1,8-Naphthyridines IX. Potent anti-inflammatory and/or analgesic activity of a new group of substituted 5-amino[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6- carboxamides, of some their Mannich base derivatives and of one novel substituted 5-amino-10-oxo-10H-pyrimido[1,2-a][1,8]naphthyridine-6-carboxamide derivative

Article abstract of DOI:10.1016/j.ejmech.2014.08.069

A new group of 5-(alkylamino)-9-isopropyl[1,2,4]triazolo[4,3-a][1,8] naphthyridine derivatives bearing a CONHR group at the 6-position (1c-g), designed to obtain new effective analgesic and/or anti-inflammatory agents, were synthesized and tested along with three new 9-alkyl-5-(4-alkyl-1-piperazinyl)- N,N-diethyl [1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamides (2b-d). Besides, a new class of analogues of compounds 1 and 2, bearing a Mannich base moiety at the 9-position (12a-d), as well as the novel N,N-diethyl-5- (isobutylamino)-8-methyl-10-oxo-10H-pyrimido[1,2-a][1,8]naphthyridine-6- carboxamide (15) were prepared and tested. Compounds 1c-g exhibited very interesting anti-inflammatory properties in rats, whereas compounds 2b-d and 15 proved to be endowed with prevalent analgesic activity frequently associated with sedative effects in mice. On the contrary, the Mannich bases 12a-d resulted inactive. The most effective (80% inhibition of oedema) and potent (threshold dose 1.6 mg kg^-1 with 31% inhibition of oedema) anti-inflammatory compound 1d did not show gastrolesive effects following 100 mg kg^-1 oral administration in rats.

Full text of DOI:10.1016/j.ejmech.2014.08.069

European Journal of Medicinal Chemistry 86 (2014) 394e405
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
1,8-Naphthyridines IX. Potent anti-inflammatory and/or analgesic
activity of a new group of substituted 5-amino[1,2,4]triazolo[4,3-a]
[1,8]naphthyridine-6-carboxamides, of some their Mannich base
derivatives and of one novel substituted 5-amino-10-oxo-10H-
pyrimido[1,2-a][1,8]naphthyridine-6-carboxamide derivative
,
,
Mario Di Braccio a *, Giancarlo Grossi a *, Silvana Alfei b, Vigilio Ballabeni c,
Massimiliano Tognolini c, Lisa Flammini c, Carmine Giorgio c, Simona Bertoni c,
Elisabetta Barocelli c
a
ꢀ
Dipartimento di Farmacia, Sezione di Chimica del Farmaco e del Prodotto Cosmetico, Universita di Genova, Viale Benedetto XV, 3, I-16132 Genova, Genova,
Italy
b
ꢀ
Dipartimento di Farmacia, Sezione di Chimica e Tecnologie Farmaceutiche e Alimentari, Universita di Genova, Via Brigata Salerno, 13, I-16147 Genova,
Italy
c
ꢀ
Dipartimento di Farmacia, Universita di Parma, Parco Area delle Scienze 27/A, I-43124 Parma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new group of 5-(alkylamino)-9-isopropyl[1,2,4]triazolo[4,3-a][1,8]naphthyridine derivatives bearing a
CONHR group at the 6-position (1ceg), designed to obtain new effective analgesic and/or anti-
inflammatory agents, were synthesized and tested along with three new 9-alkyl-5-(4-alkyl-1-
Received 11 June 2014
Received in revised form
23 August 2014
Accepted 26 August 2014
Available online 27 August 2014
piperazinyl)-N,N-diethyl [1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamides (2bed). Besides,
a
new class of analogues of compounds 1 and 2, bearing a Mannich base moiety at the 9-position (12aed),
as well as the novel N,N-diethyl-5-(isobutylamino)-8-methyl-10-oxo-10H-pyrimido[1,2-a][1,8]naph-
thyridine-6-carboxamide (15) were prepared and tested. Compounds 1ceg exhibited very interesting
anti-inflammatory properties in rats, whereas compounds 2bed and 15 proved to be endowed with
prevalent analgesic activity frequently associated with sedative effects in mice. On the contrary, the
Mannich bases 12aed resulted inactive. The most effective (80% inhibition of oedema) and potent
(threshold dose 1.6 mg kgꢀ1 with 31% inhibition of oedema) anti-inflammatory compound 1d did not
show gastrolesive effects following 100 mg kgꢀ1 oral administration in rats.
Keywords:
[1,2,4]Triazolo[4,3-a][1,8]naphthyridines
10H-pyrimido[1,2-a][1,8]naphthyridines
Anti-inflammatory
Analgesic
Gastrolesivity
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
agents, whereas the 5-(4-methyl-1-piperazinyl)derivative 2a [3],
in addition to a moderate anti-inflammatory activity, exhibited
On the basis of the pharmacological data concerning different
series of original tricyclic compounds published in previous papers
[1e5], a number of 5-amino[1,2,4]triazolo[4,3-a][1,8]naphthyr-
idine-6-carboxamides proved to be potent analgesic and/or anti-
inflammatory agents, depending on the substituents present in
the common scaffold. Namely, through experimental findings ob-
tained in rodents we were able to identify the 5-isobutylamino
compounds 1a [1] and 1b [5] as effective anti-inflammatory
very potent analgesic properties: all these triazolonaphthyridine
derivatives were devoid of acute gastrolesivity [4,5].
Compounds 1a and 2a, labelled as NF 161 and NF 177, respec-
tively, were subjected to an in vitro study which showed that their
anti-inflammatory activity reasonably derives from inhibitory ef-
fects on adhesion, evoked by several pro-inflammatory stimuli, of
human polymorphonuclear cells (PMNs) to human umbilical
vascular endothelial cells (HUVEC), as well as from their inhibition
of superoxide anion production and of myeloperoxidase release,
without involving the cyclooxygenase (COX) inhibition, in accor-
dance with the absence of acute gastrolesivity [6]. This pharma-
cological profile was interesting since it positively responds to the
* Corresponding authors.
E-mail addresses: dbraccio@unige.it (M. Di Braccio), grossig@unige.it (G. Grossi).
http://dx.doi.org/10.1016/j.ejmech.2014.08.069
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

M. Di Braccio et al. / European Journal of Medicinal Chemistry 86 (2014) 394e405
395
requirement of a reduced gastrolesivity that has been pursued,
during the past decade, through the development of various
structural classes of NSAIDs with improved gastric tolerability
mainly thanks to their ability to selectively inhibit COX-2 isoen-
zyme [7].
suitable primary amines gave good yields of 1,2-dihydro-4-
hydroxy-2-oxo-1,8-naphthyridine-3-carboxamides 4a,c,d, which
in turn were converted into 2,4-dichloroderivatives 5a,c,d, by re-
action with POCl3 at reflux for 1 h (compounds 5a,c) or with the
Vilsmeier reagent in refluxing chloroform (2h) (compound 5d).
From the subsequent cyclocondensation of the dichloroderivatives
5a,c,d with isobutyrohydrazide (Dowtherm A, 160 ꢁC, 30 min), the
5-chloro-9-isopropyl[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-
carboxamide derivatives 6a,c,d were obtained in moderate yields
along with small amounts of the corresponding 5-hydroxy ana-
logues 7a,c,d, very likely derived through hydrolysis of 5-
chloroderivatives 6a,c,d in the reaction medium (the N-ethyl car-
boxamide derivatives 4b, 5b and 6b have been previously described
by us [4]). As a last step, the treatment of 6aed with an excess of
isopropylamine (compounds 1c,d,f,g) or 3-pentylamine (com-
pound 1e) (ethanol solution, closed vessel, 140 ꢁC, 16 h) afforded
high yields of the desired compounds 1ceg.
In the present work we further investigate the structur-
eeactivity relationship within the 5-amino substituted [1,2,4]tri-
azolo[4,3-a][1,8]naphthyridine-6-carboxamide class through fine
chemical modulations of the two more interesting molecules syn-
thesized by us within these studies: 1b (which proved to be an anti-
inflammatory agent better than 1a [5]) and the best analgesic
compound 2a (Fig. 1). With this purpose, three subset of new
compounds related to compounds 1b and 2a were developed and
tested for biological activity (see Scheme 1). The first group (com-
pounds 1ceg) includes analogues of 1b in which the 9-isopropyl
group is maintained on the triazole ring, while the alkyl groups
both on the 5-amino substituent and on the 6-carboxamide func-
tion are properly varied; the second subset (compounds 2bed)
consists of close structural analogues of the 5-(4-methyl-1-
The reaction of 9-alkyl-5-chloro-N,N-diethyl[1,2,4]triazolo[4,3-
a][1,8]naphthyridine-6-carboxamides 8a [4] or 8b [3] with suitable
1-alkylpiperazines in dimethyl sulfoxide (130 ꢁC, 2 h) afforded the
corresponding 5-(4-alkyl-1-piperazinyl)derivatives 2bed in good
yields.
piperazinyl)derivative 2a.
A third subseries of compounds
(12aed) is characterized by a Mannich base moiety inserted in the
triazole ring in place of 9-alkyl group, while at the positions 5 and 6
of the [1,2,4]triazolo[4,3-a][1,8]naphthyridine scaffold are main-
tained the substituents which previously have displayed the best
pharmacological results, i.e. the 5-isopropylamino or 5-(4-methyl-
1-piperazinyl) substituents and the N,N-diethyl 6-carboxamide
group, respectively. Furthermore a single variation of the hetero-
cyclic ring fused with the 1,8-naphthyridine system was examined
by synthesizing the N,N-diethyl-5-(isobutylamino)-8-methyl-10-
On the other hand, the reaction of morpholine or 1-
methylpiperazine with ethyl bromoacetate (molar ratio 2:1, dry
benzene solution, room temperature) gave the corresponding ethyl
(dialkylamino)acetates, which were evaporated to dryness and
directly treated with an equimolar amount of hydrazine hydrate
(ethanol solution, closed vessel, 120 ꢁC, 8 h) to yield the desired
(dialkylamino)acetic acid hydrazides 9a,b (overall yields 52e58%).
The subsequent cyclocondensation of hydrazides 9a,b with the 2,4-
dichloro-N,N-diethyl-1,8-naphthyridine-3-carboxamide 10 [9]
(Dowtherm A, 160 ꢁC, 90 min), gave moderate yields of 9-(dia-
lkylamino)methyl-5-chloro-N,N-diethyl[1,2,4]triazolo[4,3-a][1,8]
naphthyridine-6-carboxamide derivatives 11a,b, which in turn, by
reaction with an excess of isopropylamine (ethanol solution, closed
vessel, 140 ꢁC, 16 h) or 1-methylpiperazine (dimethyl sulfoxide
solution, 130 ꢁC, 2 h), gave good yields of desired compounds 12a,c
or 12b,d, respectively.
Finally, an ethanolic solution of 2-chloro-N,N-diethyl-4-(iso-
butylamino)-1,8-naphthyridine-3-carboxamide 13 [10] was treated
with an excess of 30% aqueous ammonia (closed vessel, 130 ꢁC,
24 h) to give the corresponding 2-aminoderivative 14 in high yield.
This one was cyclocondensed with an excess of ethyl acetoacetate
in the presence of a catalytic amount of monohydrate p-toluene-
sulfonic acid (Dowtherm A,180 ꢁC, 20 h) to afford the expected N,N-
diethyl-5-(isobutylamino)-8-methyl-10-oxo-10H-pyrimido[1,2-a]
[1,8]naphthyridine-6-carboxamide 15 in rather low yield (30%).
The structures attributed to the compounds described in this
paperare consistent with the results of elemental analyses and IR,1H
NMR and 13C NMR spectral data (see Section 5.1.). In the 1H NMR
oxo-10H-pyrimido[1,2-a][1,8]naphthyridine-6-carboxamide
15,
with the aim to evaluate the effects of such molecular change on
the anti-inflammatory and/or analgesic activities.
All the compounds were studied in vivo for their anti-
inflammatory (in rat paw oedema test) and analgesic (in mice
writhing test) activities. The molecules endowed with a statistically
significant antinociceptive effect were tested also for the inhibition
of the spontaneous locomotor activity in mice to ascertain the
presence of sedative effects confounding writhing test data. For the
compound 1d, which proved to be the most interesting one among
all new synthesized compounds because of its marked and potent
anti-inflammatory activity without any acute gastrolesivity in rats,
the in vitro antiplatelet activity was also determined as well as
ability to inhibit NOꢀ2 production in cell cultures treated with li-
popolysaccharides (LPS).
2. Chemistry
In Scheme 1 are reported the synthetic routes to target com-
pounds 1ceg, 2bed, 12aed, and 15. Thus, the reaction of ester 3 [8]
heated in a closed vessel (160 ꢁC, 16 h) with an ethanol solution of
CH3
CH
CH3
CH
CH3
CH3
CH3
CH3
CH
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
H
CON
CON(C2H5)2
CON(C2H5)2
H
C2H5
H
1a
(NF 161)
2a
(NF 177)
1b
CH2 CH(CH3)2
CH2 CH(CH3)2
N
CH3
Fig. 1. Structures of the substituted 5-amino[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamides 1a, 1b and 2a, taken as lead compounds.

396
M. Di Braccio et al. / European Journal of Medicinal Chemistry 86 (2014) 394e405
Scheme 1. Synthetic routes to the [1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide derivatives 1ceg, 2bed, 12aed and to the N,N-diethyl-5-(isobutylamino)-8-methyl-10-
oxo-10H-pyrimido[1,2-a][1,8]naphthyridine-6-carboxamide 15.
spectra (CDCl3) of diethylamide derivatives 2bed,12aed,14 and 15,
as previously observed in the case of analogous compounds [11], the
complex pattern of CH2 signals indicates that the methylene protons
are diastereotopic, thus suggesting the chirality of these molecules
(atropisomerism due to the hindered rotation around the eCONEt2
bond because of the presence of an adjacent secondary or tertiary
alkylamino group). Particular evidence of this situation is given by
the 1H NMR spectra of the Mannich bases 12aed in which the 9-CH2
signals appear as an AB system (J ¼ 14.2 Hz) whose centres are sit-
uated in the ranges 4.47e4.52 ppm and 4.55e4.63 ppm, respec-
tively (compounds 12bed), whereas for 12a the AB system is
centred at 4.84 ppm and 4.92 ppm. On the other hand, in the case of
less hindered 5-chloroderivatives 11a,b the 9-CH2 protons are
almost isochronous and the 9-CH2 signal appears as a broad singlet
d
d
at
d 4.80 ppm (compound 11a) or a sharp singlet at d 4.63 ppm
(compound 11b), respectively.

M. Di Braccio et al. / European Journal of Medicinal Chemistry 86 (2014) 394e405
397
Table 1
Structures and pharmacological data of compounds 1ceg, 2bec, 12bec and 15.
Compd.
R
Dose
Analgesic
activitya
Anti-inflammatory
Spontaneous mice
(mg kgꢀ1 oral)
activityb (% inhibition)
locomotor activityc
(% inhibition)
1c
CH(CH3)2
CH(CH3)2
100
50
67*d
11
50*
3
Sedation
Sedation
1d
100
50
25
12.5
6.25
3.12
1.6
66*
64**d
15
e
80**
72**
57**
54**
40*
31*
31*
0
e
e
e
0.8
e
1e
1f
CH(CH3)2
CH(CH3)2
100
50
25
34
41
0
60**
35*
30*
12
12.5
e
100
50
0
0
72**
63*
25
12.5
6.25
e
e
e
70**
44**
19
1g
CH(CH3)2
100
50
25
0
e
34**
33*
9
e
2b
2c
C2H5
N(C2H5)2
N(C2H5)2
100
50
25
95**
81*
10
39**
29*
22
Sedation
Sedation
CH(CH3)2
100
50
25
12.5
6.25
100**
74**
72**
50*
31*
35*
36**
31*
8
Sedation
Sedation
Sedation
Sedation
0
2d
CH(CH3)2
N(C2H5)2
100
50
25
85**
96**
48*
13
0
Sedation
Sedation
Sedation
e
e
e
12.5
12a
N(C2H5)2
100
0
0
12b
12c
N(C2H5)2
N(C2H5)2
100
100
37
0
8
13
12d
N(C2H5)2
100
0
0
(continued on next page)

398
M. Di Braccio et al. / European Journal of Medicinal Chemistry 86 (2014) 394e405
Table 1 (continued )
Compd.
R
Dose
Analgesic
activitya
Anti-inflammatory
Spontaneous mice
locomotor activityc
(mg kgꢀ1 oral)
activityb (% inhibition)
(% inhibition)
15
100
50
25
71**d
54**
0
15
Indomethacin
10
84**e
51**e
a
Acetic acid induced writhing in mice.
Carrageenan induced rat paw oedema.
Measured as the distance travelled in 30 min.
*P < 0.05,**P < 0.01 significance as compared to controls (Student's t-test).
Data from Ref. [2].
b
c
d
e
3. Results and discussion
As regards the analogues of the lead compound 2a, all the com-
pounds showed an apparent analgesic activity in writhing test
prevailing over the anti-inflammatory activity which was exhibited
only by compounds 2c and 2b (Table 1). Actually, mice treated with
antinociceptive doses of the compounds showed reduced sponta-
neous locomotor activity with respect to control mice when evalu-
The analgesic and anti-inflammatory activities of compounds
under study (Table 1) have been evaluated in vivo at the initial dose
of 100 mg kgꢀ1 oral. The compounds exhibiting a statistically sig-
nificant activity at this dose were further tested at doses decreasing
by a factor of two, until statistically significant activity was no
longer observed.
In the subset of molecules structurally related to the com-
pound 1b the prevalence of anti-inflammatory activity over the
analgesic effect was clearly detected. In particular, derivative 1d
proved to possess very potent anti-inflammatory activity in the
ated by video-analysis (14.9
5.0
m
30 minꢀ1 and
16.8 3.6 m 30 minꢀ1 respectively travelled by treated animals
compared to 26.3 3.5 m 30 minꢀ1 for control animals), suggesting
the occurrence of confounding sedative effect. Indeed, the fine
modulation of the 4-methyl-1-piperazinyl moiety of 2a performed
in derivatives 2c and 2d as well as the replacement of 9-isopropyl
substituent with a 9-ethyl group (compound 2b) produced a fall in
analgesic activity with respect to the parent compound 2a that had
proved to possess antinociceptive effect at non-sedative doses [3,4].
Unfortunately, the Mannich bases 12aed were completely
inactive as anti-inflammatory or antinociceptive agents up to
100 mg kgꢀ1 oral dose.
carrageenan-induced paw oedema assay in rats showing
a
threshold dose of 1.6 mg kgꢀ1 oral (about 30% inhibition;
P < 0.05). Compound 1d proved to be also the most effective
compound within this series in counteracting the oedematous
response caused by carrageenan injection, showing a 80% inhi-
bition at 100 mg kgꢀ1 and being its 12.5 mg kgꢀ1 oral dose quite
equiactive with conventional NSAID indomethacin 10 mg kgꢀ1
(Table 1). Compound 1d, at the highest dose tested
(100 mg kgꢀ1os) proved to be completely devoid of acute gas-
trolesivity in rats (0/8 rats with gastric lesions) showing a more
favourable tolerability profile than indomethacin (8/8 rats with
gastric lesions). It is noteworthy that the substitution of iso-
butylamino moiety with isopropylamino group in position 5 of
the [1,2,4]triazolo[4,3-a][1,8]naphthyridine structure yielded the
compound 1d more potent and effective than the parent com-
pound 1b (Fig. 1) [5] and of the analogue 1a (Fig. 1) [1,6] previ-
ously described as interesting anti-inflammatory agents.
Finally, the N,N-diethyl-5-(isobutylamino)-8-methyl-10-oxo-
10H-pyrimido[1,2-a][1,8]naphthyridine-6-carboxamide 15 did not
exhibit any appreciable anti-inflammatory activity, whereas
showed a certain antinociceptive property being significantly active
(54% inhibition in the writhing test in mice, P < 0.01) down to the
50 mg kgꢀ1 dose without any sedative effect.
4. Conclusions
Taking into account the pharmacological data of the new com-
pounds 1ceg, 2bed, 12aed and 15 described in the present paper
(Tables 1 and 2), the following conclusions can be drawn.
Compound 1d inhibited in vitro platelet aggregation induced in
guinea pig platelet rich plasma (PRP) by arachidonic acid, U46619 [a
synthetic analogue of Thromboxane A2 (TXA2)] and ADP, showing a
non acetylsalicylic acid-like potency profile (Table 2). Such results
suggest for 1d antiplatelet activity also a COX-independent mech-
anism consistent with the findings reported for the analogue 1a (NF
161) when studied on prostaglandin E2 (PGE2) and prostacyclin
(PGI2) production by human umbilical vascular endothelial cells
(HUVEC) in response to arachidonic acid [6]. Further in vitro data
show that compound 1d, at variance with dexamethasone, does not
inhibit LPS-induced nitrite production in the murine macrophage
cell line J774, suggesting no interference with inducible nitric oxide
synthase (iNOS) pro-inflammatory activity. Indeed, compared with
cells treated with LPS in absence of any pharmacological treatment
- The overall good anti-inflammatory activities of compounds
1ceg confirms that the replacement of the 6-CON(C2H5)2 sub-
stituent of the lead compound 1a, with a properly monoalkyl
substituted 6-carboxamide group afforded very interesting anti-
inflammatory agents. The best N-alkyl substituents of the car-
boxamide group were C2H5 or C3H7 (compounds 1d,e,f),
whereas the short methyl group (compound 1c) or the branched
isopropyl group (compound 1g) afforded negative results.
- Maintaining fixed the 6-CONHC2H5 and 9-i-C3H7 substituents,
the 5-alkylamino group which proved to be the most effective
for the anti-inflammatory activity was the isopropylamino
group (compound 1d), clearly better than the isobutylamino
(lead compound 1b [5]) or 3-pentylamino (compound 1e)
groups.
(0.78
drug dexamethasone (1
nitrite production (0.39 0.09
0.05 m
g mgꢀ1 protein), conventional anti-inflammatory
mM), as expected, significantly inhibited
m
g mgꢀ1 protein; ꢀ50%; P < 0.01),
- The most active compound (1d) was a very potent anti-
inflammatory agent, which exhibited oedema inhibition values
of 80% (P < 0.01) and 72% (P < 0.01) at the 100 mg kgꢀ1 and
whereas compound 1d (100
mM) slightly reduced nitrite levels
(0.69 0.07
m
g mgꢀ1 protein; ꢀ12%; P > 0.05).

M. Di Braccio et al. / European Journal of Medicinal Chemistry 86 (2014) 394e405
399
Table 2
5. Experimental protocols
In vitro inhibitory activity of N-ethyl-9-isopropyl-5-(isopropylamino)[1,2,4]triazolo
[4,3-a][1,8]-naphthyridine-6-carboxamide 1d on platelet aggregation induced in
guinea pig PRP by ADP, arachidonic acid and U46619. Acetylsalicylic acid (ASA) was
used as reference drug.
5.1. Chemistry
Melting points were determined using a Fisher-Johns apparatus
and are uncorrected. IR spectra were recorded on a PerkineElmer
“Spectrum One” spectrophotometer (abbreviations relative to IR
bands: br ¼ broad, s ¼ strong, w ¼ weak, sh ¼ shoulder). 1H NMR
spectra were recorded partly on a Varian Gemini 200 (200 MHz)
spectrometer and partly on a Bruker WM-300 (300 MHz) spec-
trometer; chemical shifts (
thylsilane as an internal reference (
d
) are reported in ppm using tetrame-
¼ 0). Spin multiplicities are
d
given as follows: s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), dd (double doublet). J values for H-2, H-3, H-4 signals
(dd) of all triazolonaphthyridine derivatives: J2,3 ¼ J3,2 ¼ 4.7 Hz,
J2,4 ¼ J4,2 ¼ 1.7 Hz, J3,4 ¼ J4,3 ¼ 8.1 Hz. 13C NMR spectra were acquired
on a Bruker DPX spectrometer at 75.5 MHz with tetramethylsilane
as internal reference. Analyses were performed by the Laboratorio
di Microanalisi, Dipartimento di Scienze Farmaceutiche, Universita'
di Genova. Thin layer chromatograms were run on Merck silica gel
60 F254 precoated plastic sheets (layer thickness 0.2 mm). Column
chromatography was performed using Carlo Erba silica gel
(0.05e0.20 mm) or Carlo Erba neutral aluminium oxide (Brock-
mann activity I).
Compound IC50 (m
M) SDa
ADP (5.0
m
M) Arachidonic acid (100.0
m
M) U46619 (2.0 mM)
1d
281 24
24 10
45 15
89 21
>500
ASA
>500
a
IC50 ¼ compound concentration which inhibits platelet aggregation by 50%.
50 mg kgꢀ1 doses, respectively and it proved to be significantly
active down to the 1.6 mg kgꢀ1 threshold dose (inhibition 31%,
P < 0.05). It also showed significant analgesic activity (without
sedative effect) only at 50 mg kgꢀ1 dose (inhibition 64%,
P < 0.01). It is noteworthy that 1d was devoid of gastrolesive
effects at the anti-inflammatory dose equiactive with ulcero-
genic dose of indomethacin.
- Compound 1d exhibited also in vitro activity against the platelet
aggregation induced in guinea pig PRP by ADP, arachidonic acid
and U46619: it is noteworthy to point out that its antiplatelet
profile, compared with that of acetylsalicylic acid, suggests
additional mode of action other than COX-inhibition, thus con-
firming previous studies on analogous compounds [6]. The
occurrence of non COX-dependent mechanisms could be an
interesting feature since also the use of the newer NSAIDs,
namely the selective COX-2 inhibitors, has been associated with
increased cardiovascular risks raising a question on their safety
(withdrawal of rofecoxib and valdecoxib from the market) [12].
In vitro data showing the ineffectiveness of 1d in reducing ni-
trite production in LPS-treated murine macrophages seem to
rule out that the inhibition of iNOS pro-inflammatory pathway
could be one of these additional mechanisms which remain to
be clarified.
5.1.1. N-substituted-1,2-dihydro-4-hydroxy-2-oxo-1,8-
naphthyridine-3-carboxamides 4a,c,d
A mixture of 25.0 mmol (5.50 g) of ester 3 [8], 50 mL of ethanol
and an excess (10 mL) of propylamine or isopropylamine (com-
pounds 4c and 4d, respectively) or 25 mL of 33% methylamine in
absolute ethanol (compounds 4a) was heated in a closed vessel at
150 ꢁC for 16 h. After cooling the resulting suspension was
concentrated at reduced pressure. The suspended solid was
collected by filtration, washed with a little acetone and dried to give
the nearly pure compound 4 which was then crystallized from the
proper solvent. According to this procedure the following com-
pounds were obtained:
5.1.1.1. 1,2-Dihydro-4-hydroxy-N-methyl-2-oxo-1,8-naphthyridine-
3-carboxamide (4a). The reaction of 3 with methylamine yielded
5.10 g (93%) of 4a, whitish needles, m.p. 288e289 ꢁC dec, after
crystallization from ethanol. It was impossible to record the 1H
NMR spectrum of 4a due to its insolubility in all conventional sol-
vents. IR (KBr): 3250e2400 (OH þ NH), 1667 s and 1617 s (CO),
1572 s, br cmꢀ1. Anal. Calcd for C10H9N3O3 (219.20): C, 54.79; H,
4.14; N, 19.17. Found: C, 55.05; H, 4.26; N, 19.03.
- The 5-(4-alkyl-1-piperazinyl)derivatives 2bed, designed as an-
alogues of lead compound 2a [3,4], exhibited the expected good
antinociceptive properties in writhing test in mice, with the 9-
isopropyl substituted compounds 2c,d being apparently the
most potent analgesic agents. Moreover, it should be empha-
sized that all these antinociceptive effects were accompanied by
a marked sedation which could strongly confound the correct
evaluation of analgesic activity.
5.1.1.2. 1,2-Dihydro-4-hydroxy-N-propyl-2-oxo-1,8-naphthyridine-3-
carboxamide (4c). The reaction of 3 with propylamine yielded
5.75 g (93%) of 4c, whitish needles, m.p. 232e233 ꢁC, after crys-
tallization from ethanol (Lit. [13], no m.p. reported). IR (KBr):
3250e2400 (OH þ NH), 1660 s and 1614 s (CO), 1566 s, br cmꢀ1. 1H
NMR (300 MHz, DMSO-d6, ppm):
d
0.93 (t, J ¼ 7.3 Hz, 3H,
CONHCH2CH2CH3), 1.59 (m, 2H, CONHCH2CH2CH3), 3.34 (m, 2H,
CONHCH2CH2CH3; t, J ¼ 7.3 Hz, after treatment with D2O), 7.35 (dd,
J6,5 ¼ 8 Hz, J6,7 ¼ 4.8 Hz, 1H, H-6), 8.36 (dd, J5,6 ¼ 8 Hz, J5,7 ¼ 1.6 Hz,
1H, H-5), 8.69 (dd, J7,6 ¼ 4.8 Hz, J7,5 ¼1.6 Hz,1H, H-7), 10.24 (broad s,
1H, CONHCH2CH2CH3; disappeared with D2O), 12.27 (s, 1H, 1-NH;
disappeared with D2O); (4-OH signal was not detectable). Anal.
Calcd for C12H13N3O3 (247.25): C, 58.29; H, 5.30; N, 16.99. Found: C,
58.07; H, 5.45; N, 17.12.
- The replacement of the 9-alkyl substituent with a Mannich base
moiety (compounds 12aed) resulted in a nearly complete loss of
the analgesic/anti-inflammatory properties.
- Inserting a dihydropyrimidinone moiety in place of the triazole
ring in a molecule which maintained the same 5-amino and 6-
carboxamide groups of lead compound 1a, the new compound
15 was obtained which exhibited marked, although not potent,
analgesic activity (inhibition 71%, P < 0.01 and 54%, P < 0.01, at
the 100 mg kgꢀ1 and 50 mg kgꢀ1 doses, respectively). On the
contrary, compound 15 was nearly inactive in the Winter's assay
for anti-inflammatory activity.
5.1.1.3. 1,2-Dihydro-4-hydroxy-N-isopropyl-2-oxo-1,8-
naphthyridine-3-carboxamide (4d). The reaction of 3 with iso-
propylamine yielded 5.56 g (90%) of 4d, whitish needles, m.p.

400
M. Di Braccio et al. / European Journal of Medicinal Chemistry 86 (2014) 394e405
238e239 ꢁC, after crystallization from acetone (Lit. [14], no m.p.
reported). IR (KBr): 3250e2450 (OH þ NH), 1658 s and 1616 s (CO),
of 4d in CHCl3 (150 mL) was then added and the resulting mixture
was refluxed for 2 h. The final mixture was poured onto ice-water:
after hydrolysis of excess of POCl3, the mixture was carefully alkal-
ized with Na2CO3, then exhaustively extracted with CH2Cl2. The
combined extracts (dried over anhydrous Na2SO4 and evaporated to
dryness at reduced pressure) afforded a thick oil which was chro-
matographed on a silica gel column, eluting first with the mixture
CH2Cl2eEtOAc (3:1) to discard several impurities, then with the
mixture CH2Cl2eEtOAc (1:2) to recover 5d as a whitish solid (2.09 g,
49%); white needles, m.p. 182e184 ꢁC, after crystallization from
EtOAc/petroleum ether. IR (KBr): 3264 (NH), 1644 s (CO), 1586,
1558 s, br cmꢀ1 1H NMR (300 MHz, DMSO-d6, ppm):
. d 1.24 [d,
J ¼ 6.6 Hz, 6H, CONHCH(CH3)2], 4.11 [m, 1H, CONHCH(CH3)2], 7.35
(dd, J6,5 ¼ 8 Hz, J6,7 ¼ 4.8 Hz, 1H, H-6), 8.35 (dd, J5,6 ¼ 8 Hz,
J5,7 ¼ 1.6 Hz, 1H, H-5), 8.69 (dd, J7,6 ¼ 4.8 Hz, J7,5 ¼ 1.6 Hz, 1H, H-7),
10.15 [d, J ¼ 6.8 Hz, 1H, CONHCH(CH3)2; disappeared with D2O],
12.28 (s, 1H, 1-NH; disappeared with D2O); (4-OH signal was not
detectable). 13C NMR (75.5 MHz, DMSO-d6, ppm):
d 22.06, 40.81,
95.99, 109.85, 118.73, 133.35, 133.38, 154.00, 163.29, 169.45, 172.12.
Anal. Calcd for C12H13N3O3 (247.25): C, 58.29; H, 5.30; N, 16.99.
Found: C, 58.37; H, 5.51; N, 16.63.
1547 cmꢀ1. 1H NMR (200 MHz, CDCl3, ppm):
d
1.37 [d, J ¼ 6.6 Hz, 6H,
CONHCH(CH3)2], 4.39 [m,1H, CONHCH(CH3)2], 6.79 [d, J ¼ 6.8 Hz,1H,
CONHCH(CH3)2; disappeared with D2O], 7.58 (dd, J6,5 ¼ 8 Hz,
J6,7 ¼4.8 Hz,1H, H-6), 8.42 (dd, J5,6 ¼ 8 Hz, J5,7 ¼ 1.6 Hz,1H, H-5), 9.07
(dd, J7,6 ¼ 4.8 Hz, J7,5 ¼1.6 Hz, 1H, H-7). Anal. Calcd for C12H11Cl2N3O
(284.14): C, 50.72; H, 3.90; N,14.79. Found: C, 50.59; H, 4.09; N,14.96.
5.1.2. N-substituted-2,4-dichloro-1,8-naphthyridine-3-
carboxamides 5a,c
A mixture of 5.0 g of 4a (22.81 mmol) or 4c (20.22 mmol) and
50 mL of POCl3 was stirred at 110 ꢁC for 1 h. The excess of POCl3 was
removed by heating at reduced pressure and the residue was dis-
solved in warm water; the resulting dark solution (300 mL) was
cooled, carefully treated with NaHCO3 up to pH 7; CH2Cl2 (200 mL)
was added and the mixture was stirred at room temperature for
30 min, decolourized with charcoal, filtered and transferred in a
separatory funnel. The organic layer was collected and the aqueous
one was exhaustively extracted with CH2Cl2. The combined extracts
(dried over anhydrous Na2SO4 and evaporated to dryness at
reduced pressure) afforded a thick oil which was chromatographed
on a silica gel column, eluting first with the mixture CH2Cl2eEtOAc
(2:1) to discard some impurities, then with the mixture
CH2Cl2eEtOAceacetone (2:1:1) to recover the proper compound 5.
From the eluate collected, after removal of solvents, the following
compounds were obtained:
5.1.4. N-substituted-5-chloro-9-isopropyl[1,2,4]triazolo[4,3-a][1,8]
naphthyridine-6-carboxamides 6a,c,d and N-substituted-5-
hydroxy-9-isopropyl[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-
carboxamides 7a,c,d
A mixture of 8.0 mmol of 5a (2.05 g) or 5c (2.27 g) or 5d (2.27 g),
12.0 mmol (1.23 g) of isobutyrohydrazide and 10 mL of Dowtherm A
was stirred at 160 ꢁC for 30 min. After cooling, 10% aqueous Na2CO3
(50 mL) and CH2Cl2 (50 mL) were added and the mixture was further
stirred at room temperature for 30 min. After discarding some
insoluble impurities by filtration, the mixture was transferred in a
separatory funnel, then the organic layer was collected and the
aqueous one was exhaustively extracted with CH2Cl2. The combined
extracts were dried (anhydrous Na2SO4) then evaporated to dryness
at reduced pressure, and the residue was chromatographed on a
silica gel column eluting first with CH2Cl2 to remove Dowtherm A.
Then the column was eluted with the mixture CH2Cl2eEtOAc (1:1),
affording small amounts of the 5-hydroxyderivatives 7; the subse-
quent elutionwith the mixture CH2Cl2eEtOAceacetone (2:1:1) gave
the desired compounds 6. According to this procedure, there were
obtained the following compounds:
5.1.2.1. 2,4-Dichloro-N-methyl-1,8-naphthyridine-3-carboxamide
(5a). The reaction carried out with 4a gave 3.05 g (52%) of 5a, white
crystals, m.p. 231e232 ꢁC, after crystallization from acetone/pe-
troleum ether. IR (KBr): 3262 (NH), 1649 s (CO), 1589, 1548 cmꢀ1. 1H
NMR (300 MHz, DMSO-d6, ppm):
d
2.87 (d, J ¼ 4.7 Hz, 3H,
CONHCH3; s, after treatment with D2O), 7.89 (dd, J6,5 ¼ 8 Hz,
J6,7 ¼ 4.8 Hz, 1H, H-6), 8.74 (dd, J5,6 ¼ 8 Hz, J5,7 ¼ 1.6 Hz, 1H, H-5),
8.80 (broad q, 1H, CONHCH3; disappeared with D2O), 9.24 (dd,
J7,6 ¼ 4.8 Hz, J7,5 ¼ 1.6 Hz, 1H, H-7). 13C NMR (75.5 MHz, DMSO-d6,
5.1.4.1. 5-Chloro-9-Isopropyl-N-methyl[1,2,4]triazolo[4,3-a][1,8]
naphthyridine-6-carboxamide (6a) and 5-hydroxy-9-isopropyl-N-
methyl[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide (7a).
The reaction carried out with 5a afforded:
ppm):
d 25.89, 119.87, 124.50, 131.08, 134.24, 140.79, 149.01, 153.51,
155.96, 162.55. Anal. Calcd for C10H7Cl2N3O (256.09): C, 46.90; H,
2.76; N, 16.41. Found: C, 46.91; H, 2.77; N, 16.32.
Compound 7a: 0.09 g, (3.9%); white needles, m.p. 265e266 ꢁC,
after crystallization from acetone. 1H NMR was not recorded
because of the insolubility of 7a in all conventional solvents. IR
(KBr): 3170 br (NH þ OH), 1643 s (CO), 1612 cmꢀ1. Anal. Calcd for
5.1.2.2. 2,4-Dichloro-N-propyl-1,8-naphthyridine-3-carboxamide
(5c). The reaction carried out with 4c gave 3.16 g (55%) of 5c, as an
off white resinous solid; white needles, m.p. 118e119 ꢁC, after
crystallization from EtOAc/petroleum ether. IR (KBr): 3301 (NH),
C14H15N5O2 (285.31): C, 58.94; H, 5.30; N, 24.55. Found: C, 59.06; H,
.
1657 s (CO), 1595 w, 1579, 1554 cmꢀ1 1H NMR (300 MHz, CDCl3,
5.48; N, 24.61.
Compound 6a: 0.66 g, (27%); white crystals, m.p. 261e263 ꢁC,
after crystallization from EtOAc. IR (KBr): 3284 (NH), 1655 s (CO),
ppm):
d
1.06 (t, J ¼ 7.3 Hz, 3H, CONHCH2CH2CH3), 1.74 (m, 2H,
CONHCH2CH2CH3), 3.51 (m, 2H, CONHCH2CH2CH3; t, J ¼ 7.3 Hz,
after treatment with D2O), 7.44 (t, J ¼ 7.3 Hz, 1H, CONHCH2CH2CH3;
disappeared with D2O), 7.51 (dd, J6,5 ¼ 8 Hz, J6,7 ¼ 4.8 Hz, 1H, H-6),
8.28 (dd, J5,6 ¼ 8 Hz, J5,7 ¼ 1.6 Hz, 1H, H-5), 8.95 (dd, J7,6 ¼ 4.8 Hz,
1608 w, 1591 w cmꢀ1. 1H NMR (300 MHz, CDCl3, ppm):
d 1.50 [d,
J ¼ 6.8 Hz, 6H, 9-CH(CH3)2], 3.12 (d, J ¼ 4.8 Hz, 3H, CONHCH3; s, after
treatment with D2O), 4.44 [m, 1H, 9-CH(CH3)2], 7.65 (dd, 1H, H-3),
8.74e8.81 (m, 2H, H-2,4), 9.04 (broad s, 1H, CONHCH3; disappeared
J7,5 ¼ 1.6 Hz, 1H, H-7). 13C NMR (75.5 MHz, CDCl3, ppm):
d 11.55,
with D2O). 13C NMR (75.5 MHz, CDCl3, ppm):
d 21.00, 26.75, 28.10,
22.56, 41.94, 119.72, 123.43, 131.15, 134.16, 141.33, 150.62, 153.60,
155.16, 162.64. Anal. Calcd for C12H11Cl2N3O (284.14): C, 50.72; H,
3.90; N, 14.79. Found: C, 50.96; H, 3.74; N, 14.62.
118.53, 119.98, 122.51, 133.97, 136.62, 142.76, 148.24, 150.23, 157.25,
161.75. Anal. Calcd. for C14H14ClN5O (303.75): C, 55.36; H, 4.65; N,
23.06. Found: C, 55.39; H, 4.54; N, 23.34.
5.1.3. 2,4-Dichloro-N-isopropyl-1,8-naphthyridine-3-carboxamide
(5d)
To a cooled amount of 300 mmol (21.93 g) of DMF were added
dropwise 300 mmol (27.5 mL) of POCl3 and the mixture was stirred
at room temperature for 15 min; a suspension of 15.0 mmol (3.71 g)
5.1.4.2. 5-Chloro-9-Isopropyl-N-propyl[1,2,4]triazolo[4,3-a][1,8]
naphthyridine-6-carboxamide (6c) and 5-hydroxy-9-isopropyl-N-
propyl[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide (7c).
The reaction carried out with 5c afforded:

M. Di Braccio et al. / European Journal of Medicinal Chemistry 86 (2014) 394e405
401
Compound 7c: 0.11 g, (4.4%); white needles, m.p. 236e237 ꢁC,
after crystallization from acetone. IR (KBr): 3200 br (NH þ OH),
with 6a (0.61 g) and isopropylamine yielded 1c (0.57 g, 87%); yel-
low needles, m.p. 161e163 ꢁC, after crystallization from diisopropyl
ether. IR (KBr): 3290 br (NH), 1642 s (CO), 1616, 1558 cmꢀ1. 1H NMR
1658 sh and 1632 s (CO), 1605 w, 1571 w, 1543 cmꢀ1 1H NMR
.
(300 MHz, DMSO-d6, ppm):
d
0.95 (t,
J
¼
7.3 Hz, 3H,
(300 MHz, CDCl3):
d
1.33 [d, J ¼ 6.3 Hz, 6H, HNCH(CH3)2], 1.55 [d,
CONHCH2CH2CH3), 1.42 [d, J ¼ 6.8 Hz, 6H, 9-CH(CH3)2], 1.55 (m, 2H,
CONHCH2CH2CH3), 3.55 (m, 2H, CONHCH2CH2CH3; t, J ¼ 7.3 Hz,
after treatment with D2O), 4.41 [m,1H, 9-CH(CH3)2], 7.74 (dd,1H, H-
3), 8.73 (dd, 1H, H-4), 8.86 (dd, 1H, H-2), 10.19 (broad t, 1H,
CONHCH2CH2CH3; disappeared with D2O), 13.84 (broad s, 1H, OH;
disappeared with D2O). Anal. Calcd for C16H19N5O2 (313.36): C,
61.33; H, 6.11; N, 22.35. Found: C, 61.18; H, 6.23; N, 22.49.
J ¼ 6.8 Hz, 6H, 9-CH(CH3)2], 3.04 (d, J ¼ 4.7 Hz, 3H, CONHCH3, s after
treatment with D2O), 3.95 [m, 1H, HNCH(CH3)2], 4.43 [m, 1H, 9-
CH(CH3)2], 7.48 (dd, 1H, H-3), 8.45 (dd, 1H, H-4), 8.73 (dd, 1H, H-
2), 9.96 [d, 9.7 Hz, 1H, HNCH(CH3)2; disappeared with D2O], 10.57
(broad s,1H, CONHCH3, disappeared with D2O). 13C NMR (75.5 MHz,
CDCl3, ppm):
d 21.13, 24.28, 26.08, 28.09, 51.12, 98.94, 115.92,
120.60, 136.62, 144.04, 149.84, 150.03, 152.90, 155.54, 167.55. Anal.
Calcd. for C17H22N6O (326.40): C, 62.56; H, 6.79; N, 25.75. Found: C,
62.40; H, 6.91; N, 25.50.
Compound 6c: 1.20 g (45%); white crystals, m.p. 215e216 ꢁC,
after crystallization from EtOAc/petroleum ether. IR (KBr): 3278
(NH), 1656 s (CO), 1606 w, 1590 w, 1571 w cmꢀ1. 1H NMR (300 MHz,
CDCl3, ppm):
d
1.07 (t, J ¼ 7.3 Hz, 3H, CONHCH2CH2CH3), 1.54 [d,
5.1.5.2. N-Ethyl-9-isopropyl-5-(isopropylamino)[1,2,4]triazolo[4,3-a]
[1,8]naphthyridine-6-carboxamide (1d). The reaction carried out
with 6b [4] (0.64 g) and isopropylamine yielded 1d (0.58 g, 85%);
yellow needles, m.p. 148e149 ꢁC, after crystallization from diiso-
J ¼ 6.8 Hz, 6H, 9-CH(CH3)2], 1.75 (m, 2H, CONHCH2CH2CH3), 3.54
(m, 2H, CONHCH2CH2CH3; t, J ¼ 7.3 Hz, after treatment with D2O),
4.47 [m, 1H, 9-CH(CH3)2], 7.65 (dd, 1H, H-3), 8.74e8.81 (m, 2H, H-
2,4), 8.96 (broad s, 1H, CONHCH2CH2CH3; disappeared with D2O).
propyl ether. IR (KBr): 3220 br (NH), 1641 s (CO), 1616, 1556 s cmꢀ1
.
13C NMR (75.5 MHz, CDCl3, ppm):
d 11.72, 21.04, 22.57, 28.13, 41.94,
1H NMR (300 MHz, CDCl3, ppm):
d
1.30e1.34 [d þ t, 6H þ 3H,
118.52, 120.09, 122.49, 133.87, 136.57, 142.75, 148.32, 150.21, 157.26,
161.05. Anal. Calcd. for C16H18ClN5O (331.80): C, 57.92; H, 5.47; N,
21.11. Found: C, 57.66; H, 5.55; N, 20.93.
HNCH(CH3)2 þ CONHCH2CH3], 1.55 [d, J ¼ 6.8 Hz, 6H, 9-CH(CH3)2],
3.52 (m, 2H, CONHCH2CH3; q, after treatment with D2O), 3.95 [m,
1H, HNCH(CH3)2], 4.43 [m, 1H, 9-CH(CH3)2], 7.48 (dd, 1H, H-3), 8.45
(dd, 1H, H-4), 8.73 (dd, 1H, H-2), 9.97 [d, J ¼ 9.8 Hz, 1H,
HNCH(CH3)2; disappeared with D2O], 10.62 (broad s, 1H,
CONHCH2CH3, disappeared with D2O). 13C NMR (75.5 MHz, CDCl3,
5.1.4.3. 5-Chloro-N-9-diisopropyl[1,2,4]triazolo[4,3-a][1,8]naphthyr-
idine-6-carboxamide (6d) and 5-hydroxy-N-9-diisopropyl[1,2,4]tri-
azolo[4,3-a][1,8]naphthyridine-6-carboxamide (7d). The reaction
carried out with 5d afforded:
ppm):
136.60, 144.03, 149.82, 150.11, 152.99,155.54, 166.72. Anal. Calcd. for
18H24N6O (340.43): C, 63.50; H, 7.11; N, 24.69. Found: C, 63.31; H,
d 14.62, 21.14, 24.27, 28.09, 34.38, 51.16, 99.06, 115.96, 120.61,
Compound 7d: 0.10 g (4%); white needles, m.p. 284e285 ꢁC, after
crystallization from acetone. IR (KBr): 3167 br (NH þ OH), 1639 s
(CO), 1611, 1557, 1541 w cmꢀ1. 1H NMR (200 MHz, DMSO-d6, ppm):
C
7.38; N, 24.59.
d
1.23 [d, J ¼ 6.8 Hz, 6H, CONHCH(CH3)2], 1.43 [d, J ¼ 6.8 Hz, 6H, 9-
CH(CH3)2], 4.15 [m, 1H, CONHCH(CH3)2], 4.41 [m, 1H, 9-CH(CH3)2],
7.78 (dd,1H, H-3), 8.73 (dd,1H, H-4), 8.89 (dd,1H, H-2),10.15 [broad
d, 1H, CONHCH(CH3)2; disappeared with D2O], 13.30 (broad s, 1H,
OH; disappeared with D2O). Anal. Calcd for C16H19N5O2 (313.36): C,
61.33; H, 6.11; N, 22.35. Found: C, 61.20; H, 6.35; N, 22.30.
5.1.5.3. N-Ethyl-5-[(1-ethylpropyl)amino]-9-isopropyl[1,2,4]triazolo
[4,3-a][1,8]naphthyridine-6-carboxamide (1e). The reaction carried
out with 6b [4] (0.64 g) and 3-pentylamine gave 1e (0.62 g, 84%);
yellow needles, m.p. 92.5e94 ꢁC, after crystallization from petro-
leum ether. IR (KBr): 3235 br (NH), 1630 s (CO), 1606, 1556 s cmꢀ1
.
Compound 6d: 1.04 g, (39%); white crystals, m.p. 258e260 ꢁC,
after crystallization from EtOAc. IR (KBr): 3265 (NH), 1672 sh and
1650 s (CO), 1602 w, 1588 w, 1564 cmꢀ1. 1H NMR (200 MHz, CDCl3,
1H NMR (200 MHz, CDCl3, ppm):
d
0.99 [t, J ¼ 7.4 Hz, 6H,
HNCH(CH2CH3)2], 1.33 (t, J ¼ 7.2 Hz, 3H, CONHCH2CH3), 1.55e1.85
[m, 4H, HNCH(CH2CH3)2], 1.59 [d, J ¼ 6.8 Hz, 6H, 9-CH(CH3)2],
3.45e3.80 [m, 2H þ 1H, CONHCH2CH3 þ HNCH(CH2CH3)2], 4.50 [m,
1H, 9-CH(CH3)2], 7.53 (dd, 1H, H-3), 8.54 (dd, 1H, H-4), 8.75 (dd, 1H,
H-2), 10.35 [d, J ¼ 9.8 Hz, 1H, HNCH(CH2CH3)2; disappeared with
D2O], 10.53 (broad s, 1H, CONHCH2CH3, disappeared with D2O).
Anal. Calcd. for C20H28N6O (368.47): C, 65.19; H, 7.66; N, 22.81.
Found: C, 65.20; H, 7.77; N, 23.07.
ppm):
d
1.37 [d, J ¼ 6.8 Hz, 6H, CONHCH(CH3)2],1.59 [d, J ¼ 6.8 Hz, 6H,
9-CH(CH3)2], 4.40 [m,1H, CONHCH(CH3)2], 4.54 [m,1H, 9-CH(CH3)2],
7.65 (dd,1H, H-3), 8.70e8.95 [m, 3H, H-2,4 þ CONHCH(CH3)2; m, 2H
after treatment with D2O]. Anal. Calcd. for C16H18ClN5O (331.80): C,
57.92; H, 5.47; N, 21.11. Found: C, 58.08; H, 5.30; N, 21.19.
5.1.5. General procedure for the synthesis of compounds 1ceg
A mixture of 2.0 mmol of the proper 5-chloroderivative 6, 25 mL
of ethanol and an excess (5 mL) of the proper alkylamine was
heated in a closed vessel at 140 ꢁC for 16 h. After cooling the
resulting yellow fluorescent solution was evaporated to dryness in
vacuo and the residue was partitioned between CH2Cl2 (100 mL)
and 5% aqueous NaHCO3 (50 mL); the organic layer was collected
and the aqueous one was further extracted twice with CH2Cl2. The
combined organic extracts were dried (anhydrous Na2SO4) then
evaporated to dryness at reduced pressure, and the residue was
chromatographed on a silica gel column eluting with the mixture
CH2Cl2eEtOAc (1:1) to afford the pure compounds 1ceg as thick
oils which were treated with a small amount of petroleum ether to
give yellow crystalline solids, which then were crystallized from an
appropriate solvent or solvent mixture. The following compounds
were prepared in this way:
5.1.5.4. 9-Isopropyl-5-(isopropylamino)-N-propyl[1,2,4]triazolo[4,3-
a][1,8]naphthyridine-6-carboxamide (1f). The reaction carried out
with 6c (0.66 g) and isopropylamine yielded 1f (0.64 g, 90%); yellow
needles, m.p. 145e146 ꢁC, after crystallization from diisopropyl
ether/petroleum ether. IR (KBr): 3225 br (NH), 1639 s (CO), 1613,
1558 s cmꢀ1. 1H NMR (300 MHz, CDCl3,ppm):
d
1.05 (t, J ¼ 7.3 Hz,
3H, CONHCH2CH2CH3), 1.33 [d, J ¼ 6.3 Hz, 6H, HNCH(CH3)2], 1.55 [d,
J ¼ 6.8 Hz, 6H, 9-CH(CH3)2],1.72 (m, 2H, CONHCH2CH2CH3), 3.46 (m,
2H, CONHCH2CH2CH3; t, J ¼ 7.3 Hz, after treatment with D2O), 3.94
[m, 1H, HNCH(CH3)2], 4.44 [m, 1H, 9-CH(CH3)2], 7.48 (dd, 1H, H-3),
8.45 (dd, 1H, H-4), 8.72 (dd, 1H, H-2), 9.97 [d, J ¼ 9.9 Hz, 1H,
HNCH(CH3)2; disappeared with D2O], 10.65 (broad s, 1H,
CONHCH2CH2CH3, disappeared with D2O). 13C NMR (75.5 MHz,
CDCl3, ppm):
d 11.87, 21.14, 22.63, 24.26, 28.09, 41.33, 51.16, 99.18,
115.97, 120.62, 136.60, 144.04, 149.81, 150.12, 152.98, 155.54, 166.84.
Anal. Calcd. for C19H26N6O (354.46): C, 64.38; H, 7.39; N, 23.71.
Found: C, 64.20; H, 7.55; N, 23.60.
5.1.5.1. 9-Isopropyl-5-(isopropylamino)-N-methyl[1,2,4]triazolo[4,3-
a][1,8]naphthyridine-6-carboxamide (1c). The reaction carried out

402
M. Di Braccio et al. / European Journal of Medicinal Chemistry 86 (2014) 394e405
5.1.5.5. N-9-diisopropyl-5-(isopropylamino)[1,2,4]triazolo[4,3-a][1,8]
naphthyridine-6-carboxamide (1g). The reaction carried out with 6d
(0.66 g) and isopropylamine yielded 1g (0.61 g, 86%); yellowneedles,
m.p. 152e154 ꢁC, after crystallization from diisopropyl ether/pe-
m.p. 180e182 ꢁC, after crystallization from EtOAc/petroleum ether.
IR (KBr): 3343 br (OH), 1635 s (CO), 1605, 1589 w, 1556 w cmꢀ1. 1H
NMR (200 MHz, CDCl3, ppm):
3H þ 3H, N(CH2CH3)2], 1.51 and 1.59 [2 d, J ¼ 6.8 Hz, 3H þ 3H, 9-
CH(CH3)2], 2.40e3.90 [m, 17H, piperazine
d
1.25 and 1.38 [2 t, J ¼ 7.2 Hz,
troleum ether. IR (KBr): 3211 br (NH),1622 s (CO),1575,1558 s cmꢀ1
.
1H NMR (200 MHz, CDCl3, ppm):
d
1.34 [2 d overlapped, J ¼ 6.3 Hz,
CH2's þ N(CH2CH3)2 þ NeCH2CH2OH þ OH; m, 16H after treatment
with D2O], 4.45 [m,1H, 9-CH(CH3)2], 7.55 (dd,1H, H-3), 8.48 (dd,1H,
H-4), 8.74 (dd, 1H, H-2). Anal. Calcd. for C23H33N7O2 (439.56): C,
62.85; H, 7.57; N, 22.31. Found: C, 62.57; H, 7.82; N, 22.01.
6H þ 6H, HNCH(CH3)2 þ CONHCH(CH3)2], 1.57 [d, J ¼ 6.8 Hz, 6H, 9-
CH(CH3)2], 3.95 [m, 1H, HNCH(CH3)2], 4.31 [m, 1H,
CONHCH(CH3)2], 4.45 [m, 1H, 9-CH(CH3)2], 7.50 (dd, 1H, H-3), 8.47
(dd, 1H, H-4), 8.75 (dd, 1H, H-2), 10.21 [broad s, 1H, HNCH(CH3)2;
disappeared with D2O], 10.42 (broad s, 1H, CONHCH(CH3)2, dis-
appeared with D2O). Anal. Calcd. for C19H26N6O (354.46):C, 64.38; H,
7.39; N, 23.71. Found: C, 64.14; H, 7.56; N, 23.84.
5.1.7. Synthesis of (dialkylamino)acetic acid hydrazides 9a,b
Ethyl bromoacetate (8.35 g, 50 mmol) was added dropwise to a
cooled solution of 100 mmol of morpholine (8.71 g) or 1-
methylpiperazine (10.02 g) in dry benzene (20 mL) and the
mixture was stirred at room temperature for 3 h. The white pre-
cipitate (dialkylamine hydrobromide) was then removed by filtra-
tion and the filtrate, containing the ethyl (dialkylamino)acetate,
was evaporated to dryness in vacuo. To the resulting oil, dissolved
in EtOH (50 mL), hydrazine hydrate (2.50 g, 50 mmol) was added
and the mixture was heated in closed vessel at 120 ꢁC for 8 h. After
removal of solvent, the residue was treated with a little petroleum
ether to give the nearly pure hydrazides as white solids.
5.1.6. General procedure for the synthesis of compounds 2bed
A mixture of 3.5 mmol of chloroderivative 8a [4] or 8b [3], 5 mL
of dimethyl sulfoxide and an excess (5 mL) of the proper 1-
alkylpiperazine was heated at 130 ꢁC for 2 h, with stirring. Then
the resulting solution was poured into water and the mixture was
exhaustively extracted with CH2Cl2. The combined extracts were
dried (anhydrous Na2SO4), then concentrated to a small volume at
reduced pressure, and finally chromatographed on a silica gel col-
umn eluting first with the mixture CH2Cl2eEtOAc (1:1) to remove
impurities and small amounts of starting compounds. The subse-
quent elution with the mixture EtOAceacetoneeMeOH (9:9:2)
gave solid residues which were taken up in diethyl ether and
filtered to give pure compounds 2bed as off white solids which
were then crystallized from the proper solvents. The following
compounds were so obtained:
5.1.7.1. 4-Morpholinylacetic acid hydrazide (9a). Yield: 4.15 g (52%)
m.p. 99e101 ꢁC (Lit. [15] m.p. 103e104 ꢁC).
5.1.7.2. 4-Methyl-1-piperazinylacetic
acid
hydrazide
(9b).
Yield: 5.00 g (58%) m.p. 86e87 ꢁC (Lit. [16] m.p. 87e89 ꢁC).
5.1.8General procedure for the synthesis of 9-[(dialkylamino)
methyl]-5-chloro-N,N-diethyl[1,2,4]triazolo[4,3-a][1,8]
naphthyridine-6-carboxamides 11a,b
The mixture of 2,4-dichloro-N,N-diethyl-1,8-naphthyridine-3-
carboxamide 10 [9] (2.98 g, 10.0 mmol) and 4-morpholinylacetic
acid hydrazide 9a (2.39 g, 15.0 mmol) or 4-methyl-1-
piperazinylacetic acid hydrazide 9b (2.58 g, 15.0 mmol) and Dow-
therm A (10 mL) was stirred at 160 ꢁC for 1.5 h.
After cooling, 10% aqueous Na2CO3 (50 mL) and CH2Cl2 (50 mL)
were added and the mixture was further stirred at room temper-
ature for 30 min. After discarding some insoluble impurities by
filtration, the mixture was transferred in a separatory funnel, then
the organic layer was collected and the aqueous one was exhaus-
tively extracted with CH2Cl2. The combined extracts were dried
(anhydrous Na2SO4) then evaporated to dryness at reduced pres-
sure, and the residue was chromatographed on a silica gel column
eluting first with CH2Cl2 to remove Dowtherm A. Then the column
was eluted with EtOAc to remove small amounts of starting com-
pound 10 and some impurities; the subsequent elution with the
mixture EtOAceacetoneeMeOH (9:9:2) gave oily residues from
which, after treatment with a small amount of diisopropyl ether,
pure compounds 11a,b separated out as pale pink crystalline solids,
which then were crystallized from an appropriate solvent or sol-
vent mixture. According to this procedure, there were obtained the
following compounds:
5.1.6.1. N,N-9-triethyl-5-(4-methyl-1-piperazinyl)[1,2,4]triazolo[4,3-
a][1,8]naphthyridine-6-carboxamide (2b). The reaction carried out
with 8a [4] (1.16 g) and 1-methylpiperazine yielded 2b (1.17 g, 84%);
white crystals, m.p. 194e195 ꢁC, after crystallization from EtOAc. IR
(KBr): 1631 s (CO),1598, 1582, 1550 cmꢀ1. 1H NMR (300 MHz, CDCl3,
ppm):
d
1.23 and 1.35 [2 t, J ¼ 7.2 Hz, 3H þ 3H, N(CH2CH3)2], 1.50 (t,
J ¼ 7.4 Hz, 3H, 9-CH2CH3), 2.39 (s, 3H, NeCH3), 2.70e3.90 [m, 14H,
piperazine CH2's þ 9-CH2CH3 þ N(CH2CH3)2], 7.50 (dd, 1H, H-3),
8.43 (dd, 1H, H-4), 8.69 (dd, 1H, H-2). 13C NMR (75.5 MHz, CDCl3,
ppm):
d 11.98, 12.28, 13.57, 22.41, 38.75, 43.77, 46.30, 49.00e52.00
(very broad signal), 55.61, 116.29, 118.27, 121.29, 134.87, 143.66,
144.18, 148.49, 148.60, 152.42, 164.22. Anal. Calcd. for C21H29N7O
(395.51): C, 63.77; H, 7.39; N, 24.79. Found: C, 63.66; H, 7.60; N,
24.97.
5.1.6.2. N,N-diethyl-5-(4-ethyl-1-piperazinyl)-9-isopropyl[1,2,4]tri-
azolo[4,3-a][1,8]naphthyridine-6-carboxamide (2c). The reaction
carried out with 8b [3] (1.21 g) and 1-ethylpiperazine yielded 2c
(1.20 g, 81%); white crystals, m.p. 183e184 ꢁC, after crystallization
from EtOAc/diisopropyl ether. IR (KBr): 1634 s (CO), 1604, 1587 w,
1557 w cmꢀ1. 1H NMR (300 MHz, CDCl3, ppm):
d
1.14 (t, J ¼ 7.2 Hz,
3H, NeCH2CH3), 1.23 and 1.36 [2 t, J ¼ 7.2 Hz, 3H þ 3H, N(CH2CH3)2],
1.49 and 1.58 [2 d, J ¼ 6.8 Hz, 3H þ 3H, 9-CH(CH3)2], 2.30e3.74 [m,
12H, piperazine CH2's þ N(CH2CH3)2], 2.52 (q, J ¼ 7.2 Hz, 2H,
NeCH2CH3), 4.43 [m, 1H, 9-CH(CH3)2], 7.48 (dd, 1H, H-3), 8.43 (dd,
1H, H-4), 8.68 (dd, 1H, H-2). 13C NMR (75.5 MHz, CDCl3, ppm):
5.1.8.1. 5-Chloro-N,N-diethyl-9-(4-morpholinylmethyl)[1,2,4]triazolo
[4,3-a][1,8]naphthyridine-6-carboxamide (11a). Obtained from re-
action carried out with 4-morpholinylacetic acid hydrazide 9a
(1.58 g, 39%); white crystals, m.p. 161e163 ꢁC, after crystallization
from EtOAc/petroleum ether. IR (KBr): 1643 s (CO), 1599, 1585, 1555
d
12.02, 12.26, 13.57, 20.78, 21.35, 27.98, 38.76, 43.78, 52.45, 53.33,
53.36, 116.16, 118.40, 121.14, 134.91, 143.82, 144.32, 148.41, 148.45,
156.15, 164.29. Anal. Calcd. for C23H33N7O (423.56): C, 65.22; H,
7.85; N, 23.15. Found: C, 65.25; H, 7.88; N, 22.98.
w cmꢀ1 1H NMR (200 MHz, CDCl3, ppm):
. d 1.20 and 1.41 [2 t,
5.1.6.3. N,N-diethyl-5-[(4-(2-hydroxyethyl)-1-piperazinyl)]-9-
isopropyl[1,2,4]triazolo[4,3-a][1,8]-naphthyridine-6-carboxamide
(2d). The reaction carried out with 8b [3] (1.21 g) and 1-(2-
hydroxyethyl)piperazine yielded 2d (1.33 g, 86%); white crystals,
J ¼ 7.2 Hz, 3H þ 3H, N(CH2CH3)2], 2.99 (broad s, 4H, morpholine
NCH2's), 3.33 [q, J ¼ 7.2 Hz, 2H, 2H of N(CH2CH3)2], 3.55e3.97 [m,
6H, morpholine OCH2's þ 2H of N(CH2CH3)2], 4.80 (broad s, 2H, 9-
CH2N), 7.69 (dd, 1H, H-3), 8.61 (dd, 1H, H-4), 8.82 (dd, 1H, H-2).

M. Di Braccio et al. / European Journal of Medicinal Chemistry 86 (2014) 394e405
403
Anal. Calcd. for C19H23ClN6O2 (402.88): C, 56.64; H, 5.75; N, 20.86.
Found: C, 56.33; H, 5.59; N, 20.81.
heated at 130 ꢁC for 2 h, with stirring. The resulting solution was
then poured into water and the mixture was exhaustively extracted
with CH2Cl2. The combined extracts were dried (anhydrous
Na2SO4) and evaporated to dryness in vacuo to give thick oily res-
idues from which the pure compound 12b or 12d, respectively, was
obtained as described below:
5.1.8.2. 5-Chloro-N,N-diethyl-9-[(4-methyl-1-piperazinyl)methyl]
[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide
(11b).
Obtained from reaction carried out with 4-methyl-1-
piperazinylacetic acid hydrazide 9b (1.46 g, 35%); white crystals,
m.p. 106e107.5 ꢁC, after crystallization from diisopropyl ether. IR
(KBr): 1634 s (CO),1600 w,1584 w,1556 w cmꢀ1. 1H NMR (200 MHz,
5.1.10.1. N,N-diethyl-5-(4-methyl-1-piperazinyl)-9-(4-
morpholinylmethyl)[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-
carboxamide (12b). The oily residue derived from the reaction
carried out with 11a (1.21 g) was taken up in a mixture EtOAc/Et2O/
petroleum ether and let to stand at room temperature until the
sufficiently pure 12b$0.5H2O separated out as an off white solid
(1.09 g, 76%); pale pink crystals, m.p. 182e184 ꢁC, after crystalli-
zation from EtOAc/diisopropyl ether. IR (KBr): 3578 (crystallization
water), 1634 s (CO), 1602, 1583, 1552 w cmꢀ1. 1H NMR (300 MHz,
CDCl3, ppm):
d
1.17 and 1.38 [2 t, J ¼ 7.2 Hz, 3H þ 3H, N(CH2CH3)2],
2.38 (s, 3H, NeCH3), 2.61 and 2.95 (2 broad s, 4H þ 4H, piperazine
CH2's), 3.30 [q, J ¼ 7.2 Hz, 2H, 2H of N(CH2CH3)2], 3.52e3.95 [m, 2H,
2H of N(CH2CH3)2], 4.62 (s, 2H, 9-CH2N), 7.65 (dd,1H, H-3), 8.57 (dd,
1H, H-4), 8.79 (dd, 1H, H-2). Anal. Calcd. for C19H23ClN6O2 (415.93):
C, 57.76; H, 6.30; N, 23.57. Found: C, 57.52; H, 6.57; N, 23.52.
5.1.9. General procedure for the synthesis of compounds 12a,c
A mixture of 3.0 mmol of the 5-chloroderivatives 11a or 11b,
25 mL of ethanol and an excess (5 mL) of isopropylamine was
heated in a closed vessel at 140 ꢁC for 16 h. After cooling the
resulting solution was evaporated to dryness in vacuo and the
residue was partitioned between CH2Cl2 (100 mL) and 5% aqueous
NaHCO3 (50 mL); the organic layer was collected and the aqueous
one was further extracted twice with CH2Cl2. The combined organic
extracts were dried (anhydrous Na2SO4) then evaporated to dry-
ness at reduced pressure, and the oily residue was taken up in a
small amount of EtOAc/diisopropyl ether and let to stand at room
temperature until the sufficiently pure compound 12a or 12c,
respectively separated out as yellowish solid which were then
crystallized from the proper solvents. The following compounds
were prepared in this way:
CDCl3, ppm):
d
1.22 and 1.35 [2 t, J ¼ 7.2 Hz, 3H þ 3H, N(CH2CH3)2],
2.39 (s, 3H, NeCH3), 2.70e3.90 [m, 20H, piperazine
CH2's þ morpholine CH2's þ N(CH2CH3)2], 4.47 and 4.63 (AB system,
J ¼ 14.2 Hz, 1H þ 1H, 9-CH2N), 7.52 (dd, 1H, H-3), 8.44 (dd, 1H, H-4),
8.68 (dd, 1H, H-2). 13C NMR (75.5 MHz, CDCl3, ppm):
d 12.72, 13.96,
39.29, 44.20, 44.26, 46.71, 54.09, 55.02, 56.01, 67.40, 116.45, 118.88,
121.95, 135.47, 144.46, 144.67, 147.72, 148.96, 149.22, 164.52. Anal.
Calcd. for C24H34N8O2$0.5H2O (475.60): C, 60.61; H, 7.42; N, 23.56.
Found: C, 60.27; H, 7.62; N, 23.26.
5.1.10.2. N,N-diethyl-5-(4-methyl-1-piperazinyl)-9-[(4-methyl-1-
piperazinyl)methyl][1,2,4]tria-zolo[4,3-a][1,8]naphthyridine-6-
carboxamide (12d). The oily residue derived from the reaction
carried out with 11b (1.25 g) was chromatographed on a neutral
aluminium oxide column, eluting with the mixture CH2Cl2/petro-
leum ether/MeOH (6:6:1). The eluate collected, after removal of
solvents, afforded a thick oil from which the pure compound 12d
separated out as a solid after treatment with a small amount of
diisopropyl ether (0.84 g, 58%); pale pink crystals, m.p. 142e143 ꢁC,
after crystallization from EtOAc/petroleum ether. IR (KBr): 1636 s
(CO), 1605, 1586 w, 1555 w cmꢀ1. 1H NMR (300 MHz, CDCl3, ppm):
5.1.9.1. N,N-diethyl-5-(isopropylamino-9-(4-morpholinylmethyl))
[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide
(12a).
The reaction carried out with 11a (1.21 g) yielded 12a (1.18 g, 92%);
pale yellow crystals, m.p. 211e213 ꢁC, after crystallization from
EtOAc. IR (KBr): 3370 (NH), 1635 s (CO), 1601 s, 1558 w, 1532 cmꢀ1
.
1H NMR (200 MHz, CDCl3, ppm):
N(CH2CH3)2 HNCH(CH3)2], 2.76e4.00 [m, 13H, morpholine
d
1.14e1.43 [m, 12H,
d
1.22 and 1.35 [2 t, J ¼ 7.2 Hz, 3H þ 3H, N(CH2CH3)2], 2.27 and 2.39
þ
(2 s, 3H þ 3H, 2 NeCH3), 2.30e3.90 [m, 20H, 2 piperazine
CH2's þ N(CH2CH3)2], 4.52 and 4.59 (AB system, J ¼ 14.2 Hz,
1H þ 1H, 9-CH2N), 7.51 (dd, 1H, H-3), 8.43 (dd, 1H, H-4), 8.68 (dd,
CH2's þ N(CH2CH3)2 þ HNCH(CH3)2], 4.43 [d, J ¼ 9.8 Hz, 1H,
HNCH(CH3)2; disappeared with D2O], 4.84 and 4.91 (AB system,
J ¼ 14.2 Hz, 1H þ 1H, 9-CH2N), 7.56 (dd, 1H, H-3), 8.36 (dd, 1H, H-4),
8.72 (dd, 1H, H-2). Anal. Calcd. for C22H31N7O2 (425.53): C, 62.10; H,
7.34; N, 23.04. Found: C, 61.79; H, 7.56; N, 22.76.
1H, H-2). 13C NMR (75.5 MHz, CDCl3, ppm):
d 12.30, 13.54, 38.82,
43.80, 45.99, 46.33, 50.00e52.00 (very broad signal), 53.23, 54.11,
54.96, 55.60, 115.97, 118.42, 121.49, 134.99, 144.02, 144.14, 147.58,
148.55, 148.74, 164.11. Anal. Calcd. for C25H37N9O (479.63): C, 62.61;
H, 7.78; N, 26.28. Found: C, 62.92; H, 7.64; N, 26.06.
5.1.9.2. N,N-diethyl-5-(isopropylamino)-9-[(4-methyl-1-piperazinyl)
methyl][1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide (12c).
The reaction carried out with 11b (1.25 g) yielded 12c (1.04 g, 79%);
pale yellow crystals, m.p. 192e194 ꢁC, after crystallization from
5.1.11. 2-Amino-N,N-diethyl-4-(isobutylamino)-1,8-naphthyridine-
3-carboxamide (14)
EtOAc. IR (KBr): 3365 (NH), 1627 s (CO), 1600 s, 1545 s, cmꢀ1
.
1H
1.19 and 1.34 [2 t, J ¼ 7.2 Hz,
3H þ 3H, N(CH2CH3)2], 1.22 and 1.29 [2 d, J ¼ 6.3 Hz, 3H þ 3H,
HNCH(CH3)2], 2.11e3.90 [m, 13H, piperazine
A
mixture of 2-chloro-N,N-diethyl-4-(isobutylamino)-1,8-
NMR (300 MHz, CDCl3, ppm):
d
naphthyridine-3-carboxamide 13 [10] (3.35 g, 10.0 mmol),
ethanol (50 mL) and 30% aqueous NH3 (25 mL) was heated in a
closed vessel at 130 ꢁC for 24 h. After cooling, the mixture was
concentrated in vacuo to remove ethanol, then was diluted with
water (150 mL) and exhaustively extracted with CHCl3. The com-
bined extracts were dried (anhydrous Na2SO4), then evaporated to
dryness at reduced pressure to give a white solid which was taken
up in a small amount of Et2O and filtered obtaining pure compound
14 (2.40 g, 76%), white crystals, m.p. 251e252 ꢁC dec., after crys-
tallization from acetone/EtOAc. IR (KBr): 3467, 3353 and 3264
(NH2 þ NH), 1627 (CO), 1604, 1553 s, 1527 cmꢀ1. 1H NMR (200 MHz,
CH2's þ N(CH2CH3)2 þ HNCH(CH3)2], 2.28 (s, 3H, NeCH3), 4.40 [d,
J ¼ 10.3 Hz, 1H, HNCH(CH3)2; disappeared with D2O], 4.51 and 4.55
(AB system, J ¼ 14.2 Hz, 1H þ 1H, 9-CH2N), 7.46 (dd, 1H, H-3), 8.30
(dd, 1H, H-4), 8.67 (dd, 1H, H-2). 13C NMR (75.5 MHz, CDCl3, ppm):
d
12.71, 13.85, 24.07, 24.14, 39.12, 43.64, 45.95, 49.12, 53.25, 54.23,
54.92, 107.85, 116.61, 121.06, 133.78, 141.11, 143.70, 146.99, 148.50,
148.58, 164.69. Anal. Calcd. for C23H34N8O (438.58): C, 62.99; H,
7.81; N, 25.55. Found: C, 63.04; H, 7.89; N, 25.23.
CDCl3, ppm):
6H, HNCH2CH(CH3)2], 1.90 [m, 1H, HNCH2CH(CH3)2], 3.00e3.90 [m,
6H, N(CH2CH3)2 HNCH2CH(CH3)2], 4.85 [broad t, 1H,
HNCH2CH(CH3)2; disappeared with D2O], 5.23 (broad s, 2H, NH2;
d
0.80e1.35 [m, 6H, N(CH2CH3)2], 0.99 [d, J ¼ 6.6 Hz,
5.1.10. General procedure for the synthesis of compounds 12b,d
A mixture of 3.0 mmol of chloroderivative 11a or 11b, 5 mL of
dimethyl sulfoxide and an excess (5 mL) of 1-methylpiperazine was
þ

404
M. Di Braccio et al. / European Journal of Medicinal Chemistry 86 (2014) 394e405
disappeared with D2O), 7.15 (dd, J6,5 ¼ 8.2 Hz, J6,7 ¼4.3 Hz, 1H, H-6),
8,02 (dd, J5,6 ¼ 8.2 Hz, J5,7 ¼ 1.8 Hz, 1H, H-5), 8.78 (dd, J7,6 ¼ 4.3 Hz,
J7,5 ¼ 1.8 Hz, 1H, H-7). Anal. Calcd for C17H25N5O (315.42): C, 64.74;
H, 7.99; N, 22.20. Found: C, 64.41; H, 7.97; N, 22.27.
(VA), Italy]. Oedema values, obtained in the control group, were
considered arbitrarily as 100; the percentage of inhibition was
calculated from the difference in the swelling between the treated
and the control group.
5.1.12. N,N-diethyl-5-(isobutylamino)-8-methyl-10-oxo-10H-
5.2.2. Analgesic activity
pyrimido[1,2-a][1,8]naphthyridine-6-carboxamide (15)
Antinociceptive activity was studied by writhing test [18],
through the intraperitoneal injection of 0.2 mL/mouse of acetic acid
solution (0.6%) (Sigma Aldrich, Milan, Italy) 1 h after oral treatment
with the test drugs or indomethacin. Control group received 0.5%
methylcellulose. Complete extension of either hind limb was
regarded as a writhing response. The number of writhes of each
mouse was counted over a period of 30 min after the injection of
the noxious agent. The percentage of inhibition was calculated from
the difference in the writhing response between the treated and
the control group.
A mixture of compound 14 (1.89 g, 6.0 mmol), an excess of ethyl
acetoacetate (5 mL), Dowtherm A (5 mL) and monohydrate p-tol-
uenesulfonic acid (0.20 g) was heated at 180 ꢁC for 20 h with stir-
ring. After cooling, the mixture was partitioned between CH2Cl2
(100 mL) and 10% aqueous Na2CO3 (50 mL); the organic layer was
collected and the aqueous one was further extracted twice with
CH2Cl2. The combined organic extracts were dried (anhydrous
Na2SO4) then evaporated to dryness at reduced pressure. The dark
liquid residue was chromatographed on a silica gel column eluting
first with CH2Cl2 to remove Dowtherm A, then with EtOAc to
remove the excess ethyl acetoacetate and impurities and finally
with the mixture EtOAceTHF (1:1) to recover compound 15. The
eluate collected, after removal of solvents, afforded a brown thick
oil from which, after addition of a small amount of Et2O, the suffi-
ciently pure compound 15 separated out as a yellowish solid
(0.69 g, 30%); yellow crystals, m.p. 210e211 ꢁC, after crystallization
from EtOAc. IR (KBr): 3317 (NH), 1674 s (10-CO), 1620 (amide CO),
5.2.3. Spontaneous locomotor activity
Locomotor activity was measured by means of a black and white
video tracking system (Any-maze, Stoelting Co, Wood Dale, IL)
connected to a PC and operating on the principle of contrast [19].
The mouse to be tracked is placed against a contrasting background
within individual 21 ꢂ 21 ꢂ 30 cm open field (arena), the apparatus
assigns an XeY coordinate pair to the centroid of the subject and
the XeY coordinates are continuously monitored and compared to
detect motion. The locomotor activity was measured, after oral
administration of the compounds under study or vehicle, as the
distance (metres) travelled in 30 min (a measure of general
ambulation), recorded by the videocamera and analysed with the
video-tracking system that considers an animal inside a zone
whenever its centre point is within it. All experiments were con-
ducted from 9.00 to 13.00. For every compound under study, the
occurrence of statistically significant differences in the distance
travelled between the treated and the control group was indicated
as “sedation” in Table 1.
1602, 1552 w cmꢀ1 1H NMR (300 MHz, CDCl3, ppm):
. d 1.02 [d,
J ¼ 6.6 Hz, 6H, HNCH2CH(CH3)2], 1.13 and 1.30 [2 t, J ¼ 7.2 Hz,
3H þ 3H, N(CH2CH3)2], 2.00 [m,1H, HNCH2CH(CH3)2], 2.23 (s, 3H, 8-
CH3), 3.06 and 3.42 [2 m, 1H þ 1H, HNCH2CH(CH3)2], 3.29 and
3.49e3.72 [2 m, 2H þ 2H, N(CH2CH3)2], 5.51 [broad s, 1H,
HNCH2CH(CH3)2; disappeared with D2O], 6.10 (s, 1H, H-9), 7.31 (m,
1H, H-3), 8.14 (m, 1H, H-4), 8.73 (m, 1H, H-2). 13C NMR (75.5 MHz,
CDCl3, ppm): d 11.92, 13.45, 20.40, 23.72, 29.26, 38.83, 43.35, 52.75,
106.11, 106.24, 115.07, 121.39, 130.89, 144.31, 146.40, 148.49, 151.03,
161.77, 162.78, 166.73. Anal. Calcd. for C21H27N5O2 (381.48): C,
66.12; H, 7.13; N, 18.36. Found: C, 66.04; H, 7.30; N, 18.41.
5.2. Pharmacology
5.2.4. Gastrolesivity
The acute gastrolesivity of compound 1d (100 mg kgꢀ1) or
indomethacin (10 mg kgꢀ1) was evaluated by examining the
stomachs excised 5 h after oral administration in rats. The stom-
achs, fixed in 2% formalin (injection of 6 mL per stomach), were
opened along the greater curvature and mounted over a flat sur-
face. The stomachs were examined with a stereomicroscope (M8
Wild Heerbrung, Switzerland) by an observer unaware of the
treatment the rats received. Acute gastrolesivity was expressed as
the number of animals with gastric damage over the number of
treated animals.
Wistar rats (250e300 g) and Swiss mice (25e35 g) of either sex
were used. The test compounds were suspended in 0.5% methyl-
cellulose and administered at the initial dose of 100 mg kgꢀ1 by oral
gavage (1 mL 100 gꢀ1 body weight) in animals fasted overnight.
Compounds that exhibited a statistically significant effect at this
dose were further tested at doses decreasing by a factor of two. In
these experimental tests for the evaluation of analgesic and anti-
inflammatory activity, indomethacin (10 mg kgꢀ1 oral dose) was
used as reference drug. The same dose of indomethacin was used in
rats to investigate the acute gastrolesivity while in the same test the
compound 1d was studied at the oral dose of 100 mg kgꢀ1. Groups
of 8e10 animals were used and control animals were orally treated
with an equivalent volume of vehicle alone.
5.2.5. In vitro antiplatelet activity
Guinea pig blood was obtained by cardiac puncture after CO2
euthanasia and collected in plastic tubes in presence of sodium
citrate 3.8% (1 part citrate: 9 parts blood). After centrifugation for
15 min at 180 g to obtain platelet rich plasma (PRP), the remaining
blood was spinned 10 min at 2000 g to obtain platelet poor plasma
(PPP). Platelet aggregation was performed in the PAP4D aggreg-
ometer (BioData corp, USA) at 37 ꢁC under continuous stirring
(1000 rpm), following Born's turbidimetric method [20]. Aggrega-
tion was recorded as the percent change in light transmission: the
baseline was set using PRP and maximal transmission using PPP.
PRP was preincubated at 37 ꢁC for 5 min with solvent (dimethyl
sulfoxide, at the maximal final concentration 0.5%), compound 1d
or acetylsalicylic acid (ASA) before addition of platelet aggregatory
agent. Maximal aggregation was induced stimulating platelets with
All procedures and animal care were in accordance with the
European Communities Council Directive (86/609/EEC) and were
approved by the Veterinarian Department of the Italian Ministry of
Health (DL 116/92).
5.2.1. Anti-inflammatory activity
Anti-inflammatory activity was studied by inducing paw
oedema according to Winter's method [17]. 1% Carrageenan
(0.1 mL) (Sigma Aldrich, Milan, Italy) was injected subcutaneously
into the plantar surface of the rat hind paw 1 h after the oral
administration of the test compounds or indomethacin. Control
group received 0.5% methylcellulose. Paw volume was determined
immediately after the injection of phlogogen agent and again 3 h
later by means of a plethysmometer [Mod. 7140, Basile, Comerio
2
mM U46619, 5
mM ADP or 100 mM arachidonic acid. Tests were
performed within 3 h to avoid platelet inactivation. The effects of

M. Di Braccio et al. / European Journal of Medicinal Chemistry 86 (2014) 394e405
405
1d and acetylsalicylic acid were assessed as percent inhibition
Acknowledgements
compared with control sample. DMSO 0.5% did not interfere with
platelet aggregation.
The Authors thank Dr. G. Domenichini for his skilful technical
assistance in pharmacological experiments, O. Gagliardo for
elemental analyses, F. Tuberoni for I.R. spectra and Dr. R. Raggio for
1H NMR spectra. The financial support from University of Genoa
and University of Parma is gratefully acknowledged.
5.2.6. Cell cultures and nitrite determination
The murine monocyte/macrophage cell line J774 was obtained
from ATCC (Manassas, USA). J774 cell line was grown in Dulbec-
co's modified Eagle's medium (DMEM e Gibco Laboratories,
Grand Island, NY) and cultured at 37 ꢁC in humidified 5% CO2/95%
air. Culture medium was supplemented with 10% foetal bovine
serum and 2 mM glutamine. The cells were plated onto 24-well
culture plates at a density of 2.5 ꢂ 105 mLꢀ1 and allowed to
adhere overnight. Thereafter, medium was replaced with fresh
medium. J774 macrophages produced NO in response to LPS
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m
m
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m
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Results were expressed as mean
SEM. Differences between
treated and control groups were determined by Student's t-test
(*P < 0.05 or **P < 0.01 being considered as statistically significant
or highly significant, respectively).