Discovery of novel West Nile Virus protease inhibitor based on isobenzonafuranone and triazolic derivatives of eugenol and indan-1,3-dione scaffolds

Article abstract of DOI:10.1371/journal.pone.0223017

The West Nile Virus (WNV) NS2B-NS3 protease is an attractive target for the development of therapeutics against this arboviral pathogen. In the present investigation, the screening of a small library of fifty-eight synthetic compounds against the NS2-NB3 protease of WNV is described. The following groups of compounds were evaluated: 3-(2-aryl-2-oxoethyl)isoben-zofuran-1(3H)-ones; eugenol derivatives bearing 1,2,3-triazolic functionalities; and indan-1,3-diones with 1,2,3-triazolic functionalities. The most promising of these was a eugenol derivative, namely 4-(3-(4-allyl-2-methoxyphenoxy)-propyl)-1-(2-bromobenzyl)-1H-1,2,3-triazole (35), which inhibited the protease with IC₅₀of 6.86 μmol L^-1. Enzyme kinetic assays showed that this derivative of eugenol presents competitive inhibition behaviour. Molecular docking calculations predicted a recognition pattern involving the residues His⁵¹and Ser¹³⁵, which are members of the catalytic triad of the WNV NS2B-NS3 protease.

Full text of DOI:10.1371/journal.pone.0223017

RESEARCH ARTICLE
Discovery of novel West Nile Virus protease
inhibitor based on isobenzonafuranone and
triazolic derivatives of eugenol and indan-1,3-
dione scaffolds
1
,2☯
2☯
1,2☯
Andr e´ S. de Oliveira
, Poliana A. R. Gazolla , Ana Fl a´ via C. da S. Oliveira
, Wagner
2
2
2
1
´
L. Pereira , L ´ı via C. de S. Viol , Ang e´ lica F. da S. Maia , Edjon G. Santos , Italo E. P. da
SilvaID , Tiago A. de Oliveira Mendes , Adalberto M. da Silva , Roberto S. Dias , Cynthia
C. da Silva , Marcelo D. PolêtoID , R o´ bson R. Teixeira *, Sergio O. de PaulaID *
1
3
4,5
1
1
1
4
1
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1
Departamento de Biologia Geral, Universidade Federal de Vi c¸ osa, Vi c¸ osa, MG, Brazil, 2 Instituto Federal
de Educa c¸ ão, Ciência e Tecnologia do Norte de Minas Gerais, Fazenda Biribiri, MG, Brazil, 3 Departamento
de Bioqu ´ı mica Biologia Molecular, Universidade Federal de Vi c¸ osa, Vi c¸ osa, MG, Brazil, 4 Departamento de
Qu ´ı mica, Universidade Federal de Vi c¸ osa, Vi c¸ osa, MG, Brazil, 5 Instituto Federal de Educa c¸ ão, Ciência e
Tecnologia Catarinense, Araquari, SC, Brazil
☯
These authors contributed equally to this work.
*
depaula@ufv.br (SOP); robsonr.teixeira@ufv.br (RRT)
OPEN ACCESS
Citation: de Oliveira AS, Gazolla PAR, Oliveira
Abstract
AFCdS, Pereira WL, de S. Viol LC, Maia AFdS, et al.
(2019) Discovery of novel West Nile Virus protease
The West Nile Virus (WNV) NS2B-NS3 protease is an attractive target for the development
of therapeutics against this arboviral pathogen. In the present investigation, the screening of
a small library of fifty-eight synthetic compounds against the NS2-NB3 protease of WNV is
described. The following groups of compounds were evaluated: 3-(2-aryl-2-oxoethyl)isoben-
zofuran-1(3H)-ones; eugenol derivatives bearing 1,2,3-triazolic functionalities; and indan-
inhibitor based on isobenzonafuranone and triazolic
derivatives of eugenol and indan-1,3-dione
scaffolds. PLoS ONE 14(9): e0223017. https://doi.
org/10.1371/journal.pone.0223017
Editor: Graciela Andrei, Katholieke Universiteit
Leuven Rega Institute for Medical Research,
BELGIUM
1,3-diones with 1,2,3-triazolic functionalities. The most promising of these was a eugenol
derivative, namely 4-(3-(4-allyl-2-methoxyphenoxy)-propyl)-1-(2-bromobenzyl)-1H-1,2,3-
Received: April 12, 2019
-1
triazole (35), which inhibited the protease with IC50 of 6.86 μmol L . Enzyme kinetic assays
Accepted: September 11, 2019
Published: September 26, 2019
showed that this derivative of eugenol presents competitive inhibition behaviour. Molecular
51
135
docking calculations predicted a recognition pattern involving the residues His and Ser
which are members of the catalytic triad of the WNV NS2B-NS3 protease.
,
Copyright: © 2019 de Oliveira et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Introduction
The West Nile Virus (WNV) is a member of the same family as the Dengue virus (DENV),
Zika virus (ZIKV) and Yellow Fever virus (YFV), the Flaviviridae family, Flavivirus genus.
They are arboviruses that present RNA as a genome [1]. Diseases caused by Flavivirus are the
major causes of fatality in poverty-stricken regions across Africa, Asia and some parts of the
Americas. The combined potential health risk associated with arthropod-borne viruses like
DENV, WNV, and ZIKV is enormous. These arboviruses are either emerging or re-emerging
in many regions [2].
Funding: The following authors of this paper
received scholarships during this research: ASO
and AFCSO from the Instituto Federal de Educa c¸ ão,
Ciência e Tecnologia do Norte de Minas Gerais
(IFNMG – www.ifnmg.edu.br); EGS, IEPS and AMS
from the Funda c¸ ão de Amparo à Pesquisa do
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1 / 25

Discovery of novel West Nile Virus protease inhibitor
Estado de Minas Gerais (FAPEMIG – www.
fapemig.br); RSD, TAOM and SOP from the
Conselho Nacional de Desenvolvimento Cient ´ı fico e
Tecnol o´ gico (CNPQ – www.memoria.cnpq.br);
PARG, WLP, LCSV and AFSM from the
Three WNV strains are known to be capable of causing unforeseen and large epidemics,
leading to serious public health problems. Since 2004, lineages 1 and 3 have been circulating in
Europe and, since 2010, beginning in a major epidemic in Greece, lineage 2 has been circulat-
ing in several European countries. [3, 4]. The WNV crossed the Atlantic and reached the West-
ern Hemisphere in 1999, when a group of patients with encephalitis was reported in the New
York City metropolitan area. Within three years, the virus spread to Canada and Mexico, fol-
lowed by animal cases in Central and South America [5, 6]. Recently, the first human case of
WNV was reported in Brazil, with the development of encephalitis. It is possible that sporadic
cases or small groups of the WNV disease had already occurred in different regions of the
country without being properly diagnosed [7].
Coordena c¸ ão de Aperfei c¸ oamento de Pessoal de
N ´ı vel Superior (CAPES – www.capes.gov.br). The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
WNV is a genetically and geographically diverse virus. Four or five distinct WNV genetic
lines have been proposed, based on phylogenetic analyses of published isolates. Their genomes
differ from each other by about 20–25%, and are well correlated with the geographic point of
isolation [8–10]. They are enveloped viruses whose genome consists of single-stranded, posi-
tive-polarity RNA approximately 11 kb. This RNA contains a single open reading frame
encoding a precursor polyprotein, which is processed by viral and host proteases, giving rise to
three structural proteins: capsidial protein (C), envelope glycoprotein (E) and pre-membrane/
membrane protein (prM/M); and seven non-structural proteins, NS1, NS2A, NS2B, NS3,
NS4A, NS4B and NS5, which are involved in the replicative cycle of the virus[11]. Viral prote-
ase performs the cleavage of some sites: NS2A-NS2B, NS2B-NS3, NS3-NS4A and NS4B-NS5.
It also cleaves the signal sequences at the C-prM position and the NS4A-NS4B, within NS2A,
and within the NS3 itself [12, 13].
Despite the tremendous efforts invested in Flavivirus research, no clinically approved anti-
viral chemotherapeutics are available for humans, and disease treatment is limited to support-
ive care [13]. Inhibition of viral enzymes has proved to be one important approach toward the
development of antiviral therapies [2, 13–15]. Non-structural proteins encoded by these RNA
viruses are essential for their replication and maturation, and thus may offer ideal targets for
developing antiviral drugs [2]. Flavivirus genomes are translated into a single polyprotein that
needs to be cleaved by viral and host proteases. Because it processes most of the polyprotein
cleavages, viral protease is necessary and essential for virus replication [16, 17].
Considering the premises, the screening of a small library of fifty-eight synthetic com-
pounds against the NS2-NB3 protease of WNV is described in the present investigation. The
following groups of compounds were evaluated: (I) 3-(2-aryl-2-oxoethyl)isobenzofuran-1
(3H)-ones; (II) eugenol derivatives bearing 1,2,3-triazolic functionalities; and (III) indan-
1,3-diones with 1,2,3-triazolic functionalities. Fig 1 displays the general structures of the evalu-
ated compounds.
The isobenzofuran-1-(3H)-ones, 1,2,3-triazolic derivatives, eugenol, and indan-1,3-diones
are substances that have been described in the literature as being endowed with antiviral activi-
ties [8, 18–28]. Isobenzofuran-1-(3H) -one derivatives have several biological activities [18,
2
9–35], highlighting antiviral action for HIV[18]. Indan-1,3-diones derivatives have been spe-
cifically associated with antiviral activity. Studies have indicated efficient action of indan-
,3-diones against the enzyme integrase of the HIV-1 virus [36]; against the structure of
human papillomavirus (HPV) [37–39]; and also against the Hepatitis C virus (HCV) protease
1
[
40], and recently for WNV protease [41]. Eugenol has been tested against the Herpes virus
HSV), HSV-1 (five viral isolates) and HSV-2 (five viral isolates), providing complete protec-
(
tion against one isolate of each type, HSV-1 and 2, and protection between 16.5% and 87.7%
for the remaining isolates [21–23]. The triazole ring was associated with the molecules due to
the antimicrobial activity already described for 1,2,3-triazole compounds, especially studies
that demonstrated antiviral action against the Dengue virus [19, 20]. This fact prompted the
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Discovery of novel West Nile Virus protease inhibitor
Fig 1. General structures of compounds evaluated as inhibitors of WNV NS2-NB3 protease. 3-(2-aryl-2-oxoethyl)isobenzofuran-1(3H)-ones (I); eugenol derivatives
bearing 1,2,3-triazolic functionalities (II); indan-1,3-diones with 1,2,3-triazolic functionalities (III).
https://doi.org/10.1371/journal.pone.0223017.g001
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Discovery of novel West Nile Virus protease inhibitor
authors to evaluate the effect of the compounds presenting general structures (I), (II) and (III)
on the NS2-NB3 WNV protease.
Materials and methods
Synthesis
Solvents were purchased from Vetec (Rio de Janeiro, Brazil). Benzyl alcohols, pent-4-yn-1-ol,
methanesulfonyl chloride, sodium azide, triethylamine, propargyl bromide, acetophenones,
and indan-1,3-dione were procured from Sigma Aldrich (St. Louis, MO, United States) and
used as received. Eugenol was extracted via hydrodistillation from cloves purchased in the
local market in Vi c¸ osa, Minas Gerais state, Brazil, and subsequently purified by column chro-
1
13
matography (vide infra). H- and C-NMR spectra were recorded on a Varian Mercury 300
MNR Spectrometer (Varian, Palo Alto, CA, United States) at 300 MHz and 75 MHz, respec-
tively, using CDCl , C D or DMSO-d as solvents. NMR data are presented as follows: chemi-
3
6
6
6
cal shift (δ) in ppm, multiplicity, the number of protons, and J values in Hertz (Hz).
Multiplicities are shown as the following abbreviations: s (singlet), brs (broad singlet), d (dou-
blet), dap (apparent doublet), dd (doublet of a doublets), t (triplet), brd (broad doublet), ddtap
(
apparent doublet of doublets of triplets), q (quartet), quint (quintet), and m (multiplet). Some
19 13
13
signals in the C NMR spectra were described as multiplets due to the F- C coupling. IR
spectra were obtained using a Varian 660-IR equipped with GladiATR (Varian, Palo Alto, CA,
−1
USA) scanning from 4000 to 500 cm . Analytical thin-layer chromatography analysis was
conducted on aluminum-backed, pre-coated silica gel plates using different solvent systems.
TLC plates were visualized using potassium permanganate solution, phosphomolybdic acid
solution and/or UV light. Flash column chromatography was performed using silica gel 60
(
60–230 mesh). Melting points were determined using a MQAPF-302 melting point apparatus
Microquimica, Santa Catarina, Brazil) and are uncorrected. Solvents were dried using stan-
(
dard procedures described in the literature [42].
Synthesis of 3-(2-oxo-aryl)-isobenzofuran-1-(3H)-ones 1–18. The preparation of com-
pounds 1–18 (Fig 2) was carried out via ZrOCl �8H O catalyzed condensation reactions
2
2
between phthalaldehydic acid and different acetophenones as previously reported [43].
Extraction and purification of eugenol (19). Eugenol (19) was extracted via hydrodistil-
lation from dried flower buds of Eugenia caryophyllata, commonly known as cloves, purchased
in the local market in Vi c¸ osa, Minas Gerais state, Brazil. Thus, 60.0 g of cloves were mixed
with 500 mL of distilled water in a round-bottom flask which was connected to the hydrodistil-
lation apparatus. The mixture was heated for three hours. The obtained hydrolate was trans-
ferred to a separatory funnel and the aqueous layer was extracted with dichloromethane (3 x
30 mL). The organic extracts were combined and the resulting organic layer was dried over
anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting
oil was submitted to column chromatography eluted with hexane-ethyl acetate (6:1 v/v). The
described procedure afforded 7.12 g of eugenol (19), which corresponded to approximately a
12% yield in relation to the initial mass of cloves used in the extraction process.
Synthesis of 4-allyl-2-methoxy-1-(prop-2-yn-1-yloxy)benzene (20). A 50 mL round-
bottom flask was charged with eugenol (19) (1.20 g, 7.32 mmol), sodium hydroxide (0.313 g,
o
7
.83 mmol) and 25 mL of methanol. The resulting mixture was heated to 40 C and magneti-
cally stirred for 30 minutes. After this time, methanol was removed under reduced pressure
and 10.0 mL of anhydrous ethanol was added for the removal of the residual water. The etha-
nol was removed under reduced pressure. Then, the round-bottom flask, under a nitrogen
atmosphere, was charged with anhydrous acetonitrile (25.0 mL) and propargyl bromide (800
μL, 8.79 mmol) was added slowly. The mixture was magnetically stirred at room temperature
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Discovery of novel West Nile Virus protease inhibitor
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Discovery of novel West Nile Virus protease inhibitor
Fig 2. Structures of compounds 1–18, 23–49, and 51–63.
https://doi.org/10.1371/journal.pone.0223017.g002
for 18 hours. TLC analysis revealed the completion of the reaction after this time. The reaction
mixture was concentrated under reduced pressure and the residue was partitioned between
-1
2
5.0 mL of sodium hydroxide solution (0.1 mol L ) and 25 mL of diethyl ether. The layers
were separated and the aqueous phase was extracted with diethyl ether (2 x 25.0 mL). The
organic extracts were combined and the resulting organic layer was washed with brine (25.0
mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
The residue was purified by column chromatography eluted with hexane-ethyl acetate (6:1 v/
v). The described procedure afforded 1.29 g (6.37 mmol, 87% yield) of compound 20.
Synthesis of pent-4-yn-1-yl methanesulfonate (21). Pent-4-yn-1-ol (1.68 g, 20.0 mmol)
and dichloromethane (20 mL) were added to a 100 mL round-bottom flask under nitrogen
atmosphere. The mixture was cooled to -50˚C and triethylamine (5.60 mL, 40.0 mmol) was
added. After that, methanesulfonyl chloride was added slowly (2.32 mL, 30.0 mmol) to the
reaction mixture under continuous stirring. The progress of the reaction was monitored by
TLC. After completion of it, 10 mL of distilled water were added. The organic phase was
washed with 1% HCl solution (3 x 15 mL) followed by saturated aqueous NaHCO (3 x 5 mL),
3
dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue
was purified by column chromatography eluted with hexane-ethyl acetate-dicloromethane
(3:1:3 v/v) to give compound 21 in 92% yield (3.00 g, 18.0 mmol).
Synthesis 4-allyl-2-methoxy-1-(pent-4-yn-1-yloxy)benzene (22). This compound was
prepared using a similar procedure to that described for the preparation of compound 20. In
this case, the alkylating agent corresponded to compound 21. Substance 22 was obtained in
78% yield (1.64 g, 7.13 mmol) after purification by column chromatography eluted with hex-
ane-ethyl acetate (6:1 v/v).
Synthesis of compounds 23–51 exemplified by the synthesis of compound 4-((4-allyl-
2-methoxyphenoxy)methyl)-1-benzyl-1H-1,2,3-triazole (23). A 10 mL round bottom flask
was charged with 4-allyl-2-methoxy-1-(prop-2-yn-1-yloxy)benzene (20) (0.150 g, 0.740
mmol), benzyl azide (0.0990g; 0.740 mmol), sodium ascorbate (0.0590 g, 0.300 mmol), 1.00
mL of etanol and 1.00 mL of distilled water. Then, CuSO �5H O (0.0370 g, 0.150 mmol) was
4
2
added. The reaction mixture was vigorously stirred at room temperature. After completion of
the reaction, as noticed was by TLC analysis, the mixture was extracted with dichloromethane
(3 x 10.0 mL). The organic extracts were combined and the resulting organic layer was washed
with aqueous sodium carbonate saturated solution, dried over anhydrous sodium sulphate, fil-
trated and concentrated under reduced pressure. The residue was purified by silica gel column
chromatography eluted with hexane-ethyl acetate-dichloromethane (3:1:3 v/v). Compound 23
was obtained in 91% yield (0.228g, 0.680 mmol). Selected IR and NMR spectra is show at A-C
Figs in S1 File.
Compounds 24–51 were prepared using a procedure similar to that described for the syn-
1
13
thesis of 23. The structures of these compounds were confirmed by NMR ( H and C), and IR
analyses and are supported by the following data.
Data for 4-((4-allyl-2-methoxyphenoxy)methyl)-1-(4-fluorobenzyl)-1H-1,2,3-triazole
(24)
White solid, m.p. 92.1–92.7˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.39 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at D-F Figs in S1 File.
Data for 4-((4-allyl-2-methoxyphenoxy)methyl)-1-(4-chlorobenzyl)-1H-1,2,3-triazole
(25)
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Discovery of novel West Nile Virus protease inhibitor
White solid, m.p. 112.3–112.6˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.37 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at G-I Figs in S1 File.
Data for 4-((4-allyl-2-methoxyphenoxy)methyl)-1-(4-bromobenzyl)-1H-1,2,3-triazole
(26)
White solid, m.p. 119.1–120.2˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.59 (hexane-ethyl acetate-dichloro-
methane 3:1:3 v/v). Selected IR and NMR spectra is show at J-L Figs in S1 File.
f
Data for 4-((4-allyl-2-methoxyphenoxy)methyl)-1-(4-iodobenzyl)-1H-1,2,3-triazole
(27)
White solid, m.p. 129.2–131.1˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.37 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at M-O Figs in S1 File.
Data for 4-((4-allyl-2-methoxyphenoxy)methyl)-1-(4-nitrobenzyl)-1H-1,2,3-triazole
(28)
White solid, m.p. 120.9–121.9˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v) TLC: R = 0.14 (hexane-ethyl acetate-dichloro-
methane 3:1:3 v/v). Selected IR and NMR spectra is show at P-R Figs in S1 File.
f
Data for 4-((4-allyl-2-methoxyphenoxy)methyl)-1-(4-methoxybenzyl)-1H-1,2,3-triazole
(29)
White solid, m.p. 92.4–93.1˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.36 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at S-U Figs in S1 File.
Data for 4-((4-allyl-2-methoxyphenoxy)methyl)-1-(4-(trifluoromethoxy)benzyl)-1H-
,2,3-triazole (30)
1
White solid, m.p. 126.4–127.3˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.49 (hexane-ethyl acetate-dichloro-
methane 3:1:3 v/v). Selected IR and NMR spectra is show at V-W Figs in S1 File.
f
Data for 4-((4-allyl-2-methoxyphenoxy)methyl)-1-(4-(trifluoromethyl)benzyl)-1H-
,2,3-triazole (31)
1
White solid, m.p. 142.7–143.0˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.43 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at Y-AA Figs in S1 File.
Data for 4-((4-allyl-2-methoxyphenoxy)methyl)-1-(3,4-difluorobenzyl)-1H-1,2,3-tria-
zole (32)
White solid, m.p. 104.9–105.2˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.30 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at AB-AD Figs in S1 File.
Data for 4-((4-allyl-2-methoxyphenoxy)methyl)-1-(2,5-dichlorobenzyl)-1H-1,2,3-tria-
zole (33)
White solid, m.p. 87.9–88.4˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.59 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at AE-AG Figs in S1 File.
Data for 4-((4-allyl-2-methoxyphenoxy)methyl)-1-(4-methylbenzyl)-1H-1,2,3-triazole
(34)
White solid, m.p. 81.4–82,3˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.66 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at AH-AJ Figs in S1 File.
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Discovery of novel West Nile Virus protease inhibitor
Data for 4-((4-allyl-2-methoxyphenoxy)methyl)-1-(2-bromobenzyl)-1H-1,2,3-triazole
(35)
White solid, m.p. 67.5–68.6˚C, TLC: R = 0.70 (hexane-ethyl acetate-dichloromethane 3:1:3
f
v/v). Selected IR and NMR spectra is show at AK-AM Figs in S1 File.
Data for 4-((4-allyl-2-methoxyphenoxy)methyl)-1-(3-bromobenzyl)-1H-1,2,3-triazole
(36)
White solid, m.p. 97.5–97.9˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.70 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at AN-AP Figs in S1 File.
Data for 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-benzyl-1H-1,2,3-triazole (37)
White solid, m.p. 71.4–72.6˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane- (3:1:3 v/v), TLC: R = 0.16 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at AQ-AS Figs in S1 File.
Data for 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-fluorobenzyl)-1H-1,2,3-triazole
(38)
White solid, m.p. 96.4–96,8˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.21 (hexane-ethyl acetate-dichloro-
methane 3:1:3 v/v). Selected IR and NMR spectra is show at AT-AV Figs in S1 File.
f
Data for 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-chlorobenzyl)-1H-1,2,3-triazole
(39)
White solid, m.p. 81.8–82.2˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.46 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at AX-AY Figs in S1 File.
Data for 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-bromobenzyl)-1H-1,2,3-triazole
(40)
White solid, m.p. 89.4–90.3˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.46 (hexane-ethyl acetate-dichloro-
methane 3:1:3 v/v). Selected IR and NMR spectra is show at AZ-BB Figs in S1 File.
f
Data for 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-iodobenzyl)-1H-1,2,3-triazole
(41)
White solid, m.p. 98.3–99.2˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.45 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at BC-BE Figs in S1 File.
Data for 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-nitrobenzyl)-1H-1,2,3-triazole
(
42)
White solid, m.p. 79.7–80.4˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R 0.29 (hexane-ethyl acetate-dichloromethane
f
3
:1:3 v/v). Selected IR and NMR spectra is show at BF-BH Figs in S1 File.
Data for 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-methoxybenzyl)-1H-1,2,3-tria-
zole (43)
White solid, m.p. 88.9–90.4˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.22 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at BI-BK Figs in S1 File.
Data for 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-(trifluoromethoxy)benzyl)-1H-
,2,3-triazole (44)
1
White solid, m.p. 93.4–94.9˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.49 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at BL-BN Figs in S1 File.
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Discovery of novel West Nile Virus protease inhibitor
Data for 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-(trifluoromethyl)benzyl)-1H-
,2,3-triazole (45)
1
White solid, m.p. 111.3–112.5˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.50 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at BO-BQ Figs in S1 File.
Data for 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(3,4-difluorobenzyl)-1H-1,2,3-tria-
zole (46)
White solid, m.p. 97.1–97.8˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.41 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at BR-BT Figs in S1 File.
Data for 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-methylbenzyl)-1H-1,2,3-triazole
(47)
White solid, m.p. 59.6–60.9˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.42 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at BU-BX Figs in S1 File.
Data for 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(2-bromobenzyl)-1H-1,2,3-triazole
(48)
White solid, m.p. 47.8–49.1˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.61 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at BW-BZ Figs in S1 File.
Data for 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(3-bromobenzyl)-1H-1,2,3-triazole
(49)
White solid, m.p. 65.9–67.1˚C, purified by column chromatography eluted with hexane-
ethyl acetate-dichloromethane (3:1:3 v/v), TLC: R = 0.32 (hexane-ethyl acetate-dichloro-
f
methane 3:1:3 v/v). Selected IR and NMR spectra is show at CA-CC Figs in S1 File.
2
,2-di(prop-2-yn-1-yl)-1H-indene-1,3(2H)-dione (50). To a mixture of indan-1,3-dione
0.146 g, 1.00 mmol) and distilled water (40 mL), it was added potassium hydroxide (0.140 g,
.250 mmol) and propargyl bromide (0.364 mL, 4.00 mmol). The resulting mixture was mag-
(
0
o
netically stirred at 40 C for 24h. After this time, the reaction mixture was cooled down to
-1
room temperature and it was neutralized with aqueous solution of HCl 0.100 mol L . The
aqueous phase was extracted with ethyl acetate (3 x 20 mL). The organic extracts were com-
bined and the resulting organic phase was washed with brine (15 mL), dried over anhydrous
sodium sulfate, filtered, and concentrated under reduced pressure. Compound 50 was not sub-
mitted to any purification process and it was obtained in 93% yield (0.207 g, 0.930 mmol).
Synthesis of compounds 51–63 exemplified by the synthesis of 2,2-bis((1-(4-bromoben-
zyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indene-1,3(2H)-dione (51). To a 10 mL round bot-
tom-flask, containing 1.5 mL of dichloromethane and 1.5 mL of distilled water, it was added
4-bromobenzylazide (168.8 mg, 0.800 mmol), alkyne 50 (88.8 mg, 0.400 mmol), and sodium
ascorbate (60.0 mg, 0.300 mmol). Then, it was added CuSO �5H O (38.0 mg, 0.150 mmol) and
4
2
the resulting mixture was vigorously stirred at room temperature for 2 hours. After that, it was
added to the reaction mixture sodium carbonate saturated aqueous solution (20 mL) and the
resulting aqueous phase was extracted with dichloromethane (3 x 15 mL) followed by ethyl
acetate (3 x 15 mL). The organic extracts were combined and the resulting organic phase was
dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The
residue was purified by silica-gel column chromatography eluted with hexane-ethyl-acetate-
dichloromethane 3:1:3 v/v. Compound 51 was obtained in 51% yield (143.1 mg, 0.220 mmol).
Selected IR and NMR spectra is show at CD-CF Figs in S1 File.
Compounds 52–63 were synthesized employing a procedure similar to that described for
the preparation of 51. The structures of substances 52–63 are supported by the following data.
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Discovery of novel West Nile Virus protease inhibitor
Data for 2,2-bis((1-(3-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indene-1,3(2H)-
dione (52)
o
White solid, m.p. 185.6–187.2 C, purified by silica gel column chromatography eluted with
hexane-ethyl-acetate-dichloromethane (3:1:1 v/v), TLC: R = 0.08 (hexane-ethyl acetate-
f
dichloromethane 3:1:1 v/v). Selected IR and NMR spectra is show at CG-CI Figs in S1 File.
Data for 2,2-bis((1-(2-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indene-1,3(2H)-
dione (53)
o
Pale yellow solid, m.p. 178.4–179.7 C, purified by silica gel column chromatography eluted
with hexane-ethyl-acetate-dichoromethane (3:1:1 v/v), TLC: R = 0.11 (hexane-ethyl acetate-
f
dichloromethae 3:1:1 v/v). Selected IR and NMR spectra is show at CJ-CL Figs in S1 File.
Data for 2,2-bis((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indene-1,3(2H)-
dione (54)
o
White solid, m.p. 222.3–223.9 C, purified by silica gel column chromatography eluted with
hexane-ethyl-acetate-dichloromethane (3:1:1 v/v), TLC: R = 0.02 (hexane-ethyl acetate-
f
dichoromethane 3:1:1 v/v). Selected IR and NMR spectra is show at CM-CO Figs in S1 File.
Data for 2,2-bis((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indene-1,3(2H)-
dione (55)
o
White solid, m.p. 223.0–224.5 C, purified by silica gel column chromatography eluted with
hexane-ethyl-acetate-dichloromethane (3:1:1 v/v), TLC: R = 0.01 (hexane-ethyl acetate-
f
dichoromethane 3:1:1 v/v). Selected IR and NMR spectra is show at CP-CR Figs in S1 File.
Data for 2,2-bis((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indene-1,3
(
2H)-dione (56)
o
White solid, m.p. 118.5–119.8 C, purified by silica gel column chromatography eluted with
hexane-ethyl-acetate-dichloromethane (3:1:1 v/v), TLC: R = 0.01 (hexane-ethyl acetate-
f
dichloromethane 3:1:1 v/v). Selected IR and NMR spectra is show at CS-CU Figs in S1 File.
Data for 2,2-bis((1-(4-trifluoromethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-
indene-1,3(2H)-dione (57)
o
White solid, m.p. 253.7–254.5 C, purified by silica gel column chromatography eluted with
hexane-ethyl-acetate-dichloromethane (3:1:1 v/v), TLC: R = 0.02 (hexane-ethyl acetate-
f
dichloromethane 3:1:1 v/v). Selected IR and NMR spectra is show at CV-CW Figs in S1 File.
Data for 2,2-bis((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indene-1,3(2H)-
dione (58)
o
White solid, m.p. 271.2–272.4 C, purified by silica gel column chromatography eluted with
hexane-ethyl-acetate-dichloromethane (3:1:1 v/v), TLC: R = 0.01 (hexane-ethyl acetate-
f
dichloromethane 3:1:1 v/v). Selected IR and NMR spectra is show at CY-DA Figs in S1 File.
Data for 2,2-bis((1-(2,4-difluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indene-1,3
(
2H)-dione (59)
o
White solid, m.p. 178.3–179.5 C, purified by silica gel column chromatography eluted with
hexane-ethyl-acetate-dichloromethane (3:1:1 v/v), TLC: R = 0.06 (hexane-ethyl acetate-
f
dichloromethane 3:1:1 v/v). Selected IR and NMR spectra is show at DB-DD Figs in S1 File.
Data for 2,2-bis((1-(4-iodobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indene-1,3(2H)-
dione (60)
o
White solid, m.p. 211.3–212.2 C, purified by silica gel column chromatography eluted with
hexane-ethyl-acetate-dichloromethane (3:1:1 v/v), TLC: R = 0.06 (hexane-ethyl acetate 3:1:1
f
v/v). Selected IR and NMR spectra is show at DE-DG Figs in S1 File.
Data for 2,2-bis((1-(2-benzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indene-1,3(2H)-dione
(61)
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Discovery of novel West Nile Virus protease inhibitor
o
White solid, m.p. 161.5–162.8 C, purified by silica gel column chromatography eluted with
hexane-ethyl-acetate-dichloromethane (3:1:1 v/v), TLC: R = 0.05 (hexane-ethyl acetate-
f
dichloromethane 3:1:1 v/v). Selected IR and NMR spectra is show at DH-DJ Figs in S1 File.
Data for 2,2-bis((1-(4-trifluoromethylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-
indene-1,3(2H)-dione (62)
o
White solid, m.p. 243.7–244.6 C, purified by silica gel column chromatography eluted with
hexane-ethyl-acetate-dichloromethane (3:1:1 v/v), TLC: R = 0.24 (hexane-ethyl acetate-
f
dichloromethane 3:1:1 v/v). Selected IR and NMR spectra is show at DK-DM Figs in S1 File.
Data for 2,2-bis((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indene-1,3(2H)-
dione (63)
o
White solid, m.p. 170.8–171.4 C, purified by silica gel column chromatography eluted with
hexane-ethyl-acetate-dichloromethane (3:1:1 v/v), TLC: R = 0.08 (hexane-ethyl acetate-
f
dichloromethane 3:1:1 v/vSelected IR and NMR spectra is show at DN-DP Figs in S1 File.
Evaluation of the inhibitory activity of compounds 1–18, 23–49, and 51–63
on WNV NS2B-NS3 protease
In order to assess the activity of the synthesized compounds on WNV NS2B-NS3pro, recombi-
nant 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 Sys-
tems (Minneapolis, MN, United States). Compounds 1–18, 23–49, and 51–63 were dissolved
in pure dimethyl sulfoxide (DMSO); solutions were then diluted in a buffer to obtain working
solutions with a final concentration of 1% v/v DMSO. A volume of 50 μL of purified
-1
-1
NS2B-NS3pro (final concentration of 1 ng μL ) diluted in a buffer (50 mmol L Tris, 30% (v/
v) glycerol, pH 9.5) was incubated with 50 μL of each compound (final concentration of 16
-1
μmol L ) in a 96-well black plate for 30 min at 21–22˚C. After this time, the assay was initiated
-1
by addition of 50 μL of the substrate (40 mmol L –initial concentration). A solution contain-
ing a buffer and DMSO was used as negative control on the same plate. The blank contained
50 μL of the buffer and 100 μL of the substrate. The fluorescence intensity was continuously
recorded at a 360 nm excitation wavelength and at an emission wavelength of 460 nm using a
1
SpectraMax M5 microplate reader (Molecular Devices, San Jos e´ , CA, United States). Com-
pounds which effectively inhibited 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 triplicate during three iso-
lated experiments, and the statistical analyses were conducted by utilizing the multiple com-
parisons of one-way ANOVA.
Determination of IC50
The inhibitory enzymatic activity of compound 35, the one most active against the WNV
-1
-1
NS2-NB3 protease, was evaluated at eight different concentrations (66 μmol L –0.5 μmol L )
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 software 6 (GraphPad Software Inc., San Diego, CA, United States), using
four-parameter nonlinear regression analysis (Hill slope method).
Determination of Ki
-1
Three different concentrations (2, 4 and 8 μmol L ) of inhibitor 35 and five different concen-
-1
trations of substrate pERTKR-AMC (20, 40, 60, 80, 100 mmol L ) were tested in vitro against
-1
-1
the WNV protease (37.04 nmol L protein, 1 ng μL ). Fluorescence was measured in triplicate
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Discovery of novel West Nile Virus protease inhibitor
wells at an interval of 30 s. The velocity values (RFU/minute) were then calculated for each
substrate/inhibitor pair. K values were calculated with GraphPad Prism software 6 (GraphPad
i
Software Inc., San Diego, CA, United States) with non-linear regression in the competitive
inhibition mode of enzyme-kinetics.
Cytotoxicity assay
4
The cytotoxicity of compound 35 was assessed using an MTT assay [44]. VERO cells (5 x 10
cells) were seeded in 96-well plates. Each well contained 100 μL of each compound solution at
-1
different concentrations (1000, 250, 125, 63, 32, 17, 8 and 4 μmol L ). The compound was
diluted in MEM medium with 2% FBS and 1% DMSO. After 24 h of incubation at 37 C, 100
o
μL of the MTT solution (5%) was added to the wells. After 4 h at 37˚C, the MTT solution was
removed and 100 μL/well of DMSO was added to solubilize the formazan. Absorbance was
measured at 550 nm in a microplate reader (Multiskan™ GO Microplate Spectrophotometer–
1
ThermoFisher , Waltham, MA, United States). The data were analyzed and CC was deter-
50
mined using GraphPad Prism 6.
Virucidal assay
The virucidal assay was performed as described by Oliveira et al. [41].
Molecular modeling studies
Ligands were prepared in the Ligprep program (Schr o¨ dinger, New York, NY, United States)
45] employing the OPLS_2005 force field, with protonation states predicted using Epik at pH
.5 ± 2.0. The West Nile Virus protease NS2B-NS3 PDB code 2IJO [46], chosen as the receptor,
[
9
was prepared with the Protein Preparation Wizard (Schr o¨ dinger, New York, NY, United
States), with removal of all waters and addition of hydrogens based on PROPKA calculations
at pH 9.5. Docking calculations were performed with the Glide software (Schr o¨ dinger, New
York, NY, United States) [47, 48], employing the Induced Fit docking methodology [49] and
Glide SP. All the software packages used are part of the Schr o¨ dinger Release 2016–2 package
(Schr o¨ dinger, New York, NY, United States) [50]. A spherical grid with 12 Å radius was cen-
tered in the Isoleucine 123 residue so that the active site was fully included within the grid. All
residues within 5 Å from the center were considered flexible. Docking results were ranked
based on their docking score and the top ranking poses for each compound were analyzed
with the Maestro 10.6 software (Schr o¨ dinger, New York, NY, United States) [51].
Results and discussion
Synthesis of compounds 1–18, 23–49, and 51–63
The structures of the synthesized compounds are depicted in Fig 2.
As previously mentioned, the synthesis of isobenzofuran-(3H)-ones 1–18 has been previ-
ously reported [43].
For the preparation of compounds 23–49, we initially isolated eugenol (19) from dried
flower buds of Eugenia caryophyllata via hydrodistillation. Subsequently, eugenol (19) was
submitted to alkylation procedures, affording terminal alkynes 21 and 22 in yields of, respec-
tively, 87% and 78% (Fig 3).
Then, the CuAAC reactions (click reactions) between benzyl azides (ArCH N ) and termi-
2
3
nal alkynes 21 and 22 led to the formation of triazolic derivatives 23–49 (Fig 4). The click reac-
tions, in general, took less than one minute for their completion.
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Discovery of novel West Nile Virus protease inhibitor
o
o
3 3
Fig 3. Preparation of alkylated derivatives of eugenol. i) NaOH, CH OH, 40˚C; acetonitrile, r.t; ii) NaOH, CH OH, 40 C; acetonitrile, 70 C.
https://doi.org/10.1371/journal.pone.0223017.g003
A similar sequence, namely alkylation of indan-1,3-dione followed by click reaction
between compound 50 and benzyl azides, was utilized to prepared triazolic compounds 51–63
(
Fig 5).
It should be mentioned that the azides, used in the preparation of compounds 23–49 and
1–63, were obtained via the methodology previously described in the literature [52]. Once
5
prepared, the synthesized compounds were submitted to biological assays to evaluate their
inhibitory effects against the NS2-NB3 protease of WNV.
Identification of WNV protease inhibitors
The Flavivirus protease is essential for the processing of the polyprotein which generates the
viral proteins required for viral replication and maturation of infectious virions. Therefore, the
protease is an ideal target for the discovery of antivirals against Flavivirus [53]. In the present
investigation, we evaluated the inhibitory activity of fifty-eight compounds against the WNV
NS2B-NS3 protease (eighteen 3-(2-oxo-2-aryl)-isobenzofuran-1(3H)-ones, compounds 1–18;
twenty-seven derivatives of eugenol with triazole rings, compounds 23–49; thirteen derivatives
of indan-1,3-diones presenting triazole rings, compounds 51–63, Fig 2). In this regard, a puri-
fied preparation of WNV NS2B3NS3pro and the fluorogenic peptide substrate pERTKR-AMC
was utilized.
Our primary screen resulted in the identification of eighteen compounds (~31% of the eval-
uated compounds) that exhibited inhibitory effects on protease activity (Fig 6).
Subsequently, we took these eighteen compounds and conducted a secondary screening
with further validation based on their relative strengths of inhibition (at least 50%). This
resulted in the selection of compound 35, a eugenol derivative which was utilized in further
experiments.
A derivative of the natural product eugenol capable of significantly inhibiting the activity of
the WNV NS2B-NS3 protease was identified. Taking the eugenol derivatives into
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Discovery of novel West Nile Virus protease inhibitor
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Discovery of novel West Nile Virus protease inhibitor
Fig 4. Preparation of eugenol derivatives 23–49. iii) sodium ascorbate (40 mol%), CuSO
4
2 2
�5H O (20 mol%), EtOH/H O (1:1 v/v).
https://doi.org/10.1371/journal.pone.0223017.g004
consideration, we made variations in the size of the carbon chain that links eugenol moiety
and the triazole ring. Also, the substitution pattern of the aromatic ring attached to the triazolic
portion was varied. These modifications afforded a group of eugenol triazolic derivatives from
which was identified the very active compound 35. It is important to mention that further
chemical modifications can be planned regarding the eugenol and triazole fragments so that
new derivatives with improved activity may be obtained. Considering that there is no Flavivi-
rus protease inhibitor approved for pre-clinical trial [54], the exploitation of the new scaffold
herein identified, namely triazolic derivatives of eugenol, would be of considerable
importance.
Determination of IC and Ki values of the enzymatic inhibitory activity of
compound 35
50
-
1
The enzymatic inhibition was evaluated in the presence of varying concentrations (0.5 μmol L
1
to 66 μmol L ) of compound 35. A dose-response inhibition was noticed with IC value of 6.86
50
-1
μmol L (Fig 7).
The enzyme kinetic assay was conducted under five different substrate concentrations and
three varying concentrations of compound 35. We used the Michaelis-Menten equation to
find the values of VMAX and K ; and with these, the Lineweaver-Burk graph was built.
M
o
Fig 5. Synthesis of compounds 51–63. iv) aqueous KOH, then propargyl bromide, 40 C, 24h; v) sodium ascorbate (40 mol%), CuSO
4
2 2 2 2
�5H O (20 mol%), CH Cl /H O
(1:1 v/v), 2h.
https://doi.org/10.1371/journal.pone.0223017.g005
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Discovery of novel West Nile Virus protease inhibitor
-1
Fig 6. Inhibitory activity screening for WNV protease. Fifty-eight compounds were evaluated at the concentration of 16 μmol L against the WNV NS2-NB3
protease. The assays were conducted in triplicate during three isolated experiments (p value < 0.0001).
https://doi.org/10.1371/journal.pone.0223017.g006
Compound 35 showed a decrease of enzymatic VMAX and an increase in K , presenting the
M
-1
behaviour of a competitive inhibitor (Fig 8A and 8B). The K value was 3.06 (± 0.38) μmol L .
i
For compound 35, the enzyme kinetic data showed an increase in the K value, a typical
behaviour of competitive inhibitors. In addition, this compound displayed a low K value. In a
M
i
recent investigation, Balasubramanian and collaborators [53] found eight promising flavivirus
-1
protease inhibitors presenting K values within the 0.22 to 6.9 μmol L range. However, the K
i
i
value may not be analyzed individually. In the study by Balasubramanian and collaborators, a
-1
-1
compound presenting low K value (0.22 μmol L ) but displaying a CC of 29.16 μmol L
i
50
was identified [53].
For the development of new drugs, the World Health Organization (WHO) strongly rec-
ommends the use of compounds with pharmacological effects already described and sub-
stances already approved for clinical use, so that several steps can be abbreviated in the long
validation process [55]. Eugenol (19) has been safely used in in vivo experiments, and it has
Fig 7. Enzymatic inhibitory profile of compound 35.
https://doi.org/10.1371/journal.pone.0223017.g007
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Discovery of novel West Nile Virus protease inhibitor
Fig 8. Enzymatic kinetics of WNV NS2-NB3 protease in the presence of compound 35. In A, the Michaelis-Menten
plot is shown. In B, the Lineweaver-Burk plot is presented.
https://doi.org/10.1371/journal.pone.0223017.g008
been recognized as a safe, effective and inexpensive anesthetic for fish, amphibians and rats
[56, 57]. Also, the analgesic effect of eugenol (19) in different models of pain has been well doc-
umented [58–62]. A recent work with BALB/c mice used a derivative of eugenol (19) to treat
visceral Leishmaniasis. This derivative presented low cytotoxicity for macrophages as well as
for naive mice with immune-stimulatory activity. Moreover, no biochemical alterations in
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Discovery of novel West Nile Virus protease inhibitor
hepatic and renal enzymes were noticed [63]. Eugenol (19) is generally non-allergenic for
humans, although in sensitized individuals it may cause a range of tissue reactions from low-
grade local to systemic. Low concentrations of eugenol (19) are well known to exert local anti-
inflammatory, antiseptic, and anesthetic effects on dental pulp. Also, eugenol (19) may have
antibacterial effects that are beneficial for dental hygiene, being included in materials such as
toothpastes and mouthwashes [64–66]. All these features make eugenol (19), as well as its
derivatives, very interesting compounds to be explored in drug development.
Cytotoxicity assay
The cytotoxicity of compound 35 on Vero cells was investigated via the colorimetric MTT
-1
assay and the determined CC50 value was 327.20 μmol L (Fig 9). Luo and collaborators [54]
reported the best parameters for the most promising compounds against the protease of Flavi-
virus. They highlighted a compound with low cytotoxicity presenting CC superior to 300
50
-1
μmol L . Eugenol derivative 35 presented a similar CC , and it can be considered a com-
50
pound with low cytotoxicity. Therefore, although a very potent inhibitor, this compound pre-
sented considerable cytotoxicity.
Molecular modeling
Multiple targets and inhibitory mechanisms have been proposed for Flavivirus proteases so far
[53, 54, 67, 68], which have provided insightful structural information regarding the
NS2B-NS3 catalytic site, helping computational-aided drug design efforts even further. For
instance, by solving NS2B-NS3’s tridimensional structure bound to a peptide-like inhibitor,
Erbel and coworkers [69] provided fundamental information regarding the protease fold, cata-
lytic site structure and inhibition mechanisms. As found by others [46, 70–72], inhibitors
51
75
129
153
161
often bind to His or to nearby residues such as Asp , Asp , Gly and Tyr (S1 Fig),
hampering the bond of the substrate to the catalytic site.
In order to perform our own molecular docking calculations, the crystallographic structure
of the NS2B-NS3 protease was obtained from PDB 2IJO, in which NS2B-NS3 is co-crystallized
with the WNV protease inhibitor aprotinin. All molecules derived from eugenol were analyzed
for their probable three-dimensional binding conformation, binding energy, chemical groups
involved, profile of binding and identity of the involved amino acids, as observed in table A in
S1 File. The predicted recognition mechanism for compound 35 relies on interactions with
5
1
134
135
161
His , Thr , Ser and Tyr , as represented in Fig 10A, and the ligand occupies pocket S1
+
51
of the catalytic site. The NH of the His ring interacts with the triazole ring of 35 in cation-π
and π-π type interactions, and the Tyr ring interacts with the phenyl ring of 35 via π-π type
interaction, while a halogen bond can be formed between the Br atom and the OH from Ser
161
135
134
51
135
or the NH from Thr . It is important to highlight that both His and Ser are members of
the catalytic triad of the NS2B-NS3 protease, which might explain the observed competitive
inhibitory activity of 35.
In addition, compound 36 showed no inhibitory activity, despite the fact that the only dif-
ference from compound 35 is the position of a bromo substituent (orto in 35 and meta in 36).
Our calculations suggest that the formation of a halogen bond for 36 with bromo in meta
might induce a more stable conformation for the ligand in which the triazole ring interacts
153
51
with Gly instead of His and, consequently, leaves the S1 pocket free (Fig 10B). These
results suggest that the bromo substituent in orto is pivotal for a proper recognition of com-
51
pound 35, along with the cation-π interaction between His and the triazole ring, while the
methoxyphenyl ring region might be a suitable target for further improvements.
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Discovery of novel West Nile Virus protease inhibitor
Fig 9. Dose-response profile of compound 35 on Vero cells.
https://doi.org/10.1371/journal.pone.0223017.g009
Virucidal assay
To determine whether compound 35 would have antiviral activity for Flaviviruses, a virucidal
assay was performed with four serotypes of DENV, due to its close evolutionary proximity to
WNV and due to the highly conserved NS2B-NS3 fold and sequence. This assay was per-
formed by prior incubation of the compound with each viral strain, followed by its addition to
the cell layer for virus adsorption and internalization. Subsequently, the compound-virus solu-
tion was removed and antiviral action was observed through the formation of lysis plates. The
concentration of test compounds that inhibited 50% of the viral infection (EC ) was obtained
50
by nonlinear regression, leading to the calculation of the Selectivity Index (SI).The observed
results are presented in Table 1.
The compound had good SI values for DENV-1-3 and a higher value for DENV-4. All
together, the results indicate that compound 35 has significant antiviral efficacy and is a prom-
ising antiviral candidate for Flavivirus.
Conclusion
In this work, a small library of fifty-eight synthetic compounds (isobenzofuran-1(3H)-ones
and triazolic derivatives of eugenol and indandione) were screened against the WNV
NS2B-NS3 protease. By modifying the structure of the natural product eugenol to produce
1,2,3-triazolic derivatives, a compound presenting low cytotoxicity and considerable inhibitory
protease activity was identified. Compound 35 corresponds to 4-(3-(4-allyl-2-methoxyphe-
noxy)propyl)-1-(2-bromobenzyl)-1H-1,2,3-triazole. In addition, molecular docking calcula-
51
134
tions suggested that the inhibition mechanism relies on interactions between His , Thr
,
135
161
Ser and Tyr . The virucidal assay with DENV-1-4 strains indicates that compound 35 is a
promising lead compound for antiviral activity against Flavivirus. Taken together, our results
provide insightful information for further development of Flavivirus protease inhibitors via
rational drug design. Efforts towards this end are under way in our laboratories.
PLOS ONE | https://doi.org/10.1371/journal.pone.0223017 September 26, 2019
19 / 25

Discovery of novel West Nile Virus protease inhibitor
135
51
Fig 10. WNV protease docking with compound 35 and 36. The entire protease is shown in lilac, highlighting residues Ser (dark blue), His (orange),
75
and Asp (green). A. In white is compound 35. B. In gray is compound 36. Hydrogen bonds are shown in yellow, halogen bonds are shown in red, T-shape
π-π interactions are shown in cyan and cation-π interactions are shown in pink.
https://doi.org/10.1371/journal.pone.0223017.g010
Table 1. CC50, EC50 and SI values of the compound 35 in the presence of DENV-1-4.
Compound
CC50
μmol L
DENV-1
DENV-2
DENV-3
DENV-4
-
1
EC50
SI
EC50
SI
EC50
SI
EC50
SI
-
1
-1
-1
-1
μmol L
46,57
μmol L
49,20
μmol L
70,10
μmol L
14,17
35
327,20
7
7
5
23
https://doi.org/10.1371/journal.pone.0223017.t001
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Discovery of novel West Nile Virus protease inhibitor
Supporting information
S1 File. Table A. Docking of the compounds 23 to 49 with WNV protease. The entire prote-
ase is shown in lilac, highlighting residues Ser135 (dark blue), His51 (orange), and Asp75
(
green). The compound is show in red. FigsA-DP Figs. Selected IR and NMR spectra.
PDF)
(
S1 Fig. Inhibition mechanisms observed between WNV NS2B-NS3 protease cocrystallized
peptide-like molecules obtained from Protein Data Bank. A. 2YOL; B. 3E90; C. 5IDK; and
D. 2FP7.
(TIFF)
Author Contributions
Conceptualization: Andr e´ S. de Oliveira.
Formal analysis: Andr e´ S. de Oliveira, Poliana A. R. Gazolla, Ana Fl a´ via C. da S. Oliveira.
Investigation: Andr e´ S. de Oliveira, Poliana A. R. Gazolla, Ana Fl a´ via C. da S. Oliveira, Wag-
´
ner L. Pereira, L ´ı via C. de S. Viol, Ang e´ lica F. da S. Maia, Edjon G. Santos, Italo E. P. da
Silva, Tiago A. de Oliveira Mendes, Adalberto M. da Silva, Roberto S. Dias, Cynthia C. da
Silva.
Methodology: Andr e´ S. de Oliveira, Poliana A. R. Gazolla, Ana Fl a´ via C. da S. Oliveira, Wag-
´
ner L. Pereira, L ´ı via C. de S. Viol, Ang e´ lica F. da S. Maia, Edjon G. Santos, Italo E. P. da
Silva, Tiago A. de Oliveira Mendes, Adalberto M. da Silva, Roberto S. Dias, Cynthia C. da
Silva.
Project administration: R o´ bson R. Teixeira, Sergio O. de Paula.
Resources: R o´ bson R. Teixeira, Sergio O. de Paula.
Software: Tiago A. de Oliveira Mendes, Marcelo D. Polêto.
Supervision: R o´ bson R. Teixeira, Sergio O. de Paula.
Validation: Andr e´ S. de Oliveira, Poliana A. R. Gazolla, Ana Fl a´ via C. da S. Oliveira.
Visualization: R o´ bson R. Teixeira, Sergio O. de Paula.
Writing – original draft: Andr e´ S. de Oliveira, Poliana A. R. Gazolla, Ana Fl a´ via C. da S. Oli-
veira, R o´ bson R. Teixeira, Sergio O. de Paula.
Writing – review & editing: Andr e´ S. de Oliveira, Poliana A. R. Gazolla, Ana Fl a´ via C. da S.
Oliveira, Marcelo D. Polêto, R o´ bson R. Teixeira, Sergio O. de Paula.
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