Azolylthioacetamides as a potent scaffold for the development of metallo-β-lactamase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.10.038

In an effort to develop new inhibitors of metallo-β-lactamases (MβLs), twenty-eight azolylthioacetamides were synthesized and assayed against MβLs. The obtained benzimidazolyl and benzioxazolyl substituted 1–19 specifically inhibited the enzyme ImiS, and 10 was found to be the most potent inhibitor of ImiS with an IC₅₀ value of 15 nM. The nitrobenzimidazolyl substituted 20–28 specifically inhibited NDM-1, with 27 being the most potent inhibitor with an IC₅₀ value of 170 nM. Further studies with 10, 11, and 27 revealed a mixed inhibition mode with competitive and uncompetitive inhibition constants in a similar range as the IC₅₀ values. These inhibitors resulted in a 2–4-fold decrease in imipenem MIC values using E. coli cells producing ImiS or NDM-1. While the source of uncompetitive (possibly allosteric) inhibition remains unclear, docking studies indicate that 10 and 11 may interact orthosterically with Zn2 in the active site of CphA, while 27 could bridge the two Zn(II) ions in the active site of NDM-1 via its nitro group.

Full text of DOI:10.1016/j.bmcl.2017.10.038

Accepted Manuscript
Azolylthioacetamides as a potent scaffold for the development of metallo-β-
lactamase inhibitors
Yang Xiang, Ya-Nan Chang, Ying Ge, Joon S. Kang, Yi-Lin Zhang, Xiao-Long
Liu, Peter Oelschlaeger, Ke-Wu Yang
PII:
S0960-894X(17)31032-6
https://doi.org/10.1016/j.bmcl.2017.10.038
BMCL 25367
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
21 June 2017
18 September 2017
19 October 2017
Please cite this article as: Xiang, Y., Chang, Y-N., Ge, Y., Kang, J.S., Zhang, Y-L., Liu, X-L., Oelschlaeger, P.,
Yang, K-W., Azolylthioacetamides as a potent scaffold for the development of metallo-β-lactamase inhibitors,
Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.10.038
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Azolylthioacetamides as a potent scaffold for the
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development of metallo-β-lactamase inhibitors
a
a
a
b
a
a
c
Yang Xiang , Ya-Nan Chang , Ying Ge , Joon S. Kang , Yi-Lin Zhang , Xiao-Long Liu , Peter Oelschlaeger ,
a,
and Ke-Wu Yang *

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Azolylthioacetamides as a potent scaffold for the development of metallo-β-
lactamase inhibitors
a
a
a
b
a
a
c
Yang Xiang , Ya-Nan Chang , Ying Ge , Joon S. Kang , Yi-Lin Zhang , Xiao-Long Liu , Peter Oelschlaeger , and Ke-
Wu Yang *
a,
a
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Innovation Laboratory of Chemical Biology, College of
Chemistry and Materials Science, Northwest University, Xi’an 710127, P. R. China.
b
Department of Biological Sciences, California State Polytechnic University, 3801 West Temple Avenue, Pomona, CA 91768, USA.
c
Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, 309 East Second Street, Pomona, CA 91766, USA.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
In an effort to develop new inhibitors of metallo-β-lactamases (MβLs), twenty-eight
azolylthioacetamides were synthesized and assayed against MβLs. The obtained benzimidazolyl
and benzioxazolyl substituted 1-19 specifically inhibited the enzyme ImiS, and 10 was found to
be the most potent inhibitor of ImiS with an IC50 value of 15 nM. The nitrobenzimidazolyl
substituted 20-28 specifically inhibited NDM-1, with 27 being the most potent inhibitor with an
IC50 value of 170 nM. Further studies with 10, 11, and 27 revealed a mixed inhibition mode with
competitive and uncompetitive inhibition constants in a similar range as the IC50 values. These
inhibitors resulted in a 2-4-fold decrease in imipenem MIC values using E. coli cells producing
ImiS or NDM-1. While the source of uncompetitive (possibly allosteric) inhibition remains
unclear, docking studies indicate that 10 and 11 may interact orthosterically with Zn2 in the
active site of CphA, while 27 could bridge the two Zn(II) ions in the active site of NDM-1 via its
nitro group.
Keywords:
Antibiotic resistance
Metallo-β-lactamase
Inhibitor
Azolylthioacetamides
2
009 Elsevier Ltd. All rights reserved.
Following the discovery of the antibiotic penicillin in the
to a cleaved β-lactam ring. Class B enzymes, also known as
metallo-β-lactamases (MβLs), utilize either 1 or 2 equivalents of
Zn(II) for full catalytic activity to hydrolyze β-lactam
1
1
920s, β-lactam antibiotics were developed in the 1940s as
2
miracle drugs to treat bacterial infections. β-Lactams exert their
antibacterial effect by inhibiting transpeptidases (penicillin
binding proteins) that are responsible for the biosynthesis of the
6
,7
antibiotics. The MβLs are further divided into subclasses B1-
7
B3, based on amino acid sequence homology and Zn(II) content.
3
bacterial cell wall consisting of peptidoglycan. However, the
The New Delhi metallo-β-lactamase-1 (NDM-1), a B1
subclass MβL, was initially discovered in Klebsiella pneumoniae
overuse of antibiotics has resulted in a large number of bacteria
that are resistant to these drugs. The most important mechanism
of β-lactam resistance is the production of β-lactamases. β-
Lactamases render bacteria resistant to many commonly used
8
9, 10
in 2008, and has since rapidly spread globally.
The plasmid-
encoded NDM-1 gene has been shown to horizontally transfer to
other pathogenic bacteria. ImiS, a B2 subclass enzyme, has a
antibiotics,
including
penicillins,
cephalosporins
and
narrow substrate profile including carbapenems, which have been
carbapenems, by catalyzing the hydrolysis of the C-N bond of the
11, 12
3
,4
called “last resort”antibiotics.
The MβLs including NDM-1
β-lactam ring of the drugs, and thus inactivating the antibiotics.
and ImiS hydrolyze all clinical carbapenems and have grown into
9
To date, there have been more than 2,000 distinct β-
a serious threat to the health of human beings. There are no
13
lactamases identified, and these enzymes have been categorized
known clinical inhibitors of the MβLs to date.
5
into classes A−D, based on amino acid sequence homology.
Given the enormous biomedical importance of MβLs, there
has been a large amount of effort in identifying novel inhibitors.
King et al. reported that the aspergillomarasmine A is a potent
Enzymes of classes A, C, and D, also known as serine β-
lactamases (SβLs), use an active-site serine to perform a
nucleophilic attack on the β-lactam carbonyl, ultimately leading
14
15
inhibitor of NDM-1 and VIM-2.
Biphenyl tetrazoles,
∗
Corresponding author. Tel/Fax: +8629-8153-5035, E-mail: kwyang@nwu.edu.cn

1
6
17
succinic and amino acid-derived thiols also displayed
inhibitoryactivity on IMP-1. Chiou et al. reported that Ebselen
inhibits NDM-1 by forming a S-Se bond with the Cys221 residue
potential as MβL inhibitors was evaluated with the enzymes
NDM-1, ImiS and L1, which are representatives of the B1, B2
and B3 subclasses of MβLs, respectively. Also, the ability of
these inhibitors to restore the potency of existing antibiotics
against antibiotic resistant strains was evaluated. Furthermore,
molecular docking was employed in studying possible
interactions between the inhibitors and their preferred MβLs.
1
8
in the active site of the enzyme. Also, some broad-spectrum
inhibitors of the MβLs have been reported, including
mercaptophosphonates, thiomandelic acid, thiols,
mercaptothioenolates.
1
9
and
2
0
Recently, we found that the azolylthioacetamides provide a
Twenty-eight new azolylthioacetamides (Fig. 1) were
synthesized as shown in Scheme S1 in the Supplementary Data.
highly promising scaffold for the development of MβL
2
1-23
inhibitors.
Specifically, the aromatic carboxyl substituted
Firstly, the mercaptoazoles were synthesized by a previously
2
2
21
azolylthioacetamides
inhibit
ImiS,
while
the
reported method. o-Diaminobenzene reacted with NH
2
-CN or
The intermediates
amino-benzimidazole and amino-benzoxazole were acylated with
2
3
24
triazolylthioacetamides inhibit NDM-1. In addition, some of the
azolylthioacetamides exhibit broad-spectrum inhibitory activity
against CcrA, NDM-1, ImiS, and L1, representing members of
Br-CN to give amino-benzimidazoles.
2
1
chloroacetyl chloride to offer α-chloroacetamides. Finally,
under alkaline conditions, the azoles and α-chloroacetamides
were cross-linked by nucleophilic substitution to afford the target
2
1
all three subclasses. Our goal is to develop inhibitors of MβLs
and to use these inhibitors in combination with the β-lactam
antibiotics to combat infections caused by the MβL-producing
bacteria. To further explore the structure-activity relationships of
compounds with the azolylthioacetamide scaffold, twenty-eight
new compounds were synthesized and characterized. Their
21-23
products azolylthioacetamides (1-11 and 13-28).
Bifunctional
12 was synthesized by a different route. All compounds were
1
13
characterized by H and C NMR and confirmed by MS.
Fig. 1. Structures of the synthesized azolylthioacetamides. The varied atoms and substituents are shown in red and green,
respectively.
To test whether these azolylthioacetamides were inhibitors of
MβLs, enzymes from the three subclasses B1 (NDM-1), B2
concentrations causing 50% decrease of enzyme activity (IC50)
are listed in Table 1. 1-19 specifically inhibited ImiS, with 1-12
with benzimidazole (Fig. 1) exhibiting a lower IC50 value range
of 0.015-18 µM than 13-19 with benzoxazole (IC50 =29-384 µM).
5, 10, 11, 16 and 19 with an amino-triazole showed lower IC50
values than the corresponding compounds with the triazole,
oxazole or thiazole group. 10 gave the lowest IC50 value of 15 nM
against ImiS. On the other hand, 20-28 with a nitrobenzimidazole
significantly inhibited NDM-1 with an IC50 value range of 17 nM
(
ImiS) and B3 (L1) were over-expressed and purified as
25-28
previously described.
The inhibition experiments were
conducted as described in the Supplemental Data.
The inhibition studies indicated that, while none of the
azolylthioacetamides tested had any activity against L1 at
concentrations up to 500 µM (data not shown), they had specific
inhibitory activity against either NDM-1 or ImiS. The inhibitor

to 6.4 µM. This observation suggests that the nitro group of these
inhibitors may be involved in binding selectively to the Zn (II)
ion(s) in the dinuclear active site of the enzyme. 27 exhibited the
lowest IC50 value of 170 nM against NDM-1, which is similar to
the data (150 nM) of the best NDM-1 inhibitor among the
To identify the inhibition mode of the azolylthioacetamides
against MβLs, we studied inhibition kinetics of ImiS and NDM-1
with different substrate concentrations and different
concentrations of 10 and 11 as representatives of compounds
with a benzimidazole group and 27 as a representative with a
nitro substituted benzimidazole group. The concentrations of 10
were varied from 0 to 125 nM and those of 11 and 27 from 0 to
2
3
triazolylthioacetamides that we recently reported.
Table 1. IC50 values of azolylthioacetamides against metallo-β-lactamases
NDM-1 and ImiS
2
50 nM. Imipenem concentrations were varied from 0 to 150 nM.
Enzyme and inhibitors were pre-incubated for 5 min before
starting the kinetic assays. The mode of inhibition was
determined by generating Lineweaver–Burk plots, and K values
i
were obtained by fitting initial velocity versus substrate
concentration at each inhibitor concentration using SigmaPlot
IC50 (µM)
ImiS
IC50 (µM)
NDM-1
Compds
Compds
NDM-1
-
ImiS
1
2
3
4
5
6
7
8
9
0.33±0.02
0.63±0.06
18±3
15
16
17
18
19
20
21
22
23
24
25
26
-
-
384±7
-
-
-
-
-
-
-
-
-
-
-
29±1
1
2.0.
-
133±3
The Lineweaver–Burk plots of ImiS- and NDM-1-catalyzed
hydrolysis of imipenem in the absence and presence of 10, 11
and 27 are shown in Fig. 2. The results indicate that all three
compounds exhibit a competitive and uncompetitive mixed
inhibition mode. The Kic/Kiu values were determined to be 22 ±
3.0±0.1
-
378±9
0.27±0.04
1.8±0.1
-
53±1
6.4±0.2
1.3±0.1
5.9±0.4
0.50±0.09
0.73±0.09
5.1±0.2
2.5±0.3
-
-
-
-
-
-
-
4
=
(
/11 ± 2 nM (alpha = 0.52) for 10, 110 ± 50 /80 ± 30 nM (alpha
0.72) for 11, and 550 ± 90/310 ± 50 nM (alpha = 0.56) for 27
average ± standard deviation of triplicates). The K values of 27
are similar to the data of the best azolylthioacetamide inhibitor
1.2±0.2
i
1.7±0.1
23
0.94±0.07
0.015±0.009
0.077±0.01
0.39±0.07
against NDM-1 (490 nM), while the K
i
values of 10 against
ImiS are significantly (~50-fold) smaller than the data of the best
1
1
1
0
1
2
2
2
compounds (1.2 µM) that we recently reported, indicating that
the activity of this azolylthioacetamide is greatly improved. We
can currently not identify the source of the apparent
uncompetitive inhibition mode; it might be due to binding of the
compounds to allosteric sites of the enzymes. We hypothesize
that the competitive inhibition mode is due to orthosteric
inhibition, i.e., binding of the compounds to the MβLs’ active
sites.
1
1
3
4
-
-
196±4
259±8
27
28
0.17± 0.03
1.1±0.1
-
-
The substrate for NDM-1 and ImiS was imipenem; -: no inhibition at an
inhibitor concentration of 500 µM.
.
A
B
C
5
4
3
2
e+6
e+6
e+6
e+6
2
.5e+7
.0e+7
.5e+7
.0e+7
.0e+6
.0
1
8
6
4
2
0
e+7
e+6
e+6
e+6
e+6
2
1
1
5
1e+6
0
0
-.04
-.02
0.00
.02
.04
.06
-.02 0.00
.02
.04
.06
.08
.10
.12 -.15
-.10
-.05
0.00
.05
.10
.15
-5.0e+6
1/[Substrate] (nmol/mL)
-2e+6
-1e+6
1/[Substrate] (nmol/mL)
1
/[Substrate] (nmol/mL)
Fig. 2. Lineweaver–Burk plots of ImiS- and NDM-1-catalyzed hydrolysis of imipenem in the absence and presence of 10 (A), 11 (B), and 27 (C). Concentrations
of inhibitors 10/11 and 27 were 0/0 (●), 31.25/62.5 (○), 62.5/125 (▼) and 125/250 nM (▽).
The ability of the azolylthioacetamides to restore the
antimicrobial activity of imipenem against bacteria expressing
ImiS or NDM-1 was investigated by determining the minimum
inhibitory concentrations (MICs) of the antibiotic in the presence
and absence of 16 µg/mL 1-28. The results are listed in Table 2.
E. coli BL21 (DE3) harboring pET26b-NDM-1 or pET26b-ImiS
were used to evaluate these inhibitors. A significant (2-4-fold)
decrease in MIC of imipenem was observed for 1-19 with E. coli
cells expressing ImiS, while 20-28 showed no effect. Conversely,
20-28 decreased imipenem MICs with E. coli cells expressing
NDM-1, while 1-19 had no effect. The ability of these
azolylthioacetamides to partially restore the antibacterial activity
of the antibiotic is consistent with their inhibitory effect on the
MβLs. No antibacterial effect of the compounds alone against E.
coli with and without ImiS or NDM-1 plasmid was observed.

Table 2. Antibacterial activities (MICs, µg/mL) of imipenem against E. coli BL21 (DE3) with ImiS or NDM-1 in the absence
and presence of inhibitors 1-28 at a concentration of 16 µg/mL. MIC of imipenem for E. coli cells that don’t expressing MβL was
0
.125 µg/mL.
E. coli-NDM-1
E. coli-ImiS
MIC (µg/mL)
1
6
1
(
control)
Compds
MIC (µg/mL)
Compds
15
MIC (µg/mL)
Compds
MIC (µg/mL)
Compds
15
MIC (µg/mL)
1
2
3
4
5
6
7
8
9
16
16
16
16
16
16
32
16
16
16
16
16
16
16
32
16
16
16
16
8
1
2
0.5
0.5
0.5
0.5
0.5
0.25
0.5
1
16
16
17
3
0.5
17
18
4
0.25
0.5
18
19
5
19
20
6
0.5
20
21
8
7
0.5
21
1
22
8
8
0.25
0.5
22
1
23
4
9
23
1
1
1
1
1
1
0
1
2
3
4
24
4
10
11
12
13
14
0.25
0.25
0.25
0.5
24
1
25
8
25
1
26
8
26
2
27
4
27
1
28
8
0.5
28
1
To explore potential orthosteric binding modes, three typical
interact with Asn233 and Thr157 via the amide group.
Compound 10 has additional interactions with Zn2 and Lys224
via its aryl amino group (Fig. 3B). This finding is consistent with
representatives of azolylthioacetamides were docked into the
active sites of their preferred targets: 10 and 11 into CphA, (in
lieu of ImiS, which has not been crystallized, yet, and with which
it shares 96% sequence identity), and 27 into NDM-1. The
the slightly lower IC50 and K values obtained from experiment,
i
22
relative to 11. Contrary to the previous findings, the triazole
ring of compound 27 did not show any interactions with Zn1 or
Zn2 of NDM-1 in the docked conformations (Fig. 3E). Instead,
the nitro group acted as a bridging ligand of Zn1 and Zn2, which
is reminiscent of the binding mode of a micromolar inhibitor of
conformations shown in Fig.
3
are the lowest-energy
conformations of those clusters, with binding energies of -6.1, -
.9, and -10.3 kcal/mol for the CphA/10, CphA/11 and NDM-
/27 complexes, respectively. While the CphA/ImiS inhibitors
are more potent in experiment, the docking binding energy is
lower with the NDM-1 inhibitors, which is most likely due to a
second Zn(II) ion in NDM-1 resulting in stronger electrostatic
interactions in the model.
5
1
2
9
IMP enzymes recently studied. The nitro group as a zinc ligand
has been demonstrated previously in a crystal structure of Zn-
dependent carboxypeptidase A in complex with 2-benzyl-3-
30
nitropropanoic acid. Overall, 27 indicates several possible
interactions with the NDM-1 active site, including the Zn(II)
ions, Lys211, Gln123 and Asn220, that are comparable to the
binding mode of hydrolyzed ampicillin from a crystal structure
In terms of possible binding mode these docking calculations
indicate that in compounds 10 and 11, which exhibited the lowest
IC50 and K values with ImiS, the N-amino triazole may interact
i
(
Fig. 3D, PDB code 4HL2). While other conformations were
face-on with Zn2 with N---Zn(II) distances between 3.0 and 4.1
Å (Fig. 3, panels B and C). Interaction with active site Zn(II)
ions is typically the key force for inhibitor binding to MβLs, as
16
obtained from the docking study as well, the majority of the
conformations showed interactions similar to those highlighted in
the selected conformations, namely interactions of the N-amino
triazole of 10 and 11 with Zn2 of CphA and the nitro group of 27
with Zn1 and Zn2 of NDM-1.
1
5
observed for instance with biphenyl tetrazoles, succinic acids,
17
or thiol compounds. The triazoles also form hydrogen bonds
with His118 and His196 of CphA. In addition, both compounds

Fig. 3. Lowest-energy conformations of 10 and 11 docked into the active site of CphA (PDB code 2QDS) and 27 docked into the active site of NDM-1 (PDB
code 4HL2). Both enzymes are depicted as follows: backbone as cartoon in green and side chains of selected residues as licorice colored by atom (H, white; C,
cyan; N, blue; O, red; S, yellow). The Zn(II) ions are shown as purple spheres. A shows superimposition of compounds 10 and 11 in the CphA active site. 10 is
depicted as licorice colored by atom (same colors as for protein residues except C in gray), and 11 as orange sticks. B, C, D, and E are detailed views of 10, 11,
hydrolyzed ampicillin and 27, respectively, displaying key enzyme residues and indicating interactions between the inhibitors and enzyme residues with dashed
3
1
lines. All images were generated with VMD.
4
.
King, D. T. S.; Sobhanifar, N. C.; Strynadka, N. C. J. Protein Sci.
016, 25, 787.
2
In summary, twenty-eight new azolylthioacetamides were
synthesized, characterized by NMR and confirmed by MS.
5. Ambler, R. P.; Trans, P. R. Soc. Lond, B. Biol. Sci. 1980, 289,
321.
6
.
Crowder, M. W.; Spencer, J.; Vila, A. J. Acc. Chem. Res. 2006,
9, 721.
Biological
activity
evaluation
indicates
that
the
3
azolylthioacetamides with benzimidazolyl and benzoxazolyl 1-19
specifically inhibit ImiS, with the lowest IC50 value of 15 nM.
However, the azolylthioacetamides with nitrobenzimidazolyl 20-
7
8
.
.
Bebrone, C. Biochem. Pharmacol. 2007, 74, 1686.
Yong, D. M. Toleman, A. Giske, C. G.; Cho H. S.; Sundman K.;
Lee K.; Walsh T. R. Antimicrob. Agents Chemother. 2009, 53,
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2
8 significantly inhibit NDM-1, with the lowest IC50 value of 170
9
1
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Walsh, T. R. Int. J. Antimicrob. Agents. 2010, 36 (Suppl 3), S8.
nM. These inhibitors, in combination with imipenem, resulted in
a 2-4-fold decrease in imipenem MIC values with E. coli cells
expressing ImiS or NDM-1. Docking studies suggest that the N-
amino triazole of 10 and 11 interacts with Zn2 in the active site
of CphA, while, inhibitor 27 uses its nitro group to bridge the two
Zn(II) ions in the active site of NDM-1. The information gained
in this work is valuable for the further development of MβL
inhibitors.
0. Mojica, M. F.; Bonomo, R. A.; Fast, W. Curr. Drug Targets.
016, 17, 1029.
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1. Schneider, K. D.; Karpen, M. E.; Bonomo, R. A.; Leonard, D. A.;
Powers, R. A. Biochem. 2009, 48, 11840.
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2
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