1,8-Naphthyridines VII. New substituted 5-amino[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamides and their isosteric analogues, exhibiting notable anti-inflammatory and/or analgesic activities, but no acute gastrolesivity

Article abstract of DOI:10.1016/j.ejmech.2007.10.010

The [1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide derivatives 5-amino (2) or 5-alkoxy (3) substituted and the 5-amino[1,2,4]triazolo[4,3-a]quinoline-4-carboxamide derivatives (4), designed to obtain new effective analgesic and/or anti-inflammatory agents were synthesized. Ten compounds 2 and 4 showed an interesting analgesic activity: the most potent ones are 2j (36% inhibition, P < 0.05) and 4b (77% inhibition, P < 0.01) at 6.25 and 25 mg kg^-1 doses, respectively. Compounds 2i-l and 4c showed notable anti-inflammatory properties: the most potent ones are 2i (68% inhibition, P < 0.01) and 2l (42% inhibition, P < 0.05) at 12.5 and 6.25 mg kg^-1 doses, respectively. The replacement in compounds 2 of the N-substituted 5-amino substituents with similar alkoxy groups usually afforded less active compounds 3.

Full text of DOI:10.1016/j.ejmech.2007.10.010

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1665e1680
http://www.elsevier.com/locate/ejmech
Original article
1,8-Naphthyridines VII. New substituted 5-amino[1,2,4]triazolo
[4,3-a][1,8]naphthyridine-6-carboxamides and their isosteric analogues,
exhibiting notable anti-inflammatory and/or analgesic activities,
but no acute gastrolesivity
a
a
a
b
Giorgio Roma a, , Giancarlo Grossi , Mario Di Braccio , Daniela Piras , Vigilio Ballabeni ,
*
Massimiliano Tognolini b, Simona Bertoni b, Elisabetta Barocelli b
a Dipartimento di Scienze Farmaceutiche, Universita` di Genova, Viale Benedetto XV, 3, I-16132 Genova, Italy
b Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, Universita` di Parma,
Viale G.P. Usberti 27/A, I-43100 Parma, Italy
Received 27 July 2007; received in revised form 2 October 2007; accepted 8 October 2007
Available online 11 October 2007
Abstract
The [1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide derivatives 5-amino (2) or 5-alkoxy (3) substituted and the 5-amino[1,2,4]tria-
zolo[4,3-a]quinoline-4-carboxamide derivatives (4), designed to obtain new effective analgesic and/or anti-inflammatory agents were synthe-
sized. Ten compounds 2 and 4 showed an interesting analgesic activity: the most potent ones are 2j (36% inhibition, P < 0.05) and 4b (77%
inhibition, P < 0.01) at 6.25 and 25 mg kgꢀ1 doses, respectively. Compounds 2iel and 4c showed notable anti-inflammatory properties: the
most potent ones are 2i (68% inhibition, P < 0.01) and 2l (42% inhibition, P < 0.05) at 12.5 and 6.25 mg kgꢀ1 doses, respectively. The replace-
ment in compounds 2 of the N-substituted 5-amino substituents with similar alkoxy groups usually afforded less active compounds 3.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: [1,2,4]Triazolo[4,3-a][1,8]naphthyridines; Isosteric analogues; Anti-inflammatory; Analgesic; Gastrolesivity; Structureeactivity relationships
1. Introduction
derivatives 1, not only due to their notably higher anti-inflam-
matory activity (carrageenan-induced rat paw edema test), but
also due to their more potent analgesic properties (acetic acid
induced writhing test in mice) [3,4], depending on the substit-
uents. It must also be observed that, on the whole, compounds
2 show notable potency difference between their analgesic and
anti-inflammatory activities [4]: such results suggest that dif-
ferent mechanisms of action support their analgesic and anti-
inflammatory properties, respectively. The activities of the
most potent anti-inflammatory (2a,b) [3] and analgesic (2c)
[4] compounds previously described by us are reported in
Table 1.
The results of a recent in vitro study clearly indicate that
the anti-inflammatory activity of compounds 2a,c (chosen as
examples) reasonably derives from the anti-adhesive effects
of these compounds and from their inhibition of superoxide
anion production and of myeloperoxidase release, without
In the course of our studies aimed at obtaining new
anti-inflammatory and analgesic agents devoid of undesirable
side effects, properly substituted 4H-[1,2,4]triazolo[4,3-
a][1,5]benzodiazepin-5-amines 1 [1,2] and 5-amino[1,2,4]tria-
zolo[4,3-a][1,8]naphthyridine-6-carboxamides 2 [3,4] proved
to be the most interesting compounds, being both endowed
with appreciable anti-inflammatory and analgesic properties
and devoid of acute gastrolesivity and significant acute
toxicity (Fig. 1).
However, the 1,8-naphthyridine tricyclic derivatives 2 ap-
pear to be clearly more effective than the 1,5-benzodiazepine
* Corresponding author. Tel.: þ39 10 353 8374; fax: þ39 10 353 8358.
E-mail address: roma@unige.it (G. Roma).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.10.010

1666
G. Roma et al. / European Journal of Medicinal Chemistry 43 (2008) 1665e1680
R'
compounds 2deg,m,o). The 5-(dialkylamino)derivative 2l
R"'
N
N
was obtained by methylation of the previously described com-
pound 2b [3], using iodomethane in 2-butanone at reflux in the
presence of anhydrous K2CO3eKOH.
N
N
N
N
N
R
R
R
The treatment of the 5-chloroderivatives 8e [8] or 8d [4]
with a mild excess of the suitable sodium alkoxides, in anhy-
drous tetrahydrofuran at reflux, gave good yields of the 5-al-
koxyderivatives 3a or 3bef, respectively.
CON
N
R'
N
R"
R"
N
2
1
R"'
The substituted 5-amino[1,2,4]triazolo[4,3-a]quinoline-4-
carboxamides 4aed were obtained by the four-step procedure
described in Scheme 2, starting from ethyl 1,2-dihydro-4-hy-
droxy-2-oxoquinoline-3-carboxylate 9 [9] which was treated
with excess diethylamine in ethanol solution (closed vessel,
150 ꢁC) to give the corresponding diethylamide 10 (65%
yield). By heating compound 10 in refluxing POCl3, a mixture
of the 4-chloroderivative 11 (12%) and 2,4-dichloroderivative
12 (67%) was obtained, which were easily separated by col-
umn chromatography. Compound 11 was then converted into
the 2,4-dichloroderivative 12 (82%) by subsequent treatment
with refluxing POCl3. The cyclocondensation of 2,4-dichloro-
derivative 12 with formic hydrazide or isobutyrohydrazide
(Dowtherm A, 150 ꢁC) afforded the 5-chloro[1,2,4]tria-
zolo[4,3-a]quinoline-4-carboxamide derivative 13a or 13b, re-
spectively, in low yields. Finally, the treatment of 13a or 13b
with excess 1-methylpiperazine (140 ꢁC) afforded the corre-
sponding compound 4a (69%) or 4b (52%), whereas the reac-
tion of 13b with ethylamine or isobutylamine (160 ꢁC, in
a closed vessel) gave compound 4c (84%) or 4d (80%),
respectively.
Fig. 1. Structures of the substituted 4H-[1,2,4]triazolo[4,3-a][1,5]benzodiaze-
pin-5-amines 1 and 5-amino[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carbox-
amides 2.
involving the cyclooxygenase (COX) inhibition (in accordance
with the absence of acute gastrolesivity) [5].
On the basis of the very interesting pharmacological prop-
erties and the structural features of compounds 2aec, we have
now designed and synthesized several novel properly
substituted compounds 2 (2deo), their 5-alkoxy substituted
analogues 3aef, and the [1,2,4]triazolo[4,3-a]quinoline deriv-
atives 4aed, whose tricyclic system is isosteric to that of com-
pounds 2 and 3. All these compounds have been tested for
their anti-inflammatory and analgesic properties, in order to
further investigate the structureeactivity relationships (SAR)
in this structuralepharmacological field and possibly to obtain
new potent anti-inflammatory and/or analgesic agents. For the
effective compounds the acute gastrolesivity, which is the
most frequent harmful side effect in NSAIDs, was also
evaluated.
Furthermore, the compounds showing interesting and statis-
tically significant antinociceptive activity in the writhing test
were also tested for their inhibitory properties on spontaneous
mice locomotor activity, in order to verify the occurrence of
confounding CNS depressant properties of these compounds.
The results of elemental analyses, IR and 1H NMR spectral
data completely agree with the structures attributed to the
compounds described in this paper (see Section 5.1 and Table
3), and the spectral data are consistent with those previously
reported by us for analogous compounds [3,4].
2. Chemistry
3. Results and discussion
The [1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxa-
mide derivatives 2deo and 3aef were prepared through the
synthetic routes reported in Scheme 1. Thus, the methyl 1,2-
Compounds 2deo, 3aef and 4aed were tested in vivo for
their anti-inflammatory and analgesic activities at the initial
dose of 100 mg kgꢀ1. The compounds exhibiting a statistically
significant activity at this dose were further tested at doses de-
creasing by a factor of two, until statistically significant activity
was no longer observed. For the compounds showing statisti-
cally significant analgesic activity their effect on spontaneous
locomotor activity in mice (at properly chosen doses) was eval-
uated after oral administration. The corresponding pharmaco-
logical results are listed in Table 2. The compounds provided
with statistically significant anti-inflammatory and/or analgesic
activities were also tested for their acute gastrolesivity in rats (at
200 mg kgꢀ1 os dose): for all the compounds tested no acute
gastrolesive effect was detected in each of the 8 treated rats
(0/8), whereas indomethacin caused gastric ulcers in 7 of the
8 treated rats (7/8) at 10 mg kgꢀ1 os dose [4] (see Section 5.2).
Many novel compounds 2 exhibited interesting activities.
For instance, compounds 2iek,l showed statistically significant
anti-inflammatory activity down to 12.5 and 6.25 mg kgꢀ1
dihydro-4-hydroxy-2-oxo-1,8-naphthyridine-3-carboxylate
5
[6] was heated in a closed vessel (150 ꢁC) with an ethanol so-
lution of excess ethylamine or pyrrolidine to give high yields
of the corresponding amides 6a (95%) or 6b (93%), respec-
tively, which were then heated in refluxing phosphorus
oxychloride, affording good yields of the corresponding 2,4-
dichloroderivatives 7a,b. From the subsequent cyclocondensa-
tion of dichloroderivatives 7a, 7b, and 7c [7] with proper
hydrazides (Dowtherm A, 160 ꢁC) the novel 5-chloro[1,2,4]-
triazolo[4,3-a][1,8]naphthyridine-6-carboxamide derivatives
8aec, respectively, were obtained in moderate yields. Finally,
the treatment of the proper 5-chloroderivatives 8aec, 8d [4],
or 8e,f [8] with an excess of suitable amines afforded rather
high yields of the desired compounds 2dek,meo. This reac-
tion was carried out under two different heating conditions,
depending on the use of primary amines (120 ꢁC in closed ves-
sel, compounds 2hek,n) or substituted piperazines (130 ꢁC,

G. Roma et al. / European Journal of Medicinal Chemistry 43 (2008) 1665e1680
1667
Table 1
Structures and pharmacological data of the most potent anti-inflammatory (2a,b) and analgesic (2c) agents previously described by us
CH3
CH3
CH
N
N
N
N
N
CON(C2H5)2
R"
R"'
2a-c
Compound
Dose
(mg kgꢀ1 os)
Analgesic
activitya,e
(% inhibition)
Anti-
Spontaneous
mice locomotor
activityc,e
Acute
gastrolesivity
in ratsd,e
R"
N
inflammatory
activityb,f
(% inhibition)
R"'
(% inhibition)
2a
NHei-C4H9
200
100
50
100**g
100**
33
74**
70**
66**
61**
50**
27*
18
0/8
0/8
0/8
25
e
12.5
6.25
3.12
e
e
e
15
2b
NHeC2H5
200
100
50
100**
100**
20
65**
63**
63**
61**
35**
33*
9
25
e
12.5
6.25
3.12
e
e
e
13
2c
200
100
50
86**
88**
85**
90**
92**
83**
85**
39*
34**
23
e
N
NCH3
94**
100**h
99**h
74**h
19h
25
e
12.5
6.25
3.12
1.6
e
e
e
3
e
e
0.8
3
e
e
a
Acetic acid induced writhing in mice.
Carrageenan-induced rat paw edema.
Activity counted in 30 min in mice.
Number of rats showing gastric lesions.
Data from Ref. [4].
b
c
d
e
f
Data from Ref. [3] for 2a,b and from Ref. [4] for 2c.
*P < 0.05, **P < 0.01 significance as compared to controls (Student’s t-test).
Unpublished data.
g
h
doses, respectively (carrageenan-induced rat paw edema
assay). In particular, compound 2i, at 12.5 mg kgꢀ1 dose
(68% edema inhibition, P < 0.01), and compound 2l, at
6.25 mg kgꢀ1 dose (42% edema inhibition, P < 0.05), respec-
tively, appear a little more potent than the lead compound 2a
at the same doses (50%, P < 0.01, and 27%, P < 0.05 edema
inhibition, respectively) (Table 1). It is worth noting that no
compound of this group caused acute gastric damage in rats
even when orally administered at 200 mg kgꢀ1 dose.
statistically significant antinociceptive activity in the acetic
acid induced writhing test in mice. Compound 2j, the most po-
tent one as analgesic agent, produced an interesting statistically
significant protection down to the 6.25 mg kgꢀ1 dose (36%,
P < 0.05), but was found to be clearly less potent than the lead
compound 2c [4] (Table 1). It must be observed that, in the range
of the doses tested, several compounds sometimes notably in-
hibited the spontaneous locomotor activity of the treated mice,
thus exhibiting sedative effects confounding the analgesia study.
Only two compounds (2h,n) out of nine demonstrated sta-
tistically significant antinociceptive activity, not associated
with significant sedative effects, at 100 mg kgꢀ1 dose: 47%
On the other hand, compounds 2e,f,hel,n,o at 100 mg kgꢀ1
dose (inhibition range 47e100%) and seven of them also at
50 mg kgꢀ1 dose (inhibition range 54e75%) showed

1668
G. Roma et al. / European Journal of Medicinal Chemistry 43 (2008) 1665e1680
H
N
H
N
N
Cl
N
O
N
N
O
N
N
Cl
C2H5NH2
POCl3
H
H
Δ
Δ
COOCH3
CON
CON
CON(C2H5)2
C2H5
C2H5
Cl
OH
OH
7c
Cl
5
6a
7a
(CH3)2CHCONHNH2
CH3
Δ
HN
C2H5CONHNH2
Δ
Δ
C2H5
N
CH3
H
N
CH3 CH
CH3 CH
N
O
N
N
N
N
N
N
N
HN
NCH3
N
N
N
N
CON
CON(C2H5)2
H
H
OH
Δ
6b
CON
CON
Cl
POCl3
Cl
C2H5
NCH3
C2H5
8c
Cl
Δ
N
8a
2o
N
N
(CH3)2CH(CH2)2NH2
Δ
CON
C2H5
N
Cl
7b
N
N
CH3
CH3
N
CH3 CH
CH3 CH
Δ
N
N
N
(CH3)2CHCONHNH2
CON(C2H5)2
H
R"
R"'
Δ
N
N
N
N
N
N
R"
HN
2k
N
N
Compd.
R"'
CH2CH2CH CH3
CH3
CON
CON
N
NCH3
2m
2n
R"
Cl
R"'
NHCH2CH(CH3)2
8b
2m,n
CH3
CH3 CH
N
R"
R"
N
N
N
HN
N
R"O-Na+
Compd.
Compd. OR"
R"'
R"'
Δ
THF an.,
Δ
CON(C2H5)2
3b
3c
3d
3e
3f
O
NCH3
N
NCOOC2H5
2f
Cl
OC2H5
NH N NCH3
2g
8d
CH3
CH3
OCH(CH3)2
O(CH2)3CH3
NHCH2CH(OCH3)2
NHCH(CH3)2
2h
2i
CH3 CH
CH3 CH
N
N
N
N
N
N
N
N
N
OCH2CH(CH3)2
NHCH(C2H5)2
2j
CON(C2H5)2
CON(C2H5)2
R"
OR"
R"'
3b-f
2f-j
N
N
N
N
N
HN
NCH3
HN
NCH3
N
N
N
N
N
N
N
N
N
Δ
N
N
N
Δ
CON(C2H5)2
NCH3
CON(C2H5)2
CON(C2H5)2
Cl
N
NCH3
8f
CON(C2H5)2
2e
2d
Cl
8e
CH3
CH3
N
CH3 CH
CH3 CH
N
NaO
NCH3
N
N
N
N
N
N
N
N
N
N
N
CH3I
anhyd.K2CO3/KOH
THF an., Δ
CON(C2H5)2
NCH3
CON(C2H5)2
CH3
O
CON(C2H5)2 2-butanone at reflux
H
N
3a
C2H5
C2H5
2l
2b
Scheme 1. Synthetic routes to the [1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide derivatives 2deo and 3aef.

G. Roma et al. / European Journal of Medicinal Chemistry 43 (2008) 1665e1680
1669
H
N
O
CON(C2H5)2
Cl
11
H
N
H
N
O
O
(C2H5)2NH
POCl3
POCl3
COOC2H5
CON(C2H5)2
OH
OH
9
10
N
Cl
CON(C2H5)2
Cl
12
CH3
CH
CH3
N
CH3
CH CH2NH2
CH3
CH
N
N
CH3
CH3
N
CON(C2H5)2
N
N
N
Cl
H
(CH3)2CHCONHNH2
N
CH2 CH CH3
CON(C2H5)2
CON(C2H5)2
CH3
4d
Cl
Cl
13b
CH3
CH
HN
NCH3
12
CH3
N
N
C2H5NH2
N
H2NNHCHO
CON(C2H5)2
NCH3
CH3
CH
N
N
CH3
N
N
N
N
4b
N
CON(C2H5)2
CON(C2H5)2
Cl
H
N
13a
C2H5
HN
NCH3
4c
N
N
N
N
CON(C2H5)2
NCH3
4a
Scheme 2. Synthetic route to the 5-amino[1,2,4]triazolo[4,3-a]quinoline-4-carboxamide derivatives 4aed.
(P < 0.05) and 60% (P < 0.01) reduction of writhe number, re-
spectively. It is also interesting to point out that compound 2l,
at 50 mg kgꢀ1 dose, showed a notable and statistically signif-
icant antinociceptive activity (59%, P < 0.01), but not
significant and very low (22%) inhibition of spontaneous loco-
motor activity in mice.
Only three (3a,b,e) of the substituted 5-alkoxy[1,2,4]-
triazolo[4,3-a][1,8]naphthyridine-6-carboxamides
3
herein

1670
G. Roma et al. / European Journal of Medicinal Chemistry 43 (2008) 1665e1680
Table 2
Structures and pharmacological data of compounds 2deo, 3aef, and 4aed
R'
R'
R'
N
N
N
N
N
N
N
N
N
N
N
N
N
R
R
R
R
R
R
CON
CON
CON
R"
R"'
R"
R"'
OR"
2d-o
3a-f
OR00
4a-d
Analgesic
Compound
R
R
R0
Dose
Anti-inflammatory Spontaneous
R"
R"'
(mg kgꢀ1 os) activitya
activityb
(% inhibition) (% inhibition)
mice locomotor
activityc
(% inhibition)
N
N
2d
N(C2H5)2
H
e
100
14
0
e
NCH3
NCH3
N
N
N
2e
2f
N(C2H5)2
N(C2H5)2
C6H5
e
e
100
50
100**d
62*
15
0
72**
62**
e
e
e
25
CH(CH3)2
100
50
100**
75**
53*
0
83**
69**
96**
e
NCOOC2H5
e
e
e
25
12.5
29
2g
2h
N(C2H5)2
N(C2H5)2
CH(CH3)2
e
e
100
17
0
e
NH
N
NCH3
CH(CH3)2 NHeCH2CH(OCH3)2
CH(CH3)2 NHeCH(CH3)2
100
50
47*
8
0
0
e
e
e
e
25
e
2i
2j
N(C2H5)2
e
e
100
50
83**
54**
33*
0
81**
75**
75**
68**
15
98**
64*
26
25
12.5
6.25
0
e
e
N(C2H5)2
CH(CH3)2 NHeCH(C2H5)2
100
50
91**
75**
58**
49*
50**
47**
47**
45*
11
97**
84**
70**
92**
81**
e
25
12.5
6.25
3.12
36*
18
e
2k
2l
N(C2H5)2
C2H5
NHe(CH2)2CH(CH3)2
e
e
100
50
98**
75**
15
41**
41**
43*
29*
0
95**
76**
e
25
12.5
6.25
e
e
e
e
CH3
N
N(C2H5)2
CH(CH3)2
100
50
81**
59**
17
71**
67**
65**
51*
42*
0
53*
22
e
C2H5
25
12.5
6.25
3.12
e
e
e
e
e
e
2m
2n
CH(CH3)2
e
e
100
39
0
e
N
N
NCH3
CH(CH3)2 NHeCH2CH(CH3)2
100
50
60**
64*
29
0
5
N
e
e
e
e
25

G. Roma et al. / European Journal of Medicinal Chemistry 43 (2008) 1665e1680
1671
Table 2 (continued)
Compound
R
R
R0
OR00
Dose
(mg kgꢀ1 os) activitya
(% inhibition) (% inhibition)
Analgesic
Anti-inflammatory Spontaneous
R"
activityb
mice locomotor
activityc
(% inhibition)
N
N
R"'
2o
3a
3b
NHeC2H5
N(C2H5)2
N(C2H5)2
CH(CH3)2
H
e
100
50
79**
24
0
80**
e
NCH3
N
e
e
100
50
45*
20
0
55*
e
O
NCH3
NCH3
e
CH(CH3)2
e
100
50
87**
35
18
e
65*
e
O
3c
N(C2H5)2
N(C2H5)2
CH(CH3)2
CH(CH3)2
e
e
OC2H5
100
22
10
e
3d
OCH(CH3)2
100
50
11
e
24*
0
e
e
3e
N(C2H5)2
CH(CH3)2
e
O(CH2)3CH3
100
50
40*
14
3
76*
21
e
3f
N(C2H5)2
N(C2H5)2
CH(CH3)2
H
e
OCH2CH(CH3)2 100
26
0
e
4a
e
100
50
55**
58*
28
0
0
N
NCH3
NCH3
e
e
e
e
25
4b
4c
4d
N(C2H5)2
N(C2H5)2
N(C2H5)2
CH(CH3)2
e
100
50
100**
100**
77**
14
34*
0
54**
48**
21
N
25
12.5
e
e
e
CH(CH3)2 NHeC2H5
e
e
100
50
91**
43*
12
42**
43**
24*
6
39*
2
25
12.5
e
e
e
CH(CH3)2 NHeCH2CH(CH3)2
100
50
88**
29
0
14
e
e
Indomethacin
Diazepam
10
10
84**e
51**e
e
98**f
e
e
a
Acetic acid induced writhing in mice.
Carrageenan-induced rat paw edema.
Activity counted in 30 min in mice.
b
c
d
e
f
*P < 0.05, **P < 0.01 significance as compared to controls (Student’s t-test).
Data from Ref. [2].
Data from Ref. [4].
described exhibited a statistically significant antinociceptive
activity (inhibition range 40e87%, at 100 mg kgꢀ1 dose), al-
ways coupled with sedation (inhibition range of spontaneous
mice locomotor activity 55e76%, P < 0.05), whereas no com-
pound 3 afforded anti-inflammatory activity except for the
weakly active compound 3d (24% edema inhibition,
P < 0.05) at the 100 mg kgꢀ1 dose.
The N,N-diethyl[1,2,4]triazolo[4,3-a]quinoline-4-carboxa-
mide derivatives 4aed showed an, on the whole, interesting,
statistically significant antinociceptive activity (writhing
test), frequently also at not sedative doses (Table 2): in partic-
ular, the most potent compound 4b produced a 77% writhe
number reduction (P < 0.01) at a dose (25 mg kgꢀ1) not sig-
nificantly effective for the spontaneous locomotor activity in-
hibition in mice. As regards the anti-inflammatory activity of
compounds 4aed, the only interesting, but weakly potent,
compound was 4c which showed statistically significant
activity down to 25 mg kgꢀ1 dose (maximal inhibition 43%,
P < 0.01, at 50 mg kgꢀ1 dose). As for the tested compounds
2 and 3, no acute gastrolesive effect was detected in rats after
the oral administration of compounds 4aed at 200 mg kgꢀ1
dose.
In order to investigate the mechanism underlying the anti-
nociceptive activity of the compounds under study, the most
active one, compound 2c, was tested (writhing test) after pre-
treatment of mice with selective antagonists of different recep-
tors involved in pain modulation. Compound 2c was tested at
the lowest dose (3.12 mg kgꢀ1) producing the same effect
(85% inhibition, P < 0.01) shown at 200 mg kgꢀ1 dose (see
Table 1). The ineffectiveness of Naloxone [10], Atropine
[11], Mecamylamine [12], or Methysergide [13] in counteract-
ing the analgesic effect of compound 2c led us to rule out any
involvement of m-opioid, muscarinic, nicotinic and serotonine
receptors in the 2c antinociceptive activity.

1672
G. Roma et al. / European Journal of Medicinal Chemistry 43 (2008) 1665e1680
Table 3
Physical and chemical data of compounds 2deo, 3aef, 4aed
R'
R'
R'
N
N
N
N
N
N
N
N
N
N
N
N
N
R
R
R
R
R
R
CON
CON
CON
R"
R"
R"'
OR"
R"'
2d-o
3a-f
4a-d
1H NMRe (d, ppm)
Compounda Yield (%) Solventb m.p. (ꢁC) Molecular formulac IRd (cmꢀ1
)
2d
2e
2f
85
70
75
174e175 (A)
227e228 (B)
167e168 (C)
C
19H25N7O
1640 s (CO), 1602, 1583,
1549, 1508 w
1.14 and 1.28 [2t, 3H þ 3H, N(CH2CH3)2], 2.15e3.57
0
[m, 11H, piperazine CH2 s þ 3H of N(CH2CH3)2], 2.31
(s, 3H, NCH3), 3.80 [m, 1H, 1H of N(CH2CH3)2], 7.40
(dd, 1H, H-3), 8.36 (dd, 1H, H-4), 8.61 (dd, 1H, H-2),
9.35 (s, 1H, H-9)
C25H29N7O
1636 s (CO), 1600, 1585,
1550, 1507 w
1.19 and 1.31 [2t, 3H þ 3H, N(CH2CH3)2], 2.20e3.63
0
[m, 11H, piperazine CH2 s þ 3H of N(CH2CH3)2], 2.35
(s, 3H, NCH3), 3.80 [m, 1H, 1H of N(CH2CH3)2],
7.27e7.50 (m, 4H, H-3 þ 3H of C6H5), 7.70e7.85 (m,
2H, 2H of C6H5), 8.30e8.41 (m, 2H, H-2,4)
C24H33N7O3
1704 (carbamate CO), 1628 s
(amide CO), 1602, 1587,
1555, 1513 w
1.11e1.33 [m, 9H, N(CH2CH3)2 þ COOCH2CH3], 1.37
and 1.50 [2d, 3H þ 3H, 9-CH(CH3)2], 2.80e3.70 [m,
0
11H, piperazine CH2 s þ 3H of N(CH2CH3)2], 4.10
[q þ m, 3H, COOCH2CH3 þ 1H of N(CH2CH3)2], 4.37
[m, 1H, 9-CH(CH3)2], 7.55 (dd, 1H, H-3), 8.37 (dd, 1H,
H-4), 8.63 (dd, 1H, H-2)
2g
2h
44
65
231e232 (B)
161e162 (B)
C22H32N8O
3220 br (NH), 1606 s (CO),
1559, 1519
1.06 and 1.29 [2t, 3H þ 3H, N(CH2CH3)2], 1.39 and
1.47 [2d, 3H þ 3H, 9-CH(CH3)2], 2.21 (s, 3H, NCH3),
0
2.27e3.49 [m, 11H, piperazine CH2 s þ 3H of
N(CH2CH3)2], 3.77 [m, 1H, 1H of N(CH2CH3)2], 4.31
[m, 1H, 9-CH(CH3)2], 5.56f (s, 1H, NH), 7.36 (dd, 1H,
H-3), 8.50 (dd, 1H, H-4), 8.58 (dd, 1H, H-2)
1.11 and 1.32 [2t, 3H þ 3H, N(CH2CH3)2], 1.40 and
1.47 [2d, 3H þ 3H, 9-CH(CH3)2], 3.08e3.80 [m, 6H,
N(CH2CH3)2 þ NCH2CH(OCH3)2], 3.35 and 3.38 [2s,
3H þ 3H, NCH2CH(OCH3)2], 4.20 [m, 1H, 9-
CH(CH3)2], 4.60 [t, 1H, NCH2CH(OCH3)2], 5.40f (near
t, 1H, NH), 7.18 (dd, 1H, H-3), 8.13 (dd, 1H, H-4), 8.46
(dd, 1H, H-2)
C21H30N6O3
3265 (NH), 1609 s (CO),
1551, 1523
2i
2j
90
87
209e210 (B)
149e150 (C)
C
20H28N6O
3363 (NH), 1638 s (CO),
1604 s, 1532
1.03e1.35 [m, 12H, N(CH2CH3)2 þ NCH(CH3)2], 1.43
and 1.50 [2d, 3H þ 3H, 9-CH(CH3)2], 3.13e3.85 [m,
5H, N(CH2CH3)2 þ NCH(CH3)2], 4.23f (d, 1H, NH),
4.33 [m, 1H, 9-CH(CH3)2], 7.39 (dd, 1H, H-3), 8.23
(dd, 1H, H-4), 8.61 (dd, 1H, H-2)
C22H32N6O
3362 (NH), 1604 s, br (CO),
1531
0.83 and 0.92 [2t, 3H þ 3H, NCH(CH2CH3)2], 1.11 and
1.26 [2t, 3H þ 3H, N(CH2CH3)2], 1.35e1.77 [m, 4H,
NCH(CH2CH3)2], 1.41 and 1.48 [2d, 3H þ 3H, 9-
CH(CH3)2], 3.10e3.70 [m, 5H,
N(CH2CH3)2 þ NCH(CH2CH3)2], 4.31 [m, 1H, 9-
CH(CH3)2], 4.35f (d, 1H, NH), 7.38 (dd, 1H, H-3), 8.23
(dd, 1H, H-4), 8.59 (dd, 1H, H-2)
2k
73
185e187 (D)
C21H30N6O
3265 (NH), 1603 s (CO),
1549, 1520 w
0.88 and 0.91 [2 overlapped d, 6H,
NCH2CH2CH(CH3)2], 1.11 and 1.28 [2t, 3H þ 3H,
N(CH2CH3)2], 1.42 [t, 3H, 9-CH2CH3], 1.58e1.77 [m,
3H, NCH2CH2CH(CH3)2], 2.88 [m, 1H, 1H of
NCH2CH2CH(CH3)2], 3.21e3.55 [m, 6H, 3H of
N(CH2CH3)2 þ 9-CH2CH3 þ 1H of
NCH2CH2CH(CH3)2], 3.85 [m, 1H, 1H of
N(CH2CH3)2], 6.35f (d, 1H, NH), 6.80 (dd, 1H, H-3),
8.04 (dd, 1H, H-4), 8.19 (dd, 1H, H-2)

G. Roma et al. / European Journal of Medicinal Chemistry 43 (2008) 1665e1680
1673
Table 3 (continued)
Compounda Yield (%) Solventb m.p. (ꢁC) Molecular formulac IRd (cmꢀ1
)
1H NMRe (d, ppm)
2l
69
84
88
161e162 (A)
211e212 (B)
233e235 (B)
C
20H28N6O
1630 s (CO), 1599, 1582,
1549
1.09e1.32 [m, 9H, NCH2CH3 þ N(CH2CH3)2], 1.42
and 1.50 [2d, 3H þ 3H, 9-CH(CH3)2], 2.83 (s, 3H,
NCH3), 3.00e3.78 [m, 6H, NCH2CH3 þ N(CH2CH3)2],
4.36 [m, 1H, 9-CH(CH3)2], 7.21 (dd, 1H, H-3), 8.34
(dd, 1H, H-4), 8.60 (dd, 1H, H-2)
2m
2n
C22H29N7O
1637 s (CO), 1601, 1584,
1550, 1518 w
1.41 and 1.48 [2d, 3H þ 3H, 9-CH(CH3)2], 1.70e2.08
0
(m, 4H, pyrrolidine b-CH2 s), 2.20e3.90 (m, 12H,
0
0
pyrrolidine a-CH2 s þ piperazine CH2 s), 2.33 (s, 3H,
NCH3), 4.36 [m, 1H, 9-CH(CH3)2], 7.41 (dd, 1H, H-3),
8.35 (dd, 1H, H-4), 8.60 (dd, 1H, H-2)
C21H28N6O
3283 br (NH), 1606 s (CO),
1552, 1521 sh
0.92 [d, 6H, NCH2CH(CH3)2], 1.25e1.63 [m, 6H, 9-
CH(CH3)2], 1.70e2.08 [m, 5H, pyrrolidine b-
0
CH2 s þ NCH2CH(CH3)2], 2.45e3.90 [m, 6H,
0
pyrrolidine a-CH2 s þ NCH2CH(CH3)2], 4.09 [m, 1H,
9-CH(CH3)2], 6.43f (near t, 1H, NH), 6.93 (dd, 1H, H-
3), 8.20 (dd, 1H, H-4), 8.27 (dd, 1H, H-2)
2o
3a
3b
82
72
72
231e232 (B)
206e208 (B)
143e145 (A)
C20H27N7O
3251 (NH), 1661 s (CO), 1602, 1.27 (t, 3H, HNCH2CH3), 1.35 [d, 6H, 9-CH(CH3)2],
1586, 1553, 1535, 1515
2.31 (s, 3H, NCH3), 2.56 and 3.28 (2m, 4H þ 4H,
0
piperazine CH2 s), 3.51 (m, 2H, HNCH2CH3; q after
treatment with D2O), 4.09 [m, 1H, 9-CH(CH3)2], 7.43
(dd, 1H, H-3), 8.18f (near t, 1H, NH), 8.41 (dd, 1H, H-
4), 8.56 (dd, 1H, H-2)
C
C
20H26N6O2
1628 s (CO), 1607, 1593 sh,
1558, 1517 w
1.06 and 1.29 [2t, 3H þ 3H, N(CH2CH3)2], 1.75e2.45
(m, 6H, 6H of piperidine), 2.27 (s, 3H, NCH3), 2.64e
2.93 (m, 2H, 2H of piperidine), 3.12e3.57 [m, 3H, 3H
of N(CH2CH3)2], 3.84 [m, 1H, 1H of N(CH2CH3)2],
4.60 (m, 1H, piperidine OCH), 7.51 (dd, 1H, H-3), 8.42
(dd, 1H, H-4), 8.67 (dd, 1H, H-2), 9.40 (s, 1H, H-9)
1.07 and 1.28 [2t, 3H þ 3H, N(CH2CH3)2], 1.42 and
1.50 [2d, 3H þ 3H, 9-CH(CH3)2], 1.70e2.35 (m, 6H,
6H of piperidine), 2.21 (s, 3H, NCH3), 2.55e2.80 (m,
2H, 2H of piperidine), 3.12e3.35 and 3.43e3.80 [2m,
2H þ 2H, N(CH2CH3)2], 4.36 [m, 1H, 9-CH(CH3)2],
4.56 (m, 1H, piperidine OCH), 7.44 (dd, 1H, H-3), 8.39
(dd, 1H, H-4), 8.64 (dd, 1H, H-2)
23H32N6O2
1638 s (CO), 1606, 1593 sh,
1558, 1516 w
3c
3d
3e
83
75
77
124e125 (E)
153e154 (F)
111e112 (D)
C19H25N5O2
1635 s (CO), 1608, 1594,
1561 w
1.19 and 1.36 [2t, 3H þ 3H, N(CH2CH3)2], 1.50 (t, 3H,
OCH2CH3), 1.52 and 1.60 [2d, 3H þ 3H, 9-CH(CH3)2],
3.30e3.49 and 3.55e3.87 [2m, 2H þ 2H,
N(CH2CH3)2], 4.25e4.65 [m, 3H, OCH2CH3 þ 9-
CH(CH3)2], 7.54 (dd, 1H, H-3), 8.48 (dd, 1H, H-4), 8.74
(dd, 1H, H-2)
C
20H27N5O2
1636 s (CO), 1608, 1590, 1559, 1.16 and 1.36 [2t, 3H þ 3H, N(CH2CH3)2], 1.38 and
1516 w
1.44 [2d, 3H þ 3H, OCH(CH3)2], 1.52 and 1.60 [2d,
3H þ 3H, 9-CH(CH3)2], 3.20e3.45 and 3.50e3.85 [2m,
2H þ 2H, N(CH2CH3)2], 4.46 [m, 1H, 9-CH(CH3)2],
4.95 [m, 1H, OCH(CH3)2], 7.53 (dd, 1H, H-3), 8.48 (dd,
1H, H-4), 8.73 (dd, 1H, H-2)
C21H29N5O2
1639 s (CO), 1607, 1592 sh,
1560, 1516
0.98 (t, 3H, OCH2CH2CH2CH3), 1.16 and 1.32 [2t,
3H þ 3H, N(CH2CH3)2], 1.37e1.61 (m, 2H,
OCH2CH2CH2CH3), 1.48 and 1.56 [2d, 3H þ 3H, 9-
CH(CH3)2], 1.71e1.93 (m, 2H, OCH2CH2CH2CH3),
3.20e3.86 [m, 4H, N(CH2CH3)2], 4.12e4.55 [m, 3H,
OCH2CH2CH2CH3 þ 9-CH(CH3)2], 7.50 (dd, 1H, H-3),
8.44 (dd, 1H, H-4), 8.70 (dd, 1H, H-2)
3f
78
160e161 (G)
C21H29N5O2
1639 s (CO), 1612 s, 1594,
1560, 1517
1.08 [d, 6H, OCH2CH(CH3)2], 1.19 and 1.34 [2t,
3H þ 3H, N(CH2CH3)2], 1.50 and 1.58 [2d, 3H þ 3H,
9-CH(CH3)2], 2.19 [m, 1H, OCH2CH(CH3)2], 3.30e
3.88 [m, 4H, N(CH2CH3)2], 3.96e4.10 and 4.23e4.36
(2m, 1H þ 1H, OCH2CH(CH3)2], 4.43 [m, 1H, 9-
CH(CH3)2], 7.51 (dd, 1H, H-3), 8.47 (dd, 1H, H-4), 8.72
(dd, 1H, H-2)
(continued on next page)

1674
G. Roma et al. / European Journal of Medicinal Chemistry 43 (2008) 1665e1680
Table 3 (continued)
Compounda Yield (%) Solventb m.p. (ꢁC) Molecular formulac IRd (cmꢀ1
)
1H NMRe (d, ppm)
4a
4b
4c
69
52
84
233e234 (B)
145e147 (G)
159e160 (C)
C
20H26N6O
1617 s (CO), 1598, 1559,
1517 w
1.12 and 1.27 [2t, 3H þ 3H, N(CH2CH3)2], 2.17e3.56
0
[m, 11H, piperazine CH2 s þ 3H of N(CH2CH3)2], 2.34
(s, 3H, NCH3), 3.67e3.90 [m, 1H, 1H of N(CH2CH3)2],
7.45 and 7.61 (2 near t, 1H þ 1H, H-7,8), 7.85 and 8.08
(2 near d, 1H þ 1H, H-6,9), 9.07 (s, 1H, H-1)
C23H32N6O
1635 s (CO), 1598, 1559,
1524 w
1.14 and 1.29 [2t, 3H þ 3H, N(CH2CH3)2], 1.49 and
1.58 [2d, 3H þ 3H, 1-CH(CH3)2], 2.04e3.78 [m, 13H,
0
piperazine CH2 s þ N(CH2CH3)2 þ 1-CH(CH3)2], 2.36
(s, 3H, NCH3), 7.46 and 7.60 (2 near t, 1H þ 1H, H-
7,8), 8.08 and 8.15 (2 near d, 1H þ 1H, H-6,9)
1.09 and 1.27 [2t, 3H þ 3H, N(CH2CH3)2], 1.30 (t, 3H,
HNCH2CH3), 1.46 and 1.55 [2d, 3H þ 3H, 1-
CH(CH3)2], 3.00e3.78 [m, 7H,
C20H27N5O
3281 (NH), 1610 s, br (CO),
1563 sh, 1547, 1504 w
HNCH2CH3 þ N(CH2CH3)2 þ 1-CH(CH3)2], 5.21f (br
s, 1H, NH), 7.17 and 7.41 (2 near t, 1H þ 1H, H-7,8),
7.84 (near d, 2H, H-6,9)
4d
80
125e126 (H)
C22H31N5O
3270 (NH), 1612 s, br (CO),
1546
0.96 [d, 6H, NCH2CH(CH3)2], 1.10 and 1.26 [2t,
3H þ 3H, N(CH2CH3)2], 1.45 and 1.55 [2d, 3H þ 3H,
1-CH(CH3)2], 1.97 [m, 1H, NCH2CH(CH3)2], 2.82 [m,
1H, 1H of NCH2CH(CH3)2], 3.17e3.87 [m, 6H,
N(CH2CH3)2 þ 1H of NCH2CH(CH3)2 þ 1-CH(CH3)2],
5.35f (br s, 1H, NH), 7.16 and 7.40 (2 near t, 1H þ 1H,
H-7,8), 7.84 (near d, 2H, H-6,9)
a
For the substituents, see Table 2.
Crystallization solvent: A ¼ diisopropyl ether, B ¼ ethyl acetate, C ¼ ethyl acetateediisopropyl ether, D ¼ ethyl acetateepetroleum ether,
b
E ¼ dichloromethaneepetroleum ether, F ¼ dichloromethaneediisopropyl ether, G ¼ diethyl etherepetroleum ether, H ¼ diethyl etherediisopropyl ether.
c
Anal. C, H, N.
In KBr pellets. Abbreviations: s ¼ strong, w ¼ weak, br ¼ broad, sh ¼ shoulder.
d
e
In CDCl3 solutions. Abbreviations: s ¼ singlet, br s ¼ broad singlet, d ¼ doublet, dd ¼ double doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet. 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.
f
Disappeared with D2O.
As we reported above (Table 1), 2a, and 2c proved to be,
respectively, the most potent anti-inflammatory and the most po-
tent analgesic compounds 2 previously synthesized by us [3,4].
The remarkable difference between the activities of these two
compounds clearly depends on the structural diversity of their
selectively effective 5-amino substituents, whereas the 6-(dieth-
ylcarbamoyl) and 9-isopropyl substituents resulted the most ef-
fective ones for both the activities of compounds 2 [3,4]. The
pharmacological results now exhibited by the novel compounds
2deo (Table 2) are in accordance with the above conclusions.
For instance, the replacement of the 6-CON(C2H5)2 substituent
piperazinyl group) exhibits a lower but interesting analgesic
activity. Also in the case of the anti-inflammatory reference
compound 2a [3], the replacement of its 5-(isobutylamino)
substituent with the (2-dimethoxyethyl)amino group (whose
two oxygen atoms are potential hydrogen bond acceptors)
gave compound 2h (Table 2) completely devoid of anti-
inflammatory properties.
Anyway, by properly modifying the 5-(isobutylamino) sub-
stituent of compound 2a, we have now obtained a group of
new, very interesting anti-inflammatory agents showing no
acute gastrolesivity in rats (compounds 2i,j,l), the most potent
of which and of all the compounds 2 previously synthesized by
us [3,4] is the 5-(isopropylamino) substituted compound 2i,
that at 12.5 mg kgꢀ1 dose exhibited a 68% (P < 0.01) inhibi-
tion of the carrageenan-induced rat paw edema (indomethacin:
51% inhibition, P < 0.01, at 10 mg kgꢀ1 dose) (Table 2).
,
6-CON
with its cyclic analogue
, in the very potent analgesic
agent 2c (Table 1), afforded compound 2m (Table 2) completely
devoid of statistically significant analgesic activity; by perform-
ing the same replacement in compound 2a (Table 1), its notably
potent anti-inflammatory activity was eliminated (compound
2n, Table 2). Similarly, as regards the 9-substituent, the phenyl
substituted compound 2e (Table 2) has shown an analgesic
potency notably lower than that of its 9-isopropyl substituted
analogue 2c.
The
N,N-diethyl-9-isopropyl[1,2,4]triazolo[4,3-a][1,8]-
naphthyridine-6-carboxamide derivatives 2c,b,i,a, and their
corresponding compounds 3b,c,d,f, respectively, differ only in
their 5-substituents which are N-substituted amino groups or
similarly O-substituted alkoxy groups, respectively. Both the
analgesic activity of compound 3b and the anti-inflammatory
activity of compounds 3c,d,f (Table 2) are remarkably less
potent than those exhibited by the corresponding compounds
2c and 2b,i,a (Tables 1 and 2), respectively (see in particular
the anti-inflammatory activity data of compounds 3c,f).
On the other hand, the replacement, in the reference com-
pound 2c [4], of the 5-(4-methyl-1-piperazinyl) substituent
(the most effective one for the analgesic potency) with simi-
lar groups has afforded the less active compounds 2f,g,
among which 2f (whose 5-substituent is a 4-substituted-1-

G. Roma et al. / European Journal of Medicinal Chemistry 43 (2008) 1665e1680
1675
The above structural and pharmacological data seem to
clearly indicate that it is harmful to replace the 5-amino
substituent of compounds 2 with a 5-alkoxy one.
5. Experimental protocols
5.1. Chemistry
The molecular modification of compounds 2a, 2b, and 2c,
taken as leads in this study, has also concerned the synthesis of
their identically substituted isosteric analogues [1,2,4]tria-
zolo[4,3-a]quinoline-4-carboxamide derivatives 4d, 4c, and
4b respectively. In this case, the analgesic activity of com-
pound 4b is very interesting, although less potent than that
of 2c, and also 4c is rather active. As regards the anti-inflam-
matory properties, compound 4c exhibited an interesting activ-
ity, but notably less potent than that of the corresponding lead
compound 2b, whereas compound 4d, isosteric analogue of
the very potent anti-inflammatory agent 2a, did not show
any activity (Tables 1 and 2).
Melting points were determined using a FishereJohns
apparatus and are uncorrected. IR spectra were recorded on
a PerkineElmer ‘‘Spectrum One’’ spectrophotometer (abbre-
viations relative to IR bands: br ¼ broad, s ¼ strong, w ¼ weak,
1
sh ¼ shoulder). H 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), br s (broad singlet), d (doublet), t (triplet), q (quar-
tet), m (multiplet), dd (double doublet). Analyses of all new
compounds, indicated by the symbols of the elements, were
within ꢂ0.4% of the theoretical values and were performed
by the Laboratorio di Microanalisi, Dipartimento di Scienze
4. Conclusions
`
Farmaceutiche, Universita di Genova. Thin layer chromato-
On the basis of the pharmacological data of compounds
2deo, 3aef, and 4aed, reported in Table 2, the following
conclusions can be drawn.
grams 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 (Brockmann activity I).
- The herein described compounds 2i,l showed a remark-
able anti-inflammatory activity (Table 2), slightly more
potent than that of the lead compounds 2a,b (Table
1), whereas compounds 2f,i and particularly the potent
compound 2j showed notable analgesic activity (Table
2), but proved to be clearly less potent than 2c (Table
1). On the basis of the very satisfactory pharmacological
properties (among which the complete absence of acute
gastrolesivity) on the whole shown by compounds 2 pre-
viously [3,4] and now synthesized by us, we regard it
reasonable to consider this one as a very interesting
new structural class of potent anti-inflammatory and/or
analgesic agents, devoid of COX inhibitory properties
[5].
5.1.1. N-Substituted 1,2-dihydro-4-hydroxy-2-oxo-1,
8-naphthyridine-3-carboxamides (6a,b)
A mixture of 25.0 mmol (5.50 g) of ester 5 [6], 50 mL of
ethanol and an excess (10 mL) of the proper amine was heated
in a closed vessel at 150 ꢁC for 16 h. After cooling the result-
ing suspension was evaporated to dryness at reduced pressure.
The residue so obtained was taken up in water and carefully
treated with 2 N HCl down to pH 5: the solid that separated
out was collected by filtration, washed with water and dried
to give the nearly pure compound 6 which was then crystal-
lized from ethanol. According to this procedure the following
compounds were obtained.
- The pharmacological data shown by compounds 3aef
(Table 2) clearly indicate that the 5-alkoxy substituents are
notably less effective than the N-alkyl substituted 5-amino
substituents for the anti-inflammatory and/or analgesic
activities of the 5-substituted [1,2,4]triazolo[4,3-a][1,8]
naphthyridine-6-carboxamide derivatives.
- On the other hand, the first interesting pharmacological re-
sults afforded by the 5-amino[1,2,4]triazolo[4,3-a]quino-
line-4-carboxamide derivatives 4aed now tested induce
us to pursue this study.
5.1.1.1. N-Ethyl-1,2-dihydro-4-hydroxy-2-oxo-1,8-naphthyri-
dine-3-carboxamide (6a). The reaction of 5 with ethylamine
yielded 5.54 g (95%) of 6a [14], whitish needles, m.p. 250e
1
252 ꢁC. H NMR (DMSO-d6): d 1.14 (t, 3H, HNCH2CH3),
3.37 (m, 2H, HNCH2CH3; q, after treatment with D2O), 7.31
(dd, J6,5 ¼ 8 Hz, J6,7 ¼ 4.8 Hz, 1H, H-6), 8.32 (dd,
J5,6 ¼ 8 Hz, J5,7 ¼ 1.6 Hz, 1H, H-5), 8.65 (dd, J7,6 ¼ 4.8 Hz,
J7,5 ¼ 1.6 Hz, 1H, H-7), 10.13 (near t, 1H, HNCH2CH3; disap-
peared with D2O), 12.21 (s, 1H, 1-NH; disappeared with
D2O); (4-OH signal was not detectable); IR (KBr): 3200e
2550 (OH þ NH), 1663 s (2-CO), 1614 s (amide CO), 1574
s, br cmꢀ1. Anal. (C11H11N3O3) C, H, N.
As reported above, the mechanism of action underlying the
anti-inflammatory activity of compounds 2 involves their anti-
adhesive effects, as well as their inhibition of superoxide an-
ion production and of myeloperoxidase release, but not the
COX inhibition [5]. As regards the mechanism of action re-
lated to the analgesic activity of compounds 2, the further re-
sults now obtained for compound 2c seem to exclude the
involvement in its analgesic activity of the m-opioid, musca-
rinic, nicotinic and serotonine receptors, and also of a generic
sedative effect.
5.1.1.2. 4-Hydroxy-3-(pyrrolidin-1-ylcarbonyl)-1,8-naphthyri-
din-2(1H )-one (6b). The reaction of 5 with pyrrolidine
yielded 6.03 g (93%) of 6b, white needles, m.p. 262e265 ꢁC
dec.0 1H NMR (DMSO-d6): d 1.77 (m, 4H, pyrrolidine b-
0
CH2 s), 3.30 (m, 4H, pyrrolidine a-CH2 s), 7.22 (dd,
J6,5 ¼ 8 Hz, J6,7 ¼ 4.8 Hz, 1H, H-6), 8.24 (dd, J5,6 ¼ 8 Hz,
J5,7 ¼ 1.6 Hz, 1H, H-5), 8.51 (dd, J7,6 ¼ 4.8 Hz,
J7,5 ¼ 1.6 Hz, 1H, H-7), 11.84 (s, 1H, 1-NH; disappeared

1676
G. Roma et al. / European Journal of Medicinal Chemistry 43 (2008) 1665e1680
with D2O); (4-OH signal was not detectable); IR (KBr):
3200e2400 s (OH þ NH), 1635 s, br (CO), 1580 s, br cmꢀ1
evaporated to dryness at reduced pressure, and the residue was
subjected to column chromatography (silica gel for 8a,b; neu-
tral aluminium oxide for 8c), in all cases eluting first with di-
chloromethane to remove Dowtherm A. The reaction
products were then recovered eluting with ethyl acetate (com-
pound 8a,c) or with the mixture ethyl acetateetetrahydrofuran
(1:1) (compound 8b). The eluate collected, after removal of
solvents, gave an oily or solid residue from which compounds
8aec were obtained as described below.
.
Anal. (C13H13N3O3) C, H, N.
5.1.2. N-Substituted 2,4-dichloro-1,8-naphthyridine-
3-carboxamides (7a,b)
A mixture of 5.0 g of 6a (21.44 mmol) or 6b (19.28 mmol)
and 50 mL of POCl3 was stirred at 110 ꢁC for 1 h. The excess
POCl3 was removed by heating at reduced pressure and the
residue was dissolved in warm water; the resulting solution
was cooled, carefully treated with NaHCO3 up to pH 7, and
finally exhaustively extracted with dichloromethane. The com-
bined 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 with
dichloromethaneeethyl acetate (1:1). From the eluate col-
lected, after removal of solvents, the following compounds
were obtained.
5.1.3.1.
5-Chloro-N-ethyl-9-isopropyl[1,2,4]triazolo[4,3-a]
[1,8]naphthyridine-6-carboxamide (8a). The solid residue
obtained from the reaction performed with 7a was taken up
in a little diethyl ether and filtered to give the nearly pure
8a as a whitish crystalline solid (0.65 g, 34%); white crystals,
1
m.p. 223e224 ꢁC, after crystallization from ethyl acetate. H
NMR (CDCl3): d 1.26 (t, 3H, HNCH2CH3), 1.47 [d, 6H, 9-
CH(CH3)2], 3.52 (m, 2H, HNCH2CH3; q, after treatment
with D2O), 4.43 [m, 1H, 9-CH(CH3)2], 7.57 (dd,
J3,4 ¼ 8.1 Hz, J3,2 ¼ 4.7 Hz, 1H, H-3), 8.65e8.75 (m, 2H,
H-2,4), 8.87 (br s, 1H, NH; disappeared with D2O); IR
(KBr): 3255 (NH), 1667 s (CO), 1601, 1586, 1559,
1520 cmꢀ1. Anal. (C15H16ClN5O) C, H, N.
5.1.2.1.
2,4-Dichloro-N-ethyl-1,8-naphthyridine-3-carboxa-
mide (7a). The reaction carried out with 6a gave 3.59 g
(62%) of 7a, white crystals, m.p. 132e133.5 ꢁC, after crystal-
1
lization from ethyl acetate. H NMR (CDCl3): d 1.28 (t, 3H,
HNCH2CH3), 3.53 (m, 2H, HNCH2CH3; q, after treatment
with D2O), 7.05 (near t, 1H, HNCH2CH3; disappeared with
D2O), 7.47 (dd, J6,5 ¼ 8.4 Hz, J6,7 ¼ 4.2 Hz, 1H, H-6), 8.28
(dd, J5,6 ¼ 8.4 Hz, J5,7 ¼ 2 Hz, 1H, H-5), 8.93 (dd,
J7,6 ¼ 4.2 Hz, J7,5 ¼ 2 Hz, 1H, H-7); IR (KBr): 3312 s (NH),
1668 s (CO), 1598 w, 1576, 1553, 1529 cmꢀ1. Anal.
(C11H9Cl2N3O) C, H, N.
5.1.3.2. (5-Chloro-9-isopropyl[1,2,4]triazolo[4,3-a][1,8]naph-
thyridin-6-yl)(1-pyrrolidinyl)methanone (8b). The oily residue
derived from the reaction carried out with 6a, treated with a lit-
tle diisopropyl ether, afforded pure 8b as whitish solid (0.70 g,
34%); m.p. 206e208 ꢁC after crystallization from ethyl aceta-
1
teediisopropyl ether. H NMR (CDCl3): d 1.45 and 1.51 [2d,
3H þ 3H, 9-CH(CH3)2], 1.70e2.11 (m, 4H, pyrrolidine b-
0
5.1.2.2. (2,4-Dichloro-1,8-naphthyridin-3-yl)(1-pyrrolidinyl)-
methanone (7b). The reaction carried out with 6b gave
3.20 g (56%) of 7b, white crystals, m.p. 149e151 ꢁC, after
CH2 s), 3.13e3.45 and 3.60e3.91 (2 m, 2H þ 2H, pyrrolidine
0
a-CH2 s), 4.42 [m, 1H, 9-CH(CH3)2], 7.55 (dd, J3,4 ¼ 8.1 Hz,
J3,2 ¼ 4.7 Hz, 1H, H-3), 8.48 (dd, J4,3 ¼ 8.1 Hz, J4,2 ¼ 1.7 Hz,
1H, H-4), 8.71 (dd, J2,3 ¼ 4.7 Hz, J2,4 ¼ 1.7 Hz, 1H, H-2); IR
(KBr): 1651 s (CO), 1602, 1590, 1559, 1519 cmꢀ1. Anal.
(C17H18ClN5O) C, H, N.
1
crystallization from ethyl acetateepetroleum ether.0 H NMR
(CDCl3): d 1.66e2.15 (m, 4H, pyrrolidine b-CH2 s), 3.02e
3.30 (m, 2H, 2H of pyrrolidine a-CH2 s), 3.69 (t, 2H, 2H of
0
0
pyrrolidine a-CH2 s), 7.59 (dd, J6,5 ¼ 8.4 Hz, J6,7 ¼ 4.2 Hz,
1H, H-6), 8.53 (dd, J5,6 ¼ 8.4 Hz, J5,7 ¼ 2 Hz, 1H, H-5), 9.12
(dd, J7,6 ¼ 4.2 Hz, J7,5 ¼ 2 Hz, 1H, H-7); IR (KBr): 1634 s
(CO), 1596, 1573, 1542 cmꢀ1. Anal. (C13H11Cl2N3O) C, H, N.
5.1.3.3.
5-Chloro-N,N-9-triethyl[1,2,4]triazolo[4,3-a][1,8]
naphthyridine-6-carboxamide (8c). The nearly solid residue
obtained from the reaction performed with 7c and propionic
hydrazide was taken up in a little diethyl ether and filtered to
give pure 8c as a whitish crystalline solid (0.86 g, 43%); white
crystals, m.p. 176e177 ꢁC, after crystallization from ethyl ac-
etate. 1H NMR (CDCl3): d 1.10 and 1.31 [2t, 3H þ 3H,
N(CH2CH3)2], 1.45 (t, 3H, 9-CH2CH3), 3.24 [q, 2H, 2H of
N(CH2CH3)2], 3.40e3.93 [m, 4H, 2H of N(CH2CH3)2 þ 9-
CH2CH3], 7.56 (dd, J3,4 ¼ 8.1 Hz, J3,2 ¼ 4.7 Hz, 1H, H-3),
8.48 (dd, J4,3 ¼ 8.1 Hz, J4,2 ¼ 1.7 Hz, 1H, H-4), 8.70 (dd,
J2,3 ¼ 4.7 Hz, J2,4 ¼ 1.7 Hz, 1H, H-2); IR (KBr): 1639 s (CO),
1599, 1586, 1556, 1519 w cmꢀ1. Anal. (C16H18ClN5O) C, H, N.
5.1.3. N-Substituted 5-chloro[1,2,4]triazolo-
[4,3-a][1,8]naphthyridine-6-carboxamides (8aec)
A mixture of 6.0 mmol of 7a (1.62 g) or 7b (1.78 g),
9.0 mmol (0.92 g) of isobutyrohydrazide and 10 mL of Dow-
therm A (in the case of 8a or 8b), or a mixture of 6.0 mmol
of 7c [7] (1.79 g), 9.0 mmol (0.79 g) of propionic hydrazide
and 10 mL of Dowtherm A (in the case of 8c), were stirred
at 160 ꢁC for 30 min (1 h in the case of preparation of 8c). After
cooling, 10% aqueous Na2CO3 (50 mL) and dichloromethane
(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 separa-
tory funnel, then the organic layer was collected and the aque-
ous one was exhaustively extracted with dichloromethane. The
combined organic phases were dried (anhydrous Na2SO4), then
5.1.4. Synthesis of the N-substituted 5-amino[1,2,4]triazolo-
[4,3-a][1,8]naphthyridine-6-carboxamide derivatives
2dek,meo
Compounds 2dek,meo were prepared by the reaction of
the new 5-chloroderivatives 8aec or the previously described

G. Roma et al. / European Journal of Medicinal Chemistry 43 (2008) 1665e1680
1677
8def with an excess of the proper amine through the below
described experimental procedures, depending on the amine
used.
solvent was removed in vacuo and the residue was parti-
tioned between water (200 mL) and dichloromethane
(200 mL). The organic layer was collected and the aqueous
one was further extracted twice with dichloromethane. The
combined organic phases were dried (anhydrous Na2SO4)
and then evaporated to dryness in vacuo to give an oil
which was chromatographed on a silica gel column eluting
first with ethyl acetate. The first fractions of this eluate,
containing some impurities, were discarded. Compound 2l
was then recovered by eluting with the mixture ethyl acet-
ateeacetone (4:1). The eluate collected, evaporated to dry-
ness in vacuo, afforded an oil from which, after treatment
with a little diisopropyl ether, pure compound 2l separated
out as pale yellow solid.
5.1.4.1. Compounds 2hek,n. A mixture of 5.0 mmol of 8b
(1.72 g) (reaction with isobutylamine to give 2n), or
8c (1.66 g) (reaction with isopentylamine to give 2k), or
8d [4] (1.73 g) (reactions with 2,2-dimethoxyethylamine,
isopropylamine or 3-pentylamine to give 2h, 2i, or 2j, re-
spectively), 5 mL of the proper amine and 25 mL of anhy-
drous ethanol was heated at 120 ꢁC in a closed vessel for
14 h. After cooling, the resulting solution was evaporated
to dryness at reduced pressure and the residue was parti-
tioned between 10% aqueous Na2CO3 (100 mL) and di-
chloromethane (100 mL). The organic layer was collected
and the aqueous one was further extracted twice with di-
chloromethane. The combined organic phases were dried
(anhydrous Na2SO4) and then evaporated to dryness at re-
duced pressure to give a thick oil which was chromato-
graphed on a silica gel column eluting first with ethyl
acetate to discard a little amount of starting compound
and impurities, then with tetrahydrofuran to recover com-
pounds 2. The residues obtained from the fractions collected
(after removal of solvent), treated with a little diethyl ether,
afforded pure compounds 2hek,n which were crystallized
from the suitable solvents.
Data for compound 2l are reported in Table 3.
5.1.6. General procedure for the synthesis of 5-alkoxy-N,N-
diethyl[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-
carboxamide 3a and its 9-isopropyl analogues 3bef
Sodium (0.14 g, 6 mmol) was dissolved in 5 mL of proper
anhydrous alcohol (ethanol for 3c, isopropanol for 3d, n-
butanol for 3e, isobutanol for 3f) or in 3 mL of 1-methyl-
4-piperidinol (preparation of 3a,b). When a limpid solution
was obtained (in the case of butanols and 1-methyl-4-piper-
idinol, the reaction was carried out at 100 ꢁC to hasten the
dissolution of sodium), the alcohol was removed in vacuo:
the resulting solid or resinous (in the case of 1-methyl-4-
piperidinol) residue was taken up in 25 mL of anhydrous
tetrahydrofuran, then 4.0 mmol of 5-chloroderivative 8e [8]
(1.21 g, preparation of 3a) or 8d [4] (1.38 g, preparation of
3bef) were added to the mixture. The resulting turbid solu-
tion was refluxed (80 ꢁC) for 1 h with stirring. After cooling,
the solvent was removed at reduced pressure and the residue
was partitioned between water (100 mL) and dichlorome-
thane (100 mL). The organic layer was collected and the
aqueous one was further extracted twice with dichlorome-
thane. The combined organic phases were washed with
water, dried (anhydrous Na2SO4), and then evaporated to
dryness in vacuo to give oily residues from which, after treat-
ment with a little diisopropyl ether or petroleum ether, the
pure compounds 3aef separated out as crystalline whitish
solids that were crystallized from the proper solvents.
Data for compounds 3aef are reported in Table 3.
5.1.4.2. Compounds 2deg,m,o. A mixture of 5.0 mmol of 8a
(1.59 g), or 8b (1.72 g), or 8e [8] (1.52 g), or 8f [8] (1.90 g)
(reactions with 1-methylpiperazine to give 2o, 2m, 2d, or
2e, respectively), or 5.0 mmol of 8d [4] (1.73 g) (reactions
with ethyl piperazine-1-carboxylate or 1-amino-4-methylpi-
perazine to give 2f or 2g, respectively), 5 mL of the proper
amine and 5 mL of dimethyl sulphoxide was stirred at
130 ꢁC for 2 h (compounds 2d,f,m) or 4 h (compounds 2e,g)
or 5 h (compound 2o). The solution obtained was then poured
into water (200 mL) and the resulting emulsion was exhaus-
tively extracted with dichloromethane. The combined extracts,
dried (anhydrous Na2SO4) and evaporated to dryness in vacuo,
afforded an oily or nearly solid residue from which, after treat-
ment with a little diethyl ether (compounds 2deg,m) or ethyl
acetate (compound 2o) and standing, the expected compound
2 separated out as a pink-orange solid. After crystallization
from the proper solvent with charcoal, white or whitish crys-
tals were obtained.
5.1.7. N,N-Diethyl-1,2-dihydro-4-hydroxy-2-oxoquinoline-
3-carboxamide (10)
Data for compounds 2dek,meo are reported in Table 3.
A mixture of 25.0 mmol (5.83 g) of ester 9 [9], 50 mL of
ethanol and an excess (10 mL) of diethylamine was heated
in a closed vessel at 150 ꢁC for 16 h. After cooling, the result-
ing suspension was evaporated to dryness at reduced pressure.
The residue so obtained was taken up in water and carefully
treated with 2 N HCl down to pH 5; the resulting emulsion
was exhaustively extracted with chloroform and the combined
extracts were dried (Na2SO4) and evaporated to dryness at re-
duced pressure. The solid residue obtained was taken up in
a little ethyl acetate and filtered to give pure 10 (4.25 g,
65%), white crystals, m.p. 238e240 ꢁC, after crystallization
5.1.5. N,N-Diethyl-5-(N-ethyl-N-methylamino)-9-isopropyl-
[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamide (2l)
To the suspension of 3.0 mmol (1.06 g) of 2b [3] in
80 mL of 2-butanone, 24 mmol (1.34 g) of finely powdered
KOH mixed with 2.0 g of anhydrous K2CO3 were added,
then the mixture was refluxed (80 ꢁC) for 5 min. A solution
of 4.0 mmol (0.25 mL) of iodomethane in 10 mL of 2-buta-
none was then slowly added and the resulting mixture was
further refluxed for 1 h with stirring. After cooling, the

1678
G. Roma et al. / European Journal of Medicinal Chemistry 43 (2008) 1665e1680
from ethanol. 1H NMR (CDCl3): d 1.31 [near t, 6H,
N(CH2CH3)2], 3.34e3.70 [m, 4H, N(CH2CH3)2], 7.10e7.42,
7.61 and 8.13 (3m, 2H þ 1H þ 1H, H-6,7,8,9), 10.57 (br s,
1H, 1-NH; disappeared with D2O); (4-OH signal was not de-
tectable); IR (KBr): 3220e2500 (OH þ NH), 1651 s (2-CO),
1608 (amide CO), 1583 br, 1496 cmꢀ1. Anal. (C14H16N2O3)
C, H, N.
(1.22 g) and 10 mL of Dowtherm A was stirred at 180 ꢁC
for 1.5 h. In both the cases, after cooling, 10% aqueous
Na2CO3 (50 mL) and dichloromethane (50 mL) were added
and the mixture was further stirred at room temperature for
30 min. After discarding some insoluble impurities by filtra-
tion, the mixture was transferred in a separatory funnel, then
the organic layer was collected and the aqueous one was ex-
haustively extracted with dichloromethane. The combined
organic phases were dried (anhydrous Na2SO4), then evapo-
rated to dryness at reduced pressure, and the resulting resi-
due was subjected to chromatography on a silica gel
column, eluting first with the mixture dichloromethaneepe-
troleum ether (1:1) in order to remove Dowtherm A. The
subsequent elution with the mixture dichloromethaneeethyl
acetate (4:1) allowed the recovery of significant amounts
of starting compound 12 (0.79 g or 0.71 g in the case of
preparations of 13a or 13b, respectively). Finally, compound
13a or 13b was obtained eluting with the mixture ethyl
acetateetetrahydrofuran (1:1) [in the case of preparation
of 13a, the first fractions of this latter eluate (containing
a small amount of an unidentified compound) were dis-
carded]. The eluate collected, after removal of solvents,
gave an oily or solid residue from which compounds 13a,
or 13b was obtained as described below.
5.1.8. 4-Chloro-N,N-diethyl-1,2-dihydro-2-oxoquinoline-3-
carboxamide (11) and 2,4-dichloro-N,N-diethylquinoline-
3-carboxamide (12)
A mixture of 5.0 g (19.21 mmol) of 10 and 50 mL of
POCl3 was stirred at 110 ꢁC for 2 h. The excess POCl3
was removed by heating at reduced pressure and the residue
was dissolved in warm water; the resulting solution was
cooled, carefully treated with NaHCO3 up to pH 7, and
finally exhaustively extracted with dichloromethane. The
combined extracts (dried over anhydrous Na2SO4 and evap-
orated to dryness at reduced pressure) afforded a thick oil
which was chromatographed on a silica gel column, eluting
with dichloromethaneeethyl acetate (1:1). The eluate
collected, after removal of solvents, gave a solid residue
which was taken up in a little petroleum ether and filtered
to yield 3.81 g (67%) of pure 12, white crystals, m.p.
120e121 ꢁC, after crystallization from diisopropyl
ether. 1H NMR (CDCl3): d 1.09 and 1.26 [2t, 3H þ 3H,
N(CH2CH3)2], 3.14 [q, 2H, 2H of N(CH2CH3)2], 3.60
[m, 2H, 2H of N(CH2CH3)2], 7.63 and 7.76 (2 near t,
1H þ 1H, H-6,7), 7.98 and 8.15 (2 near d, 1H þ 1H, H-
5,8); IR (KBr): 1633 s (CO), 1575, 1557 cmꢀ1. Anal.
(C14H14Cl2N2O) C, H, N.
5.1.10.1. 5-Chloro-N,N-diethyl[1,2,4]triazolo[4,3-a]quinoline-
4-carboxamide (13a). The solid residue obtained from the re-
action performed with formic hydrazide was taken up in a little
diethyl ether and filtered to give the nearly pure 13a as a whit-
ish crystalline solid (0.60 g, 25%); white crystals, m.p. 201e
203 ꢁC, after crystallization from ethyl acetate. 1H NMR
(CDCl3): d 1.09 and 1.31 [2t, 3H þ 3H, N(CH2CH3)2], 3.23
[q, 2H, 2H of N(CH2CH3)2], 3.52 and 3.80 [2m, 1H þ 1H,
2H of N(CH2CH3)2], 7.59 and 7.73 (2 near t, 1H þ 1H, H-
7,8), 7.97 and 8.20 (2 near d, 1H þ 1H, H-6,9), 9.22 (s, 1H,
Chromatography was pursued eluting with the mixture
ethyl acetateetetrahydrofuran (1:1): the fraction collected,
after removal of solvents, afforded pure compound 11 as
a white solid (0.64 g, 12%), m.p. 188e190 ꢁC, after crystalli-
1
zation from ethyl acetate. H NMR (CDCl3): d 1.11 and 1.27
H-1); IR (KBr): 1630 s (CO), 1596, 1566, 1524 w cmꢀ1
Anal. (C15H15ClN4O) C, H, N.
.
[2t, 3H þ 3H, N(CH2CH3)2], 3.27 [q, 2H, 2H of
N(CH2CH3)2], 3.48 and 3.74 [2m, 1H þ 1H, 2H of
N(CH2CH3)2], 7.24 and 7.51 (2 near t, 1H þ 1H, H-6,7),
7.39 and 7.90 (2 near d, 1H þ 1H, H-5,8), 12.75 (br s, 1H,
1-NH; disappeared with D2O); IR (KBr): 3200e2400 (NH),
5.1.10.2. 5-Chloro-N,N-diethyl-1-isopropyl[1,2,4]triazolo[4,
3-a]quinoline-4-carboxamide (13b). The residue obtained
from the reaction performed with isobutyrohydrazide was
treated with a little diisopropyl ether to afford the nearly
pure 13b as a whitish crystalline solid (0.91 g, 33%); white
crystals, m.p. 166.5e168 ꢁC, after crystallization from ethyl
acetateediisopropyl ether. 1H NMR (CDCl3): d 1.09 and
1.30 [2t, 3H þ 3H, N(CH2CH3)2], 1.53 and 1.59 [2d,
3H þ 3H, 1-CH(CH3)2], 3.22 [q, 2H, 2H of N(CH2CH3)2],
3.48e3.89 [m, 3H, 2H of N(CH2CH3)2 þ 1-CH(CH3)2], 7.60
and 7.71 (2 near t, 1H þ 1H, H-7,8), 8.15 and 8.23 (2 near
d, 1H þ 1H, H-6,9); IR (KBr): 1643 s (CO), 1601, 1567,
1530 w cmꢀ1. Anal. (C18H21ClN4O) C, H, N.
1650 (2-CO), 1634
s (amide CO), 1596, 1562 sh,
1500 cmꢀ1. Anal. (C14H15ClN2O2) C, H, N.
5.1.9. Chlorination of compound 11 to compound 12
A mixture of 3.0 g (10.76 mmol) of 11 and 30 mL of POCl3
was stirred at 110 ꢁC for 2 h. After a work-up identical to the
one described above in Section 5.1.8, 2.63 g (82%) of pure di-
chloroderivative 12 was obtained.
5.1.10. 5-Chloro-N,N-diethyl[1,2,4]triazolo-
[4,3-a]quinoline-4-carboxamide derivatives 13a,b
In the case of preparation of 13a, a mixture of 8.0 mmol
of 12 (2.38 g), 16.0 mmol of formic hydrazide (0.96 g) and
10 mL of Dowtherm A was heated at 180 ꢁC for 3.5 h, with
stirring; in the case of preparation of 13b, a mixture of
8.0 mmol of 12 (2.38 g), 12.0 mmol of isobutyrohydrazide
5.1.11. Synthesis of substituted 5-amino[1,2,4]triazolo[4,3-
a]quinoline-4-carboxamide derivatives 4aed
Compounds 4aed were prepared by the reaction of the 5-
chloroderivatives 13a,b with an excess of the proper amine

G. Roma et al. / European Journal of Medicinal Chemistry 43 (2008) 1665e1680
1679
through the below described experimental procedures, depend-
ing on the amine used.
The test compounds were suspended in 0.5% methylcellu-
lose and administered at the initial dose of 100 mg kgꢀ1 by
oral gavage (1 mL 100 gꢀ1 body weight) in animals fasted
overnight. Compounds active at this dose were further tested
at doses decreasing by a factor of 2. Control animals were
orally treated with an equivalent volume of vehicle alone.
Groups of 8e10 animals were used. The ethical guidelines
for investigation of experimental pain in conscious animals
were followed and all the tests were carried out according to
the EEC ethical regulation (EEC Council 86/609; D.L. 27/
01/1992, No. 116).
5.1.11.1. N,N-Diethyl-5-(4-methyl-1-piperazinyl)[1,2,4]tria-
zolo[4,3-a]quinoline-4-carboxamide (4a) and its 1-isopropyl
derivative (4b). A mixture of 5.0 mmol of 13a (1.59 g, prepa-
ration of 4a) or 13b (1.72 g, preparation of 4b), 5 mL of
1-methylpiperazine and 5 mL of dimethyl sulphoxide was
stirred at 140 ꢁC for 3 h. The solution obtained was then
poured into water (200 mL) and the resulting emulsion was
exhaustively extracted with dichloromethane. The combined
extracts, dried (anhydrous Na2SO4) and evaporated to dryness
in vacuo, afforded an oily residue which was chromatographed
on a silica gel column, eluting first with mixture ethyl
acetateetetrahydrofuran (1:1) to remove impurities and a little
amount of starting compound, then with the mixture
dichloromethaneetriethylamine (9:1) (compound 4a) or
dichloromethaneepetroleum etheretriethylamine (6:3:1)
(compound 4b). After removal of solvents from the eluates
collected, 4a was obtained as whitish solid which was crystal-
lized from ethyl acetate, whereas the oily 4b was precipitated
as maleate (white crystals, m.p. 218e220 ꢁC) by treatment
with a solution of maleic acid in anhydrous ethanol. From
an analytical sample of this maleate, the free base 4b was ob-
tained (after treatment with aqueous NaHCO3 and extraction
with chloroform) as colourless thick oil which, after a long
standing at room temperature, became an amorphous white
solid that was crystallized from diethyl etherepetroleum ether.
5.2.1. Anti-inflammatory activity
Anti-inflammatory activity was studied by inducing paw
edema according to Winter’s method [15]. Carrageenan (1%,
0.1 mL) (SigmaeAldrich, Milan, Italy) was injected subcuta-
neously into the plantar surface of the rat hind paw 1 h after
the oral administration of the test compound. 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 (VA), Italy). Edema values, ob-
tained 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.
5.2.2. Analgesic activity
Antinociceptive activity was studied by writhing test [16],
through the intraperitoneal injection of 0.2 mL/mouse of ace-
tic acid solution (0.6%) (SigmaeAldrich, Milan, Italy) 1 h af-
ter oral treatment with the test drugs. 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 percent-
age of inhibition was calculated from the difference in the
writhing response between the treated and the control groups.
To investigate the possible mechanism underlying the anti-
nociceptive effect demonstrated by compound 2c, different
groups of mice were pretreated with Naloxone (1 mg kgꢀ1
i.p.), Atropine (5 mg kgꢀ1 i.p.), Mecamylamine (1 mg kgꢀ1
i.p.) and Methysergide (1 mg kgꢀ1 s.c.) 10 min before com-
pound 2c (3.12 mg kgꢀ1) or vehicle oral administration. All
the drugs were purchased from SigmaeAldrich, Milan, Italy.
5.1.11.2. N,N-Diethyl-5-(ethylamino)-1-isopropyl[1,2,4]tria-
zolo[4,3-a]quinoline-4-carboxamide (4c) and its 5-(isobutyla-
mino) analogue (4d). A mixture of 5.0 mmol of 13b
(1.72 g), 5 mL of ethylamine (4c) or isobutylamine (4d), and
25 mL of anhydrous ethanol was heated at 160 ꢁC in a closed
vessel for 14 h. After cooling, the resulting solution was evap-
orated to dryness at reduced pressure and the residue was par-
titioned between 10% aqueous Na2CO3 (100 mL) and
dichloromethane (100 mL). The organic layer was collected
and the aqueous one was further extracted twice with dichloro-
methane. The combined organic phases were dried (anhydrous
Na2SO4), then evaporated to dryness at reduced pressure, to
give a thick oil from which nearly pure compound 4d sepa-
rated out as a whitish solid by simple addition of a little diethyl
etherepetroleum ether, whereas, in the case of 4c, a chroma-
tography on a silica gel column, eluting with the mixture di-
chloromethaneepetroleum etheretriethylamine (5:5:1), was
necessary to obtain pure 4c.
5.2.3. Spontaneous locomotor activity
Locomotor activity was measured by means of an activity
cage (height 35 cm, width 23 cm, depth 19 cm, Model 7401,
Basile, Comerio (VA), Italy) where the bridges the animals
make or break with their paws produce random configurations
which are converted into pulses. After oral administration of
the compounds under study or vehicle, mice were placed sin-
gularly into the activity cage and locomotor activity was auto-
matically recorded at time intervals of 5 min for 90 min. Mice
spontaneous motility was evaluated considering the number of
pulses recorded in the final 30 min period. All experiments
were conducted from 9:00 to 13:00. The percentage of inhibi-
tion was calculated from the difference in the number of
pulses between the treated and the control groups.
Data for compounds 4aed are reported in Table 3.
5.2. Pharmacology
Wistar rats (250e300 g) and Swiss mice (25e35 g) of ei-
ther sex were used. During the night preceding each experi-
ment they were kept in proper cages to prevent coprophagy
at constant environmental conditions (21 ꢂ 1 ꢁC; 40 ꢂ 5% rel-
ative humidity).

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G. Roma et al. / European Journal of Medicinal Chemistry 43 (2008) 1665e1680
5.2.4. Gastrolesivity
References
The acute gastrolesivity of the test compounds was evalu-
ated by examining the stomachs excised 5 h after oral admin-
istration of the drugs (200 mg kgꢀ1) in rats. The stomachs,
fixed in 2% formalin (injection of 6 mL per stomach), were
opened along the greater curvature and mounted over a flat
surface. The stomachs were examined with a stereomicroscope
(M8 Wild Heerbrung, Switzerland) by an observer unaware of
the treatment the rats received. Acute gastrolesivity was ex-
pressed as the number of animals with gastric damage over
the number of treated animals.
[1] G. Roma, G.C. Grossi, M. Di Braccio, M. Ghia, F. Mattioli, Eur. J. Med.
Chem. 26 (1991) 489e496.
[2] G.C. Grossi, M. Di Braccio, G. Roma, V. Ballabeni, M. Tognolini,
F. Calcina, E. Barocelli, Eur. J. Med. Chem. 37 (2002) 933e944.
[3] G. Roma, M. Di Braccio, G.C. Grossi, F. Mattioli, M. Ghia, Eur. J. Med.
Chem. 35 (2000) 1021e1035.
[4] G.C. Grossi, M. Di Braccio, G. Roma, V. Ballabeni, M. Tognolini,
E. Barocelli, Eur. J. Med. Chem. 40 (2005) 155e165.
[5] C. Dianzani, M. Collino, M. Gallicchio, M. Di Braccio, G. Roma,
R. Fantozzi, J. Inflamm. 3 (2006) 4.<http://www.journal-inflammation.-
com/content/3/1/4>.
[6] G. Koller, Chem. Ber. 60 (1927) 407e410.
[7] M. Di Braccio, G. Roma, A. Balbi, E. Sottofattori, M. Carazzone, Farm-
aco 44 (1989) 865e881.
[8] M. Di Braccio, G. Roma, G.C. Grossi, M. Ghia, F. Mattioli, Farmaco 52
(1997) 49e53.
5.2.5. Data analysis
Results were expressed as means ꢂ SEM. 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).
[9] J. Reisch, R.A. Salehi-Artimani, A. Bathe, M. Muller, Monatsh. Chem.
119 (1988) 781e791.
[10] K.M. Raehal, L.M. Bohn, A.A.P.S.J. 7 (2005) E587eE591.
[11] J.C. Eisenach, Life Sci. 64 (1999) 549e554.
Acknowledgements
[12] K.K. Jain, Curr. Opin. Investig. Drugs 5 (2004) 76e81.
[13] J.A. Lopez-Garcia, Curr. Top. Med. Chem. 6 (2006) 1987e1996.
[14] E.A. Mohamed, R.M. Abdel-Rahman, Z. El-Gendy, M.M. Ismail, Comm.
Fac. Sci. Univ. Ankara Ser. B: Chem. and Chem. Engin. 40 (1e2) (1996)
1e12.
[15] C.A. Winter, E.A. Risley, G.W. Nuss, J. Pharmacol. Exp. Ther. 141
(1963) 369e376.
[16] R. Koster, M. Anderson, E.J. De Beer, Fed. Proc. 18 (1959) 412e421.
The authors thank Dr. G. Domenichini for his skilful tech-
nical assistance in pharmacological experiments, O. Gagliardo
for elemental analyses, F. Tuberoni for IR spectra and Dr. R.
Raggio for H NMR spectra. The financial support from Uni-
versity of Genoa and University of Parma is gratefully
acknowledged.
1