N-(2-(arylmethylimino)ethyl)-7-chloroquinolin-4-amine derivatives, synthesized by thermal and ultrasonic means, are endowed with anti-Zika virus activity

Article abstract of DOI:10.1016/j.ejmech.2017.01.007

Zika virus (ZIKV), an emerging Flavivirus, was recently associated with severe neurological complications and congenital diseases. Therefore, development of antiviral agents capable of inhibiting ZIKV replication is urgent. Chloroquine is a molecule with a confirmed safety history for use with pregnant women, and has been found to exhibit anti-ZIKV activity at concentrations around 10?μM. This suggests that modifications to the chloroquine structure could be promising for obtaining more effective anti-ZIKV agents. Here, we report the ability of a series of N-(2-(arylmethylimino)ethyl)-7-chloroquinolin-4-amine derivatives to inhibit ZIKV replication in?vitro. We have found that the quinoline derivative, N-(2-((5-nitrofuran-2-yl)methylimino)ethyl)-7-chloroquinolin-4-amine, 40, was the most potent compound within this series, reducing ZIKV replication by 72% at 10?μM. Compound 40 exhibits an EC₅₀value of 0.8?±?0.07?μM, compared to that of chloroquine of 12?±?3.2?μM. Good activities were also obtained for other compounds, including those with aryl groups?=?phenyl, 4-fluorophenyl, 4-nitrophenyl, 2,6-dimethoxyphenyl, 3-pyridinyl and 5-nitrothien-2-yl. Syntheses of these quinoline derivatives have been obtained both by thermal and ultrasonic means. The ultrasonic method produced comparable yields to the thermal (reflux) method in very much shorter times 30–180?s compared to 30–180?min reactions times. These results indicate that this group of compounds is a good follow-up point for the potential discovery of new drugs against the Zika disease.

Full text of DOI:10.1016/j.ejmech.2017.01.007

Accepted Manuscript
N-(2-(arylmethylimino)ethyl)-7-chloroquinolin-4-amine derivatives, synthesized by
thermal and ultrasonic means, are endowed with anti-Zika virus activity
Giselle Barbosa-Lima, Ligia S. da Silveira Pinto, Carlos R. Kaiser, James L. Wardell,
Caroline S. De Freitas, Yasmine R. Vieira, Andressa Marttorelli, José Cerbino Neto,
Patrícia T. Bozza, Solange M.S.V. Wardell, Marcus V.N. de Souza, Thiago M.L.
Souza
PII:
S0223-5234(17)30007-7
10.1016/j.ejmech.2017.01.007
EJMECH 9158
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 October 2016
Revised Date: 4 January 2017
Accepted Date: 5 January 2017
Please cite this article as: G. Barbosa-Lima, L.S. da Silveira Pinto, C.R. Kaiser, J.L. Wardell, C.S. De
Freitas, Y.R. Vieira, A. Marttorelli, J.C. Neto, P.T. Bozza, S.M.S.V. Wardell, M.V.N. de Souza, T.M.L.
Souza, N-(2-(arylmethylimino)ethyl)-7-chloroquinolin-4-amine derivatives, synthesized by thermal and
ultrasonic means, are endowed with anti-Zika virus activity, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.01.007.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
N-(2-(Arylmethylimino)ethyl)-7-chloroquinolin-
4-amine derivatives, synthesized by thermal and
ultrasonic means, are endowed with anti-Zika
virus activity.
Giselle Barbosa-Lima¥,a,b,c, Ligia S. da Silveira Pinto¥,d, Carlos R. Kaiserd, James L. Wardelle,f,
Caroline S. De Freitasa,b, Yasmine R. Vieiraa,b,c, Andressa Marttorellia,b, José Cerbino Netoc,
Patrícia T. Bozzaa, Solange M. S. V. Wardellg, Marcus V. N. de Souza#*,d,eand Thiago M. L.
Souza#,a,b,c
¥These authors contributed equally as first author
#These authors contributed equally as last author
b
aInstituto Oswaldo Cruz, Fiocruz - Fundação Oswaldo Cruz, Rio de Janeiro, Brazil; Centro de
Desenvolvimento Tecnológico em Saúde, Fiocruz - Fundação Oswaldo Cruz, Rio de Janeiro,
c
Brasil. Instituto Nacional de Infectologia Evandro Chagas, Fiocruz - Fundação Oswaldo Cruz,
d
Rio de Janeiro, Brazil; Departamento de Química Orgânica, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, Brazil; eInstituto de Tecnologia em Fármacos - Far-Manguinhos, Fiocruz
f
- Fundação Oswaldo Cruz, Rio de Janeiro, Brazil; Department of Chemistry, University of
1

ACCEPTED MANUSCRIPT
g
Aberdeen, Old Aberdeen, Scotland; CHEMSOL, 1 Harcourt Road, Aberdeen AB15 5 NY,
Scotland, UK.
ABSTRACT. Zika virus (ZIKV), an emerging Flavivirus, was recently associated with severe
neurological complications and congenital diseases. Therefore, development of antiviral agents
capable of inhibiting ZIKV replication is urgent. Chloroquine is a molecule with a confirmed
safety history for use with pregnant women, and has been found to exhibit anti-ZIKV activity at
concentrations around 10 µM. This suggests that modifications to the chloroquine structure could
be promising for obtaining more effective anti-ZIKV agents. Here, we report the ability of a
series of N-(2-(arylmethylimino)ethyl)-7-chloroquinolin-4-amine derivatives to inhibit ZIKV
replication in vitro. We have found that the quinoline derivative, N-(2-((5-nitrofuran-2-
yl)methylimino)ethyl)-7-chloroquinolin-4-amine, 40, was the most potent compound within this
series, reducing ZIKV replication by 72 % at 10 µM. Compound 40 exhibits an EC50 value of 0.8
± 0.07 µM, compared to that of chloroquine of 12 ± 3.2 µM. Good activities were also obtained
for other compounds, including those with aryl groups = phenyl, 4-fluorophenyl, 4-nitrophenyl,
2,6-dimethoxyphenyl, 3-pyridinyl and 5-nitrothien-2-yl. Syntheses of these quinoline derivatives
have been obtained both by thermal and ultrasonic means. The ultrasonic method produced
comparable yields to the thermal (reflux) method in very much shorter times 30-180 seconds
compared to 30–180 min reactions times. These results indicate that this group of compounds is
a good follow-up point for the potential discovery of new drugs against the Zika disease.
KEYWORDS. Zika virus; quinoline derivatives; sonochemical method.
2

ACCEPTED MANUSCRIPT
Introduction
Quinolinyl derivatives are found in many synthetic and natural products possessing a wide
range of biological and pharmacological activities. In particular, quinolinyl derivatives have
found importance over many years in antimalarial drug research [1–3]. Many of these quinolinyl
compounds have subsequently been used as starting points for research in other medical areas,
including as antituberculosis [4,5], anticancer [6–11] and, more recently, as anti-Zika virus
(ZIKV) agents [12].
ZIKV is a Flavivirus, which belongs to the Flaviviridae family. This family includes other
agents of clinical significance, such as dengue (DENV), West Nile (WNV) and Japanese
encephalitis (JEV) viruses. Besides being an arthropod-born virus, transmitted by mosquitoes of
the genus Aedes [13], ZIKV is also transmitted by sexual contact [14–16]. Prior to 2007 only a
few cases of ZIKV infection in humans has been confirmed [13,17]. Nevertheless, by the year of
2016, 52 countries and territories of the Americas, Africa, Asia and Pacific had reported
autochthonous ZIKV transmission [18]. A dramatic increase in neurological disorders, during
fetal development and adulthood, associated with ZIKV infection led the World Health
Organization (WHO) to declare Zika as a Public Health Emergency of International Concern
[19]. Effective antiviral drugs able to inhibit ZIKV replication are necessary. In this regard,
various molecules are under investigation, showing promising results [12,20–23], such the
anthelmintic drug niclosamide [22,23]; nucleoside/nucleotides analogs [20–22], as the anti-
hepatitis C (HCV) drug sofosbuvir, and the antimalarial substance, chloroquine [12,22]. Among
these, chloroquine is the only molecule with confirmed safety history for use with pregnant
women and fetus. However, chloroquine’s anti-ZIKV potency is around 10 µM, at least 10-times
higher than observed for compounds with antiviral activity [24,25]. While this information
3

ACCEPTED MANUSCRIPT
indicates that the chloroquine chemical structure is promising against ZIKV, medicinal
chemistry-driven approaches could lead to derivatives with improved potencies in anti-ZIKV
therapies.
In this study, we have evaluated the anti-ZIKV activity of N-(2-(arylmethylimino)ethyl)-7-
chloroquinolin-4-amine derivatives – which contain the 7-chloroquinoline unit found in both
amodiaquine and chloroquine. We have measured the ability of this family of compounds to
inhibit ZIKV replication, by reducing viral RNA levels. Indeed, we have found that the N-(2-
(arylmethylimino)ethyl)-7-chloroquinolin-4-amine derivatives could highlight potential lead
molecules for the development of novel anti-ZIKV drugs.
Results and discussion
Synthesis and characterization
Reaction of 4,7-dichloroquinoline with ethylenediamine on heating formed the N-(7-
chloroquinolin-4-yl)ethane-1,2-diamine 1. Subsequently, reactions of substituted benzaldehydes,
2-21, with 1 generated the N-(2-(arylmethylimino)ethyl)-7-chloroquinolin-4-amine derivatives,
22-41, using both thermal and ultrasonic methods in ethanol solutions (Scheme 1). Comparisons
of the yields and reaction times in the two processes are listed in Table 1. The optimum results
using ultrasonics were obtained with a frequency of 20 kHz, 50% of the maximum power output,
without pulsing and with 30-180 seconds reaction times. Comparable yields using the reflux
method were only obtained after 30–180 min reactions times because of the effects of cavitation.
Characterisation of compounds 22-41 was achieved generally from IR and NMR spectral and
HRMS (TOFF) data, and specifically for compound, 24, as its trihydrate, by X-ray
1
crystallography. Two sets of signals for the N=CH hydrogens in the H NMR spectra indicated
4

ACCEPTED MANUSCRIPT
that (E) : (Z) isomers had been formed: ratios of the isomers are given in Table 1. Some general
spectral findings are (i) the chemical shifts of the N=CH, CH2N=CH and NHCH2 protons in the
1H NMR spectra are found in the ranges 8.24–8.62, 3.83–3.97 and 3.56–3.76 ppm, respectively
and (ii) the N–H and N=C stretching vibrations in the IR spectra occur in the ranges 3019–3426
and 1526–1582 cm-1, respectively.
Scheme 1 Synthesis of N-(2-(arylmethylimino)ethyl)-7-chloroquinolin-4-amine derivatives
Conditions. (a) Ethylenediamine, 80 °C ‰ 1 h, without stirring, 135 °C ‰ 3 h, with stirring,
94% (b) EtOH, ultrasound, 30-180 seconds, 52-88%, or reflux, 30-180 minutes; 58-81% yields
(c) R= heteroaryl, EtOH, U.S. 30-120 seconds, 40-77%, or reflux, 30-60 minutes, 47-70% yields.
5

ACCEPTED MANUSCRIPT
Table 1 Comparison of classical and ultrasonic methods for the preparation of the N-(2-
(arylmethylimino)ethyl)-7-chloroquinolin-4-amine derivatives, the geometric isomeric product
ratios as determined by 1H NMR spectra and the inhibition of ZIKV replication.
ZIKV RNA
Ultrasound
Thermal
Relative proportion
between the isomers
Entry
inhibition at
10 µM (%)
Yied %/ time (s)
Yied %/ time (min)
1
43 ± 8.0
76 ± 1.8
57 ± 5.1
70 ± 2.7
59 ± 5.6
66 ± 8.1
82 ± 3.8
75 ± 3.2
-
-
-
22
23
24
25
26
27
28
52/120
74/30
69/180
80/60
76/60
80/60
64/120
58/60
69/30
76/60
80/60
74/30
81/60
59/180
-
82:18
-
93:7
85:15
86:14
79:21
29
30
31
32
33
34
52 ± 5.2
NE
83/60
88/30
74/60
74/45
59/30
77/30
80/30
65/30
78/30
71/30
68/30
73/30
84:16
82:18
72:28
-
74 ± 5.1
NE
51 ± 1.3
NE
-
69:31
35
75 ± 3.2
70/30
73/30
79:21
36
37
38
41 ± 3.1
70 ± 2.3
80 ± 6.7
60/30
64/120
40/120
74/30
53/60
47/30
85:15
76:24
86:14
6

ACCEPTED MANUSCRIPT
39
74 ± 4.2
72 ± 5.3
78 ± 3.5
47 ± 5.3
50/120
49/30
77/30
52/30
74:26
78:22
81:19
-
40
41
60/30
70/60
Chloroquine
-
-
NE – Non evaluated. These substances did not have their antiviral activity evaluated because
they were cytotoxic, reducing the cell viability in more than 80 % at 10 µM.
Anti-Zika virus activity
Considering that chloroquine has shown anti-ZIKV activity, with an EC50 of approximately 10
µM [12,22], we screened the chloroquine analogs, 22-41, for inhibition of ZIKV replication at
this concentration. Positively, almost the entire series showed improved antiviral activities when
compared to chloroquine (Table 1). Subsequently, we further examined the pharmacological
activity of the best hits against ZIKV, that is, those molecules inhibiting approximately or greater
than 75 % of virus replication. Derivatives, 22, 27, 28, 31, 35, 38, 39, 40 and 41, were selected
(Table 1). All the tested compounds had potencies around 10-times greater than did chloroquine
to inhibit ZIKV replication, with the exception of 38, which was around 5-times more potent
(Table 2). Tested derivatives were more lipophilic than chloroquine (Table 2). Among these
molecules, compound 40 was the most potent, with an EC50 value of 0.8 ± 0.07 µM, whereas this
pharmacological parameter for chloroquine was 12 ± 3.2 µM (Table 2). The medicinal chemistry
approach improved the antiviral activity of the chloroquine analogs without affecting their
cytotoxicity, because all tested molecules had similar CC50 values to that of chloroquine (Table
2). Of note, compound 38 was the least cytotoxic molecule (Table 2). The selective index (SI) –
which represents the ratio between the CC50 and EC50 values, and thus the in vitro safety – for 40
7

ACCEPTED MANUSCRIPT
was 515 (Table 2). This means that SI value for compound 40 is 15-times higher than this
parameter for chloroquine (Table 2). When compared to other compounds with antiviral activity
against ZIKV and potential clinical use, such as sofosbuvir, 40 presented a comparable SI value
[20]. Altogether, these results indicate that N-(2-(arylmethylimino)ethyl)-7-chloroquinolin-4-
amine derivatives, especially, compound 40, are endowed with an higher anti-ZIKV activity than
chloroquine.
Table 2 Potency, cytotoxicity and lipophilicity of the most active quinoline derivatives against
ZIKV replication.
Compound
EC50
CC50
SIa
cLogPb
4.14
4.10
4.31
3.38
4.16
2.91
2.91
3.48
4.13
22
27
28
31
35
38
39
40
41
1.2 ± 0.05
1.5 ± 0.03
1.5 ± 0.08
1.6 ± 0.02
1.5 ± 0.08
3.4 ± 0.08
1.5 ± 0.09
0.8 ± 0.07
1.5 ± 0.03
489 ± 16
512 ± 27
534 ± 42
487 ± 41
458 ± 36
578 ± 26
571 ± 52
412 ± 26
478 ± 34
407
341
356
304
305
170
380
515
318
8

ACCEPTED MANUSCRIPT
Chloroquine
12 ± 3.2
412 ± 24
34
5.00
aSI, selective index is determined by the ratio between CC50 and EC50 values
bCalculated using www. molinspiration.com
The structure-activity relationship (SAR) analysis indicated that the imino group is critical for
the activity since the ethylenediamine group does not present any anti-ZIKV activity. The
number, the positions, and the types of substituents attached to the aromatic ring are also critical
factors for biological activity. For example, compound, 35, with methoxy groups at C-2 and 6
was more active compound 34, with chloro groups at C-2 and 6. Compound 35 was also more
active than compounds 24, 31, 33 and 36, indicating that the number and positions of methoxy
groups are critical for anti-ZIKV activity. The importance of the substituents and position into
the aromatic ring were demonstrated by the compounds 27, 28 and 29 since fluorine 28 and nitro
27 at para-position was more active than bromine 29 at the same position. It is necessary to
mention that compound 22 without substituent also displayed good anti-ZIKV activity. In the
case of heterocyclic rings the position of the nitrogen of pyridine is critical for biological
activity. The most active compound 40 of this series indicated that the furan nucleus is more
active than thiophene nucleus 41.
9

ACCEPTED MANUSCRIPT
Scheme 2 Critical factors for the biological activity: number, the positions and the types of
substituents.
Crystal structure determination of the trihydrate of compound 24
Attempts to grow suitable crystals of compound 24 by slow evaporation from a moist ethanol
solution at room temperature led to the isolation of the trihydrate, [(24).3(H2O)]. Data for
[(24).3(H2O)] were collected at 120(2) K [26-28]. The trihydrate crystallises in the triclinic space
group, P-1, with Z= 2. The atom arrangement and numbering scheme of the trihydrate are
illustrated in Figure 1a. Features of the molecular configuration of 24 are (a) a quasi ´´T´´ shape,
see Figure 1b, with the dihedral angle between the quinoline and phenyl rings of 62.18(5)o, (b)
the methoxy substituent in the phenyl ring is sited away from the N2-C9-C10-N3 link between
the two aryl units and (c) the N2-C9-C10-N3 fragment has a syn arrangement as shown by the
dihedral angle of 72.72(14)o, which is ideal for coordinating to metals.
The intermolecular interactions in [(24).3(H2O)] are set of π—π stacking interactions and
classical O-H—O, N-H---O and O-H---N hydrogen bonds, which link the molecules of water
and 24 into a three dimensional array.
10

ACCEPTED MANUSCRIPT
(b)
(a)
Figure 1 (a) Atom arrangements and numbering scheme for [(24).3H2O], (b) view of the
conformation of molecule 24.
Experimental
Chemistry
Chemical reagents and solvents were used as obtained from Merck and Aldrich. A Multiwave
Eco-sonics QR750 ultrasonic generator (20 kHz, 750 W) equipped with a converter/transducer,
and titanium oscillator (horn diameter = 4 mm) was used for the ultrasonic irradiation. NMR
spectra were recorded at room temperature in deuterated dimethyl sulfoxide on a Bruker Avance
500 spectrometer operating at 400.00 and 500.00 MHz (1H) and 100 and 125.0 MHz (13C).
Melting points were determined on a Büchi apparatus. Infrared spectra were recorded in a
Thermo Nicolet Nexus 670 spectrometer, as potassium bromide pellets and frequencies are
expressed in cm-1. HRMS were performed on Bruker Compact QTOF mass spectrometer system.
TLC plates, coated with silica gel, were run in an ethyl acetate: methanol: triethylamine mixture
(8:1:1). The spots were developed and viewed under ultraviolet light.
11

ACCEPTED MANUSCRIPT
General ultrasonic irradiation protocol for the synthesis of the imino-quinoline
derivatives (22-41)
In a round bottom flask 25.0 mL equipped with magnetic stirrer and reflux condenser was
added 4,7-dichloroquinoline (1 g; 5 mmol) and ethylenediamine (1.17 g; 25.3 mmol). The
reaction mixture was heated at 80 °C without stirring for 1 hour. After this period, heating was
increased to 135 °C and was stirred for 3 hours. The flask containing the reaction mixture was
allowed to stand at room temperature for 12 hours. After this time, finely divided ice was added
to the precipitation of the product. The solid 1 formed was filtered and washed with distilled
water (20 mL) and ethyl ether (20 mL).
Solutions of N-(7-chloroquinolin-4-yl)ethane-1,2-diamine (100 mg) in ethanol (3 mL), and the
benzaldehyde (0.45 mmol) in ethanol (2 mL) were mixed and ultrasonically irradiated
(frequency = 50 KHz, amplitude = 50% of the maximum power output) without a pulse for 30-
180 seconds. After the ultrasonic irradiation, the resulting mixture was concentrated under
reduced pressure, the residue was washed with cold water and ether, to leave derivatives 22–41.
N-(2-(Benzylideneamino)ethyl)-7-chloroquinolin-4-amine (22) The product was obtained as
1
a white solid; m.p.: 170-174 °C; H NMR (400 MHz, DMSO) δ 8.41 (d, 1H, J = 5.4 Hz, H2);
8.35 (s, 1H, N=CH); 8.25 (d, 1H, J = 9.0 Hz, H5); 7.78 (d, 1H, J = 2.0 Hz, H8); 7.73-7.71 (m,
1H, H6); 7.45-7.42 (m, 5H, H2’/H3’/H4’/H5’/H6’); 6.60 (d, 1H, J = 5.4 Hz, H3); 3.88 (t, 2H, J =
6.0 Hz, CH2N=CH); 3.62 (q, 2H, J = 6.0, NHCH2); 13C NMR (100 MHz, DMSO) δ162.2; 151.9;
150.0; 149.1; 136.0; 133.4; 130.7; 128.6; 127.9; 127.8; 127.5; 124.1; 124.0; 117.4; 99.0; 58.8;
43.1. IR (KBr): ν = 3211 (N–H), 1574 (C=N) cm-1. HRMS m/z [M+H]+ = 310.1054, calc.
310.1033.
12

ACCEPTED MANUSCRIPT
N-(2-((2-Hydroxyphenyl)methylimino)ethyl)-7-chloroquinolin-4-amine (23), yellow solid,
1
m.p.: 161-164 °C; H NMR (400 MHz, DMSO) δ 8.54 (s, 1H, N=CH); 8.41 (d, 1H, J = 5.4 Hz,
H2); 8.24 (d, 1H, J = 9.1 Hz, H5); 7.79 (d, 1H, J = 2.2 Hz, H8); 7.48-7.43 (m, 2H, H6 and H6’);
7.39 (td, 1H, J = 9.1 Hz and J = 2.2 Hz, H5’); 7.33-7.29 (m, 1H, H4’); 6.88-6.85 (m, 1H, H3’);
13
6.62 (d, 1H, J = 5.4 Hz, H3); 3.89 (t, 2H, J = 6.0 Hz, CH2N=CH); 3.64 (m, 2H, NHCH2); C
NMR (100 MHz, DMSO) δ167.0; 160.6; 151.9; 149.9; 149.1; 133.4; 132.3; 131.6; 127.5; 124.2;
124.0; 118.6; 117.5; 116.4; 99.0; 58.7; 56.6; 43.0. IR (KBr): ν = 3232 (N–H), 1578 (C=N) cm-1.
HRMS m/z [M+H]+ = 326.1074, calc. 326.0982.
N-(2-((2-Methoxyphenyl)methylimino)ethyl)-7-chloroquinolin-4-amine (24) The product
1
was obtained as a white solid; m.p.: 108-110 °C; H NMR (400 MHz, DMSO) δ 8.62 (s, 1H,
N=CH); 8.40 (d, 1H, J = 5.3 Hz, H2); 8.24 (d, 1H, J = 9.0 Hz, H5); 7.84-7.78 (m, 2H, H8 and
H3’); 7.44-7.40 (m, 2H, H4’ and H6’); 7.06 (d, 1H, J = 9.0 Hz, H6); 6.97 (t, 1H, J = 7.4 Hz,
H5’); 6.59 (d, 1H, J = 5.3 Hz, H3); 3.88-3.85 (m, 2H, CH2N=CH); 3.75 (s, 3H, OCH3); 3.61-
3.59 (m, 2H, NHCH2); 13C NMR (100 MHz, DMSO) δ 158.3; 157.3; 151.8; 150.1; 149.1; 133.4;
132.2; 127.5; 126.6; 124.0; 123.8; 120.4; 117.4; 111.7; 99.1; 59.2; 55.5; 43.1. IR (KBr): ν = 3338
(N–H), 1581 (C=N) cm-1. HRMS m/z [M+H]+ = 340.1112, calc. 340.1138.
N-(2-((3-Nitrophenyl)methylimino)ethyl)-7-chloroquinolin-4-amine (25) The product was
1
obtained as a yellow solid; m.p.: 170-172 °C; H NMR (400 MHz, DMSO) δ 8.52 (s, 1H,
N=CH); 8.50 (s, 1H, H2’); 8.42 (d, 1H, J = 5.6 Hz, H2); 8.29 (m, 2H, H5 and H4’); 8.12 (d, 1H,
J = 7.6 Hz, H6’); 7.78 (d, 1H, J = 2.1 Hz, H8); 7.73 (t, 1H, J = 7.6 Hz, H5’); 7.47 (d, 1H, J = 9.0
Hz, H6); 6.67 (d, 1H, J = 5.6 Hz, H3); 3.93 (t, 2H, J = 6.0 Hz, CH2N=CH); 3.69 (m, 2H,
13

ACCEPTED MANUSCRIPT
NHCH2); 13C NMR (100 MHz, DMSO) δ 160.5; 160.4; 150.9; 150.5; 148.1; 137.5; 134.2; 134,0;
130.3; 125.0; 124.5; 124.2; 121.9; 117.1; 99.1; 60.5; 58.7; 43.0. IR (KBr): ν = 3213 (N–H), 1526
(C=N) cm-1. HRMS m/z [M+H]+ = 355.0956, calc. 355.0884 .
N-(2-((4-Methylphenyl)methylimino)ethyl)-7-chloroquinolin-4-amine (26) The product
was obtained as a white solid; m.p.: 180-182 °C; 1H NMR (400 MHz, DMSO) δ 8.40 (d, 1H, J =
5.4 Hz, H2); 8.30 (s, 1H, N=CH); 8.28-8.23 (m, 1H, H5); 7.78 (d, 1H, J = 2.2 Hz, H8); 7.62-7.58
(m, 2H, H2’ and H6’); 7.43 (dd, 1H, J = 8.9 Hz and J = 2.2 Hz, H6); 7.24 (d, 2H, J = 7.9 Hz, H3’
and H5’); 6.60 (d, 1H, J = 5.4 Hz, H5); 3.87-3.84 (m, 2H, CH2N=CH); 3.62-3.58 (m, 2H,
13
NHCH2); 2.33-2.32 (m, 6H, OCH3) C NMR (100 MHz, DMSO) δ 161.9; 151.8; 149.9; 149.0;
140.4; 133.3; 129.1; 127.8; 127.7; 123.9; 117.3; 98.9; 60.8; 58.7; 43.1; 20.9. IR (KBr): ν = 3219
(N–H), 1574 (C=N) cm-1. HRMS m/z [M+H]+ = 324.1276, calc. 324.1189 .
N-(2-((4-Nitrophenyl)methylimino)ethyl)-7-chloroquinolin-4-amine (27) The product was
1
obtained as a yellow solid; m.p.: 120-123 °C; H NMR (400 MHz, DMSO) δ 8.50 (s, 1H,
N=CH); 8.41 (d, 1H, J = 5.5 Hz, H2); 8.27 (m, 3H, H5, H3’ and H5’); 7.98 (d, 1H, J = 8.6 Hz,
H2’ and H6’); 7.78 (d, 1H, J = 1.9 Hz, H8); 7.44 (dd, 1H, J = 8.6 Hz and J = 1,9 Hz, H6); 6.62
13
(d, 1H, J = 5.5 Hz, H3); 3.95 (t, 2H, J = 5.8 Hz, CH2N=CH); 3.67 (m, 2H, NHCH2); C NMR
(100 MHz, DMSO) δ 160.7; 151.5; 150.1; 148.6; 148.4; 141.4; 133.4; 128.9; 128.7; 127.2;
124.1; 123.9; 123.8; 117.3; 99.0; 60.6; 58.9; 42.8. IR (KBr): ν = 3212 (N–H), 1579 (C=N) cm-1.
HRMS m/z [M+H]+ = 355.0980, calc. 355.0884.
14

ACCEPTED MANUSCRIPT
N-(2-((4-Fluorophenyl)methylimino)ethyl)-7-chloroquinolin-4-amine (28) The product was
obtained as a white solid; m.p.: 179-181 °C; 1H NMR (400 MHz, DMSO) δ 8.40 (d, 1H, J = 5.4
Hz, H2); 8.34 (s, 1H, N=CH); 8.25-8.21 (m, 1H, H5); 7.79-7.76 (m, 3H, H8, H3’ and H5’); 7.44-
7.41 (m, 1H, H6); 7.26 (t, 2H, J = 8.8 Hz, H2’ and H6’); 6.59 (d, 1H, J = 5.4 Hz, H3); 3.86 (t,
2H, J = 6.2 Hz; CH2N=CH); 3.60 (q, 2H, J = 6.2 Hz; NHCH2); 13C NMR (100 MHz, DMSO) δ
160.9; 151.8; 150.1; 149.0; 133.4; 130.2; 130.1; 127.5; 124.1; 124.0; 117.4; 115.7; 115.5; 99.0;
58.7; 43.1. IR (KBr): ν = 3215 (N–H), 1578 (C=N) cm-1. HRMS m/z [M+H]+ = 328.1026, calc.
328.0939.
N-(2-((4-Bromophenyl)methylimino)ethyl)-7-chloroquinolin-4-amine (29) The product was
obtained as a white solid; m.p.: 211-213 °C; 1H NMR (400 MHz, DMSO) δ 8.40 (d, 1H, J = 4.3
Hz, H2); 8.33 (s, 1H, N=CH); 8.24 (d, 1H, J = 8.8 Hz, H5); 7.79 (dd, 1H, J = 8.8 Hz and J = 1.8
Hz, H6); 7.68-7.63 (m, 3H, H8, H2’ and H6’); 7.47-7.43 (m, 2H, H3’ and H5’); 6.60 (d, 1H, J =
13
4.5 Hz, H3); 3.87 (t, 2H, J = 4.6 Hz; CH2N=CH); 3.63-3.60 (m, 2H, NHCH2); C NMR (100
MHz, DMSO) δ 161.1; 151.8; 151.0; 149.9; 149.0; 135.0; 133.3; 131.6; 129.7; 127.4; 124.0;
123.9; 117.3; 98.9; 58.7; 42.9; 40.6. IR (KBr): ν = 3204 (N–H), 1578 (C=N) cm-1.
N-(2-((2,3-Dihydroxyphenyl)methylimino)ethyl)-7-chloroquinolin-4-amine (30), yellow
solid. m.p.: 139-143 °C; 1H NMR (400 MHz, DMSO) δ 8.49 (s, 1H, N=CH); 8.41 (d, 1H, J = 5.4
Hz, H2); 8.26 (d, 1H, J = 9.1 Hz, H5); 7.79 (d, 1H, J = 2.2 Hz, H8); 7.51 (s, 1H, OH); 7.45 (dd,
1H, J = 9.1 Hz and J= 2.2 Hz, H6’); 6.86-6.80 (m, 2H, H4’ and H6); 6.67-6.56 (m, 2H, H5’ and
13
H3); 3.90 (t, 2H, J = 5.8 Hz, CH2N=CH); 3.64 (d, 2H, J = 5.8 Hz, NHCH2); C NMR (100
MHz, DMSO) δ 167.0; 151.8; 151.6; 149.9; 148.9; 145.9; 133.3; 127.4; 124.1; 123.9; 121.7;
15

ACCEPTED MANUSCRIPT
117.9; 117.4; 117.4; 117.3; 98.9; 55.4; 42.9. IR (KBr): ν = 3379 (N–H), 1575 (C=N) cm-1.
HRMS m/z [M+H]+ = 342.1029, calc. 342.0931.
N-(2-((2-Hydroxy-3-methoxyphenyl)methylimino)ethyl)-7-chloroquinolin-4-amine
(31)
1
The product was obtained as a yellow solid; m.p.: 122-123 °C; H NMR (400 MHz, DMSO) δ
8.57 (s, 1H, N=CH); 8.52 (s, 1H, H8); 8.42 (d, 1H, J = 5.6 Hz, H2); 8.28 (d, 1H, J = 8.9 Hz, H5);
7.81-7.80 (m, 1H, H8’); 7.51-7.47 (m, 1H, H4’); 7.03-6.95 (m, 1H, H9’); 6.78 (d, 1H, J = 8.9 Hz,
H6); 6.65 (d, 1H, J = 5.6 Hz, H3); 3.92 (s, 2H, CH2N=CH); 3.76 (d, 5H, J = 1.8 Hz; OCH3 and
NHCH2); 13C NMR (100 MHz, DMSO) δ 167.0; 151.5; 151.0; 150.4; 147.9; 133.8; 126.6; 124.3;
124.0; 123.0; 118.2; 117.6; 117.2; 114.6; 98.9; 58.2; 56.0; 55.6; 42.9. IR (KBr): ν = 3426 (N–H),
1575 (C=N) cm-1. HRMS m/z [M+H]+ = 356.1184, calc. 356.1088.
N-(2-((2,4-Dichlorophenyl)methylimino)ethyl)-7-chloroquinolin-4-amine (32), white solid;
1
m.p.: 134-137 °C; H NMR (400 MHz, DMSO) δ 8.58 (s, 1H, N=CH); 8.42-8.40 (m, 1H, H2);
8.25 (t, 1H, J = 7.1 Hz, H5); 7.96-7.92 (m, 1H, H6’); 7.79 (dd, 1H, J = 7.9 Hz and J = 2.2 Hz,
H8); 7.69-7.67 (m, 1H, H3’); 7.56-7.45 (m, 2H, H6 and H5’); 6.61 (t, 1H, J = 6.4 Hz, H3); 3.97-
13
3.93 (m, 2H, CH2N=CH); 3.66-3.63 (m, 2H, NHCH2); C NMR (100 MHz, DMSO) δ157.5;
135.8; 134.6; 133.8; 131.5; 131.4; 129.3; 129.2; 129.2; 127.8; 124.2; 124.0; 117.3; 99.0; 98.6;
60.3; 58.8; 42.7; 40.7. IR (KBr): ν = 3208 (N–H), 1581 (C=N) cm-1. HRMS m/z [M+H]+ =
378.0353, calc. 378.0253.
N-(2-((2,5-Dimethoxyphenyl)methylimino)ethyl)-7-chloroquinolin-4-amine
(33)
The
1
product was obtained as a yellow solid; m.p.: 103-106 °C; H NMR (400 MHz, DMSO) δ 8.57
16

ACCEPTED MANUSCRIPT
(s, 1H, N=CH); 8.40 (d, 1H, J = 5.3 Hz, H2); 8.24 (d, 1H, J = 8.9 Hz, H5); 7.77 (s, 1H, H8); 7.46
(m, 1H, H6); 7.34 (s, 1H, H6’); 6.99 (s, 2H, H3’ and H4’); 6.61 (d, 1H, J = 5.3 Hz, H3); 3.87-
3.85 (m, 2H, CH2N=CH); 3.75-3.69 (m, 6H, OCH3); 3.62 (m, 2H, NHCH2); 13C NMR (100
MHz, DMSO) δ 157.1; 153.0; 152.7; 151.4; 150.2; 148.6; 133.4; 127.1; 124.3; 124.0; 124.0;
118.1; 117.3; 113.2; 110.2; 99.0; 59.1; 55.9; 55.3. IR (KBr): ν = 3377 (N–H), 1582 (C=N) cm-1.
HRMS m/z [M+H]+ = 370.1229, calc. 370.1244.
N-(2-((2,6-Dichlorophenyl)methylimino)ethyl)-7-chloroquinolin-4-amine (34) The product
1
was obtained as a white solid; m.p.: 179-185 °C; H NMR (400 MHz, DMSO) δ 8.49 (s, 1H,
N=CH); 8.40 (d, 1H, J = 5.4 Hz, H2); 8.24 (d, 1H, J = 9.0 Hz, H5); 7.78 (d, 1H, J = 2.1 Hz, H8);
7.52-7.37 (m, 4H, H6/H5’/H3’/H4’); 6.58 (d, 1H, J = 5.4 Hz, H3); 3.97 (t, 2H, J = 5.7 Hz,
CH2N=CH); 3.65 (q, 2H, J = 5.7, NHCH2); 13C NMR (100 MHz, DMSO) δ158.2; 157.6; 151.7;
149.8; 148.9; 133.5; 133.3; 131.1; 128.9; 128.7; 127.3; 124.0; 123.9; 117.4; 98.8; 61.2; 58.9;
42.4. IR (KBr): ν = 3211 (N–H), 1575 (C=N) cm-1. HRMS m/z [M+H]+ = 378.0333, calc.
378.0253.
N-(2-((2,6-Dimethoxyphenyl)methylimino)ethyl)-7-chloroquinolin-4-amine
(35)
The
1
product was obtained as a yellow solid; m.p.: 71-73 °C; H NMR (400 MHz, DMSO) δ 8.40 (s,
1H, N=CH); 8.22 (d, 1H, J = 9.0 Hz, H5); 7.78 (d, 1H, J = 2.2 Hz, H8); 7.44 (dd, 1H, J = 9.0 Hz
and J = 2.2 Hz, H6); 7.34-7.28 (m, 2H, H2 and H4’); 6.64 (d, 2H, J = 8.4 Hz, H3’ and H5’);
3.66-3.64 (m, 6H, OCH3); 3.83 (t, 2H, J = 5.7 Hz, CH2N=CH); 3.56 (q, 2H, J = 5.7, NHCH2);
13C NMR (100 MHz, DMSO) δ158.9; 157.2; 151.8; 150.0; 149.0; 133.4; 131.4; 127.4; 124.0;
117.5; 113.2; 104.3; 99.0; 60.1; 55.7; 42.9. IR (KBr): ν = 3339 (N–H), 1574 (C=N) cm-1.
17

ACCEPTED MANUSCRIPT
N-(2-((3,4,5-Trimethoxyphenyl)methylimino)ethyl)-7-chloroquinolin-4-amine (36) The
1
product was obtained as a white solid; m.p.: 92-93 °C; H NMR (400 MHz, DMSO) δ 8.43 (d,
1H, J = 5.6 Hz, H2); 8.30 (d, 1H, J = 9.0 Hz, H5); 8.24 (s, 1H, N=CH); 7.80 (d, 1H, J = 2.2 Hz,
H8); 7.48 (dd, 1H, J = 2.2 Hz and J = 9.0 Hz, H6); 7.03-7.02 (m, 2H, H2’ and H6’); 6.67 (d, 1H,
J = 5.6 Hz, H3); 3.87-3.84 (m, 2H, J = 6.1 Hz, CH2N=CH); 3.79-3.76 (m, 9H, OCH3); 3.68-3.65
(m, 2H, NHCH2); 13C NMR (100 MHz, DMSO) δ 191.9; 161.9; 153.3; 153.0; 139.6; 131.5;
124.6; 124.3; 117.1; 106.7; 105.0; 99.2; 60.1; 58.8; 55.8; 43.2. IR (KBr): ν = 3019 (N–H), 1582
(C=N) cm-1. HRMS m/z [M+H]+ = 400.1423, calc. 400.1350.
N-(2-((Pyridin-2-yl)methylimino)ethyl)-7-chloroquinolin-4-amine (37) The product was
obtained as a white solid; m.p.: 130-133 °C; 1H NMR (400 MHz, DMSO) δ 8.61 (d, 1H, J = 4.8
Hz, H3’); 8.40 (d, 1H, J = 5.4 Hz, H2); 8.35 (s, 1H, N=CH); 8.24 (d, 1H, J = 9.0 Hz, H5); 7.96
(d, 1H, J = 7.5 Hz, H6’); 7.87 (td, 1H, J = 7.5 Hz and J = 1.3 Hz, H5’); 7.77 (d, 1H, J = 2.2 Hz,
H8); 7.46-7.42 (m, 2H, H6 and H4’); 7.61 (d, 1H, J = 5.4 Hz, H3); 3.95 (t, 2H, J = 6.1 Hz,
13
CH2N=CH); 3.67-3.62 (m, 2H, NHCH2); C NMR (100 MHz, DMSO) δ 163.1; 153.9; 151.8;
149.9; 149.2; 149.0; 136.7; 133.3; 127.4; 125.1; 124.0; 123.9; 120.4; 117.3; 99.0; 58.5; 42.8. IR
(KBr): ν = 3237 (N–H), 1580 (C=N) cm-1. HRMS m/z [M+H]+ = 311.1081, calc. 311.0985.
N-(2-((Pyridin-3-yl)methylimino)ethyl)-7-chloroquinolin-4-amine (38) The product was
obtained as a white solid; m.p.: 160-162 °C; 1H NMR (500 MHz, DMSO) δ 8.86 (d, 1H, J = 1.5
Hz, H5’); 8.63 (dd, 1H, J = 4.8 Hz and J = 1.5 Hz, H4’); 8.41-8.40 (m, 2H, N=CH and H2); 8.24
(d, 1H, J = 9.0 Hz, H5); 8.10 (d, 1H, J = 9.0 Hz, H6); 7.78 (d, 1H, J = 2.2 Hz, H8); 7.48-7.42 (m,
3H, H2’, H3’ and H3); 3.90 (t, 2H, J = 6.0 Hz, CH2N=CH); 3.64 (q, 2H, J = 6.0 Hz; NHCH2);
18

ACCEPTED MANUSCRIPT
13C NMR (125 MHz, DMSO) δ 160.1; 151.9; 151.4; 150.0; 149.5; 149.1; 134.5; 133.4; 131.4;
127.5; 124.1; 123.9; 123.9; 117.4; 99.1; 59.0; 42.9. IR (KBr): ν = 3216 (N–H), 1579 (C=N) cm-1.
HRMS m/z [M+H]+ = 311.1068, calc. calc. 311.0985.
N-(2-((Pyridin-4-yl)methylimino)ethyl)-7-chloroquinolin-4-amine (39) The product was
obtained as a white solid; m.p.: 186-188 °C; 1H NMR (400 MHz, DMSO) δ 8.66 (d, 2H, J = 5.9
Hz, H3’ and H5’); 8.41 (d, 1H, J = 5.4 Hz, H2); 8.38 (s, 1H, N=CH); 8.23 (d, 1H, J = 9.0 Hz,
H5); 7.78 (d, 1H, J = 2.2 Hz, H8); 7.65 (d, 2H, J = 5.9 Hz, H2’ and H6’); 7.45-7.42 (m, 1H, H6);
6.61 (d, 1H, J = 5.4 Hz, H3); 3.93 (t, 2H, J = 6.2 Hz, CH2N=CH); 3.64 (q, 2H, J = 6.2 Hz;
NHCH2); 13C NMR (100 MHz, DMSO) δ 160.5; 151.3; 149.7; 149.4; 148.5; 141.9; 132.8; 126.9;
123.5; 123.4; 121.2; 116.8; 98.5; 58.3; 42.2. IR (KBr): ν = 3212 (N–H), 1577 (C=N) cm-1.
HRMS m/z [M+H]+ = 311.0972, calc. 311.0985.
N-(2-((5-Nitrofuran-2-yl)methylimino)ethyl)-7-chloroquinolin-4-amine (40) The product
was obtained as a black solid; m.p.: 236-237 °C; 1H NMR (400 MHz, DMSO) δ 8.43 (d, 1H, J =
5.6 Hz, H2); 8.31-8.26 (m, 2H, H5 and N=CH); 7.81 (d, 1H, J = 2.2 Hz, H8); 7.75 (d, 1H, J = 3.9
Hz, H4’); 7.49 (dd, 1H, J = 8.9 Hz and J = 2.2 Hz, H6); 7.23 (d, 1H, J = 3.9 Hz, H5’); 6.66 (d,
13
1H, J = 5.6 Hz, H3); 3.94 (t, 2H, J = 5.8, CH2N=CH); 3.70-3.65 (m, 2H, NHCH2); C NMR
(100 MHz, DMSO) δ 152.1; 151.1; 150.6; 150.5; 147.5; 133.9; 126.3; 124.4; 124.1; 117.2;
117.1; 116.3; 113.8; 99.0; 58.8; 42.8. IR (KBr): ν = 3238 (N–H), 1574 (C=N) cm-1. HRMS m/z
[M+H]+ = 345.0751, calc. 345.0676.
19

ACCEPTED MANUSCRIPT
N-(2-((5-Nitrothiophen-2-yl)methylimino)ethyl)-7-chloroquinolin-4-amine
(41)
The
product was obtained as a brown solid; m.p.: 116-118 °C; 1H NMR (400 MHz, DMSO) δ 8.50 (s,
1H, N=CH); 8.40 (d, 1H, J = 5.4 Hz, H2); 8.23 (d, 1H, J = 9.0 Hz, H5); 8.10 (d, 1H, J = 4.2 Hz,
H4’); 7.78 (d, 1H, J = 2.1 Hz, H8); 7.51 (d, 1H, J = 4.2 Hz, H5’); 7.46-7.43 (m, 1H, H6); 6.60 (d,
1H, J = 5.4 Hz, H3); 3.91-3.88 (m, 2H, CH2N=CH); 3.65-3.61 (m, 2H, NHCH2); 13C NMR (100
MHz, DMSO) δ 155.9; 151.8; 151.7; 149.9; 148.9; 148.3; 133.3; 130.2; 130.1; 127.3; 124.1;
123.9; 117.3; 99.0; 58.4; 42.5. IR (KBr): ν = 3218 (N–H), 1581 (C=N) cm-1. HRMS m/z
([M+H]+ = 361.0545, calc. 361.0448.
Biological assays
Cells
African green monkey kidney cells (Vero) were cultured in DMEM (Life Technologies, Grand
Island, NY). Aedes albopictus cells (C6/36) were grown in L-15 medium supplemented with
0.3% tryptose phosphate broth, 0.75 g/L sodium bicarbonate, 1.4 mM glutamine, and
nonessential amino acids. The culture medium of the cell types was supplemented with 10 %
fetal bovine serum (FBS; HyClone, Logan, Utah), 100 U/mL penicillin and 100 µg/mL
streptomycin. Mammals cells were kept at 37 °C in 5% CO2, whereas mosquito cells were
maintained at 26 °C.
Virus
ZIKV strain used is representative of the virus circulating in Brazil [20]. ZIKV was grown in
C6/36 cells, tittered by 50 % endpoint of ZIKV cytopathic effect in tissue cultures (TCID50) and
further passaged at the multiplicity of infection (MOI) of 0.01 [29]. After 9 days post-infection,
20

ACCEPTED MANUSCRIPT
cells were lysed by freezing and thawing and centrifuged at 1,500 x g at 4 °C for 20 min to
remove cellular debris. Infectious virus titers were determined by TCID50/mL in Vero cells and
stored at -70 °C for further studies.
Cytotoxity assay
Monolayers of 2 x 104 Vero cells in 96-well culture plates were incubated with the compounds
at different concentrations for 72 h. Then, 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-
Tetrazolium-5-Carboxanilide (XTT) at 5 mg/ml was added to DMEM in the presence of 0.01 %
N-methyl-dibenzopirazina methyl sulfate (PMS). After incubation for 4 h at 37°C, the plates
were read in a spectrophotometer at 492 nm and 620 nm [30]. The 50 % cytotoxic concentration
(CC50) was calculated by linear regression analysis of the dose–response curves generated from
the data.
Antiviral Assay
Monolayers of Vero cells (2 x 104 cell/well) in 96-well plates were infected with ZIKV at MOI
of 0.1 for 1 h at 37 ºC. Cells were washed to remove residual viruses and various concentrations
of the compounds were added. After 24 h, viruses in the supernatant were harvested, RNA was
extracted and ZIKV RNA was quantified by RT-PCR (see molecular biology analysis below).
For comparison, the reference compound chloroquine was used as a positive control. Linear
regression of the dose-response curve was performed to determine the 50 % inhibitory effect on
viral replication (EC50) for the tested and reference compounds.
Molecular Biology analysis
21

ACCEPTED MANUSCRIPT
Total RNA from the culture supernatant was extracted using QIAamp Viral RNA Mini Kit
(Qiagen®), according to manufacturer’s instructions. Quantitative RT-PCR was performed
using QuantiTect Probe RT-PCR Kit (Quiagen®) in an ABI PRISM 7300 Sequence Detection
System (Applied Biosystems). Amplifications were carried out in 25 µL reaction mixtures
containing 2× reaction mix buffer, 50 µM of each primer, 10 µM of probe and 5 µL of RNA
template. Primers, probes and cycling conditions recommended by the Centers for Disease
Control and Prevention (CDC) were used to detect the ZIKV [17]. The standard curve method
was employed for virus quantification.
Statistical analyses
The dose-response curves used to calculate the EC50 and CC50 values and the % of ZIKV
replication inhibition were generated by Excel for Windows. All of the experiments were
performed at least three times, and the results are displayed as mean ± standard error of the mean
(SEM).
Conclusion
In this study we report the synthesis of a series of twenty N-(2-(arylmethylimino)ethyl)-7-
chloroquinolin-4-amine derivatives obtained by thermal and ultrasonic means. The ultrasonic
procedures are simple, safe and with short reaction times. Furthermore, we report the anti-ZIKV
activity of these derivatives, which represent an improvement of chloroquine chemical structure
towards the development of future anti-ZIKV therapies. Compound 40 was the most potent in
inhibiting ZIKV replication, without affecting the cytotoxicity when compared to chloroquine.
This study also reveals the importance of the number, the positions, and the types of substituents,
22

ACCEPTED MANUSCRIPT
as: phenyl, 4-fluorophenyl, 4-nitrophenyl and 2,6-dimethoxyphenyl, attached to the aromatic ring
as being critical factors for the biological activity. The 3-pyridinyl, indicating that the positions
of N atom attached to pyridinyl ring haven’t significant change at the activity and 5-nitro
-thiophen and -furan substituents indicated that the furan nucleus are more active than thiophene
nucleus. These factors can be critical for the biological activity and a good starting point to the
discovery of new prototypes against Zika.
AUTHOR INFORMATION
Corresponding Author
*Marcus V. N. de Souza
E-mail address: marcos_souza@far.fiocruz.br
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript. The authors (Giselle B. Lima and Ligia S. da S. Pinto;
Thiago M. L. Souza and Marcus V. N. de Souza) contributed equally.
ACKNOWLEDGMENT
We thank the Dr Ana Bispo de Fillips for critical discussion. Thanks are due to the the Oswaldo
Cruz Institute and Evandro Chagas Clinical Research Institute.
23

ACCEPTED MANUSCRIPT
Funding: This work was supported by grants from PAPES/Fiocruz (www.ioc.fiocruz.br), CNPq
(www.cnpq.br) and Faperj (www.faperj.br). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
REFERENCES
[1]
[2]
[3]
W. Cunico, C.A. Cechinel, H.G. Bonacorso, M.A.P. Martins, N. Zanatta, M.V.N. de
Souza, I.O. Freitas, R.P.P. Soares, A.U. Krettli, Antimalarial activity of 4-(5-
trifluoromethyl-1h-pyrazol-1-yl)-chloroquine analogues, Bioorg. Med. Chem. Lett. 16
(2006) 649–653.
A.A. Andrade, F. de P. Varotti, I.O. de Freitas, M.V.N. de Souza, T.R.A. Vasconcelos, N.
Boechat, A.U. Krettli, Enhanced activity of mefloquine and artesunic acid against
plasmodium falciparum in vitro and p. berghei in mice by combination with ciprofloxacin,
Eur. J. Pharmacol. 558 (2007) 194–198.
F. de P. Varotti, A.C.C. Botelho, A.A. Andrade, R.C. de Paula, E.M.S. Fagundes, A.
Valverde, L.M.U. Mayer, J.S. Mendonça, M.V.N. de Souza, N. Boechat, A.U. Krettli,
Synthesis, antimalarial activity, and intracellular targets of mefas, a new hybrid compound
derived from mefloquine and artesunate, Antimicrob. Agents Chemother. 52 (2008)
3868–3874.
[4]
[5]
M.V.N. de Souza, K.C. Pais, C.R. Kaiser, M.A. Peralta, M. de L Ferreira, M.C.S.
Lourenço, Synthesis and in vitro antitubercular activity of a series of quinoline
derivatives, Bioorg. Med. Chem. 17 (2009) 1474–1480.
R.S.B. Gonçalves, C.R. Kaiser, M.C.S. Lourenço, M.V.N. de Souza, J.L. Wardell,
24

ACCEPTED MANUSCRIPT
S.M.S.V Wardell, A.D. da Silva, Synthesis and antitubercular activity of new mefloquine-
oxazolidine derivatives, Eur. J. Med. Chem. 45 (2010) 6095–6100.
[6]
[7]
M. de L.F. Bispo, C.C. de Alcantara, M.O. de Moraes, C. do Ó Pessoa, F.A.R. Rodrigues,
C.R. Kaiser, S.M.S.V. Wardell, J.L. Wardell, M.V.N. de Souza, A new and potent class of
quinoline derivatives against cancer, Monatsh. Chem. 146 (2015) 2041–2052.
V. Facchinetti, F.A. Guimarães, M.V.N. de Souza, C.R.B. Gomes, M.C.B.V. de Souza,
J.L. Wardell, S.M.S.V. Wardell, T.R.A. Vasconcelos, Synthesis of novel ethyl
(substituted)phenyl-4-oxothiazolidin-3-yl)-1-ethyl-4-oxo-1,4-dihydroquinoline-3-
carboxylates as potential anticancer agents, J. Heterocycl. Chem. 52 (2015) 1245–1252.
[8]
R.C. Montenegro, L.V. Lotufo, M.O. de Moraes, C. do Ó. Pessoa, F.A.R. Rodrigues, M.
de L.F. Bispo, L.N. de F. Cardoso, C.R. Kaiser, M.V.N. de Souza, Synthesis and
antitumoral evaluation of 7-chloro-4-quinolinylhydrazones derivatives, Med. Chem. 7
(2011) 599–604.
[9]
R.C. Montenegro, L.V. Lotufo, M.O. de Moraes, C. do O Pessoa, F.A.R. Rodrigues, M. de
L.F. Bispo, B.A. Freire, C.R. Kaiser, M.V.N. de Souza, Cytotoxic activity of
polysubstituted 7-chloro-4-quinolinylhydrazone derivatives, Lett Drug Des Discov. 9
(2012) 251-256.
[10] F.A.R. Rodrigues, I. da S. Bomfim, B.C. Cavalcanti, C. do Ó. Pessoa, J.L. Wardell,
S.M.S.V Wardell, A.C. Pinheiro, C.R. Kaiser, T.C.M. Nogueira, J.N. Low, L.R. Gomes,
M.V.N. de Souza, Design, synthesis and biological evaluation of (e)-2-(2-
arylhydrazinyl)quinoxalines, a promising and potent new class of anticancer agents,
Bioorg. Med. Chem. Lett. 24 (2014) 934–939.
25

ACCEPTED MANUSCRIPT
[11] F.A.R. Rodrigues, I. da S. Bomfim, B.C. Cavalcanti, C. Pessoa, R.S.B. Goncalves, J.L.
Wardell, S.M.S.V. Wardell, M.V.N. de Souza, Mefloquine-Oxazolidine derivatives: a new
class of anticancer agents, Chem. Biol. Drug Des. 83 (2014) 126–131.
[12] R. Delvecchio, L.M. Higa, P. Pezzuto, A.L. Valadao, P.P. Garcez, F.L. Monteiro, E.C.
Loiola, S. Rehen, L. Campanati, R.S. de Aguiar, A. Tanuri, Chloroquine inhibits zika
virus infection in different cellular models, Cold Spring Harbor Labs Journals, 2016
doi: http://dx.doi.org/10.1101/051268.
[13] D. Musso, D.J. Gubler, Zika virus. Clin. Microbiol. Rev. 29 (2016) 487–524.
[14] D. Musso, C. Roche, E. Robin, T. Nhan, A. Teissier, V.M. Cao-Lormeau, Potential sexual
transmission of zika virus. Emerg. Infect. Dis. 21 (2015) 359–361.
[15] D.T. Deckard, W.M. Chung, J.T. Brooks, J.C. Smith, S. Woldai, M. Hennessey, N. Kwit,
P. Mead, Male-to-male sexual transmission of zika virus, Morb. Mortal. Wkly. Rep. 65
(2016) 372–374.
[16] E. D’Ortenzio, S. Matheron, X. de Lamballerie, B. Hubert, G. Piorkowski, M. Maquart, D.
Descamps, F. Damond, Y. Yazdanpanah, I. Leparc-Goffart, Evidence of sexual
transmission of zika virus. N. Engl. J. Med. 374 (2016) 2195–2198.
[17] R.S. Lanciotti, O.L. Kosoy, J.J. Laven, J.O. Velez, A.J. Lambert, A.J. Johnson, S.M.
Stanfield, M.R. Duffy, Genetic and serologic properties of zika virus associated with an
epidemic, Emerg. Infect. Dis. 14 (2008) 1232–1239.
[18] WHO. WHO | Zika situation report.
[19] WHO. WHO | WHO Director-General summarizes the outcome of the Emergency
26

ACCEPTED MANUSCRIPT
Committee regarding clusters of microcephaly and Guillain-Barré syndrome
http://www.who.int/mediacentre/news/statements/2016/emergency-committee-zika-
microcephaly/en/ (accessed Aug 30, 2016).
[20] C.Q. Sacramento, G.R. de Melo, N. Rocha, L.V.B. Hoelz, M. Mesquita, C.S. de Freitas,
N. Fintelman-Rodrigues, A. Marttorelli, A.C. Ferreira, G. Barbosa-Lima, M.M. Bastos, E.
de M. Volotao, D.A. Tschoeke, L. Leomil, F.A. Bozza, P.T. Bozza, N. Boechat, F.L.
Thompson, A.M.B. de Filippis, K. Bruning, T. Souza, The clinically approved antiviral
drug sofosbuvir impairs brazilian zika virus replication; Cold Spring Harbor Labs
Journals, 2016 doi: http://dx.doi.org/10.1101/061671.
[21] M. Onorati, Z. Li, F. Liu, A.M.M. Sousa, N. Nakagawa, M. Li, M.T. Dell’Anno, F.O.
Gulden, S. Pochareddy, A.T.N. Tebbenkamp, W. Han, M. Pletikos, T. Gao, Y. Zhu, C.
Bichsel, L. Varela, K. Szigeti-Buck, S. Lisgo, Y. Zhang, A. Testen, X.B. Gao, J. Mlakar,
M. Popovic, M. Flamand, S.M. Strittmatter, L.K. Kaczmarek, E.S. Anton, T.L. Horvath,
B.D. Lindenbach, N. Sestan, Zika virus disrupts phospho-tbk1 localization and mitosis in
human neuroepithelial stem cells and radial glia. Cell Rep. 2016 doi:
10.1016/j.celrep.2016.08.038.
[22] N.J. Barrows, R.K. Campos, S.T. Powell, K.R. Prasanth, G. Schott-Lerner, R. Soto-
Acosta, G. Galarza-Muñoz, E.L. McGrath, R. Urrabaz-Garza, J. Gao, P. Wu, R. Menon,
G. Saade, I. Fernandez-Salas, S.L. Rossi, N. Vasilakis, A. Routh, S.S. Bradrick, S.S.M.A.
Garcia-Blanco, A screen of fda-approved drugs for inhibitors of zika virus infection. Cell
Host Microbe 20 (2016) 259–270.
[23] M. Xu, E.M. Lee, Z. Wen, Y. Cheng, W.K. Huang, X. Qian, J. Tcw, J. Kouznetsova, S.C.
27

ACCEPTED MANUSCRIPT
Ogden, C. Hammack, F. Jacob, H.N. Nguyen, M. Itkin, C. Hanna, P. Shinn, C. Allen, S.G.
Michael, A. Simeonov, W. Huang, K.M. Christian, A. Goate, K.J. Brennand, R. Huang,
M. Xia, G.L. Ming, W. Zheng, H. Song, H. Tang, Identification of small-molecule
inhibitors of zika virus infection and induced neural cell death via a drug repurposing
Screen. Nat. Med. 2016 doi: 10.1038/nm.4184.
[24] E.P. Acosta, C. Flexner, Antiviral agents (nonretroviral), in: Goodman & Gilman’s - The
Pharmacological Basis of Therapeutics, New York, 2011, pp 1593–1622.
[25] C. Flexner, Antiretroviral Agents and Treatment of HIV Infection, in: Goodman and
Gilman’s - The Pharmacological Basis of Therapeutics, New York, 2011, pp 1623–1664.
[26] Data were collected at 100(2)K with Mo-Ka radiation using an Rigaku Saturn724+ (2x2
bin mode) of the UK National Crystallographic Service (NCS), based at the University of
Southampton. Data collection, data reduction and unit cell refinement were achieved with
CrystalClear-SM Expert 3.1 b27 [20a]. Correction for absorption was achieved by an
empirical absorption correction using spherical harmonics, implemented in SCALE3
ABSPACK scaling algorithm. The program MERCURY [20b] were used in the
preparation of the Figures. SHELXL97 [20c] and PLATON [20d] were used in the
calculation of molecular geometry. The structures were solved by direct methods using
SHELXS-97 [20c] and fully refined by means of the program SHELXL-97 [20c].
Difference map peaks provided positions for water hydrogens and hydrogen attached to
N(2). The coordinates, along with isotropic displacement parameters, were fully refined.
All other hydrogen atoms were placed in calculated positions.
[27] (a) CrystalClear-SM Expert. Rigaku Corporation, Tokyo, Japan, 2012, (b) Mercury 3.3.1
28