Diaryl-substituted thiosemicarbazone: A potent scaffold for the development of New Delhi metallo-β-lactamase-1 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104576

The superbug infection caused by New Delhi metallo-β-lactamase (NDM-1) has become an emerging public health threat. Inhibition of NDM-1 has proven challenging due to its shuttling between pathogenic bacteria. A potent scaffold, diaryl-substituted thiosemicarbazone, was constructed and assayed with metallo-β-lactamases (MβLs). The obtained twenty-six molecules specifically inhibited NDM-1 with IC₅₀ 0.038–34.7 μM range (except 1e, 2e, and 3d), and 1c is the most potent inhibitor (IC₅₀ = 0.038 μM). The structure-activity relationship of synthetic thiosemicarbazones revealed that the diaryl-substitutes, specifically 2-pyridine and 2-hydroxylbenzene improved inhibitory activities of the inhibitors. The thiosemicarbazones exhibited synergistic antimycobacterial actions against E. coli-NDM-1, resulted a 2–512-fold reduction in MIC of meropenem, while 1c restored 16–256-, 16-, and 2-fold activity of the antibiotic on clinical isolates ECs, K. pneumonia and P. aeruginosa harboring NDM-1, respectively. Also, mice experiments showed that 1c had a synergistic antibacterial ability with meropenem, reduced the bacterial load clinical isolate EC08 in the spleen and liver. This work provided a highly promising scaffold for the development of NDM-1 inhibitors.

Full text of DOI:10.1016/j.bioorg.2020.104576

Bioorganic Chemistry 107 (2021) 104576
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Diaryl-substituted thiosemicarbazone: A potent scaffold for the
development of New Delhi metallo-β-lactamase-1 inhibitors
Jia-Qi Lia, Le-Yun Suna, Zhihui Jiangb, Cheng Chena, Han Gaoa, Jia-Zhu Chigana,
,
*
Huan-Huan Dinga, 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 Department of Pharmacy, General Hospital of Southern Theatre Command of PLA, Guangzhou 510010, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The superbug infection caused by New Delhi metallo-β-lactamase (NDM-1) has become an emerging public
health threat. Inhibition of NDM-1 has proven challenging due to its shuttling between pathogenic bacteria. A
potent scaffold, diaryl-substituted thiosemicarbazone, was constructed and assayed with metallo-β-lactamases
(MβLs). The obtained twenty-six molecules specifically inhibited NDM-1 with IC50 0.038–34.7 µM range (except
1e, 2e, and 3d), and 1c is the most potent inhibitor (IC50 = 0.038 µM). The structure-activity relationship of
synthetic thiosemicarbazones revealed that the diaryl-substitutes, specifically 2-pyridine and 2-hydroxylbenzene
improved inhibitory activities of the inhibitors. The thiosemicarbazones exhibited synergistic antimycobacterial
actions against E. coli-NDM-1, resulted a 2–512-fold reduction in MIC of meropenem, while 1c restored 16–256-,
16-, and 2-fold activity of the antibiotic on clinical isolates ECs, K. pneumonia and P. aeruginosa harboring NDM-1,
respectively. Also, mice experiments showed that 1c had a synergistic antibacterial ability with meropenem,
reduced the bacterial load clinical isolate EC08 in the spleen and liver. This work provided a highly promising
scaffold for the development of NDM-1 inhibitors.
Antibiotic resistance
Thiosemicarbazone
Inhibitor
Metallo-β-lactamases
NDM-1
1. Introduction
clinic, such as clavulanic acid, tazobactam, avibactam, and sulbactam
[7,8]. So far, many MβLs inhibitors have been reported, such as β-lactam
β-Lactam antibiotics, as the most important and frequently used
antimicrobial agents, constituting more than 50% of the antibiotics
prescribed worldwide [1]. However, the overuse of antibiotics has
exacerbated the emergence of bacterial resistance and caused a serious
public health threat [2]. Bacteria resist antibiotics in many ways, and the
most important way is the production of β-lactamases, which catalyze
the cleavage of β-lactam rings of the antibiotics [3]. β-Lactamases have
been divided into A-D categories according to the amino acid homolo-
gous sequences [4]. Class A, C and D enzymes are called serine-β-lac-
tamases (SβLs), which use a catalytic mechanism where an active site
serine nucleophilically attacks the β-lactam carbonyl [5]. Class B en-
zymes, also called metallo-β-lactamase (MβL), are divided into B1, B2
and B3 subclasses according to amino acid sequence and Zn(II) content
[6].
analogues [9], thiols [10–12], carboxylic acids [13,14], ebselen [15,16],
rhodanines [17–19], and cyclic boronates [20–22]. However, there is no
MβL inhibitor for clinic purposes to date.
New Delhi metallo-β-lactamase-1 (NDM-1), a B1 subclasses MβL,
hydrolyzes almost all β-lactam antibiotics, even meropenem or imipe-
nem [23]. NDM-1 is plasmid-encoded, which facilitates its rapid trans-
mission and makes inhibiting it a daunting challenge [24]. The clinical
pathogenic bacteria with NDM-1, such as Carbapenem-Resistant
Enterobacteriaceae (CRE), K. pneumonia, and P. aeruginosa, are serious
resistant. Effective inhibitors of NDM-1 have been sought after exten-
sively. Recently, some inhibitors of NDM-1 have been reported [25–28],
including fungal natural product aspergillomarasmine A (AMA) [26],
which restores the antibacterial activity of meropenem against
K. pneumoniae expressing NDM-1, 1,4,7-triazacyclononane-1,4,7-tria-
cetic acid (NOTA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-
acetic acid (DOTA), which exhibits synergistic antibacterial ability
To combat antibiotic resistance, an ideal strategy is to develop
β-lactamase inhibitors. Currently, the SβLs inhibitors have been used in
* Corresponding author.
E-mail address: kwyang@nwu.edu.cn (K.-W. Yang).
https://doi.org/10.1016/j.bioorg.2020.104576
Received 30 September 2020; Received in revised form 16 December 2020; Accepted 16 December 2020
Available online 22 December 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.

J.-Q. Li et al.
Bioorganic Chemistry 107 (2021) 104576
with meropenem on the bacteria harboring NDM-1 [27]. Besides, piro-
indoline-thiadiazole (SIT-Z5) has been reported to be an effective in-
hibitor of NDM-1 [28].
more than 63% inhibitory activities against NDM-1, but less than 35%
on the other MβLs. Therefore, NDM-1 was focused on inhibition studies
for the remainder of this work.
The thiosemicarbazones have been reported to have a wide phar-
macological activity, including antibacterial, antiviral, antiparasitic,
antifungal, antimalarial, and anticancer [29–33]. Recently, several thi-
osemicarbazones have been used for clinical trials to treat cancer (see
Fig. 1). 3-aminopyridinecarboxaldehyde thiosemicarbocyclic ketone (3-
AP, Phase II) irreversibly inhibits ribonucleotide reductase through
metal chelating. [34,35]. Also, di-2-pyridylketone 4-cyclohexyl-4-
methyl-3-thiosemicarbazone (DpC, Phase I) and (E)-N’-(6,7-dihy-
droquinolin-8(5H)-ylidene)-4-(pyridin-2-yl)piperazine-1-carbothiohy-
drazide (COTI-2, Phase I) have been used for the treatment of resistant
cancers and gynecological malignant tumors [36]. Furthermore, the
thiosemicarbazone has been reported to inhibit urease and tyrosinase by
metal complexation [37]. The binding of the thiosemicarbazone to
metal enlightened us that it may be used for the development of the MβL
inhibitors. Our goal is to develop the MβL inhibitors with a new back-
bone and fight resistant bacterial infections by β-lactam antibiotics
combined with these compounds (see Fig. 2).
The inhibitor concentrations causing 50% decrease of enzyme ac-
tivity (IC50) of thiosemicarbazones 1a-1k, 2a-2h, and 3a-3g against
NDM-1 were determined. The substrate (meropenem) concentration is
40 μM. The IC50 data (Table 1) indicated that most thiosemicarbazones
(except 1e, 2e, 3d, 3f, 3g) had strong inhibitory efficacy against NDM-1,
exhibiting an IC50 value in the 0.038–25.7 µM range, and 1c was the
most effective inhibitor (IC50 = 0.038 µM). Interesting is that the 2-pyr-
idine and 2-hydroxyphenyl substituted derivatives had an IC50 in the
range of 0.038–5.48 and 0.12–13.4 µM, respectively, which is smaller
than the IC50 of the 4-hydroxyphenyl substituted compounds
(15.4–34.7 µM), suggesting that the N and OH at ortho-position of
benzene ring bound to Zn(II) at enzymatic active sites. A structur-
e–activity relationship (SAR) was revealed through analysis the IC50
data, which is that the R1 substitutes 2-pyridine and 2-hydroxylbenzene
significantly improved the inhibitory activities of thiosemicarbazones
against NDM-1 as compared to the same substitutes at 4-position, and
the aromatic R2 substitutes make the inhibitors have better activities
than the aliphatic substitutes.
In this work, twenty-six thiosemicarbazones (Fig. 2) were designed,
synthesized, and characterized by 1H and 13C NMR (see supporting in-
formation) and confirmed by HRMS. The inhibitory activities of these
compounds were evaluated with five MβLs NDM-1, VIM-2, IMP-1, ImiS,
L1. The inhibitory mode was investigated by thermodynamics, enzy-
matic kinetics, and inductively coupled mass spectrometry assays [26].
Also, the synergistic antibacterial activity of these thiosemicarbazones
and meropenem against antibiotic-resistant strains were evaluated.
Moreover, the antibacterial activity of 1c combined with meropenem
was also studied in vivo.
Due to the best inhibitory potency of thiosemicarbazone 1c, we
assayed the time- and concentration-dependent inhibitions of it. Fig. 4
showed that the residual activity of NDM-1 decreased with the extension
of the compound incubation with NDM-1, and the thiosemicarbazone
showed the maximum inhibition after incubating with NDM-1 for about
30 min (Fig. 4a). Also, as increasing the thiosemicarbazone concentra-
tions (6.25–500 nM), the residual activity of NDM-1 decreased, indi-
cating that 1c is a time- and concentration-dependent inhibitor of NDM-
1. The time-course of inactivation of NDM-1 by EDTA with a concen-
tration in the range of 0.63–10 µM was assayed. As shown in Fig. 4c, the
inhibition model of EDTA against NDM-1 is similar to that observed with
1c (Fig. 4b). Also, the inhibition curves of NDM-1 by 1c in the presence
of Zn (II) ions (1.0 µM) were assayed. The concentrations of 1c ranged
from 6.25 to 500 nM. As shown in Fig. 4d, the inhibitory activity of the
compound on NDM-1 was affected by addition of the metals, implying
that 1c inhibits NDM-1 by removing the Zn (II) ions at active sites of the
enzyme.
2. Results and discussion
2.1. Synthesis of thiosemicarbazones
Primary amines reacted with CS2 under basic condition overnight,
iodine was added and stirred for 30 min to give thiocyanates. In iso-
propanol, thiocyanates further reacted with hydrazine hydrate to form
thiosemicarbazides, which reacted with aldehydes at 100 ◦C for 2 h to
offer the final products thiosemicarbazones 1a-1k, 2a-2h, and 3a-3g.
The primary amines chosen have aromatics and aliphatics, including
ethylamine, cyclohexylamine, N-butylamine, isobutylamine, aniline, o-
toluidine, p-toluidine, 4-butylbenzenamine, 4-chlorobenzenamine, 4-
methoxybenzenamine, and 2-ethylbenzenamine.
To further study the inhibition mechanism of thiosemicarbazone 1c
against NDM-1, dose-dependent, dialysis, Zn(II) supplement, and Zn(II)
content determination experiments were performed. In the dose-
dependent experiments, the concentration of meropenem is 40 µM,
concentrations of 1c are in the range of 0–1.0 µM. The concentration of
Zn(II) ions in the deionized water used in the dialysis experiments was
determined by ICP-MS to be <0.001 nM. As shown in Fig. 5a, the in-
crease of 1c concentration resulted in the decrease of residual activity of
NDM-1, when 1c concentration is up to 1.0 µM, the residual activity is
less than 5%, revealing that the thiosemicarbazone is a dose-dependent
inhibitor of NDM-1. For the dialysis experiments, NDM-1 and 1c (10 µM)
were incubated for 1 h in advance, the processed samples (2 mL) were
put into a 10 kDa cut-off dialysis bag and dialyzed with deionized water
for 24 h, and the residual activities of NDM-1 were determined. The
result (Fig. 5b) shows that the activity of NDM-1 does not change after
dialysis. Afterward, the NDM-1 sample after dialysis was incubated with
ZnCl2 (1–100 µM) for 30 min, and the residual activity of NDM-1 was
2.2. Activity evaluation
To investigate whether the synthetic thiosemicarbazones were the
inhibitors of MβLs, the activity evaluation was conducted on an Agilent
UV8453 spectrometer using meropenem (40 µM, monitoring at 300 nm)
as substrate. The thiosemicarbazones tested concentrations ranged from
0 to 80 µM.
Percent inhibition of thiosemicarbazones on MβLs NDM-1, VIM-2,
IMP-1, ImiS, and L1 using meropenem (40 µM) as substrate. Fig. 3
showed that the 2-pyridine substituted thiosemicarbazones exhibited
Fig. 1. Structures of thiosemicarbazones.
2

J.-Q. Li et al.
Bioorganic Chemistry 107 (2021) 104576
Fig. 2. Structures of synthetic thiosemicarbazones.
Fig. 3. Percent inhibition of thiosemicarbazones (5 µM) on MβLs NDM-1, VIM-2, IMP-1, and L1 using meropenem (40 µM) as substrate.
assayed. As shown in Fig. 5c, the concentration of Zn(II) increased, the
activity of NDM-1 was restored, confirming that the thiosemicarbazone
inhibits NDM-1 through metal chelating.
thiosemicarbazones specifically inhibit NDM-1.
2.3. Inhibition assay of NDM-1 by ITC
Furthermore, the dialyzed NDM-1 sample was acidized with
concentrated nitric acid, and the Zn(II) content in protein was deter-
mined by inductively coupled mass spectrometry (ICP-MS). The con-
centrations of 1c are in the range of 0–20 µM. The results (Fig. 5d)
revealed that NDM-1 was inactivated by 1c and lose 1.2 equivalents of
Zn(II) compared with the control group, suggesting that 1c chelated out
the Zn(II) in the active site of NDM-1, therefore exhibited inhibitory
activity. Meanwhile, the percent inhibition of 1c, 2c, 3b and EDTA
against four MβLs was determined. As shown in Fig. 5e, unlike EDTA,
The thermodynamic inhibition of 1c on NDM was investigated by
isothermal titration calorimetry (ITC) on MicroCal-ITC200 in single in-
jection mode at 25 ◦C [38]. The concentrations of NDM-1 and mer-
openem were 0.1 and 500 µM, respectively. NDM-1 and different
concentrations of 1c (1, 2.5, 5, and 10 µM) were incubated for 1 h in
advance. A single 38
containing sample cell (210
meropenem hydrolysis with NDM-1 by 1c is shown in Fig. 6. Also,
μ
L of meropenem were titrated into the NDM-1-
μ
L). ITC monitored inhibition progress of
3

J.-Q. Li et al.
Bioorganic Chemistry 107 (2021) 104576
when it is used combined with most thiosemicarbazones (except 3a, 3f,
and 3g), resulted in a 2–512-fold reduction in MICs, while the inhibitors
with aromatic R2 groups exhibited stronger synergistic antibacterial
ability than that with aliphatic R2 substrates, and 1c had the strongest
ability, reflecting the consistency of the ability to inhibit NDM-1.
Moreover, the synergistic antibacterial effect of thiosemicarbazone
and meropenem (MER) against eight clinical pathogenic bacteria pro-
ducing NDM-1 were assayed. The collected MIC data (Table 2B) shown
that 1c exhibited strong activity on all clinical bacteria tested, resulting
in a 512-, 16–128-, 16-, and 2-fold reduction in MIC of meropenem
against E. coli, EC01-EC10, K. pneumonia, and P. aeruginosa, respectively,
confirming that activity response of the antibiotic is due to the inhibition
of thiosemicarbazone to NDM-1 in living bacterial cells.
Table 1
Inhibitory activity (IC50, µM) of thiosemicarbazone derivatives against MβL
NDM-1.
Compd.
IC50
Compd.
IC50
R1 = 2-pyridine R2 = aromatic
R1 = 2-pyridine R2 = aliphatic
1a
1b
1c
1d
1e
1f
0.063
0.51
0.038
0.11
NA
1h
1i
1.53
5.42
2.48
3.56
1j
1k
0.57
0.27
1g
R1 = 2-hydroxyphenyl
2a
2b
2c
2d
6.98
0.47
0.12
0.28
2e
2f
NA
0.42
8.64
13.4
To verify the synergistic antibacterial effect of thiosemicarbazone
and meropenem (MER) against E. coli-NDM-1, the zone of inhibition
experiment was studied. As shown in Fig. 7, the inhibitory zones used in
combination of 1c and meropenem are much larger than that of mer-
openem alone, and also, increasing the amount of inhibitor could
improve the bacteriostatic ability of meropenem.
2g
2h
R1 = 4-hydroxyphenyl
3a
3b
3c
3d
17.8
15.4
19.8
NA
3e
3f
25.7
>80
>80
3g
Given the excellent inhibitory activity of 1c, we investigated the
synergistic effect of 1c and meropenem at different concentrations on
E. coli-NDM-1. The concentrations of MER and 1c are in the range of
0–256 and 0–64 µg/ml, respectively. It can be observed in Fig. 8a that 1c
alone had no inhibition for the growth of bacteria at a concentration up
to 64 µg/mL, but significantly reduced the MIC of MER in combination
with the antibiotic, indicating that thiosemicarbazone restored the
antibacterial activity of MER by inhibiting NDM-1.
aNDM-1 and the thiosemicarbazones (0–80 µM) were preincubated for 1 h. NA:
data is not available.
overlayed heat flow curves of meropenem (50 μM) hydrolysis by NDM-1
(25 nM) in the absence and presence of 1c with different concentrations
(0, 6.25, 25, 200, and 500 nM) are shown in Fig. S1. It can be observed
that with the increase of the thiosemicarbazone concentration, the
thermopower (dq/dt) and the total heat (Q) released (Fig. 6, inset)
decreased gradually, confirming that 1c is a dose-dependent inhibitor of
NDM-1, similar to earlier reported the thermodynamic inhibition of
ebselen on NDM-1 [38].
Next, 1c was chosen to conduct the dose-dependent antibacterial
assay against E. coli-NDM-1 and clinical pathogenic bacteria EC08. It is
observed in Fig. 8b that with the increase of inhibitor concentration, the
MIC values of MER decreased. When the concentration of 1c is up to 64
µg/mL, MIC of MER against E. coli-NDM-1 and EC08 decreased 512 and
256-fold, respectively.
2.4. Antibacterial activity assays in vitro
The sterilization efficiency of 1c in combination with MER was tested
by time-killing kinetics, EC08 was used for the assay, the concentration
of inhibitor and MER was 16 and 32 µg/mL, respectively. It can be
observed in Fig. 8c that MER alone cannot kill EC08, but MER combi-
nation with 1c rapidly killed the bacteria as shown in Fig. 8d.
The minimum inhibitory concentration (MIC) was measured to
investigate the synergistic antibacterial of thiosemicarbazones 1a-1k,
2a-2h, and 3a-3g combined with meropenem against E. coli-NDM-1
[39]. The E. coli BL21 (DE3) cells with and without NDM-1 were used for
assays of the inhibitors (16 μg/mL). The enhancement of the antibac-
terial effect of meropenem on E. coli NDM-1 can be observed in Table 2A
Fig. 4. Time- and concentration-dependent inhibition of NDM-1 by thiosemicarbazone 1c and EDTA. Monitoring of NDM-1 inhibition by 1c (5 µM) for 60 min (a)
and by 1c (6.25–500 nM) for 120 min (b), and by EDTA (0.63–10 nM) for 120 min (c), and by 1c (6.25–500 nM) in the presence of Zn(II) ions for 120 min (d).
4

J.-Q. Li et al.
Bioorganic Chemistry 107 (2021) 104576
Fig. 5. The inhibitory mode of thiosemicarbazone 1c on NDM-1. a, the IC50 curve of 1c inhibits NDM-1; b, dialysis experiments proved that the activity of NDM-1 did
not change after dialysis; c, addition of excess ZnCl2 restores activity of NDM-1 after inactivation; d, depletion of Zn(II) in NDM-1 was confirmed by ICP-MS; e,
percent inhibitions of EDTA, 1c, 2c and 3b (10 µM) on MβLs.
the same time, the inhibitory activity of these compounds against NDM-
1 was affected by Zn(II) ions.
2.6. Cytotoxicity assay
The potential toxicity of enzyme inhibitors is a principal problem for
clinical medical applications. The thiosemicarbazones 1c, 2c, and 3b
with different working concentrations (3.13, 6.25, 12.5, 25, 50, and 100
μ
M) were subjected to a cytotoxicity assay with mouse fibroblast cells
(L929) with previously reported method [39]. As shown in Fig. 10, more
than 60% of the cells tested maintained viability in the presence of the
inhibitors at a concentration of up to 50 µM, suggesting that these thi-
osemicarbazones have certain cytotoxicity.
2.7. Antibacterial activity assays in vivo
The antibacterial activity of thiosemicarbazone 1c in combination
with MER in vivo against clinical pathogenic bacteria EC08, a mice
infection model was established according to the previous method [40].
In short, Kunming mice were infected intraperitoneally with a sublethal
dose of EC08. After 2 h, mice were injected intraperitoneally with a
single dose of the drug. The effects of monotherapy and combination
treatment of 1c and MER on bacterial load in the liver and spleen were
determined. Fig. 11 showed that monotherapy of 1c did not affect
bacterial growth compared with the control, and MER alone slightly
limited bacterial growth in vivo. But the combination therapy of 1c and
meropenem reduced the bacterial load in the liver and spleen of mice.
Antibacterial activity assays in vivo indicated thiosemicarbazone can
cooperate with meropenem to fight the clinical challenge of
carbapenem-resistant pathogens harboring-NDM-1 (such as EC08).
Fig. 6. Overlayed heat flow curves of meropenem (500
μM) hydrolysis by
NDM-1 (0.1
μM) in the absence and presence of thiosemicarbazone 1c at
different concentrations (0, 1, 2.5, 5, and 10 µM).
2.5. Metal suppression experiments
To identify the effect of Zn(II) ions on thiosemicarbazone inhibitory
activity, we performed the metal suppression experiments. We assayed
bacterial growth of E. coli cells harboring NDM-1 following treatment
with EDTA or 1c in the presence of Zn(II) ions. As shown in Fig. 9, when
Zn(II) ions (240 μM), meropenem (MER) (80 μM), the inhibitors (240
μ
M) EDTA or 1c were used alone, a fluctuation of 85–92% of the bac-
3. Conclusions
terial growth rates was obtained. By contrast, when MER was used in
combination with EDTA or 1c, the bacterial growth rate is less than 20%.
While when Zn(II) ions were pre-cultured with the combination of EDTA
or 1c, 83–85% of the bacterial growth rates were obtained. These data
indicate that EDTA or 1c used alone did not affect the growth of bac-
teria, while the combination of these inhibitors with MER inhibited it, at
Twenty-six thiosemicarbazones were synthesized and characterized
by 1H and 13C NMR and HRMS. The biochemical evaluation revealed
that all compounds (except 1e, 2e and 3d) strong inhibit NDM-1, with
an IC50 value in the range of 0.038–5.42, 0.12–13.4, and 15.4–34.7 µM
for the 2-pyridine, 2-hydroxyphenyl, and 4-hydroxyphenyl substituted
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J.-Q. Li et al.
Bioorganic Chemistry 107 (2021) 104576
Table 2
MIC of meropenem combined with thiosemicarbazones (16
μ
g/mL) against E. coli-NDM-1a (A), and meropenem against clinical strains harboring NDM-1, E. coli, K.
pneumonia, P. Aeruginosa, and ECs in the presence of thiosemicarbazone 1c (16
μ
g/mL) (B).
A
Compd.
E. coli-NDM-1
Compd.
E. coli-NDM-1
Compd.
E. coli-NDM-1
MER
1a
1b
1c
128
4
1i
64
64
64
64
32
4
2g
2h
3a
3b
3c
3d
3e
3f
64
1j
64
8
1k
2a
2b
2c
2d
2e
2f
128
64
0.25
4
1d
1e
16
8
64
1f
8
16
16
32
32
1g
1h
8
128
128
16
3g
B
Compd.
E. coli
EC01
EC04
EC07
EC08
EC10
K. pneumonia
P. aeruginosa
MER
128
64
4
32
2
64
1
128
1
128
2
32
2
64
32
MER+1c
0.25
a
The MIC of meropenem alone against E. coli cells without NDM-1 is 0.125
μ
g/mL.
4. Experimental
4.1. Materials
1H and 13C NMR spectra were recorded on a Japan Electronics 400
MHz MRI. Chemical shifts are given in parts per million (ppm) on the
delta scale. 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. Determination of enzyme
inhibitory activity of compounds on Agilent 8453 UV–Vis spectrometer.
4.2. Synthesis of thiosemicarbazones (1a-1k, 2a-2h and 3a-3g)
[41–43]
Primary amine (5 mmol) was added to 15 mL of the mixed solution of
ethanol and water (1:1). Then, CS2 (5 eq) and triethylamine (1 eq) were
added to the solution, and the solution was stirred at room temperature
overnight. Iodine elemental (dissolved in acetonitrile, 1 eq) was slowly
added to the mixture and stirred until yellow precipitated out. After
further reaction for 30 min, the organic solvent was concentrated and
extracted with n-hexane. The organic phase was washed with saturated
brine and dried over anhydrous sodium sulfate. Concentrating on the
organic phase, and 10 mL of isopropanol was added. In an ice bath,
hydrazine hydrate was slowly added to isothiocyanate-containing iso-
propanol and stirred for 10 min until the precipitate was completely
precipitated. The precipitate was filtered and dried under vacuum.
The dried thiosemicarbazone and the corresponding aldehyde (1 eq)
were dissolved into the mixture of water and ethanol (1:1). Pyrrole (0.1
eq) was added to the solution as a catalyst. The solution was refluxed at
100 ◦C for 3–4 h, then cooled to room temperature, and allowed to stand
overnight until crystals precipitated. The precipitated crystals were
filtered and dried to give the product (1a-1k, 2a-2h, and 3a-3g).
Fig. 7. The resistance of E. coli producing NDM-1 were tested by inhibition
zone experiments. Meropenem (MER): MH with meropenem only; MER + 1c
(16 µg/mL), and MER + 1c (64 µg/mL). MER concentration used was 32
μ
g/mL.
molecules, respectively, 1c is the most potent inhibitor (IC50 = 0.038
µM), and 3f and 3g had IC50 values more than 80 µM. Analysis of the IC50
data reveals a structure–activity relationship, which is that the R1 sub-
stitutes 2-pyridine and 2-hydroxylbenzene significantly improved
inhibitory activities of the inhibitors against NDM-1 as compared to the
same substitutes at 4-position, and the aromatic R2 substitutes make the
inhibitors have better activities than the aliphatic substitutes. MIC tests
demonstrated that the thiosemicarbazones restored a 2–512-fold anti-
microbial activity of meropenem on E. coli expressing NDM-1, specif-
ically 1c restored 16–256-, 16-, 2-fold activity of the antibiotic on
clinical isolates ECs, K. pneumonia and P. aeruginosa harboring NDM-1,
respectively. Mice experiments showed that 1c had synergistic anti-
bacterial efficacy with meropenem, reduced the load of clinical bacteria
EC08 in the spleen and liver after a single intraperitoneal dose. These
studies indicated that the thiosemicarbazone is an effective scaffold for
the development of NDM-1 inhibitors.
4.2.1. (E)-4-phenyl-1-(pyridin-2-ylmethylene)thiosemicarbazide (1a)
White solid, yield 74%. 1H NMR (400 MHz, DMSO) δ: 11.99 (s, 1H),
10.21 (s, 1H), 8.55 (d, J = 4.8 Hz, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.17 (s,
1H), 7.81 (td, J = 7.9, 1.5 Hz, 1H), 7.52 (d, J = 8.1 Hz, 2H), 7.42–7.30
(m, 3H), 7.19 (t, J = 7.4 Hz, 1H). 13C NMR (101 MHz, DMSO) δ: 176.50,
153.24, 149.42, 143.15, 139.03, 136.56, 128.19, 126.17, 125.62,
124.32, 120.70. HRMS (ESI) m/z: 279.0689 (Calcd. for [M + Na]+
279.0674).
6

J.-Q. Li et al.
Bioorganic Chemistry 107 (2021) 104576
Fig. 8. a, Representative heat plots of microdilution checkerboard assay for the combination of Meropenem (MER); b, dose-dependent antibacterial assays of the
thiosemicarbazone (0–64 (64 µg/ mL) against EC08 and E. coli-NDM-1; c, time kill kinetics curves for MER (32 μg/ mL) and 1c (64 µg/ mL) monotherapy and
combination therapy against EC08 during a 24 h incubation; d, the picture of the bacterial growth after 24 h.
Fig. 10. Percent cell viability (relative to without thiosemicarbazones of L929
mouse fibroblastic cells in the presence of 1c, 2c, and 3b at concentrations of
3.13, 6.25, 12.5, 25, 50, and 100 µM.
J = 7.3 Hz, 2H), 1.16 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz, DMSO) δ:
177.65, 153.44, 149.35, 142.72, 141.24, 137.55, 136.53, 129.35,
128.38, 127.20, 125.97, 124.19, 120.55, 24.16, 14.28. HRMS (ESI) m/z:
307.1021 (Calcd. for [M + Na]+ 307.0988).
Fig. 9. Growth of E. coli cells-NDM-1 in the presence of MER (80
trol), inhibitors (240 M) EDTA or thiosemicarbazone 1c, combination of MER
with the inhibitors, and the combination with additional Zn(II) ions. Zn(II) ions
were from ZnCl2 (240 M).
μM, as con-
μ
4.2.3. (E)-4-(4-chlorophenyl)-1-(pyridin-2-ylmethylene)thiosemicarbazide
(1c)
μ
White solid, yield 84%. 1H NMR (400 MHz, DMSO) δ 12.11 (s, 1H),
10.29 (s, 1H), 8.59 (d, J = 4.7 Hz, 1H), 8.43 (d, J = 8.0 Hz, 1H), 8.21 (s,
1H), 7.85 (t, J = 7.7 Hz, 1H), 7.61 (d, J = 8.7 Hz, 2H), 7.48–7.36 (m,
3H). 13C NMR (101 MHz, DMSO) δ: 176.52, 153.15, 149.46, 143.48,
138.02, 136.59, 129.64, 128.10, 124.41, 120.75. HRMS (ESI) m/z:
4.2.2. (E)-4-(2-ethylphenyl)-1-(pyridin-2-ylmethylene)thiosemicarbazide
(1b)
White solid, yield 76%. 1H NMR (400 MHz, DMSO) δ: 12.00 (s, 1H),
10.13 (s, 1H), 8.67–8.53 (m, 1H), 8.44 (d, J = 7.5 Hz, 1H), 8.20 (s, 1H),
7.82 (t, J = 7.5 Hz, 1H), 7.43–7.35 (m, 1H), 7.34–7.21 (m, 4H), 2.61 (d,
7

J.-Q. Li et al.
Bioorganic Chemistry 107 (2021) 104576
Fig. 11. The antibacterial activity of thiosemicarbazone 1c in combination with MER against EC08 in vivo. Groups of mice infected by EC08 were treated with a
single dose of meropenem (10 mg/kg), 1c (2 mg/kg), a combination of MER (10 mg/kg) and 1c (2 mg/kg), and EC08 (as control) by intraperitoneal injection.
Bacterial load in the liver (a) and spleen (b) was determined by selective plating.
313.0291 (Calcd. for [M + Na]+ 313.0285).
(q, J = 12.6 Hz, 2H), 1.12 (q, J = 12.4 Hz, 1H).13C NMR (101 MHz,
DMSO) δ: 176.00, 153.30, 149.44, 142.47, 136.58, 124.19, 120.45,
52.98, 31.84, 25.09. HRMS (ESI) m/z: 285.1169 (Calcd. for [M + Na]+
285.1144).
4.2.4. (E)-1-(pyridin-2-ylmethylene)-4-p-tolylthiosemicarbazide (1d)
White needle crystal, yield 82%. 1H NMR (400 MHz, DMSO) δ: 11.98
(s, 1H), 10.18 (s, 1H), 8.58 (d, J = 4.5 Hz, 1H), 8.44 (d, J = 8.1 Hz, 1H),
8.19 (s, 1H), 7.84 (t, J = 8.5 Hz, 1H), 7.45–7.35 (m, 3H), 7.18 (d, J = 8.2
Hz, 2H), 2.32 (s, 3H). 13C NMR (101 MHz, DMSO) δ: 176.55, 153.28,
149.40, 143.00, 136.54, 134.80, 128.66, 126.09, 124.28, 120.68, 20.69.
HRMS (ESI) m/z: 293.0850 (Calcd. for [M + Na]+ 293.0831).
4.2.9. (E)-4-ethyl-1-(pyridin-2-ylmethylene)thiosemicarbazide (1i)
White needle crystal, yield 92%. 1H NMR (400 MHz, DMSO) δ: 11.64
(s, 1H), 8.70 (t, J = 5.8 Hz, 1H), 8.55 (d, J = 4.8 Hz, 1H), 8.25 (d, J = 8.0
Hz, 1H), 8.09 (s, 1H), 7.83 (t, J = 7.7 Hz, 1H), 7.43–7.26 (m, 1H), 3.61
(p, J = 7.0 Hz, 2H), 1.16 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO)
δ: 176.99, 153.44, 149.40, 142.12, 136.51, 124.09, 120.23, 38.47,
14.60. HRMS (ESI) m/z: 231.0700 (Calcd. for [M + Na]+ 231.0675).
4.2.5. (E)-4-(4-butylphenyl)-1-(pyridin-2-ylmethylene)thiosemicarbazide
(1e)
White needle crystal, yield 78%. 1H NMR (400 MHz, DMSO) δ: 11.99
(s, 1H), 10.19 (s, 1H), 8.58 (d, J = 6.0 Hz, 1H), 8.44 (d, J = 7.9 Hz, 1H),
8.19 (s, 1H), 7.84 (t, J = 8.5 Hz, 1H), 7.45–7.30 (m, 3H), 7.19 (d, J = 8.3
Hz, 2H), 2.67–2.53 (m, 2H), 1.56 (p, J = 7.5 Hz, 2H), 1.32 (dt, J = 14.8,
7.3 Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, DMSO) δ:
176.47, 153.30, 149.40, 142.99, 139.75, 136.55, 128.00, 126.09,
124.29, 120.67, 34.44, 33.27, 21.80, 13.88. HRMS (ESI) m/z: 335.1327
(Calcd. for [M + Na]+ 335.1301).
4.2.10. (E)-4-butyl-1-(pyridin-2-ylmethylene)thiosemicarbazide (1j)
White solid, yield 65%. 1H NMR (400 MHz, DMSO) δ: 11.62 (s, 1H),
8.66 (t, J = 5.6 Hz, 1H), 8.56 (d, J = 4.5 Hz, 1H), 8.26 (d, J = 7.9 Hz,
1H), 8.09 (s, 1H), 7.84 (t, J = 7.7 Hz, 1H), 7.40–7.32 (m, 1H), 3.57 (q, J
= 6.6 Hz, 2H), 1.58 (p, J = 7.5 Hz, 2H), 1.32 (h, J = 7.4 Hz, 2H), 0.91 (t,
J = 7.3 Hz, 3H). 13C NMR (101 MHz, DMSO) δ: 177.18, 153.44, 149.39,
142.11, 136.52, 124.09, 120.23, 43.37, 31.04, 19.67, 13.89. HRMS
(ESI) m/z: 237.1167 (Calcd. for [M + H]+ 237.1168).
4.2.6. (E)-1-(pyridin-2-ylmethylene)-4-o-tolylthiosemicarbazide (1f)
White solid, yield 88%. 1H NMR (400 MHz, DMSO) δ: 10.09 (s, 1H),
8.54 (d, J = 4.4 Hz, 1H), 8.39 (d, J = 8.0 Hz, 1H), 8.15 (s, 1H), 7.79 (t, J
= 8.4 Hz, 1H), 7.40–7.28 (m, 1H), 7.28–7.14 (m, 4H), 2.20 (s, 3H). 13C
NMR (101 MHz, DMSO) δ: 177.27, 153.41, 149.37, 142.75, 138.06,
136.55, 135.68, 130.17, 128.90, 126.95, 126.05, 124.21, 120.57, 17.85.
HRMS (ESI) m/z: 293.0852 (Calcd. for [M + Na]+ 293.0831).
4.2.11. (E)-4-isobutyl-1-(pyridin-2-ylmethylene)thiosemicarbazide (1 k)
White solid, yield 56%. 1H NMR (400 MHz, DMSO) δ: 11.64 (s, 1H),
8.66 (t, J = 5.9 Hz, 1H), 8.56 (d, J = 4.9 Hz, 1H), 8.26 (d, J = 8.1 Hz,
1H), 8.10 (s, 1H), 7.84 (td, J = 7.8, 1.6 Hz, 1H), 7.37 (ddd, J = 7.3, 4.9,
0.9 Hz, 1H), 3.44–3.38 (m, 2H), 2.04 (dq, J = 13.6, 6.8 Hz, 1H), 0.89 (d,
J = 6.8 Hz, 7H). 13C NMR (101 MHz, DMSO) δ: 177.55, 153.42, 149.41,
142.18, 136.56, 124.11, 120.24, 50.95, 27.89, 20.19. HRMS (ESI) m/z:
237.1190 (Calcd. for [M + H]+ 237.1168).
4.2.7. (E)-4-(4-methoxyphenyl)-1-(pyridin-2-ylmethylene)
thiosemicarbazide (1 g)
White solid, yield 75%. 1H NMR (400 MHz, DMSO) δ: 11.96 (s, 1H),
10.16 (s, 1H), 8.58 (d, J = 4.4 Hz, 1H), 8.44 (d, J = 8.0 Hz, 1H), 8.18 (s,
1H), 7.83 (t, J = 8.3 Hz, 1H), 7.38 (t, J = 7.8 Hz, 3H), 6.94 (d, J = 8.9 Hz,
2H), 3.77 (s, 3H). 13C NMR (101 MHz, DMSO) δ: 176.86, 157.18,
153.34, 149.41, 142.91, 136.56, 131.92, 127.83, 124.28, 120.68,
113.41, 55.34. HRMS (ESI) m/z: 309.0794 (Calcd. for [M + Na]+
309.0781).
4.2.12. (E)-1-(2-hydroxybenzylidene)thiosemicarbazide (2a)
White solid, yield 95%. 1H NMR (400 MHz, DMSO) δ: 11.38 (s, 1H),
9.87 (s, 1H), 8.37 (s, 1H), 8.11 (s, 1H), 7.91 (d, J = 6.3 Hz, 2H),
7.30–7.13 (m, 1H), 6.94–6.71 (m, 2H). 13C NMR (101 MHz, DMSO) δ:
177.75, 156.53, 139.78, 131.25, 126.88, 120.46, 119.43, 116.17. HRMS
(ESI) m/z: 196.0515 (Calcd. for [M + H]+ 196.0539).
4.2.13. (E)-1-(2-hydroxybenzylidene)-4-(2-ethylphenyl)thiosemicarbazide
(2b)
4.2.8. (E)-4-cyclohexyl-1-(pyridin-2-ylmethylene)thiosemicarbazide (1h)
White solid, yield 88%. 1H NMR (400 MHz, DMSO) δ: 11.64 (s, 1H),
8.56 (d, J = 4.7 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.18 (d, J = 8.5 Hz,
1H), 8.10 (s, 1H), 7.84 (t, J = 7.7 Hz, 1H), 7.40–7.33 (m, 1H), 4.21 (dtt,
J = 12.1, 8.6, 3.8 Hz, 1H), 1.87 (d, J = 10.0 Hz, 2H), 1.73 (d, J = 12.7
Hz, 2H), 1.61 (d, J = 12.2 Hz, 1H), 1.45 (q, J = 1.8, 10.8 Hz, 2H), 1.28
Light yellow solid, yield 72%. 1H NMR (400 MHz, DMSO) δ: 11.72 (s,
1H), 9.89 (s, 2H), 8.48 (s, 1H), 8.06 (d, J = 7.3 Hz, 1H), 7.25 (ddd, J =
24.1, 9.7, 5.0 Hz, 5H), 6.88 (d, J = 8.1 Hz, 1H), 6.82 (t, J = 7.5 Hz, 1H),
2.60 (q, J = 7.5 Hz, 2H), 1.15 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz,
DMSO) δ: 176.99, 156.59, 140.95, 137.69, 131.25, 129.17, 128.33,
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Bioorganic Chemistry 107 (2021) 104576
126.91, 125.87, 119.33, 116.12, 24.19, 14.28. HRMS (ESI) m/z:
300.1157 (Calcd. for [M + H]+ 300.1165).
4.2.21. (E)-1-(4-hydroxybenzylidene)-4-(4-chlorophenyl)
thiosemicarbazide (3b)
White soild, yield 74%. 1H NMR (400 MHz, DMSO) δ: 11.75 (s, 1H),
10.03 (s, 1H), 9.94 (s, 1H), 8.08 (s, 1H), 7.73 (d, J = 8.6 Hz, 2H), 7.64 (d,
4.2.14. (E)-1-(2-hydroxybenzylidene)-4-(4-chlorophenyl)
thiosemicarbazide (2c)
J = 8.8 Hz, 2H), 7.41 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 8.7 Hz, 2H). 13
C
White solid, yield 69%. 1H NMR (400 MHz, DMSO) δ: 11.86 (s, 1H),
10.04 (d, J = 45.0 Hz, 2H), 8.51 (t, J = 9.0 Hz, 1H), 8.19–7.96 (m, 1H),
7.63 (d, J = 7.5 Hz, 2H), 7.41 (d, J = 8.3 Hz, 2H), 7.34–7.13 (m, 1H),
6.87 (dt, J = 15.0, 7.7 Hz, 2H). 13C NMR (101 MHz, DMSO) δ: 175.77,
156.75, 140.50, 138.24, 131.52, 129.24, 127.99, 127.41, 127.16,
120.29, 119.32, 116.15. HRMS (ESI) m/z: 328.0256 (Calcd. for [M +
Na]+ 328.0282).
NMR (101 MHz, DMSO) δ: 175.46, 159.65, 143.76, 138.24, 129.63,
129.20, 127.98, 127.35, 124.98, 115.66. HRMS (ESI) m/z: 328.0258
(Calcd. for [M + Na]+ 328.0282).
4.2.22. (E)-1-(4-hydroxybenzylidene)-4-p-tolylthiosemicarbazide (3c)
White soild, yield 69%. 1H NMR (400 MHz, DMSO) δ: 11.61 (s, 1H),
9.92 (s, 2H), 8.07 (s, 1H), 7.73 (d, J = 8.5 Hz, 2H), 7.44 (d, J = 8.2 Hz,
2H), 7.16 (d, J = 8.1 Hz, 2H), 6.81 (d, J = 8.5 Hz, 2H), 2.30 (s, 3H). 13
C
4.2.15. (E)-1-(2-hydroxybenzylidene)-4-p-tolylthiosemicarbazide (2d)
White solid, yield 74%. 1H NMR (400 MHz, DMSO) δ: 11.72 (s, 1H),
9.98 (s, 2H), 8.50 (d, J = 6.5 Hz, 1H), 8.09 (s, 1H), 7.43 (d, J = 8.0 Hz,
2H), 7.30–7.05 (m, 3H), 6.86 (dt, J = 14.4, 7.6 Hz, 2H), 2.30 (s, 3H). 13C
NMR (101 MHz, DMSO) δ: 175.87, 156.65, 140.05, 136.67, 134.42,
131.36, 128.59, 127.17, 125.76, 120.38, 119.31, 116.12, 20.69. HRMS
(ESI) m/z: 308.0808 (Calcd. for [M + Na]+ 308.0828).
NMR (101 MHz, DMSO) δ: 175.59, 159.54, 143.28, 136.68, 134.39,
129.54, 128.59, 125.73, 125.11, 115.65, 20.71. HRMS (ESI) m/z:
308.0815 (Calcd. for [M + Na]+ 308.0828).
4.2.23. (E)-1-(4-hydroxybenzylidene)-4-(4-butylphenyl)thiosemicarbazide
(3d)
White solid, yield 82%. 1H NMR (400 MHz, DMSO) δ: 11.60 (s, 1H),
9.91 (s, 2H), 8.06 (s, 1H), 7.72 (d, J = 8.6 Hz, 2H), 7.46 (d, J = 8.2 Hz,
2H), 7.17 (d, J = 8.2 Hz, 2H), 6.80 (d, J = 8.6 Hz, 2H), 2.58 (t, J = 7.6
Hz, 2H), 1.57 (p, J = 8.0, 7.6 Hz, 2H), 1.31 (dt, J = 14.3, 7.3 Hz, 2H),
0.91 (d, J = 14.6 Hz, 3H). 13C NMR (101 MHz, DMSO) δ: 175.48,
159.51, 143.22, 139.30, 136.84, 129.50, 127.88, 125.67, 125.08,
115.60, 34.43, 33.29, 21.81, 13.88. HRMS (ESI) m/z: 328.1476 (Calcd.
for [M + H]+ 328.1478).
4.2.16. (E)-1-(2-hydroxybenzylidene)-4-(4-butylphenyl)thiosemicarbazide
(2e)
White solid, yield 82%. 1H NMR (400 MHz, DMSO) δ: 11.73 (s, 1H),
9.98 (s, 2H), 8.50 (s, 1H), 8.24–8.03 (m, 1H), 7.46 (d, J = 8.1 Hz, 2H),
7.30–7.05 (m, 3H), 7.05–6.71 (m, 2H), 2.58 (d, J = 7.1 Hz, 2H), 1.55 (d,
J = 6.8 Hz, 2H), 1.44–1.17 (m, 2H), 0.91 (q, J = 8.2, 7.1 Hz, 3H). 13
C
NMR (101 MHz, DMSO) δ: 175.81, 156.67, 140.02, 139.36, 136.85,
131.35, 127.92, 127.17, 125.69, 120.38, 119.31, 116.12, 34.47, 33.31,
21.84, 13.90. HRMS (ESI) m/z: 328.1465 (Calcd. for [M + H]+
328.1478).
4.2.24. (E)-1-(4-hydroxybenzylidene)-4-o-tolylthiosemicarbazide (3e)
White solid, yield 76%. 1H NMR (400 MHz, DMSO) δ: 11.62 (s, 1H),
9.89 (s, 1H), 9.84 (s, 1H), 8.06 (s, 1H), 7.72 (d, J = 8.7 Hz, 2H),
7.34–7.29 (m, 1H), 7.27 (dd, J = 6.6, 2.3 Hz, 1H), 7.25–7.16 (m, 2H),
6.80 (d, J = 8.7 Hz, 2H), 2.24 (s, 3H). 13C NMR (101 MHz, DMSO) δ:
176.42, 159.45, 142.93, 138.26, 135.42, 130.11, 129.43, 128.73,
126.63, 125.94, 125.24, 115.63, 17.95. HRMS (ESI) m/z: 308.0822
(Calcd. for [M + Na]+ 308.0828).
4.2.17. (E)-1-(2-hydroxybenzylidene)-4-o-tolylthiosemicarbazide (2f)
White solid, yield 76%. 1H NMR (400 MHz, DMSO) δ: 11.73 (s, 1H),
9.90 (s, 2H), 8.48 (s, 1H), 8.08 (d, J = 6.8 Hz, 1H), 7.37–7.09 (m, 5H),
6.94–6.71 (m, 2H), 2.24 (s, 3H). 13C NMR (101 MHz, DMSO) δ: 161.22,
141.18, 122.82, 120.00, 115.85, 114.69, 113.29, 111.71, 111.25,
110.53, 105.05, 103.91, 100.70, 2.50. HRMS (ESI) m/z: 308.0827
(Calcd. for [M + Na]+ 308.0828).
4.2.25. (E)-1-(4-hydroxybenzylidene)-4-cyclohexylthiosemicarbazide (3f)
White solid, yield 85%. 1H NMR (400 MHz, DMSO) δ: 11.23 (s, 1H),
9.88 (s, 1H), 7.97 (s, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.60 (d, J = 8.6 Hz,
2H), 6.80 (d, J = 8.6 Hz, 2H), 4.18 (ddt, J = 15.3, 11.0, 4.7 Hz, 1H),
1.92–1.82 (m, 2H), 1.77–1.66 (m, 2H), 1.60 (d, J = 12.4 Hz, 1H), 1.42
(qd, J = 12.1, 2.8 Hz, 2H), 1.28 (q, J = 12.4 Hz, 2H), 1.13 (q, J = 12.3
Hz, 1H). 13C NMR (101 MHz, DMSO) δ: 175.40, 159.35, 142.64, 129.16,
125.13, 115.64, 52.55, 32.01, 25.26, 25.03. HRMS (ESI) m/z: 278.1340
(Calcd. for [M + H]+ 278.1322).
4.2.18. (E)-1-(2-hydroxybenzylidene)-4-cyclohexylthiosemicarbazide (2g)
White solid, yield 85%. 1H NMR (400 MHz, DMSO) δ: 11.35 (s, 1H),
9.92 (s, 1H), 8.38 (s, 1H), 7.92 (dd, J = 19.8, 8.0 Hz, 2H), 7.22 (t, J = 7.6
Hz, 1H), 6.85 (dd, J = 16.9, 8.1 Hz, 2H), 4.31–4.03 (m, 1H), 1.91–1.10
(m, 10H). 13C NMR (101 MHz, DMSO) δ: 175.64, 156.50, 139.39,
131.14, 126.74, 120.45, 119.30, 116.12, 52.63, 31.94, 25.02. HRMS
(ESI) m/z: 300.1142 (Calcd. for [M + Na]+ 300.1141).
4.2.26. (E)-1-(4-hydroxybenzylidene)-4-ethylthiosemicarbazide (3g)
White solid, yield 82%. 1H NMR (400 MHz, DMSO) δ: 11.23 (s, 1H),
9.86 (s, 1H), 8.37 (d, J = 5.7 Hz, 1H), 7.96 (s, 1H), 7.62 (d, J = 8.6 Hz,
2H), 6.79 (d, J = 8.6 Hz, 2H), 3.58 (p, J = 6.9 Hz, 2H), 1.14 (t, J = 7.1
Hz, 3H). 13C NMR (101 MHz, DMSO) δ: 176.41, 159.29, 142.32, 129.08,
125.31, 115.63, 38.29, 14.82. HRMS (ESI) m/z: 224.0855 (Calcd. for [M
+ H]+ 224.0852).
4.2.19. (E)-1-(2-hydroxybenzylidene)-4-ethylthiosemicarbazide (2h)
White solid, yield 79%. 1H NMR (400 MHz, DMSO) δ: 11.35 (s, 1H),
9.88 (s, 1H), 8.41 (d, J = 30.7 Hz, 2H), 7.93 (d, J = 7.5 Hz, 1H), 7.20 (d,
J = 7.1 Hz, 1H), 7.02–6.68 (m, 2H), 3.66–3.45 (m, 2H), 1.14 (dd, J =
8.7, 5.2 Hz, 3H). 13C NMR (101 MHz, DMSO) δ: 176.58, 156.44, 139.17,
131.09, 126.75, 120.55, 119.30, 116.13, 38.38, 14.76. HRMS (ESI) m/z:
224.0852 (Calcd. for [M + H]+ 224.0852).
4.2.20. (E)-1-(4-hydroxybenzylidene)-4-(2-ethylphenyl)thiosemicarbazide
(3a)
4.3. Determination of IC50 and percent inhibition
White solid, yield 82%. 1H NMR (400 MHz, DMSO) δ: 11.62 (s, 1H),
9.86 (d, J = 23.1 Hz, 2H), 8.06 (s, 1H), 7.72 (d, J = 8.6 Hz, 2H),
7.39–7.11 (m, 4H), 6.80 (d, J = 8.6 Hz, 2H), 2.61 (q, J = 7.5 Hz, 2H),
1.15 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz, DMSO) δ: 176.77, 159.44,
142.92, 140.95, 137.72, 129.40, 128.31, 126.89, 125.86, 125.25,
115.63, 24.20, 14.29. HRMS (ESI) m/z: 322.0976 (Calcd. for [M + Na]+
322.0985).
NDM-1, VIM-2, IMP-1, ImiS, and L1 were over-expressed and puri-
fied. The concentration of the substrate (meropenem) was 40 µM. 10
mM thiosemicarbazones (1a-1k, 2a-2h, and 3a-3g) were dissolved in
DMSO and diluted to final concentration at a range of 0–80 µM (the
content of DMSO after dilution is less than 5%) to determine the IC50
value. The percent inhibition rates were determined at an inhibitor
concentration of 5 µM. Measuring antibiotic hydrolysis rates on the
Agilent UV8453 spectrometer and determine three times in parallel.
9

J.-Q. Li et al.
Bioorganic Chemistry 107 (2021) 104576
4.4. Isothermal titration calorimetry (ITC) assays
4.8. Metal suppression experiments
The thermodynamic inhibition of 1c against NDM-1 was determined
by the ITC experiment on a Malvern MicroCal iTC 200 instruments.
NDM-1 and different concentrations of 1c (1, 2.5, 5, and 10 µM) were
In metal suppression experiments, bacterial growth of E. coli cells
harboring NDM-1 following treatment with EDTA or 1c in the presence
of Zn(II) ions was assayed. The concentration of MER was 80
μM, and
incubated for 1 h in advance. A single 38
μL of meropenem were titrated
concentration of Zn(II) ions and inhibitors was 240 μM. E. coli cells with
into the NDM-1-containing sample cell (210
μL). The final concentration
OD600 of 0.4–0.6 were diluted 84-fold, 3 mL inhibitor (or 3 mL combi-
nation of inhibitor and ZnCl2), 3 mL MER and 6 mL bacteria were mixed
and incubated at 37 ◦C for 16 h. The OD600 values of E. coli cells were
measured on Agilent UV8453 spectrometer. The OD600 value of the
control group is considered to be 100% bacterial growth rate. The
bacterial growth rate of other groups was calculated according to the
measured OD600 values. The average results were plotted as the mean ±
SD of the three experiments.
of NDM-1 is 100 nM. Heat flow was recorded as a function of time and
the experimental data were processed by MicroCal Analysis Launcher
Origin 7 software.
4.5. Determination of MIC
The minimum inhibitory concentration (MIC) of the compounds or
meropenem alone and
a combination of meropenem and thio-
semicarbazones were determined using the broth micro-dilution method
and the Clinical and Laboratory Standards Institute (CLSI) method. The
solutions of E. coli containing NDM-1 and clinical isolates containing
NDM-1 (EC01-EC08, K. pneumonia, and P. aeruginosa) were cultivated to
OD600 = 0.4–0.6 and were diluted 84-fold. The above clinical isolates
are from the General Hospital of the Southern War Zone of the Chinese
people’s Liberation Army and the Health Science Center at Xi’an Jiao-
tong University (Xian, China). Thiosemicarbazones were dissolved in
4.9. Cytotoxicity assay
A cytotoxicity of inhibitors 1c, 2c and 3b to mouse fibro-blast cells
(L929) was assayed. The cells with a density of 1.0 × 104 cells/well in
100 μL of culture medium were seeded into 96-well plates and main-
tained for 24 h. Then solutions of inhibitors 1c, 2c and 3b with work
concentrations (3.13, 6.25, 12.5, 25, 50, and 100 M) were added to 96-
well plates, respectively, and incubated for another 48 h. Six wells
containing only cells suspended in a mixture of 99 L of complete me-
μ
DMSO and diluted to a concentration of 64
than 5%). Antibiotic solutions were prepared as 0.25–128
solutions (dilute in multiples of 2). 50 µL inhibitor (final concentration,
μ
g/mL (DMSO content is less
μ
μg/mL stock
dium and 1 μL of DMSO were used as the control for investigating cell-
viability. Three wells containing only the complete medium were used
as the blank control. Following that, the medium was removed, and 100
16 μg/ml), 50 µL antibiotics with different concentrations (0.25–128 μg/
mL) and 100 µL bacterial solution was mixed and incubated at 37 ◦C for
16–24 h. All experiments were measured in parallel three times.
μ
L of fresh culture medium and 10 μL of CCK8 were added to each well.
After incubation for 4 h, the 96-well plates were then vigorously shaken
to solubilize the formed product and the absorbance at a wavelength of
490 nm was read on a Microplate Reader and analyzed. All experiments
were conducted in triplicate.
4.6. Time-dependent kill assay
To further explore the synergistic antibacterial effect of 1c and
meropenem against clinical bacteria EC08, a time-dependent kill
experiment was studied. In short, bacteria were incubated and diluted to
4.10. Mice experiments
the concentration of ~ 107 CFU/mL. EC08 was treated with 1c (16
μ
g/
g/
The mice experiment was approved by the Animal Medicine Com-
mittee of Xi’an Jiaotong University, and all experimental steps complied
with the Guidelines for Care and Use of Laboratory Animals of Xi’an
Jiaotong University. Animals were given the sterile ultrapure water and
standard commercial diet quantitatively. The mice were randomly
divided into four groups, and male and female were cultured separately,
with six mice in each group. The bacteria were cultured to an OD600
value of 0.8, and 150 µL of bacterial solution was injected into the
abdominal cavity of mice. After infection for 2 h, the mice infected group
were conducted intraperitoneal treatment with MHB, thio-
semicarbazone 1c (2 mg/kg), and meropenem (10 mg/kg), a combina-
tion of meropenem (10 mg/kg) and 1c (2 mg/kg). The weight of liver
and spleen of each mice were kept the same and added with 1 mL of
sterile PBS to homogenate for 3–5 min. The slurries were dilute serially,
placed 10 µL on LB-agar plates and counted the number of colonies.
mL), meropenem (32
mL) and 1c (16
μg/mL) and a combination of meropenem (32
μ
μ
g/mL). At different time intervals (0–24 h), aliquots of
bacterial suspensions were taken to determine the number of bacterial
colonies by agar plates.
4.7. Determination of Zn(II) content
The concentrations of Zn(II) ions in the buffer used in the kinetic
experiments and in the deionized water used in the dialysis experiments,
and the ability of 1c to chelate Zn(II) from purified NDM-1 were
analyzed by ICP-MS. The purified NDM-1 was diluted to a concentration
of 20 µM. Different concentrations of thiosemicarbazone 1c (2.5, 5, 10,
and 20 µM) and NDM-1 were incubated at 4 ◦C for 1 h. The processed
samples (2 mL) were put into a 10 kDa cut-off dialysis bag and dialyzed
with deionized water for 24 h. Deionized water was changed every 6 h
during dialysis. Next, samples were added 5% concentrated nitric acid
and heated for 10 min. Samples were diluted to the measured concen-
tration (NDM-1 at a final concentration of 4 µM) by adding deionized
water. Zn(II) content of processed samples was analyzed by ICP-MS. The
samples tested were transferred by nebulization into the ICP MS spec-
trometer. Quantitative analysis was performed in triplicate for each
sample with 60 sweeps per reading using the peak-hopping mode with a
50 ms/AMU dwell time for each element. Instrument settings were: rf
power (1600 W), integration time (35 s), collision gas (40Ar), RPQ
voltage (25 V) and sample flow rate (4 r.p.m.). Isotope abundance was
determined by integrating peak areas using the NexION software pro-
gram, and the data was represented graphically using Microsoft Excel. A
calibration curve with four standards and a correlation coefficient of
greater than 0.999 was generated using Zn reference solutions.
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.
Acknowledgements
This work was supported by the grants (22077100 and 2019KW-068
to K.W.Y.) from National Natural Science Foundation of China and
Shaanxi Province International Cooperation Project and grant
(201710010013 to Z.H.J.) from the Science and Technology Program of
Guangzhou, China.
10

J.-Q. Li et al.
Bioorganic Chemistry 107 (2021) 104576
beta-lactamases: enhancement of activity of multiple antibiotics against isogenic
strains expressing single beta-lactamases, Antimicrob. Agents Chemother. 64
(2020) e00212–e220.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104576.
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metallo-beta-lactamase, serine-beta-lactamase and penicillin-binding protein
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