Hydroxamic acid with benzenesulfonamide: An effective scaffold for the development of broad-spectrum metallo-β-lactamase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104436

Given that β-lactam antibiotic resistance mediated by metallo-β-lactamases (MβLs) seriously threatens human health, we designed and synthesized nineteen hydroxamic acids with benzenesulfonamide, which exhibited broad-spectrum inhibition against four teste

Full text of DOI:10.1016/j.bioorg.2020.104436

Bioorganic Chemistry 105 (2020) 104436
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Hydroxamic acid with benzenesulfonamide: An effective scaffold for the
development of broad-spectrum metallo-β-lactamase inhibitors
Jia-Qi Li, Cheng Chen, Min Yao, Le-Yun Sun, Han Gao, Jiazhu Chigan, Ke-Wu Yang*
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
A R T I C L E I N F O
A B S T R A C T
Keywords:
Given that β-lactam antibiotic resistance mediated by metallo-β-lactamases (MβLs) seriously threatens human
health, we designed and synthesized nineteen hydroxamic acids with benzenesulfonamide, which exhibited
broad-spectrum inhibition against four tested MβLs ImiS, L1, VIM-2 and IMP-1 (except 6, 13 and 18 on IMP-1,
and 18 on VIM-2), with an IC50 value in the range of 0.6–9.4, 1.3–27.4, 5.4–43.7 and 5.2–49.7 µM, respectively,
and restored antibacterial activity of both cefazolin and meropenem, resulting in a 2–32-fold reduction in MIC of
the antibiotics. Compound 17 shows reversible competitive inhibition on L1 with a Ki value of 2.5 µM and
significantly reduced the bacterial load in the spleen and liver of mice infected by E. coli expressing L1. The
docking studies suggest that 17 tightly binds to the Zn(II) of VIM-2 and CphA by the oxygen atoms of sulfon-
amide group, but coordinates with the Zn(II) of L1 through the oxygen atoms of hydroxamic acid group. These
studies reveal that the hydroxamic acids with benzenesulfonamide are the potent scaffolds for the development
of MβL inhibitors.
Antibiotic resistance
Metallo-β-lactamases
Inhibitor
Benzenesulfonamide
Hydroxamic acid
1. Introduction
covalent mechanisms which can be targeted by β-lactamase inhibitors.
So far, some SβL inhibitors are clinically available to overcome bacterial
Since people discovered penicillin in the 1920s and used it to fight
bacteria [1], the β-lactam antibiotics have been developed as one of the
most commonly and extremely important clinical antibacterial drugs for
the treatment of bacterial infections [2]. However, the overuse of these
antibiotics has exacerbated the production of resistant bacteria, which
has become a critical public health threat [3]. Bacteria resistant to an-
tibiotics in several ways, and the most common way is that bacteria
hydrolyze antibiotics by the production of β-lactamases, which catalyze
the cleavage of β-lactam rings of the antibiotics [2,4]. Due to differences
in amino acid sequence homology, these enzymes have been categorized
into classes A–D [5]. Class A, C and D enzymes are called serine β-lac-
tamases (SβLs). These enzymes use a catalytic mechanism in which the
active site of serine residue nucleophilically attacks the β-lactam
carbonyl group, causing the β-lactam ring fracture. Class B enzymes, also
called as metallo-β-lactamases (MβLs), are Zn(II)-dependent and utilize
Zn(II) ions to catalyze the hydrolysis of the β-lactam ring. Based on
amino acid sequence homology, MβLs are further divided into subclasses
B1, B2 and B3 [6,7].
resistance through the combination with β-lactam antibiotics, such as
clavulanic acid, sulbactam, tazobactam and avibactam [8,9], but there is
no MβL inhibitor available [3]. To prevent the hydrolysis of β-lactam
antibiotics by MβLs, a number of classes of MβL inhibitors have been
reported, such as β-lactam analogues [10], thiols [11,12], carboxylic
acids [13,14], ebselen [15], rhodanines [16], cyclic boronates [17] and
others [18–21]. However, only few reports are on broad-spectrum in-
hibitors of MβLs. The broad-spectrum MβL inhibitor is the compound
that inhibits several MβLs, even MβLs from several subclasses.
Hydroxamic acids have been extensively studied to matrix metal-
loproteinase targets, when combined with Zn(II) ions in the active
centers of these enzymes, causing the enzyme to lose its catalytic activity
[22]. For example, hydroxamic acid (SAHA, vorinostat; Fig. 1A), inhibit
class I and II histone deacetylases (HDACs) by chelating Zn(II) ions at the
active site and SAHA has been approved by Food and Drug Adminis-
tration (FDA) for the treatment of T-cell lymphoma [23,24]. Hydroxa-
mic acids have also been studied for inhibition of some MβLs [25,26].
Recently, we reported that the benzenesulfonamide exhibits strong
enzymatic inhibition activity against ImiS [27]. This enlightened us to
SβLs utilize an active-site serine inactivating the β-lactams in
* Corresponding author.
E-mail address: kwyang@nwu.edu.cn (K.-W. Yang).
https://doi.org/10.1016/j.bioorg.2020.104436
Received 29 June 2020; Received in revised form 10 September 2020; Accepted 26 October 2020
Available online 29 October 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.

J.-Q. Li et al.
Bioorganic Chemistry 105 (2020) 104436
cross linking the hydroxamic acid and sulfonamide group to develop
MβLs inhibitors. Our goal is to develop broad-spectrum MβL inhibitor
and use it in combination with β-lactam antibiotics to fight resistant
bacterial infections.
ImiS, monitoring at 300 nm) and cefazolin (for VIM-2, IMP-1 and L1,
monitoring at 265 nm) as substrates. The concentration of substrate was
40 µM, and the concentrations of inhibitor were in the range of 0–20 µM,
respectively [30–32]. All experimental data were obtained by the Agi-
lent UV8453 spectrometer.
Towards this goal, nineteen hydroxamic acids with benzenesulfo-
namides 1–18 and DSN (shown in Fig. 1) were designed, synthesized,
The synthetic compounds 1–18 were divided into three categories
(Fig. 1), 1–8 m-substituted with 3-phenylsulfonamido, 9–13 o-
substituted with 2-phenylsulfonamido, and 14–18 p-substituted with 4-
phenylsulfonamido. Percent inhibition showed in Fig. S1 indicated that
the hydroxamic acids synthesized exhibited 63–98% inhibition against
ImiS, 14–18 exhibited >70% inhibitory activity on L1, and 17 showed
broad-spectrum inhibition on VIM-2, IMP-1, ImiS and L1, with an in-
hibition rate >70%.
1
characterized by H and 13C NMR and confirmed by HRMS (see Sup-
porting Information). Inhibitory activity of the hydroxamic acids was
evaluated with four MβLs from three subgroups of B1 (VIM-2 and IMP-
1), B2 (ImiS) and B3 (L1), and the inhibitory mode was investigated. The
antibacterial activity of antibiotics in combination with the inhibitors
against drug-resistant bacteria was assayed in vitro (MIC) and in vivo
(mice experiments). Also, the interaction of hydroxamic acids with the
target enzymes was studied by molecular docking.
Hydroxamic acids concentrations causing 50% decrease in enzyme
activity (IC50) were determined. As shown in Table 1, the hydroxamic
acids exhibited broad-spectrum inhibition against four tested MβLs ImiS,
L1, VIM-2 and IMP-1 (except 6, 13 and 18 on IMP-1, and 18 on VIM-2),
with an IC50 value in the range of 0.6–9.4, 1.3–27.4, 5.4–43.7 and
5.2–49.7 µM, respectively. Also, the m- and p-substituted hydroxamic
acids had better inhibitory activities against VIM-2 and L1, respectively,
than the other substituted categories, and 17 was found to be the most
potent inhibitor, with an IC50 value in the range of 1.3–9.9 µM, which is
similar to the data (IC50 = 0.11–9.3 µM) of the benzenesulfonamide
derivatives against ImiS [27]. In addition, for L1, 17 exhibited better
inhibitory activity (IC50 = 1.3 µM) than 12 (IC50 = 7.8 µM), and then
than 4 (IC50 = 12.7 µM). The analysis of the structure–activity rela-
tionship revealed that the m- and p-substituted sulfonyl group improved
the inhibitory activity of the hydroxamic acids against VIM-2 and L1,
respectively.
2. Results and discussion
2.1. Synthesis of hydroxamic acid
The hydroxamic acids with benzenesulfonamides 1–18 and DSN
were synthesized based on two strategies: first, adjust the position (o-, m-
, p-) of phenylsulfonamide relative to N-hydroxybenzamide, to define
the optimal position where hydroxamic acids bind the active site of
MβLs, which confers the best inhibitory effect; and second, adjust
different substituent groups on p-position of phenylsulfonamide to test
the effect on hydroxamic acid inhibitory activity. The hydroxamic acids
were synthesized by previously reported methods [28,29]. Briefly,
aminobenzoic acids reacted with benzenesulfonyl chlorides in K2CO3
aqueous solution, and the resulting mixture was acidified with HCl to
give the benzenesulfonamide benzoic acids [28]. In the presence of the
N, N-carbonyldiimidazole (CDI), the benzoic acids reacted with hy-
droxylamine hydrochloride for 24 h to afford the desired hydroxamic
acids with benzenesulfonamides [29].
Given the broad-spectrum potency of hydroxamic acids against
MβLs, the residual activities of four MβLs in the presence of inhibitor 17
at different concentrations were assayed, the generated inhibition
curves (Fig. 2) shown that the inhibitor exhibited >80% inhibition
(>20% residual activity) against VIM-2, IMP-1, ImiS and L1 at a con-
centration of 10, 40, 80 and 5 µM, respectively.
2.2. Activity evaluation of hydroxamic acids
To test if these hydroxamic acids were broad-spectrum inhibitors of
MβLs, we selected VIM-2, IMP-1, ImiS and L1 as the representatives of
MβLs for the test. The antibiotics were selected to be meropenem (for
2.3. Inhibition mode of hydroxamic acids
Next, compound 17 was selected to determine Ki value and to
Fig. 1. Structures of the synthesized hydroxamic acids (1–18 and DSN) and the reported hydroxamic acid (SAHA).
2

J.-Q. Li et al.
Bioorganic Chemistry 105 (2020) 104436
Table 1
Inhibitory activity (IC50, μM) of hydroxamic acids against four enzymes as the representatives of MβLs.
Compd.
B1
VIM-2a
B2
ImiSb
B3
L1a
Compd.
B1
VIM-2a
B2
ImiSb
B3
L1a
IMP-1a
IMP-1a
m-substituted
o-substituted
1
7.4
7.4
0.9
0.6
0.7
3.2
7.3
2.5
3.7
0.9
6.5
16.7
19.2
16.3
13.6
12.7
15.1
27.4
6.2
9
15.4
17.5
7.45
9.4
14.1
29.9
19.8
47.8
> 50
0.8
0.73
1.2
4.6
7.7
8.3
2
8.4
14.5
5.2
10
19.4
12.8
7.8
3
5.4
11
4
7.8
7.8
12
5
25.4
21.7
11.4
10.1
16.9
> 50
23.1
> 50
49.7
12.5
> 50
> 50
13
43.7
13.4
6
p-substituted
7
14
15
16
17
18
15.4
17.2
9.7
19.8
33.4
32.7
9.8
1.0
0.9
1.6
5.7
9.4
1.8
5.2
6.7
1.3
5.5
8
10.1
9.75
> 50
DSN
SAHA
9.9
> 50
> 50
a
The antibiotic used was cefazolin.
b
The antibiotic used was meropenem.
Fig. 2. Inhibition curves of hydroxamic acid 17 against VIM-2, IMP-1, ImiS,
and L1.
identify the inhibition mode of the hydroxamic acids against MβLs.
Cefazolin as the substrate of L1 for kinetic studies. The concentrations of
substrate and the inhibitor were varied between 20–100 µM and 0–80
µM, respectively. After pre-incubation of the enzyme and inhibitor for 3
h, the hydrolysis rates of substrate were determined in triplicate. The
inhibition mode was assayed by generating Lineweaver–Burk plots, and
Ki values were obtained by fitting initial velocity versus substrate con-
centration at each inhibitor concentration using SigmaPlot 12.0. The
Lineweaver-Burk plots of cefazolin hydrolysis catalyzed by L1 in the
absence and presence of hydroxamic acid 17 are shown in Fig. 3. The
Lineweaver-Burk plots suggest that the compound is a competitive in-
hibitor. The Ki value of 17 was determined to be 2.5 µM.
Fig. 3. Lineweaver–Burk plots of L1 catalyzed hydrolysis of cefazolin in the
○
absence and presence of 17. Inhibitor concentrations were 0 (●), 1.25 ( ), 2.5
(▾), 5 (▽), and 10 µM (■).
N’,N’’-triacetic acid (NOTA) and 17 (final concentration is 50
μM),
respectively, then dialyzed versus specific buffer. After the dialysate was
centrifuged, deionized water was added to make up the solution to the
volume before centrifugation and the residual activity of the enzyme
was determined. It is clearly observed in Fig. 6 that there is no significant
change in enzyme activity before and after treatment with 5% DMSO.
When the inhibitors were EDTA and NOTA, the enzyme activity was not
restored after treatment. However, it is worth noting that the residual
activity of L1 treated by 17 was only 10%, but the activity was restored
to 60% after dialysis and centrifugation, indicating that the inhibition of
17 on L1 is reversible.
We assayed the time-dependent inhibition of the hydroxamic acid on
MβL, and the inhibition curves are shown in Fig. 4. The inhibitory effect
of 17 on L1 reached the maximum (>82% inhibition) at a concentration
of 10 μM in ~250 min (Fig. 4A), and as the inhibitor concentration
increased from 0 to 5 µM, the residual activity of the enzyme decreased
(Fig. 4B), implying that the compound displays a slow-binding or slow
tight-binding mode.
Considering the effect of Zn(II) concentration on the inhibitory
ability of hydroxamic acids with potential metal-chelating motify, in-
hibition tests of VIM-2 and L1 were performed at three different Zn(II)
2.4. Antibacterial activity assays in vitro
concentrations (0, 1 and 100 μM). As shown in Fig. 5, the presence of
additional free Zn(II) ions has little effect on the IC50 value which is in
the range of 0.67–1.32 µM for L1 and 5.50–6.90 µM for VIM-2, and in-
hibitor 17 does not lose its inhibitory ability at a high Zn(II) ions con-
To further study the synergistic antibacterial activity of the
hydroxamic acids, we determined MICs of antibiotics in the absence and
presence of hydroxamic acids (16 µg/mL) as previously reported method
[27]. E. coli-DH10B cells harboring VIM-2, IMP-1, ImiS, or L1 were used
in these assays. The data listed in Table 2A indicated that the
hydroxamic acids tested decreased MICs of cefazolin against E. coli
centration up to 100 μM.
To further study the action mechanism of the hydroxamic acids with
MβL, the L1 enzyme was treated with EDTA, 1,4,7-triazacyclononane-N,
3

J.-Q. Li et al.
Bioorganic Chemistry 105 (2020) 104436
Fig. 4. Time-inhibition curves of L1 by hydroxamic acid 17 at 10 μM (A), and at a concentration between 0.01 and 10 µM (B).
150
100
50
before dialysis
after dialysis
0
7
1
A
A
O
S
T
T
D
O
M
E
N
D
Fig. 6. Residual activity of L1 inhibited by hydroxamic acid 17, NOTA and
EDTA, before and after dialysis and centrifugation. The concentration of in-
hibitors used was 20 µM.
antibacterial effect of cefazolin. At a dose of 128 μg/mL of 14 and 17, the
MICs of antibiotic were reduced 64 and 128 folds, respectively. In
addition, the inhibitor itself does not affect growth of the bacteria, even
at a concentration up to 1024 μg/mL.
The antibacterial ability of antibiotics combined with hydroxamic
acids was further studied by the inhibition zone experiment. It can be
observed in Fig. 7, the combination of cefazolin (CEF) with 14 or 17
resulted in a bigger inhibition area compared to the antibiotic alone, and
the inhibitory area of 17 is slightly larger than that of 14, indicating that
17 had a better effect of restoring antibacterial ability of the antibiotics.
To further probe the action mechanism of hydroxamic acids, metal
suppression experiments were performed. Bacterial growth of E. coli
cells harboring L1 following treatment with NOTA, EDTA or 17 in the
presence of Zn(II) ions was assayed. As shown in Fig. 8, when cefazolin
Fig. 5. The dose-inhibition curves of hydroxamic acid 17 on L1 (A) and VIM-2
(B) obtained at three different Zn(II) ion concentrations (0, 1 and 100 M).
μ
producing L1, and 17 was found to have the largest effect, resulting in a
32-fold reduction in MIC of the antibiotic. 1, 3–4, 7–9, 11 and 16–17
decreased 2-fold MIC of cefazolin on E. coli producing VIM-2 or IMP-1. In
addition to 11–13 and 16–18, other compounds decreased 2–4-fold MIC
of meropenem on E. coli with ImiS. Analysis of these MIC data and the
corresponding IC50 values listed in Table 1 indicates that the inhibitors
7–9, 12 and 14–18 and 6 have synergistic antibacterial effect in com-
bination with antibiotics against the E. coli with L1 and E. coli with ImiS,
respectively.
(CEF) (36 μM), the inhibitors (240 μM) EDTA, NOTA or 17 were used
alone, the bacterial growth rates fluctuated around 95% compared with
the negative control. By contrast, when CEF was used in combination
with EDTA, NOTA or 17, the bacterial growth rate decreased sharply.
While when Zn(II) ions were pre-cultured with the combination of EDTA
or NOTA, the bacterial growth rate rose again and fluctuated around
83%, close to the case that the compounds were used alone. However,
the addition of Zn(II) ions basically did not affect the antibacterial ac-
tivity of CEF in combination with 17. These data indicate that the
growth of bacteria is affected little by the use of these enzyme inhibitors
alone, while the combination of these inhibitors with CEF inhibited the
growth of bacteria effectively. In addition, the activity of these inhibitors
was affected in the presence of additional Zn(II) ions, but did not for
hydroxamic acid 17, implying the different action mechanism.
It is worth noting that the p-substituted hydroxamic acids signifi-
cantly increased the antimicrobial effect of cefazolin against E. coli
expressing L1, resulting in an 8–32-fold reduction in MIC. In view of the
excellent broad-spectrum inhibitory effect of p-substituted compounds,
a dose-dependent MIC assay of 14 and 17 against bacteria expressing L1
was performed. The MIC data listed in Table 2B showed that increasing
the dose of inhibitors would lead to a gradual increase in the
4

J.-Q. Li et al.
Bioorganic Chemistry 105 (2020) 104436
Table 2
MICs of cefazolin and meropenem combined with hydroxamic acids (16 μg/mL) against E. coli Bl21 (EC) producing MβLs (A), and MICs of cefazolin (16 μg/mL)
combined with 14 (0–128
μ
g/mL) or 17 (0–128
μ
g/mL) against the L1 E. coli (B).a
A
Inhibitors
EC-VIM-2b
EC-IMP-1b
EC-ImiSc
EC-L1b
Inhibitors
EC-VIM-2b
EC-IMP-1b
EC-ImiSc
EC-L1b
Blank
128
64
32
16
32
16
16
32
32
32
16
16
32
16
8
8
8
8
8
4
8
8
8
8
32
16
16
16
16
16
16
8
11
64
32
32
32
32
32
16
16
32
32
32
16
16
16
8
16
8
1
2
3
4
5
6
7
8
9
10
B
12
128
128
128
128
128
64
128
64
13
16
2
14
64
15
8
2
128
128
64
16
16
16
16
16
16
2
17
1
18
128
128
128
2
128
128
128
8
DSN
SAHA
32
32
4
16
Compd./
μg/mL
0
4
8
8
16
2
64
1
128
1
14
32
32
17
1
0.5
0.25
a
The MICs of cefazolin and imipenem alone against E. coli cells without MβLs are 1 and 0.125
The antibiotic used was cefazolin.
μ
g/mL, respectively.
b
c
The antibiotics used was meropenem.
Fig. 8. Growth of E. coli cells harboring L1 in the presence of CEF (36
control), inhibitors (240 M) EDTA, NOTA or hydroxamic acid 17, combination
of CEF with the inhibitors, and the combination with additional Zn(II) ions. Zn
(II) ions were from ZnCl2 (240 M).
μM, as
Fig. 7. E. coli cells producing L1 were tested by agar disk diffusion assays. CEF:
MH with cefazolin only; CEF + 14: MH with cefazolin and 14; CEF + 17: MH
with cefazolin and 17. The concentration of antibiotic and inhibitors used was
μ
μ
32 and 128 μg/mL, respectively.
restore cefazolin activity in vivo [27]. In short, the systemic infection
model of Kunming mice was infected intraperitoneally with a sublethal
dose of E. coli BL21 cells harboring L1. After infection of the bacteria for
2 h, a single dose of the drug was injected intraperitoneally, and after 24
h of treatment, the effects of combination and monotherapy treatment of
17 and cefazolin on bacterial load in the liver and spleen were exam-
ined. The assay results are shown in Fig. 10. It can be observed that the
number of colonies treated with the compound alone and the blank
control group are not much different, 17 alone had no effect on bacterial
burden. But compared with cefazolin monotherapy, co-administration
of 17 resulted in a significant reduction of the bacterial load detected
in the liver (Fig. 10A) and spleen (Fig. 10B), indicating that the
hydroxamic acid 17 had synergistic antibacterial effect in vivo.
2.5. Cytotoxicity study
In order to determine the cytotoxicity of hydroxamic acids, we
selected 1, 4, 14 and 17 as representative compounds, and premixed
compounds at different concentrations (6.25, 12.5, 25, 50, 100, and 200
μ
M) with mouse fibroblasts. Fig. 9 shows that even in the case of an
inhibitor concentration of up to 200
μM, >80% of the cells tested
remained viable, indicating that these hydroxamic acids have low
cytotoxicity.
2.6. Antibacterial activity assays in vivo
We selected 17 as a representative compound in combination with
cefazolin to treat infected mice to test whether hydroxamic acids can
5

J.-Q. Li et al.
Bioorganic Chemistry 105 (2020) 104436
atom of the hydroxamic acid group also forms three hydrogen bonds
with Phe156 (2.6 Å), Thr157 (3.4 Å) and Gly160 (1.9 Å), respectively.
For L1/17 (Fig. 11C), the hydroxamic acid acted as Zn(II) binding group
(ZBG), one oxygen atom of it tightly bound to two Zn(II) (1.9 Å for Zn2
and 1.8 Å for Zn1) and forms two hydrogen bonds with His121 (3.2 Å)
and His116 (3.2 Å), while another oxygen atom binds to one Zn(II) (2.5
Å for Zn314) and forms a hydrogen bond with His263 (3.1 Å). These
studies reveal that the relatively broad-spectrum activity of compound
17 stems from an ability to utilizes different binding modes to different
enzyme, the inhibitor utilizes sulfonamide as ZBG to inhibit VIM-2 and
ImiS, but use hydroxamic acid as ZBG to inhibit L1.
The hydroxamic acids 4, 12 and 17 were selected as the represen-
tative of m-, o- and p-substituted derivatives to determine the lowest
interaction energy of the complex (enzyme/inhibitor) by molecular
docking. As shown in Table S1, the lowest binding energy of complexes
of 4, 12, and 17 with VIM-2 (-7.77, ꢀ 7.35, and ꢀ 6.95 kcal/mol), CphA
(-8.29, ꢀ 8.23, and ꢀ 8.03 kcal/mol), and L1 (ꢀ 7.11, ꢀ 7.24, and ꢀ 7.53
kcal/mol) is completely consistent with the corresponding IC50 values of
the inhibitors on three enzymes listed in Table 1. These data support the
structure–activity relationship (SAR) gained by enzyme kinetic assays.
Fig. 9. Percent cell viability (relative to without hydroxamic acid) of L929
mouse fibroblastic cells in the presence of 1, 4, 14 and 17 at concentrations of
6.25, 12.5, 25, 50, 100, and 200 µM.
2.7. Docking study
3. Conclusions
In an effort to explore the binding mode, 17 was selected to dock into
the active sites of VIM-2, CphA (in place of ImiS that has not been
crystallized yet and shares 96% sequence identity with CphA), and L1 as
the representative of B1-B3 subclasses MβLs, and the conformations are
shown in Fig. 11. The lowest interaction energy of VIM-2/17, CphA/17,
and L1/17 complexes was calculated to be ꢀ 6.95, ꢀ 8.04, and ꢀ 7.53
kcal/mol, respectively, indicating that 17 fitted very tightly to the
substrate binding site of MβLs.
In summary, nineteen hydroxamic acids with benzenesulfonamides
1–18 and DSN were synthesized and characterized by 1H and 13C NMR
and HRMS. Biochemical evaluation revealed that hydroxamic acids
exhibited broad-spectrum inhibition against four tested MβLs ImiS, L1,
VIM-2 and IMP-1 (except 6, 13 and 18 on IMP-1, and 18 on VIM-2), with
an IC50 value in the range of 0.6–9.4, 1.3–27.4, 5.4–43.7 and 5.2–49.7
µM, respectively. Analysis of structure–activity relationship revealed
that the m- and p-substituted sulfonyl group improved the inhibitory
activity of the hydroxamic acids against VIM-2 and L1, respectively.
Compound 17 shows reversible competitive inhibition on L1 with a Ki
value of 2.5 µM. The MIC assays indicated that all hydroxamic acids
restored antibacterial activity of both cefazolin and meropenem against
E. coli producing L1, resulting in a 2–32-fold reduction in MIC of the
antibiotics. Mice experiments showed that 17 and cefazolin had syner-
gistic antibacterial efficacy in mice infected by E. coli producing L1 and
significantly reduced the bacterial load in the spleen and liver of mice.
In the complex VIM-2/17 (Fig. 11A), one of oxygen atoms of the
sulfonamide group is coordinated with two Zn(II) ions (2.2 Å for Zn1 and
1.8 Å for Zn2), and binds to His179 (3.1 Å), while another oxygen atom
forms two hydrogen bonds with His179 (3.2 Å) and Asn210 (2.1 Å).
Also, one oxygen atom of hydroxamic acid group forms a hydrogen bond
with Asn148 (1.9 Å). In complex CphA/17 (Fig. 11B), one oxygen atom
of the sulfonamide group is coordinated with Zn(II) (2.3 Å), and another
one binds His196 (1.9 Å) and Asn233 (2.4 Å). In addition, one oxygen
Fig. 10. Hydroxamic acid restores cefazolin activity in vivo. Groups of mice infected with L1 E. coli BL21 cells were treated with a single dose of cefazolin (10 mg/kg),
inhibitor 17 (10 mg/kg), a combination of cefazolin (10 mg/kg) and 17 (10 mg/kg), and E. coli-L1 (as control) by intraperitoneal injection. Bacterial load in the
spleen (A) and liver (B) was determined by selective plating.
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J.-Q. Li et al.
Bioorganic Chemistry 105 (2020) 104436
Fig. 11. Lowest-energy molecular docking conformations of 17 docked into the active catalytic site of VIM-2 (11A), CphA (11B) and L1 (11C). The enzyme backbone
is shown as a cartoon in rainbow, and hydroxamic acid is shown as sticks colored by element (H, white; C, light-blue; N, blue; O, red; S, yellow; Cl, green). The dotted
line indicates the interaction between inhibitor and enzyme residues. All figures were generated with PyMOL. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
The docking studies suggest that the relatively broad-spectrum activity
of 17 stems from an ability to utilizes different binding modes to
different enzyme, the inhibitor utilizes sulfonamide as ZBG to inhibit
VIM-2 and ImiS, but use hydroxamic acid as ZBG to inhibit L1. Cyto-
toxicity tests showed low toxicity of hydroxamic acids to mouse fibro-
blasts L929 cells. These studies demonstrate that the hydroxamic acids
with benzenesulfonamide are the potent scaffolds for the development
of MβLs inhibitors.
from Shanghai Aladdin Biochemical Technology Co., Ltd. Benzene-
sulfonyl chlorides were purchased from SAIN Chemical Technology
(Shanghai) Co., Ltd-Energy Chemical. Analytical Thin Layer Chroma-
tography (TLC) was performed on silica gel GF254 plates and monitored
at an ultraviolet wavelength of 254 nm. 1H and 13C NMR spectra of
compounds were determined on a Bruker Avance III 400 MHz NMR
spectrometer. Chemical shifts are given in parts per million (ppm) on the
delta scale. The peak patterns are reported as singlet (s), doublet (d),
triplet (t), quartet (q), doublet (dd), doublet of doublet of doublets (ddd)
and multiplet (m). The spectra were recorded with TMS as internal
standard. Coupling constants (J) were reported in Hertz (Hz). Mass
spectra were obtained on a micro TOF-Q (BRUKER) mass spectrometer.
Determination of inhibitory activity of compounds on enzyme was
performed on Agilent 8453 UV–Vis spectrometer.
4. Experimental
4.1. Materials
Aminobenzoic acids and N, N-carbonyldiimidazole were purchased
7

J.-Q. Li et al.
Bioorganic Chemistry 105 (2020) 104436
4.2. General procedure for preparation of hydroxamic acids (1–18 and
(Calcd for [M+Na]+ 345.0516 m/z).
DSN) [28,29]
4.2.7. 3-(4-tert-butylphenylsulfonamido)-N-hydroxybenzamide (7)
White solid, yield 63%. 1H NMR (400 MHz, DMSO) δ 11.17 (s, 1H),
10.48 (s, 1H), 9.03 (s, 1H), 7.71 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 11.2 Hz,
3H), 7.42–7.19 (m, 3H), 1.25 (s, 9H). 13C NMR (101 MHz, DMSO) δ
163.86, 156.09, 138.29, 136.86, 134.06, 129.33, 126.61, 126.30,
121.83, 118.54, 34.97, 30.81. HRMS (ESI) m/z: 371.1021 (Calcd for
[M+Na]+ 371.1036 m/z).
The mixed solution of 3-aminobenzoic acid (1 g, 7.3 mmol) and
benzene sulfonyl chloride (1 eq) was slowly added with an aqueous
solution of 1 mol/L Na2CO3 drop-wise until the solution is clear, reacted
at room temperature for 4 h. After the completion of the reaction
monitored through TLC, 1 mol/L HCl was slowly added to the mixture
and stirred until a large amount of precipitated out and the precipitate
was collected [28].
Anhydrous tetrahydrofuran (THF) (5 mL) was added to the mixture
of CDI (4.5 mmol, 1.5 eq) and carboxylic acid (3.0 mmol) and was
stirred for 1 h. Hydroxylamine hydrochloride (417 mg, 6 mmol) was
added and stirred at room temperature overnight (about 16 h). The re-
action mixture was diluted with 5% aq. KHSO4 (30 mL) and extracted
with EtOAc (2 × 30 mL) and brine (30 mL) and washed. The organic
phase was retained and dried with Na2SO4 and concentrated in vacuo to
give the product [29].
4.2.8. 3-(4-bromophenylsulfonamido)-N-hydroxybenzamide (8)
Light orange solid, yield 72%. 1H NMR (400 MHz, DMSO) δ
7.80–7.59 (m, 4H), 7.53 (s, 1H), 7.39 (d, J = 7.1 Hz, 1H), 7.35–7.09 (m,
2H). 13C NMR (101 MHz, DMSO) δ 163.79, 138.80, 137.96, 134.13,
132.59, 129.48, 128.81, 127.08, 122.78, 122.47, 119.35. HRMS (ESI)
m/z: 392.9514 (Calcd for [M+Na]+ 392.9515 m/z).
4.2.9. N-hydroxy-2-(phenylsulfonamido)benzamide (9)
Light orange solid, yield 53%. 1H NMR (400 MHz, DMSO) δ 11.56 (s,
1H), 11.47 (s, 1H), 9.41 (s, 1H), 7.79 (d, J = 7.3 Hz, 2H), 7.63 (t, J = 7.4
Hz, 1H), 7.58–7.51 (m, 3H), 7.51–7.41 (m, 2H), 7.09 (t, J = 8.1 Hz, 1H).
13C NMR (101 MHz, DMSO) δ 165.22, 138.86, 138.13, 133.52, 132.52,
129.58, 127.83, 126.86, 123.60, 119.50, 118.98. HRMS (ESI) m/z:
315.0409 (Calcd for [M+Na]+ 315.0410 m/z).
4.2.1. N-hydroxy-3-(phenylsulfonamido)benzamide (1)
Light orange solid, yield 52%. 1H NMR (400 MHz, DMSO) δ 7.72 (d,
J = 7.1 Hz, 2H), 7.59–7.45 (m, 4H), 7.32–7.14 (m, 3H). 13C NMR (101
MHz, DMSO) δ 163.85, 139.41, 138.09, 134.03, 133.14, 129.43, 129.31,
126.73, 122.41, 122.14, 119.05. HRMS (ESI) m/z: 315.0401 (Calcd for
[M+Na]+ 315.0410 m/z).
4.2.10. N-hydroxy-2-(4-methylphenylsulfonamido)benzamide (10)
White solid, yield 57%. 1H NMR (400 MHz, DMSO) δ 11.55 (s, 1H),
11.42 (s, 1H), 9.40 (s, 1H), 7.67 (d, J = 8.1 Hz, 2H), 7.52 (dd, J = 17.4,
7.9 Hz, 2H), 7.43 (t, J = 7.6 Hz, 1H), 7.34 (d, J = 8.0 Hz, 2H), 7.07 (t, J
= 7.4 Hz, 1H), 2.32 (s, 3H). 13C NMR (101 MHz, DMSO) δ 165.25,
143.96, 138.28, 136.02, 132.50, 130.00, 127.80, 126.91, 123.40,
119.24, 118.71, 21.08. HRMS (ESI) m/z: 329.0539 (Calcd for [M+Na]+
329.0566 m/z).
4.2.2. N-hydroxy-3-(4-methylphenylsulfonamido)benzamide (2)
Light orange solid, yield 72%. 1H NMR (400 MHz, DMSO) δ 11.16 (s,
1H), 10.39 (s, 1H), 9.02 (s, 1H), 7.64 (d, J = 8.3 Hz, 2H), 7.51 (s, 1H),
7.33 (d, J = 7.9 Hz, 3H), 7.30–7.20 (m, 2H), 2.32 (s, 3H). 13C NMR (101
MHz, DMSO) δ 163.90, 143.52, 138.23, 136.59, 134.03, 129.87, 129.30,
126.80, 122.22, 122.00, 118.87, 21.07. HRMS (ESI) m/z: 329.0544
(Calcd for [M+Na]+ 329.0566 m/z).
4.2.3. N-hydroxy-3-(4-nitrophenylsulfonamido)benzamide (3)
Light orange solid, yield 72%. 1H NMR (400 MHz, DMSO) δ 11.19 (s,
1H), 10.79 (s, 1H), 9.04 (s, 1H), 8.37 (d, J = 9.0 Hz, 2H), 8.00 (d, J = 9.0
Hz, 2H), 7.52 (t, J = 1.8 Hz, 1H), 7.41 (dt, J = 7.7, 1.3 Hz, 1H), 7.32 (t, J
= 7.8 Hz, 1H), 7.24 (ddd, J = 8.0, 2.2, 1.1 Hz, 1H). 13C NMR (101 MHz,
DMSO) δ 163.68, 150.02, 144.83, 137.37, 134.19, 129.58, 128.38,
124.86, 123.02, 122.84, 119.57. HRMS (ESI) m/z: 360.0244 (Calcd for
[M+Na]+ 360.0261 m/z).
4.2.11. N-hydroxy-2-(4-nitrophenylsulfonamido)benzamide (11)
White solid, yield 54%. 1H NMR (400 MHz, DMSO) δ 11.46 (s, 2H),
9.37 (s, 1H), 8.34 (d, J = 8.5 Hz, 2H), 8.00 (d, J = 8.5 Hz, 2H), 7.50 (dd,
J = 13.6, 5.4 Hz, 3H), 7.23–7.10 (m, 1H). 13C NMR (101 MHz, DMSO) δ
164.92, 150.14, 144.19, 136.95, 132.55, 128.55, 128.01, 124.88,
124.63, 120.98, 120.80. HRMS (ESI) m/z: 360.0250 (Calcd for
[M+Na]+ 360.0261 m/z).
4.2.12. 2-(4-chlorophenylsulfonamido)-N-hydroxybenzamide (12)
Light orange solid, yield 54%. 1H NMR (400 MHz, DMSO) δ 11.49 (d,
J = 48.1 Hz, 2H), 9.41 (s, 1H), 7.77 (d, J = 7.6 Hz, 2H), 7.61 (d, J = 7.6
Hz, 2H), 7.55 (d, J = 7.0 Hz, 1H), 7.47 (s, 2H), 7.12 (s, 1H). 13C NMR
(101 MHz, DMSO) δ 164.88, 138.20, 137.49, 132.31, 129.51, 128.62,
127.72, 123.79, 119.91, 119.49. HRMS (ESI) m/z: 349.0002 (Calcd for
[M+Na]+ 349.0020 m/z).
4.2.4. 3-(4-chlorophenylsulfonamido)-N-hydroxybenzamide (4)
White solid, yield 55%. 1H NMR (400 MHz, DMSO) δ 11.20 (s, 1H),
10.56 (s, 1H), 9.05 (s, 1H), 7.75 (d, J = 8.5 Hz, 2H), 7.63 (d, J = 8.6 Hz,
2H), 7.52 (s, 1H), 7.39 (d, J = 7.7 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.22
(d, J = 7.8 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 163.78, 138.25,
138.06, 137.79, 134.11, 129.64, 129.45, 128.71, 122.75, 122.50,
119.33. HRMS (ESI) m/z: 349.0003 (Calcd for [M+Na]+ 349.0020 m/z).
4.2.13. 2-(4-acetamidophenylsulfonamido)-N-hydroxybenzamide (13)
White solid, yield 43%. 1H NMR (400 MHz, DMSO) δ 11.44 (d, J =
81.7 Hz, 2H), 10.32 (s, 1H), 9.40 (s, 1H), 7.70 (s, 4H), 7.58–7.39 (m,
3H), 7.07 (s, 1H), 2.05 (s, 3H). 13C NMR (101 MHz, DMSO) δ 169.21,
165.13, 143.59, 138.47, 132.42, 128.96, 128.21, 127.84, 123.29,
119.45, 118.95, 118.73, 24.24. HRMS (ESI) m/z: 372.0645 (Calcd for
[M+Na]+ 372.0625 m/z).
4.2.5. 3-(4-acetamidophenylsulfonamido)-N-hydroxybenzamide (5)
White solid, yield 65%. 1H NMR (400 MHz, DMSO) δ 11.66 (s, 1H),
10.93 (s, 2H), 9.58 (s, 1H), 8.20 (d, J = 11.7 Hz, 4H), 8.02 (s, 1H), 7.86
(d, J = 7.1 Hz, 1H), 7.79 (t, J = 7.3 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H),
2.57 (s, 3H). 13C NMR (101 MHz, DMSO) δ 169.20, 163.88, 143.35,
138.33, 133.98, 132.91, 129.25, 128.04, 122.35, 121.96, 119.00,
118.66, 24.24. HRMS (ESI) m/z: 372.0646 (Calcd for [M+Na]+
372.0625 m/z).
4.2.14. N-hydroxy-4-(phenylsulfonamido)benzamide (14)
1
White solid, yield 73%. H NMR (400 MHz, DMSO) 11.07 (s, 1H),
4.2.6. N-hydroxy-3-(4-methoxyphenylsulfonamido)benzamide (6)
White solid, yield 65%. 1H NMR (400 MHz, DMSO) δ 7.71 (d, J = 8.8
Hz, 2H), 7.53 (s, 1H), 7.35 (d, J = 7.5 Hz, 1H), 7.29 (t, J = 7.7 Hz, 1H),
7.23 (d, J = 8.2 Hz, 1H), 7.05 (d, J = 8.9 Hz, 2H), 3.78 (s, 3H). 13C NMR
(101 MHz, DMSO) δ 162.61, 138.41, 134.03, 131.09, 129.30, 129.02,
122.19, 121.93, 118.85, 114.56, 55.75. HRMS (ESI) m/z: 345.0514
10.71 (s, 1H), 8.99 (s, 1H), 7.84–7.79 (m, 2H), 7.58 (dt, J = 22.8, 7.8 Hz,
5H), 7.15 (d, J = 8.7 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 163.83,
140.52, 139.48, 133.28, 129.54, 128.27, 128.02, 126.80, 118.60. HRMS
(ESI) m/z: 315.0413 (Calcd for [M+Na]+ 315.0410 m/z).
8

J.-Q. Li et al.
Bioorganic Chemistry 105 (2020) 104436
4.2.15. N-hydroxy-4-(4-methylphenylsulfonamido)benzamide (15)
White solid, yield 63%. 1H NMR (400 MHz, DMSO) δ 11.04 (s, 1H),
10.59 (s, 1H), 8.94 (s, 1H), 7.68 (d, J = 7.6 Hz, 2H), 7.60 (d, J = 7.5 Hz,
determined, respectively, using the broth micro-dilution method. The
solutions of E. coli containing MβLs (VIM-2, IMP-1, ImiS and L1) were
cultivated to OD600 = 0.4–0.6, and were diluted 84-fold. Cefazolin was
dissolved in MH medium to prepare 4096, 2048, 1024, 512, 256, 128,
64 and 32 (for E. coli-VIM-2) and 64, 32, 16, 8, 4, 2, 1 and 0.5 µg/mL (for
E. coli-IMP-1 and L1), and meropenem was dissolved in MH medium to
prepare 64, 32, 16, 8, 4, 2, 1 and 0.5 µg/mL (for E. coli-ImiS) stock so-
lutions, respectively. Compounds 1–18 and DSN were dissolved in MH
solution containing 20% DMSO and diluted to a concentration of 64 µg/
mL, 50 µL inhibitor, 50 µL antibiotics with different concentrations and
100uL bacterial solution was mixed and incubated at 37℃ for 16–24 h.
Furthermore, 14 and 17 were prepared 512, 256, 128, 64, 32 and 16 µg/
mL stock solutions, and was diluted to 200 µL with 50 µL antibiotics
solution and 100 µL bacterial solution and incubated for 16–24 h.
2H), 7.35 (d, J = 7.7 Hz, 2H), 7.13 (d, J = 7.5 Hz, 2H), 2.32 (s, 3H). 13
C
NMR (101 MHz, DMSO) δ 163.90, 143.72, 140.65, 136.62, 129.97,
128.27, 127.90, 126.89, 118.46, 21.11. HRMS (ESI) m/z: 329.0550
(Calcd for [M+Na]+ 329.0566 m/z).
4.2.16. N-hydroxy-4-(4-nitrophenylsulfonamido)benzamide (16)
White solid, yield 72%. 1H NMR (400 MHz, DMSO) δ 11.08 (s, 1H),
10.94 (s, 1H), 8.96 (s, 1H), 8.38 (d, J = 8.3 Hz, 2H), 8.04 (d, J = 8.3 Hz,
2H), 7.64 (d, J = 8.2 Hz, 2H), 7.16 (d, J = 8.2 Hz, 2H). 13C NMR (101
MHz, DMSO) δ 163.65, 150.08, 144.77, 139.71, 128.64, 128.41, 124.92,
119.21. HRMS (ESI) m/z: 360.0248 (Calcd for [M+Na]+ 360.0261 m/z).
4.2.17. 4-(4-chlorophenylsulfonamido)-N-hydroxybenzamide (17)
Light orange solid, yield 74%. 1H NMR (400 MHz, DMSO) δ 11.07 (s,
1H), 10.75 (s, 1H), 8.97 (s, 1H), 7.79 (d, J = 7.6 Hz, 2H), 7.64 (t, J = 7.7
Hz, 4H), 7.14 (d, J = 7.7 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 163.51,
139.90, 138.04, 137.94, 129.48, 128.50, 128.09, 118.67. HRMS (ESI)
m/z: 348.9999 (Calcd for [M+Na]+ 349.0020 m/z).
4.6. Cytotoxicity assays
A cytotoxicity of inhibitors 1, 4, 14 and 17 to mouse fibro-blast cells
(L929) was assayed [16]. The cells with a density of 1.0 × 104 cells/well
in 100
μL of culture medium were seeded into 96-well plates and
maintained for 24 h. Then solutions of inhibitors 1, 4, 14 and 17 with
work concentrations were added to 96-well plates, respectively, and
incubated for another 48 h. Six wells containing only cells suspended in
4.2.18. 4-(4-acetamidophenylsulfonamido)-N-hydroxybenzamide (18)
Light orange solid, yield 44%. 1H NMR (400 MHz, DMSO) δ 10.82 (d,
J = 16.0 Hz, 2H), 10.31 (s, 1H), 8.94 (s, 1H), 7.76–7.68 (m, 4H), 7.60 (d,
J = 8.7 Hz, 2H), 7.12 (d, J = 8.7 Hz, 2H), 2.05 (s, 3H). 13C NMR (101
MHz, DMSO) δ 169.16, 163.84, 143.39, 140.70, 132.93, 128.16, 127.73,
118.73, 118.42, 24.22. HRMS (ESI) m/z: 372.0648 (Calcd for [M+Na]+
372.0625 m/z).
a mixture of 99 μL of complete medium 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 μL 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.
4.2.19. 3-(1-(dimethylamino)naphthalene-5-sulfonamido)-N-
hydroxybenzamide (DSN)
Yellow solid, yield 34%. 1H NMR (400 MHz, DMSO) δ 10.92 (s, 1H),
8.43 (d, J = 8.5 Hz, 1H), 8.36 (d, J = 8.6 Hz, 1H), 8.22 (d, J = 7.2 Hz,
1H), 7.67–7.56 (m, 3H), 7.51–7.46 (m, 1H), 7.26 (dd, J = 18.5, 6.2 Hz,
3H), 2.78 (s, 6H). 13C NMR (101 MHz, DMSO) δ 163.82, 151.62, 138.10,
134.53, 133.94, 131.76, 130.45, 129.94, 129.60, 129.08, 128.46,
124.27, 123.65, 122.67, 119.28, 118.57, 115.47, 45.15. HRMS (ESI) m/
z: 408.0966 (Calcd for [M+Na]+ 408.0988 m/z).
4.7. Metal suppression experiments
In metal suppression experiments, bacterial growth of E. coli cells
harboring L1 following treatment with NOTA, EDTA or 17 in the pres-
ence of Zn(II) ions was assayed. The concentration of CEF was 36
μM,
and concentration of Zn(II) ions and inhibitors was 240
μM. E. coli cells
with OD600 of 0.4–0.6 were diluted 84-fold, 3 mL inhibitor (or 3 mL
combination of inhibitor and ZnCl2), 3 mL CEF and 6 mL bacteria were
mixed and incubated at 37℃ 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.
4.3. Determination of IC50
VIM-2, IMP-1, ImiS and L1 were over-expressed and purified
[30–32], and were selected as representative MβLs for IC50 determina-
tion. hydroxamic acids 1–18 and DSN were dissolved with DMSO to
ensure that final concentrations of DMSO is less than 5% during pre-
mixing, and MβLs sample and antibiotics were dissolved in 30 mM Tris
(pH 7.0). All inhibitors were premixed with the enzyme between 0 and
80 µM and measured, and the antibiotics concentration was 40 µM. The
hydrolysis rate of antibiotics was measured on an Agilent UV8453
spectrometer, IC50 values were determined by plotting a linear fit of
inhibitor concentration and percent inhibition.
4.8. Mice experiments
The mice experiment was approved by the Animal Medicine Com-
mittee of Xi’an Jiaotong University, and all experimental steps were in
compliance 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 200 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 PBS, hydroxamic acid
17, and cefazolin, a combination of cefazolin and 17. The injection
concentration is 10 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 5 µL on LB-agar
plates and counted the number of colonies.
4.4. Determination of Ki
At an antibiotic concentration of 20, 40, 60, 80 and 100 µM, the
enzyme hydrolysis rates at different inhibitor concentrations were
measured and the data were collated using Sigma Plot 12.0 to generate a
Lineweaver-Burk plot to determine the hydroxamic acid inhibition
mode and Ki value. The hydrolysis rates of antibiotics were obtained by
Agilent UV8453 spectrometer.
4.5. Determination of MIC
The minimum inhibitory concentration (MIC) of compounds, anti-
biotics alone and the combination of antibiotics and inhibitors were
9

J.-Q. Li et al.
Bioorganic Chemistry 105 (2020) 104436
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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.
[15] C. Chen, Y. Xiang, K.W. Yang, Y.Z. Zhang, W.M. Wang, J.P. Su, et al., A protein
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Acknowledgements
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This work was supported by grants from National Natural Science
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