Synthesis, antiplatelet and in silico evaluations of novel N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-carbohydrazides

Article abstract of DOI:10.1016/j.bmc.2009.03.053

This paper describes the synthesis, antiplatelet and theoretical evaluations of 10 N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-carbohydrazides (2a-j). These compounds were synthesized, characterized and screened for their in vitro antiplatelet

Full text of DOI:10.1016/j.bmc.2009.03.053

Bioorganic & Medicinal Chemistry 17 (2009) 3713–3719
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, antiplatelet and in silico evaluations of novel
N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-carbohydrazides
b
a
b
Alessandro K. Jordão a, Vitor F. Ferreira a, , Emerson S. Lima , Maria C. B. V. de Souza , Eduardo C. L. Carlos ,
*
Helena C. Castro c, Reinaldo B. Geraldo c, Carlos R. Rodrigues c, Maria C. B. Almeida a, Anna C. Cunha a,
*
a Universidade Federal Fluminense, Departamento de Química Orgânica, Outeiro de São João Batista, s/n°, Niterói, 24020-141 Rio de Janeiro, Brazil
b Universidade Federal do Amazonas, Faculdade de Ciências Farmacêuticas, Rua Alexandre Amorin, 330, Manaus, 69010-300 Amazonas, Brazil
c Universidade Federal Fluminense, Departamento de Biologia Celular e Molecular, LABioMol, Niterói, 24020-141 Rio de Janeiro, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper describes the synthesis, antiplatelet and theoretical evaluations of 10 N-substituted-phenyla-
mino-5-methyl-1H-1,2,3-triazole-4-carbohydrazides (2a–j). These compounds were synthesized, charac-
terized and screened for their in vitro antiplatelet profile against human platelet aggregation using
arachidonic acid, adrenaline and ADP as agonists. Among NAH derivatives 2a–j, the compounds 2a, 2c,
2e, 2g and 2h were the most promising molecules with significant antiplatelet activity.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 12 January 2009
Revised 23 March 2009
Accepted 25 March 2009
Available online 1 April 2009
Keywords:
Triazoles
Diazo compounds
Antiplatelet properties
In silico evaluation
1. Introduction
disease. The mechanism of action of ASA is related to its capacity to
permanently inactivate the platelet PGHS by acetylation of the
The platelets play a major role in the pathogenesis of thrombotic
disorders that are activated by a number of metabolic pathways.1–3
These pathways lead to the production of pro-aggregatory mole-
cules such as thromboxane A2 (TXA2) and adenosine diphosphate
(ADP) that are released from platelet granules. These molecules am-
plify platelet responses to collagen and recruitadditional platelets to
the site of injury.2–5 In addition to these agonists, there are adrena-
line and noradrenaline that act on the alpha-adrenergic receptor in
human platelets with a weaker pro-aggregatory profile but increas-
ing the platelet sensitivity to other agonists such as thrombin.
Adrenaline and noradrenaline are involved in stress situations that
in this new millennium might contribute on causing heart attacks,
stroke and vascular incidents.6
Among the physiological agonists that activate platelets, arachi-
donic acid metabolites are of particular relevance (Fig. 1).7,8 Arachi-
donic acid is metabolized to TXA2 and 12-hydroxyeicosatetraenoic
(12-HETE) through two catalytic pathways mediated by cyclooxy-
genase (a prostaglandin endoperoxide H synthase—PGHS), and
lipoxygenase, respectively.7,8 The increasing of TXA2 concentration
has been linked to cardiovascular diseases including acute myocar-
dial ischemia and heart failure. Currently, the inhibition of TXA2 pro-
duction by acetylsalicylic acid (ASA), also known as aspirin, is widely
usedto reducetheriskof myocardialinfarctionandthromboembolic
serine aminoacid residue.9,10 However, collateral effects and resis-
tance to aspirin are fully described in literature.7,8,11
The antiplatelet therapy has been widely recognized as of
potential medical application to reduce the incidence of stroke,
heart attacks and death from vascular causes in individuals with
symptomatic atherothrombotic diseases.12 Stroke is responsible
for at least 5–7 million deaths and 50–58 millions stroke related-
disability. According to World Health Organization, 87% of these
deaths were in low-income and middle-income countries and
without intervention, the number of global deaths should rise to
6.5 million in 2015 and to 7.8 million in 2030.13
Recently, we described the synthesis of N-substituted-phenyl-
1,2,3-triazole-4-acylhydrazones with antiplatelet activity.14 These
compounds inhibited the arachidonic acid-induced platelet aggre-
gation in rabbit and human citrated platelet rich plasma.14 From
this study we identified the antiplatelet compound N0-[(40-bro-
mo-phenyl)methylene)]-1-(p-chlorophenyl)-1H-1,2,3-triazole-4-
carbohydrazide (1) (Fig. 1), which presented a similar profile to
aspirin without the undesired gastric ulceration symptoms. Studies
also showed that hydrazone14,16–19 group may play an important
role for the antithrombotic activity.
In the search for new triazole derivatives with antiplatelet
activity and to perform a structure–activity relationship study,
we describe herein the synthesis and antiplatelet evaluation of
new substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-car-
bohydrazides (2), in which the 4-bromophenyl moiety (subunit
* Corresponding authors. Tel.: +55 21 26292364; fax: +55 21 26292135 (A.C.C.).
E-mail address: annac@vm.uff.br (A.C. Cunha).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.053

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A. K. Jordão et al. / Bioorg. Med. Chem. 17 (2009) 3713–3719
COOH
Arachidonic Acid
COOH
OCOMe
Br
COX-1
O
N
AAS
COOH
O
O
N
H
N
H
?
N
1
Me
N
OH
TxA Synthase
COOH
PGH2
Cl
O
HO
OH
Tromboxane A 2 (TXA2)
TXA2 released by platelets binds to platelet
recept or stostimulate platelet activation
GPIIb/IIIa
Exposition
PLATELET AGGREGATION
Figure 1. Arachidonic acid (AA) metabolism involved in the platelets aggregation process. Arachidonic acid is metabolized through cyclooxygenase (COX) and thromboxane
A2 (TXA2) synthase generating PGH2 and thromboxane A2 (TXA2), respectively. Aspirin (AAS) is able to inhibit COX. The feasible targets in the AA-pathway (COX and TXA2-
synthase) of N0-[(40-bromo-phenyl)methylene)]-1-(p-chlorophenyl)-1H-1,2,3-triazole-4-carbohydrazide (1) are boxed.
Differents aromatic rings
A
Br
Ar
O
O
N
N
N
H
N
H
H
N
N
H
N
N
1
Me
Me
N
N
N
Structural Modifications
Br
2
,
,
Ar
=
S
B
H
N
R
NO2
Cl
,
O
O
R = H and Cl
Presence of anitrogen bridge between
the azaheterocyclic and the phenyl ring
Figure 2. Design of N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-carbohydrazides (2a–j).
A) of 1 was replaced by a different aromatic ring (e.g., pyridyl, bro-
mothienyl, furyl and nitrofuryl) moiety (Fig. 2). We also modified
the nature of the subunit B to investigate among other features
the contribution of the spacer N–H between the azaheterocyclic
for the biological activity of this new series of compounds. Finally,
these derivatives were submitted to an in silico oral biodisponibil-
ity screening, and drugscore and druglikeness evaluation to ana-
lyze their overall potential to qualify for a drug, also comparing
them to some current antiplatelets drugs (i.e., acetylsalicylic acid,
clopidogrel, cilostazol and tirofiban).
bethoxy-1H-1,2,3-triazoles 3a–b were prepared in moderated
yields by the condensation of ethyl 2-diazoacetoacetate with
substituted phenylhydrazine hydrochlorides, according to our pre-
vious report.15 These compounds were easily converted into the
corresponding carbohydrazides 4a–b, on treatment with hydrazine
hydrate in refluxing ethanol15 (Scheme 1 and Table 1).
The N-acylheteroarylhydrazones (NAH) 2a–h were prepared by
the condensation of the compounds 4a–b with the suitable aromatic
aldehydes (Route I) in ethanol using hydrochloric as catalyst (Route
I), while the 5-nitrofuran derivatives 2i–j were obtained by the con-
densationof thecorresponding compounds 4a–bwithcommercially
available 5-nitro-2-furfurylidene diacetate in a mixture of ethanol/
sulfuric acid 50% (Route II). The yields of 2a–j are listed in Table 1.
The E and Z adducts of the reaction of hydrazine derivatives 4a–b
with aldehydes were in equilibrium with the corresponding hydra-
zones 2a–j, through the intermediate hemiaminals 5a–j (Scheme
2). To assure the diastereomeric ratio of the NAH derivatives 2, which
2. Results and discussion
2.1. Chemistry
The synthesis of new 4-acylhydrazone derivatives 2a–j is
shown in Scheme 1. The ethyl N-substituted-phenylamino-4-car-

A. K. Jordão et al. / Bioorg. Med. Chem. 17 (2009) 3713–3719
3715
O
Ar
H
O
O
ROUTE I
ArCHO/ HCl cat.
EtOH
N C
N
N
H
N
O
Me
N
N
N NH2
H
N
NH2NH2 80%
EtOH, reflux
N
Me
N
Me
Me
N
N
N
H
N
N
H
4a, R1 = H
, R1 =Cl
H
4b
R
R
R
2a, R = H and A r = Ph
2b, R = Cl and A r = Ph
, R = H and A r = pyrid-4"-yl
2d, R = Cl and A r = pyrid-4"-yl
2e, R = H and Ar = 5" -bromo-thien-2"-yl
2f, R = Cl and Ar = 5" -bromo-thien-2"-yl
3a
3b
, R1 = H
, R1 = Cl
ROUTE II
2c
OAc
OAc
H
EtOH/ H2SO4 50%
O
O2N
2g, R = H and Ar = fur-2"-yl
2h, R = Cl and Ar = fur-2"-yl
NO2
O
O
N C
N
N
H
H
N
Me
N
2i, R = H
2j, R = Cl
N
H
R
Scheme 1. Synthetic pathways used for 2a–j.
initially screened at 100
l
g mlꢀ1 for inhibitory effects on human
Table 1
Yields and mps for NAH derivatives 2a–j
platelets aggregation using arachidonic acid as agonist (Fig. 3).
Interestingly, almost all compounds presented a significant inhib-
itory profile (P50%) at this concentration, except for 2a, 2g and
2i. Thus, these data suggest the maintenance of an antiplatelet pro-
file in these derivatives molecular structure (Fig. 3).
Then we determined the experimental half-maximal inhibi-
tory concentration (IC50) of all derivatives to compare with aspi-
rin, the most used antiplatelet drug current on the market.
Interestingly the determination of IC50 on arachidonic acid-in-
duced platelet aggregation assays showed three different levels
Compound
R
Ar
Mp (°C)
Yield (%)
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
Cl
H
Cl
H
Cl
H
Cl
H
Cl
Phenyl
200–202
250–252
115–125
140–145
100–103
180–182
212–214
165–166
175–176
235–237
50
67
94
70
77
82
60
85
89
87
Phenyl
Pyrid-400-yl
Pyrid-400-yl
500-Bromo-thien-200-yl
500-Bromo-thien-200-yl
Fur-200-yl
Fur-200-yl
500-Nitrofur- 200-yl
500-Nitrofur- 200-yl
of antiplatelet activity that included lower (8.8–9.1
similar (32.2–35.5
g mlꢀ1), or higher (111.5–229.9 g mlꢀ1) val-
ues than that of aspirin (32.5 0.2
g mlꢀ1) (Table 2). The best
profile (8.8–9.1
g mlꢀ1) was observed for compounds 2c, 2e,
l
g mlꢀ1),
2j
l
l
l
l
was essential for the analysis of the biological results, we deter-
mined the stereochemistry of the imine double bond in this series.
A detailed analysis of the 1H NMR spectra of these derivatives 2a–j
allowed us to detect only the presence of one N–H signal, which
was attributed to the (E)-diastereomer, on the basis of nuclear Over-
hauser (NOE) experiment. For compound 2i, the irradiation of N–H
hydrogen signal at 10.22 ppm resulted in a NOE-enhancement of
N@CH hydrogen signal at 8.54 ppm (14.45%). Despite the possibility
of formation of two diastereoisomers we only noticed the (E)-isomer
that was identified and isolated from the reaction.
2f and 2h, which infer that the ring moiety substitution seems
to be more important than R position to this inhibitory activity
since both 2e and 2f present different R substituents and same
ring moiety (bromothienyl) (Table 2). However R position is also
significant to this acid arachidonic-induced platelet aggregation
inhibitory activity as this profile may be negatively (i.e., 2d) or
positively affected (i.e., 2b, 2h and 2j) when the ring is replaced
with the addition of chlorine at R position compared to 2a (Table
2). The maintenance of hydrogen at R position and the presence
of furyl, phenyl or nitrofuryl moiety generated the less active
compounds (2a, 2g and 2i) against arachidonic acid-induced
platelet aggregation (Table 2). However the heterogeneous elec-
tronic distribution in the ring moiety caused by the heteroatom
presence (i.e., oxygen, sulfur and nitrogen) apparently still fa-
vours the interaction of the derivative with the platelet target
when compared to a phenyl ring (i.e., 2a).
2.2. Biological assays and Structure–Activity Relationship (SAR)
evaluations
To confirm the potentiality of the 4-acylhydrazone moiety for
designing new antiplatelet agents, the NAH derivatives (2a–j) were
Ar
H
H
O
Ar
N
O
N
O
O
N
N
N
H
H
N
H
NHNH2
N
N
Ar
OH
H
N
N
H
Me
N
N
N
N
Me
N
Me
+
Me
ArCHO
and
N
N
N
N
N
5a-j
H
N
H
N
H
H
2a-j
E-diastereomer
R
R
R
R
Z-diastereomer
4a-b
Scheme 2. Equilibrium of E- and Z-hydrazones through intermediate hemiaminals.

3716
A. K. Jordão et al. / Bioorg. Med. Chem. 17 (2009) 3713–3719
inhibition of common steps such as the integrin aIIbb3 (GPIIb/IIIa),
which is expressed on the platelet surface and a final common
pathway of platelet aggregation caused by any agonist6 is initially
discarded since these derivatives present significantly different
degree of activity against arachidonic acid and ADP-induced plate-
let aggregation assays.
The experimental results of adrenaline-induced platelet aggre-
gation assays showed that most of the derivatives were able to sig-
nificantly inhibit this process at the screening concentration
(100 l
g mlꢀ1) except for 2c, 2d, 2f and 2j. However these less
active compounds were the most active against the human platelet
aggregation induced by arachidonic acid. Once more these data
reinforce the hypothesis of a different target, other than that of
aspirin. The IC50 determination revealed 2a and 2i as mixed ADP/
adrenaline antagonists whereas 2g acted as a more specific adren-
aline antagonist (Table 2). The structure–activity analysis of the
derivatives structure suggested that the chemical requirements
for establishing a significant inhibitory activity against adrena-
line-induced platelet aggregation are different than those appar-
ently required for other agonists (i.e., arachidonic acid).
Apparently a molecule with a smaller or less polar ring with no
substitution at R position is preferred for displaying a higher inhib-
itory activity (IC50 < 10 l
g mlꢀ1), except for 2h.
2.3. In silico oral biodisponibility—molecular modeling
approach
The N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-
carbohydrazides (2a–j) were submitted to an in silico evaluation
using a molecular modeling approach. Since a good absorption
after oral administration is obligatory for antiplatelet medical use
purpose, we analyzed these derivatives according to the rule-of-
five developed by Lipinski et al. (Table 2).20 The rule-of-five
theoretically indicates if a chemical compound could be an orally
active drug in humans. The rule states that the most ‘druglike’ mol-
ecules present clogP 6 5, molecular weight (MW) 6 500, and num-
ber of hydrogen bond acceptors 6 10 and donors 6 5. Molecules
violating more than one of these rules may have problems with
bioavailability. The results showed that all compounds of the
N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-carbo-
hydrazides (2a–j) fulfilled the Lipinski ‘rule-of-five’ (Table 2).
Importantly, according to the theoretical analysis of the lipophilic-
ity (clogP), the most active inhibitors (IC50 < 10 l
g mlꢀ1) of arachi-
donic acid-pathway (2c, 2e, 2f and 2h), ADP-pathway (2a and 2i)
or adrenaline-pathway (2a, 2e, 2h and 2i) were sufficiently hydro-
phobic for penetrating the biological membranes in Lipinski rules,
except for 2f and 2i.
Figure 3. Inhibitory effect (%) of the N-substituted-phenylamino-5-methyl-1H-
1,2,3-triazole-4-carbohydrazides (2a–j) (100 l
g mlꢀ1) on in vitro platelet aggrega-
Herein, we also calculated the druglikeness and drugscore val-
ues for these derivatives to analyze their overall potential to qual-
ify for a drug including the comparison with some drugs currently
in use in therapy against atherothrombotic disease (i.e., ASA, clop-
idogrel, cilostazol and tirofiban) (Fig. 4). The druglikeness value is
calculated based on the occurrence frequency of every one of the
fragments of the analyzed molecules and compared to commercial
drugs and non-druglike compounds. In the Osiris program, the
occurrence frequency of each fragment is determined within the
collection created by shredering 3300 traded drugs as well as
15,000 commercially available chemicals (Fluka) yielding a com-
plete list of all available fragments. In this case, positive values
point out that the molecule contains predominantly the better
fragments, which are frequently present in commercial drugs but
not in the non-druglike collection of Fluka compounds. The drug-
score combines druglikeness, clogP, logS, molecular weight and
toxicity risks in one handy value that may be used to judge the
drug potential of a compound. Interestingly we noticed the com-
pounds with a positive druglikeness (1.27–5.01) and drugscore
tion of human citrated platelet-rich plasma induced by adrenaline (ADR), arachi-
donic acid (ARC), or adenosine diphosphate (ADP). ADR, ARC and ADP are the
positive aggregatory controls without the derivatives.
Given the redundance of discrete pathways leading to platelet
aggregation, some known antiplatelet drugs such as aspirin inhibit
TXA2-mediated, adrenaline-mediated and ADP-mediated platelet
aggregation, whereas leave the activity of other platelet agonists
such as thrombin largely unaffected.6 Interestingly our evaluation
of these derivatives using ADP or adrenaline as agonists showed
different results from that observed for aspirin (Table 2).
Surprisingly only 2a and 2i showed a significant antiplatelet
profile (>50%) against ADP-induced platelet aggregation at the
screening concentration (100
NAH derivatives presented an IC50 significantly higher (136.4–
337.5
g mlꢀ1) than that of aspirin (21.5 2.4 g mlꢀ1) (Table 2).
l
g mlꢀ1) (Fig. 3). In fact most of the
l
l
These results may possibly suggest a different target for these
derivatives than that of aspirin (COX1). Despite of that, the

A. K. Jordão et al. / Bioorg. Med. Chem. 17 (2009) 3713–3719
3717
Table 2
Comparison of biological and theoretical features of NAH derivatives 2a–j including experimental half-maximal inhibitory concentration (IC50 in
l
g mlꢀ1) on in vitro human
platelet aggregation assays using arachidonic acid, adenosine diphosphate (ADP) and adrenaline as agonist and theoretical oral biodisponibility (Lipinski rule-of-five) predicted by
using a molecular modeling approach
Compound
R
Ar
Experimental IC50
Arachidonic acid ADP
(l
g mlꢀ1
)
Theoretical oral biodisponibility (Lipinski rule-of-five)a
Adrenaline
HBA
HBD
Molecular weight
cLogP
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
Cl
H
Cl
H
Cl
H
Cl
H
Cl
—
Phenyl
229.9 19.8
32.2 2.1
8.8 0.1
33.1 0.8
9.1 0.2
9.1 0.1
111.5 1.7
8.9 0.1
19.04 3.3
180.4 4.3
277.8 54.5
168.6 5.4
316.9 59.9
332.5 6.6
337.5 35.7
261.0 22.3
11.59 5.9
136.4 4.3
21.5 2.4
9.6 0.25
28.9 4.2
161.1 5.5
144.6 1.9
13.4 0.5
144.2 8.8
20.1 4.1
7.8 0.7
7
7
8
8
7
7
8
8
2
2
2
2
2
2
2
2
2
2
1
320.35
354.80
321.34
355.79
405.28
439.72
310.31
344.76
355.31
389.75
180.16
4.07
4.69
2.99
3.61
4.88
5.49
3.17
3.79
3.52
4.14
1.43
Phenyl
Pyrid-400-yl
Pyrid-400-yl
500-Bromo-thien-200-yl
500-Bromo-thien-200-yl
Fur-200-yl
Fur-200-yl
500-Nitrofur- 200-yl
500-Nitrofur- 200-yl
—
169 18.8
35.5 0.5
32.5 0.2
8.9 0.66
146.9 1.4
11.7 0.5
11
11
4
2j
ASA
Acetylsalicylic acid (ASA) also known as aspirin was used as an antiplatelet control.
a
For good theoretical oral biodisponibility - number of hydrogen bond acceptors (HBA) 6 10 and donors (HBD) 6 5, clogP 6 5 and molecular weight 6 500.
(IC50 < 20 l
g mlꢀ1) of arachidonic acid, ADP and/or adrenaline-
pathways (2a, 2c, 2e, 2g and 2h) to be further in vitro and
in vivo investigated. Interestingly, as the antiplatelet profile of
these compounds was different from aspirin and compound 1 it
suggests other platelet targets for these derivatives than
cyclooxygenase.
4. Experimental
Chemical reagents and all solvents used in this study were pur-
shed from Merck AG (Darmstadt, Germany) and VETEC LTDA (Rio
de Janeiro, Brazil). Melting points were determined with a Fisher-
Johns instrument and are uncorrected. Infrared (IR) spectra were
recorded on Perkin–Elmer FT-IR, model 1600 senes spectropho-
tometer in KBr pellets (Ontario, Canada). NMR spectra, unless
otherwise stated, were obtained in deuterated Me2SO-d6 using a
Varian Unity Plus 300 MHz spectrometer (Palo Alto, California,
US). Chemical shifts (d) are expressed in ppm and the coupling con-
stants (J) in hertz. The progress of all reactions was monitored by
TLC performed on 2.0 cm ꢁ 6.0 cm aluminium 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. Microanalyses were performed using a Perkin–
Elmer Model 2400 instrument and all values were within 0.4%
of the calculated compositions.
The 4-carbethoxy triazoles 3a–b were prepared by a known
procedure described in the literature.15
Figure 4. Comparison of the theoretical druglikeness and drugscore values of the
N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-carbohydrazides (2a–j)
with current antiplatelet drugs on the market or described in the literature.
Positive values are expected for more safe and active molecules.
4.1. General procedure for the preparation of the N-substituted-
phenylamino-5-methyl-1H-1,2,3-triazole-4-carbohydrazides 2a–h
To a solution of hydrazide derivatives 4a–b (0.50 mmol) in
15 ml of EtOH, was added an equimolar amount of the appropriate
aromatic aldehydes in the presence of catalytic amount of aqueous
HCl (37%). The reaction was stirred for 2 h, at room temperature.
Then, the solvent was evaporated and the precipitate was collected
by filtration, washed with cold water and dried under vacuum. The
NAH derivates 2a–h were purified in flash chromatography column
using 50% n-hexane/EtOAc as mixture eluent.
(0.15–0.31) values were similar or even better than some of the
drugs currently used on the market (Fig. 4). The theoretical studies
also pointed these derivatives as free of irritant and antireproduc-
tive effects (not shown). Although the Osiris risk alerts are not a
fully reliable toxicity prediction, the theoretical low-toxicity profile
of these compounds reinforces further synthetic and biological
exploration for the development of new antiplatelet drugs.
3. Conclusion
4.1.1. 5-Methyl-N0-(phenylmethylene)-1-(phenylamino)-1H-
1,2,3-triazole-4-carbohydrazide 2a
In summary, based on our biological and theoretical results we
identified some of the synthesized 1,2,3-triazole NAH derivatives
as potential lead compounds and significant inhibitors
IR (KBr) mmax (cmꢀ1) 3226 and 3033 (N–H), 1675 (C@O), 1602
(C@N); 1H NMR (300.00 MHz, DMSO-d6) d 2.47 (s, 3H, CH3),
6.48–6.51 (m, 2H), 6.91–6.96 (m, 1H), 7.24–7.29 (m, 2H), 7.45–

3718
A. K. Jordão et al. / Bioorg. Med. Chem. 17 (2009) 3713–3719
7.47 (m, 3H), 7.71–7.73 (m, 2H), 8.58 (s, 1H, N@CH), 10.23 (br s, 1H,
N–H), 12.15 (br s, 1H, NH–N) ppm. 13C NMR (75 MHz, DMSO-d6) d
8.1 (CH3), 112.9 (C-20 and C-60), 121.4 (C-40), 127.1 (C-300 and C-500),
128.8 (C-200 and C-600), 129.4 (C-30 and C-50), 130.0 (C-400), 134.4 (C-
100), 136.2 (C-4 or C-5), 138.4 (C-4 or C-5), 146.1 (C-10), 148.1
(N@CH), 157.1 (C@O) ppm. Anal. Calcd for C17H16N6O: C, 63.74;
H, 5.03; N, 26.23. Found: C, 63.20; H, 5.00; N, 26.10.
2H, J = 9.0), 8.62 (s, 1H, N@CH), 10.36 (br s, 1H, N–H), 12.16 (br s,
1H, NH–N) ppm. 13C NMR (75 MHz, DMSO-d6) d 8.3 (CH3), 114.8
(C-20 and C-60), 114.9 (C-400), 125.4 (C-40), 129.5 (C-30 and C-50),
131.5 (C-300), 131.6 (C-200), 136.3 (C-4 or C-5), 138.8 (C-4 or C-5),
141.1 (C-100), 142.6 (N@CH), 145.1 (C-10), 157.1 (C@O) ppm. Anal.
Calcd for C15H12BrClN6OS: C, 40.97; H, 2.75; N, 19.11. Found: C,
41.39; H, 3.01; N, 19.02.
0
4.1.2. 1-(4-Chlorophenylamino)-5-methyl-N0-
4.1.7. N -[(Fur-200-yl)methylene]-5-methyl 1-(phenylamino)-1H-
(Phenylmethylene)-1H-[1,2,3]-triazole-4-carbohydrazide 2b
IR (KBr) mmax (cmꢀ1) 3292 and 3025 (N–H), 1641 (C@O), 1608
(C@N); 1H NMR (300.00 MHz, DMSO-d6) d 2.47 (s, 3H, CH3), 6.53
(d, 2H, J = 8.8), 7.3 (d, 2H, J = 8.8), 7.45–7.47 (m, 3H), 7.70–7.73
(m, 2H), 8.58 (s, 1H, N@CH), 10.36 (br s, 1H, N–H), 12.10 (br s,
1H, NH–N) ppm. 13C NMR (75 MHz, DMSO-d6) d 8.1 (CH3), 114.6
(C-20 and C-60), 125.1 (C-40), 127.1 (C-300 and C-500), 128.8 (C-200
and C-600), 129.3 (C-30 and C-50), 130.0 (C-400), 134.3 (C-100), 136.3
(C-4 or C-5), 138.4 (C-4 or C-5), 145.0 (C-10), 148.1 (N@CH),
156.9 (C@O) ppm. Anal. Calcd for C17H15ClN6O: C, 57.55; H, 4.26;
N, 23.69. Found: C, 57.29; H, 4.63; N, 22.94.
[1,2,3]-triazole-4-carbohydrazide 2g
IR (KBr) mmax (cmꢀ1) 3489 and 3204 (N–H), 1663 (C@O), 1610
(C@N); 1H NMR (300.00 MHz, DMSO-d6) d 2.46 (s, 3H, CH3),
6.49–6.51 (m, 2H), 6.63 (dd, 1H, J = 3.2; 1.9), 6.90 (d, 1H, J = 3.4),
6.94–6.96 (m, 1H), 7.23–7.29 (m, 2H), 7.84 (d, 1H, J = 1.5), 8.47
(s, 1H, N@CH), 10.19 (br s, 1H, N–H), 12.10 (br s, 1H, NH–N)
13
ppm. C NMR (75 MHz, DMSO-d6) d 8.1 (CH3), 112.2 (C-300),
112.9 (C-20 and C-60), 113.3 (C-200), 121.4 (C-40), 129.4 (C-30 and
C-50), 136.2 (C-4 or C-5), 145.1 (C-400), 138.4 (C-4 or C-5), 137.8
(N@CH), 146.1 (C-10), 149.5 (C-100), 156.9 (C@O) ppm. Anal. Calcd
for C15H14N6O2: C, 58.06; H, 4.55; N, 27.08. Found: C, 58.05; H,
4.32; N, 27.28.
4.1.3. 5-Methyl-1-(phenylamino)-N0-[(pyrid-400-yl)methylene]-
1H-[1,2,3]-triazole-4-carbohydrazide 2c
4.1.8. 1-(4-Chlorophenylamino)-N0-[(fur-200-yl)methylene]-5-
methyl-1H-1,2,3-triazole-4-carbohydrazide 2h
IR (KBr) mmax (cmꢀ1) 3413 and 3220 (N–H), 1671 (C@O), 1602
(C@N); 1H NMR (300.00 MHz, DMSO-d6) d 2.48 (s, 3H, CH3),
6.50–6.53 (m, 2H), 6.91–6.97 (m, 1H), 7.24–7.29 (m, 2H), 7.94 (d,
2H, J = 6.2), 8.64 (s, 1H, N@CH), 8.79 (d, 2H, J = 6.2), 10.30 (br s,
1H, N–H), 12.64 (br s, 1H, NH–N) ppm. 13C NMR (75 MHz, DMSO-
d6) d 8.2 (CH3), 112.9 (C-20 and C-60), 121.4 (C-40), 122.4 (C-200
and C-600), 129.4 (C-30 and C-50), 135.9 (C-4 or C-5), 139.1 (C-4 or
C-5), 145.9 (C-100), 146.1 (C-10), 146.1 (C-300 and C-500), 146.2
(N@CH), 157.0 (C@O) ppm. Anal. Calcd for C17H15ClN6O: C, 63.74;
H, 5.03; N, 26.23. Found: C, 63.20; H, 5.00; N, 26.10.
IR (KBr) mmax (cmꢀ1) 3437 and 3222 (N–H), 1672 (C@O), 1592
(C@N); 1H NMR (300.00 MHz, DMSO-d6) d 2.46 (s, 3H, CH3), 6.53
(d, 2H, J = 8.8), 6.63 (dd, 1H, J = 3.4; 1.9), 6.89 (d, 1H, J = 3.4), 7.29
(d, 2H, J = 8.8), 7.83 (d, 1H, J = 1.2), 8.47 (s, 1H, N@CH), 10.35 (br
s, 1H, N–H), 12.11 (br s, 1H, NH–N) ppm. 13C NMR (75 MHz,
DMSO-d6) d 8.0 (CH3), 112.1 (C-300), 113.2 (C-200), 114.6 (C-20 and
C-60), 125.1 (C-40), 129.2 (C-30 and C-50), 136.2 (C-4 or C-5), 137.7
(N@CH), 138.3 (C-4 or C-5), 144.9 (C-10), 145.0 (C-400), 149.5 (C-
100), 156.8 (C@O) ppm. Anal. Calcd for C15H13ClN6O2: C, 52.26; H,
3.80; N, 24.38. Found: C, 51.87; H, 3.57; N, 24.09.
4.1.4. 1-(4-Chlorophenylamino)-5-methyl-N0-[(pyrid-400-
yl)methylene]-1H-[1,2,3]-triazole-4-carbohydrazide 2d
IR (KBr) mmax (cmꢀ1) 3399 and 3236 (N–H), 1672 (C@O), 1584
(C@N); 1H NMR (300.00 MHz, DMSO-d6) d 2.48 (s, 3H, CH3), 6.55
(d, 2H, J = 9.0), 7.31 (d, 2H, J = 8.8), 7.94 (d, 2H, J = 6.4), 8.64 (s,
1H, N@CH), 8.78 (d, 2H, J = 6.4), 10.46 (br s, 1H, N–H), 12.64 (br
s, 1H, NH–N) ppm. 13C NMR (75 MHz, DMSO-d6) d 8.1 (CH3),
114.6 (C-20 and C-60), 122.3 (C-200 and C-600), 125.1 (C-40), 129.2
(C-30 and C-50), 135.9 (C-4 or C-5), 139.1 (C-4 or C-5), 144.0
(N@CH), 144.9 (C-10), 145.9 (C-100), 146.1 (C-300 and C-500), 157.0
(C@O) ppm. Anal. Calcd for C16H14ClN7O: C, 54.01; H, 3.97; N,
27.56. Found: C, 54.40; H, 4.00; N, 27.40.
4.2. General procedure for the preparation of the N-
substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-
carbohydrazides 2i–j
A solution of 5-nitro-2-furfurylidene diacetate (0.4 mmol) in a
mixture of EtOH (10.0 ml) with a 50% aqueous solution of sulfuric
acid (1.0 ml) was heated for 1–2 min on a steam bath and chilled to
the room temperature, and then 0.4 mmol of the 4a–b were added.
The resulting mixture was stirred for 2 h at room temperature and
then was poured into crushed ice. The insoluble product was fil-
tered off and purified by column chromatography using 50% n-hex-
ane/EtOAc as mixture eluent.
4.1.5. N0-[(500-Bromothien-200-yl)methylene]-5-methyl-1-
(phenylamino)-1H-[1,2,3]-triazole-4-carbohydrazide 2e
IR (KBr) mmax (cmꢀ1) 3472 and 3208 (N–H), 1663 (C@O), 1610
(C@N); 1H NMR (300.00 MHz, DMSO-d6) d 2.45 (s, 3H, CH3),
6.51–6.54 (m, 2H), 6.91–6.96 (m, 1H), 7.25 (d, 2H, J = 3.9), 7.27
(d, 2H, J = 3.9), 7.25–7.33 (m, 2H), 8.67 (s, 1H, N@CH), 10.35 (br s,
1H, N–H), 12.18 (br s, 1H, NH–N) ppm. 13C NMR (75 MHz, DMSO-
d6) d 8.1 (CH3), 112.9 (C-20 and C-60), 114.4 (C-400), 121.2 (C-40),
129.4 (C-30 and C-50), 131.0 (C-300), 131.1 (C-200), 135.8 (C-4 or C-
5), 138.2 (C-4 or C-5), 140.8 (C-100), 141.9 (N@CH), 145.8 (C-10),
157.0 (C@O) ppm. Anal. Calcd for C15H13BrN6OS: C, 44.45; H,
3.23; N, 20.74. Found: C, 44.28; H, 3.40; N, 20.39.
4.2.1. N0-[(500-Nitrofur-200-yl)methylene]-1-(phenylamino)-5-
methyl-1H-1,2,3-triazole-4-carbohydrazide 2i
IR (KBr) mmax (cmꢀ1) 3300 and 3147 (N–H), 1660 (C@O), 1601
(C@N); 1H NMR (300.00 MHz, DMSO-d6) d 2.48 (s, 3H, CH3),
6.50–6.52 (m, 2H), 6.92–6.96 (m, 1H), 7.26–7.29 (m, 2H), 7.25 (d,
1H, J = 3.9), 7.78 (d, 1H, J = 3.9), 8.54 (s, 1H, N@CH), 10.22 (br s,
1H, N–H) ppm. 13C NMR (75 MHz, DMSO-d6) d 8.1 (CH3), 112.9
(C-20 and C-60), 114.7 (C-200), 115.1 (C-300), 121.4 (C-40), 129.4 (C-
30 and C-50), 135.7 (N@CH), 135.8 (C-4 or C-5), 139.0 (C-4 or C-5),
146.0 (C-10), 151.8 (C-100), 151.8 (C-400), 157.2 (C@O) ppm. Anal.
Calcd for C15H13N7O4: C, 50.71; H, 3.69; N, 27.59. Found: C,
50.10; H, 3.50; N, 27.30.
4.1.6. N0-[(500-Bromothien-200-yl)methylene]-1-(4-
chlorophenylamino)-5-methyl-1H-[1,2,3]-triazole-4-
carbohydrazide 2f
4.2.2. N0-[(500-Nitrofur-200-yl)methylene]-1-(4-chlorophenylamino)-
5-methyl-1H-1,2,3-triazole-4-carbohydrazide 2j
IR (KBr) mmax (cmꢀ1) 3439 and 3298 (N–H), 1642 (C@O), 1602
(C@N); 1H NMR (300.00 MHz, DMSO-d6) d 2.43 (s, 3H, CH3), 6.50
(d, 2H, J = 9.0), 7.24 (d, 2H, J = 3.9), 7.26(d, 2H, J = 3.9), 7.29 (d,
IR (KBr) mmax (cmꢀ1) 3273 and 3100 (N–H), 1639 (C@O), 1590
(C@N); 1H NMR (300.00 MHz, DMSO-d6) d 2.47 (s, 3H, CH3), 6.54
(d, 2H, J = 8.8), 7.25 (d, 1H, J = 3.9), 7.31 (d, 2H, J = 9.0), 7.78 (d,

A. K. Jordão et al. / Bioorg. Med. Chem. 17 (2009) 3713–3719
3719
2H, J = 3.9), 8.53 (s, 1H, N@CH), 10.38 (br s, 1H, N–H) ppm. 13C NMR
(75 MHz, DMSO-d6) d 8.2 (CH3), 114.6 (C-20 and C-60), 114.7 (C-200),
115.1 (C-300), 125.2 (C-40), 129.3 (C-30 and C-50), 135.8 (N@CH),
135.9 (C-4 or C-5), 139.1 (C-4 or C-5), 144.9 (C-10), 151.8 (C-100),
151.9 (C-400), 157.2 (C@O) ppm. Anal. Calcd for C15H12ClN7O4: C,
46.22; H, 3.10; N, 25.16. Found: C, 46.00; H, 3.20; N, 25.20.
that is determined within the collection of traded drugs and within
the supposedly non-druglike collection of Fluka compounds and
the drug-score was related to topological descriptors, fingerprints
of molecular druglikeness, structural keys and other properties as
clogP and molecular weights.22
Acknowledgements
4.3. Platelet aggregation assays
This work was supported by the Brazilian agencies FAPERJ and
CNPq. Fellowships granted to UFF, by CAPES, CNPq, CNPq-PIBIC,
CNPq-REDOXOMA and FAPERJ are gratefully acknowledged. The
authors have declared no conflict of interest.
Platelet aggregation was monitored by measuring light transmis-
sion according Born method21 using a aggregometer (Qualiterm, PA-
04). Firstly, human platelet-rich plasma (PRP) was prepared from
whole blood centrifugation at 120g for 5 min at room temperature,
and the platelet count was adjusted to 3.0 ꢁ 105/
lL to standardize
References and notes
the aggregation study, by adding homologous platelet-poor plasma
obtained by centrifugation of the blood at 1500g for 5 min. Measure-
mentof plateletaggregationwascompletedwithin3 hof bloodsam-
pling. For the screening assays, the PRP was preincubated at 37 °C for
2 min with solvent (DMSO, final concentration 1%) or with the NAH
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derivatives (100
the platelet agonists. Maximal aggregation was obtained stimulat-
ing platelets with ADP (10
mol lꢀ1), adrenaline (0.3 mol lꢀ1) or
arachidonic acid (10
mol lꢀ1). The effects of NAH derivatives were
l l
g mlꢀ1 or 1–500 g mlꢀ1) before the addition of
l
l
l
expressed as inhibitory effect (%) compared with control samples
containing the inducer only. DMSO (1%) did not interfere signifi-
cantly with platelet aggregation.
13. World Health Organization (WHO) http://www.who.int/cardiovascular_
diseases/en/. Accessed in 2008.
4.4. In silico oral biodisponibility
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(http://www.molinspiration.com). The theoretical oral bioavail-
ability ranking of chemical compounds can be estimated using
the Lipinski0s ‘rule-of-five’, since it describes molecular properties
important for a drug pharmacokinetics in the human body, includ-
ing their absorption, distribution, metabolism and excretion
(ADME). The active compound must present at least three of four
rules: H-bond donors (HBD) 6 5, H-bond acceptors (HDA) 6 10,
molecular mass (MM) 6 500, and the calculated logP (clogP) 6 5.20
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ris Property Explorer (http://www.organic-chemistry.org). Drug-
likeness is based on the occurrence frequency of each fragment
17. Fraga, A. G. M.; Rodrigues, C. R.; Miranda, A. L. P.; Barreiro, E. J.; Fraga, C. A. M.
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