Zirconium catalyzed synthesis of 2-arylidene Indan-1,3-diones and evaluation of their inhibitory activity against NS2B-NS3 WNV protease

Article abstract of DOI:10.1016/j.ejmech.2018.02.037

A simple and efficient Knoevenagel procedure for the synthesis of 2-arylidene indan-1,3-diones is herein reported. These compounds were prepared via ZrOCl2·8H2O catalyzed reactions of indan-1,3-dione with several aromatic aldehydes and using water as the

Full text of DOI:10.1016/j.ejmech.2018.02.037

Accepted Manuscript
Zirconium catalyzed synthesis of 2-arylidene Indan-1,3-diones and evaluation of their
inhibitory activity against NS2B-NS3 WNV protease
Ana Flávia C. da S. Oliveira, Ana Paula M. de Souza, André S. de Oliveira, Milene L.
da Silva, Fabrício M. de Oliveira, Edjon G. Santos, Ítalo Esposti P. da Silva, Rafaela
S. Ferreira, Filipe S. Villela, Felipe T. Martins, Daniel H.S. Leal, Boniek G. Vaz,
Róbson R. Teixeira, Sergio O. de Paula
PII:
S0223-5234(18)30167-3
10.1016/j.ejmech.2018.02.037
EJMECH 10214
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 November 2017
Revised Date: 2 February 2018
Accepted Date: 10 February 2018
Please cite this article as: Ana.Flá.C.da.S. Oliveira, A.P.M. de Souza, André.S. de Oliveira, M.L. da
Silva, Fabrí.M. de Oliveira, E.G. Santos, Í.Esposti.P. da Silva, R.S. Ferreira, F.S. Villela, F.T. Martins,
D.H.S. Leal, B.G. Vaz, Ró.R. Teixeira, S.O. de Paula, Zirconium catalyzed synthesis of 2-arylidene
Indan-1,3-diones and evaluation of their inhibitory activity against NS2B-NS3 WNV protease, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.02.037.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
ZIRCONIUM CATALYZED SYNTHESIS OF 2-ARYLIDENE INDAN-1,3-
DIONES AND EVALUATION OF THEIR INHIBITORY ACTIVITY
AGAINST NS2B-NS3 WNV PROTEASE
Ana Flávia C. da S. Oliveira, ‡ [a,b] Ana Paula M. de Souza, ‡ [c] André S. de Oliveira, ‡ [a,b]
Milene L. da Silva,[c] Fabrício M. de Oliveira,[d] Edjon G. Santos,[a] Ítalo Esposti P. da
Silva,[a] Rafaela S. Ferreira,[e] Filipe S. Villela,[e] Felipe T. Martins,[f] Daniel H. S. Leal,[g]
Boniek G. Vaz,[f] Róbson R. Teixeira,*[c] and Sergio O. de Paula*[a]
[a] Departamento de Biologia Geral, Universidade Federal de Viçosa, Av. P.H. Rolfs, S/N,
36570-900 – Viçosa (Brazil).
[b] Instituto Federal de Educação, Ciência e Tecnologia do Norte de Minas, 39101-000 –
Diamantina (Brazil).
[c] Departamento de Química, Universidade Federal de Viçosa, Av. P.H. Rolfs, S/N, 36570-900
– Viçosa (Brazil).
[d] Instituto Federal de Minas Gerais (IFMG), Campus Ouro Branco Rua Afonso Sardinha, 90,
36420-000, Ouro Branco (Brazil).
[e] Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Av.
Antônio Carlos 6627, 31270901 – Belo Horizonte (Brazil).
[f] Instituto de Química, Universidade Federal de Goiás, Av. Esperança, s/n, Campus
Samambaia, 74690-970, Goiânia (Brazil).
[g] Centro Universitário Norte do Espírito Santo, Universidade Federal do Espírito Santo,
Rodovia BR 101 Norte, Km 60 - Bairro Litorâneo, 29932-900 - São Mateus (Brazil).
‡These authors contributed equally.
*Corresponding Authors
Sérgio Oliveira de Paula (depaula@ufv.br): Departamento de Biologia Geral, Universidade
Federal de Viçosa, Av. P.H. Rolfs, S/N, 36570-900 – Viçosa (Brazil), Tel./Fax: +55-31-3899-
2589; and Róbson Ricardo Teixeira (robsonr.teixeira@ufv.br): Universidade Federal de Viçosa,
Av. P.H. Rolfs, S/N, 36570-900 – Viçosa (Brazil), Tel./Fax: +55-31-3899-3209.
KEYWORDS: antiviral agents; West Nile Virus; indan-1,3-dione; Knoevenagel condensation.
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ABSTRACT: A simple and efficient Knoevenagel procedure for the synthesis of 2-arylidene
indan-1,3-diones is herein reported. These compounds were prepared via ZrOCl2·8H2O
catalyzed reactions of indan-1,3-dione with several aromatic aldehydes and using water as the
solvent. The 2-arylidene indan-1,3-diones were obtained with 53%-95% yield within 10-45
minutes. The synthesized compounds were evaluated as inhibitors of the NS2B-NS3 protease of
West Nile Virus (WNV). It was found that hydroxylated derivatives impaired enzyme activity
with varying degrees of effectiveness. The most active hydroxylated derivatives, namely 2-(4-
hydroxybenzylidene)-1H-indene-1,3(2H)-dione (14) and 2-(3,4-dihydroxybenzylidene)-1H-
indene-1,3(2H)-dione (17), were characterized as noncompetitive enzymes inhibitors, with IC50
values of 11 ꢀmol L-1 and 3 ꢀmol L-1, respectively. Docking and electrostatic potential surfaces
investigations provided insight on the possible binding mode of the most active compounds
within an allosteric site.
INTRODUCTION
The formation of carbon-carbon bonds (single, double and triple) is one of the most important
aspects in organic chemistry. Several methodologies have been developed to achieve the
formation of these bonds, and the Knoevenagel condensation is a synthetically useful carbon-
carbon forming transformation [1-3]. This reaction involves condensation between activated
methylene- and carbonyl-containing compounds. The Knoevenagel reaction has been explored in
organic synthesis for the preparation of several classes of compounds, such as coumarines which
are important intermediates in the synthesis of pharmaceuticals, cosmetics, and perfumes [4, 5].
In addition, this reaction has been used in the total synthesis of natural compounds such as the
illudalane sesquiterpene illudinine [6]. Typically, the Knoevenagel condensation is carried out in
the presence of weak bases such as ethylenediamine, piperidine or ammonium salts. Over the
years, several modifications of this reaction have been reported, including the use of Lewis acids
[7].
Zirconyl chloride is a Lewis acid that has drawn much attention of organic chemists because of
its water stability, low toxicity, and commercial availability. As a consequence, this oxysalt has
been applied in several organic transformations such as oxidation of alcohols [8], esterification
of carboxylic acids [9], synthesis of 2-aryloxazolines and bis-oxazolines [10], preparation of
enaminones and enamino esters [11], among others [11-22]. As can be noticed, Zirconyl chloride
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is a useful catalyst that can be utilized in a variety of transformations, such as the Knoevenagel
methodology presented in this investigation which resulted in the discovery of indan-1,3-dione
derivatives endowed with Flavivirus protease inhibitory activity.
The Flavivirus genus encompasses a group of enveloped RNA arthropod-borne viruses
(arboviruses) responsible for important human and animal diseases caused by virus like Yellow
Fever Virus (YFV), Zika Virus (ZIKV), Dengue Virus (DENV), West Nile Virus (WNV),
Japanese Encephalitis Virus (JEV), Tick-Borne Encephalitis Virus (TBEV), and Kyasanur Forest
Disease Virus (KFDV) [23].
WNV infection represents a serious burden to human and animal health because of the virus
capability to cause unforeseen and large epidemics. Cases of WNV infection were limited to
European, Asian and African countries until 1999, when the virus reached the Western
Hemisphere [24, 25]. Within three years, the virus spread to Canada and Mexico, followed by
animal and human cases in Central and South America [26-28].
WNV is an enveloped virus whose genome consists of a single-stranded RNA with positive
polarity and approximately 11 kb. It contains a single open reading frame encoding a precursor
polyprotein, which is processed by viral and host proteases, giving rise to three structural
proteins, namely capsidial protein (C), envelope glycoprotein (E) and pre-membrane/membrane
protein (prM/M). The RNA also encodes seven non-structural proteins, NS1, NS2A, NS2B, NS3,
NS4A, NS4B and NS5, which are involved in the replicative cycle of the virus[29].
Viral protease is responsible for the cleavage of some sites, including NS2A-NS2B, NS2B-
NS3, NS3-NS4A, NS4B-NS5. It also cleaves the signal sequences at the C-prM position and the
NS4A-NS4B, within NS2A, and within the NS3 itself [30, 31].
Viral enzymes are important targets for therapeutic intervention and they have been explored
towards the development of antiflavivirals [31-33]. As a consequence, research groups have
identified reversible and irreversible inhibitors for Flavivirus proteases [34-38].
Within this context, we report herein a simple and efficient zirconium catalyzed Knovenagel
procedure for the synthesis of 2-arylidene-indan-1,3-diones. The synthesized compounds were
evaluated as inhibitors of the NS2B-NS3 protease of West Nile Virus (WNV) and the results are
discussed. This investigation was conducted in order to assess the possibility of using these
derivatives as antiviral for the treatment of diseases caused by Flaviridae viruses. It was found
that the hydroxylated derivatives impaired enzyme activity with varying degrees of effectiveness.
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Docking and electrostatic potential surfaces investigations provided insights about the way the
most active compounds act by inhibiting the activity of this enzyme.
RESULTS AND DISCUSSION
Synthesis of 2-arylidene-indan-1,3-diones: The reaction of indan-1,3-dione and 4-
chlorobenzaldehyde was chosen as the model reaction for optimization of the catalytic system
and reaction conditions. All the reactions were performed in air and the results are shown in
Table 1.
Table 1. Knoevenagel condensation reactions of 4-chlorobenzaldehyde and indan-1,3-dione
under different conditions
Reagent
ratio
Catalyst
loading
Heating
source
Temperature
(°C)
Reaction
Yield
(%)
Entry
Solvent
time (min)
(in mmol) (mol%)
1
Oil bath
Oil bath
MW
1:1
1:1.2
1:1
1:1
1:1
1:1
1:1
1:1
1:1
1:1
4
4
4
4
4
4
4
2
8
-
free
85
85
85
85
85
40
r.t.
85
85
85
70
33
31
36
68
70
86
83
84
66
27
2
free
90
3
free
30
4
Oil bath
MW
water
water
water
water
water
water
water
65
5
30
6
Oil bath
Oil bath
Oil bath
Oil bath
Oil bath
120
480
110
30
7
8
9
10
420
MW = microwave irradiation; r.t. = room temperature
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We started the optimization process running a solvent free reaction at 85 oC, using 1 mmol of
each reagent, 4 mol% of ZrOCl2ꢀ8H2O, and oil bath conventional heating. After 70 min, the 4-
chlorobenzylidene indan-1,3-dione (1) was obtained with 33% yield after recrystallization (Entry
1). Increasing the amount of 4-chlorobenzaldehyde (1.2 mmol, Entry 2) did not improve the
yield. Compound 1 was obtained with 36% yield when the reaction was conducted under
microwave irradiation and in the absence of solvent (Entry 3). When water was used as solvent,
the yield of the reaction was substantially increased (Entries 4 and 5) compared to the reactions
performed in the absence of solvents. For the sack of simplicity, we decided to continue the
optimization process employing conventional heating, despite the shorter reaction time under
microwave irradiation. Decreasing reaction temperature (Entries 6 and 7) resulted in longer
reaction times albeit with better yields compared to the reaction condition of Entry 4. By
reducing the catalyst loading to 2 mol%, compound 1 was obtained in 84% yield after 110
minutes (Entry 8). Doubling the amount of catalyst to 8 mol% (Entry 9), the reaction time was
reduced and the yield was about the same compared to reaction of Entry 4; however, the reaction
work up was troublesome. To verify the impact of the zirconium catalyst, we also run the
reaction in its absence (Entry 10). As expected, the reaction time was substantially increased and
the product was obtained only with 27% yield, reinforcing the importance of the catalytic effect
of ZrOCl2ꢀ8H2O.
Since reaction conditions of entry 4 afforded compound 1 with synthetically useful yield,
simple workup and shorter period of time (compared to other reactions herein conducted with
conventional heating), they were after applied in order to investigate the scope of the zirconium
catalyzed Knoevenagel reaction to prepare 2-arylidene indan-1,3-diones. Fifteen compounds
were synthesized (Scheme 1) and obtained in synthetically useful yields (53%−95%) within
10−45 minutes (see Table 1S in the Supporting Information). These indan-1,3-dione derivatives
comprised compounds containing electron-donating and electron withdrawing groups attached to
the aromatic ring of arylidene portion. A heterocyclic derivative (compound 9) was also
prepared. It should be mentioned that interesting heterocycle scaffolds can be accessed from
compounds containing active methylene moieties via condensation of them with aromatic
aldehydes as described, for instance, by Elmuradov and co-workers[39].
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Scheme 1. Structures of indan-1,3-diones 2−16.
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The derivative 2-(3,4-dihydroxybenzylidene)-1H-indene-1,3(2H)-dione (17) was prepared, in
58% yield, via dimethylation of compound 13 using BBr3 (Scheme 2).
H3CO
OCH3
HO
OH
Ò
Ò
1) BBr3, CH2Cl2, 0 o
2) H2O
C
r.t.
O
O
13
17
( )
(
)
Scheme 2. Preparation of compound 17.
The identities of substances 1−17 were confirmed by IR and NMR spectroscopy as well as
mass spectrometry. In the IR spectrum, two bands, one observed within 1709−1731 cm-1 and the
other within 1655−1688 cm-1 range, were attributed to the carbonyl groups. Two signals at
13
approximately 190 ppm in the C NMR spectra confirmed the presence of the carbonyl groups
in the structures of compounds 1−17. The substitution patterns of the aromatic rings in the
arylidene portions were compatible with the 1H NMR spectra.
We were able to obtain a monocrystal of compound 15. Considering that X-ray information
can be useful in terms of structure activity-relationship, we have also determined its crystal
structure. A summary of crystal data collect and processing is presented in the Supporting
Information (Table 2s). Compound 15 has crystallized in the centrosymmetric monoclinic space
group P21/c with just one molecule in the asymmetric unit (Figure 1), which is almost planar
(root mean square deviation of all non-hydrogen atoms is 0.0532 Å, with the largest atom
deviation from the least-square plane of 0.202(5) Å for C18).
Figure 1. 30% ellipsoid plot for non-hydrogen atoms of compound 15 in its crystal structure.
Hydrogens were drawn as arbitrary radius spheres. Atom labeling scheme is arbitrary.
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The crystal packing of 15 is mainly stabilized through the formation of one-dimensional chains
assembled with 21-axis symmetry related molecules along the crystallographic axis b. In these
chains, there is one classical intermolecular hydrogen bond between the hydroxyl group (donor)
and one carbonyl oxygen (acceptor), besides one non-classical hydrogen bond involving a CH
aromatic moiety (donor) and one methoxy oxygen (acceptor) (Figure 2).
Figure 2. One-dimensional chain found in the crystal packing of compound 15. Just the
hydrogen bonding (black lines) donor and acceptor atoms were labeled only once.
Biological activity assays and Computational analyses: The evaluation of the inhibitory
effect of compounds 1−17 on NS2B-NS3 WNV enzyme was carried out via a fluorogenic
substrate assay. Initially, the enzyme was incubated for 30 minutes with the compounds under
evaluation. Subsequently, reactions were initiated with the addition of the substrate
pERTKRAMC. Among the evaluated indan-1,3-dione derivatives, compounds 14 and 17 showed
inhibitory behavior, with reduction of 41% and 100% of the enzymatic activity, respectively, at
the concentration of 16 ꢀmol L−1 (Figure 3). These compounds present, as a common structural
feature, a hydroxyl group at the para position of the aromatic ring of the arylidene moiety.
Moreover, compound 17, which displayed the best inhibitory response, possesses an additional
hydroxyl group at the meta position. Previous investigations conducted with HIV protease have
demonstrated that hydroxyl groups would be important for inhibiting the replication of this virus.
Effective HIV protease inhibitors had a hydroxyl group that replaced the water molecule from
the catalytic active-center. In this case, the hydroxyl group established hydrogen bonding with
catalytically active aspartates[40]. Tomlinson and Watowich demonstrated the involvement of
the hydroxyl group in DENV protease inhibition. Significant interactions occurred between
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hydroxyl groups of inhibitors with preserved residues that constitute the catalytic triad (His51,
Asp75, Ser135) of the protease[41].
Figure 3. Antiviral screening for WNV protease: seventeen indan-1,3-dione derivatives were
evaluated in at the concentration of 16 ꢀmol L-1 against WNV NS2-NB3 protease. p value <
0.0001. ** indicates statistical significance. The assays were conducted in triplicated on three
isolated experiments.
In order to obtain the inhibitory concentration of the compounds that reduces activity by 50%
(IC50), enzymatic inhibition of the NS2-NB3 WNV protease was evaluated under varying
concentrations of the most active compounds 14 and 17. Both these indan-1,3-dione derivatives
showed dose-response inhibition with IC50 values of 11 ꢀmol L-1 and 3 ꢀmol L-1, respectively.
With the aim of verifying the possible mechanism of inhibition by these compounds, enzyme
kinetic assays were conducted under varying substrate concentrations and three concentrations of
1
each compound (2, 4 and 8 ꢀmol L− ). Compounds 14 and 17 promoted a significant decrease in
the maximum enzymatic velocity without changes in KM values as depicted in Figure 4. The
Lineweaver-Burk plot presented lines intersecting on the x-axis for both compounds. Ki values
1
1
were 12.31 ꢀmol L− and 1.25 ꢀmol L− for 14 and 17, respectively. This inhibition profile
suggests a noncompetitive inhibition mode of action, in which the inhibitor and the substrate can
bind simultaneously to an enzyme molecule.
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Figure 4. Enzymatic kinetics of WNV NS2-NB3 protease in the presence of compounds 14 and
17. In A and C are shown the Michaelis-Menten plots for compounds 14 and 17, respectively. In
B and D are presented the Lineweaver-Burk plots for compounds 14 and 17, respectively.
To propose likely binding modes for compounds 14 and 17, we turned to molecular docking
calculations. Other investigations have already identified noncompetitive inhibitors for the
Flavivirus proteases, with inhibitor binding to allosteric sites with low Ki and IC50 values[42-46].
Taking into account the noncompetitive mode of inhibition, the possible binding site region was
defined by analogy with an allosteric site previously characterized for the homologous Dengue
virus type 2 protease[45, 47]. Docking studies were performed with the Induced Fit methodology
of Glide[48, 49], therefore accounting for protein flexibility in the presence of ligands. A
consistent binding mode was proposed for both compounds, in which ligand orientation and
complex stability seem to be guided by several hydrogen bond interactions (Figure 5, Table 4).
The para-hydroxyl group is predicted to interact with residues Trp89 and Ile147, while one of
the carbonyl groups interacts with Gln167. The meta-hydroxyl group, present only in 17,
hydrogen bonds to Gly124. These results provide a possible explanation for the importance of
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meta and para phenyl hydroxyl groups for effective inhibition of NS2-NB3 WNV protease, in
agreement with the SAR data here reported.
Figure 5. Docking predicted binding modes for compounds 14 (A) and 17 (B) to WNV protease.
The protease is shown as cartoon, with residues involved in hydrogen bonds (Trp89, Gly124,
Ile147 and Gln169) and the central residue in the grid (Ile123) highlighted as sticks and colored
by atom. Hydrogen bonds are highlighted as yellow dashes.
Table 4. Predicted hydrogen bond interactions between WNV protease and
compounds 14 and 17.
Ligand atom
Compound Interacting residue and atom Distance(Å)a
2.8
3.2
2.8
2.9
3.2
3.1
3.1
14
17
14
17
14
17
17
p-OH
Ile147, backbone carbonyl
Trp89, backbone NH
p-OH
Indandione carbonyl
m-OH
Gln167, backbone NH
Gly124, backbone NH
adistances between donor and acceptor atoms.
Considering that only two compounds (14 and 17) showed activity against WNV NS2-NS3
protease and based on the hypothesis that this activity could be due, in part, to the presence of the
hydroxyl groups (acting as, for example, H-bond forming groups), we decided to investigate the
role of such groups through some computational calculations. These two compounds are not the
only ones with H-bond forming groups attached to the phenyl ring of arylidene portion.
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Compounds 4, 5, 8, 10, 15 and 16, for example, also contain small H-bond forming groups
attached to the phenyl ring (nitro-, fluoro-, hidroxi-, cyano-, hidroxi- and hidroxi- groups,
respectively). Besides that, compounds 6, 12 and 13 are only methoxy-substituted, while
compound 11 is a dimethylamino-compound. Therefore, all of these groups could be H-bond
forming. However, the bulky substituents in 6 and 11 to 13 could hinder these compounds from
interacting with the active site of WNV NS2-NS3 protease. Since compounds 8, 15 and 16 were
considerably less active than 14 and 17 (probably due to steric effects of the methyl groups) and
all the remaining compounds showed no activity, we decided to treat the compounds 2
(unsubstituted; included only for comparison), 4, 5, 8, 10 and 14 to 17 with computational
methods.
Considering that the crystal structure of compound 15 exhibits a periplanar relation between
the indan-1,3-dione and arylidene moieties, we performed a conformational analysis around the
torsion between these ring systems (the C2-C10-C11-C12 dihedral angle in Figure 1).
Calculations were performed based on two methods: gas phase and water implicit solvation. For
both methods, we used DFT functional B3LYP and basis set 6-31G(d,p) for compounds 14, 15
and 17. The resulting calculations showed the same periplanar relation between the indan-1,3-
dione and arylidene groups as the most stable conformations (torsions very close to zero or
180°). For computational simplicity reasons, the remaining calculations were performed
exclusively in gas phase.
Compounds 2, 4, 5, 8, 10 and 14 to 17 were fully optimized in gas phase from periplanar
conformations at the B3LYP/6-31G(d,p) level. Molecular electrostatic potential (MEP) surfaces
were generated for all the resulting optimized geometries, as shown in Figure 6. We can see from
these calculations that, for all compounds, both carbonyl groups show local negative electrostatic
potentials. However, important differences are observed regarding positive electrostatic
potentials. For compounds 2, 5 and 10, weak positive potentials are well spread over the
surfaces. For the nitrocompound 4, the positive potentials are mainly over the indan-1,3-dione
system. Compounds 4, 5 and 10 show also negative potentials over the nitro, fluoro and cyano
functional groups. Finally, active compounds 8 and 14 to 17 show positive potentials over the
hydrogens of hydroxyl groups. In compounds 8, 15 and 16, steric effects of methoxy groups
could hinder better interactions with target macromolecules. Thus, compounds 14 and 17, the
most bioactives of the tested set, are the only ones to exhibit positive potentials and with no
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hindrance. The values of the potentials for these two molecules, higher than the other ones,
indicates that the electrostatic interactions between each of them and target macromolecules
would be more efficient than the other compounds [49,50].
Figura 6. MEP surfaces mapped from total electron density for compounds 2, 4, 5, 8, 10 and 14
to 17. Electrostatic potentials are displayed on a 0.002 a.u. isodensity surface. The limits of
electrostatic potentials for each molecule are under surfaces. Potential increases in the following
order: red (most negative) → orange → yellow → green → blue (most positive).
In the present investigation, the most bioactive compounds 14 and 17, along with compounds
8, 15 and 16 (which showed negligible inhibitory effect), are the only phenolic ones. This
observation strengthens the hypothesis that this functional group represents an important
structural feature for protease inhibition by indan-1,3-diones.
It should be mentioned that derivatives of indan-1,3-diones presenting antiviral activity for
Human Papillomavirus (HPV) [50-52], Human Immunodeficiency Virus (HIV) [53] and
Hepatitis C Virus (HCV) [54] have been described. Concerning HIV and HCV, the effects of the
indan-1,3-dione derivatives occurred in viral replication, against HIV integrase and HCV
protease. HCV is also a member of the Flaviviridae family.
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The effect of compounds 14 and 17 was also assessed against Vero cell line. It was noticed that
Vero cells morphology and viability were unaffected by treatment with compounds. The
cytotoxicity concentration of the compounds that reduces cell viability by 50% (CC50)
corresponded to 89,72 µmol L-1 and 267,60 µmol L-1 for 14 and 17 respectively, demonstrating
the high inhibitory potential of these derivatives. Lyu, Rhim and Park evaluated the cytotoxicity
of flavonoids on Vero cells and they considered as low cytotoxic compounds those presenting
CC50> 50 µmol L-1, which indicates that the compounds did not affect the growth of Vero cells
[55].
To prove the potential antiviral efficacy of compounds 14 and 17, we tested these active indan-
1,3-diones in cellular assays against all serotypes of dengue virus, which are members of
Flaviviridae family. The virucidal assay was conducted by prior incubation of the virus with the
evaluated compounds. After incubation, the mixture containing the virus and thecompound under
evaluation was added to the VERO cell line for a period to allow viral adsorption and
internalization. Subsequently, the mixture was replaced by a semi solid medium and the
development of lysis plates on VERO cells was verified. This assay was conducted using
different concentrations of compounds against DENV-1-4 viruses. The concentration of test
compounds that inhibited 50% of viral infection (EC50) was obtained by nonlinear regression,
leading to the calculation of the Selectivity Index (SI). These values are summarized in Table 3S.
Compound 14, despite having antiviral activity, did not present relevant SI values, whereas for
compound 17 high SI values were observed, 34, 13, 147 and 9 DENV-1, DENV-2, DENV-3 and
DENV- viruses, respectively. The results indicate that the compounds have significant antiviral
efficacy and are promissing antiviral canditates.
CONCLUSIONS
It was herein described the preparation of a series of 2-arylidene indan-1,3-diones via
zirconium catalyzed Knoevenagel condensation. From our point of view, the reported
methodology presents the following advantages: i) it does not require the use of toxic solvents;
ii) the catalyst is easy to handle, is commercially available, and presents low cost; iii) the
reactions are simple to run, affords compounds in synthetically useful yields, and can be
conducted in an open-air flask. Biological assays revealed that among the prepared compounds,
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two of them (compounds 14 and 17) display inhibitory activity against the NS2B-NS3 WNV
protease, without any cytotoxic effect on Vero cells. The viral protease utilized in the biological
assays is a very important enzyme for Flaviviral replication. Based on data herein described, it is
clear that the compounds are non-competitive inhibitors with low IC50 and Ki values. To the best
of our knowledge, this is the first report describing anti-WNV activity of 2-arylidene indan-1,3-
dione derivatives. The potential antiviral efficacy of these compounds was demonstrated by
virucidal assay against the four serotypes of dengue virus, a Flaviviridae family member like
WNV.
EXPERIMENTAL SECTION
Reagents: All reagents were purchased from commercial sources (Sigma Aldrich - St. Louis,
MO, US and Vetec - Rio de Janeiro, Brazil) and were employed as received. Solvents were
procured from Vetec (Rio de Janeiro, Brazil) and were used as received. The 1H (300 MHz) and
13C NMR (75 MHz) spectra were recorded on a Varian Mercury 300 instrument ((Varian, Palo
Alto, California, US) 300), using CDCl3 and DMSO-d6 as solvents. Hydrogen nuclear magnetic
resonance (NMR) data are presented as follows: chemical shift (δ) in ppm, number of hydrogen
atoms, multiplicity, J values in Hertz (Hz). Multiplicities are shown as the following
abbreviations: s (singlet), d (doublet), dap (apparent doublet), tap (apparent triplet), ddap
(apparent double of a doublet) m (multiplet). Infrared spectra (IR) were obtained employing the
equipment Agilent 660-IR (Santa Clara, California) with accessory GladiATR. High resolution
mass spectra were recorded on a Q-Exactive (Thermo Scientific, Bremen, Germany). The spectra
were acquired using the following conditions. Ionization source: Electron spray (+) and (-);
Spray voltage: 3.5 kV; Capillary temperature: 275 °C; Sheath gas: 5 (arbitrary units); Auxiliary
gas: 0 (arbitrary units). For the mass spectrometry analyses, the samples were prepared as
follows: a mass of 1 mg of the compound to be analyzed was dissolved in 1 mL of acetonitrile.
Then, the solution was diluted with 1 mL of methanol so that the final concentration
corresponded to 1 ppm. The resulting solution was directly injected in the Q-Exactive equipment
at 5 µmL min-1. The spectra were recorded in full MS mode. Melting points are uncorrected and
were determined using MQAPF-301 melting point apparatus (Microquimica, Rio de Janeiro,
Brazil). Low resolution mass spectra were obtained on a SHIMADZU GCMS-QP5050A
instrument (Kyoto, Japan) by direct injection using the following temperature program: 40 °C
15

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min-1 until temperature reaches 60 °C, and then 80 °C min-1 until temperature reaches 300 °C;
the detector temperature was 280 °C. Analytical thin layer chromatography analyses were carried
out on TLC plates recovered with 60GF254 silica gel. Single-crystal X-ray diffraction data for
compound 15 were acquired using a Bruker-AXS Kappa Duo diffractometer with an APEX II
CCD detector. MoKα radiation from an IꢁS micro-source with multilayer optics was employed.
After diffraction images collect and treatment, the crystallographic softwares were used as
follows: SHELXS-97 [56] for structure solving, SHELXL-97 [56] for structure refinement,
ORTEP-3 [57] and MERCURY [58] for structure analysis and preparation of artworks.
Hydroxyl hydrogen followed a riding model for its coordinates and isotropic atomic
displacement parameter, as well as all other CH hydrogens, even though it was firstly identified
from the difference Fourier electron density map. Crystal structure of compound 15 was
deposited with the Cambridge Crystallographic Data Centre (see Table 3 for deposit number).
Synthesis:
Synthesis of 2-(4-chlorobenzylidene)-1H-indene-1,3(2H)-dione (1): In a typical procedure, a
round-bottomed flask (10 mL) was charged with indan-1,3-dione (150 mg, 1 mmol), 4-
chlorobenzaldehyde (143 mg, 1 mmol), ZrOCl2ꢀ8H2O (12 mg, 4 mol%) and 3.00 mL of distilled
water. The reaction mixture was heated to 85 °C and kept under magnetic stirring for 65 minutes.
The progress of reaction was monitored by TLC analysis. After the completion of the reaction,
the mixture was vacuum filtered and the residue washed with ice-cold ethanol. Compound 1 was
obtained as a yellow solid in 68% yield (182 mg, 0.677 mmol) after recrystallization from
dichloromethane-ethanol (1:1 v/v). Structure of 1 is supported by the following data.
1
Mp 176.3-177.8 ºC. IR (ATR): 3092, 3061, 1725, 1685, 1573, 1072, 733, 427 cm-1. H NMR
(300 MHz, CDCl3) δ: 7.47 (2H, d, J = 8.4 Hz), 7.81-7.83 (3H, m), 7.99-8.01 (2H, m), 8.41 (2H,
d, J = 8.4 Hz). 13C NMR (75 MHz, CDCl3) δ: 123.39, 123.41, 129.1, 129.4, 131.5, 135.3, 135.5,
139.5, 140.1, 142.5, 145.1, 188.9, 189.9. HRMS (M+H+): Calculated for C16H10ClO2,
269.03693; found: 269.03619.
Synthesis of derivatives 2-16: A similar procedure to that described for the preparation of 1 was
utilized to synthesize compounds 2-16. The structures of them are supported by the following
data.
2-benzylidene-1H-indene-1,3(2H)-dione (2): Yellow solid, Mp 150.6-150.8 ºC. IR (ATR):
3066, 3026, 1726, 1681, 1609, 1584, 1564, 732, 683 cm-1. 1H NMR (300 MHz, CDCl3) δ: 7.48-
16

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13
7.58 (3H, m), 7.78-7.84 (2H, m), 7.89 (1H, s), 7.98-8.04 (2H, m), 8.44-8.46 (2H, m). C NMR
(75 MHz, CDCl3) δ: 123.3, 123.4, 128.8, 129.2, 133.1, 133.2, 134.1, 135.1, 135.3, 140.0, 142.5,
146.9, 188.9, 190.2. HRMS (M+H+): Calculated for C16H11O2, 235.07590; found: 235.07530.
2-(4-bromobenzylidene)-1H-indene-1,3(2
H
)-dione (3): Yellow solid, Mp 169.7-170.3 ºC. IR
1
(ATR): 3088, 3055, 1725, 1685, 1574, 1069, 504 cm-1. H NMR (300 MHz, CDCl3) δ: 7.63
(2H,d, J = 8.7 Hz), 7.79 (1H, s), 7.81-7.83 (2H, m), 7.99-8.02 (2H, m), 8.32 (2H, d, J = 8.7 Hz).
13C NMR (75 MHz, CDCl3) δ: 123.41, 123.43, 128.3, 129.6, 131.9, 132.1, 135.4, 135.5, 140.1,
142.5, 145.2, 188.9, 189.9. HRMS (M+H+): Calculated for C16H10BrO2, 312.98642; found:
312.98538.
2-(4-nitrobenzylidene)-1H-indene-1,3(2H)-dione (4): Yellow solid, Mp 233.1-233.6 ºC. IR
(ATR): 3109, 3076, 1732, 1688, 1566, 1514, 1345, 857 cm-1. 1H NMR (300 MHz, DMSO-d6) δ:
7.93 (1H, s), 7.96-8.04 (4H, m), 8.33 (2H, d, J = 9.0 Hz), 8.58 (2H, d, J = 9.0 Hz). 13C NMR (75
MHz, DMSO-d6) δ: 123.79, 123.86, 132.9, 134.6, 136.7, 136.8, 138.9, 140.2, 142.0, 142.6,
149.3, 188.6, 189.2. MS m/z (%): 279 ([M+], 43), 262 (76), 249 (7) 232 (100), 221 (6), 205 (17),
193 (6), 176 (63), 165 (19), 151 (24), 126 (5), 104 (41), 88 (22), 76 (56), 63 (12), 50 (55).
2-(4-fluorobenzylidene)-1H-indene-1,3(2H)-dione (5): Yellow solid, Mp 181.7 ºC. IR
(ATR): 3073, 3039, 1729, 1689, 1578, 1199, 834 cm-1. 1H NMR (300 MHz, CDCl3) δ: 7.18 (2H,
tap, J = 8.7 Hz), 7.79 (1H, s), 7.80-7.83 (2H, m), 7.96-8.03 (2H, m), 8.52 (2H, ddap, J1 = 8.7 Hz,
13
2
J2 = 5.6 Hz). C NMR (75 MHz, CDCl3) δ: 116.1 (d, JC-F = 21.8 Hz), 123.32, 123.34, 128.6,
4
129.6 (d, JC-F = 3.0 Hz), 135.3, 135.4, 136.9 (d,3JC-F = 9.0 Hz), 140.0, 142.4, 145.4, 165.6 (d,
1JC-F = 255.8 Hz), 189.1, 190.1. HRMS (M+H+): Calculated for C16H10FO2, 253.06648; found:
253.06578.
2-(4-methoxybenzylidene)-1
H
-indene-1,3(2
H)-dione (6): Yellow solid, Mp 155.3-156.8 ºC.
1
IR (ATR): 3093, 3043, 2981, 1718, 1681, 1247, 1268, 1177, 1020, 1511, 833, 730 cm-1. H
NMR (300 MHz, CDCl3) δ: 3.90 (3H, s),7.00 (2H, d, J = 9.0 Hz), 7.76-7.80 (2H, m), 7.83 (1H,
s), 7.93-8.00 (2H, m), 8.53 (2H, d, J = 9.0 Hz). 13C NMR (75 MHz, CDCl3) δ: 55.6, 114.4,
123.0, 126.4, 126.5, 134.8, 135.0, 137.1, 139.9, 142.3, 146.8, 164.0, 189.4, 190.7. HRMS
(M+H+): Calculated for C17H13O3, 265.08647; found: 265.08572.
2-(benzo[
d
][1,3]dioxol-5-ylmethylene)-1
H
-indene-1,3(2
H)-dione (7): Yellow solid, Mp
1
207.1-207.8 ºC. IR (ATR): 3099, 3075, 2908, 1715, 1673, 1562, 1276, 929, 724 cm-1. H NMR
(300 MHz, CDCl3) δ: 6.09 (2H, s),6.93 (1H, d, J = 8.4 Hz), 7.77-7.82 (4H, m), 7.94-8.00 (2H,
17

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m), 8.51 (1H, d, J = 1.5 Hz). 13C NMR (75 MHz, CDCl3) δ: 102.1, 108.6, 112.8, 123.13, 123.10,
126.8, 128.1, 132.9, 134.9, 135.1, 139.9, 142.4, 146.9, 148.1, 152.4, 189.3, 190.6. HRMS
(M+H+): Calculated for C17H11O4, 279.06573; found: 279.1590.
2-(4-hydroxy-3-methoxybenzylidene)-1H-indene-1,3(2H)-dione (8): Yellow solid, Mp
215.6-216.4 ºC. IR (ATR): 3535-3316 (broad band), 3085, 2974, 1719, 1679, 1572, 1278, 1255,
1154, 1017, 731 cm-1. 1H NMR (300 MHz, DMSO-d6) δ: 3.90 (3H, s), 6.93 (1H, d, J = 8.4 Hz),
7.72 (1H, s), 7.86-7.94 (5H, m), 8.68 (1H, d, J = 1.8 Hz). 13C NMR (75 MHz, DMSO-d6) δ: 56.1,
116.1, 117.2, 123.1, 123.2, 125.4, 125.5, 132.1, 135.8, 135.9, 139.6,142.1, 147.3, 147.8, 153.7,
189.6, 190.4. HRMS (M+H+): Calculated for C17H13O4, 281.08138; found: 281.08072.
2-(furan-2-ylmethylene)-1H-indene-1,3(2H)-dione (9): Yellow solid, Mp 202.1-203.4 ºC. IR
(ATR): 3138, 3099, 1725, 1681, 1585, 1462, 1346, 1158, 733 cm-1. 1H NMR (300 MHz, CDCl3)
13
δ: 6.68-6.80 (1H, m), 7.75-7.81 (4H, m), 7.95-7.98 (2H, m), 8.54-8.61 (1H, m). C NMR (75
MHz, CDCl3) δ: 114.6, 122.9, 123.1, 124.8, 129.3, 134.9, 135.1, 140.4, 142.3, 149.0, 151.4,
188.9, 190.1. HRMS (M+H+): Calculated for C14H9O3, 225.05517; found: 225.05455.
4-((1,3-dioxo-1
H-inden-2(3H)-ylidene)methyl)benzonitrile (10): Yellow solid, Mp 233.6-
1
235.2 ºC. IR (ATR): 3099, 3053, 2226, 1726, 1684, 1586, 842, 730 cm-1. H NMR (300 MHz,
DMSO-d6) δ: 7.88 (1H, s), 7.95-8.06 (6H, m), 8.53 (2H, d, J = 8.4 Hz). 13CNMR (75 MHz,
DMSO-d6) δ: 114.4, 118.8, 123.7, 123.8, 132.4, 132.7, 133.9, 136.6, 136.7, 137.2, 140.1, 142.5,
142.7, 188.6, 189.2. HRMS (M+H+): Calculated for C17H10NO2, 260.07115; found: 260.07053.
2-(4-(dimethylamino) benzylidene)-1H-indene-1,3(2H)-dione (11): Red solid, Mp 180.1-
181.5 ºC. IR (ATR): 3080, 3032, 2927, 1709, 1665, 1613, 1555, 1518, 1330, 1186, 815, 722 cm-
1. 1H NMR (300 MHz, CDCl3) δ: 3.11 (6H, s), 6.71 (2H, d, J = 9.1 Hz), 7.67-7.71 (2H, m), 7.75
(1H, s), 7.87-7.94 (2H, m), 8.50 (2H, d, J = 9.1 Hz). 13C NMR (75 MHz, CDCl3) δ: 40.0, 111.3,
121.9, 122.4, 122.9, 134.0, 134.3, 137.9, 139.8, 142.2, 147.5, 153.9, 189.9, 191.7. HRMS
(M+H+): Calculated for C18H16NO2, 278.11810; found: 278.11746.
2-(3,4,5-trimethoxybenzylidene)-1H-indene-1,3(2H)-dione (12): Orange solid, Mp 185.4-
185.6 ºC. IR (ATR): 3099, 3002, 2972, 2936, 1715, 1678, 1561, 1499, 1307, 1240, 1223, 1128,
732 cm-1. 1H NMR (300 MHz, CDCl3) δ: 3.98 (3H, s),3.99 (6H, s), 7.78-7.81 (3H, m), 7.95 (2H,
s), 7.97-7.99 (2H, m). 13C NMR (75 MHz, CDCl3) δ: 56.3, 61.0, 112.0, 123.1, 123.2, 127.7,
128.4, 135.0, 135.2, 139.8, 142.4, 143.0, 147.3, 152.8, 189.4, 190.4. HRMS (M+H+): Calculated
for C19H17O5, 325.10760; found: 325.10675.
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2-(3,4-dimethoxybenzylidene)-1H-indene-1,3(2H)-dione (13): Yellow solid, Mp 205.0-
205.4 ºC. IR (ATR): 3088, 3010, 2972, 1712, 1671, 1565, 1505, 1271, 1247, 1141, 1019, 736
1
cm-1. H NMR (300 MHz, CDCl3) δ: 3.97 (3H, s),4.07 (3H, s), 6.95 (1H, d, J = 8.4 Hz), 7.71-
7.83 (4H, m), 7.95-7.99 (2H, m), 8.83 (1H, dap, J = 1,5 Hz). 13C NMR (75 MHz, CDCl3) δ: 56.1,
110.6, 115.5, 122.9, 123.0, 126.4, 126.9, 131.3, 134.8, 135.0, 139.8, 142.3, 147.4, 148.8, 153.9,
189.7, 190.7. HRMS (M+H+): Calculated for C18H15O4, 295.09703; found: 295.09650.
2-(4-hydroxybenzylidene)-1
H-indene-1,3(2
H)-dione (14): Yellow solid, Mp 235.8-236.0 ºC.
1
IR (ATR): 3059, 1715, 1655, 1547, 1505, 1204, 737 cm-1. H NMR (300 MHz, DMSO-d6) δ:
6.92 (2H, d, J = 8.7 Hz), 7.72 (1H, s), 7.86-7.92 (4H, m), 8.50 (2H, d, J = 8.7 Hz), 10.86 (1H, s).
13C NMR (75 MHz, DMSO-d6) δ: 116.4, 123.1, 123.2, 125.0, 125.6, 135.8, 135.9, 138.0, 139.6,
142.0, 146.7, 163.7, 189.4, 190.4. HRMS (M-H-): Calculated for C16H9O3, 249.05517; found:
249.05568.
2-(4-hydroxy-3,5-dimethoxybezylidene)-1H-indene-1,3(2H)-dione (15): Orange solid, Mp
222.4-223.1 ºC. IR (ATR): 3545-3197 (broad band), 3093, 3010, 2941, 1712, 1667, 1565, 1503,
1318, 1227, 1083, 732 cm-1. 1H NMR (300 MHz, DMSO-d6) δ: 3.88 (6H, s), 7.74 (1H, s), 7.85-
7.93 (4H, m), 8.13 (2H, s). 13 CNMR (75 MHz, DMSO-d6) δ: 56.5, 113.2, 123.1, 123.2, 124.2,
125.7, 135.8, 135.9, 139.6, 142.2, 143.1, 147.8, 147.9, 189.7, 190.4. HRMS (M+H+): Calculated
for C18H15O5, 311.09195; found: 311.09131.
2-(3-hydroxy-4-methoxybenzylidene)-1H-indene-1,3(2H)-dione (16): Yellow solid, Mp
219.0-219.2 ºC. IR (ATR): 3546-3288, 3072, 2989, 1718, 1681, 1574, 1498, 1276, 1219, 1137,
734 cm-1. 1H NMR (300 MHz, DMSO-d6) δ: 3.89 (3H, s), 7.09 (1H, d, J = 8.6 Hz), 7.67 (1H, s),
13
7.87-7.95 (5H, m), 8.28 (1H, dap, J = 1.9 Hz). C NMR (75 MHz, DMSO-d6) δ: 56.3, 112.2,
120.3, 123.2, 126.4, 126.6, 130.0, 135.9, 136.1, 139.7, 142.2, 146.7, 146.9, 153.7, 189.3, 190.3.
HRMS (M+H+): Calculated for C17H13O4, 281.08138; found: 281.08052.
Synthesis of 2-(3,4-dihydroxybenzylidene)-1H-indene-1,3(2H)-dione (17): To a bitubulated
round bottomed flask (150 mL), under a nitrogen atmosphere, it was added compound 13 (200
mg, 0.679 mmol) along with 5.0 mL of anhydrous dichloromethane. The resulting mixture was
kept under magnetic stirring and cooled in an ice bath for 40 minutes. Then, 2.7 mL of 1.00 mol
L-1 solution of BBr3 in dichloromethane was added dropwise. After the addition, the reaction
mixture was kept under stirring for 24 hours at room temperature. Subsequently, 10.0 mL of
distilled water were added and a brown precipitate formed. The resulting mixture was transferred
19

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to a separatory funnel and the aqueous phase was extracted with ethyl acetate (3 x 30.0 mL). The
organic extracts were combined, and the resulting organic phase was dried over anhydrous
sodium sulfate, filtered and concentrated under reduced pressure. Compound 17 was obtained as
a yellow solid after washing with dichloromethane and acetone in 58% yield (105 mg, 0.394
mmol).
Mp 238.0-239.4 ºC. IR (ATR): 3492-3109, 3093, 3039, 1720, 1666, 1555, 1384, 1182, 730
cm-1. 1H NMR (300 MHz, CDCl3) δ: 6.89 (1H, d, J = 8.1 Hz),7.64 (1H, s), 7.83 (1H, dd, J1 = 8.1
Hz, J2 = 1.8 Hz), 7.86-7.94 (4H, m), 8.32 (1H, dap, J = 1.8 Hz), . 13C NMR (75 MHz, CDCl3) δ:
116.2, 121.0, 123.1, 125.2, 125.5, 130.7, 135.7, 135.9, 139.6, 142.1, 145.7, 147.3, 153.0, 189.4,
190.5. HRMS (M-H-): Calculated for C16H9O4, 265.05008; found: 265.05060.
Biological assays
Evaluation of the activity of compounds 1−17 against WNV NS2B-NS3 protease:
Recombinant WNV NS2B-NS3 protease (catalog number SE-2907, already purified and
activated) and fluorescent substrate pERTKR-AMC (catalog number ES013) were purchased
from (R & D Systems, Minneapolis, MN, USA,). Compounds 1−17 were dissolved in pure
DMSO; solutions were then diluted in buffer to obtain working solutions with a final
concentration of DMSO of 1% v/v. A volume of 50 µL of the purified protease (final
concentration of 1 ng µL-1) diluted in buffer (50 mmol L-1 Tris, 30% (v/v) glycerol, pH 9.5) was
incubated with 50 µL of DMSO solution of each indan-1,3-dione derivative (final concentration
of 16 µmol L-1) in a 96-well black plate for 30 min at 21−22 °C. After this time, the assay was
initiated by addition of 50 µL of the substrate (40 mmol L-1 - initial concentration). A solution
containing buffer and DMSO was used as negative control on the same plate. The blank
contained 50 µL buffer and 100 µL of the substrate. The fluorescence intensity was continuously
recorded on a wavelength of excitation of 360 nm and an emission wavelength of 460 nm using
SpectraMax® M5 reader (Molecular Devices). Compounds, which effectively inhibit the enzyme
were selected for further biological assays. Analyses were performed using Microsoft Excel
(Microsoft Office Software) and GraphPad Prism 6 (GraphPad Software Inc.). The assays were
conducted in triplicated on three isolated experiments and the statistical analyses were conducted
by utilizing the multiple comparisons of one-way ANOVA.
Determination of IC50 values: The inhibitory enzymatic activity of compounds 14 and 17, the
most active ones against the WNV NS2-NB3 protease, were evaluated at eight different
20

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concentrations (66 µmol L-1−0.5 µmol L-1) using the protease assay as described above.
Fluorescence was measured in triplicate wells at intervals of 30 s for 5 min in three independent
experiments. IC50 values were calculated using GraphPad Prism 6 GraphPad Software Inc.),
using the four-parameter nonlinear regression analysis (Hill slope method).
Determination of Ki values: Three different concentrations (2, 4 and 8 µmol L-1) of inhibitors
14 and 17 and five different concentrations of substrate pERTKR-AMC (20, 40, 60, 80, 100
mmol L-1) were tested in the in vitro against WNV protease (37.04 nmol L-1 protein, 1 ng µL-1 ).
Fluorescence was measured in triplicate wells at an interval of 30 s. The velocity values
(RFU/minute) were then calculated for each substrate/inhibitor pair. Ki values were calculated
with GraphPad Prism software 6 (GraphPad Software Inc.) with non-linear regression at
competitive inhibition mode of enzyme-kinetics.
Cytotoxicity assay: The cytotoxicity of compounds 14 and 17 was assessed using MTT
assay[59]. VERO cells (5 x 104 cells) were seeded in 96-well plates. Each well contained 100 µL
of each compound solution at different concentrations (1000, 250, 125, 63, 32, 17, 8 and 4 µmol
L-1). The compounds were diluted in MEM medium with 2% FBS and 1% DMSO. After 24 h of
o
incubation at 37 C, 100 µL of a MTT solution (5%) was added to the wells. After 4 h at 37 ºC,
the MTT solution was removed and 100 µL/well of DMSO was added to solubilize the
formazan. Absorbance was measure at 550 nm was measured in a microplate reader
(Multiskan™ GO Microplate Spectrophotometer – ThermoFisher®). The data were analyzed and
CC50 was determined using GraphPad Prism 6 (GraphPad Software Inc.).
Virucidal assay: VERO cells (8 x 105 cells per well) were seeded in 24-well plates. 100 ꢁL of
viral suspension (50-100 PFU) were incubated with 100 ꢁL of varying concentrations of the
tested compounds (3, 6, 12, 25, 50 and 100 ꢁmol L-1) and incubated at 37 ° C for 1 h. The
medium was aspirated from the plates and 100 ꢁL of the mixture contaning the virus and the
compound mixture was added to the cell monolayer. The plates were incubated for 1 hour under
shaking for a better viral distribution. After this time, the viral suspension mixed at the various
concentrations of the evaluated compound was aspirated and 1500 ꢁL of a 3% CMC solution in
DMEM (twice concentrated) medium supplemented with 2% FBS and 1% PSA was added to
each well. Plates were incubated for 5-6 days. After this period, the medium was removed and
the cells fixed by adding formaldehyde 20% for 30 minutes at 37 ºC and stained by adding 2 to 3
drops of 5% violet crystal dye for 40 minutes at room temperature on a mechanical stirrer. After
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this time, the dye was aspirated and the plates were placed to dry at room temperature and
quantified by visualization of lysis plates. The data were analyzed and EC50 was determined
using GraphPad Prism 6 (GraphPad Software Inc.). With the value of CC50 and EC50 in hands, it
was possible to calculate the value of the selectivity index (SI), which corresponds to the ratio
CC50/EC50.
Computational analyses
Conformational analyzes, optimizations and electrostatic potential calculations: Molecules
were prepared in the program GaussView 5.0.8 [60]. Conformational analyses and geometries
optimizations were performed with the software Gaussian 09W [61]. Implicit solvation
calculations were run using the IEF-PCM model. Total electron density and molecular
electrostatic potential surfaces were generated from Gaussian outputs with GaussView.
Docking studies: Ligands were prepared in the program Ligprep [62] employing the
OPLS_2005 force field, with protonation states predicted with Epik at pH 9.5 ± 2.0. The West
Nile virus protease NS2B-NS3 PDB code 2FP7[63], chosen as the receptor, was prepared with
the Protein Preparation Wizard (Schrödinger Release 2015-3: Schrödinger Suite 2015-3 Protein
Preparation Wizard; Epik version 3.3, Schrödinger, version 6.8) [64], with removal of all waters
and addition of hydrogens based on PROPKA calculations at pH 9.5. Docking calculations were
performed with the software Glide (Small-Molecule Drug Discovery Suite 2015-4: Glide,
version 6.8) [48] employing the Induced Fit docking methodology [49] and Glide SP. The grid
,
was centered in the amino acid Isoleucine 123, and had 12 Å dimensions. All residues within 5 Å
from Ile123 were considered as flexible. Docking results were ranked based on their docking
score and the top ranking poses for each compound were analyzed with the software PyMOL
[65].
AUTHOR CONTRIBUTIONS
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript. AFCSO and ASO were responsible for all in vitro assays
with WNV protease with the assistance of EGS and IEPS under the supervision of SOP. APMS
performed the synthesis of the compounds with the assistence of MLS under the supervision of
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RRT. RSF and FTM performed the docking studies. DHSL conducted the molecular electrostatic
potential analysis. BGV was responsible for the acquisition of high resolution mass spectra. FTM
performed the acquisition and interpretation of crystallographic data.
FUNDING SOURCES
This work was supported by National Counsel of Technological and Scientific Development
(CNPq), Foundation for Supporting Research in the state of Minas Gerais (FAPEMIG) and by
the National Program for Academic Cooperation (PROCAD) of the Coordination for the
Improvement of Higher Education Personnel-CAPES/Brazil.
ACKNOWLEDGMENT
D. H. S. Leal would like to thank the Computational Chemistry Group from Universidade
Federal de Lavras for the use of softwares (Gaussian and GaussView) and computers in this
work.
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TABLE OF CONTENTS GRAPHIC
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Inhibitory Activity against NS2B-NS3 WNV protease by 2-Arylidene Indan-1,3-diones synthesis
by Zirconium Catalyzed
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