Synthesis and biological evaluation of 1,8-naphthyridin-4(1H)-on-3- carboxamide derivatives as new ligands of cannabinoid receptors

Article abstract of DOI:10.1016/j.bmc.2004.01.035

Cannabinoid receptors have been studied extensively in view of their potential functional role in several physiological and pathological processes. For this reason, the search for new potent, selective ligands for subtype CB receptors, CB₁ and CB₂, is still of great importance, in order to investigate their role in various physiological functions. The present study describes the synthesis and the biological properties of a series of 1,8-naphthyridine derivatives, characterised by the presence of some important structural requirements exhibited by other classes of cannabinoid ligands, such as an aliphatic or aromatic carboxamide group in position 3, and an alkyl or arylalkyl substituent in position 1. These compounds were assayed for binding both to the brain and to peripheral cannabinoid receptors (CB₁ and CB₂). The results obtained indicate that the naphthyridine derivatives examined possess a greater affinity for the CB₂ receptor than for the CB₁ receptor. In particular, derivatives 6a and 7a possess an appreciable affinity for the CB₂ receptor, with K _i values of 5.5 and 8.0 nM respectively; also compounds 4a, 5a and 8a exhibit a good CB₂ affinity, with K_i values in the range of 10-44 nM. Furthermore, compounds 3g-i and 18 revealed a good CB ₂ selectivity, with a CB₁/CB₂ ratio >20.

Full text of DOI:10.1016/j.bmc.2004.01.035

Bioorganic & Medicinal Chemistry 12 (2004) 1921–1933
Synthesis and biological evaluation of
1,8-naphthyridin-4(1H)-on-3-carboxamide derivatives
as new ligands of cannabinoid receptors
Pier Luigi Ferrarini,a,* Vincenzo Calderone,b Tiziana Cavallini,a Clementina Manera,a
Giuseppe Saccomanni,a Luca Pani,c Stefania Ruiuc and Gian Luigi Gessad
aDipartimento di Scienze Farmaceutiche, via Bonanno 6, 56126 Pisa, Italy
bDipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, via Bonanno 6, 56126 Pisa, Italy
cNeuroscienze S.c.a.r.l., Zona Industriale Macchiareddu, 09010 Uta, Cagliari, Italy
d
`
‘B.B. Brodie’ Dipartimento di Neuroscienze, Universita di Cagliari, Cittadella Universitaria,
SS 554 km 4.500, 09042 Monserrato, Italy
Received 25 July 2003; accepted 23 January 2004
Abstract—Cannabinoid receptors have been studied extensively in view of their potential functional role in several physiological
and pathological processes. For this reason, the search for new potent, selective ligands for subtype CB receptors, CB1 and CB2, is
still of great importance, in order to investigate their role in various physiological functions. The present study describes the
synthesis and the biological properties of a series of 1,8-naphthyridine derivatives, characterised by the presence of some important
structural requirements exhibited by other classes of cannabinoid ligands, such as an aliphatic or aromatic carboxamide group in
position 3, and an alkyl or arylalkyl substituent in position 1. These compounds were assayed for binding both to the brain and to
peripheral cannabinoid receptors (CB1 and CB2). The results obtained indicate that the naphthyridine derivatives examined possess
a greater affinity for the CB2 receptor than for the CB1 receptor. In particular, derivatives 6a and 7a possess an appreciable affinity
for the CB2 receptor, with Ki values of 5.5 and 8.0 nM respectively; also compounds 4a, 5a and 8a exhibit a good CB2 affinity, with
Ki values in the range of 10–44 nM. Furthermore, compounds 3g–i and 18 revealed a good CB2 selectivity, with a CB1/CB2 ratio
>20.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
(Fig. 1) is the prototypical example. These compounds
led to the identification and characterization of two
subtypes of G-protein-coupled membrane cannabinoid
receptors, termed CB1,3,6,7 and CB2,8 primarily present
in the nervous system and in the immune system
respectively. These receptors exert an action either in
the central nervous system, by the control of cognition,
memory and motor function and perception of pain, or
in peripheral systems, in particular in the urogenital,
gastrointestinal and cardiovascular ones. Furthermore,
the CB2 receptors are widely expressed in immune cells,
B cells and natural killer cells.
The use of Cannabis preparations for medicinal pur-
poses has been known about for a thousand years;
however, its utility in recent years has been limited
because of its psychoactive properties. Following the
identification of Á9-tetrahydrocannabinol1 (Á9-THC,
Fig. 1) as the principal psychoactive constituent of
marijuana, other cannabimimetic compounds were dis-
covered, including endogenous arachidonyl-ethanol-
amide (anandamide)2 (Fig. 1) and compounds with
structures differing quite dramatically from that of the
‘classic’ cannabinoid compounds, such as bicyclic can-
nabinols3,4 typified by CP55940 (Fig. 1), cannabimimetic
indoles, pyrroles and indenes,5 of which WIN-55,212-2
Recently, there has been renewed interest in cannabi-
noids as anticancer agents. In particular Ajulemic acid
(AJA, dimethylheptyl-THC-11-oic acid) (Fig. 1), a syn-
thetic analogue of THC has proved to be equipotent or
more potent than THC in several anti-inflammatory
bioassays, presenting significant antitumor effects.9,10
* Corresponding author. Fax: +39-050-40517; e-mail: ferrarini@
farm.unipi.it
0968-0896/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.01.035

1922
P. L. Ferrarini et al. / Bioorg. Med. Chem. 12 (2004) 1921–1933
Figure 1. Molecular structures of cannabinoid CB1 and CB2 receptor ligands.
Moreover AJA is without any psychoactive effects and
has a favourable toxicity profile. It has been reported
that its antineoplastic effects are mediated primarily
through actions on CB2 receptors.10 As regards selective
ligands to cannabinoid subtype receptors, the arylpyr-
azole derivatives SR14171611 and SR14452812 (Fig. 1)
have been reported to be CB1- and CB2-selective ligands,
respectively, with an antagonist activity. Furthermore, a
novel 1,2-dihydroquinoline-3-carboxamide (JTE-907)13
(Fig. 1), a selective ligand for the CB2 receptor, has
recently been synthesized. At present, the research and
development of new potent, selective ligands for CB1
and/or CB2 is still of great importance for the investi-
gation the role of the two subtypes of cannabinoid
receptors in various physiological functions. In parti-
cular, new potent, selective CB2 receptor ligands are
important to understand certain effects of cannabinoids,
such as their immunosuppressant and anti-inflamma-
tory activities.14,15
On the basis of the above considerations, the present
paper describes the synthesis and the biological proper-
ties of a series of 1,8-naphthyridine derivatives with a
general structure A (Fig. 2), variously substituted on the
heterocyclic nucleus. These compounds are character-
ized by the presence of certain important structural
requirements shown by other classes of cannabinoid
ligands, such as an aliphatic or aromatic carboxamide
group in position 3, present in quinolone derivatives like
JTE-90713 (Fig. 1) and in arylpyrazole derivatives
Figure 2. General structure of 1,8-naphthyridine derivatives.

P. L. Ferrarini et al. / Bioorg. Med. Chem. 12 (2004) 1921–1933
1923
like SR-14452812 (Fig. 1) and an alkyl or arylalkyl sub-
stituent in position 1, present in aminoalkylindole deri-
vatives like WIN-55,212-25 (Fig. 1) and in SR-14452812
(Fig. 1).
and then with 4-(2-chloroethyl)morpholine hydrochlo-
ride (Scheme 1).
Analogously, the 3-carboxycyclohexylamide derivatives
4a–8a and the 3-carboxybenzylamide derivatives 4e–8e
and 4l were prepared from the 1,8-naphthyridine deri-
vatives 2a, 23 or 2l, respectively, with the appropiate
benzylchloride or alkylchloride (Scheme 2).
2. Chemistry
The compounds described in this study are shown in
Table 1, and their synthetic methods are outlined in
Schemes 1–5. 1,8-Naphthyridin-3-carboxamide deriva-
tives with a general structure A (Fig. 2) were prepared
from 7-methyl-1,8-naphthyridin-4(1H)-on-3-carboxylic
acid ethyl ester (1) which was obtained following the
synthethic route described in literature.16 The heating of
ester 1 with the appropiate amine in a sealed tube pro-
vided the carboxamide derivatives 2a–l (Scheme 1). The
1-ethylmorpholine derivatives 3a–l were obtained by
treatment of 2a–l in anhydrous DMF with NaH for 1 h
To confirm that the alkylation reaction leads to the 1-
position substituted derivatives, the N-cyclohexyl-1-
benzyl-7-methyl-1,8-naphthyridin-4(1H)-on-3-carboxy-
amide (4a) was also prepared following a different syn-
thetic route, as described in Scheme 3. The ethyl ester 1
was firstly N-alkylated, as reported above, to obtain the
1-benzyl derivative 9 and was then transformed into the
expected cyclohexylamide derivative 4a. The reaction of
the 1-benzyl-7-methyl-1,8-naphthyridin-4(1H)-on-3-car-
boxylic acid ethyl ester (9) with aqueous 10% sodium
Table 1. Radioligand binding data of 1,8-naphthyridine derivatives 2a, 2e, 3a–l, 4a,e–8a,e, 4l, 15, 16, 18, 25a,b and 26a–d
Receptor affinity(nM)
Selectivity ratio
a
b
Compd
R
R1
R2
n
KiCB1
KiCB2
KiCB1/KiCB2
2a
2e
3a
3b
3c
3d
3e
3f
H
H
cyclohexyl
benzyl
cyclohexyl
morph
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1000
1000
1000
1000
1000
1000
1000
1000
Ethylmorph
Ethylmorph
Ethylmorph
Ethylmorph
Ethylmorph
Ethylmorph
Ethylmorph
Ethylmorph
Ethylmorph
Ethylmorph
benzyl
100ꢀ8
10
9
1000
117ꢀ15
1000
CH2cyclohexyl
N-CH3pipz
benzyl
4-CH3-cyclohexyl
cyclopentyl
cycloheptyl
isopentyl
p-Cl-benzyl
cyclohexyl
benzyl
1000
537ꢀ2
475ꢀ2
5
30
2
2
0
0
4
ꢀ217.9
3g
3h
3i
1000
50ꢀ4
22ꢀ22
50ꢀ3
1000
560ꢀ33
1000
1000
5.5
2
3l
4a
4e
4l
12 ꢀ13
7
10ꢀ0.5
13
benzyl
benzyl
1000
1000
1000
1000
p-Cl-benzyl
cyclohexyl
benzyl
cyclohexyl
benzyl
cyclohexyl
benzyl
cyclohexyl
benzyl
5a
5e
6a
6e
7a
7e
8a
8e
o-F-benzyl
o-F-benzyl
p-F-benzyl
p-F-benzyl
n-hexyl
n-hexyl
n-butyl
n-butyl
2
ꢀ17
08
44ꢀ24.7
600ꢀ60
5.5ꢀ0.4
65.3ꢀ6
8.0ꢀ0.211.9
325ꢀ2
17.5ꢀ1
1000
1000
2
2.7
7
15ꢀ1.8
457ꢀ40
95ꢀ3
1000
5
15
3
2
ꢀ10.4
62
1000
15
16
18
25a
25b
26a
26b
26c
Ethylmorph
Ethylmorph
Ethylmorph
Ethylmorph
benzyl
Ethylmorph
benzyl
n-hexyl
benzyl
NH2
NH2
Cl
CH3
CH3
CH3
CH3
CH3
CH3
0
0
0
1
1
1
1
1
1
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
cyclohexyl
cyclohexyl
benzyl
25ꢀ1.8
40
2
1000
729ꢀ821.4
1000
benzyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
530ꢀ50
1000
1000
26d
SR141716A
SR144528
n-butyl
1000
1.8ꢀ00.75
514ꢀ30
0.0035
250
70ꢀ10
0.28btc00.4
a Affinity of compounds for CB1 receptor was evaluated using mouse cerebellum membranes and [3H]-CP 55,940.
b Affinity of compounds for CB2 receptor was assayed using mouse spleen homogenate and [3H]-CP 55,940. Ki values were obtained from five
indipendent experiments carry out in triplicate and are expressed as the meanꢀstandard error.

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P. L. Ferrarini et al. / Bioorg. Med. Chem. 12 (2004) 1921–1933
Scheme 1. (a) RNH2; (b) DMF, NaH, 4-(2-chloroethyl)morpholine hydrochloride.
Scheme 2. (a) DMF, NaH, RCl.
hydroxide at reflux gave the 3-carboxylic acid derivative
10. This compound was refluxed in Dowtherm A in the
presence of copper chromite as a catalyst to afford 1-
benzyl-7-methyl-1,8-naphthyridin-4(1H)-one (11), which
had previously been synthesised by following a different
method.17
As described in Scheme 4, starting from the 7-acetam-
ido-1,8-naphthyridin-4(1H)-on-3-carboxylic acid ethyl
ester 12,18 analogously to the procedure described
above, was transformed into the 3-carboxyamide deri-
vatives 13 and 14 by reaction in a sealed tube with
the appropriate amine. Under these conditions, also the

P. L. Ferrarini et al. / Bioorg. Med. Chem. 12 (2004) 1921–1933
1925
Scheme 3. (a) DMF, NaH, C6H5CH2Cl. (b) cyclohexylamine. (c) 10% NaOH (d) Dowtherm A, copper chromite.
hydrolysis of the acetamido group takes place. The
reaction of 13 and 14 in anhydrous DMF and NaH with
4-(2-chloroethyl)morpholine hydrochloride afforded the
1,8-naphthyridine derivatives 15 and 16 (Scheme 4).
Diazotization of N-cyclohexyl-7-amino-1,8-naphthyr-
idin-4(1H)-on-3-carboxyamide (14), carried out in aqu-
eous 37% hydrochloric acid at ꢁ5 ꢂC during the
addition of the NaNO2, and then at 40 ꢂC for 3 h, gave
the corresponding 7-chloro derivative 17. This last
compound was then converted into the corresponding
1-ethylmorpholine derivative 18 by reaction with 4-(2-
chloroethyl)morpholine hydrochloride under the same
conditions described above. The reaction of 7-methyl-2,3-
dihydro-1,8-naphthyridine-4(1H)-one (19)19 with glyoxylic
acid at 100 ꢂC for 1 h afforded the 3-methylcarboxylic
acid 20 (Scheme 5) which, by reaction with anhydrous
EtOH and 96% sulphuric acid at 80 ꢂC, was converted into
the ethylester 21, which had previously been synthesised by
following a different method.20 The heating of ester 21
with benzylamine or cyclohexylamine in a sealed tube
provided the corresponding 3-methylcarboxamide
derivatives 22 and 23 (Scheme 5). Lastly 22 and 23, by
reaction with the appropriate benzylchloride or alkyl-
chloride in anhydrous DMF and NaH, afforded the
desired 1,8-naphthyridine derivatives 25a,b and 26a–d.
in mouse spleen homogenate, respectively. For the pur-
poses of comparison, and as references values, we have
also included results for the two prototypical cannabi-
noid ligands, SR141716A and SR144528.21
4. Results and discussion
The 1,8-naphthyridine derivatives examined have in
common the presence in the heterocyclic nucleus of a
substituent of various kinds on the nitrogen in position
1, an amido substituent in position 3 and a substituent
in position 7.
4.1. CB1 Receptor affinities
An examination of the results shown in Table 1 reveals
that for the 7-methyl-3-carboxyamido compounds (2a,e,
3a–l, 4a,e,l and 5a,e–8a,e), the absence of a substituent
on the nitrogen in position 1 determined a poor affinity
(2a and 2e Ki>1000). Likewise, the compounds with an
ethylmorpholino group in position 1, regardless of the
nature of the carboxyamido substituent in position 3,
exhibited a poor affinity, with Ki values >1000. The
only exceptions were the 3f and 3h derivatives, with a
modest Ki value (Ki=537 and 560 nM respectively). The
presence of an n-alkyl substituent (n-butyl or n-hexyl),
with the simultaneous presence of an aliphatic carbox-
yamide, such as cyclohexylamide, in position 3, deter-
mined a fair affinity, which proved to be higher for the
n-hexyl-substituted compound (7a Ki=95 nM and 8a
Ki=262 nM). Analogously, for 3-carboxycyclohexyl-
amide derivatives, the presence in position 1 of a benzyl
3. Biology
Affinities of the 1,8-naphthyridine derivatives 2a,e, 3a–l,
4a,e–8a,e, 4l, 15, 16, 18, 25a,b and 26a–d for CB1 and
CB2 receptors were assessed by competition experiments
with [3H]-CP 55,940 in mouse cerebral membranes and

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P. L. Ferrarini et al. / Bioorg. Med. Chem. 12 (2004) 1921–1933
Scheme 4. (a) C6H5CH2NH2. (b) DMF, NaH, 4-(2-chloroethyl)morpholine hydrochloride. (c) cyclohexylamine. (d) NaNO2, HCl.
group, whether substituted or not, led to compounds
with a fair affinity. In particular, the p-fluoro benzyl
derivative (6a) proved to be the compound with the
highest affinity in this series towards the CB1 receptor, with
a Ki value of 15 nM. Furthermore, it appeared to be clear
that 3-carboxycyclohexylamide derivatives generally
possess a higher affinity towards CB1 compounds than
3-benzyl carboxyamide compounds. The substitution of
the methyl in position 7 with an amino group or an
atom of chlorine did not lead to an improvement in the
affinity. Lastly, the presence of a methylene spacer
between the heterocyclic nucleus and the carboxyamidic
group in position 3, produced a markedly negative effect
on the affinity of these compounds for the CB1 receptor,
as confirmed by a comparison of compounds 26b–d
with 4a, 7a and 8a.
vatives possess a significant affinity, with Ki values of
5.5 and 8.0 nM, respectively; also compounds 4a, 5a
and 8a possess a good affinity, with values of Ki in the
range of 10-44 nM. Among the 3-carboxycyclohex-
ylamide compounds, the N-ethylmorpholino derivatives
possess a lower affinity than the N-benzyl or N-alkyl ones.
For the N-alkyl derivatives, the n-hexyl group proved to
be more effective than the n-butyl. For the N-benzyl deri-
vatives, the presence of an atom of fluorine on the benzyl
increases the affinity, above all of the substitution is the
para position. Unlike findings for the CB1 receptor, the
substitution of the methyl in position 7 of the naph-
thyridine nucleus with the NH2 group determines a
decrease in the affinity, as confirmed by a comparison of
16 with 3a, and of 15 with 3e, while the presence in this
position of an atom of chlorine determines an approxi-
mately 4-fold increase in the affinity, as can be seen from
a comparison of 18 with 3a. Lastly, in line with findings
for the CB1 receptor, the presence of a methylene spacer
between the naphthyridine nucleus and the carbox-
yamido group leads to a marked decrease in the affinity.
4.2. CB2 Receptor affinities
The results obtained indicate that, as had previously
been found for the CB1 receptor, the naphthyridine
derivatives without any substituents on the nitrogen in
position 1 exhibit a poor affinity towards the CB2
receptor (2a and 2e Ki>1000). Furthermore, an impor-
tant result to be underlined is that 3-carboxycyclohex-
ylamide derivatives possess a higher affinity than 3-
carboxybenzylamide compounds, analogously to results
for the CB1 receptor. In particular, the 6a and 7a deri-
In conclusion, the results obtained show that the naph-
thyridine derivatives examined generally exhibit a
higher affinity for the CB2 receptor than for the CB1
receptor. Furthermore, some of these compounds
showed a good CB2 selectivity, such as 3g–i and 18, for
which the CB1/CB2 ratio is >20.

P. L. Ferrarini et al. / Bioorg. Med. Chem. 12 (2004) 1921–1933
1927
Scheme 5. (a) glyoxylic acid. (b) EtOH, H2SO4. (c) C6H5CH2NH2. (d) cyclohexylamine. (e) DMF, NaH, RCl.
It is also possible to hypothesise that in order to obtain a
good affinity on the CB2 receptor, the presence is necessary
in position 1 of a n-alkylic or benzylic substituent, which
may be substituted, and in position 3, of a carboxyamide
of an aliphatic nature, such as the cyclohexylamide
directly linked to the 1,8-naphthyridine nucleus (Fig. 2).
rected. IR spectra in Nujol mulls were determined on an
ATI Mattson Genesis Series FTIR spectrometer. 1H
NMR spectra were recorded with a Bruker AC-200
spectrometer in d units from TMS as an internal
standard. Analytical TLC was carried out on Merck
0.2mm precoated silica-gel glass plates (60 F-524)
and location of spots was detected by illumination
with a UV lamp. Elemental analyses (C, H, N) were
withinꢀ0.4% of the theoretical values and were per-
formed on a Carlo Erba elemental analyzer model 1106
apparatus.
In the light of these results, it is clear that this series of
naphthyridine derivatives represents a new class of het-
erocyclic derivatives, acting as ligands of cannabinoid
receptors. The study of these compounds deserves to be
developed in order to be optimise their affinity and
selectivity towards the cannabinoid receptor subtypes.
5.1.2. General procedure for the synthesis of 7-methyl-
1,8-naphthyridin-3-carboxyamide derivatives (2a–l). A
mixture of 1 mmol of 7-methyl-1,8-naphthyridin-4(1H)-
on-3-carboxylic acid ethyl ester (1) and 10 mmol of the
appropriate amine was heated in a sealed tube at 120 ꢂC
(2a, c, e–h, l) or at 160 ꢂC (2b, d, i) for 24 h. After cool-
ing, the reaction mixture was treated with ethyl ether to
give a solid residue which was collected by filtration
and purified by crystallization (2a, c–l) or by flash-
5. Experimental
5.1. Chemistry
5.1.1. General information. All melting points were
taken on a Kofler hot stage apparatus and are uncor-

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P. L. Ferrarini et al. / Bioorg. Med. Chem. 12 (2004) 1921–1933
chromatography, eluting with ethyl acetate/methanol,
10:1 (2b) to obtain the title compounds.
H, 6.32; N, 15.49%; found: C, 66.59; H, 6.55; N,
15.51%.
5.1.3. N-Cyclohexyl-7-methyl-1,8-naphthyridin-4(1H)-on-
3-carboxamide (2a). Yield 85%; mp 302–304 ꢂC (ethyl
acetate).1H NMR (DMSO) d 10.31 (d, 1H, NH), 8.74 (s,
1H, H2), 8.43 (d, 1H, H5), 7.25 (d, 1H, H6), 3.90 (brs,
1H, CH), 2.57 (s, 3H, CH3), 1.81–1.06 (m, 10H, cyclo-
hexyl). Anal. C16H19N3O2 (MW 285.34): C, 67.35;
H, 6.71; N, 14.74%; found: C, 67.01; H, 6.32; N,
14.50%.
5.1.10. N-Cyclohepthyl-7-methyl-1,8-naphthyridin-4(1H)-
on-3-carboxamide (2h). Yield 70%; mp 270–272 ꢂC
(ethyl acetate). H NMR (DMSO) d: 9.93 (d, 1H, NH),
8.61 (s, 1H, H2), 8.49 (d, 1H, H5), 7.42(d, 1H, H 6), 4.15
(m, 1H, CH), 2.62 (s, 3H, CH3), 1.85–1.56 (m, 12H,
cycloheptyl). Anal. C17H21N3O2 (MW 299.36): C, 68.20;
H, 7.07; N, 14.05%; found: C, 68.02; H, 7.05; N,
14.04%.
1
5.1.4. 7-Methyl-N-(morpholin-4-yl)-1,8-naphthyridin-
4(1H)-on-3-carboxamide (2b). Yield 29%; mp 243–
245 ꢂC (ethyl acetate).1H NMR (CDCl3) d 10.75 (brs,
1H, NH), 8.61 (s, 1H, H2), 8.48 (d, 1H, H5), 7.43 (d, 1H,
H6), 3.68 (m, 4H, morpholine), 2.87 (m, 4H, morpho-
line), 2.63 (s, 3H, CH3). Anal. C14H16N4O3 (MW
288.30): C, 58.33; H, 5.59; N, 19.44%; found: C, 58.42;
H, 5.76; N, 19.53%.
5.1.11. N-(1 - Ethylpropyl) - 7 - methyl - 1,8 - naphthyridin-
4(1H)-on-3-carboxamide (2i). Yield 81%; mp 213–
215 ꢂC (toluene). 1H NMR (DMSO) d: 9.73 (d, 1H,
NH), 8.63 (s, 1H, H2), 8.49 (d, 1H, H5), 7.43 (d, 1H,
H6), 3.83 (m, 1H, CH), 2.62 (s, 3H, CH3), 1.48 (m, 4H,
CH2), 0.85 (m, 6H, CH3). Anal. C15H19N3O2 (MW
277,33): C, 65.91; H, 7.01; N, 15.38%; found: C, 65.67;
H, 6.99; N, 15.32%.
5.1.5. N-(Cyclohexylmethyl)-7-methyl-1,8-naphthyridin-
4(1H)-on-3-carboxamide (2c). Yield 82%; mp 262–
5.1.12. N-(p - Chlorbenzyl) - 7 - methyl - 1,8 - naphthyridin-
4(1H)-on-3-carboxamide (2l). Yield 61%; mp 280–
283 ꢂC (ethyl acetate).1H NMR (CDCl3) d 8.95 (s, 1H,
H2), 8.59 (d, 1H, H5), 7.30 (m, 5H, Ar+H6), 4.66 (d,
2H, CH2), 2.68 (s, 3H, CH3). Anal. C17H14ClN3O2
(MW 327.76): C, 62.30; H, 4.31; N, 12.82%; found: C,
62.15; H, 4.11; N, 12.50%.
264 ꢂC (cyclohexane). H NMR (DMSO) d 9.89 (t, 1H,
1
NH), 8.62(s, 1H, H ), 8.47 (d, 1H, H5), 7.43 (d, 1H,
2
H6), 3.40 (m, 1H, CH,), 3.18 (m, 2H, NCH2), 2.62 (s,
3H, CH3), 1.88–1.34 (m, 10H, cyclohexyl). Anal.
C17H21N3O2 (MW 299.36): C, 68.23; H, 7.07; N,
14.05%; found: C, 68.50; H, 7.05; N, 14.11%.
5.1.13. General procedure for the synthesis of N1-substi-
tuted 1,8-naphthyridine derivatives (3a–l, 4a–8a, 4e–8e,
4l). 1.25 mmol of NaH was added to a solution of 1
mmol of 7-methyl-1,8-naphthyridine-3-carboxamide
derivatives 2a–l in 10 mL of dry N,N-dimethylforma-
mide. After 1 h the appropriate chloride (1 mmol) was
added and the mixture was stirred for 24 h at room
temperature for compounds 3c, d, f, g, i, 4e–8e and 4l or
at 50 ꢂC for compounds 3a, b, e, h, l and 4a–8a. The
products were then obtained by the following work-up:
in the case of 3a–l, the solvent was evaporated in vacuo
and the solid obtained was treated with water and col-
lected by filtration (3c, d, f, g, i) or extracted with
chloroform (3a, b, e, h, l) and then the combinated
extracts were washed with water, dried (magnesium
sulphate) and evaporated to dryness in vacuo. For
compounds 4a–8a, 4e–8e and 4l, the reaction mixture
was treated with water and then the precipitate formed
was collected by filtration.
5.1.6. 7-Methyl-N-(4-methylpiperazin-1-yl)-1,8-naphthyr-
idin-4(1H)-on-3-carboxamide (2d). Yield 64%; mp 235–
237 ꢂC (ethyl acetate).1H NMR (DMSO) d 10.73 (brs,
1H, NH), 8.61 (s, 1H, H2), 8.48 (d, 1H, H5), 7.42(d, 1H,
H6), 2.85 (m, 4H, piperazine), 2.62 (s, 3H, CH3), 2.50
(m, 4H, piperazine), 2.19 (s, 3H, NCH3). Anal.
C15H19N5O2 (MW 301.34): C, 59.80H, 6.36; N, 23.24%;
found: C, 60.11; H, 6.45; N, 23.38%.
5.1.7. N-Benzyl-7-methyl-1,8-naphthyridin-4(1H)-on-3-
carboxamide (2e). Yield 70%; mp 278–280 ꢂC (ethyl
acetate).1H NMR (DMSO) d 10.21 (t, 1H, NH), 8.66 (s,
1H, H2), 8.47 (d, 1H, H5), 7.41 (d, 1H, H6), 7.30 (brs,
5H, Ar), 4.55 (d, 2H, CH2), 2.62 (s, 3H, CH3). Anal.
C17H15N3O2 (MW 293.30): C, 69.62; H, 5.13; N,
14.33%; found: C, 68.02; H, 5.05; N, 14.04%.
5.1.8. 7-Methyl-N-(4-methylcyclohexyl)-1,8-naphthyri-
din-4(1H)-on-3-carboxamide (2f). Yield 56%; mp 260–
270 ꢂC (cyclohexane). H NMR (DMSO) d 10.55 and
5.1.14. N-Cyclohexyl - 7 - methyl - 1 - (2 - morpholin-4-yl-
ethyl)-1,8-naphthyridin-4(1H)-on-3-carboxamide
1
10.16 (2d, 1H, NH), 8.75 and 8.74 (2s, 1H, H2), 8.48
and 8.43 (2d, 1H, H5), 7.29 and 7.25 (2d, 1H, H6), 4.10
(3a).
Yield 30%; mp 191–195 ꢂC (trituration with ethyl
1
0
0
(m, 1H, H1 ), 3.70 (m, 1H, H4 ), 2.62 (s, 3H, CH3), 2.49
(s, 3H, CH3), 1.87–1.68 (m, 8H, cyclohexyl). Anal.
C17H21N3O2 (MW 299.37): C, 68.20; H, 7.07; N,
14.05%; found: C, 68.40; H, 7.15; N, 14.01%.
ether). H NMR (CDCl3) d 10.15 (t, 1H, NH), 8.91 (s,
1H, H2), 8.65 (d, 1H, H5), 7.30 (d, 1H, H6), 4.61 (t, 2H,
CH2N), 4.15 (brs, 1H, CH) 3.67 (m, 4H, morph), 2.78
(t, 2H, NCH2), 2.68 (s, 3H, CH3), 2.54 (m, 4H, morph),
2,00–1.26 (m, 10H, cyclohexyl). Anal. (C22H30N4O3)
(MW 398.49): C, 66.31; H, 7.59; N, 14.06%; found: C,
66.51; H, 7.83; N, 14.42%.
5.1.9. N-Cyclopentyl-7-methyl-1,8-naphthyridin-4(1H)-
on-3-carboxamide (2g). Yield 68%; mp 280–283 ꢂC
1
(ethyl acetate). H NMR (DMSO) d 9.88 (d, 1H, NH),
8.61 (s, 1H, H2), 8.48 (d, 1H, H5), 7.42(d, 1H, H 6), 4.23
(m, 1H, CH), 2.62 (s, 3H, CH3), 1.98–1.44 (m, 8H,
cyclopentyl). Anal. C15H17N3O2 (MW 271.31): C, 66.40;
5.1.15. 7-Methyl-N-morpholin-4-yl-1-(2-morpholin-4-yle-
thyl)-1,8 - naphthyridin - 4(1H) - on - 3 - carboxamide (3b).
Yield 25%; mp 146–148 ꢂC [flash chromatography elut-

P. L. Ferrarini et al. / Bioorg. Med. Chem. 12 (2004) 1921–1933
1929
ing with ethyl acetate/toluene/diethylamine (10:4:1)]. 1H
NMR (CDCl3) d 10.82(s, 1H, NH), 8.92(s, 1H, H 2),
5.1.21. N-Cycloheptyl-7-methyl-1-(2-morpholin-4-ylethyl)-
1,8-naphthyridin-4(1H)-on-3-carboxamide (3h). Yield
67%; mp 174–177 ꢂC (ethyl acetate). 1H NMR (DMSO)
8.64 (d, 1H, H5), 7.32(d, 1H, H ), 4.62(t, 2H, NCH 2),
6
3.89 (m, 4H, morph), 3.66 (m, 4H, morph), 3.00 (m, 4H,
morph), 2.77 (t, 2H, NCH2), 2.69 (s, 3H, CH3), 2,53 (m,
4H, morph). Anal. (C20H27N5O4) (MW 401.46): C,
59.84; H, 6.78; N, 17.44%; found: C, 59.51; H, 7.02; N,
17.62%.
d 9.93 (d, 1H, NH), 8.92(s, 1H, H ), 8.55 (d, 1H, H5),
2
7.45 (d, 1H, H6), 4.79 (t, 2H, CH2N), 4.15 (m, 1H, CH),
3.48 (m, 4H, morph), 2.68 (t, 2H, CH2N), 2.64 (s, 3H,
CH3), 2.50 (m, 4H, morph), 1.87–1.57 (m, 12H, cyclo-
heptyl). Anal. (C23H32N4O3) (MW 412,50): C, 66.96; H,
7.82; N, 13.58%; found: C, 66.72; H, 7.80; N, 13.54%.
5.1.16. N-(Cyclohexylmethyl)-7-methyl-1-(2-morpholin-
4-ylethyl) - 1,8 - naphthyridin - 4(1H) - on - 3 - carboxamide
(3c). Yield 85%; mp 161–162 ꢂC (ethyl acetate). 1H
NMR (DMSO) d 9.95 (t, 1H, NH), 8.93 (s, 1H, H2),
8.55 (d, 1H, H5), 7.47 (d, 1H, H6), 4.66 (t, 2H,
CH2N), 3.49 (m, 4H, morph), 3.40 (m, 1H, CH), 3.20
(t, 2H, CH2N), 2.64 (s, 3H, CH3), 2.62 (t, 2H,
CH2N), 2.45 (m, 4H, morph), 1.90–1.68 (m, 10H,
cyclohexyl). Anal. (C23H32N4O3) (MW 412.52): C,
66.99; H, 7.77; N, 13.59%; found: C, 66.72; H, 7.79; N,
13.64%.
5.1.22. N-(1-Ethylpropyl)-7-methyl-1-(2-morpholin-4-yl-
ethyl)-1,8-naphthyridin-4(1H)-on-3-carboxamide (3i).
Yield 86%; mp 140–142 ꢂC (cyclohexane). 1H NMR
(DMSO) d 9.65 (d, 1H, NH), 8.92(s, 1H, H ), 8.54 (d,
2
1H, H5), 7.46 (d, 1H, H6), 4.66 (t, 2H, CH2N), 3.85 (m,
1H, CH), 3.48 (m, 4H, morph), 2.66 (t, 2H, CH2N), 2.64
(s, 3H, CH3), 2.49 (m, 4H, morph), 1.53 (m, 4H, CH2),
0.87 (m, 6H, CH3). Anal. (C21H30N4O3) (MW 386.48):
C, 65.28; H, 7.77; N, 14.50%; found: C, 65.11; H, 7.80;
N, 14.44%.
5.1.17. 7-Methyl-N-(4-methylpiperazin-1-yl)-1-(2-morph-
olin-4-ylethyl)-1,8-naphthyridin-4(1H)-on-3-carboxamide
(3d). Yield 51%; mp 182–185 ꢂC (cyclohexane). 1H
5.1.23. N-(p-Chlorbenzyl)-7-methyl-1-(2-morpholin-4-yl-
ethyl)-1,8-naphthyridin-4(1H)-on-3-carboxamide (3l).
Yield 61%; mp 132–134 ꢂC (trituration with ethyl
1
NMR (CDCl3) d 10.78 (t, 1H, NH), 8.92(s, 1H, H ),
ether). H NMR (CDCl3) d 10.27 (t, 1H, NH), 8.92 (s,
2
8.64 (d, 1H, H5), 7.31 (d, 1H, H6), 4.61 (t, 2H, NCH2),
3.66 (m, 4H, morph), 3.04 (m, 4H, piperazine), 2.78 (t,
2H, NCH2), 2.68 (s, 3H, CH3), 2.53 (m, 4H, morph),
2,50 (m, 4H, piperazine), 2.35 (s, 3H, NCH3). Anal.
(C21H30N6O3) (MW 414.50): C, 60.84; H, 7.30; N,
20.28%; found: C, 61.05; H, 7.45; N, 20.53%.
1H, H2), 6.64 (d, 1H, H5), 7.30 (m, 5H, Ar+H6), 4.62
(m, 4H, CH2N+CH2Ar), 3.67 (m, 4H, morph), 2.79 (t,
2H, NCH2), 2.68 (s, 3H, CH3), 2.54 (m, 4H, morph).
Anal. (C23H25N4O3Cl) (MW 440.92): C, 62.65; H, 5.71;
N, 12.71%; found: C, 62.43; H, 5.93; N, 12.52%.
5.1.24. 1-Benzyl-N-cyclohexyl-7-methyl-1,8-naphthyridin-
4(1H)-on-3-carboxamide (4a). Yield 57%; mp 194–
5.1.18. N-Benzyl-7-methyl-1-(2-morpholin-4-ylethyl)-1,8-
naphthyridin-4(1H)-on-3-carboxamide (3e). Yield 61%;
196 ꢂC (cyclohexane). H NMR (DMSO) d 9.85 (d, 1H,
1
mp 154–157 ꢂC (trituration with ethyl ether). H NMR
NH), 9.07 (s, 1H, H2), 8.55 (d, 1H, H5), 7.48 (d, 1H,
H6), 7.32(m, 5H, Ph), 5.80 (s, 2H, CH 2Ph), 3.85 (m, 1H,
CH), 2.64 (s, 3H, CH3), 1.85–1.31 (m, 10H, cyclohexyl).
Anal. (C23H25N3O2) (MW 375,46): C, 73.57; H, 6.71; N,
11.19%; found: C, 73.65; H, 6.69; N, 11.15%.
1
(CDCl3) d 10.25 (t, 1H, NH), 8.94 (s, 1H, H2), 8.64 (d,
1H, H5), 7.34 (m, 6H, Ar+H6), 4.60 (m, 4H,
CH2N+CH2Ar), 3.67 (m, 4H, morph), 2.79 (t, 2H,
NCH2), 2.68 (s, 3H, CH3), 2.55 (m, 4H, morph). Anal.
(C23H26N4O3) (MW 406.47): C, 67.96; H, 6.45; N,
13.78%; found: C, 68.12; H, 6.54; N, 13.58%.
5.1.25. N-Cyclohexyl-1-(o-fluorobenzyl)-7-methyl-1,8-
naphthyridin-4(1H)-on-3-carboxamide (5a). Yield 63%;
mp 205–208 ꢂC (cyclohexane). 1H NMR (DMSO) d 9.85
(d, 1H, NH), 9.08 (s, 1H, H2), 8.54 (d, 1H, H5), 7.46 (d,
1H, H6), 7.42(m, 4H, Ph), 5.81 (s, 2H, CH 2Ph), 3.85 (m,
1H, CH), 2.60 (s, 3H, CH3), 1.87–1.23 (m, 10H, cyclo-
hexyl). Anal. (C23H24FN3O2) (MW 393,45): C, 70.21;
H, 6.15; N, 10.68%; found: C, 69.94; H, 6.35; N,
10.64%.
5.1.19. 7-Methyl-N-(4-methylcyclohexyl)-1-(2-morpho-
lin-4-ylethyl)-1,8-naphthyridin-4(1H)-on-3-carboxamide
1
(3f). Yield 79%; H NMR (DMSO) d 10.18 and 9.78
(2d, 1H, NH), 8.94 and 8.92 (2s, 1H, H2), 8.58 and 8.53
(2d, 1H, H5), 7.65 (d, 1H, H6), 4.66 (t, 2H, CH2N), 4.15
(m, 1H, CH), 3.49 (m, 4H, morph), 2.64 (s, 3H, CH3),
2.49 (t, 2H, CH2N), 2.44 (t, 4H, morph), 1.68–1.87 (m,
12H, cyclohexyl+CH3). Anal. (C23H32N4O3) (MW
412.52): C, 66.99; H, 7.77; N, 13.59%; found: C, 67.26;
H, 7.80; N, 13.54%.
5.1.26. N-Cyclohexyl-1-(p-fluorobenzyl)-7-methyl-1,8-
naphthyridin-4(1H)-on-3-carboxamide (6a). Yield 64%;
mp 222–224 ꢂC (ethyl acetate). 1H NMR (DMSO) d
9.85 (d, 1H, NH), 9.10 (s, 1H, H2), 8.54 (d, 1H, H5),
7.48 (d, 1H, H6), 7.18 (m, 4H, Ph), 5.76 (s, 2H, CH2Ph),
3.82(m, 1H, CH), 2.65 (s, 3H, CH 3), 1.86-1.23 (m, 10H,
cyclohexyl). Anal. (C23H24FN3O2) (MW 393,45): C,
70.21; H, 6.15; N, 10.68%; found: C, 69.94; H, 6.12; N,
10.64%.
5.1.20. N-Cyclopentyl-7-methyl-1-(2-morpholin-4-yl-
ethyl)-1,8-naphthyridin-4(1H)-on-3-carboxamide (3g).
Yield 63%; mp 208–210 ꢂC (ethyl acetate). H NMR
1
(DMSO) d 9.85 (d, 1H, NH), 8.93 (s, 1H, H2), 8.55 (d,
1H, H5), 7.47 (d, 1H, H6), 4.66 (t, 2H, CH2N), 4.25 (m,
1H, CH), 3.48 (m, 4H, morph), 2.67 (t, 2H, CH2N), 2.64
(s, 3H, CH3), 2.48 (m, 4H, morph), 1.88–1.22 (m, 8H,
cyclopentyl). Anal. (C21H28N4O3) (MW 384.47): C,
65.62; H, 7.29; N, 14.58%; found: C, 65.39; H, 7.32; N,
14.52%.
5.1.27. N-Cyclohexyl-1-hexyl-7-methyl-1,8-naphthyridin-
4(1H)-on-3-carboxamide (7a). Yield 47%; m.p 110–
111 ꢂC (petroleum ether 60–80 ꢂC). 1H NMR (DMSO) d

1930
P. L. Ferrarini et al. / Bioorg. Med. Chem. 12 (2004) 1921–1933
9.85 (d, 1H, NH), 8.93 (s, 1H, H2), 8.54 (d, 1H, H5),
7.47 (d, 1H, H6), 4.53 (t, 2H, CH2N), 3.85 (m, 1H, CH),
2.65 (s, 3H, CH3), 1.81–1.28 (m, 18H, cyclohex-
yl+hexyl) 0.85 (m, 3H, CH3). Anal. (C22H31N3O2)
(MW 369.50): C, 71.51; H, 8.48; N, 11.37%; found: C,
71.51; H, 8.44; N, 11.33%.
5.1.34. N-(p-Chlorbenzyl)-1-benzyl-7-methyl-1,8-naph-
thyridin-ꢂ4(1H)-on-3-carboxamide (4l). Yield 71%; m.p
1
177–178 C (cyclohexane). H NMR (DMSO) 10.10 (t,
1H, NH), 9.12(s, 1H, H 2), 8.54 (d, 1H, H5), 7.48 (d, 1H,
H6), 7.37 (m, 9H, Ar), 5.80 (s, 2H, NCH2) 4.56 (d, 2H,
NCH2), 2.64 (s, 3H, CH3). Anal. (C24H20ClN3O2) (MW
417.89): C, 68.98; H, 4.82; N, 10.06%; found: C, 68.83;
H, 5.12; N, 10.43%.
5.1.28. 1-Butyl-N-cyclohexyl-7-methyl-1,8-naphthyridin-
4(1H)-on-3-carboxamide (8a). Yield 44%; mp 156–
ꢂ
1
158 C (cyclohexane). H NMR (DMSO) d 9.90 (d, 1H,
NH), 8.93 (s, 1H, H2), 8.54 (d, 1H, H5), 7.47 (d, 1H,
H6), 4.54 (t, 2H, CH2N), 3.85 (m, 1H, CH), 2.66 (s, 3H,
CH3), 1.81–0.87 (m, 14H, cyclohexyl+butyl), 0.90 (t,
3H, CH3). Anal. (C20H27N3O2) (MW 341.44): C, 70.35;
H, 7.97; N, 12.31%; found: C, 70.51; H, 7.39; N,
12.33%.
5.1.35. Ethyl 1-benzyl-7-methyl-1,8-naphthyridin-4(1H)-
on-3-carboxylate (9). NaH (0.0870 g, 1.81 mmol, 50%
in mineral oil) was added to a solution of 7-methyl-1,8-
naphthyridine 1 (0.350 g, 1.5 mmol) in 10 mL of dry
DMF. After 1 h benzyl chloride (0.13 mL, 1 mmol) was
added and the mixture was stirred for 24 h at rt The
solution was evaporated in vacuo and the addition of
ethyl ether caused the precipitation of the title com-
pound as a pure solid: 0.396 g (yield 81%); mp 140–
5.1.29. 1-Benzyl-N-benzyl-7-methyl-1,8-naphthyridin-
4(1H)-on-3-carboxamide (4e). Yield 56%; mp 174–
ꢂ
1
141 C. H NMR (DMSO) d 8.96 (s, 1H, H2), 8.44 (d,
1H, H5), 7.41 (d, 1H, H6), 7.36 (m, 5H, Ph), 5.69 (s, 2H,
CH2Ph), 4.23 (q, 2H, CH2), 2.59 (s, 3H, CH3), 1.27 (t,
3H,CH3). Anal. C19H18N2O3 (MW 322.35): C, 70.79; H,
5.63; N, 8.69%; found: C, 70.50; H, 5.60; N, 8.70%.
176 ꢂC (cyclohexane). H NMR (DMSO) 10.18 (t, 1H,
1
NH), 9.14 (s, 1H, H2), 8.54 (d, 1H, H5), 7.47 (d, 1H,
H6), 7.30 (m, 10H, Ar), 5.80 (s, 2H, NCH2) 4.56 (d, 2H,
NCH2), 2.64 (s, 3H, CH3), Anal. (C24H21N3O2) (MW
383.44): C, 75.18; H, 5.52; N, 10.96%; found: C, 74.87;
H, 5.33; N, 10.60%.
5.1.36. 1-Benzyl-N-cyclohexyl-7-methyl-1,8-naphthyridin-
4(1H)-on-3-carboxamide (4a). A mixture of the 7-
methyl-1,8-naphthyridine derivative 9 (0.130 g, 0.40
mmol) and cyclohexylamine (0.50 mL, 4.0 mmol) was
heated in a sealed tube at 120 ꢂC for 24 h. After cooling
the reaction mixture was treated with ethyl ether to give
4a as a pure solid: 0.115 g (yield 76.6%).
5.1.30. N-Benzyl-1-(o-fluorobenzyl)-7-methyl-1,8-naph-
thyridin-ꢂ4(1H)-on-3-carboxamide (5e). Yield 60%; mp
1
193–195 C (cyclohexane). H NMR (DMSO) 10.25 (t,
1H, NH), 9.08 (s, 1H, H2), 8.61 (d, 1H, H5), 7.29 (m,
8H, Ar+H6), 7.08 (m, 2H, Ar), 5.74 (s, 2H, NCH2) 4.68
(d, 2H, NCH2), 2.69 (s, 3H, CH3), Anal.
(C24H20FN3O2) (MW 401.44): C, 71.81; H, 5.02; N,
10.47%; found: C, 71.57; H, 5.36; N, 10.10%.
5.1.37. 1-Benzyl-7-methyl-1,8-naphthyridin-4(1H)-on-3-
carboxylic acid (10). A mixture of ethyl 1-benzyl-7-
methyl-1,8-naphthyridin-4(1H)-on-3-carboxylate (0.300
g, 0.93 mmol) in 4 mL of 10% sodium hydroxide solu-
tion was refluxed for 1.5 h. After cooling the solid was
filtered and the pH of the solution was adjusted to 4
with aqueous 37% hydrochloric acid to obtain the title
compound as a pure solid: 0.167 g (yield 63%); mp 235–
5.1.31. N-Benzyl-1-(p-fluorobenzyl)-7-methyl-1,8-naph-
thyridin-4(1H)-on-3-carboxamide (6e). Yield 77%; mp
206–209 ꢂC (cyclohexane). H NMR (DMSO) 10.20 (t,
1
1H, NH), 9.03 (s, 1H, H2), 8.62(d, 1H, H ), 7.32(m,
5
8H, Ar+H6), 7.01 (m, 2H, Ar), 5.65 (s, 2H, NCH2) 4.67
(d, 2H, NCH2), 2.70 (s, 3H, CH3). Anal.
(C24H20FN3O2) (MW 401.44): C, 71.81; H, 5.02; N,
10.47%; found: C, 71.77; H, 5.26; N, 10.25%.
237 ꢂC. H NMR (DMSO) d: 9.34 (s, 1H, H2), 8.62(d,
1
1H, H5), 7.59 (d, 1H, H6), 7.32(m, 5H, Ph), 5.85 (s, 2H,
CH2Ph), 2.68 (s, 3H, CH3). Anal. C17H14N2O3 (MW
294.30): C, 69.38; H, 4.79; N, 9.52%; found: C, 69.00;
H, 4.45; N, 9.50%.
5.1.32. N-Benzyl-1-hexyl-7-methyl-1,8-naphthyridin-4(1H)-
on-3-carboxamide (7e). Yield 70%; mp 140–142 ꢂC
1
(cyclohexane). H NMR (DMSO) d 10.22 (t, 1H, NH),
5.1.38. 1-Benzyl-7-methyl-1,8-naphthyridin-4-(1H)-one
(11). A mixture of 1-benzyl-7-methyl-1,8-naphthyridin-
4(1H)-on-3-carboxylic acid (0.108 g, 0.35 mmol) and 10
mg of copper chromite in 4 mL of Dowtherm A was
refluxed for 2h. Filtration while hot and addition of
hexane caused the precipitation of the pure product:
0.030 g (yield 33%); mp 120–123 ꢂC. 1H NMR (DMSO)
d: 8.38 (d, 1H, H5), 8.26 (s, 1H, H2), 8.32(m, 6H,
H6+Ph), 6.15 (d, 1H, H3), 5.57 (s, 2H, CH2Ph), 2.60 (s,
3H, CH3). Anal. C16H14N2O (MW 250.29): C, 76.78;
H, 5.64; N, 11.19%; found: C, 76.50; H, 5.60; N,
11.30%.
8.99 (s, 1H, H2), 8.53 (d, 1H, H5), 7.47 (d, 1H, H6), 7.33
(m, 5H, Ar) 4.57 (m, 4H, 2CH2N), 2.64 (s, 3H, CH3),
1.79 (m, 2H, hexyl), 1.28 (brs, 6H, hexyl), 0.83 (m,
3H, CH3). Anal. (C23H27N3O2) (MW 377.48): C, 73.18;
H, 7.21; N, 11.13%; found: C, 73.08; H, 7.15; N,
10.89%.
5.1.33. N-Benzyl-1-butyl-7-methyl-1,8-naphthyridin-
4(1H)-on-3-carboxamide (8e). Yield 44%; mp 140–
143 ꢂC (cyclohexane). 1H NMR (DMSO) d 10.22 (t, 1H,
NH), 8.98 (s, 1H, H2), 8.53 (d, 1H, H5), 7.47 (d, 1H,
H6), 7.33 (m, 5H, Ar) 4.55 (m, 4H, 2CH2N), 2.65 (s, 3H,
CH3), 1.78 (m, 2H, butyl), 1.28 (m, 2H, butyl), 0.91 (t,
3H, CH3). Anal. (C21H23N3O2) (MW 349.43): C, 72.18;
H, 6.63; N, 12.03%; found: C, 71.95; H, 6.99; N,
11.95%.
5.1.39. 7-Amino-N-benzyl (13) and 7-Amino-N-cyclo-
hexyl-1,8-naphthyridin-4(1H)-on-3-carboxamide (14). A
mixture of ethyl 7-amino-1,8-naphthyridin-4(1H)-on-3-
carboxylate (0.276 g, 1 mmol) and the appropriate

P. L. Ferrarini et al. / Bioorg. Med. Chem. 12 (2004) 1921–1933
1931
amine (10 mmol) was heated in a sealed tube at 120 ꢂC
for 24 h. After cooling the reaction mixture was treated
with ethyl ether to give a solid residue which was col-
lected by filtration and purified by crystallization. 13:
5.1.42. 7-Chloro-N-cyclohexyl-1-(2-morpholin-4-ylethyl)-
1,8-naphthyridin -4(1H)-on-3-carboxamide (18). NaH
(0.053 g, 1.10 mmol, 50% in mineral oil) was added to a
solution of the 7-chloro-1,8-naphthyridine derivative 17
(0.167 g, 0.55 mmol) in 10 mL of dry DMF. After 1 h,
the 4-(2-chloroethyl)-morpholine hydrochloride (0.102
g, 0.55 mmol) was added and the mixture was stirred for
24 h at 50 ꢂC. The solvent was evaporated in vacuo and
the solid obtained was washed with water, collected by
filtration and purified by crystallization from cyclohex-
ane: 0.162g (yield 70%); mp 188–190 ꢂC. 1H NMR
(DMSO) d: 9.80 (d, 1H, NH), 8.96 (s, 1H, H2), 8.66 (d,
1H, H5), 7.66 (d, 1H, H6), 4.62(t, 2H, CH 2N), 3.87 (m,
1H, CH), 3.47 (m, 4H, morph), 2.66 (t, 2H, CH2N), 2.46
(m, 4H, morph), 1.88–1.23 (m, 10H, cyclohexyl). Anal.
C21H27N4O3Cl (MW 418.92): C, 60.21; H, 6.50; N,
13.37%; found: C, 60.53; H, 6.49; N, 13.34%.
0.190 g (yield 57%); mp 260–263 ꢂC (ethyl acetate). H
1
NMR (DMSO) d: 11.80 (brs, 1H, OH), 10.48 (d, 1H,
NH), 8.38 (s, 1H, H2), 8.09 (d, 1H, H5), 7.32(m,
5H,Ar), 7.12(brs, 2H, NH 2), 6.56 (d, 1H, H6), 4.52(d,
2H, CH2). Anal. C16H14N4O2 (MW 294.31): C, 65.31;
H, 4.76; N, 19.05%; found: C, 63.43; H, 5.02; N,
19.23%. 14: 0.260 g (yield 79%); mp 275–278 ꢂC (ethyl
acetate). 1H NMR (DMSO) d: 11.80 (brs, 1H, OH),
10.14 (d, 1H, NH), 8.33 (s, 1H, H2), 8.10 (d, 1H, H5),
7.10 (s, 2H, NH2), 6.55 (d, 1H, H6), 3.90 (brs, 1H, CH),
1.84–1.29 (m, 10H, cyclohexyl). Anal. C15H18N4O2
(MW 286.33): C, 62.92; H, 6.34; N, 19.57%; found: C,
63.15; H, 6.32; N, 19.86%.
5.1.40. 7-Amino-N-benzyl (15) and 7-amino-N-cyclo-
hexyl-1-(2-morpholin-4-ylethyl)-1,8-naphthyridin-4(1H)-on-
3-carboxamide (16). NaH (0.090 g 1.82mmol, 50% in
mineral oil) was added to a solution of 7-amino-1,8-
naphthyridine derivative 13 or 14 (0.91 mmol) in 10 mL
of dry DMF. After 1 h, 4-(2-chloroethyl)-morpholine
hydrochloride (0.170 g, 0.91 mmol) was added and the
mixture was stirred for 24 h at 50 ꢂC. The solvent was
evaporated in vacuo and the solid was dissolved in
chloroform; the solution was washed with water and
evaporated under reduced pressure. Addition of ethyl
ether caused the precipitation of the title compounds as
5.1.43. (4-Hydroxy-7-methyl-1,8-naphthyridin-3-yl)acetic
acid (20). A mixture of 7-methyl-2,3-dihydro-1,8-naph-
thyridine-4(1H)-one 19 (0.500 g, 3,09 mmol) and
glyoxylic acid (0.830g, 9,02mmol) in 10 mL of anhydrous
ethanol and KOH (0.700 g, 12,47 mmol) was refluxed for
1 h. After cooling, the suspension was filtered and the pH
of the filtrate was adjusted to 3 with aqueous 37% hydro-
chloric acid, to obtain a solid which was collected by
filtration, washed with H2O and crystallized from ethanol:
(0.31 g, yield 46%); mp dec 315 ꢂC. 1H NMR (DMSO) d
12.00 (brs, 1H, OH), 8.33 (d, 1H, H5), 7.92(s, 1H, H ),
2
7.25 (d, 1H, H6), 3.37 (s, 2H, CH2), 2.57 (s, 3H, CH3).
Anal. C11H10N2O3 (MW 218.21): C, 60.55; H, 4.62; N,
12.84%; found: C, 60.11; H, 5.00; N, 12.60%.
pure solids. 15: 0.156 g (yield 42%); mp 192–195 ꢂC. H
1
NMR (DMSO) d 10.44 (brs, 1H, NH), 8.68 (s, 1H, H2),
8.14 (d, 1H, H5), 7.33 (m,5H Ar), 7.21 (brs, 2H, NH2),
6.58 (d, 1H, H6), 4.53 (m, 4H, 2NCH2), 3.51 (m, 4H,
morph), 2.63 (m, 2H, NCH2), 2.42 (m, 4H, morph). Anal.
C22H25N5O3 (MW 407.46): C, 64.86; H, 6.14; N, 17.20%;
found: C, 65.02; H, 6.43; N, 17.53%. 16: 0.142 g (yield
5.1.44. Ethyl (4-hydroxy-7-methyl-1,8-naphthyridin-3-
yl)acetate (21). A solution of (4-hydroxy-7-methyl-1,8-
naphthyridin-3-yl)acetic acid 20 (2,94 mmol) in 50 mL
of anhydrous ethanol and 1 mL of 98% sulphuric acid
was heated at 80 ꢂC for 4 h. After cooling, the pH was
adjusted to 8 with a saturated solution of NaHCO3, and
the solution was evaporated in vacuo to obtain a solid
residue which was washed with H2O, collected by fil-
tration and crystallized from ethyl acetate: 0.48 g, yield
39%); mp 171–174 ꢂC. H NMR (CDCl3) d 10.15 (brs,
1
1H, NH), 8.62(s, 1H, H ), 8.14 (d, 1H, H5), 7.18 (brs,
2
2H, NH2), 6.58 (d, 1H, H6), 4.50 (m, 2H, NCH2), 3.90
(brs, 1H, CH), 3.50 (m, 4H, morph), 2.62 (m, 2H,
NCH2), 2.48 (m, 4H, morph), 1.95–1.20 (m, 10H,
cyclohexyl). Anal. C21H29N5O3 (MW 399.48): C, 63.14;
H, 7.32; N, 17.53%; found: C, 63.34; H, 7.53; N,
17.78%.
67%; mp 228–230 ꢂC. H NMR (DMSO) d 10.30 (brs,
1
1H, OH), 8.61 (d, 1H, H5), 7.83 (s, 1H, H2), 7.19 (d, 1H,
H6), 4.20 (q, 2H, CH2), 3.59 (s, 2H, CH2), 2.67 (s, 3H,
CH3), 1.28 (t, 3H, CH3). Anal. C13H14N2O3 (MW
246.26): C, 63.40; H, 5.73; N, 11.38%; found: C, 63.29;
H, 6.01; N, 11.35%.
5.1.41. 7-Chloro-N-cyclohexyl-1,8-naphthyridin-4(1H)-on-
3-carboxamide (17). Sodium nitrite (1.14 g, 16.6 mmol)
was added portionwise to a cooled solution (ꢁ5 ꢂC) of
7-amino-N-cyclohexyl-1,8-naphthyridin-4(1H)-on-3-
carboxamide (0.952g, 3.33 mmol) in 115 mL of con-
centrated hydrochloric acid. After standing for 3 h at
40 ꢂC, the mixture was poured over crushed ice and the
pH was adjusted to 4–5 with aqueous concentrated
ammonium hydroxide. The solid was collected by
filtration, washed with water and purified by flash
chromatography, using ethyl acetate as the eluant to
obtain the title compound: 0.54 g (yield 52%); mp
266–268 ꢂC. 1H NMR (DMSO) d 9.80 (d, 1H, NH),
8.65 (s, 1H, H2), 8.59 (d, 1H, H5), 7.60 (d, 1H, H6), 3.85
(m, 1H, CH), 1.95–1.31 (m, 10H, cyclohexyl). Anal.
C15H16ClN3O2 (MW 305.76): C, 58.92; H, 5.27; N,
13.74%; found: C, 58.61; H, 5.25; N, 13.71%.
5.1.45. N-Benzyl-2-(4-hydroxy-7-methyl-1,8-naphthyridin-
3-yl)acetamide (22) and N-cyclohexyl-2-(4-hydroxy-7-
methyl-1,8-naphthyridin-3-yl)acetamide (23). A mixture
of ethyl ester 21 (0.300 g, 1.20 mmol) and 12 mmol of
the appropriate amine in a sealed tube was heated at
120 ꢂC for 24 h. After cooling, the reaction mixture was
treated with ethyl ether to give a residue which was col-
lected by filtration and purified by crystallization. 22:
0.320 g, yield 87%; mp dec 314–316 ꢂC (ethanol). H
1
NMR d 12.0 (br, 1H, OH), 1.87 (d, 2H, H5+NH), 7.90
(s, 1H, H2), 7.25 (m, 6H, H6+Ar), 4.24 (d, 2H, CH2),
3.32(s, 2H, CH 2), 2.56 (s, 3H, CH3). Anal. C18H17N3O2
(MW 307.34): C, 70.34; H, 5.58; N, 13.67%; found: C,
70.00; H, 5.68; N, 13.70%. 23: 0.250g, yield 70%; mp

1932
P. L. Ferrarini et al. / Bioorg. Med. Chem. 12 (2004) 1921–1933
dec 320 ꢂC (ethanol). H NMR d 12.0 (br, 1H, OH),
8.34 (d, 1H, H5), 7.79 (brs, 2H, H2+NH), 7.25 (d, 1H,
H6), 3.46 (m, 1H, CH), 3.23 (s, 2H, CH2), 2.57 (s, 3H,
CH3), 1.80–1.10 (m, 10H, cyclohexyl). Anal. C17H21N3O2
(MW 299.37): C, 68.20; H, 7.07; N, 14.04%; found: C,
68.50; H, 7.10; N, 14.16%.
5.1.51. N-Cyclohexyl-2-(1-hexyl-7-methyl-1,8-naphthyri-
din-4(1H)-on-3-yl)acetamide (26c). Yield 27%; mp 123–
1
ꢂ
1
125 C (cyclohexane). H NMR d 8.59 (d, 1H, H5), 7.84
(s, 1H, H2), 7.20 (brs, 1H, NH), 7.22 (d, 1H, H6), 4.38 (t,
2H, CH2), 3.77 (m, 1H, CH), 3.43 (s, 2H, CH2), 2.66 (s,
3H, CH3), 1.95–1.10 (m, 18H, cyclohexyl+(CH2)4),
0.98 (t, 3H, CH3). Anal. C23H33N3O2 (MW 383.53): C,
72.06; H, 8.67; N, 10.97%; found: C, 71.79; H, 8.65; N,
10.80%.
5.1.46. General procedure for the synthesis of N1-substi-
tuted-2-(4-hydroxy-7-methyl-1,8-naphthyridin-3-yl)aceta-
mide derivatives (25a,b and 26a–d). 1.2mmol of NaH
was added to a stirred solution of 1 mmol of N-sub-
stituted-2-(4-hydroxy-7-methyl-1,8-naphthyridin-3-yl)-
acetamide derivatives 22 or 23 in 10 mL of dry N,N-
dimethylformamide at 50 ꢂC for compounds 25a and
26a or at room temperature for compounds 25b and
26b–d. After 1 h, the appropriate chloride or bromide (1
mmol) was added, and the mixture was stirred for 24 h
at 50 ꢂC for compounds 25a and 26a or at room temp-
erature for compounds 25b and 26b–d. The products
were then obtained by the following work-up: in the
case of 26a, the solvent was evaporated in vacuo and the
solid was washed with water and collected by filtration,
whereas for the other compounds (25b and 26a–d), the
reaction mixture was treated with water and the pre-
cipitate formed was collected by filtration.
5.1.52. N-Cyclohexyl-2-(1-butyl-7-methyl-1,8-naphthyri-
din-4(1H)-on-3-yl)acetamide (26d). Yield 65%; mp 177–
ꢂ
1
179 C (cyclohexane). H NMR d 8.60 (d, 1H, H5), 7.85
(s, 1H, H2), 7.20 (brs, 1H, NH), 7.23 (d, 1H, H6), 4.37 (t,
2H, CH2), 3.75 (m, 1H, CH), 3.43 (s, 2H, CH2), 2.66 (s,
3H, CH3), 1.93–1.05 (m, 14H, cyclohexyl+(CH2)2),
0.89 (t, 3H, CH3). Anal. C21H29N3O2 (MW 355.47): C,
70.95; H, 8.22; N, 11.83%; found: C, 70.64; H, 8.55; N,
11.56%.
5.2. Biology
5.2.1. General information. Male CD1 mice weighing
20–25 g, (Charles River, Calco, LC, Italy) were housed
in animal care quarters maintained at 22ꢀ2 ꢂC on a 12-
h light/dark cycle, and food and water were available ad
libitum. All experimental protocols were accepted by
the Ethical Committee at the University of Cagliari and
performed in strict accordance with the E.C. regulation
for care and use of experimental animals (EEC Nꢂ86/
609).
5.1.47. N-Benzyl-2-[7-methyl-1-(2-morpholin-4-ylethyl)-
1,8-naphthyridinꢂ-4(1H)-on-3-yl]acetamide (25a). Yield
1H, H5), 8.12(s, 1H, H ), 7.29 (m, 6H, H6+Ar), 4.47
1
25%; mp 60–63 C (cyclohexane). H NMR d 8.40 (d,
2
(m, 2H, NCH2), 4.27 (d, 2H, CH2), 3.55 (m, 4H,
morph), 3.30 (s, 2H, CH2), 2.61 (m, 2H, NCH2), 2.58 (s,
3H, CH3). Anal. C24H28N4O3 (MW 420.50): C, 68.50;
H, 6.71; N, 13.32%; found: C, 68.30; H, 7.00; N,
13.29%.
[3H]-CP-55,940 (specific activity 180 Ci/mmol) was pur-
chased from New England Nuclear (Boston, MA,
USA). CP 55,940 was obtained from Tocris Cookson
Ltd (Bristol, UK). For biochemical experiments, drugs
were dissolved in dimethyl-sulphoxide, (DMSO).
DMSO concentration in the different assays never
exceeded 0.1% (v/v) and was without any effects on
radioligand binding.
5.1.48. N-Benzyl-2-(1-benzyl-7-methyl-1,8-naphthyridin-
4(1H)-on-3-yl)acetamide (25b). Yield 86%; mp 172–
ꢂ
1
174 C (cyclohexane). H NMR d 8.58 (d, 1H, H5), 7.92
(s, 1H, H2), 7.80 (br, 1H, NH), 7.27 (m, 6H, H6+Ar),
5.63 (s, 2H, NCH2), 4.39 (d, 2H, NCH2), 3.50 (s, 2H,
CH2), 2.67 (s, 3H, CH3). Anal. C25H23N3O2 (MW
397.47): C, 75.56; H, 5.83; N, 10.57%; found: C, 75.21;
H, 5.92; N, 10.19%.
5.2.2. Tissue preparation. Mice were killed by cervical
dislocation and the brain (minus cerebellum) and spleen
were rapidly removed and placed on an ice-cold plate.
After thawing, tissues were homogenized in 20 vol. (w/
v) of ice-cold TME buffer (50 mM Tris–HCl, 1 mM
EDTA and 3.0 mM MgCl2, pH 7.4).The homogenates
were centrifuged at 1,086ꢃg for 10 min at 4 ꢂC, and the
resulting supernatants were centrifuged at 45,000ꢃg for
30 min in a Beckman SW41 swing-out rotor, at 4 ꢂC.
5.1.49. N-Cyclohexyl-2-[7-methyl-1-(2-morpholin-4-yl-
ethyl)-1,8-naphthyridin-4(1H)-on-3-yl] acetamide (26a).
Yield 50%; mp 95–98 ꢂC (cyclohexane). 1H NMR d 8.39
(d, 1H, H5), 8.08 (s, 1H, H2), 7.79 (d, 1H, NH), 7.29 (d,
1H, H6), 4.46 (m, 2H, NCH2), 3.49 (m, 5H, CH+morph),
3.23 (s, 2H, CH2), 2.61 (m, 2H, NCH2), 2.59 (s, 3H, CH3),
2.40 (m, 4H, morph), 1.83–1.05 (m, 10H, cyclohexyl).
Anal. C23H32N4O3 (MW 412.52): C, 66.96; H, 7.82; N,
13.58%; found: C, 66.70; H, 8.10; N, 13.29%.
5.2.3. Binding study at CB1 and CB2 receptors. [3H]-CP-
55,940 binding was performed by a modification of the
method previously described.22 Briefly, the membranes
(30–80 mg of protein) were incubated with 0.5–1 nM of
[3H]-CP55940 for 1 h at 30 ꢂC in a final volume of 0.5
mL of TME buffer containing 5 mg/mL of fatty acid-
free bovine serum albumin (BSA). Non-specific binding
was estimated in the presence of 10 mM of CP55940. All
binding studies were performed in disposable glass tubes
pre-treated with Sigma-Cote (Sigma Chemical Co. Ltd.,
Poole, UK), in order to reduce non-specific binding.
The reaction was terminated by rapid filtration through
5.1.50. N-Cyclohexyl-2-(1-benzyl-7-methyl-1,8-naphthyr-
idin-4(1H)-on-3-yl)acetamide (26b). Yield 76%; mp
207–209 ꢂC (cyclohexane). H NMR d 8.61 (d, 1H, H5),
1
7.92(s, 1H, H ), 7.33 (brs, 5H, Ar), 7.23 (d, 1H, H6),
2
5.61 (s, 2H, NCH2), 3.70 (m, 1H, CH), 3.40 (s, 2H,
CH2), 2.67 (s, 3H, CH3), 1.91–1.12(m, 10H, cyclo-
hexyl). Anal. C24H27N3O2 (MW 389.49): C, 74.03; H,
6.99; N, 10.79%; found: C, 73.61; H, 7.11; N, 10.35%.

P. L. Ferrarini et al. / Bioorg. Med. Chem. 12 (2004) 1921–1933
1933
Whatman GF/C filters presoaked in 0.5% poly-
ethyleneimine (PEI) using a Brandell 96-sample har-
vester (Gaithersburg, MD, USA). Filters were washed
five times with 4 mL aliquots of ice cold Tris HCl buffer
(pH 7.4) containing 1 mg/mL BSA The filter-bound
radioactivity was measured in a liquid scintillation
counter (Tricarb 2100, Packard, Meridien, USA) with 4
mL of scintillation fluid (Ultima Gold MV, Packard).
Protein determination was performed by means of the
Bradford23 protein assay, using BSA as a standard in
accordance with the protocol of the supplier (Bio-Rad,
Milan, Italy).
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Prism program. IC50 values were derived from the
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