Halogen-substituted triazolethioacetamides as a potent skeleton for the development of metallo-β-lactamase inhibitors

Article abstract of DOI:10.3390/molecules24061174

Metallo-β-lactamases (MβLs) are the target enzymes of β-lactam antibiotic resistance, and there are no effective inhibitors against MβLs available for clinic so far. In this study, thirteen halogen-substituted triazolethioacetamides were designed and synthesized as a potent skeleton of MβLs inhibitors. All the compounds displayed inhibitory activity against ImiS with an IC₅₀ value range of 0.032–15.64 μM except 7. The chlorine substituted compounds (1, 2 and 3) inhibited NDM-1 with an IC₅₀ value of less than 0.96 μM, and the fluorine substituted 12 and 13 inhibited VIM-2 with IC₅₀ values of 38.9 and 2.8 μM, respectively. However, none of the triazolethioacetamides exhibited activity against L1 at inhibitor concentrations of up to 1 mM. Enzyme inhibition kinetics revealed that 9 and 13 are mixed inhibitors for ImiS with K_i values of 0.074 and 0.27μM using imipenem as the substrate. Docking studies showed that 1 and 9, which have the highest inhibitory activity against ImiS, fit the binding site of CphA as a replacement of ImiS via stable interactions between the triazole group bridging ASP120 and hydroxyl group bridging ASN233.

Full text of DOI:10.3390/molecules24061174

molecules
Communication
Halogen-Substituted Triazolethioacetamides as a
Potent Skeleton for the Development of
Metallo-β-Lactamase Inhibitors
Yilin Zhang 1,*, Yong Yan 1, Lufan Liang 1, Jiefeng 1, Xuejun Wang 1, Li Li 1 and Kewu Yang 2
1
College of Biology Pharmacy and Food Engineering, Shangluo University, Shangluo 726000, China;
yananan@yeah.net (Y.Y.); lianglouyv@163.com (L.L.); jiefeng_ab123@163.com (J.F.);
xuejunwangd@163.com (X.W.); Lily8028@163.com (L.L.)
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College
2
of Chemistry and Materials Science, Northwest University, Xi’an 710127, China; kwyang@nwu.edu.cn
*
Correspondence: 233049@slxy.edu.cn; Tel.: +86-15091567430
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 1 January 2019; Accepted: 23 March 2019; Published: 25 March 2019
Abstract: Metallo-
and there are no effective inhibitors against M
halogen-substituted triazolethioacetamides were designed and synthesized as a potent skeleton of
Ls inhibitors. All the compounds displayed inhibitory activity against ImiS with an IC50 value
range of 0.032–15.64 M except . The chlorine substituted compounds ( and ) inhibited NDM-1
with an IC50 value of less than 0.96 M, and the fluorine substituted 12 and 13 inhibited VIM-2 with
IC50 values of 38.9 and 2.8 M, respectively. However, none of the triazolethioacetamides exhibited
activity against L1 at inhibitor concentrations of up to 1 mM. Enzyme inhibition kinetics revealed
that and 13 are mixed inhibitors for ImiS with Ki values of 0.074 and 0.27 M using imipenem as the
substrate. Docking studies showed that and , which have the highest inhibitory activity against
β-lactamases (M
βLs) are the target enzymes of
β-lactam antibiotic resistance,
βLs available for clinic so far. In this study, thirteen
Mβ
µ
7
1,
2
3
µ
µ
9
µ
1
9
ImiS, fit the binding site of CphA as a replacement of ImiS via stable interactions between the triazole
group bridging ASP120 and hydroxyl group bridging ASN233.
Keywords: halogen-substitutedtriazolethioacetamides; MβLs; inhibitor
1. Introduction
β
-Lactam antibiotics containing a four membered
accounting for over 65% of injectable antibiotics in clinic [
by the overuse of antibiotics has become a threat to global public health. The main mechanisms of
resistance to -lactam antibiotics is the expression of -lactamase from bacteria, which can catalyze the
hydrolysis of the C-N bond of the -lactam ring to deactivate the antibiotics [ ]. So far, there have
been more than 2000 distinct -lactamases identified [ ]. Based on amino acid sequence homology,
-lactamases can be divided into four classes: Classes A, C and D, known as serine- -lactamases
(SBLs), can restore the efficacy of -lactam antibiotics by a successful clinic combination drug therapy
with SBL inhibitors (sulbactam, tazobactam and clavulanic acid) [ ]; Class B enzymes, known as
metallo- -lactamases (M Ls), display activity by one or two Zn ions in the active sites, which are
further divided into subclasses B1-B3 [ ]. Although a number of promising inhibitor molecules
have been reported in recent years, there are no effective inhibitors against M Ls available for
clinical therapy [ ]. More seriously, new Delphi metallo- -lactamase 1 (NDM-1), which were first
identified from pathogenic bacterium in 2008 and classified as B1 subclass M Ls, greatly aggravates
the challenge to treat bacterial infection in clinic because of the ability to hydrolyze almost all -lactam
β
-lactam ring are a major class of antibiotics,
]. However, antibacterial resistance caused
1
β
β
β
2,3
β
4
β
β
β
5,6
β
β
6,7
β
8
β
β
β
Molecules 2019, 24, 1174; doi:10.3390/molecules24061174
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1174
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antibiotics [9–11]. Obviously, lack of knowledge about catalytic mechanisms of NDM-1 has delayed
the development of clinical inhibitors [12].
In 2016, Mojica et al. reported that the pace of the development of MβLs inhibitors has slowed,
which was mainly because a shallow, relatively featureless active site and few scaffolds that can
selectively attach to the active site [13]. Mollard et al. reported that thiomandelic acid was capable
of potential broad spectrum M
1.28 M for the S-isomer. Moreover, the thiol group can bind the two zinc ions which is the active
site of M Ls [14]. The Weide et al. research group developed more than 200 sulftriazole derivatives,
and there were 31 compounds which had inhibitory activity against VIM-2 with Ki values ranging
from 0.01–0.39 M [15]. The N-methyl mercaptosulfhydrazole compounds substituted containing
trifluoromethyl could inhibit M Ls in competing mode, the enzyme activity test showed that the
βLs inhibitors with Ki values of 0.09 µM for rthiomandelic acid and
µ
β
µ
β
mercapto group was the necessary group to produce the activity [16].
In our group’s preliminary work, we found that the azolylthioacetamides scaffold has potential
inhibition for M
effectively inhibit ImiS and NDM-1 [17
broad-spectrum inhibitory activity against all three subclasses of M
want to develop new azolylthioacetamides by conjugation of the triazole and thioacetamides company
with halogen to find effective M Ls inhibitors. To further explore the structure-activity relationships
of the azolylthioacetamide compounds, thirteen halogen-substituted triazolethio-acetamides were
synthesized and eleven of that were novel compounds. Their potential as M Ls inhibitors were
evaluated with the enzymes VIM-2, NDM-1, ImiS and L1, which are representatives of the B1, B2 and
B3 subclasses of M Ls. Also, molecule docking was adopted to explore the binding method between
β
Ls, and the aryl-substituted thioacetamides containing the triazole group can
20]. In addition, four of the azolylthioacetamides exhibit
Ls [17]. Based on the research, we
–
β
β
β
β
inhibitors and MβLs.
2. Results and Discussion
The thirteen halogen-substituted triazolethioacetamides were designed and synthesized as listed
in Figure 1 and the synthetic procedures were available in Scheme 1. Briefly, the crosslink between
2-(5-mercapto-4H-1,2,4-triazol-3-yl) phenol (s4) and R-substituted-2-chloro-N-phenyl -acetamide
(
N1-13) gave the corresponding target compounds (
1–13). All compounds were characterized by
1H and 13C NMR and confirmed by MS.
Figure 1. The halogen-substituted triazolethioacetamides.

Molecules 2019, 24, 1174
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Scheme 1. Synthetic route of halogen-substituted triazolethioacetamides.
To test whether these halogen-substituted triazolethioacetamides were inhibitors of the M
three subclasses M Ls B1(VIM-2 and NDM-1), B2 (ImiS), and B3 (L1) were expressed and purified
as previously described [21 24]. The inhibition studies indicated that the halogen-substituted
βLs,
β
–
triazolethioacetamides had inhibitory activity against NDM-1, VIM-2 and ImiS, but no activity was
observed against L1 at inhibitor concentrations up to 1 mM. The inhibitor concentrations causing
a 50% decrease of enzyme activity (IC50) was listed in Table 1. As a result, the halogen-substituted
triazolethioacetamides
while 12 showed a slightly higher IC50 value of 15.64
fluorine at the p-position of anilino group. In particular,
against ImiS, and gave the lowest IC50 value of 32 nM. On the other hand,
with an IC50 value ranging from 0.17 to 0.96 M. Meanwhile, 12 and 13 gave the suitable IC50 value of
38.9 and 2.8 M to inhibit VIM-2. This observation suggests that the species, location and amount of
halogen substitution can affect the inhibitory activity of triazolethioacetamides in varying degrees. On
the whole, triazolethioacetamides containing chlorine ( ) increase the inhibitory potency slightly
on average compared to those containing fluorine ( 12), but the presence of trifluoromethoxy (13) is
beneficial to increase the inhibitory activity. The chlorine or fluorine substitution on the 2-position (
and ) of anilino group exhibited higher potency than that on the 3-position ( and 10) and 4-position
and 11). Furthermore, replacement of two hydrogen at the anilino group with chlorine ( and ) or
fluorine (12) relatively reduces the inhibitory activity, especially compound 12 with two fluorine at
2,5-position of anilino group. Dramatically, replacing hydrogen at the 2, 4-position with nitro ( ) does
not change their activities significantly. exhibited the lowest IC50 value of 170 nM against NDM-1,
1–
10 and 13 specifically inhibited ImiS exhibiting an IC50 value of less than 1
M and 11 nearly had no inhibitory activity with a
and exhibited higher inhibitory activities
inhibited NDM-1
µM,
µ
1–
3
9
1
1–3
µ
µ
1–8
9–
1
9
2
(3
4
5
6–8
2
which is similar to the data (160 nM) of the best NDM-1 inhibitor among the triazolylthioacetamides
that we recently reported [18].

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Table 1. IC50 values of azolylthioacetamides against MβLs (µM).
Compds
ImiS
NDM-1
VIM-2
1
1
2
3
4
5
6
7
8
9
10
11
12
13
0.032 ± 0.004
0.045 ± 0.002
0.051 ± 0.006
0.27 ± 0.02
0.16 ± 0.03
0.78 ± 0.08
0.20 ± 0.04
0.26 ± 0.03
0.072 ± 0.005
0.98 ± 0.04
>50
0.96 ± 0.07
-
0.17 ± 0.02
-
-
-
-
-
-
-
-
0.37 ± 0.05
-
-
-
-
-
-
-
-
-
-
-
-
15.64 ± 3
38.9 ± 5
0.23 ± 0.09
2.8 ± 0.3
1–: no inhibition at an inhibitor concentration of 1 mM.
To identify the inhibition mode of the halogen-substituted azolylthioacetamides against M
typical representatives and 13 for ImiS were chosen to determine Ki values. Lineweaver Burk plots
of ImiS catalyzed hydrolysis of imipenem in the absence and presence of inhibitors are displayed
in Figure 2. Compounds and 13 exhibited Ki values of 0.074 and 0.27 M, respectively, which are
βLs,
9
−
9
µ
slightly larger than their correlative IC50 values. The analysis also demonstrated that all the compounds
employed the same partially mixed inhibition type.
Figure 2. Lineweaver–Burk plot of ImiS catalyzed hydrolysis of imipenem in the absence and presence
of 9 (A) and 13 (B). Inhibitor concentrations were 0 µM (•), 0.25 µM (#), 0.5 µM (H), and 1.0 µM (5).
Molecular docking analysis was achieved from three comparable conformations (out of 50) docked
into corresponding target protein for four representative halogen-substituted triazolethioacetamides.
Because there is no high-resolution crystal structure of ImiS available, the very closely related (96%
sequence identity) CphA was used instead. The lowest-energy conformations were shown in Figure 3,
with the binding energies of
CphA/13, NDM-1/2 and VIM-2/13 complexes, respectively.
−
7.6,
−
7.47,
−
7.75,
−
6.99 and
−
5.87 kcal/mol for the CphA/1, CphA/9,

Molecules 2019, 24, 1174
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Figure 3. Low energy conformations of 1, 9 and 13 docked into the active site of CphA (PDB code
2QDS) ( ), 2 docked into NDM-1 (PDB code 4EYL) ( ) and 13 docked into VIM-2 (PDB code 4NQ2)
). The enzyme backbone is shown as cartoon in green, and the Zn(II) ions are shown as magenta
A
–C
D
(
E
spheres. Colored sticks by element were used to show ligand (C, yellow; N, blue; O, red; S, sand; Cl,
viridis; F:cyan; H, grey) and the residue side chains (C, cyan; N, blue; O, red; S, yellow; H, grey). The
interactions between the inhibitors and enzyme residues were displayed by dashed lines.
Docking studies reveal that
1 and 9, which exhibited the lowest IC50 values with ImiS, have
similar binding patterns with residue side chains in CphA (Figure 3A,B). The triazole group both
interact with ASP120 at distances of less than 3.48 Å, and hydroxyl group of 1 and 9 interact with
ASN233 at average distances of 2.11 and 3.25 Å, which may result a 0.13 kcal/mol more favorable
binding energy relative to
with Zn(II) ions as and
9
9
. Compound 13 with trifluoromethoxy group did not bind ASP120 together
did, but via three different interaction modes to get the lowest bonding
1
energy for CphA with distances between 1.89 and 3.21 Å, the interactions are the amide carbonyl group
and ASP264, the trifluoromethoxy group and GLN68, the hydroxyl group interact with ASN233and
H196. This may explain why although the docking binding energy of CphA/
1
(
−
7.6 kcal/mol) and
and showed
CphA/ 7.47 kcal/mol) complexes are larger than CphA/13 7.75 kcal/mol),
9
(
−
(
−
1
9
more inhibitory potency against CphA. However, the docking mode of VIM-2/13 complex continued
to change, one is the amide carbonyl group form hydrogen bonds with ASN210 and ARG 205, the
other is that the interaction between the triazole group and H116, and, in addition, the hydroxyl group
that interacts with ASP118. In compound 2, the hydroxyl group coordinates at Zn(II) ions of NDM-1
in comparable distances of 3.59 Å, the hydroxyl group and triazole group both interact with ASP 124.
The docking results reveal that the triazole ring of these compounds plays an important role in the
interactions with the close residue side chains, meanwhile there is rarely any direct interaction with
active site Zn(II) ions as seen in previous findings, which may be due to the introduction of halogen,
nitro and trifluoromethoxy influence the relectrostatic interactions between ligand and target protein.
It was interesting to observe that the compounds had different activity against VIM-2 and NDM-1,
even though the active sites of NDM-1and VIM-2 are quite similar. There are two differences between
the enzymes which may be significant for the inhibitor binding MβLs: (1) The distance between Zn(II)

Molecules 2019, 24, 1174
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ion and Asp124 which interacts with the hydroxyl group and the triazole ring of
2
in NDM-1, is bigger
in VIM-2 (16.12 Å, PDB code 4NQ2) than in NDM-1 (2.12 Å, PDB code 4EYL). Thus it is impossible to
establish effective interaction among the hydroxyl group, Asp124 and Zn(II) ion in VIM-2. (2) VIM-2
has a histidine at position 116 and a aspartic acid at position 118, which are the co-interacting amino
acids of active center Zn(II) ion and 13. However, NDM-1 has a histidine and valine at the same
corresponding position, so 13 is difficult to show activity against NDM-1.
Methyl group is the ioisosteric surrogate of chlorine. If the chlorine of compound 1 was replaced
by methyl group, the methyl substituted triazolethioacetamides only inhibit NDM-1 with an IC50
value of 0.16 µM [18], which is significantly higher than that of compound 1, but no inhibitory activity
was observed against ImiS. From Figure 3, it is obvious to observe that the triazolethioacetamides are
embedded in the active pocket of ImiS in folding mode, and binded to the NDM-1 active center in
a relatively stretched state. The methyl group will increase steric hindrance of inhibitor molecules,
and weaken the ability to combine ImiS. Thus, hindrance of substituents can not only change the
conformation of triazolethioacetamides, but also affect the interaction between triazolethioacetamides
and MβLs.
3. Materials and Methods
3.1. General Information
General chemicals were purchased from TCI (Tokyo Chemical Industry, Tokyo, Japan) and were
used without further purification. All antibiotics used were purchased from Sigma-Aldrich (St. Louis,
MO, USA).1H NMR and 13C NMR spectra were recorded on a NMR spectra were recorded with a
Bruker DRX 600 MHz spectrometer (Bruker Daltonics Inc., Billerica, MA, USA). The peaks patterns
are indicated as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet doublet; m, multiplet.
The spectra were recorded with TMS as internal standard. Coupling constants (J) were reported in
hertz (Hz). Chemical shifts were given in part per million (ppm) on the delta scale. Analytical Thin
Layer Chromatography (TLC) was carried out on silica gel F254 plates with visualization by ultraviolet
radiation. HRMS spectra were recorded on a Bruker MicrOTOF-Q II (Bruker Daltonics Inc., Billerica,
MA, USA) mass spectrometer. Inhibition studies were performed on an Agilent-8453 UV-visible
spectrometer (Santa Clara, CA, USA).
3.2. Synthesis and Characterization
Briefly, R-substituted-2-chloro-N-phenylacetamide (N1–13) and the intermediate 2-(5-mercapto
-4H-1,2,4-triazol-3-yl)phenol (s4) were prepared as previously reported [17]. A solution of
2-(5-mercapto-4H-1,2,4-triazol-3-yl) phenol (s4) (3 mmol) and NaOH (3.6 mmol) dissolved in
H2O (15 mL) was added in a 50 mL three-neck round bottomed flask, kept stirring for 30 min.
N-substituted-2-chloroacetamides (N1-13) (3 mmol) in hot ethanol (5 mL) was added drop wise to
the mix solution, and the slurry was stirred at reflux for 6 h. The resultung solution was cooled and
neutralized with HCl (5M) to pH 7.0. The resulting white precipitate (1–13) was filtered off, washed
with water (3
was shown in the Supplementary Data.
×
80 mL), and dried in vacuo. The spectrogram information for the target compounds
3.3. Determination of IC50 and Ki values
The inhibition studies were carried out on an Agilent UV 8453 spectrophotometer at 25 ◦C using
cefazolin as substrate of CcrA, NDM-1, and L1 and imipenem as substrate of ImiS. Inhibitors
were dissolved in DMSO and then diluted with Tris-HCl, pH 7.0. The substrate concentrations were
varied between 25 and 400 M, and inhibitor concentrations were varied between 125 nM and 1 mM.
1–13
µ
The enzyme and inhibitor were pre-incubated for 30 min before starting the kinetic experiments.
The IC50 values for all analyzed compounds were calculated based on the kinetic data. The mode

Molecules 2019, 24, 1174
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of inhibition was determined by generating Lineweaver-Burk plots of the data [23], and the Ki was
determined by replotting the data for slope and intercept versus substrate concentration.
3.4. Docking Calculations
Inhibitors were docked into the active sites of NDM-1 (PDB code 4EYL), CphA (PDB code 2QDS),
VIM-2 (PDB code 4NQ2). The program AutoDock 4.2 [25] was used for molecule docking analysis.
The flexible ligand was docked into each rigid monomeric receptor using a grid box with equal space
of 0.375 Å per grid and center of the one or two active-site Zn(II) ions. Fifty conformations were
generated for each complex. The rest of the parameters were set at their default values and all docking
calculations were performed without constraints. Binding energies were calculated via the Lamarckian
genetic algorithm and conformations that constitute each cluster were defined by a root mean square
deviation tolerance.
4. Conclusions
In summary, we have successfully developed a potent skeleton as MβLs inhibitors, and thirteen
halogen-substituted triazolethioacetamides were synthesized and characterized by NMR and MS.
Biological activity assays reveal that the triazolethioacetamides have special potency to inhibit ImiS
with the lowest IC50 value of 32 nM, and compound 1–3 with chlorine group display mix inhibition
against NDM-1 with a IC50 range from 170 to 960 nM. Meanwhile, 12 with two fluorine group and 13
with trifluoromethoxy group show certain inhibitory activity against VIM-2 in vitro. Docking studies
reveal that the triazolethioacetamides, which can form stable interactions with the triazole bridging
ASP120, and the phenolichydroxyl group interacting with ASN233 in CphA, promote the inhibitory
activity against ImiS. The identification of thirteen halogen-substituted triazolethioacetamides which
show that a mix mode of inhibition provides potent information for the further development of
inhibitors against MβLs.
Supplementary Materials: The 1H NMR,13C NMR and HRMS spectra data of all the target compounds were
provided in supporting information.
Author Contributions: The manuscript was written through contributions of all authors. Y.Z. designed and
conducted the experiments, analyzed the data, prepared the original draft and funding acquisition; Y.Y. provided
synthesis and software support, reviewed and edited the draft; L.L. (Lufan Liang) and J.F. completed the synthesis
assay and enzyme kinetic experiments; X.W. and L.L. (Li Li) conducted Docking assay; K.Y. provided the enzymes
used in the experiment.
Funding: This work was supported by the Natural Science Foundation of Shaanxi Province (2018JQ2070), the
Scientific Research Plan Project by Educational Department of Shaanxi Province (18JK0250), the Natural Science
Foundation of Shangluo University (16SKY021) and the National Student’s Innovation and Entrepreneurship
Training Program (201711396005).
Acknowledgments: Thanks all the funds mentioned above for their support. Inhibition study was carried out
in key laboratory of synthetic and natural functional molecule chemistry of ministry of education at Northwest
University. All the enzymes used in the study were donated from Ke-wu Yang.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: All the target compounds halogen-substituted triazolethioacetamides are available from the
authors, the synthetic raw materials are purchased from the reagent company and the metallo-beta-lactamase
used in the manuscript were provided from Professor Ke-wu Yang.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).