General role of the amino and methylsulfamoyl groups in selective cyclooxygenase(COX)-1 inhibition by 1,4-diaryl-1,2,3-triazoles and validation of a predictive pharmacometric PLS model

Article abstract of DOI:10.1016/j.ejmech.2015.02.049

A novel set of 1,4-diaryl-1,2,3-triazoles were projected as a tool to study the effect of both the heteroaromatic triazole as a core ring and a variety of chemical groups with different electronic features, size and shape on the catalytic activity of the

Full text of DOI:10.1016/j.ejmech.2015.02.049

European Journal of Medicinal Chemistry 94 (2015) 252e264
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
General role of the amino and methylsulfamoyl groups in selective
cyclooxygenase(COX)-1 inhibition by 1,4-diaryl-1,2,3-triazoles and
validation of a predictive pharmacometric PLS model
,
,
Maria Grazia Perrone a 1, Paola Vitale a 1, Andrea Panella a, Cosimo G. Fortuna b,
,
*
Antonio Scilimati a
a
ꢀ
Dipartimento di Farmacia e Scienze del Farmaco, Universita degli Studi di Bari “A.Moro”, Via E. Orabona 4, 70125 Bari, Italy
Dipartimento di Scienze Chimiche, Universita degli Studi di Catania, Viale A. Doria 6, 95125 Catania, Italy
b
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel set of 1,4-diaryl-1,2,3-triazoles were projected as a tool to study the effect of both the hetero-
aromatic triazole as a core ring and a variety of chemical groups with different electronic features, size
and shape on the catalytic activity of the two COX isoenzymes. The new triazoles were synthesized in fair
to good yields and then evaluated for their inhibitory activity towards COXs arachidonic acid conversion
Received 31 July 2014
Received in revised form
23 February 2015
Accepted 24 February 2015
Available online 28 February 2015
catalysis. Their COXs selectivity was also measured.
A predictive pharmacometric Volsurf plus
model, experimentally confirmed by the percentage (%) of COXs inhibition at the concentration of 50
m
M
and IC50 values of the tested compounds, was built by using a number of isoxazoles of known COXs
inhibitory activity as a training set. It was found that two compounds {4-(5-methyl-4-phenyl-1H-1,2,3-
triazol-1-yl)benzenamine (18) and 4-[1-(4-methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-yl]benzen-
Keywords:
1,2,3-Triazoles
COX inhibition
Predictive pharmacometric model
Volsurf plus
amine (19)} bearing an amino group (NH2) are potent and selective COX-1 inhibitors (IC50 ¼ 15 and 3
mM,
respectively) and that the presence of a methylsulfamoyl group (SO2CH3) is not a rule to have a Coxib. In
fact, 4-(4-methoxyphenyl)-5-methyl-1-[4-(methylsulfonyl)phenyl]-1H-1,2,3-triazole (23) has COX-1
IC50 ¼ 23
mM and was found inactive towards COX-2.
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
Moreover, COX-1, being predominantly localized in microglia,
emerged as a prominent player in CNS pathologies with a marked
neuroinflammatory component [3e7]. Then, COX-1 high selective
inhibition rather than COX-2 (by Coxibs, i.e. celecoxib) is more
likely to reduce neuroinflammation. Hence, COX-1 inhibitors have
been further investigated, in combination with other specific drugs,
as part of a therapeutic protocol aimed to prevent and control
neurodegenerative diseases with pronounced inflammation, very
often first step of neurodegeneration mechanisms [8].
In addition, the use of COX-1 as a theranostic target in many
human health problems, is becoming more and more attractive,
since it has been proven that its selective inhibition is not
responsible of stomach damage [9], the main heavy adverse side
effect of NSAIDs.
As a consequence, the development of selective COX-1 in-
hibitors could be quite important for the treatment and visualiza-
tion of diseases in which the COX-1 isoenzyme is overexpressed [1].
Very few selective COX-1 inhibitors are currently known, and
among those we focused our attention on the diarylheterocycle
scaffold.
In the last two decades, the interest around the cyclooxygenases
was focused on the inducible isoform COX-2, trying to exalt ther-
apeutic over adverse side effects and because deemed solely
responsible of inflammation processes in respect to the “constitu-
tive” isoenzyme COX-1 [1]. In recent years, the attention around the
COX-1 has been growing ever more, especially since it was
demonstrated that COX-1 isoform, but not COX-2, is overexpressed
in several human pathologies. Particularly, COX-1 is expressed at
high levels from the early to advanced stages of human epithelial
ovarian cancer, skin and colon, and seems to play a specific key role
also in cancer progression. As a consequence, COX-1 might be a
novel biomarker for early ovarian cancer detection [2], as well as in
other cancer types [1].
* Corresponding author.
E-mail address: antonio.scilimati@uniba.it (A. Scilimati).
M. G. Perrone and P. Vitale have equally contributed to this project.
1
http://dx.doi.org/10.1016/j.ejmech.2015.02.049
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

M.G. Perrone et al. / European Journal of Medicinal Chemistry 94 (2015) 252e264
253
Over the past decade, starting from our lead P6 [10], [3-(5-
chlorofuran-2-yl)-5-methyl-4-phenylisoxazole] (Fig. 1), some dia-
rylheterocycles as COX-1 inhibitors have been prepared. Isoxazole
nucleus was deeply investigated by linking to the diarylheterocycle
several groups with different features to better identify the COX-1
active site requirements for its efficacious, potent and selective
inhibition [11,12].
Triazole has already been used to prepare COXs inhibitors. Most
triazoles are known to possess remarkable biological properties
such as anti-allergic [13], antibacterial [14], anti-HIV [15] activities
and are valuable tools in drug discovery.
adjacent atoms of a central heterocycle (Fig. 1). We recently pub-
lished new COX-1 inhibitors belonging to the class of diary-
lheterocycles having the aryl groups positioned at C3 and C5 of the
isoxazole ring (3t and 3u, Fig. 2) and not at C3/C4 as usual. In spite of
the greater distance between the two aryls, they retain a fair
amount of activity as COX-1 inhibitors [22].
On the other hand, 1,2,3-triazoles and their derivatives have
emerged as powerful pharmacophores [23], gaining interest for
their various biological activities [24e26] as chemotherapeutic
agents [27,28], potent antimicrobial [29], anti-inflammatory
[30,31], local anaesthetic [32], anticonvulsant [33], antineoplastic
[34], antimalarial [35] and antiviral activity [36]. Considering such a
biological importance of 1,2,3-triazoles, a new series of the diary-
lhetherocycles having the aryl groups positioned on N1 and C4 of
the triazole nucleus was projected to explore further productive
and selective interactions with COXs active site.
In addition, a database in which the COX inihibitor activity of
eleven isoxazoles, previously published by us [12], was generated
with the aim to perform a Quantitative StructureeActivity Rela-
tionship (QSAR) investigation using the Volsurf plus (Volsurfþ)
program. VolSurf plus is an automatic procedure to convert infor-
mation coded into the 3D GRID Molecular Interaction Fields into
128 physico-chemically relevant molecular descriptors [18,19]. The
molecular descriptors obtained refer to molecular size and shape,
hydrophilic and hydrophobic properties, hydrogen bonding,
amphiphilic moments, critical packing parameters. Moreover,
pharmacokinetic descriptors related to solubility, metabolic sta-
bility and cell permeability are generated. A complete list of the
VolSurf plus descriptors is reported elsewhere [19,37].
Most efforts have been made to develop suitable synthesis of
1,2,4-triazoles endowed with biological activity.
FK881 [3-methoxy-1,5-bis(4-methoxyphenyl)-1H-1,2,4-triaz-
ole] (Fig. 1) is one of the most studied selective COX-1 inhibitors
having the 1,2,4-triazole as a central ring [16]. In view of the various
pharmacological properties of the triazole nucleus [17], herein we
describe a new series of 1,4-diarylheterocycles bearing the 1,2,3-
triazole (regioisomer of 1,2,4-triazole, chemical portion of FK881),
as a core ring. The novel 1,2,3-triazoles were designed by using a
Volsurf plus [18,19] approach, that allowed also to predict their
activity as potent and selective COX-1 inhibitors. In order to
demonstrate the validity of the Volsurf plus derived pharmaco-
metric model, we prepared and tested the predicted novel 1,4-
diaryl-1,2,3-triazoles towards both COX isoenzymes. The
structure-inhibitory activity model by Volsurf plus was build also as
an attempt to rationalize our previous experimental findings.
The COX inhibitory activity evaluation data confirmed the in
silico predictions and revealed that in the diarylheterocycle series,
the introduction of the 1,2,3-triazole nucleus is not responsible of
the COX inhibition; on the contrary, the presence of the NH2 group
somewhere in the molecule is crucial in the selective interaction
with COX-1 binding site, as recently found in the isoxazole scaffold
[12].
A partial least squares regression (PLS) was performed using
VolSurf plus, and the model was validated using the Leave One Out
(LOO) method.
The eleven compounds of training set and their biological data
used to generate the pharmacometric PLS model are listed in
Table 1.
A two component PLS model based on the 128 VolSurf plus
descriptors was obtained with overall R2 0.90 and Q2 0.63. The PLS
score plot is depicted in Fig. 3; the background is coloured ac-
cording to the activity value: blue for active and red (in web
version) from inactive. As it is possible to notice, three active
compounds previously synthesized were selected in order to verify
the goodness of the model and they are, as expected, in the blue
region of the plot. The PLS recomputed versus experimental plot of
the resulting model is shown in Fig. 4. A significant discrimination
between active and inactive compounds was obtained. Compounds
are color-coded by their activity values, using a scale from red
(actives) to blue (inactives) (in web version).
2. Results and discussion
2.1. Design
The 1,2,3-triazole possesses favorable properties in the medici-
nal chemistry field. It has a moderate dipole character, hydrogen
bonding capability, rigidity and remarkable metabolic stability [20].
In addition, 1,2,3-triazole nucleus has been used to reduce the lip-
ophilicity of some Coxibs [21].
COX-1 inhibitors belonging to the diarylheterocycle class are
usually tricyclic compounds possessing two aryls bonded to the
Fig. 2. Diarylheterocycles as COX-1 selective inhibitors (3t and 3u) with two aryls
positioned at the C3 and C5 of the isoxazole ring. COXs IC50 values are by Human Whole
Blood Assay (HWBA) [22].
Fig. 1. P6 and FK881 chemical structure.

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M.G. Perrone et al. / European Journal of Medicinal Chemistry 94 (2015) 252e264
Table 1
Structures of the eleven compounds used to generate the QSAR model.a
Compound code
Structure
COX-1 IC50 (% Inhib.)b
39 M (89)
Compound
COX-1 IC50 (% Inhib.)b
P6
m
MPA7
MPA9
PM32
PM41
MPA15
27 mM (61)
MPA8
(39)
(7)
MPA4
1.1
m
M (94)
(37)
(11)
(32)
MPA10
MPA13
30 m
M (55)
(17)
MPA35
4 mM (78)
a
Inhibitory activity on oCOX-1 and oCOX-2 was determined by using a colorimetric COX (ovine) inhibitor screening assay kit [12].
The percentage (%) of inhibition at 50 mM is reported in the square brackets.
b
Fig. 3. PLS score plot t1-t2.

M.G. Perrone et al. / European Journal of Medicinal Chemistry 94 (2015) 252e264
255
Fig. 4. PLS recomputed versus experimental on t3.
Since the model seems to clearly separate active from inactive
compounds, eighteen new 1,2,3-triazoles were designed and pro-
jected into the model for activity prediction. A PLS model was ob-
tained in which the first three components explained more than
94.7% of variance.
Fig. 5AeB shows the 3D PLS model after projection of the test-
set, and yellow points (in web version) indicate the predicted
compounds. Two different prospectives of the same model are re-
ported. In Fig. 5A, some compounds of the training set were
selected to evidence the validity of the model. In particular, three
isoxazoles reported as active [12] and two reported as inactive were
selected. In Fig. 5B, selected yellow point are reported, two pre-
dicted as active compounds (MPA 73 and MPA 317, Table 2), that are
clustered with the active compounds of the training set (Table 1),
and one predicted as inactive (MPA 333, Table 2). Probably, the first
component (red axis) is discriminant for the activity of 1,2,3-
triazoles. In order to demonstrate the validity of the model all the
eighteen compounds of the test set were synthesized.
Fig. 5. AeB. Discriminant PLS t1-t2-t3 score plot reporting the projection of the test-
set onto the training set PLS model.
2.2. Chemistry
from substituted aryl amines by using t-BuONO and moist NaN3 in
t-BuOH at room temperature [41] (Scheme 1).
We recently reported the regioselective “one-pot” preparation
of 3,4-diarylisoxazoles from reaction of arylnitrile oxides and the
sodium enolates of 1-arylpropan-2-one, easily prepared by treating
the ketones with NaH in thermodynamic conditions [12,22]. Pre-
viously, we also described an efficient two-step procedure for the
synthesis of 1-aryl-1H-1,2,3-triazoles, based on the reaction of
arylazides with the lithium enolate of the acetaldehyde, followed
by the dehydration of the hydroxy-triazoline intermediates, under
basic conditions [38]. In continuation of our interest in both
developing novel heterocycles synthetic methodologies [39,40] and
to also study their biological activity, we prepared a series of novel
1,4-diaryl-1,2,3-triazoles by reaction of different arylazides with
the enolates of various 1-arylpropan-2-ones.
1,3-Diaryl-5-methyl-1,2,3-triazoles 6e16 were prepared “one-
pot” by 1,3-dipolar cycloaddition of the arylazides (1e5) to sodium
1-arylpropan-2-one enolates (Scheme 2), obtained, as previously
reported by us, by reacting the ketones with NaH at 0 ꢀC. Specif-
ically, 1-(furan-2-yl)propan-2-one sodium enolate reacted with
arylazides 3 and 4 to give 14 and 15 (Scheme 2, Table 2).
1-(4-Methoxyphenyl)-5-methyl-4-phenyl-1H-1,2,3-triazole (8)
was demethylated by using BBr3 in CH2Cl2 to prepare the corre-
sponding phenol 17 (Scheme 3).
Reduction reaction of 9 and 11 in the presence of SnCl2 and HCl
led to the preparation of 4-(5-methyl-4-phenyl-1H-1,2,3-triazol-1-
yl)benzenamine (18) and 4-(1-(4-methoxyphenyl)-5-methyl-1H-
All the arylazides were prepared in fair to good yields (40e80%)

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M.G. Perrone et al. / European Journal of Medicinal Chemistry 94 (2015) 252e264
Table 2
1,2,3-triazole-4-yl)benzenamine (19) (Scheme 4).
COX inhibitory activity of triazoles 6e23.a,b
10 and 12 CH3S- moiety was oxidized by m-chloroperox-
ybenzoic acid (mCPBA) to the corresponding methylsulfonyl
affording 20 and 21 (Scheme 5).
Triazole 14 was treated with N-chlorosuccinimide in dry DMF to
afford the chlorinated derivative 22 (Scheme 6).
Compound 23 was prepared by sulphur atom oxidation of tri-
azole 16 in the presence of mCPBA (Scheme 7).
Compound
Ar
R1
COX-1
% Inhibition
COX-2
% Inhibition
(IC50)
(IC50
)
6 (MPA 85)
7 (MPA 86)
8 (MPA 66)
9 (MPA 71)
C6H5
C6H5
C6H5
C6H5
Cl
F
OCH3
NO2
SCH3
OCH3
OCH3
OCH3
OCH3
NO2
SCH3
OH
NH2
n.a.
n.a.
26
n.a.
n.a.
20
n.a.
25
n.a.
n.a.
n.a.
n.a.
56 (15
100 (3
21
20
45
n.a.
n.a.
35
n.a.
14
n.a.
n.a.
n.a.
n.a.
40
n.a.
26
n.a.
n.a.
20
n.a.
n.a.
n.a.
2.3. COX inhibitory activity
All 1,2,3-triazoles (6e23) have been tested to exploit their
inhibitory behaviour towards both COX-1 and COX-2 isoenzymes
(Table 1). A halogen such as Cl and F in 6 and 7, respectively, did not
confer any activity to the molecules. It also occurred in the case of 9
in which the halogen is replaced by the NO2, a strong electron-
withdrawing group (EWG) endowed with -M, -I electronic fea-
tures. An unexpected behaviour was found when the phenyl of 9
was replaced by the furanyl giving 15. In the series of isoxazoles, the
replacement of a phenyl with furanyl or even better with a 5-
chlorofuran-2-yl increases the COX-1 selectivity. Instead, the tri-
10 (MPA 125)
11 (MPA 302)
12 (MPA 333)
13 (MPA 375)
14 (MPA 80)
15 (MPA 87)
16 (MPA 387)
17 (MPA 106)
18 (MPA 73)
19 (MPA 317)
20 (MPA 129)
21 (MPA 335)
22 (MPA 295)
23 (MPA 394)
C6H5
4-NO2C6H5
4-CH3SC6H5
4-CH3OC6H5
furan-2-yl-
furan-2-yl-
4-CH3OC6H5
C6H5
C6H5
4-H2NC6H5
C6H5
m
m
M)
M)
OCH3
SO2CH3
OCH3
OCH3
SO2CH3
4-CH3SO2C6H5
5-chlorofuran-2-yl-
4-CH3OC6H5
azole 15 becomes a weak COX-2 inhibitor (40% inhibition at 50
NO2 of 9 replaced by SCH3 provides 10 that has a little COX-2
inhibitory activity (14% inhibition at 50 M), both compounds are
mM).
55 (23
m
M)
m
a
6e23 were screened for their inhibitory activity on COX-1 and COX-2 at 50 mM
not active on COX-1. In our previous investigations, the methoxy
was found to be a “key group” in the interaction with COX-1 active
site [11]. In fact, the para-methoxyphenyl ring of a pyrazole deriv-
ative (MPA 14, Fig. 7) is oriented toward the apex of the COX-1
active site and forms hydrophobic interactions with amino acid
residues Phe 518, Met 522, and Ile 523, whereas its methoxy sub-
stituent lies within van der Waals contact range of the side chains of
Phe 381, Leu 384, Tyr 385, and Trp 387. Notably, the oxygen atom of
the para-methoxy group forms two contact points to the Trp 387
side chain. Trp 387 is located at the top of the COX-1 active site near
the catalytic Tyr 385 residue and has been shown to be critical for
the proper positioning of arachidonic acid within the active site to
yield the cyclooxygenase product, prostaglandin G2.
by using a colorimetric COX (ovine) inhibitor screening assay kit.
b
n.a. ¼ not active: no inhibitory activity was observed at 50
mM.
A number of others COX-1 selective inhibitors such as mofe-
zolac, SC-560 (Fig. 6) and FK881 (Fig. 1) have this group in their
chemical structure and in all cases the corresponding molecules are
Scheme 1. Reagents and conditions: (a) t-BuONO, moist NaN3, r.t [41].
Scheme 2. Reagents and conditions: (a) NaH, THF, 0 ꢀC.

M.G. Perrone et al. / European Journal of Medicinal Chemistry 94 (2015) 252e264
257
Scheme 3. Reagents and conditions: (a) BBr3, dry CH2Cl2, r.t.
Scheme 4. Reagents and conditions: (a) SnCl2/HCl, EtOH, reflux, 4 h.
Scheme 5. Reagents and conditions: (a) mCPBA, dry CH2Cl2.
Scheme 6. Reagents and conditions: (a) N-Chlorosuccinimide, dry DMF, 0 ꢀC.
Scheme 7. Reagents and conditions: (a) mCPBA, dry CH2Cl2.

258
M.G. Perrone et al. / European Journal of Medicinal Chemistry 94 (2015) 252e264
Fig. 6. Mofezolac, SC-560 and MPA 14 chemical structures and their respective COXs IC50 values.
Fig. 7. Concentration-response curves for the inhibition of COX-1 activity by P6 (:; IC50 ¼ 39
2.4
m
M), 18 ( ; IC50 ¼ 15
4.91
m
M), 19 (C; IC50 ¼ 3
0.1 mM) 23 (△;
▪
IC50 ¼ 23 2.4 M) obtained by a colorimetric COX (ovine) assay kit. IC50 values represent the mean SEM of three separate experiments.
m
potent and selective COX-1 inhibitors.
An OCH3 in the para position of the triazole N1-phenyl of 8,
inhibits the 26 and 35% (at 50 mM) of the catalytic activity of COX-1
active site. The corresonding isoxazole bearing the NH2 (MPA 4
and MPA 35MPA35, Table 1), forms strong H-bond with COX-1 Ser
530, which adopts a “down” position during the flexible docking
simulation. It does not matter if the NH2 is bound to the C5-iso-
xazole (MPA 4MPA4, Table 1) or to the phenyl at C4-isoxazole (MPA
35, Table 1). In fact, compounds 18 (MPA 73) and 19 (MPA 317)
bearing the NH2 are endowed with COX-1 inhibitory activity and
selectivity as also predicted by the Volsurf plus model.
and COX-2, respectively. In the series of the 1,2,3-triazoles the OCH3
presence does not heavily affect the COXs activity and does not
determine COX-1 selectivity. The demethylation of 8 (R1 ¼ OCH3) to
17 (R1 ¼ OH), determines a complete loss of COX-1 inhibition, a
residual COX-2 inhibition is observed (26% inhibition at 50 mM). The
inactivity is also found when the C4-phenyl ring of 8 is substituted
with a furan ring (14), whereas the introduction of a chlorine on the
furyl (22) confers approximately a doubling of the inhibitory ac-
The three triazole-nitrogen atoms do not seem to play a sig-
nificant role, it is not recognized as for the isoxazole ring [12]. In
silico investigations have shown that the first interactions are H-
bonding formation between the nitrogen and oxygen of the iso-
xazole and COX-1 Tyr 355 [located at the COX entrance, together
with Arg 120 and Glu 524 separate the membrane-binding domain
(lobby) and the core of the catalitic domain] that during energetic
stabilization, a rearrangement of the inhibitor in the active site
ultimately results in a stable complex with Ser 530 residue, posi-
tioned in the middle of the long hydrophobic channel forming the
COXs active site. In particular, a final and stable H-bonding is
formed between the furanyl-oxygen atom and the Ser 530-
hydroxyl of the COX-1 (Ser 530 is the amino acid acetylated by
aspirin during the interaction of acetyl salycilic acid and COXs).
Such a positioning of P6 and its analogues in the COX-1 active site is
one of the reasons of their inhibitory potency and selectivity. Based
on the COXs inhibition data (Table 2), the 1,2,3-triazole moiety is
not able to establish productive interactions, as it is the case of
FK881, a 1,2,4,-triazole.
tivity which increases to 45% at 50 mM. Compounds 20 and 21 have
been prepared to verify if also in the class of 1,2,3-triazole de-
rivatives the SO2Me, typical pharmacophore feature of COX-2 in-
hibitors, at the para-position of one of the aryl ring, confers COX-2
selectivity [42]. It is known that SO2CH3 is present in most of the
Coxibs and is allocated in the COX-2 site pocket not accessible in
COX-1 for the presence on the entrance of Ile 523. In our case the
methylsulfamoyl substituent is found to be detrimental for COX-2
selectivity (20). Unexpectedly, it confers a slight selectivity for
COX-1 in 21 (20% inhibition at 50 mM). It seems that the low COX-1
inhibitory activity of 8, 11, 13, and 20 should not be related to the
electronic density and/or steric hindrance of the R1 group (OCH3)
linked to the N1-triazole. The high COX-1 inhibitory potency of 23
seems due to a synergic effect of OCH3 and SO2CH3 groups,
particularly linked to their position in the molecule. Compound 21,
bearing the same groups of its regioisomer 23, is almost inactive.
The NH2 group is determinant in the interaction with COX-1
By considering the percentage inhibition of COXs by the novel

M.G. Perrone et al. / European Journal of Medicinal Chemistry 94 (2015) 252e264
259
triazoles, even when in the molecule is present a furyl, it seems that
the triazole as core ring is not able to form H-bonds similar to the
isoxazole.
Most depends upon molecule volume, electronic and steric
features of the substituents present on one or both the aryls linked
to the triazole. It seems that is also important for a specific sub-
stituent the which aryl is bonded. One aryl is linked to triazole-N1,
whereas the second to the triazole-C4.
a Varian Mercury-VX Spectrometer and chemical shifts are re-
ported in parts per million (
). 19F NMR spectra were recorded by
d
using CFCl3 as internal standard. Absolute values of the coupling
constant are reported. FT-IR spectra were recorded on a Per-
kineElmer 681 spectrometer. Thin-layer chromatography (TLC)
was performed on silica gel sheets with fluorescent indicator, the
spots on the TLC were observed under ultraviolet light. Chroma-
tography was conducted by using silica gel 60 with a particle size
In particular, the presence on N1-aryl of R1 ¼ Cl, F, OH, NO2 or
SCH3 (Scheme 2 and 3, Table 2) renders the molecule unable to
inhibit COX-1.
A p-OCH3 on the same N1-aryl allows the compounds to inhibit
COX-1, also depending on the substitution on C4-aryl. If such a
substitution is NO2, p-OCH3 or p-SO2CH3 there is a recovery of COX-
1 mediated catalysis inhibition. The less hindered furanyl gives
compounds still not 14 or poor active 15 endowed with a fair COX-2
distribution 40e63 mm and 230-400 ASTM. GCeMS analyses were
performed on an HP 5995C model and elemental analyses on an
Elemental Analyzer 1106-Carlo Erba-instrument. ESI-MS analyses
were performed on an Agilent 1100 LC/MSD trap system VL. All
synthesized compounds were analyzed by HPLC analysis per-
formed on an Agilent 1260 Infinity instrument equipped with a
1260 DAD VL þ detector, and their purity is higher than 95%.
Structure elucidation of the 1,4-diaryl-1,2,3-triazoles was per-
formed by 2D NOESY spectra recorded on a Bruker 600 MHz: as
mentioned above, with careful attention to the spatial connectivity
revealed by the correlation between diagnostic methyl and proton
signals of both aromatic rings. The 2D data of compound 8 and 14
(Figs. 8 and 9) were then used to definitively attribute chemical
structures to 1,4-diaryl-1,2,3-triazoles 6e23.
inhibition (40% at 50
molecule (22) a COX-1 inhibition (45% at 50
m
M). When the chloro-furanyl is present in the
M) is observed. Once
m
again the exchange chlorineehydrogen replacement is detrimental
for the potency and selectivity of COX inhibitors [22].
A selective and potent COX-1 inhibitions (Table 1, Fig. 7) were
instead obtained when on the para-position of N1-aryl there is
SO2CH3 and the C4-aryl is an anisole (23), or even better when R1 is
OCH3 on N1-phenyl and to the triazole-C4 is an aniline (19), or R1 of
the N1-phenyl is a NH2 and C4-aryl is phenyl (18). In fact, the three
4.1.2. General procedure for the preparation of arylazides 1e5 [41]
To a solution of moist NaN3 (6mmol/300 mL water) in t-BuOH
COX-1 IC50 values are 23, 3 and 15
m
M, respectively.
(2 mL) the opportune arylazide, dissolved in t-BuOH (3 mL), was
added dropwise. The reaction mixture was stirred overnight at
r.t. The reaction products were extracted with EtOAc. The organic
phase was dried over anhydrous Na2SO4 and then the solvent was
evaporated under reduced pressure. The product was isolated by
column chromatography (silica gel Hexane/EtOAc 8:2). All spec-
troscopic data are in accordance with published findings [44].
Antiproliferative activity of compound 18 and 19 was also
measured in MCF-7 human breast adenocarcinoma MCF-7 and
SKOV-3 cell lines. No cytotoxic effect was observed [43].
3. Conclusion
A novel set of 1,2,3-triazoles with a variety of chemical groups
with different electronic properties, size and shape were projected
to test the effect of replacing the isoxazole, pyrazole and 1,2,4-
triazole by the triazole as a core ring in diarylheterocycles COXs
inhibitors. The adopted strategy also involved a pharmacometric
molecular modeling to project active derivatives. Suitable synthetic
strategies were developed to obtain the designed compounds,
prepared in fair to good yields. Then, the novel 1,2,3-triazoles were
evaluated for their capability to inhibit COXs catalytic activity.
The pharmacometric predictive model was built by using as a
training set a number of isoxazoles of known COXs inhibitory ac-
tivity. Experiments confirmed the prediction power of the model
developed by using Volsurf plus. It was found that compounds
bearing a NH2 group would be particulary potent and selective
COX-1 inhibitor. In fact, 18 and 19 are the most potent and selective
4.1.3. General procedure for the preparation of triazoles 6e13
To a stirring suspension of NaH (1 eq.) in THF (3 mL) kept at 0 ꢀC
a solution of the proper ketone (0.5 eq.) in THF (3 mL) was added
dropwise (Scheme 2, Table 2). The reaction mixture was stirred at
0 ꢀC for 1 h. Then, a solution of the opportune arylazide (0.5 eq.) in
THF (3 mL) was added dropwise and the reaction mixture was
stirred at r.t. till the disappearance of the arylazide (18h). The re-
action mixture was quenched by adding aqueous NH4Cl, and the
reaction product was extracted three times with ethyl acetate. The
combined organic phases were dried over anhydrous Na2SO4 and
then evaporated under vacuum.
4.1.3.1. 1-(4-Chlorophenyl)-5-methyl-4-phenyl-1H-1,2,3-triazole (6).
The product was isolated as a yellow solid (14% yield) by column
chromatography (silica gel, hexane/EtOAc ¼ 7:3); mp: 138e140 ꢀC;
COX-1 inhibitors with IC50 values of 15 and 3
mM, respectively.
Another interesting and unexpected result derived from the COXs
inhibitory activity and COX-1 selectivity of 23 bearing the SO2CH3
group, the classical “key group” known to achieve COX-2 selectivity.
1H NMR (300 MHz, CDCl3):
d 7.77e7.74 (m, 2H, aromatic protons),
7.56e7.53 (m, 2H, aromatic protons), 7.51e7.36 (m, 5H, aromatic
protons), 2.48 ppm (s, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d
145.3,
It inhibited only the COX-1 (IC50 of 23
COX-2 catalytic activity.
In order to improve our knowledge on selective COXs inhibition
(particularly COX-1 selective inhibition), investigations of inhibitor-
protein interactions by pharmacometric modeling/X-ray/NMR/ESR
measurements will be the subsequent step.
m
M) leaving unchanged the
135.8, 135.1, 131.4, 130.0, 129.8, 129.0, 128.2, 127.5, 126.7, 10.5. FT-IR
(KBr): 3057, 1503, 1459, 1445, 1406, 1384, 1253, 1120, 1095, 1072,
1007, 838, 768, 717, 695 cmꢁ1; GCeMS (70 eV) m/z (rel.int.): 271
[(Mþ þ 2), 1], 269 (Mþ, 4), 241 (100), 206 (63), 191 (5), 178 (8), 165
(85), 115 (15), 103 (65), 89 (15), 77 (27).
4.1.3.2. 1-(4-Fluorophenyl)-5-methyl-4-phenyl-1H-1,2,3-triazole (7).
4. Experimental
The product was isolated as a yellow oil (20% yield) by column
chromatography (silica gel, hexane/EtOAc
¼
9:1). 1H NMR
4.1. Chemistry
(300 MHz, CDCl3): 7.78e7.75 (m, 2H, aromatic protons), 7.52e7.46
d
(m, 4H, aromatic protons), 7.41e7.35 (m, 2H, aromatic protons),
4.1.1. General methods
7.30e7.22 (m, 2H, aromatic protons), 2.46 (s, 3H, CH3). 13C NMR
Melting points taken on Electrothermal apparatus were uncor-
(75 MHz, CDCl3):
d
163.2 (d, 1J19Fe13C ¼ 250 Hz), 145.1, 132.7 (d,
rected. 1H NMR and 13C NMR spectra were recorded on 300 MHz on
3J19Fe13C ¼ 7.9 Hz), 131.5, 130.0, 129.0, 128.1, 127.6, 127.4, 116.9 (d,

260
M.G. Perrone et al. / European Journal of Medicinal Chemistry 94 (2015) 252e264
Fig. 8. 2D NOESY experiment for compound 8.
Fig. 9. 2D NOESY experiment of compound 14.
2J19Fe13C ¼ 23 Hz), 10.4. 19F NMR (376 MHz, CDCl3):
d
¼ - 111.37. FT-
(m, 2H, aromatic protons), 3.89 (s, 3H, OCH3), 2.45 (s, 3H, CH3). 13
NMR (75 MHz, CDCl3): 160.5, 144.8, 131.7, 130.2, 129.5, 129.0,
C
IR (KBr): 3051, 1604, 1518, 1444, 1415, 1383, 1236, 1157, 1115, 1091,
841, 820, 769, 720, 698 cm-1. GCeMS (70 eV) m/z (rel. int.): 253 (Mþ,
3), 225 (100), 210 (4), 196 (4), 183 (85), 148 (18), 122 (8), 103 (41).
d
128.0, 127.4, 126.9, 114.8, 55.9, 10.4. FT-IR (KBr): 3053, 2967, 2932,
1610, 1519, 1444, 1307, 1257, 1020, 841, 775, 697 cmꢁ1. GCeMS
(70 eV) m/z (rel. int.): 265 (Mþ, 2), 237 (100), 222 (53), 207 (5), 194
(66), 165 (12), 152 (22), 103 (20). The unambiguous structure
elucidation of this product was achieved by 2D NOESY spectrum:
in, the diagnostic cross-peaks between the methyl protons
(2.4 ppm) and both the aromatic proton signals (7.4 and 7.8 ppm,
respectively) were evidenced (Fig. 8).
4.1.3.3. 1-(4-Methoxyphenyl)-5-methyl-4-phenyl-1H-1,2,3-triazole
(8). The product was isolated as a yellow solid (48% yield) by col-
umn chromatography (silica gel, hexane/EtOAc
132.0e135.0 ꢀC. 1H NMR (300 MHz, CDCl3):
7.79e7.78 (m, 2H,
aromatic protons), 7.76e7.34 (m, 5H, aromatic protons), 7.08e7.03
¼
9:1); mp:
d

M.G. Perrone et al. / European Journal of Medicinal Chemistry 94 (2015) 252e264
261
4.1.3.4. 5-Methyl-1-(4-nitrophenyl)-4-phenyl-1H-1,2,3-triazole (9)
[45]. The product was isolated as a yellow solid (70% yield) by
column chromatography (silica gel, hexane/EtOAc ¼ 7:3); mp:
(300 MHz, CDCl3): d 7.52e7.51 (m, 1H, furyl proton); 7.41e7.36 (m,
2H, aromatic protons); 7.07e7.02 (m, 2H, aromatic protons);
6.83e6.82 (m, 1H, furyl proton); 6.53e6.51 (m, 1H, furyl proton);
3.88 (s, 3H, OCH3); 2.49 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3):
197.4e200.2 ꢀC. 1H NMR (300 MHz, CDCl3):
d 8.49e8.44 (m, 2H,
aromatic protons); 7.81e7.75 (m, 4H, aromatic protons); 7.53e7.48
(m, 2H, aromatic protons); 7.43e7.39 (m, 1H, aromatic proton); 2.58
d 160.6, 147.3, 142.2, 137.9, 130.0, 129.1, 126.9, 114.9, 111.5, 106.9,
55.9, 9.8. FT-IR (KBr): 3130, 2919, 1610, 1518, 1463, 1384, 1302, 1255,
1020, 838 cmꢁ1. GCeMS (70 eV) m/z (rel. int.): 255 (Mþ,94) 227
(66), 212 (65),198 (52),184 (100),167 (27),115 (52), 92 (54), 77 (77),
64 (43). The unambiguous structure elucidation of this product was
achieved by 2D NMR spectroscopy (Fig. 9), as evidenced by the
diagnostic cross-peaks between the methyl protons (2.49 ppm) and
the proton signals of both the aromatic rings (7.40 and 6.84 ppm,
respectively).
(s, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d 148.0, 146.2, 141.4, 130.9,
129.7, 129.1, 128.5, 127.6, 125.6, 125.4, 10.8. FT-IR (KBr): 3093, 2922,
1615, 1599, 1523, 1503, 1346, 1268, 863, 855 cmꢁ1. GCeMS (70 eV)
m/z (rel. int.): 280 (Mþ, 6), 252 (44), 206 (100), 191 (14), 178 (19),
165 (43), 115 (15), 103 (37).
4.1.3.5. 5-Methyl-1-[4-(methylthio)phenyl]-4-phenyl-1H-1,2,3-
triazole (10). The product was isolated as an orange solid (35%
yield) by column chromatography (silica gel, hexane/EtOAc ¼ 7:3);
4.1.3.10. 4-(Furan-2-yl)-5-methyl-1-(4-nitrophenyl)-1h-1,2,3-
triazole (15). The product was isolated as a yellow oil (7% yield) by
column chromatography (silica gel, HEXANE/EtOAc 7:3); 1H NMR
mp: 149.1e153.0 ꢀC. 1H NMR (300 MHz, CDCl3):
d 7.79e7.76 (m, 2H,
aromatic protons); 7.51e7.40 (m, 7H, aromatic protons); 2.55 (s, 3H,
SCH3); 2.47 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d
145.1, 141.2,
(300 MHz, CDCl3): d 8.47e8.43 (m, 2H, aromatic protons);
133.4, 131.6, 129.9, 129.0, 128.0, 127.5, 127.0, 125.8, 15.8, 10.5. FT-IR
(KBr): 3053, 2919, 2851, 1610, 1504, 1438, 1402, 1384, 1258, 1094,
1074, 820, 837, 771, 716, 698 cmꢁ1. GCeMS (70 eV) m/z (rel. int.):
281 (Mþ, 4), 253 (68), 238 (100), 220 (14), 206 (99), 178 (15), 165
(48), 150 (9), 130 (8), 115 (11), 103 (44).
7.79e7.74 (m, 2H, aromatic protons); 7.55e7.54 (m, 1H, furyl pro-
ton); 6.90e6.88 (m, 1H, furyl proton); 6.56e6.55 (m, 1H, furyl
proton); 2.64 (s, 3H, CH3). ESI-MS: m/z (%): C13H10N4O3, 293
(M þ Na)þ.
4.1.3.11. 4-(4-Methoxyphenyl)-5-methyl-1-[4-(methylthio)phenyl]-
1H-1,2,3-triazole (16). The product was isolated as a white solid
(33% yield) by crystallization from hexane/CHCl3); mp:
4.1.3.6. 1-(4-Methoxyphenyl)-5-methyl-4-(4-nitrophenyl)-1H-1,2,3-
triazole (11). The product was isolated as a brown solid (95% yield)
by column chromatography (silica gel, hexane/EtOAc ¼ 7:3); mp:
184.9e185 ꢀC; 1H NMR (300 MHz, CDCl3):
d 7.70e7.68 (m, 2H, ar-
225e230 ꢀC. 1H NMR (300 MHz, CDCl3):
d
8.36e8.33 (m, 2H, aro-
omatic protons); 7.44e7.37 (m, 4H, aromatic protons); 7.03e6.99
(m, 2H, aromatic protons); 3.86 (s, 3H, OCH3); 2.55 (s, 3H, SCH3);
matic protons); 8.01e7.97 (m, 2H, aromatic protons); 7.43e7.40 (m,
2H, aromatic protons), 7.09e7.07 (m, 2H, aromatic protons); 3.90 (s,
2.44 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d 159.5, 144.9, 141.1,
3H, OCH3); 2.52 (s, 3H, CH3). 13C NMR (75 MHz, DMSO-d6):
d
160.8,
133.5, 129.2, 128.7, 126.9, 125.7, 124.2, 114.4, 55.6, 55.5, 10.5. FT-IR
(KBr): 2920, 2834, 1640, 1614, 1510, 1384, 1247, 1384, 1247, 1177,
1026, 832, 528 cmꢁ1. GCeMS (70 eV) m/z (rel. int.): 311 (20), 283
(37), 268 (48), 236 (100), 152 (12), 77 (11). HRMS (ESI) Calcd for
147.2, 142.6, 138.3, 131.6, 128.9, 127.5, 127.1, 124.4, 115.0, 55.9, 10.7.
ESI-MS: m/z (%): C16H14N4O3, 333 (M þ Na)þ. FT-IR (KBr): 3432,
2965, 2921, 1601, 1564, 1519,1503, 1488, 1337, 1254, 1169, 835,
717 cmꢁ1
.
C
17H18N3OS: 312,1171 [MþH]þ, Found: 312,1156 [MþH]þ.
4.1.3.7. 1-(4-Methoxyphenyl)-5-methyl-4-[4-(methylthio)phenyl]-
1H-1,2,3-triazole (12). The product was isolated as a white solid
(50% yield) by column chromatography (silica gel, hexane/
EtOAc ¼ 7:3); mp: 184e186 ꢀC; 1H NMR (300 MHz, CDCl3):
4.1.3.12. Synthesis of 4-(5-methyl-4-phenyl-1H-1,2,3-triazol-1-yl)
phenol (17) [44]. To a stirred solution of 1-(4-methoxyphenyl)-5-
methyl-4-phenyl-1H-1,2,3-triazole (8) (83 mg, 0.31 mmol) in dry
CH2Cl2 (5 mL) kept at ꢁ50 ꢀC, BBr3 (1 mL, 0.31 mmol) was added.
The stirred reaction mixture was kept at room temperature for 1 h.
Then, NaHCO3 (5 mL) was added and the aqueous phase extracted
three times with EtOAc. The combined organic extracts were dried
over anhydrous Na2SO4 and the solvent was removed under
reduced pressure. The product was isolated as a white solid (51%
yield) by column chromatography (silica gel, hexane/EtOAc ¼ 1:1);
d
7.72e7.68 (m, 2H, aromatic protons); 7.42e7.26 (m, 4H, aromatic
protons); 7.25e7.04 (m, 2H, aromatic protons); 3.89 (s, 3H, OCH3);
2.53 (s, 3H, SCH3); 2.43 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d
159.6, 145.7, 141.2, 140.7129.1, 128.7, 128.7125.6, 123.3, 114.3, 55.3,
44.5, 10.5. FT-IR (KBr): 2960, 2923, 2851, 1519, 1304, 1259,
1149,1092, 1016, 838, 803 cm-1. GCeMS (70 eV) m/z (rel. int.): 311
(Mþ,8), 283 (100), 268 (79), 253 (9), 240 (31), 221 (14), 207 (7), 195
(12), 165 (7), 152 (21),134 (14), 120 (9), 103 (6), 92 (11), 77 (12), 64
(8).
mp: 200e203 ꢀC. 1H NMR (300 MHz, CDCl3):
d 10.04 (OH, exchange
with D2O); 7.75e7.74 (m, 2H, aromatic protons); 7.72e7.48 (m, 2H,
aromatic protons); 7.47e7.37 (m, 3H, aromatic protons), 6.97e6.94
(m, 2H, aromatic protons); 2.38 (s, 3H, CH3). 13C NMR (75 MHz,
4.1.3.8. 1,4-Bis-(4-methoxyphenyl)-5methyl-1H-1,2,3-triazole (13).
The product was isolated as a brown solid (41% yield) by column
chromatography (silica gel, hexane/EtOAc ¼ 6:4); mp: 162e163 ꢀC;
DMSO-d6): d 159.1,143.8,132.1,131.1,129.5,128.2,128.1,127.5,127.3,
116.6, 10.5. ESI-MS: m/z (%): C15H13N3O, 274 (M þ Na)þ. FT-IR (KBr):
3445 (broad), 2923, 2852, 1637, 1513, 1494, 1456, 1076, 843,
1H NMR (300 MHz, CDCl3):
d
7.74e7.64 (m, 2H, aromatic protons);
780 cmꢁ1
.
7.46e7.37 (m, 2H, aromatic protons); 7.29e7.20 (m, 4H, aromatic
protons); 3.88 (s, 3H, OCH3); 3.85 (s, 3H, OCH3); 2.41 (s, 3H, CH3). 13
NMR (75 MHz, CDCl3): 160.5, 159.5, 144.7, 129.5, 128.7, 126.9,
124.3, 114.8, 114.4, 56.0, 55.7, 55.4, 10.4. FT-IR (KBr): 2955, 2934,
2837, 1617, 1520, 1467, 1439, 1247, 1176, 1117, 1049, 1024, 834 cmꢁ1
C
4.1.4. General procedure for the synthesis of 4-(5-methyl-4-phenyl-
1H-1,2,3-triazol-1-yl)benzenamine (18) and 4-[1-(4-
d
methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-yl]benzenamine (19)
To a stirred solution of stannous chloride (0.72 mmol) in HCl 37%
(1 mL) was added dropwise 5-methyl-1-(4-nitrophenyl)-4-phenyl-
1H-1,2,3-triazole (9) (0.18 mmol) or 1-(4-methoxyphenyl)-5-
methyl-4-(4-nitrophenyl)-1H-1,2,3-triazole (11) (0.18 mmol) dis-
solved in absolute EtOH (12 mL). The stirred reaction mixture was
kept at 80 ꢀC for 4 h. Then 10% NaOH (10 mL) was added to the
reaction mixture till pH ¼ 12, and the aqueous phase was extracted
.
GCeMS (70 eV) m/z (rel. int.): 295 (15), 267 (100), 266 (95), 224
(25), 211 (20), 92 (22), 77 (25).
4.1.3.9. 4-(Furan-2-yl)-1-(4-methoxyphenyl)-5-methyl-1h-1,2,3-
triazole (14). The product was isolated as a brown oil (45% yield) by
column chromatography (silica gel, hexane/EtOAc ¼ 7:3); 1H NMR

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M.G. Perrone et al. / European Journal of Medicinal Chemistry 94 (2015) 252e264
three times with EtOAc. The combined organic extracts were dried
over anhydrous Na2SO4 and the solvent removed under reduced
pressure.
2H, aromatic protons); 3.90 (s, 3H, OCH3); 3.10 (s, 3H, SCH3); 2.50 (s,
3H, CH3). 13C NMR (75 MHz, CDCl3):
160.8, 142.9, 139.4, 137.3,
131.4, 129.0, 128.1, 127.7, 127.0, 115.0, 55.9, 44.7, 10.6. FT-IR (KBr):
3421,2961, 2924, 2852, 2361, 2343, 1734, 1609, 1518, 1458, 1303,
1148, 1091, 1016, 802 cmꢁ1. ESI-MS: m/z (%): C17H17N3O3S, 366
(M þ Na)þ.
d
4.1.4.1. 4-(5-Methyl-4-phenyl-1H-1,2,3-triazol-1-yl)benzenamine
(18) [45]. The product was isolated as a brown solid (58% yield) by
column chromatography (silica gel, petroleum ether/EtOAc ¼ 8:2);
mp: 181e184 ꢀC. 1H NMR (300 MHz, CDCl3):
d
7.79e7.76 (m, 2H,
4.1.5.3. 4-(4-Methoxyphenyl)-5-methyl-1-[4-(methylsulphonyl)
phenyl]-1H-1,2,3-triazole (23). The product was isolated as a white
solid (60% yield) by column chromatography (silica gel, hexane/
EtOAc ¼ 3:7); mp: 240.9e241.5 ꢀC; 1H NMR (300 MHz, CDCl3):
aromatic protons); 7.51e7.31 (m, 3H, aromatic protons); 7.28e7.23
(m, 2H, aromatic protons), 6.91e6.78 (m, 2H, aromatic protons);
4.95 (NH2, exchange with D2O); 2.44 (s, 3H, CH3). 13C NMR (75 MHz,
CDCl3):
115.3, 10.4. FT-IR (KBr): 3419, 3347, 3237, 1645,1607, 1521, 1492,
1442, 1308, 1257, 1170, 1134, 1073, 830, 771, 720, 704, 697 cmꢁ1
d
147.9, 144.6, 131.9, 130.1, 128.9, 127.9, 127.3, 127.3, 126.9,
d 8.19e8.15 (m, 2H, aromatic protons); 7.81e7.77 (m, 2H, aromatic
protons); 7.71e7.66 (m, 2H, aromatic protons); 7.04e7.00 (m, 2H,
aromatic protons); 3.13 (s, 3H, OCH3); 2.53 (s, 3H, SO2CH3); 1.25 (s,
.
GCeMS (70 eV) m/z (rel. int.): 250 (2), 222 (100), 204 (75), 180 (31),
3H, CH3). 13C NMR (75 MHz, CDCl3):
d 159.6, 145.0, 141.1, 133.5,
145 (12), 118 (9), 103 (15), 89 (6), 65 (15). HRMS (ESI) Calcd for
C
129.2, 128.7, 127.0, 125.7, 124.2, 114.4, 55.6, 38.4, 10.5. ESI-MS: m/z
(%): C16H14N4O3, 366 (M þ Na)þ. HRMS (ESI) Calcd for C17H18N3O3S:
344,1069 [MþH]þ, Found: 344,1073 [MþH]þ.
15H15N4: 251.1291 [MþH]þ, Found: 251.1292 [MþH]þ.
4.1.4.2. 4-[1-(4-Methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-yl]
benzenamine (19). The product was isolated as a brown solid (30%
yield) by column chromatography (silica gel, hexane/EtOAc ¼ 3:7);
4.1.6. Synthesis of 4-(5-chlorofuran-2-yl)-1-(4-methoxyphenyl)-5-
methyl-1H-1,2,3-triazole (22)
mp: 165e166 ꢀC; 1H NMR (300 MHz, CDCl3):
d
7.58e7.55 (m, 2H,
A mixture of 4-(furan-2-yl)-1-(4-methoxyphenyl)-5-methyl-
1H-1,2,3-triazole (14) (65 mg, 0.255 nmol) in anhydrous DMF
(4 mL) was stirred at 0 ꢀC under inert atmosphere. The reaction
mixture was allowed to reach room temperature, then N-chlor-
osuccinimide (36 mg, 0.273 mmol) was added at 10 min intervals
partitioned in three times. The reaction mixture was monitored by
TLC (silica gel, hexane/EtOAc ¼ 8:2) and stirred until the disap-
pearance of 14 (approximately 5 h). The reaction mixture was
quenched by adding H2O. The reaction product was extracted three
times with ethyl acetate. The combined organic phases were dried
over anhydrous Na2SO4 and the solvent evaporated under vacuum.
The product was isolated as a yellow oil (45% yield) by column
aromatic protons); 7.42e7.39 (m, 2H, aromatic protons); 7.06e7.03
(m, 2H, aromatic protons), 6.80e6.77 (m, 2H, aromatic protons);
3.88 (s, 3H, OCH3); 3.48 (NH2, exchange with D2O); 2.40 (s, 3H, CH3).
13C NMR (75 MHz, CDCl3):
d 160.4, 146.3, 145.1, 129.7, 129.0, 128.6,
126.9, 122.1, 115.0, 114.8, 55.8, 22.9. ESI-MS: m/z (%): C16H16N4O, 303
(M þ Na)þ. FT-IR (KBr): 2956, 2918, 2850, 2581, 1610, 1513, 1463,
1254, 1096, 1027, 833 cm-1. HRMS (ESI) Calcd for C16H17N4O:
281,1402 [MþH]þ, Found: 281,1395 [MþH]þ.
4.1.5. General procedure for the synthesis of 5-methyl-1-[4-
(methylsulfonyl)phenyl]-4-phenyl-1H-1,2,3-triazole (20) and 1-(4-
methoxyphenyl)-5-methyl-4-[4-(methylsulfonyl)phenyl]-1H-1,2,3-
triazole (21) and 4-(4-methoxyphenyl)-5-methyl-1-(4-
chromatography (silica gel, hexane/EtOAc
¼
8:2); 1H NMR
(300 MHz, CDCl3): 7.40e7.36 (m, 2H, aromatic protons);
d
methylsulfonylphenyl)-1H-1,2,3-triazole (23)
m-Chloroperoxybenzoic acid (0.26 mmol) was added to an ice-
cold stirred solution of 5-methyl-1-[4-(methylthio)phenyl]-4-
7.08e7.04 (m, 2H, aromatic protons); 6.83e6.81 (m, 1H, furyl pro-
ton); 6.30e6.29 (m, 1H, furyl proton); 3.89 (s, 3H, OCH3); 2.48 (s,
3H, CH3). 13C NMR (75 MHz, CDCl3):
d 160.7, 146.8, 137.0, 136.2,
phenyl-1H-1,2,3-triazole
(10)
(0.13
mmol)
or
1-(4-
130.2, 129.0, 126.9, 114.9, 109.0, 108.2, 55.9, 9.8. FT-IR (KBr): 2918,
2850, 2360, 2343, 1703, 1519, 1256, 1216, 836 cm-1. GCeMS (70 eV)
m/z (rel. int.): 289 (Mþ,14), 261 (23), 246 (21), 226 (100), 218 (26),
198 (52), 183 (40), 155 (24), 113 (12), 92 (19), 77 823), 64 (15). HRMS
(ESI) Calcd for C14H13ClN3O2: 290,0696 [MþH]þ, Found: 290,0692
[MþH]þ.
methoxyphenyl)-5-methyl-4-[4-(methylthio)phenyl]-1H-1,2,3-
triazole (12) or 4-(4-methoxyphenyl)-5-methyl-1-[4-(methylthio)
phenyl]-1H-1,2,3-triazole (16) in dry CH2Cl2 (5 mL). The reaction
mixture was stirred and slowly allowed to reach room temperature.
Then, the reaction mixture was stirred for 1 h. Water (5 mL) was
added and the aqueous phase extracted three times with EtOAc.
The combined organic extracts were dried over anhydrous Na2SO4
and the solvent removed under reduced pressure.
4.2. Computational methods
4.2.1. VolSurf plus descriptors
4.1.5.1. 5-Methyl-1-[4-(methylsulfonyl)phenyl]-4-phenyl-1H-1,2,3-
triazole (20). The product was isolated as a white solid (42% yield)
by column chromatography (silica gel, hexane/EtOAc ¼ 3:7); mp:
The interaction of molecules with biological membranes is
mediated by surface properties such as shape, electrostatic forces,
H-bonds and hydrophobicity. Therefore, the GRID [17] force field
was chosen to characterize potential polar and hydrophobic inter-
action sites around target molecules by the water (OH2), the hy-
drophobic (DRY), and the carbonyl oxygen (O) and amide nitrogen
(N1) probe. The information contained in the MIF is transformed
into a quantitative scale by calculating the volume or the surface of
the interaction contours. The VolSurf plus procedure is as follows: i)
in the first step, the 3D molecular field is generated from the in-
teractions of the OH2, the DRY, O and N1 probe around a target
molecule; ii) the second step consists in the calculation of de-
scriptors from the 3D maps obtained in the first step. The molecular
descriptors obtained, called VolSurf plus descriptors, refer to mo-
lecular size and shape, to hydrophilic and hydrophobic regions and
to the balance between them, to molecular diffusion, LogP, LogD, to
the “charge state” descriptors, to the new 3D pharmacophoric
231e234 ꢀC. 1H NMR (300 MHz, CDCl3):
d 8.18e8.16 (m, 2H, aro-
matic protons); 7.81e7.52 (m, 4H, aromatic protons); 7.50e7.47 (m,
2H, aromatic protons), 7.43e7.40 (m, 1H, aromatic protons); 3.14 (s,
3H, SO2CH3); 2.56 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d 146.0,
141.5, 140.8, 131.0, 129.3, 129.1, 128.4, 127.6, 125.9, 44.7, 10.7. FT-IR
(KBr): 3025, 3010, 2958, 1505, 1446, 1385, 1300, 1155, 788, 746,
692 cmꢁ1. GCeMS (70 eV) m/z (rel. int.): 313 (2), 285 (12), 207 (15),
206 (100), 205 (11), 204 (16), 178 (11), 165 (24), 103 (21).
4.1.5.2. 1-(4-Methoxyphenyl)-5-methyl-4-[4-(methylsulfonyl)
phenyl]-1H-1,2,3-triazole (21). The product was isolated as a white
solid (80% yield) by crystallization from hexane/EtOAc; mp:
241e242 ꢀC; 1H NMR (300 MHz, CDCl3):
d
8.07e7.99 (m, 4H, aro-
matic protons); 7.43e7.40 (m, 2H, aromatic protons); 7.09e7.06 (m,

M.G. Perrone et al. / European Journal of Medicinal Chemistry 94 (2015) 252e264
263
descriptors and to some descriptors on some rilevant ADME
properties. In total VolSurf plus uses 128 descriptors [46e48]; iii)
finally, chemometric tools (PCA [49], PLS [50,51]) are used to create
relationships of the VolSurf plus descriptor matrix with ADME
properties. The scheme of the VolSurf plus programme steps and a
detailed definition of Volsurf plus descriptors have recently been
reported [52].
Cicco for her contribution to the synthesis of some of novel target
compounds and to Dr. Francesco Cannito and to Consorzio Inter-
universitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici
(C.I.R.C.M.S.B.) Bari for HR-MS analysis.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.02.049.
4.3. Biology
4.3.1. Cyclooxygenase inhibition studies
References
The target compounds 6e23 were evaluated for their ability to
inhibit ovine COX-1/COX-2 enzyme (percent inhibition at 50 mM).
[1] M.G. Perrone, A. Scilimati, L. Simone, P. Vitale, Selective COX-1 inhibition: a
therapeutic target to be reconsidered, Curr. Med. Chem. 17 (2010)
3769e3805.
Inhibition of the enzyme was determined using a colorimetric COX
(ovine) inhibitor screening assay kit (Catalog No. 760111, Cayman
Chemicals, Ann Arbor, MI, USA) following the procedure described
in the catalog according to the manufacturer's instructions.
COX is a bifunctional enzyme exhibiting both cyclooxygenase
and peroxidase activity. The COX component converts arachidonic
acid to a hydroperoxide (PGG2), and the peroxidase component
reduces the endoperoxide to the corresponding alcohol (PGH2), the
precursor of PGs, thromboxanes, and prostacyclins. The Colori-
metric COX Inhibitor Screening Assay measures the peroxidase
component of overall cyclooxygenases activity. The peroxidase
activity is assayed colorimetrically by monitoring the appearance of
oxidized N,N,N0,N'-tetramethyl-p-phenylenediamine (TMPD) at
590 nm. 6e23 were dissolved in a minimum volume of DMSO to
prepare to stock solutions.
[2] M.G. Perrone, P. Malerba, J. Uddin, P. Vitale, A. Panella, B.C. Crews, C.K. Daniel,
K. Ghebreselasie, M. Nickels, M.N. Tantawy, H.C. Manning, L.J. Marnett,
A. Scilimati, PET radiotracer [18F]-P6 selectively targeting COX-1 as a novel
biomarker in ovarian cancer: preliminary investigation, Eur. J. Med. Chem. 80
(2014) 562e568.
€
[3] J.M. Schwab, R. Beschorner, R. Meyermann, F. Gozalan, H.J. Schluesener,
Persistent accumulation of cyclooxygenase-1-expressing microglial cells and
macrophages and transient upregulation by endothelium in human brain
injury, J. Neurosurg. 96 (2002) 892e899.
[4] O. Pepicelli, E. Fedele, M. Berardi, M. Raiteri, G. Levi, A. Greco, M.A. Ajmone-
Cat, L. Minghetti, Cyclooxygenase-1 and -2 differently contribute to prosta-
glandin E2 synthesis and lipid peroxidation after in vivo activation of N-
methyl-d-aspartate receptors in rat hippocampus, J. Neurochem. 93 (2005)
1561e1567.
[5] R. Calvello, M.A. Panaro, M.L. Carbone, A. Cianciulli, M.G. Perrone, P. Vitale,
P. Malerba, A. Scilimati, Novel selective COX-1 inhibitors suppress neuro-
inflammatory mediators in LPS-stimulated N13 microglial cells, Pharmacol.
Res. 66 (2012) 137e148.
€
ꢁ
[6] E. Candelario-Jalil, A.C. de Oliveira, S. Graf, H.S. Bhatia, M. Hüll, E. Munoz,
B.L. Fiebich, Resveratrol potently reduces prostaglandin E2 production and
free radical formation in lipopolysaccharide-activated primary rat microglia,
J. Neuroinflamm. 4 (2007) 25e37.
4.3.2. Antiproliferative assay
Determination of cell growth was performed using the MTTas-
say at 48 h. On day 1,30,000 cells/well were seeded into 96-well
[7] S.H. Choi, R. Langenbach, F. Bosetti, Genetic deletion or pharmacological in-
hibition of cyclooxygenase-1 attenuate lipopolysaccharide-induced inflam-
matory response and brain injury, FASEB J. 22 (2008) 1491e1501.
[8] J.R. Barrio, N. Satyamurthy, S. Huang, A. Petric, G.W. Small, V. Kepe, Dissecting
molecular mechanisms in the living brain of dementia patients, Acc. Chem.
Res. 42 (2009) 842e850.
[9] M.G. Perrone, D.D. Lofrumento, P. Vitale, F. De Nuccio, V. La Pesa, A. Panella,
R. Calvello, A. Cianciulli, M.A. Panaro, A. Scilimati, Selective Cyclooxygenase-1
inhibition by P6 and gastrotoxicity: preliminary investigation, Pharmacology
(2015) 22e28.
plates in a volume of 100
mL. On day 2, the various drugs concen-
tration (0.1e100 M) were added. In all the experiments, the drug
m
solvent (DMSO) was added in each control to evaluate a possible
solvent cytotoxicity. After the established incubation time with
drugs, MTT (0.5 mg/mL) was added to each well, and after 3 h in-
cubation at 37 ꢀC, the supernatant was removed. The formazan
crystals were solubilized using 100 mL of DMSO and the absorbance
[10] L. Di Nunno, P. Vitale, A. Scilimati, S. Tacconelli, P. Patrignani, Novel synthesis
of 3,4-diarylisoxazole analogues of valdecoxib: reversal cyclooxygenase-2
selectivity by sulfonamide group removal, J. Med. Chem. 47 (2004)
4881e4890.
values at 570 nm was determined on the microplate reader Victor 3
from PerkinElmer Life Sciences.
[11] M.G. Perrone, P. Vitale, P. Malerba, A. Altomare, R. Rizzi, A. Lavecchia, C. Di
Giovanni, E. Novellino, A. Scilimati, Diarylheterocycle core ring features effect
in selective COX-1 inhibition, Chem. Med. Chem. 7 (2012) 629e641.
[12] P. Vitale, M.G. Perrone, P. Malerba, A. Lavecchia, A. Scilimati, Selective COX-1
inhibition as a target of theranostic novel diarylisoxazoles, Eur. J. Med. Chem.
74 (2014) 606e618.
[13] V.S. Kristi, A.W. Barbara, Trends in Cancer Therapy: Role of Monoclonal An-
tibodies, Sem. Oncol. Nurs. 19 (2003) 169e209.
[14] D.R. Buckle, D.J. Outred, C.J.M. Rockell, H. Smith, B.A. Spicer, Studies on 1,2,3-
4.4. Statistical analysis
The IC50 values of the compounds reported in Fig. 7 and Table 2
were determined by nonlinear curve fitting using the GraphPad
Prism program (GraphPad Prism Software (Windows version),
Graph-Pad Software, Inc., San Diego, CA, USA) and are the
mean SEM from three separate experiments.
triazoles.
(Piperazinylalkoxy)-[1]-benzopyrano-[2,3-d]-1,2,3-triazol-9(1H)-
ones with combined H1-antihistamine and mast cell stabilizing properties,
J. Med. Chem. 29 (1986) 2262e2267.
Acknowledgements
[15] M.J. Genin, D.A. Allwine, D.J. Anderson, M.R. Barbachyn, D.E. Emmert,
S.A. Garmon, D.R. Graber, K.C. Grega, J.B. Hester, D.K. Hutchinson, J. Morris,
R.J. Reischer, C.W. Ford, G.E. Zurenko, J.C. Hamel, R.D. Schaadt, D. Stapert,
B.H. Yagi, Substituent effects on the antibacterial activity of nitrogen-carbon-
linked (azolylphenyl)oxazolidinones with expanded activity against the
fastidious gram-negative organisms Haemophilus influenzae and Moraxella
catarrhalis, J. Med. Chem. 43 (2000) 953e970.
[16] J. Imanishi, Y. Morita, E. Yoshimi, K. Kuroda, T. Masunaga, K. Yamagami,
M. Kuno, E. Hamachi, S. Aoki, F. Takahashi, K. Nakamura, S. Miyata, Y. Ohkubo,
S. Mutoh, Pharmacological profile of FK881 (ASP6537), a novel potent and
selective cyclooxygenase-1 inhibitor, Biochem. Pharmacol. 82 (2011)
746e754.
[17] S.P. Bathula, R. Vadla, Bioactivity of 1, 4-Disubstituted-1,2,3-Triazoles as
cytotoxic agents against the various Human Cell Lines, Asian J. Pharm. Clin.
Res. 4 (2011) 66e67.
[18] G. Cruciani, P. Crivori, P.A. Carrupt, B. Testa, Molecular Fields in quantitative
structure-permeation relationships: the VolSurf approach, Theochem 503
(2000) 17e30.
Thanks are due to Laboratorio di Ricerca per la Diagnostica dei
Beni Culturali (University of Bari) and to C.I.N.M.P.I.S. Consortium
for NMR facilities. Thanks are due also to (a) Public Research Lab-
oratories Networks of Regione Puglia-APQ Research Programme
‘Integrated renewable energy generation from the regional agro-
industrial sector. Project Code 01’. (Intervento cofinanziato dal-
l’Accordo di Programma Quadro in materia di Ricerca Scientifica e
II Atto Integrativo e PO FESR 2007e2013, Asse I, Linea 1.2-PO FSE
2007e2013 Asse IV ‘‘Investiamo nel vostro futuro’’); (b) MIUR
(Rome e Italy) for Progetti di Ricerca Industriale nell'ambito del
Programma Operativo Nazionale R&C 2007e2013 e Project “Rea-
search, Application, Innovation, Services in Bioimaging (R.A.I.SE.)”
code PON01_03054. The authors would like to thank Dr. Luciana

264
M.G. Perrone et al. / European Journal of Medicinal Chemistry 94 (2015) 252e264
[19] The VolSurfþ Manual available at: http://www.moldiscovery.com/docs/
vsplus/.
[20] H.C. Kolb, K.B. Sharpless, The growing impact of click chemistry on drug
discovery, Drug Discov. Today 8 (2003) 1128e1137.
ring, J. Chem. Soc. Perkin Trans. 1 (1998) 1677e1684.
[36] L. Zhou, A. Amer, M. Korn, R. Burda, J. Balzarini, E. De Clercq, E.R. Kern,
P.F. Torrence, Synthesis and antiviral activities of 1,2,3-triazole functionalized
thymidines: 1,3-dipolar cycloaddition for efficient regioselective diversity
generation, Antivir. Chem. Chemother. 16 (2005) 375e383.
[37] C.G. Fortuna, V. Barresi, G. Berellini, G. Musumarra, Design and synthesis of
trans 2-(furan-2-yl)vinyl heteroaromatic iodides with antitumour activity,
Bioorg. Med. Chem. 16 (2008) 4150e4159.
[38] L. Di Nunno, A. Scilimati, Decomposition of arylazides by THF/n-butyllithium-
II-isolation of 1-aryl-4,5-dihydro-5-hydroxy-1H-1,2,3-triazoles, Tetrahedron
42 (1986) 3913e3920.
[39] P. Vitale, A. Scilimati, Five-membered ring heterocycles by reacting enolates
with dipoles, Curr. Org. Chem. 17 (2013) 1986e2000.
[40] P. Vitale, A. Scilimati, Functional 3-arylisoxazoles and 3-aryl-2-isoxazolines
from reaction of aryl nitrile oxides and enolates: synthesis and reactivity,
Synthesis 45 (2013) 2940e2948.
[41] J. Das, S.N. Patil, R. Awasthi, C.P. Narasimhulu, S. Trehan, An easy access to aryl
azides from aryl amines under neutral conditions, Synthesis 11 (2005)
1801e1806.
[42] J.J. Talley, Selective inhibitors of cyclooxygenase-2 (COX -2), Prog. Med. Chem.
36 (1999) 201e234.
[43] T. Liu, X. Dong, N. Xue, R. Wu, Q. He, B. Yang, Y. Hu, Synthesis and biological
evaluation of 3,4-diaryl-5-aminoisoxazole derivatives, Bioorg. Med. Chem. 17
(2009) 6279e6285.
[21] F. Wuest, X. Tang, T. Kniess, J. Pietzsch, M. Suresh, Synthesis and cyclo-
oxygenase
inhibition
of
various
(aryl-1,2,3-triazole-1-yl)-meth-
anesulfonylphenyl derivatives, Bioorg. Med. Chem. 17 (2009) 1146e1151.
[22] P. Vitale, S. Tacconelli, M.G. Perrone, P. Malerba, L. Simone, A. Scilimati,
A. Lavecchia, M. Dovizio, E. Marcantoni, A. Bruno, P. Patrignani, Synthesis,
pharmacological characterization and docking analysis of a novel family of
diarylisoxazoles as highly selective COX-1 inhibitors, J. Med. Chem. 56 (2013)
4277e4299.
[23] Y. Bourne, H.C. Kolb, Z. Radi, K.B. Sharpless, P. Taylor, P. Marchot, Freeze-frame
inhibitor captures acetylcholinesterase in a unique conformation, Proc. Natl.
Acad. Sci. U. S. A. 101 (2004) 1449e1454.
[24] D.R. Buckle, D.J. Outred, C.J. Rockell, H. Smith, B.A. Spicer, Studies on v-tri-
azoles. antiallergic 9-oxo-1H,9H-benzopyrano[2,3-d]-v-triazoles, J. Med.
Chem. 26 (1983) 251e254.
[25] M.F. Mady, G.E.A. Awad, K.B. Jørgensen, Ultrasound-assisted synthesis of novel
1,2,3-triazoles coupled diaryl sulfone moieties by the CuAAC reaction, and
biological evaluation of them as antioxidant and antimicrobial agents, Eur. J.
M. Chem. 84 (2014) 433e443.
ꢂ
[26] R. Alvarez, S. Velazquez, A. San Felix, S. Aquaro, E.D. Clercq, C.F. Perno,
A. Karlsson, J. Balzarini, M.J. Camarasa, 1,2,3-Triazole-[20,50-bis-O-(tert-butyl-
dimethylsilyl)-beta-D-ribofuranosyl]-30-spiro-500-(400-amino-100,200-oxathiole-
200,200-dioxide) (TSAO) analogues: synthesis and anti-HIV-1 activity, J. Med.
Chem. 37 (1994) 4185e4194.
[44] K.D. Grimes, A. Gupte, C.C. Aldrich, Copper(II)-catalyzed conversion of aryl/
heteroaryl boronic acids, boronates, and trifluoroborates into the corre-
sponding azides: substrate scope and limitations, Synthesis
9 (2010)
[27] Y.S. Sanghvi, B.K. Bhattacharya, G.D. Kini, S.S. Matsumoto, S.B. Larson,
W.B. Jolley, R.K. Robins, G.R. Revankar, Growth inhibition and induction of
cellular differentiation of human myeloid leukemia cells in culture by car-
bamoyl congeners of ribavirin, J. Med. Chem. 33 (1990) 336e344.
[28] M. Journet, D. Cai, J.J. Kowal, R.D. Larsen, Highly efficient and mild synthesis of
variously 5-substituted-4-carbaldehyde-1,2,3-triazole derivatives, Tetrahe-
dron Lett. 42 (2001) 9117e9118.
1441e1448.
[45] A. Da Settimo, O. Livi, G. Biagi, G. Primofiore, G. Masoni, 1,2,3-Triazole de-
rivatives of aryloxyalanoic acids, Farm. Sci. 37 (1982) 728e739.
[46] P. Crivori, G. Cruciani, P.A. Carrupt, B. Testa, Predicting blood-brain barrier
permeation from three-dimensional molecular structure, J. Med. Chem. 43
(2000) 2204e2216.
[47] G. Cruciani, M. Meniconi, E. Carosati, I. Zamora, R. Mannhold, VOLSURF: a tool
for drug ADME-properties prediction, in: Drug Bioavailability. Estimation of
Solubility, Permeability and Absorption, Meth. and Princ. in Med. Chem, vol.
18, Wiley-VCH publishers, 2003, pp. 406e419.
[29] M.D. Chen, S.J. Lu, G.P. Yuag, S.Y. Yang, X.L. Du, Synthesis and antibacterial
activity of some heterocyclic b-enamino ester derivatives with 1,2,3-triazole,
Heterocycl. Comm. 6 (2000) 421e426.
[30] S. Shafi, M.M. Alam, N. Mulakayala, C. Mulakayala, G. Vanaja, A.M. Kalle,
R. Pallu, M.S. Alam, Synthesis of novel 2-mercapto benzothiazole and 1,2,3-
triazole based bis-heterocycles: their anti-inflammatory and anti-noceptive
activities, Eur. J. Med. Chem. 49 (2012) 324e333.
[31] N. Upmanyu, J.K. Gupta, K. Shah, P. Mishra, Synthesis of new 1,2,4-triazoles as
anti-inflammatory and anti-nociceptive agents, Pharm. Chem. J. 45 (2011)
433e439.
[32] K.M. Banu, A. Dinakar, C. Ananthanarayanan, Synthesis, characterization,
antimicrobial studies and pharmacological screening of some substituted
1,2,3-triazoles, Indian J. Pharm. Sci. 4 (1999) 202e205.
[33] L.P. Guan, Q.H. Jin, G.R. Tian, K.Y. Chai, Z.S. Quan, Synthesis of some quinoline-
2(1H)-one and 1,2,4-triazol-[4,3-a]-quinoline derivatives as potent anticon-
vulsants, J. Pharm. Pharm. Sci. 10 (2007) 254e262.
[48] G. Berellini, G. Cruciani, R. Mannhold, Pharmacophore, drug metabolism, and
pharmacokinetics models on non-peptide angiotensin ii AT1, AT2 and mixed
AT1/AT2 receptor antagonists, J. Med. Chem. 48 (2005) 4389e4399.
[49] R. Mannhold, G. Berellini, E. Carosati, P. Benedetti, Use of MIF-based VolSurf
descriptors in physicochemical and pharmacokinetic studies, in: G. Cruciani
(Ed.), Molecular Interaction Fields in Drug Discovery, vol. 27, Wiley-vch Verlag
GmbH & Co. KGaA, Weinheim, 2005, pp. 173e193.
€
€
[50] S. Wold, M. Sjostrom, in: B.R. Kowalski (Ed.), Chemometrics: Theory and
Application, ACS Symposium Series, Washington, 1977, pp. 243e282.
[51] S. Wold, C. Albano, W.J. Dunn III, U. Edlund, K. Esbensen, P. Geladi, S. Hellberg,
€
€
E. Johansson, W. Lindberg, M. Sjostrom, Multivariate data analysis in chem-
istry, in: B.R. Kowalski (Ed.), Chemometrics: MaThematics and Statistics in
Chemistry, 1984, pp. 17e96.
[34] A. Passannanti, P. Diana, P. Barraja, F. Mingoia, A. Lauria, G. Cirrincione, Pyrrolo
[2,3-d][1,2,3]triazoles as potential antineoplastic agents, Heterocycles 48
(1998) 1229e1235.
[35] M. Jilino, F.G. Stevens, Antitumour polycyclic acridines. Part 5. Synthesis of
7H-pyrido[4,3,2-kl]acridines with exploitable functionality in the pyridine
[52] V. Barresi, C. Bonaccorso, G. Consiglio, L. Goracci, N. Musso, G. Musumarra,
C. Satriano, C.G. Fortuna, Modeling, design and synthesis of new heteroaryl
ethylenes active against MCF-7 breast cancer cell-line, Mol. Biosyst. 9 (2013)
2426e2429.