Inhibition of Zika virus replication by synthetic bis-naphthoquinones

Article abstract of DOI:10.21577/0103-5053.20190071

Zika virus (ZIKV) is a mosquito-borne pathogen which is a current global public health concern. There are currently no approved vaccines or antivirals against ZIKV infection. Taking into account that naphthoquinones have shown promising antiviral activity

Full text of DOI:10.21577/0103-5053.20190071

http://dx.doi.org/10.21577/0103-5053.20190071
J. Braz. Chem. Soc., Vol. 00, No. 00, 1-10, 2019
Printed in Brazil - ©2019 Sociedade Brasileira de Química
Article
Inhibition of Zika Virus Replication by Synthetic Bis-Naphthoquinones
a
Daniel T. G. Gonzaga,a,b Rafaela S. P. Gomes,c Roberta K. F. Marra,a Fernando C. da Silva,
Max W. L. Gomes,c Davis F. Ferreira,d,e Raissa M. A. Santos,a Ana M. V. Pinto,f
Norman Arthur Ratcliffe,g Claudio C. Cirne-Santos,c Caroline S. Barros,c Vitor F. Ferreira*,a
and Izabel Christina N. P. Paixão *,c
aInstituto de Química, Universidade Federal Fluminense, 24020-141 Niteroi-RJ, Brazil
bInstituto Biomédico, Centro Universitário Estadual da Zona Oeste, 23070-200 Rio de Janeiro-RJ, Brazil
cInstituto de Biologia, Universidade Federal Fluminense, 24020-141 Niteroi-RJ, Brazil
dInstituto de Microbiologia Prof. Paulo de Goes, Universidade Federal do Rio de Janeiro,
21941-599 Rio de Janeiro-RJ, Brazil
eDepartment of Molecular and Structural Biochemistry, North Carolina State University,
120 W Broughton Dr, Raleigh, 27607 NC, USA
fUnidade de Farmácia, Universidade Federal Fluminense, 24210-130 Niteroi-RJ, Brazil
gDepartment of Biociences, College of Science Swansea University, SA2 8PP Swansea, UK
Zika virus (ZIKV) is a mosquito-borne pathogen which is a current global public health concern.
There are currently no approved vaccines or antivirals against ZIKV infection. Taking into account
that naphthoquinones have shown promising antiviral activity, the aim of this study was to describe
the screening of two bis-naphthoquinones series against ZIKV. Twenty seven compounds were
evaluated against ZIKV usingVero cells. The findings showed that among the compounds analyzed
four were promising. Compound 3,3’-((2-nitrophenyl)methylene)bis(2-hydroxynaphthalene-
1,4-dione) containing the nitro group at the ortho position showed the best selectivity index,
followed by compound 3,3’-(4-chlorophenylmethylene)bis(naphthalene-1,2,4-triyl triacetate) with
the chlorophenylmethylene radical. These results demonstrate that these bis-naphthoquinones are
largely effective in inhibiting the replication of ZIKV.
Keywords: Zika virus, Flavivirus, arbovirus, antiviral, bis-naphthoquinones
Introduction
ZIKV belongs to the virus family Flaviviridae and its
name derives from the Zika Forest of Uganda, where the
virus was first isolated in 1947 from a Rhesus macaque.5 In
recent years, ZIKV has been rapidly spreading in tropical
South America and French Polynesia despite attempts to
stop the spread of infection vectors. At present, Brazil is
the country with the highest number of reported cases of
infection, as well as of cases of microencephaly.6,7
In the process of controlling the spread of the
disease caused by the virus, there are several approaches
ranging from mosquito control,8 to interrupt the chain of
transmission, to attacking the virus via multiple therapeutic
agents.Vaccine development also has great potential but so
far there are limited prospects of vaccine production in the
short term. The Butantan Institute in Brazil in association
with National Institutes of Health (NIH) has announced
The spread of the disease caused by the Zika virus
(ZIKV) has become a serious public health problem of
global proportions. The disease is transmitted to people
through the bite of infected mosquitoes, Aedes aegypti and
Aedes albopictus.1 Usually, the disease has moderate effects
and symptoms disappear within a few days. Many people
have no or only mild symptoms. However, ZIKV infection
during pregnancy can cause a severe malformation of the
fetus, known as microcephaly, as well as other serious
brain damage.2,3 Some recent research4 also suggests that
Guillain-Barré Syndrome, an uncommon sickness of the
nervous system, is also associated with Zika virus infection.
*e-mail: izabeluff@gmail.com, vitorferreira@id.uff.br

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Inhibition of Zika Virus Replication by Synthetic Bis-Naphthoquinones
J. Braz. Chem. Soc.
Figure 1. Approved drugs that have been used against ZIKV.
the development of a vaccine that may go to human trials
in 5 years.9
quinone methide.24 Among the compound inserted in the
class of naphthoquinones, can be cited the droserone (6)
which reduced the production of viral particle of measles
virus to 50% (half maximal effective concentration,
EC50) at a concentration of 2 µM,25 2-aminomethyl-
3-hydroxy-1,4-naphthoquinone (7) which showed
promising antiviral activity against Herpes simplex
virus type-1 (HSV-1) presenting EC50 of 0.83 µM and
showed activity comparable to acyclovir, the antiviral
currently used clinically, which had EC50 of 1.09 µM,26
the pyranaphthoquinone (8) that exhibited EC50 of 0.3114
µM for dengue virus replication in mammalian cells27 and
the naphthoquinone trimer (9) which was inhibitor of
hepatitis B virus (HBV) and had EC50 of 0.009 µM and
selective index above 3000 (Figure 2).28
Alternatively, since there is no drug approved for the
treatment of Zika infection, drug repurposing screening
of pharmaceuticals or biopharmaceuticals as therapeutic
or prophylactic agents has recently been explored.10-12
There are still no approved drugs for specific therapy to
prevent or treat ZIKV infection.13 The search for new uses
of drugs already on the market can be a good strategy in
view of the safety proven mainly in the case of pregnant
women. However, the discovery of molecules with
different structures and mechanisms of action is also of
great importance. Indeed, recently, several drugs have been
evaluated against ZIKV with promising results (Figure 1),
as for example chloroquine 1a, hydroxychloroquine 1b,14
7-Deaza-2’-C-methyladenosine (2),15,16 gemcitabine (3)17
and bithionol (4).18 Elfiky19 compared by molecular docking
several inhibitors of the viral polymerases of ZIKV and
hepatitis C virus (HCV) and found ribavirin and sofosbuvir
(Figure 1) are possibly effective in regulating the life cycle
of these viruses.19 More recently, Sacramento et al.20 have
demonstrated that the antiviral sofosbuvir (5), used against
the HCV, which is also in the Flaviviridae, inhibits ZIKV
ribonucleic acid (RNA) polymerase, targeting conserved
amino acid residues.
Several previous studies21,22 indicated that compounds
containing a naphthoquinone moiety play key roles in
the respiratory cycle, electron transport chain, blood
clotting and the carboxylation of glutamate in cells. The
mechanism of action of naphthoquinones may involves the
generation of reactive oxygen species (ROS) induced by
bioreduction of the quinone nucleus by specific enzymes
to generate reactive oxygen species inside virus.23 Other
possible mechanism of action include intercalation,
induction of breaks in the deoxyribonucleic acid (DNA)
chain and bioreductive alkylation via formation of
Figure 2. Examples of antiviral naphthoquinones.
It was shown that bis-hydroxy-1,4-naphthoquinones
(10) have promising antimicrobial activity. de Araújo

Vol. 00, No. 00, 2019
Gonzaga et al.
3
et al.29 reported that these compounds have moderate
activities against Leishmania amazonensis and Leishmania
braziliensis promastigotes without significant toxicity. The
compound 10a presents high potency against Leishmania
amazonensis with EC50 of 0.3 0.1 µM while 10b was
more active against Leishmania braziliensis with EC50 of
0.9 0.08 µM (Figure 3).
The physical and spectroscopic data for 10a, 10b-e and
10g-o were previously reported by Tisseh and Bazgir,30
which developed some efficient method for production
bis-benzoquinonylmethanes.31
3,3’-(o-Tolylmethylene)bis(2-hydroxynaphthalene-
1,4-dione) (10f)
Yellow solid; yield 68%; mp 177-178 ºC; infrared (IR)
(KBr) ν / cm-1 558, 579, 627, 653, 644, 675, 693; 1H NMR
(500 MHz, DMSO-d6) d 8.00 (2H, dd, J 7.1 and 1.7 Hz),
7.95 (2H, dd, J 7.1 and 1.7), 7.81 (2H, td, J 9.0, 7.1 and
1.7), 7.76 (2H, td, J 9.0, 7.1 and 1.7 Hz), 7.21 (1H, dd, J 6.5
and 2.3 Hz), 7.10-7.11 (3H, m), 6.05 (1H, s), 2.26 (1H,
s); 13C NMR (125 MHz, DMSO-d6) d 19.5, 36.44, 123.5,
125.7, 126.0, 126.1, 126.5, 129.2, 129.9, 130.4, 132.7,
133.5, 135.1, 135.9, 139.4, 156.6, 181.5, 183.9; HRMS
(electrospray ionization, ESI) m/z, calcd. for C28H18O6Na+
[M + Na]+: 473.1001, found: 473.0980, ∆ = 2.1 ppm.
Figure 3. Example of leishmanicidal bis-hydroxy-1,4-naphthoquinones.
The aim of this work is to demonstrate the antiviral
potential of bis-naphthoquinones on the replication
of ZIKV that has reached a large number of people in
the population and has brought serious complications
especially in pregnant women.
General procedure for preparation of bis(naphthalene-
1,2,4-triyl triacetates) (13a-n)
In a 50 mL round bottom flask, 1 mmol of
bis(2-hydroxynaphthalene-1,4-diones) (12), 7.2 g of
zinc metallic, 15 mL of acetic anhydride and pyridine
catalytic were added. The solution was stirred for 72 h
in room temperature and the progress of the reaction
was followed by TLC. In the next step, the solution was
filtered, extracted with chloroform and the product was
purified by column chromatography, using hexane/ethyl
acetate as the eluent.
Experimental
Chemistry
Reagents were purchased from Sigma-Aldrich and were
used without further purification. Column chromatography
was performed with silica gel 60 (Merck 70-230 mesh).
Analytical thin layer chromatography (TLC) was performed
with silica gel plates (Merck, TLC silica gel 60 F254), and
the plots were visualized using UV light or aqueous solutions
of ammonium sulfate. Yields refer to chromatographically
and spectroscopically homogeneous materials.
Melting points were obtained on a Fischer-Johns
apparatus and were uncorrected. Infrared spectra were
measured using KBr pellets on a PerkinElmer model
1420 FT-IR spectrophotometer, calibrated relative to the
1601.8 cm-1 absorbance of polystyrene.
Nuclear magnetic resonance (NMR) spectra were
recorded on a Varian Unity Plus VXR (500 MHz)
instrument in dimethyl sulfoxide (DMSO-d6) or CDCl3
solutions. The chemical shift data were reported in units of
d (ppm) downfield from tetramethylsilane or the solvent,
either of which were used as an internal standard; coupling
constants (J) are reported in hertz and refer to apparent
peak multiplicities.
3,3’-(Phenylmethylene)bis(naphthalene-1,2,4-triyl
triacetate) (13a)
White solid; yield 83%; mp 222-223 ºC; IR (KBr)
ν / cm-1 1767, 1603, 1496, 1426, 1365, 1163, 1098,
1
1035, 908, 871, 759, 733, 699, 605, 585, 545; H NMR
(500 MHz, CDCl3) d 1.85 (s, 6H), 2.02 (s, 6H), 2.37 (s,
6H), 6.27 (s, 1H), 7.18-7.24 (m, 5H), 7.46-7.57 (m, 6H),
7.68 (d, J 7.8 Hz, 2H); 13C NMR (125 MHz, CDCl3) d 19.9,
20.5, 20.6, 42.0, 121.7, 121.9, 122.1, 125.6, 126.1, 127.0,
127.1, 127.2, 127.5, 128.7, 136.9, 139.0, 139.1, 144.5,
150.7, 167.0, 168.0, 169.0; HRMS (ESI) m/z, calcd. for
C39H32O12Na [M + Na]+: 715.1786, found: 715.1792,
∆ = 0.8 ppm.
3,3’-(2-Chlorophenylmethylene)bis(naphthalene-1,2,4-triyl
triacetate) (13c)
The high-resolution mass spectra (electrospray
ionization, HRESIMS) were obtained using a QTOF Micro
(Waters) mass spectrometer.
White solid; yield 78%; mp 168-169 ºC; IR (KBr)
ν / cm-1 2935, 1770, 1604, 1496, 1428, 1365, 1165,
1098, 1037, 909, 870, 760, 733, 699, 584, 545; 1H NMR

4
Inhibition of Zika Virus Replication by Synthetic Bis-Naphthoquinones
J. Braz. Chem. Soc.
(500 MHz, CDCl3) d 1.84 (s, 6H), 1.94 (s, 6H), 2.33 (s,
6H), 6.53 (s, 1H), 7.11-7.19 (m, 2H), 7.29-7.32 (m, 2H),
7.41-7.54 (m, 7H), 7.70-7.76 (m, 2H); 13C NMR (125 MHz,
CDCl3) d 19.9, 20.6, 20.8, 40.2, 121.9, 122.0, 122.2, 124.8,
126.0, 127.2, 127.2, 127.5, 127.6, 127.6, 128.6, 129.5,
133.4, 134.9, 136.9, 136.9, 150.3, 166.8, 167.9, 175.6;
HRMS (ESI) m/z, calcd. for C39H31ClO12Na [M + Na]+:
749.1397, found: 749.1402, ∆ = 0.7 ppm.
3,3’-(3-Methylphenylmethylene)bis(naphthalene-1,2,4-triyl
triacetate) (13g)
White solid; yield 75%; mp 245-246 ºC; IR (KBr)
ν / cm-1 1763, 1606, 1425, 1366, 1195, 1168, 1099, 1027,
958, 934, 871, 760, 742, 677, 640, 606, 586, 546; 1H NMR
(500 MHz, CDCl3) d 1.18 (s, 6H), 1.97 (s, 6H), 2.15 (s, 3H),
2.29 (s, 6H), 6.16 (s, 1H), 6.89-7.05 (m, 4H), 7.37-7.48 (m,
6H), 7.76 (d, J 7.8 Hz, 2H); 13C NMR (125 MHz, CDCl3)
d 19.7, 20.3, 20.4, 21.1, 41.7, 114.5, 121.7, 121.9, 125.6,
125.9, 126.7, 126.9, 126.9, 127.3, 127.4, 128.4, 130.4,
136.7, 137.9, 138.7, 144.3, 166.6, 167.7, 168.8; HRMS
(ESI) m/z, calcd. for C40H34O12Na [M + Na]+: 729.1945,
found: 729.1948, ∆ = 0.4 ppm.
3,3’-(3-Chlorophenylmethylene)bis(naphthalene-1,2,4-triyl
triacetate) (13d)
White solid; yield 98%; mp 167-168 ºC; IR (KBr)
ν / cm-1 1774, 1640, 1597, 1505, 1477, 1427, 1366,
1165, 1098, 1037, 1025, 924, 906, 872, 767, 744, 676,
642, 606, 587, 549; 1H NMR (500 MHz, CDCl3) d 1.88
(s, 6H), 2.03 (s, 6H), 2.34 (s, 6H), 6.19 (s, 1H), 7.14 (s,
3H), 7.25 (s, 1H), 7.44-7.55 (m, 6H), 7.74 (d, J 8.0 Hz,
2H); 13C NMR (125 MHz, CDCl3) d 19.9, 20.6, 20.8,
40.2, 121.9, 122.0, 122.3, 124.8, 126.0, 127.2, 127.2,
127.6, 127.6, 128.6, 129.5, 133.4, 134.9, 136.9, 136.9,
150.3, 166.8, 167.9, 175.6; HRMS (ESI) m/z, calcd. for
C39H31ClO12Na [M + Na]+: 749.1402, found: 749.1396,
∆ = 0.8 ppm.
3,3’-(4-Methylphenylmethylene)bis(naphthalene-1,2,4-triyl
triacetate) (13h)
White solid; yield 83%; mp 233-235 ºC; IR (KBr)
ν / cm-1 1769, 1605, 1513, 1426, 1366, 1164, 1097, 1025,
1
918, 880, 814, 758, 745, 675, 605, 588, 543; H NMR
(500 MHz, CDCl3) d 1.83 (s, 6H), 1.99 (s, 6H), 2.26 (s,
3H), 2.34 (s, 6H), 6.20 (s, 1H), 6.99 (d, J 8.1 Hz, 2H), 7.09
(2H, d, J 8.1 Hz, 2H), 7.42-7.53 (m, 6H), 7.73 (d, J 7.8 Hz,
2H); 13C NMR (125 MHz, CDCl3) d 19.7, 20.2, 20.4, 21.0,
41.5, 121.7, 121.9, 125.7, 125.9, 126.9, 127.3, 129.0, 129.6,
135.7, 136.0, 136.7, 138.8, 144.2, 150.5, 166.7, 167.8,
168.9; HRMS (ESI) m/z, calcd. for C40H34O12Na [M + Na]+:
729.1932, found: 729.1948, ∆ = 0.4 ppm.
3,3’-(4-Chlorophenylmethylene)bis(naphthalene-1,2,4-triyl
triacetate) (13e)
White solid; yield 78%; mp 140-141 ºC; IR (KBr)
ν / cm-1 1773, 1605, 1491, 1427, 1366, 1165, 1098, 1058,
1025, 919, 872, 829, 767, 746, 678, 634, 605, 586, 539;
1H NMR (500 MHz, CDCl3) d 1.87 (s, 6H), 2.03 (s, 6H),
2.35 (s, 6H), 6.19 (s, 1H), 7.15-7.17 (m, 4H), 7.44-7.55 (m,
6H), 7.73 (d, J 8.0 Hz, 2H); 13C NMR (125 MHz, CDCl3)
d 19.7, 20.2, 20.4, 41.1, 121.8, 121.9, 125.0, 125.9, 127.0,
127.1, 127.5, 128.5, 131.0, 132.6, 136.7, 137.6, 138.7,
144.4, 166.8, 167.7, 168.8; HRMS (ESI) m/z, calcd. for
C39H31ClO12Na [M + Na]+: 749.1404, found: 749.1402,
∆ = 0.5 ppm.
3,3’-(4-Acetoxyphenylmethylene)bis(naphthalene-1,2,4-triyl
triacetate) (13i)
White solid; yield 70%; mp 285-286 ºC; IR (KBr)
ν / cm-1 2938, 1766, 1639, 1607, 1507, 1423, 1367, 1193,
1167, 1099, 1026, 943, 923, 908, 884, 767, 746, 684, 637,
605, 588, 549; 1H NMR (500 MHz, CDCl3) d 1.87 (s, 6H),
2.03 (s, 6H), 2.23 (s, 3H), 2.34 (s, 6H), 6.23 (s, 1H), 6.90 (d,
J 8.7 Hz, 2H), 7.23 (d, J 8.7 Hz, 2H), 7.39-7.54 (m, 6H), 7.73
(d, J 7.9 Hz, 2H); 13C NMR (125 MHz, CDCl3) d 19.7, 20.3,
20.5, 21.0, 41.3, 121.6, 121.8, 122.0, 125.9, 127.0, 127.4,
130.6, 136.5, 136.7, 138.7, 144.4, 149.5, 166.8, 167.8, 169.0,
169.3; HRMS (ESI) m/z, calcd. for C41H34O14Na [M + Na]+:
773.1836, found: 773.1847, ∆ = 1.4 ppm.
3,3’-(2-Methylphenylmethylene)bis(naphthalene-1,2,4-triyl
triacetate) (13f)
White solid; yield 75%; mp 155-157 ºC; IR (KBr)
ν / cm-1 1770, 1605, 1489, 1456, 1366, 1196, 1167, 1098,
1057, 1024, 918, 871, 760, 742, 677, 640, 606, 586, 546;
1H NMR (500 MHz, CDCl3) d 1.72 (s, 6H), 1.96 (s, 6H),
2.12 (s, 3H), 2.33 (s, 6H), 6.22 (s, 1H), 7.03-7.11 (m,
3H), 7.44-7.53 (m, 7H), 7.74 (dd, J 12.3 and 4.5 Hz, 2H);
13C NMR (125 MHz, CDCl3) d 19.7, 19.8, 20.6, 20.7, 40.2,
121.9, 122.0, 122.0, 122.2, 126.2, 126.6, 127.0, 127.1,
127.2, 127.3, 127.5, 127.5, 130.5, 131.6, 136.9, 136.9,
167.8; HRMS (ESI) m/z, calcd. for C40H34O12Na [M + Na]+:
729.1921, found: 729.1948, ∆ = 0.9 ppm.
3,3’-(3-Acetoxyphenylmethylene)bis(naphthalene-1,2,4-triyl
triacetate) (13j)
White solid; yield 82%; mp 178-179 ºC; IR (KBr) ν/ cm-1
1763, 1606, 1487, 1428, 1365, 1194, 1163, 1098, 1025, 920,
1
870, 744, 678, 608, 584, 549, 531; H NMR (500 MHz,
CDCl3) d 1.88 (s, 6H), 2.04 (s, 6H), 2.17 (s, 3H), 2.34 (s, 6H),
6.25 (s, 1H), 6.90-6.93 (m, 2H), 7.11 (d, J 8.3 Hz, 1H), 7.19
(d, J 8.3 Hz, 1H), 7.43-7.54 (m, 6H), 7.76 (d, J 7.8 Hz, 2H);
13C NMR (125 MHz, CDCl3) d 19.7, 20.3, 20.5, 21.0, 41.3,

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Gonzaga et al.
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120.3, 121.8, 122.0, 123.0, 125.0, 125.9, 127.0, 127.4, 129.2,
132.4, 136.7, 138.7, 140.7, 144.5, 150.7, 166.8, 167.8, 169.0,
169.4; HRMS (ESI) m/z, calcd. for C41H34O14Na [M + Na]+:
773.1844, found: 773.1847, ∆ = 0.4 ppm.
ν / cm-1 1780, 1604, 1509, 1430, 1368, 1199, 1179, 1100,
1041, 919, 876, 836, 768; H NMR (500 MHz, CDCl3)
1
d 1.82 (s, 6H), 1.99 (s, 6H), 2.29 (s, 6H), 6.14 (1H, s),
7.09-7.12 (m, 4H), 7.38-7.49 (m, 6H), 7.65 (d, J 7.9 Hz,
2H); 13C NMR (125 MHz, CDCl3) d 19.9, 20.5, 20.6, 41.0,
115.4 (d, J 21.2 Hz), 122.0, 122.1, 125.4, 126.0, 127.2,
127.3, 127.7, 131.6, 135.0, 136.9, 138.9, 144.5, 161.8 (d,
J 244.6 Hz), 167.0, 167.8, 169.0; HRMS (ESI) m/z, calcd.
for C39H31FO12Na [M + Na]+: 733.1686, found: 733.1698,
∆ = 1.6 ppm.
3,3’-(2,3,4,5,6-Pentafluorophenylmethylene)
bis(naphthalene-1,2,4-triyl triacetate) (13k)
White solid; yield 42%; mp 140-141 ºC; IR (KBr)
ν / cm-1 2974, 1779, 1638, 1606, 1523, 1503, 1427, 1370,
1198, 1180, 1100, 1041, 993, 980, 769; 1H NMR (500 MHz,
CDCl3) d 1.94 (s, 6H), 2.04 (s, 6H), 2.30 (s, 6H), 6.26
(s, 1H), 7.42-7.50 (m, 6H), 7.66-7.69 (m, 2H); 13C NMR
(125 MHz, CDCl3) d 19.7, 20.0, 20.4, 32.0, 114.1, 121.8,
121.9, 121.9, 122.0, 125.6, 127.1, 127.2, 127.7, 136.6,
138.6, 141.4, 144.0, 147.6, 166.9, 167.4, 168.1; HRMS
(ESI) m/z, calcd. for C39H27F5O12Na [M + Na]+: 805.1299,
found: 805.1321, ∆ = 2.7 ppm.
Cells and viruses
The African Green monkey kidney cell line (VERO-
ATCCCCL81) obtained from theAmerican Type Collection
were grown and maintained in Dulbecco’s Modified
Eagle Medium (DMEM, Gibco BRL, Life Technologies)
containing 50 µg mL−1 gentamicin and supplemented with
5% heat-inactivated and certified Gibco fetal calf serum
(FCS). ZIKVMR766 was kindly provided by Dr Davis
Fernandes Ferreira, Universidade Federal do Rio de Janeiro,
RJ, Brazil.
3,3’-(4-Bromophenylmethylene)bis(naphthalene-1,2,4-triyl
triacetate) (13l)
White solid; yield 60 %; mp 296-297 ºC; IR (KBr)
ν / cm-1 3073, 2974, 2937, 2045, 1777, 1636, 1605, 1504,
1488, 1456, 1425, 1404, 1368, 1310, 1275, 1201, 1193,
1180, 1100, 1072, 1040, 1012, 940, 920, 904, 887, 809,
766, 754, 744; 1H NMR (500 MHz, CDCl3) d 1.82 (s, 6H),
1.98 (s, 6H), 2.30 (s, 6H), 6.12 (1H, s), 7.07 (d, J 8.7 Hz,
2H), 7.27 (d, J 8.7 Hz, 2H), 7.39-7.50 (m, 6H), 7.68 (d,
J 8.1 Hz, 2H); 13C NMR (125 MHz, CDCl3) d 19.8, 20.3,
20.5, 120.7, 121.8, 121.9, 125.0, 125.9, 127.0, 127.1, 127.5,
131.5, 136.8, 138.2, 138.7, 144.4, 166.8, 167.7, 168.8;
HRMS (ESI) m/z, calcd. for C39H31BrO12Na [M + Na]+:
795.0900, found: 795.0897, ∆ = 0.4 ppm.
Cytotoxicity assay
The cytotoxic effects of all compounds was assayed
by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide, MTT),32 modified by Pinto et al.33 Vero cells,
3 × 103 mL, were grown in 96 well microplates at 37 °C
under a 5% CO2 humidified atmosphere.After 24 h, the cells
were treated with different concentrations of compounds
(25, 50, 100, 200 or 400 µM) and maintained under the same
conditions as above described and untreatedVero cells were
used as controls. After 72 h incubation, the supernatants
were discarded and MTT (Sigma Aldrich), 100 µL of
5 mg mL−1 in DMEM medium, was added to each well and
re-incubated for 3 h at 37 °C in 5% CO2. Then, the medium
was discarded and DMSO (100 µL) added following
re-incubation for another 30 min. Optical densities were
measured at 520 nm on a microplate reader (Thermo
Plate TP-Reader). The compound concentration required
to reduce the optical density of MTT was calculated by
linear regression and results expressed as 50% cytotoxic
concentration (CC50). The experiments were performed in
quadruplicate and for three times independently.
3,3’-(2,4-Dichlorophenylmethylene)bis(naphthalene-1,2,4-
triyl triacetate) (13m)
White solid; yield 97%; mp 180-181 ºC; IR (KBr)
ν / cm-1 2973, 2934, 1774, 1637, 1605, 1587, 1559, 1502,
1472, 1452, 1425, 1368, 1276, 1179, 1099, 1040, 918, 876,
1
825, 769, 751, 708, 676, 641, 607; H NMR (500 MHz,
CDCl3) d 1.85 (s, 6H), 1.93 (s, 6H), 2.29 (s, 6H), 6.40 (s,
1H), 7.07 (dd, J 7.8 and 1.5 Hz, 1H), 7.28-7.33 (m, 2H),
7.48-7.52 (m, 6H), 7.65-7.72 (m, 2H); 13C NMR (125 MHz,
CDCl3) d 19.9, 20.0, 20.6, 39.7, 122.0, 122.1, 122.2, 124.4,
126.0, 127.2, 127.4, 127.7, 127.8, 129.1, 133.8, 134.1,
135.5, 136.0, 136.9, 137.0, 166.6, 167.2, 167.8; HRMS
(ESI) m/z, calcd. for C39H30Cl2O12Na [M + Na]+: 783.0997,
found: 783.1012, ∆ = 1.9 ppm.
Screening of antiviral activity of compounds by plaque
reduction assay
3,3’-(4-Fluorophenylmethylene)bis(naphthalene-1,2,4-triyl
triacetate) (13n)
An antiviral screening test was performed with Vero
cells (2 × 104) grown in 24 well microplates inoculated
White solid; yield 53%; mp 167-168 ºC; IR (KBr)

6
Inhibition of Zika Virus Replication by Synthetic Bis-Naphthoquinones
J. Braz. Chem. Soc.
with ZIKV (MOI 0.1), as described previously34 with some
modifications.33 After 2 h virus adsorption at 37 °C in 5%
CO2, the supernatant was discarded and the cells were
washed for removal of the non-adherent viral particles
followed by addition of 40 µM of different compounds
(10a-o) and (13a-n) in order to identify if any of these
compounds had the ability to inhibit the replication of
ZIKV. Then, the infected and treated plaques with the
respective compounds were re-incubated for 120 h at 37 °C
in 5% CO2. The cell monolayer was then fixed and stained
with 1% crystal violet in 10% formalin. The compound
concentration required to reduce the viral plaque number
by 50% (EC50) in the treated cells compared to untreated
ones was performed as described by Pinto et al.33
(RT-PCR) was performed using the Bio Gene Zika Virus
PCR kit in the CFX96™ IVD Real-Time PCR system. The
results were evaluated taking into consideration the viral
RNA quantification in relation to the triplicate positive
control of three independent experiments. To determine the
inhibition characteristic of the compounds, we used Ribavirin
as a control.
Statistical analysis
The data were analyzed by the Tukey’s test using the
GraphPad Instat version 3 program.36 A p value of < 0.05
was considered statistically significant.
Results
Extraction and quantification of viral RNA by real time-PCR
Synthesis
The compounds which showed high inhibition of viral
replication were subjected to the real-time polymerase chain
reaction (PCR) assay. For this, 5 × 105 Vero cells were grown
in 24-well plates in DMEM medium supplemented with 5%
fetal bovine serum (FBS) and subsequently infected with
ZIKV (MOI 0.1) for 1 h.After this time, the cells were washed
and the compounds 10o, 13e, 13h, 13j and 13k were added
at concentrations of 1.25, 2.5, 5.0 or 10 µM and incubated
for four days. Subsequently, the supernatants were collected
for the extraction of viral RNA by Bio GeneViral DNA/RNA
Extraction, as described by Ogura et al.35 The real time PCR
The bis-2-hydroxynaphthalene-1,4-diones or bis-
hydroxy-1,4-naphthoquinones (10) can be easily prepared
by reaction of 2 equivalents of lawsone (11) and
arylaldehydes (12) (Scheme 1), using the procedure
described by Tisseh and Bazgir,30 with yields ranging from
50-98%. In the next step the bis(naphthalene-1,2,4-triyl
triacetates) (13a-n) were prepared by one-pot reductive
acetylation of 10a-n in the presence of zinc metallic, acetic
anhydride and pyridine as a catalyst with yields ranging
from 42-97%.
Scheme 1. Synthetic route to prepare compounds 10 and 13.

Vol. 00, No. 00, 2019
Gonzaga et al.
7
The aim of this study was to screen the bis-
naphthoquinones series 10 and 13 for their potential
activity against ZIKV.
due to the acetylation of the molecules. In this way, we
identified those compounds which to perform the antiviral
assays at concentrations well below the toxic concentration
while maintaining approximately > 99% of cell viability.
The compounds with the most promising anti-ZIKV
effects were found to be 10o, 13e, 13h, 13j and 13k which
were able to inhibit > 99% ZIKV replication (Table 1).
The naphthoquinone derivatives (10o, 13e, 13h, 13j and
13k) which showed 100% inhibition (Table 1) were also
evaluated by RT-PCR on infected and treated cells. These
compounds were chosen to be used in the determination
of the mechanism of ZIKV inhibition and were selected
to perform RT-PCR studies in a dose response curve, with
concentrations ranging from 1.25 to 10 µM (Figure 4).
The results showed that the compounds were still
capable of inhibiting the replication of ZIKV by more
than 90% with the lower concentration tested confirming
our previous findings, except for the concentration 13k at
1.25 µM.
After determining the inhibitory potential of the
compounds, the plaque reduction assays of the EC50 values
(Table 2) were investigated in Vero cell culture. Different
compound concentrations were tested (from 0.65 to 20 µM)
for 10o, 13e, 13h, 13j and 13k, which showed the greatest
inhibitory potential for ZIKV replication. The results
demonstrate that all derivatives showed potent inhibition
of ZIKV replication with EC50 values ranging from 0.62 to
1.38 µM. However, when cytotoxicity is considered, the 10o
derivative gave the highest result with a selectivity index
(SI) value of 1664 µM. Ribavirin was used as control and
presented an EC50 of 2.98 µM.
Evaluation of the anti-ZIKV activity of the compounds
To observe the toxicity of the naphthoquinones
compounds studied, MTT assays were performed in Vero
cells using concentrations of the compounds ranging from
25 to 400 µM and incubated for 24 to 48 h to analyze the
cytopathic effect. The results in Table 1 show that the series
of thirteen compounds (10a-o) have CC50 values from 2876
to 992 µM. However, the series with fourteen compounds
(13a-n) showed lower CC50 values from 549 to 63 µM,
Table 1. Cytotoxicity (CC50) and anti-ZIKV profile (PI: percentage of
inhibition ZIKV replication) of compounds of series 10 and 13
Compound
10a
10c
10d
10e
10f
CC50a / µM
981
PIb / %
0
0
1255
1325
1721
1103
1482
1465
2128
2876
992
0
0
0
10g
10h
10i
80
0
0
10j
0
10k
10l
0
1176
1604
1635
2297
527
0
10m
10n
10o
13a
13c
13d
13e
13f
0
50
> 99
0
Discussion
Several research groups are seeking new compounds
to inhibit ZIKV infection, which has been a major public
health problem since the outbreak of severe cases in the
February 2016, with this virus as an important public
health emergency of international concern, a large amount
of data was generated in ZIKV infections. This includes
details of the pathogenesis of ZIKV, transmission routes,
diagnosis, vaccines and antiviral development.37 The long
persistence of ZIKV infection, lasting for a few weeks to
months, and the decrease in viremia emphasis the urgent
need of studies of substances with antiviral potential against
these arboviruses.38,39
278
60
70
> 99
55
0
428
483
121
13g
13h
13i
549
505
> 99
0
231
13j
132
> 99
> 99
0
13k
13l
63
130
13m
238
0
In the present study, the inhibitory potential of
naphthoquinone-derived compounds on ZIKV virus
replication was evaluated. Initially, studies of the inhibition
of viral replication inVero cells using 20 µM concentrations
of a panel of twenty seven naphthoquinone derivatives
13n
365
0
aConcentration that reduced 50% cytotoxic when compared to untreated
controls; b PI: percentage of ZIKV inhibition at 40 µM of compounds. The
mean values standard deviations are representative of three independent
experiments.

8
Inhibition of Zika Virus Replication by Synthetic Bis-Naphthoquinones
J. Braz. Chem. Soc.
Figure 4. Effect of compounds 10o (a), 13e (b), 13h (c) 13j (d) and 13k (e) at concentrations of 1.25, 2.5, 5 and 10 µM on ZIKV genomic RNA by
quantitative PCR. Statistical analysis was performed using Tukey’s test.*p < 0.05, **p < 0.01.
Table 2. Cytotoxicity (CC50), anti-ZIKV profile (EC50) and selectivity
index (SI) for compounds 10o, 13e, 13h, 13j and 13k
mortality in cases of ZIKV infection. Studies to evaluate
the true inhibition potential were initiated with dose-
dependent response tests using different concentrations
Compound
10o
CC50a / µM
2297
483
EC50b / µM
1.38
SIc
1664
743
454
213
69
of the derivatives in Veri cells infected with ZIKV, aiming
the analysis of the mechanism of action.41,42 The results
demonstrated that the naphthoquinone compounds are able
to inhibit viral replication by 50% at very low doses. For
example, compound 10o has an EC50 of 1.38 µM while
compound 13e has an EC50 in a nanomolar concentration
of 0.65 µM, demonstrating their great potential to inhibit
ZIKV. Considering that Ribavirin has been widely used by
several groups as a positive control and has been presented
as a possible treatment strategy for arbovirus infection, bis-
naphthoquinones have an even more potent inhibition profile.
The present study results on the cytotoxicity of
the naphthoquinones showed that the non-acetylated
derivatives had lower cytotoxicities than the acetylated
derivatives with some of the former compounds having 4
times less cytotoxicity than the latter (Table 1). However,
in contrast to non-acetylated compounds which are less
toxic, we observed that a greater number of acetylates
13e
0.65
13h
505
1.11
13j
132
0.62
13k
63
0.91
aConcentration that reduced 50% cytotoxic when compared to untreated
b
controls; concentration that reduced 50% of ZIKV replication when
compared to infected controls; cselectivity index was defined as the ratio
between CC50 and EC50 and represents the safety for in vitro assays. The
mean values standard deviations are representative of three independent
experiments.
was undertaken and the results identified five derivatives
able to inhibit > 99% ZIKV replication. Considering that
currently there are no drugs approved for the treatment of
ZIKV infection,40 it is essential to study compounds with
high capacity to inhibit viral replication and that have low
toxicity may be a good strategy to reduce morbidity and

Vol. 00, No. 00, 2019
Gonzaga et al.
9
showed a high capacity to inhibit the replication of ZIKV.
Moreira et al.43 identified acetylated isolapachol (14) as a
potent and selective antiplasmodium agent, corroborating to
the idea that acetylation may be one of the factors increasing
biological activity (Figure 5).
assessed by the viral plaques assay, low concentrations
of the compounds were able to significantly inhibit the
replication of the ZIKV particles. Additionally we have
shown that the compounds were also able to inhibit viral
RNA synthesis. Therefore, several workers have identified
compounds capable of inhibiting the replication of ZIKV
by different mechanisms of action45 but studies are required
to detect compounds not only capable of inhibiting viral
replication but also with the ability of reducing morbidity
and mortality in cases of new outbreaks.
Figure 5. Acetylated isolapachol (14) with antiplasmodium activity.43
Conclusions
There are currently no drugs approved for the treatment
of ZIKV-infections, not even for reduce the neuronal
damage possibly suffered due to infection.44 A previous
study11 also describe several other compounds having
promising effects against ZIKV replication, but more time
is required for defining and analyzing the mechanisms
involved. Furthermore, Barrows et al.13 examined 774 Food
DrugAdministration (FDA)-approved drugs for anti-ZIKV
activity and demonstrated that at least 20 tested positive
for viral inhibition, including mycophenolate mofetil (15)
and sertraline (16), which reduced viral infection in vitro
(Figure 6).
Our findings showed that among the naphthoquinone
compounds analyzed, five were particularly promising.
Compound10owitho-nitroshowedthebestselectivityindex,
followed by compound 13e with chlorophenylmethylene
radical. The antiviral activity observed may give us the hope
that these compounds can be exploited therapeutically for
new urgently needed antiviral therapies and their effects
will be tested in rodent models for ZIKV infection therapy.
Supplementary Information
Supplementary information is available free of charge
at http://jbcs.sbq.org.br as PDF file.
Acknowledgments
The authors thank the Brazilian agencies CNPq,
CAPES, and FAPERJ and FIOCRUZ for the HRMS. V.
F. F., F. C. S. and I. C. N. P. P. are recipients of CNPq
research fellowships; D. T. G. G., R. K. F. M. and N. A. R.
received FAPERJ research fellowships. C. C. C. S. thanks
to CAPES for the Postdoc fellowship and C. S. B. thanks
FAPERJ for the Postdoc fellowship. The authors would
like to acknowledge for the technical support of Thereza
Elizabeth P. P. Garcia.
Figure 6. Compounds mycophenolate mofetil (15) and sertraline (16)
with effects against ZIKV.45
The guaranteed standard for using a drug for antiviral
therapy is in its selectivity index (SI), which consists of
the ratio between a 50% cytotoxic concentration (CC50) of
the cells and the 50% inhibitory concentration (EC50) of
the virus. The ideal drug should have an SI between 100
and 1000. The results demonstrate that the CC50 values
combined with the EC50 values generate an index above 100
for almost all the compounds except for 13k.As can be seen
in Table 2, compound 10o showed a higher SI 1664 µM
followed by 13e which, although much lower, still had a
SI of 743 µM.
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