Discovery of a new family of heterocyclic amine linked plastoquinone analogs for antimicrobial evaluation

Article abstract of DOI:10.1002/ddr.21591

A series of aminobenzoquinones, denoted as PQ analogs (PQ1-13), were synthesized by employing a green methodology approach using water as solvent developed by Tandon et al. Subsequently, in vitro antimicrobial potential of all PQ analogs was evaluated in a panel of seven bacterial strains (three gram positive and four gram negative bacteria) and three fungi. The antifungal profile of all PQ analogs indicated that four analogs (while PQ2, PQ9, and PQ10 were effective against Candida tropicalis, PQ11 is effective against Candida albicans) have potent antifungal activity. The results revealed that PQ9 showed similar antibacterial activity against Staphylococcus epidermidis compared clinically prevalent antibacterial drugs cefuroxime. PQ11 exhibited the highest antibacterial activity against S. epidermidis, which was about fourfold better than that of cefuroxime. Owing to their outstanding activities, PQ9 and PQ11 were chosen for a further investigation for biofilm and cytotoxicity evaluation. Based on the tests performed, there was a significant positive correlation between inhibition of the biofilm attachment and time. In addition, PQ9 and PQ11 showed cytotoxic effects at high concentrations on Balb/3T3, HaCaT, HUVEC, and NRK-52E cells (>24 and >18 μg/mL, respectively). Thus, two analogs (PQ9 and PQ11) were identified as the hits with the strong antibacterial efficiency against the S. epidermidis with low MIC values.

Full text of DOI:10.1002/ddr.21591

Received: 7 May 2019
Revised: 30 July 2019
Accepted: 31 July 2019
DOI: 10.1002/ddr.21591
R E S E A R C H A R T I C L E
Discovery of a new family of heterocyclic amine linked
plastoquinone analogs for antimicrobial evaluation
Amaç F. Tuyun1
| Mahmut Yıldız2 | Nilüfer Bayrak3 | Hatice Yıldırım3
|
Emel Mataracı Kara4 | Ayse T. Jannuzzi5 | Berna Ozbek Celik4
1Engineering Sciences Department,
Abstract
Engineering Faculty, Istanbul University-
Cerrahpasa, Istanbul, Turkey
A series of aminobenzoquinones, denoted as PQ analogs (PQ1-13), were synthesized
by employing a green methodology approach using water as solvent developed by
Tandon et al. Subsequently, in vitro antimicrobial potential of all PQ analogs was
evaluated in a panel of seven bacterial strains (three gram positive and four gram
negative bacteria) and three fungi. The antifungal profile of all PQ analogs indicated
that four analogs (while PQ2, PQ9, and PQ10 were effective against Candida
tropicalis, PQ11 is effective against Candida albicans) have potent antifungal activity.
The results revealed that PQ9 showed similar antibacterial activity against Staphylo-
coccus epidermidis compared clinically prevalent antibacterial drugs cefuroxime.
PQ11 exhibited the highest antibacterial activity against S. epidermidis, which was
about fourfold better than that of cefuroxime. Owing to their outstanding activities,
PQ9 and PQ11 were chosen for a further investigation for biofilm and cytotoxicity
evaluation. Based on the tests performed, there was a significant positive correlation
between inhibition of the biofilm attachment and time. In addition, PQ9 and PQ11
showed cytotoxic effects at high concentrations on Balb/3T3, HaCaT, HUVEC, and
NRK-52E cells (>24 and >18 μg/mL, respectively). Thus, two analogs (PQ9 and
PQ11) were identified as the hits with the strong antibacterial efficiency against the
S. epidermidis with low MIC values.
2Chemistry Department, Gebze Technical
University, Gebze, Kocaeli, Turkey
3Chemistry Department, Engineering Faculty,
Istanbul University-Cerrahpasa, Istanbul,
Turkey
4Pharmaceutical Microbiology Department,
Pharmacy Faculty, Istanbul University,
Istanbul, Turkey
5Pharmaceutical Toxicology Department,
Pharmacy Faculty, Istanbul University,
Istanbul, Turkey
Correspondence
Amaç F. Tuyun, Engineering Sciences
Department, Engineering Faculty, Istanbul
University-Cerrahpasa, Avcilar 34320, Istanbul,
Turkey.
Email: aftuyun@gmail.com, aftuyun@istanbul.
edu.tr
Funding information
Scientific Research Projects Coordination Unit
of Istanbul University, Grant/Award Number:
Project number: FBA-2016-22584
K E Y W O R D S
aminobenzoquinone, antibacterial activity, antibiofilm activity, antifungal activity, cytotoxicity,
green chemistry, piperazines, piperidines
in 31 patients in a U.S. hospital have at least one HAI on any single
1
| INTRODUCTION
day (CDCP, 2016; ECDPC, 2013). Staphylococcus epidermidis and
Enterococcus faecalis are two gram positive bacteria becoming major
nosocomial pathogens. S. epidermidis is a primary cause of HAIs
related to implanted prosthetic medical devices (i.e., heart valves,
artificial joints, and cerebrospinal fluid shunt) due to the capable of
forming biofilms and colonization in these devices (Namvar et al.,
2014). E. faecalis is accounted for nosocomial infections such as
bloodstream, surgical site, and urinary tract (Nallapareddy
et al., 2006).
Nosocomial infections that allude to healthcare-associated infections
(HAIs) and hospital-acquired infections lead to inconvenience like
pain, staying in the hospital for a long time, consistent disability, mor-
bidity, and even mortality affecting hundreds of millions of patients in
the world (ECDPC, 2014; WHO, 2011). According to the reports of
European Centre for Disease Prevention and Control (ECDC) and the
Centers for Disease Control and Prevention (CDC), about 80,000
patients (one in 18 patients) in a European hospital approximately one
Drug Dev Res. 2019;1–12.
wileyonlinelibrary.com/journal/ddr
© 2019 Wiley Periodicals, Inc.
1

2
TUYUN ET AL.
One of the two important mechanisms is that quinones can react
Moon, Cho, & Yun, 2004; Yuk et al., 2000), lipoxy-genase inhibitory
(Richwien & Wurm, 2004), human monoamine oxidase (MOA) inhibi-
tory (Cerqueira, Netz, Diniz, do Canto, & Follmer, 2011), and anti-
platelet activities (Lien et al., 2002; Yuk et al., 2000). On the other
hand, introducing amino substituents into the 1,4-quinone moiety,
named aminoquinones, significantly improves biological properties
which are of great importance in pharmaceutical chemistry (Egleton
et al., 2014; Pingaew et al., 2015; Prachayasittikul et al., 2014; Xu
et al., 2012). Aminoquinones are generally synthesized by the
(a) reaction of hydroquinone and amines (Ikeda, Wakabayashi, &
Nakane, 1991; Niedermeyer, Mikolasch, & Lalk, 2005), (b) nucleophilic
addition reactions of 1,4-quinones with amines (Ryu et al., 2005;
Valderrama et al., 2016), and (c) nucleophilic substitution reactions of
the methoxy or halogen derivative of 1,4-quinones with amines in dif-
ferent media (Delarmelina et al., 2015; Kacmaz et al., 2018; Pingaew
et al., 2015; Ryu, Nho, Jin, Oh, & Choi, 2014; Tandon, Kumar, Mis-
hra, & Shukla, 2012; Tandon & Maurya, 2009).
with the thiol group of glutathione, which can act as electrophiles.
The core structure of quinones could undergo one or two electron
reduction to the corresponding semiquinones or hydroquinones,
respectively. While a semiquinone occurs by a one-electron reduction,
hydroquinone occurs by a two-electron reduction. The second one is
the production of the semiquinones by a one-electron reduction
(Castro, Mariani, Panek, Eleutherio, & Pereira, 2008; Gutierrez, 2000;
Widhalm & Rhodes, 2016). Recent years have witnessed respectable
progress in the development of biologically active 1,4-quinones based
on natural compounds (Johnson-Ajinwo et al., 2018; Nain-Perez et al.,
2017; Nain-Perez, Barbosa, Maltha, & Forlani, 2017). 1,4-Quinone
moiety constitutes the fundamental framework of versatile natural or
synthetic quinone molecules such as plastoquinone (PQ) (Kawamukai,
2018), mitomycin
C
(Begleiter, 2000; Sugiura, 1961), and
thymoquinone (Collett et al., 2010; El-Dakhakhany, 1963; Glamoclija
et al., 2018) since they have extensively found ample and numerous
applications in the design and synthesis of pharmacologically active
agents exhibiting a wide range of biological activities such as antican-
cer (Wellington, 2015; Wellington, Kolesnikova, Nyoka, & McGaw,
2019), antibacterial (Janeczko, Demchuk, Strzelecka, Kubinski, &
Maslyk, 2016; Jordao et al., 2013), antifungal (Ryu, Oh, Choi, & Kang,
2014; Shrestha et al., 2017), anti-HIV (Alfadhli et al., 2016; Ilina et al.,
2002), antimalarial (Carneiro et al., 2016; Pingaew et al., 2015), anti-
allergic (Lien, Huang, Teng, Wang, & Kuo, 2002), antiinflammatory
(Tandon, Chhor, Singh, Rai, & Yadav, 2004), antithrombotic (Jin, Ryu,
A literature survey reveals that Tandon et al. have delineated on a
number of heterocyclic amine core linked 1,4-quinones (I-III in
Figure 1) with their antibacterial and antifungal activities by a green
methodology approach using water as the solvent (Tandon, Maurya,
Verma, Kumar, & Shukla, 2010). Davids et al. have discovered the het-
erocyclic amine core linked 1,4-quinones (IV and V in Figure 1) with
their impressive antiproliferative activity against the three cancer cell
lines (Davids et al., 2012). Abenquines are natural products containing
amino acid residues and the first total synthesis of abenquines has
FIGURE 1 Design of target molecules
(PQ1-13) based on pharmacologically
interesting heterocyclic amine core linked
1,4-quinones from literature

TUYUN ET AL.
3
been achieved by Nain-Perez and coworkers (Nain-Perez, Barbosa,
Maltha, & Forlani, 2016). Nain-Perez et al. have shown abenquines
and their analogs’ cytotoxicity activities against six human tumor cell
lines and nonmalignant mouse fibroblasts (Nain-Perez, Barbosa,
Rodriguez-Hernandez, Kramell, et al., 2017). Additionally, the same
group has also used this class of structures as a model for the synthe-
sis of new herbicides targeting photosynthesis (Nain-Perez, Barbosa,
Maltha, Giberti, & Forlani, 2017). Inspired by the above findings and
as a part of our ongoing program for the generation of biologically
active analogs, we thought of designing the newer heterocyclic amine
linked PQ analogs by the introducing of heterocyclic amine core
linked into the 1,4-quinone to ascertain their antimicrobial profiles.
Some of the biologically active compounds containing 1,4-quinone
moiety and heterocyclic amine, which provide the basic
pharmacophore in designing our target compounds are shown in
Figure 1.
[4.5]decane, 4-benzylpiperidine, morpholine, thiomorpholine, ethyl
piperazine-1-carboxylate, 1-phenylpiperazine, and 1-(4-nitrophenyl)
piperazine at around 50–60 ꢀC for 6–12 hr using water as solvent,
target molecules, namely PQ analogs (PQ1-13), were obtained as
shown in Scheme 1 according to the developed method by Tandon
et al. (2010). Among used heterocyclic amines, substitution reaction
of the precursor (2) with 2-methylpiperidine failed to produce any
product. Futile efforts by changing the reaction conditions did not
yield the desired product possibly due to steric hindrance of the
bulky ortho substituent as the spatial arrangement of atoms or func-
tional groups near the reacting site hindered the reaction from pro-
ceeding. PQ1, PQ2, PQ9, and PQ10 were prepared before by our
group (Tuyun & Yıldız, 2018), while the other PQ analogs are novel
molecules.
The structures of all PQ analogs (PQ1-13, Table 1) were determined
on the basis of their analytical and spectroscopic data (FTIR, 1H NMR,
13C NMR, and MS). 13C NMR spectra of the PQ analogs showed the
downfield shifts of two quaternary carbon in the region between
180 and 184 ppm indicated the nonequivalent carbonyl carbons within
the quinone moiety. In 13C NMR spectra, two carbon atoms in the
methyl groups of quinone moiety (C-5 and C-6) were displayed at around
12 ppm. The protons of methyl groups attached to quinone moiety
appeared at around 2 ppm. In the mass spectra of all PQ analogs, the
molecular ions were observed with the base peaks corresponding to the
molecular weights of the analogs. Further, in the FTIR, the absorption sig-
nal at around 1,650 cm−1 indicated the presence of the carbonyl group.
Additionally, the structures of the PQ7 (1894854) and PQ12 (1894852)
were further confirmed by the single crystal diffraction (Figure 2) (for
details, please see the Supplementary file).
With the rapid emergence of resistant bacteria in the world, the
effectiveness of antibiotics saving millions of lives is endangered.
Research and development of novel antimicrobial drugs are the vital
roles that must be adopted to fight the emergence and spread of anti-
biotic resistance in worldwide. In this context, our laboratory is dedi-
cated to synthesizing pharmacologically active substances based on
1,4-quinone moiety (Bayrak et al., 2017; Tuyun et al., 2015; Yildirim
et al., 2017). Encouraged by all these facts and in continuation of our
previous work on the discovery of pharmacologically important PQ
analogs employing 2,3-dimethyl-1,4-benzoquinone moiety of PQ as a
core structure linked with heterocyclic amines, in this article, we
report the design, synthesis, antimicrobial evaluation, antibiofilm eval-
uation, cytotoxicity, and structure–activity relationship (SAR) of het-
erocyclic amine linked PQ analogs.
2.2 | Determination of the in vitro antibacterial and
antifungal activity
2
| RESULTS AND DISCUSSION
2.1 | Chemical synthesis
After synthesis and characterization, we studied the in vitro antimicro-
bial activity of PQ analogs (PQ1-13) against three gram positive bac-
teria (Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis
ATCC 12228, and Enterococcus faecalis ATCC 29212), four gram neg-
ative bacteria (Pseudomonas aeruginosa ATCC 27853, Escherichia coli
ATCC 25922, Klebsiella pneumoniae ATCC 4352, and Proteus mirabilis
ATCC 14153), and three fungi (Candida albicans ATCC 10231, Candida
parapsilosis ATCC 22019, and Candida tropicalis ATCC 750) by the
microbroth dilutions technique using the Clinical Laboratory Stan-
dards Institute (CLSI) recommendations (CLSI, 1997; CLSI, 2006). The
1,4-Quinones react with various amines to generate amino-1,4-quinone
compounds in different media with nucleophilic substitution reactions or
nucleophilic addition reactions by oxidative addition pathway. However,
this conversion has already been affected from the reaction medium and
it is found that the reaction takes place smoothly in water with high
yields as a solvent instead of other solvents. When the precursor (2) was
stirred with different heterocyclic amines (2.2 equivalent) viz. pyrrolidine,
piperidine, hexamethyleneimine, 2-methylpiperidine, 3-methylpiperidine,
4-methylpiperidine, 4-piperidinecarboxamide, 1,4-dioxa-8-azaspiro
SCHEME 1 Synthesis of the
chlorinated PQ analogs (PQ1-13):
(i) HNO3/HCl, 10 min, 90 ꢀC;
(ii) substituted heterocyclic amines,
H2O, 50–60 ꢀC, 6–18 hr

4
TUYUN ET AL.
TABLE 1 In vitro antibacterial activity results of the PQ analogs (PQ1-13)
Microorganisms
Gram positive bacteria (MIC, μg/mL)
ID
n
1
2
3
2
2
2
2
2
2
2
2
2
2
X
R1
H
H
H
H
H
H
H
H
H
H
H
H
H
R2
H
R3
Staphylococcus aureus
Staphylococcus epidermidis
Enterococcus faecalis
PQ1
CH
CH
CH
CH
CH
CH
C
H
–
312.5
78.12
1,250
1,250
1,250
625
19.53
625
9.76
19.53
2.44
625
–
625
312.5
625
625
–
PQ2
H
H
–
PQ3
H
H
625
9.76
78.12
9.76
–
PQ4
CH3
H
H
PQ5
CH3
PQ6
H
CONH2
OCH2CH2O
CH2Ph
–
625
625
–
PQ7
H
PQ8
CH
O
H
19.53
625
–
PQ9
H
156.25
156.25
–
PQ10
PQ11
PQ12
PQ13
Amikacin
Cefuroxime
S
H
–
N
H
COOEt
Ph
625
–
N
H
–
N
H
4-NO2-Ph
–
–
–
–
128
–
–
9.8
Cefuroxime-Na
1.2
–
–
FIGURE 2 ORTEP drawings of PQ7 and PQ12
MIC (minimum inhibitory concentration) values were determined by
comparison with standard agents.
625 μg/mL. Additionally, PQ3 and PQ4 had the same inhibitory activity
against P. mirabilis and E. coli with the MIC value at 625 μg/mL, respec-
tively. Some of the PQ analogs exhibited moderate activity against gram
positive bacteria. PQ7 and PQ10 showed good activity against
S. epidermidis with the MIC value of 19.53 μg/mL (twofold less potent
compared reference standard cefuroxime). As shown in Table 1, among
the all PQ analogs (PQ1-13), the activity of the PQ11 was the best and
most potent activity against S. epidermidis which was about fourfold
better than that of cefuroxime. Notably, PQ11, completely inhibited
the growth of S. epidermidis, was tested at the MIC level of
2.44 μg/mL. PQ9 had the same inhibitory activity against S. epidermidis
as that of cefuroxime (MIC = 9.76 μg/mL). PQ9 and PQ10 possessed
moderate activity against E. faecalis which had the MIC values
The antimicrobial assay results of all the PQ analogs (PQ1-13) are
given in Tables 1 and 2. Concerning the antibacterial activity, the gram
positive bacteria were more susceptible to PQ analogs than the gram
negative ones. Generally, the results showed that some compounds dis-
played varying effects on the growth of the tested gram positive bacte-
rial strains. The test-cultures E. coli, K. pneumoniae, and P. mirabilis were
appeared as resistant to the most synthesized compounds between the
studied concentrations at 2500–1.22 μg/mL. The results showed that
the analogs (PQ1, PQ5, and PQ8-13) exhibited no antibacterial activity
against the gram negative bacteria. PQ2-4, PQ6, and PQ7 had a
weak activity against P. aeruginosa with the MIC value at

TUYUN ET AL.
5
TABLE 2 In vitro antifungal activity results of the PQ analogs (PQ1-13)
Microorganisms
Fungi (MIC, μg/mL)
ID
n
1
2
3
2
2
2
2
2
2
2
2
2
2
X
R1
H
H
H
H
H
H
H
H
H
H
H
H
H
R2
H
R3
Candida albicans
Candida parapsilosis
Candida tropicalis
39.06
9.76
PQ1
PQ2
PQ3
PQ4
PQ5
PQ6
PQ7
PQ8
PQ9
PQ10
PQ11
PQ12
PQ13
CH
CH
CH
CH
CH
CH
C
H
78.12
39.06
156.25
–
156.25
78.12
78.12
312.5
312.5
625
H
H
H
H
78.12
156.25
312.5
312.5
156.25
156.25
19.53
19.53
156.25
312.5
625
CH3
H
H
CH3
–
H
CONH2
OCH2CH2O
CH2Ph
–
625
H
78.12
625
156.25
78.12
78.12
78.12
156.25
–
CH
O
H
H
78.12
78.12
19.53
–
S
H
–
N
H
COOEt
Ph
N
H
N
H
4-NO2-Ph
156.25
–
–
Amphotericin B
Clotrimazole
0.5
1
4.9
–
–
156.25 μg/mL very close to that of Amikacin. The most potent analogs
against S. aureus in the series were PQ4 and PQ6; but these were eight-
analogs was investigated by analyzing the structural features that are
essential for activity. The results obtained from the antimicrobial activity
study are quite interesting since they present some correlation against
S. epidermidis. PQ1 with the 5-membered ring containing only one nitro-
gen atom had low antibacterial activity against S. epidermidis
(MIC = 312.5 μg/mL). With the ring expansion from a 5-membered to a
7-membered ring containing only one nitrogen atom, the best MIC value
was for a 6-membered ring in the selected PQ1-3 against S. epidermidis.
When a methyl or an amide group was added in the 3- or 4-position of
the 6-membered ring (PQ4-6), the activity dramatically decreased. Lead-
ing the remarkable increase has been noticed for PQ analogs containing
ethylenedioxy group in the 4-position of the 6-membered ring (PQ7). For
the analogs by the introduction of the second heteroatom into the het-
erocyclic amine such as sulfur or oxygen to afford PQ9 or PQ10, PQ9
had the same activity with the control drug against S. epidermidis. PQ11,
both replacing the heteroatom with a nitrogen atom and having an ester
group in the 4-position on the 6-membered ring, had the best activity
against S. epidermidis. It is thus evident that PQ analogs containing
6-membered ring with additional heteroatom favor the inhibitory activity
against S. epidermidis.
fold less potent (MIC
= 9.76 μg/mL) than reference standard
cefuroxime-Na. Evaluation of the antifungal activity of the PQ analogs
exhibited that most of the PQ analogs (PQ1-13) possessed activity
against C. albicans, C. parapsilosis, and C. tropicalis with MIC values of
between 9.76 and 625 μg/mL. PQ2 was the most potent analog against
C. tropicalis (MIC = 9.76 μg/mL) while PQ11 was the most potent ana-
log against C. albicans (MIC = 19.53 μg/mL). PQ9 and PQ10 were also
effective against C. tropicalis at the MIC level of 19.53 μg/mL (Table 2).
According to the results, two analogs (PQ9 and PQ11) were the most
effective analogs and chosen for further investigation against the stan-
dard S. epidermidis and C. albicans. For that reason, we investigated
the potential antimicrobial activity against each of 20 clinically
obtained strains of Candida spp. and Staphyloccocus spp. Susceptibil-
ity testing demonstrated that the MIC ranges for PQ9 and PQ11
were 78.12–625 and 39.06–625 μg/mL for Candida spp. and 19.53–
>2,500 and 9.76–>2,500 μg/mL for Staphyloccocus spp.,
respectively.
2.3 | Structure–activity relationship study for
antibacterial and antifungal activity
2.4 | Determination of the in vitro antibiofilm
activity
The aim of using versatile heterocyclic amines was to investigate the
effects of the structural features of the PQ analogs on the antimicrobial
activity. A structure–activity relationship (SAR) of this class of the PQ
Because of the only biofilm which susceptible to studied active mole-
cules were S. epidermidis and C. albicans, we performed both biofilm

6
TUYUN ET AL.
FIGURE 3 Inhibition of Candida albicans and Staphylococcus epidermidis: (a) Surface attachment to the wells contained 1/10 × MIC of
molecules and an inoculum of 1 × 107 cfu/200 μL, incubated for 2, 4, or 6 hr for C. albicans (a1) and for S. epidermidis (a2) incubated for 1, 2, or
4 hr at 37 ꢀC; (b) Biofilm formation in each well contained 1 ×, 1/10 ×, or 1/100 × MIC of molecules and an inoculum of 5 × 105 cfu/200 μL,
incubated for 24 hr at 37 ꢀC (b1 for C. albicans and b2 for S. epidermidis). Control bars indicate microorganisms without molecules, accepted as
100%. Six wells were used for each molecule. Each experiment is representative of two independent tests, and the error bars indicate the
standard deviations. All differences between the control and the molecules treated biofilms were statistically significant (p < .001)
attachment and inhibition of biofilm formation assays with them.
Based on the tests performed, there was a significant positive correla-
tion between inhibition of the biofilm attachment and time for both
analogs (PQ9 and PQ11), especially for S. epidermidis for PQ9. In addi-
tion, they showed inhibitory activity against biofilm formation at 24 hr
according to their concentrations (Figure 3). Importantly, the results
showed that both analogs (PQ9 and PQ11) are able to reduce biofilm
mass, ranging from around 30% to 60%, at 1 × MIC, 1/10 × MIC, and
1/100 × MIC, respectively (Figure 3, b1 and b2).
fibroblasts), HUVEC (Human umbilical vein endothelial cells), HaCaT
(human keratinocytes), and NRK-52E (normal rat kidney cells) after
24 hr exposure. Our results demonstrated that the analog PQ9 did
not affect cell viability at tested concentrations below 12.5 μg/mL
and showed significant concentration-dependent cytotoxic activity
in the concentration range of 12.5–100 μg/mL after 24 hr exposure
on tested cell lines (p < .05), (Figure 4a). While the analog PQ11 did
not show cytotoxic effects tested concentrations below 6.25 μg/mL
and exhibited significant concentration-dependent cytotoxic activ-
ity in the concentration range of 6.25–100 μg/mL after 24 hr expo-
sure (p < .05), (Figure 4b). Based on the IC50 values, the analog
PQ11 is found to have greater cytotoxic activity than the analog
PQ9 for all of the tested cell lines (p < .05). HaCaT were the most
sensitive cells to both of the compounds with IC50 values of
24.36 μg/mL of PQ9 and 18.14 μg/mL of PQ11. While Balb/3T3
were the least affected cell line with IC50 values of 51.76 μg/mL of
PQ9 and 33.91 μg/mL of PQ11. IC50 values of PQ9 and PQ11 were
36.76 and 26.41 μg/mL on HUVEC, respectively. PQ9 and PQ11
affected the cell viability of NRK-52E cells similarly to the other cell
lines with IC50 values of 36.17 μg/mL and 22.73 μg/mL, respec-
tively. Taken together, these provide the foundation for further
investigation on whether the analogs PQ9 and PQ11 could be used
Regarding the control bars, we could say that microorganisms in
biofilm could alter physiological properties from their planktonic coun-
terparts. When a cell switches to the biofilm mode of growth, it
undergoes a phenotypic shift in behavior in which large suites of
genes are differentially regulated (Donlan, 2001; Ghannoum
&
O'Toole, 2004; Harrison, Ceri, Stremick, & Turner, 2004; Sanchez
et al., 2019). So, in these kinds of studies, variation in the results could
be observed. For this reason, six wells for each molecule in each
experiment and two repetitions were performed.
2.5 | Cytotoxic evaluation with MTT assay
Due to their significant inhibitory activity potentials against
S. epidermidis, the cytotoxic effects of PQ9 and PQ11 analogs were
determined by using MTT assay on Balb/3T3 (mouse embryonic

TUYUN ET AL.
7
cytotoxic potential than PQ9 for all of the cell lines that we used.
Taken together, these findings provide the foundation for further
investigation on whether PQ9 and PQ11 can be used for their
antibacterial properties at the concentrations without substantial
cytotoxicity. The analogs are more selective for the antibacterial activ-
ity, especially for gram positive bacteria than fungi. PQ analogs (PQ9
and PQ11) appear as the lead molecules as potent antibacterial agents
for further studies, that is, improving the biological activity and the
mechanism of action of these analogs against bacterial and fungal
strains. Therefore, these analogs could be applied in the future to con-
trol S. epidermidis infections one of the major HAIs.
4
| EXPERIMENTAL SECTION
4.1 | Chemistry
Melting points (mp) were determined using capillary tubes on a Buchi
B-540 melting point apparatus and were uncorrected. All
corresponding substituted heterocyclic amines, reagents and solvents
employed in the synthesis process were obtained from commercial
suppliers and used without further purification unless otherwise
noted. Merck DC-plates (aluminum based, silica gel 60 F254) were
used for analytical thin layer chromatography (TLC) purchased from
Merck KGaA. Plates were viewed by UV light (254 nm). Silica gel
60 (Merck, 63–200 μm particle sized, 60–230 mesh) was used for col-
umn chromatographic separations. Proton nuclear magnetic reso-
nance (1H NMR) and carbon nuclear magnetic resonance (13C NMR)
FIGURE 4 Cytotoxicity of PQ9 and PQ11 compounds on
Balb/3T3 (mouse embryonic fibroblasts), HUVEC (human umbilical
vein endothelial cells), HaCaT (human keratinocytes), and NRK-52E
(normal rat kidney cells) after 24 hr exposure with MTT assay. Cells
were treated with PQ9 and PQ11 at 1.5625, 3.125, 6.25, 12.5, 18.75,
25, 37.5, 50, 62.5, 75, and 100 μg/mL concentrations. Data are
presented as the mean SD (n = 3)
spectra were obtained on
a
VarianUNITY INOVA spectrometers
(500 MHz for 1H NMR and 125 MHz for 13C NMR) in CDCl3 refer to
the solvent signal center at δ 7.19 and δ 76.0 ppm. The chemical shifts
(δ) and coupling constants (J) are expressed in parts per million (ppm)
and hertz (Hz), respectively. Standard abbreviations indicating multi-
plicity were used as follows: s (singlet), br s (broad singlet), d (doublet),
t (triplet), q (quartet), dt (doublet of triplets), td (triplet of doublets), qd
(quartet of doublets), and m (multiplet). FTIR spectra were recorded as
ATR on either a Thermo Scientific Nicolet 6700 spectrometer or an
Alpha T FTIR spectrometer. Mass spectra were run by a BRUKER
Microflex LT by MALDI (Matrix Assisted Laser Desorption Ionization)-
TOF technique via the addition of 1,8,9-anthracenetriol (DIT,
dithranol) as the matrix. Data for the single crystal compounds were
obtained with Bruker APEX II QUAZAR three-circle diffractometer.
Indexing was performed using APEX2 (APEX2, 2014). Data integration
and reduction were carried out with SAINT (SAINT, 2013). Absorption
correction was performed by a multi-scan method implemented in
SADABS (SADABS, 2012). The Bruker SHELXTL (SHELXTL, 2000)
software package was used for structures solution and structures
refinement. Aromatic C-bound and N-bound hydrogen atoms were
positioned geometrically and refined using a riding mode. Crystal
structure validations and geometrical calculations were performed
using the Platon software (Spek, 2009). Mercury software (Macrae
et al., 2006) was used for visualization of the .cif files. The precursor
(2) was synthesized using the reported method in the literature (Ryu &
for their antibacterial properties at the concentrations without sub-
stantial cytotoxicity.
3
| CONCLUSIONS
In the present study, the synthesis and in vitro the antibacterial and
antifungal activity profile of a library of heterocyclic amine linked PQ
analogs have been reported. Two analogs (PQ7 and PQ10) had the
inhibitory activity against S. epidermidis, these were twofold less
potent than the reference standard of cefuroxime. Among the most
promising antibacterial compounds, PQ11 showed better antibacterial
activity than clinically prevalent antibacterial drug cefuroxime against
S. epidermidis, while PQ9 had similar activity. These two analogs (PQ9
and PQ11) effectively decreased biofilm formations against C. albicans
and S. epidermidis and had a better profile than control at different
concentrations and time. Thus, SAR study showed that the introduc-
tion of the second heteroatom, such as nitrogen or oxygen atom, on
the six-membered ring played an important role in the antibacterial
activity. Evaluation of cytotoxicity with nontumor cell lines showed
that IC50 values are >24 μg/mL for PQ9 while >18.14 μg/mL for
PQ11. Similar to their antibacterial activities, PQ11 exhibited more

8
TUYUN ET AL.
Lee, 2006). PQ1, PQ2, PQ9, and PQ10 have been prepared according
to the literature (Tuyun & Yıldız, 2018).
(500 MHz, CDCl3) δ (ppm): 0.82 (d, J = 6.3 Hz, 3H, CH3Piperidine),
1.03–1.12 (m, 1H, CHPiperidine), 1.62–1.69 (m, 2H, CH2Piperidine),
1.71–1.81 (m, 2H, CH2Piperidine), 1.92 (d, J = 2.44 Hz, 3H, CH3), 1.98
(d, J = 2.44 Hz, 3H, CH3), 2.69–2.75 (m, 1H, NCH2Piperidine), 3.02–3.08
(m, 1H, NCH2Piperidine), 3.48–3.57 (m, 2H, NCH2Piperidine). 13C NMR
(125 MHz, CDCl3) δ (ppm): 12.6, 13.1 (CH3), 19.0, 26.2, 32.1, 32.7,
52.0, 59.1 (CH2Piperidine), 118.4, 138.5, 141.2, 148.7 (Cq), 180.1, 184.1
(>C═O). MS (MALDI TOF) m/z: 267 [M]+. Anal. Calcd. for
4.2 | General procedure for the preparation of
heterocyclic amine linked plastoquinone analogs
(PQ1-13) (Tandon et al., 2010)
A suspension of the appropriate heterocyclic secondary amines
(1.10 mmol, 2.2 equiv) and 2,3-dichloro-5,6-dimethyl-1,4-benzo-
quinone 2 (0.1025 g, 0.50 mmol) in H2O (10 mL) was stirred at
50–60 ꢀC for 6–18 hr until consumption of the 1,4-benzoquinone.
The reaction mixture was cooled to ambient temperature. After evap-
oration of the solvent, the residue was dissolved with CHCl3 (50 mL),
and the solution was washed sequentially with water (3 × 30 mL). The
organic layer was dried over Na2SO4, filtered, and concentrated under
reduced pressure, and the residue was purified by means of column
chromatography on silica gel to give target compounds.
C14H18ClNO2 (267.75).
4.2.5 | 2-Chloro-5,6-dimethyl-3-(4-methylpiperidin-
1-yl)-1,4-benzoquinone (PQ5)
Following general procedure by applying 4-methylpiperidine
(0.1091 g, 1.10 mmol), the crude residue was purified by column chro-
matography to furnish PQ5 as a purple solid. Yield: 32%, mp
76–78 ꢀC. FTIR (ATR) υ (cm−1): 2,945, 2,904, 2,863 (CHaliphatic), 1,655
(>C═O). 1H NMR (500 MHz, CDCl3) δ (ppm): 0.91 (d, J = 6.8 Hz, 3H,
CH3Piperidine), 1.31 (qd, J = 12.7 and 3.9 Hz, 2H, CH2Piperidine),
1.49–1.59 (m, 1H, CHPiperidine), 1.61–1.66 (m, 2H, CH2Piperidine), 1.92
(d, J = 1.5 Hz, 3H, CH3), 1.98 (d, J = 1.0 Hz, 3H, CH3), 3.10 (td,
J = 13.2 and 2.4 Hz, 2H, NCH2Piperidine), 3.59 (dt, J = 13.2 and 2.4 Hz,
2H, NCH2Piperidine). 13C NMR (125 MHz, CDCl3) δ (ppm): 12.6, 13.1
(CH3), 21.9, 30.6, 35.1, 51.9 (CH2Piperidine), 118.5, 138.5, 141.2, 148.8
(Cq), 180.1, 184.1 (>C═O). MS (MALDI TOF) m/z: 267 [M]+. Anal.
Calcd. for C14H18ClNO2 (267.75).
4.2.1 | 2-Chloro-5,6-dimethyl-3-(pyrrolidin-1-yl)-
1,4-benzoquinone (PQ1)
The title analog (PQ1) was prepared according to the literature and
identified by comparing its spectral properties to those reported for
the analog in the literature (Tuyun & Yıldız, 2018).
4.2.2 | 2-Chloro-5,6-dimethyl-3-(piperidin-1-yl)-
1,4-benzoquinone (PQ2)
4.2.6 | 1-(2-Chloro-4,5-dimethyl-
3,6-dioxocyclohexa-1,4-dien-1-yl)piperidine-
4-carboxamide (PQ6)
The title analog (PQ2) was prepared according to the literature and
identified by comparing its spectral properties to those reported for
the analog in the literature (Tuyun & Yıldız, 2018).
Following general procedure by applying 4-piperidinecarboxamide
(0.1410 g, 1.10 mmol), the crude residue was purified by column chro-
matography to furnish PQ6 as a dark purple solid. Yield: 38%, mp
175–176 ꢀC. FTIR (ATR) υ (cm−1): 3,423, 3,315, 3,215 (NHamide),
2,956, 2,917, 2,849 (CHaliphatic), 1,660 (>C═O). 1H NMR (500 MHz,
CDCl3) δ (ppm): 1.80–1.93 (m, 4H, CH2Piperidine), 1.93 (d, J = 1.0 Hz,
3H, CH3), 1.99 (d, J = 1.0 Hz, 3H, CH3), 2.31–2.39 (m, 1H, CHPiperidine),
3.15 (td, J = 13.7 and 2.4 Hz, 2H, NCH2Piperidine), 3.66 (d, J = 13.7 Hz,
2H, NCH2Piperidine), 5.55 (br s, 1H, NH), 5.70 (br s, 1H, NH). 13C NMR
4.2.3 | 2-(Azepan-1-yl)-3-chloro-5,6-dimethyl-
1,4-benzoquinone (PQ3)
Following general procedure by applying hexamethyleneimine
(0.1091 g, 1.10 mmol), the crude residue was purified by column chro-
matography to furnish PQ3 as a dark purple oil. Yield: 24%. FTIR
(ATR) υ (cm−1): 2,924, 2,854 (CHaliphatic), 1,660 (>C═O). 1H NMR
(500 MHz, CDCl3) δ (ppm): 1.59–1.63 (m, 4H, CH2), 1.69–1.74 (m, 4H,
CH2), 1.92 (t, J = 1.5 Hz, 3H, CH3), 1.98 (t, J = 1.5 Hz, 3H, CH3),
3.50–3.54 (m, 4H, NCH2). 13C NMR (125 MHz, CDCl3) δ (ppm): 12.7,
13.1 (CH3), 27.4, 29.1, 29.7, 54.4 (CH2Azepan), 117.9, 138.7, 140.9,
150.4 (Cq), 180.1, 184.9 (>C═O). MS (MALDI TOF) m/z: 267 [M]+.
Anal. Calcd. for C14H18ClNO2 (267.75).
(125 MHz, CDCl3)
δ (ppm): 12.6, 13.1 (CH3), 29.7, 42.2, 50.9
(CH2Piperidine), 120.0, 138.8, 141.2, 148.4 (Cq), 176.8, 180.2, 183.9
(>C═O). MS (MALDI TOF) m/z: 267 [M-2CH3]+. Anal. Calcd. for
C14H17ClN2O3 (296.75).
4.2.7 | 2-Chloro-5,6-dimethyl-3-(1,4-dioxa-
4.2.4 | 2-Chloro-5,6-dimethyl-3-(3-methylpiperidin-
1-yl)-1,4-benzoquinone (PQ4)
8-azaspiro[4.5]decan-8-yl)-1,4-benzoquinone (PQ7)
Following general procedure by applying 1,4-dioxa-8-azaspiro[4.5]dec-
ane (0.1575 g, 1.10 mmol), the crude residue was purified by column
chromatography to furnish PQ7 as a dark red solid. Yield: 50%, mp
94–95 ꢀC. FTIR (ATR) υ (cm−1): 2,949, 2,919, 2,863 (CHaliphatic), 1,654
(>C═O). 1H NMR (500 MHz, CDCl3) δ (ppm): 1.77 (t, J = 5.9 Hz, 4H,
Following general procedure by applying 3-methylpiperidine
(0.1091 g, 1.10 mmol), the crude residue was purified by column chro-
matography to furnish PQ4 as a dark purple oil. Yield: 35%. FTIR
(ATR) υ (cm−1): 2,927, 2,848 (CHaliphatic), 1,659 (>C═O). 1H NMR

TUYUN ET AL.
9
CH2), 1.93 (s, 3H, CH3), 1.98 (s, 3H, CH3), 3.46 (t, J = 5.4 Hz, 4H, NCH2),
3.92 (s, 4H, OCH2). 13C NMR (125 MHz, CDCl3) δ (ppm): 11.6, 12.1
(CH3), 35.0 (CH2), 48.4 (NCH2piperidine), 63.4 (OCH2), 105.7 (Cpiperidine),
118.6, 137.7, 140.2, 147.5 (Cq), 179.2, 182.9 (>C═O). MS (MALDI TOF)
m/z: 312 [M+H]+. Anal. Calcd. for C15H18ClNO4 (311.76).
4.2.12 | 2-Chloro-5,6-dimethyl-
3-(4-phenylpiperazin-1-yl)-1,4-benzoquinone (PQ12)
Following general procedure by applying 1-phenylpiperazine (0.1785 g,
1.10 mmol), the crude residue was purified by column chromatography to
furnish PQ12 as a dark brown solid. Yield: 70%, mp 118–120 ꢀC. FTIR
(ATR) υ (cm−1): 2,959, 2,917, 2,841 (CHaliphatic), 1,658 (>C═O). 1H NMR
(500 MHz, CDCl3) δ (ppm): 1.93 (d, J = 1.0 Hz, 3H, CH3), 1.98 (d, J = 1.0 Hz,
3H, CH3), 3.22 (t, J = 4.9 Hz, 4H, NCH2), 3.57 (t, J = 4.9 Hz, 4H, NCH2),
6.79–6.82 (m, 1H, CHaromatic), 6.80 (t, J = 7.3 Hz, 1H, CHaromatic), 6.86 (d,
J = 7.8 Hz, 2H, CHaromatic), 7.17–7.22 (m, 2H, CHaromatic). 13C NMR
(125 MHz, CDCl3) δ (ppm): 11.6, 12.1 (CH3), 49.2, 49.9 (CH2piperidine),
115.5, 119.3, 128.2, 137.8, 140.3, 146.8, 150.1 (CHaromatic and Cq), 179.0,
182.8 (>C═O). MS (MALDI TOF) m/z: 331 [M + H]+. Anal. Calcd. for
C18H19ClN2O2 (330.81).
4.2.8 | 2-(4-Benzylpiperidin-1-yl)-3-chloro-
5,6-dimethyl-1,4-benzoquinone (PQ8)
Following general procedure by applying 4-benzylpiperidine (0.1928 g,
1.10 mmol), the crude residue was purified by column chromatography to
furnish PQ8 as a dark purple oil. Yield: 33%. FTIR (ATR) υ (cm−1): 3,026
(CHaromatic), 2,918, 2,848 (CHaliphatic), 1,659 (>C═O). 1H NMR (500 MHz,
CDCl3) δ (ppm): 1.38 (qd, J = 12.2 and 3.9 Hz, 2H, CH2Piperidine), 1.60–1.66
(m, 2H, CH2Piperidine), 1.66–1.74 (m, 1H, CHPiperidine), 1.89–1.92 (m, 3H,
CH3), 1.95–1.97 (m, 3H, CH3), 2.50 (d, J = 6.8 Hz, 2H, CH2), 3.05 (td,
J = 13.2 and 2.0 Hz, 2H, NCH2Piperidine), 3.56–2.62 (m, 2H, NCH2Piperidine),
7.06–7.09 (m, 2H, CHAromatic), 7.09–7.13 (m, 1H CHAromatic), 7.21
(t, J = 7.8 Hz, 2H CHAromatic). 13C NMR (125 MHz, CDCl3) δ (ppm): 12.6,
13.1 (CH3), 33.1, 37.8, 43.2, 51.8 (CH2Piperidine and CH2), 118.7, 125.9,
128.2, 129.2, 138.5, 140.1, 141.2, 148.7 (CHaromatic and Cq), 180.1, 184.1
(>C═O). MS (MALDI TOF) m/z: 343 [M]+. Anal. Calcd. for C20H22ClNO2
(343.85).
4.2.13 | 2-Chloro-5,6-dimethyl-3-(4-(4-nitrophenyl)
piperazin-1-yl)-1,4-benzoquinone (PQ13)
Following general procedure by applying 1-(4-nitrophenyl)piperazine
(0.2280 g, 1.10 mmol), the crude residue was purified by column chro-
matography to furnish PQ13 as a dark purple solid. Yield: 67%, mp
184–185 ꢀC. FTIR (ATR) υ (cm−1): 2,915 (CHaliphatic), 1,661 (>C═O). 1
H
NMR (500 MHz, CDCl3) δ (ppm): 1.95 (d, J = 1.0 Hz, 3H, CH3), 2.00
(s, 3H, CH3), 3.47–3.49 (m, 4H, NCH2), 3.57–3.61 (m, 4H, NCH2), 6.79
(d, J = 9.3 Hz, 2H, CHaromatic), 8.07 (d, J = 9.3 Hz, 2H, CHaromatic). 13C
4.2.9 | 2-Chloro-5,6-dimethyl-3-morpholino-
1,4-benzoquinone (PQ9)
NMR (125 MHz, CDCl3)
δ (ppm): 11.6, 12.1 (CH3), 47.0, 49.3
The title analog (PQ9) was prepared according to the literature and
identified by comparing its spectral properties to those reported for
the analog in the literature (Tuyun & Yıldız, 2018).
(CH2piperidine), 112.1, 119.9, 124.9, 138.0, 140.4, 146.5, 153.8
(CHaromatic and Cq), 179.0, 182.8 (>C═O). MS (MALDI TOF) m/z:
375 [M]+. Anal. Calcd. for C18H18ClN3O4 (375.81).
4.2.10 | 2-Chloro-5,6-dimethyl-3-thiomorpholino-
1,4-benzoquinone (PQ10)
4.3 | Antimicrobial activity
4.3.1 | Determination of minimum inhibitory
The title analog (PQ10) was prepared according to the literature and
identified by comparing its spectral properties to those reported for
the analog in the literature (Tuyun & Yıldız, 2018).
concentrations (MIC)
Antimicrobial activities against Staphylococcus aureus ATCC 29213, Staph-
ylococcus epidermidis ATCC 12228, Escherichia coli ATCC 25922, Klebsiella
pneumoniae ATCC 4352, Pseudomonas aeruginosa ATCC 27853, Proteus
mirabilis ATCC 14153, Enterococcus faecalis ATCC 29212, Candida albicans
ATCC 10231, Candida parapsilosis ATCC 22019, and Candida tropicalis
ATCC 750 were determined by the microbroth dilution technique using
the Clinical Laboratory Standards Institute (CLSI) recommendations (CLSI,
1997; CLSI, 2006). Mueller–Hinton broth for bacteria and RPMI-1640
medium for the yeast strain were used as the test media. Serial twofold
dilutions ranging from 2,500 μg/mL to 1.2 μg/mL were prepared in the
media. The inoculum was prepared using a 4–6 hr broth culture of each
bacteria and 24 hr culture of yeast strains adjusted to a turbidity equivalent
of a 0.5 McFarland standard, diluted in broth media to give a final concen-
tration of 5 × 105 cfu/mL for bacteria and 0.5 × 103 to 2.5 × 103 cfu/mL
for yeast in the test tray. The trays were covered and placed in plastic bags
to prevent evaporation. The trays containing Mueller–Hinton broth were
incubated at 35 ꢀC for 18–20 hr while the trays containing RPMI-1640
4.2.11 | Ethyl 4-(2-chloro-4,5-dimethyl-
3,6-dioxocyclohexa-1,4-dien-1-yl)piperazine-
1-carboxylate (PQ11)
Following general procedure by applying ethyl piperazine-1-carboxylate
(0.1740 g, 1.10 mmol), the crude residue was purified by column chro-
matography to furnish PQ11 as a purple oil. Yield: 60%. FTIR (ATR) υ
(cm−1): 2,915, 2,848 (CHaliphatic), 1,659, 1,649 (>C═O). 1H NMR
(500 MHz, CDCl3) δ (ppm): 1.21 (t, J = 7.3 Hz, 3H, CH3), 1.93 (d,
J = 1.0 Hz, 3H, CH3), 1.98 (d, J = 1.5 Hz, 3H, CH3), 3.35–3.37 (m, 4H,
NCH2), 3.52–3.54 (m, 4H, NCH2), 4.10 (q, J = 7.3 Hz, 2H, OCH2). 13C
NMR (125 MHz, CDCl3) δ (ppm): 11.6, 12.1, 13.7 (CH3), 28.7, 49.8
(NCH2piperidine), 60.6 (OCH2), 119.7, 138.0, 140.3, 147.0 (Cq), 154.5,
179.1, 182.8 (>C═O). MS (MALDI TOF) m/z: 326 [M]+. Anal. Calcd. for
C15H19ClN2O4 (326.78).

10
TUYUN ET AL.
medium were incubated at 35 ꢀC for 46–50 hr. The MIC was defined as
the lowest concentration of compound, giving complete inhibition of visi-
ble growth. As a control, antimicrobial effects of the solvents were investi-
gated against test microorganisms. The results were evaluated according
to the values of the controls.
vein endothelial cells) and 5 × 103 cells/well for NRK-52E (normal rat
kidney cells) were plated into 96 microplates and incubated overnight
before to start treatments. Compound stocks were dissolved in
dimethyl sulfoxide (DMSO) (Bioshop Canada, Ontario, Canada) and
stocks were diluted with the medium before to use. After changing
medium, compounds were added into wells at different concentra-
tions (1.5625–100 μg/mL). DMSO concentration was 0.25% in the
final culture medium and negative control cells received 0.25%
DMSO. Positive control wells received 1% TritonX-100. After 24 hr
exposure, cells were incubated with 5 mg/mL MTT (Santa Cruz Bio-
technology, Santa Cruz, CA) containing medium for 3 hr at 37 ꢀC in
the dark. Then the medium was discarded and MTT formazan dis-
solved in 100 μL DMSO. Optical density (OD) of the solutions was
measured at 590 nm using a microplate reader (Biotek, Epoch, Winoo-
ski, VT). All experiments were made as three independent experiments
and performed in triplicates. Cell viability calculated according to the
following formula:
According to the antimicrobial activity results, we also studied
in vitro activities of the PQ9 and PQ11 against each of 20 clinically
obtained strains of Candida spp. and Staphyloccocus spp. by the micro-
broth dilution technique as described by the CLSI recommendations
(CLSI, 1997; CLSI, 2006).
4.3.2 | Determination of antibiofilm activities
Biofilm attachment and inhibition of biofilm formation assays were per-
formed as previously described method with some modifications
(Mataraci & Dosler, 2012). 1/10 × MIC concentrations of molecules
were added to the 24 hr biofilm and plates were incubated 1, 2, and
4 hr for S. epidermidis and 2, 4, and 6 hr for C. albicans at 37 ꢀC; mole-
cules at 1× 1/10 × and 1/100 × MIC concentrations were added to the
24 hr biofilm and plates were incubated 24 hr at 37 ꢀC, respectively.
Six wells were used for each molecule. The positive controls were
microorganisms in TSB-glucose without molecules. After the incubation,
wells were washed with PBS solutions and measured at OD595 nm.
Cell viability (percentage of negative control) = [(ODsample − ODpositive
control)/(ODnegative control − ODpositive control)] × 100.
ACKNOWLEDGMENTS
This work was financially supported by Scientific Research Projects
Coordination Unit of Istanbul University (Project number: FBA-
2016-22584) for supplying the equipment and materials. The crystal-
lographic data have been deposited at the Cambridge Crystallographic
Data Centre, and CCDC reference numbers are 1894854 for the PQ
analog (PQ7) and 1894852 for the PQ analog (PQ12). The data can be
obtained available free of charge from http://www.ccdc.cam.ac.uk/
conts/retrieving.html or from the Cambridge Crystallographic Data
Centre (CCDC), Cambridge, UK; fax: +44 (0)1223336033; email:
deposit@ccdc.cam.ac.uk. We also gratefully thank Sadık Metin
Ceyhan for valuable suggestions on X-ray diffraction analysis from the
Department of Chemistry in the Faculty of Science at Gebze Techni-
cal University.
4.3.3 | Statistical analysis
All experiments were performed in two independent assays. One way
ANOVA-Bonferroni's multiple comparison test was used to compare
differences between control and antimicrobials treated biofilms.
p value <.001 was considered as statistically significant.
4.4 | Cytotoxicity
4.4.1 | Cell cultures
Balb/3T3 mouse embryonic fibroblasts, HUVEC Human umbilical vein
endothelial cells, HaCaT human keratinocytes, and NRK-52E normal
rat kidney cells were purchased from American Type Culture Collec-
tion (ATCC). All of the cells were incubated in Dulbecco's modified
Eagle's medium (GIBCO Laboratories, Grand Island, NY) supplemented
with 10% fetal bovine serum (GIBCO Laboratories) and 1% anti-
biotic/antimycotic solution (Invitrogen Carlsbad, CA) in a humidified
atmosphere of 95% air and 5% CO2 at 37 ꢀC. The cells were passaged
routinely when they reached 80% confluency.
AUTHOR CONTRIBUTIONS
A.F.T. designed the research, and A.F.T., M.Y., N.B., and
H.Y. performed the synthetic work. E.M.K. and B.Ö.Ç. coordinated
the biological research, E.M.K. and A.T.J. undertook the main biologi-
cal experiments. All authors discussed, edited, and approved the final
version.
CONFLICT OF INTEREST
4.4.2 | Cell treatments and MTT assay
The authors declare no conflict of interest.
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
assay was used to determine cytotoxic effects of compounds PQ9
and PQ11 after 24 hr exposure following the protocol described by
van Meerloo et al. (van Meerloo, Kaspers, and Cloos, 2011) with minor
modifications. 1 × 104 cells/well for Balb/3T3 (mouse embryonic
fibroblasts), HaCaT (human keratinocytes), HUVEC (Human umbilical
ORCID
Amaç F. Tuyun
https://orcid.org/0000-0001-5698-1109
https://orcid.org/0000-0003-3988-6120
Hatice Yıldırım

TUYUN ET AL.
11
Gutierrez, P. L. (2000). The metabolism of quinone-containing alkylating
agents: Free radical production and measurement. Frontiers in Biosci-
ence, 5, D629–D638.
Harrison, J. J., Ceri, H., Stremick, C., & Turner, R. J. (2004). Differences in
biofilm and planktonic cell mediated reduction of metalloid oxyanions.
FEMS Microbiology Letters, 235(2), 357–362.
REFERENCES
Alfadhli, A., Mack, A., Harper, L., Berk, S., Ritchie, C., & Barklis, E. (2016).
Analysis of quinolinequinone reactivity, cytotoxicity, and anti-HIV-1
properties. Bioorganic & Medicinal Chemistry, 24(21), 5618–5625.
APEX2, version 2014.1-1, Bruker, 2014, Bruker AXS Inc., Madison, WI.
Bayrak, N., Yildirim, H., Tuyun, A. F., Kara, E. M., Celik, B. O., Gupta, G. K.,
… Nasiri, H. R. (2017). Synthesis, computational study, and evaluation
of in vitro antimicrobial, antibiofilm, and anticancer activities of new
sulfanyl aminonaphthoquinone derivatives. Letters in Drug Design &
Discovery, 14(6), 647–661.
Begleiter, A. (2000). Clinical applications of quinone-containing alkylating
agents. Frontiers in Bioscience, 5, E153–E171.
Carneiro, P. F., Pinto, M. C. R. F., Marra, R. K. F., da Silva, F. D.,
Resende, J. A. L. C., Silva, L. F. R. E., … Ferreira, V. F. (2016). Synthesis
and antimalarial activity of quinones and structurally-related oxirane
derivatives. European Journal of Medicinal Chemistry, 108, 134–140.
Ikeda, T., Wakabayashi, H., Nakane, M. (1991). Benzoquinone antiallergy
and antiinflammatory agents. Patent.
Ilina, T. V., Semenova, E. A., Pronyaeva, T. R., Pokrovskii, A. G.,
Nechepurenko, I. V., Shults, E. E., … Tolstikov, G. A. (2002). Inhibition
of HIV-1 reverse transcriptase by aryl-substituted naphto- and anthra-
quinones. Doklady Biochemistry and Biophysics, 382, 56–59.
Janeczko, M., Demchuk, O. M., Strzelecka, D., Kubinski, K., & Maslyk, M. (2016).
New family of antimicrobial agents derived from 1,4-naphthoquinone.
European Journal of Medicinal Chemistry, 124, 1019–1025.
Jin, Y. R., Ryu, C. K., Moon, C. K., Cho, M. R., & Yun, Y. P. (2004). Inhibitory effects
of j78, a newly synthesized 1,4-naphthoquinone derivative, on experimental
thrombosis and platelet aggregation. Pharmacology, 70(4), 195–200.
Johnson-Ajinwo, O. R., Ullah, I., Mbye, H., Richardson, A., Horrocks, P., &
Li, W. W. (2018). The synthesis and evaluation of thymoquinone ana-
logues as anti-ovarian cancer and antimalarial agents. Bioorganic &
Medicinal Chemistry Letters, 28(7), 1219–1222.
Jordao, A. K., Novais, J., Leal, B., Escobar, A. C., dos Santos, H. M.,
Castro, H. C., & Ferreira, V. F. (2013). Synthesis using microwave irra-
diation and antibacterial evaluation of new N,O-acetals and N,S-
acetals derived from 2-amino-1,4-naphthoquinones. European Journal
of Medicinal Chemistry, 63, 196–201.
Kacmaz, A., Acar, E. T., Atun, G., Kaya, K., Sigirci, B. D., & Bagcigil, F.
(2018). Synthesis, electrochemistry, DFT calculations, antimicrobial proper-
ties and X-ray crystal structures of some NH- and/or S-substituted-1,-
4-quinones. ChemistrySelect, 3(30), 8615–8623.
Kawamukai, M. (2018). Biosynthesis and applications of prenylquinones.
Bioscience Biotechnology and Biochemistry, 82(6), 963–977.
Lien, J. C., Huang, L. J., Teng, C. M., Wang, J. P., & Kuo, S. C. (2002). Syn-
thesis of 2-alkoxy 1,4-naphthoquinone derivatives as antiplatelet,
antiinflammatory, and antiallergic agents. Chemical & Pharmaceutical
Bulletin, 50(5), 672–674.
Macrae, C. F., Edgington, P. R., McCabe, P., Pidcock, E., Shields, G. P.,
Taylor, R., … van De Streek, J. (2006). Mercury: Visualization and anal-
ysis of crystal structures. Journal of Applied Crystallography, 39,
453–457.
Mataraci, E., & Dosler, S. (2012). In vitro activities of antibiotics and anti-
microbial cationic peptides alone and in combination against
methicillin-resistant Staphylococcus aureus biofilms. Antimicrobial
Agents and Chemotherapy, 56(12), 6366–6371.
Castro, F. A. V., Mariani, D., Panek, A. D., Eleutherio, E. C. A.,
&
Pereira, M. D. (2008). Cytotoxicity mechanism of two naph-
thoquinones (Menadione and Plumbagin) in Saccharomyces cerevisiae.
PLoS One, 3(12), e3999.
Cerqueira, E. C., Netz, P. A., Diniz, C., do Canto, V. P., & Follmer, C. (2011).
Molecular insights into human monoamine oxidase (MAO) inhibition
by 1,4-naphthoquinone: Evidences for menadione (vitamin K3) acting
as a competitive and reversible inhibitor of MAO. Bioorganic & Medici-
nal Chemistry, 19(24), 7416–7424.
Clinical and Laboratory Standards Institute (CLSI), Reference Method for
Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved
Standard–Second Edition, Wayne, PA, 1997.
Clinical and Laboratory Standards Institute (CLSI), Methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically,
Wayne, PA, 2006.
Collett, N. P., Amin, A. R. M. R., Bayraktar, S., Pezzuto, J. M., Shin, D. M.,
Khuri, F. R., … Kucuk, O. (2010). Cancer prevention with natural com-
pounds. Seminars in Oncology, 37(3), 258–281.
Davids, H., Theunissen, R., Chakravorty, S., Mohammed, R., Frost, C., van
Otterlo, W. A. L., & de Koning, C. B. (2012). Aminonaphthoquinones as
potential anti-breast cancer agents. African Journal of Pharmacy and
Pharmacology, 6(45), 3102–3112.
Delarmelina, M., Daltoe, R. D., Cerri, M. F., Madeira, K. P., Rangel, L. B. A.,
Lacerda, V., … Greco, S. J. (2015). Synthesis, antitumor activity and
docking of 2,3-(substituted)-1,4-naphthoquinone derivatives con-
taining nitrogen, oxygen and sulfur. Journal of the Brazilian Chemical
Society, 26(9), 1804–1816.
Donlan, R. M. (2001). Biofilm formation: A clinically relevant microbiologi-
cal process. Clinical Infectious Diseases, 33(8), 1387–1392.
Egleton, J. E., Thinnes, C. C., Seden, P. T., Laurieri, N., Lee, S. P.,
Hadavizadeh, K. S., … Russell, A. J. (2014). Structure–activity relation-
ships and colorimetric properties of specific probes for the putative
cancer biomarker human arylamine N-acetyltransferase 1. Bioorganic &
Medicinal Chemistry, 22(11), 3030–3054.
Nain-Perez, A., Barbosa, L. C. A., Maltha, C. R. A., & Forlani, G. (2016). First
total synthesis and phytotoxic activity of Streptomyces sp. metabolites
abenquines. Tetrahedron Letters, 57(16), 1811–1814.
Nain-Perez, A., Barbosa, L. C. A., Maltha, C. R. A., & Forlani, G. (2017). Natural
abenquines and their synthetic analogues exert algicidal activity against
bloom-forming cyanobacteria. Journal of Natural Products, 80(4), 813–818.
Nain-Perez, A., Barbosa, L. C. A., Maltha, C. R. A., Giberti, S., & Forlani, G.
(2017). Tailoring natural abenquines to inhibit the photosynthetic elec-
tron transport through interaction with the D1 protein in photosystem
II. Journal of Agricultural and Food Chemistry, 65(51), 11304–11311.
Nain-Perez, A., Barbosa, L. C. A., Rodriguez-Hernandez, D., Kramell, A. E.,
Heller, L., & Csuk, R. (2017). Natural abenquines and synthetic ana-
logues: Preliminary exploration of their cytotoxic activity. Bioorganic &
Medicinal Chemistry Letters, 27(5), 1141–1144.
El-Dakhakhany, M. (1963). Studies on the chemical constitution of Egyp-
tian N. sativa L. seeds. Planta Medica, 11, 465–470.
European Centre for Disease Prevention and Control (ECDPC). 2013. Retrieved
from https://ecdc.europa.eu/en/publications-data/point-prevalence-
survey-healthcare-associated-infections-and-antimicrobial-use-0.
European Centre for Disease Prevention and Control (ECDPC). 2014. Anti-
microbial resistance and healthcare-associated infections’, in: Annual
epidemiological report 2014.
Ghannoum, M., & O'Toole, G. A. (2004). Microbial biofilms. Washington,
DC: ASM Press.
Nallapareddy, S. R., Singh, K. V., Sillanpaa, J., Garsin, D. A., Hook, M.,
Erlandsen, S. L., & Murray, B. E. (2006). Endocarditis and biofilm-
associated pili of Enterococcus faecalis. The Journal of Clinical Investiga-
tion, 116, 2799–2807.
Namvar, A. E., Bastarahang, S., Abbasi, N., Ghehi, G. S.,
Farhadbakhtiarian, S., Arezi, P., … Chermahin, S. G. (2014). Clinical
Glamoclija, U., Padhye, S., Spirtovic-Halilovic, S., Osmanovic, A., Veljovic, E.,
Roca, S., … Završnik, D. (2018). Synthesis, biological evaluation and docking
studies of benzoxazoles derived from thymoquinone. Molecules, 23(12),
Article Number: 3297.

12
TUYUN ET AL.
characteristics of Staphylococcus epidermidis: A systematic review.
GMS Hygiene and Infection Control, 9(3), Doc23.
nitrogen and sulfur containing hetero-1,4-naphthoquinones as potent
antifungal and antibacterial agents. European Journal of Medicinal
Chemistry, 45(6), 2418–2426.
Niedermeyer, T. H. J., Mikolasch, A., & Lalk, M. (2005). Nuclear amination cata-
lyzed by fungal laccases: Reaction products of p-hydroquinones and pri-
mary aromatic amines. The Journal of Organic Chemistry, 70(6), 2002–2008.
Pingaew, R., Prachayasittikul, V., Worachartcheewan, A., Nantasenamat, C.,
Prachayasittikul, S., Ruchirawat, S., & Prachayasittikul, V. (2015). Novel
1,4-naphthoquinone-based sulfonamides: Synthesis, QSAR, anticancer
and antimalarial studies. European Journal of Medicinal Chemistry, 103,
446–459.
Prachayasittikul, V., Pingaew, R., Worachartcheewan, A., Nantasenamat, C.,
Prachayasittikul, S., Ruchirawat, S., & Prachayasittikul, V. (2014). Synthe-
sis, anticancer activity and QSAR study of 1,4-naphthoquinone deriva-
tives. European Journal of Medicinal Chemistry, 84, 247–263.
The Centers for Disease Control and Prevention (CDCP), 2016. Retrieved
from https://www.cdc.gov/hai/data/portal/progress-report.html.
Tuyun, A. F., Bayrak, N., Yildirim, H., Onul, N., Kara, E. M., & Celik, B. O. (2015).
Synthesis and in vitro biological evaluation of aminonaphthoquinones and
benzo[b]phenazine-6,11-dione derivatives as potential antibacterial and
antifungal compounds. Journal of Chemistry, 2015, 1–8.
Tuyun, A. F., & Yıldız, M. (2018). Synthesis and characterization of new
cyclic aminobenzoquinones. Journal of the Turkish Chemical Society
Section A: Chemistry, 5(2), 371–380.
Valderrama, J. A., Delgado, V., Sepulveda, S., Benites, J., Theoduloz, C.,
Calderon, P. B., & Muccioli, G. G. (2016). Synthesis and cytotoxic activ-
ity on human cancer cells of novel Isoquinolinequinone-amino acid
derivatives. Molecules, 21(9), Article Number: 1199.
Richwien, A.,
& Wurm, G. (2004). Influence of 2-aryl-3-halogen/3--
hydroxy-1,4-naphthoquinones with salicylic and cinnamic acid partial
structures on the arachidonic acid cascade. Pharmazie, 59(3), 163–169.
Ryu, C. K., & Lee, J. Y. (2006). Synthesis and antifungal activity of
6-hydroxycinnolines. Bioorganic & Medicinal Chemistry Letters, 16(7),
1850–1853.
Ryu, C. K., Lee, S. K., Han, J. Y., Jung, O. J., Lee, J. Y., & Jeong, S. H. (2005).
Synthesis and antifungal activity of 5-arylamino-4,7-dioxobenzo[b]thio-
phenes. Bioorganic & Medicinal Chemistry Letters, 15(10), 2617–2620.
Ryu, C. K., Nho, J. H., Jin, G., Oh, S. Y., & Choi, S. J. (2014). Synthesis of benzofuro
[6,7-d]thiazoles, benzofuro[7,6-d]thiazoles and 6-arylaminobenzo[d]thia-
zole-4,7-diones as antifungal agent. Chemical & Pharmaceutical Bulletin, 62
(7), 668–674.
van Meerloo, J., Kaspers, G. J., & Cloos, J. (2011). Cell sensitivity assays:
The MTT assay. Methods in Molecular Biology, 731, 237–245.
Wellington, K. W. (2015). Understanding cancer and the anticancer activi-
ties of naphthoquinones—A review. RSC Advances, 5(26), 20309–
20338.
Wellington, K. W., Kolesnikova, N. I., Nyoka, N. B. P., & McGaw, L. J.
(2019). Investigation of the antimicrobial and anticancer activity of
aminonaphthoquinones. Drug Development Research, 80, 138–146.
World Health Organization (WHO). (2011). Report on the burden of
endemic health care-associated infection worldwide. Geneva, Switzer-
land: World Health Organization.
Ryu, C. K., Oh, S. Y., Choi, S. J., & Kang, D. Y. (2014). Synthesis of antifun-
gal evaluation of 2H-[1,2,3]Triazolo[4,5-g]isoquinoline-4,9-diones.
Chemical & Pharmaceutical Bulletin, 62(11), 1119–1124.
Widhalm, J. R., & Rhodes, D. (2016). Biosynthesis and molecular actions of
specialized 1,4-naphthoquinone natural products produced by horti-
cultural plants. Horticulture Research, 3, 16046.
SADABS, version 2012/2, Bruker, 2012, Bruker AXS Inc., Madison, WI.
SAINT, version 8.34A, Bruker, 2013, Bruker AXS Inc., Madison, WI.
Sanchez, M. C., Romero-Lastra, P., Ribeiro-Vidal, H., Llama-Palacios, A.,
Figuero, E., Herrera, D., & Sanz, M. (2019). Comparative gene expression
analysis of planktonic Porphyromonas gingivalis ATCC 33277 in the presence
of a growing biofilm versus planktonic cells. BMC Microbiology, 19(1), 58.
SHELXTL, version 6.14, Bruker, 2000, Bruker AXS Inc., Madison, WI.
Shrestha, J. P., Baker, C., Kawasaki, Y., Subedi, Y. P., de Paul, N. N. V.,
Takemoto, J. Y., & Chang, C. W. T. (2017). Synthesis and bioactivity
investigation of quinone-based dimeric cationic triazolium amphiphiles
selective against resistant fungal and bacterial pathogens. European
Journal of Medicinal Chemistry, 126, 696–704.
Xu, K., Xiao, Z. Y., Tang, Y. B., Huang, L., Chen, C. H., Ohkoshi, E., &
Lee, K. H. (2012). Design and synthesis of naphthoquinone derivatives
as antiproliferative agents and 20S proteasome inhibitors. Bioorganic &
Medicinal Chemistry Letters, 22(8), 2772–2774.
Yildirim, H., Bayrak, N., Tuyun, A. F., Kara, E. M., Celik, B. O., & Gupta, G. K.
(2017). 2,3-Disubstituted-1,4-naphthoquinones containing an arylamine
with trifluoromethyl group: Synthesis, biological evaluation, and compu-
tational study. RSC Advances, 7(41), 25753–25764.
Yuk, D. Y., Lee, H. Y., Ryu, C. K., Hong, J. T., Kang, W. S., Yoo, H. S., &
Yun, Y. P. (2000). Studies on the antithrombotic and antiplatelet activi-
ties of NQ304,
a newly synthesized naphthoquinone derivative.
Arzneimittel-Forschung, 50(3), E254–E259.
Spek, A. L. (2009). Structure validation in chemical crystallography. Acta
Crystallographica Section D, 65, 148–155.
Sugiura, K. (1961). Antitumor activity of mitomycin C. Cancer Chemother-
apy Reports, 13, 51–65.
Tandon, V. K., Chhor, R. B., Singh, R. V., Rai, S. J., & Yadav, D. B. (2004).
Design, synthesis and evaluation of novel 1,4-naphthoquinone deriva-
tives as antifungal and anticancer agents. Bioorganic & Medicinal Chem-
istry Letters, 14(5), 1079–1083.
Yuk, D. Y., Ryu, C. K., Hong, J. T., Chung, K. H., Kang, W. S., Kim, Y., …
Yun, Y. P. (2000). Antithrombotic and antiplatelet activities of
2-chloro-3-[4-(ethylcarboxy)-phenyl]-amino-1,4-naphthoquinone
(NQ12), a newly synthesized 1,4-naphthoquinone derivative. Biochem-
ical Pharmacology, 60(7), 1001–1008.
SUPPORTING INFORMATION
Tandon, V. K., Kumar, S., Mishra, N. N., & Shukla, P. K. (2012). Micelles cat-
alyzed chemo- and regio-selective one pot and one step synthesis of
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
2,3,5,6-tetrakis(alkyl
and
arylsulfanyl)-1,4-benzoquinones
and
2,5-diaminosubstituted-1,4-benzoquinones "in-water" and their bio-
logical evaluation as antibacterial and antifungal agents. European Jour-
nal of Medicinal Chemistry, 56, 375–386.
How to cite this article: Tuyun AF, Yıldız M, Bayrak N, et al.
Discovery of a new family of heterocyclic amine linked
plastoquinone analogs for antimicrobial evaluation. Drug Dev
Res. 2019;1–12. https://doi.org/10.1002/ddr.21591
Tandon, V. K., & Maurya, H. K. (2009). ‘On water’: Unprecedented
nucleophilic substitution and addition reactions with 1,4-quinones in
aqueous suspension. Tetrahedron Letters, 50(43), 5896–5902.
Tandon, V. K., Maurya, H. K., Verma, M. K., Kumar, R., & Shukla, P. K.
(2010). ‘On water’ assisted synthesis and biological evaluation of