Free radical rearrangement synthesis and microbiological evaluation of novel 2-sulfoether-4-quinolone scaffolds as potential antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2018.05.021

To develop novel antibacterial agents, 2-sulfoether-4-quinolone scaffolds were synthesized by a free radical process and evaluated for their antibacterial abilities. Excellent activities against Gram-positive bacteria were observed, among which compounds 3m, 3n, 3p and 3t possessed the lowest MICs against both S. aureus and B. cereus (0.8 μM and 1.61 μM, respectively). The structure-activity relationship (SAR) showed that: (i) the antibacterial activity was related to the substituent, such as 2-SCH₃ = 2-SCH₂CH₃ > 2-S(=O)CH₃ > 2-OH, 8-Br > 7-Br > 6-Br; (ii) -CF₃ increased the antibacterial activity; (iii) the di-substituted group performed the better activity. The DNA supercoiling inhibitory analysis confirmed their fluoroquinolone characters. The docking showed that compound 3n was nicely bound into the DNA-gryase complex via extensive interactions, including conventional hydrogen bonds, halogen bonds and hydrophobic interactions. The microscopy analysis of compound 3n against S. aureus exhibited the damages on the cell wall construction, which may facilitate the penetration into Gram-positive bacteria.

Full text of DOI:10.1016/j.ejmech.2018.05.021

European Journal of Medicinal Chemistry 154 (2018) 144e154
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Free radical rearrangement synthesis and microbiological evaluation
of novel 2-sulfoether-4-quinolone scaffolds as potential antibacterial
agents
a
a
,
c
a
a
a
a
Kehan Xu , Shaosheng He , Shichao Chen , Guoliang Qiu , Jingbo Shi , Xinhua Liu ,
b
b,
*
a, **
Xiaoyu Wu , Jing Zhang , Wenjian Tang
School of Pharmacy, Department of Medical Microbiology, Anhui Institute of Innovative Drugs, Anhui Medical University, Hefei, 230032, China
Anhui Prevention and Treatment Center for Occupational Disease, Anhui No. 2 Province People's Hospital, Hefei, 230022, China
Lujiang County People's Hospital, Anhui, Lujiang, 231500, China
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 February 2018
Received in revised form
To develop novel antibacterial agents, 2-sulfoether-4-quinolone scaffolds were synthesized by a free
radical process and evaluated for their antibacterial abilities. Excellent activities against Gram-positive
bacteria were observed, among which compounds 3m, 3n, 3p and 3t possessed the lowest MICs
7
May 2018
against both S. aureus and B. cereus (0.8
SAR) showed that: (i) the antibacterial activity was related to the substituent, such as 2-SCH
SCH CH > 2-OH, 8-Br > 7-Br > 6-Br; (ii) -CF increased the antibacterial activity; (iii) the
> 2-S(¼O)CH
m
M and 1.61
m
M, respectively). The structure-activity relationship
Accepted 14 May 2018
(
3
¼ 2-
2
3
3
3
di-substituted group performed the better activity. The DNA supercoiling inhibitory analysis confirmed
their fluoroquinolone characters. The docking showed that compound 3n was nicely bound into the
DNA-gryase complex via extensive interactions, including conventional hydrogen bonds, halogen bonds
and hydrophobic interactions. The microscopy analysis of compound 3n against S. aureus exhibited the
damages on the cell wall construction, which may facilitate the penetration into Gram-positive bacteria.
© 2018 Elsevier Masson SAS. All rights reserved.
Keywords:
Quinolone
Free radical process
DNA gyrase
Antibacterial
Electron microscope
1. Introduction
The nitrogen-containing heterocycles are important building
blocks in the development of medicinal chemistry [7e9], among of
The treatment of infections is facing a major problem caused by
antimicrobial resistance (AMR) of the pathogen bacteria, and
sometimes even worse, for instance, Mycobacterium tuberculosis and
Staphylococcus aureus have a strong vitality and the rapid multi-
antibiotics-resistance (MDR) development capability [1e3]. In
contrast, new antibacterial agents are currently being developed at a
much slower pace than the growing need. With diminishing avail-
able pharmaceutical options, to more effectively counter the threat
of AMR, the number of innovative antibiotics must be increased. It
could be addressed by identifying new mechanisms, origins and
distribution of AMR through the combination of computational
modeling and advanced biological tools [4], or by generating new
chemicals that can extend the life of existing antibiotics [5,6].
which the quinolones are one of the most early studied and
modified [10]. The bacterial type II topoisomerase, including DNA
gyrase (also named topoisomerase II) and topoisomerase IV, is the
major target of quinolones. Both enzymes are able to remove pos-
itive or negative supercoiling. DNA gyrase prefer to perform the
intramolecular reactions to resolve chromosomal DNA supercoils,
while the topoisomerase IV is prone to the intermolecular re-
actions, facilitating the decatenation of the linked DNA molecules
[11,12]. Thus, both enzymes are the lethal targets of quinolones. As
entirely synthetic drugs, the therapeutic 4-quinolone have been
studied and modified extensively, resulting four generations with
enhanced activity against gastrointestinal infections, respiratory
and urinary tract diseases [13e15]. The most efficient approach to
seek for novel 4-quinolone antibacterial agents is to synthesize new
analogues or modify existing drugs, in combination with the
analysis and improvement of the pharmacokinetics and metabolic
properties [9,10,16e20]. Since the modification based on 2-
substituted-4-quinolone are still not fully investigated [21], which
*
Corresponding author.
** Corresponding author.
E-mail addresses: hfzj2552@163.com (J. Zhang), ahmupharm@126.com
(
W. Tang).
https://doi.org/10.1016/j.ejmech.2018.05.021
223-5234/© 2018 Elsevier Masson SAS. All rights reserved.
0

K. Xu et al. / European Journal of Medicinal Chemistry 154 (2018) 144e154
145
prompt us to further understand this type of scaffold.
Table 1
The chemical structures of compounds 3a‒3t, 4 and 5.
The quinolone structural motifs are pharmacologically active
substances for drug discovery since many of them have been shown
to exhibit excellent biological and pharmacological activities
[
22,23], such as antiparasitic [24e26], antimicrobial [27,28], anti-
viral [29] and antitumor activity [30,31]. Furthermore, the hetero-
cyclic compounds with nitrogen and sulfur atoms have been
identified to possess the most comprehensive spectrum of biolog-
ical activities [32,33], for which we put our attempts on the syn-
thesis of new heterocyclic derivatives combining both known
active quinolone and sulfur atom in one molecular frame. The 2-
substituted-4-quinolone was obtained by a free radical process
and a series of 2-methylthioquinolin-4(1H)-one derivatives were
synthesized to explore the effects of 2-substituents group on
antibacterial activity (Fig. 1). Most of the compounds possessed
excellent antibacterial activities against Gram-positive bacteria,
Compound
R
1
R
2
R
3
R
4
R
5
3
3
a
b
SCH
SCH
SCH3
3
H
H
H
H
H
H
H
H
H
H
H
H
CF
CF
CF
H
CF
H
H
H
H
H
CF
H
H
H
CH
H
OCH
H
H
Br
CF
OCF
H
H
H
CN
H
NO
H
CF
CF
CF
3
H
CF
H
H
H
H
H
H
Br
H
H
H
H
H
H
CF
H
H
H
H
H
H
H
H
3
3
3c
3d
Cl
CH
H
OCH
H
Br
H
H
H
H
Cl
CF
3
OCH
H
F
Cl
H
Cl
H
SCH
SCH
SCH
SCH
SCH
SCH
SCH
SCH
SCH
SCH
SCH
SCH
SCH
SCH
SCH
SCH
SCH
3
3
3
3
3
3
3
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
e
f
g
h
3
3
3
among which the most potent compound 3n with 2-SCH
3
and CF
3
3i
3
3
3
3
3
3
3
j
k
l
m
n
o
p
di-substitution was used to study the preliminary mechanism of
antibacterial activity against S. aureus.
CH
3
3
3
3
3
3
2
. Results and discussion
3
3
2.1. Chemistry
3q
3
3r
3s
3t
4
5
2
Twenty 2-methylthioquinolin-4(1H)-ones (3a‒3t, as shown in
3
Table 1) were obtained from its corresponding acetoacetanilide (1)
Scheme 1) according to the literature method [33,34]. The inter-
mediate 2-(bis(methylthio)methylene)-3-oxo-N-phenyl-
S¼OCH
3
3
3
(
OH
H
butanamide (2) were synthesized by the nucleophilic reaction of
active methylene group of acetoacetanilide (1) with carbon bisul-
fide in the present of K CO and subsequent methylation with
2 3
dimethyl sulfate. The thermal cyclization of compound 2 gave the
title compound 3 in o-dichlorobenzene.
methylthio of quinolone 3a was hydrolyzed in 50% sulfuric acid to
afford the hydrolysis product 5 (Scheme 3).
2.2. Crystal structure analysis
Surprisingly, the cyclization doesn't produce 4-methylthio-2-
quinolone, but forms 2-methylthio-4-quinolone. The cyclization
could be a free radical process (Scheme 2), that is, the themolysis of
compound 2 generates a radical intermediate (2a) by removing
methanethiol, and accompanying with the cleavage of C-N bond,
and then the electrophilic addition of alkene free radical to
nitrogen-atom produces an intermediate 2b. Finally, the title
compound 3 was obtained from the rearrangement reaction of
ortho-H-migration and carbonyl addition of the intermediate 2b.
This offers an efficient synthetic way to this kind of 2-
thiosubstituted-4-quinolone.
The structure of compound 3a was determined by X-ray crys-
tallography. Crystal data of 3a: Clear light colorless crystals ob-
ꢀ
tained from EtOH/H
NO S$H
a ¼ 4.6964(6), b ¼ 16.416(2),
2
O, yield, 68%; mp 155e157 C;
C
13
H
9
F
3
2
2
O, M ¼ 318.29, Monoclinic, space group P1 21/n 1;
c
¼
18.198(2) (Å);
a
¼ 90,
3
b
¼ 94.200(13),
g
¼ 90, V ¼ 1399.2(3) Å , T ¼ 293.48(10) K, Z ¼ 4,
3
Dc ¼ 1.511 g/cm , F(000) ¼ 652, Reflections collected/Independent
reflections ¼ 2743/1436, Data/restraints/parameters ¼ 2743/1/195,
2
2
Goodness of fit on F ¼ 1.045, Fine, R
1
¼0.0480, wR(F ) ¼ 0.1248.
Crystal data of 3j: Clear light colorless crystals obtained from
Further, compounds 3a‒3t were characterized by NMR spectra,
high resolution mass spectra (HRMS) and X-ray crystallography.
The 2-methylthio-4-quinolone core for each molecule was readily
ꢀ
EtOH, yield, 57%; mp 173e174 C;
12
C H
10BrNO
‒1; a ¼ 4.4810(4),
¼ 101.267(11), ¼ 92.790(8),
2 3 2
S$CH CH OH,
M ¼ 358.25, Triclinic, space group
P
b ¼ 9.7061(16), c ¼ 17.5903(19) (Å);
a
b
confirmed by observation of the C-atom signals at
d(C)
3
g
¼ 102.661(11), V ¼ 728.71(16) Å , T ¼ 293(2) K, Z ¼ 2,
1
72e175 C(4-C¼O), 153e159 C(2), while the
d
(C) of 2-carbonyl C-
3
Dc ¼ 1.633 g/cm , F(000) ¼ 364, Reflections collected/Independent
reflections ¼ 4986/2650, Data/restraints/parameters ¼ 2650/1/185,
atom signal is no more than 170 for the 4-methylthio-2-quinolone
core [34,35].
2
2
Goodness of fit on F ¼ 1.040, Fine, R
1
¼0.0367, wR(F ) ¼ 0.1022.
To observe the effect of 2-methylthio of 4-quinolone on the
antibacterial activity, the oxidation product 4 was obtained from
the quinolone 3a using hydrogen peroxide in AcOH. Further, the 2-
The structures of compounds 3a and 3j were determined using
MoK
a
radiation (
l
¼ 0.71073 Å), which are both 4-quinolones
rather than 2-quinolones as shown in Fig. 2. The 2-substituted
group is methylthio, which is consistent with it in reference [35].
Crystallographic data (excluding structure factors) for the structure
had been deposited with the Cambridge Crystallographic Data
Center as supplementary publication Nos. CCDC 1581290 and CCDC
1821733 for compounds 3a and 3j, respectively.
2.3. Biological activity
In order to determine the antimicrobial potential of the syn-
Fig. 1. The general design strategy in this study.
thetic quinolone derivatives, they were initially tested against a

146
K. Xu et al. / European Journal of Medicinal Chemistry 154 (2018) 144e154
Scheme 1. Synthesis of compounds 3a¡3t.
ꢀ
Reagents and conditions: (i) carbon bisulfide/K
2
CO
3
/TBAB; (ii) dimethyl sulfate; (iii) o-dichlorobenzene/180 C.
Scheme 2. Proposed reaction mechanism for formation of compound 3.
Scheme 3. Synthesis of compounds 4 and 5.
Reagents and conditions: (iv) 30% hydrogen peroxide/AcOH; (v) 50% sulfuric acid, 100 C.
ꢀ
panel of bacteria and fungi in Mueller-Hinton broth (MHB),
including two Gram-positive bacteria: Staphylococcus aureus and
Bacillus cereus; two Gram-negative bacteria: Pseudomonas aerugi-
nosa and Escherichia coli; and one fungi: Candida albicans, respec-
tively. As shown in Table 2, these compounds had no antimicrobial
activity against Gram-negative bacteria and fungi, with high MIC
four compounds were able to inhibit the growth of S. aureus
effectively at the MIC or higher concentrations. Once the concen-
tration drop down to half or one fourth of the MICs, they could only
slow down the growing rate of S. aureus during logarithmic period,
while the growth could be recovered after being incubated for
longer time (Fig. 3).
values ꢁ 200
mM. In contrast, the moderate to strong level of anti-
bacterial activity against Gram-positive bacteria were observed.
2.4. SAR analysis
Compounds 3m, 3n, 3p and 3t performed best against both
S. aureus and B. cereus with the MIC values at 0.8
m
M and 1.61
m
M,
The structure-activity relationship (SAR) analysis showed that
the antibacterial activity was related to the position and the
amount of the substituent in the quinolone scaffold: (i) the activity
varied with the substituent groups at 2-position (MICs of com-
pounds 3a with 2-SCH , 3k with 2-SCH CH , 4 with 2-S(¼O)CH
respectively, which is comparable with the commercial agent
gentamicin, ofloxacin and tetracycline (Table 2). Additionally, the
MBC values of the potent compounds were measured, among
which 3n, 3p and 3t were the most active compounds against both
3
2
3
3
S. aureus and B. cereus, with MBC values at 6.25
m
M (Table 3). Here
and 5 with 2-OH were 6.25, 50
m
M; 6.25, 25
m
M; 12.5, 3.125
m
M; and
the Methicillin-resistant Staphylococcus aureus (MRSA) was also
included. However, all the tested compounds were inactive against
MRSA (Table 3).
>200, >200 mM against S. aureus and B. cereus, respectively); and
the antibacterial activity of Br-substituted compounds varied ac-
cording to the substituent position, e.g. compounds at 8-position >
We further investigated the growth inhibition effects of com-
pounds 3m, 3n, 3p and 3t on S. aureus. The results showed that all
7-position > 6-position, with the MIC ¼ 6.25, 12.6 and 25
mM for 3h,
3i and 3j, respectively; (ii) Compounds with CF substituent
3

K. Xu et al. / European Journal of Medicinal Chemistry 154 (2018) 144e154
147
2.6. Molecular docking
The mode of action of ciprofloxacin on S. aureus DNA gyrase had
been studied in great detail and the crystal structure are available
for the substrate-bound enzyme [36]. Here we tried to understand
the mechanism of the antibacterial ability of these compounds by
molecular docking using the gyrase-DNA-ciprofloxacin complex
structure (PDB code: 2XCT) as model. Compound 3n were docked
into the ciprofloxacin binding site and fit very well in the pocket
formed by the gyrase-DNA complex (Fig. 4). It was found that
compound 3n could mimic the nucleotide located at the nick of the
broken dsDNA, with the sulfur atom mimicking the phosphate and
the quinolone ring mimicking the ribose and forming strong
p-p
contacts with DG-8 and DG-9, with the distance at ~3.9 Å and 3.6 Å,
respectively (Fig. 4B and C). The 2-methylthio substituent of qui-
nolone ring introduced the
p-interaction between compound 3n
and neighboring pyrimidine ring of DG-8 and DG-9 as well. Our
modifications at the 5- and 8-position introduced extensive
halogen bonding interactions between the trifluoromethyl group
and the gyrase-DNA complex, where the nucleotide DG-8, DC-13
and residue Arg458 were involved (Fig. 4D). Besides, three con-
ventional hydrogen bonds were formed, including two hydrogen
bonds formed between the introduced 8-position trifluoromethyl
group, the 4-ketone group and the nucleotide DG-9, and one
hydrogen bond between the nitrogen of the ligand and residue
Arg458 from the DNA gyrase (Fig. 4C). Additionally, 4-carbonyl and
2
þ
3
-carbonyl groups anchor Mn with distances at 2.5 Å and 2.7 Å
(Fig. 4D), respectively. This result revealed that the modification
based on the quinolone does not affect the binding mode of phar-
macophore. In brief, the introduction of 2-methylthio substituent of
quinolone ring increased the hydrophobic interaction of compound
3n with the nucleotides and an extra hydrogen bond (3.2 Å) was
observed (Fig. 4C). Besides, extensive halogen interactions with
gyrase-DNA complex were observed based on the introduction of
trifluoromethyl groups (Fig. 4C and D). Together, these additional
interactions may explain the excellent antibacterial activity of
compound 3n and other compounds with di-substituent contain-
ing -CF
.7. Transmission electron microscope
On the basis of the observation that these compounds were able
3
.
2
to inhibit the growth of Gram-positive bacteria and had no effects
on Gram-negative bacteria, we hypothesized that the modifications
on these derivatives might affect the function of bacterial cell wall,
which is one of the most apparent discrepancies between Gram-
positive and negative bacteria [37]. Transmission electron micro-
scopy (TEM) of S. aureus showed significant changes of the bacteria
before and after the treatment of compound 3n. As shown in
Fig. 5A, normal S. aureus forms a typical coccus with complete cell
membrane and cell wall surrounded at the outmost (Fig. 5B), with a
few abnormal bacteria possibly due to the damage during sample
preparation. After being treated with compound 3n at concentra-
Fig. 2. ORTEP drawing of the crystal structures of compounds 3a and 3j.
showed good antibacterial activity, except for compound 3o with
both CF
substituent behave mediocrely; (iii) Compounds 3m, 3n, 3p, 3q and
t with di-substituent containing CF generally showed the best
antibacterial activity, with the compound 3o was an exception.
3 3
and OCH substituent; while all compounds with Br
3
3
2.5. DNA supercoiling activity
tion of 0.8 mM for 24 h, most of the bacteria present unusual vari-
ations. Around 30% of the bacteria showed a fluffy surface which
indicate the swelling of the cell wall region (Fig. 5C), and cell wall
damage was observed in a certain proportion of the cell, which was
indicated by the emerging of solute loss and blemishes on the cell
wall (Fig. 5D and E). Furthermore, a larger proportion, around 50%
of the bacteria have lost the cell wall protection, leading to the
shrink of the cell morphology and possibly the cell death in the end
(Fig. 5F). The observed cell morphology changes are consistent with
our speculation that the bactericidal function of compound 3n may
also affect the cell wall architecture of the Gram-positive bacteria,
and the underlying mechanism is still need to be known.
Quinolones are synthetic compounds affecting DNA replication
by targeting the DNA gyrase subunit A. To investigate the substi-
tution of sulfoether and trifluoromethyl group on the fluo-
roquinolone targeting DNA gyrase, the inhibitory effects of
compounds with relatively high antibacterial activity were
measured against the enzyme from S. aurus. It was revealed that
some of the compounds displayed greater inhibitory activities in
comparison with the positive control ciprofloxacin (Table 4), which
confirmed the fluoroquinolone character of the synthesized
compounds.

148
K. Xu et al. / European Journal of Medicinal Chemistry 154 (2018) 144e154
Table 2
MIC (
M)a of 4-quinoline scaffolds against bacteria and fungi.b
m
Compoundc
Gram-positive
Gram-positive
fungus
SA
BC
PA
EC
CA
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
5
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
6.25
3.125
>200
>200
>200
>200
>200
6.25
12.5
25
6.25
3.125
0.80
0.80
>200
0.80
0.80
12.5
>200
0.80
12.5
>200
0.80
0.125
0.0325
/
50
50
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
1.0
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
0.125
0.50
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
200
>200
200
200
>200
>200
>200
>200
>200
/
>200
>200
>200
>200
>200
>200
200
200
25
12.5
1.60
1.60
>200
1.60
3.125
6.25
>200
1.60
3.125
>200
1.60
0.80
1.60
/
s
t
A
B
C
D
E
4.0
2.0
/
/
/
/
0.25
/
0.2
1.0
m
m
g/mL
g/mL
/
/
a
b
c
MICs representing mean values of at least three replicates.
SA: Staphylococcus aureus, BC: Bacillus cereus, PA: Pseudomonas aeruginosa, EC: Escherichia coli, CA: Candida albican.
A: Gentamicin, B: Ofloxacin, C: Tetracycline, D: Amphotericin B, E: Fluconazole.
Table 3
MIC ( M) and MBC (
m
m
M)a of 4-quinoline scaffolds against Gram-positive bacteria.b
Compoundc
SA
BC
MRSA
MIC
MBC
MIC
MBC
MIC
3
3
3
3
3
3
3
3
3
3
3
4
a
b
h
k
l
m
n
p
q
r
6.25
3.125
6.25
6.25
3.125
0.80
0.80
0.80
0.80
12.5
0.80
12.5
0.80
0.125
0.0325
25
6.25
25
25
25
12.5
6.25
25
6.25
>200
6.25
50
50
50
>200
25
12.5
1.61
1.61
1.61
3.125
6.25
1.61
3.125
1.6
>200
>200
>200
50
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
100
25
12.5
6.25
25
6.25
>200
6.25
50
3.2
1.6
3.2
t
A
B
C
3.2
0.5
0.125
0.8
1.6
200
a
b
c
MICs and MBCs representing mean values of at least three replicates.
SA: Staphylococcus aureus, BCe: Bacillus cereus, MRSA: Methicillin-resistant Staphylococcus aureus.
A: Gentamicin, B: Ofloxacin, C: Tetracycline.
3
. Conclusion
antibacterial activity was related to the substituent position, such
as 2-SCH CH > 2-S(¼O)CH > 2-OH (MICs ¼ 6.25, 6.25,
¼ 2-SCH
12.5 and > 200 M for 3a, 3k, 4 and 5), 8-Br > 7-Br > 6-Br
(MICs ¼ 6.25, 12.6 and 25 M for 3h, 3i and 3j); (ii) CF substitu-
3
2
3
3
In summary, a series of 2-methylthioquinolin-4(1H)-one de-
m
rivatives were synthesized by a free radical process and evaluated
for their antibacterial activities. Most of the compounds showed
potential antimicrobial activity against Gram-positive bacteria,
among which the compounds 3m, 3n, 3p and 3t had the lowest
m
3
ent improved antibacterial activity, while Br substituent showed
moderate activity; (iii) Compounds 3m, 3n, 3p, 3q and 3t with di-
substituent showed the best antibacterial activity. The DNA
supercoiling inhibitory effects of the potent compounds were
consistent with classical fluoroquinolone, performing as a DNA
gyrase inhibitor. The docking resulted in the appropriate confor-
mation for all the potent compounds in the DNA-gryase complex.
MICs against both S. aureus and B. cereus (0.8 mM and 1.61 mM,
respectively), and the growth inhibition assay on S. aureus showed
that they were able to inhibit the growth of S. aureus effectively at
the MIC and higher concentrations. SAR showed: (i) the

K. Xu et al. / European Journal of Medicinal Chemistry 154 (2018) 144e154
149
Fig. 3. S. aureus growth inhibition assay. Titration curves showing the effect of different concentrations of compounds 3m (A), 3n (B), 3p (C), 3t (D) on the growth of S. aureus. Each
OD point presented is the average values of three tests and all experiments are internally controlled.
Table 4
4.1.1. General procedures for synthesis of 3-Acetyl-4-(methylthio)
quinolin-2(1H)-one derivatives 3aꢂ3m
Inhibitory effects of the title compounds against S. aureus
DNA gyrase.a
To a solution of acetoacetanilide 1 (5.0 mmol) in DMF (10 mL),
anhydrous potassium carbonate (15 mol, 2.0 g) and tetrabuty-
lammonium bromide (0.16 g; 0.5 mmol) were added. The mixture
was stirred for 30 min at room temperature then carbon disulfide
Compound
IC50 (mg/mL)
3
3
3
3
3
3
n
q
p
t
k
r
0.71 ± 0.11
0.79 ± 0.09
2.11 ± 0.18
2.69 ± 0.61
5.11 ± 0.55
25.1 ± 2.34
4.95 ± 0.56
(5.0 mmol, 0.30 mL) was added and the stirring was continued for
1
h. Then dimethyl sulfate (15 mol, 1.5 mL) was dropped to the
mixture over a period of 20 min and stirring was continued for
4 h at room temperature. The dark red solution was poured onto
crushed ice and the precipitate was filtered and washed thoroughly
using cold methanol to obtain compound 2, which is directly used
to next reaction without further purification.
ciprofloxacin
a
DNA gyrase supercoiling activity.
A solution of the ketene dithioacetal 2 in o-dichlorobenzene
Compound 3n localized nicely in the complex via extensive in-
teractions, including conventional hydrogen bonds, halogen bonds
and hydrophobic interactions. Moreover, the microscopy analysis of
compound 3n against S. aureus displayed the cell wall destruction,
suggesting that the active compounds may facilitate the penetra-
tion into bacteria. Together, these results would be very useful for
the SAR study and further optimization on 2-sulfoether-4-
quinolone scaffolds in the future.
(
10 mL) was heated under reflux for ca. 4 h. The reaction solution
was left to cool at room temperature overnight and the crystalline
product was filtered and washed trice with Et O. The product was
recrystallized from o-xylene to give the quinolone 3.
2
4
(
(
2
.1.1.1. 3-Acetyl-2-methylthio-6-(trifluoromethyl)quinolin-4(1H)-one
ꢀ
1
3a). White powder, yield, 45%; m.p. 155‒157 C;
H NMR
300 MHz, DMSO‑d 11.14 (s, 1H, NH), 8.35 (s, 1H), 8.01 (s, 2H),
6
) d
13
.68 (s, 3H, SCH
3 3 6
), 2.52 (s, 3H, COCH ); C NMR (75 MHz, DMSO‑d )
4
. Methods and materials
d
198.6 (CO), 173.4 (C-4), 158.1 (C-2), 141.8 (C-9), 128.5 (C-7), 125.5
(
C-6), 125.1 (C-3), 124.8 (C-5), 124.5 (CF
3
), 122.7 (C-10), 120.1 (C-8),
þ
4.1. Chemistry
31.6 (COCH
C H F NO S: 302.0457; found: 302.0458.
3
), 15.0 (SCH
3
). TOF-HRMS: m/z [M þ H] calcd for
13 11 3 2
All reagents were purchased from commercial sources and were
used without further purification. Melting points (uncorrected)
4.1.1.2. 3-Acetyl-2-methylthio-7-(trifluoromethyl)quinolin-4(1H)-
ꢀ
1
were determined on a XT4MP apparatus (Taike Corp., Beijing,
one (3b). White powder, yield, 50%; m.p. 143‒144 C; H NMR
(300 MHz, DMSO‑d
10.94 (s,1H, NH), 8.29 (d, J ¼ 8.3 Hz, 2H), 7.68
(d, J ¼ 8.5 Hz, 1H), 2.67 (s, 3H, SCH
(150 MHz, DMSO‑d 198.7 (CO), 173.7 (C-4), 158.9 (C-2), 139.6 (C-
9), 132.2 (C-8), 127.4 (C-7), 125.0 (C-6), 123.2 (CF ), 120.5 (C-3), 119.9
). TOF-HRMS: m/z [M þ
1
13
China). H NMR and C NMR spectra were recorded on Bruker AV-
00 or AV-300 MHz instruments in CDCl or DMSO‑d . Chemical
shifts are reported in parts per million ( ) downfield from the signal
6
) d
13
6
3
6
3 3
), 2.53 (s, 3H, COCH ); C NMR
d
6
) d
of tetramethylsilane (TMS) as internal standards. Coupling con-
stants are reported in Hz. The multiplicity is defined by s (singlet),
d (doublet), t (triplet), or m (multiplet). High resolution mass
spectra (HRMS) were obtained on an Agilent 1260-6221 TOF mass
spectrometry. Column and thin-layer chromatography (CC and TLC,
resp.) were performed on silica gel (200e300 mesh) and silica gel
GF254 (Qingdao Marine Chemical Factory) respectively.
3
(C-10), 116.6 (C-5), 32.0 (COCH
3
), 15.1 (SCH
3
þ
H] calcd for C13
H F
11 3
NO
2
S: 302.0457; found: 302.0456.
4.1.1.3. 3-Acetyl-8-chloro-2-(methylthio)quinolin-4(1H)-one
(3c).
Yellow powder, yield, 45%; m.p. 133‒135 C; H NMR (300 MHz,
DMSO‑d
8.16 (d, J ¼ 6.8 Hz, 1H, NH), 7.92 (d, J ¼ 7.7 Hz, 1H), 7.44
ꢀ
1
6
) d

150
K. Xu et al. / European Journal of Medicinal Chemistry 154 (2018) 144e154
Fig. 4. Molecular docking of compound 3n in DNA gyrase A from S. aureus. A. Overview of compound 3n docking with the DNA gyrase A in complex with a dsDNA (PDB: 2XCT). The
protein was shown as surface and the hydrophobicity of the protein was illustrated by gradient color from blue (low) to white (high). DNA was shown as cartoon and colored in
green and the ligand was colored in grey. B. Overview of compound 3n mimicking the nucleotide in the nick of the broken dsDNA. The distances between the compound and
neighboring nucleotides were illustrated with red dashed lines. The sulphur atom that could mimic the phosphorus on the DNA backbone was indicated by black arrow. C and D.
Close-up of the interactions between compound 3n and the DNA Gyrase A–dsDNA complex. The residues involved in the interactions were shown as sticks. The metal ion was
shown as sphere and colored in purple. The hydrogen bonds, halogen bonds and metal-receptor interactions were indicated by black, brown and red dash lines, respectively. The
structures are all displayed using pymol (Schr o€ dinger, LLC). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this
article.)
(
t, J ¼ 7.9 Hz,1H), 2.67 (s, 3H, SCH
3
), 2.58 (s, 3H, COCH
3
). TOF-HRMS:
(C-9), 129.2 (C-6), 125.7 (C-5), 124.5 (C-7), 118.4 (C-10), 116.5 (C-3),
þ
m/z [M þ H] calcd for C12
H
11ClNO
2
S: 268.0194; found: 268.0192.
112.6 (C-8), 56.7 (OCH
[
3
), 31.6 (COCH
3 3
), 13.8 (SCH ). TOF-HRMS: m/z
M þ H]þ calcd for C13
H14NO S: 264.0689; found: 264.0691.
3
4
.1.1.4. 3-Acetyl-8-methyl-2-(methylthio)quinolin-4(1H)-one (3d).
ꢀ
1
Light yellow powder, yield, 47%; m.p. 105‒107 C; H NMR
4.1.1.7. 3-Acetyl-6-methoxy-2-(methylthio)quinolin-4(1H)-one (3g).
ꢀ
1
(
300 MHz, DMSO‑d
6
)
d
9.34 (s, 1H, NH), 8.02 (d, J ¼ 7.7 Hz, 1H), 7.58
), 2.60 (s,
); C NMR (150 MHz, DMSO‑d
198.0 (CO), 174.8 (C-4), 157.5 (C-2), 137.3, 133.4, 126.2,
25.2, 124.2, 123.2, 118.2, 31.6 (COCH ), 16.1 (CH ), 13.9 (SCH ). TOF-
S: 248.0740; found:
White powder, yield, 57%; m.p. 234‒236 C; H NMR (300 MHz,
DMSO‑d
10.85 (s, 1H, NH), 7.77 (d, J ¼ 9.0 Hz, 1H), 7.53 (d,
J ¼ 2.8 Hz, 1H), 7.33 (dd, J ¼ 9.0, 2.9 Hz, 1H), 3.85 (s, 3H, OCH ), 2.63
); C NMR (150 MHz, DMSO‑d
198.7 (CO), 173.7 (C-4), 156.2 (C-2), 154.7, 133.9, 126.1, 122.0, 120.0,
(
3
d, J ¼ 7.0 Hz, 1H), 7.32 (t, J ¼ 7.6 Hz, 1H), 2.69 (s, 3H, SCH
3
6
) d
13
H, COCH
3
), 2.56 (s, 3H, Ar-CH
3
6
/
3
13
CDCl
1
3
)
d
(s, 3H, SCH
3
), 2.52 (s, 3H, COCH
3
6
)
3
3
3
d
þ
HRMS: m/z [M þ H] calcd for C13
H14NO
2
118.7, 105.0, 55.4 (OCH
3
), 31.5 (COCH
3 3
), 14.8 (SCH ). TOF-HRMS: m/z
2
48.0741.
[M þ H]þ calcd for C13
H14NO S: 264.0689; found: 264.0689.
3
4
.1.1.5. 3-Acetyl-6-methyl-2-(methylthio)quinolin-4(1H)-one
(3e).
4.1.1.8. 3-Acetyl-8-bromo-2-(methylthio)quinolin-4(1H)-one
(3h).
Yellow powder, yield, 53%; m.p. 157‒159 C; H NMR (300 MHz,
DMSO‑d
ꢀ
1
ꢀ
1
White powder, yield, 52%; m.p. 204‒205 C; H NMR (600 MHz,
DMSO‑d
6
)
d
10.81 (s, 1H 1H, NH), 7.90 (s, 1H), 7.70 (d, J ¼ 8.4 Hz, 1H),
), 2.51 (s, 3H, COCH ),
); C NMR (150 MHz, DMSO‑d
199.2 (C¼O),
6
)
d
9.29 (s, 1H, NH), 8.31e8.14 (m, 1H), 8.09 (d, J ¼ 7.6 Hz,
7
2
.52 (dd, J ¼ 8.4, 1.6 Hz, 1H), 2.64 (s, 3H, SCH
3
3
1H), 7.38 (t, J ¼ 7.9 Hz, 1H), 2.67 (s, 3H, SCH
3
), 2.59 (s, 3H,COCH
3
).
13
þ
.41 (s, 3H, Ar-CH
3
6
)
d
TOF-HRMS: m/z [M þ Na] calcd for C12
H
10BrNNaO S: 333.9508;
2
174.6 (C-4),156.1 (C-2),137.9 (C-9),134.3 (C-6),134.1 (C-7),125.3 (C-
found: 333.9504.
5
), 124.9 (C-10), 119.8 (C-8), 118.6 (C-3), 32.0 (COCH
3
), 21.2 (Ar-CH
3
),
S:
þ
15.3 (SCH
3
). TOF-HRMS: m/z [M þ H] calcd for C13
H14NO
2
4
.1.1.9. 3-Acetyl-7-bromo-2-(methylthio)quinolin-4(1H)-one
(3i).
Light yellow powder, yield, 60%; m.p. 166‒167 C; H NMR
300 MHz, DMSO‑d
10.75 (s, 1H, NH), 8.07 (d, J ¼ 1.3 Hz, 1H), 8.01
(d, J ¼ 8.6 Hz, 1H), 7.54 (dd, J ¼ 8.6, 1.5 Hz, 1H), 2.64 (s, 3H, SCH ),
198.3 (CO),
ꢀ
1
2
48.0740; found: 248.0742.
(
6
) d
4
.1.1.6. 3-Acetyl-8-methoxy-2-(methylthio)quinolin-4(1H)-one (3f).
3
ꢀ
1
13
White powder, yield, 40%; m.p. 175‒177 C; H NMR (300 MHz,
DMSO‑d
9.65 (s, 1H, NH), 7.71e7.62 (m, 1H), 7.34 (d, J ¼ 4.4 Hz,
H), 4.01 (s, 3H, OCH
NMR (75 MHz, DMSO‑d
3 6
2.51 (s, 3H, COCH ); C NMR (150 MHz, DMSO‑d ) d
6
)
d
173.6 (C-4), 157.2 (C-2), 140.0, 127.4, 127.3, 125.6, 123.8, 120.6, 119.5,
13
þ
2
3
), 2.64 (s, 3H, SCH
3
), 2.53 (s, 3H, COCH
3
);
C
31.5 (COCH
3
), 14.7 (SCH
3
). TOF-HRMS: m/z [M þ Na] calcd for
6
)
d
198.1 (CO), 174.3 (C-4), 156.9 (C-2), 147.5
C
12
H10BrNNaO
2
S: 333.9508; found: 333.9508.

K. Xu et al. / European Journal of Medicinal Chemistry 154 (2018) 144e154
151
Fig. 5. Microscopic analysis of S. aureus treated with compound 3n at the MIC concentration. A. Normal growing S. aureus with the treatment of solvent DMSO as a control; B. The
percentage of abnormal S. aureus as a function of compound 3n, each dataset presented is the average values calculated from three views. C. S. aureus performed cell wall distortion;
D and E. S. aureus performed apparent cell wall damage; F: S. aureus performed cell wall lost.
4
.1.1.10. 3-Acetyl-6-bromo-2-(methylthio)quinolin-4(1H)-one (3j).
J ¼ 8.3 Hz, 1H), 7.83 (d, J ¼ 8.3 Hz, 1H), 3.32 (s, 3H, SCH
3
), 2.69 (s, 3H,
10ClF NO S:
ꢀ
1
þ
Yellow powder, yield, 50%; m.p. 172‒174 C; H NMR (300 MHz,
DMSO‑d
10.97 (s, 1H, NH), 8.17 (d, J ¼ 2.0 Hz, 1H), 7.85 (dd,
J ¼ 8.8, 2.3 Hz, 1H), 7.78 (d, J ¼ 8.8 Hz, 1H), 2.65 (s, 3H, SCH ), 2.51 (s,
198.4 (CO), 172.8 (C-4),
57.0 (C-2), 138.4 (C-9), 135.0 (C-7), 127.3 (C-5), 126.5 (C-8), 120.9
COCH
3
). TOF-HRMS: m/z [M þ H] calcd for C13
H
3
2
6
)
d
336.0067; found: 336.0065.
3
13
3
1
H, COCH
3
); C NMR (75 MHz, DMSO‑d
6
)
d
4.1.1.14. 3-Acetyl-5,8-ditrifluoromethyl-2-(methylthio)quinolin-
ꢀ
4(1H)-one (3n). Light yellow powder, yield, 45%; m.p. 139‒141 C;
1
(
C-10), 119.5 (C-3), 117.0 (C-6), 31.6 (COCH
3
), 14.9 (SCH
3
). TOF-
H NMR (300 MHz, CDCl3)
d
16.43 (s,1H, NH), 8.08 (d, J ¼ 7.9 Hz,1H),
þ
HRMS: m/z [M þ Na] calcd for C12
H
10BrNNaO
2
S: 333.9508;
7.89 (d, J ¼ 7.9 Hz, 1H), 3.00 (d, J ¼ 2.7 Hz, 3H, SCH ), 2.73 (d,
3
13
found: 333.9508.
J ¼ 2.7 Hz, 3H, COCH ); C NMR (150 MHz, DMSO‑d )
6
d
210.3 (CO),
3
1
60.1 (C-4), 145.5 (C-2), 129.2, 129.1, 129.0, 124.1, 124.0, 123.5 (q,
4
.1.1.11. 3-Acetyl-2-ethylthio-6-(trifluoromethyl)quinolin-4(1H)-one
CF
3
), 123.3 (q, CF
3
), 119.7, 117.8, 31.5 (COCH
3
), 13.4 (SCH
3
). TOF-
ꢀ
1
þ
(3k). White powder, yield, 54%; m.p. 158‒159 C; H NMR
HRMS: m/z [M þ Na] calcd for C14
9 6 2
H F NNaO S: 392.0150; found:
(
300 MHz, DMSO‑d
6
)
d
11.61 (s, 1H, NH), 8.35 (s, 1H), 8.04 (d,
), 3.25 (q,
392.0147.
J ¼ 8.8 Hz, 1H), 7.95 (d, J ¼ 8.7 Hz, 1H), 3.35 (s, 3H, COCH
3
13
J ¼ 7.4 Hz, 2H, SCH
150 MHz, DMSO‑d
), 128.9 (C-7), 125. 3 (C-5), 124.9 (CF
20.3 (C-8), 31.8 (COCH ), 26.7 (SCH
2
CH
d
3
), 1.24 (t, J ¼ 7.3 Hz, 3H SCH
199.7 (CO), 173.8 (C-4), 153.3 (C-2), 142.3 (C-
), 123.5 (C-3), 123.0 (C-10),
CH ), 14.0 (SCH CH ). TOF-
NNaO S: 338.0433;
2
3
CH ); C NMR
4.1.1.15. 3-Acetyl-2-methylthio-8-methoxy-5-(trifluoromethyl)quino-
lin-4(1H)-one (3o). White powder, yield, 48%; m.p. 275‒276 C; H
NMR (300 MHz, DMSO‑d6)
ꢀ
1
(
9
1
6
)
3
d
9.75 (s, 1H, NH), 7.76 (d, J ¼ 8.6 Hz, 1H),
3
þ
2
3
2
3
7.40 (d, J ¼ 8.6 Hz, 1H), 4.09 (s, 3H, OCH
3
), 2.65 (s, 3H, SCH
3
), 2.47 (s,
þ
HRMS: m/z [M þ Na] calcd for C14
H
12
F
3
2
3H, COCH ). TOF-HRMS: m/z [M þ H] calcd for C H F NO S:
3
14 13
3
3
found: 338.0438.
332.0563; found: 332.0562.
4
.1.1.12. 3-Acetyl-2-methylthio-6-(trifluoromethoxyl)quinolin-4(1H)-
4.1.1.16. 3-Acetyl-2-methylthio-6-cyano-7-(trifluoromethyl)quinolin-
ꢀ
1
ꢀ
1
one (3l). White powder, yield, 50%; m.p. 164‒166 C; H NMR
300 MHz, CDCl
15.56 (s, 1H, NH), 8.07 (s, 1H), 7.82 (d, J ¼ 9.1 Hz,
H), 7.56 (d, J ¼ 8.9 Hz, 1H), 2.97 (d, J ¼ 3.3 Hz, 3H, SCH ), 2.72 (d,
198.4 (CO),
4(1H)-one (3p). Yellow powder, yield, 40%; m.p. 182‒184 C;
NMR (300 MHz, CDCl 15.73 (s, 1H, NH), 8.76 (s, 1H), 8.17 (s, 1H),
3.00 (s, 3H, SCH ), 2.76 (s, 3H, COCH
DMSO‑d 198.0 (CO), 160.0 (C-4), 136.6 (C-2), 134.2, 129.2 (q, CF
H
(
1
3
)
d
3
) d
13
3
3
3
); C NMR (150 MHz,
),
), 14.8
NaO S:
13
J ¼ 3.4 Hz, 3H, COCH
3
); C NMR (150 MHz, DMSO‑d
6
)
d
6
)
d
3
173.0 (C-4), 157.1 (C-2), 144.69, 144.68, 138.1, 125.7, 121.0, 120.2 (q,
124.9, 123.1, 121.3, 120.7, 119.5, 115.3 (CN), 102.5, 31.5 (COCH
3
þ
OCF
3
), 119.3, 116.1, 31.5 (COCH
3
), 14.8 (SCH
3
). TOF-HRMS: m/z [M þ
(SCH
3
). TOF-HRMS: m/z [M þ Na] calcd for C14
H
9
F
3
N
2
2
þ
Na] calcd for C13H F NNaO S: 340.0226; found: 340.0226.
10 3 3
349.0229; found: 349.0224.
4
.1.1.13. 3-Acetyl-8-chloro-2-methylthio-5-(trifluoromethyl)quino-
4.1.1.17. 3-Acetyl-8-fluoro-2-methylthio-5-(trifluoromethyl)quinolin-
ꢀ
lin-4(1H)-one (3m). Light yellow powder, yield, 54%; m.p. 206‒
2
4(1H)-one (3q). Light yellow powder, yield, 46%; m.p. 169‒171 C;
ꢀ
1
1
08 C; H NMR (300 MHz, DMSO‑d
6
)
d
9.64 (s, 1H, NH), 8.06 (d,
H NMR (300 MHz, DMSO‑d
6
)
d
16.33 (s, 1H, NH), 7.80 (dd, J ¼ 8.1,

152
K. Xu et al. / European Journal of Medicinal Chemistry 154 (2018) 144e154
4
.4 Hz, 1H), 7.40 (t, J ¼ 8.6 Hz, 1H), 2.97 (s, 3H, SCH
3
), 2.73 (s, 3H,
NO S:
4.3. Biology
þ
COCH
3
). TOF-HRMS: m/z [M þ H] calcd for C13
H
10
F
4
2
3
20.0363; found: 320.0360.
4.3.1. Determination of minimum inhibitory concentrations (MICs)
and minimum bactericidal concentrations (MBC)
The MICs and MBCs of compounds were determined in Mueller-
Hinton (MH) broth according to the methodology of the Clinical
Laboratory Standards Institute (CLSI), using the micro-broth dilu-
tion method in 96-well micro-test plates [36]. The bacterial inoc-
ulation was prepared by growing colonies from MH agar in MH
4
.1.1.18. 3-Acetyl-8-chloro-2-methylthio-6-nitroquinolin-4(1H)-one
ꢀ
1
(3r). Yellow powder, yield, 56%; m.p. 204‒207 C; H NMR
(
300 MHz, CDCl
3
)
d
11.30 (s, 1H, NH), 9.19 (d, J ¼ 2.4 Hz, 1H), 8.64 (d,
J ¼ 2.4 Hz, 1H), 3.07 (s, 3H, SCH
3
), 2.82 (s, 3H, COCH
3
). TOF-HRMS:
þ
m/z [M þ H] calcd for C12
H10ClN O S: 313.0044; found: 313.0042.
2 4
broth. The final test concentration of compounds ranged from 0.2 to
5
200
m
M and bacterial inocula of 10 CFU/mL. The MICs were
4.1.1.19. 3-Acetyl-2-(methylthio)quinolin-4(1H)-one
(3s). White
recorded as the lowest concentration of the test compound inhib-
ꢀ
1
powder, yield, 43%; m.p. 230‒231 C; H NMR (300 MHz, DMSO‑d
d
6
)
ꢀ
iting visual growth, after incubation at 37 C for 18e20 h. MICs
10.87 (s, 1H, NH), 8.16e8.05 (m, 1H), 7.80 (d, J ¼ 8.2 Hz, 1H),
), 2.52 (s,
198.7 (CO),174.2
were performed in at least duplicate and the mean MIC is reported.
To determine MBC, 30 mL from each well, where no growth was
7
3
.74e7.66 (m, 1H), 7.39 (t, J ¼ 7.3 Hz, 1H), 2.65 (s, 3H, SCH
3
13
3 6 3
H, COCH ); C NMR (150 MHz, DMSO‑d /CDCl ) d
detected, the compounds with concentration ꢁ MIC was spread on
(
(
C-4), 156.3 (C-2), 139.5, 132.4, 125.1, 124.9, 124.4, 119.6, 118.3, 31.6
COCH
ꢀ
MH agar plates and incubated for 24 h at 37 C. The lowest com-
þ
3
), 14.9 (SCH
H12NO S: 234.0583; found: 234.0583.
12 2
3
). TOF-HRMS: m/z [M þ H] calcd for
pound concentration resulting in no growth on the agar plate
represented the MBC. Each sample was tested in triplicate and each
experiment was repeated three times.
C
4
.1.1.20. 3-Acetyl-8-chloro-2-methylthio-6-(trifluoromethyl)quino-
lin-4(1H)-one (3t). Light yellow powder, yield, 50%; m.p. 168‒
ꢀ
1
4.3.2. Inhibition of bacterial growth
The effect of concentrations ranging from 0.5 to 4 times MIC of
the four most active compounds on the growth of S. aureus was
quantified after 0, 4, 8, 12, 16, 20 and 24 h incubation at 37 C. At
each time point, an aliquot (100 mL) was pipetted and measured for
1
70 C; H NMR (300 MHz, CDCl
3
)
d
15.74 (s, 1H 1H,NH), 8.44 (s, 1H),
), 2.79 (s, 3H,COCH ). TOF-
ClF NNaO S: 357.9887;
7.98 (d, J ¼ 1.7 Hz, 1H), 2.98 (s, 3H,SCH
3
3
þ
HRMS: m/z [M þ Na] calcd for C13
H
9
3
2
ꢀ
found: 357.9882.
the A600nm. The experiment was carried out in three biologically
independent assays and each sample was tested in triplicate.
4
.1.2. Synthesis of 3-Acetyl-2-methylsulfoxy-6-(trifluoromethyl)
quinolin-4(1H)-one (4)
To a solution of the quinolone 3a (300 mg, 1.0 mmol) in glacial
acetic acid (10 mL) was added 30% hydrogen peroxide (0.3 mL,
4.4. DNA gyrase assays
3
.0 mmol). The reaction mixture was stirred at room temperature
for 24 h. The reaction mixture was diluted with ice-water (50 mL).
The solid precipitated was filtered, washed with water, dried, and
crystallized from ethanol to obtain compound 4 (120 mg, 44%).
According to the manufacturer's protocol, the relaxed pBR322
DNA (Inspiralis Ltd) as a substrate was used to assay DNA super-
coiling activity. The reaction mixture (20 mL) contained 35 mM
ꢀ
1
White powder, m.p. 219‒221 C; H NMR (300 MHz, DMSO‑d
6
)
2
Tris‒HCl (pH 7.5), 24 mM KCl, 4 mM MgCl , 2 mM dithiothreitol,
d
12.24 (s, 1H, NH), 8.56e8.38 (m, 2H), 8.12 (d, J ¼ 8.7 Hz, 1H), 2.99
1.8 mM spermidine, 1 mM ATP, 6.5% glycerol, 0.1 mg/mL bovine
serum albumin (BSA), 12.5 ng/mL relaxed pBR322, and 5 U of DNA
gyrase (New England BioLabs). The reaction mixture was incubated
at 37 C for 60 min with the title compound, following by the
3
termination by adding the loading dye (3.5 mL) and 20 mL CHCl /
(
d, J ¼ 1.4 Hz, 3H, SCH ), 2.63 (d, J ¼ 1.5 Hz, 3H, COCH ).
3
3
ꢀ
4
4
.1.3. Synthesis of 3-acetyl-2-hydroxy-6-(trifluoromethyl)quinolin-
(1H)-one (5)
isoamyl alcohol (24:1). After a brief vortex, the blue aqueous phase
was analyzed by electrophoresis in 1% agarose gel. One unit of
supercoiling activity was defined as the amount of DNA gyrase
required to supercoil 0.5 mg of plasmid in 1 h. The IC50 value was
defined as the drug concentration that reduced the enzymatic ac-
tivity observed with drug-free controls by 50% [39].
A suspension of the quinolone 3a (1.0 mmol, 300 mg) in 50%
sulfuric acid (10 mL) was heated under reflux for 4 h. Then, the
mixture was left to cool and diluted with ice-cold water (100 mL).
The solid precipitate was filtered, washed with water and crystal-
lized from ethanol to afford compound 5 (115 mg, 42%). White
ꢀ
1
6
powder, m.p. 304‒306 C; H NMR (300 MHz, DMSO‑d ) d 12.23 (s,
1
2
2
H, NH), 8.28 (s, 1H), 7.96e7.88 (m,1H), 7.66 (s, 1H), 6.09 (s, 1H, OH),
þ
3
.60 (s, 3H, COCH ). TOF-HRMS: m/z [M þ H] calcd for C12
9 3 3
H F NO :
4.5. Molecular docking study
72.0529; found: 272.0531.
A structure based in silico procedure was applied to study the
4
.2. Crystallographic studies
binding mode of the active compounds into DNA-gyrase from
S. aureus. The CDOCKER of Discovery Studio 2017R2 (DS) was
conducted to explain SAR of a series of compounds and to further
improve the design of more effective DNA-gyrase inhibitors. The
target enzyme (PDB ID: 2XCT) was prepared with Prepare Protein of
DS to ensure the integrity. The title compounds were processed by
Full Minimization of the Small Molecular in DS and docked into the
ligand binding site of the protein using CDOCKER. The docking
results for each compound were extracted and analyzed in both
CDOCKER and Pymol. The potent compounds with the lowest
-CDOCKER_INTERACTION_ENERGY value among the output con-
figurations were selected to analyze the interactions with target
enzyme in details and all the results were displayed using Pymol.
X-ray single-crystal diffraction data for compounds 3a and 3j
was collected on a Bruker SMART APEX CCD diffractometer at 294 K
using CuK radiation ( scan mode. The
¼ 0.71073 Å) by the
a
l
u
program SAINT was used for integration of the diffraction profiles.
The structure was solved by direct methods using the SHELXS
program of the SHELXTL package and refined by full-matrix least-
squares methods with SHELXL [38]. The corrections for LP factors
were applied. All non-hydrogen atoms of compound 3a were
refined with anisotropic thermal parameters. All hydrogen atoms
were generated theoretically onto the parent atoms and refined
isotropically with fixed thermal factors.

K. Xu et al. / European Journal of Medicinal Chemistry 154 (2018) 144e154
153
4
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K.K. Brown, C.J. Lewis, E.W. May, M.R. Saunders, O. Singh, C.E. Spitzfaden,
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Type IIA topoisomerase inhibition by a new class of antibacterial agents,
Nature 466 (2010) 935e940.
The bacterial strain S. aureus ATCC 6538 was grown overnight at
ꢀ
37 C on MullereHinton Agar. The bacteria were harvested and the
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7
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[
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4 4
and the bacteria were post-fixed in 1% OsO for 2 h. The OsO were
then pipetted out into an osmium waste bottle and the bacteria
were washed in PBS (pH 7.2) for three times. Fixed microbial pellets
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heterocycle-fused quinolinone derivatives: a review, Eur. J. Med. Chem. 97
The authors declare no competing financial interest.
(2015) 397e408.
[
24] J.M. Kraus, H.B. Tatipaka, S.A. McGuffin, N.K. Chennamaneni, M. Karimi, J. Arif,
C.L.M.J. Verlinde, F.S. Buckner, M.H. Gelb, Second generation analogues of the
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Acknowledgments
Financial support was provided by the Natural Science Funda-
tion of Anhui provincial Department of Education (KJ2017A180,
KJ2017A831, 12925KJ2018B12), and Research Fund for the Doctoral
Scientific Research of Anhui Medical University (XJ201615). We are
grateful to Professor Q. H. Song (The University of Science and
Technology of China) for helpful discussion.
[
25] F.S. Buckner, M.T. Bahia, P.K. Suryadevara, K.L. White, D.M. Shackleford,
N.K. Chennamaneni, M.A. Hulverson, J.U. Laydbak, E. Chatelain, I. Scandale,
C.L.M.J. Verlinde, S.A. Charman, G.I. Lepesheva, M.H. Gelb, Pharmacological
characterization, structural studies, and in vivo activities of anti-chagas dis-
ease lead compounds derived from Tipifarnib, Antimicrob. Agents Chemother.
56 (2012) 4914e4921.
[
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26] Y.L. Fan, X.W. Cheng, J.B. Wu, M. Liu, F.Z. Zhang, Z. Xu, L.S. Feng, Anti-
plasmodial and antimalarial activities of quinolone derivatives: an overview,
Eur. J. Med. Chem. 146 (2018) 1e14.
27] S. Sarveswari, V. Vijayakumar, R. Siva, R. Priya, Synthesis of 4-hydroxy-2(1H)-
quinolone derived chalcones, pyrazolines and their antimicrobial, in silico
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quinolones: synthesis, antimicrobial evaluation, interaction with DNA, com-
bination with topoisomerase IV and penetrability into cells, Eur. J. Med. Chem.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.05.021.
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