2,3-Disubstituted-1,4-naphthoquinones containing an arylamine with trifluoromethyl group: Synthesis, biological evaluation, and computational study

Article abstract of DOI:10.1039/c7ra00868f

Antibacterial and antifungal organic compounds are becoming increasingly important for biomedical applications. This study deals with the synthesis, characterization of structures, in silico PASS prediction, and the discovery of antibacterial and antifungal properties based on new sulfanyl-1,4-naphthoquinone derivatives containing an arylamine with a trifluoromethyl group at different positions, which can be further applied in drug discovery and development. The in vitro antimicrobial potential of the newly synthesized compounds was evaluated in a panel of seven bacterial strains (three Gram-positive and four Gram-negative bacteria) and one yeast, with an additional study on antibiofilm activities. The compounds (5b and 5e) were identified as having strong antibacterial efficiency against the human-originated pathogen S. epidermidis, with minimal inhibitory concentration values (4.88 and 2.44 μg mL^-1, respectively). The toxicity of both compounds (5b and 5e) was studied in detail to compare these compounds with Cefuroxime (a clinically proven drug). The antibacterial activity of the compound 5f was equal to that of Cefuroxime. Moreover, three compounds (5b, 5e, and 5f) exhibited excellent antibacterial activity, and 5b and 5e were two and four times more active than the reference antimicrobial compound (Cefuroxime), respectively. For this reason, these three compounds (5b, 5e, and 5f) are being considered as promising antibacterial agents. In addition, docking studies were used to better rationalize the action and prediction of the binding modes of these compounds.

Full text of DOI:10.1039/c7ra00868f

RSC Advances
View Article Online
View Journal | View Issue
PAPER
2,3-Disubstituted-1,4-naphthoquinones containing
an arylamine with trifluoromethyl group: synthesis,
biological evaluation, and computational study†
Cite this: RSC Adv., 2017, 7, 25753
a
a
b
Emel Mataracı Kara,c
*
*
Hatice Yıldırım,
Nilufer Bayrak,
Amac Fatih Tuyun,
¨
Berna O¨ zbek Çelikc and Girish Kumar Gupta
d
Antibacterial and antifungal organic compounds are becoming increasingly important for biomedical
applications. This study deals with the synthesis, characterization of structures, in silico PASS prediction,
and the discovery of antibacterial and antifungal properties based on new sulfanyl-1,4-naphthoquinone
derivatives containing an arylamine with a trifluoromethyl group at different positions, which can be
further applied in drug discovery and development. The in vitro antimicrobial potential of the newly
synthesized compounds was evaluated in a panel of seven bacterial strains (three Gram-positive and four
Gram-negative bacteria) and one yeast, with an additional study on antibiofilm activities. The compounds
(5b and 5e) were identified as having strong antibacterial efficiency against the human-originated
pathogen S. epidermidis, with minimal inhibitory concentration values (4.88 and 2.44 mg mLÀ1
,
respectively). The toxicity of both compounds (5b and 5e) was studied in detail to compare these
compounds with Cefuroxime (a clinically proven drug). The antibacterial activity of the compound 5f was
equal to that of Cefuroxime. Moreover, three compounds (5b, 5e, and 5f) exhibited excellent
antibacterial activity, and 5b and 5e were two and four times more active than the reference
antimicrobial compound (Cefuroxime), respectively. For this reason, these three compounds (5b, 5e, and
5f) are being considered as promising antibacterial agents. In addition, docking studies were used to
better rationalize the action and prediction of the binding modes of these compounds.
Received 20th January 2017
Accepted 18th April 2017
DOI: 10.1039/c7ra00868f
rsc.li/rsc-advances
malaria, other pathogens, and infections that have been
causing the majority of deaths since 2010.1a,2
1. Introduction
Infectious diseases are among the most serious threats of the
“top global health risk factors”, just like the high risk factors of
chemical spills and nuclear accidents.1b,c,2,3 WHO has, therefore,
provided effective global strategies for the control of major
infectious diseases that are being dubbed as the “infectious
diseases control programs”, which are necessary in order to
help build these health systems.4
The fact that the resistance of bacterial and fungal patho-
gens leads to an ever-increasing global public health threat
reects the widespread and growing demand for new antibac-
terial and antifungal compounds, which are among the most
prevalently used drugs.5 Unfortunately, very few new classes of
antibacterial drugs have been registered for clinical practice in
the past 50 years.6 Not surprisingly, this has recently caused
a rapidly growing interest in the synthesis of new and efficient
antibacterial and antifungal compounds that may be drug
candidates for reducing infections caused by pathogens.7
The antimicrobial activities of an increasing number of
quinone compounds are being investigated for the discovery of
new antibacterial and antifungal compounds.7,8 A series of
sulfanyl-1,4-naphthoquinone derivatives was synthesized and
potent antifungal activity was exhibited in vitro by one
Although there has been a decline in the incidence of infectious
diseases, there is no place in the world where infectious
diseases are an insignicant cause of illness and death.1,2 The
number of deaths caused by pathogens and parasites have
shown a minor decline of only 1% per year. Over 10 million
people have died from infections since 1990.1a,2 The World
Health Organization (WHO) estimates 13 million deaths from
infectious diseases in 2050.2 People in low- and middle-income
countries suffer from diseases such as HIV/AIDS, tuberculosis,
aChemistry Department, Engineering Faculty, Istanbul University, Avcilar, 34320,
Istanbul, Turkey. E-mail: hyildirim@istanbul.edu.tr; Fax: +90 212 473 7180; Tel:
+90 212 473 7070
bEngineering Sciences Department, Engineering Faculty, Istanbul University, Avcilar,
34320, Istanbul, Turkey. E-mail: auyun@gmail.com; auyun@istanbul.edu.tr; Fax:
+90 212 473 7180; Tel: +90 212 473 7070
cPharmaceutical Microbiology Department, Pharmacy Faculty, Istanbul University,
Beyazit, 34116, Istanbul, Turkey
dDepartment of Pharmaceutical Chemistry, Maharishi Markandeshwar College of
Pharmacy, Maharishi Markandeshwar University, Mullana, Ambala 133207,
Haryana, India
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra00868f
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 25753–25764 | 25753

View Article Online
RSC Advances
Paper
compound, compared to the clinically proven antifungal drug
uconazole, against S. schenckii, and the clinically proven
drug amphotericin-B against T. mentagraphytes.9 1,4-Naph-
thoquinones containing an amino group have been used as
antibacterials, antimalarials, antituberculars, larvicides and
molluscicides, herbicides, and fungicides in many medical and
biological applications.10–14
A series of phenylamino-1,4-
naphthoquinones was synthesized to investigate their anti-
tumor effects against cancer cell lines and healthy broblasts,
and it was reported that the presence of a chlorine atom in the
acceptor quinone nucleus and/or the presence of a methyl
group at the nitrogen atom of the donor phenylamino group
induced changes in cytotoxic activity.15
Since the triuoromethyl group (–CF3) is one of the most
important substituents in organic chemistry because of its
interesting stereoelectronic prole,16 our attention was turned
to the naphthoquinone compounds containing the –CF3 on the
arylamines at the o-, m-, and p-positions, in light of previous
studies.17 The ÀCF3 has an effect based on the different electron
density distributions on the reactivity of the molecule. The
efficiency of –CF3 on the physiological activity is a very signi-
cant topic in pharmaceutical studies. The physiological prole
is dependent on the position of –CF3 within the bioactive
molecules.16
Fig.
1
Structures of some nitro free and nitro substituted
aminonaphthoquinones.17
A series of naphthoquinone derivatives has been synthesized
and tested for their biological activity against human African
trypanosomiasis, normally known as sleeping sickness,
a dangerous and oen ignored parasitic disease.17 The activities
of some compounds have been compared to evaluate the
importance of the position of the –CF3 group on the phenyl
amine ring and its substitution that caused by the inhibition of
T. brucei cell proliferation. Compounds shown in Fig. 1 with
–CF3 (electron withdrawing group) at different positions on the
phenyl amine ring showed T. brucei inhibitory activity with
average cytotoxicity. It was found that the presence of a chlorine
atom instead of a second arylamine led to an increase in the
activity. Additionally, changing the –CF3 from the para position
to the ortho position led to an increase in the activity. It was
reported that the reason for this enhancement in T. brucei
inhibitory activity might have been the strong electron with-
drawing groups (–CF3, –NO2, and Cl) within the structure;
replacing the –CF3 with the methyl group at different positions
led to activity loss. In this study, it can be clearly seen that the
substitution patterns (2nd and 4th positions) on aniline and
monoamino- or diamino-substituted compounds affect the T.
brucei inhibitory activity.17
Sulfanyl aminonaphthoquinone and aminonaphthoquinone
derivatives have important applications in medicinal chemistry
as biologically active substances. Among them, some most
striking examples are shown in Fig. 2. Tandon et al. reported the
synthesis of some sulfanyl aminonaphthoquinone compounds
(I–IV) and their biological evaluation. Some of these compounds
showed signicant antibacterial activity.7c,d Very recently,
another research study on the synthesis of the sulfanyl aryla-
mino quinone compounds was reported by Ryu et al. Some of
the tested compounds (V and VI) completely inhibited fungal
growth when compared to the Candida species.8a Tandon et al.
Fig. 2 Structures of some important sulfanyl aminonaphthoquinones
with biological activity in literature.
synthesized other series of sulfanyl aminonaphthoquinone
and aminonaphthoquinone derivatives, and the synthesized
compounds were tested for antibacterial and antifungal activi-
ties. Particularly, sulfanyl aminonaphthoquinone derivatives
containing the arylsulfanyl group did not exhibit any signicant
activity (VII). In this study, the antifungal activity was observed
to decrease when the 2,3-disubstituted moiety was altered by
the phenyl group.8b
There are several factors that affect the biological evaluation
of the quinone structures, which are listed as follows: (a)
a naphthoquinoidal moiety; (b) an aromatic ring such as
aniline; (c) the presence and position of substituents on the
phenyl amine ring, and insertion of additional electronegative
atoms (Cl, S, N etc.).15,17,18 Encouraged by all these facts, and in
25754 | RSC Adv., 2017, 7, 25753–25764
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
characteristic absorptions around 3275–3346 cmÀ1 (NH), 1657–
1676 cmÀ1 (C]O), 3419–3433 (OH) cmÀ1, 2851–2978 cmÀ1
(CHaliphatic) and 3024–3076 cmÀ1 (CHaromatic). The 1H NMR
spectra of 5a–k and 6 (CDCl3-d1) exhibited the NH signal as
a singlet between 7.64–7.93 ppm in the aromatic region and at
5.86–8.09 ppm corresponding to the aromatic protons. The
signals associated with the other functional groups all appeared
in the expected regions. Outstandingly, the presence of two peaks
for the carbonyl groups (–C]O) in 13C NMR spectra was the
proof of the two substituents (amino and sulfanyl groups)
attached to the naphthoquinone core. There was only one peak
for the carbonyl group in the 13C NMR spectrum of compound 6
because of the symmetry.
Fig. 3 Structures of the sulfanyl derivatives of 1,4-naphthoquinone
based on aryl amines containing ÀCF3 group.
order to continue our previous studies, which were mainly
about the synthesis of biologically active 1,4-naphthoquinone
2.2. The prediction of biological activity based on spectra
structures, we chose our compounds by (i) using sulfanyl The prediction of biological activity based on the spectral
derivatives of arylamino-1,4-naphthoquinone as the core analysis of the synthesized compounds in the present
structure; (ii) using arylamine ring containing the CF3 group at communication was easily carried out by using the freely
different positions (o-, m-, and p-); (iii) using different types of available website with the internet version of PASS and Phar-
thiols, as shown in Fig. 3. From this point of view, profound maExpert: http://www.pharmaexpert.ru/PASSOnline/.20,21 The
pharmacological effects of related compounds in previous activity spectrum for a molecule is a list of biological activity for
studies gave us clues regarding the synthesis, characterization, which the probability of the compound being active (Pa) and the
and evaluation of some new sulfanyl-1,4-naphthoquinone probability of the compound being inactive (Pi) are calculated.
derivatives containing an arylamine with the –CF3 at different Pa and Pi values are independent and their values vary from 0 to
positions, as antimicrobial agents.
1. For example, the PASS prediction results for one of the
compound (5e) are summarized in Table 1.
It was found that for antibacterial and anti-infective activity,
the compounds had Pa nearly equal to 0.2. These ndings also
showed some good experimental results and can be considered
to be valuable while planning the experiment, but some
important factors should be taken into account such as the
particular interest in some kind of activity, desirable novelty,
the available facilities for experimental testing, etc.
2. Results and discussion
2.1. The synthesis of substituted naphthoquinones
Herein, the synthesis of 3-arylamino-1,4-naphthoquinone
derivatives (3a–c) was carried out via the reactions of 2,3-
dichloro-1,4-naphthoquinone (1) and primary arylamines (2a–c)
containing an electron withdrawing substituent (–CF3) on the
rings at the o-, m-, and p-positions as the starting materials in
different mediums.17,19,27 Unlike the synthesis of 3a and 3b re-
2.3. The antimicrobial activity
ported previously in the scientic literature, compound 3c was All the newly synthesized sulfanyl 1,4-naphthoquinone deriva-
obtained in low yields in DMF in the presence of K2CO3 (10%). tives containing an arylamine with the –CF3 at different posi-
The synthesis of new sulfanyl 1,4-naphthoquinone derivatives tions (5a–k) and 2,3-disulfanyl 1,4-naphthoquinone (6) were
containing an arylamine with the –CF3 at different positions evaluated for their in vitro antibacterial and antifungal activi-
(5a–k) was achieved at room temperature via the reaction of 3- ties. The antimicrobial assay results for all the newly synthe-
arylamino-1,4-naphthoquinone derivatives (3a–c) with a variety sized 1,4-naphthoquinone structures (5a–k and 6) are given in
of thiols (4a–d) in CH2Cl2 in the presence of Et3N (1.1 eq.) as Table 2.
a base (shown in Scheme 1) in high yields.8 Additionally,
In vitro activities of the studied molecules against planktonic
compound 6 was obtained as a side-product from the reaction cells are summarized in Table 2. Concerning the antibacterial
of 2-chloro-3-((4-(triuoromethyl)phenyl)amino)naphthalene- activity, the results revealed that some compounds displayed
1,4-dione (3a) with butane-2-thiol (4a). Among the synthesized varying effects on the growth of the tested Gram-positive and
compounds, the sulfanyl 1,4-naphthoquinone derivatives (5a–k) Gram-negative bacterial strains. As indicated in Table 2, among
and the side product (6) are new, while the arylamino-1,4- the synthesized compounds of 5b–g and 6, they generally
naphthoquinone derivatives (3a–c) have been reported in showed potent antibacterial activity against some pathogenic
earlier studies.17,19,27 New sulfanyl 1,4-naphthoquinone deriva- strains that were tested. Actually, the activities of compounds 5b
tives containing an arylamine with the –CF3 at different posi- and 5e were superior as compared to that of Cefuroxime: 5b and
tions (5a–k) were obtained in the range of 30–85% yield as 5e completely inhibited the growth of S. epidermidis, and were
orange and red powders or oils. Spectroscopic methods like one tested at the MIC level of 4.88 and 2.44 mg mLÀ1, respectively. In
dimensional NMR techniques (1H, 13C), FTIR spectra and mass addition to these, the activity of compound 5f was equal to that
spectrometry were applied to conrm the structures of obtained of Cefuroxime. In general, most of the tested compounds
compounds. The FTIR spectra of 5a–k and 6 showed some showed better antibacterial activity against the Gram-positive
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 25753–25764 | 25755

View Article Online
RSC Advances
Paper
Scheme 1 Synthetic pathway for the preparation of new sulfanyl 1,4-naphthoquinone derivatives substituted with arylamines containing the
ÀCF3 at o-, m-, and p-positions.
bacteria compared to the Gram-negative ones. The results (5a–b, 5e–f, and 5h–k) exhibited no antibacterial activity against
revealed that compounds 5d and 5g exhibited moderate activity the Gram-negative bacteria. Regarding the antifungal activity of
against Gram-positive bacteria. The test-cultures P. aeruginosa, the tested compounds, only 2 compounds (5j and 5k) were able
E. coli, and S. aureus appeared non-effective to most of the to induce appreciable growth inhibitory activity against C.
synthesized compounds. Some of the synthesized compounds albicans.
(5d–e, 5g and 5j) possessed activity against E. faecalis, which had
To evaluate the importance of the position of the –CF3 on the
MIC values between 312.5–625 mg mLÀ1. In addition to E. fae- phenyl amine ring with respect to the biological efficiency, the
calis, the compounds (5d and 5g) possessed activity against S. activities of all the synthesized compounds were compared
epidermidis, which had the MIC value of 156.2 mg mLÀ1. 2,3- (Scheme 1). The decrease in the antibacterial activities of the
Disulfanyl 1,4-naphthoquinone (6) was only able to induce compounds with the –CF3 at the p-positions on the phenyl-
appreciable growth inhibitory activity against S. epidermidis, amine ring, (5a, 5d, 5g, and 5j) was the proof of an important
which had the MIC value of 78.12 mg mLÀ1. The compounds factor for the antibacterial activity. In terms of the structure–
25756 | RSC Adv., 2017, 7, 25753–25764
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
Table 1 PASS prediction results for compound (5e)
Pa
Pi
Activity
0.625 0.042 TP53 expression enhancer
0.589 0.094 Membrane permeability inhibitor
0.426 0.027 Pyruvate decarboxylase inhibitor
0.336 0.001 CDC25 phosphatase inhibitor
0.285 0.201 Aspartyltransferase inhibitor
0.233 0.173 Anti-infective
0.173 0.070 Protein synthesis inhibitor
0.179 0.144 Phosphatidylcholine-sterol O-acyltransferase inhibitor
0.158 0.045 Antibacterial, ophthalmic
0.138 0.025 Antibiotic anthracycline-like
activity relationship, the sulfanyl 1,4-naphthoquinone deriv-
atives containing an arylamine with the –CF3 at the m-posi-
tions (5b, 5e, 5h, and 5k) showed, in general, more potent
antibacterial activities than the other sulfanyl 1,4-naph-
thoquinone derivatives with an arylamine containing the –CF3
at the p-positions (5a, 5d, 5g, and 5j). The sulfanyl 1,4-naph-
thoquinone derivatives containing an arylamine with the –CF3
at the o-positions (5c, 5f, and 5i) showed, in general, more
potent antibacterial activity than other sulfanyl 1,4-naph-
thoquinone derivatives with an arylamine containing the –CF3
at the p-position, but unfortunately, not better than other
sulfanyl 1,4-naphthoquinone derivatives with an arylamine
containing the –CF3 at the m-positions. The replacement of
a halogen with the different types of sulfanyl derivatives led to
variable types of activities. However, aliphatic substitutions
like sec-butylthio and 2-hydroxypropylthio moieties of the
compounds (5b–f) in comparison to aralkyl or heteroaryl
substitutions (5g, 5h, and 5k) may contribute partially toward
biological potency with the additional effect of the position of
–CF3. Moreover, this particular modication does not signi-
cantly affect the antifungal activity. Indeed, the aralkyl deriv-
atives (5j and 5k) showed moderate inhibition effect against C.
albicans; this leads to the conclusion that the moiety may be
a factor affecting the antimicrobial activity of the synthesized
sulfanyl 1,4-naphthoquinone derivatives. In this study, when
the in vitro antimicrobial activities of all the newly synthesized
sulfanyl 1,4-naphthoquinone derivatives were investigated,
three molecules (5b, 5e, and 5f) had strong antibacterial effi-
ciencies against S. epidermidis, which is usually considered as
a common human originated pathogen.
The cytotoxic effects of the two compounds (5b and 5e) were
obtained as the IC50 values, which represent the molar drug
concentrations required to cause 50% inhibition of cell
viability. Consequently, all tested compounds signicantly
induced dose-dependent loss of viability in HepG2 cells aer
24 h. The IC50 values (5b: 19.54 mg mLÀ1, 5e: 10.18 mg mLÀ1) for
the compounds were four times higher than the MIC results
shown in Fig. 4 and 5.
2.4. Antibiolm activity
A biolm is a community of microbes embedded in a self-
produced polymeric matrix, which adheres to
a surface.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 25753–25764 | 25757

View Article Online
RSC Advances
Paper
biolm organisms are far more resistant to antimicrobial agents
than the organisms in the suspension (up to 1000-fold
increase).22
When we considered the antibiolm activities of the anti-
bacterial molecules 5b and 5e against the S. epidermidis biolms,
the MBEC values were 5000 and 1250 mg mLÀ1, respectively. The
MBEC/MIC ratio, which is one of the important parameters for
choosing antibiotics in the treatment of biolm associated
infections, was found to be 1024 and 512 fold, respectively, in
contrast to many antibiotics (up to 10 000 fold).22a
Since the only biolm susceptible to the studied active mole-
cules was S. epidermidis, both the biolm attachment and inhi-
bition of the biolm formation assays were performed. When
these tests were carried out, 5b inhibited the biolm attachment
according to the time, and it showed a signicant inhibition
activity against the biolm formation at the 24th hour according to
its varying concentrations (Fig. 6). In contrast to these ndings,
there was an inhibition in the biolm attachment of 5e at the end
of the 2nd hour; following that, as the number of cells increased
within the environment, this activity was decreased at the 4th hour.
The biolms are not affected by “therapeutically achievable
concentrations” of antimicrobial agents. The antibiolm ther-
apies are generally focused on the inhibition of the biolm
formation.22b For this purpose, the inhibitions of bacterial
attachment to the surfaces were investigated; the inhibition of
the biolm production at the MIC or subMIC values of mole-
cules were investigated as well. 5b and 5e were able to inhibit
the attachment of bacteria at the MIC or subMIC values, and the
biolm formation aer 24 hours was approximately 50% (at 1/
10Â MIC p < 0.001). Since inhibiting a mature biolm can be
very difficult, the inhibition of the biolm formation in the early
stages may seem to be more applicable and advantageous.
Fig. 4 The effect of the compound 5b on the % inhibition at different
concentrations.
Fig. 5 The effect of the compound 5e on the % inhibition at different
concentrations.
2.5. Docking studies
Microbial cells that grow in biolms behave quite differently
from their planktonic counterparts. The bacteria on the biolm
structures are protected from the environmental conditions,
antimicrobial agents, and host immune responses, and the
In silico approaches like docking studies are currently gaining
much attention to pre-access the probable mechanism for the
action and design of new templates towards more potential
antibacterial
agents.21f
The
signicant
experimental
Fig. 6 The inhibition of S. epidermidis. (A) The surface attachment to the wells containing 1/10Â MIC of molecules and an inoculum of 1 Â 107
cfu per 200 mL, incubated for 1, 2, or 4 h at 37 ꢀC. (B) The biofilm formation in each well containing 1Â, 1/10Â, or 1/100Â MIC of molecules and an
inoculum of 5 Â 105 cfu per 200 mL, incubated for 24 h at 37 ꢀC. The control bars indicating bacteria without the molecule of interest are
accepted as 100%. Six wells were used for each molecule. Each experiment is representative of the two independent tests, and the error bars
indicate the standard deviations. All the differences between the control and molecule-treated biofilms are statistically significant (p < 0.001).
25758 | RSC Adv., 2017, 7, 25753–25764
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
antibacterial activities of the compounds 5b and 5e gave us (catalytic site residue, ligand to mevalonate C1 carboxyl) and Ser
a hint to perform molecular docking studies to understand the 141 (ATP binding motif residue) polar active side chains indi-
protein–ligand interactions. The docking was performed via the cate the binding affinity of the compounds with the protein.
Molegro Virtual Docker 2010.4.1.0 program23 for the active site This could be further investigated under the topic of MDD
of Staphylococcus epidermidis mevalonate diphosphate decar- inhibitors.25
boxylase (PDB ID: 4DPT) in order to predict the binding mode
and support the biological results.24
3. Conclusion
Mevalonate diphosphate decarboxylase (MDD) catalyzed the
formation of isopentenyl 5-diphosphate in an ATP-dependent The sulfanyl 1,4-naphthoquinone derivatives, containing an
irreversible reaction and was therefore an attractive target for arylamine with the –CF3 at the different positions (5a–k), and
the inhibitor development that could lead to the new antimi- 2,3-disulfanyl 1,4-naphthoquinone (6) were synthesized via the
crobial agents.24b In the case of compound 5b, it revealed the nucleophilic substitution of aminonaphthoquinones (3a–c)
MolDock score of À72.77 and formed three interactions, shown with 1.1 eq. of aryl- and alkylthiols (4a–d) in CH2Cl2 in the
as green dotted lines (Fig. 7), which were expressed as hydrogen presence of Et3N (1.1 eq.) as a base at the room temperature.8 All
bonds formed with the oxygen atom of the C]O (naph- of the newly synthesized compounds (5a–k and 6) were tested
thoquinone) moiety at the position 4 with Arg 144 and Ala 14, for their antimicrobial activities. Based on the testing data for
˚
˚
with distances of 2.59 A and 3.19 A, respectively and one the presented compounds, the in vitro antimicrobial activities
hydrogen bond formed between the –NH– moiety at the posi- were evaluated against the different Gram-positive and Gram-
˚
tion of 3 and Asn 12 of distance 3.35 A.
negative bacterial strains in addition to the antifungal activi-
In the case of the compound 5e, it revealed the MolDock ties. The antibacterial prole of the synthesized compounds
score of À78.46 and formed the main interactions as depicted (5a–k) indicated that compounds (5b–k) had potent antibacte-
in Fig. 8, expressed as one hydrogen bond formed with the rial activities. Among the most promising antibacterial
oxygen atom of the C]O (naphthoquinone) moiety at the compounds, 5b and 5e exhibited better antibacterial activity
position 4, and bonds with the oxygen atom of the 2-hydrox- than clinically prevalent antibacterial drug Cefuroxime against
ypropylthio group with Ala 284 and Ser 141, with distances of S. epidermidis, while 5f had similar activity. The compounds (5b,
˚
˚
3.14 A and 2.93 A, respectively.
5e, and 5f) are therefore lead compounds as potent antibacterial
Compound 6 revealed the MolDock score of À56.46. The agents for further studies. The tested compounds (5b and 5e)
supposed Moldock score was found to be positively correlated signicantly induced dose-dependent loss of viability in HepG2
with the experimental result values, where the antibacterial cells aer 24 h. The IC50 values (5b: 19.54 mg mLÀ1, 5e: 10.18 mg
activities against S. epidermidis of the compounds of 5e, 5b and mLÀ1) for the compounds were four times higher, compared to
6 decrease from le to right. Interactions with the Arg 144 the MIC results. These results suggest that the sec-butylthio and
2-hydroxypropylthio moieties with the additional effect of the
position of –CF3 are promising leads for the development of
antibacterial agents. 5b and 5e were able to inhibit the attach-
ment of bacteria at the MIC or subMIC values and the biolm
formation aer 24 hours was approximately 50%, at 1/10Â MIC
(p < 0.001). The molecular docking studies mentioned above
further helped in supporting the experimental results. These
experimental ndings and structures with the additional efforts
will pave the way for more potent biologically active derivatives
containing the quinone chromophore.
Fig. 7 The binding mode of compound 5b in S. epidermidis mevalo-
nate diphosphate decarboxylase [PDB ID: 4DPT].
4. Experimental
4.1. Chemistry
All of the reagents were obtained from the commercial suppliers
and were used without further purication unless otherwise
noted. Petroleum ether had a boiling range of 40–60 ꢀC.
Analytical thin layer chromatography (TLC) plates were
purchased from Merck KGaA (silica gel 60 F254) based on Merck
DC-plates (aluminum based). Visualization of the chromato-
gram was performed by UV light (254 nm). Column chromato-
graphic separations were carried out using silica gel 60 (Merck,
1
63–200 mm particle size, 60–230 mesh). H NMR and 13C NMR
spectra were recorded with the VarianUNITY INOVA spectrom-
eters with 500 MHz frequency for 1H and 125 MHz frequency for
Fig. 8 The binding mode of compound 5e in S. epidermidis meval-
onate diphosphate decarboxylase [PDB ID: 4DPT].
1
13C NMR in ppm (d). H NMR spectra and 13C NMR spectra in
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 25753–25764 | 25759

View Article Online
RSC Advances
Paper
CDCl3 were referenced to the solvent signals centered at d 7.19 1.61–1.48 m, 4H (–CH2–); 1.24–1.22 dd, J: 6.84, 2.93 Hz, 6H
and d 76.0 ppm, respectively. Standard abbreviations indicating (–CH3); 0.96–0.93 td, J: 7.32, 2.44 Hz, 6H, (–CH3). 13C NMR (125
multiplicity were used as follows: s (singlet), br s (broad singlet), MHz, CDCl3) d (ppm): 178.3 (–C]O); 148.1 (Cq); 132.4, 125.9
d (doublet), t (triplet), and m (multiplet). Coupling constants J (–CHarom); 132.1 (Cq); 44.5 (–CH–); 29.8 (–CH2–); 20.2, 20.1, 10.4,
were given in Hz. FTIR spectra were recorded as ATR on either 10.3 (–CH3). MS (ESI+) m/z (%): 334 (100, [M]+), 335 (71, [M +
a Thermo Scientic Nicolet 6700 spectrometer or Alpha T FTIR H]+). Anal. calcd for C18H22O2S2 (334.50).
spectrometer. Mass spectra were obtained on either a Thermo
4.1.1.2. 2-(Sec-butylthio)-3-(3-(triuoromethyl)phenylamino)-
Finnigan LCQ Advantage MAX MS/MS spectrometer equipped naphthalene-1,4-dione (5b). This compound was synthesized
with ESI (electrospray ionization) sources. The purity of the from 2-chloro-3-((3-(triuoromethyl)phenyl)amino)naphthalene-
sulfanyl 1,4-naphthoquinone derivatives was obtained by HPLC 1,4-dione (3b) and butane-2-thiol (4a) as a red powder by using
analysis. HPLC was performed on a Shimadzu/DGU-20A5 HPLC the general procedure. Yield: 0.045 g, mp: 109–110 ꢀC, 39%. FTIR
apparatus tted with a 25 cm Chiralpac AD-H chiral column. (ATR) n (cmÀ1): 3275 (–NH), 3076 (–CHarom), 2967, 2929
The melting points (mp) were determined with a Buchi B-540 (–CHaliphatic), 1662 (–C]O). 1H NMR (500 MHz, CDCl3) d (ppm):
melting point apparatus, but were uncorrected. The 3- 8.09–8.08 d, J: 7.81 Hz, 1H (–CHarom); 8.01–8.00 d, J: 7.81 Hz, 1H
arylamino-1,4-naphthoquinone
derivatives
(3a–c)
were (–CHarom); 7.78 s, 1H (–NH); 7.69–7.65 td, J: 7.32, 0.97 Hz, 1H
prepared via the reaction between the 2,3-dichloro-1,4- (–CHarom); 7.62–7.59 t, J: 7.32 Hz, 2H, (–CHarom); 7.38–7.35 t, J:
naphthoquinone (1) and triuoromethyl substituted aryl 7.81 Hz, 1H (–CHarom); 7.32–7.30 d, J: 7.81 Hz, 1H (–CHarom); 7.09–
amines (2a–c) according to the literature reported previously 7.08 d, J: 7.81 Hz, 1H, (–CHarom); 2.98–2.93 m, 1H (–CH–); 1.36–
and the references cited therein.17,19,26,27 All of the synthesized 1.23 m, 2H (–CH2–); 0.97–0.95 d, J: 6.83 Hz, 3H (–CH3); 0.69–0.66
sulfanyl 1,4-naphthoquinone derivatives (5a–k) and side t, J: 7.32 Hz, 3H (–CH3). 13C NMR (125 MHz, CDCl3) d (ppm):
product (6) are new.
180.2, 179.1 (–C]O); 143.7, 137.8, 132.3, 129.7, 127.8, 124.4,
4.1.1. General procedure for synthesis of the sulfanyl 1,4- 119.9, 118.9, 118.1 (Cq); 133.6, 132.0, 126.0, 125.7 (–CHarom); 43.2
naphthoquinone derivatives (5a–k). The 3-arylamino-1,4- (–CH–); 28.7 (–CH2–); 19.3, 10.1 (–CH3). MS (ESI+) m/z (%): 406
naphthoquinone derivatives (3a–c) and thiols in CH2Cl2 were (100, [M + H]+). Anal. calcd for C21H18F3NO2S (405.43).
stirred at room temperature using Et3N. The resulting solution
4.1.1.3. 2-(Sec-butylthio)-3-((2-(triuoromethyl)phenyl)amino)-
was extracted with 100 mL chloroform, and then washed with naphthalene-1,4-dione (5c). This compound was synthesized
water (4 Â 100 mL) and dried over calcium chloride. The solvent from 2-chloro-3-((2-(triuoromethyl)phenyl)amino)naphthalene-
was removed in vacuo. The residue was subjected to column 1,4-dione (3c) and butane-2-thiol (4a) as a red oil by using the
chromatography on silica gel using suitable solvents to give the general procedure. Yield: 0.039 g, 70%. FTIR (ATR) n (cmÀ1): 3328
products.
(–NH), 3072 (–CHarom), 2963, 2922, 2872 (–CHaliphatic), 1664 (–C]
4.1.1.1. 2-(Sec-butylthio)-3-(4-(triuoromethyl)phenylamino)- O). 1H NMR (500 MHz, CDCl3) d (ppm): 8.08–8.07 dd, J: 7.32, 1.46
naphthalene-1,4-dione (5a). This compound was synthesized Hz, 1H (–CHarom); 8.00–7.98 dd, J: 7.32, 1.46 Hz, 1H (–CHarom);
from 2-chloro-3-((4-(triuoromethyl)phenyl)amino)naphthalene- 7.91 s, 1H (–NH); 7.68–7.65 td, J: 7.81, 0.98 Hz, 1H (–CHarom);
1,4-dione (3a) and butane-2-thiol (4a) as a red oil by using the 7.61–7.58 m, 2H, (–CHarom); 7.43–7.40 t, J: 7.81 Hz, 1H (–CHarom);
general procedure. Yield: 0.030 g, 52%. FTIR (ATR) n (cmÀ1): 3323 7.19–7.16 t, J: 7.81 Hz, 1H (–CHarom); 6.90–6.88 d, J: 8.30 Hz, 1H,
(–NH), 3030 (CHarom), 2967, 2929, 2875 (CHaliphatic), 1659 (C]O). (–CHarom); 3.07–3.02 m, 1H (–CH–); 1.42–1.26 m, 2H (–CH2–);
1H NMR (500 MHz, CDCl3) d (ppm): 8.10–8.08 dd, J: 7.81, 0.98 Hz, 0.99–0.98 d, J: 6.34 Hz, 3H (–CH3); 0.73–0.70 t, J: 7.32 Hz, 3H
1H (–CHarom); 8.03–8.01 dd, J: 7.81, 0.98 Hz, 1H (–CHarom); 7.75 (–CH3). 13C NMR (125 MHz, CDCl3) d (ppm): 180.1, 178.8 (–C]O);
bs, 1H (–NH); 7.70–7.66 td, J: 7.33, 1.47 Hz, 1H (–CHarom); 7.63– 144.9, 132.2, 130.8, 129.8, 125.3, 118.9 (Cq); 133.5, 125.9, 125.7,
7.60 td, J: 7.81, 1.47 Hz, 1H (–CHarom); 7.51–7.49 d, J: 8.30 Hz, 2H 124.0, 123.4 (–CHarom); 43.3 (–CH–); 28.9 (–CH2–); 19.3, 10.0
(–CHarom); 6.99–6.97 d, J: 8.30 Hz, 2H (–CHarom); 3.02–2.98 q, J: (–CH3). MS (ESI+) m/z (%): 406 (100, [M + H]+). Anal. calcd for
6.83 Hz, 1H (–CH–); 1.38–1.28 m, 2H (–CH2–); 0.99–0.97 d, J: 6.83 C21H18F3NO2S (405.43).
Hz, 3H (–CH3); 0.71–0.68 t, J: 7.32 Hz, 3H (–CH3). 13C NMR (125
4.1.1.4. 2-(2-Hydroxypropylthio)-3-(4-(triuoromethyl)phenyl-
MHz, CDCl3) d (ppm): 180.2, 179.1 (C]O), 143.4, 140.5 (Cq), amino)naphthalene-1,4-dione (5d). This compound was synthe-
133.6, 132.1, 126.0, 125.7, 120.5 (CHarom), 132.2, 129.7, 124.7 (Cq), sized from 2-chloro-3-((4-(triuoromethyl)phenyl)amino)
43.3 (–CH–); 28.8 (–CH2–); 19.4, 10.1 (–CH3). MS (ESIÀ) m/z (%): naphthalene-1,4-dione (3a) and 3-mercaptobutan-2-ol (4b)
402 (100, [M À 3H]+), 403 (22, [M À 2H]+). Anal. calcd for as an orange powder by using the general procedure. Yield:
C21H18F3NO2S (405.43).
Additionally,
(triuoromethyl)phenyl)amino)naphthalene-1,4-dione (3a) and (–CHaliphatic), 1669 (–C]O). 1H NMR (500 MHz, CDCl3)
0.085 g, mp: 139–140 ꢀC, 73%. FTIR (ATR) n (cmÀ1): 3419
6
was also obtained from 2-chloro-3-((4- (–OH), 3336 (–NH), 3030 (–CHarom), 2978, 2961, 2929
butane-2-thiol (4a) by using the general procedure.
d (ppm): 8.10–8.08 dd, J: 7.81, 1.46 Hz, 1H (–CHarom); 8.03–
8.01 dd, J: 7.32, 0.98 Hz, 1H (–CHarom); 7.86 s, 1H (–NH); 7.71–
7.68 td, J: 7.32, 1.47 Hz, 1H (–CHarom); 7.64–7.61 td, J: 7.32,
1.46 Hz, 1H (–CHarom); 7.53–7.51 d, J: 8.78 Hz, 2H (–CHarom);
7.05–7.03 d, J: 8.79 Hz, 2H (–CHarom); 3.64–3.60 m, 1H (–CH–);
2.77–2.73 dd, J: 13.18, 2.93 Hz, 1H (–CH2–); 2.48–2.43 dd, J:
13.66, 8.78 Hz, 1H (–CH2–); 1.06–1.05 d, J: 6.34 Hz, 3H (–CH3).
4.1.1.1.1 2,3-Bis(sec-butylthio)naphthalene-1,4-dione (6). Red
oil, yield: 0.035 g, 37%. FTIR (ATR) n (cmÀ1): 2967, 2925, 2875
(–CHaliphatic), 1661 (C]O). 1H NMR (500 MHz, CDCl3) d (ppm):
7.99–7.98 dd, J: 5.37, 2.93 Hz, 2H (–CHarom); 7.62–7.61 dd, J:
5.86, 2.93 Hz, 2H (–CHarom); 4.04–4.00 q, J: 6.83 Hz, 2H (–CH–);
25760 | RSC Adv., 2017, 7, 25753–25764
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
13C NMR (125 MHz, CDCl3) d (ppm): 181.4, 180.3 (–C]O); 8.29 Hz, 2H (–CHarom); 6.09–6.08 dd, J: 2.93, 1.95 Hz, 1H
135.1, 133.5, 127.4, 127.1 (–CHarom); 146.2, 142.1, 133.5, (–CHarom); 5.88–5.87 d, J: 3.42 Hz, 1H (–CHarom); 3.91 s, 2H
130.8, 126.2, 122.5 (Cq); 66.5 (–CH–); 43.6 (–CH2–); 22.1 (–CH2–). 13C NMR (125 MHz, CDCl3) d (ppm): 179.7, 178.9 (–C]
(–CH3). MS (ESI+) m/z (%): 429 (100, [M + Na]+), 407 (52, [M]+). O); 149.7, 144.8, 141.2, 140.9 (Cq); 133.7, 132.3, 132.0, 129.6,
Anal. calcd for C20H16F3NO3S (407.41).
125.9, 125.8, 121.0 (–CHarom); 124.8, 117.4, 109.5, 107.2 (Cq);
4.1.1.5. 2-(2-Hydroxypropylthio)-3-(3-(triuoromethyl)phenyl- 29.4 (–CH2–). MS (ESI+) m/z (%): 452 (27, [M + Na]+), 451 (100, [M
amino)naphthalene-1,4-dione (5e). This compound was synthe- + Na À 1]+). Anal. calcd for C22H14F3NO3S (429.41).
sized from 2-chloro-3-((3-(triuoromethyl)phenyl)amino)
4.1.1.8. 2-(Furan-2-ylmethylthio)-3-(3-(triuoromethyl)phenyl-
naphthalene-1,4-dione (3b) and 3-mercaptobutan-2-ol (4b) amino)naphthalene-1,4-dione (5h). This compound was synthe-
as a red oil by using the general procedure. Yield: 0.076 g, sized from 2-chloro-3-((3-(triuoromethyl)phenyl)amino)
65%. FTIR (ATR) n (cmÀ1): 3433 (–OH), 3346 (–NH), 3050 naphthalene-1,4-dione (3b) and furan-2-ylmethanethiol (4c) as
(–CHarom), 2980, 2930 (–CHaliphatic), 1664 (–C]O). 1H NMR a red oil by using the general procedure. Yield: 0.08 g, 66%.
(500 MHz, CDCl3) d (ppm): 8.09–8.07 d, J: 7.81 Hz, 1H FTIR (ATR) n (cmÀ1): 3308 (–NH), 3072 (–C–Oether), 2928
(–CHarom); 8.01–7.99 d, J: 7.32 Hz, 1H (–CHarom); 7.91 s, 1H (–CHaliphatic), 1668 (–C]O). 1H NMR (500 MHz, CDCl3) d (ppm):
(–NH); 7.70–7.66 td, J: 7.81, 1.46 Hz, 1H (–CHarom); 7.62–7.59 8.11–8.10 d, J: 7.81 Hz, 1H (–CHarom); 7.99–7.97 d, J: 7.80 Hz, 1H
td, J: 7.32, 1.47 Hz, 1H (–CHarom); 7.41–7.35 t, J: 7.81 Hz, 1H (–CHarom); 7.71–7.67 td, J: 7.81, 1.46 Hz, 1H (–CHarom); 7.66 s, 1H
(–CHarom); 7.37–7.35 t, J: 7.81 Hz, 1H (–CHarom); 7.25 s, 1H (–NH); 7.62–7.59 td, J: 7.81, 1.46 Hz, 1H (–CHarom); 7.35–7.34 m,
(–CHarom); 7.16–7.15 d, J: 7.32 Hz, 1H (–CHarom); 3.62–3.58 m, 2H (–CHarom); 7.13–7.12 m, 2H (–CHarom); 7.03–7.01 m, 1H
1H (–CH–); 2.71–2.68 dd, J: 13.67, 3.42 Hz, 1H (–CH2–); 2.43– (–CHarom); 6.10–6.09 dd, J: 3.41, 1.47 Hz, 1H (–CHarom); 5.87–
2.39 dd, J: 13.67, 8.79 Hz, 1H (–CH2–); 1.05–1.03 d, J: 6.35 Hz, 5.86 d, J: 3.41 Hz, 1H (–CHarom); 3.88 s, 2H (–CH2–). 13C NMR
3H (–CH3). 13C NMR (125 MHz, CDCl3) d (ppm): 181.5, 180.4 (125 MHz, CDCl3) d (ppm): 179.6, 179.0 (–C]O); 149.8, 145.7,
(–C]O); 146.7, 139.6, 133.5, 130.7, 129.4, 126.4, 122.0, 120.1, 141.2, 138.5, 132.4, 129.6, 128.1, 125.0, 120.5, 118.7, 115.9 (Cq);
117.4 (Cq); 135.1, 133.3, 127.4, 127.1 (–CHarom); 66.4 (–CH–); 133.7, 131.9, 126.0, 125.8, 109.5, 107.2 (–CHarom); 29.6 (–CH2–).
43.8 (–CH2–); 22.1 (–CH3). MS (ESIÀ) m/z (%): 407 (16, [M]+), MS (ESIÀ) m/z (%): 428 (100, [M À H]+). Anal. calcd for
406 (35, [M À H]+), 405 (100, [M À 2H]+). Anal. calcd for
C
22H14F3NO3S (429.41).
4.1.1.9. 2-((Furan-2-ylmethyl)thio)-3-((2-(triuoromethyl)phenyl)-
C
20H16F3NO3S (407.41).
4.1.1.6. 2-((2-Hydroxypropyl)thio)-3-((2-(triuoromethyl)phenyl)- amino)naphthalene-1,4-dione (5i). This compound was synthesized
amino)naphthalene-1,4-dione (5f). This compound was synthesized from 2-chloro-3-((2-(triuoromethyl)phenyl)amino)naphthalene-
from 2-chloro-3-((2-(triuoromethyl)phenyl)amino)naphthalene- 1,4-dione (3c) and furan-2-ylmethanethiol (4c) as a red oil by
1,4-dione (3c) and 3-mercaptobutan-2-ol (4b) as a red powder by using the general procedure. Yield: 0.07 g, 85%. FTIR (ATR) n
using the general procedure. Yield: 0.059 g, mp: 85–87 C, 66%. (cmÀ1): 3294 (–NH), 2922 (–CHaliphatic), 1676 (–C]O). 1H NMR (500
ꢀ
FTIR (ATR) n (cmÀ1): 3423 (–OH), 3328 (–NH), 3075 (–CHarom), MHz, CDCl3) d (ppm): 8.09–8.07 dd, J: 7.81, 0.98 Hz, 1H (–CHarom);
2966, 2919 (–CHaliphatic), 1667 (–C]O). 1H NMR (500 MHz, CDCl3) 7.97–7.95 dd, J: 7.81, 0.98 Hz, 1H (–CHarom); 7.75 s, 1H (–NH); 7.68–
d (ppm): 8.09–8.07 dd, J: 7.32, 0.98 Hz, 1H (–CHarom); 8.01–7.99 dd, 7.65 td, J: 7.32, 1.46 Hz, 1H (–CHarom); 7.60–7.56 m, 2H (–CHarom);
J: 7.81, 0.97 Hz, 1H (–CHarom); 7.93 s, 1H (–NH); 7.70–7.66 td, J: 7.39–7.36 t, J: 7.80 Hz, 1H (–CHarom); 7.21–7.17 q, J: 7.32 Hz, 1H
7.81, 1.46 Hz, 1H (–CHarom); 7.62–7.60 m, 2H (–CHarom); 7.47–7.44 (–CHarom); 7.12–7.10 d, J: 2.44 Hz, 1H (–CHarom); 6.82–6.80 d, J: 7.81
t, J: 7.80 Hz, 1H (–CHarom); 7.26–7.23 t, J: 7.80 Hz, 1H (–CHarom); Hz, 1H (–CHarom); 6.07–6.06 dd, J: 3.44, 1.96 Hz, 1H (–CHarom);
7.00–6.99 d, J: 7.80 Hz, 1H (–CHarom); 3.62–3.54 m, 1H (–CH–); 5.86–5.85 d, J: 3.90 Hz, 1H (–CHarom); 3.91 s, 2H (–CH2–). 13C NMR
2.96 s, 1H (–OH); 2.78–2.75 dd, J: 13.67, 2.96 Hz, 1H (–CH2–); 2.42– (125 MHz, CDCl3) d (ppm): 179.6, 178.7 (–C]O); 149.7, 145.9,
2.38 dd, J: 13.67, 8.79 Hz, 1H (–CH2–); 1.04–1.03 d, J: 6.35 Hz, 3H 141.2, 132.4, 130.9, 129.5, 125.5, 116.6, 116.1 (Cq); 133.7, 131.9,
(–CH3). 13C NMR (125 MHz, CDCl3) d (ppm): 180.3, 178.8 (–C]O); 131.8, 125.9, 125.7, 124.5, 124.0 (–CHarom); 29.4 (–CH2–). MS (ESI+)
146.2, 132.2, 131.1, 129.5, 125.6 (Cq); 133.8, 132.1, 126.2, 125.9, m/z (%): 452 (100, [M + Na]+), 430 (22, [M + H]+). Anal. calcd for
124.9, 124.4 (–CHarom); 65.0 (–CH–); 42.7 (–CH2–); 20.7 (–CH3). MS C22H14F3NO3S (429.41).
(ESI+) m/z (%): 408 (100, [M + H]+), 430 (54, [M + Na]+). Anal. calcd
for C20H16F3NO3S (407.41).
4.1.1.10. 2-(Phenethylthio)-3-(4-(triuoromethyl)phenylamino)-
naphthalene-1,4-dione (5j). This compound was synthesized from
4.1.1.7. 2-(Furan-2-ylmethylthio)-3-(4-(triuoromethyl)phenyl- 2-chloro-3-((4-(triuoromethyl)phenyl)amino)naphthalene-1,4-
amino)naphthalene-1,4-dione (5g). This compound was synthe- dione (3a) and 2-phenylethanethiol (4d) as a red powder by
sized
from
2-chloro-3-((4-(triuoromethyl)phenyl)amino) using the general procedure. Yield: 0.074 g, mp: 85–87 ꢀC, 57%.
naphthalene-1,4-dione (3a) and furan-2-ylmethanethiol (4c) as FTIR (ATR) n (cmÀ1): 3321 (–NH), 3272, 3060, 3025 (–CHarom);
a red powder by using the general procedure. Yield: 0.037 g, mp: 2930 (–CHaliphatic), 1662 (–C]O). 1H NMR (500 MHz, CDCl3)
91–92 ꢀC, 30%. FTIR (ATR) n (cmÀ1): 3334 (–NH), 3104 (–C– d (ppm): 8.07–8.05 dd, J: 7.33, 1.46 Hz, 1H (–CHarom); 8.00–7.98
1
O–ether), 2964, 2918, 2851 (–CHaliphatic), 1659 (–C]O). H NMR dd, J: 7.32, 1.47 Hz, 1H (–CHarom); 7.69–7.66 td, J: 7.32, 1.47 Hz,
(500 MHz, CDCl3) d (ppm): 8.10–8.08 dd, J: 7.81, 1.46 Hz, 1H 1H (–CHarom); 7.64 bs, 1H (–NH); 7.63–7.60 td, J: 7.33, 1.46 Hz,
(–CHarom); 7.99–7.97 dd, J: 7.81, 1.46 Hz, 1H (–CHarom); 7.70– 1H (–CHarom); 7.50–7.48 d, J: 8.78 Hz, 2H (–CHarom); 7.06–7.03 t,
7.67 td, J: 7.32, 1.46 Hz, 1H (–CHarom); 7.66 bs, 1H (–NH); 7.62– J: 7.33 Hz, 2H (–CHarom); 6.98–6.96 d, J: 7.32 Hz, 1H (–CHarom);
7.59 td, J: 7.81, 1.47 Hz, 1H (–CHarom); 7.48–7.47 d, J: 8.30 Hz, 2H 6.94–6.92 d, J: 8.30 Hz, 4H (–CHarom); 2.88–2.85 t, J: 7.32 Hz, 2H
(–CHarom); 7.12–7.11 t, J: 0.98 Hz, 1H, (–CHarom); 6.93–6.91 d, J: (–CH2–); 2.66–2.63 t, J: 7.81 Hz, 2H (–CH2–). 13C NMR (125 MHz,
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 25753–25764 | 25761

View Article Online
RSC Advances
Paper
CDCl3) d (ppm): 180.0, 178.9 (–C]O); 142.5, 140.5, 138.5, 134.2
(Cq); 133.5, 132.3, 132.0, 127.5, 127.3, 125.9, 125.7 (–CHarom);
129.6, 125.4, 124.7, 120.2 (Cq); 35.3, 33.5 (–CH2–). MS (ESIÀ) m/
z (%): 453 (20, [M]+), 452 (48, [M À H]+), 451 (100, [M À 2H]+).
Anal. calcd for C25H18F3NO2S (453.48).
4.2.2. Determination of antibiolm activity. Measurements
of the antimicrobial susceptibilities of the bacterial and C.
albicans biolms were assessed using a minimum biolm
eradication concentration (MBEC) assay, which was performed
with the stated mentioned techniques as in the following
modications.30 The 24 h biolms were grown in a 96 well tissue
culture, the microtiter plates were washed three times with 250
mL PBS solutions and air-dried. Serial two-fold dilutions ranging
from 10 000 to 625 mg mLÀ1 for the molecules were prepared in
cation adjusted Mueller–Hinton broth (CAMHB). Following
that, 200 mL of each sample concentration were added to each
corresponding well and the plates were incubated for 24 h at
37 ꢀC. Aer the incubation, the antibiotics were gently aspi-
rated, the plates were washed, thoroughly scraped, and the
contents of each well were incubated in a sonicating water bath
for 5 minutes to disrupt the biolms. 100 mL samples were
plated on TSA and the colonies were counted aer 24 h of
4.1.1.11. 2-(Phenethylthio)-3-(3-(triuoromethyl)phenylamino)-
naphthalene-1,4-dione (5k). This compound was synthesized from
2-chloro-3-((3-(triuoromethyl)phenyl)amino)naphthalene-1,4-
dione (3b) and 2-phenylethanethiol (4d) as a red powder by
using the general procedure. Yield: 0.05 g, mp: 93–95 ꢀC, 39%.
FTIR (ATR) n (cmÀ1): 3308 (–NH), 3063, 3025 (–CHarom), 2931
(–CHaliphatic), 1657 (–C]O). 1H NMR (500 MHz, CDCl3) d (ppm):
8.06–8.05 dd, J: 7.32, 1.46 Hz, 1H (–CHarom); 7.99–7.97 dd, J:
7.32, 0.97 Hz, 1H (–CHarom); 7.68–7.65 td, J: 7.32, 1.47 Hz, 1H
(–CHarom); 7.66 s, 1H (–NH); 7.61–7.58 td, J: 7.80, 1.46 Hz, 1H
(–CHarom); 7.36–7.33 t, J: 7.81 Hz, 1H (–CHarom); 7.31–7.29 d, J:
7.81 Hz, 1H (–CHarom); 7.15 s, 1H, (–CHarom); 7.06–7.03 m, 3H
(–CHarom); 6.98–6.93 m, 3H (–CHarom); 2.86–2.83 t, J: 7.33 Hz,
2H (–CH2–); 2.63–2.60 t, J: 7.32 Hz, 2H (–CH2–). 13C NMR (125
MHz, CDCl3) d (ppm): 179.9, 179.0 (–C]O); 143.1, 138.6, 138.0,
132.4, 129.6, 127.9, 125.4, 124.0, 119.9, 118.6, 117.8 (Cq); 133.6,
132.0, 127.5, 127.3, 125.9, 125.7 (–CHarom); 35.2, 33.6 (–CH2–).
MS (ESIÀ) m/z (%): 453 (25, [M]+), 452 (100, [M À H]+). Anal.
calcd for C25H18F3NO2S (453.48).
ꢀ
incubation at 37 C. MBEC was dened as the lowest concen-
tration of molecules where the microorganism failed to regrow
aer the exposure.
Biolm attachment and inhibition of the biolm formation
assays were performed as in the previously described method
with some further modications.30 1/10Â MIC of molecules
were added to the 24 h biolm and the plates were incubated for
ꢀ
1, 2 and 4 h at 37 C; molecules at 1Â, 1/10Â and 1/100Â MIC
concentrations were added to the 24 h biolm, and plates were
incubated for 24 h at 37 ꢀC, respectively. Six wells were used for
each molecule. The positive controls were microorganisms in
TSB-glucose without the molecules of interest. Aer the incu-
bation, wells were washed with PBS solutions and measured at
OD595 nm.
4.2. In vitro antimicrobial activity
4.2.1. Determination of minimum inhibitory concentra-
tions (MIC). Antibacterial activities against the three Gram-
positive bacteria (Staphylococcus aureus ATCC 29213, Staphylo-
coccus epidermidis ATCC 12228, and Enterococcus faecalis ATCC
29212) and four Gram-negative bacteria (Pseudomonas aerugi-
nosa ATCC 27853, Escherichia coli ATCC 25922, Klebsiella pneu-
moniae ATCC 4352, and Proteus mirabilis ATCC 14153) and the
antifungal activity against a yeast Candida albicans ATCC 10231
were determined by the microbroth dilutions technique using
the Clinical Laboratory Standards Institute (CLSI) recommen-
dations.28,29 Mueller–Hinton broth for bacteria and RPMI-1640
medium for the yeast strain were used as the test media.
Serial two-fold dilutions ranging from 2500 mg mLÀ1 to 0.61 mg
mLÀ1 were prepared in the medium. Inoculum was prepared
using a 4–6 h broth culture for each bacteria type and 24 hours
of culture of yeast strains were adjusted to a turbidity equivalent
to the 0.5 McFarland standard, diluted in broth media to give
4.2.3. Statistical analysis. All of the experiments were per-
formed in two independent assays. In the determination of MIC
and MBEC values, when the results were different in the both
experiments, we made another test for the nal result. In the
assays of biolm attachment and the inhibition of biolm
formation, results were presented as mean Æ standard devia-
tion of two independent experiments. One way ANOVA-Bon-
ferroni's multiple comparison test was used to compare the
differences between the control and the antimicrobial treated
biolms. P value < 0.001 was considered as a statistically
signicant parameter.
4.3. Cell culture and cytotoxicity assays
a nal concentration of 5 Â 105 cfu mLÀ1 for the bacteria and 5 Compounds 5b and 5e were assayed for in vitro cytotoxicity
 103 cfu mLÀ1 for the yeast in the test tray. The trays were against the hepatocellular carcinoma (HepG2) cell line, which
covered and placed into plastic bags to prevent evaporation. The was kindly provided by the Department of Pharmaceutical
trays containing Mueller–Hinton broth were incubated at 35 ꢀC Toxicology in the Faculty of Pharmacy at Istanbul University.
for 18–20 h, while the trays containing RPMI-1640 medium were HepG2 cells were grown in Eagle's Minimum Essential Medium
incubated at 35 ꢀC for 46–50 h. The MIC was dened as the (EMEM) (Gibco), and were supplemented with 10% FBS (Gibco),
lowest concentration of compound, where the complete inhi- and 100 units per mL penicillin G and 100 units per mL strep-
ꢀ
bition of visible growth can be seen. As the control, the anti- tomycin under the humidied, 5% CO2 atmosphere at 37 C.
microbial effects of the solvents were investigated against the For the assays, the cells were seeded as 1 Â 104 cells per well in
test microorganisms. According to these values of the control 96 well plates and were allowed 24 h to ensure the attachment.
groups, the results were evaluated. The MIC values of the Following the 24 h, the cells were incubated either in the
compounds are given in Table 2.
absence or presence of serial dilutions of test compounds for
25762 | RSC Adv., 2017, 7, 25753–25764
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
a further 24 h. Aer the 24 h, cell viability was determined by
XTT (Xenometrix) (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-
2H-tetrazolium-5-carboxanilide) assay in accordance with the
manufacturer's protocol.31 In each assay, three replicates for
each concentration were used and this process, which used
three replicates was repeated four times. The cytotoxic effects of
each compound were obtained as IC50 values, which represent
the molar drug concentrations required to cause 50% inhibition
of cell viability.
7 (a) A. Riffel, L. F. Medina, V. Stefani, R. C. Santos, D. Bizani
and A. Brandelli, Braz. J. Med. Biol. Res., 2002, 35, 811–818;
(b) C. K. Ryu, R. E. Park, M. Y. Ma and J. H. Nho, Bioorg.
Med. Chem. Lett., 2007, 17, 2577–2580; (c) V. K. Tandon,
H. K. Maurya, D. B. Yadav, A. Tripathi, M. Kumar and
P. K. Shukla, Bioorg. Med. Chem. Lett., 2006, 16, 5883–5887;
(d) V. K. Tandon, H. K. Maurya, M. K. Verma, R. Kumar
and P. K. Shukla, Eur. J. Med. Chem., 2010, 45, 2418–2426.
8 (a) C. K. Ryu, J. A. Choi and S. H. Kim, Arch. Pharmacal Res.,
1998, 21, 440–444; (b) V. K. Tandon, H. K. Maurya,
N. N. Mishra and P. K. Shukla, Eur. J. Med. Chem., 2009,
44, 3130–3137; (c) V. K. Tandon, D. B. Yadav, H. K. Maurya,
A. K. Chaturvedi and P. K. Shukla, Bioorg. Med. Chem.,
2006, 14, 6120–6126.
4.4. Docking studies
The molecules were built using ChemSketch 12.0.1 and con-
verted to 3D structure from the 2D structures, and further 3D
structures were energetically minimized and saved as MDL Mol
File (*.mol2). The docking was performed by Molegro Virtual
Docker 2010.4.1.0 program.23 Staphylococcus epidermidis
mevalonate diphosphate decarboxylase [PDB ID: 4DPT] was
taken as the target enzyme for the rationalization of the anti-
bacterial activity.24 The polar hydrogen atoms, partial atomic
charges and solvation parameters were added. The MolDock
optimization search algorithm with a maximum of 100 runs was
used for the calculations, while all the other parameters were
kept as default. All the spatial structures were examined
manually, and the best one was attained for further use.
9 V. K. Tandon, H. K. Maurya, A. Tripathi, G. B. ShivaKeshava,
P. K. Shukla, P. Srivastava and D. Panda, Eur. J. Med. Chem.,
2009, 44, 1086–1092.
10 R. F. Silver and H. L. Holmes, Can. J. Chem., 1968, 46, 1859–
1864.
11 I. Oeriu, Biokhimiia, 1963, 28, 380–383.
12 B. Prescott, J. Med. Chem., 1969, 12, 181–182.
13 E. M. Hodnett, C. Wongwiechintana, W. J. Dunn 3rd and
P. Marrs, J. Med. Chem., 1983, 26, 570–574.
14 N. G. Clark, Pestic. Sci., 1984, 15, 235–240.
15 J. Benites, J. A. Valderrama, K. Bettega, R. C. Pedrosa,
P. B. Calderon and J. Verrax, Eur. J. Med. Chem., 2010, 45,
6052–6057.
16 S. V. Druzhinin, E. S. Balenkova and V. G. Nenajdenko,
Tetrahedron, 2007, 63, 7753–7808.
17 B. S. Samant and C. Chakaingesu, Bioorg. Med. Chem. Lett.,
2013, 23, 1420–1423.
18 V. K. Tandon, H. K. Maurya, D. B. Yadav, A. Tripathi,
M. Kumar and P. K. Shukla, Bioorg. Med. Chem. Lett., 2006,
16, 5883–5887.
Acknowledgements
This work was nancially supported by the Scientic Research
Projects Coordination Unit of Istanbul University (Project
number: 53374) in the supply of the equipment and materials.
We also gratefully thank to Prof. Dr Buket Alpertunga and Res.
Assist. Ay¸se Tarbın Jannuzzi for providing us the hepatocellular
carcinoma (HepG2) cell lines and valuable suggestions from the
Department of Pharmaceutical Toxicology in the Faculty of
Pharmacy at Istanbul University.
19 A. F. Tuyun, N. Bayrak, H. Yildirim, N. Onul, E. M. Kara and
B. O. Celik, J. Chem, 2015, 645902.
20 Website: http://www.ibmc.msk.ru/PASS.
21 (a) V. V. Poroikov, D. A. Filimonov, Y. V. Borodina,
A. A. Lagunin and A. Kos, J. Chem. Inf. Comput. Sci., 2000,
40, 1349–1355; (b) V. V. Poroikov and D. A. Filimonov, J.
Comput.-Aided Mol. Des., 2002, 16, 819–824; (c)
A. Geronikaki, E. Babaev, J. Dearden, W. Dehaen,
D. Filimonov, I. Galaeva, V. Krajneva, A. Lagunin,
F. Macaev, G. Molodavkin, V. Poroikov, S. Pogrebnoi,
V. Saloutin, A. Stepanchikova, E. Stingaci, N. Tkach,
L. Vlad and T. Voronina, Bioorg. Med. Chem., 2004, 12,
6559–6568; (d) A. Geronikaki, A. Lagunin, V. Poroikov,
D. Filimonov, D. Hadjipavlou-Litina and P. Vicini, SAR
QSAR Environ. Res., 2002, 13, 457–471; (e) R. K. Goel,
V. Kumar and M. P. Mahajan, Bioorg. Med. Chem. Lett.,
2005, 15, 2145–2148; (f) G. K. Gupta, V. Saini, R. Khare and
V. Kumar, Med. Chem. Res., 2014, 23, 4209–4220; (g)
A. Kumar, P. Lohan, D. K. Aneja, G. K. Gupta, D. Kaushik
and O. Prakash, Eur. J. Med. Chem., 2012, 50, 81–89.
22 (a) R. M. Donlan and J. W. Costerton, Clin. Microbiol. Rev.,
2002, 15, 167; (b) P. Jorge, A. Lourenco and M. O. Pereira,
Biofouling, 2012, 28, 1033–1061.
References
1 (a) C. Dye, Philos. Trans. R. Soc., B, 2014, 369; (b) M. L. Cohen,
Nature, 2000, 406, 762–767; (c) A. S. Fauci, Clin. Infect. Dis.,
2001, 32, 675–685.
2 World Health Organization, Mortality and Global Health
Estimates, Geneva, Switzerland, 2013.
3 K. F. Smith, M. Goldberg, S. Rosenthal, L. Carlson, J. Chen,
C. Chen and S. Ramachandran, J. R. Soc., Interface, 2014, 11,
20140950.
4 World Health Organization, Global Tuberculosis Control,
Geneva, Switzerland, 2010.
5 (a) M. N. Gwynn, A. Portnoy, S. F. Rittenhouse and
D. J. Payne, Ann. N. Y. Acad. Sci., 2010, 1213, 5–19; (b)
D. Sanglard, Curr. Opin. Microbiol., 2002, 5, 379–385; (c)
M. L. Cohen, Science, 1992, 257, 1050–1055; (d) H. S. Gold
and R. C. Moellering Jr, N. Engl. J. Med., 1996, 335, 1445–
1453.
6 (a) A. R. M. Coates and Y. Hu, Br. J. Pharmacol., 2007, 152,
1147–1154; (b) K. Lewis, Nature, 2012, 485, 439–440.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 25753–25764 | 25763

View Article Online
RSC Advances
Paper
23 R. Thomsen and M. H. Christensen, J. Med. Chem., 2006, 49, 27 A. Mital, M. Sonawane, S. Bindal, S. Mahlavat and V. Negi,
3315–3321. Der Pharma Chem., 2010, 2(3), 63–73.
24 (a) M. L. Barta, D. A. Skaff, W. J. McWhorter, 28 Clinical and Laboratory Standards Institute, Reference
T. J. Herdendorf, H. M. Miziorko and B. V. Geisbrecht, J.
Biol. Chem., 2011, 286, 23900–23910; (b) D. A. Skaff,
W. J. McWhorter, B. V. Geisbrecht, G. J. Wyckoff and
H. M. Miziorko, Arch. Biochem. Biophys., 2015, 566, 1–6.
25 (a) D. V. Krepkiy and H. M. Miziorko, Biochemistry, 2005, 44,
2671–2677; (b) D. Krepkiy and H. M. Miziorko, Protein Sci.,
2004, 13, 1875–1881; (c) N. E. Voynova, Z. J. Fu,
Method for Broth Dilution Antifungal Susceptibility Testing of
Yeasts, CLSI, Wayne, PA, 2th edn, 1997, Approved Standard
M27-A2.
29 Clinical and Laboratory Standards Institute, Methods for
dilution antimicrobial susceptibility tests for bacteria that
grow aerobically, CLSI, Wayne, PA, 7th edn, 2006, Approved
standard M7-A7.
K. P. Battaile, T. J. Herdendorf, J. J. P. Kim and 30 E. Mataraci and S. Dosler, Antimicrob. Agents Chemother.,
H. M. Miziorko, Arch. Biochem. Biophys., 2008, 480, 58–67. 2012, 56, 6366–6371.
26 N. P. Buu-Hoi, R. Royer and M. Hubert-Habart, Recl. Trav. 31 http://www.aniara.com/Resources/Xenometrix-Literature.aspx.
Chim. Pays-Bas, 1954, 73, 188–192.
25764 | RSC Adv., 2017, 7, 25753–25764
This journal is © The Royal Society of Chemistry 2017