Evaluation of Biological Activities of Quinone-4-oxoquinoline Derivatives Against Pathogens of Clinical Importance

Abstract

Background
Microbial resistance has become a worldwide public health problem, and may lead to morbidity and mortality in affected patients.

Objective
Therefore, this work aimed to evaluate the antibacterial activity of quinone-4-oxoquinoline derivatives.

Method
These derivatives were evaluated against Gram-positive and Gram-negative bacteria by their antibacterial activity, anti-biofilm, and hemolytic activities and by in silico assays.

Results
The quinone-4-oxoquinoline derivatives presented broad-spectrum antibacterial activities, and in some cases were more active than commercially available reference drugs. These compounds also inhibited bacterial adhesion and the assays revealed seven non-hemolytic derivatives. The derivatives seem to cause damage to the bacterial cell membrane and those containing the carboxyl group at the C-3 position of the 4-quinolonic nucleus were more active than those containing a carboxyethyl group.

Conclusion
The isoquinoline-5,8-dione nucleus also favored antimicrobial activity. The study showed that the target of the derivatives must be a non-conventional hydrophobic allosteric binding pocket on the DNA gyrase enzyme.

Full text

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Evaluation of Biological Activities of Quinone-4-oxoquinoline Derivatives
Against Pathogens of Clinical Importance
Francislene Juliana Martins1, Fernanda Savacini Sagrillo2, Rafaelle Josianne Vinturelle Medeiros1,
Alan Gonçalves de Souza2, Amanda Rodrigues Pinto Costa2, Juliana Silva Novais3,4, Leonardo
Alves Miceli5, Vinícius Campos2, Agnes Marie Sá Figueiredo6, Anna Claudia Cunha2, Natalia
Lidmar von Ranke5, Murilo Lamim Bello5, Bárbara Abrahim-Vieira5, Alessandra De Souza5,
Norman Ratcliffe7, Fernanda da Costa Santos Boechat2, Maria Cecília Bastos Vieira de Souza2,*,
Carlos Rangel Rodrigues5,*and Helena Carla Castro1,*
1Biology Institute, Postgraduate Program in Science and Biotechnology, Federal Fluminense University, Niterói, Rio
de Janeiro, Brazil; 2Department of Organic Chemistry, Chemistry Institute, Federal Fluminense University, Niterói,
Rio de Janeiro, Brazil; 3Medical School, Postgraduate Program in Pathology, Federal Fluminense University, Niterói,
Rio de Janeiro, Brazil; 4Faculty of Pharmacy, Estácio de Sá University (UNESA), São Gonçalo, Rio de Janeiro, Brazil;
5Department of Drugs and Medicines, Faculty of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro,
Brazil; 6 Department of Medical Microbiology, Paulo Goes Institute of Microbiology, Federal University of Rio de
Janeiro, Rio de Janeiro, Brazil; 7Department of Biosciences, College of Science Swansea University, SA2 8PP, UK
Abstract: Background: Microbial resistance has become a worldwide public health problem and
may lead to morbidity and mortality in affected patients.
Objective: Therefore, this work aimed to evaluate the antibacterial activity of quinone-4-
oxoquinoline derivatives.
Methods: These derivatives were evaluated against Gram-positive and Gram-negative bacteria by
their antibacterial activity, anti-biofilm, and hemolytic activities and in silico assays.
Results: The quinone-4-oxoquinoline derivatives presented broad-spectrum antibacterial activities
and, in some cases, were more active than commercially available reference drugs. These com-
pounds also inhibited bacterial adhesion, and the assays revealed seven non-hemolytic derivatives.
The derivatives seem to cause damage to the bacterial cell membrane, and those containing the car-
boxyl group at the C-3 position of the 4-quinolonic nucleus were more active than those containing
a carboxyethyl group.
Conclusion: The isoquinoline-5,8-dione nucleus also favored antimicrobial activity. The study
showed that the target of the derivatives must be a non-conventional hydrophobic allosteric binding
pocket on the DNA gyrase enzyme.
A R T I C L E H I S T O R Y
Received: December 16, 2021
Revised: March 02, 2022
Accepted: March 17, 2022
DOI:
10.2174/1568026622666220504124710
Keywords: Drug resistance, Antibacterial agents, Quinone derivatives, 4-Oxoquinolines, Gram-positive bacterial infections,
Gram-negative bacterial infections.
1. INTRODUCTION
At the beginning of the 20th century, bacterial infections
already represented about a third of the infectious diseases
*Address correspondence to these authors at the Department of Organic
Chemistry, Chemistry Institute, Federal Fluminense University, Niterói,
Rio de Janeiro, Brazil; E-mail: mceciliabvs@gmail.com (M.C.B.V. Souza);
Department of Drugs and Medicines, Faculty of Pharmacy, Federal
University of Rio de Janeiro, Rio de Janeiro, Brazil; E-mail: rangelfarma-
cia@gmail.com (C.R. Rodrigues); Biology Institute, Postgraduate Program
in Science and Biotechnology, Federal Fluminense University, Niterói, Rio
de Janeiro, Brazil; E-mail: hcastrorangel@yahoo.com.br (H.C. Castro)
that decimated the world population. The introduction of
antimicrobial agents, such as sulfonamides and penicillin,
and later synthetic compounds like oxoquinolines, repre-
sented a breakthrough in science. However, along with the
overuse of these antimicrobials, microorganisms emerged
capable of resisting pharmacological treatments [1-4].
The high incidence of antibiotic-resistant bacteria has
become a serious world health problem, especially in inten-
sive care units. This leads not only to high morbidity but
also mortality, with the latter reaching ca 69% for patients
infected by β-lactamase and carbapenemase-producing bac-

974 Current Topics in Medicinal Chemistry, 2022, Vol. 22, No. 12 Martins et al.
teria. The United States Centers for Disease Control and
Prevention estimates that around 23,000 annual deaths from
infections occur due to resistant microorganisms, and this
number may rise to millions in the coming decades [5-11].
Altogether, the irrational use of antimicrobials for hu-
man or veterinary treatment for disease control or as growth
promoters and their release into the environment, the in-
crease in numbers of immunocompromised individuals, and
the lack of hygiene and late diagnosis of infections are all
factors that contribute to the increase of bacterial resistance.
In addition, the low permeability of some drugs, such as the
beta-lactams and aminoglycosides, can reduce their effec-
tiveness against intracellular pathogens, allowing the devel-
opment of recurrent infections [12-18].
The emergence of multiresistant bacteria has led to the
search for new antimicrobials [19]. These include oxoquino-
lines that are used in clinical practice and can act on Gram-
positive and Gram-negative bacteria by interfering in the
activity of the enzymes, DNA Gyrase, and Topoisomerase
IV [20]. Due to their biological properties, these compounds
have been widely studied, showing that substitutions at the
C-6 and C-8 positions of the 4-oxoquinoline skeleton lead to
the development of novel derivatives, such as fluoro-4-
oxoquinolines [1, 7, 20, 21].
Another class of compounds with established biological
properties and potential as new drugs are naphthoquinones,
which are aromatic compounds with antibacterial, antifun-
gal, antiparasitic, and antiviral activities. Among the proba-
ble mechanisms of cytotoxicity attributed to pharmacophore
1,4-naphthoquinone is the interference with the transport of
electrons in the respiratory chain and the production of reac-
tive oxygen species [22-24].
One strategy for producing new antimicrobials derived
from 4-oxoquinolines includes modification of existing drug
structures. This process aims at developing derivatives to
which bacteria have limited or no resistance. This method
highlights molecular hybridization, based on the incorpora-
tion of two or more pharmacophore units into a single mole-
cule. This technique provides new compounds with com-
plementary activities and/or that act on multiple pharmaco-
logical targets and/or substances in which some units can
counteract side effects caused by others [25, 26]. For exam-
ple, 4-oxoquinoline derivatives have already been shown to
be active against pathogens of clinical importance [27].
Fig. (1) shows the structures of 4-oxoquinoline and 1,4-
naphthoquinone nuclei, including Fluoro-4-oxoquinoline
ciprofloxacin, an antibacterial agent used in the clinic [20],
and the 1,4-naphthoquinone derivative, which can inhibit P.
aeruginosa biofilm formation with a better profile than
ciprofloxacin [22]. Thus, hybrids containing these two types
of nuclei in their structures may have the potential to present
an antibacterial profile that should be further investigated.
Nowadays, in silico approaches play a vital role in the
molecular interactions and safety assessment of new drugs.
In this context, machine learning and QSAR models are
alternatives to animal testing. Also, in silico studies are be-
ing endorsed by regulatory agencies, as they are typically
based on human data, with an enhancement of interspecies
transferability [28, 29].
Due to the immediate need for new antimicrobial drugs,
this paper aimed to synthesize innovative quinone-4-
oxoquinoline derivatives and evaluate their antibacterial,
biofilm formation inhibition, and hemolytic properties as
well as their structure-function-activities.
2. MATERIALS AND METHODS
2.1. Chemistry
Quinone-4-oxoquinoline derivatives were synthesized
and provided by the Department of Organic Chemistry at
Federal Fluminense University. Compounds (1a-c) and (3a-
c) and the following synthetic precursor compounds (4a-c)
were obtained as described in previous work (Fig. 2) [30].
Compounds (2a-c) are new to the literature (see Supporting
Information).
2.2. Biological Activities
2.2.1. Bacterial Strains
For the experiments, 10 reference bacterial species from
the American Type Culture Collection (ATCC) were used,
provided by the National Institute of Quality and Health
Fig. (1). General structures of 4-oxoquinoline and 1,4-naphthoquinone nuclei and examples of their respective derivatives with antibacterial
activity.

Evaluation of Biological Activities of Quinone-4-oxoquinoline Derivatives Current Topics in Medicinal Chemistry, 2022, Vol. 22, No. 12 975
Control: Staphylococcus aureus ATCC 25923, Staphylococ-
cus epidermidis ATCC 1222, Staphylococcus simulans
ATCC 27851, Pseudomonas aeruginosa ATCC 27853,
Escherichia coli ATCC 25922, Enterococcus faecalis
ATCC 29312, Serratia marcescens ATCC 14756, Proteus
mirabilis ATCC 15290, and Enterobacter cloacae ATCC
23355. Also, a hospital sample of ST239 Staphylococcus
aureus BMB 9393 (MRSA) was used, a strain widely dis-
seminated in Brazilian hospitals and whose genome was
sequenced by Costa et al. [31].
2.2.2. Disk Diffusion Method
The antibacterial susceptibility assay was performed by
diffusion of the test substance in Mueller-Hinton agar from
filter paper impregnated with the sample at 5 mg mL-1. A
bacterial suspension corresponding to the 0.5 scale of
MacFarland (CLSI, 2015) was prepared, which was spread
onto the surface of the solid agar. Dimethylsulfoxide PA
(DMSO) was used as a negative control, while ciprofloxacin
and vancomycin were used as positive controls. The plates
were incubated at 37 °C for 24 h and then checked for the
presence of halos from growth inhibition around the discs.
The diameters of the growth inhibition halos were measured
in mm, using a caliper [32].
2.2.3. Determination of Minimum Inhibitory Concentra-
tion (MIC)
The derivatives that were active in the previous assay
had their antibacterial activity analyzed quantitatively for
MIC determination. The tests were carried out in triplicate
using the microdilution technique in Mueller-Hinton broth
(Kasvi, Brazil), using 96-well flat-bottom microtiter plates
(Kasvi, Curitiba, Brazil). The derivatives were diluted with
DMSO and evaluated in the range of 256 μg mL-1 to 1 μg
mL-1. The bacterial suspension was prepared at a concentra-
tion corresponding to the turbidity of the MacFarland 0.5
scale and diluted appropriately to obtain a final concentra-
tion of 5 x 105 CFU mL-1. Each well of the microplate re-
ceived 100 μL aliquot of the derivative in concentrations
defined by serial dilution and 100 μL of the adjusted bacte-
rial inoculum. The microplate was maintained at 37 °C for
24 h. After that, the MIC was defined as the lowest concen-
tration capable of inhibiting the visible growth of the bacte-
rium, verified by the development of turbidity. The drugs
ciprofloxacin and vancomycin were used as positive con-
trols at concentrations ranging between 64 to 2 μg mL-1
against Gram-negative and Gram-positive bacteria, respec-
tively, and DMSO was used as a negative control at the
maximum concentration of 5 % [33].
2.2.4. Effect of Quinone-4-oxoquinoline Derivatives on
Biofilm Development
To evaluate the interference in the formation of bacterial
biofilms, the S. aureus BMB 9393 (MRSA) species, a
strong biofilm producer, was used. The bacteria were cul-
tured in Tryptic Soy Broth (TSB) (Kasvi, Brazil) with 1 %
glucose and under agitation (250-300 revolutions per mi-
nute) to stimulate biofilm formation [34]. All assays de-
scribed below were performed in triplicate.
2.2.4.1. Interference in the Initial Stages of Biofilm
Formation
After 20 h incubation at 37 °C, the bacterial inoculum
was diluted at 1: 100 in TSB, containing subinhibitory con-
centrations (1/2 x MIC, 1/4 x MIC, or 1/8 x MIC) of the
active derivatives against S. aureus diluted in DMSO. Ali-
quots of 200 μL of these mixtures were transferred to 96-
well inert polystyrene microtiter plates (Nunc, Roskilde,
Denmark) and incubated at 37 °C for 24 h. The supernatants
were then removed, and the wells were washed with dis-
tilled water, dried at 65 °C for 1 h, and the wells stained
with crystal violet PA. The optical density of the biofilms
was measured in a microplate reader at 570 nm (SpectraMax
190, Microplate Reader, Molecular Devices, Sunnyvale,
CA). The drug vancomycin in the proportions of 1/2 x MIC,
1/4 x MIC, or 1/8 x MIC was used as a positive control and
2 % DMSO as a negative control.
2.2.4.2. Interference in Preformed Biofilms
To evaluate the interference of the compounds with the
preformed (mature) biofilms, the bacterial inoculum was
diluted at 1:100, as previously described, and transferred to
the wells of the microplate in aliquots of 200 μL without the
addition of the derivatives or the control drug. The plate was
incubated at 37 °C for 24 h, and then the supernatant was
replaced by dilutions of the derivatives or vancomycin in the
proportions of 1 x MIC, 2 x MIC, and 4 x MIC in TSB cul-
Fig. (2). Structures of quinone-4-oxoquinoline derivatives (1a-c, 2a-c, and 3a-c), and synthetic precursor compounds (4a-c).

976 Current Topics in Medicinal Chemistry, 2022, Vol. 22, No. 12 Martins et al.
ture medium supplemented with 5% glucose. The plate was
reincubated for a further 24 h and stained with crystal violet,
and optical density readings of the biofilms were measured
as described previously.
2.2.5. Evaluation of Hemolytic Activity
The hemolytic activity test is one parameter used to
evaluate the hemocompatibility of substances by the degree
of erythrocyte lysis and release of hemoglobin. Hemolytic
activity was investigated according to an adaptation pro-
posed by Parnham and Wetzig [35].
Blood samples containing 3.2% sodium citrate anticoag-
ulant were washed three times in 1 M phosphate-buffered
saline (PBS) at 210 x g for 10 min and finally resuspended
in 5 mL of PBS.
The derivatives or vancomycin or ciprofloxacin at 200
μg mL-1 were incubated at 37 °C in a water bath with the
erythrocyte suspension for 3 h. Hemoglobin release was
quantified after centrifugation and verified using a Spec-
troMax 190 Microplate Reader at 540 nm. Complete hemol-
ysis was obtained using Triton X-100 (positive control). A
percentage of hemolysis of less than 10 % characterizes
adequate hemocompatibility and low toxicity of the deriva-
tives [36]. The experiments were performed in triplicate.
2.2.6. Evaluation of the Change in Plasma Membrane
Permeability
Derivatives that were active against both Gram-positive
and Gram-negative bacteria were evaluated for their ability
to interfere with plasma membrane integrity by the follow-
ing tests. Strains of S. aureus ATCC 25923 and P. aeru-
ginosa ATCC 27853 were used, and the derivatives were
tested at the concentrations of 1 x MIC, 2 x MIC, and 4 x
MIC.
2.2.6.1. Evaluation of Protein Extravasation
Bacterial cells were collected in the exponential growth
phase, centrifuged at 400 x g for 15 min, the supernatant
was discarded, and the pellet was washed twice and resus-
pended in PBS (pH = 7.4). The cell density was adjusted to
108 cells mL-1. Derivatives at concentrations of 1 x MIC, 2
x MIC, or 4 x MIC were incubated at 37 °C for 60 min with
the bacterial inoculum. Subsequently, the suspension was
centrifuged at 13400 x g for 15 min. Bradford's reagent was
added to the supernatant in the proportion of 20 %, and the
optical density was read at 595 nm. Bacterial cells without
the derivatives acted as negative controls in the experiment
(Adapted from Zhou et al. [37]).
2.2.7. Crystal Violet Assay for Verifying Interference with
Membrane Integrity
Bacterial cells (S. aureus ATCC 25923 and P. aerugino-
sa ATCC 27853) were collected in the exponential growth
phase, centrifuged at 4500 x g at 4 °C for 5 min; the super-
natant was discarded, and the pellet was washed twice and
resuspended in PBS buffer (pH = 7.4). The cell density was
adjusted to 108 cells mL-1. Derivatives at concentrations of 1
x MIC, 2 x MIC, and 4 x MIC were incubated at 37 °C for
30 min with bacteria and then centrifuged at 9300 x g for 5
min; the supernatant was discarded, and the pellet was re-
suspended with 10 μg mL-1 of crystal violet in PBS buffer.
This suspension was incubated at 37 °C for 10 min, centri-
fuged at 13,400 x g for 15 min, and the optical density of the
supernatant was read at 590 nm. The crystal violet alone in
PBS buffer (10 μg mL-1) was considered a positive control,
and the untreated bacteria incubated with the dye was a neg-
ative control (Adapted from Devi et al. [38] Masłyk et al.
[39]).
2.2.8. Statistical Analysis
The results obtained in evaluating the hemolytic activity
and the change in plasma membrane permeability were ana-
lyzed by variance analysis (ANOVA) followed by the Tuk-
ey test and T-test for independent samples, respectively,
with the aid of Statistical Package for Social Sciences v.14.0
for Windows. For the evaluation of protein extravasation,
data were expressed as mean ± standard deviation, and the
calculated mean values for the derivatives were compared to
the negative control (PBS) and positive control (Triton X-
100). For the crystal violet assay, the data were expressed as
mean ± standard deviation, and the mean values calculated
were compared to the negative control (bacteria not submit-
ted to treatment and incubated with Bradford's reagent or
crystal violet) and positive control (crystal violet alone). In
both trials, the level of significance was < 5 %, and the
graphs were constructed using the Origin 6.0 software.
2.2.9. In silico Pharmacokinetic and Toxicity
To assess the quinone-4-oxoquinoline derivatives for
potential oral administration, Absorption, Distribution,
Metabolism, and Elimination (ADMET) parameters were
evaluated through solubility in water, permeability, Absorp-
tion, Distribution, Metabolism, and Elimination (ADMET)
plasma protein binding, and inhibition of metabolic enzy-
mes. Risk assessment was achieved by Ames mutagenicity,
cardiotoxicity, and acute toxicity analyses. All derivatives
and vancomycin and ciprofloxacin were evaluated using
machine learning and QSAR models implemented in
ADMET predictorTM version 9.5 (Simulations Plus, Inc.,
Lancaster, CA, USA).
2.2.10. Molecular Modeling
As the quinone-4-oxoquinoline derivatives were active
against both Gram-positive and Gram-negative organisms
and the derivatives structures resembled the known oxo-
quinolines bound to the bacterial DNA gyrase, we per-
formed a molecular dynamics approach to test the DNA
gyrase as a possible binding target. This enzyme was pre-
sent in both Gram-positive and negative forms.
Therefore, a system was built with the S. aureus DNA
Gyrase (PDB: 5CDQ) with a high concentration of com-
pound 3b (one of the most active compounds tested). The
system was constructed according to the method recently
developed by Privat et al. [40]. The initial system built for

Evaluation of Biological Activities of Quinone-4-oxoquinoline Derivatives Current Topics in Medicinal Chemistry, 2022, Vol. 22, No. 12 977
the molecular dynamics (MD) is presented in Fig. (3). This
approach allows the derivative 3b to freely interact with the
enzyme during the MD trajectory and only dock in the sites
that present an affinity.
The 3b derivative model was built using the Spartan'10
software package (WaveFunction Inc., Irvine, CA, USA),
with Hartree-Fock (HF) ab initio calculation at a single
point using a 6-31G* basis set [41], performed in the most
stable conformer obtained using the Merck Molecular Force
Field (MMFF94) [42], followed by geometry optimizations
with the semi-empirical method Recife Model 1 (RM1)
[43]. We also applied a repulsive potential between the lig-
ands to prevent their association [44]. It is important to men-
tion that the repulsive potential only alters ligand-ligand
interactions in the system, thereby keeping all other interac-
tions in the system unperturbed.
2.2.11. Molecular Dynamics
Molecular dynamic simulations were carried out by
AMBER 18.0 with the PMEMD version (Particle Mesh
Ewald Molecular Dynamics). The molecular systems were
thermalized with gradual heating of 30 K every 10 ps up to
300 K. The thermalization was performed in a canonical
ensemble employing the Langevin thermostat. Next, the
systems were equilibrated for 10 ps at 300 K under 1 atm
pressure employing the Berendsen barostat [45]. Finally, the
molecular dynamics production was performed over 150 ns
with a time step for integration of 2 fs under a canonical
ensemble and periodic boundary conditions. Throughout all
MD simulations, the SHAKE [46] algorithm was employed
on atoms covalently bonded to a hydrogen atom.
To obtain robust results, we performed four independent
replicates of molecular dynamics trajectories for each sys-
tem (MD1, MD2, MD3, MD4), using different initial ran-
dom seeds for each simulation [47].
The Cpptraj were used to compute the root-mean-square
deviations (RMSD) and the distance variation of the ligands
during the molecular dynamics trajectories. Trajectories
visual inspections were performed using the VMD program
(Visual Molecular Dynamics version 1.9.2) [48].
3. RESULTS
3.1. Biological Activity
The disc diffusion method showed that the derivatives
were active against S. aureus (2a-c, and 3a-c), S. epidermid-
is (2b-c and 3a-c), S. simulans (2a-c, and 3a-c), S. aureus
BMB 9393 (MRSA) (1a-c, 2a-c, and 3a-c), P. aeruginosa
(1a-c, 2a-c, and 3a-c), and E. coli (1b, 2c, and 3a-c) at 5 mg
mL-1 concentration. The synthetic precursor compounds (4a-
c) were not active in these working conditions. The results
in Table 1 show diameters in mm of growth inhibition halos.
Fig. (3). Structure of the initial system proposed for molecular dynamics, which comprises the S. aureus DNA Gyrase (PDB: 5CDQ) in solu-
tion with 20 derivatives 3b. The water molecules were removed for better visualization. (A higher resolution / colour version of this figure is
available in the electronic copy of the article).

978 Current Topics in Medicinal Chemistry, 2022, Vol. 22, No. 12 Martins et al.
The results also show no activity of any of the derivatives
against E. faecalis, K. pneumoniae, S. marcescens, P. mira-
bilis, and E. cloacae. Results are shown in Table 1.
Derivatives exhibiting halos of inhibition of microbial
growth (Table 1) were evaluated using an assay for MIC
determination (Table 2).
The assay showed that derivatives 2a-c and 3a-b were
active against S. aureus and S. aureus MRSA BMB 9393. In
contrast to S. epidermidis, 2b, 2c, 3a-c inhibited the growth
of this microorganism. The 2a-c and 3a-c derivatives also
showed activity against S. simulans. It is worth mentioning
that 3b presented MIC close to that of the reference drug
vancomycin against S. aureus, S. epidermidis (4 μg mL-1
/7.3μM), and S. simulans (1 μg mL-1 /1.8 μM). In addition,
2c also presented MIC close to vancomycin versus S. simu-
lans (2 μg mL-1 /4.4 μM).
In the analysis of the effect against Gram-negative mi-
croorganisms, 1a, 1b, 2a-c, and 3a-c were active against P.
aeruginosa, while 1b, 2c, and 3a-c were active against E.
coli. It is important to note that derivative 2c presented MIC
close to that of the drug ciprofloxacin against P. aeruginosa
(1 μg mL-1 /2.2 μM).
Derivatives that showed antibacterial activity against S.
aureus MRSA (Table 2, 2a-c, 3a, and 3b) were evaluated
for interference in the formation of bacterial biofilms at
concentrations of 1/2 MIC, 1/4 MIC, and 1/8 MIC. To eval-
uate interference in the formation of biofilms in vitro by S.
aureus MRSA, optical densities following crystal violet
Table 1. Evaluation of antibacterial activity of the derivatives against test bacteria, using the disk diffusion method.
Compounds
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
VAN
CIP
Microorganism
S. aureus
0
0
0
9
10
6
10
9
6
0
0
0
15
NP
S. epidermidis
0
0
0
0
6
8
8
9
6
0
0
0
18
NP
S. simulans
0
0
0
9
6
9
4
10
8
0
0
0
20
NP
Methicillin resistant
S. aureus (MRSA)
9
7
6
8
9
9
12
11
9
0
0
0
14
NP
E. faecalis
0
0
0
0
0
0
0
0
0
0
0
0
19
NP
P. aeruginosa
11
9
8
11
10
11
18
17
10
0
0
0
NP
29
E. coli
0
6
0
0
0
6
12
6
7
0
0
0
NP
36
K. pneumoniae
0
0
0
0
0
0
0
0
0
0
0
0
NP
27
S. marcescens
0
0
0
0
0
0
0
0
0
0
0
0
NP
28
P. mirabilis
0
0
0
0
0
0
0
0
0
0
0
0
NP
28
E. cloaceae
0
0
0
0
0
0
0
0
0
0
0
0
NP
40
Results expressed in mm. VAN = Vancomycin. CIP = Ciprofloxacin. NP = Not performed.
Table 2. Determination of minimum inhibitory concentration (MIC) of derivatives.
Microorganism
Derivatives μg mL-1 (μM)
1a
1b
1c
2a
2b
2c
3a
3b
3c
VAN
CIP
S. aureus
NP
NP
NP
64
(164.8)
128
(297.4)
16
(35.5)
16
(31.8)
4
(7.3)
> 256
(452.6)
2
(1.4)
NP
S. epidermidis
NP
NP
NP
NP
64
(148.7)
16
(35.5)
64
(127.1)
4
(7.3)
64
(113.2)
2
(1.4)
NP
S. simulans
NP
NP
NP
16
(41.2)
8
(18.6)
2
(4.4)
16
(31.8)
1
(1.8)
128
(226.3)
2
(1.4)
NP
Methicillin-
resistant S. aureus
(MRSA)
> 256
(614.8)
> 256
(558.3)
> 256
(535.0)
128
(329.6)
128
(297.4)
16
(35.5)
16
(31.8)
8
(14.6)
> 256
(452.6)
2
(1.4)
NP
P. aeruginosa
128
(307.4)
256
(558.3)
> 256
(535.0)
32
(82.4)
16
(37.2)
1
(2.2)
4
(7.9)
4
(7.3)
256
(452.6)
NP
0,5
(1.5)
E. coli
NP
128
(279.2)
NP
NP
NP
4
(8.9)
8
(15.9)
4
(7.3)
128
(226.3)
NP
0,5
(1.5)
Results expressed in μg mL-1 and (μM). VAN = Vancomycin. CIP = Ciprofloxacin. NP = Not performed.

Evaluation of Biological Activities of Quinone-4-oxoquinoline Derivatives Current Topics in Medicinal Chemistry, 2022, Vol. 22, No. 12 979
staining were initially performed to verify if the derivative
concentrations used would interfere with bacterial growth
(Fig. 4).
Fig. (4). Evaluation of the concentration of the derivatives
interfering with bacterial growth of S. aureus MRSA. OD: Optical
density. C-: negative control. Van: vancomycin.
Derivative 2a, at concentrations of 1/2 MIC, and 1/4
MIC, interfered with bacterial growth. Even though this is
an important fact, since this compound can reduce the bacte-
rial population even in subinhibitory concentrations, such
concentrations were not considered in the evaluation of bio-
film formation.
Fig. (5) shows the data concerning the interference in the
process of bacterial biofilm formation when the derivatives
were incubated together with the S. aureus MRSA.
Fig. (5). Effect of quinone-4-oxoquinoline derivatives in the
process of bacterial biofilm formation of S. aureus BMB 9393
(MRSA). C-: negative control. Van: vancomycin. Data were
expressed as mean ± standard deviation. We used ANOVA
followed by Tukey’s test. * Means were statistically different from
the negative control (p< 0.05). (A higher resolution / colour ver-
sion of this figure is available in the electronic copy of the article).
Derivatives 2a and 2b inhibited adhesion of S. aureus
MRSA by 94.1% ± 1.3% and 93.2% ± 2.5 % at the concen-
trations of 16 μg mL-1 and 64 μg mL-1, respectively. In addi-
tion, 3a, 2c and 2b inhibited up to 52.6% ± 9.8%, 45.3% ±
4.5%, and 44.6% ± 9.7% bacterial adhesion at concentra-
tions of 2 μg mL-1, 8 μg mL-1, and 32 μg mL-1, respectively.
Derivative 3b did not reduce adhesion at the concentrations
evaluated.
The results for vancomycin were not different from the
negative control at concentrations of 1/4 x MIC and 1/8 x
MIC (p < 0.05) and inhibited only 6.6% of the biofilm for-
mation in the concentration of 1/2 x MIC. None of the de-
rivatives was able to interfere with the preformed bacterial
biofilms.
The derivatives were also evaluated for their hemolytic
properties. The data obtained for the derivatives, vancomy-
cin, and ciprofloxacin were statistically different from the
positive and negative controls (p < 0.05). Derivatives 1a,
1b; 2a, 2c; 3a-c and the drugs were considered non-
hemolytic at 200 μg mL-1. 1c and 2b caused more than 10%
hemolysis (10.71% ± 0.5% and 10.33% ± 0.6%, respective-
ly) when evaluated at the concentration of 200 μg mL-1 and
after 3 h of incubation (Fig. 6).
Fig. (6). In vitro evaluation of hemocompatibility of the quinone-4-
oxoquinolinic derivatives. C-: negative control (PBS). C+: positive
control (Triton X-100 a 1%). Van: vancomycin. Cip: Ciprofloxa-
cin. Data were expressed as mean ± standard deviation. We used
ANOVA followed by Tukey’s test. Means followed by different
letters were statistically different (p< 0.05).
The Bradford reagent assay was performed with the de-
rivatives active against P. aeruginosa and S. aureus, and the
results can be seen in Fig. (7). Analysis of the data obtained
for derivatives showed that, for P. aeruginosa (Fig. 7a) and
S. aureus (Fig. 7b), the value of all means differed statisti-
cally from the result obtained with the negative control (p <
0.05). In addition, treatment with all derivatives revealed an
increased concentration of released proteins by the bacteria
due to interference with the permeability of the cell mem-
brane since there was a statistically significant difference
between the 1 x MIC, 2 x MIC, and 4 x MIC concentrations.
For ciprofloxacin and vancomycin, no relationship was re-
ported between increased derivative concentration and pro-
tein released by the cells (Fig. 7).

980 Current Topics in Medicinal Chemistry, 2022, Vol. 22, No. 12 Martins et al.
(a)
(b)
Fig. (7). Evaluation of protein extravasation by the bacterial cells using Bradford's reagent. (a): Assay performed with P. aeruginosa ATCC
27853. (b): Assay performed with S. aureus ATCC 25923. Ciprofloxacin. Van: Vancomycin. C-: microorganism untreated. Data were ex-
pressed as mean ± standard deviation. We used an independent-samples T-test to evaluate the differences within a group. Means followed
by different letters were statistically different (p < 0.05).
(a) (b)
Fig. (8). Evaluation of the permeability of the microbial membrane using crystal violet bacterial uptake. (a): Assay performed with P.
aeruginosa ATCC 27853. (b): Assay performed with com S. aureus ATCC 25923. CIP: Ciprofloxacin. Van: Vancomycin. C-:
microorganism not treated. Data were expressed as mean ± standard deviation. We used an independent-samples T-test to evaluate the
differences within a group. Means followed by different letters were statistically different (p < 0.05).
Janeczko et al. [49] observed an increase in the uptake
of crystal violet dye when the cells underwent some disturb-
ance in cell membrane integrity, and this uptake was con-
centration-dependent. This assay was also performed against
both bacteria, and the data can be analyzed in Fig. (8). The
data showed that for both bacteria species, the crystal violet
uptake in derivative-incubated bacteria was significantly
higher than the negative control (p < 0.05).
Compound 2a promoted the highest increase in crystal
violet uptake, reaching 74.7% and 83.1% in the concentra-
tion of 4 x MIC for P. aeruginosa and S. aureus, respective-
ly. These results are related to the concentration of the de-
rivative used, confirming that these compounds promoted
perturbation in cell membrane permeability. For vancomy-
cin (Fig. 8b), the increase in concentration also promoted
the increase of crystal violet uptake, but the same was not
observed for ciprofloxacin (Fig. 8a). Also, the uptake was
significantly lower than that of the derivatives (p < 0.05).
3.2. In silico Pharmacokinetic and Toxicity
In the simulated pharmacokinetic parameters of the qui-
none-4-oxoquinoline derivatives (2a-c and 3a-c), solubility
was critical for these series since all derivatives presented
low solubility. The chosen excipients should be further stud-
ied. Effective human jejunal permeability analyses showed
that all derivatives presented a good permeability.
Taking into account the potential drug-drug interactions
(DDI) associated with antimicrobials, all the quinone-4-
oxoquinoline derivatives were submitted to P-glycoprotein
(P-gp), Organic Anion Transporting Polypeptide 1B1
(OATP1B1), Organic Cation Transporter 2 (OCT2) trans-
porter, and human cytochrome systems inhibition analyses.
All derivatives presented P-gp and OATP1B1 inhibition
potential, including vancomycin. None of the derivatives
were OCT2 inhibitors, as well as vancomycin and ciprof-
loxacin. Besides, P-gp substrate potential was attributed to

Evaluation of Biological Activities of Quinone-4-oxoquinoline Derivatives Current Topics in Medicinal Chemistry, 2022, Vol. 22, No. 12 981
2b and 3b derivatives, vancomycin, and ciprofloxacin. Re-
garding the CYP enzymes, only 2b and 2c were inhibitors of
CYP2C9, whereas 3c derivative and ciprofloxacin were
inhibitors of CYP3A4 cytochrome as described in the litera-
ture [50].
In the toxicity evaluation, only 2b and 2c derivatives
presented potential mutagenicity based on the Ames test.
None of the derivatives showed inhibition of the potassium
channel, the human Ether-à-go-go Related Gene (hERG)
product, responsible for the repolarization of the cardiac ac-
tion potential. Likewise, no derivatives presented acute tox-
icity risk since LD50 values were higher than 300 mg kg-1.
3.3. MD Results
We conducted four independent replicates of molecular
dynamics trajectories (MD1, MD2, MD3, and MD4) with
compound 3b and the S. aureus NA Gyrase (PDB: 5CDQ)
to evaluate the possible target and mechanism of action of
the compounds. The proposed MD was built based on the
recently proven method of Privat et al. [40] to predict poten-
tial binding sites and mechanism of action [40]. This ap-
proach allows the derivative 3b to freely interact with the
enzyme during the MD trajectory and only dock in the sites
that present an affinity.
The four replicates of MD presented a stable trajectory,
as shown in Fig. (S1) (Supporting Information). From the
MD trajectories, the 3b derivative showed interactions with
an allosteric site, lying at the interface between the GyrB
Topoisomerase-Primase (TOPRIM) domain and the GyrA
winged-helix domain (WHD) (Fig. 9). The pocket is formed
mainly by M27, I30, M179, R342, and P343 belonging to
GyrA and R630 and E634 belonging to GyrB (Fig. 9) (nu-
meration corresponding to S. aureus DNA Gyrase). Since
DNA Gyrase is a symmetric enzyme composed of two pairs
of GyrA and GyrB, there are two hydrophobic allosteric
binding pockets, namely 1 and 2 (Fig. 10).
From the four molecular dynamics simulations per-
formed with the 3b derivative and S. aureus DNA Gyrase,
only in one MD, the 3b did not finish its trajectories near the
allosteric hydrophobic binding pocket (MD1). However, in
all other three simulations (MD2, MD3, and MD4), the 3b
compound completed hydrophobic interactions with the
main residues from binding pockets 1 and 2, such as M27,
I30, M179, and P343. The MD2, MD3, and MD4 showed,
respectively, one, three, and four numbers of 3b molecules
placed next to the referred allosteric site. Fig. (11) shows the
trajectory of each 3b ligand that performed interaction with
the allosteric pocket, and Table 3 shows the time that each
ligand remained bound to the pocket.
We observed that in MD3 and MD4, two ligands inter-
acted, respectively, with each allosteric pocket, 1 and 2,
indicating a good affinity towards this region. The most
representative structure of the ligand docked into the pock-
ets showed hydrogen bonds (HB) with residues GyrA P343
and GyrB E634, both involved in resistance appearing muta-
tions. In MD3, 3b had two HB with GyrB E634, whereas in
MD4, 3b formed just one HB with GyrA P343. These find-
ings suggest that the DNA Gyrase enzyme is a new qui-
none-4-oxoquinoline derivatives target. Figs. (9 and 10)
show the 3b derivatives interaction with these allosteric
binding pockets 1 and 2.
In addition to these two ligands docked in the pocket, in
DM3 and MD4, it is noted that one and two ligands docked
close to the pocket, respectively, and indeed interacted with
some of its conserved residues, as indicated in Figs. (12 and
13), suggesting that the region surrounding the pocket might
be attractive for the ligand 3b interaction.
4. DISCUSSION
The disk-diffusion method showed that the evaluated de-
rivatives were active against Gram-positive and Gram-
negative bacteria. Rau et al. [24] synthesized 5 hetero-1,4-
naphthoquinones containing thiol groups substituted with
alkanoic acid residues at the 2- or 2,3-positions of the naph-
thoquinone ring and found growth inhibition halos that var-
ied between 12 and 16 mm. Not one of the derivatives tested
was active against Gram-negative bacteria, in contrast to the
present study.
Regarding the data obtained in the assay for MIC deter-
mination, it is important to consider that derivatives 2a-c
containing the carboxyl group at the C-3 position of the 4-
quinolonic nucleus were more active than 1a-c with a car-
boxyethyl group at the same position. Also, substances 3a
and 3b were more active than substances 2a and 2b, which
Table 3. Representation of the time each ligand remained bound to the allosteric binding site. The allosteric binding site 1 and 2
are comprised of GyrA and GyrB residues. This table only includes the ligands docked into the allosteric site and does not
include the ligands that performed interactions with conserved residues from the pocket.
Molecular Dynamics
Ligands
Allosteric Site
Time (ns)
MD 2
Lig1409
1
140
MD 3
Lig1407
1
90
MD 3
Lig1409
2
120
MD 4
Lig1398
1
130
MD 4
Lig1409
2
30

982 Current Topics in Medicinal Chemistry, 2022, Vol. 22, No. 12 Martins et al.
(a)
(b)
Fig. (9). The crystal structure of S. aureus DNA Gyrase enzyme and DNA (PDB: 5CDQ) at two different angles displays the hydrophobic
allosteric binding pocket. a) Orthogonal view of the DNA gyrase enzyme (dark gray), DNA (green), and the hydrophobic allosteric binding
pockets 1 and 2 featured in green spheres. The allosteric binding pocket in detach shows its main residues. b) A label about the domains in
both angles showing DNA in green, WHD and first α-helix in light blue, TOPRIM domain in magenta, fluoro-4-oxoquinoline binding site in
red, DNA intercalator binding site in yellow, and hydrophobic allosteric binding pockets 1 and 2 featured in light green. (A higher resolution
/ colour version of this figure is available in the electronic copy of the article).

Evaluation of Biological Activities of Quinone-4-oxoquinoline Derivatives Current Topics in Medicinal Chemistry, 2022, Vol. 22, No. 12 983
(a)
(b)
(c)
Fig. (10). The crystal structure of S. aureus DNA Gyrase enzyme and DNA (PDB: 5CDQ) at two different angles displays the hydrophobic
allosteric binding pocket. Orthogonal view of the complex in carton (a) and surface (b) representations showing the enzyme (gray), the
DNA (orange), and the hydrophobic allosteric binding pockets 1 and 2 (green). In (c), the sequence alignment of DNA Gyrase from some
key pathogenic bacteria strains shows the conservative residues with S. aureus residue numbers above. (A higher resolution / colour version
of this figure is available in the electronic copy of the article).
Fig. (11) contd…

984 Current Topics in Medicinal Chemistry, 2022, Vol. 22, No. 12 Martins et al.
Fig. (11). Distance of 3b ligands and the conserved residue Pro343 from the allosteric pocket vs. time in the MD performed. This figure only
shows the ligands trajectory distance that interacted with the allosteric pocket for more than 50 ns.
(a)
(b)
(c)
(d)
Fig. (12). An enlarged view of the allosteric binding site 2 with the 3b compound final binding mode after the 150 ns dynamic simulations
near the pocket region, with the main residues that compose the hydrophobic allosteric pocket shown as sticks. Carbon in green, oxygen in
red, nitrogen in blue, and hydrogen in white. (a): In MD3, one of the 3b compound’s final trajectory locations is nearest the allosteric hy-
drophobic binding pocket 2. 3b compound is shown as sticks. Carbon in pink, oxygen in red, nitrogen in blue, and hydrogen in white. Resi-
dues of the pocket are identified by white labels. (b): In MD4, one of 3b compounds’ final trajectory location is nearest the allosteric hydro-
phobic binding pocket 2. 3b compound is shown as sticks. Carbon in yellow, oxygen in red, nitrogen in blue, and hydrogen in white. Resi-
dues of the pocket are identified by white labels. Hydrogen bond identified in green dashed lines. (c): In MD4, one of the 3b compound’s
final trajectory locations is nearest the allosteric hydrophobic binding pocket 2. 3b compound is shown as sticks. Carbon in yellow, oxygen
in red, nitrogen in blue, and hydrogen in white. Residues of the pocket are identified by white labels. (d): In MD4, one of the 3b com-
pound’s final trajectory locations is nearest the allosteric hydrophobic binding pocket 2. 3b compound is shown as sticks. Carbon in yellow,
oxygen in red, nitrogen in blue, and hydrogen in white. Residues of the pocket are identified by white labels. (A higher resolution / colour
version of this figure is available in the electronic copy of the article).

Evaluation of Biological Activities of Quinone-4-oxoquinoline Derivatives Current Topics in Medicinal Chemistry, 2022, Vol. 22, No. 12 985
(a)
(b)
(c)
(d)
Fig. (13). An enlarged view of the allosteric binding site 1 with 3b compound final binding mode after the 150 ns dynamic simulations,
which ended up near the pocket region, with the main residues, which compose the hydrophobic allosteric pocket, as shown in sticks. Car-
bon in green, oxygen in red, nitrogen in blue, and hydrogen in white. (a): In MD2, only 3b compound’s final trajectory location is nearest
the allosteric hydrophobic binding pocket 1. 3b compound is shown as sticks. Carbon in light blue, oxygen in red, nitrogen in blue, and hy-
drogen in white. Residues of the pocket are identified by white labels. (b): In MD 8, the 3b compound’s final trajectory location is nearest
the allosteric hydrophobic binding pocket 1. 3b compound is shown as sticks. Carbon in pink, oxygen in red, nitrogen in blue, and hydrogen
in white. Residues of the pocket are identified by white labels. Hydrogen bond identified in green dashed lines. (c): In MD3, one of the 3b
compound’s final trajectory locations is nearest the allosteric hydrophobic binding pocket 1. 3b compound is shown as sticks. Carbon in
pink, oxygen in red, nitrogen in blue, and hydrogen in white. Residues of the pocket are identified by white labels. (d): In MD4, one of the
3b compound’s final trajectory locations is nearest the allosteric hydrophobic binding pocket 1. 3b compound is shown as sticks. Carbon in
yellow, oxygen in red, nitrogen in blue, and hydrogen in white. Residues of the pocket are identified by white labels in white. Residues of
the pocket are identified by white labels. (A higher resolution / colour version of this figure is available in the electronic copy of the article).
might be related to the isoquinoline-5,8-dione nucleus pre-
sent in these substances, while type 2 substances have the
1,4-naphthoquinone nucleus.
S. aureus is considered the most frequently isolated hu-
man pathogen in clinical infections [51]. Methicillin-
resistant S. aureus infections (MRSA) remain for decades as
the major cause of health-care-related infection, with some
clones rapidly transmitted in hospitals and the community in
several countries [51, 52]. Furthermore, in the future, van-
comycin will lose its effectiveness against such microorgan-
isms [53]. In the present study, 5 of the 9 derivatives evalu-
ated were active against methicillin-resistant S. aureus (MIC
128 to 8 μg mL-1).
Ramos-Peralta et al. [23] verified a MIC value for naph-
thoquinones isolated from natural sources as 7.8 μg mL-1
and MIC ranging from 16 to 64 μg mL-1 for synthetic naph-
thoquinones (2-hydroxy-1,4-naphthoquinone derivatives
with cyano and 4-chlorophenyl C4 groups) against S. aureus
from nosocomial infections. In the present study, compound
3b was the most active against S. aureus with a MIC value
of 4 μg mL-1 and could represent a promising alternative to
the treatment of infections by S. aureus.
In 2017, Moreira et al. [54] evaluated 2-hydroxy-3-
phenylsulfanylmethyl-[1,4]-naphthoquinones against S. au-
reus, S. epidermidis, S. simulans, and E. coli. The data
showed that the most active compounds had MIC of 32 μg
mL-1, 16 μg mL-1, 32 μg mL-1, and 256 μg mL-1 for the re-
spective microorganisms. The results obtained in the present
work for the quinone-4-oxoquinoline derivatives were more
promising since MIC values ranging from 1 μg mL-1 to 4 μg
mL-1 were observed (Table 2).
Ferretti et al. [55] synthesized and evaluated the antimi-
crobial activity of oxoquinoline derivatives against S. au-
reus, Bacillus subtilis, E. coli, P. aeruginosa, and Shigella
sonnei. The MIC for the most active compounds were 3.12
μg mL-1, 1.56 μg mL-1, 1.52 μg mL-1, 50 μg mL-1, and 3.12
μg mL-1, respectively. The authors concluded that the func-
tionalization of the nitrogen atom affected antimicrobial
activity. In the present work, the quinone-4-oxoquinoline
derivatives were more active than the derivatives synthe-

986 Current Topics in Medicinal Chemistry, 2022, Vol. 22, No. 12 Martins et al.
sized by Ferretti et al. [55] against P. aeruginosa, with MIC
values of 1, 4, 4, and 16 μg mL-1 for derivatives 2c, 3a, 3b,
and 2b, respectively. This bacterium has great importance in
nosocomial infections since, due to additional mechanisms
of intrinsic resistance, it can become rapidly multidrug-
resistant [56, 57].
In addition, P. aeruginosa can form bacterial biofilms in
medical devices and/or artificial organs and the lungs of
patients with cystic fibrosis. This bacterial biofilm guaran-
tees the formation of physical and chemical barriers against
the host immune response, which in the case of patients with
cystic fibrosis, can increase their death rate [58-61]. This
complex network is formed when bacterial cells are fixed on
a biotic or abiotic surface, form microcolonies, and produce
a polymeric extracellular matrix, which may be composed
of proteins, sugars, and extracellular nucleic acids [58, 62].
Derivatives 2a-c, 3a, and 3b were evaluated for interfer-
ence in the formation of bacterial biofilms at concentrations
of 1/2 MIC, 1/4 MIC, and 1/8 MIC. The data showed ex-
pressive results for derivatives 2a and 2b that inhibited the
adhesion of S. aureus MRSA by more than 90% at concen-
trations of 16 μg mL-1 and 64 μg mL-1, respectively.
However, none of the derivatives interfered with the pre-
formed bacterial biofilm.
Wu et al. [59] described the difficulty in eliminating in-
fections in which the bacterial biofilm is already established
and requires intensive and aggressive antimicrobial inter-
ventions. In vitro data showed that biofilm eradication re-
quires concentrations 10 to 1000 times greater than that es-
tablished as the MIC capable of acting on the planktonic
form of the bacteria. The current treatment to combat this
type of infection requires combination therapy with drugs of
different action mechanisms. Due to this problem, innova-
tive compounds are required to inhibit the adhesion of the
microorganisms before biofilm formation [63].
Moreira et al. [54] observed that 2-hydroxy-3-phenyl-
sulfanylmethyl-[1,4]-naphthoquinone derivatives interfered
with the formation of bacterial biofilms and the mature bio-
films. However, the derivative 15 only inhibited 34 ± 2.4%
of biofilm formation, a result lower than that for the deriva-
tives in the present research. The quinone-4-oxoquinoline
derivatives, therefore, contribute positively to the inhibition
of the processes of biofilm formation.
Evaluation of the hemocompatibility of the quinone-4-
oxoquinolinic derivatives showed that derivatives 1a, 1b;
2a, 2c; 3a-c did not cause hemolysis at a concentration of
200 μg mL-1. Moreira et al. [64] synthesized and evaluated
the antiparasitic activity of three semi-synthesized naphtho-
quinones structurally related to lapachol and lapachone. The
compound designated 1 and derived from lapachol did not
induce lysis of erythrocytes up to 200 μM. The authors con-
cluded that naphthoquinones do not cause hemolysis and
genotoxicity. These data are also in accord with those of de
Sena-Pereira et al. [65], in which the three 2-hydroxy-1,4-
naphthoquinone derivatives tested were non-hemolytic.
Likewise, α and β-2,3-dihydrofuranonephtoquinones exhib-
ited no important hemolysis up to 50 μg mL-1 [66].
Naphthoquinones may be considered toxic, but modifi-
cations in their structures contribute to improved biological
activity and reduced toxicity [65].
Some compounds are capable of causing damage to the
cell membrane of microorganisms, and the extravasation of
intracellular material is an indicator of alteration in the per-
meability of this structure. The Bradford assay for quantifi-
cation of proteins in solution is a simple, fast, and adequate-
ly sensitive method, which allows the determination of these
macromolecules in the extracellular component after com-
ing into contact with the test substance [67-69].
The assays performed in this study showed that the eval-
uated derivatives interfered with the permeability of the cell
membrane of P. aeruginosa and S. aureus since there was
an increase in the concentration of proteins released by these
bacteria. Janeczko et al. [49] found that 1,4-naphthoquinone
derivatives interfered with the integrity of the cell mem-
brane of Candida albicans, with the release of intracellular
components that absorb electromagnetic energy at the wave-
length of 260 nm and proteins with absorption at 595 nm.
In the assay for evaluating the interference with the per-
meability of the microbial membrane by using crystal violet,
the results corroborated the findings of a study conducted by
Janeczko et al. [49] since the increase in this uptake was
concentration-dependent.
Masłyk et al., [39] after evaluating arylcyanomethylene
quinone oxime against C. albicans, concluded that this crys-
tal violet assay is useful for the verification of cell mem-
brane damage, with the uptake of this compound increasing
from 12 to 31% after yeast treatment with a concentration of
4 x MIC.
Regarding the evaluation of in silico pharmacokinetics
and toxicity, the findings suggest that all derivatives pre-
sented a good pharmacokinetic and toxicity profile [70],
highlighting the 2a and 3a that showed better toxicity re-
sults.
Among the quinone-4-oxoquinoline derivatives, the
compounds with the most activity against both Gram-
positive and Gram-negative bacteria were compounds 3b,
2c, and 3a, as highlighted by their antibacterial profiles
(Table 2).
Such observation prompted us to perform a computa-
tional simulation to study the DNA Gyrase as a potential
target since the DNA Gyrase is a common target for oxo-
quinolines in both Gram-positive and Gram-negative [71-76].
According to our dynamics simulation results, the 3b de-
rivative does not interact with oxoquinolines known binding
region, which is responsible for the stabilization of the dou-
ble-stranded cleaved DNA-enzyme complex. We observed
that the 3b derivative showed interactions with other known
domains within the Gyrase enzyme, such as the TOPRIM
domain and the ATPase domain. Therefore, this feature
could represent a new alternative to treatment since the ac-
tivity of derivatives should not be affected by traditional
mutations related to oxoquinoline resistance, known as the
oxoquinoline resistance determining region (QRDR) [77].

Evaluation of Biological Activities of Quinone-4-oxoquinoline Derivatives Current Topics in Medicinal Chemistry, 2022, Vol. 22, No. 12 987
However, the quinone-4-oxoquinoline derivatives could
be causing DNA-enzyme cleavage complex stabilization,
similar to fluoro-4-oxoquinolines, but aiming at a different
region not directly interacting with the DNA molecule, as
observed previously by Chan et al. [78], and an allosteric
site targeted by thiophene compounds as reported by
Klostermeier [79].
This allosteric binding pocket is mainly hydrophobic by
nature and opens itself as a groove toward the outer side of
the enzyme, lying at the interface between the GyrB TO-
PRIM domain and the GyrA winged-helix domain (WHD)
(Fig. 9). Interestingly, the four residues R342, P343, R630,
and E634, that comprise the allosteric binding pocket, are
highly conserved across key pathogenic bacteria strains and
even more appealing; they cannot be found in human topoi-
somerase II [78].
Interestingly, in two out of the four simulations, MD3
and MD4, the 3b derivative showed HB with residues GyrA
P343 and GyrB E634, both involved in resistance appearing
mutations. In MD3, 3b realized two HB with GyrB E634,
whereas in MD4, 3b formed just one HB with GyrA P343.
These findings also suggested that the DNA Gyrase enzyme
is a new quinone-4-oxoquinoline derivatives target.
CONCLUSION
The quinone-4-oxoquinoline derivatives were found to
be active against six bacterial species, including Gram-
positive and Gram-negative microorganisms. Derivatives 3b
and 2c were promising when evaluated against Gram-
positive bacteria, with MIC values close to the MIC values
observed for antibacterial in clinical use. It is worth men-
tioning that five of the nine derivatives evaluated were ac-
tive against MRSA. In relation to Gram-negative microor-
ganisms, eight derivatives were active against P.
aeruginosa, and five were active against E. coli. Derivative
2c presented as an important antibacterial compound with
MIC close to that of the reference drug (ciprofloxacin)
against P. aeruginosa. It should be noted that six derivatives
had a broad-spectrum antibacterial activity.
The compounds were promising in inhibiting bacterial
adhesion and preventing biofilm formation since 2a and 2b
inhibited biofilm formation by up to 94.1% and 93.2% at
concentrations of 16 μg mL-1 and 64 μg mL-1 respectively.
Neither one of the derivatives was active against the pre-
formed biofilm.
The evaluation of the data obtained by the hemolysis test
showed that seven derivatives were considered non-
hemolytic at 200 μg mL-1.
Assays for verification of the change in cell membrane
permeability have shown that the derivatives seem to induce
perturbation in the integrity of this structure with increased
release of proteins into the extracellular medium and in-
creased uptake of crystal violet by the bacterial cells.
The derivatives containing the carboxyl group at the C-3
position of the 4-quinolonic nucleus were more active than
those containing a carboxyethyl group. In addition, the iso-
quinoline-5,8-dione nucleus also favored antimicrobial ac-
tivity. Interestingly, compounds 2a-2c were more effective
than 1a-1c, which indicated that the acid functional group in
2a-2c is important for antimicrobial activity. Therefore, acid
derivatives of 3a-3c should be further explored by using
different synthetic and/or purification strategies since the
attempt to synthesize them at a high purity degree is not
feasible.
The quinone-4-oxoquinoline possible target proposed for
the derivatives is the DNA gyrase enzyme due to their oxo-
quinoline structure and broad-spectrum activity. The mech-
anism of action was assessed by several molecular dynamic
simulations, with a crystallographic structure of S. aureus
DNA gyrase obtained in PDB as 5CDQ. The molecular dy-
namics results showed that the 3b compound has an affinity
for a specific hydrophobic pocket located between GyrA
and GyrB subunits, near to the DNA molecule but not inter-
acting with it. Since DNA gyrase is a symmetric enzyme,
there are two pockets in each enzyme, referred to as pocket
1 and pocket 2. Such allosteric binding pockets are com-
posed of highly conserved residues, such as P343, R630,
and E634, and interactions with that region result in a simi-
lar stabilization of the DNA-enzyme cleavage complex ob-
served by the fluoro-4-oxoquinolines main binding site in-
hibition effects.
Those findings suggest that derivatives have the same
target as oxoquinolines in DNA gyrase but a different
mechanism of action that does not overlap with bacterial
resistance mutations to fluoro-4-oxoquinolines.
LIST OF ABBREVIATIONS
ADME = Absorption, Distribution, Metabolism, and
Elimination
DDI = Drug-drug interactions
DMSO = Dimethylsulfoxide
HB = hydrogen bonds
HF = Hartree-Fock
MD = Molecular dynamics
MIC = Minimum Inhibitory Concentration
MRSA = Methicillin-Resistant Staphylococcus au-
reus
OATP1B1 = Organic Anion Transporting Polypeptide
1B1
OCT2 = Organic Cation Transporter 2
PBS = Phosphate-buffered saline
P-gp = P-glycoprotein
RM1 = Recife Model 1
RMSD = Root-mean-square deviations
TSB = Tryptic Soy Broth
VMD = Visual Molecular Dynamics

988 Current Topics in Medicinal Chemistry, 2022, Vol. 22, No. 12 Martins et al.
AUTHORS' CONTRIBUTIONS
Francislene Juliana Martins: study concept or design, da-
ta collection, data analysis or interpretation, and writing the
paper; Fernanda Savacini Sagrillo: study concept or design,
data collection, data analysis or interpretation, and writing
the paper; Rafaelle Josianne Vinturelle Medeiros: data col-
lection; Alan Gonçalves de Souza: data collection; Amanda
Rodrigues Pinto Costa: data collection; Juliana Silva No-
vais: data collection; Leonardo Alves Miceli: data collec-
tion, and writing the paper; Vinícius Campos: data collec-
tion; Agnes Marie Sá Figueiredo: supervision; Anna Claudia
Cunha: supervision; Natalia Lidmar von Ranke: data collec-
tion, and writing the paper; Murilo Lamim Bello: supervi-
sion; Bárbara Abrahim-Vieira: data collection; Alessandra
De Souza: data collection; Norman Ratcliffe: review, and
editing; Fernanda da Costa Santos Boechat: supervision;
Maria Cecília Bastos Vieira de Souza: study concept or de-
sign, and supervision; Carlos Rangel Rodrigues: study con-
cept or design, and supervision, and Helena Carla Castro:
study concept or design, and supervision.
ETHICS APPROVAL AND CONSENT TO PARTICI-
PATE
Not applicable.
HUMAN AND ANIMAL RIGHTS
No Animals/Humans were used for studies that are basis
of this research.
CONSENT FOR PUBLICATION
Not applicable.
AVAILABILITY OF DATA AND MATERIALS
The authors confirm that the data supporting the findings
of this study are available within the article.
FUNDING
This study was financially supported by Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(Grant no. 309703/2019-0) and Fundação Carlos Chagas
Filho de Amparo à Pesquisa do Estado do Rio de Janeiro
(Grant no. FAPERJ-E-26/202.447/2019).
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
The authors would like to thank Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq),
Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES), Fundação Carlos Chagas Filho de
Amparo à Pesquisa do Estado do Rio de Janeiro, and
Universidade Federal Fluminense for the support provided
for this research.
SUPPLEMENTARY MATERIAL
Supplementary material is available on the publisher’s
website along with the published article.
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