Docking of anti-HIV-1 oxoquinoline-acylhydrazone derivatives as potential HSV-1 DNA polymerase inhibitors

Article abstract of DOI:10.1016/j.molstruc.2014.05.081

Although there are many antiviral drugs available for the treatment of herpes simplex virus (HSV) infections, still the synthesis of new anti-HSV candidates is an important strategy to be pursued, due to the emergency of resistant HSV strains mainly in human immunodeficiency virus (HIV) co-infected patients. Some 1,4-dihydro-4-oxoquinolines, such as PNU-183792 (1), show a broad spectrum antiviral activity against human herpes viruses, inhibiting the viral DNA polymerase (POL) without affecting the human POLs. Thus, on an ongoing antiviral research project, our group has synthesized ribonucleosides containing the 1,4-dihydro-4-oxoquinoline (quinolone) heterocyclic moiety, such as the 6-Cl derivative (2), which is a dual antiviral agent (HSV-1 and HIV-1). Molecular dynamics simulations of the complexes of 1 and 2 with the HSV-1 POL suggest that structural modifications of 2 should increase its experimental anti-HSV-1 activity, since its ribosyl and carboxyl groups are highly hydrophilic to interact with a hydrophobic pocket of this enzyme. Therefore, in this work, comparative molecular docking simulations of 1 and three new synthesized oxoquinoline-acylhydrazone HIV-1 inhibitors (3-5), which do not contain those hydrophilic groups, were carried out, in order to access these modifications in the proposition of new potential anti-HSV-1 agents, but maintaining the anti-HIV-1 activity. Among the docked compounds, the oxoquinoline-acylhydrazone 3 is the best candidate for an anti-HSV-1 agent, and, in addition, it showed anti-HIV-1 activity (EC₅₀ = 3.4 ± 0.3 μM). Compounds 2 and 3 were used as templates in the design of four new oxoquinoline-acylhydrazones (6-9) as potential anti-HSV-1 agents to increase the antiviral activity of 2. Among the docked compounds, oxoquinoline-acylhydrazone 7 was selected as the best candidate for further development of dual anti-HIV/HSV activity.

Full text of DOI:10.1016/j.molstruc.2014.05.081

Accepted Manuscript
Docking of anti-HIV-1 Oxoquinoline-Acylhydrazone Derivatives as Potential
HSV-1 DNA Polymerase Inhibitors
Julliane Diniz Yoneda, Magaly Girão Albuquerque, Kátia Zaccur Leal,
Fernanda da Costa Santos, Pedro Netto Batalha, Leonardo Brozeguinnia, Peter
R. Seidl, Ricardo Bicca de Alencastro, Anna Cláudia Cunha, Maria Cecília B.V.
de Souza, Vitor F. Ferreira, Viveca A. Giongo, Cláudio Cirne-Santos, Izabel
C.P. Paixão
PII:
S0022-2860(14)00595-X
DOI:
http://dx.doi.org/10.1016/j.molstruc.2014.05.081
MOLSTR 20680
Reference:
Journal of Molecular Structure
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 February 2014
30 May 2014
30 May 2014
Please cite this article as: J.D. Yoneda, M.G. Albuquerque, K.Z. Leal, F.d.C. Santos, P.N. Batalha, L. Brozeguinnia,
P.R. Seidl, R.B.d. Alencastro, A.C. Cunha, M.B.V. de Souza, V.F. Ferreira, V.A. Giongo, C. Cirne-Santos, I.C.P.
Paixão, Docking of anti-HIV-1 Oxoquinoline-Acylhydrazone Derivatives as Potential HSV-1 DNA Polymerase
Inhibitors, Journal of Molecular Structure (2014), doi: http://dx.doi.org/10.1016/j.molstruc.2014.05.081
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Docking of anti-HIV-1 Oxoquinoline-Acylhydrazone Derivatives
as Potential HSV-1 DNA Polymerase Inhibitors
Julliane Diniz Yoneda a,b*, Magaly Girão Albuquerque c*, Kátia Zaccur Leal a,d, Fernanda da Costa Santos a,e, Pedro
Netto Batalha a, Leonardo Brozeguinnia a, Peter R. Seidl f, Ricardo Bicca de Alencastro c, Anna Cláudia Cunha a,e
,
Maria Cecília B. V. de Souza a,e, Vitor F. Ferreira a,e, Viveca A. Giongo g, Cláudio Cirne-Santos g, Izabel C.P. Paixão g
a Programa de Pós-Graduação em Química, Instituto de Química, Universidade Federal Fluminense, Niterói, RJ, Brazil.
b
Departamento de Química, Instituto de Ciências Exatas, Universidade Federal Fluminense, Pólo Universitário de
Volta Redonda, Volta Redonda, RJ, Brazil. (jullianeyoneda@id.uff.br)
c
Departamento de Química Orgânica, Instituto de Química, Universidade Federal do Rio de Janeiro, Centro de
Ciências Matemáticas e da Natureza, Rio de Janeiro, RJ, Brazil. (magaly@iq.ufrj.br, bicca@iq.ufrj.br)
d
Departamento de Físico-Química, Instituto de Química, Universidade Federal Fluminense, Niterói, RJ, Brazil.
(kzleal@gmail.com)
e Departamento de Química Orgânica, Instituto de Química, Universidade Federal Fluminense, Niterói, RJ, Brazil.
(fernand@vm.uff.br, annac@vm.uff.br, mceciliabvs@gmail.com, cegvito@vm.uff.br)
f
Departamento de Processos Orgânicos, Escola de Química, Universidade Federal do Rio de Janeiro, Centro de
Tecnologia, Rio de Janeiro, RJ, Brazil. (pseidl@eq.ufrj.br)
g
Programa de Pós-graduação em Biologia das Interações/ Programa de Pós-graduação em Neurociências, Instituto de
Biologia, Universidade Federal Fluminense, Rio de Janeiro, RJ, Brazil. (ipaixao@vm.uff.br, vgiongo@gmail.com,
claudiocirne@gmail.com)
* Corresponding author: Departamento de Química Orgânica, Instituto de Química, Centro de Ciências Matemáticas
e da Natureza, Universidade Federal do Rio de Janeiro. Centro de Tecnologia, Bloco A, Sala 609, Ilha do Fundão,
Cidade Universitária, 21949-900, Rio de Janeiro, RJ, Brazil. Phone/Fax: (+55) 21-3938-7132. E-mail:
magaly@iq.ufrj.br; jullianeyoneda@id.uff.br

ABSTRACT
Although there are many antiviral drugs available for the treatment of herpes simplex virus (HSV) infections, still the
synthesis of new anti-HSV candidates is an important strategy to be pursued, due to the emergency of resistant HSV
strains mainly in human immunodeficiency virus (HIV) co-infected patients. Some 1,4-dihydro-4-oxoquinolines, such
as PNU-183792 (1), show a broad spectrum antiviral activity against human herpes viruses, inhibiting the viral DNA
polymerase (POL) without affecting the human POLs. Thus, on an ongoing antiviral research project, our group has
synthesized ribonucleosides containing the 1,4-dihydro-4-oxoquinoline (quinolone) heterocyclic moiety, such as the 6-
Cl derivative (2), which is a dual antiviral agent (HSV-1 and HIV-1). Molecular dynamics simulations of the complexes
of 1 and 2 with the HSV-1 POL suggest that structural modifications of 2 should increase its experimental anti-HSV-1
activity, since its ribosyl and carboxyl groups are highly hydrophilic to interact with a hydrophobic pocket of this
enzyme. Therefore, in this work, comparative molecular docking simulations of 1, 2 and three new synthesized
oxoquinoline-acylhydrazone HIV-1 inhibitors (3-5), which do not contain those hydrophilic groups, were carried out, in
order to access these modifications in the proposition of new potential anti-HSV-1 agents, but maintaining the anti-
HIV-1 activity. Among the docked compounds, the oxoquinoline-acylhydrazone 3 is the best candidate for an anti-
HSV-1 agent, and, in addition, it showed anti-HIV-1 activity (EC50 = 3.4 0.3 μM). Compounds 2 and 3 were used as
templates in the design of four new oxoquinoline-acylhydrazones (6-9) as potential anti-HSV-1 agents to increase the
antiviral activity of 2. Among the docked compounds, oxoquinoline-acylhydrazone 7 was selected as the best candidate
for further development of dual anti-HIV/HSV activity.
Keywords: acylhydrazone; dual antiviral agents; docking; HIV-1; HSV-1; quinolone; molecular modeling.

1. INTRODUCTION
In humans, the herpes simplex virus (HSV) causes mucocutaneous lesions, which are uncomfortable to the
patient and may represent a life risk for immunocompromised individuals [1]. This virus exists in two forms: HSV-1
(associated to the oral disease) and HSV-2 (associated to the genital disease). At present, there is no cure for their
effects, only treatments which can accelerate the scarring process of the formed vesicles or diminish the period between
two consecutive disease manifestations.
HSV infections were one of the first diseases to be treated by means of antiviral agents. Among them, analogs
of the natural nucleosides (e.g., guanosine) were introduced in the antiviral therapy, in 1960, targeting the viral DNA
polymerase (POL), since it is a key enzyme in the viral replication cycle [2]. An alternative target for anti-herpes viral
therapy is the viral helicase/primase complex [3], but in this work we will focus on the POL inhibitors. Guanosine
analogs, such as aciclovir (ACV), ganciclovir, and penciclovir, act as viral POL inhibitors, in a competitive manner [3].
In fact, these nucleoside analogs are pro-drugs, which must be phosphorylated to the monophosphate form first by viral
kinases, and then further phosphorylated to the triphosphate form, by cellular kinases before they become active
inhibitors [4].
On the other hand, foscarnet (a pyrophosphate analogue), an antiviral drug available for the treatment of HSV
and HIV (human immunodeficiency virus) resistant infections, directly inhibits POL by binding to a site on the HSV
POL (EC: 2.7.7.7) or HIV reverse transcriptase (RT) (HIV RT has a POL domain, EC: 2.7.7.49) [5], and it is not
dependent on activation (phosphorylation) by viral or cellular kinases [6]. Interesting, computational studies comparing
the 3D structures of the polymerase domains of Escherichia coli POL I and HIV-1 RT, in spite of low sequence
similarity, showed conserved residues that may represent a similar structure-function feature in all polymerases [7].
Moreover, considerable progress has been made in the development of potential drugs with dual (or multiple) antiviral
activities, such as agents targeting HIV/HSV co-infection [8-12].
One of the limitations of the antiviral drugs available for the herpes treatment today is that they are active
against only some of the eight human herpes viruses (HHV). However, some 1,4-dihydro-4-oxoquinolines, like PNU-
183792 (1, Figure 1), inhibit six of the eight HHVs, showing a broad spectrum antiviral activity profile against human
herpes viruses [13]. In addition, these non-nucleoside inhibitors [13-16] are selective because they inhibit the viral
POLs without affecting the human POLs [4].
The broad spectrum antiviral activity of PNU-183792-like compounds can be explained by the fact that they
seem to interact with a region within the domain III (residues 805-845) of the viral POL, which is extremely conserved
in many herpes viruses [4], and they do not need phosphorylation by viral kinases [17]. Besides, they are also active
against viruses resistant to ACV [13].
Although there are many antiviral drugs available for the treatment of HSV infections, the synthesis of new
anti-HSV candidates is still an important strategy to be pursued [18, 19], due to the emergency of resistant HSV strains
[20] mainly in HIV/HSV co-infected patients [21]. Thus, our group has synthesized ribonucleosides containing the 1,4-
dihydro-4-oxoquinoline heterocyclic moiety, such as the 6-Cl substituted ribonucleoside (2, Figure 1), as new anti-
HSV-1 candidates [17,22].

O
N
O
O
O
Cl
OH
N
H
N
O
N
Cl
HO
O
CH
2
( )
3
1
( ) PNU-183792
OH
OH
O
O
R
N
Cl
N
H
N
NO
2
O
N
H
R =
3
( )
4
( )
5
( )
Figure 1. Chemical structures of PNU-183792 (1), [14] the 6-Cl substituted ribonucleoside (2), [17,22] and the
oxoquinoline-acylhydrazones 3, 4, and 5 (in blue, the common 1,4-dihydro-4-oxoquinoline heterocyclic moiety).
The 6-Cl substituted ribonucleoside (2) inhibits HSV-1 replication, has low citotoxicity and high selective
index [17,22]. It inhibits the HSV-1 POL activity but, differently from ACV, according to an uncompetitive mechanism,
i.e., the inhibitor binds to the substrate (DNA) and enzyme (POL) complex [17]. Both the 6-Cl substituted
ribonucleoside (2) and its aglycone moiety, which was also tested, inhibit the HSV-1 POL activity, showing that the
main subunit responsible for the inhibition is the 6-Cl-1,4-dihydro-4-oxoquinoline ring (colored in blue in Figure 1)
[17]. This result is in line with the previously mentioned studies of Wathen for the 1,4-dihydro-4-oxoquinolines [13].
However, the design and development of new drugs is a multistage and very expensive process, which must be
properly planned and controlled [23]. In this context, molecular modeling is a useful tool for the analysis of the
molecular details of compounds involved in biological processes, explaining the structure-activity relationship of
molecules candidates to drug prototypes. Interesting docking and molecular dynamics studies have been developed
focusing in a natural product targeting the HSV-1 DNA POL [24], but not including the substrate structure.
We have recently demonstrated that some structural modifications should increase the anti-HSV-1 activity of
the ribonucleoside derivative mentioned earlier [25]. Besides, PNU-183792 is more active than 2, according to
experimental data from literature [7,22] (Table 1). Therefore, in this work, we use molecular modeling simulations,
such as molecular docking, in order to rationalize these modifications and help our synthesis group to plan new
potential anti-HSV-1 agents with anti-HIV activity.
Table 1. Anti-HSV-1 activity for compounds 1 [14] and 2 [22] compared to ACV.
#
1
EC50 (µM)
3.3
#
2
EC50 (µM)
1.5
ACV
3.9
ACV
1.25
The present study is based on the model proposed by Liu and co-workers [26]. According to this model, PNU-
183792 (1, Figure 1), a compound which also contains a 1,4-dihydro-4-oxoquinoline heterocyclic moiety, binds to the
double-stranded DNA (dsDNA) and POL complex, but does not bind to POL or to dsDNA alone [26]. This is in
according to the mechanism of action proposed for the 6-Cl substituted ribonucleoside inhibitor (2, Figure 1). In
contrast to other nucleoside inhibitors, such as ACV, and like PNU-183792, 2 is not dependent on viral or cellular
phosphorylation events to generate an active form and does not form covalent complexes with the HSV POL [17].

In the Liu’s model, PNU-183792 replaces the incoming nucleotide and dislocates the template base from the
active site [26]. The oxoquinoline ring of PNU-183792 stacks against the primer-3’-end and the template, and also
interacts with the active site residues of the HSV POL [26]. The p-Cl-phenyl group binds to a hydrophobic pocket lined
by residues Phe820, Gln617, Gln618, and Val823 [26].
Our group has synthesized the oxoquinoline-acylhydrazones 3, 4, and 5 (Figure 1), which are active against
HIV-1 in peripheral blood mononuclear cells (PBMC), according to the results in Section 3. Other acylhydrazones are
reported as HIV-1 inhibitors, such as BBNH that inhibits both the RT POL and the RNaseH activities with similar
potency (IC50 ≈ 3 µM) [27]. In addition, the 6-Cl substituted ribonucleoside (2) is a dual antiviral agent, since it also
inhibits HIV-1 replication targeting the RT enzyme [28].
Possibly, the inhibition mechanism of the oxoquinoline-acylhydrazones (3-5) is similar to that of the 6-Cl
substituted ribonucleoside (2) and PNU-183792 (1) because the active aglycone moiety of 2 is common to all of them
(Figure 1). Because the oxoquinoline-acylhydrazones (3-5), which shows activity against HIV-1, are structurally related
to PNU-183792 (anti-HSV-1) and to the 6-Cl substituted ribonucleoside (2) (dual antiviral activity, anti-HSV-1 [17]
and anti-HIV-1 [28]), these molecules are being used as a starting point for the proposition of new structures targeting
the HSV POL enzyme, but maintaining the anti-HIV-1 activity.
2. MATERIALS AND METHODS
2.1. Molecular Modeling
The structure of the HSV-1 POL was obtained from the Protein Data Bank [29] (PDB ID: 2GV9, chain B)
[26]. Since some residues near to domain III are missing, this structure was reconstructed by comparative modeling,
using the automated module of the Swiss-Model server [30]. The dsDNA-POL complex was created in the Swiss Pdb-
Viewer program [31]. The 2GV9 structure, reconstructed by comparative modeling, was superposed to a theoretical
model of the POL (PDB ID: 1B1F) [32].
The docking simulations were carried out using the residue module of the Surflex-Dock v.2.2 program [33,34].
The active site was generated selecting important residues from region III (i.e., Gln617, Gln618, Lys811, Asn815,
Tyr818, Phe820, Val823, and Asp888), according to the Liu’s model [26], with a radii of 3 Å related to them; from the
template residues T6-T10; and from the primer residues P19-21. This selection gave better results, leading to a
conformation and orientation for PNU-183792, used as a reference compound, closer to that proposed in the Liu’s
model.
Before docking, the structures of all the studied compounds, obtained by systematic conformational search in
the Spartan program (Wavefunction, Inc.), were optimized using a B3LYP density functional with a 6-31G(d,p) basis
set in the Gaussian 98 package [35]. Charges were calculated by fitting the electrostatic potential to a grid of points
selected, according to the CHELPG scheme [36].
The docking studies were performed testing all tools available in the Surflex-Dock program to generate the
binding mode according to the Liu’s model, in which the residue-based approach showed the best performance [33,34].
In the case of PNU-183792, from the twenty structures generated by the docking output, we selected the one with the
closest binding mode (conformation and orientation) to that proposed for this ligand in the Liu’s model [26]. In the case
of the other compounds, we selected those with the closest binding mode to the one selected for PNU-183792. For each
selected pose, the ligand structure was optimized inside the active site of the protein, using the Surflex-Dock program.
The partial atomic charges were recalculated according to the CHELPG scheme, using the Gaussian 98 program.
The final ligand-dsDNA-POL complexes obtained were submitted to the LPC/CSU server
(http://ligin.weizmann.ac.il/cgi-bin/lpccsu/LpcCsu.cgi) [37] for automatic interactions analysis. The WebLab

ViewerLite program (Molecular Simulations, Inc.) was used for structure visualization.
In addition, the 2D structure of the compounds were analyzed on the OSIRIS Property Explorer program
(Thomas Sander, Actelion Pharmaceuticals Ltd.; http://www.organic-chemistry.org/prog/peo/) in order to calculate the
fragment-based drug-likeness score, which is based on the occurrence frequency of each fragment in the individual
structure that are associated with drug-like (3300 traded drugs) and non-drug-like (15000 commercially available Fluka
chemicals) databases.
2.2. Synthesis
The 3-acylhydrazone-oxoquinolines 3, 4, and 5 were synthesized as previously described for our research
group [38]. These analogues were prepared by reaction between the carbohydrazide and suitable aromatic aldehydes in
ethanol, using hydrochloric acid as catalyst. The 5-nitrofuran derivative 5 was obtained by the condensation with
commercially available (5-nitrofuran-2-yl)methylene diacetate in a mixture of ethanol and sulfuric acid 50%.
(3) (E)-4-oxo-N'-(4-chlorobenzylidene)-1,4-dihydroquinoline-3-carbohydrazide:
1
White solid. Yield 95%. Mp > 300ºC; H-NMR (500.00 MHz, DMSO-d6) δ 13.32 (s, 1H, CONHN), 8.85 (s,
1H, H-2), 8.46 (s, 1H, H-1’), 8.31 (dd, 1H, J= 8.3; 1.1 Hz, H-5), 7.81 (ddd, 1H, J= 8.3; 8.3; 1.1 Hz, H-7), 7.78 (d, 2H,
J= 8.3 Hz, H-4’ and H-6’), 7.75 (d, 1H, J= 8.3 Hz, H-8), 7.54 (dd, 1H, J= 8.3; 8.3 Hz, H-6), 7.52 (d, 2H, J= 8.3 Hz, H-3’
and H-7’); 13C-NMR (125.0 MHz, DMSO-d6) δ 175.8 (C-4), 161.2 (C=ONHN), 146.1 (C-1’), 144.0 (C-2), 138.9 (C-
8a), 134.2 (C-5’), 133.2 (C-2’), 132.7 (C-7), 128.6 (C-3’ and C-7’ or C-4’ and C-6’), 128.5 (C-3’ and C-7’ or C-4’ and
C-6’), 125.7 (C-4a), 125.2 (C-5), 125.0 (C-6), 119.0 (C-8), 109.8 (C-3), Mass calcd. for C17H12ClN3O2 325.0618; MS
(ESI+) m/z found 326.0687 [M+H+].
(4) (E)-4-oxo-N'-(pyrid-4’-ylmethylene)-1,4-dihydroquinoline-3-carbohydrazide:
Yellow solid. Yield 90%. Mp > 300ºC; 1H-NMR (500.00 MHz, DMSO-d6) δ 13.49 (s, 1H, CONHN), 8.87 (s,
1H, H-2), 8.67 (d, 2H, J= 6,0 Hz, H-4’ and H-5’), 8.48 (s, 1H, H-1’), 8.31 (dd, 1H, J= 8.2; 1.1 Hz, H-5), 7.81 (ddd, 1H,
J= 8.2; 8.2; 1.1 Hz, H-7), 7.76 (d, 1H, J= 8.2 Hz, H-8), 7.68 (d, 2H, J= 6.0 Hz, H-3’and H-6’), 7.54 (ddd, 1H, J= 8.2;
8.2; 1.1 Hz, H-6); 13C-NMR (125.0 MHz, DMSO-d6) δ 175.9 (C-4), 161.6 (C=ONHN), 150.0 (C-4’ and C-5’), 145.2
(C-1’), 144.4 (C-2), 141.5 (C-2’), 138.9 (C-8a), 132.9 (C-7), 125.8 (C-4a), 125.3 (C-5 and C-6), 120.9 (C-3’ and C-6’),
119.1 (C-8), 109.7 (C-3); Mass calcd. for C16H12N4O2 292.0960; MS (ESI+) m/z found 293.1035 [M+H+].
(5) (E)-4-oxo-N'-[(5-nitrofuran-2-yl)methylene]-1,4-dihydroquinoline-3-carbohydrazide:
Yellow solid. Yield 84%. Mp > 300ºC; 1H-NMR (500.00 MHz, DMSO-d6) δ 13.50 (s, 1H, CONHN), 12.96 (d,
1H, J= 6.6 Hz, H-1), 8.86 (d, 1H, J= 6.6 Hz, H-2), 8.51 (s, 1H, H-1’), 8.31 (dd, 1H, J= 7.2; 1.1 Hz, H-5), 7.81 (ddd, 1H,
J= 8.3; 7.2; 1.1 Hz, H-7), 7.76 (d, 1H, J= 3.8 Hz, H-4’), 7.75 (dd, 1H, J= 7.2; 1.1 Hz, H-8), 7.54 (ddd, 1H, J= 8.3; 7.2;
1.1 Hz, H-6), 7.17 (d, 1H, J= 3.8 Hz, H-3’); 13C-NMR (125.0 MHz, DMSO-d6) δ 175.8 (C-4), 161.7 (C=ONHN), 151.8
(C-2’ and C-5’), 144.4 (C-2), 138.8 (C-8a), 135.5 (C-1’), 132.9 (C-7), 125.7 (C-4a), 125.2 (C-5 and C-6), 119.0 (C-8),
114.3 (C-4’), 114.9 (C-3’), 109.5 (C-3), Mass calcd. for C15H10N4O5 326.0651; MS (ESI+) m/z found 327.0718
[M+H+].
2.3. Biological Tests
2.3.1. Cells and virus
Peripheral blood mononuclear cells (PBMCs) from healthy human donors were obtained by density gradient
centrifugation (Histopaque; Sigma, St. Louis, USA) from buffy coat preparations. PBMCs were resuspended in RPMI
1640 (LGC Bio, São Paulo, Brazil) supplemented with 10% FBS (Hyclone, Logan, USA), penicillin and streptomycin
(CultiLab, São Paulo, Brazil), 2 mM glutamine and 10 mM HEPES (Sigma, St. Louis, USA) and stimulated with 5
mg/mL of phytohemagglutinin (PHA; Sigma, St. Louis, USA) for two to three days. They were further maintained in

culture medium containing 5 U/mL of recombinant human interleukin-2 (Sigma, St. Louis, USA). HIV-1 isolate Ba-L
(R5-tropic, subtype B) [39] was used to infect cells. Virus isolates were prepared in PHA-activated PBMCs from
healthy human donors.
2.3.2. Screening of in vitro anti-HIV-1 activity
This assay followed the procedures previously described [39] with minor modifications. Peripheral blood
mononuclear cells (PBMCs) were initially exposed to viral suspensions containing 5-10 ng/mL of (Ba-L/R2) HIV-1
p24 Ag during 2-3 h. Cells were washed, resuspended in complete medium, plated in 96-well culture plates (2.0 x 105
cells/well) in triplicate, and treated with different concentrations (µM) of compounds. After seven days at 37°C in 5%
CO2, viral replication was assessed by measuring the HIV-1 p24 Ag presence in culture supernatants by an ELISA
capture assay (ZeptoMetrix Co., Buffalo, USA).
3. RESULTS AND DISCUSSION
The results for activity against HIV-1 in PBMC for the synthesized oxoquinoline-acylhydrazones 3, 4, and 5
(Figure 1), according to the biological tests described in Section 2.3, are shown in Table 2. The p-Cl-phenyl is the most
potent (3, EC50 = 3.5 0.3 μM), followed by the 5-nitro-2-furanyl (5, EC50 = 56 4.1 μM) and the 4-pyridyl (4, EC50
100 5.5 μM).
=
Table 2. Results of in vitro antiviral activity against HIV-1 in peripheral blood mononuclear cells (PBMC) for the
oxoquinoline-acylhydrazones 3, 4, and 5 (structures in Figure 1).
#
3
4
5
% of inhibition at 50 µM
99 2.1
EC50 (µM)
3.4 0.3
100 5.5
56 4.1
10 1.1
40 1.8
In order to evaluate the potential binding mode of 3, 4, and 5 on the active site of the HSV-1 POL in complex
with the dsDNA substrate, compounds 3, 4, and 5 along with 1 (PNU-183792) were docked into the dsDNA-POL
complex, using the Surflex-Dock program. The best poses (Figure 2) are those in which the compounds show a binding
mode (conformation and orientation) more similar to that proposed for PNU-183792 in the Liu’s model, where the p-
Cl-phenyl group of 1 points in the direction of Val823, a very important residue for the activity of compounds like
PNU-183792 with the HSV-1 POL [4,25].
Figure 3 shows a close view of the superimposition of the docking output structures of compounds 3, 4, and 5
to PNU-183792 (1). According to Figure 3, compound 3 is found in an overall spatial arrangement more similar to that
of PNU-183792 (including the p-Cl-phenyl group of both, 1 and 3) than 4 or 5, indicating that its anti-HSV-1 activity
must be tested.
The ligand-protein interaction analysis of compounds 3, 4, 5, and PNU-183792 (1), performed with the LPC
server, showed that although the three compounds have thirteen interactions in common to that presented by PNU-
183792, compound 3 is the one showing interactions with Thr887 (Table 3, Figure 4), near to Asp888 (Figure 4),
considered an important residue from region III, according to the Liu’s model [26]. Figure 4 shows compound 3 and
PNU-183792 (1) in the active site with the morpholinyl ring of PNU-183792 (1) posed in accord to the Liu’s model.

Figure 2. Best docking poses for compounds 3 (green), 4 (magenta), 5 (blue), and PNU-183792 (1) (yellow) close to
Val823 (orange) into the HSV-1 DNA polymerase in complex with the substrate (dsDNA).
Figure 3. Superimposition of the docking output structures of compounds 3, 4, and 5 (gray) to PNU-183792 (1) (white).

Table 3. Interactions of compounds 3, 4, 5, and PNU-183792 (1) with residues of HSV-1 DNA polymerase in complex
with dsDNA. The protein residues considered most important by the Liu’s model are in bold (2006), while P20 and P21
are the primer residues from DNA.
Residue ID
Ile504
3
4
5
1
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Gly616
Gln617
Gln618
Ile619
X
X
Gln640
Ser720
Pro723
Lys811
Val812
Cys814
Asn815
Ser816
Tyr818
Gly819
Phe820
Val823
His825
Gly826
Thr887
P20
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
P21
Common to 1
13 13 13

Figure 4. PNU-183792 (1) (white), compound 3 (gray), and some of the most important residues in the Liu’s model
(Asn815 and Thr887) and other residues close by (Lys811, Tyr818, and Asp888).
The docking results for compounds 1, 3, 4, and 5 were used to propose four new oxoquinoline-acylhydrazone
derivatives (6-9, Figure 5), using the concepts of structural simplification, molecular hybridization, and increasing
conformational flexibility, considering compounds 2 (anti-HIV-1 and anti-HSV-1) and 3 (anti-HIV-1) as the reference
structures, in order to increase interactions of the new proposed compounds with residues (from the enzyme and/or the
primer/template DNA) in the POL active site.
All the proposed structures (6-9) will keep a chlorine atom at position 6 because they result from structural
modifications earlier proposed for the 6-Cl substituted ribonucleoside (2). Structural simplification will be achieved by
replacing the glycoside group of 2 (because it is highly polar, hydrophilic and it is not essential for anti-HSV-1 or anti-
POL activities) by hydrogen (6) or alkyl groups (methyl, 7; ethyl, 8). Molecular hybridization will be achieved by
merging the aglycone and p-Cl-benzylidene-hydrazine groups of 2 and 3, respectively, in all proposed structures (6-9).
The conformational flexibility of 7 (R = Me) (this compound has already been synthesized [38]) will be increased by
reduction of the N=C bond in the design of 9. Maybe, the free bond rotation of 9 could lead to a conformation and
orientation more similar to that of PNU-183792 (1).

Cl
O
N
O
O
O
Cl
N
OH
N
H
N
H
HO
group not
essential for
inhibitory
3
( ) anti-HIV-1
O
(EC50 = 3.4 M)
Structural
μ
simplification
activity
(HSV-1 and
polymerase)
and
OH
OH
Molecular
hybridization
2
( ) anti-HIV-1 (RT) and
anti-HSV-1 (DNA POL)
Cl
O
O
EC50 = 1.5 M (HSV-1)
μ
Cl
N
K = 0.1 M (DNA POL)
μ
i
N
H
Increasing
conformational
flexibility
N
6
7
8
R = H ( ), Me ( ), Et ( )
R
O
Cl
O
H
N
Cl
N
H
N
R
9
R = Me ( )
Figure 5. Structural modifications proposed for the 6-Cl substituted ribonucleoside (2) and compound 3 in the design of
the new four oxoquinoline-acylhydrazone derivatives 6 (R=H), 7 (R=Me), 8 (R=Et), and 9 (R=Me).
Figure 6. Drug-likeness scores (OSIRIS Property Explorer) for PNU-183792 (1), 6-Cl substituted ribonucleoside (2),
oxoquinoline-acylhydrazones (3, 4, and 5) and the new proposed oxoquinoline-acylhydrazones (6, 7, 8, and 9).

The fragment based drug-likeness profile of all compounds (1-9) performed on the OSIRIS Property Explorer
program is shown in Figure 6. Among the synthesized oxoquinoline-acylhydrazones (3-5), only 3 (the most potent
compound against HIV-1, Table 2) has a drug-likeness score higher than that of PNU-183792 (1) (Figure 6). Among the
new proposed oxoquinoline-acylhydrazones (6-9), 7 and 8 showed the greatest drug-likeness, even greater than PNU-
183792 (1) and 3, indicating that they are good drug candidates. The 6-Cl substituted ribonucleoside (2) was the only
compound that showed a negative drug-likeness score (Figure 6), indicating that, in fact, the proposed structural
modifications are interesting in order to turn it into a better drug candidate.
The docking poses for the new proposed oxoquinoline-acylhydrazones 6, 7, 8, and 9 with dsDNA-POL
superposed to PNU-183792 (1) are shown in Figure 7. Proposed derivatives 6 (R=H) and 7 (R=Me) had a spatial
arrangement closer to that obtained in the docking result of PNU-183792, while 8 (R=Et) had its orientation completely
inverted, probably because the replacement of the methyl by ethyl group imposed a steric hindrance. Compound 9 (the
reduced analog of 7, R=Me) showed two possible poses, probably due to its higher conformational freedom: one with
its methyl group superposed on the morpholinyl ring of PNU-183792 (pose 9a) and the other with its methyl group
superposed on the methyl group of PNU-183792 (pose 9b).
Figure 7. Docking results for the new proposed oxoquinoline-acylhydrazones (6, 7, 8, and 9, in gray) with HSV-1 DNA
polymerase superposed to PNU-183792 (1, in white).

The analysis of the interactions between the docking output structures and the POL (Table 4) show that the
compounds 6 and 7 are the ones for which interactions as that of PNU-183792 (1) increased the most. They also keep
the same interactions made by compound 3. Compound 6 gained five interactions (compared to compound 3) with POL
residues, two of which are common to PNU-183792, whereas compound 7 gained four interactions, three of which are
common to PNU-183792. As mentioned before, compound 8 shows a completely inverted orientation compared to
PNU-183792, while compound 9 (poses a and b) shows a superimposition to PNU-183792 not so good as those of
compounds 6 and 7 (Figure 7).
Table 4. Interactions of the new proposed oxoquinoline-acylhydrazones (6, 7, 8 and 9) and PNU-183792 (1) with
residues of HSV-1 DNA polymerase in complex with dsDNA (T6 is a template and P18, P20, and P21 are primer
residues from DNA; X* and *** represent interactions gained and lost, respectively, compared to compound 3).
Residue ID
6
7
8
9a 9b
1
Ile504
Lys534
Gly616
Gln617
Gln618
Ile619
Gln640
Ser720
Tyr722
Pro723
Lys811
Val812
Cys814
Asn815
Ser816
Tyr818
Gly819
Phe820
Val823
His825
Gly826
Leu827
Thr887
T6
X
X
X
X
X
X
X*
X* X* X*
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
***
X
***
X
X* X*
X*
X
X
X
X
X
X
X
X
X
X
X
X* X*
X* X*
X*
X*
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
***
X
X
X
X
X
X
X
X
X
X
X
X
X
X
***
***
X
X
X
X
***
X*
X
X
X
X
*** X
X*
P18
X*
X
P20
X
X
X
X
X
X
X
X
X
X
P21
X
Common to 1 15 16 13 14 14

Accordingly, we are proposing the synthesis of the new proposed oxoquinoline-acylhydrazones 7 (Figure 5),
which has a high drug-likeness score (Figure 6) and shows interaction with residues Gly616, Tyr722, Pro723, and
Cys814. These interactions are important because they are maintained by PNU-183792 (1) during 8 ns of molecular
dynamics simulations [25]. Besides, the methyl group in 7 makes it more interesting than structure 6, due to a “push-
pull” effect which seems to be important in the molecular dynamics simulation studies [25].
4. CONCLUSIONS
According to our docking studies, oxoquinoline-acylhydrazone 3, the most potent compound against HIV-1
(EC50 = 3.4 0.3 μM), is a good candidate to be an anti-HSV-1 agent. In order to increase the anti-HSV-1 activity of
the 6-Cl substituted ribonucleoside 2, but maintaining its anti-HIV-1 activity, we propose four new oxoquinoline-
acylhydrazones (6-9) as good candidates to anti-HSV-1 agents. Among them, 7 was selected as the best candidate to
synthesis and further development of dual anti-HIV/HSV agents.
ACKNOWLEDGEMENTS
JDY held a graduate fellowship from CAPES and FCS from FAPERJ. RBA has a fellowship from FAPERJ.
PRS, MGA, MCBVS, RBA and VFF have research fellowships from the CNPq. We would also like to thank FAPERJ
for funding support.
REFERENCES
[1] S.N. Travassos, C.M.A.F. Nogueira, N.C.F. Wanke, Herpesvírus – Tratamentos, An. Bras. Dermatol. 63 (1988) 19-
23.
[2] E. de Clercq, Highlights in the Discovery of Antiviral Drugs: A Personal Retrospective, J. Med. Chem. 53 (2010)
1438-1450.
[3] S. K. Weller, R. D. Kuchta, The DNA Helicase-Primase Complex as a Target for Herpes Viral Infection, Expert
Opin. Ther. Targets. 17 (2013) 1119-1132.
[4] D.R. Thomsen, N.L. Oien, T.A. Hopkins, M.L. Knechtel, R.J. Brideau, M.W. Wathen, F.L. Homa, Amino-acid
Changes within Conserved Region III of the Herpes Simplex Virus and Human Cytomegalovirus DNA Polymerases
Confer Resistance to 4-Oxo-Dihydroquinolines, a Novel Class of Herpesvirus, Antiviral Agents, J. Virol. 77 (2003)
1868-1876.
[5] C.A. Shaw-Reid, B. Feuston, V. Munshi, K. Getty, J. Krueger, D.J. Hazuda, M.A. Parniak, M.D. Miller, D. Lewis,
Dissecting the Effects of DNA Polymerase and Ribonuclease H Inhibitor Combinations on HIV-1 Reverse
Transcriptase Activities, Biochemistry 44 (2005) 1595-1606.
[6] C.S. Crumpacker, Mechanism of Action of Foscarnet Against Viral Polymerases, Am. J. Med. 92 (1992) S3-S7
(Suppl. 1).
[7] P. N. S. Yadav, J. S. Yadav, E. Arnoldg, M. J. Modak, A Computer-Assisted Analysis of Conserved Residues in the
Three-dimensional Structures of the Polymerase Domains of Escherichia coli DNA Polymerase I and HIV-1 Reverse
Transcriptase, J. Biol. Chem. 269 (1994) 716-720.
[8] C.F. Perno, E. Balestra, S. Aquaro, S. Panti, A. Cenci, G. Lazzarino, B. Tavazzi, D. Di Pierro, J. Balzarini, R. Calio.
Potent Inhibition of Human Immunodeficiency Virus and Herpes Simplex Virus Type
1 by 9-(2-
Phosphonylmethoxyethyl)Adenine in Primary Macrophages is Determined by Drug Metabolism, Nucleotide Pools, and
Cytokines. Mol Pharmacol., 50 (1996) 359-66.
[9] L. Rossi, S. Serafini, L. Cappellacci, E. Balestra, G. Brandi, G.F. Schiavano, P. Franchetti, M. Grifantini, C.F.
Perno, M. Magnani. Erythrocyte-Mediated Delivery of a New Homodinucleotide Active Against Human

‘Immunodeficiency Virus and Herpes Simplex Virus. J Antimicrob Chemother., 47 (2001) 819-27.
[10] A.A. Abdel-Hafez, H.A. Elsherief, M. Jo, M. Kurokawa, K. Shiraki, T. Kawahata, T. Otake, N. Nakamura, M.
Hattori. Synthesis and Evaluation of Anti-HIV-1 and Anti-HSV-1 Activities of 4H-[1,2,4]-Triazolo[1,5-A]Pyrimidin-5-
One Derivatives. Arzneimittelforschung. 52 (2002) 833-9.
[11] J. Balzarini, G. Andrei, E. Balestra, D. Huskens, C. Vanpouille, A. Introini, S. Zicari, S. Liekens, R. Snoeck, A.
Holy, C.F. Perno, L. Margolis, D. Schols, A Multi-Targeted Drug Candidate with Dual Anti-HIV and Anti-HSV
Activity, PLoS Pathog. 7 (2013) e1003456.
[12] A.M. Chamoun-Emanuelli, M. Bobardt, B. Moncla, M.K. Mankowski, R.G. Ptak, P. Gallay, Z. Chen. Evaluation
of PD 404,182 as an Anti-HIV and Anti-Herpes Simplex Virus Microbicide. Antimicrob Agents Chemother. 58 (2014)
687-97.
[13] M.W. Wathen, Non-Nucleoside Inhibitors of Herpesviruses, Rev. Med. Virol. 12 (2002) 167-178.
[14] R.J. Brideau, M.L. Knechtel, A. Huang, V.A. Vaillancourt, E.E. Vera, N.L. Oien, T.A. Hopkins, J.L. Wieber, K.F.
Wilkinson, B.D. Rush, F.J. Schwende, M.W. Wathen, Broad-Spectrum Antiviral Activity of PNU-183792, a 4-Oxo-
Dihydroquinoline, Against Human and Animal Herpesviruses, Antivir. Res. 54 (2002) 19-28.
[15] N.L. Oien, R.J. Brideau, T.A. Hopkins, J.L. Wieber, M.L. Knechtel, J.A. Shelly, R.A. Anstadt, P.A. Wells, R.A.
Poorman, A. Huang, V.A. Vaillancourt, T.L. Clayton, T.A. Tucker, M.W. Wathen, Broad Spectrum Antiherpes
Activities of 4-Hydroxyquinoline Carboxamides, a Novel Class of Herpesvirus Polymerase Inhibitors, Antimicrob.
Agents Chemother. 46 (2002) 724-730.
[16] C.B. Hartline, E.A. Harden, S.L. Williams-Aziz, N.L. Kushner, R.J. Brideau, E.R. Kern, Inhibition of Herpesvirus
Replication by a Series of 4-Oxo-Dihydroquinolines with Viral Polymerase Activity, Antivir. Res. 65 (2005) 97-105.
[17] T.M.L. Souza, M.C.B.V. de Souza, V.F. Ferreira, C.V.B.S. Canuto, I.P. Marques, C.F.L. Fontes, I.C.P.P.
Frugulhetti, Inhibition of HSV-1 Replication and HSV DNA Polymerase by the Chloroxoquinolinic Ribonucleoside 6-
Chloro-1,4-Dihydro-4-Oxo-1-(β-D-Ribofuranosyl)Quinoline-3-Carboxylic Acid and its Aglycone, Antivir. Res. 77
(2008) 20-27.
[18] E. de Clercq, Antiviral Drugs: Current State of the Art, J. Clin. Virol. 22 (2001) 73-89.
[19] R. Narang, B. Narasimhan, S. Sharma, A Review on Biological Activities and Chemical Synthesis of Hydrazide
Derivatives, Curr. Med. Chem. 19 (2012) 596-612.
[20] F. Morfin, D. Thouvenot, Herpes Simplex Virus Resistance to Antiviral Drugs, J. Clin. Virol. , 26 (2003) 29-37.
[21] L. M. Bush, K. Talledo-Thais, A. Casal-Fernandez, M. T. Perez, Resistant Herpes Simplex Virus Infection and
HIV: A Potential Diagnostic and Therapeutic Dilemma, LabMedicine 42 (2011) 452-457.
[22] C.V.B.S. Canuto, C.R.B. Gomes, I.P. Marques, L.V. Faro, F.C. Santos, I.C.P.P. Frugulhetti, T.M.L. Souza, G.A.
Romeiro, V.F. Ferreira, M.C.B.V. Souza, Synthesis and Anti-HSV-1 Activity of 1,4-Dihydro-4-Oxoquinoline
Ribonucleosides, Lett. Drug Des. Discov. 4 (2007) 404-409.
[23] H. Kubinyi, QSAR: Hansch Analysis and Related Approaches, VCH, Weinheim, Germany, 1993.
[24] A. Grover, V. Agrawal, A. Shandilya, V. S. Bisaria, D. Sundar, Non-Nucleosidic Inhibition of Herpes Simplex
Virus DNA Polymerase: Mechanistic Insights into the Anti-Herpetic Mode of Action of Herbal Drug Withaferin A,
BMC Bioinformatics. 12 (2011) S22.
[25] J.D. Yoneda, M.G. Albuquerque, K.Z. Leal, P.R. Seidl, R.B. Alencastro, Molecular Modeling Studies of 1,4-
Dihydro-4-Oxoquinoline Ribonucleosides with Anti-HSV-1 Activity, J. Mol. Struct. 1006 (2011) 536-541.
[26] S. Liu, J.D. Knafels, J.S. Chang, G.A. Waszak, E.T. Baldwin, M.R. Deibel, D.R. Thomsen, F.L. Homa, P.A. Wells,
M.C. Tory, R.A. Poorman, H. Gao, X. Qiu, A.P. Seddon, Crystal Structure of Herpes Simplex 1 Virus DNA
Polymerase, J. Biol. Chem. 281 (2006) 18193-18200.

[27] N. Sluis-Cremer, D. Arion, M.A. Parniak, Destabilization of the HIV-1 Reverse Transcriptase Dimer upon
Interaction with N-Acyl Hydrazone Inhibitors, Mol. Pharmacol. 62 (2002) 398-405.
[28] T.M.L. Souza, C.C. Cirne-Santos, D.Q. Rodrigues, C.M. Abreu, A. Tanuri, V.F. Ferreira, I.P. Marques, M.C.B.V.
de Souza, C.F.L. Fontes, I.C.P.P. Frugulhetti, D.C. Bou-Habib, The Compound 6-Chloro-1,4-Dihydro-4-Oxo-1-(β-D-
Ribofuranosyl) Quinoline-3-Carboxylic Acid Inhibits HIV-1 Replication by Targeting the Enzyme Reverse
Transcriptase, Curr. HIV Res. 6 (2008) 209-217.
[29] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne, The
Protein Data Bank, Nucleic Acids Research. 28 (2000) 235-242.
[30] K. Arnold, L. Bordoli, J. Kopp, T. Schwede, The Swiss-Model Workspace: a Web-Based Environment for Protein
Structure Homology Modeling, Bioinformatics. 22 (2006) 195-201.
[31] N. Guex, M.C. Peitsch, Swiss-Model and the Swiss-PdbViewer: An Environment for Comparative Protein
Modeling, Electrophoresis. 18 (1997) 2714-2723.
[32] L. Huang, K.K. Ishii, H. Zuccola, A.M. Gehring, C.B.C. Hwang, J. Hogle, D.M. Coen, The Enzymological Basis
for Resistance of Herpesvirus DNA Polymerase Mutants to Acyclovir: Relationship to the Structure of Alpha-Like
DNA Polymerases, Proc. Natl. Acad. Sci. 96 (1999) 447-452.
[33] A.N. Jain, Surflex: Fully Automatic Flexible Molecular Docking Using a Molecular Similarity-Based Search
Engine, J. Med. Chem. 46 (2003) 499-511.
[34] A.N. Jain, Surflex-Dock 2.1: Robust Performance from Ligand Energetic Modeling, Ring Flexibility, and
Knowledge-Based Search, J. Comput-Aided Mol. Des. 21 (2007) 281-306.
[35] Frisch, M.J. et al. Gaussian 98. Gaussian, Inc., Pittsburg, PA, 1998.
[36] C.M. Breneman, K.B. Wiberg, Determining Atom-Centered Monopoles from Molecular Electrostatic Potentials,
The Need for High Sampling Density in Formamide Conformational Analysis, J. Comp. Chem. 11 (1990) 361-373.
[37] V. Sobolev, A. Sorokine, J. Prilusky, E.E. Abola, M. Edelman, Automated Analysis of Interatomic Contacts in
Proteins, Bioinformatics. 15 (1999) 327-332.
[38] F.C. Santos, H.C. Castro, M.C.S. Lourenço, P.A. Abreu, P.N. Batalha, A.C. Cunha, G.S.L. Carvalho, C.R.
Rodrigues, C.A. Medeiros, S.D. Souza, V.F. Ferreira, M.C.B.V. de Souza, Tuberculosis: Finding a New Potential
Antimycobacterium Derivative in a Aldehyde-Arylhydrazone-Oxoquinoline Series, Curr. Microbiol. 65 (2012) 455-
460.
[39] C.C. Cirne-Santos, T.M. Souza, V.L. Teixeira, C.F. Fontes, M.A. Rebello, L.R. Castello-Branco, C.M. Abreu, A.
Tanuri, I.C. Frugulhetti, D.C. Bou-Habib, The Dolabellane Diterpene Dolabelladienetriol is a Typical Noncompetitive
Inhibitor of HIV-1 Reverse Transcriptase Enzyme, Antivir. Res. 77 (2008) 64-71.

Graphical abstract
In this work, we use molecular modeling simulations, such as molecular docking, in order to rationalize structural modifications which
should increase the anti-HSV-1 activity of a ribonucleoside containing the 1,4-dihydro-4-oxoquinoline heterocyclic moiety.

Highlights
. Compound 3 is a good candidate to be an anti-HSV-1 agent.
. Four compounds were proposed as good candidates to anti-HSV-1 agents.
. Compound 7 was selected as the best candidate to synthesis and further studies.