1,8-Naphthyridines VI. Synthesis and anti-inflammatory activity of 5-(alkylamino)-N,N-diethyl[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamides with a new substitution pattern on the triazole ring

Article abstract of DOI:10.1016/j.ejmech.2007.04.016

On the basis of the good anti-inflammatory properties shown by the 9-alkyl-N,N-dialkyl-5-(alkylamino)[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamides 1, a series of analogues of such compounds, in which the 9-alkyl substituent was replaced by an ester or amide group (compounds 3a-i), was prepared and tested (inhibition of carrageenan-induced paw edema in the rat). Also two 5-(N-alkyl,N-acylamino) derivatives (compounds 4a,b) were synthesized and evaluated for the same purpose. Even though the general trend for these new [1,2,4]triazolo[4,3-a][1,8]naphthyridine derivatives was a decrease in activity compared with compounds 1, some of the new synthesized compounds exhibited still good anti-inflammatory properties.

Full text of DOI:10.1016/j.ejmech.2007.04.016

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European Journal of Medicinal Chemistry 43 (2008) 584e594
http://www.elsevier.com/locate/ejmech
Original article
1,8-Naphthyridines VI. Synthesis and anti-inflammatory activity of
5-(alkylamino)-N,N-diethyl[1,2,4]triazolo[4,3-a][1,8]naphthyridine-
6-carboxamides with a new substitution pattern on the triazole ring
a
a
a
Mario Di Braccio a, , Giancarlo Grossi , Giorgio Roma , Daniela Piras ,
*
Francesca Mattioli b, Marzia Gosmar b
a Dipartimento di Scienze Farmaceutiche, Universita` di Genova, Viale Benedetto XV, 3, 16132 Genova, Italy
b Dipartimento di Medicina Interna, Sezione di Farmacologia Clinica, Universita` di Genova, Viale Benedetto XV, 2, 16132 Genova, Italy
Received 23 February 2007; received in revised form 19 April 2007; accepted 26 April 2007
Available online 21 May 2007
Abstract
On the basis of the good anti-inflammatory properties shown by the 9-alkyl-N,N-dialkyl-5-(alkylamino)[1,2,4]triazolo[4,3-a][1,8]naphthyri-
dine-6-carboxamides 1, a series of analogues of such compounds, in which the 9-alkyl substituent was replaced by an ester or amide group
(compounds 3aei), was prepared and tested (inhibition of carrageenan-induced paw edema in the rat). Also two 5-(N-alkyl,N-acylamino)
derivatives (compounds 4a,b) were synthesized and evaluated for the same purpose. Even though the general trend for these new [1,2,4]tria-
zolo[4,3-a][1,8]naphthyridine derivatives was a decrease in activity compared with compounds 1, some of the new synthesized compounds
exhibited still good anti-inflammatory properties.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: [1,2,4]Triazolo[4,3-a][1,8]naphthyridine-6-carboxamide derivatives; Anti-inflammatory activity
1. Introduction
More recently, we have prepared and tested a new series of
[1,2,4]triazolo[4,3-a][1,8]naphthyridine derivatives 2 analogue
to compounds 1 in which the monoalkylamino group at posi-
tion 5 was replaced by a dialkylamino one. In this series, on
the basis of the pharmacological results reported above for
compounds 1, the substituents at positions 6 and 9 were
always CON(C2H5)2 and i-C3H7, respectively [2].
The pharmacological results afforded by compounds 2 were
satisfactory. Several members of this series displayed a fairly
good anti-inflammatory activity, even though inferior to that of
the most active compounds 1; in addition, some derivatives
showed very interesting analgesic properties (acetic acid in-
duced writhing test in mice). In particular, compound 2h
(Fig. 1) exhibited a very potent analgesic activity (85% inhibi-
tion of the acetic acid induced writhing in the mouse down to
3.12 mg/kg dose) [2].
We previously described the synthesis of a series of new
original anti-inflammatory agents having the 5-(monoalkyl-
amino)[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide
structure 1 [1].
Fourteen out of the twenty-four compounds prepared ex-
hibited a statistically significant anti-inflammatory activity,
when tested for the inhibitory properties against the carra-
geenan-induced paw edema in the rat. The best results were
obtained when R ¼ C2H5, R0 ¼ C2H5 or i-C4H9 and R00 ¼ C2H5
or i-C3H7. In particular, compounds 1c (R ¼ C2H5, R0 ¼ C2H5,
R00 ¼ i-C3H7) and 1n (R ¼ C2H5, R0 ¼ i-C4H9, R00 ¼ i-C3H7)
resulted the most effective anti-inflammatory agents (edema
inhibition ¼ 61% at 25 mg/kg p.o.) [1].
On this basis, in order to explore the potentiality of this new
class of analgesic/anti-inflammatoryagents, we haveconsidered
* Corresponding author. Tel.: þ39 10 3538352; fax: þ39 10 3538358.
E-mail address: dbraccio@unige.it (M. Di Braccio).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.04.016

M. Di Braccio et al. / European Journal of Medicinal Chemistry 43 (2008) 584e594
585
CH3
CH
CH3
R"
N
N
N
N
N
N
N
N
N
CON(C2H5)2
CONR2
H
H
N
R'
R'
1
1c : R' = C2H5
1n : R' = i-C4H9
CH3
CH
CH3
CH3
CH3
CH
N
N
N
N
N
N
N
N
N
CON(C2H5)2
CON(C2H5)2
R
N
N
CH3
R'
2
2h
Fig. 1. Structures of 9-alkyl-N,N-dialkyl-5-(alkylamino)[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamides 1 (e.g. 1c,n) and 5-(dialkylamino)-N,N-diethyl-9-
isopropyl[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamides 2 (e.g. 2h).
interesting to change the type of substitution at the position 9 of
the triazolonaphthyridine scaffold by introducing in this posi-
tion an ester or a substituted amide group directly or through
a one carbon spacer. This project bases its rationale on the
well known importance of the COOH and related groups in
the structures of the most known NSAIDs. Therefore we carried
out the synthesis of new compounds, represented by the general
structure 3 (Fig. 2), where the amide group in position 6 was al-
ways CON(C2H5)2, and the amino groups in position 5 were
NHeC2H5, NHeC4H9, or NHei-C4H9.
The preparation of ester-hydrazides 6aec was performed
using a method analogous to that employed by O’Callaghan
[3] for the synthesis of compound 6b, with work-up variations
to get an easier recovery of desired hydrazides. Thus, a suitable
diester (diethyl oxalate 5a, diethyl malonate 5b, or diethyl
2-methylmalonate 5c) was treated with hydrazine hydrate in
ethanol at room temperature overnight, using a 3:1 molar excess
of ester versus hydrazine to limit, as far as possible, the bishy-
drazide formation. After removal of the solvent, a little amount
of insoluble bishydrazide was removed by filtration and the
desired compounds 6aec were separated from the excess ester
through partition between water and diethyl ether, throwing
away the organic phase and collecting the aqueous one. After
removal of the water at reduced pressure, compounds 6aec
were obtained in moderate yields (27e36%).
We also prepared compounds 4a and 4b, in which an amide
functionality was obtained by involving the 5-amino group of
two of the most active compounds of the series 1.
2. Chemistry
The reaction of ethyl 2-chloro-2-oxoacetate with an ex-
cess of suitable amines (in dichloromethane solution, room
temperature, 30 min) afforded the corresponding ethyl
N,N-dialkyloxamates 7aec, which were in turn treated
with hydrazine hydrate in refluxing ethanol (2 h) to give
the desired N,N-dialkyl-2-hydrazino-2-oxoacetamides 8aec
(yields: 75e87%); similarly, the N-substituted ethyl malona-
mates 9a [4] or 9b [5], treated with hydrazine hydrate in
The synthetic strategy used to prepare compounds 3 con-
sisted in introducing the ester or amide moieties into proper
bifunctional hydrazides (compounds 6aec, 8aec, or 10a,b)
(Scheme 1), which were in turn cyclocondensed with suit-
able 1,8-naphthyridine derivatives (compounds 11a,b or 12)
to give, in one or two steps, the desired compounds 3
(Scheme 2).

586
M. Di Braccio et al. / European Journal of Medicinal Chemistry 43 (2008) 584e594
X
cyclocondensation afforded unsatisfactory yields of the ex-
N
R' = C2H5, C4H9, i-C4H9
pected compounds, these bifunctional hydrazides were more
conveniently treated with 12 [6] to give the intermediate chlor-
oderivatives 13aee. The experimental conditions of this step
were properly optimized, depending on the kind of hydrazide:
heating in Dowtherm A at 150 ꢀC for short time (20e45 min)
in the case of preparation of 13a,b,d or for 2 h in the case of
the sterically hindered compound 13c; heating in a mixture of
Dowtherm Aepyridine (1:1) to solubilize 10b at 160 ꢀC for
1.5 h, in the case of preparation of 13e. The yields ranged
from 52e64% (13a,b,d) to 15e19% (13e,c), respectively.
The subsequent reaction of chloroderivatives 13aee with
n-butylamine or isobutylamine [excess amine in ethanol,
130 ꢀC in closed vessel for 8 h (preparation of 3eeg), or ex-
cess amine in dimethyl sulphoxide at 130 ꢀC for 3 h (prepara-
tion of 3h,i)] afforded the desired compounds 3eei in good
yields (73e89%).
N
N
N
N
X = COOC2H5,CH2COOC2H5, CHCOOC2H5
CH3
CON(C2H5)2
or CONR2, CH2CONHR"
in which: R = C2H5, C3H7, i-C3H7
H
R'
R" = i-C4H9, 2-thiazolyl
3
CH3
C2H5
CH3 CH
N
N
N
N
N
N
N
N
CON(C2H5)2
C2H5
CON(C2H5)2
N
N
CH2 CH CH3
CH3
4a
4b
CO
CO
CH3
Finally, the reaction of 1b [1] with nicotinoyl chloride hy-
drochloride, in anhydrous toluene, in the presence of triethyl-
amine (120 ꢀC, 3 h) afforded 4a in moderate yield (40%),
while the treatment of 1n [1] with acetic anhydride at
140 ꢀC for 8 h gave a good yield (85%) of 4b (Scheme 2).
The structures attributed to the compounds described in this
paper are consistent with the results of elemental analyses and
N
Fig. 2. Structures of compounds 3 and 4a,b.
refluxing ethanol (2 h), afforded the corresponding N-
substituted 3-hydrazino-3-oxopropanamides 10a,b (yields:
60e74%), respectively (Scheme 1).
The cyclocondensation of 11a [6] or 11b [7] with ester-
hydrazides 6aec in excess (Dowtherm A, 150 ꢀC, 20 min)
gave fairly good yields (41e62%) of compounds 3aed. Since
in the case of amide-hydrazides 8aec and 10a,b the
1
IR and H NMR spectral data (see Section 5).
In the 1H NMR spectra (CDCl3) of compounds 3aei, 13aee,
4a,b, as previously observed in the case of compounds 1 [1]
and 2 [2], 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 C-6eCONEt2 bond). Particular
1
evidence of this situation is given by the H NMR spectra of
9-acetate and 9-acetamide derivatives (compounds 3b,c and
3gei, 13d,e, respectively) in which the 9-CH2 signals appear
as an AB system (J ¼ 18 Hz) whose centers are situated in
the ranges d 4.40e4.48 and 4.48e4.66, respectively (5-amino-
derivatives 3b,c,g,h), whereas for 5-chloroderivatives 13d,e the
AB system is centered at d 4.68 and 4.74 or 5.00 and 5.10,
respectively. On the other hand, in the case of 3i the 9-CH2
protons are almost isochronous and the AB pattern appears as
a nearly singlet at d 4.88.
R
R
R
Compd.
.
N2H4 H2O
COOC2H5
+
CONHNH2
COOC2H5
5a,6a
5b,6b
5c,6c
5a-c
6a-c
CH2COOC2H5
CHCOOC2H5
CH3
.
N2H4 H2O
COCl
+
CONR2
CONR2
COOC2H5
7a-c
HNR2
COOC2H5
CONHNH2
8a-c
3. Pharmacological results and discussion
NR2
Compd.
Compounds 3aei, 4a,b and 13 were tested in vivo for their
anti-inflammatory activity (carrageenan-induced paw edema
test in the rat). All the compounds were administered orally
and assayed at the initial dose of 200 mg/kg. Compounds which
exhibited a statistically significant activity at this dose were fur-
ther tested at doses decreasing by a factor of two. The results of
the pharmacological evaluation are listed in Table 1.
All the tested compounds did not induce direct signs of tox-
icity or mortality in the animals subjected to experiment. Four
of the twelve tested compounds showed a statistically signifi-
cant anti-inflammatory activity in carrageenan-induced paw
edema test in the rat. At the 200 mg/kg dose the degree of pro-
tection was 65% for 3c, 60% for 3e, 51% for 4a and 35% for
7a,8a
7b,8b
7c,8c
N(C2H5)2
N(C3H7)2
N(i-C3H7)2
Compd. NHR
H
CONHR
CONHR
.
N2H4 H2O
9a,10a
N
CH2COOC2H5
CH2CONHNH2
i-C4H9
H
9a,b
10a,b
N
S
9b,10b
N
Scheme 1. Synthesis of bifunctional hydrazides 6aec, 8aec and 10a,b.

M. Di Braccio et al. / European Journal of Medicinal Chemistry 43 (2008) 584e594
587
X
X
Compd.
R'
N
i-C4H9
COOC2H5
N
N
N
Cl
CON(C2H5)2
3a
3b
N
N
X
CONHNH2
+
C2H5
CH2COOC2H5
CH2COOC2H5
Δ
CON(C2H5)2
i-C4H9
i-C4H9
3c
3d
6a-c
NHR'
NHR'
CHCOOC2H5
CH3
6a : X = COOC2H5
11a,b
3a-d
6b : X = CH2COOC2H5
6c : X = CHCOOC2H5
11a : R' = C2H5
11b : R' = i-C4H9
CH3
X
Compd.
X
CON(C2H5)2
CON(C3H7)2
CON(i-C3H7)2
8a, 13a
8b, 13b
8c, 13c
N
N
N
N
Cl
CON(C2H5)2
N
N
+
X
CONHNH2
H
Δ
10a, 13d
10b, 13e
CH2CON
CON(C2H5)2
i-C4H9
8a-c, 10a,b
Cl
H
Cl
CH2CON
S
12
R'NH2
13a-e
N
Δ
R'
Compd.
X
X
N
N
i-C4H9
i-C4H9
CON(C2H5)2
CON(C3H7)2
3e
3f
N
N
H
n-C4H9
CH2CON
3g
3h
3i
i-C4H9
H
CON(C2H5)2
NHR'
3e-i
i-C4H9
CH2CON
CH2CON
i-C4H9
H
i-C4H9
S
N
C2H5
COCl
N
N
N
N
N
Δ
CON(C2H5)2
C2H5
R"
N
N
N
4a
CO
N
N
N
CON(C2H5)2
N
H
R'
CH3
CH CH3
N
1b,n
N
(CH3CO)2O
N
N
1b: R' = R" = C2H5
1n: R' = i-C4H9, R" = i-C3H7
CON(C2H5)2
Δ
N
CH2 CH CH3
CH3
4b
CO
CH3
Scheme 2. Synthetic routes to compounds 3aei and 4a,b.
3f. The most active compound was 3c that produced a statisti-
cally significant inhibition of the carrageenan-induced paw
edema also at the 100 mg/kg dose (35%), whereas the inhibi-
tion was not statistically significant at 50 mg/kg (21%). The
other three compounds did not display a statistically signifi-
cant activity when tested at the 100 mg/kg dose. From the
above pharmacological results the following remarks can be
made.
ꢁ With compounds bearing an ester group in position 9 the best
result was shown by the ethyl 9-acetate 3c [good and statis-
tically significant inhibition of paw edema at 200 mg/kg

588
M. Di Braccio et al. / European Journal of Medicinal Chemistry 43 (2008) 584e594
Table 1
Anti-inflammatory activity of compounds 3aei, 13c and 4a,b
X
X
X
N
N
N
N
N
N
N
N
N
N
N
N
N
CON(C2H5)2
CON(C2H5)2
CON(C2H5)2
R'
R"
Cl
NHR'
4a,b
3a-i
13c
Compound
R0
X
R00
Dose (mg/kg p.o.)
Anti-inflammatory activity in rata
Edema (mL) (mean ꢂ SD)
Inhibition (%)
3a
3b
3c
i-C4H9
C2H5
COOC2H5
CH2COOC2H5
CH2COOC2H5
e
e
e
200
200
200
100
50
233 ꢂ 560
348 ꢂ 61
102 ꢂ 45
190 ꢂ 46
228 ꢂ 24
254 ꢂ 42
20
0
i-C4H9
65**
35*
21
CH3
CHCOOC2H5
3d
3e
3f
i-C4H9
i-C4H9
i-C4H9
n-C4H9
e
e
e
e
200
12
CON(C2H5)2
CON(C3H7)2
200
100
200
117 ꢂ 31
204 ꢂ 32
190 ꢂ 40
60**
30
35*
H
CH2CON
3g
200
200
200
350 ꢂ 38
239 ꢂ 44
238 ꢂ 45
0
18
18
i-C4H9
H
CH2CON
CH2CON
3h
i-C4H9
e
i-C4H9
H
S
3i
i-C4H9
e
N
CO
4a
C2H5
C2H5
200
100
200
200
6
143 ꢂ 57
199 ꢂ 30
276 ꢂ 164
204 ꢂ 49
47 ꢂ 27
51**
31
N
4b
13c
i-C4H9
i-C3H7
COCH3
e
5
30
e
CON(i-C3H7)2
Indomethacin
92**
**P < 0.01, *P < 0.05 (Student t-test versus controls).
a
Carrageenan paw edema test (control value: 290 ꢂ 93 mL).
(65%), partially maintained at 100 mg/kg (35%)]. The ethyl
9-carboxylate 3a and ethyl 9-(2-propanoate) 3d did not show
any significant activity even at 200 mg/kg. Surprisingly,
when the 5-(isobutylamino) group in the ethyl 9-acetate 3c
was replaced with the 5-(ethylamino) one (compound 3b),
a complete disappearance of the activity was observed.
This result is in contrast with what previously observed [1]
for compounds of type 1 in which the 5-(ethylamino) deri-
vative 1c (Fig. 1) was as active as the corresponding
5-(isobutylamino) analogue 1n.
ꢁ In compounds bearing an amide group in position 9 the best
results were obtained with the 9-carboxamide derivatives 3e
and 3f. The edemainhibitionat 200 mg/kgwas goodand sta-
tistically significant (60%) for the N,N-diethyl derivative 3e,
whereas the N,N-dipropyl analogue 3f resulted only moder-
ately active (35%). Also the N,N-diisopropyl 5-chloro-9-
carboxamide 13c showed a similar level of edema inhibition
(30%), even if not statistically significant. The N-monosub-
stituted 9-acetamides 3gei resulted almost inactive.
31% (not statistically significant) at 100 mg/kg], whereas
the acetyl derivative 4b was nearly inactive.
4. Conclusions
Compounds 3aei, 4a,b and 13aee are novel chemically in-
teresting [1,2,4]triazolo[4,3-a][1,8]naphthyridine derivatives
in which a second acyl moiety has been introduced in addition
to the pre-existent N,N-diethyl 6-carboxamide group. Contrary
to our expectations, the introduction of an additional acyl
group did not improve the anti-inflammatory properties of
the new synthesized compounds. Nevertheless, some represen-
tative members of the series (3c,e,f and 4a) still maintain
a good activity.
5. Experimental protocols
5.1. Chemistry
ꢁ As far as the amides 4a and 4b are concerned, the nicoti-
noyl derivative 4a showed fairly good anti-inflammatory
properties [51% of edema inhibition at 200 mg/kg and
Melting points were determined using a FishereJohns
apparatus and are uncorrected. IR spectra were recorded
on a PerkineElmer ‘‘Spectrum One’’ spectrophotometer

M. Di Braccio et al. / European Journal of Medicinal Chemistry 43 (2008) 584e594
589
(abbreviations relative to IR bands: s ¼ strong, br ¼ broad,
w ¼ weak, sh ¼ shoulder). 1H NMR spectra were recorded
on a Varian Gemini 200 (200 MHz) spectrometer, using
(CH3)4Si as an internal reference (d ¼ 0), and chemical shifts
are reported in parts per million. Spin multiplicities are given
as follows: s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), dd (double doublet). Analyses of all new com-
pounds (Table 2) were within ꢂ0.4% of the theoretical values
and were performed by the Laboratorio di Microanalisi, Dipar-
According to this procedure the following compounds were
prepared.
5.1.1.1. Ethyl 2-hydrazino-2-oxoacetate (6a). Yield 4.52 g
(29%), white needles, m.p. 50e52 ꢀC (lit. [8]: m.p. 51e
53 ꢀC).
5.1.1.2. Ethyl 3-hydrazino-3-oxopropanoate (6b). Yield 6.23 g
(36%), white needles, m.p. 72e74 ꢀC (lit. [3]: m.p. 68e
69 ꢀC).
`
timento di Scienze Farmaceutiche, Universita di Genova. Thin
layer chromatograms were run on Merck silica gel 60 F254 pre-
coated plastic sheets (layer thickness 0.2 mm). Column chro-
matography was performed using Carlo Erba silica gel
(0.05e0.20 mm) or Carlo Erba neutral aluminium oxide
(Brockmann activity I).
5.1.1.3. Ethyl 3-hydrazino-2-methyl-3-oxopropanoate (6c).
1
Yield 5.12 g (27%), white needles, m.p. 86e87 ꢀC. H NMR
(CDCl3): d 1.20 (t, 3H, CH2CH3), 1.37 (d, 3H, CHCH3),
3.22 (q, 1H, CHCH3), 3.60e4.00 (broad signal, 2H,
CONHNH2; disappeared with D2O), 4.11 (q, 2H, CH2CH3),
7.74 (br s, 1H, CONHNH2; disappeared with D2O). IR
(KBr): 3288 and 3212 (NH), 1733 (ester CO), 1648 s (hydra-
5.1.1. General procedure for the synthesis of compounds
6aec
zide CO), 1544, 1459 cmꢃ1
.
A mixture of 0.36 mol of the proper diester [diethyl oxalate
(54.60 g), diethyl malonate (57.60 g) or diethyl 2-methylmalo-
nate (62.64 g)], 0.12 mol (6.00 g) of hydrazine hydrate and
40 mL of ethanol was stirred at room temperature overnight.
The final mixture was concentrated at reduced pressure and
cooled at 4 ꢀC so that the bishydrazide separated out as a white
solid. This solid was removed by filtration and the filtered solu-
tion was transferred into a separatory funnel and partitioned bet-
ween water and diethyl ether. The aqueous phase was collected
whereas the organic layer containing the excess diester was
extracted twice with water and discarded. The entire aqueous
phasewas then evaporated to dryness at reduced pressure afford-
ing a sticky residue from which, after addition of a little metha-
nol/diethyl ether (1:1), the desired monohydrazide separated out
as a white solid which was recovered by filtration and dried.
5.1.2. General procedure for the synthesis of N,N-dialkyl-2-
hydrazino-2-oxoacetamides 8aec
A one-pot procedure was used starting from ethyl 2-
chloro-2-oxoacetate and proper dialkylamines to give the in-
termediate ethyl N,N-dialkyloxamates 7aec, which in turn
were treated with hydrazine hydrate to give the desired
N,N-dialkyl-2-hydrazino-2-oxoacetamides 8aec. Thus, to an
ice-cooled solution of 0.10 mol of the proper dialkylamine
[diethylamine (7.30 g), dipropylamine (10.10 g) or diisopro-
pylamine (10.10 g)] in 20 mL of dichloromethane was care-
fully added 0.04 mol (5.44 g) of ethyl 2-chloro-2-oxoacetate
diluted in 20 mL of dichloromethane: an exothermic reaction
evolving white fumes occurred. The mixture was stirred at
Table 2
Elemental analyses of compounds 6aec, 10a,b, 3aei, 13aee and 4a,b
Compound
Molecular formula
Analysis (calcd./found)
C%
H%
N%
S%
6a
C4H8N2O3
C5H10N2O3
C6H12N2O3
C7H15N3O2
C6H8N4O2S
36.36, 36.15
41.09, 41.31
44.99, 45.25
48.54, 48.31
35.99, 36.26
61.15, 60.80
60.29, 60.29
61.95, 60.78
62.71, 63.08
62.85, 62.62
64.21, 64.06
63.55, 63.81
63.55, 63.53
57.48, 57.10
56.64, 56.42
58.53, 58.56
58.53, 58.65
57.62, 57.66
51.41, 51.43
64.70, 65.05
65.05, 64.96
6.10, 6.30
6.90, 7.06
7.55, 7.80
8.73, 8.87
4.03, 4.21
6.84, 6.92
6.58, 6.79
7.09, 7.40
7.32, 7.43
7.57, 7.51
7.98, 8.20
7.78, 7.92
7.78, 7.85
5.87, 6.06
5.75, 5.67
6.32, 5.97
6.32, 6.27
6.04, 6.34
4.09, 4.38
6.11, 6.25
7.60, 7.38
21.20, 21.44
19.17, 18.92
17.49, 17.21
24.26, 24.04
27.98, 27.64
20.37, 20.60
21.09, 21.10
19.70, 19.50
19.08, 19.39
22.31, 22.35
20.97, 20.82
21.62, 21.48
21.62, 21.77
23.32, 23.12
20.86, 20.78
19.50, 19.34
19.50, 19.57
20.16, 19.82
22.09, 21.85
22.01, 21.73
19.80, 19.79
e
6b
6c
e
e
10a
10b
3a
e
16.01, 15.80
C
21H28N6O3
e
3b
3c
C20H26N6O3
C22H30N6O3
C23H32N6O3
e
e
3d
3e
e
C23H33N7O2
C25H37N7O2
e
e
3f
3g
C
C
24H35N7O2
24H35N7O2
e
3h
3i
e
6.67, 6.35
C23H28N8O2S
C19H23ClN6O2
C21H27ClN6O2
C21H27ClN6O2
13a
13b
13c
13d
13e
4a
e
e
e
C
20H25ClN6O2
e
7.22, 7.43
C19H18ClN7O2S
C24H27N7O2
C23H32N6O2
e
e
4b

590
M. Di Braccio et al. / European Journal of Medicinal Chemistry 43 (2008) 584e594
room temperature for 30 min, then poured into cold water
(150 mL) and, after careful addition of NaHCO3 up to pH
7, was kept stirring at room temperature for further 30 min.
The organic layer was then collected and the aqueous one
was extracted twice with dichloromethane. The combined or-
ganic phase was then dried (Na2SO4) and evaporated to dryness
at reduced pressure to give the desired ethyl N,N-dialkyloxa-
mates 7aec as colourless oils. To these oils, dissolved in etha-
nol (15 mL), 0.04 mol (2.00 g) of hydrazine hydrate was added
and the mixture was refluxed for 2 h with stirring. The resulting
colourless solutions were then evaporated to dryness at reduced
pressure to give the crude compounds 8aec as waxy oils which
were used in the subsequent synthetic step without further
purification.
(50 mL) were added to the mixture which was stirred for
30 min at room temperature. The mixture was filtered to re-
move a reddish insoluble solid, then transferred into a separa-
tory funnel. The organic layer was collected and the aqueous
one was extracted twice with dichloromethane. The combined
organic extracts (dried over Na2SO4 and decolourized with
charcoal) were evaporated to dryness at reduced pressure to
give an orange liquid which was chromatographed on an alu-
minium oxide column, eluting with dichloromethane until
Dowtherm A was completely removed. The desired com-
pounds 3aed were then recovered eluting with tetrahydrofu-
ran. The eluate collected, after removal of solvent, afforded
a pale orange thick oil from which compounds 3aed were ob-
tained as reported below.
According to this procedure the following compounds were
prepared.
5.1.4.1. Ethyl 6-[(diethylamino)carbonyl]-5-(isobutylamino)
[1,2,4]triazolo[4,3-a][1,8]naphthridine-9-carboxylate 3a. The
solid residue derived from reaction carried out with 1.67 g of
11b [7] and 1.30 g of ethyl 2-hydrazino-2-oxoacetate 6a, was
taken up in a little diethyl ether and filtered to afford the
pure 3a (0.85 g, 41%), whitish solid, m.p. 217e218 ꢀC, after
crystallization from ethyl acetate with charcoal. 1H NMR
(CDCl3): d 1.01 and 1.03 [2d, 3H þ 3H, NCH2CH(CH3)2],
1.23 and 1.35 [2t, 3H þ 3H, N(CH2CH3)2], 1.47 (t, 3H,
COOCH2CH3), 2.16 [m, 1H, NCH2CH(CH3)2], 2.67 [m, 1H,
1H of NCH2CH(CH3)2], 3.27e3.57 [m, 4H, 3H of
N(CH2CH3)2 þ 1H of NCH2CH(CH3)2], 4.08 [m, 1H, 1H of
N(CH2CH3)2], 4.58 (q, 2H, COOCH2CH3), 6.75e6.89 [m,
2H, H-3 þ NH; dd (d ¼ 6.83), J3,4 ¼ 8.3 Hz, J3,2 ¼ 4.6 Hz,
1H, H-3, after treatment with D2O], 8.13 (dd, J4,3 ¼ 8.3 Hz,
J4,2 ¼ 1.5 Hz, 1H, H-4), 8.40 (dd, J2,3 ¼ 4,6 Hz, J2,4 ¼ 1.5 Hz,
1H, H-2). IR (KBr): 3264 (NH), 1744 (ester CO), 1604 s
5.1.2.1. N,N-Diethyl-2-hydrazino-2-oxoacetamide (8a). Yield
5.54 g (87%) (lit. [9]: m.p. 83e85 ꢀC).
5.1.2.2. 2-Hydrazino-2-oxo-N,N-dipropylacetamide (8b). Yield
5.90 g (79%).
5.1.2.3. 2-Hydrazino-N,N-diisopropyl-2-oxoacetamide (8c).
Yield 5.60 g (75%).
5.1.3. General procedure for the synthesis of N-substituted
3-hydrazino-3-oxopropanamides 10a,b
A mixture of 15 mmol of the proper N-substituted ethyl ma-
lonamate 9a or 9b, 15 mmol (0.75 g) of hydrazine hydrate and
100 mL of ethanol was refluxed for 2 h with stirring. From the
resulting colourless solution, compounds 10a,b were obtained
as described below.
(amide CO), 1545, 1463 cmꢃ1
.
5.1.3.1. 3-Hydrazino-N-isobutyl-3-oxopropanamide 10a. The
solution derived from reaction carried out with 2.80 g of
N-isobutyl ethyl malonamate 9a (prepared through the reac-
tion of ethyl 3-chloro-3-oxopropanoate with excess isobutyl-
amine in dichloromethane at room temperature; also a new
preparation has been reported [4]) was concentrated at reduced
pressure and diluted with a little petroleum ether so that 10a
separated out as a white solid (1.56 g, 60%), m.p. 71e72 ꢀC,
after crystallization from diisopropyl ether.
5.1.4.2. Ethyl [6-[(diethylamino)carbonyl]-5-(ethylamino)
[1,2,4]triazolo[4,3-a][1,8]naphthyridin-9-yl]acetate 3b. The
solid residue obtained from reaction carried out with 1.53 g
of 11a [6] and 1.44 g of ethyl 3-hydrazino-3-oxopropanoate
6b, was treated with a little diethyl ether so that 3b
separated out as a crystalline solid (1.12 g, 56%), whitish crys-
tals, m.p. 152.5e153 ꢀC, after crystallization from ethyl ace-
tate/petroleum ether with charcoal. 1H NMR (CDCl3):
d
1.03e1.49 [m, 12H, N(CH2CH3)2 þ 5-NCH2CH3 þ
COOCH2CH3], 3.12 (m, 1H, 1H of 5-NCH2CH3) 3.33e3.62
[m, 4H, 3H of N(CH2CH3)2 þ 1H of 5-NCH2CH3], 3.96 [m,
1H, 1H of N(CH2CH3)2], 4.13 (q, 2H, COOCH2CH3), 4.44
and 4.52 (AB system, J ¼ 18 Hz, 1H þ 1H, 9-CH2COO),
5.90 (br s, 1H, NH; disappeared with D2O), 7.04
(dd, J3,4 ¼ 8.3 Hz, J3,2 ¼ 4.6 Hz, 1H, H-3), 8.18 (dd,
J4,3 ¼ 8.3 Hz, J4,2 ¼ 1.5 Hz, 1H, H-4), 8.32 (dd,
J2,3 ¼ 4.6 Hz, J2,4 ¼ 1.5 Hz, 1H, H-2). IR (KBr): 3293 (NH),
5.1.3.2. 3-Hydrazino-3-oxo-N-(2-thiazolyl)propanamide 10b.
The solution derived from reaction carried out with 3.21 g of
N-(2-thiazolyl) ethyl malonamate 9b [5] was cooled down to
room temperature so that 10b crystallized as white needles
(2.23 g, 74%), m.p. 198e199 ꢀC, after recrystallization from
ethanol (lit. [10]: m.p. 200 ꢀC).
5.1.4. General procedure for the synthesis of compounds
3aed
1743 (ester CO), 1607 s (amide CO), 1547, 1476 cmꢃ1
.
A mixture of 5.0 mmol of compound 11a [6] or 11b [7],
10.0 mmol of the proper ester-hydrazide 6a, 6b or 6c and
6 mL of Dowtherm A was stirred at 150 ꢀC for 20 min. After
cooling, dichloromethane (100 mL) and 10% aqueous Na2CO3
5.1.4.3. Ethyl [6-[(diethylamino)carbonyl]-5-(isobutylamino)
[1,2,4]triazolo[4,3-a][1,8]naphthyridin-9-yl]acetate 3c. The
solid residue obtained from reaction carried out with 1.67 g
of 11b [7] and 1.44 g of ethyl 3-hydrazino-3-oxopropanoate

M. Di Braccio et al. / European Journal of Medicinal Chemistry 43 (2008) 584e594
591
6b, was taken up in a little diethyl ether to give pure 3c
(1.33 g, 62%), whitish crystals, m.p. 199e200 ꢀC, after crys-
tallization from ethyl acetate/petroleum ether with charcoal.
solvent, afforded a pale orange thick oil from which compounds
13aed were obtained as reported below.
1H NMR (CDCl3):
d
1.01 and 1.03 [2d, 3H þ 3H,
5.1.5.1. 5-Chloro-N,N,N0,N0-tetraethyl[1,2,4]triazolo[4,3-a]
[1,8]naphthyridine-6,9-dicarboxamide 13a. The pink solid
residue obtained from reaction carried out (20 min) with
1.27 g of N,N-diethyl-2-hydrazino-2-oxoacetamide 8a, was
taken up in a little diethyl ether/petroleum ether and filtered
to obtain pure 13a (1.05 g, 52%), white crystals, m.p. 198e
199 ꢀC, after crystallization from ethyl acetate/petroleum ether
NCH2CH(CH3)2], 1.15 and 1.17 [2t, 3H þ 3H, N(CH2CH3)2],
1.31 (t, 3H, COOCH2CH3), 2.04 [m, 1H, NCH2CH(CH3)2],
2.72 [m, 1H, 1H of NCH2CH(CH3)2], 3.22e3.51 [m, 4H, 3H
of N(CH2CH3)2 þ 1H of NCH2CH(CH3)2], 3.91e4.18 [m,
1H, 1H of N(CH2CH3)2], 4.11 (q, 2H, COOCH2CH3), 4.40
and 4.48 (AB system, J ¼ 18 Hz, 1H þ 1H, 9-CH2COO), 6.30
(br s, 1H, NH; disappeared with D2O), 6.96 (dd, J3,4 ¼ 8.3 Hz,
J3,2 ¼ 4.6 Hz, 1H, H-3), 8.17 (dd, J4,3 ¼ 8.3 Hz, J4,2 ¼ 1.5 Hz,
1H, H-4), 8.24 (dd, J2,3 ¼ 4.6 Hz, J2,4 ¼ 1.5 Hz, 1H, H-2). IR
(KBr): 3257 (NH), 1743 (ester CO), 1608 s (amide CO), 1548,
1
with charcoal. H NMR (CDCl3): d 1.16, 1.20, 1.40 and 1.43
[4t,
3H þ 3H þ 3H þ 3H,
6-CON(CH2CH3)2 þ 9-CON
(CH2CH3)2], 3.09e3.42, 3.46e3.71 and 3.77e4.04 [3m,
4H þ 2H þ 2H, 6-CON(CH2CH3)2 þ 9-CON(CH2CH3)2], 7.68
(dd, J3,4 ¼ 8.1 Hz, J3,2 ¼ 4.8 Hz, 1H, H-3), 8.58 (dd,
J4,3 ¼ 8.1 Hz, J4,2 ¼ 1.6 Hz, 1H, H-4), 8.74 (dd, J2,3 ¼ 4.8 Hz,
J2,4 ¼ 1.6 Hz, 1H, H-2). IR (KBr): 1647 s, br (CO), 1602, 1587
1469 cmꢃ1
.
5.1.4.4. Ethyl 2-[6-[(diethylamino)carbonyl]-5-(isobutylamino)
[1,2,4]triazolo[4,3-a][1,8]naphthyridin-9-yl]propanoate 3d.
The solid residue obtained from reaction carried out with
1.67 g of 11b [7] and 1.58 g of ethyl 3-hydrazino-2-methyl-
3-oxopropanoate 6c, was taken up in a little diethyl ether
and filtered to give pure 3d (1.00 g, 45%), whitish crystals,
m.p. 192e193 ꢀC, after crystallization from ethyl acetate/
w, 1556 w, 1521 w, 1508 cmꢃ1
.
5.1.5.2.
5-Chloro-N6,N6-diethyl-N9,N9-dipropyl[1,2,4]tria-
zolo[4,3-a][1,8]naphthyridine-6,9-dicarboxamide 13b. The
thick oily residue obtained from reaction carried out (20 min)
with 1.50 g of N,N-dipropyl-2-hydrazino-2-oxoacetamide 8b,
was dissolved in a little diethyl ether and allowed to stand until
pure 13b separated out as a pink crystalline solid (1.38 g, 64%);
after crystallization from ethyl acetate/petroleum ether with
charcoal, white crystals (m.p. 172e173 ꢀC) were obtained. 1H
NMR (CDCl3): d 0.77 [t, 3H, 3H of N(CH2CH2CH3)2], 1.12
and 1.21 [2t, 3H þ 3H, N(CH2CH3)2], 1.41 [t, 3H, 3H of
N(CH2CH2CH3)2], 1.48e2.00 [m, 4H, N(CH2CH2CH3)2],
2.96e3.94 [m, 8H, N(CH2CH3)2 þ N(CH2CH2CH3)2], 7.67
(dd, J3,4 ¼ 8.1 Hz, J3,2 ¼ 4.8 Hz, 1H, H-3), 8.58 (dd,
J4,3 ¼ 8.1 Hz, J4,2 ¼ 1.6 Hz, 1H, H-4), 8.75 (dd, J2,3 ¼ 4.8 Hz,
J2,4 ¼ 1.6 Hz, 1H, H-2). IR (KBr): 1643 s, br (CO), 1603, 1588
1
diisopropyl ether with charcoal. H NMR (CDCl3): d 0.90e
1.30 [m, 12H, N(CH2CH3)2 þ NCH2CH(CH3)2], 1.35 [t, 3H,
COOCH2CH3], 1.85e2.16 [m, 1H, NCH2CH(CH3)2], 1.95
[d, 3H, 9-CH(CH3)COOCH2CH3], 2.70 [m, 1H, 1H of
NCH2CH(CH3)2],
3.20e3.60
[m,
4H,
3H
of
N(CH2CH3)2 þ 1H of NCH2CH(CH3)2], 3.80e4.21 [m, 1H,
1H of N(CH2CH3)2], 4.05 [q, 2H, COOCH2CH3], 4.68 [q,
1H, 9-CH(CH3)COOCH2CH3], 6.49 (near d, 1H, NH; disap-
peared with D2O), 6.88 (dd, J3,4 ¼ 8.3 Hz, J3,2 ¼ 4.6 Hz, 1H,
H-3), 8.04e8.28 (m, 2H, H-2,4). IR (KBr): 3277 (NH), 1736
(ester CO), 1608 s (amide CO), 1542, 1463 cmꢃ1
.
w, 1557 w, 1520 w, 1505 cmꢃ1
.
5.1.5.3. 5-Chloro-N6,N6-diethyl-N9,N9-diisopropyl[1,2,4]tria-
zolo[4,3-a][1,8]naphthyridine-6,9-dicarboxamide 13c. The
thick oily residue obtained from reaction carried out (2 h)
with 1.50 g of N,N-diisopropyl-2-hydrazino-2-oxoacetamide
8c, was dissolved in a little diethyl ether and allowed to stand
until pure 13c separated out as a pink crystalline solid (0.40 g,
19%); after crystallization from ethyl acetate with charcoal,
5.1.5. General procedure for the synthesis of 9-substituted
5-chloro-N,N-diethyl[1,2,4]triazolo[4,3-a][1,8]
naphthyridine-6-carboxamides 13aed
A mixture of 5.0 mmol (1.49 g) of dichloroderivative 12 [6],
8.0 mmol of the proper amide-hydrazide 8aec or 10a and 6 mL
of Dowtherm A was stirred at 150 ꢀC for the time reported be-
low for each case. After cooling, dichloromethane (100 mL)
and 10% aqueous Na2CO3 (50 mL) were added to the mixture
which was stirred for 30 min at room temperature. The mixture
was filtered to remove a reddish insoluble solid, then was trans-
ferred into a separatory funnel. The organic layer was collected
and the aqueous one was extracted twice with dichloromethane.
The combined organic extracts (dried over Na2SO4 and decol-
ourized with charcoal) were evaporated to dryness at reduced
pressure to give an orange liquid which was chromatographed
on a silica gel column. The elution was carried out first with di-
chloromethane to remove the Dowtherm A, then with dichloro-
methane/ethyl acetate (1:1) to remove a small amount of
starting compound 12 and finally with ethyl acetate to recover
compounds 13aed. The eluate collected, after removal of
1
white crystals (m.p. 236e237 ꢀC) were obtained. H NMR
(CDCl3): d 1.07 and 1.20 [2d, 3H þ 3H, NCH(CH3)2], 1.21
and 1.41 [2t, 3H þ 3H, N(CH2CH3)2], 1.67 and 1.74 [2d,
3H þ 3H, NCH(CH3)2], 3.35 [q, 2H, 2H of N(CH2CH3)2],
3.52e3.97 [m, 4H, 2H of N(CH2CH3)2 þ NCH(CH3)2 þ
NCH(CH3)2], 7.67 (dd, J3,4 ¼ 8.1 Hz, J3,2 ¼ 4.8 Hz, 1H, H-3),
8.56 (dd, J4,3 ¼ 8.1 Hz, J4,2 ¼ 1.6 Hz, 1H, H-4), 8.74 (dd,
J2,3 ¼ 4.8 Hz, J2,4 ¼ 1.6 Hz, 1H, H-2). IR (KBr): 1647 s
(CO), 1599, 1582 w, 1551 w, 1516 w, 1501 cmꢃ1
.
5.1.5.4. 5-Chloro-N,N-diethyl-9-[2-(isobutylamino)-2-oxoethyl]
[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide 13d.
Thethick oilyresidue obtainedfromreactioncarried out (45 min)
with 1.39 g of N-isobutyl-3-hydrazino-3-oxopropanamide 10a,

592
M. Di Braccio et al. / European Journal of Medicinal Chemistry 43 (2008) 584e594
wastakenupinalittlediethyletheranddiisopropyl etherandkept
at 4 ꢀC so that pure 13d separated out as a pink-orange crystalline
solid (1.19 g, 57%); after crystallization from ethyl acetate/petro-
leum ether with charcoal, a whitish crystalline solid (m.p. 185e
186 ꢀC) was obtained. 1H NMR (CDCl3): d 0.86 and 0.87 [2d,
3H þ 3H, NCH2CH(CH3)2], 1.14 and 1.36 [2t, 3H þ 3H,
N(CH2CH3)2], 1.76 [m, 1H, NCH2CH(CH3)2], 3.08 [t, 2H,
NCH2CH(CH3)2], 3.28 [q, 2H, 2H of N(CH2CH3)2], 3.56 and
3.84 [2m, 1H þ 1H, 2H of N(CH2CH3)2], 4.68 and 4.74 (AB sys-
tem, J ¼ 18 Hz, 1H þ 1H, 9-CH2CON), 7.29 (br s, 1H, NH; dis-
appeared with D2O), 7.60 (dd, J3,4 ¼ 8.1 Hz, J3,2 ¼ 4.8 Hz, 1H,
H-3), 8.51 (dd, J4,3 ¼ 8.1 Hz, J4,2 ¼ 1.6 Hz, 1H, H-4), 8.70 (dd,
J2,3 ¼ 4.8 Hz, J2,4 ¼ 1.6 Hz, 1H, H-2). IR (KBr): 3339 (NH),
1690 and 1682 sh (secondary amide CO), 1634 s (tertiary amide
for 8 h. After cooling, the resulting solution was evaporated
to dryness at reduced pressure and the residue was partitioned
between dichloromethane and aqueous 5% NaHCO3; the or-
ganic phase was then collected and the aqueous one was fur-
ther extracted twice with dichloromethane. The combined
organic extracts (dried over Na2SO4), after removal of solvent,
afforded an oily or nearly solid residue from which com-
pounds 3eeg were recovered as described below for each
case.
Compounds 3h,i: A mixture of 3.0 mmol of the proper
compound 13, 7 mL of isobutylamine and 7 mL of dimethyl
sulphoxide was heated at 130 ꢀC for 3 h. The resulting solu-
tion was poured into ice-water and the mixture was subjected
to an exhaustive extraction with chloroform. The combined or-
ganic extracts (dried over Na2SO4), after removal of solvent,
afforded an oily or nearly solid residue from which com-
pounds 3h,i were recovered as described below for each case.
According to the above procedures the following com-
pounds were prepared.
CO), 1601 w, 1588 w, 1548, 1483 cmꢃ1
.
5.1.6. 5-Chloro-N,N-diethyl-9-[2-oxo-2-(2-thiazolylamino)
ethyl][1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-
carboxamide 13e
A mixture of 9.0 mmol (2.68 g) of dichloroderivative 12
[6], 11.0 mmol (2.20 g) of 3-hydrazino-3-oxo-N-(2-thiazolyl)-
propanamide 10b, 20 mL of Dowtherm A and 20 mL of dry
pyridine was stirred at 160 ꢀC for 1.5 h. After cooling,
150 mL of diethyl ether and 150 mL of aqueous 6 N HCl
were added and the mixture was stirred at room temperature
for 30 min. The acidic aqueous phase was collected whereas
the ethereal one was extracted with aqueous 6 N HCl, then dis-
carded. The entire aqueous phase was cooled with ice, treated
with concentrated aqueous ammonia up to pH 9 and finally
extracted with chloroform. The organic phase, dried over
Na2SO4 and evaporated to dryness at reduced pressure, af-
forded a dark oil which was subjected to column chromatog-
raphy on a silica gel column. The elution was carried out
first with ethyl acetate to remove some impurities, then with
the mixture dichloromethane/acetone (1:1) to recover 13e.
The eluate collected, after removal of solvents, gave a solid
residue from which, by adding a little ethyl acetate, pure com-
pound 13e (0.60 g, 15%) separated out as a whitish crystalline
solid, m.p. 262e263.5 ꢀC, after crystallization from dichloro-
methane/ethyl acetate. 1H NMR (CDCl3): d 1.21 and 1.41 [2t,
3H þ 3H, N(CH2CH3)2], 3.36 [q, 2H, 2H of N(CH2CH3)2],
3.63 and 3.88 [2m, 1H þ 1H, 2H of N(CH2CH3)2], 5.00 and
5.10 (AB system, J ¼ 18 Hz, 1H þ 1H, 9-CH2CON), 6.99 (d,
J ¼ 3.6 Hz, 1H, thiazolyl H-50), 7.51 (d, J ¼ 3.6 Hz, 1H, thia-
zolyl H-40), 7.63 (dd, J3,4 ¼ 8.1 Hz, J3,2 ¼ 4.8 Hz, 1H, H-3),
8.58 (dd, J4,3 ¼ 8.1 Hz, J4,2 ¼ 1.6 Hz, 1H, H-4), 8.69 (dd,
J2,3 ¼ 4.8 Hz, J2,4 ¼ 1.6 Hz, 1H, H-2), 12.28 (br s, 1H, NH;
disappeared with D2O). IR (KBr): 3190 (NH), 1686 (second-
ary amide CO), 1627 s (tertiary amide CO), 1602, 1587 w,
5.1.7.1. N,N,N0,N0-Tetraethyl-5-(isobutylamino)[1,2,4]triazolo
[4,3-a][1,8]naphthyridine-6,9-dicarboxamide 3e. The oily
residue derived from the reaction carried out with 13a
(1.21 g) and isobutylamine was treated with a little diethyl
ether so that pure compound 3e separated out as a crystalline
solid (1.17 g, 89%); whitish crystals, m.p. 193e194 ꢀC, after
1
crystallization from ethyl acetate/petroleum ether. H NMR
(CDCl3): d 1.04 [d, 6H, NCH2CH(CH3)2], 1.13, 1.21, 1.35
and 1.39 [4t, 3H þ 3H þ 3H þ 3H, 6-CON(CH2CH3)2 þ 9-
CON(CH2CH3)2], 2.12 [m, 1H, NCH2CH(CH3)2], 2.90 [m,
1H, 1H of NCH2CH(CH3)2], 3.19 [q, 2H, 2H of 9-
CON(CH2CH3)2], 3.35e3.56, 3.60e3.77 and 3.95 [3m,
4H þ 2H þ 1H, 4H of 6-CON(CH2CH3)2 þ 2H of 9-CON(-
CH2CH3)2 þ 1H of NCH2CH(CH3)2], 5.46 (br s, 1H, NH; dis-
appeared with D2O), 7.11 (dd, J3,4 ¼ 8.3 Hz, J3,2 ¼ 4.6 Hz,
1H, H-3), 8.21 (dd, J4,3 ¼ 8.3 Hz, J4,2 ¼ 1.5 Hz, 1H, H-4),
8.56 (dd, J2,3 ¼ 4.6 Hz, J2,4 ¼ 1.5 Hz, 1H, H-2). IR (KBr):
3325 (NH), 1627 s (CO), 1606, 1540, 1505 w cmꢃ1
.
5.1.7.2. N6,N6-Diethyl-5-(isobutylamino)-N9,N9-dipropyl[1,2,4]
triazolo[4,3-a][1,8]naphthyridine-6,9-dicarboxamide 3f. The
oily residue obtained from the reaction carried out with 13b
(1.29 g) and isobutylamine were treated with a little diethyl
ether so that pure compound 3f separated out as a crystalline
solid (1.04 g, 74%); whitish crystals, m.p. 177e178 ꢀC, after
crystallization from ethyl acetate/diisopropyl ether with
charcoal. 1H NMR (CDCl3):
d 0.72 [t, 3H, 3H of
N(CH2CH2CH3)2], 1.04 [d, 6H, NCH2CH(CH3)2], 1.10 and
1.16 [2t, 3H þ 3H, N(CH2CH3)2], 1.35 [t, 3H, 3H of
N(CH2CH2CH3)2], 1.50e1.73 and 1.76e1.94 [2m, 2H þ 2H,
N(CH2CH2CH3)2], 2.10 [m, 1H, NCH2CH(CH3)2], 2.92 [m,
1H, 1H of NCH2CH(CH3)2], 3.12 [t, 2H, 2H of N(CH2CH2
CH3)2], 3.32e3.71 [m, 6H, 3H of N(CH2CH3)2 þ 2H of
N(CH2CH2CH3)2 þ 1H of NCH2CH(CH3)2], 3.95 [m, 1H,
1H of N(CH2CH3)2], 5.33 (br s, 1H, NH; disappeared with
D2O), 7.14 (dd, J3,4 ¼ 8.3 Hz, J3,2 ¼ 4.6 Hz, 1H, H-3), 8.20
(dd, J4,3 ¼ 8.3 Hz, J4,2 ¼ 1.5 Hz, 1H, H-4), 8.57 (dd,
1556 s, 1482 cmꢃ1
.
5.1.7. General procedure for the synthesis of 9-substituted
5-(alkylamino)-N,N-diethyl[1,2,4]triazolo[4,3-a][1,8]
naphthyridine-6-carboxamides 3eei
Compounds 3eeg: A mixture of 3.0 mmol of the proper
compound 13, 7 mL of isobutylamine or n-butylamine and
15 mL of ethanol was heated in a closed vessel at 130 ꢀC

M. Di Braccio et al. / European Journal of Medicinal Chemistry 43 (2008) 584e594
593
J2,3 ¼ 4.6 Hz, J2,4 ¼ 1.5 Hz, 1H, H-2). IR (KBr): 3318 (NH),
1637 s (CO), 1608, 1536 cmꢃ1
out first with ethyl acetate to remove a little amount of starting
compound 13e, then with acetone to recover 3i. The eluate
collected, after removal of solvent afforded a yellowish solid
that was taken up in a little diethyl ether and filtered to yield
pure 3i (1.05 g, 73%); pale-yellow crystals, m.p. 184e186 ꢀC
dec, after crystallization from ethyl acetate/diisopropyl ether
with charcoal. 1H NMR (CDCl3): d 1.06 [d, 6H, NCH2
CH(CH3)2], 1.17 and 1.34 [2t, 3H þ 3H, N(CH2CH3)2], 2.11
[m, 1H, NCH2CH(CH3)2], 2.68 [m, 1H, 1H of NCH2
CH(CH3)2], 3.22e3.54 [m, 4H, 3H of N(CH2CH3)2 þ 1H of
NCH2CH(CH3)2], 4.05 [m, 1H, 1H of N(CH2CH3)2], 4.88
(near s, 2H, 9-CH2CON), 6.67 (br s, 1H, 5-NHCH2; disap-
peared with D2O), 6.80e6.93 (m, 1H, H-3), 6.85 (d,
J ¼ 3.6 Hz, 1H, thiazolyl H-50), 7.37 (d, J ¼ 3.6 Hz, 1H, thia-
zolyl H-40), 8.09e8.27 (m, 2H, H-2,4), 12.10 (br s, 1H,
CONH; disappeared with D2O). IR (KBr): 3263 and 3204
(NH), 1697 w and 1667 w (secondary amide CO), 1607 s (ter-
.
5.1.7.3. 5-(Butylamino)-N,N-diethyl-9-[2-(isobutylamino)-2-
oxoethyl][1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxa-
mide 3g. The nearly solid residue obtained from the reaction
carried out with 13d (1.25 g) and n-butylamine were taken
up in a little diethyl ether and the crystalline solid that sepa-
rated out was recovered by filtration to give pure 3g (1.08 g,
79%); whitish crystals, m.p. 213e214 ꢀC, after crystallization
1
from ethyl acetate with charcoal. H NMR (CDCl3): d 0.86e
1.08 [m, 9H, NCH2CH(CH3)2 þ NCH2CH2CH2CH3], 1.18 and
1.34 [2t, 3H þ 3H, N(CH2CH3)2], 1.48 (m, 2H, NCH2
CH2CH2CH3), 1.71e1.95 [m, 3H, NCH2CH2CH2CH3 þ
NCH2CH(CH3)2], 2.95 [m, 1H, 1H of NCH2CH(CH3)2],
3.14 (t, 2H, NCH2CH2CH2CH3), 3.30e3.52 [m, 4H, 3H of
N(CH2CH3)2 þ 1H of NCH2CH(CH3)2], 4.00 [m, 1H, 1H of
N(CH2CH3)2], 4.47 and 4.63 (AB system, J ¼ 18 Hz,
1H þ 1H, 9-CH2CON), 6.38 (near d, 1H, 5-NHCH2; disap-
peared with D2O), 6.98 (dd, J3,4 ¼ 8.3 Hz, J3,2 ¼ 4.6 Hz, 1H,
H-3), 7.93 (near t, 1H, CONHCH2; disappeared with D2O),
8.17 (dd, J4,3 ¼ 8.3 Hz, J4,2 ¼ 1.5 Hz, 1H, H-4), 8.26 (dd,
J2,3 ¼ 4.6 Hz, J2,4 ¼ 1.5 Hz, 1H, H-2). IR (KBr): 3329 br
(NH), 1685 sh and 1671 (secondary amide CO), 1607 s (ter-
tiary amide CO), 1552, 1471 cmꢃ1
.
5.1.8. N,N,9-Triethyl-5-[ethyl(pyridin-3-ylcarbonyl)
amino][1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-
carboxamide 4a
A mixture of 4.0 mmol (1.30 g) of 1b [1], 20.0 mmol
(3.54 g) of nicotinoyl chloride hydrochloride, 50 mL of anhy-
drous toluene and 20 mL of triethylamine was heated at
120 ꢀC for 3 h, with stirring. The mixture was then evaporated
to dryness at reduced pressure and the residue partitioned be-
tween dichloromethane and 10% aqueous Na2CO3. The or-
ganic phase was collected and the aqueous one was
extracted twice with dichloromethane. The combined organic
extracts, dried over Na2SO4 and evaporated to dryness at re-
duced pressure, afforded a thick oil which was chromato-
graphed on an aluminium oxide column. The elution was
performed first with dichloromethane to remove some impuri-
ties, then with the mixture dichloromethane/triethylamine
(9:1) to recover 4a. The eluate collected, after removal of sol-
vents, gave a thick oil from which, after addition of a little di-
ethyl ether and standing, pure 4a separated out as a pink solid
(0.71 g, 40%); whitish crystals, m.p. 203e204 ꢀC, after crys-
tallization from ethyl acetate/petroleum ether. 1H NMR
tiary amide CO), 1548, 1463 cmꢃ1
.
5.1.7.4. N,N-Diethyl-5-(isobutylamino)-9-[2-(isobutylamino)-
2-oxoethyl][1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carbox-
amide 3h. The oily residue obtained from the reaction carried
out with 13d (1.25 g) and isobutylamine were subjected to
chromatography on a silica gel column eluting first with ace-
tone to remove a little amount of starting compound 13d, then
with the mixture dichloromethane/methanol (9:1) to recover
3h. The eluate collected, after removal of solvent gave an
oil that was treated with a little ethyl ether to afford pure 3h
(1.09 g, 80%); whitish crystals, m.p. 194e195 ꢀC, after crys-
tallization from ethyl acetate with charcoal. 1H NMR
(CDCl3): d 0.85e1.15 [m, 12H, 5-NCH2CH(CH3)2 þ CON
HCH2CH(CH3)2], 1.19 and 1.33 [2t, 3H þ 3H, N(CH2
CH3)2], 1.85 and 2.09 [2m, 1H þ 1H, 5-NCH2CH
(CH3)2 þ CONHCH2CH(CH3)2], 2.70 [m, 1H, 1H of 5-
NCH2CH(CH3)2], 3.15 [t, 2H, CONHCH2CH(CH3)2], 3.24e
3.52 [m, 4H, 3H of N(CH2CH3)2 þ 1H of 5-NCH2CH(CH3)2],
4.04 [m, 1H, 1H of N(CH2CH3)2], 4.48 and 4.66 (AB system,
J ¼ 18 Hz, 1H þ 1H, 9-CH2CON), 6.40 (near d, 1H, 5-
NHCH2; disappeared with D2O), 6.99 (dd, J3,4 ¼ 8.3 Hz,
J3,2 ¼ 4.6 Hz, 1H, H-3), 7.87 (near t, 1H, CONHCH2; disap-
peared with D2O), 8.19 (dd, J4,3 ¼ 8.3 Hz, J4,2 ¼ 1.5 Hz, 1H,
H-4), 8.26 (dd, J2,3 ¼ 4.6 Hz, J2,4 ¼ 1.5 Hz, 1H, H-2). IR
(KBr): 3271 br (NH), 1684 sh and 1665 (secondary amide
(CDCl3):
d
1.11 and 1.20e1.65 [t þ m, 3H þ 9H,
N(CH2CH3)2 þ 5-NCH2CH3 þ 9-CH2CH3], 3.11e4.20 and
4.42e4.65 [2m, 7H þ 1H, N(CH2CH3)2 þ 5-NCH2CH3 þ 9-
CH2CH3], 6.90e7.05 and 7.40e8.95 (2m, 7H, H-
2,3,4 þ pyridyl H’s). IR (KBr): 1645 s (CO), 1611, 1587,
1562 w, 1482 w cmꢃ1
.
5.1.9. 5-[Acetyl(isobutyl)amino]-N,N-diethyl-9-isopropyl
[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide 4b
A mixture of 3.0 mmol (1.15 g) of 1n [1] and 10 mL of ace-
tic anhydride was stirred at 140 ꢀC for 8 h. The mixture was
then poured into ice-water and the resulting solution was
treated with NaHCO3 up to pH 7, then exhaustively extracted
with chloroform. The thick oil obtained from the combined
extracts (dried over Na2SO4 and evaporated to dryness at re-
duced pressure) was chromatographed on an aluminium oxide
CO), 1607 s (tertiary amide CO), 1548, 1466 cmꢃ1
.
5.1.7.5. N,N-Diethyl-5-(isobutylamino)-9-[2-oxo-2-(2-thiazolyl-
amino)ethyl][1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide
3i. The oily residue obtained from the reaction carried out
with 13e (1.33 g) and isobutylamine were subjected to chro-
matography on a silica gel column. The elution was carried

594
M. Di Braccio et al. / European Journal of Medicinal Chemistry 43 (2008) 584e594
column. The elution was carried out first with dichlorome-
thane to remove some impurities, then with ethyl acetate to re-
cover 4b. From the eluate collected, after removal of solvent,
a thick oil was obtained from which, after addition of a little
diisopropyl ether, pure 4b separated out as a white solid
(1.08 g, 85%), m.p. 137e138 ꢀC, after crystallization from dii-
Paw volume, as determined by measuring the amount of water
displaced after immersing the paw to the level of the lateral
malleolus, was recorded immediately after the carrageenan in-
jection, and 2 h later. The difference between the two values
was taken as edema volume. The percent inhibition of the
edema in treated rats as compared to controls was calculated.
Indomethacin (6 mg/kg p.o.) was used as positive control.
1
sopropyl ether. H NMR (CDCl3): d 0.80e0.95, 1.00e1.15,
1.25e1.40 and 1.52e1.90 [4m, 19H, 5-NCH2CH(CH3)2 þ
N(CH2CH3)2 þ 9-CH(CH3)2 þ 5-NCH2CH(CH3)2], 2.00 (s,
3H, COCH3), 3.05e4.10 [m, 6H, N(CH2CH3)2 þ 5-NC
5.2.2. Data analysis
Results were calculated as mean ꢂ SD. Differences be-
tween treated and control groups were determined by Stu-
dent’s t-test (*P < 0.05 or **P < 0.01 being considered as
statistically significant or highly significant, respectively).
H2CH(CH3)2], 4.54 [m, 1H, 9-CH(CH3)2], 7.61 (dd, J3,4
8.3 Hz, J3,2 ¼ 4.6 Hz, 1H, H-3), 8.17 (dd, J4,3 ¼ 8.3 Hz, J4,2
¼
¼
1.5 Hz, 1H, H-4), 8.82 (dd, J2,3 ¼ 4.6 Hz, J2,4 ¼ 1.5 Hz, 1H,
H-2). IR (KBr): 1671 (CO), 1632 s (CO), 1609, 1587, 1558
.
w, 1514 w cmꢃ1
Acknowledgements
5.2. Pharmacology
The financial support from MIUR (Ministero dell’Istru-
`
zione, dell’Universita e della Ricerca, Rome, Italy) is grate-
fully acknowledged.
The carrageenan-induced paw edema test [11] was per-
formed in groups of five male Sprague-Dawley rats (120e
150 g). Animals were purchased from Harlan (Correzzana,
Italy) and allowed to acclimate in our animal facility for one
week before use. They were housed in an air-conditioned
room (22 ꢂ 2 ꢀC, relative humidity 50 ꢂ 10%) with a 12-h
light/dark cycle, kept on bedding chips (Harlan), and had
free access to tap water and rat chow (TRM, Harlan). Rats
were fasted for 15 h before treatment, but had always free ac-
cess to water.
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