Antiplatelet properties of novel N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone derivatives

Article abstract of DOI:10.1016/S0968-0896(03)00055-5

This paper describes the design, synthesis and pharmacological evaluation of new N-acylhydrazone (NAH) compounds, belonging to the N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone class (2a-p). Classical heteroaromatic ring bioisosterism strategies wer

Full text of DOI:10.1016/S0968-0896(03)00055-5

Bioorganic & Medicinal Chemistry 11 (2003) 2051–2059
Antiplatelet Properties of Novel N-Substituted-phenyl-1,2,3-
triazole-4-acylhydrazone Derivatives
Anna C. Cunha,a Juliana M. Figueiredo,a Jorge L. M. Tributino,a Ana L. P. Miranda,a
Helena C. Castro,a,b Russolina B. Zingali,b Carlos A. M. Fraga,a,d
Maria Cecılia B. V. de Souza,c Vitor F. Ferreirac and Eliezer J. Barreiroa,d,
*
a
´
Laboratorio de Avaliac¸a˜o e Sıntese de Substancias Biotivas (LASSBio), Faculdade de Farmacia,
´
ˆ
´
Universidade Federal do Rio de Janeiro, PO Box 68006, 21944-971, Rio de Janeiro, RJ, Brazil
b
´
Laboratorio de Hemostase e Venenos (LabHemoVen), Departamento de Bioquımica Medica,
´
´
Instituto de Cieˆncias Biomedicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
´
Departamento de Quımica Orgaˆnica, Instituto de Quımica, Universidade Federal Fluminense, Niteroi, RJ, Brazil
c
´
´
´
Instituto de Quımica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
d
´
Received 25 March 2002; accepted 27 December 2002
Abstract—This paper describes the design, synthesis and pharmacological evaluation of new N-acylhydrazone (NAH) compounds,
belonging to the N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone class (2a–p). Classical heteroaromatic ring bioisosterism
strategies were applied to the previously reported N-phenylpyrazolyl-4-acylhydrazone derivative 1, elected as lead-compound due to
its important anti-aggregating profile on arachidonic acid induced platelet aggregation (IC50=24ꢀ0.5 mM), from which emerge this
new series 2. These new compounds 2a–p were readily synthesized, characterized and tested on platelet aggregation assays induced
by collagen (5 mg/mL), ADP (5 mM) and arachidonic acid (100 mM) in rabbit citrated platelet-rich plasma. Compounds 2b, 2d, and
2h were found to be the most potent, exhibiting a significant antiplatelet activity on arachidonic acid- and collagen-induced platelet
aggregation. In addition, these new antiplatelet agents are free of gastric ulcerogenic effect and presented discrete anti-inflammatory
and analgesic properties. The N-para-chlorophenyltriazolyl-4-acylhydrazone compound 2h produced the highest inhibitory effect
on collagen (IC50=21.6ꢀ0.4 mM) and arachidonic acid-induced platelet aggregation (IC50=2.2ꢀ0.06 mM), suggesting that the
nature of the substituent on the phenyl ring of the N-heteroaromatic system of NAH moiety may be an important structural
requirement for the improvement of antiplatelet activity, in comparison with lead-series 1.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
effective way to prevent thrombotic events.5 However,
long-term treatment using ASA causes adverse gastro-
intestinal side effects and reduces prostaglandin I2
levels, an important vasodilator and antiplatelet eicosa-
noid.1,4,5 Other antiplatelet agents acting by different
mechanisms of action have been developed and descri-
bed, but the demand still remains for innovative anti-
thrombotic drugs, which are safer than those currently
adopted for clinical use.6ꢁ8
Thromboxane A2 (TXA2) and its metabolic precursor,
prostaglandin endoperoxide H2 (PGH2), are potent
inducers of platelet aggregation and vasoconstriction
and are also involved in several pathological processes
including thromboembolic diseases.1,2 Several classes of
compounds such as nonsteroidal anti-inflammatory
drugs (NSAIDS) have been used in the treatment of
various circulatory disorders.3,4 Acetylsalicylic acid
(ASA), the most popular antiplatelet drug and COX-1
inhibitor, inhibits biosynthesis of PGH2 and conse-
quently reduces TXA2 level in platelets, resulting in an
In our laboratory various N-acylheteroarylhydrazones
(NAH) have been synthesized and were found to be
very effective in antiplatelet activity on arachidonic acid
induced platelet aggregation in rabbit citrated platelet-
rich plasma.9ꢁ12 The N-phenylpyrazolyl-4-acylhydra-
zone derivative (1) was the most active in this series
(IC50=24ꢀ0.5 mM) (Fig. 1).12,13 In an effort to optimize
*Corresponding author. Fax: +55-21-2562-6644; e-mail: eliezer@
pharma.ufrj.br; website: http://www.farmacia.ufrj.br/lassbio
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00055-5

2052
A. C. Cunha et al. / Bioorg. Med. Chem. 11 (2003) 2051–2059
Chemistry
The synthesis of the new N-substituted-phenyl-1,2,3-tri-
azole-4-acylhydrazone derivatives 2a–p (Scheme 1) was
initiated from the N-substituted triazole-4-carbox-
aldehydes 3a–c prepared in good yields, employing the
reaction of diazomalonaldehydes with appropriate
amines in acetic acid, according to the procedure
described by Arnold and coworkers.14 Converting the
aldehyde function of 3a–c to the corresponding methyl
ester 4a–c by oxidation with manganese dioxide in the
presence of sodium cyanide in methanol,15 we were able
to obtain the corresponding acylhydrazides inter-
mediates 5a–c by treatment with hydrazine hydrate in
ethanol at reflux. Finally, the new NAH compounds 2a–p
were prepared in good yields by condensing compounds
5a–c with the suitable aromatic aldehydes in ethanol
using hydrochloric acid as catalyst (Scheme 1).
All new acylhydrazone derivatives (2a–p) were obtained
as stable solid as illustrated in Table 1.
In order to assure the diastereomeric ratio of the
N-acylarylhydrazone derivatives (2a–p), essential to the
complete understanding of the biological results, the
next step in this work was to determine the relative
configuration of the imine double bond in this series. A
1
detailed analysis of the H NMR spectra of these new
Figure 1. Design concept of N-substituted-phenyl-1,2,3-triazole-4-
acylhydrazone derivatives.
derivatives (2a–p) allowed us to detect only the presence
of one hydrogen signal, which was attributed to the
(E)-diastereomer. The assignment of (E)-configuration to
the hydrazone double bond was performed by the chemi-
cal shift comparison with previous data obtained in our
laboratory9ꢁ11,16 and is in agreement with data from Kar-
abatsos22 and Fuchs.23 In fact the –CH¼N hydrazonic
the antiplatelet activity of structurally simple azahetero-
cyclic N-acylhydrazone derivatives, we describe in this
paper a new class of N-substituted-phenyl-1,2,3-tri-
azole-4-acylhydrazone derivatives 2a–p, structurally
designed by applying classical heteroaromatic ring bio-
isosterism strategy to the previously described anti-
platelet class 1.13
Considering that the N-phenylpyrazolyl moiety in ser-
ies (1) which has the NAH chain with identical ortho-
substituents, represented by C-3 and C-5 hydrogens of
the pyrazolyl ring, we decided to investigate the eventual
effect of ‘desymmetrization’ around this pharmacophoric
chain constructing the isosteric N-substituted-phenyl-
1,2,3-triazole system of compounds 2a–p.
This isosteric ring replacement maintained a similar
lipophilic character of the N-heteroaromatic ring,
represented by the presence of 6-p electrons, con-
nected to an additional phenyl ring in both series
(Fig. 1).
In addition, we also decided to investigate the influence
of the aryl substituent of the imine function on anti-
platelet activity by introducing different aromatic rings
such as 2-furyl (2e, 2j, and 2p), para-phenyl substituted
(2a–c, 2f–h and 2l–n) and 3,5-ditert-butyl-4-hydroxy-
phenyl rings (2d, 2f, and 2e) (Fig. 1). Furthermore, we
also modified the electronic and lipophilic contributions
of the N-phenyl ring by introducing two different halo-
gen atoms as para-substituents (Fig. 1).
Scheme 1. (a) NaCN, MnO2, MeOH, rt, 5–6 h (71–85%); (b)
.
NH2NH2 H2O 80%, EtOH, reflux, 5–6 h (68–84%); (c) ArCHO,
EtOH, HCl (cat.), reflux (32–98%).

A. C. Cunha et al. / Bioorg. Med. Chem. 11 (2003) 2051–2059
Table 1. N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone derivatives 2a–p
2053
Compd
R
R1
Yield (%)
Mp (ꢂC)
Molecular formulaa
Molecular weight
R (cmꢁ1
)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2l
2m
2n
2o
2p
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
F
H
N(CH3)2
42
95
93
78
58
88
60
84
76
76
98
56
71
32
92
>250
>250
>250
C16H13N5O
C18H18N6O
C16H12BrN5O
C24H29N5O2
C14H11N5O2
C16H12ClN5O
C18H17ClN6O
C16H11BrClN5O
C24H28ClN5O2
C14H10ClN5O2
C16H12FN5O
C18H17FN6O
C16H11BrFN5O
C24H28FN5O2
C14H10FN5O2
291.31
334.38
370.21
419.52
281.27
325.76
368.82
404.65
453.97
315.72
309.30
352.37
388.20
437.51
299.26
3400 (N–H), 1673(C¼O)
3428 (N–H), 1666 (C¼O)
3400(N–H), 1680 (C¼O)
3424 (N–H), 1671 (C¼O)
3400 (N–H), 1672 (C¼O)
3453 (N–H), 1682 (C¼O)
3446 (N–H), 1671 (C¼O)
3453 (N–H), 1682 (C¼O)
3445 (N–H), 1658 (C¼O)
3449 (N–H), 1667 (C¼O)
3457 (N–H), 1672 (C¼O)
3446 (N–H), 1671 (C¼O)
3448 (N–H), 1677 (C¼O)
3448 (N–H), 1650 (C¼O)
3445 (N–H), 1665 (C¼O)
Br
—
—
H
N(CH3)2
>250
211–212
>250
>250
>250
>250
>250
>250
250–251
>250
Br
—
—
H
F
F
F
F
N(CH3)2
Br
—
248–249
>250
—
aThe analytical results for C, H, N were within ꢀ0.4% of calculated values.
proton in the (E)-isomer occurs at 8.25–8.71 ppm and
the iminic carbon atom appears at 145.2–150.7 ppm in
the 13C NMR spectra suggesting the (E)-isomer.23,24
100 mM concentration revealed the compounds 2b
(R=H and R1=N(CH3)2), 2d (R=H) and 2h (R=Cl
and R1=Br) as those with the most important anti-
platelet profiles on the arachidonic acid induced assay
(89, 82 and 100%, respectively). It is interesting to
observe that the introduction of a para-halogen sub-
stituent in the N-phenyl ring of 2b (cf. 2g and 2m) and 2d
(cf. 2i and 2o) was deleterious to antiplatelet activity in
the AA-induced assay. In contrast, the presence of the
para-chloro instead of the para-fluoro substituent in the
N-phenyl ring of the para-bromophenyl NAH deriva-
tives [i.e., 2c (13%), 2h (100%) and 2n (13%)] furnished
Results and Discussion
These new derivatives 2a–p were next screened in order
to evaluate their effects on in vitro rabbit platelet
aggregation induced by arachidonic acid (AA)
(100 mM), collagen (5 mg/mL) or adenosine 5-diphos-
phate (ADP) (5 mM) (Table 2).17 The initial screening at
Table 2. Effect of N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone derivatives 2a–p (100 mM) and indomethacin (10 mM) on in vitro platelet
aggregation of rabbit citrated platelet-rich plasma induced by arachidonic acid, collagen and ADP
Compd
R
R1
Arachidonic acid (200 mM)
Collagen (5 mg/mL)
ADP (5 mM)
n
Aggregation (%) Inhibition (%)
n
Aggregation (slope) Inhibition (%)
n
Aggregation (%) Inhibition (%)
Control
DMSO
Indomethacin
2a
2b
2c
2d
2e
2f
2g
2h
2I
2j
—
—
—
H
H
H
H
H
Cl
—
—
—
5
5
4
3
3
3
4
3
4
4
4
3
3
3
3
4
3
4
100ꢀ3
94ꢀ3
0ꢀ0
—
6
100*
7
89*
13
82*
16
20
7
100*
8
11
17
9
13
6
2
5
5
4
3
4
3
3
3
3
3
3
6
3
3
3
3
3
5
7.5ꢀ0.8
6.9ꢀ1.0
0.3ꢀ0.1
7.3ꢀ1.0
4.9ꢀ0.8
7.4ꢀ1.1
2.5ꢀ0.5
6.0ꢀ1.1
7.5ꢀ0.6
7.4ꢀ0.7
1.6ꢀ0.2
6.2ꢀ1.2
7.2ꢀ0.5
7.3ꢀ0.4
7.5ꢀ0.5
7.4ꢀ0.7
5.8ꢀ1.5
7.5ꢀ0.4
—
8
96*
0
59*
7
69*
35*
0
5
5
4
3
6
3
3
3
3
3
6
3
3
3
3
4
3
3
100ꢀ2
97ꢀ2
96ꢀ2
76ꢀ7
91ꢀ5
100ꢀ3
86ꢀ5
72ꢀ8
100ꢀ2
50ꢀ5
96ꢀ8
88ꢀ9
55ꢀ7
85ꢀ3
65ꢀ3
92ꢀ5
100ꢀ1
98ꢀ3
—
3
4
24
9
0
14
28*
0
50*
2
12
45*
25
35*
8
H
N(CH3)2
Br
92ꢀ5
11ꢀ2
87ꢀ5
18ꢀ6
84ꢀ3
80ꢀ5
93ꢀ2
0ꢀ0
—
—
H
Cl N(CH3)2
2
Cl
Cl
Cl
F
F
F
Br
—
—
H
80*
40*
1
2
0
1
23
0
92ꢀ5
89ꢀ3
83ꢀ7
91ꢀ5
87ꢀ4
94ꢀ6
98ꢀ5
2l
2m
2n
2o
2p
N(CH3)2
Br
—
F
F
0
2
—
n, number of independent experiments in triplicate. Results are expressed as meanꢀSEM. *p <0.05 (Student’s t-test).

2054
A. C. Cunha et al. / Bioorg. Med. Chem. 11 (2003) 2051–2059
the most potent compound in this assay. The 2-furyl
NAH derivatives 2e, 2j and 2p presented a poor activity
in the AA-induced assay (16, 11 and 14%, respectively),
while 2j was active at the screening concentration in the
ADP-induced test (45%), contrasting with the original
series represented by compound 1 (Table 2).
(100 mmol/kg, po), using indomethacin as standard. In
addition, the acetic acid-induced mice abdominal con-
strictions test was used to investigate the analgesic pro-
file of these NAH derivatives. The results obtained
indicated that only compound 2h exhibited a discrete
antiinflammatory/analgesic activity, inhibiting 30.5% of
the edema formation and 23.1% of the induced
abdominal constrictions. Moreover, a weak analgesic
effect was also observed for compound 2b (18.9%).
Finally, the gastric mucosa was investigated, indicating
an absence of any ulcerative behavior for these three
NAH derivatives, based on the lack of any kind of
lesions on the animals stomachs (Table 4).18,19
Compounds 2b, 2d, and 2h also showed an important
antiplatelet profile in collagen-induced platelet aggrega-
tion (59, 69 and 80%, respectively) (Table 2). These
active compounds (2b, 2d, and 2h) were not able to
inhibit neither the ADP-induced platelet aggregation
(Table 2) nor the aggregatory activity of U-46619 (not
shown). This last evidence indicated that the antiplatelet
activity was not due to an effect at TXA2 receptor level.
The constitutive isoform of COX-1 present in the plate-
lets is blocked by acetylsalicylic acid (ASA) and indo-
methacin, preventing the formation of TXA2, thereby
inhibiting arachidonic acid and collagen but not ADP-
induced rabbit platelet aggregation.19ꢁ21 Compounds
2b, 2d, and 2h exhibited a similar ASA profile, suggest-
ing that they also modulate TXA2 formation.19 For this
reason, we decided to investigate the effect of the most
potent antiplatelet derivative 2h on the TXA2 produc-
tion. The observed results for thromboxane production
by platelets in response to collagen induction measuring
the bioformation of TXB2, a stable metabolite of TXA2,
in the presence or absence of compound 2h, confirmed
that the production of TXA2 is effectively suppressed by
this compound (Fig. 2).
The antiplatelet potency of compounds 2b, 2d, and 2h
was confirmed by determination of IC50 values obtained
from concentration–response curves (Table 3). The
N-para-chlorophenyl isoster 2h was the most potent
antiplatelet compound in the AA- and collagen-induced
tests (IC50=2.2ꢀ0.06 and 21.6ꢀ0.1 mM; respectively)
(Table 3). From data illustrated in Table 3, we can
conclude that compound 2h was much more potent (27-
fold) than the N-nor-para-chlorophenyl analogue 2b,
which presented an IC50=96ꢀ8 mM on the AA-induced
assay. Compound 2h was also more potent as an
antiplatelet agent than the lead-derivative
1
(IC50=24ꢀ0.5 mM), representing an optimization of
this initial series. Interestingly, the NAH derivative 2d
presented an IC50 value (21ꢀ2 mM) similar to com-
pound 1 IC50 value (Table 3).
The analysis of the structure–activity relationships sug-
gests that the triazole ring in the new series of N-sub-
stituted-phenyl-1,2,3-triazole-4-acylhydrazone
(NAH)
The pharmacological investigation of the anti-inflam-
matory properties of compounds 2b, 2d, and 2h were
next assayed in the carrageenan induced rat paw edema
class (2a–p) is not directly involved in the establishment of
the antiplatelet activity showed by compound 2h. This
hypothesis is brought about due to the results of com-
pound 2e on platelet aggregation assay. Derivative 2e,
which has only the isosteric replacement of the pyrazole
system from the lead-derivative 1 by the triazole ring, did
not present any significant activity on arachidonic induced
platelet aggregation at 100 mM, when compared to the
lead-derivative 1 (16 Â 100%, respectively). Therefore, it
seems reasonable to conclude that, within this series, the
nature of the substituents introduced in N-phenyl ring and
arylhydrazone moiety could be crucial to determine the
potential of the biological activity. Hence, the most sig-
nificant antiplatelet profile of the isoster compound 2h
(R=Cl and R1=Br) could be correlated with the lipo-
philic influence and inductive electron attracting proper-
ties of halogenated substituents on the biological activity.
Table 3. IC50 (mM) of N-substituted-phenyl-1,2,3-triazole-4-acyl-
hydrazone derivatives 2b, 2d, and 2h (0.1–100 mM) on arachidonic acid
(AA) and collagen-induced platelet aggregation
Compd
R
R1
IC50 (mM)
Collagen
AA
2b
2d
H
H
Cl
—
N(CH3)2
96ꢀ10
21ꢀ2
32ꢀ3
73ꢀ8
—
Br
—
2h
Indomethacin
2.2ꢀ0.06
0.6ꢀ0.02
21.6ꢀ0.1
3.1ꢀ0.9
IC50 values are determined by nonlinear regression (% of inhibition Â
log concentration) using the Microcal Origin program (Version 4.0).
Table 4. Effects of N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone derivatives 2b, 2d, and 2h (100 mmol/kg) and indomethacin (100 mmol/kg)
on carrageenan-induced rat paw edema assay and abdominal constrictions induced by acetic acid (0.6%, ip) in mice
Compd
R
R1
Carrageenan-induced rat paw edema
Abdominal constrictions induced by acetic acid Ulcerogenic effects
n
Volume variation (mL) Inhibition (%)
n
Constrictions count
Inhibition (%)
Vehicle control
2b
2d
2h
—
H
H
Cl
—
— 10
N(CH3)2 10
491.4ꢀ38.6
579.0ꢀ18.5
482.3ꢀ29.5
341.3ꢀ43.5
228.1ꢀ31.9
—
n.s.
n.s.
30.5*
53.5*
10
14
13
12
10
79.0ꢀ4.9
60.7ꢀ4.2
69.8ꢀ4.7
64.0ꢀ4.1
38.7ꢀ4.9
—
19.0
11.7
23.1
51.0*
—
—
—
—
Br
—
7
10
9
Indomethacin
+++++
Compounds are administered po. Results are expressed as meanꢀSEM. n, number of animals; *p<0.05 (Student’s t-test); n.s., non significant.
Percentage of inhibition obtained by comparison with vehicle control group.

A. C. Cunha et al. / Bioorg. Med. Chem. 11 (2003) 2051–2059
2055
General procedure for the preparation of 4-formyltriazoles
3b–c
A solution of diazomalonaldehyde (0.482 g) in methanol
(1.0 mL) and acetic acid (0.7 mL) was added dropwise to
a stirring solution of p-chloro or p-fluoro aniline
(368 mmol) in methanol (9 mL). The stirring was con-
tinued for 1 h at room temperature and a colored solid
was formed which was collected by filtration and
washed with cold water and dried under vacuum. The
triazole was suspended in acetyl acetate (20 mL), and
heated in a boiling water-bath and then an aqueous
solution of HCl (0.1 N, 10 mL) was added. After cooling
the solution, the organic phase was separated and washed
with an aqueous solution of sodium bicarbonate (15 mL).
After drying over anhydrous sodium sulfate, the solvent
was removed under reduced pressure to furnish crude
triazole, which were purified by short column flash chro-
matography using 80% n-hexane–ethyl acetate as eluent.
The following triazoles 3b–c were thus prepared.
Figure 2. Effects of NAH derivative 2h (100 mM) and indomethacin
(10 mM) on platelets TXB2 formation induced by collagen. Results are
expressed as meanꢀSEM for n=3 independent experiments. Basal=
unstimulated platelets. Control=platelets stimulated by collagen
5 mg/mL. Control was employed as 100% of TXB2 formation.
*p<0.05 (Student’s t-test).
(p-Chlorophenyl)-4-formyl-1,2,3-triazole (3b). Derivative
3b was obtained as a yellow solid in 76% yield mp
155–156 ꢂC. H NMR (200 MHz, CDCl3) d 10.2 (s, 1H,
1
CHO), 7.79 (d, 2H, H20 and H60, J=8.4 Hz), 7.64 (d,
2H, H30 and H50, J=8.4 Hz); 13C NMR (50 MHz,
CDCl3) d 186.0 (C¼O), 146.0 (C4), 137.6 (C40), 133.9
(C10), 130.7 (C30 and C50), 126.2 (C5), 122.5 (C20 and
C60). IR (KBr) cmꢁ1 1705 (n C¼O).
Conclusions
In summary, from the present study it is evident that
the most potent antiplatelet compound is 2h, which
presents a similar profile to ASA without the unde-
sired gastric ulceration. Therefore, this series of NAH
derivatives represents a novel family of antiplatelet
agents.
1-(p-Fluorophenyl)-4-formyl-1,2,3-triazole (3c). Deriva-
tive 3c was obtained as a yellow solid in 78% yield, mp
150–152 ꢂC. H NMR (200 MHz, CDCl3) d 10.2 (s, 1H,
1
CHO), 7.78 (dd, 2H, H20 and H60, J=4.5 and 9.0 Hz),
7.32 (dd, 2H, H30 and H50, J=7.8 and 9.0 Hz); 13C
NMR (50 MHz, CDCl3) d 185.2 (C¼O), 146.4 (C4),
165.3 (d, C40, J=255 Hz), 131.7 (C10), 125.7 (C5), 123.3
(d, C20 and C60, J=15 Hz), 117.3 (d, C30 and C50,
J=30 Hz). IR (KBr) cmꢁ1 1699 (n C¼O).
Experimental
Chemistry
Melting points were determined with a Quimis 340
apparatus and are uncorrected. 1H NMR spectra, unless
otherwise stated, were obtained in deuterated dimethyl-
sulfoxide containing ca. 1% tetramethylsilane as an
internal standard using a Bruker AC 200 spectrometer
at 200 MHz. Splitting patterns are as follows: s, singlet;
d, doublet; dd, double doublet; br, broad; m, multi-
plet. 13C NMR spectra were obtained using the same
spectrometer described above at 50 MHz, using deut-
erated dimethylsulfoxide as internal standard. IR
spectra were obtained using a Bruker IFS66 spectro-
photometer employing potassium bromide plates.
Microanalysis data were obtained using a Perkin–
Elmer 240 analyzer, using a Perkin–Elmer AD-4 bal-
ance. The progress of all reactions was monitored by
TLC performed on 2.0 Â 6.0 cm aluminum sheets
precoated with silica gel 60 (HF-254, E. Merck) to a
thickness of 0.25 mm. The developed chromatograms
were viewed under ultraviolet light at 254 nm. E.
Merck silica gel (60–200 mesh) was used for column
chromatography. The diazomalonaldehyde used in
synthesis was prepared by a known procedure descri-
bed in the literature.14
General procedure for the preparation of methyl
N-substituted-phenyl-4-carbethoxy-1,2,3-triazoles (4a–c)
To a mixture of 1.00 mmol of 4-formyltriazoles 3a–c in
methanol (30 mL) was added 0.08 g (1.67 mmol) of
sodium cyanide and 0.44 g of activated manganese
dioxide (7.91 mmol). The reaction was stirred at room
temperature for 6 h and the suspension was filtered
through Celite1 and treated with dichloromethane (3 Â
20 mL). The organic layers were joined, dried over
anhydrous sodium sulfate, filtered and evaporated
under reduced pressure to furnish crude methyl esters
4a–c, which were purified by column flash chromato-
graphy using 80% n-hexane–ethyl acetate as eluent.
1-Phenyl-4-carbethoxy-1,2,3-triazole (4a). Derivative 4a
was obtained as yellow solid in 71% yield, mp 115 ꢂC.
1H NMR (200 MHz, DMSO-d6) d 9.54 (s, 1H, H5),
8.03–7.97 (m, 2H, H20 and H60), 7.68–7.51 (m, 3H, H30-
H50), 3.92 (s, 3H, OCH3); 13C NMR (50 MHz, DMSO-
d6) d 160.5 (C¼O), 136.0 (C10), 139.5 (C4), 129.4 (C30
and C50), 129.3 (C40), 127.2 (C5), 120.5 (C20 and C60),
51.9 (OCH3). IR (KBr) cmꢁ1 1734 (n C¼O).

2056
A. C. Cunha et al. / Bioorg. Med. Chem. 11 (2003) 2051–2059
1 - (p - Chlorophenyl) - 4 - carbethoxy - 1,2,3 - triazole (4b).
Derivative 4b was obtained as yellow solid in 85%
(50 MHz, DMSO-d6) d 161.7 (d, C40, J=244.8 Hz),
158.7 (C¼O), 142.6 (C4), 132.7 (C10), 124.3 (C5), 122.7
(d, C20 and C60, J=8.7 Hz), 116.5 (d, C30 and C50,
J=23.3 Hz). IR (KBr) cmꢁ1 3327 and 3117 (n N–H),
1661 (n C¼O).
ꢂ
1
yield, mp 174–178 C. H NMR (200 MHz, DMSO-d6)
d 9.54 (s, 1H, H5), 8.0 (d, 2H, H20 and H60, J=8.8 Hz),
7.67 (d, 2H, H30 and H50, J=8.8 Hz), 3.88 (s, 3H,
OCH3); 13C NMR (50 MHz, DMSO-d6) d 160.7 (C¼O),
140.6 (C4), 139.6 (C4), 135.3 (C10), 134.6 (C40), 130.0
(C30 and C50), 125.3 (C5), 121.8 (C20 and C60), 52.3
(OCH3). IR (KBr) cmꢁ1 1707 (n C¼O).
General procedure for the preparation of the N-sub-
stituted-phenyl-1,2,3-triazole-4-acylhydrazone derivatives
2a–p
1 - (p - Fluorophenyl) - 4 - carbethoxy - 1,2,3 - triazole (4c).
Derivative 4c was obtained as yellow solid in 79%
yield, mp 186 ꢂC. 1H NMR (200 MHz, DMSO-d6) d
9.50 (s, 1H, H5), 8.02 (dd, 2H, H20 and H60, J=8.8 and
4.8 Hz), 7.46 (dd, 2H, H30 and H50, J=8.8 and 8.7 Hz),
3.90 (s, 3H, OCH3); 13C NMR (50 MHz, DMSO-d6) d
162.5 (d, C40, J=245.5 Hz), 160.9 (C¼O), 140.0 (C4),
133.1 (C10), 127.9 (C5), 123.4 (d, C20 and C60,
J=8.9 Hz), 117.2 (d, C30 and C50, J=23.2 Hz), 52.5
(OCH3). IR (KBr) cmꢁ1 1707 (n C¼O).
An equimolar amount of appropriates aromatic alde-
hydes was added to a solution of hydrazide derivatives
5a–c (0.50 mmol) in 15 mL of ethanol, in the presence of
a catalytic amount of hydrochloric acid. The reaction
was stirred for 4–6 h, at reflux and the solvent was
evaporated and the colored precipitate was collected by
filtration, washed with cold water, ethyl ether and dried
under vacuum to give the desired N-acylhydrazone
derivatives 2a–p.
Benzylidene-1H-1-(phenyl)-1,2,3-triazole-4-carbohydra-
zide (2a). Derivative 2a was obtained as a white solid
by condensation of 5a with benzaldehyde. 1H NMR
(200 MHz, DMSO-d6) d 12.22 (s, 1H, N–H), 9.47 (s, 1H,
H5), 8.59 (s, 1H, N¼CH), 8.0 (d, 2H, H20 and H60,
J=7.7 Hz), 7.45–7.74 (m, 6H, H30, H40, H50, H200, H400
and H600); 13C NMR (50 MHz, DMSO-d6) d 156.6
(C¼O), 149.3 (N¼CH), 143.4 (C4), 136.9 (C10), 130.9
(C300 and C500), 130.6 (C400), 129.9 (C30 and C50), 129.5
C40), 126.5 (C5), 127.9 (C200 and C600), 121.2 (C20 and
C60).
General procedure for preparation of the N-substituted-
phenyl-4-carbohydrazide-1,2,3-triazole 5a–c
A solution of the appropriate methyl ester derivatives
4a–c (3.70 mmol) and 3.7 mL of 80% hydrazine mono-
hydrate in 10 mL of ethanol, was stirred at reflux for
5–6 h. The reaction mixture was then concentrated
under reduced pressure and the colored solid which
formed was collected by filtration, washed with cold
water and dried under vacuum to give the desired
hydrazides 5a–c.
(40 - N,N - Dimethylaminobenzylidene) - 1H - 1 - (phenyl) -
1,2,3-triazole-4-carbohydrazide (2b). Derivative 2b was
obtained as a yellow solid by condensation of 5a with 4-
1-Phenyl-1H-1,2,3-triazole-4-carbohydrazide (5a). Deri-
vative 5a was obtained as a yellow solid in 77% yield by
reaction of methyl ester 4a with hydrazine mono-
1
N,N-dimethylaminobenzaldehyde. H NMR (200 MHz,
hydrate, mp 186 ꢂC. H NMR (200 MHz, DMSO-d6) d
DMSO-d6) d 11.89 (br, 1H, N–H), 9.43 (s, 1H, H5), 8.43
(s, 1H, N¼CH), 7.67–7.53 (m, 3H, H30, H40 and H50),
8.03–7.98 (m, 2H, H20 and H60), 7.54 (d, 2H, H200 and
H600, J=8.9 Hz), 6.77 (d, 2H, H300 and H500, J=8.9 Hz),
2.99 (s, 6H, N(CH3)2); 13C NMR (50 MHz, DMSO-d6)
d 155.3 (C¼O), 151.5 (C400), 149.3 (N¼CH), 142.9 (C4),
136.2 (C10), 129.7 (C30 and C50), 129.0 (C40), 128.4 (C200
and C600), 125.1 (C5), 121.4 (C100), 120.4 (C20 and C60),
111.6 (C300 and C500), 40.0 (N(CH3)2).
1
9.90 (br, 1H, N–H), 9.28 (s, 1H, H5), 7.99–7.95 (m, 2H,
H20 and H60), 7.67–7.49 (m, 2H, H30 and H50) 4.58 (br,
2H, NH2); 13C NMR (50 MHz, DMSO-d6) d 158.9
(C¼O), 142.7 (C4), 136.1 (C10), 129.9 (C30 and C50),
129.1 (C40), 124.2 (C5),120.4 (C20 and C60). IR (KBr)
cmꢁ1 3318 and 3151 (n N–H), 1671 (n C¼O).
1-(4-Chlorophenyl)-1H-1,2,3-triazole-4-carbohydrazide
(5b). Derivative 5b was obtained in 68% yield by reac-
tion of methyl ester 4b with hydrazine monohydrate, as
a yellow solid, mp 230–233 ꢂC. 1H NMR (200 MHz,
DMSO-d6) d 9.72 (1H, s, N–H), 9.22 (s, 1H, H5), 7.99
(d, 2H, H20 and H20, J=8.9 Hz), 7.64 (d, 2H, H30 and
H50, J=8.9 Hz) 4.52 (br, 2H, NH2); 13C NMR
(50 MHz, DMSO-d6) d 158.9 (C¼O), 142.9 (C4), 135.0
(C10), 133.3 (C40), 129.7 (C30 and C50), 124.3 (C5), 122.0
(C20 and C60). IR (KBr) cmꢁ1 3285 and 3087 (n N–H),
1661 (n C¼O).
(40 -Bromobenzylidene)-1H-1-(phenyl)-1,2,3-triazole-4-
carbohydrazide (2c). Derivative 2c was obtained as a
yellow solid by condensation of 5a with 4-bromo-
1
benzaldehyde. H NMR (200 MHz, DMSO-d6) d 12.3
(s, 1H, N–H), 9.49 (s, 1H, H5), 8.56 (s, 1H, N¼CH),
8.00 (d, 2H, H20 and H60, J=7.6 Hz), 7.69 (d, 2H, H200
and H600, J=7.9 Hz) 7.65 (d, 2H, H30 and H50,
J=7.9 Hz); 13C NMR (50 MHz, DMSO-d6) d 156.6
(C¼O), 147.9 (N¼CH), 143.3 (C4), 136.8 (C10), 134.1
(C100), 132.3 (C300 and C500), 129.9 (C30 and C50), 129.6
(C200 and C600), 129.0 (C40), 126.4 (C5), 124.0 (C400),
121.1 (C20 and C60).
1-(4-Fluorophenyl)-1H-1,2,3-triazole-4-carbohydrazide
(5c). Derivative 5c was obtained in 84% yield by reac-
tion of methyl ester 4c with hydrazine monohydrate, as
a yellow solid, mp 236–240 ꢂC. 1H NMR (200 MHz,
DMSO-d6) d 9.74 (br, 1H, N–H), 9.22 (s, 1H, H5), 8.20–
7.93 (m, 2H, H20 and H60), 7.44 (dd, 2H, H30 and H50,
J=8.9 and 8.9 Hz), 4.52 (br, 2H, NH2); 13C NMR
(30 - 50 - Di - tert - butyl - 40 - hydroxybenzylidene) - 1H - 1 -
(phenyl)-1,2,3-triazole-4-carbohydrazide (2d). Derivative
2d was obtained as a yellow solid by condensation of 5a
with 30-50-di-tert-butyl-40-hidroxybenzylideno. 1H NMR

A. C. Cunha et al. / Bioorg. Med. Chem. 11 (2003) 2051–2059
2057
(200 MHz, DMSO-d6) d 11.94 (s, 1H, N–H), 9.44 (s,
1H,H5), 8.49 (s, 1H, N¼CH), 8.00 (d, 2H, H20 and H60,
J=7.9 Hz), 7.66–7.44 (m, 2H, H30, H40 and H50), 7.47
(s, 2H, H200 and H600), 5.56 (s, 1H, OH), 1.41 (s,18H,
6CH3) ppm; 13C NMR (50 MHz, DMSO-d6) d 156.8
(C¼O), 156.1 (C400), 150.6 (N¼CH), 143.2 (C4), 139.7
(C300 and C500), 134.1 (C10), 133.3 (C100), 130.4 (C30 and
C50), 129.2 (C40), 126.2 (C5), 124.5 (C200 and C600), 121.0
(C20 and C60), 35.0 (C(CH3)3), 30.7 (6 CH3).
(30 -50 -Di-tert-butyl-40 -hydroxybenzylidene)-1H-1-(p-
chlorophenyl)-1,2,3-triazole-4-carbohydrazide (2i). Deri-
vative 2i was obtained as a white solid by condensation
of 5b with 30-50-di-tert-butyl-40-hydroxybenzaldehyde.
1H NMR (200 MHz, DMSO-d6) d 11.94 (s, 1H, N–H),
9.44 (s, 1H, H5), 8.48 (s, 1H, N¼CH), 8.04 (d, 2H, H20
and H60, J=8.8 Hz), 7.69 (d, 2H, H30 and H50,
J=8.8 Hz), 7.47 (s, 2H, H200 and H600), 5.56 (s, 1H, OH),
1.48 (s, 18H, 6CH3); 13C NMR (50 MHz, DMSO-d6)
156.8 (C¼O), 156.1 (C400), 150.7 (N¼CH), 143.5 (C4),
139.7 (C300 and C500), 134.1 (C10), 133.3 (C100), 130.4 (C30
and C50), 126.2 (C5), 124.5 (C200 and C600), 122.7 (C20
and C60), 35.0 (C(CH3)3), 30.7 (6 CH3).
(20 -Furylidene)-1H-1-(phenyl)-1,2,3-triazole-4-carbohy-
drazide (2e). Derivative 2e was obtained as a white
solid by condensation of 5a with furfural. 1H NMR
(200 MHz, DMSO-d6) d 12.26 (s,1H, N–H), 9.47 (s,1H
H5), 8.50 (s, 1H, N¼CH), 7.99 (d, 2H, H20 and H60,
J=7.6 Hz), 7.85 (d, 1H, H400, J=1.8 Hz), 7.66–7.49 (m,
3H, H30, H40 and H50), 6.90 (d, 1H, H200, J=3.4 Hz),
6.64 (dd, 1H, H300, J=3.4 and 1.8 Hz); 13C NMR
(50 MHz, DMSO-d6) d 155.8 (C¼O), 145.2 (N¼CH),
149.9 (C100), 145.2 (N¼CH), 142.6 (C4), 138.2 (C4¼),
136.2 (C10), 129.9 (C30 and C50), 129.2 (C40), 125.8 (C5),
120.5 (C20 and C60), 113.5 (C200), 112.2 (C300).
(20 -Furylidene)-1H-1-(p-chlorophenyl)-1,2,3-triazole-4-
carbohydrazide (2j). Derivative 2j was obtained as a
1
brown solid by condensation of 5b with furfural. H
NMR (200 MHz, DMSO-d6) d 12.25 (s, 1H, N–H), 9.51
(s, 1H, H5), 8.47 (s, 1H, N¼CH), 8.05 (d, 2H, H20 and
H60, J=8.9 Hz), 7.87 (d, 1H, H400, J=1.7 Hz), 7.71 (d,
2H, H30 and H50, J=8.9 Hz), 6.95 (d, 1H, H200,
J=3.3 Hz), 6.65 (1H, dd, H300, J=3.3 and 1.7 Hz); 13C
NMR (50 MHz, DMSO-d6) d 156.3 (C¼O), 149.9 (C100),
145.7 (N¼CH), 143.3 (C4), 138.8 (C400), 134.1 (C10),
130.4 (C30 and C50), 126.5 (C5), 122.7 (C20 and C60),
114.1 (C200), 112.7 (C300).
(Benzylidene)-1H-1-(p-chlorophenyl)-1,2,3-triazole-4-car-
bohydrazide (2f). Derivative 2f was obtained as a white
solid by condensation of 5b with benzaldehyde. 1H
NMR (200 MHz, DMSO-d6) d 12.24 (s, 1H, N–H), 9.50
(s, 1H,H5), 8.59 (s, 1H, N¼CH), 8.05 (d, 2H, H20 and
H60, J=8.8 Hz), 7.72 (d, 2H, H30 and H50, J=8.8 Hz),
7.73–7.69 (m, 2H, H200 and H600), 7.48–7.46 (m, 3H,
H300, H400 and H500); 13C NMR (50 MHz, DMSO-d6) d
156.3 (C¼O), 149.3 (N¼CH), 143.3 (C4), 135.5 (C40),
134.7 or 134.1 (C10 or C100), 130.7 (C300 and C500), 130.4
(C400), 129.3 (C30 and C50), 127.9 (C200 and C600), 126.5
(C5), 122.7 (C20 and C60).
(Benzylidene)-1H-1-(p-fluorophenyl)-1,2,3-triazole-4-car-
bohydrazide (2l). Derivative 2l was obtained as a white
solid by condensation of 5c with benzaldehyde. 1H
NMR (200 MHz, DMSO-d6) d 12.4 (s,1H, N–H), 9.6 (s,
1H, H5), 8.71 (s, 1H, N¼CH), 8.18 (dd, 2H, H20 and
H60, J=8.9 and 4.5 Hz), 7.87–7.84 (m, 2H, H30 and
H50), 7.65–7.58 (m, 5H, H200-H600); 13C NMR (50 MHz,
DMSO-d6) d 162.5 (d, J=246 Hz, C40), 156.5 (C¼O),
149.2 (N¼CH), 143.2 (C4), 134.6 (C100), 133.2 (C10),
130.7 (C300 and C500), 129.4 (C400), 127.7 (C200 and C600),
126.6 (C5), 123.5 (d, C20 and C60, J=8.9 Hz), 117.3 (d,
C30 and C50, J=23.2 Hz).
(40-N,N-Dimethylaminobenzylidene)-1H-1-(p-chlorophe-
nyl)-1,2,3-triazole-4-carbohydrazide (2g). Derivative 2g
was obtained as a white solid by condensation of 5b
with 4-N,N-dimethylaminobenzaldehyde. 1H NMR
(200 MHz, DMSO-d6) d 12.06 (s, 1H, N–H), 9.60 (s, 1H,
H5), 8.54 (s, 1H, N¼CH), 8.17 (d, 2H, H20 and H60,
J=5.8 Hz), 7.83 (d, 2H, H30 and H50, J=8.7 Hz, m),
7.66 (d, 2H, H200 and H600, J=8.7 Hz), 6.89 (d, 2H, H300
and H500, J=8.7 Hz), 3.10 (s, 6H, N(CH3)2); 13C NMR
(50 MHz, DMSO-d6) d 155.9 (C¼O), 152.2 (C400), 150.0
(N¼CH), 143.6 (C4), 135.4 (C40), 134.2 (C10), 130.5 (C30
and C50), 129.2 (C200 and C600), 126.2 (C5), 121.9 (C100),
122.8 (C20 and C60), 112.3 (C300 and C500), 40.4
(N(CH3)2).
(40 -N,N-Dimethylaminebenzylidene)-1H-1-(p-fluorophe-
nyl)-1,2,3-triazole-4-carbohydrazide (2m). Derivative 2m
was obtained as a white solid by condensation of 5c
with 4-N,N-dimethylaminobenzaldehyde. 1H NMR
(200 MHz, DMSO-d6) d 11.89 (s, 1H, N–H), 9.40 (s, 1H,
H5), 8.43 (s, 1H, N¼CH), 8.06 (d, 2H, H20 and H60,
J=8.4 and 4.5 Hz), 7.51 (d, 2H, H30 and H50, J=8.7 and
8.1 Hz), 7.47 (d,002H, H200 and H600, J=8.5 Hz), 6.77 (d,
2H, H300 and H5 , J=8.5 Hz), 2.98 (s, 6H, N(CH3)2); 13
C
NMR (50 MHz, DMSO-d6) d 162,6 (d, C40, J=245 Hz),
155,4 (C¼O), 151,5 (C400), 149,3 (N¼CH), 142,9 (C4),
132.8 (C10), 128.5 (C200 and C600), 125.6 (C5), 122.9 (d,
C20 and C60, J=8.9 Hz), 121.4 (C100), 116.7 (d, C30 and
C50, J=23.3 Hz), 111.7 (C300 and C500), 40.4 (N(CH3)2).
(4-Bromobenzylidene)-1H-1-(p-chlorophenyl)-1,2,3-tria-
zole-4-carbohydrazide (2h). Derivative 2h was obtained
as a white solid by condensation of 5b with 4-bromo-
1
benzaldehyde. H NMR (200 MHz, DMSO-d6) d 12.34
(s, 1H, N–H), 9.52 (s, 1H, H5), 8.56 (s, 1H, N¼CH),
8.05 (d, 2H, H20 and H60, J=8.8 Hz), 7.71 (d, 2H, H30
and H50, J=8.8 Hz), 7.68 (d, H200 and H600, J=7.9 Hz),
7.65 (d, H300 and H500, J=7.9 Hz); 13C NMR (50 MHz,
DMSO-d6) d 156.5 (C¼O), 147.9 (N¼CH), 143.0 (C4),
135.6 (C40), 134.2 or 134.1 (C10 or C100), 132.5 (C300 and
C500), 130.5 (C30 and C50), 126.1 (C5), 124.0 (C400), 122.9
(C20 and C60).
(4-Bromobenzylidene)-1H-1-(p-fluorophenyl)-1,2,3-tri-
azole-4-carbohydrazide (2n). Derivative 2n was obtained
as a white solid by condensation of 5c with 4-bromo-
1
benzaldehyde. H NMR (200 MHz, DMSO-d6) d 12.01
(s, 1H, N–H), 9.32 (s, 1H, H5), 8.55 (s, 1H, N¼CH),
8.06–7.99 (m, 2H, H20 and H60), 7.46 (dd, 2H, H30 and
H50, J=8.8 and 8.8 Hz), 7.69 (d, 2H, H200 and H600,
J=7.9 Hz), 7.69 (d, 2H, H300 and H500, J=7.9 Hz); 13C

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A. C. Cunha et al. / Bioorg. Med. Chem. 11 (2003) 2051–2059
NMR (50 MHz, DMSO-d6) d 156.8 (C¼O), 143.1 (C4),
162.6 (d, C40, J=245 Hz), 133.4 or 133.3 (C10 or C100),
132.3 (C300 and C500), 129.5 (C200 and C600), 126.6 (C5),
123.9 (C400), 123.7 (d, C20 and C60, J=8.9 Hz), 117.2 (d,
C30 and C50, J=23.2 Hz).
(2 mM) and indomethacin (50 mM) were added to halt
TXB2 formation. Finally, platelets were centrifuged at
13,000g  5 min and the supernatant was assayed for
TXB2 using EIA kits (Biotrak, Amershan).
Anti-inflammatory activity. The anti-inflammatory
activity was determined in vivo using the carrageenan
induced rat paw edema test according to Ferreira.18
Albino rats of both sexes (150–200 g) were used. Com-
pounds were orally administered (100 mmol/kg, 0.1 mL/
20 g animal weight) as a suspension in 5% arabic gum
(Sigma) in saline (vehicle). Control animals received
equal volume of the vehicle. One hour after, the animals
were then injected with either 0.1 mL of 1% carrageenan
solution in saline (0.1 mg/paw) or sterile saline (NaCl
0.9%), into the subplantar surface of one of the hind
paws, respectively. The paw volumes were measured using
a glass plethysmometer coupled to a peristaltic pump,
3 h after the subplantar injection. The edema was calcu-
lated as the volume difference between the carrageenan
and saline-treated paw. Indomethacin (100 mmol/kg)
was used as standard in the same conditions. Antiin-
flammatory activity was expressed as percentage of
inhibition of the edema when compared with vehicle
control group. The stomachs of the animals were also
verified to estimate ulcerogenic effects. After the assays,
the stomachs are opened across the great curvature,
rinsed with saline and observed in a stereomicroscope
for ulcers. A score of 2 and 5 were attributed for punc-
tual and haemorragic lesions, respectively.
(30-50-Di-tert-butyl-40-hidroxybenzylidene)-1H-1-(p-fluoro-
phenyl)-1,2,3-triazole-4-carbohydrazide (2o). Derivative
2o was obtained as a brown solid by condensation of 5c
with 30-50-di-tert-butyl-40-hidroxybenzaldehyde. 1H
NMR (200 MHz, DMSO-d6) d 11.2 (s, 1H, N–H),
9.42 (1H, s, H5), 8,49 (s, 1H, N¼CH), 8.03 (dd, 2H,
H20 and H60, J=6.9 and 4.6 Hz), 7.53–7.36 (m, 2H,
H30 and H50), 7.47 (s, 2H, H200 and H600), 1.48 (s,
18H, 6CH3), 5.60 (s, 1H, s, OH); 13C NMR
(50 MHz, DMSO-d6) 156.8 (C¼O), 156.1 (C400), 150.6
(N¼CH), 143.4 (C4), 139.7 (C300 and C500), 133.3 or
133.2 (C10), 133.3 or 133.2 (C100), 124.5 (C200 and
C600), 126.3 (C5), 123.5 (d, C20 and C60, J=8.9 Hz),
117.3 (d, C30 and C50, J=23.1 Hz), 35.0 (C(CH3)3),
30.7 (6 CH3).
(20 -Furylidene)-1H-1-(p-fluorophenyl)-1,2,3-triazole-4-
carbohydrazide (2p). Derivative 2p was obtained as a
brown solid by condensation of 5c with furfural. 1H
NMR (200 MHz, DMSO-d6) d 12,4 (s, 1H, N–H), 9.60
(s, 1H, H5), 8.60 (s, 1H, N¼CH), 8.17 (d, 2H, H20 and
H60, J=9.0 and 4.5 Hz), 7.99 (d, 1H, H400, J=1.8 Hz),
7.61 (d, 2H, H30 and H50, J=9.9 and 9.0 Hz), 7.06 (d,
1H, H200, J=3.3 Hz), 6.77 (dd, 1H, H300, J=3.3 and
1.8 Hz); 13C NMR (50 MHz, DMSO-d6) d 162.4 (d, C40,
J=246 Hz), 156.3 (C¼O), 149.9 (C100), 145.8 (N¼CH),
143.2 (C4), 138.8 (C400), 133.2 (C10), 126.5 (C5), 123.5
(d, C20 and C60, J=8.9 Hz), 117.3 (d, C30 and C50,
J=23.2 Hz), 114.1 (C200), 112.8 (C300).
Analgesic activity. The analgesic activity was deter-
mined in vivo by using the abdominal constriction test
induced by acetic acid 0.6% (0.1 mL/10 g) in mice.19
Albino swiss mice of both sexes (18–23 g) were used.
Compounds were administered orally (100 mmol/kg;
0.1 mL/20 g) as a suspension in 5% arabic gum in saline
(vehicle). Indomethacin (100 mmol/kg) was used as the
standard drug under the same conditions. Acetic acid
solution was administered ip 1 h after administration of
the NAH compounds 2a–p. Ten minutes after ip injection
of the acetic acid solution, the number of constrictions
per animal was recorded for 20 min. Control animals
received an equal volume of vehicle. Analgesic activity
was expressed as percentage of inhibition of constric-
tions when compared with the vehicle control group.
Pharmacology
Antiplatelet activity. Blood was withdrawn from rabbit
ear central artery and mixed with 3.8% trisodium citrate
(9:1 v/v). Platelet-rich plasma (PRP) was prepared by
centrifugation at 500g  10 min at room temperature.
The platelet-poor plasma (PPP) was prepared by cen-
trifugation of the pellet at 2000g  10 min at room
temperature. Platelet aggregation was monitored by the
turbidimetric method of Born and Cross17 in a Chrono-
Log aggregometer. PRP (300 mL) was incubated at
37 ꢂC for 1 min with continuous stirring at 900 rpm.
Platelet aggregation was induced by ADP (5 mM), col-
lagen suspension (5 mg/mL) or AA (100 mM). Com-
pounds or vehicle DMSO (0.5% v/v) were incubated
with the PRP samples 5 min before addition of the
aggregating agent. DMSO presented neither pro- nor
antiplatelet aggregation activity. Indomethacin (10 mM)
was used as standard. The platelet aggregation was
expressed as percentage of aggregation for ADP and
AA and as the maximum rate of aggregation (slope) for
collagen. All concentrations values were expressed as
final concentration in PRP.
Statistical analysis. All results are expressed as mean-
ꢀSEM for n animals or experiments per group. Data
were statistically analyzed by Student’s t-test for a sig-
nificance level of *p<0.05.
Acknowledgements
Thanks are due to CNPq (BR.), FAPERJ (BR.), Pronex
(BR.) and FUJB (BR.), CAPES (BR.) for financial
support and fellowships. We also thank the analytical
center of NPPN-UFRJ (BR.) for the spectroscopic
data.
Platelet TXB2 formation. Washed platelets were incu-
bated with test compounds (100 mM) for 5 min and
treated with collagen (5 mg/mL) for 5 min. EDTA

A. C. Cunha et al. / Bioorg. Med. Chem. 11 (2003) 2051–2059
2059
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