Ebselen bearing polar functionality: Identification of potent antibacterial agents against multidrug-resistant Gram-negative bacteria

Article abstract of DOI:10.1016/j.bioorg.2019.103286

Antibiotic-resistant bacteria has become one of the greatest challenges to global human health today. Innovative strategies are needed to identify new therapeutic leads to tackle infections of drug-resistant Gram-negative bacteria. We herein synthesize a series of EB analogues to investigate their antibacterial activities. Select polar functionality at N-terminus of EB exhibited higher activities against multi-drug-resistant Gram-negative pathogens, including E. coli, P. aeruginosa and K. pneumoniae. EB analogue 4g and 4i exhibited potent antibacterial activities against E. coli-ESBL (MIC = 1–4 μg/mL) and E. coli producing NDM-1 (MIC = 4–32 μg/mL), which is superior to the traditional antibiotics (cefazolin, imipenem). Furthermore, the time-kill kinetics studies and the inhibition zone tests indicated that analogue 4i effectively and rapidly cause death of E. coli-ESBL and E. coli-NDM-1. Additionally, accumulation assays and SEM images showed that 4i could permeate bacterial membranes, leading to an irregular cell morphology. Importantly, bacterial resistance for analogue 4i was difficult to induce against E. coli-ESBL. EB analogues here reported low cytotoxicity against L-929 cells and mice model in vivo. We believe that EB analogues with polar functionality could play a pivotal role in the development of novel antibacterial agents in eradicating multi-drug-resistant Gram-negative pathogens infections.

Full text of DOI:10.1016/j.bioorg.2019.103286

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Ebselen bearing polar functionality: Identification of potent antibacterial agents
against multidrug-resistant Gram-negative bacteria
Cheng Chen, Kewu Yang
PII:
S0045-2068(19)30636-4
DOI:
https://doi.org/10.1016/j.bioorg.2019.103286
YBIOO 103286
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Ebselen bearing polar functionality: Identification of potent antibacterial agents
against multidrug-resistant Gram-negative bacteria
Cheng Chen and Kewu Yang*
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of
Education, Chemical Biology Innovation Laboratory, College of Chemistry and Materials
Science, Northwest University, 1 Xuefu Avenue, Xi'an 710127, P. R. China.
*Corresponding author: E-mail: kwyang@nwu.edu.cn; Tel: +8629-8153-5035
ABSTRACT: antibiotic-resistant bacteria has become one of the greatest challenges to global
human health today. Innovative strategies are needed to identify new therapeutic leads to
tackle infections of drug-resistant Gram-negative bacteria. We herein synthesize a series of
EB analogues to investigate their antibacterial activities. Select polar functionality at
N-terminus of EB exhibited higher activities against multi-drug-resistant Gram-negative
pathogens, including E. coli, P. aeruginosa and K. pneumoniae. EB analogue 4g and 4i
exhibited potent antibacterial activities against E. coli-ESBL (MIC = 1-4 µg/mL) and E. coli
producing NDM-1 (MIC = 4-32 µg/mL), which is superior to the traditional antibiotics
(cefazolin, imipenem). Furthermore, the time-kill kinetics studies and the inhibition zone tests
indicated that analogue 4i effectively and rapidly cause death of E. coli-ESBL and E.
coli-NDM-1. Additionally, accumulation assays and SEM images showed that 4i could
permeate bacterial membranes, leading to an irregular cell morphology. Importantly, bacterial
resistance for analogue 4i was difficult to induce against E. coli-ESBL. EB analogues here
reported low cytotoxicity against L-929 cells and mice model in vivo. We believe that EB
analogues with polar functionality could play a pivotal role in the development of novel
antibacterial agents in eradicating multi-drug-resistant Gram-negative pathogens infections.
KEYWORDS: Gram-negative pathogens, drug discovery, Ebselen analogues, antibacterial
activity.
1
. Introduction

The continuing and increasing prevalence of multidrug-resistant (MDR) Gram-negative
bacteria (GNB) has become a tremendous threat to public health worldwide, these bacteria,
including Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and
Escherichia coli, are called antibiotic-resistant “priority pathogens” released by World Health
Organization (WHO) in 2017 [1-3]. The dissemination of
Beta-Lactamases (ESBL)-producing organisms and Carbapenem-resistant Enterobacteriaceae
CRE) are the major causes of healthcare-associated bacterial infection, such as those in the
Extended Spectrum
(
surgical site, urinary tract, intra-abdominal and bloodstream region [4, 5]. In particular, CRE
involves the most worrisome evolution of the antibiotic resistance crisis, which is almost
resistant to all available antibiotics [6, 7]. Currently, the clinically most important
carbapenemases in Enterobacteriaceae consist of the class A enzymes of the K. pneumoniae
carbapenemases (KPC) type, the zinc-dependent class B metallo-β-lactamases (MβLs) of the
VIM, IMP, and NDM types and class D carbapenemases of the OXA-48 type [8, 9]. These
types of CRE are structurally and mechanistically different from each other, making it
challenging to identify the antibacterial agent that is active against all types of CRE [10].
Gram-negative bacteria (GNB) have two cellular membranes, and the lipopolysaccharide
-coated outer membrane impermeable to most small molecules is one of the largest of
challenges in the discovery of new Gram-negative antibacterial agents [3, 11]. In addition,
there is a limited understanding of this type of compounds that can be accumulated in
Gram-negative pathogens. One useful strategy for identifying the novel antimicrobials is to
carefully monitor and tune these physicochemical properties of small-molecule that increase
accumulation in GNB and enable broad antibacterial coverage [12]. The “eNTRy rules”
reported by Richter et al showed the ability of diverse compounds to accumulate in E. coli led
to the development of predictive guidelines for compound accumulation, which suggested
that compounds are most likely to accumulate if they contain a primary amine, low
globularity and relatively rigid. These guidelines were then successfully applied to convert a
natural product that is active only against Gram-positive bacteria, into an antibiotic with
activity against multi-drug-resistant Gram-negative pathogens [13].
In previous disclosures, Ebselen (EB) has antioxidant, anti-inflammatory and
cytoprotective properties, and the safety of EB in humans has been demonstrated by three

different clinical trials investigating the treatment of stroke patients [14-16]. Moreover, EB
has been shown to have excellent antimicrobial activity against clinical multidrug-resistant
Gram-positive pathogens, including Methicillin-resistant S. aureus (MRSA) and
Vancomycin-resistant Enterococcus faecium (VRE) [17]. Lu et. al. revealed that EB, a
substrate of mammalian thioredoxin reductase (TrxR) but an irreversible inhibitor of bacterial
TrxR, blocks the electron transfer via TrxR and displays selectively antibacterial activity
toward GSH-negative bacteria, with no inhibitory effect on GSH-positive bacteria, for
example, Escherichia coli. [18] Additionally, Chiou et al and our previous studies showed
that EB is a potent covalent inhibitor of NDM-1 binding the Cys208 at active site of enzyme,
in combination β-lactams to combat effectively clinically isolates producing NDM-1. [8, 19]
Ngo et al also reported that EB and its sulfur analogues (Ebsulfur) showed potent antibacterial
efficacy against MRSA and also exhibited broad-spectrum antifungal activity.[20, 21]
However, EB did not show potent antimicrobial activity against Gram-negative pathogens,
which might be due to its reduced ability to cross outer membrane barrier or the efflux pump
rather than lack of target of EB inside Gram-negative bacteria [22]. Inspired by the above
information, we herein synthesized a series of EB-based analogues appending various polar
functionality at the N-terminus to further investigate antibacterial activities of these EB
analogues in fighting against some of the most problematic Gram-negative bacteria in the
clinic.
2
2
. Results and discussion
.1 Chemistry
To gain insights into the relationship that exists between physicochemical properties and
antibacterial activity against GNB, we targeted the N-terminus of the EB scaffold for
optimization through the incorporation of polar functionality with the goal of increasing
accumulation of new EB small molecules. We initiated this study with a series of amidation
for 2-(chloranylidene)-selanyl)benzoyl chloride (3) using small, different polar amines
(Scheme 1). The synthetic procedure involved preparation of acyl chloride through reflux of
disselenide and SOCl at 85°C and recrystallization using cyclohexane, then acyl chloride was
2
dropwise added to solution of the appropriate amine and trimethylamine (TEA) at 0°C to

furnish the desired amidation product. Through this procedure, we synthesized Ebselen 4a
and eight EB analogues 4b-g in 56-75% yield (Scheme 1). Analogues 4e and 4f with
Boc-group were deprotected using hydrochloric acid in methanol to afford 4h (86% yield)
and 4i (92% yield), respectively. Ebselen and eight EB analogues we reported here had ClogP
values with a range of 1.16 - 4.41, all EBs synthesized were advanced to antibacterial
investigation.
O
O
O
O
(a)
OH
(b)
(c)
OH
Cl
N R
Se
NH2
Se 2
SeCl
1
2
3
4a-4i
O
O
O
O
O
N
N
Se
N
Se
N
Se
4b
4c
4a
O
O
N
O
O
O
O
N
Se
N
H
O
N
N
Se
N
O
Se
H
4
d
4e
4h
4
f
O
O
O
+
-
+
-
N
Se
NH3 Cl
N
Se
OH
N
Se
NH3 Cl
4
g
4i
Scheme 1. Synthetic route and structures of EB analogues. Reagents and conditions: (a) (1)
NaNO , HCl (aq), 0℃, 30min; (2) Na Se H O, 0℃~60℃, 6h; (b) SOCl , DMF, reflux 3h; (c)
2
2
2,
2
2
amine (excess)/TEA.
2
.2. Antimicrobial activity evaluation
Following chemical synthesis of these EB analogues bearing polarity at the N-terminus,
EB 4a-i were firstly assayed for antibacterial activities against Gram-positive strains: S.
aureus, methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecium (VRE).
Antibacterial activity was determined via microdilution minimum inhibitory concentration
(MIC) assays that were performed in 96-well plates [23]. The tested compounds were
screened in MIC assays using 2-fold serial dilutions (0.125 to 128 µg/mL) in three

independent experiments. MIC data (Table 1) indicated that the EB analogues display
-32-fold stronger antibacterial effect than the vancomycin as control (MIC = 2-256 μg/mL)
4
on three Gram-positive strains tested, with an MIC value ranging from 0.25 to 64 μg/mL, the
data previously reported also indicated EB against S. aureus ATCC strains with a MIC range
of 0.125-7.8 μg/mL [17, 20, 22], and 4g and 4i were found to be the most potent compounds
(MIC = 0.25-16 μg/mL). More importantly, both 4g and 4i with ClogP reducing appendage
exhibited higher antibacterial property than the control (EB, 4a).
Subsequently, to further investigate whether EB analogues with various polarity
enhanced antibacterial activities against multi-drug-resistant Gram-negative strains, five
clinical isolates Extended Spectrum Beta-Lactamases E. coli (E. coli-ESBL), Extended
Spectrum Beta-Lactamases K. pneumonia (K. pneumonia-ESBL), P. aeruginosa and K.
pneumonia-NDM-1 were used for evaluation of the EB analogues. MIC data (Table 1)
showed that the 4g and 4i had the lowest ClogP (1.16 and 1.27), and both compounds were
found to be effectively inactive against all Gram-negative strains tested with a MIC range of
1
-64 μg/mL. Particularly, for E. coli-ESBL, 4i proved to be the best MIC value of 1 μg/mL,
being 256-fold more potent than the antibiotic cefazolin (MIC = 256 μg/mL). Nevertheless,
for the other EB analogues with different ClogP values, the enhanced antibacterial activities
against Gram-negative bacteria were not to be observed. Additionally, a comparison of Clog
P for all EB analogues indicates that antibacterial activity increased with increased polarity
attached to the N-terminus of EB (Fig. S1). We then assayed the antibacterial activity of the
EB analogues against clinical isolates E. coli producing NDM-1 (EC01-EC24) and other five
E. coli expressing MβLs, indicating that 4i with low globularity, relatively rigid (rotatable
bonds = 3), and primary amine showed strong antibacterial effect against clinically isolates
and E. coli expressing MβLs, with a best MIC value of 4 μg/mL and 0.5 μg/mL for EC08,
and E. coli expressing IMP-1 (Table 3), which are 256-2500-fold higher efficiency than the
cefazolin (10000 μg/mL) and imipenem (128 μg/mL). The MIC50/90 value for 4i against all
Gram-negative pathogens tested were 2.56 and 16.32 µg/mL, respectively. However, 4h did
not present antibacterial activity towards Gram-negative bacteria (GNB), probably because 4h
with sterically encumbered primary amine is not high accumulators, which is not in line with

the eNTRy rules [13]. Additionally, MH buffer (pH ~ 7.4) was used in antibacterial assays,
h (pKa ~ 5) may exist in the uncharged form, so as to decrease accumulation on the surface.
4
Table 1. MICs (μg/mL) of EB 4a and analogues 4b-i against multi-drug-resistant
Gram-positive and Gram-negative bacteria, using vancomycin (Van) and cefazolin (Czo) as
control.
Strains
ClogPa
4a
4b
4c
4d
4e
4.41
1
4f
4g
1.16
0.25
8
4h
4i
Van Czo
3.7
2.9
1.95
0.25
64
2.3
3.04
2
2.37 1.27
-
2
-
S. aureus
MRSA
0.5
64
64
0.5
64
64
0.25
32
1
0.25
-
-
G+
64
64
64
64
32
4
8
2
128
256
-
VRE
32
64
16
32
-
E. coli
>
>
>
>
128 >128
128 >128
64
64
>128 >128
4
>128
512
-
ESBLa
E. coli
64
>64 >128 >128
4
8
>256
>128
>128
>128
1
4
-
-
-
-
256
256
512
512
-
ESBLb
G-
K. pneumonia
128 >128 >64
64
>128 >64
-
ESBL
K. pneumonia
NDM-1
P. aerugions
128 >128 >128 >128 >128 >128
32
16
-
>128 >128 >128 >128 >128 >128
64
16
Notes: all the clinical isolates MRSA, VRE, E. coli-ESBLa, b, K. pneumonia-ESBL, K. pneumonia
NDM-1, P. aerugions from the Health Science Center at Xi’an Jiaotong University China. All these
-
isolates were identified by VITEK2 Compact (bioMérieux, France) and 16S rRNA gene sequencing.
PCR and nucleotide sequencing were employed to screen for the presence of NDM genes. a: all ClogP
calculated from ChemBioDraw Ultra 14.0. G+: Gram-positive bacteria, G-: Gram-negative bacteria.
Table 2. Antibacterial activities (MIC, μg/mL) of EB 4a and analogues 4b-i, cefazolin (Czo)
and imipenem (IMI) against clinical isolates EC01-EC24 (E. coli producing NDM-1) and E.
coli expressing MβLs.
Strains 4a
EC01
4b
4c
4d
4e
4f
4g
4h
4i
Czo
IMI
>128
>128
128
128
>128
>128
32
>128
8
5000
128

EC04
EC06
EC07
EC08
EC10
EC23
EC24
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>64
64
128
>64
64
>128
>128
>128
>128
>128
>128
>128
>64
>128
>128
>128
128
16
16
16
8
128
16
4
100000 64
>128
>64
128
128
64
>128
>128
>128
>128
>128
>128
>64
2500
5000
10000
2500
10000
5000
256
64
32
4
128
128
256
64
64
>128
>128
64
>128
>128
>128
>64
32
8
32
16
8
>128
>64
4
64
E. coli- >64
NDM-1
>64
4
1
128
E. coli- >64
CcrA
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
4
2
2
2
2
>64
1
128
256
128
-
64
E. coli- >64
VIM-2
>64
1
128
64
E. coli- >64
IMP-1
>128
>128
>64
0.5
1
E. coli- >64
ImiS
128
32
E. coli- >64
L1
0.5
64
Notes: all the clinical isolates E. coli producing NDM-1 (EC01-24) from the Health Science Center at
Xi’an Jiaotong University, China.
2
.2. Uptake of EB analogues by ESBL-E. coli
To investigate the uptake of EB analogues by ESBL-E. coli cells, ICP-AES (inductively
coupled plasma absorbance emission spectroscopy) studies were carried out. Experiments to
investigate the accumulation of selenium (Se) in the ESBL-E. coli were conducted at high
−
8
concentrations. As shown in Fig. 1, accumulation of 4g and 4i uptake to 8 × 10 μg per cell
about 2 h, which is obvious higher than other analogues. The presence of a primary amine or
polar group is clearly important for accumulation in E. coli. The above accumulation results
are consistent with their antibacterial activity.

Fig. 1. ICP-AES data for the uptake of Se by ESBL-E. coli after exposure to different EB
analogues. Se contents per cell are expressed as Se (μg) per cell.
2
.3. Time-dependent killing
To determine the bactericidal rate of EB analogues against Gram-negative bacteria, the
time-kill kinetics of analogue 4i was performed [24]. E. coli-ESBL and EC08 were grown to
early exponential phase and challenged with 4i (2×, 4×, 8×MIC), and cefazolin (2×MIC). As
shown in Fig. 2 and S2, 4i had excellent bactericidal activity against E. coli-ESBL and EC08,
showing superior activity compared with that of cefazolin in killing early exponential phase
populations. Moreover, 4i (8×MIC) killed early exponential phase E. coli-ESBL and EC08 at
6
h, whereas cefazolin (2×MIC) did not kill E. coli-ESBL and EC08 at 24 h. The cefazolin
samples were cloudy after 10 h treatment, but the both samples treated with 4i were clear (Fig.
S3). These results suggest that 4i rapidly kills both E. coli-ESBL and E. coli producing
NDM-1.
Fig. 2. Time-dependent killing curves. E.coli-ESBL were grown to early exponential phase
and challenged with 4i at 2×MIC, 4×MIC and 8×MIC, and cefazolin (Czo) at 2×MIC.

2
.4. Zeta potential (ζ) measurement
To obtain deeper insights into the interactions between EB analogues and E. coli-ESBL,
Zeta potential (ζ) was employed to this study [25]. As shown in Fig. 3, after incubation with all
analogues, only the ζ potential of E. coli-ESBL by treated with analogue 4h and 4i became
more positive compared with other EB analogues. Clearly, the interaction of 4h and 4i with E.
coli-ESBL was influenced obviously by the ionic strength, indicating that the accumulation of
4
h and 4i to E. coli-ESBL was dominated by electrostatic interactions. 4i showed the
electrostatic interactions with negatively charged lipopolysaccharide of an outer membrane, so
as to promote the accumulation of it on the cell surface and then enter into cell through bacterial
porins. Though 4h also accumulates on the surface of cell by electrostatic interactions, and
make zeta potential of cell became more positive, it is difficult to cross the porins due to
sterically encumbered primary amine, and could not exhibit antibacterial activity.
Fig. 3. Zeta potentials (ζ) of E. coli-ESBL in the absence and presence of all EB analogue, final
measurements were performed in 1 mM PBS solution at 25 °C.
2
.5. Inhibition zone tests
Thenceforward, to further elucidate the antibacterial effect of 4i against different
Gram-negative bacteria including: E. coli producing NDM-1, E. coli-ESBL, P. aeruginosa and
K. Pneumoniae-NDM-1, the inhibition zone tests were carried out [26]. As shown in Fig. S4,
there are gradually increasing diameter of inhibition zones for the antibacterial test with the
concentration of 4i raising. It clearly demonstrated that the introduction of a primary amine
makes EB analogue 4i had excellent antibacterial properties against the multidrug-resistant
Gram-negative bacteria.

2
.6. SEM characterization
We next advanced to be direct visualization of the morphological changes of bacteria in
the presence of EB analogues using field-emission SEM [27, 28]. As shown in Fig. 4, the
control group exhibited a smooth cell surface and clear bacterial edges. After treatment with 4i,
catastrophic structural damages to the E. coli-ESBL and P. aeruginosa were visualized, and
almost all the bacteria were collapsed and fused. SEM images showed an irregular cell
morphology caused by 4i possibly through permeabilizing bacterial membranes and then
targeting related sulfhydryl-dependent enzymes of thioredoxin system in bacteria.[29] These
results were consistent with antibacterial experiments.
Fig. 4. SEM images of E. coli-ESBL and P. aeruginosa before (a, c) and after (b, d) treatment
with analogue 4i, bar scare: 5 μm.
2
.7. Bacterial resistance studies
Subsequently, we evaluate the ability of analogue 4i to suppress the development of
resistance against Gram-negative E. coli-ESBL through resistance selection studies after a
prolonged passage at sub-inhibitory concentrations. The control antibiotic cefazolin (Czo)
was chosen for E. coli-ESBL. Only a 2-fold change was detected in the MIC of 4i against E.
coli-ESBL after 20 passages. However, antibiotic Czo displayed a 64-folds increase in the

MIC (Fig. 5). These results indicated that analogue 4i had major advantages compared to
conventional antibiotic and induced less bacterial resistance.
Fig. 5. Resistance acquisition curves during serial passage with the subinhibitory
b
concentration of 4i or Czo against E. coli-ESBL . MIC test was performed every two
passages.
2
.7. Cytotoxicity assays
The potential toxicity of candidates is a major concern for development of clinically useful
of broad-spectrum antibacterial agents. To verify the safety of EB analogues, analogue 4g and
4
i were selected to a cytotoxicity assay with L929 cells at various concentrations (3.5, 7, 14,
8, 56, 112 μg/mL) [30]. As shown in Fig. 6, both compounds exhibited relatively low
2
cytotoxicity against L929 cell, with a cell viability of 70% or higher at a concentration up to
8 μg/mL, which is higher than the effective antibacterial concentration. Furthermore, the
median lethal dose (LD ), a helpful pointer of the substance’s acute toxicity, was established
2
5
0
after intraperitoneal injection in Kunming mice 20−22 g followed by an observation period of
2 h. The LD50 values in mice was determined to be 61 mg/kg for 4i (Fig. 7), suggesting a
relatively low toxicity of EB analogue 4i in vitro and in vivo.
7

Fig. 6. Cytotoxicity assay in L929 cells were treated with EB analogues 4g and 4i at a
concentration ranging from 3.5 to 112 µg/mL. DMSO was used as a negative control.
Fig. 7. Dose-response for EB analogue 4i administrated by intraperitoneal injection (i.p.)
route. mice were observed up to 72 h (n = 6 per group).
3
. Conclusions
In summary, a series of EB analogues 4a-i with various polarity were synthesized and
evaluated. Analogue 4g and 4i, with the lowest ClogP (1.16 and 1.27), were found to have
excellent antibacterial activity against the multidrug-resistant Gram-negative bacteria,
particularly E. coli-ESBL (MIC = 1-4 µg/mL) and carbapenem-resistant pathogens E. coli
producing NDM-1 (MIC = 4-32 µg/mL), being more potent than the traditional antibiotics

(cefazolin and imipenem). The MIC50/90 values for 4i against all Gram-negative pathogens
tested was 2.56 and 16.32 µg/mL, respectively Furthermore, the time-kill kinetics studies,
accumulation assays and SEM images revealed that the analogue 4i could permeate bacterial
membranes, leading to an irregular cell morphology and rapid death of E. coli-ESBL and E.
coli-NDM-1. Moreover, EB analogues did not induce bacteria to develop resistance. Also, EB
analogues tested exhibited low cytotoxicity against L-929 cells and mice model in vivo. These
studies demonstrated that the EB analogues with polarity present a novel scaffold for the
development of antibacterial agents in combating multi-drug-resistant Gram-negative
pathogens infections.
4
. Material and methods
4
.1. Chemistry
1
13
H and C NMR spectra were recorded on a Bruker Avance III 400-MHz spectrometer. Chemical
shifts are given in parts per million (ppm) on the delta scale. The peak patterns are reported as
singlet (s), doublet (d), triplet (t), quartet (q), doublet doublet (dd), and mul-tiplet (m). The spectra
were recorded with TMS as internal standard. Coupling constants (J) were reported in Hertz (Hz).
Mass spectra were obtained on a micro TOF-Q (BRUKER) mass spectrometer. The reactions were
followed by thin-layer chromatography (TLC) on glass packed precoated silica gel plates and
visualized in an iodine chamber or with a UV lamp. Flash column chromatography was performed
using silica gel (200–300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. Activity
evaluation of inhibitors was performed on an Agilent 8453 UV-Vis spectrometer.
A solution of 2-(chloroseleno)benzoyl chloride (1 mmol) in dry ether (10 mL) was added
dropwise over 30 min to a stirred solution of the appropriate amine (1.2 mmol) and
triethylamine (3.5 mmol) in dry DCM (10 mL) at 0°C. The reaction mixture was stirred at
room temperature overnight. The solvent was removed under reduced pressure and the
residue was washed with water (20 mL), and extracted with DCM (3 × 10 mL). The
combined organic extracts were dried over anhydrous MgSO , the solvent removed was under
4
reduced pressure, and the crude product was purified by flash column chromatography on

silica gel (eluents: 10–50% ethyl acetate in petroleum gradient) [31]. All EB analogues except
4
c and 4e have been reported.
4
.1.1. 2-Phenylbenzo[d][1,2]selenazol-3(2H)-one (4a).
1
Gray solid, yield 80%, H NMR (400 MHz, DMSO-d ) δ 8.26 (d, J = 8.0 Hz, 1H), 7.90 (d, J =
6
7
.5 Hz, 1H), 7.68 – 7.60 (m, 3H), 7.49 – 7.42 (m, 3H), 7.26 (t, J = 7.3 Hz, 1H). 13C NMR
(
101 MHz, DMSO-d ) δ 165.42, 140.48, 139.73, 132.37, 129.59, 128.23, 126.75, 126.55,
6
+
1
2
26.04, 125.01, 121.08. HRMS (ESI): m/z Calcd for: C H NOSe [M+Na] 297.9747; found
1
3
9
97.9729.
4
.1.2. 2-(furan-2-ylmethyl)Benzo[d][1,2]selenazol-3(2H)-one (4b).
1
White solid, yield 65%, H NMR (400 MHz, DMSO-d ) δ 8.01 (d, J = 8.0 Hz, 1H), 7.84 (d, J
6
=
7.7 Hz, 1H), 7.65 – 7.57 (m, 2H), 7.42 (t, J = 7.3 Hz, 1H), 6.45 – 6.42 (m, 2H), 4.93 (s, 2H).
1
3
C NMR (101 MHz, DMSO-d ) δ 166.65, 151.58, 143.75, 142.48, 139.74, 131.98, 128.26,
6
+
1
3
27.89, 126.16, 111.17, 109.26, 40.09. HRMS (ESI): m/z Calcd for: C H NO Se [M+Na]
12 9 2
01.9696; found 301.9646.
4
.1.3. 2-(3-methylisoxazol-5-yl)Benzo[d][1,2]selenazol-3(2H)-one (4c).
1
White solid, yield 65%, H NMR (400 MHz, DMSO-d ) δ 8.09 (d, J = 8.0 Hz, 1H), 7.92 (d, J
6
1
3
=
7.7 Hz, 1H), 7.72 (t, J = 8.3 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.01 (s, 1H), 2.44 (s, 3H). C
NMR (101 MHz, DMSO-d ) δ 171.48, 165.97, 159.48, 139.76, 133.79, 128.28, 127.77,
6
+
1
26.85, 126.79, 96.12, 12.84. HRMS (ESI): m/z Calcd for: C H N O Se [M+Na] 302.9649;
1
1
8
2
2
found 302.9639.
4
.1.4. 2-(3-morpholinopropyl)Benzo[d][1,2]selenazol-3(2H)-one (4d).
1
White solid, yield 65%, H NMR (400 MHz, Chloroform-d) δ 8.03 (d, J = 7.8 Hz, 1H), 7.65 –
7
–
1
.55 (m, 2H), 7.42 (d, J = 8.1 Hz, 1H), 3.92 (t, J = 6.7 Hz, 2H), 3.73 (t, J = 4.7 Hz, 4H), 2.49
2.39 (m, 6H), 1.92 (m, 2H). 13C NMR (101 MHz, Chloroform-d) δ 167.46, 138.10,
31.93,128.42, 127.46, 126.06, 123.91, 66.88, 54.79, 53.35, 42.71, 26.59. HRMS (ESI): m/z
+
Calcd for: C H N O Se [M+Na] 349.0431; found 349.0367.
1
4
18
2
2

4
.1.5. Tert-butyl (4-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)phenyl)carbamate (4e).
1
Grey solid, yield 86%, H NMR (400 MHz, DMSO-d ) δ 8.15 (d, J = 8.0 Hz, 1H), 7.91 – 7.84
6
(
m, 1H), 7.70 – 7.61 (m, 1H), 7.57 – 7.40 (m, 5H), 1.49 (s, 9H). 13C NMR (101 MHz,
DMSO-d ) δ 165.33, 153.23, 139.53, 137.83, 134.22, 132.38, 129.07, 128.23, 126.58, 125.78,
6
+
1
4
18.99, 79.63, 28.59. HRMS (ESI): m/z Calcd for: C H N O Se [M+Na] 413.0380; found
1
8
19
2
3
13.0375.
4
.1.6. Tert-butyl (2-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)ethyl)carbamate (4f).
1
White solid, yield 75%, H NMR (400 MHz, Chloroform-d) δ 8.03 (d, J = 7.8 Hz, 1H), 7.69
(d, J = 8.0 Hz, 1H), 7.58 (td, J = 7.7, 1.3 Hz, 1H), 7.45 – 7.38 (m, 1H), 3.95 (t, J = 5.7 Hz,
1
3
2
1
H), 3.44 (q, J = 5.9 Hz, 2H), 1.41 (s, 9H). C NMR (101 MHz, Chloroform-d) δ 170.11,
55.98, 138.40, 132.04, 128.67, 126.20, 124.29, 45.84, 28.38, 8.65. HRMS (ESI): m/z Calcd
+
for: C H N O Se [M+H] 343.0561; found 343.0526.
1
4
19
2
3
4
.1.7. 2-(2-hydroxyethyl)Benzo[d][1,2]selenazol-3(2H)-one (4g).
1
White solid, yield 62%, H NMR (400 MHz, DMSO-d ) δ 8.04 (d, J = 8.0 Hz, 1H), 7.82 (d, J
6
=
7.7 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.40 (t, J = 7.3 Hz, 1H), 3.81 (t, J = 5.2 Hz, 2H), 3.63
(
q, J = 5.1 Hz, 2H). 13C NMR (101 MHz, DMSO-d ) δ 167.06, 140.66, 131.77, 128.11,
6
-
1
2
27.64, 126.08, 126.01, 60.89, 46.56. HRMS (ESI): m/z Calcd for: C H NO Se [M-H]
9 9 2
41.9720; found 241.9768.
4
.1.8. 2-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)ethan-1-aminium chloride (4h).
1
White solid, yield 86%. H NMR (400 MHz, D O) δ 8.08 (d, J = 8.0 Hz, 1H), 7.94-7.86 (m,
2
2
H), 7.73-7.67 (m, 2H), 7.49 (t, J = 7.5 Hz, 1H), 7.12 (d, J = 9.0 Hz, 2H). 13C NMR (101
MHz, DMSO-d ) δ 165.56, 140.91, 140.26, 132.07, 130.09, 128.53, 127.99, 127.50, 126.34,
6
+
1
2
25.67, 124.46. HRMS (ESI): m/z Calcd for: C H N OSe [M+H] 291.0031; found
1
3
11
2
91.0060.
4
.1.9. 4-(3-oxobenzo[d][1,2]selenazol-2(3H)-yl)benzenaminium chloride (4i).

1
White solid, yield 92%. H NMR (400 MHz, D O) δ 7.84 (t, J = 8.7 Hz, 1H), 7.68 – 7.59 (m,
2
1
3
2
H), 7.48 – 7.41 (m, 1H), 4.08 (t, J = 5.8 Hz, 2H), 3.14 (q, J = 7.3 Hz, 2H). C NMR (101
MHz, D O) δ 169.59, 132.67, 128.24, 127.52, 126.46, 126.04, 124.81, 41.95, 39.48. HRMS
2
+
(
ESI): m/z Calcd for: C H N OSe [M+H] 243.0031; found 243.0042.
9
11
2
4
.2. MIC determination
A single colony of clinical isolates S. aureus, MRSA, VRE, E. coli-ESBL, P. aeruginosa, K.
Pneumoniae and EC 01-24 producing NDM-1 on LB agar plates was transferred to 5 mL of
Mueller-Hinton (MH) liquid medium and grown at 37 °C overnight. The bacterial cells were
collected by centrifugation (4,000 rpm for 10 min). After discarding the supernatant, the
pelleted cells were re-suspended in MH medium and diluted to an OD600 of 0.5. MIC values
were determined by using the CLSI guidelines [32]. The MIC was interpreted as the lowest
concentration of the drug that completely inhibited the visible growth of bacteria after
incubating plates for at least 16 h at 37 °C. Each inhibitor was tested in triplicate.
4
.3. Time-dependent killing
A single colony of clinical isolates E. coli-ESBL and EC08 on LB agar plates was transferred
to 5 mL of Mueller-Hinton (MH) liquid medium and grown at 37 °C overnight. Bacteria were
then treated with cefazolin (2×MIC) and 4i at 2×, 4×, 8×MIC, respectively, in culture tubes at
3
7℃, 225 rpm. At different times (0-24h) intervals 20 μL of aliquots from the suspension was
subjected to 10-fold serial dilution in 1×PBS. 10 μL of the dilution was plated on solid agar
plates and incubated at 37℃ for 16-18h. The bacterial colonies were counted and results
represented in log10 (CFU/mL) use the origin 8.0 [24].
4
.4. Zeta potential measurements
The E. coli in PBS was incubated with 10 mM all EB analogue 4a-i at 37 °C for 10 min. After
that, the unbound compound was removed with centrifugation (8000 rpm for 5 min). The
obtained pellets were washed with PBS solution, respectively. After centrifugation (8000 rpm
for 5 min), the pellets were resuspended in 1 mL PBS and kept on ice. The specimens were
prepared for zeta potential measurements. The E. coli incubated without EB analogues as the
control group was conducted under the same condition.

4
.5. Uptake of all EB analogues by ESBL-E. coli and determination of intracellular Se
contents
Uptake and cellular accumulation of all EB analogues by ESBL-E. coli was determined by
measuring bacterial cell Se content by ICP-AES as follows. ESBL-E. coli cultures were
grown to OD600 = 1.0 in LB broth, washed in PBS, and then resuspended in PBS to
8
9
approximately 10 ~10 CFU/mL. EB analogues 4a-i was added to cells at 20 μM, and then 5
mL samples of culture were harvested at 60 min after EB analogues addition. The untreated
cells were also taken for comparison. Samples were centrifuged for 20 min to obtain cell
pellets to discard unbound extracellular EB analogues. To prepare cell material for ICP-AES,
cell pellets were resuspended in 0.5 mL nitric acid (69% (w/v) and then placed in a sonicator
bath for 30 min to completely dissolve cells. The resulting digest was then diluted to a final
volume of 5 mL with diluted nitric acid, and then samples were analyzed on IRIS Advantage
(Thermo Scientific) inductively-coupled plasma-atomic emission spectrophotometer. Levels
of Se in the samples were determined by a calibration curve using multielement standard
solutions containing 0.1, 0.2, 5 and 10 mg/L Se.
4
.6. Inhibition zone tests
EC08 producing NDM-1, E. coli-ESBL, P. aeruginosa and K. Pneumoniae-NDM-1 was
administered to the liquid medium, and cultured on it by air bath oscillator for 5 h. The
rotating speed was 150 rpm, the temperature was 37 °C. Then, bacteria were vaccinated
to solid medium. After drilling holes on the Agar plate using the oxford ring, and 10 μL the
different concentration of compound 4i (1-1024 μg/mL) was added, then developed under the
incubator for 16-18 h at 37 °C and observed the bacteriostatic circle.
4
.7. SEM characterization
To directly visualize the morphological changes of E. coli-ESBL and P. aeruginosa by
compound 4i, SEM characterization was employed to this study. After the operation
according to antibacterial experiments, the mixture of cells and 4i was centrifuged (3500 g for
1
0 min). The supernatants were removed, and the bacterial pellets were fixed with 0.5%

glutaraldehyde in PBS at room temperature for 30 min. Then, 2 μL aliquots of bacterial
suspensions were transferred onto clean silicon slices. As soon as the specimens became dried,
0
.1% glutaraldehyde was added for further fixation for 3 h. Next, the specimens were washed
with sterile water twice and then dehydrated with increasing concentrations of ethanol (40%
for 30 min, 70% for 30 min, 90% for 30 min, and 100% for 30 min). At last, the dried
specimens were coated with platinum for SEM measurements.
4
.8. Cytotoxicity assays
A cytotoxicity assay was performed to evaluate the toxicity of analogues 4g and 4i to mouse
fibro-blast cells (L929). The cells were seeded into 96-well plates at cell density of 1.0 × 104
cells/well in 100 μL of culture medium and maintained for 24 h. Then solutions of compound
4
g and 4i with different concentrations were added to 96-well plates respectively, and
incubated for another 48 h. Six wells containing only cells suspended in a mixture of 99 μL of
complete medium and 1 μL of DMSO were used as the control for investigating cell-viability.
Six wells containing only the complete medium were used as the blank control. Following
that, the medium was removed. Finally, 100 μL of fresh culture medium and 10 μL of Cell
Counting Kit solution (purchased from 7Sea) were added to each well. After incubation for 4
h, the 96-well plates were then vigorously shaken to solubilize the formazan product and the
absorbance at a wavelength of 450 nm was read on a Microplate Reader and analyzed. All
experiments were conducted in triplicate.
4
.9. Determination of Median lethal dose (LD )
50
Kunming mice weighing 20-22 g were obtained from Experimental Animal Center, Health
Science Center of Xi'an Jiaotong University. The animals were kept in a room temperature.
For each compound, the animals were randomly divided into 7 groups of 6 animals. Each
group of animals are administrated different doses of compound tested (10-120 mg/kg). The
animals were observed during 72 h after administering the test compound. The geographic
mean of the least dose that killed mice (LD ) and the highest dose that did not induced
1
00
mortality (LD ) was taken as the LD .
0
50

Acknowledgements
This work was supported by grants 21572179 (to K.W.Y.) from the National
Natural Science Foundation of China.
Conflicts of interest
None
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Ebselen bearing polar functionality: Identification of potent antibacterial agents
against multidrug-resistant Gram-negative bacteria
Cheng Chen and Kewu Yang*
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of
Education, Chemical Biology Innovation Laboratory, College of Chemistry and Materials
Science, Northwest University, 1 Xuefu Avenue, Xi'an 710127, P. R. China.
*Corresponding author: E-mail: kwyang@nwu.edu.cn; Tel: +8629-8153-5035
Graphical Abstract
O
O
N
N
-
-
-
ClogP reducing appendage
simplified structure
improved properties
Se
Se
high accumulators
EB analogues
NH3+(ClogP = 1.27)
Ebselen (EB, ClogP = 3.7)
=
MIC > 128 g/mL
MIC = 1-2 g/mL (E. coli-ESBL)
g/mL (E. coli-NDM-1)
(
E. coli-ESBL and E.coli-NDM-1)
4

Highlights
·
Select polar functionality at N-terminus of EB exhibited higher activities against
multi-drug-resistant Gram-negative pathogens, including E. coli-ESBL and MBLs, P.
aeruginosa and K. Pneumoniae.
·
EB analogue 4i with lower ClogP (1.27) exhibited potent antibacterial activities against E.
coli-ESBL (MIC = 1 µg/mL) and EC08 (E. coli producing NDM-1) (MIC = 4 µg/mL),
which proved to be more potent than cefazolin and imipenem.
·
·
The time-kill kinetics studies and the inhibition zone test indicated that analogue 4i
effectively and rapidly cause death of E. coli-ESBL and E. coli-NDM-1.
The accumulation assay and SEM images showed 4i could permeabilize bacterial
membranes, leading to an irregular cell morphology.
·
·
Importantly, bacterial resistance for analogue 4i was difficult to induce against E. coli-ESBL.
EB analogues here reported low cytotoxicity against L-929 cells and mice model in vivo.