Synthesis, biological evaluation and molecular docking studies of 3-(triazolyl)-coumarin derivatives: Effect on inducible nitric oxide synthase

Article abstract of DOI:10.1016/j.ejmech.2012.10.010

A series of 3-(triazolyl)-coumarins were synthesized and tested as anti-inflammatory agents. It was possible to infer that these compounds do not alter the interaction of LPS with TLR-4 or TLR-2, as the intracellular pathways involved in the TNF-α secretion and COX-2 activity were not affected. Nevertheless, the compounds inhibited iNOS-derived NO production, without affecting the eNOS activity. The outcome of the docking studies showed that π?π interactions with the heme group are important for the iNOS inhibition, thus making compound 3c a promising lead. Moreover, the efficacy of this compound was visualized by the reduced number of neutrophils in the LPS-inflamed subcutaneous tissue. Together, biological and docking data show that triazolyl-substituted coumarins, that can act on iNOS, are a good scaffold to be explored.

Full text of DOI:10.1016/j.ejmech.2012.10.010

European Journal of Medicinal Chemistry 58 (2012) 117e127
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological evaluation and molecular docking studies of 3-(triazolyl)-
coumarin derivatives: Effect on inducible nitric oxide synthase
,
a
a
b,
Hélio A. Stefani a , Karina Gueogjan , Flávia Manarin , Sandra H.P. Farsky
*
*
,
Julio Zukerman-Schpectorc , Ignez Caracelli , Sergio R. Pizano Rodrigues , Marcelo N. Muscará ,
Simone A. Teixeira f, José R. Santin b, Isabel D. Machado b, Simone M. Bolonheis b, Rui Curi g,
Marco A. Vinolo g
,
d
e
f
*
a Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brazil
b Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brazil
c Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil
d BioMat, Departamento de Física, Universidade Federal de São Carlos, São Carlos, SP, Brazil
e Programa de Pós-graduação em Biotecnologia, Universidade Federal de São Carlos, São Carlos, SP, Brazil
f Departamento de Farmacologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil
g Departamento de Fisiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3-(triazolyl)-coumarins were synthesized and tested as anti-inflammatory agents. It was
Received 22 August 2012
Received in revised form
1 October 2012
Accepted 7 October 2012
Available online 17 October 2012
possible to infer that these compounds do not alter the interaction of LPS with TLR-4 or TLR-2, as the
intracellular pathways involved in the TNF-
theless, the compounds inhibited iNOS-derived NO production, without affecting the eNOS activity. The
outcome of the docking studies showed that interactions with the heme group are important for
a secretion and COX-2 activity were not affected. Never-
p/p
the iNOS inhibition, thus making compound 3c a promising lead. Moreover, the efficacy of this
compound was visualized by the reduced number of neutrophils in the LPS-inflamed subcutaneous
tissue. Together, biological and docking data show that triazolyl-substituted coumarins, that can act on
iNOS, are a good scaffold to be explored.
Keywords:
Coumarin
Anti-inflammatory
Neutrophils
Ó 2012 Elsevier Masson SAS. All rights reserved.
Molecular docking
3-(Triazolyl)-coumarins
Click chemistry
iNOS
1. Introduction
resulting in a dramatic rise in the number of circulating neutrophils
prompted to migrate to the site of the lesion [2]. Therefore, at the
Inflammation is a complex host response to injury, coordinated
by a range of chemical mediators, which are generated by activated
plasmatic systems, and secreted or produced at the site of injury by
resident or recruited cells. Neutrophils are produced and matured
in the bone marrow, and about 1011 cells are delivered into circu-
lation each day in humans to maintain hemostasis [1]. Neverthe-
less, in the earlier phases of acute inflammatory reactions, this large
pool of neutrophils in the bone marrow is rapidly mobilized,
focus of inflammation, neutrophils play a central role in innate
immunity by engulfing invading microorganisms by phagocytosis
and destroying them. In this context, they produce reactive oxygen
intermediate species, release proteolytic enzymes, and secrete
chemical mediators, such as arachidonic acid metabolites, cyto-
kines and nitric oxide (NO) [3].
Neutralization of the offending insult ideally prompts the
resolution of inflammation. Indeed, exacerbated or non-regulated
neutrophil responses, together with inappropriate resolution,
contribute to persisting tissue damage that underlies many
inflammatory diseases. Therefore, anti-inflammatory drugs must
be efficient to halt the exacerbated reaction, and neutrophils must
be drug-target cells, considering their relevance to the evolution of
the process [3].
* Corresponding authors.
E-mail addresses: hstefani@usp.br (H.A. Stefani), sfarsky@usp.br (S.H.P. Farsky),
juliozs@gmail.com (J. Zukerman-Schpector).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.010

118
H.A. Stefani et al. / European Journal of Medicinal Chemistry 58 (2012) 117e127
NO is physiologically produced by constitutive nitric oxide
With this subunit synthesized, the 3-(trimethylsilylethynyl)
coumarin 2 was reacted with various organic azides via click
chemistry to form the corresponding 3-(1,2,3-triazolyl)coumarin
derivatives in good yields [27]. This CuI-promoted reaction was run
in the presence of PMDTA (1,1,4,7,7-pentamethyl-diethylenetri-
amine) and TBAF (Tetra-n-butylammonium fluoride) (Scheme 1)
[28].
synthases (NOS), which are Ca2þ-dependent enzymes called NOS I
or neuronal NOS (nNOS) and NOS III or endothelial NOS (eNOS). An
inducible NOS (iNOS) isoform, whose activity is Ca2þ-independent,
provides larger amounts of NO than eNOS in several pathological
circumstances, including inflammation [4]. NOS catalyzes the
conversion of the amino acid L-arginine to NO and L-citrulline [5], in
a reaction that requires molecular oxygen, nicotinamide adenine
dinucleotide phosphate (NADPH), tetrahydrobiopterin (BH4),
calmodulin, flavin adenine dinucleotide (FAD), and flavin mono-
nucleotide (FMN) as cofactors [6]. NO produced by the eNOS
activity results in vasodilation and modulatory effects on immune
responses [7], regardless of whether the NO produced by nNOS
activity exerts important beneficial neuronal effects [8]. Moreover,
the gas produced by iNOS is a pro-inflammatory agent and actively
mediates septic shock, asthma, rheumatoid arthritis, chronic
obstructive pulmonary disease (COPD) and other inflammatory
diseases. The reaction of NO and superoxides generated during
inflammation results in the formation of peroxynitrite, which is one
of the most powerful oxidant agents, leading to vascular and
cellular events of inflammatory reactions [9]. Therefore, inhibition
of iNOS-derived NO appears to be a promising strategy for the
treatment of inflammatory diseases.
Natural or synthetic coumarins, are a versatile class of hetero-
cycles and constitute a relevant class of pharmacological agents
presenting a wide range of different activities [10]. Their anti-
inflammatory properties include the inhibition of matrix metal-
loproteinase actions, phagocytosis mechanisms and the transcrip-
tion factors involved in the mRNA expression of inflammatory
proteins, as well as the inhibition of the mRNA expression and
activities of enzymes, such as iNOS and cyclooxygenase-2 (COX-2)
[11,12]. Moreover, coumarin-derivatives also have been shown to be
pharmacologically useful as anti-coagulants [13], anti-HIV agents
[14], anti-oxidants [15], anti-tumorals [16] and free radical scav-
engers [17] and shown to have extensive applications in industrial
areas [18]. Novobiocin, currently on the market, is a coumarin-
derived antibiotic used as a competitive inhibitor of the bacterial
ATP binding gyrases B subunit, blocking the negative supercoiling
of relaxed DNA [19].
The alkyl azides gave lower yields, while the electron-rich and
neutral aryl azides gave high yields of the 1,2,3-triazoles, ranging
from 82% to 90%. The structure and purity of all obtained products
were confirmed by 1H and 13C NMR spectroscopy.
2.2. Biological activity
2.2.1. 3-(Triazolyl)-coumarins derivatives compounds only reduced
NO production by LPS-stimulated neutrophils
In order to elucidate the anti-inflammatory effects of
compounds 3a, 3b, 3c and 3g, the secretion of tumor necrosis factor
(TNF-a) and prostaglandin E2 (PGE2) and the production of NO by
neutrophils were quantified in basal or Escherichia coli lipopoly-
saccharide (LPS)-stimulated conditions. All of the experimental
procedures failed to cause necrosis or apoptosis, and the viability of
neutrophil suspensions was always above 90% at the end of assays
(Figure 1 of supplementary data). Therefore, all of the actions of
coumarin compounds did not reflect neutrophil toxicity.
The data presented in Fig. 1A show that none of the tested
molecules inhibited the secretion of PGE2, which was induced by
LPS stimulation. Nevertheless, compounds 3b and 3g induced the
secretions of PGE2 at basal conditions. Additionally, all of the
compounds did not reduce the TNF-a secretion in basal or LPS-
stimulated conditions and compound 3a treatment enhanced the
levels of the cytokine in comparison to the data obtained in vehicle-
treated cells (Fig. 1B). It is noteworthy that the inhibitory effect on
TNF-
of 3-(triazolyl)-coumarin derivative compounds, as higher doses of
compounds, up to a concentration of 1000 M, did not inhibit the
a and PGE2 production was not dependent on inefficient doses
m
secretion of chemical mediators evoked by LPS stimulation (data
not shown). In fact, all compounds significantly inhibited NO
production by LPS-stimulated neutrophils. Except for compound
The synthesis of 1,2,3-triazoles is one of the most studied topics
in organic chemistry due to their applications in the chemical
industry, increasing use in medicinal chemistry and bioactive
molecules [20,21], although the 1,2,3-triazole ring does not occur in
nature. There are many examples including anti-HIV activity [22],
antimicrobial activity against Gram positive bacteria [23], selective
b3 adrenergic receptor agonist [24], by means of triazole
compounds.
In this context, considerable efforts have been made to develop
synthetic strategies to join two structural units, such as a coumarin
scaffold and a triazole moiety, in order to make a new potential
therapeutic agent.
3a, the inhibitory effect was induced with a concentration of 10 mM
(Fig. 1C).
2.2.2. 3-(Triazolyl)-coumarin derivative compounds reduced the
activity and expression of iNOS, but not eNOS
As all of the studied compounds reduced the production of NO
by LPS-stimulated neutrophils, assays were subsequently con-
ducted to address the mechanism involved in this effect. The data
presented in Fig. 2A and B show that all of the 3-(triazolyl)-
coumarin derivatives blocked the activity of iNOS in LPS-stimulated
neutrophils, which caused a marked decrease in the total NOS
activity.
In this manuscript, the synthesis, biological activity and the in
silico docking studies of a series of triazolyl substituted coumarins,
used to rationalize the pharmacological results, are presented.
It is important to emphasize that eNOS is only expressed natu-
rally in granulocyte precursors in the bone marrow, where
expression is markedly decreased during the maturation process
[29,30,31]. Therefore, it has been proposed that circulating
neutrophils do not express eNOS, and express low and high levels of
nNOS and iNOS after LPS stimulation, respectively. Western blot
assays carried out with cytoplasmic proteins collected from basal or
LPS-stimulated neutrophils corroborated these data (Fig. 2C) and
showed that the 3-(triazolyl)-coumarin derivatives reduced NO
production by altering iNOS in neutrophils. In order to investigate
whether the synthesized molecules could affect the activity or
expression of eNOS, their actions on NO production by endothelial
cells were investigated. It is noteworthy that the cultured micro-
vascular endothelial cells used in this assay only express eNOS in
2. Results and discussion
2.1. Chemistry
As shown in Scheme 1, 3-bromocoumarin, was prepared by
reacting coumarin with a mixture of oxoneÒ and hydrobromic acid
in dichloromethane at room temperature, then treating the product
with Et3N, as described in the literature [25]. To introduce the
terminal alkyne in the coumarin ring the 3-bromocoumarin was
submitted to a Sonogashira cross-coupling reaction [26].

H.A. Stefani et al. / European Journal of Medicinal Chemistry 58 (2012) 117e127
119
TMS
TMS
Br
PdCl2/PPh3
i) Oxone, 2 N HBr, CH2Cl2, r.t.
ii) Et3N, CH2Cl2, r.t.
O
CuI, Et3N
O
- 70%
O
O
O
O
CH3CN, 45 o
C
2
1
- 90%
R
N
R
N3
N
N
CuI (1 eq.), PMDTA (1.2 eq.)
TBAF (1.2 eq.), THF, r.t.
O
O
3a-g
N
N
N
N
N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
3d- 65%
O
3b-
3a- 63%
70%
3c- 82%
NO2
N
N
N
N
N
N
N
N
N
O
3f- 70%
O
O
O
O
O
3g-
3e-
70%
90%
Scheme 1. Synthesis of 3-(1,2,3-triazolyl)coumarins from 3-alkynylcoumarins and organic azides.
the absence of stimulation [32]. The data presented in Fig. 2D show
that only compound 3a evoked a slight reduction in NO levels and
that the other compounds did not alter the basal NO production by
eNOS. The efficacy of the assay was confirmed by the reduced levels
2.3. Docking studies
In order to rationalize the described biological results, docking
studies of compounds 3a, 3b, 3c and 3g were undertaken. The
program GOLD 5.0.1 [33aec] with the GoldScore fitness function,
which uses bond strengths, was used for the docking calculations.
These were performed by removing the water molecules, adding
the hydrogen atoms using the facilities within GOLD and keeping
the heme group and the tetrahydrobiopterin cofactor. The protein
binding sites were defined using those occupied by the co-
crystallized ligand AT2906 for 3e7g (iNOS) and 327864 for 3eah
(eNOS), as described in the next section. Full flexibility was allowed
for all docked compounds. The program Accelrys Discovery StudioÒ
v3.1 [33d] was used to analyze the poses (orientations/conforma-
tions) and interactions, and to present the results.
of NO in the supernatant of cells treated with
L-NG-nitroarginine
methyl ester ( -NAME), a non-specific NOS inhibitor.
L
2.2.3. 3-(Triazolyl)-coumarin derivative compounds inhibited iNOS
gene and protein expression
As compound 3c presented a higher inhibitory action on iNOS
activity, it was employed in the following assays to investigate the
mechanism of action of 3-(triazolyl)-coumarin derivative
compounds on the enzyme. The data obtained show that molecule
3c reduced the cytoplasmic expression of iNOS (Fig. 3A) and the
gene expression of iNOS in LPS-stimulated neutrophils (Fig. 3B).
2.2.4. Compound 3c reduced neutrophil recruitment to the
inflammatory site
2.3.1. Redocking
To establish the parameters to be used in the calculations,
redocking calculations were performed with two crystallographic
complexes obtained from the Protein Data Bank (PDB) [34]. These
complexes were 3e7g [35], which is the structure of human
inducible Nitric oxide synthase (iNOS) complexed with ethyl 4-[(4-
methylpyridin-2-yl)amino]piperidine-1-carboxylate (ligand 9 in
The in vivo anti-inflammatory effect of compound 3c was tested
in an air pouch model of inflammation. The data presented in Fig. 4
show the equivalent inhibitory potency of aminoguanidine,
a specific iNOS inhibitor, and compound 3c on neutrophil migration
into the subcutaneous dorsal inflamed tissue of rats evoked by
a local injection of LPS.
ꢀ
reference [30]), with a cavity radius of 11.645 A, and endothelial

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H.A. Stefani et al. / European Journal of Medicinal Chemistry 58 (2012) 117e127
Fig. 1. The effects of 3-(triazolyl)-coumarin derivative compounds on (A) PGE2, (B) TNF-
a
secretion and (C) NO production by neutrophils. Neutrophils (1 ꢀ 106) were incubated for
18 h with culture medium (basal) or LPS (5
m
g/mL) simultaneously with each 3-(triazolyl)-coumarin derivative or a control. PGE2 and TNF-
a
concentrations were quantified by ELISA
ꢁ
and NO2 levels by Griess reaction. Results express the mean ꢂ s.e.m. of 4 independent assays. #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. respective basal values; *P < 0.05,
**P < 0.01 and ***P < 0.001 vs. respective values in the control group.
Fig. 2. Effects of 3-(triazolyl)-coumarin derivative compounds on (A) total and (B) iNOS activity; (C) on NOS expressions on LPS-stimulated neutrophils and (D) on NO production by
cultured microvascular endothelial cells. Neutrophils (1 ꢀ 106) were incubated for 18 h with LPS (5
mg/mL) and with each 3-(triazolyl)-coumarin derivative compound or control.
NOS activity and expression were measured by enzymatic assay and western blot, respectively. Confluent microvascular endothelial cells were incubated for 48 h with L-NAME
ꢁ
(100
m
M), each 3-(triazolyl)-coumarin derivative compound or control. NO2 concentration was quantified by Griess reaction. Results express the mean ꢂ s.e.m. of 4 independent
assays. #P < 0.05 and ##P < 0.01 and ###P < 0.001 vs. control.

H.A. Stefani et al. / European Journal of Medicinal Chemistry 58 (2012) 117e127
121
Fig. 3. Effects of compound 3c on iNOS expression (A) and gene expression (B) by LPS stimulated neutrophils. Neutrophils (1 ꢀ 106) were incubated for 18 h with LPS (5
m
g/mL) and
with compound 3c or control. Protein and mRNA levels were measured by western blot and real-time PCR, respectively. Results express the mean ꢂ s.e.m. of 4 independent assays.
*P > 0.05 vs. respective control.
Fig. 4. Effects of compound 3c and aminoguanidine on the neutrophil influx into the subcutaneous tissue of rats (air pouch) inflamed by LPS. Animals were treated with compound
3c, aminoguanidine or control, via the oral route, simultaneously to the injection of LPS (1.5 mg/mL) into the pouch. Four hours later, the number of leukocytes in the inflammatory
exudate was quantified using a Neubauer chamber and dyed smears. A dotted line indicates the number of neutrophils in the non-inflamed tissue. Results express mean ꢂ s.e.m. of
3 animals in each group. ##P < 0.01 vs. control.
Nitric oxide synthase (eNOS), which is 3eah [35] complexed with
Figs. 5 and 6 show that compound 15 has in eNOS a similar
orientation to that in iNOS and, on the other hand, compound 9
presents a quite different orientation for eNOS than for iNOS, which
has the ring moiety positioned far away from the heme group in
(3s,5e)-3-propyl-3,4-dihydrothieno[2,3-f][1,4]oxazepin-5(2h)-
imine (ligand 15 in reference [30]), with a cavity radius of 12.418 A
ꢀ
used.
Redocking results showed that the binding modes obtained by
GOLD were almost identical for the two crystallographic crystal
complexes (Figure S1 and Table S1 for 3e7g and Figure S4 and
Table S4 for 3eah).
eNOS, preventing it from forming pep interactions with the heme
group. These results are in good agreement with the experimental
results (Table 1) where it was shown that compound 15 inhibited
eNOS while 9 did not.
In addition, cross-docking calculations were performed, that is,
compound 15 was docked in iNOS and compound 9 in eNOS. The
results in iNOS are shown in Fig. 5, where it can be seen that both
molecules are oriented in a very similar way, that is, with the aryl
rings almost parallel to the heme group.
These results are in good agreement with the experimental
results of Garcin et al. [35], which are shown in Table 1, where the
data of IC50 are collated.
2.3.2. Docking of the coumarinic compounds
As the redocking results showed that the parameters were
adequate, these were used for the docking calculations.
It can be seen in Fig. 7(a), where the predicted poses for the four
coumarinic compounds are shown, that compounds 3a, 3b and 3g
place the aromatic moiety almost perpendicular to the heme group,
whilst the phenyl ring of compound 3c is almost parallel to the
Fig. 5. Cross-docking in iNOS (3e7g). (a) The relative orientation of ligands 9 and 15, in relation to the heme group. (b) A different view, without the heme group.

122
Table 1
H.A. Stefani et al. / European Journal of Medicinal Chemistry 58 (2012) 117e127
IC50 values (
m
M) e data from Garcin et al. [35].
ligand
iNOS
eNOS
9
15
0.35
0.04
58
0.2
heme group. As compound 9 and 3c are good inhibitors of iNOS,
their poses in the binding site of 3e7g, a scheme of which shows
that there is not much room for ligands (Fig. 8), were super-
imposed, showing that they are quite similar (Fig. 7(b)). Therefore,
a detailed analysis was performed.
The pep interactions (the heme ring labeling is shown in Fig. 9)
for both compounds are collated in Table 2. A complete set of data,
together with some 2D diagrams, can be found in Tables S1, S2 and
S3, for ligands 9, 15 and 3c in iNOS, respectively.
Fig. 9 shows the binding site of iNOS with ligand 3c inside it,
occupying almost the same space as ligands 9 and 15 did (in the
cross-docking studies), and with the phenyl moiety parallel to the
heme group. The three ligands created interactions with Glu377,
which is located in front of the cavity, and with Pro350, which is at
the back of the cavity. As the phenyl moieties are almost parallel to
Fig. 8. Scheme of the binding site of iNOS.
A complete set of interactions involving ligands 9, 15 and 3c
together with 2D diagrams are shown in Tables S4eS6. It can be
seen that this situation is quite different from that for iNOS, as all
ligands are positioned with their coumarin moiety perpendicular to
the heme group. This result suggests that the compounds described
in this work would not be eNOS inhibitors, as was experimentally
shown. The same applies to ligand 9 as the cross-docking shown in
Fig. 6 (see also Figure S6) suggests that it adopts an inverted
orientation as compared to ligand 15, and, as such, it should not be
an eNOS inhibitor. The results are shown in Table 2 and summa-
rized in Fig. 13. It is worth pointing out that ligand 15, which is an
the heme group, a series of
p/p interactions were observed, as
well as CH/ interactions (see Table 2 and Table S3).
p
The rather small binding site (see Figure S5, where a surface
representation of the cavity with ligands 9 and 3c is presented)
meant that ligands 3a, 3b and 3g could not accommodate the
coumarin moiety parallel to the heme group, rather it was posi-
tioned perpendicular to it so that the
p/p interactions were lost,
and with the aliphatic chain sticking out of the cavity, as shown in
Fig. 7(a) and 10 and Tables S7, S8 and S9. A schematic summary of
these results is shown in Fig. 11.
Fig. 12 shows the docking results for eNOS together with the
crystallographic ligand 15.
inhibitor of eNOS, makes only one p/p interaction, and ligands 9
and 3c make none.
Fig. 6. Cross-docking in eNOS (3eah). (a) The relative orientation of ligand 9 and 15, in relation to the heme group. (b) A different view, without the heme group.
Fig. 7. (a) Docking poses of coumarins 3a, 3b, 3g (stick) and 3c (ball and stick, light blue) in iNOS. (b) Ligands 3c and 9 in iNOS. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)

H.A. Stefani et al. / European Journal of Medicinal Chemistry 58 (2012) 117e127
123
Fig. 9. Binding site of iNOS with the surface occupied by ligands 9 and 15 (in the cross-
Fig. 10. Ligand 3a in the iNOS cavity, showing the aliphatic moiety coming out.
docking) is shown in purple with ligand 3c inside it. (For interpretation of the refer-
ences to color in this figure legend, the reader is referred to the web version of this
article.)
compounds did not affect eNOS activity, which may represent
a desirable effect of the coumarin compounds. Low amounts of NO
produced by eNOS exerts beneficial effects on vasculature, such as
vasodilation and the modulation of leukocyteeendothelial inter-
actions [38]. On the other hand, high levels of NO generated by
iNOS expression and activity are involved in the genesis of
a diversity of inflammatory diseases [39], and the development of
drugs to target iNOS has been searched for the pharmaceutical
industry [40e42].
3. Conclusions
The actions of 3-(triazolyl)-coumarin derivative compounds
were initially tested regarding the ability of neutrophils to produce
chemical mediators after LPS stimulation. LPS binds to Toll-like
receptor 4 (TLR-4) expressed on phagocyte cell membranes, and
induces intracellular signaling resulting in gene and protein
expression of inflammatory molecules [36]. These actions are
mainly mediated by the activation of the MYD-88 intracellular
pathway and p38 phosphorylation, which activates nuclear tran-
scription factors, such as nuclear factor kappa B (NFkB) and
activating-protein-1 (AP-1) [36]. Based on data obtained here, it is
possible to infer that the 3-(triazolyl)-coumarins derivatives do not
alter the interaction of LPS with TLR-4, which are the intracellular
pathways involved in the TNF-a secretion and gene and protein
expressions of COX-2, and COX-2 activity, as well.
Data from the literature have shown that different classes of
coumarin compounds inhibit TNF-
a or PGE2 secretions by affecting
NF B nuclear translocation and inhibiting the phosphorylation of
k
kinases p38, JNK1/2, PKC in LPS-stimulated macrophages and
mononuclear cell lineages [37]. The chemical structure of the
compounds studied in the present work differs from those by the
insertion of the 1,2,3-triazole ring in the position 3 of the coumarin
molecule, which may be responsible for the distinct mechanism of
action.
Differently from results detected for TNF-a and PGE2, data pre-
sented here clearly showed that all of the coumarin compounds
affected NO production, by reducing iNOS gene and protein
expressions and iNOS activity, as well. It is noteworthy that the
Table 2
p/p interactions for ligands 9, 15 and 3c in iNOS.
Centroids (Ct)
ꢀ
Interaction
Distance (A)
Receptor
Ligand
Ligand 9
p
/
p
p
3.93
4.18
Heme e Ct B
Heme e Ct AB
Ct 1
Ct 1
p/
Ligand 15
p
/
p
p
3.66
3.16
Heme e Ct B
Heme e Ct C
Ct 1
Ct 1
p/
Fig. 11. Orientation of the ligands in relation to the heme group. (a) Ligands 9, 15 and
3c are active and located with the phenyl rings parallel to the heme group, whilst in (b)
3a, 3b and 3g are perpendicular to the heme, with the aliphatic moiety out of the
binding cavity.
Ligand 3c
p/
p
p
3.65
4.03
Heme e Ct B
Heme e Ct AB
Ct 1
Ct 1
p/

124
H.A. Stefani et al. / European Journal of Medicinal Chemistry 58 (2012) 117e127
using UV light. Silica gel column chromatography was performed
on silica gel (Merck e 230e400 mesh). NMR spectra were collected
on a Bruker DPX300 (300 MHz) spectrometer. Chemical shifts were
given in parts per million (ppm) downfield from the internal
reference tetramethylsilane standard; coupling constants (J value)
were given in Hertz (Hz). Melting points were measured by a BÜCHI
Melting Point B-545 and were uncollected. GCeMS spectra were
measured on a Shimadzu GCMS-QP5050A. Analytical HPLC spectra
were reported using an Agilent 1100 with a UV detector measuring
absorbance at 210 nm. All compounds were found to be >95% pure
by HPLC analysis.
Fig. 12. Docking poses of coumarins 3a, 3b, 3c and 3g and ligand 15 in eNOS.
4.2. Synthesis of 3-bromo-2H-cromen-2-one
Coumarin (2.92 g, 20 mmol), oxone (14.8 g, 24 mmol), CH2Cl2
(80 mL) and HBr (22 mL, 44 mmol) were added to a two-necked
round-bottomed flask under nitrogen, with magnetic stirring. The
solution was left at room temperature for 2 h. After that, Et3N
(8.7 mL, 60 mmol) was added dropwise, after which the solution
was followed by GC. After consumption of the entire amount of
coumarin, the mixture was extracted with two 50 mL portions of
ethyl acetate. The organic phase was dried with MgSO4 and
concentrated under a vacuum, giving an orange solid. 3-Bromo-
2H-chromen-2-one (1): Yield 90%. 1H NMR (CDCl3, 300 MHz)
The efficacy of compound 3c on the in vivo inflammatory reac-
tion was visualized by the reduced number of neutrophils in the
LPS-inflamed subcutaneous tissue, equivalent to that evoked by
aminoguanidine, an iNOS inhibitor used as a positive control. The
migration of neutrophils to the inflamed area is a hallmark of an
innate inflammatory response and it must be halted in order to
control the development of the process [43]. Together, the data
obtained here show that triazolyl-substituted coumarins may
represent a class of anti-inflammatory molecules by affecting iNOS
enzyme and blocking the undesirable progress of the innate host
response.
d
(ppm) 7.27e7.36 (m, 2H), 7.45 (d, J ¼ 7.62 Hz), 7.57 (t, J ¼ 7.06 Hz,
1H), 8.10 (s, 1H). 13C NMR (CDCl3, 75 MHz)
d
(ppm) 111.8, 116.8,
The docking results show that the 3-(triazolyl)coumarin is
a good scaffold to be explored, and that substituents that can
perform p/p interactions with the heme group are important. This
119.3,125.0,127.1,132.0,144.4,153.2,157.0. EM m/z (%) 224 (69),196
(20), 145 (28), 89 (100), 63 (37). M. p ¼ 110e111 ꢃC (Lit) (Kim e Park,
2004).
makes ligand 3c a very promising compound as it showed a very
good docking result for iNOS and not eNOS, therefore suggesting it
as a selective agent for iNOS. Moreover, due to the small volume of
binding sites, the use of aliphatic substituents showed not to be
a good choice.
4.3. General procedure for Sonogashira cross-coupling
3-Bromo-2H-cromen-2-one (0.45 g, 2 mmol), PdCl2 (0.028 g,
8 mol%), CuI (0.030 g, 8 mol%) and PPh3 (0.084 g, 16 mol%) in
acetonitrile (20 mL) were added to a two-necked round-bottomed
flask equipped with a reflux condenser under N2. After this, Et3N
(0.84 mL, 6 mmol) and acetylene (0.34 mL, 2.4 mmol) were added.
The reaction mixture was stirred at 45 ꢃC, and was monitored by
TLC and GC analysis. After the consumption of the starting material,
the mixture was diluted with ethyl acetate (20 mL) and washed
with brine (2 ꢀ 20 mL). The organic phase was separated, dried
over MgSO4 and concentrated under a vacuum. The residue was
purified by flash chromatography (ethyl acetate/hexane 10:90). 3-
((Trimethylsilyl)ethynyl)-2H-chromen-2-one (2): Yield 70%. 1H
4. Experimental section
4.1. Chemistry
All of the reactions were carried out under inert gas or with
a CaCl2 tube and the reaction mixtures were stirred magnetically.
All of the reagents and solvents were purchased from commercial
suppliers and were used without further purification, unless
otherwise noted. Reaction products were monitored by TLC using
0.25 mm E. Merck silica gel plates (60 F254) and were visualized
NMR (CDCl3, 300 MHz) d (ppm) 0.27 (s, 9H), 7.28e7.32 (m, 2H), 7.45
(d, J ¼ 1.8 Hz, 1H), 7.52 (t, J ¼ 8.5 Hz, 1H), 7.91 (s, 1H). 13C NMR
(CDCl3, 75 MHz)
d (ppm) 0.0 (3C), 98.3, 102.3, 113.1, 116.9, 118.9,
125.0, 127.9, 132.5, 146.1, 153.6, 159.3. EM m/z (%) 242 (30), 227
(100), 211 (1), 199 (22).
4.4. General procedure for the azideeacetylene cycloaddition
reactions
In a two-necked round-bottomed flask containing CuI (0.096 g,
0.5 mmol), a solution of the 3-((trimethylsilyl)ethynyl)-2H-chro-
men-2-one (0.155 g, 0.5 mmol) dissolved in THF (2 mL) and an
organic azide (0.6 mmol) was added, using PMDETA (0.125 mL,
0.6 mmol) as a base. Next, TBAF was added dropwise (0.6 mL,
0.6 mmol). The product was extracted with a sat. aq. solution of
NH4Cl (20 mL) and ethyl acetate (3 ꢀ 50 mL). The organic phase was
washed with H2O (3 ꢀ 30 mL), dried over MgSO4, filtered and then
concentrated under a vacuum. The product was purified by flash
chromatography and eluted with ethyl acetate/hexane (15:85). 3-
(1-Hexyl-1H-1,2,3-triazol-4-yl)-2H-chromen-2-one (3a): Yield
Fig. 13. The relative orientation of ligands 9, 15 and 3c in iNOs and eNOS binding sites.
63%. 1H NMR (CDCl3, 300 MHz)
d
(ppm) 0.89 (t, J ¼ 6.7 Hz, 3H),

H.A. Stefani et al. / European Journal of Medicinal Chemistry 58 (2012) 117e127
125
1.24e1.45 (m, 6H), 1.97 (t, J ¼ 6.7 Hz, 2H), 4.42 (t, J ¼ 7.2 Hz, 2H),
7.28e7.39 (m, 2H), 7.54 (t, J ¼ 7.8 Hz, 1H), 7.63 (d, J ¼ 6.9 Hz, 1H),
a multi-well plate reader (SpectraMax 190, Moꢁlecular Devices, INC,
USA). The results were reported as mM of NO2 .
8.38 (s, 1H), 8.74 (s, 1H). 13C NMR (CDCl3, 75 MHz)
d (ppm) 13.9,
22.3, 26.1, 30.2, 31.1, 50.5, 116.4, 118.3, 119.3, 123.8, 124.7, 128.3,
131.5, 137.1, 140.5, 153.1, 159.5. EM m/z (%) 297 (32), 212 (31), 185
(100), 115 (21). IV (cmꢁ1) 1716, 1605, 1527, 1430, 920. HRMS (ESI-
4.6.4. Nitric oxide synthase gene expression e real time PCR
Total RNA was obtained from 1.0 ꢀ 107 neutrophils using Trizol
reagentÔ (Invitrogen Life Technologies, Rockville, MD, USA), as
previously described by Chomczynski and Sacchi [46]. Total RNA
TOF) m/z calcd. for
C17H19N3NaO2 320.1374, found 320.1410.
M.p. ¼ 102e103 ꢃC.
(3
mg) was reverse transcribed to cDNA using the reverse tran-
scriptase RevertaidÔ M-MuLV. Expression of iNOS was evaluated
by real-time PCR [47] using a Rotor Gene 3000 (Corbett Research,
Mortlake, Australia) and SYBR Green as a fluorescent dye. The
following primers were used: iNOS sense: 50-cgagttgtg-
gattgttcttcgc-30; iNOS antisense: 50-cgtctcttcccgtggcaaagc-30;
GAPDH sense: 50-gatgctggtgctgagtatgtcg-30; and GAPDH antisense:
50-gtggtgcaggatgcattgctga-30. Quantification of gene expression
was carried out using the method described by Liu and Saint [48].
4.5. Neutrophil isolation
Neutrophils were obtained from adult male Wistar rats 4 h after
an intra-peritoneal injection of 10 mL of 1% sterile oyster glycogen
solution in phosphate-buffered saline (PBS; Sigma, St. Louis, MO,
USA). Following this period, the animals were anesthetized with
ketamine (80 mg/kg) and xylazine (8 mg/kg) and the cells were
collected by rinsing the abdominal cavity with 10 mL of sterile PBS.
All procedures were performed according to the protocols
approved by the local committee for Ethical Surveillance in Animal
Experimentation (COBEA) for the proper care and use of experi-
mental animals.
4.6.5. Nitric oxide synthase (NOS) activity
Neutrophils were incubated with compounds 3b, 3g, 3c or 3a in
the presence or absence of LPS for 18 h and NOS activity was
evaluated according to Teixeira et al. [49]. Briefly, cell samples were
homogenized in 5 volumes of cold incubation buffer (50 mM Trise
HCl buffer, pH 7.4; BioAgency, São Paulo, Brazil) containing phe-
nylmethylsulfonyl fluoride (PMSF, 1 mM; Sigma, St. Louis, MO, USA)
4.6. Microvascular endothelial cell culture
Primary cultures of microvascular endothelial cells (PMECs)
were obtained from the rat cremaster muscle using the method
described by Chen et al. [44], and modified by Lotufo et al. [45]. Rats
were anesthetized and non-coagulated by intra-peritoneal injec-
tion of heparin (1000 units per animal). The animals were exsan-
guinated by cutting the bilateral carotid arteries. Subsequently, the
cremaster muscles were isolated, cut into pieces of approximately
2 mm ꢀ 2 mm, and two pieces were placed into each well in a 24
well plate to be cultured in DMEM supplemented with 20% fetal calf
serum and 1% gentamicin. After 48 h, the tissues were discarded
and the medium changed. When the cells achieved confluence,
they were sub-cultured with trypsin-EDTA solution into a 6 well
plate, following which the cells were ready to use.
and L-citrulline (1 mM; Sigma, St. Louis, MO, USA). The cell
homogenates were incubated at 37 ꢃC for 30 min in the presence of
NADPH (1 mM; Sigma, St. Louis, MO, USA), CaCl2 (2 mM; Synth,
Diadema, Brazil), FAD (2
(2 M; Sigma, St. Louis, MO, USA), BH4 (2
USA), calmodulin (3.7 U) and -arginine containing 100,000 dpm of
[2,3,4,5-3H] -arginine monohydrochloride (1 M; GE HealthCare,
m
M; Sigma, St. Louis, MO, USA), FMN
m
mM; Sigma, St. Louis, MO,
L
L
m
UK). Pharmacological controls of enzymatic activity were carried
out in parallel and consisted of the omission of CaCl2 and the
addition of either EGTA (1 mM; Sigma, St. Louis, MO, USA) or L-
NAME (1 mM; Sigma, St. Louis, MO, USA) to the incubation medium.
The protein content of the samples was determined using the
Bradford colorimetric method, and the activity of NOS was
expressed as pmol L-citrulline produced/min/mg of protein.
4.6.1. Neutrophil treatments
Neutrophils were incubated (18 h; 37 ꢃC, 5% CO2) in the presence
4.6.6. Nitric oxide synthase (NOS) expression e western blot
Cell samples were homogenized in TRIS buffer (50 mM, pH 7.4)
containing PMSF protease inhibitor mixture (Sigma, St. Louis, MO,
or absence of 5
026:B6, Sigma, St. Louis, MO, USA) and simultaneously treated with
compounds 3b, 3g, 3c or 3a (10 or 100 M, dissolved in Hanks’
mg/ml lipopolysaccharide of E. coli (LPS; serotype
m
USA). Homogenate proteins (50 mg) were separated by SDS-PAGE
balanced salt solution; HBSS). Control cells were LPS-stimulated
and treated with control (HBSS).
(7% polyacrylamide) according to Laemmli [50] and wet-
transferred onto a nitrocellulose membrane. Non-specific binding
sites were blocked with 0.2% casein and membranes were incu-
bated overnight with primary mouse monoclonal antibody against
iNOS (500 ng/mL; BD Transduction Laboratories, USA), primary
mouse monoclonal anti-eNOS (500 ng/mL; BD Transduction Labo-
ratories, USA) or primary mouse monoclonal anti-nNOS (500 ng/
mL; BD Transduction Laboratories, USA). Following incubation with
secondary horseradish peroxidase-conjugated antibodies, a chemi-
luminescent assay (HRP SuperSignalWestPico; Pierce, USA) was
used to detect immunoreactive bands. The intensities of the bands
were estimated by densitometric analysis and were compared to
4.6.2. Endothelial cells treatments
Endothelial cells were incubated in the absence of LPS with
100
10e100
m
M coumarin compounds or
L-NAME (unspecific NOS inhibitor;
m
M) for 48 h (37 ꢃC, 5% CO2).
4.6.3. Inflammatory mediators
Neutrophils were incubated with compounds 3b, 3g, 3c or 3a in
the presence or absence of LPS for 18 h and the supernatant was
collected and kept frozen (ꢁ80 ꢃC) until analysis. TNF-
a and PGE2
were quantified using enzyme-linked immunosorbent assay
(ELISA; DuoSet R&D Systems, Minneapolis, MN, USA; Cayman
Chemical, Ann Arbor, MI, USA, respectively) kits according to the
manufacturer’s specifications. The results were expressed as pg/ml.
Nitrite levels in culture supernatant were used as a measure-
ment of NO production by neutrophils and endothelial cells.
the intensity of b-actin expression.
4.6.7. In vivo leukocyte migration: air pouch model
Animals were anesthetized and 10 mL of sterile air was injected
subcutaneously into the dorsal region. Seven days after, the pouch
was refilled with 10 mL of air. On the 10th day following the first air
injection, compound 3c (50 mg/kg dissolved in 1 mL of sterile PBS
solution) or aminoguanidine (50 mg/kg dissolved in 1 mL of sterile
PBS solution) was administered into the intra-peritoneal cavity.
Control animals received the same volume of sterile PBS solution.
Supernatants (100 mL) were incubated for 10 min with 100 mL of
Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine
dihydrochloride in 5% phosphoric acid; both from Sigma, St. Louis,
MO, USA) and absorbances at 550 nm were determined using

126
H.A. Stefani et al. / European Journal of Medicinal Chemistry 58 (2012) 117e127
J. Cancer Res. Ther. 3 (2007) 86e91;
Simultaneously, 1 h before or after treatments, LPS (1.5 mg/mL,
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injection, the animals were re-anesthetized and sacrificed. The
pouches were washed with 2 mL of PBS solution and the total
leukocyte number in the harvested material was determined using
Neubauer chambers. Differential cell counts were performed on
smears stained with May Grunwald/Giemsa dye (SigmaeAldrich,
St. Louis, MO, USA).
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for fellowships to HAS, KG, IC, SHPF and JZS (300613/2007-5,
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