N-acylhydrazones confer inhibitory efficacy against New Delhi metallo-β-lactamase-1

Article abstract of DOI:10.1016/j.bioorg.2021.105138

The expression of β-lactamases, especially metallo-β-lactamases (MβLs) in bacteria is one of the main causes of drug resistance. In this work, an effective N-acylhydrazone scaffold as MβL inhibitor was constructed and characterized. The biological activity assays indicated that the synthesized N-acylhydrazones 1–11 preferentially inhibited MβL NDM-1, and 1 was found to be the most effective inhibitor with an IC₅₀ of 1.2 μM. Analysis of IC₅₀ data revealed a structure–activity relationship, which is that the pyridine and hydroxylbenzene substituents at 2-position improved inhibition of the compounds on NDM-1. ITC and enzyme kinetics assays suggested that it reversibly and competitively inhibited NDM-1 (K_i = 0.29 ± 0.05 μM). The synthesized N-acylhydrazones showed synergistic antibacterial activities with meropenem, reduced 4–16-fold MIC of meropenem on NDM-1- producing E. coli BL21 (DE3), while 1 restored 4-fold activity of meropenem on K. pneumonia expressing NDM-1 (NDM-K. pneumoniae). The mice experiments suggested that 1 combined meropenem to fight against NDM-K. pneumoniae infection in the spleen and liver. Cytotoxicity assays showed that 1 and 2 have low cytotoxicity. This study offered a new framework for the development of NDM-1 inhibitors.

Full text of DOI:10.1016/j.bioorg.2021.105138

Bioorganic Chemistry 114 (2021) 105138
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
N-acylhydrazones confer inhibitory efficacy against New Delhi
metallo-β-lactamase-1
,
,
Han Gaoa 1, Jia-Qi Lia 1, Peng-Wei Kanga, Jia-Zhu Chigana, Huan Wanga, Lu Liua, Yin-Sui Xua,
Le Zhaib *, Ke-Wu Yanga
,
,*
a Key Laboratory of Synthetic and Natural Functional Molecule of the Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an
710127, PR China
b Shaanxi Key Laboratory of Phytochemistry, College of Chemistry and Chemical Engineering, Baoji University of Arts and Sciences, Baoji 72101, Shaanxi Province, PR
China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The expression of β-lactamases, especially metallo-β-lactamases (MβLs) in bacteria is one of the main causes of
drug resistance. In this work, an effective N-acylhydrazone scaffold as MβL inhibitor was constructed and
characterized. The biological activity assays indicated that the synthesized N-acylhydrazones 1–11 preferentially
inhibited MβL NDM-1, and 1 was found to be the most effective inhibitor with an IC50 of 1.2 µM. Analysis of IC50
data revealed a structure–activity relationship, which is that the pyridine and hydroxylbenzene substituents at 2-
position improved inhibition of the compounds on NDM-1. ITC and enzyme kinetics assays suggested that it
reversibly and competitively inhibited NDM-1 (Ki = 0.29 ± 0.05 µM). The synthesized N-acylhydrazones showed
synergistic antibacterial activities with meropenem, reduced 4–16-fold MIC of meropenem on NDM-1- producing
E. coli BL21 (DE3), while 1 restored 4-fold activity of meropenem on K. pneumonia expressing NDM-1 (NDM-
K. pneumoniae). The mice experiments suggested that 1 combined meropenem to fight against NDM-
K. pneumoniae infection in the spleen and liver. Cytotoxicity assays showed that 1 and 2 have low cytotoxicity.
This study offered a new framework for the development of NDM-1 inhibitors.
Metallo-β-lactamases
Drug-resistance
N-acylhydrazone
NDM-1
Inhibitor
1. Introduction
antibiotics, resulting in bacteria multiple-drug resistance [7]. By
contrast, B2 subclass enzymes have a narrow range of substrates,
β-Lactam antibiotics account for more than 50% of global antibiotic
prescriptions and are the most important antibiotics [1]. However, the
emergence of bacterial resistance has been aggravated by the excessive
use of antibiotics and seriously threatens public health [2]. There are
many factors for bacterial resistance to antibiotics, among which the
production of enzymes to hydrolyze β-lactam rings is the most important
factor [3]. β-Lactamases can be classified into A-D categories based on
the homology of amino acid sequences. Class A, C and D enzymes are
called serine-β-lactamases (SβLs) [4]. They can use the serine residues at
the active site to attack the carbonyl group on the β-lactam ring
nucleophilically and catalyze the hydrolysis of antibiotics [5]. Class B
enzymes, also known as metallo-β-lactamases (MβLs), can be classified
into B1, B2 and B3 subclasses on the basis of Zn(II) content and amino
acid sequence [6].
including carbapenems known as one of the “last resort” [8]. In order to
fight against bacterial resistance, an essential strategy is to develop
β-lactamases inhibitors to restore the curative effect of existing β-lactam
antibiotics. At present, the combination of β-lactam antibiotics and their
inhibitors, such as sulbactam, has been successfully applied to treat SβLs
mediated bacterial infections. However, so far, there is no clinical
application of MβLs inhibitor. Hence, there is an urgent need to develop
MβLs inhibitors.
In the light of the importance of MβLs in biomedicine, great efforts
have been made to develop these enzymes inhibitors, such as carboxylic
acids [9,10], ebselen [11,12], thiols [13–15], β-lactam analogues [16],
rhodanines [17–19], and cyclic boronates [20–22].
The B1 subclass MβL New Delhi metallo-β-lactamase-1 (NDM-1) can
catalyze the hydrolysis of almost all β-lactam antibiotics [23]. The NDM-
1 gene is encoded by plasmid, which is conducive to its rapid
MβLs catalyze the hydrolysis of almost all clinical β-lactam
* Corresponding authors.
E-mail address: kwyang@nwu.edu.cn (K.-W. Yang).
1
Authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.105138
Received 26 April 2021; Received in revised form 25 June 2021; Accepted 27 June 2021
Available online 30 June 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

H. Gao et al.
Bioorganic Chemistry 114 (2021) 105138
transmission. Therefore, inhibition of NDM-1 activity is a serious chal-
lenge [24]. The clinical strains producing NDM-1, such as P. aeruginosa,
K. pneumonia and Carbapenem-Resistant Enterobacteriaceae (CRE), are
highly resistant to antibiotics. Compounds that can effectively inhibit
the activity of NDM-1 have been widely searched. The thio-
semicarbazones have a wide range of pharmacological activities,
including anticancer, antimalarial, antifungal, antiparasitic, antibacte-
rial and antiviral [25–29]. 3-Aminopyridinecarboxaldehyde thio-
semicarbocyclic ketone (3-AP, structure is shown Fig. 1) can inhibit
ribonucleotide reductase by metal chelation [30,31]. Some thio-
semicarbazone derivatives have been reported as effective inhibitors of
NDM-1 [32]. Recently, our studies indicated that the diaryl-substituted
thiosemicarbazones (TSC1, TSC2, structures are shown in Fig. 1) spe-
cifically inhibited NDM-1 with an IC50 value between 0.038 and 25.7
µM. The structure–activity relationship revealed that the inhibitory ef-
fect of thiosemicarbazones could be enhanced when 2-hydroxylbenzene
and 2-pyridine were used as substituents [50]. In addition, it has been
reported that thiosemicarbazone inhibits the activity of tyrosinase and
urease through metal complexation [33]. Hydrazide has a structure
similar to thiosemicarbazone. At present, hydrazides have been reported
to have effective antifungal, antibacterial, antiparasitic and antitumor
properties [34–37]. Also, some aryl-substituted hydrazides (Fig. 1) have
been reported as effective inhibitors of some metalloenzymes, such as
copper dependent amine oxidase (Cu-AOs) [38]. Recently, sulfonyl
hydrazone and hydrazide-containing compounds have been reported to
be MβLs inhibitors [39–41]. Based on the information and our studies on
thiosemicarbazones, it is reasonable to speculate thatthe N-acylhy-
drazones have the potential as effective MβLs inhibitors.
2. Results and discussion
2.1. Synthesis and activity evaluation of N-acylhydrazones
Benzoic acid was esterified to offer methyl benzoate, which is
condensed with hydrazine hydrate to be converted into benzoyl hy-
drazide. The intermediate and aldehyde were refluxed in ethanol to give
the final products.
To test whether these N-acylhydrazones were MβLs inhibitors, the
activity evaluation of N-acylhydrazones on MβLs was conducted on an
Agilent UV8453 spectrometer, using cefazolin (40 µM) as substrate for
VIM-2 and meropenem (40 µM) for NDM-1, IMP-1, ImiS and L1. The
concentration of inhibitors tested was 20 µM. Hydrolysis of meropenem
and cefazolin was monitored at 300 and 265 nm, respectively.
The inhibition percentage of N-acylhydrazones on NDM-1, VIM-2,
IMP-1, ImiS and L1 is defined as the enzyme activity not premixed with
the inhibitors (100%) minus the residual activity of the enzyme pre-
mixed with the inhibitors. As shown in Fig. 3, eleven of these compounds
had inhibitory effect on NDM-1 by more than 74%, especially 1 showed
96% inhibition.
The fifty percent inhibitory concentrations (IC50) of N-acylhy-
drazones 1–15 on four MβLs were determined on an Agilent UV8453
spectrometer. The inhibitor concentrations varied from 0 to 20
μM.
Meropenem (40
μ
M) was used as the substrate of NDM-1, IMP-1, ImiS
M) was used as the substrate of VIM-2. Hy-
and L1, and cefazolin (40
μ
drolysis of meropenem and cefazolin was monitored at 300 and 265 nm,
respectively. The IC50 data (Table 1) showed that NDM-1 was strongly
inhibited by all N-acylhydrazones tested with IC50 values between 1.2
and 15.2 µM, and 1 was the most potent inhibitor (IC50 = 1.2 µM). For
ImiS, 1, 6, 8 and 11 showed potency with IC50 values ranging from 3.5 to
In this work, fifteen N-acylhydrazones were designed, synthesized
(structures are shown in Fig. 2). 1H, 13C NMR (see supporting infor-
mation) and HRMS were used to characterize and confirm the structures
of the synthesized compounds. Five MβLs L1, ImiS, IMP-1, VIM-2 and
NDM-1 were used to evaluate the inhibitory activities of these com-
pounds. The inhibition mode of N-acylhydrazone 1 on NDM-1 was
studied by enzyme kinetics, dialysis and thermodynamics experiments
[42]. In addition, the antibacterial activities of these compounds in
combination with meropenem were evaluated. Furthermore, the anti-
bacterial activity of inhibitor 1 in vivo in collaboration with meropenem
was also evaluated. Cytotoxicity of the compounds against Vero E6 cells
was tested.
16.9
9.5
μ
M. Compounds 6 and 8 inhibited L1 with IC50 values of 8.1 and
μ
M, and 9 exhibited an IC50 value of 8.2 M against VIM-2. The IC50
μ
data revealed a structure–activity relationship, which is that, the pyri-
dine and hydroxylbenzene at 2-position improve inhibition of N-acyl-
hydrazones on NDM-1 significantly. Based on the preference of tested N-
acylhydrazones to NDM-1, we focused on the study of inhibition of
NDM-1 in the remainder of this work.
Due to the best inhibitory effect, N-acylhydrazone 1 was chosen to
perform time- and concentration-dependent inhibition studies on NDM-
1. It can be observed in Fig. 4 that, with the prolongation of incubation
time of the inhibitor and NDM-1, the residual activity of enzyme
decreased gradually, and 1 showed the maximum inhibition after the
incubation for about 15 min. In order to identify that the parent com-
pound or its hydrolyzate exhibited inhibitory effect, we tested the
Fig. 1. Structures of thiosemicarbazone (A) and hydrazide inhibitors (B).
2

H. Gao et al.
Bioorganic Chemistry 114 (2021) 105138
Fig. 2. Structures of the synthesized N-acylhydrazones.
Fig. 3. Percent inhibition of 20 µM N-acylhydrazones 1–15 on MβLs. Meropenem was used as substrate of NDM-1, IMP-1, ImiS and L1. Cefazolin was used as
substrate of VIM-2.
inhibitory activities of the commercial isoniazid and salicylaldehyde on
NDM-1, respectively. The results shown that the residual activity of the
enzyme had no significant change in the presence of both compounds
(Fig. 4A), suggesting that N-acylhydrazone 1 inhibited NDM-1.
Furthermore, the residual enzyme activity decreased as compound 1
respectively. The Lineweaver–Burk plots are shown in Fig. 5. The result
indicates that 1 is a competitive inhibitor of NDM-1 (Ki = 0.29 ± 0.05
µM).
Dose-dependent and dialysis experiments were carried out for
further investigation of the mechanism by which N-acylhydrazone 1
inhibits NDM-1 activity. The concentrations of 1 varied from 0.1 to 20
µM, and meropenem concentration was 40 µM in the dose-dependent
experiments. It can be observed that the residual enzyme activity
decreased gradually as the concentration of 1 increased, and the residual
enzyme activity was less than 10% when the concentration of 1 was up
to 10 µM (Fig. 6A), suggesting that the N-acylhydrazone is a dose-
dependent inhibitor. In the dialysis experiments, NDM-1 was premixed
with 1 (10 µM) for 30 min, and 2 mL of the premixed sample was placed
in a 10 kDa cut-off dialysis bag. The residual enzyme activities were
determined after dialysis for 2 h with deionized water. As shown in
Fig. 6B, NDM-1 activity recovered after dialysis, suggesting that the N-
acylhydrazone is a reversible enzyme inhibitor.
concentration (0.25–5 μM) increased (Fig. 4B), suggesting that the N-
acylhydrazone is a time- and concentration-dependent NDM-1 inhibitor.
2.2. Inhibition mode assay
For the purpose of identifying the inhibition mode of N-acylhy-
drazones against NDM-1, 1 was chosen to determine Ki value. The
concentrations of 1 and meropenem were in the range of 0–2.5 and
10–60 µM, respectively. After measuring the hydrolysis rates three times
in parallel, the Lineweaver–Burk plots were generated to assay the in-
hibition model of N-acylhydrazone 1. The measuring instrument and
analysis software are Agilent UV8453 spectrometer and SigmaPlot 12.0,
3

H. Gao et al.
Bioorganic Chemistry 114 (2021) 105138
the binding of N-acylhydrazones to protein is different from to free Zn
Table 1
Inhibitory activity (IC50
, μM) of N-acylhydrazones against four MβLs in 30 mM
(II) ions.
Tris-HCl, pH 7.0.a
B1
NDM-1b
B2
ImiSb
B3
L1b
2.3. Inhibition assay of NDM-1 by ITC
Compd.
VIM-2c
Isothermal titration calorimetry (ITC) is an ideal approach to mea-
sure heat change during ligand-target binding. The main principle of ITC
is to monitor the time derivative of heat change during a biomolecular
reaction or binding event by titrating one solution into another. ITC
provides thermodynamic properties, is sensitive, has a fast response
time, and does not require any labelling chemistry [43]. Pan-assay
interference compounds (PAINS) are promiscuous compound classes
that produce false positive hits in high-throughput screenings, and
hydroxyphenylhydrazones have been reported to exhibit PAINS-like
behavior [44].
1
1.2 ± 0.1
1.5 ± 0.2
2.0 ± 0.1
1.9 ± 0.1
4.0 ± 0.2
2.4 ± 0.1
3.1 ± 0.2
7.4 ± 0.5
2.7 ± 0.1
15.2 ± 0.3
3.6 ± 0.1
10.6 ± 0.5
2
3
4
5
6
3.5 ± 0.3
8.1 ± 0.5
9.5 ± 0.6
7
8
16.9 ± 0.5
9
8.2 ± 0.3
10
11
12
13
14
15
13.6 ± 0.6
To identify whether the N-acylhydrazones are real enzyme in-
hibitors, ITC was used to investigate the inhibition of NDM-1 by N-
acylhydrazone 1 in the single injection mode of MicroCal-ITC200 at
25 ◦C [43]. Prior to testing, different concentrations of 1 (125, 250, 500,
and 800 µM) and NDM-1 were incubated for 30 min. Meropenem and
a
Blank space represents that percent inhibition is under 50% at a concen-
tration of 20
μ
M.
b
The antibiotic used was meropenem. cThe antibiotic used was cefazolin.
NDM-1 concentrations were 100 and 0.1 µM, respectively. 30
μ
L mer-
Prior to testing, the enzymes and inhibitors were incubated for 30 min.
openem was titrated into the sample pool (270
μL) containing NDM-1 in
the single injection mode. The inhibition of N-acylhydrazone 1 on NDM-
1 catalytic meropenem hydrolysis was monitored by ITC (Fig. 8). It can
be observed that, with the inhibitor concentration increased, the thermo
Moreover, the NDM-1 samples after dialysis were acidified with
concentrated nitric acid, and inductively coupled mass spectrometry
(ICP-MS) was used to determine the Zn(II) content in protein. N-acyl-
hydrazone 1 and EDTA concentrations were 20 µM. As shown in Fig. 6C,
compared with the control group, EDTA inactivated NDM-1 and made
the enzyme lost 1.7 equivalents of Zn(II), indicating that EDTA exhibited
inhibitory activity by removing Zn(II) from the active site of the enzyme.
However, the Zn(II) content of NDM-1 premixed with compound 1 was
the same as that of the control group, revealing that compound 1
inhibited NDM-1 in a different way than EDTA, without removing Zn(II)
ions from the enzyme active site.
To investigate the inhibitory mechanism of N-acylhydrazones on
MβLs, the absorbance changes of 1, 13, 2 and 14 before and after mixing
with Zn(II) ions (in ZnCl2 solution) were monitored on Agilent UV8453
spectrometer, respectively. N-acylhydrazones (40 µM) and ZnCl2 (0–80
µM) were incubated for 1 min before testing. As shown in Fig. 7A, the
absorbance of 1 decreased at 290 and 335 nm but increased at 380 nm as
Zn(II) ion concentration increased. Similarly, the absorbance of 2
decreased at 290 nm, while increased at 380 nm (Fig. 7C). However, the
absorbance of 13 (Fig. 7B) and 14 (Fig. 7D) remained unchanged under
the same experimental conditions, indicating that the pyridine and
hydroxylbenzene at 2-position significantly improved the binding abil-
ity of N-acylhydrazones to Zn(II) ions from metal complex.
In order to characterize the binding of N-acylhydrazones to MβLs,
UV–Vis absorbance of 1, 13, 2, and 14 were monitored on an Agilent
UV8453 spectrometer within 1 h of premixing with NDM-1. As shown in
Fig. 7, the combination of all of these compounds with NDM-1 did not
resulted in absorption changes, which are different from the combina-
tion of the inhibitors with Zn(II) ions (in ZnCl2 solution), implying that
Fig. 5. Lineweaver–Burk plots of meropenem hydrolysis catalyzed by NDM-1
with and without N-acylhydrazone 1. The concentrations of 1 were 0 (●),
○
0.25 ( ), 0.5 (▾), 1 (▽), 2.5 µM (■).
Fig. 4. Time-dependent inhibition of NDM-1 by 5 µM N-acylhydrazone 1, intermediates isoniazid and salicylaldehyde, respectively (A), and inhibition curves of
NDM-1 by the inhibitor at a concentration between 0.25 and 5 µM (B).
4

H. Gao et al.
Bioorganic Chemistry 114 (2021) 105138
Fig. 6. The inhibitory mode assays of the N-acylhydrazone 1 on NDM-1. Residual activities of NDM-1 after premixing with compound 1 at different concentrations
(A). Residual activities of NDM-1 before and after dialysis reverse 1 (B). ICP-MS determination of Zn(II) consumption in NDM-1 samples (C).
Fig. 7. The UV–Vis monitoring of N-acylhydrazones 1 (A), 13 (B), 2 (C), and 14 (D) in the presence of Zn(II) ions (0–80 µM) and these inhibitors in the presence of
20
μ
M NDM-1 (E-H), respectively. The concentration of N-acylhydrazones was 40 µM.
5

H. Gao et al.
Bioorganic Chemistry 114 (2021) 105138
BL21 (DE3) cells with and without NDM-1. It can be observed in
Table 2A that the combination with N-acylhydrazone led to a 4–16-fold
reduction in MICs of MER on NDM-1-producing E. coli BL21 (DE3), and
compounds 1, 4 and 8 exhibited the strongest effect.
Therefore, further investigation of the antibacterial activity of N-
acylhydrazone 1 combined with MER against seven clinical strains
expressing NDM-1 was carried out. As shown in Table 2B, the MICs
indicated that 1 exhibited synergy with MER against K. pneumonia
expressing NDM-1, leading to a 4-fold reduction in MER MIC. The results
listed in Table 2C show that the inhibitor concentration increased from
16 to 64 μg/mL resulted in 4–16-fold decreased in MIC of MER on the
NDM-1-producing K. pneumonia, but only 2-fold decreased in MIC of
MER on ECs. This difference of antibacterial effect may be related to the
expression of β-lactamases in different strains and species specificity
[46].
To further confirm the antibacterial effect of N-acylhydrazone 1
combined with MER, the inhibitory zone experiment was carried out on
E. coli producing NDM-1 [50]. The result (Fig. 9) showed that the
inhibitory zone (diameter, 18 mm) of combination of 1 and MER are
larger than that of 1 alone (16 µg/mL, less than 6 mm) and MER alone
(13 mm), confirming that the N-acylhydrazone has synergistic antibac-
terial effect with MER.
Fig. 8. Overlayed heat flow curves of NDM-1 (0.1 μM) catalyzed meropenem
(100 μM) hydrolysis with and without N-acylhydrazone 1 at different concen-
trations (0, 125, 250, 500, and 800 µM).
power (dq/dt) decreased gradually, which confirmed the dose-
dependent inhibition of N-acylhydrazone 1 on NDM-1 from thermody-
namics. Furthermore, the addition of 1 did not lead to a change in the
total heat (Q) liberated from NDM-1 catalytic meropenem hydrolysis
(Fig. 8, inset). For a typical reversible inhibitor, the Q values remained
unchanged in the presence or absence of inhibitor, likely because the
formation of the enzyme–inhibitor complex (EI) that slowed down
substrate hydrolysis was reversible [43]. These results indicated that the
inhibitory mode of NDM-1 by N-acylhydrazone 1 was reversible, similar
to that of D-captopril [43].
In view of the effective antibacterial activity of N-acylhydrazone 1
combined with MER, the typical thermograms of microdilution check-
erboard experiment were investigated on E. coli (Fig. 10A) and E. coli
producing NDM-1(Fig. 10B). The MER concentrations were varied from
0 to 256 µg/mL, and the concentrations of N-acylhydrazone 1 were
varied from 0 to 128 µg/mL. As shown in Fig. 10, 1 (64 µg/mL) had
antibacterial activity against two tested strains, but no synergistic effect
with MER against E. coli at a concentration of 16 µg/mL. When com-
pound 1 combined with MER against E. coli harboring NDM-1, the MIC
of MER significantly reduced, suggesting that the antibacterial effect of
MER was restored due to the inhibition of NDM-1 by the N-
acylhydrazone.
2.4. Antibacterial activity assays in vitro
The synergistic antibacterial effect of N-acylhydrazones 1–11 in
combination with meropenem (MER) against E. coli expressing NDM-1
was studied by measuring the minimum inhibitory concentration
(MIC) [45]. The inhibitors (16 μg/mL) were evaluated using the E. coli
Table 2
MICs of MER combined with N-acylhydrazones 1–11 (16
μ
g/mL) on E. coli expressing NDM-1a (A), and MER combined with N-acylhydrazone 1 on clinical strains
expressing NDM-1 K. pneumonia and ECs (B). MICs of MER combined with different concentrations of 1 on clinical strains expressing NDM-1 K. pneumonia and ECs
(EC01-EC24) (C).
A
Compd.
E. coli-NDM-1
Compd.
E. coli-NDM-1
MER
128
8
6
16
32
8
1
2
3
4
5
7
32
32
8
8
9
32
32
16
10
11
32
B
Compd.
EC01
EC04
EC06
EC07
EC08
EC24
K. pneumonia
MER
128
64
64
32
128
64
128
64
128
64
128
64
256
64
MER + 1
C
Compd.
MER
MER + 1(16
μg/mL)
MER + 1(32
μ
g/mL)
MER + 1(64 μg/mL)
EC01
128
64
64
32
64
64
64
64
64
64
32
64
64
64
64
64
64
32
64
64
64
64
16
EC04
EC06
128
128
128
128
256
EC07
EC08
EC24
K. pneumonia
a
The MIC of MER as a single agent for E. coli not expressing NDM-1 was 0.125 μg/mL. The MICs of N-acylhydrazones 12–15 were not determined because their
percent inhibitions were less than 50%.
6

H. Gao et al.
Bioorganic Chemistry 114 (2021) 105138
after infection, a single dose of compound 1 and MER was injected into
mice intraperitoneally. The efficacy of compound 1 and MER as a single
agent and combined drugs in the remedy of the liver and spleen bacterial
infections was observed. It is observed in Fig. 11 that compound 1 had
no effect on bacterial growth as a single agent, and monotherapy of MER
inhibit the growth of bacteria slightly, but the bacterial load in liver and
spleen of mice was reduced by compound 1 combined with MER. The in
vivo antibacterial experiments indicated that compound 1 could
combine with MER to fight against NDM-1-carrying carbapenem-resis-
tant pathogens (such as NDM-K. pneumoniae).
2.6. Cytotoxicity assay
In an effort to evaluate the toxicity of N-acylhydrazones, a cytotox-
icity assay was performed according to the previously reported method
[48]. Vero E6 cells were used to assess the cytotoxicity of inhibitors 1
and 2 at different concentrations (10, 25, 50, 100, 200, and 400 μM). As
Fig. 9. The inhibitory zone experiment was used to detect the resistance of
E. coli expressing NDM-1. MER: MH with MER only; MER + 1: MH with MER
shown in Fig. 12, in the presence of N-acylhydrazones 1 and 2 concen-
trations up to 400 µM, the cell viability of Vero E6 cells exceeded 80%,
suggesting that these hydrazides have low cytotoxicity.
and 1; 1: MH with 1 only. The concentrations of MER and 1 were 32 and 16 μg/
mL, respectively.
3. Conclusions
2.5. Antibacterial activity assays in vivo
Fifteen N-acylhydrazones were synthesized and identified by 1H, 13
C
To test whether the N-acylhydrazones can restore the activity of MER
in vivo, the antibacterial effect of inhibitor 1 combined with MER against
K. pneumonia harboring NDM-1 (NDM-K. pneumoniae) was assayed in
mice according to the previous method [47]. A sublethal dose of NDM-
K. pneumoniae was injected into Kunming mice intraperitoneally. At 2 h
NMR and HRMS. The biological activity assays indicated that eleven of
these compounds exhibited inhibitory activity against NDM-1 with IC50
values ranging from 1.2 to 15.2 μM, and compound 1 is the most potent
inhibitor (IC50 = 1.2 µM). N-acylhydrazones 6 and 8 also showed ability
to inhibit ImiS and L1 with IC50 values ranging from 3.5 to 16.9 µM. The
Fig. 10. Representative thermograms of MER combined with N-acylhydrazone 1 against E. coli (A) and E. coli producing NDM-1 (B).
Fig. 11. The antibacterial effect of N-acylhydrazone 1 combined with MER on NDM-K. pneumoniae in vivo. A single dose of MHB (as control), 1 (2 mg/kg), MER (10
mg/kg), and a combination of MER (10 mg/kg) and 1 (2 mg/kg) were injected intraperitoneally into the mice groups infected with NDM-K. pneumoniae, respectively.
Bacterial load in the liver (A) and spleen (B) was determined by selective plating.
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H. Gao et al.
Bioorganic Chemistry 114 (2021) 105138
1H), 8.80 (d, J = 6.0 Hz, 2H), 8.62 (d, J = 4.6 Hz, 1H), 8.49 (s, 1H), 8.00
(d, J = 7.9 Hz, 1H), 7.92–7.80 (m, 3H), 7.48–7.36 (m, 1H). 13C NMR
(101 MHz, DMSO-D6) δ 162.02, 153.00, 150.47, 149.67, 149.30,
140.29, 137.04, 124.76, 123.24, 121.67, 120.20. HRMS (ESI) m/z:
249.0745 (Calcd. for [M + Na]+ 249.0747).
(E)-N’-(pyridin-2-ylmethylene)picolinohydrazone (3)
White solid, yield 85%. 1H NMR (400 MHz, DMSO d6) δ 9.10 (s, 1H),
8.80–8.74 (m, 1H), 8.65 – 8.56 (m, 1H), 8.47 (s, 1H), 8.28 (d, J = 7.4 Hz,
1H), 8.00 (d, J = 7.7 Hz, 1H), 7.87 (t, J = 7.3 Hz, 1H), 7.64–7.50 (m,
1H), 7.45–7.37 (m, 1H). 13C NMR (101 MHz, DMSO-D6) δ 162.24,
153.21, 152.67, 149.74, 148.87, 137.13, 135.80, 129.12, 124.78,
123.86, 120.30. HRMS (ESI) m/z: 249.0745 (Calcd. for [M + Na]+
249.0747).
(E)-N’-(2-hydroxybenzylidene)picolinohydrazone (4)
1
Fig. 12. Percent cell viability of Vero E6 cells (relative to without N-acylhy-
drazone) in the presence of 1 and 2 at concentrations of 10, 25, 50, 100, 200,
and 400 µM.
White solid, yield 91%. H NMR (400 MHz, DMSO d6) δ 12.28 (s,
1H), 11.19 (s, 1H), 9.11 (d, J = 1.7 Hz, 1H), 8.82–8.73 (m, 1H), 8.66 (s,
1H), 8.29 (d, J = 8.0 Hz, 1H), 7.57 (t, J = 6.8 Hz, 2H), 7.35–7.25 (m,
1H), 6.95 (d, J = 8.4 Hz, 2H). 13C NMR (101 MHz, DMSO-D6) δ 161.50,
157.54, 152.51, 148.71, 135.53, 131.66, 129.40, 128.72, 123.69,
119.45, 118.72, 116.50. HRMS (ESI) m/z: 264.0742 (Calcd. for [M +
Na]+ 264.0743).
IC50 data revealed a structure–activity relationship, that is, pyridine and
hydroxylbenzene substituents at 2-position improved inhibition of N-
acylhydrazones on NDM-1 significantly. ITC assays and enzyme kinetics
assays showed that compound 1 reversibly and competitively inhibited
NDM-1 (Ki = 0.29 ± 0.05 µM). MIC tests demonstrated that compounds
1–11 reduced 4–16-fold MIC of meropenem against NDM-1-producing
E. coli BL21 (DE3), while 1 reduced 4-fold MIC of meropenem against
NDM-K. pneumoniae. Mice experiments suggested that 1 could cooperate
with meropenem to fight against NDM-K. pneumoniae infection in the
spleen and liver. Cytotoxicity assay showed that the inhibitors have low
cytotoxicity. This work revealed that the N-acylhydrazone is an effective
scaffold for NDM-1 inhibitor development.
(E)-N’-(pyridin-2-ylmethylene)pyrazine-2-carbohydrazone (5)
White solid, yield 71%. 1H NMR (400 MHz, DMSO d6) δ 9.29 (s, 1H),
8.94 (d, J = 2.5 Hz, 1H), 8.80 (dd, J = 2.3, 1.6 Hz, 1H), 8.69 (s, 1H), 8.62
(d, J = 4.6 Hz, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.89 (td, J = 7.8, 1.5 Hz,
1H), 7.43 (ddd, J = 7.3, 4.9, 0.9 Hz, 1H). 13C NMR (101 MHz, DMSO-D6)
δ 159.90, 153.19, 149.99, 149.58, 148.02, 144.46, 144.33, 143.37,
136.94, 124.64, 120.12. HRMS (ESI) m/z: 250.0696 (Calcd. for [M +
Na]+ 250.0699).
(E)-N’-(2-hydroxybenzylidene)pyrazine-2-carbohydrazone (6)
1
White solid, yield 82%. H NMR (400 MHz, DMSO d6) δ 12.64 (s,
4. Experiments
1H), 11.29 (s, 1H), 9.27 (s, 1H), 8.91 (d, J = 2.5 Hz, 1H), 8.84 (s, 1H),
8.78 (d, J = 2.4 Hz, 1H), 7.50 (dd, J = 7.7, 1.5 Hz, 1H), 7.34–7.27 (m,
1H), 6.97–6.87 (m, 2H). 13C NMR (101 MHz, DMSO-D6) δ 159.51,
157.71, 150.43, 148.00, 144.30, 143.42, 131.74, 129.83, 119.44,
118.60, 116.55. HRMS (ESI) m/z: 265.0693 (Calcd. for [M + Na]+
265.0696).
4.1. Materials
1H and 13C NMR spectra were recorded on a Japan Electronics 400
MHz MRI. The peak patterns are recorded 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 con-
stants (J) were reported in Hertz (Hz). Mass spectra were obtained on a
micro TOF-Q (BRUKER) mass spectrometer.
(E)-N’-(pyridin-2-ylmethylene)furan-2-carbohydrazone (7)
1
White solid, yield 72%. H NMR (400 MHz, DMSO d6) δ 12.12 (s,
1H), 8.60 (d, J = 4.5 Hz, 1H), 8.49 (s, 1H), 7.96 (d, J = 6.3 Hz, 2H), 7.86
(t, J = 7.5 Hz, 1H), 7.46–7.27 (m, 2H), 6.80–6.65 (m, 1H). 13C NMR
(101 MHz, DMSO-D6) δ 154.45, 153.24, 149.59, 148.19, 146.18,
136.95, 124.49, 120.04, 115.60, 112.29. HRMS (ESI) m/z: 238.0584
(Calcd. for [M + Na]+ 238.0587).
4.2. Synthesis of N-acylhydrazones (1–15) [49,50]
(E)-N’-(2-hydroxybenzylidene)furan-2-carbohydrazone (8)
The corresponding carboxylic acid (0.01 mol) was dissolved in
MeOH (20 mL), and added the concentrated sulfuric acid (10 mL) to the
system and reacted at 85–95 ◦C for 2 h. The solvent was removed after
the reaction and adjusted the pH to alkali by adding 20% K2CO3. The
organic phase was extracted with ethyl acetate, dried with anhydrous
sodium sulfate, and the organic solvent was spin-dried to obtain an oily
liquid as the target product, which was dissolved in EtOH (20 mL),
added hydrazine hydrate (0.05 mol) dropwise and heated to reflux at
80 ◦C for 6–9 h and precipitate out.
1
White solid, yield 79%. H NMR (400 MHz, DMSO d6) δ 12.14 (s,
1H), 11.15 (s, 1H), 8.65 (s, 1H), 7.97 (s, 1H), 7.54 (d, J = 7.2 Hz, 1H),
7.37–7.25 (m, 2H), 6.92 (t, J = 8.7 Hz, 2H), 6.72 (s, 1H). 13C NMR (101
MHz, DMSO-D6) δ 157.40, 148.17, 146.13, 131.48, 129.34, 119.43,
118.80, 116.44, 115.32, 112.25. HRMS (ESI) m/z: 253.0580 (Calcd. for
[M + Na]+ 253.0584).
(E)-N’-(pyridin-2-ylmethylene)thiophene-2-carbohydrazone (9)
1
White solid, yield 85%. H NMR (400 MHz, DMSO d6) δ 12.07 (s,
1H), 8.67 – 8.58 (m, 1H), 8.50 – 7.85 (m, 5H), 7.48–7.37 (m, 1H),
7.28–7.18 (m, 1H). 13C NMR (101 MHz, DMSO-D6) δ 149.71, 147.82,
144.61, 137.08, 135.21, 132.42, 129.48, 128.30, 126.95, 124.45,
120.12. HRMS (ESI) m/z: 254.0356 (Calcd. for [M + Na]+ 254.0359).
(E)-N’-(pyridin-2-ylmet1hylene)benzohydrazone (10)
The dried hydrazide and the corresponding aldehyde (1 eq) were
dissolved in EtOH and refluxed at 100 ◦C for 3–4 h, then cooled to room
temperature and the crystals precipitated, which are filtered and dried
to obtain the final products (1–15).
(E)-N’-(2-hydroxybenzylidene)isonicotinohydrazone (1)
1
White solid, yield 82%. H NMR (400 MHz, DMSO d6) δ 12.11 (s,
1H), 8.61 (d, J = 4.0 Hz, 1H), 8.51 (s, 1H), 7.95 (td, J = 25.2, 23.9, 7.6
Hz, 4H), 7.57 (dt, J = 27.5, 7.0 Hz, 3H), 7.46–7.35 (m, 1H). 13C NMR
(101 MHz, DMSO-D6) δ 163.52, 153.34, 149.58, 148.14, 136.94,
133.23, 132.06, 128.63, 127.81, 124.49, 120.01. HRMS (ESI) m/z:
248.0796 (Calcd. for [M + Na]+ 248.0794).
White solid, yield 94%. H NMR (400 MHz, DMSO d6) δ 12.32 (s,
1H), 11.14 (s, 1H), 8.80 (d, J = 6.0 Hz, 2H), 8.69 (s, 1H), 7.86 (d, J = 6.1
Hz, 2H), 7.59 (dd, J = 7.7, 1.5 Hz, 1H), 7.34–7.27 (m, 1H), 6.99 – 6.87
(m, 2H). 13C NMR (101 MHz, DMSO-D6) δ 161.49, 157.66, 150.52,
149.17, 140.09, 131.87, 129.42, 121.67, 119.56, 118.81, 116.60. HRMS
(ESI) m/z: 264.0740 (Calcd. for [M + Na]+ 264.0743).
(E)-2-hydroxy-N’-(pyridin-2-ylmethylene)benzohydrazone (11)
(E)-N’-(pyridin-2-ylmethylene)isonicotinohydrazone (2)
1
1
White solid, yield 95%. H NMR (400 MHz, DMSO d6) δ 12.33 (s,
White solid, yield 79%. H NMR (400 MHz, DMSO d6) δ 12.28 (s,
8

H. Gao et al.
Bioorganic Chemistry 114 (2021) 105138
◦
1H), 11.15 (s, 1H), 8.80 (s, 2H), 8.69 (s, 1H), 7.87 (s, 2H), 7.59 (dd, J =
7.6, 1.2 Hz, 1H), 7.33–7.26 (m, 1H), 6.99–6.85 (m, 2H). 13C NMR (101
MHz, DMSO-D6) δ 161.43, 157.61, 150.44, 149.15, 140.01, 131.80,
129.38, 121.61, 119.48, 118.73, 116.53. HRMS (ESI) m/z: 264.0740
(Calcd. for [M + Na]+ 264.0743).
4 C with deionized water as dialysate for 24 h. After 10 min of acid-
olysis with concentrated nitric acid, the dialyzed sample was diluted to
4 µM for ICP-MS determination.
4.6. Isothermal titration calorimetry (ITC) test
(E)-N’-(4-methoxybenzylidene)isonicotinohydrazone (12)
1
White solid, yield 64%. H NMR (400 MHz, DMSO d6) δ 11.97 (s,
In the single injection mode of Malvern MicroCal-ITC200, isothermal
titration calorimetry (ITC) was used to investigate the inhibition of
NDM-1 by N-acylhydrazone 1 at 25 ◦C [43]. Prior to testing, NDM-1 was
incubated with different concentrations of 1 (125, 250, 500, and 800
1H), 8.86–8.71 (m, 2H), 8.41 (s, 1H), 7.82 (d, J = 4.7 Hz, 2H), 7.70 (d, J
= 8.3 Hz, 2H), 7.02 (d, J = 8.3 Hz, 2H), 3.80 (s, 3H). 13C NMR (101 MHz,
DMSO-D6) δ 161.49, 161.13, 150.37, 148.99, 140.66, 129.00, 126.61,
123.23, 121.59, 114.44, 55.37. HRMS (ESI) m/z: 256.1083 (Calcd. for
[M + H]+ 256.1081).
µM) for 30 min. 30
(270
μL meropenem was titrated into the sample pool
μ
L) containing NDM-1 in the single injection mode. Meropenem
(E)-N’-(pyridin-4-ylmethylene)isonicotinohydrazone (13)
and NDM-1 concentrations were 100 and 0.1 µM, respectively. The
change of heat flow was recorded over time and the collected data were
treated by MicroCal Analysis Launcher Origin 7 software.
1
White solid, yield 88%. H NMR (400 MHz, DMSO d6) δ 12.35 (s,
1H), 8.80 (d, J = 5.9 Hz, 2H), 8.66 (d, J = 5.8 Hz, 2H), 8.45 (s, 1H), 7.83
(d, J = 5.9 Hz, 2H), 7.69 (d, J = 5.9 Hz, 2H). 13C NMR (101 MHz, DMSO-
D6) δ 162.13, 150.42, 146.71, 141.27, 140.20, 121.69, 121.24. HRMS
(ESI) m/z: 249.0744 (Calcd. for [M + Na]+ 249.0747).
4.7. MIC determination
(E)-N’-(4-hydroxybenzylidene)isonicotinohydrazone (14)
The synergistic antibacterial effect of N-acylhydrazones in combi-
nation with meropenem (MER) was investigated by measuring the
minimum inhibitory concentration (MIC) [45]. The clinical strains are
from the Health Science Center at Xi’an Jiaotong University (Xian,
China). The strain used is E. coli BL21 (DE3), the sequence of NDM-1
expressed is truncated, the promoter is T7, and the Q37-R270
sequence of the enzyme is listed below:
1
White solid, yield 83%. H NMR (400 MHz, DMSO d6) δ 11.90 (s,
1H), 10.02 (s, 1H), 8.77 (d, J = 5.4 Hz, 2H), 8.37 (s, 1H), 7.82 (d, J = 5.4
Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H). 13C NMR
(101 MHz, DMSO-D6) δ 161.41, 159.81, 150.36, 149.45, 140.74,
129.20, 125.06, 121.60, 115.86. HRMS (ESI) m/z: 264.0745 (Calcd. for
[M + Na]+ 264.0743).
(E)-N’-(pyridin-3-ylmethylene)isonicotinohydrazone (15)
QQMETGDQRFGDLVFRQLAPNVWQHTSYLDMPGFGAVASN-
GLIVRDGGRVLVVDTAWTDDQTAQILNWIKQEINLPVALAVV-
THAHQDKMGGMDALHAAGIATYA-
1
White solid, yield 75%. H NMR (400 MHz, DMSO d6) δ 12.25 (s,
1H), 8.89 (s, 1H), 8.80 (d, J = 5.9 Hz, 2H), 8.63 (d, J = 6.1 Hz, 1H), 8.52
(s, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 5.9 Hz, 2H), 7.50 (dd, J =
7.9, 4.8 Hz, 1H). 13C NMR (101 MHz, DMSO-D6) δ 161.80, 151.05,
150.42, 148.98, 146.33, 140.30, 133.65, 129.99, 124.11, 121.59. HRMS
(ESI) m/z: 249.0742 (Calcd. for [M + Na]+ 249.0747).
NALSNQLAPQEGMVAAQHSLTFAANGWVEPA-
TAPNFGPLKVFYPGPGHTSDNITVGIDGTDIAFGG-
CLIKDSKAKSLGNLGDADTEHYAASAR-
AFGAAFPKASMIVMSHSAPDSRAAITHTARMADKLR
The solutions of EC01-EC24, K. pneumonia and E. coli producing
NDM-1 were cultured to OD600 = 0.5. After that, the culture solution was
diluted 84-fold. Under the condition that the final DMSO content was
less than 5%, N-acylhydrazones were dissolved in DMSO and diluted to
4.3. Determination of percent inhibition and IC50
Five MβLs L1, ImiS, IMP-1, VIM-2 and NDM-1 were over-expressed
and purified according to the methods previously reported [51–54].
64
μg/mL. Antibiotic solutions were diluted in a 2-fold gradient to
Meropenem (40
μ
M) was used as the substrate of NDM-1, IMP-1, ImiS
prepare stock solutions of 0.25–128 μg/mL. 100 µL bacterial solution,
and L1, and cefazolin (40
μ
M) was used as the substrate of VIM-2. Under
50 µL MER with different concentrations and 50 µL inhibitors (16 μg/ml)
the condition that the final DMSO content was less than 5%, 10 mM N-
acylhydrazones (1–15) were dissolved in DMSO and diluted to final
concentrations of 0–20 µM to determine the IC50 values. The concen-
tration of N-acylhydrazones used to determine the percent inhibition
was 20 µM. On an Agilent UV8453 spectrometer, the antibiotics hy-
drolysis rates were measured in triplicate, and the average values were
recorded.
were incubated at 37 ◦C for 16–24 h. Each experiment was performed in
triplicate.
4.8. Inhibition zone tests
E. coli producing NDM-1 was inoculated into liquid medium (10 mL,
pH = 7.5). Then, the culture solution was placed in an air bath oscillator
(150 rpm, 37 ◦C) for 5 h. After that, the culture solution was taken out
4.4. Determination of Ki
and inoculated on the solid medium. 10 μL MER, MER + 1 and 1 were
added to three holes drilled on agar plate using Oxford ring, respec-
To identify the inhibition mode of N-acylhydrazone against NDM-1,
kinetic experiments were performed. The concentrations of N-acylhy-
drazone 1 and substrate (meropenem) are 0–2.5 µM (0, 0.25, 0.5, 1 and
2.5 µM) and 10–60 µM (10, 20, 40 and 60 µM), respectively. NDM-1 was
premixed with different concentrations of compound 1 for 30 min.
Under different concentrations of meropenem, the hydrolysis rates of
NDM-1 were determined and draw Lineweaver–Burk plots, and the Ki
value was determined. The obtained experimental data were measured
three times in parallel and fitted by SigmaPlot 12.0.
tively. The concentrations of MER and 1 were 32 and 16 μg/mL,
respectively. The agar plate was cultured in 37 ◦C incubator for 16–18 h,
and the inhibition zone was observed.
4.9. Mice experiments
The mice test was approved by the Animal Medicine Committee of
Xi’an Jiaotong University, and all experimental steps were complied
with a Guideline for Care and Use of Laboratory Animals. A quantitative
standard commercial diet and sterile ultrapure water were given to the
animals. Mice were divided into four groups randomly with six mice in
each group. 150 µL bacterial suspension (OD600 = 0.8) was injected into
Kunming mice intraperitoneally. At 2 h after infection, MHB, 2 mg/kg N-
acylhydrazone 1, 10 mg/kg meropenem, and 2 mg/kg N-acylhydrazone
1 combined with 10 mg/kg meropenem were intraperitoneally injected
into the infected mice groups, respectively. After 24 h infection, all
experimental mice were sacrificed, 0.1 g spleen and liver were taken
4.5. Determination of Zn(II) content
In the experiment to determine the Zn(II) content of the enzyme after
premixing with the inhibitors, the concentration of NDM-1, N-acylhy-
drazone 1 and EDTA was 20 µM. Before dialysis, NDM-1 was incubated
with inhibitors for 1 h [14]. After that, the premixed solution was added
into the MD34-14–5 cut-off dialysis tube (8–14 kDa), and dialyzed at
9

H. Gao et al.
Bioorganic Chemistry 114 (2021) 105138
[12] C. Chen, Y. Xiang, K.W. Yang, Y.J. Zhang, W.M. Wang, J.P. Su, Y. Ge, Y. Liu,
A protein structure-guided covalent scaffold selectively targets the B1 and B2
subclass metallo-beta-lactamases, Chem. Commun. 54 (2018) 4802–4805.
[13] S. Liu, L. Jing, Z.J. Yu, C.Y. Wu, Y.X. Zheng, E. Zhang, Q. Chen, Y.M. Yu, L. Guo,
Y. Wu, G.B. Li, ((S)-3-Mercapto-2-methylpropanamido)acetic acid derivatives as
metallo-beta-lactamase inhibitors: Synthesis, kinetic and crystallographic studies,
Eur. J. Med. Chem. 145 (2018) 649–660.
from each group and homogenized in 1 mL sterile PBS. Organ homog-
enates were serially diluted in PBS, and plated on LB-agar for colony
count.
4.10. Cytotoxicity assay
[14] P. Hinchliffe, M.M. Gonzalez, M.F. Mojica, J.M. Gonzalez, V. Castillo, C. Saiz,
M. Kosmopoulou, C.L. Tooke, L.I. Llarrull, G. Mahler, R.A. Bonomo, A.J. Vila,
J. Spencer, Cross-class metallo-beta-lactamase inhibition by bisthiazolidines
reveals multiple binding modes, Proc. Natl. Acad. Sci. U. S. A. 113 (2016)
E3745–E3754.
A cytotoxicity assay was performed to evaluate the toxicity of N-
acylhydrazones 1 and 2 on Vero E6 cells. The density of the cell sus-
pension was diluted to 1.0 × 105 cells/mL. 100
μL of the same density
[15] J. Brem, S.S. van Berkel, D. Zollman, S.Y. Lee, O. Gileadi, P.J. McHugh, T.R. Walsh,
M.A. McDonough, C.J. Schofield, Structural basis of metallo-beta-lactamase
inhibition by captopril stereoisomers, Antimicrob. Agents Chemother. 60 (2016)
142–150.
cell suspension was added to 96-well plates and cultured for 24 h. After
that, N-acylhydrazones 1 and 2 at concentrations of 10, 25, 50, 100, 200,
and 400 μM were added to 96-well plates, respectively, and cultured for
24 h. The cell viability was investigated using six wells of suspension
cells contained only in a mixture of 1 L of DMSO and 99 L of complete
medium as controls. Three wells containing only the complete medium
were used as blank controls. The medium was then discarded, and 10
[16] M.I. Abboud, M. Kosmopoulou, A.P. Krismanich, J.W. Johnson, P. Hinchliffe,
J. Brem, T.D.W. Claridge, J. Spencer, C.J. Schofield, G.I. Dmitrienko,
Cyclobutanone Mimics of Intermediates in Metallo-beta-Lactamase Catalysis,
Chem. – Eur. J. 24 (2018) 5734–5737.
μ
μ
μL
[17] D. Zhang, M.S. Markoulides, D. Stepanovs, A.M. Rydzik, A. El-Hussein, C. Bon,
J. Kamps, K.D. Umland, P.M. Collins, S.T. Cahill, D.Y. Wang, F. von Delft, J. Brem,
M.A. McDonough, C.J. Schofield, Structure activity relationship studies on
rhodanines and derived enethiol inhibitors of metallo-beta-lactamases, Bioorg.
Med. Chem. 26 (2018) 2928–2936.
of CCK8 and 100 μL of fresh medium were added to each well. The 96-
well plates were shaken to dissolve the resulting product after incuba-
tion for 4 h and the absorbance at 490 nm was read on the Microplate
Reader for analysis.
[18] Y. Xiang, C. Chen, W.M. Wang, L.W. Xu, K.W. Yang, P. Oelschlaeger, Y. He,
Rhodanine as a potent scaffold for the development of broad spectrum metallo-
beta-lactamase inhibitors, ACS Med. Chem. Lett. 9 (2018) 359–364.
[19] J. Brem, S.S. van Berkel, W. Aik, A.M. Rydzik, M.B. Avison, I. Pettinati, K.
D. Umland, A. Kawamura, J. Spencer, T.D.W. Claridge, M.A. McDonough, C.
J. Schofield, Rhodanine hydrolysis leads to potent thioenolate mediated metallo-
beta-lactamase inhibition, Nat. Chem. 6 (2014) 1084–1090.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[20] A. Parkova, A. Lucic, A. Krajnc, J. Brem, K. Calvopina, G.W. Langley, M.
A. McDonough, P. Trapencieris, C.J. Schofield, Broad Spectrum beta-Lactamase
Inhibition by a Thioether Substituted Bicyclic Boronate, ACS Infect. Dis. 6 (2020)
1398–1404.
Acknowledgements
[21] O. Lomovskaya, R. Tsivkovski, K. Nelson, D. Rubio-Aparicio, D.X. Sun, M. Totrov,
M.N. Dudley, Spectrum of beta-lactamase inhibition by the cyclic boronate
QPX7728, an ultrabroad-spectrum beta-lactamase inhibitor of serine and metallo-
beta-lactamases: enhancement of activity of multiple antibiotics against isogenic
strains expressing single beta-lactamases, Antimicrob. Agents Chemother. 64
(2020), e00212-20.
This work was supported by the grants (to K.W.Y.) from National
Natural Science Foundation of China (22077100) and Shaanxi Province
International Cooperation Project (2019KW-068).
[22] J. Brem, R. Cain, S. Cahill, M.A. McDonough, I.J. Clifton, J.C. Jimenez-Castellanos,
M.B. Avison, J. Spencer, C.W.G. Fishwick, C.J. Schofield, Structural basis of
metallo-beta-lactamase, serine-beta-lactamase and penicillin-binding protein
inhibition by cyclic boronates, Nat. Commun. 7 (2016) 12406.
Appendix A. Supplementary material
[23] A.U. Khan, L. Maryam, R. Zarrilli, Structure, genetics and worldwide spread of New
Delhi metallo-beta-lactamase (NDM): a threat to public health, BMC Microbiol. 17
(2017) 12.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105138.
[24] T.R. Walsh, Emerging carbapenemases: a global perspective, Int. J. Antimicrob.
Agents 36 (2010) S8–S14.
References
[25] C.R. Nishida, P.R.O. de Montellano, Bioactivation of antituberculosis thioamide
and thiourea prodrugs by bacterial and mammalian flavin monooxygenases, Chem.
Biol. Interact. 192 (2011) 21–25.
[1] K. Bush, G.A. Jacoby, Updated functional classification of beta-lactamases,
Antimicrob. Agents Chemother. 54 (2010) 969–976.
[26] B. Sarkanj, M. Molnar, M. Cacic, L. Gille, 4-Methyl-7-hydroxycoumarin antifungal
and antioxidant activity enhancement by substitution with thiosemicarbazide and
thiazolidinone moieties, Food Chem. 139 (2013) 488–495.
[2] I. Sultan, S. Rahman, A.T. Jan, M.T. Siddiqui, A.H. Mondal, Q.M.R. Haq,
Antibiotics, Resistome and Resistance Mechanisms: A Bacterial Perspective, Front.
Microbiol. 9 (2018) 2066.
[27] V.F.S. Pape, S. Toth, A. Furedi, K. Szebenyi, A. Lovrics, P. Szabo, M. Wiese,
G. Szakacs, Design, synthesis and biological evaluation of thiosemicarbazones,
hydrazinobenzothiazoles and arylhydrazones as anticancer agents with a potential
to overcome multidrug resistance, Eur. J. Med. Chem. 117 (2016) 335–354.
[28] P. Heffeter, V.F.S. Pape, E.A. Enyedy, B.K. Keppler, G. Szakacs, C.R. Kowol,
Anticancer thiosemicarbazones: chemical properties, interaction with iron
metabolism, and resistance development, Antioxid. Redox Signal. 30 (2019)
1062–1082.
[3] M.W. Crowder, J. Spencer, A.J. Vila, Metallo-beta-lactamases: novel weaponry for
antibiotic resistance in bacteria, Acc. Chem. Res. 39 (2006) 721–728.
[4] A. Krajnc, J. Brem, P. Hinchliffe, K. Calvopina, T.D. Panduwawala, P.A. Lang,
J. Kamps, J.M. Tyrrell, E. Widlake, B.G. Saward, T.R. Walsh, J. Spencer, C.
J. Schofield, Bicyclic Boronate VNRX-5133 Inhibits Metallo- and Serine-beta-
Lactamases, J. Med. Chem. 62 (2019) 8544–8556.
[5] Z.X. Hu, G. Periyannan, B. Bennett, M.W. Crowder, Role of the Zn-1 and Zn-2 sites
in metallo-beta-lactamase L1, J. Am. Chem. Soc. 130 (2008) 14207–14216.
[6] G. Garau, I. Garcia-Saez, C. Bebrone, C. Anne, P. Mercuri, M. Galleni, J.M. Frere,
O. Dideberg, Update of the standard numbering scheme for class B beta-lactamases,
Antimicrob. Agents Chemother. 48 (2004) 2347–2349.
[29] K.L. Summers, A structural chemistry perspective on the antimalarial properties of
thiosemicarbazone metal complexes, Mini-Rev. Med. Chem. 19 (2019) 569–590.
[30] J. Murren, M. Modiano, C. Clairmont, P. Lambert, N. Savaraj, T. Doyle, M. Sznol,
Phase I and pharmacokinetic study of triapine, a potent ribonucleotide reductase
inhibitor, administered daily for five days in patients with advanced solid tumors,
Clin. Cancer Res. 9 (2003) 4092–4100.
[7] M.N. Lisa, J. Moran-Barrio, M.F. Guindon, A.J. Vila, Probing the Role of Met221 in
the Unusual Metallo-beta-lactamase GOB-18, Inorg. Chem. 51 (2012)
12419–12425.
[31] S. Attia, J. Kolesar, M.R. Mahoney, H.C. Pitot, D. Laheru, J. Heun, W. Huang,
J. Eickhoff, C. Erlichman, K.D. Holen, A phase 2 consortium (P2C) trial of 3-ami-
nopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) for advanced
adenocarcinoma of the pancreas, Invest. New Drugs 26 (2008) 369–379.
[32] B. Zhao, X.H. Zhang, T.T. Yu, Y. Liu, X.L. Zhang, Y.F. Yao, X.J. Feng, H.M. Liu, D.
Q. Yu, L.Y. Ma, S.S. Qin, Discovery of thiosemicarbazone derivatives as effective
New Delhi metallo-beta-lactamase-1 (NDM-1) inhibitors against NDM-1 producing
clinical isolates, Acta Pharm. Sin. B 11 (2021) 203–221.
[8] P.S. Mercuri, R. Esposito, S. Bletard, S. Di Costanzo, M. Perilli, F. Kerff, M. Galleni,
Mutational Effects on Carbapenem Hydrolysis of YEM-1, a New Subclass B2
Metallo-beta-Lactamase from Yersinia mollaretii, Antimicrob. Agents Chemother.
64 (2020), e00105-20.
[9] T. Christopeit, T.J.O. Carlsen, R. Helland, H.K.S. Leiros, Discovery of novel
inhibitor scaffolds against the Metallo-beta-lactamase VIM-2 by Surface Plasmon
Resonance (SPR) based fragment screening, J. Med. Chem. 58 (2015) 8671–8682.
[10] A.L.Y. Chen, P.W. Thomas, A.C. Stewart, A. Bergstrom, Z.S. Cheng, C. Miller, C.
R. Bethel, S.H. Marshal, C.V. Credille, C.L. Riley, R.C. Page, R.A. Bonomo, M.
W. Crowder, D.L. Tierney, W. Fast, S.M. Cohen, Dipicolinic acid derivatives as
inhibitors of New Delhi metallo-beta-lactamase-1, J. Med. Chem. 60 (2017)
7267–7283.
[33] O.A. Chaves, M.R.D. Santos, M.C.C. de Oliveira, C.M.R. Sant’Anna, R.C. Ferreira,
A. Echevarria, J.C. Netto-Ferreira, Synthesis, tyrosinase inhibition and
transportation behavior of novel beta-enamino thiosemicarbazide derivatives by
human serum albumin, J. Mol. Liq. 254 (2018) 280–290.
[34] M.F. El Shehry, S.Y. Abbas, A.M. Farrag, S.I. Eissa, S.A. Fouad, Y.A. Ammar,
Design, synthesis and biological evaluation of quinoxaline N-propionic and O-
[11] J.C. Chiou, S.B. Wan, K.F. Chan, P.K. So, D.D. He, E.W.C. Chan, T.H. Chan, K.
Y. Wong, J. Tao, S. Chen, Ebselen as a potent covalent inhibitor of New Delhi
metallo-beta-lactamase (NDM-1), Chem. Commun. 51 (2015) 9543–9546.
10

H. Gao et al.
Bioorganic Chemistry 114 (2021) 105138
propionic hydrazide derivatives as antibacterial and antifungal agents, Med. Chem.
[44] J.B. Baell, J.W.M. Nissink, Seven Year Itch: Pan-Assay Interference Compounds
(PAINS) in 2017-Utility and Limitations, ACS Chem. Biol. 13 (2018) 36–44.
[45] Y. Liu, C. Chen, L.Y. Sun, H. Gao, J.B. Zhen, K.W. Yang, Meta-substituted
benzenesulfonamide: a potent scaffold for the development of metallo-beta-
lactamase ImiS inhibitors, RSC Med. Chem. 11 (2020) 259–267.
Res. 27 (2018) 2287–2296.
[35] A.K.A. Kumar, Y.D. Bodke, G. Sambasivam, P.S. Lakra, Design, synthesis, and
evaluation of novel hydrazide hydrochlorides of 6-aminopyrazolo 1,5-a
pyrimidine-3-carboxamides as potent Aurora kinase inhibitors, Monatshefte für
Chemie – Chem. Month. 148 (2017) 1767–1780.
[46] A.M. Hansen, G. Bonke, W.F.J. Hogendorf, F. Bjorkling, J. Nielsen, K.T. Kongstad,
D. Zabicka, M. Tomczak, M. Urbas, P.E. Nielsen, H. Franzyk, Microwave-assisted
solid-phase synthesis of antisense acpP peptide nucleic acid-peptide conjugates
active against colistin- and tigecycline-resistant E-coli and K. pneumoniae, Eur. J.
Med. Chem. 168 (2019) 134–145.
[36] W. Ahmed, M. Rani, I.A. Khan, A. Iqbal, K.M. Khan, M.A. Haleem, M.K. Azim,
Characterisation of hydrazides and hydrazine derivatives as novel aspartic protease
inhibitors, J. Enzyme Inhib. Med. Chem. 25 (2010) 673–678.
[37] J.W. Liang, S.L. Li, S. Wang, W.Q. Li, F.H. Meng, Synthesis and biological
evaluation of novel (E)-N’-benzylidene hydrazides as novel c-Met inhibitors
through fragment based virtual screening, J. Enzyme Inhib. Med. Chem. 35 (2020)
468–477.
[47] C. Cheng, L.Y. Sun, H. Gao, P.W. Kang, J.Q. Li, J.B. Zhen, K.W. Yang, Identification
of Cisplatin and Palladium(II) Complexes as Potent Metallo-beta-lactamase
Inhibitors for Targeting Carbapenem-Resistant Enterobacteriaceae, ACS Infect. Dis.
6 (2020) 975–985.
[38] M. Artico, F. Corelli, S. Massa, G. Stefancich, L. Avigliano, O. Befani, G. Marcozzi,
S. Sabatini, B. Mondovi, Inhibition of copper-dependent amine oxidases by some
hydrazides of pyrrol-1-ylbenzoic and pyrrol-1-ylphenylacetic acids, J. Med. Chem.
31 (1988) 802–806.
[48] J.P. Su, J.Y. Liu, C. Chen, Y.J. Zhang, K.W. Yang, Ebsulfur as a potent scaffold for
inhibition and labelling of New Delhi metallo-beta-lactamase-1 in vitro and in vivo,
Bioorg. Chem. 84 (2019) 192–201.
[39] M.M. Shaaban, H.M. Ragab, K. Akaji, R.P. McGeary, A.A. Bekhit, W.M. Hussein, J.
L. Kurz, B.H. Elwakil, S.A. Bekhit, T.M. Ibrahim, M.A. Mahran, A.A. Bekhit, Design,
synthesis, biological evaluation and in silico studies of certain aryl sulfonyl
hydrazones conjugated with 1,3-diaryl pyrazoles as potent metallo-beta-lactamase
inhibitors, Bioorg. Chem. 105 (2020), 104386.
[49] L. Zhai, Y.L. Zhang, J.S. Kang, P. Oelschlaeger, L. Xiao, S.S. Nie, K.W. Yang,
Triazolylthioacetamide: A Valid Scaffold for the Development of New Delhi
Metallo-beta-Lactmase-1 (NDM-1) Inhibitors, ACS Med. Chem. Lett. 7 (2016)
413–417.
[50] J.Q. Li, L.Y. Sun, Z. Jiang, C. Chen, H. Gao, J.Z. Chigan, H.H. Ding, K.W. Yang,
Diaryl-substituted thiosemicarbazone: A potent scaffold for the development of
New Delhi metallo-β-lactamase-1 inhibitors, Bioorg. Chem. 107 (2021), 104576.
[51] H.Z. Gao, Q. Yang, X.Y. Yan, Z.J. Wang, J.L. Feng, X. Yang, S.L. Gao, L. Feng,
X. Cheng, C. Jia, K.W. Yang, Exploring antibiotic resistant mechanism by
microcalorimetry, J. Therm. Anal. Calorim. 107 (2012) 321–324.
[40] S. Siemann, D.P. Evanoff, L. Marrone, A.J. Clarke, T. Viswanatha, G.I. Dmitrienko,
N-arylsulfonyl hydrazones as inhibitors of IMP-1 metallo-beta-lactamase,
Antimicrob. Agents Chemother. 46 (2002) 2450–2457.
[41] L. Gavara, F. Verdirosa, A. Legru, P.S. Mercuri, L. Nauton, L. Sevaille, G. Feller,
D. Berthomieu, F. Sannio, F. Marcoccia, S. Tanfoni, F. De Luca, N. Gresh,
M. Galleni, J.-D. Docquier, J.-F. Hernandez, 4-(N-Alkyl- and -Acyl-amino)-1,2,4-
triazole-3-thione Analogs as Metallo-β-Lactamase Inhibitors: Impact of 4-Linker on
Potency and Spectrum of Inhibition, Biomolecules 10 (2020) 1094.
[42] A.M. King, S.A. Reid-Yu, W.L. Wang, D.T. King, G. De Pascale, N.C. Strynadka, T.
R. Walsh, B.K. Coombes, G.D. Wright, Aspergillomarasmine A overcomes metallo-
beta-lactamase antibiotic resistance, Nature 510 (2014) 503–506.
[43] Y.J. Zhang, W.M. Wang, P. Oelschlaeger, C. Chen, J.E. Lei, M. Lv, K.W. Yang, Real-
Time Monitoring of NDM-1 Activity in Live Bacterial Cells by Isothermal Titration
Calorimetry: A New Approach To Measure Inhibition of Antibiotic-Resistant
Bacteria, ACS Infect. Dis. 4 (2018) 1671–1678.
[52] X. Yang, L. Feng, K.Z. Xu, H.Z. Gao, C. Jia, C.C. Liu, J.M. Xiao, L. Zhai, L.S. Zhou, K.
W. Yang, Exploring antibiotic resistant mechanism by microcalorimetry II
Determination of thermokinetic parameters of imipenem hydrolysis with metallo-
beta-lactamase ImiS, J. Therm. Anal. Calorim. 110 (2012) 945–948.
[53] Y.L. Zhang, K.W. Yang, Y.J. Zhou, A.E. LaCuran, P. Oelschlaeger, M.W. Crowder,
Diaryl-Substituted Azolylthioacetamides: Inhibitor Discovery of New Delhi
Metallo-beta-Lactamase-1 (NDM-1), ChemMedChem 9 (2014) 2445–2448.
[54] T. Christopeit, K.W. Yang, S.K. Yang, H.K.S. Leiros, The structure of the metallo-
beta-lactamase VIM-2 in complex with a triazolylthioacetamide inhibitor, Acta
Crystall., Sect. F: Struct. Biol. Commun. 72 (2016) 813–819.
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