1,2,3-Triazolyl-4-oxoquinolines: A feasible beginning for promising chemical structures to inhibit oseltamivir-resistant influenza A and B viruses

Article abstract of DOI:10.1016/j.bmc.2015.11.028

We described the synthesis of a new congener series of 1,2,3-triazolyl-4-oxoquinolines and evaluated their ability to inhibit oseltamivir (OST)-resistant influenza strains. Oxoquinoline derivative 1i was the most potent compound within this series, inhibiting 94% of wild-type (WT) influenza neuraminidase (NA) activity. Compound 1i inhibited influenza virus replication with an EC₅₀ of 0.2 μM with less cytotoxicity than OST, and also inhibited different OST-resistant NAs. These results suggest that 1,2,3-triazolyl-4-oxoquinolines represent promising lead molecules for further anti-influenza drug design.

Full text of DOI:10.1016/j.bmc.2015.11.028

Accepted Manuscript
1,2,3-Triazolyl-4-oxoquinolines: a feasible beginning for promising chemical
structures to inhibit Oseltamivir-resistant influenza A and B viruses
Fernanda da C.S. Boechat, Carolina Q. Sacramento, Anna C. Cunha, Fernanda
S. Sagrillo, Christiane M. Nogueira, Natalia Fintelman-Rodrigues, Osvaldo
Santos-Filho, Cecília S. Riscado, Luana da S.M. Forezi, Letícia V. Faro,
Leonardo Brozeguini, Isakelly P. Marques, Vitor F. Ferreira, Thiago Moreno L.
Souza, Maria Cecília B.V. de Souza
PII:
S0968-0896(15)30153-X
http://dx.doi.org/10.1016/j.bmc.2015.11.028
BMC 12676
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 September 2015
11 November 2015
21 November 2015
Please cite this article as: Boechat, F.d.C.S., Sacramento, C.Q., Cunha, A.C., Sagrillo, F.S., Nogueira, C.M.,
Fintelman-Rodrigues, N., Santos-Filho, O., Riscado, C.S., Forezi, L.d.S.M., Faro, L.V., Brozeguini, L., Marques,
I.P., Ferreira, V.F., Souza, T.M.L., de Souza, M.C.B.V., 1,2,3-Triazolyl-4-oxoquinolines: a feasible beginning for
promising chemical structures to inhibit Oseltamivir-resistant influenza A and B viruses, Bioorganic & Medicinal
Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.11.028
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Bioorganic & Medicinal Chemistry
1,2,3-Triazolyl-4-oxoquinolines: a feasible beginning for promising chemical
structures to inhibit Oseltamivir-resistant influenza A and B viruses
Fernanda da C. S. Boechata,#, Carolina Q. Sacramentob,c,d,#, Anna C. Cunhaa, Fernanda S. Sagrilloa,
Christiane M. Nogueiraa, Natalia Fintelman-Rodriguesb,c,d, Osvaldo Santos-Filhob, Cecília S. Riscadoa,
Luana da S. M. Forezia, Letícia V. Faroa, Leonardo Brozeguinia, Isakelly P. Marquesa, Vitor F. Ferreiraa,
Thiago Moreno L. Souzab,c,d,┼,* and Maria Cecília B. V. de Souzaa,┼,
*
a Universidade Federal Fluminense, Instituto de Química - Outeiro de São João Batista, s/nº.Campus do Valonguinho, Centro, Niterói – RJ, CEP: 24020-150,
Brazil
b Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Vírus Respiratórios, NIC-WHO, Rio de Janeiro, RJ, CEP 21041-360, Brazil
c Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Imunofarmacologia, Rio de Janeiro, RJ, CEP 21041-360, Brazil
d Fundação Oswaldo Cruz, Centro de Desenvolvimento Tecnológico em Saúde, Rio de Janeiro, RJ, CEP 21041-360, Brazil
# These authors contributed equally as first authors
* These authors contributed equally as last authors
┼ Corresponding authors
* Tel: +55 21 26292148
E-mail address: mceciliabvs@gmail.com
ARTICLE INFO
ABSTRACT
We described the synthesis of
a new congener series of 1,2,3-triazolyl-4-
Article history:
Received
Received in revised form
Accepted
oxoquinolines and evaluated their ability to inhibit oseltamivir (OST)-resistant influenza strains.
Oxoquinoline derivative 1i was the most potent compound within this series, inhibiting 94 % of
wild-type (WT) influenza neuraminidase (NA) activity. Compound 1i inhibited influenza virus
replication with an EC50 of 0.2 µM with less cytotoxicity than OST, and also inhibited different
OST-resistant NAs. These results suggest that 1,2,3-triazolyl-4-oxoquinolines represent
promising lead molecules for further anti-influenza drug design.
Available online
Keywords:
1,2,3-triazole
4-oxoquinoline
2015 Elsevier Ltd. All rights reserved.
influenza viruses
neuraminidase inhibitors
oseltamivir carboxylate
antiviral resistance
1. Introduction
membrane and are released via viral neuraminidase (NA)
activity.5
Acute respiratory infections have a great impact on public
health because they are a major cause of morbidity and
Strategies to control influenza virus infections include
vaccination and antiviral drugs.6,7 The existence of multiple
zoonotic hosts,8 the time required to produce vaccines against
novel viruses, the costs of vaccine production and its
recommendation only for groups of patients at high risk for
serious influenza-related illnesses represent major limitations for
the use of vaccines.9 In contrast, anti-influenza drugs are now
recommended for clinical use whenever possible because the
most effective time frame for treatment is approximately 2.5 days
after the onset of illness.10 Moreover, because the antigenic
characteristics of viral strains that might cause future pandemic
outbreaks are unpredictable, the stockpiling of anti-influenza
drugs is a key issue in pandemic preparedness.11,12
mortality.1
Influenza
virus,
a
negative-sense-RNA
orthomixovirus,2 is the most important etiologic agent of severe
acute respiratory infections (SARI). Influenza virus causes both
seasonal infections and pandemic outbreaks.3 To enter host cells,
influenza binds to sialic acid residues on glycoproteins localized
in the cellular plasma membrane. This is followed by endocytosis
and the fusion of the viral envelope with the endocytic membrane
in a manner that is dependent on the viral protein M2.4 Next,
viral ribonucleoproteins (RNP) composed of the RNA
polymerase complex, viral RNA, nucleoprotein (NP) and nuclear
export proteins (NEP) are released into the cytoplasm and
transported to the cell nucleus, where transcription and
replication of the viral genome occur. Then, viral proteins are
trafficked to the host cell plasma membrane for the assembly of
new viruses. These particles bud through the cellular plasma
Virtually all circulating strains of influenza are resistant to the
adamantanes (M2-channel blockers). Thus, neuraminidase
inhibitors (NAIs) such as oseltamivir (OST), zanamivir,

peramivir and laninamivir constitute the only licensed class of
drugs available for clinical use against influenza (Figure 1).13
OST is the most used anti-influenza drug because it is orally
administered and is licensed to more countries than the other
drugs.14 However, approximately 1-2 % of the circulating strains
of influenza A(H1N1)pdm09 virus are resistant to OST, and
OST-resistant viruses may also cause primary infections.15,16
Therefore, the identification of novel molecules endowed with
the ability to inhibit OST-resistant strains of influenza is pivotal
because it may increase the number of options available to fight
this virus infection in the future.
Figure 1. Structures of Neuraminidase Inhibitors (NAIs) approved for clinical use.
Quinolones and triazolic derivatives have been largely
explored due to multiple biological properties. With respect to
these radicals as antivirals, they have been proven to be active
against HIV,17,18 HSV,19 HCV20, 21 and influenza.22, 23
acid residues of both WT and OST-resistant NAs. Our results
suggest that the chemical structure of 1,2,3-triazolyl-4-
oxoquinolines is promising for the development of novel anti-
influenza drugs.
In this work, we synthesized new 4-oxoquinoline derivatives
1a-j in which the core quinolone was connected to a 1,2,3-
triazole nucleus and investigated their ability to inhibit influenza
virus replication and the NA activity of OST-resistant strains of
influenza. Importantly, our most effective compound inhibited
multi-resistant strains of influenza. The triazolic ring and the
cyclohexenyl radical were found to be critical for anti-influenza
activity and allowed the compound to bind to conserved amino
2. Chemistry
The synthesis of 1,2,3-triazolyl-4-oxoquinoline derivatives
1a-j was initiated with the treatment of meta- or para-nitroaniline
with diethyl ethoxymethylenemalonate (EMME) to obtain
enamine-type derivatives that were then cyclized in refluxing
diphenyl ether.19
Scheme 1. General synthetic route to obtain azidoquinolones 4a-b: A: 1) EMME, EtOH, reflux, 24h; 2) Diphenyl Ether,
250ºC, 6h; B: K2CO3, DMF, EtBr, 80ºC, 24h; C: 1) Iron, NH4Cl 0,05M; 2) NaNO3, HCl, 30min; 3)NaN3, 15 min.
As mentioned above, NAIs are the only class of anti-influenza
drug in clinical use. Although this class is effective against all
NA types, drug-resistant influenza viruses have been
described.15,16 We initially performed NA inhibition assays
with a single dose of 320 triazolic compounds. The molecules
were tested against different NA types (or isoforms), either WT
or OST-resistant enzymes. Ideally, high values for the ratio
between the inhibitory activities against the OST-resistant and
WT enzymes would indicate promising hits. We identified 8
promising compounds with ratios above 0.8. Among these, two
1,2,3-triazolyl-4-oxoquinoline derivatives showed ratios above
1.0 (Table S1), suggesting that they are more effective against the
OST-resistant NA than the WT. Therefore, in this work we will
further examine the mechanism of inhibition of these 1,2,3-
triazolyl-4-oxoquinoline derivatives (hits 3 and 6 from Table S1),
while the other hits will be studied in future works. All 1,2,3-
triazolyl-4-oxoquinoline derivatives analyzed in this study are
shown in Table 1 and named henceforth as compounds 1a-j.
Compounds 1e and 1i were the most potent against the NA
activity, reaching inhibitions equal to 89.0 and 94.8 %,
respectively (Table 1).
The corresponding 1,4-dihydro-4-oxo-quinolines
2 were
submitted to an alkylation reaction with ethyl bromide, thereby
affording the desired N-ethyl 4-oxoquinolines 3.24 After the
chemical reduction of these compounds with iron and aqueous
ammonium chloride solution, the diazonium salts of the amino-4-
oxoquinolines were reacted with sodium azide, leading to desired
azidoquinolones 4a-b in quantitative yield (Scheme 1).25
The azido-4-oxoquinolines 2a and 2b allowed us to study their
application in the Huisgen “click-chemistry” reaction using
copper sulfate and ascorbic acid as the catalysts and
dimethylformamide as the solvent at 50 ºC25 (Table 1).
Their reactions with different alkynes resulted in the coupled
inedited 4-oxoquinolines 1a-j, with 79-98 % yields after
purification (Table 1).
All the structures of the new compounds 1a-j were confirmed
by spectral data (1H and 13C-NMR spectra) and by high
resolution mass spectrometry analysis.
3. Results and Discussion
3.1. Anti-influenza NA activity of 1,2,3-triazolyl-4-oxoquinoline
derivatives.

Table 1. Scheme summarizing the synthesis of 4-oxoquinoline 1a-j moiety, R1 substituents coupled to triazole ring located in
positions C-6 or C-7 of the 4-oxoquinolines, the yield of each derivative after purification and their anti-influenza NA activity.
Influenza H3N2 NA
#
R1
Triazole position
Yield (%)
inhibition at 50 μM (%)
C-6
C-7
C-6
C-7
C-6
C-7
C-6
C-7
88
85
83
79
97
93
95
90
59.1 ± 3.2
62.3 ± 1.1
56.5 ± 1.4
57.4 ± 2.2
89.0 ± 1.2
56.3 ± 3.3
0
1a
1b
1c
1d
1e
1f
1g
1h
0
C-6
C-7
98
95
94.8 ± 2.1
44.5 ± 2.3
1i
1j
influenza A and B. We can see, Table 2, that compound 1i
showed some advantages over OST in the inhibition of resistant
strains of influenza. Although OST was more potent than
compound 1i in the inhibition of WT strains, compound 1i IC50
values suffered only marginal changes in the presence of
resistance mutations to OST (Table 2). That is, the ratios of 1i`s
IC50 values for OST-resistant NAs over the WT counterpartners
were 3.0, 0.13 and 1.4 for influenza A(H1N1)pdm09, A/H3N2
and B, respectively (Table 2). This indicates a marginal effect of
OST-related resistance mutations towards compound 1i´s ability
to inhibit influenza A and B NAs (Table 2). The reference
compound, OST, inhibited the NA activity of the antiviral
resistant mutants of Influenza A(H1N1) [H275Y], A(H3N2)
[E119V] and B [R152K] with higher IC50 values when compared
to WT enzymes, these values increased by 27-, 380- and 5.3-fold,
respectively (Table 2). Therefore, our data indicate that
compound 1i chemical structure may be a promising one to
develop novel broad spectrum anti-influenza compounds able to
impair the NA activity of OST-resistant strains.
For comparison, OST inhibits the NA activity at the same
concentration of the tested compounds by 100 %. Interestingly,
1e and 1i have similar R1 substituents including a phenyl or
cyclohexenyl ring, respectively; which is bound to the triazolic
moiety at carbon 6 on the 4-oxoquinoline ring (Table 1). The
change of the triaolic moiety position compromised the anti-
influenza activity, probably because it interferes with the pattern
by which our compound interacts with its targets. More
interestingly, changes in R1 radical, which led to a decrease in the
steric space occupied by our compounds, could even abolish their
antiinfluenza activity (Table 1). Considering that these
compounds are very similar, being molecule 1i is slightly more
potent than 1e, subsequent experiments were performed with the
former compound.
3.2. Potency of 1,2,3-triazolyl-4-oxoquinoline derivative 1i
against NA.
To evaluate the potency of compound 1i, we measured its ability
to inhibit WT and OST-resistant NAs from circulating strains of
Table 2. Potency of the compound 1i and the reference compound OST on WT and OST-resistant influenza strains.
IC50
IC50 change (times)
Influenza strains
A/H3N2 WT
A/H3N2 E119V
A/H1N1 WT
A/H1N1 H275Y
B WT
1i (µM)
OST (nM)
0.15 ± 0.032
4.19 ± 0.16
0.21 ± 0.011
79.94 ± 3.2
16.00 ± 2.9
85.00 ± 5.4
1i
OST
NA
19.90 ± 1.3
2.60 ± 0.8
3.50 ± 0.9
10.60 ± 0.9
22.00 ± 1.1
30.00 ± 1.6
NA
0.13
NA
27.00
NA
3.00
NA
380.00
NA
B R152K
1.40
5.30

3.3. Insights into the docking site of compound 1i and its
pharmacophore group.
NAs (Table 2). Thus, we next performed in silico docking studies
of compound 1i with WT and OST-resistant NAs (Table S2) to
get insight on the pharmacophore group of this molecule and its
predicted binding sites. Compound 1i docked in the active site
cleft of all tested isoforms of NA, either WT or resistant to OST
(Figure 2). The docking sites of compound 1i and OST partially
overlapped (Figure 2A). The cyclohexenyl substituent shown
above to be critical for the anti-influenza activity had a singular
We showed above that the presence of the phenyl or
cyclohexenyl ring linked to triazolic moiety bound to carbon C-6
of the 4-oxoquinoline derivatives was critical to inhibit influenza
NA activity (Table 1) and compound 1i (cyclohexenyl
substituent) was able to inhibit OST-resistant influenza A and B
Figure 2. In silico analysis of compound 1i bound to different isoforms of influenza NA enzyme. The crystal structure of WT N1
isoform with the compound 1i (yellow) and OST (green) docked in the active site cleft (A). The cyclehexenyl radical of compound 1i is
projected to an area of NA enzyme which is not occupied by OST (B). 2D schematic figures showing compound 1i docking to different NA
isoforms are displayed as following: WT N1 (C), OST-resistant N1 (D), WT N2 (E), OST-resistant N2 (F) and WT influenza B NA (G). The
types of interaction between compound 1i and the NAs are indicated, hydrogen bounds are represented by doted lines. The red numbers, next
the amino acid residues surrounding the triazolic ring and the cyclehexenyl moiety, represent in how many different types of influenza NA
these residues are conserved (please see also Figure S1).

projection towards an area of the NA not occupied by OST
(Figure 2A and B), suggesting a possible mechanism on the
inhibition of OST- resistant strains. Figures 1C-G reveal multiple
points of interaction between our compound and the amino acids
in the active site of the NA. Moreover, the critical groups for
biological activity (i.e., the triazolic moiety at position 6 and the
cyclohexenyl ring) were nestled by very conserved amino acid
residues among various influenza NA types (Figure 2C-G and
Figure S1). These conserved amino acid residues are found in
influenza viruses infecting either humans or animals (Figures 2
and S1).26 Because the pharmacophore group of compound 1i
docked in conserved areas of the NA in versatile ways, it is
difficult to elicit specific amino acids to perform site-direct
mutagenesis assays to further evaluate the particular contribution
of amino acid residues to the interaction between compound 1i
and the influenza A and B NAs. Nevertheless, subsequent
passages of the influenza virus in the presence of the compound
1i are ongoing to evaluate whether influenza mutants resistant to
compound 1i may emerge.
Table 4. Potency against influenza replication and
cytotoxicity of compound 1i.
Compound
1i
EC50 (µM)
0.20 ± 0.01
0.03 ± 0.0023
CC50 (µM)
566 ± 89.5
321 ± 26
SIa
2,830
10,700
Oseltamivir
a SI, selective index is determined by the ratio between CC50 and EC50 values.
4. Conclusions
Novel 4-oxoquinoline derivatives containing 1,2,3-triazolyl
substituents 1a-j were successfully synthesized and fully
characterized. Compounds 1e and 1i were the most potent in
inhibiting influenza virus NA enzymes. In addition to the
inhibition of susceptible strains of influenza A and B, they also
inhibited OST-resistant strains. Compound 1i binds to the cleft of
the active site in all WT and OST-resistant NA isoforms. The
cyclohexenyl radical projects into an area of the NA that is not
occupied by OST and is critical for compound 1i antiviral
activity. Some of the amino acid residues required for the
docking of compound 1i are conserved, and changes in these
residues could reduce virus fitness. These results indicate that
the chemical structure of our synthesized oxoquinoline analogues
are interesting prototypes for further development of novel anti-
influenza drugs.
For comparisons, all the interactions of compound 1i and OST
with their targets are displayed in Table 3. Compound 1i bound
to the different NAs with free energies comparable to those
observed for OST (Table 3). Nevertheless, compound 1i is
endowed with the ability to inhibit isoforms of NA that OST
binds without achieving any antiviral effects (Table 2).
OST interacts with the different NAs using more hydrogen
bonds than compound 1i (Table 3). However, compound 1i was
more versatile in interacting with the different NAs than OST, as
judged by multiple steric hindrances with different amino acids
residues (Table 3). Altogether, our data suggest that the 4-
oxoquinolines moiety is important for compound 1i docking,
while the triazole ring and the cyclohexenyl radical are required
for antiviral activity. It is important to note that our
computational prediction (Table 3) revealed that OST interacted
with the NAS as described in the literature.27,28
5. Experimental
5.1. Chemistry
5.1.1. Synthesis of ethyl-1-ethyl-7-azido-4-oxo-1,4-
dihydroquinoline-3-carboxylate (4b).
The new compound 4b was synthesized as described
previously by us to obtain the derivative 4a.25 Reaction of 1-
ethyl-3-carbethoxy-7-amino-4-oxo-1,4-dihydroquinoline
with
nitrous acid (generated in situ from NaNO3 and HCl) followed by
the addition of sodium azide afforded the new compound 4b at a
yield of 84%. 4b: 1-ethyl-3-carbethoxy-7-azido-4-oxo-1,4-
3.4. Potency of compound 1i against influenza replication and its
cytotoxicity.
1
dihydroquinoline (84%) mp 143-145ºC; H NMR (300.00 MHz,
DMSO-d6, internal standard: Me4Si), δ 8.55 (s, 1H, H-2), 8.53 (d,
1H, J=8.5 Hz, H-5), 7.15 (dd, 1H, J= 8,5; 1,8 Hz, H-6), 6.96 (d,
1H, J= 1.8 Hz, H-8), 4.40 (q, 2H, J= 7,1 Hz, NCH2CH3), 4.22 (q,
2H, J= 7.1 Hz, OCH2CH3), 1.54 (t, 3H, J= 7.1 Hz, NCH2CH3),
and 1.42 (t, 3H, J=7.1 Hz, OCH2CH3); 13C NMR (75.0 MHz,
DMSO-d6) δ 172.0 (C-4), 164.4 (CO2CH2CH3), 149.2 (C-2),
144.1 (C-8a), 139.7 (C-6), 128.7 (C-4a), 125.3 (C-7), 116.2 (C-
8), 110.4 (C-5), 106.6 (C-3), 59.7 (OCH2CH3), 47.8 (NCH2CH3),
14.2 (OCH2CH3), and 14.1 (NCH2CH3); HRMS (ESI) m/z:
calculated for C14H14N4O3 [M+H+] 287.1144, and found
287.1143.
Next, we evaluated the anti-influenza activity and cytotoxicity
of compound 1i cell-based assays. The EC50 for compounds 1i
was 0.2 ± 0.01 M, while for OST it was 0.03 ± 0.0023 M
(Table 4). Compound 1i was less cytotoxic than OST, CC50
values of 566 ± 89.5 µM and 321 ± 26 M, respectively, were
obtained (Table 4). Selective index (SI) values were calculated
based on the ratio between the CC50 and the EC50 values. SI
values for OST and compound 1i were 10,700 and 2,830,
respectively (Table 4). Although OST’ SI value is higher than the
one observed for compound 1i, our molecule is still very safe to
be used in vitro. For comparisons, compound 1i was more potent
than other triazolic derivatives described in the literature using
cell-based assays, including the zanamivir analogues.22 These
results indicate that compound 1i is endowed with a very
important margin between anti-influenza activity and
cytotoxicity. These information, together with the anti-influenza
activity over OST-resistant strains and the apparent ability to
bind into conserved amino acid residues on NA, indicate that the
chemical structure of compound 1i is promising and may be of
interest for further development of novel anti-influenza drugs.
5.1.2. Synthesis of ethyl 1-ethyl-(1,2,3-triazol-1’-
yl)-4-oxo-1,4-dihydroquinoline-3-carboxylates (1a-
j).
CuSO4.5H2O (17.5 mg, 0.07 mmol) and ascorbic acid (29.6
mg, 0.15 mmol) were added to a solution of azidoquinolone (10a
or 10b, 100 mg, 0.37 mmol) and the desired alkyne (0.55 mmol)
in DMF (10.0 mL).

Table 3. Compound 1i and OST with WT and OST interactions during docking.
Compound 1i
OST
Binding
energy
(kcal/mol)
No. of
hydrogen
bonds
Amino acid
residues involved
in H-bounding
Binding
energy
(kcal/mol)
No. of
hydrogen
bonds
Neuraminidase
Types
No. of
bumps
Amino acid residues involved in
bumps
Amino acid residues involved in
H-bounding
No. of Amino acid residues involved
bumps
in bumps
Arg 118, 153, 224; Glu 119, 227,
277; Leu 134; Asp 151; Trp 178,
295; Ser 179, 246; Ile 222; Pro 245;
Asn 247, 347; Ala 346; Tyr 406
Trp 178; Ile 222; Arg 224; Ser
246; Glu 276, 277; Asn 294;
Tyr 406
Arg 118, 152, 292, 371; Glu 119;
Asp 151
N1 WT
-6,44
-7,49
2
4
Arg 152, Asn 294
18
17
-7,44
-7,98
6
7
8
Glu 119, 227, 277; Leu 134; Arg
156, 224, 292; Trp 178; Ser 179;
Asn 221, 247, 294; Ile 222; Gly 244;
Pro 245; Tyr 347, 406
Asp 151; Trp 178; Ile 222;
Arg 224; Ser 246; Glu 276,
277; Asn 294; Tyr 406
Asp 151; Arg 152;
Ser 246
Arg 118, 152, 292, 371; Glu 119;
Tyr 347
N1 H275Y
9
Glu 119, 227, 276, 277; Leu 134;
Asp 151; Arg 152; Trp 178, 295; Ser
179, 245, 247; Ile 222; Thr 225; Ala
246; Gly 346; Tyr 406
Arg 118, 224; Trp 178; Ile
222; Ala 246; Glu 276, 277;
Asn 294; His 347; Tyr 406
Arg 224, 292; Asn
294; His 347
Glu 119; Asp 151; Arg 152, 282,
371
N2 WT
N2 E119V
N B
-7,59
-7,65
-7,60
5
5
4
17
13
17
-8,57
-8,20
-7,99
6
10
7
10
Asp 151; Arg 152; Trp 179; Ser 179;
Ile 222; Leu 223; Glu 227, 276; Ala
246; Thr 247; Asn 294, 346
Arg 224, 292, 371;
Asn 347
Arg 118, 152, 292, 371; Asp 151;
Trp 178; Glu 227; Tyr 406
Leu 134; Arg 156, 224; Ile
222; Glu 276, 277
6
Arg 146, 149, 222, 291, 373; Asp
148; Trp 176, 407; Ser 177, 247,
248; Lys 219; Ile 220; Leu 221; Gly
242; Glu 275; His 438
Trp 176; Ile 220; Arg 222;
Ala 244; Glu 274; Asn 293;
Tyr 408
Arg 115; Ala 244;
Tyr 408
Arg 115, 149, 291, 373; Glu 116;
Asp 148
7

The resulting solution was stirred for 2 h (monitored by TLC)
at 50ºC. After this time, the reaction mixture was poured into ice,
leading to a yellow solid that was purified by flash column
chromatography (CH2Cl2/EtOH as the gradient). 1a: ethyl 1-
ethyl-6-[4’-(2”-hydroxypropan-2”-yl)-1H-1,2,3-triazol-1’-yl]-4-
oxo-1,4-dihydroquinoline3-carboxylate (88%), mp 175-177ºC;
1H NMR (300.00 MHz, DMSO-d6, internal standard: Me4Si), δ
8.77 (s, 1H, H-5’), 8.75 (s, 1H, H-2), 8.66 (d, 1H, J= 2.4 Hz, H-
5), 8.33 (dd, 1H, J= 9.1; 2.4; H-7), 8.06 (d, 1H, J= 9.1; H-8), 5.26
(s, 1H, C(CH3)2OH), 4.48 (q, 2H, J= 6.8 Hz, OCH2CH3), 4.25 (q,
2H, J= 7.0 Hz, NCH2CH3), 1.56 (s, 6H, C(CH3)2OH), 1.40 (t, 3H,
J= 6.8 Hz, OCH2CH3), and 1.30 (t, 3H, J= 7.0 Hz, NCH2CH3);
13C NMR (75.0 MHz, DMSO-d6) δ 172.2 (C-4), 164.3
(CO2CH2CH3), 157.0 (C-4’), 149.2 (C-2), 137.9 (C-8a), 133.4
(C-6), 129.0 (C-4a), 124.2 (C-7), 119.0 (C-5’), 119.3 (C-8),
116.6 (C-5), 110.3 (C-3), 66.9 (C(CH3)2OH), 59.8 (NCH2CH3),
48.1 (OCH2CH3), 30.4 (C(CH3)2OH), 14.3 (OCH2CH3), and 14.2
(NCH2CH3); ESI-FTICRMS: m/z calculated for C19H22N4O4
[M+H] 371.1873, found 371.1870; 1b: ethyl 1-ethyl-7-[4’-(2”-
hydroxypropan-2”-yl)-1H-1,2,3-triazol-1’-yl]-4-oxo-1,4-
9.46 (s, 1H, H-5’), 8.74 (s, 1H, H-2), 8.73 (d, 1H, J=2.7 Hz, H-
5), 8.37 (dd, 1H, J= 9.1; 2.7 Hz, H-7), 8.10 (d, 1H, J= 9,1 Hz, H-
8), 7.99 (d, 2H, J= 7,1 Hz, H-2” and H-6”), 7.51 (t, 2H, J= 7,3
Hz, H-3” and H-5”), 7.40 (t, 1H, J= 7.1 Hz, H-4”), 4.48 (q, 2H,
J= 7,1 Hz, OCH2CH3), 4.27 (q, 2H, J= 7.1 Hz, NCH2CH3), 1.44
(t, 3H, J= 7.1 Hz, OCH2CH3), and 1.32 (t, 3H, J=7.1 Hz,
NCH2CH3); 13C NMR (75.0 MHz, DMSO-d6) δ 172.1 (C-4),
164.2 (CO2CH2CH3), 149.0 (C-2), 147.4 (C-4’), 138.1 (C-8a),
133.1 (C-6), 131.5 (C-1”), 130.0 (C-4a), 128.7 (C-3” and C-5”),
128.1 (C-4”), 125.2 (C-2” and C-6”), 124.1 (C-7), 119.3 (C-8),
116.7 (C-5), 115.0 (C-5’), 110.4 (C-3), 59.6 (NCH2CH3), 48.0
(OCH2CH3), 14.2 (OCH2CH3), and 14.1 (NCH2CH3); ESI-
FTICRMS: m/z calculated for C22H20N4O3 [M+H] 389.1535,
found 389.1532; 1f: ethyl 1-ethyl-7-(4’-phenyl-1H-1,2,3-triazol-
1’-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (93%) mp 127-
129ºC; 1H NMR (300.00 MHz, DMSO-d6, internal standard:
Me4Si) δ 9.52 (s, 1H, H-5’), 8.76 (s, 1H, H-2), 8.46 (d, 1H, J=8.7
Hz, H-5), 8.09 (dd, 1H, J= 8.7; 1.6 Hz, H-6), 8.26 (d, 1H, J= 1.6
Hz, H-8), 7.97 (dd, 2H, J= 8.2; 1.2 Hz, H-2” and H-6”), 7.54 (t,
2H, J= 7.2 Hz, H-3” and H-5”), 7.43 (t, 1H, J= 7.2 Hz, H-4”),
4.52 (q, 2H, J= 7.0 Hz, OCH2CH3), 4.25 (q, 2H, J= 7.0 Hz,
NCH2CH3), 1.46 (t, 3H, J= 7.0 Hz, OCH2CH3), and 1.31 (t, 3H,
J=7.0 Hz, NCH2CH3); 13C NMR (75.0 MHz, DMSO-d6) δ 171.9
(C-4), 164.0 (CO2CH2CH3), 149.4 (C-2), 147.3 (C-4’), 139.2 (C-
8a and C-7), 129.3 (C-1”), 128.8 (C-3” and C-5”), 128.3 (C-4”),
127.4 (C-4a), 125.1 (C-2” and C-6”), 128.7 (C-5), 119.9 (C-5’),
116.2 (C-6), 108.1 (C-3), 107.5 (C-8), 59.6 (NCH2CH3), 47.8
(OCH2CH3), 14.0 (OCH2CH3), and 13.9 (NCH2CH3); ESI-
FTICRMS: m/z calculated for C22H20N4O3 [M+H] 389.1535,
found 389.1538; 1g: ethyl 1-ethyl-6-[4’-(hydroxymethyl)-1H-
1,2,3-triazol-1’-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylate
(95%) mp 220-221ºC; 1H NMR (300.00 MHz, DMSO-d6,
internal standard: Me4Si) δ 8.85 (s, 1H, H-5’), 8.75 (s, 1H, H-2),
8.66 (d, 1H, J= 2.7 Hz, H-5), 8.32 (dd, 1H, J= 9.3; 2.7, H-7), 8.07
(d, 1H, J= 9.3; H-8), 5.35 (t, 1H, J=5.5 Hz, CH2OH), 4.63 (d, 2H,
J= 5.5 Hz, CH2OH), 4.48 (q, 2H, J= 7.1 Hz, OCH2CH3), 4.25 (q,
2H, J= 7.3 Hz, NCH2CH3), 1.40 (t, 3H, J= 7.1 Hz, OCH2CH3),
and 1.30 (t, 3H, J= 7.3 Hz, NCH2CH3); 13C NMR (75.0 MHz,
DMSO-d6) δ 172.2 (C-4), 164.4 (CO2CH2CH3), 149.3 (C-4’),
149.2 (C-2), 138.0 (C-8a), 133.4 (C-6), 129.0 (C-4a), 124.3 (C-
7), 121.2 (C-5’), 119.4 (C-8), 116.8 (C-5), 110.3 (C-3), 59.8
(NCH2CH3), 54.9 (CH2OH), 48.2 (OCH2CH3), 14.4 (OCH2CH3),
and 14.3 (NCH2CH3); ESI-FTICRMS: m/z calculated for
C17H18N4O4 [M+H] 343.1328, found 343.1324; 1h: ethyl 1-ethyl-
7-[4’-(hydroxymethyl)-1H-1,2,3-triazol-1’-yl]-4-oxo-1,4-
dihydroquinoline-3-carboxylatye (85%), mp 179-181ºC; 1H
NMR (300.00 MHz, DMSO-d6, internal standard:Me4Si) δ 8.92
(s, 1H, H-5’), 8.75 (s, 1H, H-2), 8.40 (d, 1H, J= 8.8 Hz, H-5),
8.07 (dd, 1H, J= 8.8; 1.7; H-6), 8.20 (d, 1H, J= 1.7; H-8), 4.52 (q,
2H, J= 7.1 Hz, OCH2CH3), 4.24 (q, 2H, J= 7.1 Hz, NCH2CH3),
1.56 (s, 6H, C(CH3)2OH), 1.41 (t, 3H, J= 7.1 Hz, OCH2CH3), and
1.30 (t, 3H, J= 7.1 Hz, NCH2CH3); 13C NMR (75.0 MHz,
DMSO-d6) δ 172.2 (C-4), 164.3 (CO2CH2CH3), 157.3 (C-4’),
149.7 (C-2), 139.7 (C-8a), 139.5 (C-7), 127.4 (C-4a), 128.7 (C-
5), 119.4 (C-5’), 116.3 (C-6), 110.8 (C-3), 107.3 (C-8), 67.0
(C(CH3)2OH), 59.8 (NCH2CH3), 48.0 (OCH2CH3), 30.5
(C(CH3)2OH), 14.3 (OCH2CH3), and 14,2 (NCH2CH3); ESI-
FTICRMS: m/z calculated for C19H22N4O4 [M+H] 371.1873,
found 371.1876; 1c: ethyl 1-ethyl-6-[4’-(1”-hydroxycyclohexyl)-
1H-1,2,3-triazol-1’-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylate
(83%), mp 228-230ºC; 1H NMR (300.00 MHz, DMSO-d6) δ 8.77
(s, 1H, H-5’), 8.74 (s, 1H, H-2), 8.66 (d, 1H, J=2.4 Hz, H-5),
8.32 (dd, 1H, J= 9.2; 2.4 Hz, H-7), 8.05 (d, 1H, J= 9.2 Hz, H-8),
4.48 (q, 2H, J= 6.8 Hz, OCH2CH3), 4.25 (q, 2H, J= 7.1 Hz,
NCH2CH3), 2.03-1.48 (m, 10H, H-2” to H-6”), 1.40 (t, 3H, J= 6.8
Hz, OCH2CH3), and 1.30 (t, 3H, J=7.1 Hz, NCH2CH3); 13C NMR
(75.0 MHz, DMSO-d6) δ 172.1 (C-4), 164.3 (CO2CH2CH3),
156.8 (C-4’), 149.1 (C-2), 137.9 (C-8a), 133.4 (C-6), 124.1 (C-
7), 120.0 (C-4a), 119.4 (C-5’), 119.3 (C-8), 116.6 (C-5), 110.3
(C-3), 67.8 (C-1”), 59.7 (NCH2CH3), 48.1 (OCH2CH3), 37.5 (C-
2” and C-6”), 25.1 (C-4”), 21.6 (C-3” and C-5”), 14.3
(OCH2CH3), and 14.2 (NCH2CH3); ESI-FTICRMS: m/z
calculated for C22H26N4O4 [M+H] 411.2227, found 411.2222; 1d:
ethyl 1-ethyl-7-[4’-(1”-hydroxycyclohexyl)-1H-1,2,3-triazol-1’-
yl]-4-oxo-1,4-dihydroquinoline-3-carboxylate (79%) mp 105-
107ºC; 1H NMR (300.00 MHz, DMSO-d6, internal standard:
Me4Si) δ 8.93 (s, 1H, H-5’), 8.74 (s, 1H, H-2), 8.41 (d, 1H, J=8.8
Hz, H-5), 8.07 (dd, 1H, J= 8.8; 1.8 Hz, H-6), 8.20 (d, 1H, J= 1.8
Hz, H-8), 4.52 (q, 2H, J= 7.1 Hz, OCH2CH3), 4.25 (q, 2H, J= 7.0
Hz, NCH2CH3), 2.02-1.49 (m, 10H, H-2” to H-6”), 1.46 (t, 3H,
J= 7.1 Hz, OCH2CH3), and 1.30 (t, 3H, J=7.1 Hz, NCH2CH3);
13C NMR (75.0 MHz, DMSO-d6) δ 172.0 (C-4), 164.2
(CO2CH2CH3), 157.1 (C-4’), 149.4 (C-2), 139.6 (C-7), 139.4 (C-
8a), 128.5 (C-5), 127.2 (C-4a), 119.6 (C-5’), 116.0 (C-6), 110.1
(C-3), 107.1 (C-8), 67.9 (C-1”), 59.5 (NCH2CH3), 47.7
(OCH2CH3), 37.3 (C-2” and C-6”), 24.8 (C-4”), 21.2 (C-3” and
C-5”), 14.0 (OCH2CH3), and 13.9 (NCH2CH3); ESI-FTICRMS:
m/z calculated for C22H26N4O4 [M+H] 411.2227, found
411.2226; 1e: ethyl 1-ethyl-6-(4’-phenyl-1H-1,2,3-triazol-1’-yl)-
4-oxo-1,4-dihydroquinoline-3-carboxylate (97%), mp 237-239ºC;
1H NMR (300.00 MHz, DMSO-d6, internal standard: Me4Si) δ
1
dihydroquinoline-3-carboxylate (90%) mp 223-225ºC; H NMR
(300.00 MHz, DMSO-d6, internal standard: Me4Si) δ 9.00 (s, 1H,
H-5’), 8.75 (s, 1H, H-2), 8.41 (d, 1H, J= 8.6 Hz, H-5), 8.21 (d,
1H, J= 1.7; H-8), 8.07 (dd, 1H, J= 8.6; 1.7, H-6), 4.66 (s, 1H,
CH2OH), 4.51 (q, 2H, J= 7.1 Hz, OCH2CH3), 4.25 (q, 2H, J= 7.1
Hz, NCH2CH3), 1.42 (t, 3H, J= 7.1 Hz, OCH2CH3), and 1.30 (t,
3H, J= 7.1 Hz, NCH2CH3); 13C NMR (75.0 MHz, DMSO-d6) δ
172.0 (C-4), 164.4 (CO2CH2CH3), 149.5 (C-4’), 149.7 (C-2),
139.5 (C-8a), 128.8 (C-5), 127.4 (C-4a), 124.3 (C-7), 121.6 (C-
5’), 116.3 (C-6), 110.8 (C-3), 107.5 (C-8), 59.8 (NCH2CH3), 54.9
(CH2OH), 48.0 (OCH2CH3), 14.3 (OCH2CH3), and 14.2
(NCH2CH3); ESI-FTICRMS: m/z calculated for C17H18N4O4
[M+H] 343.1328, found 343.1326; 1i: ethyl 1-ethyl-6-(4’-
cyclohexenyl-1H-1,2,3-triazol-1’-yl)-4-oxo-1,4-
1
dihydroquinoline-3-carboxylate (98%) mp 230-231ºC; H NMR
(300.00 MHz, DMSO-d6, internal standard: Me4Si) δ 8.94 (s, 1H,
H-5’), 8.73 (s, 1H, H-2), 8.67 (d, 1H, J=2.5 Hz, H-5), 8.30 (dd,
1H, J= 9.1; 2.5 Hz, H-7), 8.06 (d, 1H, J= 9.1 Hz, H-8), 6.58 (t,
1H, J= 3.6 Hz, H-2”), 4.47 (q, 2H, J= 7.0 Hz, OCH2CH3), 4.26
(q, 2H, J= 7.1 Hz, NCH2CH3), 1.96-1.74 (m, 8H, H-3” to H-6”),
1.42 (t, 3H, J= 7.0 Hz, OCH2CH3), and 1.31 (t, 3H, J=7.1 Hz,

NCH2CH3); 13C NMR (75.0 MHz, DMSO-d6) δ 172.2 (C-4),
164.2 (CO2CH2CH3), 149.0 (C-2), 149.1 (C-4’), 137.9 (C-8a),
133.3 (C-6), 129.0 (C-4a), 127.0 (C-1”), 124.5 (C-2”), 124.0 (C-
7), 119.3 (C-8), 118.0 (C-5’), 116.5 (C-5), 110.3 (C-3), 59.7
(NCH2CH3), 48.1 (OCH2CH3), 24.6 (C-6”), 25.7 (C-3”), 21.9 (C-
4”), 21.7 (C-5”),14.3 (OCH2CH3), and 14.2 (NCH2CH3); ESI-
FTICRMS: m/z calculated for C22H24N4O3 [M+H] 393.1758,
found 393.1754; 1j: ethyl 1-ethyl-7-(4’-cyclohexenyl-1H-1,2,3-
triazol-1’-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (95%),
mp 235-237ºC; 1H NMR (300.00 MHz, DMSO-d6, internal
standard: Me4Si) δ 9.02 (s, 1H, H-5’), 8.75 (s, 1H, H-2), 8.41 (d,
1H, J=8.7 Hz, H-5), 8.17 (d, 1H, J= 1,6 Hz, H-8), 8.05 (dd, 1H,
J= 8.7; 1.6 Hz, H-6), 6.59 (t, 1H, J= 3.6 Hz, H-2”), 4.48 (q, 2H,
J= 6.8 Hz, NCH2CH3), 4.25 (q, 2H, J= 7.0 Hz, OCH2CH3), 2.44-
2.42 (m, 2H, H-3” or H-6”), 2.22-2.10 (m, 2H, H-3” or H-6”),
1.80-1.72 (m, 2H, H-4” or H-5”), 1.67-1.64 (m, 2H, H-4” or H-
5”), 1.40 (t, 3H, J= 6.8 Hz, NCH2CH3), and 1.30 (t, 3H, J=7.0
Hz, NCH2CH3); 13C NMR (75.0 MHz, DMSO-d6) δ 172.3 (C-4),
164.5 (CO2CH2CH3), 149.6 (C-4’), 149.4 (C-2), 139.8 (C-7 and
C-8a), 128.6 (C-5), 127.8 (C-4a), 127.2 (C-1”), 124.7 (C-2”),
117.7 (C-5’), 116.0 (C-6), 110.2 (C-3), 107.1 (C-8), 59.7
(NCH2CH3), 47.8 (OCH2CH3), 25.5 (C-6”), 24.4 (C-3”), 21.6 (C-
4”), 21.5 (C-5”), 14.1 (OCH2CH3), and 14.0 (NCH2CH3); ESI-
FTICRMS: m/z calculated for C22H24N4O3 [M+H] 393.1758,
found 393.1755.
5.2.3. Influenza titration.
MDCKs plated in 96-well plates (5x104 cells/well) were
infected with serial 10-fold dilutions of the supernatants from the
yield-reduction assays described above for 1 h at 37ºC and 5 %
CO2. Then, viruses were washed out and the cells were incubated
for 72 h. After this period of time, influenza-induced cytopathic
effects (CPE) were scored by TCID50.30,31
5.2.4. NA inhibition assay.
To evaluate the ability of the compounds to inhibit the NA
activity of the influenza strains described in Table S1, we
performed cell-free based assays using the NA-Starkit (Life
Technologies, CA) according to the manufacturer’s instructions.
Briefly, the NA activity of the different influenza virus strains
was tittered. Next, the NA activity was measured in the presence
of different concentrations of the compounds to determine the
enzyme inhibition. The concentration able to inhibit 50% of
influenza`s NA activity (IC50) was calculated using non-linear
regression. For comparison, every assay was performed with
OST carboxylate as a positive control.
5.3. In silico docking.
Docking of the compound 1i or OST with different NAs
(Table S4) was performed using the ArgusLab 4.0.1 software
(Planaria Software LLC).32 The crystal structures of WT and
OST-resistant NAs were obtained from the Protein Data Bank
(PDB, www.rcsb.org).33 PDB accession numbers and the degree
of resolution of the x-ray crystals are shown in Table S2. We
selected these files because they already have an OST molecule
bound in the crystal structure of the NA. To our knowledge, no
OST-resistant influenza B NA structure is deposited in the PDB.
Before docking, the structures of the proteins were cleaned by
removing the water molecules and external ligands, with the
exception of OST. The modified structure was saved in the .pdb
format to be used in all docking studies. The structure of
compound 1i was designed using Accelrys Draw 4.1 software
(Accelrys, Inc.) and improved using Accelrys® Discovery Studio
3.5 software to add hydrogens and optimize the compound
geometry (UFF Molecular Mechanics method).34 The optimized
1i molecule file was saved in .mol format for further docking
studies. The docking between the ligand 1i and each NA enzyme
isoform was performed using the “Dock a Ligand” option in the
Arguslab software. The area occupied by amino acid residues
involved in the docking of OST was considered to be the center
of the actual 1i bind site (Table S2). A spacing of 0.4 Å between
the grid points was used and the ligand was assumed to be
flexible and the protein rigid. A maximum of 150 poses were
allowed in the analysis, and each docking run was repeated three
times to obtain the best results. “ArgusDock” and “Dock” were
chosen as the docking engine for the simulations and calculation
type, respectively. Displays of the 2D interactions between
compound 1i and different NAs were obtained with
Accelrys® Discovery Studio 3.5 software.35 OST was docked
over the original reference compound found in these protein X-
ray structures as a control. Poses with lower free-energy and with
differences smaller than 3 Å between the two docked OST
structures were considered to be reliable.
5.2. Cells and viruses.
Madin-Darby canine kidney epithelial cells (MDCKs) were
cultured in Dulbecco's Modified Eagle Medium (DMEM; Life
Technologies, Grand Island, NY) supplemented with 10 % fetal
bovine serum and antibiotics (100 U/mL penicillin and 100
μg/mL streptomycin) at 37ºC and 5 % CO2. Both wild-type and
NAI-resistant influenza A and B virus strains (Table S3) were
propagated in MDCKs and stored at -70ºC. The influenza strains
displayed in Table S3 were kindly donated by Dr. Larisa V.
Gubareva and Dr. Alexander I. Klimov from the Centers for
Disease Control (CDC), Atlanta, to the Brazilian National
Influenza Center (NIC) at FIOCRUZ, Rio de Janeiro. These
viruses are reference strains used for laboratory-based
surveillance to monitor the antiviral susceptibility of circulating
influenza viruses to NAIs.
5.2.1. Cytotoxicity assay.
Monolayers of 2 x 104 MDCKs 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 the
DMEM in the presence of 0.01 % N-methyl-dibenzopirazin
methyl sulfate (PMS). After incubation for 4 h at 37°C, the plates
were read in a spectrophotometer at 492 nm and 620 nm.29 The
50 % cytotoxic concentration (CC50) was calculated by linear
regression analysis of the dose–response curves generated from
the data.
5.2.2. Yield Reduction Assay.
Monolayers of MDCK cells (2 x 105 cell/well) in 24-well
plates were infected with influenza at an MOI of 0.05 for 1 h at
37ºC. Cells were washed to remove residual viruses and various
concentrations of the compounds were added. After 24 h, viruses
in the supernatant were harvested and titrated by end-point 50%
cell culture infective dose (TCID50/mL) using MDCK cells. For
compression, the reference compound OST carboxylate (kindly
donated by Hoffman-La Roche Inc., Basel, Switzerland) 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.
5.3.1. Alignment of Genetically Diverse
Neuraminidases.
To compare the relevant residues found in the docking
analysis with their equivalents on the sequenced NA isoforms
found in nature, we created alignments using Mega 6.06
software. DNA sequences were aligned by Clustal W (1.6), and
amino acid residues were predicted. We have chosen to align the
complete genome segment of influenza A NA isoforms N1 – N9

14. Jefferson, T.; Jones, M.; Doshi, P.; Spencer, E. A.; Onakpoya, I.;
Heneghan, C. J. BMJ. 2014, 348, g2545.
(NCBI codes and influenza strains are listed in Table S4) and
analyzed specific regions containing the amino acid residues
involved in the 1i and NA interactions.
15. Fry, A. M.; Gubareva, L. V. J. Infect. Dis. 2012, 206, 145-7.
16. Hurt, A. C.; Hardie, K.; Wilson, N. J.; Deng, Y. M.; Osbourn, M.;
Leang, S. K.; Lee, R. T.; Iannello, P.; Gehrig, N.; Shaw, R.; Wark,
P.; Caldwell, N.; Givney, R. C.; Xue, L.; Maurer-Stroh, S.;
Dwyer, D. E.; Wang, B.; Smith, D. W.; Levy, A.; Booy, R.; Dixit,
R.; Merritt, T.; Kelso, A.; Dalton, C.; Durrheim, D.; Barr, I. G. J.
Infect. Dis. 2012, 206, 148-57.
17. Souza, T. M. L.; Rodrigues, D. Q.; Ferreira, V. F.; Marques, I. P.;
Santos, F. C.; Cunha, A. C.; Souza, M. C. B. V.; Frugulhetti, I. C.
P. P.; Bou-Habib, D. C.; Fontes, C. F. L. Current HIV Research.
2009, 7, 327-335.
5.4. Statistical Analyses.
The dose-response curves used to calculate the IC50, EC50 and
CC50 values 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).
Acknowledgments
18. Wang, Z.; Wu, B.; Kuhen, K. L.; Bursulaya, B.; Nguyen, T. N.;
Nguyen, D. G.; He, Y. Bioorg. Med. Chem. Lett. 2006, 16, 4174-
7.
We thank the Conselho Nacional de Desenvolvimento
19. Duffin, G. F.; Kendall, J. D. J. Chem. Soc. 1948, 70,893–94
20. Kumar, D. V.; Rai, R.; Brameld, K. A.; Somoza, J. R.;
Rajagopalan, R.; Janc, J. W.; Xia, Y. M.; Ton, T. L.; Shaghafi, M.
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Científico
e
Tecnológico (CNPq), Coordenação de
Aperfeiçoamento de Pessoal Docente (CAPES) and Fundação de
Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for
financial support and fellowships.
21. Wittine, K.; Stipković Babić, M.; Makuc, D.; Plavec, J.; Kraljević
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Graphical Abstract

1,2,3-Triazolyl-4-oxoquinolines: a feasible
beginning for promising chemical structures to
inhibit Oseltamivir-resistant influenza A and B
viruses
Leave this area blank for abstract info.
Fernanda da C. S. Boechata,#, Carolina Q. Sacramentob,c,d,#, Anna C. Cunhaa, Fernanda S. Sagrilloa, Christiane
M. Nogueiraa, Natalia Fintelman-Rodriguesb,c,d, Osvaldo Santos-Filhob, Cecília S. Riscadoa, Luana da S. M.
Forezia, Letícia V. Faroa, Leonardo Brozeguinia, Isakelly P. Marquesa, Vitor F. Ferreiraa, Thiago Moreno L.
Souzab,c,d,┼,* and Maria Cecília B. V. de Souzaa,┼,
Universidade Federal Fluminense, Instituto de Química - Outeiro de São João Batista, s/nº.Campus do
*
a
Valonguinho, Centro, Niterói – RJ, CEP: 24020-150, Brazil
b
Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Vírus Respiratórios, NIC-WHO, Rio de
Janeiro, RJ, CEP 21041-360, Brazil
c
Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Imunofarmacologia, Rio de Janeiro, RJ,
CEP 21041-360, Brazil
d Fundação Oswaldo Cruz, Centro de Desenvolvimento Tecnológico em Saúde, Rio de Janeiro, RJ, CEP 21041-
360, Brazil
# These authors contributed equally as first authors
* These authors contributed equally as last authors
┼ Corresponding authors
We described the synthesis of a new congener series of 1,2,3-triazolyl-4-oxoquinolines and evaluated their ability to inhibit oseltamivir
(OST)-resistant influenza strains. Oxoquinoline derivative 1i was the most potent compound within this series, inhibiting 94 % of wild-type
(WT) influenza neuraminidase (NA) activity. Compound 1i inhibited influenza virus replication with an EC50 of 0.2 µM with less cytotoxicity
than OST, and also inhibited different OST-resistant NAs. These results suggest that 1,2,3-triazolyl-4-oxoquinolines represent promising lead
molecules for further anti-influenza drug design.