N-thiadiazole-4-hydroxy-2-quinolone-3-carboxamides bearing heteroaromatic rings as novel antibacterial agents: Design, synthesis, biological evaluation and target identification

Article abstract of DOI:10.1016/j.ejmech.2019.112022

Due to the occurrence of antibiotic resistance, bacterial infectious diseases have become a serious threat to public health. To overcome antibiotic resistance, novel antibiotics are urgently needed. N-thiadiazole-4-hydroxy-2-quinolone-3-carboxamides are a potential new class of antibacterial agents, as one of its derivatives was identified as an antibacterial agent against S. aureus. However, no potency-directed structural optimization has been performed. In this study, we designed and synthesized 37 derivatives, and evaluated their antibacterial activity against S. aureus ATCC29213, which led to the identification of ten potent antibacterial agents with minimum inhibitory concentration (MIC) values below 1 μg/mL. Next, we performed bacterial growth inhibition assays against a panel of drug-resistant clinical isolates, including methicillin-resistant S. aureus, and cytotoxicity assays with HepG2 and HUVEC cells. One of the tested compounds named 1-ethyl-4-hydroxy-2-oxo-N-(5-(thiazol-2-yl)-1,3,4-thiadiazol-2-yl)-1,2-dihydroquinoline-3-carboxamide (g37) showed 2 to 128-times improvement compared with vancomycin in term of antibacterial potency against the tested strains (MICs: 0.25–1 μg/mL vs. 1–64 μg/mL) and an optimal selective toxicity (HepG2/MRSA, 110.6 to 221.2; HUVEC/MRSA, 77.6–155.2). Further, comprehensive evaluation indicated that g37 did not induce resistance development of MRSA over 20 passages, and it has been confirmed as a bactericidal, metabolically stable, orally active antibacterial agent. More importantly, we have identified the S. aureus DNA gyrase B as its potential target and proposed a potential binding mode by molecular docking. Taken together, the present work reports the most potent derivative of this chemical series (g37) and uncovers its potential target, which lays a solid foundation for further lead optimization facilitated by the structure-based drug design technique.

Full text of DOI:10.1016/j.ejmech.2019.112022

Journal Pre-proof
N-thiadiazole-4-hydroxy-2-quinolone-3-carboxamides bearing heteroaromatic rings
as novel antibacterial agents: Design, synthesis, biological evaluation and target
identification
Wenjie Xue, Xueyao Li, Guixing Ma, Hongmin Zhang, Ya Chen, Johannes Kirchmair,
Jie Xia, Song Wu
PII:
S0223-5234(19)31180-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.112022
EJMECH 112022
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 August 2019
Revised Date: 22 December 2019
Accepted Date: 29 December 2019
Please cite this article as: W. Xue, X. Li, G. Ma, H. Zhang, Y. Chen, J. Kirchmair, J. Xia, S. Wu, N-
thiadiazole-4-hydroxy-2-quinolone-3-carboxamides bearing heteroaromatic rings as novel antibacterial
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N-Thiadiazole-4-Hydroxy-2-Quinolone-3-Carboxamides Bearing Heteroaromatic
Rings as Novel Antibacterial Agents: Design, Synthesis, Biological Evaluation and
Target Identification
a
a
b
b
c
Wenjie Xue , Xueyao Li , Guixing Ma , Hongmin Zhang , Ya Chen , Johannes
c,d,e
a,*
a,*
Kirchmair , Jie Xia and Song Wu
a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Department of New Drug Research and Development, Institute of Materia Medica, Chinese
Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China;
b
Department of Biology, Guangdong Provincial Key Laboratory of Cell Microenvironment
and Disease Research, Shenzhen Key Laboratory of Cell Microenvironment and
SUSTech-HKU Joint Laboratories for Matrix Biology, Southern University of Science and
c
Technology, Shenzhen 518055, China; Center for Bioinformatics (ZBH), Department of
Computer Science, Faculty of Mathematics, Informatics and Natural Sciences, Universität
d
Hamburg, Hamburg, Germany. ; Department of Chemistry, Faculty of Mathematics and
e
Natural Sciences, University of Bergen, Bergen, Norway; Computational Biology Unit
(CBU), University of Bergen, Bergen, Norway.
*
Correspondence should be addressed to J.X. (jie.william.xia@hotmail.com) or S.W.
(ws@imm.ac.cn)
Dr. Jie Xia & Prof. Song Wu
Institute of Materia Medica, Chinese Academy of Medical Sciences
No. 2 Nanwei Road
Beijing 100050, China

Abstract
Due to the occurrence of antibiotic resistance, bacterial infectious diseases have become a
serious threat to public health. To overcome antibiotic resistance, novel antibiotics are
urgently needed. N-thiadiazole-4-hydroxy-2-quinolone-3-carboxamides are a potential new
class of antibacterial agents, as one of its derivatives was identified as an antibacterial agent
against S. aureus. However, no potency-directed structural optimization has been
performed. In this study, we designed and synthesized 37 derivatives, and evaluated their
antibacterial activity against S. aureus ATCC29213, which led to the identification of ten
potent antibacterial agents with minimum inhibitory concentration (MIC) values below 1
µg/mL. Next, we performed bacterial growth inhibition assays against a panel of
drug-resistant clinical isolates, including methicillin-resistant S. aureus, and cytotoxicity
assays with HepG2 and HUVEC cells. One of the tested compounds named
1
-ethyl-4-hydroxy-2-oxo-N-(5-(thiazol-2-yl)-1,3,4-thiadiazol-2-yl)-1,2-dihydroquinoline-3-
carboxamide (g37) showed 2 to 128-times improvement compared with vancomycin in
term of antibacterial potency against the tested strains (MICs: 0.25 to 1 µg/mL vs. 1 to 64
µg/mL) and an optimal selective toxicity (HepG2/MRSA, 110.6 to 221.2; HUVEC/MRSA,
7
7.6-155.2). Further, comprehensive evaluation indicated that g37 did not induce resistance
development of MRSA over 20 passages, and it has been confirmed as a bactericidal,
metabolically stable, orally active antibacterial agent. More importantly, we have identified
the S. aureus DNA gyrase B as its potential target and proposed a potential binding mode
by molecular docking. Taken together, the present work reports the most potent derivative

of this chemical series (g37) and uncovers its potential target, which lays a solid foundation
for further lead optimization facilitated by the structure-based drug design technique.
Keywords: antibiotic resistance, antibacterial agent, MRSA, DNA gyrase B, molecular
docking

1
. Introduction
Antibiotic resistance has been declared a serious threat to public health worldwide in 2001
and is still on the rise [1]. The bacteria resistant to most antibiotics in clinical use are
mainly ESKAPE species, i.e. E. faecium, S. aureus, K. pneumoniae, A. baumannii, P.
aeruginosa and Enterobacter [2, 3]. Among them, the Gram-positive bacteria represented
by methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecium (VRE)
have been categorized as serious threats by the US Centers for Disease Control and
Prevention in 2013 [4]. The situation becomes even worse as the number of new drugs
approved for the treatment of drug-resistant bacterial infections is on the decline in recent
years [5, 6]. For these reasons, the discovery of novel resistance-breaking antibacterial
agents is of utmost importance.
N-thiadiazole-4-hydroxy-2-quinolone-3-carboxamides represent an important chemical
series that shows a wide range of pharmacological activities. Several derivatives have been
reported as anticancer agents [7], fat storage regulators [8], strand transfer inhibitors of
HIV integrase [9] and anti-tuberculosis agents [10-12]. Compound 11 is a derivative with a
methyl group at the position 1 of the 1,2-dihydroquinoline and an ethyl group at the
position 5 of the thiadiazol (cf. Figure 1). Peternel L. et al. have discovered that this
compound is able to inhibit S. aureus ATCC 25923 and not toxic to the ChoK1 cell line,
with a TC /MIC ratio of 20.7 [13]. To the best of our knowledge, current pipelines for
50
antibiotics research and development do not cover this chemical series. In addition, no
structural modification that improves the potency of compound 11 against S. aureus or
other gram-positive bacteria has been reported. Although the other reported derivatives

might be repurposed for antibiotics against gram-positive bacteria, they are limited to the
structures with alkyl groups at the above-mentioned positions.
Figure. 1. (a) Antibacterial compound 11 with a desired safety margin and (b) its core
scaffold, i.e., N-thiadiazole-4-hydroxy-2-quinolone-3-carboxamide.
In order to develop N-thiadiazole-4-hydroxy-2-quinolone-3-carboxamides as a new
class of antibacterial agents, we herein performed structural modification of compound 11
(mainly with aromatic or heteroaromatic rings at the position 5 of the thiadiazol) and
determined the MICs (minimum inhibitory concentrations) of 37 new derivatives against S.
aureus ATCC 29213. Then, we tested the derivatives that showed higher potencies than
compound 11 for their antibacterial activity spectrum and cytotoxicity to HepG2 and
HUVEC cells and selected the most promising derivative. Moreover, we evaluated it for its
propensity to develop bacterial resistance, its mode of action (i.e. bactericidal or
bacteriostatic), in vitro metabolic stability, in vivo pharmacokinetic profile and in vivo
antibacterial efficacy. Lastly, we identified its potential target by cheminformatics analysis
and proposed a binding mode by molecular docking, which can make structure-based lead
optimization become feasible in the future.
2
. Results and discussions
2
.1. Molecular design

It should be noted that structural modification with the aim to improve the antibacterial
potency of the compound 11 has never been reported up to now. As a pioneering work, we
designed 37 new structures (cf. Table 1) iteratively by taking into account bioassay results
and the commercial availability of the starting materials. At first, we designed three
derivatives with alkyl groups, i.e. the methyl, ethyl and propyl groups at position 1 of the
1
,2-dihydroquinoline, while leaving position 5 of the thiadiazol unsubstituted. Once the
optimal alkyl group was determined, we designed four classes of compounds with different
substituents at position 5 of the thiadiazol. Since this position of compound 11 was
substituted with an ethyl group, it was natural to design derivatives with alkyl groups in
various carbon-atom lengths. Thus, we designed the first class of derivatives that were
substituted with methyl, ethyl, n-propyl, n-butyl, n-pentyl and n-hexyl groups. As
cycloalkyl groups may show similar properties to the alkyl groups, we introduced three
cycloalkyl groups (cyclopropyl, cyclopentyl and cyclohexyl groups) to the position, which
constituted the second class of derivatives. In order to enhance structural novelty, we
further designed the other two classes of derivatives by respectively introducing aromatic
rings (benzene rings unsubstituted and those substituted with fluoro, chloro, bromo,
difluoromethoxy, nitro, methyl, methoxy, ethoxyl and dimethylamino groups at the ortho-,
meta- or para- positions), and heteroaromatic rings (pyridin-2-yl, pyridin-3-yl, pyridin-4-yl,
thiophen-2-yl, 1-methyl-1H-pyrazol-3-yl, thiazol-4-yl, thiazol-2-yl groups) to position 5 of
the thiadiazol.
2
.2. Chemistry

The designed molecules, i.e. N-thiadiazol-4-hydroxy-2-quinolone-3-carboxamides,
were synthesized according to the route described in Scheme 1, which included the
reactions between the 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acids (c1-c3)
and the substituted aminothiadiazoles (f1-f35).
Scheme 1. Reagents and conditions: (i) NaH, 0 °C, DMF, R -X (X=I), (ii) NaH, DMF,
2
diethyl malonate, (iii) 2.8 M HCl in CH COOH, 60 °C, (iv) TFA, 80 °C, 8 h, (v) EDCI,
3
HOBt, DIPEA, DMF, r.t..
The synthesis of the 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acids (c1-c3)
started with the isatoic anhydrides (a). Firstly, the isatoic anhydrides were alkylated in the
presence of both sodium hydride and the alkyl iodide. The resulting N-alkylated anhydrides
were treated with diethyl malonate and sodium hydride to yield the N-alkylated
4
-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylates (b1-b3). These esters were then
transformed into the acids by the hydrolysis reaction in a solution of hydrochloric acid
dissolved in acetic acid. The 2-amino-1,3,4-thiadiazoles (f1-f35) were prepared by the
reactions between the nitriles (d1-d35) and the thiosemicarbazide (e) dissolved in
trifluroacetic acid (TFA) [14]. The end products (11 and g1-g37) were obtained by the
cross-linking reactions between the 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylic
acids (c1-c3) and the 2-amino-1,3,4-thiadiazoles (f1-f35). The chemical structures were

1
13
validated by high resolution mass spectrometry (HRMS), H NMR and C NMR (cf.
Figure S1).
2
.3. Preliminary structure-antibacterial activity relationship
We tested all the synthesized derivatives for their antibacterial activity (i.e. MICs)
against S. aureus ATCC29213 (i.e. a wild-type and methicillin-sensitive standard strain) by
the broth microdilution method. The chemical structures of 37 derivatives (11 and g1-g37)
and their MIC values for S. aureus ATCC 29213 are listed in Table 1. The MIC values
range from 100 µg/mL to 0.1 µg/mL, indicating that all the derivatives inhibit the growth of
S. aureus ATCC 29213. Encouragingly, ten out of the 37 newly designed and synthesized
derivatives (g1-g37) were more potent than compound 11 (MIC: 1.56 µg/mL), with MIC
values lower than 1 µg/mL. The MIC values of five derivatives even reached up to 0.1
µg/mL. The data demonstrate that our strategy for molecular design was effective to
improve antibacterial potency.
As we mention in the subsection of “Molecular design”, we initially designed,
synthesized and tested three derivatives with different alkyl groups at position 1 of the
1
,2-dihydroquinoline (g1-g3). As compound g2 with the ethyl group was more potent than
the others (MIC: 1.56 µg/mL; cf. Table 1), we decided to preserve the ethyl group at
position 1 of the 1,2-dihydroquinoline when performing further modification at position 5
of the thiadiazol.
Among other derivatives with the ethyl group at R , the first class covered those with
2
alkyl groups as R . With the exception of g9, all compounds of this class (g4-g9) exhibited
1
strong antibacterial activity, with MIC values lower than 1 µg/mL. The MIC values were

0
.78 µg/mL for the derivatives with the methyl group (g4) or the ethyl group (g5), 0.39
µg/mL for the derivatives with the n-propyl- group (g6) and 0.1 µg/mL for the derivatives
with the n-butyl- (g7) or the n-pentyl (g8) group, respectively (cf. Table 1). For this class of
derivatives, it was concluded that (i) the introduction of the alkyl groups in a length within
five carbon atoms to the 5-position of the thiadiazol was favorable for antibacterial activity,
(ii) the length of the alkyl groups affected the antibacterial activity, and four or five
carbon-atom length led to the optimal potency.
The second class of derivatives were those with the cycloalkyl substituents, including
g10 (the cyclopropyl group), g11 (the cyclopentyl group) and g12 (the cyclohexyl group).
Their MIC values were greater than that of g2, indicating that the introduction of the
cycloalkyl groups results in a decrease of antibacterial activity.
The third class of derivatives started with g13, i.e. the derivative with the benzene ring.
Compound g13 was as potent as the compounds bearing alkyl groups (MIC: 0.39 µg/mL; cf.
Table 1). This outcome was rather encouraging because no aromatic ring had been reported
as
a
substituent
at
position
5
of
the
thiadiazol
for
N-thiadiazole-4-hydroxy-2-quinolone-3-carboxamides so far. As a follow-up, we designed
and synthesized 17 derivatives with a variety of substituted benzene rings (g14-g30)
through parallel synthesis. The bioassay data suggested a preliminary structure-activity
relationship (SAR) for this class of derivatives: (i) All the substitutions in the benzene ring
reduced the antibacterial activity. The introduction of the electron-withdrawing groups
seemed to be more detrimental to antibacterial activity than the electron-donating groups
(e.g. the methoxy group). For instance, the derivative with the fluoro- group at the ortho-,

meta- or para- position was not as potent as that with the methoxy group at the same
position. As shown in Table 1, g27 (MIC: 12.5 µg/mL), g23 (MIC: 6.25 µg/mL) and g14
(MIC: 25 µg/mL) are less potent than g29 (MIC: 3.12 µg/mL), g25 (MIC: 1.56 µg/mL) and
g20 (MIC: 3.12 µg/mL), respectively. (ii) Whether the substituent of the benzene ring was
an electron-donating group or an electron-withdrawing group, the meta- position for
substitution seemed to be better than the other two positions. For instance, the derivatives
with the fluoro, the methoxy or the ethoxyl group at the meta- position (i.e. g23, g25, g26)
showed better antibacterial activity than those with the substituents at the ortho- and the
para- positions (i.e. g27/g14, g29/g20, g30/g21).
Seven derivatives with different heteroaromatic rings (g31-g37) belong to the fourth
class of derivatives. Among them, g31 (the pyridin-2-yl group) and g37 (the thiazol-2-yl
group) showed four-fold improvement in antibacterial activity compared with g13 (phenyl
group), with a MIC value as low as 0.1 µg/mL. The compounds g32 and g36 were also
potent antibacterial compounds (MIC: less than 1 µg/mL). To be specific, g32 (pyridin-3-yl
group) displayed the activity equivalent to g13 (MIC: 0.39 µg/mL), while g36 (thiazol-4-yl
group) was slightly weaker (MIC: 0.78 µg/mL) than g13.
Table 1. Chemical structures of the synthesized compounds, their antibacterial activity in
terms of MICs (µg/mL) for S. aureus ATCC29213 and GyrB inhibitory activity in term of
IC (µM).
5
0
Compound
R1
R2
S. aureus
IC for GyrB
50

a
ID
ATCC29213
1.56
(µM, mean ± SD)
11
CH
3
3
-
-
0.10±0.01
g1
g2
g3
H
H
H
CH
6.25
1.56
3.12
0.19±0.03
0.11±0.01
0.46±0.03
CH
CH
CH -
2
(CH2)2-
3
3
g4
g5
g6
g7
CH
CH
CH
CH
3
3
3
3
CH
CH
CH
CH
2
2
2
2
-
-
-
-
0.78
0.78
0.39
0.1
0.24±0.01
0.45±0.01
0.27±0.01
0.99±0.01
g8
g9
CH
CH
CH
3
3
3
CH
CH
CH
2
2
2
-
-
-
0.1
0.47±0.02
0.35±0.02
0.58±0.10
12.5
3.12
g10
g11
g12
CH
3
3
CH
2
2
-
-
6.25
12.5
0.55±0.01
0.55±0.06
CH
CH
g13
g14
g15
g16
g17
g18
CH
CH
CH
CH
CH
CH
3
3
3
3
3
3
CH
CH
CH
CH
CH
CH
2
2
2
2
2
2
-
-
-
-
-
-
0.39
25
1.72±0.10
2.30±0.04
1.46±0.20
2.19±0.24
1.42±0.02
1.61±0.16
12.5
12.5
12.5
50

g19
g20
g21
g22
CH
CH
CH
CH
3
3
3
3
CH
CH
CH
CH
2
2
2
2
-
-
-
-
6.25
3.12
100
0.64±0.07
1.14±0.14
2.30±0.11
2.66±0.13
3.12
g23
g24
g25
CH
CH
CH
3
3
3
CH
CH
CH
2
2
2
-
-
-
6.25
12.5
1.56
1.81±0.25
1.75±0.03
2.17±0.05
g26
CH
3
CH
2
-
3.12
1.69±0.06
g27
g28
CH
3
3
CH
2
2
-
-
12.5
50
0.99±0.16
3.18±0.11
CH
CH
g29
g30
g31
CH
CH
CH
3
3
3
CH
CH
CH
2
2
2
-
-
-
3.12
12.5
0.1
0.76±0.01
2.35±0.08
0.42±0.05
g32
CH
3
CH
2
-
0.39
0.24±0.02

g33
CH
3
CH
2
-
1.56
0.24±0.03
g34
g35
CH
3
3
CH
2
2
-
-
3.12
3.12
0.22±0.05
0.49±0.04
CH
CH
g36
g37
CH
3
CH
2
-
-
0.78
0.1
0.16±0.03
0.15±0.02
CH
CH
3
2
a
mean, the average of the duplicate; SD, standard deviation.
.4. Activity spectrum against a panel of gram-positive bacteria
Following the discovery of ten compounds that potently inhibited methicillin-sensitive
2
S. aureus (MSSA) ATCC 29213 (MIC: less than 1 µg/mL), we tested them against other
nine strains of MRSA, including clinical isolates (15-1, 15-2, 15-3, 15-6, 15-7, 15-8, 15-9,
ATCC 700699 and ATCC 700788), with compound 11 as a reference and vancomycin as a
positive control. Compared with their potency for MSSA (0.1 to 0.78 µg/mL), all new
derivatives showed decreased potency for the tested MRSA strains, with MIC values
ranging from 0.25 µg/mL to >64 µg/mL (cf. Table 2). Among them, the MIC values of the
derivatives with the aromatic rings except for g36 ranged from 0.25 to 2 µg/mL, while the
MIC values of those with the alkyl groups except for g8 were between 0.5 to >64 µg/mL.
The above data indicate that the introduction of the aromatic rings at position 5 of the
thiadiazol is more favorable for antibacterial activity against MRSA strains than the
substitutions with the alkyl groups. This observed structure-activity relationship was further
validated by the bioassay against other Gram-positive bacteria, including
methicillin-sensitive S. epidermidis (MSSE), methicillin-resistant S. epidermidis (MRSE),

E. faecalis (EFA), E. faecium (EFM) and vancomycin-resistant E. faecalis (VRE). For
these bacteria, the MIC values of most derivatives with aromatic rings (g13, g31 and g37)
are lower than those with alkyl groups (g4-g7).
Among derivatives with alkyl groups (including compound 11), the most potent
compound was g8. Its MIC values for the tested bacterial strains were between 0.5 and 2.0
µg/mL and hence lower than those of vancomycin (MICs: 1.0 to 64.0 µg/mL). Importantly,
g8 remained active against VRE and two vancomycin-intermediate S. aureus (VISA)
strains, i.e. ATCC700699b and ATCC700788b. Among those derivatives with the aromatic
rings, g31 and g37 were the only two compounds that showed better antibacterial activity
than vancomycin for most of the tested bacterial strains. g37 seemed to be a bit more potent
(MICs: 0.25-1 µg/mL) than g31 (MICs: 0.5-1 µg/mL). In particular, g37 was able to inhibit
the growth of the three VRE strains at the concentration of 0.5 µg/mL. while the MIC value
of g31 for these strains was 1 µg/mL.
2
.5. Cytotoxicity to HepG2 and HUVEC cells
Since the essence of antibacterial therapy is selective toxicity [13], i.e. to kill or inhibit
bacteria while not to harm patients, mammalian cell toxicity is a common metric for
antibacterial drug discovery. In this study, we tested g8 (with alkyl groups), g31 and g37
(
with the aromatic/heteroaromatic rings) for their cytotoxicity against two cell lines, i.e.
human hepatocellular carcinoma (HepG2) cells and human umbilical vein endothelial cells
HUVEC) using the sulforhodamine B (SRB) assay [15]. As shown in Table 3, the IC50
(
values of the three compounds were greater than 50 µg/mL for HepG2 cells and over 25
µg/mL for HUVEC cells. To determine the compound with the highest safety margin, we

calculated the selectivity indexes (SIs) for the three compounds by considering both the
IC and MIC values for the MRSA strains. The SI values of g37 for both HepG2 and
5
0
HUVEC were 110.6 to 221.2 and 77.6 to 155.2, respectively, and hence greater than those
of g8 and g31 (HepG2: 52.1 to 104.2 and 63.2 to 126.4; HUVEC: 41.9 to 83.8 and 26.5 to
5
3). According to the safety margins, we selected g37 for further bioassay.
2
.6. Comprehensive evaluation of g37
2
.6.1. Propensity to develop bacterial resistance
In order to study the propensity of bacterial strains to develop resistance to g37, we
treated the bacterial strain MRSA 15-1 with the sub-lethal concentration (0.5×MIC) of g37
to make it potentially develop resistance. The way to study resistance development had
been commonly used in antibacterial drug discovery [16-18]. Then, we determined the MIC
value of g37 against the bacterial strain selected by the compound treatment. The
compound treatment and the MIC determination were repeated for 20 passages. Figure 2
and Table S1 show the MIC variability of g37 during 20 passages of the test, with
vancomycin as a comparison. Clearly, the sub-lethal level of vancomycin induced the
development of bacterial resistance to vancomycin, as the MIC value increased from 1
µg/mL to 8 µg/mL. Encouragingly, the MIC values of g37 remained unchanged (0.5 µg/mL)
during 20 passages, which indicated that g37 did not induce the development of
drug-resistant bacterial strain.

Table 2. Antibacterial activity spectrum of ten potent derivatives (MIC: less than 1 µg/mL for S. aureus ATCC29213) and compound 11.
MIC (µg/mL)
a
Bacterium
Strain
1
2
4
2
1
g4
2
g5
2
2
2
2
1
1
1
2
2
2
2
2
2
2
2
2
4
2
8
8
8
g6
2
2
2
2
4
2
4
4
4
2
2
2
4
2
4
4
2
4
8
8
8
g7
2
2
2
2
4
2
2
4
4
2
2
2
2
2
2
2
2
2
8
8
8
g8
0.5
1
g13
2
2
g31
0.5
1
g32
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1
g36
4
4
g37 Vancomycin
15-1
15-2
15-3
15-6
0.5
0.5
0.5
0.5
0.5
0.5
0.25
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1
1
1
4
2
0.5
0.5
0.5
0.5
1
1
0.5
0.5
1
4
1
4
2
0.5
1
4
1
MRSA
15-7
4
4
4
1
1
1
5-8
5-9
4
2
0.5
0.25
2
0.5
1
4
1
4
4
4
1
b
ATCC700699
4
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
1
0.5
0.5
1
4
4
b
ATCC700788
4
1
2
1
4
8
15-3
15-4
15-1
4
1
1
2
8
2
MSSE
MRSE
2
1
1
1
1
4
2
4
1
1
1
1
4
1
15-2
4
1
1
1
1
8
2
15-3
15-1
15-2
15-1
15-2
4
1
1
1
1
4
2
4
2
1
1
>64
>64
>64
>64
>64
>64
>64
4
2
EFA
4
1
1
1
4
2
4
1
1
1
8
1
EFM
4
1
2
1
4
1
2
ATCC51299
32
32
32
1
1
1
32
32
32
0.5
0.5
0.5
32
64
64
VRE
ATCC700802
ATCC51575
1
1
1
1
1
1
a
:
MRSA, methicillin-resistant S. aureus; MSSE, methicillin-sensitive S. epidermidis; MRSE, methicillin-resistant S. epidermidis; EFA, E.
faecalis; EFM, E. faecium; VRE, vancomycin-resistant E. faecalis.
b
:
vancomycin intermediate sensitive S. aureus (VISA)

Table 3. Cytotoxicity of three compounds to HepG2 and HUVEC cells.
a
b
Compound
ID
IC (µM
,
mean ± SD)
SI
5
0
HepG2
52.1±12.9
63.2±7.5
55.3±8.4
0.009±0.0039
HUVEC
41.9±5.4
26.5±6.6
HepG2
HUVEC
41.9-83.8
26.5-53
77.6-155.2
n.d.
g8
g31
g37
52.1-104.2
63.2-126.4
110.6-221.2
38.8±1.8
c
paclitaxel
0.0013+0.0001
n.d.
a
values represent cytotoxicity after 72-hour treatment with compounds. mean, the average
of duplicate values; SD, standard deviation.
b
selectivity index, IC /MIC
MRSAs
n.d., not determined.
5
0
c
Figure 2. The MIC variability of g37 (▲) and vancomycin (■) against MRSA 15-1 during
2
0 passages of the resistance development assay, with vancomycin as a comparison.
2
.6.2. Time-kill kinetics analysis
In order to determine whether our lead compound g37 was bactericidal or
bacteriostatic, we studied its time-kill kinetics against MRSA 15-1 at five concentrations,
i.e. 0.5×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC. As shown in Figure 3, at 4×MIC (2 µg/mL)
and 8×MIC (4 µg/mL) concentrations, g37 reduced the starting log CFU/mL (bacteria
1
0
concentration) by more than 3 log units over 24 h. Therefore, our lead compound g37 was

bactericidal. As a comparison, we also performed the time-kill kinetics study for
vancomycin. The result showed that also vancomycin was bactericidal as the starting
bacterial concentration decreased by more than 3 log units after 24 h of treatment with
vancomycin at concentrations of 4×MIC (4 µg/mL) and 8×MIC (8 µg/mL). The time-kill
curves also showed differences between g37 and vancomycin. (i) According to the
variability of log CFU/mL (4×MIC and 8×MIC), g37 showed a lower bactericidal rate
1
0
than vancomycin, but it had an equivalent effect to vancomycin on reducing the bacterial
concentration after 24 h. (ii) Treated at lower concentrations (1×MIC and 2×MIC), the
bacterial log CFU/mL was more significantly reduced by g37.
1
0
Figure 3. Time-kill kinetics for the compound g37 and vancomycin against MRSA 15-1
within 24 h. The compound concentrations used in the assays were 0.5×MIC, 1×MIC,
2
×MIC, 4×MIC, 8×MIC. Each data point is the average of three measurements.
.6.3. In vitro metabolic stability and in vivo pharmacokinetic profile
2
Metabolic stability of small molecules affects in vivo pharmacokinetic profile and thus
in vivo efficacy. As our systemic infection model was based on mice, we firstly tested the

metabolic stability of g37 by incubating the compound with the plasma or liver microsomes
from mice. As shown in Table 4, the estimated t (i.e. half-life) value in mouse plasma was
1/2
5
03.11 min, indicating g37 was stable in mouse plasma. Besides, the value of t1/2 and CLint
i.e. intrinsic clearance) in liver microsomes was estimated as 35.6 min and 38.9
µL/min/mg proteins, respectively, which indicated g37 was a compound of slow clearance
19]. Encouraged by the stable in vitro metabolism, we then performed a preliminary
(
[
pharmacokinetic study in mice. The result of the pharmacokinetic study is shown as a curve
that describes the concentration viability of g37 in plasma along with the sampling time
after the compound was orally administered at a dose of 10 mg/kg (cf. Figure S3). We
obtained the pharmacokinetic parameters from the curve that (1) the maximum
concentration (Cmax) of the compound in plasma (including the free state and the bound
state) was 827.1 ng/mL, a concentration a bit greater than its MIC value for the bacterial
strain (0.5 µg/mL), (2) the time to reach the maximum concentration (Tmax) was 1.0 h, (3)
the t1/2 value of this compound was favorable (2.3 h) and the mean residence time during
the sampling time was 2.7 h (cf. Table 4). All the above data demonstrated that g37 had
relatively good pharmacokinetic properties.

Table 4. In vitro metabolic stability and in vivo pharmacokinetics profile of the compound
g37.
in vitro metabolic stability (mouse)
mouse pharmacokinetic profile
liver
microsomes
stability
plasma
stability
parameter
10mg/kg (p.o.)
AUC (h•ng/mL)
2539
2620
2.7
last
t1
/2
5
03.11
/
35.6
38.9
AUC (h•ng/mL)
inf
(
min)
MRT (h)
last
t (h)
2.3
1
/2
CLint
µL/min/mg)
Tmax(h)
Cmax(ng/mL)
1
(
827.1
2
.6.4. In vivo antibacterial efficacy
In light of the favorable biological and pharmacokinetic properties of g37, we were
curious to see whether the compound would show in vivo antibacterial efficacy. Prior to the
efficacy study, we determined the minimum lethal dose (MLD) for our Kunming mice as 5
7
×
10 CFU/mL, by the intraperitoneal injection of the MRSA 15-1 inoculum (0.5 mL) at
7
6
5
three different doses, i.e. 5×10 CFU/mL, 5×10 CFU/mL and 5×10 CFU/mL (cf. Table
S2). As the first attempt, we treated the mouse systematic infection model with the
compound according to a common dosing schedule for oral administration of antibiotics,
i.e. dosing twice after bacterial infection [20, 21]. Unfortunately, the compound did not
protect the mice from death, even at each dose up to 50 mg/kg (cf. Table S3).
Antibiotic prophylaxis, the administration of antibiotics before surgery or a dental
procedure, is used in patients who are faced with high risks of bacterial infections [22, 23].
Inspired by this idea, we carried out prophylactic administration at 12 h and 6 h before the
injection of the MRSA inoculum. Here, the interval of the prophylactic administration was
determined as 6 h, because we would like to keep it consistent with the double-dosing

schedule for the treatment. Then we injected the MRSA inoculum (0.5 mL) at the MLD
intraperitoneally to the mice that had been pre-treated by the compound. Subsequently, we
orally administered the compound twice, i.e. at 0 h and 6 h post-infection, at the same dose
as the prophylactic administration. A significant dose-effect relationship of g37 was
observed from Figure 4a, i.e. a higher dose led to a higher survival rate. All the treated
groups demonstrated the improvement in survival rates compared with the vehicle control
(
i.e. the untreated group, survival rate: 12.5%). The survival rates were 87.5%, 50%, 37.5%
and 25%, when treated with the compound at the doses of 9 mg/kg, 3 mg/kg, 1 mg/kg and
.33 mg/kg, respectively. Linezolid was used as a positive control and administered in the
0
same way as g37 (including the prophylactic administration). As shown in Figure 4b, both
groups of mice treated with linezolid at the doses of 9 mg/kg and 3 mg/kg survived the
infection. As a blank control, the uninfected group of mice had a survival rate of 100%. In
addition, we treated the uninfected mice with g37 according to the same dosing schedule
(including the prophylactic administration) at each dose as high as 30 mg/kg. Since no
death was observed (cf. Table S3), the compound g37 had a wide therapeutic window.
In order to uncover potential causes of the different effects between the dosing
schedules with and without prophylactic administration, we tested mouse plasma protein
binding (PPB) of g37 and found that it was rather high (99.9%). Due to the high PPB, it
seems that both pharmacokinetics and pharmacodynamics would be potentially affected by
the dosing schedules [24]. Apart from this potential cause, more studies will be performed
in the near future to understand the observation.

7
Figure 4. The survival rates of the mice infected with 5×10 CFU/mL of MRSA15-1
within 7 days after the treatment of the compound g37 (a) or linezolid (b) at four doses
ranging from 9 mg/kg to 0.33 mg/kg. The curve for the vehicle control, i.e. the mice that
were not treated with the compound is also shown.
2
.7. Target Identification
2
.7.1. Inhibition of S. aureus GyrB and DNA supercoiling by g37
It was noted that the chemical series studied in this work contained a 2-quinolone
scaffold, a bioisostere to coumarins [25]. The natural antibiotic with a coumarin core, i.e.
novobiocin, exhibits antibacterial activity by inhibiting the ATPase activity of DNA gyrase
B (GyrB) [26]. As the 4-hydroxyl coumarin-3-carbamoyl scaffold of novobiocin is
structurally related to the 4-hydroxy-2-oxo-1,2-dihydroquinoline of g37 (cf. Table S4),
we hypothesized that g37 may inhibit GyrB as well. GyrB, as a subunit of DNA gyrase,

functions to provide energy for DNA supercoiling via catalyzing the hydrolysis of ATP. The
ATPase inhibition causes a failure of DNA supercoiling and hence inhibits or kills bacteria
[
27].
We retrieved all the available GyrB inhibitors (Gram-positive bacteria) from the
ChEMBL database 25 (accessed in Apr. 2019)[28, 29] and calculated their structural
similarity with g37, i.e. Tanimoto coefficients based on MACCS fingerprints. 31 potent
GyrB inhibitors (IC : less than 1 µM) were found to be structurally related to g37 [30, 31],
5
0
with Tanimoto coefficients of 0.75 and higher (cf. Table S4). This strengthened the
hypothesis that g37 may target GyrB.
To validate our hypothesis, we measured the production of ADP before and after the
treatment of S. aureus Gyrase with different concentrations of g37. A clear dose-response
relationship was observed and the IC was determined as 0.145 µM (cf. Figure 5a, the IC
5
0
50
of the positive control, i.e. novobiocin, was 0.015 µM, which was consistent with an earlier
report [32]). Since GyrB inhibitors eventually impair DNA supercoiling, we tested the
effect of g37 on that process by agarose gel electrophoresis. As shown in Figure 5b, g37
clearly inhibited the production of the supercoiled DNA in a dose-response manner. The
above bioassays confirmed S. aureus GyrB as a potential target of g37.

Figure 5. Target validation of g37. (a) The concentration-dependent ATPase inhibition of S.
aureus Gyrase B. The IC value is 0.145 µM. (b) The effect of the compound g37 on the
50
DNA supercoiling at serial concentrations, shown by agarose gel electrophoresis. R, the
relaxed DNA; S, the supercoiled DNA.
As a follow up, we determined the IC values of all the other derivatives against S.
5
0
aureus GyrB. As their IC values were between 0.10 µM and 3.18 µM (cf. Table 1), the
50
whole series of compounds were targeting S. aureus GyrB. Apart from that, we noted that (i)
all the potent antibacterial compounds (MIC: less than 1 µg/mL) potently inhibited GyrB
(
IC : less than 1 µM) as well, with g13 as the only exception. (ii) For the weak
50
antibacterial compounds (MIC: greater than 1 µg/mL), however, they were not necessarily
weak GyrB inhibitors. For instance, the compounds including g1-g3, g9-g12 and g33-g35
showed moderate antibacterial potency but potent GyrB inhibition. The observations
uncovered the potential relationship between enzymatic inhibition and antibacterial activity.
To be specific, GyrB inhibition is an important factor for antibacterial activity, but the
increase of GyrB inhibition does not ensure strong antibacterial potency.
The homology of GyrB to the E subunit of Topoisomerase IV, i.e. ParE [27] prompted
us to measure the effect of g37 on ParE. As a result, no inhibitory activity was observed (cf.

Figure S3) for g37, with the IC value greater than 100 µM (the IC value of the positive
5
0
50
control, i.e. novobiocin, was 7.9 µM, which was close to an earlier report [32] ).
2
.7.2 Potential binding mode of g37 to S. aureus Gyrase B
We performed molecular docking to derive a plausible binding mode of g37 to the ATP
binding site of GyrB using the crystal structure of S. aureus GyrB bound to novobiocin
PDB code: 4URO). Since Zhang, J. et al. highlighted that the cocrystallized water
(
molecule labeled as wat46 was potentially involved in ligand recognition [33], we retained
the same water molecule during the docking simulations. Due to the similarity of g37 and
novobiocin in the core scaffold, we hypothesized that the 2-quinolone scaffold may interact
with GyrB in the same way as the coumarin core. The most plausible binding pose is
shown in Figure 6. Figure 6a shows g37 and novobiocin share a similar binding mode, in
particular the core scaffold, while Figure 6b shows the details of the binding mode. Similar
to the coumarin core, the carbanyl group of the 2-quinolone scaffold forms hydrogen bonds
with Arg144. In addition, the 2-quinolone scaffold interacts with GyrB via electrostatic
interactions with Arg84 and hydrophobic interaction with Pro87. Apart from the similar
interactions, g37 also binds to GyrB through a few unique interactions. To be specific, (i)
the thiadiazole may interact with Asp81, Thr173 and Gly85 via the conserved water
molecule. Also, it forms the hydrophobic interaction with Ile86 and the electrostatic
interaction with Glu58. (ii) The thiazol-2-yl group at the terminus is located at a small and
hydrophobic sub-pocket and forms the hydrophobic interaction with Ile86 and the
electrostatic interaction with Asp81. This plausible binding mode generally complies with

the bioassay data, which showed that the substitutions of the thiazol-2-yl group caused
minor variations in GyrB inhibition instead of significant changes.
Figure 6. Potential binding modes of g37 to the ATP binding site of S. aureus GyrB (PDB
code: 4URO) as derived by molecular docking with OEDocking. Images were created with
Discovery Studio 2017. (a) Surface representation of the ATP binding site where g37 is
located. Color codes: blue, g37; orange, novobiocin. (b) Detailed view of interactions
between g37 and GyrB. The interacting atoms and g37 are shown in stick representations.
3
. Conclusion
Antibiotic resistance has become a threat to public health worldwide [34]. It is well
known that one of the potential solutions to the antibiotic resistance crisis is the use of
alternative drugs structurally different from those in clinic to treat bacterial infections.
Compound 11, identified by Peternel L. et al. during a high-throughput screening campaign,
seems to represent a new class of antibacterial agents. However, no follow-up work has
been reported on this compound so far.
Starting from compound 11, in this work, we preliminarily explored the SAR of the
N-thiadiazole-4-hydroxy-2-quinolone-3-carboxamides by the design, synthesis of 37 new

derivatives and in vitro evaluation of their antibacterial activities against S. aureus ATCC
9213. Through the efforts, we identified ten derivatives with better antibacterial activity
2
than the reported compound 11. Next, we focused on the determination of the most
promising compound from the ten derivatives by considering both antibacterial activity
spectrum and mammalian cell toxicity. Firstly, we tested them against a panel of
Gram-positive bacteria including drug-resistant clinical isolates (e.g. MRSA). Most
derivatives showed good antibacterial activity, of which the potencies of g8, g31 and g37
were not lower than that of the marketed antibacterial drug vancomycin for any of the
tested bacterial strains. Following that, we tested the three compounds for their cytotoxicity
to HepG2 and HUVEC cells and demonstrated that g37 had the best safety among all the
tested compounds.
As a drug discovery project, we comprehensively evaluated g37 by performing a
resistance development assay, time-kill kinetics analysis, in vitro metabolic stability test, in
vivo pharmacokinetic profile and an in vivo antibacterial efficacy assay. We demonstrate
that g37 (i) did not induce the resistance development of MRSA 15-1 over 20 passages, (ii)
was bactericidal rather than bacteriostatic, (iii) was metabolically stable in either mouse
plasma or mouse liver microsomes, (iv) showed generally good pharmacokinetic properties
in mice, (v) was orally effective in treating the mice infected with MRSA when it was
prophylactically administered and was not toxic at a dose as high as 30 mg/kg. These data
indicate that g37 is worthy of further development.
Based on visual inspection of chemical structures and cheminformatics analysis, we
proposed that bacterial GyrB was likely to be a target of g37. We then confirmed

experimentally that g37 inhibited the ATPase activity of S. aureus GyrB and thus, DNA
supercoiling. By testing all the other derivatives against S. aureus GyrB, we preliminarily
uncovered the potential relationship between enzyme inhibition and antibacterial activity.
By molecular docking, we derived a plausible binding mode of g37 to S. aureus Gyrase B.
To the best of our knowledge, this is the first report of a potential target for
N-thiadiazole-4-hydroxy-2-quinolone-3-carboxamides. This interesting discovery may help
the structure-guided optimization of g37.
In summary, this paper describes the comprehensive use of chemical synthesis, in vitro
and in vivo bioassays as well as cheminformatics analysis to discover a promising
antibacterial agent and its potential target. g37 is the most potent compound of this
chemical series that we have tested and its target has never been reported so far.
Nevertheless, the high PPB of the compound somewhat limits its broad application.
Therefore, we will continue to optimize the pharmacokinetic properties of this compound
while aiming to maintain its favorable potency and safety profile.
4
4
4
. Experimental
.1. Chemistry
.1.1. General Methods
The process of the chemical reaction was monitored by analytical thin-layer
chromatography on the silica gel plates GF254 (Yantai Chemical Industry Research
1
13
Institute, China). Both H NMR (500 MHz) and C NMR (100 MHz or 125 MHz) spectra
were measured on the Bruker spectrometer (Varian Mercury, USA). The chemical shifts
were recorded as the δ value (ppm), using tetramethylsilane as the internal standard. HRMS