonylethanolamide
(AEA) and their synthetic derivatives,31 indoles (typified by
WIN-55,212-2), pyrazoles,32 and indenes.33
We have previously reported the synthesis and the binding
activity at mouse cannabinoid receptors of a series of 1,8-
naphthyridin-4(1H)-on-3-carboxamide derivatives whose general
structure is A (Figure 1).34
Cannabinoids are present in Indian hemp, Cannabis satiVa
L., and have been used since antiquity as medicinal agents.1
Interest in cannabinoid pharmacology has rapidly increased since
the discovery of the endocannabinoid system (ECS), which
includes cannabinoid receptors, the endocannabinoids (anan-
damide2 and 2-arachidonoylglycerol3), metabolizing enzymes
(fatty acid amide hydrolase4 and monoglyceride lipase5), and a
specific cellular uptake system (the anandamide transporter
protein6).
To date, two distinct cannabinoid receptors, CB1 and CB2,
have been identified in mammalian tissues and cloned.7,8 These
receptors belong to the superfamily of G-protein-coupled
receptor (GPCR) seven-transmembrane receptors, which nega-
tively regulate adenylate cyclase. The CB1 receptor is mainly
located in the central nervous system, with the highest density
in the cerebellum, the basal ganglia, the substantia nigra pars
compacta, and some regions of the globus pallidus. It is also
present in peripheral organs such as the adrenal glands, bone
marrow, lung, testis, and uterus.9 Unlike CB1, the CB2 receptor
is limited essentially to the cells associated with the immune
system, like spleen, thymus, and tonsils.10 Because of the
virtually exclusive peripheral expression of CB2 and its presence
only in microglial cells in the central nervous system (CNS),
selective CB2 ligands should be devoid of the undesired central
nervous system effects typical of (-)-trans-∆9-tetrahydrocan-
nabinol (∆9-THC), the major psychoactive component of
Cannabis satiVa L.11
The finding of endogenous agonists at these receptors, the
endocannabinoids, opened new therapeutic possibilities through
the modulation of the activity of the CB receptor. Moreover,
* To whom correspondence should be addressed. For C.M.: phone,
++39 050 2219554; fax, ++39 050 2219605; e-mail, manera@farm.unipi.it.
For T.T.: phone, +39 050 2219572; fax, ++39 050 2219605; e-mail,
tuccinardi@farm.unipi.it.
The binding results showed that the naphthyridine derivatives
generally exhibit a higher affinity for the CB2 than for the CB1
receptor, and for some of these compounds the Ki(CB1)/Ki(CB2)
† Universita` di Pisa.
‡ Universita` di Cagliari.
§ Universita` di Firenze.
10.1021/jm0603466 CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/02/2006

5948 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Manera et al.
model predicted an affinity lower than 13 nM, whereas for one
compound a poor affinity was calculated.
Furthermore, our studies suggested that some features of the
1,8-naphthyridine derivatives did not seem to be important for
interaction with the CB2 receptor. In particular, the methyl group
(R3) did not seem to interact strongly with any lipophilic residues
of the CB2 receptor, and the N8 atom of the naphthyridine ring
played a secondary role, since it did not interact with any polar
residue (see Figure 3).
Figure 1. General structure of 1,8-naphthyridin-4(1H)-on-3-carbox-
amide derivatives.
The virtual screening in the CB2 receptor of compounds in
which the methyl group (R3) was removed or substituted with
a chlorine atom and of compounds in which the naphthyridin-
4-one ring was substituted with a quinolin-4-one as its central
nucleus revealed that these ligands seemed to maintain good
CB2 affinity, and in some cases the affinity seemed to be greater
than that of their methyl-substituted and naphthyridine ana-
logues.
In light of these considerations, we synthesized new quinolin-
4(1H)-on-3-carboxamide derivatives and some new 1,8-naph-
thyridin-4(1H)-on-3-carboxamide in which the R3 methyl group
was removed or substituted with a chlorine atom.
Finally, all the 1,8-naphthyridine derivatives tested could form
an intramolecular H bond between the carbonylic oxygen and
the amidic NH, creating a pseudocycle planar with the naph-
thyridine ring, and our studies suggested that this interaction
was quite strong.35
ratio was higher than 20. Furthermore, some of these compounds
exhibit a CB2 affinity value in the nanomolar range.34
We recently constructed the three-dimensional models of the
CB1 and CB2 receptors by means of a molecular modeling
procedure, and a series of CB2 ligands were docked into both
receptors, showing that the CB2 model was reliable and
predictive.35
The docking study of structure A naphthyridine derivatives34
highlighted the ligand-receptor interactions that determine an
increase in affinity and selectivity. In particular, this analysis
suggested that the preservation of good CB2/CB1 selectivity and
the improvement of the CB2 affinity seemed to require (i) the
presence of a nonaromatic R2 substituent capable of interacting
in the CB2 receptor with the nonconserved residue F5.46(197)
and (ii) a lipophilic R1 substituent with an H bond acceptor
atom capable of interacting in the CB2 receptor with the
nonconserved S3.31(112).35
Bearing this in mind, new 7-methyl-1,8-naphthyridin-4(1H)-
on-3-carboxamide derivatives were synthesized and tested. For
eight compounds in this series, the virtual screening in the CB2
To verify the ability of our CB2 model to discriminate
between active and inactive ligands and also to verify whether
the formation of a planar pseudocycle was important for
Table 1. Radioligand Binding Data of Compounds 6-20, 24-26, 29, 33, 38-40
Ki (nM)
predicted
Ki(CB2) (nM)
a,c
b,c
compd
R1
R2
R3
CB1
CB2
Ki(CB1)/Ki(CB2)
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
24
25
26
benzyl
4-methylcyclohexyl
4-methylcyclohexyl
4-methylcyclohexyl
cycloheptyl
cycloheptyl
cycloheptyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
4-methylcyclohexyl
cyclohexyl
cyclohexyl
methyl
methyl
methyl
methyl
methyl
methyl
cloro
cloro
cloro
methyl
methyl
methyl
H
H
H
methyl
methyl
methyl
o-fluorobenzyloxy
Cl
H
H
Cl
NT
NT
NT
6
4
28
4.3
11
19
23
9.5
3.6
4.0
5.8
7.8
2.7
p-fluorobenzyl
o-fluorobenzyl
benzyl
p-fluorobenzyl
o-fluorobenzyl
benzyl
p-fluorobenzyl
o-fluorobenzyl
1-ethyl-4-phenylpip
phenethyl
p-methoxybenzyl
p-fluorbenzyl
benzyl
ethylmorph
o-fluorobenzyl
ethylmorph
benzyl
o-fluorobenzyl
ethylmorph
ethylmorph
benzyl
8.7 ( 1.6
37.5 ( 5.4
143.2 ( 9.1
4.3 ( 0.6
149.4 ( 1.8
463.6 ( 1.1
495.0 ( 39.4
171.2 ( 12.3
>1000
1.4 ( 0.1
8.4 ( 0.3
5.1 ( 1.3
1.0 ( 0.1
13.4 ( 4.7
24.6 ( 4.7
21.4 ( 1.0
18.1 ( 2.7
>1000
3.6
63.6
9.3
7.8
2956.9
4.5
12.1
73.7
102.8
57.8
1013.7
777.4
1763.6
1312.6
125.4
70.0
39.4
17.9
0.69
>1000
>1000
16.3 ( 1.2
35.8 ( 2.1
13.0 ( 1.4
48.6 ( 12.0
67.2 ( 11.6
>1000
>62
>28
29
>21
>15
384.1 ( 25.3
>1000
>1000
>1000
>1000
>1000
>1000
>1000
29
33
38
39
NT
NT
4-methylcyclohexyl
cyclohexyl
cyclohexyl
>1000
NT
>1000
>1000
40.5 ( 7.7
NT
4.8 ( 0.4
3.3 ( 0.4
>25
>210
>303
40
ethylmorph
cyclohexyl
SDEP
4132
4232
ACEA
JWH-133
ethylmorph
benzyl
cyclopentyl
cyclohexyl
methyl
methyl
>1000
50 ( 4
>20
13
0.03
7.0
127 ( 10
3.9 ( 0.2
458.0( 15.1
10 ( 0.5
120.8 ( 14.5
65 ( 8.7
a Affinity of compounds for CB1 receptor was evaluated using mouse brain membranes and [3H]CP-55,940. b Affinity of compounds for CB2 receptor
was evaluated using mouse spleen and [3H]CP-55,940. c NT ) not tested because of insolubility in the solvent normally used in binding assays.

Carboxamide DeriVatiVes as Agonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 5949
Scheme 1. Synthesis of N1-Substituted
Scheme 3. Synthesis of 1-(o-Fluorbenzyl)-7-
(o-fluorobenzyloxy)-1,8-naphthyridin-4(1H)-on-3-carboxamide
Derivative 29a
1,8-Naphthyridin-4(1H)-on-3-carboxamide Derivatives 6-20a
a Reagents and conditions: (i) NaNO2, H2SO4, 4 h, room temp; (ii)
o-fluorobenzyl chloride, NaH, DMF, 72 h, 80 °C.
Scheme 4. Synthesis of 7-Chloro-1,8-naphthyridin-
4(1H)-on-3-carboxamide Derivative 33a
a Reagents and conditions: (i) DMF, NaH, R3Cl; (ii) MeOH, H2, Pd/C,
3 h.
Scheme 2. Synthesis of N1-Substituted 4-Hydroxy-7-methyl-
1,2,3,4-tetrahydro-1,8-naphthyridin-3-carboxamides 24-26a
a Reagents and conditions: (i) 4-methylcyclohexylamine, 120 °C, 24 h;
(ii) NaNO2, HCl, 40 °C, 3 h; (iii) 4-(2-chloroethyl)morpholine, NaH, DMF,
50 °C, 24 h.
Scheme 5. Synthesis of N-Substituted Quinolin-4(1H)-on-3-
Carboxamides 38-40a
a Reagents and conditions: (i) NaBH4, room temp, 12 h.
interaction inside the CB2 receptor, we synthesized and tested
some new compounds characterized by the presence of a
hydroxy group in position 4 of the naphthyridine nucleus, instead
of the carbonyl oxygen atom, and by partial removal of the
aromaticity of the cycle (for which, as shown in Table 1, virtual
screening predicted a CB2 affinity greater than 750 nM).
Chemistry
The compounds described in this study are shown in Table
1, and their syntheses are outlined in Schemes 1-5. The
treatment of carboxamide derivative 1, 2, 3, 4,34 and 5 in
anhydrous DMF with NaH for 1 h and then with the appropriate
benzyl chloride or arylalkyl chloride or 4-(2-chloroethyl)-
morpholine provided the desired 1,8-naphthyridin-4-one deriva-
tives 6-20 (Scheme 1). 1-(2-Chloroethyl)-4-phenylpiperazine,
which was needed to prepare 15, was synthesized following
the method reported in the literature.36 The carboxamide 5 was
obtained by dehalogenation of 434 with H2 in the presence of
Pd/C as a catalyst.
a Reagents and conditions: (i) cyclohexylamine, 120 °C, 24 h; (ii) NaH,
DMF, R1Cl, 50 or 80 °C, 24 h.
N1-substituted 4-hydroxy-7-methyl-1,2,3,4-tetrahydro-1,8-naph-
thyridin-3-carboxamides 24-26 (Scheme 2). As reported in
Scheme 3, the diazotization of compound 2734 with NaNO2 in
aqueous 96% sulfuric acid gave the 7-hydroxy derivative 28,
which, by reaction with o-fluorobenzyl chloride under the same
conditions described above, gave N-cyclohexyl-1-(o-fluorben-
zyl)-7-(o-fluorobenzyloxy)-1,8-naphthyridin-4(1H)-on-3-car-
boxamide (29).
The reaction of the 1,8-naphthyridin-4-one derivatives 21-
2334 with sodium borohydride in anhydrous ethanol gave the

5950 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Manera et al.
Table 2. Effect of 1,8-Naphthyridine Derivatives 7 and 8 on [35S]GTPγS Binding in Mouse Brain Membranes
compd
R1
R2
R3
EC50 a (nM)
Emax a (%)
7
8
p-fluorobenzyl
o-fluorobenzyl
4-methylcyclohexyl
4-methylcyclohexyl
methyl
methyl
27 ( 4.7*
138 ( 21**
204 ( 24
172 ( 9.6
161 ( 2.3
187 ( 19
WIN-55,212-2
a Data are the mean ( SEM of at least four experiments, each performed in duplicate. Compound-mediated [35S]GTPγS binding data represent percentage
of stimulation over basal values (set as 100%). Emax and EC50 were determined by nonlinear regression curve fit (GraphPad Prism). ANOVA: F(2,8) )
21.97, P < 0.002; (/) P < 0.01 with respect to WIN-55212,2 and 7; (//) P < 0.05 with respect to WIN-55212,2 (Newman-Keuls test).
The 7-acetamido-1,8-naphthyridin-4(1H)-on-3-carboxylic acid
ethyl ester 3037 was heated at 120 °C in a sealed tube with
4-methylcyclohexylamine (Scheme 4). Under these conditions,
the hydrolysis of the acetamido group also takes place, and thus,
the 7-amino-3-carboxamide derivative 31 was obtained. Dia-
zotization of this compound carried out in aqueous 37%
hydrochloride acid afforded the 7-chloro derivative 32, which,
by reaction with 4-(2-chloroethyl)morpholine at 50 °C, gave
the desired compound 33 (Scheme 4). As reported in Scheme
5, the reaction of quinolin-4(1H)-on-3-carboxylic acid ethyl ester
34 or 3538 in a sealed tube with cychlohexylamine at 120 °C
afforded the corresponding 3-carboxamide derivatives 36 or 37,
respectively, which by treatment with NaH and then with benzyl
chloride or 4-(2-chloroethyl)morpholine gave the desired com-
pounds 38-40.
is clear from a comparison of compounds 7-11 with the
corresponding 3-carboxycyclohexylamide derivatives previously
studied.34
Furthermore, the substitution of the methyl group in position
7 of the 1,8-naphthyridine nucleus with an atom of chlorine, or
the lack of any substituent in the same position, reduces the
CB1 receptor affinity, as can be seen from a comparison of
compounds 12-14, 18-20, and 33 with the corresponding
7-methyl-1,8-naphthyridine derivatives previously studied.34
Finally, the 4-hydroxy-1,2,3,4-tetrahydro-1,8-naphthyridine
derivatives 24-26 and the quinolin-4(1H)-one derivatives 39
and 40 lack any affinity toward the CB1 receptor, with a Ki
value greater than 1000.
These results show that some of the compounds studied
possess an interesting affinity at the CB1 receptor. In particular,
the 1,8-naphthyridine derivatives 7, 8, and 10 exhibit a consider-
able affinity but are not selective for this receptor.
Results and Discussion
Influence of CB1 Ligands on [35S]GTPγS Binding. Among
all compounds showing a high affinity for CB1 receptors, 7 and
8 were selected with the aim of investigating their agonistic or
antagonistic functions at the CB1 receptor by means of a [35S]-
GTPγS binding assay, using mouse brain membranes. This assay
provides a functional measure of the interaction of the receptor
and the G protein. The first step in the activation of intracellular
signaling by the G-protein-coupled receptor is the induction of
an exchange of GDP for GTP on the guanine nucleotide binding
site of the R subunit of a heterotrimeric G protein. The effects
of various cannabinoid receptor agonists on GDP-GTP exchange
can be determined from agonist-induced binding of the non-
hydrolyzable GTP analogue [35S]GTPγS.40,41
WIN-55,212-2, a CB1 receptor agonist, was used as the
reference compound. [35S]GTPγS binding was stimulated in a
concentration-dependent and saturable manner by 7, 8, and
WIN-55,212-2 (see Table 2). Maximal stimulation (Emax) of
[35S]GTPγS binding by WIN-55,212-2 was 187 ( 19%, with
an EC50 of 204 ( 24 nM. The Emax values for 7 and 8 were
172 ( 9.6% and 161 ( 2.3%, respectively, with no significant
difference from the Emax produced by WIN-55,212-2 (ANO-
VA: F(2,8) ) 1.04, P ) 0.4068). The EC50 values for 7 and 8
were 27 ( 4.7 and 138 ( 21 nM, respectively, both of which
were more potent than WIN-55,212-2 (F(2,8) ) 21.97, P <
0.002]. These findings clearly indicate that 7 and 8 are agonists
at the CB1 receptor. Furthermore, we may hypothesize that the
other 1,8-naphthyridin-4(1H)-on-3-carboxamide derivatives 6,
9-20, 29, and 33, which are structurally analogous to 7 and 8,
possess the same kind of activity.
Pharmacology. Affinities at CB1 and CB2 receptors for the
1,8-naphthyridin-4(1H)-on-3-carboxamide derivatives 6-20,
24-26, 29, and 33 and for the quinolin-4(1H)-on-3-carboxamide
derivatives 38-40 were determined by measuring their ability
to displace [3H]CP-55,940 from its binding site in a membrane
preparation from mouse brain (minus cerebellum) and mouse
spleen homogenate, respectively. [3H]CP-55,940 binding was
carried out following a modified version of the method
previously described.39 The results of these determinations are
summarized in Table 1. The Ki values of ACEA and JWH-
133, as reference compounds at the CB1 and CB2 receptors,
respectively, are also included in Table 1.
Finally, the question of the 1,8-naphthyridine-4-one deriva-
tives functionality at the CB1 and CB2 receptors was investigated
by using a [35S]GTPγ binding assay40,41 and functional studies
on human basophils, respectively.42,43
CB1 Receptor Affinity. The results reported in Table 1 for
the 1,8-naphthyridin-4(1H)-on-3-carboxamide derivatives 6-20,
29, and 33 show that, as previously reported,34 the compounds
with an ethylmorpholino group in position 1 (20 and 33),
regardless of the nature of the carboxyamido substituent in
position 3 and of the substituent in position 7 of the heterocyclic
nucleus, exhibited a poor affinity, with Ki values greater than
1000. Analogously, compounds 15, 16, and 17, which bear in
position 1 of the naphthyridine nucleus a 1-ethyl-4-phenylpip-
erazinyl group, a phenethyl group, and a p-methoxybenzyl
group, respectively, possess a very low affinity, with Ki values
greater than 1000. The presence in position 1 of a benzyl group,
whether substituted or not, led to compounds with an interesting
affinity. In particular, the p-fluorobenzyl derivatives 7 and 10
showed the highest affinity toward the CB1 receptor, with Ki
of 8.7 and 4.3 nM, respectively.
CB2 Receptor Affinity. The results obtained indicate that,
in agreement with previous report,34 the N-benzyl-1,8-naphthy-
ridine derivatives possess a higher affinity than the N-ethyl-
morpholino derivatives, as is clear from a comparison of
compounds 7, 8, 18, and 19 with 20 and 33. For the N-benzyl-
1,8-naphthyridine derivatives (7-14, 17-19), the presence of
an atom of fluorine on the benzyl increases the affinity, above
all if the substitution is in the para position. In particular, the
p-fluorobenzyl-1,8-naphthyridine derivatives 7 (Ki ) 1.4 nM)
As regards the structural modifications in position 3 of the
1,8-naphthyridine nucleus, the substitution of the carboxycy-
clohexylamide group with a carboxy-4-methylcyclohexylamide
group or a carboxycyclohepthylamide group leads to compounds
that exhibit an increase in affinity toward the CB1 receptor, as

Carboxamide DeriVatiVes as Agonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 5951
and 10 (Ki ) 1.0 nM) proved to be the compounds with the
highest affinity in this series. Furthermore, the N-phenethyl-
1,8-naphthyridine derivative 16 showed a good affinity, with a
Ki of 16.3 nM. In contrast, the compound bearing a 1-ethyl-4-
phenylpiperazinyl group in position 1 of the naphthyridine
nucleus (15) possesses a very low affinity, with a Ki value
greater than 1000.
As had previously been found for the CB1 receptor, the
substitution of the carboxycyclohexylamide group in position
3 of the 1,8-naphthyridine nucleus with a carboxy-4-methyl-
cyclohexylamide or a carboxycyclohepthylamide group deter-
mines an increase in the affinity toward the CB2 receptor, as
confirmed by a comparison of compounds 7-11 with the
corresponding 3-carboxycyclohexylamide derivatives previously
studied.34
Furthermore, the substitution of the methyl group in position
7 of the 1,8-naphthyridine nucleus with an atom of chlorine
(12-14 and 33), or the lack of any substituent in the same
position (18-20), generally determines the maintenance or an
increase in the affinity (except for compounds 13 and 19, which
showed a 4-fold and 5-fold decrease in affinity compared with
the methyl analogues34).
As in the case of the CB1 receptor, 4-hydroxy-1,2,3,4-
tetrahydro-1,8-naphthyridine derivatives 24-26 exhibit a poor
affinity toward the CB2 receptor, with Ki values greater than
1000.
Finally, compounds 39 and 40 in which the naphthyridin-4-
one system was substituted by the quinoline-4-one, possess a
remarkable affinity, as suggested by virtual screening, with Ki
values of 4.8 and 3.3 nM, respectively.
Very recently, quinolin-4-one derivatives,44 with structures
similar to those of 39 and 40, were synthesized; however, our
compounds were characterized by different substituents in
positions 1, 3, and 7 of the heterocyclic nucleus.
Figure 2. Expression of CD203c by human basophils activated with
anti-IgE (1 µg/mL) is reduced by compounds 39 (A) and 41 (B) in a
dose-related fashion. The inhibitory effect of compounds 39 and 41
on basophil CD203c expression is reverted by SR 144528 (100 nM),
a CB2 antagonist, but not by AM 251 (100 nM), a CB1 antagonist. The
values are the mean ( SEM of six independent experiments performed
in triplicate: (//) P < 0.001.
Most compounds showed a good affinity for the CB2 receptor.
In particular, the 1,8-naphthyridin-4-one derivatives 7-11 and
the quinolin-4-one derivatives 39 and 40 possess a very high
affinity, with Ki values less than 10 nM.
As for the selectivity toward the CB2 receptor, the 1,8-
naphthyridine derivatives 16-19 and 33 show good selectivity,
with Ki(CB1)/Ki(CB2) > 21. Furthermore, quinolin-4-one de-
rivatives 39 and 40 exhibited very significant CB2 receptor
selectivity, with Ki(CB1)/Ki(CB2) greater than 210 and greater
than 303, respectively, which were higher than reports for the
analogous compounds (Ki(CB1)/Ki(CB2) ) 143 for the best
compound).44
Test for CB2 Functionality. To assess the functionality of
the studied compounds at CB2 receptors, functional studies on
human basophils were performed. Activation of CB2 receptors
is known to down-regulate the immunological activation of
guinea pig mast cells and human basophils.42,43
N-Cyclohexyl-1-benzylquinolin-4(1H)-on-3-carboxamide (39)
and N-cyclopentyl-7-methyl-1-(2-morpholin-4-ylethyl)-1,8-
naphthyridin-4(1H)-on-3-carboxamide (41),34 which is structur-
ally analogous to the 1,8-naphthyridin-4(1H)-on-3-carboxamide
derivatives studied in this paper, were used for functional
studies. Human basophils, pretreated with these compounds (1
nM to 1 µM, 30 min of preincubation, 37 °C), showed a reduced
expression of CD203c in response to immunological stimulation
(anti-IgE 1 µg/mL, 30 min, 37 °C). The inhibition was reversed
by the selective CB2 antagonist SR 144528 (SR, 100 nM, 30
min of preincubation, 37 °C) but not by AM251 (AM, 100 nM,
30 min of preincubation, 37 °C), a selective CB1 antagonist (see
Figure 2).
These results show that compounds 39 and 41 exert a CB2-
mediated inhibitory action on immunological human basophil
activation. Furthermore, we hypothesize that both the quinolin-
4(1H)-on-3-carboxamide 40 and the 1,8-naphthyridin-4(1H)-
on-3-carboxamide derivatives reported in this work possess the
same kind of activity.
Molecular Modeling. With the AUTODOCK 3.0 program,45
the compounds shown in Table 1 were docked into the CB2
receptor, and their activities were predicted on the basis of the
published computational model35 (see Experimental Section for
details). As indicated by the SDEP value (0.69) reported in Table
1, there was quite a good correlation between the experimental
and the calculated Ki. Furthermore, all the ligands with a CB2
affinity higher than 1000 nM were predicted to have an affinity
higher than 750 nM.
As suggested by our model, the compound with the best CB2
affinity was 10. The docking showed that the CB2 binding
pocket was delimited by TM3, TM4, TM5, and TM6, and the
cycloheptyl substituent of compound 10 was directed toward
the intracellular side of the receptor, interacting with W5.43-
(194) and F5.46(197) (see Figure 3). As for the p-F-benzyl
group, it interacted in a lipophilic pocket formed by L3.27-
(108), P4.60(168), and L4.61(169), and the fluorine atom formed
an H bond with S3.31(112).
The docking of compound 40 (see Figure 4), the most CB2
selective ligand in this series, also revealed that the presence
of the chlorine atom might contribute to the increase in CB2
affinity and CB2 selectivity, since it might interact with the
nonconserved S6.58(268) (aspartate in the CB1 receptor).

5952 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Manera et al.
Figure 3. Compound 10 docked into the CB2 receptor model.
Figure 5. Compounds 42 (magenta) and 25 (sky-blue) docked into
the CB2 receptor model.
the values predicted by the docking study. In particular,
compound 10, which presented p-fluorobenzyl and carboxycy-
cloheptylamide substituents bound in the 1 and 3 positions of
the 1,8-naphthyiridine-4-one nucleus, showed a high CB2 affinity
with a Ki value of 1 nM.
The substitution of the naphthyridine-4-one nucleus with the
quinoline-4-one system determined a general increase in CB2
affinity. For compound 40, the good CB2 affinity was also
accompanied by a high selectivity toward the CB2 receptor, and
the docking studies suggested that the interaction of the chlorine
atom in position 7 of the heterocyclic nucleus with the
nonconserved residue S6.58(268) in the CB2 receptor seemed
to be one of the reasons for the high selectivity value.
Finally, the low affinity shown by the new 4-hydroxytet-
rahydro-1,8-naphthyridine derivatives confirmed the hypothesis
about the fundamental role of the presence of a planar
pseudocycle with the naphthyridine nucleus obtained by an
intramolecular H bond between the carbonylic oxygen and the
amidic NH.
Figure 4. Compound 40 docked into the CB2 receptor model.
Finally, the docking of the inactive compounds 24-26
revealed that the lack of planarity determines a different position
of the central lipophilic core, determining weaker interactions
with the receptor. As shown in Figure 5, the central core of 26,
compared with the position of the naphthyridine ring of
N-cyclohexyl-7-methyl-1-benzyl-1,8-naphthyridin-4(1H)-on-3-
carboxamide (42),34 was shifted further way from TM3 and
directed toward TM5, determining weaker interactions with
M6.55(265) and L3.27(107); furthermore, this arrangement
determined a weaker interaction of the cyclohexyl ring with
F5.46(197), at a distance of 4.7 Å (while for 42 the distance is
4.0 Å).
These results provide an interesting addition to currently
available structure-activity relationships for cannabinoid agonist
ligands, opening up a new field of research for designing new
cannabinoid receptor agonists characterized by a high selectivity
toward the CB2 receptor.
Experimental Section
Chemistry. Melting points were determined on a Kofler hot stage
apparatus and are uncorrected. IR spectra in Nujol mulls were
recorded on an ATI Mattson Genesis series FTIR spectrometer.
1H NMR spectra were recorded with a Bruker AC-200 spectrometer
in δ units from TMS as an internal standard. Mass spectra were
obtained with a Hewlett-Packard MS system 5988. Elemental
analysis results (C, H, N) were within (0.4% of theoretical values
and were performed on a Carlo Erba elemental analyzer model 1106
apparatus.
General Procedure for the Synthesis of N1-Substituted 1,8-
Naphthyridine Derivatives (6-20 and 33). An amount of 1.2
mmol of NaH was added to a solution of 1 mmol of 5 or of
7-methyl- (1, 2, and 334) or of 7-chloro-1,8-naphthyridine-3-
carboxamide derivative (434 and 32) in 10 mL of dry N,N-
dimethylformamide. After 1 h, the appropriate chloride (1 mmol)
was added and the mixture was stirred for 24 h at room temperature
for compounds 6-11, 15, 18, and 19 or at 50 °C for compounds
12-14, 16, 17, and 33 or at 50 °C for 48 h for 20. The reaction
mixture, after cooling in the cases of 12-14, 17, and 33, was treated
Conclusions
In the present study, by means of a structure-based approach,
we tried to improve the activity and selectivity of 1,8-
naphthyridin-4(1H)-on-3-carboxamide derivatives, which had
proved to be a new class of CB2 ligands.34
For this purpose, following the suggestion obtained from the
docking of ligands into a CB2 receptor model,35 new 1,8-
naphthyridin-4(1H)-on-3-carboxamide derivatives and quinolin-
4(1H)-on-3-carboxamide derivatives were designed, synthesized,
and tested on the CB1 and CB2 receptors.
Some of these compounds showed a good selectivity toward
the CB2 receptor and a high CB2 affinity, in agreement with

Carboxamide DeriVatiVes as Agonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 5953
with water, and the precipitate formed was collected by filtration,
whereas for 16, the solvent was evaporated in vacuo and the solid
obtained was treated with water and collected by filtration.
N-(4′-Methylcyclohexyl)-1-benzyl-7-methyl-1,8-naphthyridin-
4(1H)-on-3-carboxamide (6). Yield 0.310 g, 80%; mp 239-241
3H, Ar), 4.70 (m, 2H, CH2), 3.80 (m, 1H, CH), 3.03 (m, 4H,
piperazinyl), 2.73 (m, 2H, CH2), 2.66 (s, 3H, CH3), 2.60 (m, 4H,
piperazinyl), 1.85-1.08 (m, 10H, cyclohexyl). Anal. (C28H35N5O2)
C, H, N
N-Cyclohexyl-7-methyl-1-phenethyl-1,8-naphthyridin-4(1H)-
on-3-carboxamide (16). Purified by flash chromatography (ethyl
acetate/hexane, 2:3), yield 0.110 g, 28%; mp 148-150 °C (crystal-
1
°C (crystallized from ethyl acetate); MS m/z 389 (M+); H NMR
δ 10.20, 9.75 (2d, 1H, NH), 9.08, 9.06 (2s, 1H, H2), 8.52, 8.60
(2d, 1H, H5), 7.48 (d, 1H, H6), 7.33 (m, 5H, Ar), 5.80 (s, 2H, CH2),
4.12, 3.69 (2m, 1H, CH), 2.63 (s, 3H, CH3), 2.20-0.86 (m, 12H,
cyclohexyl + CH3). Anal. (C24H27N3O2) C, H, N.
1
lized from hexane); MS m/z 389 (M+); H NMR δ 9.86 (d, 1H,
NH), 8.85 (s, 1H, H2), 8.54 (d, 1H, H5), 7.48 (d, 1H, H6), 7.24 (m,
5H, Ar), 4.76 (m, 2H, CH2), 3.90 (m, 1H, CH), 3.10 (m, 2H, CH2),
2.69 (s, 3H, CH3), 1.85-1.23 (m, 10H, cyclohexyl). Anal.
(C24H27N3O2) C, H, N.
N-(4′-Methylcyclohexyl)-1-(p-fluorobenzyl)-7-methyl-1,8-naph-
thyridin-4(1H)-on-3-carboxamide (7). Yield 0.270 g, 66%; mp
1
167-169 °C (crystallized from n-hexane); MS m/z 407 (M+); H
N-Cyclohexyl-7-methyl-1-(4-methoxybenzyl)-1,8-naphthyri-
din-4(1H)-on-3-carboxamide (17). Purified by flash chromatog-
raphy (ethyl acetate/hexane, 2: 1), 0.283 g, 70%; mp 170-172 °C
NMR δ 10.18, 9.77 (2d, 1H, NH), 9.12, 9.10 (2s, 1H, H2), 8.59,
8.52 (2d, 1H, H5), 7.50 (m, 3H, Ar + H6), 7.16 (m, 2H, Ar), 5.77
(s, 2H, CH2), 4.18, 3.70 (2m, 1H,CH), 2.65 (s, 3H, CH3), 1.98-
0.86 (m, 12H, cyclohexyl + CH3). Anal. (C24H26FN3O2) C, H, N.
N-(4′-Methylcyclohexyl)-1-(o-fluorobenzyl)-7-methyl-1,8-naph-
thyridin-4(1H)-on-3-carboxamide (8). Yield 0.245 g, 60%; mp
1
(crystallized from hexane); MS m/z 405 (M+); H NMR: δ 9.90
(d, 1H, NH), 9.05 (s, 1H, H2), 8.54 (d, 1H, H5), 7.47 (d, 1H, H6),
7.35 (d, 2H, Ar), 6.89 (d, 2H, Ar), 5.70 (s, 2H, CH2), 3.80 (m, 1H,
CH), 3.70 (s, 3H, OCH3), 2.67 (s, 3H, CH3), 1.90-1.30 (m, 10H,
cyclohexyl). Anal. (C24H27N3O3) C, H, N.
1
183-185 °C (crystallized from n-hexane); MS m/z 407 (M+); H
NMR δ 10.18, 9.77 (2d, 1H, NH), 9.09, 9.07 (2s, 1H, H2), 8.58,
8.52 (2d, 1H, H5), 7.46 (d, 1H, H6), 7.36-7.14 (m, 4H, Ar), 5.81
(s, 2H, CH2), 4.15, 3.70 (2m, 1H, CH), 2.61 (s, 3H, CH3), 2.00-
0.86 (m, 12H, cyclohexyl + CH3). Anal. (C24H26FN3O2) C, H, N.
N-Cycloheptyl-1-benzyl-7-methyl-1,8-naphthyridin-4(1H)-on-
3-carboxamide (9). Yield 0.225 g, 58%; mp 198-200 °C (crystal-
N-Cyclohexyl-1-(p-fluorbenzyl)-1,8-naphthyridin-4(1H)-on-3-
carboxamide (18). Yield 0.185 g, 49%; mp 193-195 °C (crystal-
1
lized from hexane); MS m/z 379 (M+); H NMR δ 9.80 (d, 1H,
NH), 9.15 (s, 1H, H2), 8.90 (dd, 1H, H7), 8.67 (dd, 1H, H5), 7.62
(m, 1H, H6), 7.37 (m, 2H, Ar), 7.15 (m, 2H, Ar), 5.81 (s, 2H, CH2),
3.85 (m, 1H, CH), 1.86-1.23 (m, 10H, cyclohexyl). Anal. (C22H22-
FN3O2) C, H, N.
1
lized from cyclohexane); MS m/z 389 (M+); H NMR δ 9.88 (d,
1H, NH), 9.07 (s, 1H, H2), 8.55 (d, 1H, H5), 7.48 (d, 1H, H6), 7.33
(m, 5H, Ar), 5.79 (s, 2H, CH2), 4.15 (m, 1H, CH), 2.64 (s, 3H,
CH3), 1.85-1.39 (m, 12H, cycloheptyl). Anal. (C24H27N3O2) C,
H, N.
N-Cycloheptyl-1-(p-fluorobenzyl)-7-methyl-1,8-naphthyridin-
4(1H)-on-3-carboxamide (10). Yield 0.300 g, 75%; mp 194-196
°C (crystallized from hexane); MS m/z 407 (M+); 1H NMR δ 9.91
(d, 1H, NH), 9.10 (s, 1H, H2), 8.55 (d, 1H, H5), 7.48 (m, 3H, Ar
+H6), 7.16 (m, 2H, Ar), 5.77 (s, 2H, CH2), 4.15 (m, 1H, CH),
2.64 (s, 3H, CH3), 1.85-1.39 (m, 12H, cycloheptyl). Anal. (C24H26-
FN3O2) C, H, N.
N-Cycloheptyl-1-(o-fluorbenzyl)-7-methyl-1,8-naphthyridin-
4(1H)-on-3-carboxamide (11). Yield 0.255 g, 63%; mp 188-189
°C (crystallized from cyclohexane); MS m/z 407 (M+); 1H NMR δ
9.90 (d, 1H, NH), 9.07 (s, 1H, H2), 8.55 (d, 1H, H5), 7.46 (d, 1H,
H6), 7.32-7.14 (m, 4H, Ar), 5.81 (s, 2H, CH2), 4.15 (m, 1H, CH),
2.61 (s, 3H, CH3), 1.85-1.39 (m, 12H, cycloheptyl). Anal. (C24H26-
FN3O2) C, H, N.
N-Cyclohexyl-1-benzyl-7-chloro-1,8-naphthyridin-4(1H)-on-
3-carboxamide (12). Yield 0.275 g, 70%; mp 258-260 °C
(crystallized from hexane); MS m/z 395 (M+); 1H NMR δ 9.72 (d,
1H, NH), 9.12 (s, 1H, H2), 8.66 (d, 1H, H5), 7.67 (d, 1H, H6), 7.32
(m, 5H, Ar), 5.73 (s, 2H, CH2), 3.85 (m, 1H, CH), 1.85-1.32 (m,
10H, cyclohexyl). Anal. (C22H22ClN3O2) C, H, N.
N-Cyclohexyl-1-benzyl-1,8-naphthyridin-4(1H)-on-3-carbox-
amide (19). Yield 0.200 g, 55%; mp 181-183 °C (crystallized from
hexane); MS m/z 361 (M+); 1H NMR δ 9.95 (d, 1H, NH), 9.12 (s,
1H, H2), 8.90 (dd, 1H, H7), 8.70 (dd, 1H, H5), 7.70 (m, 1H, H6),
7.29 (m, 5H, Ar), 5.84 (s, 2H, CH2), 3.85 (m, 1H, CH), 1.86-1.30
(m, 10H, cyclohexyl). Anal. (C22H23N3O2) C, H, N.
N-Cyclohexyl-1-(2-morpholin-4-yl-ethyl)-1,8-naphthyridin-
4(1H)-on-3-carboxamide (20). Yield 0.155 g, 40%; mp 142-144
°C (crystallized from hexane); MS m/z 384 (M+); 1H NMR δ 9.84
(d, 1H, NH), 8.98 (s, 1H, H2), 8.90 (dd, 1H, H7), 8.68 (dd, 1H,
H5), 7.60 (m, 1H, H6), 4.66 (t, 2H, CH2), 3.86 (m, 1H, CH), 3.49
(m, 4H, morpholine), 2.70 (m, 2H, CH2), 2.44 (m, 4H, morpholine),
1.85-0.82 (m, 10H, cyclohexyl). Anal. (C21H28N4O3 ) C, H, N.
N-(4′-Methylcyclohexyl)-7-chloro-1-(2-morpholin-4-yl-ethyl)-
1,8-naphthyridin-4(1H)-on-3-carboxamide (33). Purified by flash
chromatography (ethyl acetate/hexane, 1: 1), yield 0.130, 30%; mp
191-193 °C (crystallized from cyclohexane); MS m/z 432 (M+);
1H NMR δ 10.20, 10.00 (2d, 1H, NH), 8.65, 8,61 (2s, 1H, H2),
8.20, 8,18 (2d, 1H, H5), 6.87 (d, 1H, H6), 4.51 (m, 2H, CH2), 4.00
(m, 1H, CH), 3.51 (m, 4H, morpholine), 2.67 (m, 2H, CH2), 2.44
(m, 4H, morpholine), 1.89-0.76 (m, 12H, cyclohexyl + CH3). Anal.
(C22H29ClN4O3 ) C, H, N.
N-Cyclohexyl-1,8-naphthyridin-4(1H)-on-3-carboxamide (5).
A solution of 7-chloronaphthyridine 434 (0.40 g, 1.31 mmol) in
methanol (20 mL) was submitted to hydrogenation in the presence
of 10% Pd/C (0.04 g) at room pressure and temperature for 3 h.
The catalyst was filtered off, and the solvent was evaporated to
dryness under reduced pressure to give a residual solid, which was
purified by flash chromatography (ethyl acetate) and crystallized
from hexane to give 5 (0.110 g, 31%): mp 215-218 °C; 1H NMR
δ 9.85 (d, 1H, NH), 8.84 (dd, 1H, H7), 8.68 (s, 1H, H2), 8.65 (dd,
1H, H5), 7.55 (m, 1H, H6), 3.85 (m, 1H, CH), 1.86-1.22 (m, 10H,
cyclohexyl). Anal. (C15H17N3O2) C, H, N.
General Procedure for the Preparation of N1-Substituted
4-Hydroxy-7-methyl-1,2,3,4-tetrahydro-1,8-naphthyridin-3-car-
boxamides 24-26. NaBH4 (0.30 g, 8 mmol) was added to a
solution of the appropiate 7-methyl-1,8-naphthyridin-4(1H)-on-3-
carboxamide derivatives 21-2334 (0.38 mmol) in absolute ethanol
(7.5 mL), and the mixture was stirred at room temperature for 12
h. The organic solvent was evaporated from the reaction mixture
under reduced pressure to obtain a residue, which was treated with
H2O. In the cases of 24 and 26, the solid precipitate obtained was
collected by filtration and purified by crystallization from hexane,
whereas for 25 the mixture was extracted with chloroform, the
N-Cyclohexyl-7-chloro-1-(p-fluorbenzyl)-1,8-naphthyridin-
4(1H)-on-3-carboxamide (13). Purified by flash chromatography
(ethyl acetate/hexane, 5:6), yield 0.100 g, 25%; mp 198-200 °C
1
(crystallized from cyclohexane); MS m/z 413 (M+); H NMR δ
9.72 (d, 1H, NH), 9.14 (s, 1H, H2), 8.65 (d, 1H, H5), 7.68 (d, 1H,
H6), 7.44 (m, 2H, Ar), 7.18 (m, 2H, Ar), 5.71 (s, 2H, CH2), 3.89
(m, 1H, CH), 1.90-1.17 (m, 10H, cyclohexyl). Anal. (C22H21-
ClFN3O2) C, H, N.
N-Cyclohexyl-7-chloro-1-(o-fluorbenzyl)-1,8-naphthyridin-
4(1H)-on-3-carboxamide (14). Yield 0.300 g, 73%; mp 198-200
°C (crystallized from cyclohexane); MS m/z 413 (M+); 1H NMR δ
9.72 (d, 1H, NH), 9.11 (s, 1H, H2), 8.65 (d, 1H, H5), 7.67 (d, 1H,
H6), 7.41-7.12 (m, 4H, Ar), 5.76 (s, 2H, CH2), 3.85 (m, 1H, CH),
1.98-1.32 (m, 10H, cyclohexyl). Anal. (C22H21ClFN3O2) C, H, N.
N-Cyclohexyl-7-methyl-1-[2-(4-phenylpiperazin-1-yl)ethyl]-
1,8-naphthyridin-4(1H)-on-3-carboxamide (15). Purified by flash
chromatography (ethyl acetate/hexane/triethylamine, 10:1:0.2), yield
0.120 g, 25%; mp 147-149 °C (crystallized from hexane); MS
m/z 473 (M+); 1H NMR δ 9.87 (d, 1H, NH), 8.94 (s, 1H, H2), 8.53
(d, 1H, H5), 7.46 (d, 1H, H6), 7.18 (m, 2H, Ar), 6.90-6.71 (m,

5954 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Manera et al.
organic solution was dried (MgSO4) and evaporated to dryness
under reduced pressure, and the crude solid was purified by
crystallization from cyclohexane.
NH), 8.64 (m, 2H, H2 + H5), 7.62 (d, 1H, H6), 3.80 (m, 1H, CH),
1.97-1.00 (m, 9H, cyclohexyl), 0.89, 0.92 (2d, 3H, CH3). Anal.
(C16H18ClN3O2 ) C, H, N.
N-Cyclohexyl-1-(o-fluorbenzyl)-4-hydroxy-7-methyl-1,2,3,4-
tetrahydro-1,8-naphthyridin-3-carboxamide (24). Yield 0.170 g,
N-Cyclohexyl-quinolin-4(1H)-on-3-carboxamide (36) and N-Cy-
clohexyl-7-chloroquinolin-4(1H)-on-3-carboxamide (37). A mix-
ture of 1 mmol of quinoline-3-carboxylic acid ethyl ester 34 or
3538 and 10 mmol of cyclohexylamine in a sealed tube 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 purified by crystallization from ethyl acetate. 36: yield
1
87%; MS m/z 397 (M+); H NMR δ 7.82 (m, 1H, NH), 7.40 (d,
1H, H5), 7.30-7.08 (m, 4H, Ar), 6.44 (d, 1H, H6), 5.63 (d, 1H,
OH), 4.95-4.69 (m, 3H, CH2 + H4), 3.56 (m, 1H, CH), 3.30 (m,
2H, 2H2), 2.23 (s, 3H, CH3), 1.68-1.11 (m, 11H, cyclohexyl +
H3). Anal. (C23H28FN3O2 ) C, H, N.
4-Hydroxy-N-(4-methylcyclohexyl)-1-(2-morpholin-4-ylethyl)-
7-methyl-1,2,3,4-tetrahydro-1,8-naphthyridin-3-carboxamide (25).
Yield 0.100 g, 61%; MS m/z 416 (M+).1H NMR δ 7.81 (m, 1H,
NH), 7.34 (d, 1H, H5), 6.35 (d, 1H, H6), 5.55 (m, 1H, OH), 4.61
(m, 1H, H4), 3.78 (m, 1H, CH), 3.57-3.33 (m, 8H, morpholine +
NCH2 + 2H2), 2.45 (m, 6H, morpholine + CH2), 2.22 (s, 3H, CH3),
1.70-1.05 (m, 13H, cyclohexyl + CH3 + H3). Anal. (C23H36N4O3
) C, H, N.
1
0.230 g, 88%; mp 112-114 °C; H NMR δ 10.10 (d, 1H, NH),
8.72 (s, 1H, H2), 8.24 (d, 1H, Ar), 7.70 (m, 2H, Ar), 7.47 (m, 1H,
Ar), 3.82 (m, 1H, CH), 1.86-1.31 (m, 10H, 5CH2). Anal.
(C16H18N2O2 ) C, H, N. 37: yield 0.275 g, 90%; mp 132-135 °C;
1H NMR δ 10.18 (d, 1H, NH), 8.72 (s, 1H, H2), 8.21 (d, 1H, Ar),
7.69 (s, 1H, Ar), 7.40 (d, 1H, Ar), 3.81 (m, 1H, CH), 1.85-1.08
(m, 10H, cyclohexyl). Anal. (C16H17ClN2O2 ) C, H, N.
General Procedure for the Synthesis of N1-Substituted N-Cy-
clohexylquinoline-3-carboxamide Derivatives 38-40. NaH (4.36
mmol, 50% in mineral oil) was added to a hot solution (50 °C) of
N-cyclohexylquinoline-3-carboxamide derivatives 36 or 37 (0.92
mmol) in 9.2 mL of dry DMF. After 1 h, 4-(2-chloroethyl)-
morpholine hydrochloride or benzyl chloride (0.92 mmol) was
added, and the mixture was stirred for 24 h at 50 °C (38 and 40)
or at 80 °C (39). After the mixture was cooled (3-5 °C), the
addition of water caused the precipitation of the title compounds,
which were purified by crystallization.
N-Cyclohexyl-1-benzyl-4-hydroxy-7-methyl-1,2,3,4-tetrahydro-
1,8-naphthyridin-3-carboxamide (26). Yield 0.080 g, 52%; MS
1
m/z 379 (M+); H NMR: δ 7.48 (d, 1H, H5), 7.31 (m, 5H, Ar),
6.48 (m, 1H, H6), 6.00 (d, 1H, OH), 4.92 (m, 3H, CH2 + H4), 3.74
(m, 1H, CH), 3.40 (m, 2H, 2H2), 2.57 (m, 1H, H3), 2.38 (s, 3H,
CH3), 1.84-0.83 (m, 10H, 5CH2). Anal. (C23H29N3O2 ) C, H, N.
N-Cyclohexyl-7-hydroxy-1,8-naphthyridin-4(1H)-on-3-car-
boxamide (28). Sodium nitrite (0.55 g, 8.0 mmol) was added
portionwise to a cooled solution (-10 °C) of 7-amino-1,8-
naphthyridine-3-carboxamide 2734 (0.44 g, 1.6 mmol) in 7 mL of
concentrated sulfuric acid. After standing for 4 h at room temper-
ature, the mixture was poured over crushed ice and the pH was
adjusted to 8 with aqueous concentrated ammonium hydroxide. The
solid obtained was collected by filtration, washed with water, and
purified by crystallization from toluene to obtain 28 (0.490 g,
N-Cyclohexyl-1-(2-morpholin-4-ylethyl)-quinolin-4(1H)-on-3-
carboxamide (38). Yield 0.180 g, 50%; mp 169-170 °C (crystal-
lized from ethyl acetate); MS m/z 383 (M+); 1H NMR δ 10.04 (d,
1H, NH), 8.80 (s, 1H, H2), 8.34 (d, 1H, Ar), 7.90 (m, 2H, Ar),
7.52 (m, 1H, Ar), 4.58 (m, 2H, CH2), 3.90 (m, 1H, CH), 3.49 (m,
4H, morpholine), 2.65 (m, 2H, CH2), 2.42 (m, 4H, morpholine),
1.88-1.22 (m, 10H, cyclohexyl). Anal. (C22H29N3O3 ) C, H, N.
N-Cyclohexyl-1-benzylquinolin-4(1H)-on-3-carboxamide (39).
Yield 62%; mp 239-240 °C (crystallized from hexane); MS m/z
1
94%): mp 303-305 °C; H NMR δ 10.49 (d, 1H, NH), 8.45 (s,
1H, H2), 8.10 (d, 1H, H5), 6.25 (d, 1H, H6), 3.78 (m, 1H, CH),
1.82-1.26 (m, 10H, cyclohexyl). Anal. (C15H17N3O3 ) C, H, N.
N-Cyclohexyl-1-(o-fluorobenzyl)-7-(o-fluorobenzyloxy)-1,8-
naphthyridin-4(1H)-on-3-carboxamide (29). NaH (0.05 g, 1.08
mmol, 50% in mineral oil) was added to a solution of 7-hydroxy-
1,8-naphthyridine 28 (0.25 g, 0.87 mmol) in 6 mL of dry DMF.
After 1 h, 2-fluorobenzyl chloride (0.125 g, 0.87 mmol) was added
and the mixture was stirred for 3 days at 80 °C. After the mixture
was cooled, water was added and the solid obtained was collected
by filtration, purified by flash chromatography (ethyl acetate/hexane,
1:1), and crystallized from cyclohexane to give 29 (0.130 g, 30%):
mp 198-200 °C; MS m/z 503 (M+); 1H NMR δ 9.94 (d, 1H, NH),
9.03 (s, 1H, H2), 8.52 (d, 1H, H5), 7.47-7.10 (m, 8H, 2 Ar), 7.03
(d, 1H, H6), 5.80 (s, 2H, CH2), 5.43 (s, 2H, CH2), 3.85 (m, 1H,
CH), 1.32 (m, 10H, cyclohexyl). Anal. (C29H27F2N3O3 ) C, H, N.
N-(4-Methylcyclohexyl)-7-amino-1,8-naphthyridin-4(1H)-on-
3-carboxamide (31). A mixture of 1,8-naphthyridine-3-carboxylic
acid ethyl ester 3037 (0.276 g, 1 mmol) and 4-methylcyclohexy-
lamine (1.130 g, 10 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
purified by crystallization from ethyl acetate to obtain 31 (0.250 g,
1
360 (M+); H NMR δ 10.05 (d, 1H, NH), 8.97 (s, 1H, H2), 8.54
(d, 1H, Ar), 7.61-7.15 (m, 8H, Ar), 5.48 (s, 2H, CH2), 3.98 (m,
1H, CH), 2.02-1.44 (m, 10H, cyclohexyl). Anal. (C23H24N2O2 )
C, H, N.
N-Cyclohexyl-7-chloro-1-(2-morpholin-4-ylethyl)-quinolin-
4(1H)-on-3-carboxamide (40). Yield 0.200 g, 52%; mp 229-231
°C (crystallized from ethyl acetate); MS m/z 417 (M+). 1H NMR δ
10.00 (d, 1H, NH), 8.80 (s, 1H, H2), 8.35 (d, 1H, Ar), 8.05 (m,
1H, Ar), 7.60 (m, 1H, Ar), 4.50 (s, 2H, CH2), 3.80 (m, 1H, CH),
3.47 (m, 4H, morpholine), 2.45 (m, 6H, CH2 + morpholine), 1.40-
1.05 (m, 10H, cyclohexyl). Anal. (C22H28ClN3O3 ) C, H, N.
Pharmacology. Male DBA/J2 mice (Charles River, Como, Italy),
weighing 20-25 g, were maintained on ad libitum food and water
and were used in all experiments. [3H]CP-55,940 (168 Ci/mmol)
and [35S]GTPγS (1250 Ci/mmol) were purchased from Perkin-
Elmer Life Science (Boston, MA). [3H]CP-55,940 and WIN-55,-
212-2 were obtained from Tocris (Ballwin, MO). Guanosine 5′-
diphosphate (GDP) and guanosine 5′-O-(3-thiotriphosphate) (GTPγS)
were obtained from Sigma/RBI (St. Louis, MO). For biochemical
experiments, drugs were dissolved in dimethyl sulfoxide (DMSO).
DMSO concentration in the different assays never exceeded 0.1%
(v/v) and had no effects on [3H]CP-55,940 binding and [35S]GTPγS
binding assay.
1
83%): mp 198-200 °C; H NMR δ 10.45, 10.18 (2d, 1H, NH),
8.33 (s, 1H, H2), 8.13 (d, 1H, H5), 7.12 (s, 2H, NH2), 6.55 (d, 1H,
H6), 3.80 (m, 1H, CH), 1.86-0.85 (m, 12H, cyclohexyl + CH3).
Anal. (C16H20N4O2 ) C, H, N.
[3H]CP-55,940 Binding Assay. Mice were sacrificed by de-
capitation, and the brain (minus cerebellum) and spleen were rapidly
removed and placed on an ice-cold plate. After thawing, tissues
were homogenated in 20 volumes (w/v) of ice-cold TME buffer
(50 mM Tris-HCl, 1 mM EDTA, and 3 mM MgCl2, pH 7.4). The
homogenates were centrifuged at 1000g for 10 min at 4 °C, and
the resulting supernatants were centrifuged at 45000g for 30 min
at 4 °C. Aliquots of membranes were frozen at -80 °C until the
day of experiment.
N-(4-Methylcyclohexyl)-7-chloro-1,8-naphthyridin-4(1H)-on-
3-carboxamide (32). Sodium nitrite (0.34 g, 5.0 mmol) was added
portionwise to a cooled solution (-5 °C) of 7-amino-1,8-naphthy-
ridine-3-carboxamide 31 (0.3 g, 1.0 mmol) in 54.5 mL of
concentrated hydrochloric acid. The mixture was stirred for 3 h at
40 °C and, after cooling, was poured over crushed ice. The pH
was adjusted to 4-5 with aqueous concentrated ammonium
hydroxide. The solid obtained was collected by filtration, washed
with water, and purified by flash chromatography (ethyl acetate/
hexane, 1:1) to obtain 32 (0.100 g, 31%): mp 271-273 °C
The Bradford46 protein assay was used for protein determination
using bovine serum albumin (BSA) as a standard in accordance
with the supplier protocol (Bio-Rad, Milan, Italy).
1
(crystallized from ethyl acetate); H NMR δ 10.00, 9.63 (2d, 1H,

Carboxamide DeriVatiVes as Agonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 5955
[3H]CP-55,940 binding was carried out following a modified
version of the method previously described.39 Briefly, the brain or
spleen membranes (40-60 µg of protein) were incubated for 1 h
at 30 °C with [3H]CP-55,940 (0.5 nM) in a final volume of 0.5 mL
of TME buffer containing 5 mg/mL BSA. Nonspecific binding was
determined in the presence of 10 µM [3H]CP-55,940. Incubation
was terminated by rapid filtration through Whatman GF/C filters
pretreated with 0.5% (w/v) polyethyleneimine (PEI), using a
Brandell 24-sample harvester (Gaithersburg, MD). Filters were
washed three times with ice-cold Tris-HCl buffer (pH 7.4)
containing 1 mg/mL BSA. Filter-bound radioactivity was counted
in a liquid scintillation counter (Packard Tricarb1600 TR, Packard,
Meridien, CT), using 3 mL of scintillation fluid (Ultima Gold
Packard, MV, Meridien, CT).
weeks. They gave explicit informed consent to their enrollment in
this study. About 400 mL of venous blood was collected from each
of them; 64 mL of a citrate solution (CPD) was added as an
anticoagulant. The blood was centrifuged at 3500 rpm (11 min, 20
°C) in a slow-stop centrifuge (Sorvall RC 12 BP, Kendro Laboratory
Products). Plasma was removed by an automatic press (NPBI
Compomat 64). After 24 h of gentle stirring in a platelet incubator
(Helmer) at 22 °C to reduce cell stress, the buffy coat was
centrifuged at 900 rpm (9 min, 20 °C). Platelet-rich plasma was
removed by the same automatic press. An amount of 30 mL of the
residual leukocyte-rich preparation was diluted 1:4 with a buffer
with the following composition: 20 mM HEPES, 130 mM NaCl,
5 mM KCl, 5 IU/mL sodium heparin, 1.5 mg/ml bovine serum
albumin (BSA), at pH 7.4 (washing buffer). Aliquots of 10 mL
were then carefully layered over 10 mL of Ficoll-Paque in 30 mL
conical tubes (25 mm diameter) and centrifuged at room temperature
at 420g. After removal of the supernatant plasma, the basophil-
rich Ficoll-Paque layer was separated and the neutrophil-rich buffy
coat was discarded. The suspension was washed twice with the
washing buffer and centrifuged at 200g at 20 °C for 10 min. The
pellets were then resuspended in a calcium-free maintenance buffer
composed of 20 mM HEPES, 130 mM NaCl, 5 mM KCl, 5 mM
Na3EDTA, 1.5 mg/mL bovine serum albumin (BSA), pH 7.4, and
were further processed as described below. Upon isolation, cell
viability, determined by trypan blue exclusion, was always greater
than 95%. The procedure used, as a result of the low handling of
basophils, also prevented their aspecific activation, as might have
occurred using high-purifying procedures, such as specific antibody-
coated magnetic beads. Before the experiments were started,
samples from each basophil-rich leukocyte preparation were chal-
lenged for their ability to respond to anti-IgE by the flow cytometric
assay described below. Poorly responsive preparations were
discarded.
Flow Cytometric Analysis. Basophil-rich leukocyte pellets were
labeled with a saturating concentration of anti-IgE fluoresceine
isothiocyanate (FITC)-conjugated antibodies and anti-CD203c
phycoerythrin (PE)-conjugated antibodies. The fluorescent antibod-
ies were incubated with the pellets for 20 min at 4 °C. The cells
were then washed with buffer, centrifuged at 200g for 10 min at
room temperature, and then resuspended in buffer. After the lysis
of residual erythrocytes, the leukocyte suspensions were analyzed
by a flow cytometer (Coulter XL, Coulter Cytometry, Hialeah, FL).
Because the separation technique provides a leukocyte preparation
with no more than 70% basophils, it was necessary to sort the
basophil-related events using appropriate electronic gates. Basophils
were recognized by their high expression of membrane-bound IgE
resulting in a high signal related to FITC fluorescence (emission
peak at 530 nm). IgE-negative cells were then gated out by
electronic subtraction. The fluorescent signal of PE (emission peak
at 575 nm) was used to characterize activated and nonactivated
cells. Human basophils before activation showed a low expression
of CD203c, which was strongly up-regulated after the activation
of the cells.
[3H]CP-55,940 displacement curves were plotted using serial
dilutions ranging from 10-9 to 10-3 M unlabeled compounds and
[3H]CP-55,940 (0.5 nM). Independent experiments were repeated
on membrane preparations from at least three different experiments.
The calculation of the IC50 (the concentration that inhibits 50%
of specific radioligand binding) was performed by nonlinear curve
fitting of the concentration-effect curves using the GraphPad Prism
program, San Diego, CA. The F-test was used to determine the
best approximation of a nonlinear curve fitting to a one- or two-
site model (P < 0.05). IC50 values were converted to Ki values by
means of the Cheng and Prusoff equation.47
[35S]GTPγS Binding Assay. Brain tissue (minus cerebellum)
was suspended in 20 volumes of cold centrifugation buffer (50 mM
Tris-HCl, 3 mM MgCl2, 1 mM EDTA, pH 7.4) and homogenized
using a homogenizer system (Glas-Col, Terre Haute, IN). The
homogenate was centrifuged at 48000g for 10 min at 4 °C. The
pellet was then resuspended in the same buffer, homogenized, and
centrifuged as previously. The final P2 pellet was subsequently
resuspended in assay buffer (50 mM Tris-HCl, 3 mM MgCl2, 0.2
mM EGTA, 100 mM NaCl, pH 7.4), homogenized, and diluted to
a concentration of ∼2 mg/mL with assay buffer. Membrane aliquots
were then stored at -80 °C until use.
[35S]GTPγS binding is measured as previously described.40
Briefly, mouse brain membranes (5-10 µg of protein) were
incubated with compounds for 60 min at 30 °C in assay buffer
containing 0.1% BSA in the presence of 0.05 nM [35S]GTPγS and
30 µM GDP in a final volume of 1 mL. The reaction was terminated
by rapid filtration using a Packard Unifilter-GF/B, washed 2 times
with 1 mL of ice-cold 50 mM Tris-HCl, pH 7.4 buffer, and dried
for 1 h at 30 °C. The radioactivity on the filters was counted in a
liquid microplate scintillation counter (TopCount NXT, Packard,
Meridien, CT) using 30 µL of scintillation fluid (Microscint 20,
Packard, Meridien, CT).
Stock solution of compounds were prepared in DMSO and were
then diluted in assay buffer. The final concentration of DMSO was
<0.01%, which had no effect on basal or stimulated [35S]GTPγS
binding. Concentration-effect curves were determined by incubating
membranes with various concentrations of compounds (1-5000
nM) in the presence of 0.05 nM [35S]GTPγS and 30 µM GDP.
Nonspecific binding was measured in the presence of 10 µM
unlabeled GTPγS. Basal binding was assayed in the absence of
agonist and in the presence of GDP. Stimulation by the agonist
was defined as a percentage increase above basal levels (i.e.,
{[dpm(agonist) - dpm(no agonist)]/dpm(no agonist)} × 100).
Nonlinear regression analysis of concentration-response data was
performed using Prism 2.0 software (GraphPad Prism program, San
Diego, CA) to calculate Emax (maximal stimulation over basal levels)
and EC50 (concentration of agonist to obtain 50% of the maximal
effect) values.
Docking Studies. With the Macromodel program,48 the ligands
were submitted to a conformational search of 1000 steps with an
energy window, for saving the structure, of 10 kJ/mol. The
algorithm used was the Monte Carlo method with MMFFs as the
force field and a distance-dependent dielectric constant of 1.0. The
ligands were then minimized using the conjugated gradient method
until a convergence value of 0.05 kcal Å-1 mol-1, using the same
force field and dielectric constant used for the conformational
search.
Automated docking was carried out by means of the program
AUTODOCK 3.0.45 AUTODOCK TOOLS49 was used to identify
the torsion angles in the ligands, to add the solvent model and to
assign partial atomic charges (Gasteiger for the ligands and Kollman
for the receptors). The regions of interest used by AUTODOCK
were defined by considering the previously published WIN-55,-
212-2 docked into the CB2 receptor35 as the central group of a grid
of 54, 50, and 52 points in the x, y, and z directions. A grid spacing
of 0.375 Å and a distance-dependent function of the dielectric
Data are reported as the mean ( SEM of three to six experiments,
performed in triplicate. Data were statistically evaluated by one-
way analysis of variance followed by the Newman-Keuls test for
multiple comparisons.
Preparation of Basophil-Rich Leukocyte Samples. Twenty
healthy donors were recruited in the transfusion unit of Careggi
General Hospital (Florence). The subjects did not suffer from
allergic diseases and had not taken any drug during the previous 4

5956 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Manera et al.
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Figure 6. Plot of the average estimated binding free energy of the
chosen cluster vs experimental binding energy. Ligands with a
morpholinic group are indicated by 9, while all the others are indicated
by O.
constant were used for the energy map calculations. Because all
the compounds can form an intramolecular H bond and our previous
study suggested that the interaction was quite strong,35 this H bond
was also considered to be maintained during interaction in the
binding site. For this reason, during the AUTODOCK protocol,
we blocked the torsions involved in this intramolecular bond, to
prevent the loss of this interaction.
With the Lamarckian genetic algorithm, all docked compounds
were subjected to 100 runs of the AUTODOCK search, in which
the default values of the other parameters were used. Cluster
analysis was performed on the docked results using an rms tolerance
of 1.0 Å, and the cluster with the best average of estimated free
energy was chosen.
To predict the binding affinity of the ligands, we used the
correlation between the calculated and experimental binding free
energy obtained in our previous paper.35 Figure 6 shows a plot of
experimental binding energy versus the average estimated binding
free energy used for the prediction calculation. It differs slightly
from the data of our published paper because in this study the test
set previously used to validate the predictivity of the model was
incorporated in the training set.
Supporting Information Available: Elemental analysis results
for all target compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
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