Systematic research of H2dedpa derivatives as potent inhibitors of New Delhi Metallo-β-lactamase-1

Article abstract of DOI:10.1016/j.bioorg.2020.103965

New Delhi Metallo-β-lactamase-1 (NDM-1), a Zn (II)-dependent enzyme, can catalyze the hydrolysis of almost all β-lactam antibiotics including carbapenems, resulting in bacterial antibiotic resistance, which threatens public health globally. Based on our finding that H₂dedpa is as an efficient NDM-1 inhibitor, a series of H₂dedpa derivatives was systematically prepared. These compounds exhibited significant activity against NDM-1, with IC₅₀ values 0.06–0.94 μM. In vitro, compounds 6k and 6n could restore the activity of meropenem against Klebsiella pneumoniae, Escherichia coli and Proteus mirabilis possessing either NDM or IMP. In particular, the activity of meropenem against E. coli producing NDM-4 could be improved up to 5333 times when these two compounds were used. Time–kill cell-based assays showed that 99.9% of P. mirabilis were killed when treated with meropenem in combination with compound 6k or 6n. Furthermore, compounds 6k and 6n were nonhemolytic (HC₅₀ > 1280 μg/mL) and showed low toxicity toward mammalian (HeLa) cells. Mechanistic studies indicated that compounds 6k and 6n inhibit NDM-1 by chelating the Zn²⁺ ion of the enzyme.

Full text of DOI:10.1016/j.bioorg.2020.103965

BioorganicChemistry101(2020)103965
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Systematic research of H2dedpa derivatives as potent inhibitors of New
Delhi Metallo-β-lactamase-1
De-Yun Cuia,1, Yi Yanga,1, Meng-Meng Baia,1, Jiang-Xue Hanb,1, Cong-Cong Wanga,
⁎
Hong-Tao Konga, Bo-Yuan Shena, Da-Chao Yana, Chun-Ling Xiaob, Yi-Shuang Liub,
,
⁎⁎
En Zhanga,c,
a School of Pharmaceutical Sciences, Institute of Drug Discovery and Development, Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of
China, Zhengzhou University, Zhengzhou 450001, PR China
b Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
c Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou 450001, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
New Delhi Metallo-β-lactamase-1 (NDM-1), a Zn (II)-dependent enzyme, can catalyze the hydrolysis of almost all
β-lactam antibiotics including carbapenems, resulting in bacterial antibiotic resistance, which threatens public
health globally. Based on our finding that H2dedpa is as an efficient NDM-1 inhibitor, a series of H2dedpa
derivatives was systematically prepared. These compounds exhibited significant activity against NDM-1, with
IC50 values 0.06–0.94 μM. In vitro, compounds 6k and 6n could restore the activity of meropenem against
Klebsiella pneumoniae, Escherichia coli and Proteus mirabilis possessing either NDM or IMP. In particular, the
activity of meropenem against E. coli producing NDM-4 could be improved up to 5333 times when these two
compounds were used. Time–kill cell-based assays showed that 99.9% of P. mirabilis were killed when treated
with meropenem in combination with compound 6k or 6n. Furthermore, compounds 6k and 6n were non-
hemolytic (HC50 > 1280 μg/mL) and showed low toxicity toward mammalian (HeLa) cells. Mechanistic studies
indicated that compounds 6k and 6n inhibit NDM-1 by chelating the Zn2+ ion of the enzyme.
Antibiotic resistance
New Delhi Metallo-β-lactamase-1
Metallo-β-lactamase inhibitor
Picolinic acid
Enterobacteriaceae
1. Introduction
are SBLs. These enzymes can covalently bind to and hydrolyze a β-
lactam via a nucleophilic serine residue. Class B β-lactamases are MBLs
Antibiotics, including β-lactams, macrolides, and fluoroquinolones,
have reduced the burden of common infectious diseases and become
indispensable in many medical treatments. β-Lactams, such as peni-
cillins and cephalosporins, are the most clinically important types of
antibiotic in the treatment of infection caused by bacterial pathogens
[1]. However, extended spectrum β-lactamases have emerged, resulting
in widespread bacterial resistance to β-lactam antibiotics. Carbape-
nems, which are seen as the last-resort antibiotics against β-lactam-
resistant bacterial infections, are threatened by carbapenemases [2].
Based on Ambler amino acid sequence homologies, β-lactamases
can be divided into classes A, B, C and D [3,4]. β-Lactamases can also be
divided into two types according to their type of active site—serine β-
lactamases (SBLs) and metallo-β-lactamases (MBLs). Classes A, C and D
[3]. Because of the high degree of sequence diversity and varied
structures of their active sites, MBLs have been further grouped into
three subclasses, B1–B3. Subclass B2 contains mono-zinc ion in the
active site and can hydrolyze carbapenems [5]. However, the ability of
class B2 enzymes to hydrolyze penicillins and cephalosporins is lower
[6]. The active sites of subclass B1 and B3 enzymes contain two zinc
ions; these enzymes can hydrolyze all β-lactam antibiotics other than
monobactams [7]. Subclass B3 is encoded by a chromosomal gene
[8,9].
Subclass B1 MBLs are the most clinically significant MBLs. New
Delhi Metallo-β-lactamase-1 (NDM-1), a B1 subclass enzyme, was first
isolated from urine of a Swedish patient who had received antibiotic
treatment in India in 2008 [10]. The blaNDM-1 gene is easily
⁎ Corresponding author at: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China (Yi-
Shuang Liu).
⁎⁎ Corresponding author at: School of Pharmaceutical Sciences, Institute of Drug Discovery and Development, Key Laboratory of Advanced Pharmaceutical
Technology, Ministry of Education of China, Zhengzhou University, Zhengzhou 450001, PR China (En Zhang).
E-mail addresses: rememberfrost@163.com (Y.-S. Liu), zhangen@zzu.edu.cn (E. Zhang).
1 These authors contributed equally to this work and should be considered co-first authors.
https://doi.org/10.1016/j.bioorg.2020.103965
Received 14 February 2020; Received in revised form 3 May 2020; Accepted 20 May 2020
Availableonline22May2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

D.-Y. Cui, et al.
BioorganicChemistry101(2020)103965
Fig. 1. Structures of selected New Delhi Metallo-β-
lactamase-1 (NDM-1) inhibitors.
transmissible on plasmids and can coexist with genes coding for other
resistance mechanisms in bacteria. NDM-1 has rapidly spread world-
wide, leading to the emergence of multidrug-resistant bacteria [11].
Several inhibitors of SBLs, such as clavulanic acid and sulbactam,
have been used in clinical therapy as co-drugs in which the inhibitors
are combined with a β-lactam antibiotic to restore antibacterial activity
[12]. However, approved SBL inhibitors have no effect against MBLs,
and there are no drugs approved as MBL inhibitors [13]. Several groups
of MBL inhibitors have been reported [14–18], e.g., thiols [19,20],
boronic acid [21], thiazole-4-carboxylates [22], dicarboxylic acids
[23], and zinc chelators [24] that can remove or sequestrate the zinc
from the active site of MBLs. A natural product isolated from the fungus
Aspergillus versicolor, aspergillomarasmine A (AMA) (Fig. 1), which
2. Results and discussion
2.1. Synthesis and characterization
The synthesis of various substituted H2dedpa derivatives 6a–6p is
depicted in Scheme 1. Briefly, the pyridine bromide 3 was first prepared
by partial reduction of pyridine-2,6-dicarboxylate followed by bromi-
nation of alcohol 2. 1,2-Disubstituted ethylenediamines (4a–4p) were
obtained by reductive amination of aldehydes with ethylenediamine or
substitution of halides with ethylenediamine. Then, 4a–4p were cou-
pled with 3 to give esters 5a–5p. Finally, the target compounds 6a–6p
were generated by ester hydrolysis.
exhibited excellent activity against NDM-1 (IC50 = 4.0
1.0 μM) and
2.2. Inhibition assays of NDM-1
restored the activity of meropenem (MEM) against Klebsiella pneumo-
niae harboring NDM-1 in a mouse infection model, was reported in
2014 [25]. Through equilibrium dialysis and 1H NMR studies, it was
determined that the inhibitory mechanism of AMA was to sequester the
zinc ion of NDM-1 in the active site [26]. 2,6-Dipicolinic acid can also
chelate the zinc of NDM-1. On the basis of fragment-based drug dis-
covery, inhibitor 1 (Fig. 1), a dipicolinic acid derivative, was identified
In an initial inhibitory activity screening, purified NDM-1 enzyme
was used to analyze the half-maximal inhibitory concentration (IC50
)
values of the H2dedpa derivatives (Table 1) [40]. Alkyl (6a–6c), sub-
stituted aryl (6d–6l), and substituted aromatic heterocycle (6m–6p)
variants all inhibited NDM-1 activity and the IC50 values were all <
1 μM (0.06–0.94 μM). With elongation of the alkyl chain, the IC50
values of 6a–6c decreased from 0.23 to 0.42 μM. The IC50 values of
various aryl substituted forms (6d–6l) ranged between 0.06 and
0.94 μM. Para-alkyl substituted compounds showed the best activities
(6e: 0.08 μM, 6j: 0.06 μM). Pyridine (6m, 6n) and thiophene (6o, 6p)
derivatives also had good activities. Among the 16 compounds, 6j
as a potent NDM-1 inhibitor (IC50 = 80
2 nM) and this inhibitor was
relatively selective toward MBLs over other zinc-containing enzymes
[27]. In 2018, [S,S]-ethylenediamine-N,N′-disuccinic acid (EDDS)
(Fig. 1), which is produced by bacteria and has a similar structure to
AMA, showed similar ability to inhibit NDM-1 (IC50 = 0.18 μM).
Bacteria possessing NDM-1 became susceptible to imipenem when it
was combined with EDDS, both in vivo and in vitro [28]. Part metal
chelation inhibitors not only showed high enzyme inhibitory activity
but also could dramatically improve the efficiency of antibiotics when
these inhibitors were used as adjuvant in cellular assays [25,27–28].
Some metal chelators have been successfully used in clinical or
preclinical therapy [29]. Deferiprone (Fig. 2), an iron chelating drug, is
used for the treatment of transfusional iron overload and to treat su-
perficial siderosis, a rare neurodegenerative disease [30,31]. The
copper chelator D-penicillamine (Fig. 2) can effectively protect against
catecholamine-induced myocardial injury [32]. Metal chelators also
have antitumor effects, and may be applied in antibody-based radio-
tracers. H2dedpa (Fig. 2) was reported as an excellent acyclic chelator
for use as a 68Ga positron-emission tomography imaging agent [33]. In
our continuous efforts to develop small antibacterial peptide mimics
showed the lowest IC50 value, 0.06
0.01 μM.
2.3. Antibacterial activity
As these H2dedpa derivatives inhibited purified NDM-1 in vitro, they
may restore the antibacterial capacity of carbapenem antibiotics against
bacteria producing NDM-1. To test this hypothesis, microdilution broth
minimum inhibitory concentrations (MIC) for meropenem (MEM)
combined with compounds 6j, 6k and 6n were determined (Table 2).
Measurements were conducted according to Clinical and Laboratory
Standards Institute (CLSI) guidelines.
The MICs of MEM alone were in the range 80–160 μg/mL, and no
effect was observed with the three compounds used alone
(MIC > 64 μg/mL). However, when MEM and the compounds were
combined, bacteria became susceptible. Notably, these three com-
pounds could enhance the activity of MEM against Klebsiella pneumo-
niae, K. oxytoca, Enterobacter aerogenes, Escherichia coli and Proteus
mirabilis carrying the NDM-1 gene. The MIC of MEM against P. mirabilis
harboring the blaNDM-1 gene, which is resistant to several types of
widely used clinical antibiotic including cephalosporin, aminoglyco-
side, carbapenem, polymyxin, and tetracycline (detailed drug resistance
information and MICs of antibiotics shown in Table S1), was improved
nearly 2560-fold. The MICs of MEM combined with the inhibitors
against carbapenem-resistant Enterobacteriaceae producing NDM-1 are
listed in Table S3. All three inhibitors made MEM effective against these
strains.
[34–36],
dithiocarbamates
[37,38]
and
H2dedpa
(IC50 = 0.17
0.04 μM) [39] with good activity against NDM-1 at
both the enzyme and cellular levels were developed. Here, based on this
finding, a series of H2dedpa derivatives was synthesized and their in-
hibitory activities toward NDM-1 evaluated.
Rejoicing in the above achievements, MIC values were also de-
termined for E. coli and K. pneumoniae isolates producing other types of
NDM and IMP. As shown in Table 3, the activity of MEM plus the
H2dedpa-based compounds against resistant bacterial strains possessing
NDM-5, NDM-7 or IMP was lower than that toward bacteria harboring
Fig. 2. Structures of selected metal chelators.
2

D.-Y. Cui, et al.
BioorganicChemistry101(2020)103965
Scheme 1. Synthesis of H2dedpa derivatives 6a–6p. (a: NaBH4, CH3OH, r.t.; b: PBr3, CHCl3, 0 °C; c: RCHO, CH3OH, N2 then NaBH4 or RBr, DMF; d: 3, Na2CO3,
CH3CN; e: 6 M HCl, reflux or LiOH THF/H2O, r.t.).
NDM-1, but the bacteria were rendered susceptible. However, inter-
estingly, the compounds improved the efficiency of MEM against E. coli
containing NDM-4 as much as 5120-fold. This E. coli shows resistance to
the main types of antibiotic used in the clinic, such as penicillin, qui-
nolone, cephalosporin, aminoglycoside, carbapenem, polymyxin, and
tetracycline (detailed drug resistance information and MICs of anti-
biotics against these strains is shown in Table S2). The MICs of MEM
combined with other inhibitors against carbapenem-resistant En-
terobacteriaceae producing other types of NDM or IMP are listed in Table
S4. Efficiency of MEM against these strains was observed for most of the
inhibitors.
Table 2
MICs (μg/mL) of MEM toward bacteria producing NDM-1.a
bacteria
6j
6k
6n
MEMb
K. Pneumoniae
K. oxytoca
E. aerogenes
E. coli
1
2
2
80
0.5
0.5
0.5
0.25
0.06
0.25
0.5
80
0.25
0.25
0.06
80
0.25
0.06
160
160
P. mirabilis
a
b
Inhibitor concentration 32 μg/mL.
MEM used alone.
2.4. Hemolytic activity
The toxicity of compounds 6a–6p toward red blood cells was
evaluated (Fig. 3). [41] The half-value of hemolysis (HC50) of all
compounds except 6b, 6c and 6j was > 1280 μg/mL, which illustrated
that these compounds were nonhemolytic. Compound 6j, which
showed the lowest IC50 values in in vitro inhibition experiments, also
displayed the lowest HC50 value (~100 μg/mL).
Table 3
MICs (μg/mL) of MEM toward bacteria producing other NDM types or IMP.a
Bacteria
Enzyme
6j
6k
6n
Meropenem
E. coli
NDM-4
NDM-5
NDM-5
NDM-7
IMP
0.03
0.25
2
0.03
2
0.03
2
160
160
80
K. Pneumoniae
E. coli
2
2
E. coli
1
2
2
80
K. Pneumoniae
E. coli
2
16
2
16
2
80
2.5. Cytotoxicity
IMP
1
80
HeLa cells were treated with compound 6k to further estimate cy-
a
Inhibitor concentration 32 μg/mL.
totoxicity. The inhibition rates of cells and fluorescence microscopy
Table 1
IC50 values of compounds 6a–6p against NDM-1 (μM).
No.
IC50
No.
IC50
No.
IC50
No.
IC50
6a
6e
6i
0.23
0.08
0.94
0.59
0.03
0.02
0.32
0.26
6b
6f
0.28
0.64
0.06
0.46
0.06
0.14
0.01
0.06
6c
6g
6k
6o
0.42
0.30
0.11
0.21
0.07
0.05
0.01
0.03
6d
6h
6l
0.34
0.50
0.17
0.37
0.03
0.09
0.01
0.04
6j
6m
6n
6p
3

D.-Y. Cui, et al.
BioorganicChemistry101(2020)103965
Fig. 3. HC50 values of compounds 6a–6p against red blood cells. A blue star indicates HC50 > 1280 µg/mL. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
6n at 32 μg/mL were combined with MEM at 0.25 μg/mL (8 × the
MIC), the colony counts after 24 h were all 0 colony-forming units
(CFU)/mL. After 6 h, the colony count for MEM + NOTA was 101 CFU/
mL, while for MEM + compound 6k or 6n it was 0 CFU/mL.
2.7. Test of zinc homeostasis
We assumed that compounds 6k and 6n could bind Zn2+ in the
active site of NDM-1 and thus inhibit the activity of the enzyme. To test
this idea, zinc suppression experiments were designed using P. mirabilis
containing NDM-1. Zinc ions were provided by adding ZnCl2. The re-
sults are shown in Fig. 7. When compounds MEM, NOTA, 6k and 6n
were tested alone, bacterial growth was approximately 90%, similar to
that in the blank control group, indicating no antibacterial activity.
However, when MEM was combined with NOTA, 6k or 6n, bacterial
Fig. 4. Inhibition rates of HeLa cells by compound 6k.
growth plummeted to 5.77%, 2.69% and 3.66% respectively. When
bacteria were treated with these drug combinations plus Zn2+, bac-
terial growth was restored to similar levels to that in the controls. These
after 24 h are shown in Figs. 4 and 5 respectively. The inhibition rates
after treatment with 1024, 512, 256, 128, and 64 μg/mL of compound
6k were all < 50%. This result indicated low toxicity of compound 6k
toward mammalian cells. The treated HeLa cells, visualized by the
LIVE/DEAD staining method [42], exhibited almost exclusively green
fluorescence at 64 μg/mL (6k), similar to the negative control. How-
ever, cells treated with the positive control (Triton-X) displayed nearly
all red fluorescence (Fig. 5). These results indicated that the MBL in-
hibitors 6k exerted low toxicity against HeLa cells.
data demonstrate that compounds 6k and 6n inhibit NDM-1 by che-
lating Zn2+
.
2.8. Docking analysis
To verify the possible binding pattern between compound 6k, 6n
and NDM-1 enzyme, Auto Dock Vina software was used to interface the
inhibitors with the NDM-1 crystal structure (PDB code 4EYL). Virtual
docking results are shown in Fig. 8. The CDOCK ENERGY of compound
6k, 6n with NDM-1 was −42.8926 and −97.8209 respectively. The
center of active site of NDM-1 enzyme has two zinc ions [43]. Zn1 could
coordinate with three Histidine residue (His120, His122 and His189),
the other Zn2 could form coordination bonds with His250, Cys208 and
Asp124. The carbonyl of one 6k carboxylic acid bound with two zinc
ions of NDM-1 via coordinate bond, the carbonyl of the other carboxylic
acid formed two hydrogen bonds with Lys 211 and one fluorine atom
on the 2,6-difluoro benzene bound with Asn 220 (Fig. 8c). In compound
2.6. Bactericidal kinetics
Because of the difference in bactericidal efficacy of compounds in
quiescent conditions and in certain growth conditions, time–kill cell-
based assays were performed to confirm the efficiency of compounds 6k
and 6n against P. mirabilis containing the blaNDM-1 gene (Fig. 6). NOTA
was used as a positive control. As shown in Fig. 6a, when NOTA, 6k or
4

D.-Y. Cui, et al.
BioorganicChemistry101(2020)103965
Fig. 5. Fluorescence microscopy images of HeLa cells after treatment with compound 6k for 24 h and staining with calcein AM and propidium iodide. (a–c) Non-
treated cells (negative control); (d–f) cells treated with compound 6k (64 μg/mL); (g–i) cells treated with 0.1% Triton X-100 (positive control).
6n, the hydroxyl of one of carboxylic acid bound with Zn2 of NDM-1
via coordinate bond. (Fig. 8d). These results support the hypothesis that
compounds 6k and 6n inhibit NDM-1 by binding with the Zn2+ ion.
shifts (δ) are expressed in ppm, and coupling constants J are given in
hertz. High-resolution mass spectra (HRMS) data were obtained by ESI
on a TOF mass analyzer. Analytical thin layer chromatography (TLC)
was performed on glass plates pre-coated with silica gel to monitor the
reactions. Column chromatography was performed on silica gel
(300–400 mesh).
3. Experimental
3.1. General information
3.2. General procedure for the synthesis compounds 2–3[44]
Chemicals were commercially available and were used without
further purification unless otherwise stated. All solvents were dried
according to standard procedures. The 1H NMR and 13C NMR spectra
were recorded at 25 °C in CDCl3, D2O, DMSO‑d6 or CD3OD at 400 and
100 MHz, respectively, with TMS as the internal standard. Chemical
3.2.1. Methyl 6-(hydroxymethyl)picolinate (2)
NaBH4 (724.95 mg, 19.16 mmol) was added to the solution of di-
methyl pyridine-2,6-dicarboxylate (1.00 g, 5.12 mmol) in MeOH
(50 mL) at 0 °C. The solution was stirred for 5 h at 0 °C. When the
Fig. 6. Time-dependent killing of P. mirabilis by meropenem (MEM) plus compound 6k or 6n. (a) P. mirabilis containing blaNDM-1 were grown to early exponential
phase and challenged with drugs. (b) Photographs after 24 h. MEM was used at 8 × the MIC.
5

D.-Y. Cui, et al.
BioorganicChemistry101(2020)103965
3.2.2. Methyl 6-(bromomethyl)picolinate (3)
NDM-1 containing P.mirabilis
PBr3 (909.07 μL, 9.57 mmol) was added dropwise to the solution of
2 (800 mg, 4.79 mmol) in chloroform (10 mL) at 0 °C. The solution was
stirred at room temperature for 5 h. When the reaction was completed,
saturated sodium bicarbonate was added to adjust pH to 8, extracted
with dichloromethane (40 mL × 4). The organic phases was washed
with brine (5 mL × 3). The organic phases were dried over anhydrous
MgSO4, and evaporated to dryness to give light yellow solid 3 (991 mg).
Yield: 90%.
100
80
60
40
3.3. General procedure for the synthesis compounds 4a-4p
20
Method A:
0
A series of alkyl bromide (18.30 mmol) was added to ethylenedia-
mine (555, 56 μL, 8.32 mmol) in DMF (20 mL). The solution was stirred
at room temperature for 28 h. The solvent was removed under reduced
pressure to obtain a white solid. The white solid was washed with sa-
turated NaOH and water. The crude product was recrystallized using
ethanol and water to give the target product 4a-4c. Yield: 50–55%.
6k
MEM+6k
6n
MEM
control
NOTA
MEM+6n
MEM+6k+Zn
MEM+6n+Zn
NOTA+MEM
NOTA+MEM+Zn
a
Fig. 7. Metal suppression experiments. MEM, meropenem. Control, no drug
treatment.
Method B:
reaction was completed, the solvent was evaporated. Saturated NaHCO3
solution was added and extracted with dichloromethane (30 mL × 3).
The organic phases were washed with water (5 mL × 3), and brine
(5 mL × 2). The organic phases were dried over MgSO4, and evapo-
rated to dryness to give white solid 2 (686 mg). Yield: 80%.
Ethylenediamine (311 μL, 4.67 mmol) in MeOH (10 mL) was added
dropwise to the solution of a series of aldehyde (9.34 mmol) in MeOH
(25 mL). The solution was refluxed for 12 h at 75 °C. Then, the reaction
mixture was evaporated in vacuum to obtain oil product. The crude
Fig. 8. (a, b) Low energy conformations of compound 6k (a) and 6n (b) docked into the NDM-1 (PDB code 4EYL). The space represented by the enzyme is colorful
and the NDM-1 selected residues are displayed by different color sticks (C, dark grey; N, blue; O, red; H, grey; F, light blue). The compound 6k and 6n were showed as
sticks colored by atom (C, yellow; N, blue; O, red; H, grey; F, light blue). (c, d) Locally-enlarged stereoscopic view of compounds 6k (c) and 6n (d) bound to NDM-1.
The enzyme backbone is displayed for the green ribbon. Associations with protein residues are indicated by dashed lines. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article.)
6

D.-Y. Cui, et al.
BioorganicChemistry101(2020)103965
product dissolving in MeOH (10 mL) was added to NaBH4 (1.5 mmol) at
0 °C, and the solution was stirred at room temperature. When the re-
action was completed (monitored by TLC), the reaction mixture was
treated in saturated sodium bicarbonate to adjust pH to 8 and extracted
three times with dichloromethane. The combined organic phase was
dried over anhydrous MgSO4, and the solvent was then removed under
vacuum. The residue was purified by flash column chromatography to
give the product 4d-4p. Yield: 70–85%.
3.3.10. N,N-bis(4-(tert-butyl)benzyl)ethane-1,2-diamine (4j) [49]
Method B. Yield: 71%. 1H NMR (400 MHz, CDCl3) δ 7.34 (d,
J = 8.2 Hz, 4H), 7.23 (d, J = 8.1 Hz, 4H), 3.74 (s, 4H), 2.77 (s, 4H),
1.63 (s, 2H), 1.31 (s, 18H). 13C NMR (100 MHz, CDCl3) δ 149.80,
137.46, 127.84, 125.27, 53.57, 48.86, 34.45, 31.39.
3.3.11. N,N-bis(2,6-difluorobenzyl)ethane-1,2-diamine (4k)
Method B. Yield: 72%. 1H NMR (400 MHz, CDCl3) δ 7.19 (tt,
J = 8.3, 6.5 Hz, 2H), 6.90–6.81 (m, 4H), 3.87 (s, 4H), 2.70 (s, 4H), 1.62
(s, 2H). 13C NMR (100 MHz, CDCl3) δ 161.79 (dd, J = 247.3, 8.8 Hz),
128.70 (t, J = 10.4 Hz), 116.00 (t, J = 20.3 Hz), 111.14 (dd,
J = 19.1,7.1 Hz), 48.09, 40.40 (t, J = 2.8 Hz).
3.3.1. N,N-dihexylethane-1,2-diamine (4a) [45]
Method A. Yield: 52%. 1H NMR (400 MHz, CDCl3) δ 2.71 (s, 4H),
2.59 (t, J = 7.2 Hz, 4H), 1.60 (s, 2H), 1.47 (q, J = 7.3, 6.9 Hz, 4H),
1.30 (d, J = 8.8 Hz, 12H), 0.88 (t, J = 6.7 Hz, 6H). 13C NMR
(100 MHz, CDCl3) δ 50.08, 49.61, 31.79, 30.17, 27.06, 22.62, 14.03.
3.3.12. N,N-bis(2,4-difluorobenzyl)ethane-1,2-diamine (4l)
Method B. Yield: 77%. 1H NMR (400 MHz, CDCl3) δ 7.29 (td,
J = 8.0, 7.6, 6.0 Hz, 2H), 6.95–6.66 (m, 4H), 3.78 (s, 4H), 2.73 (s, 4H),
1.59 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 162.06 (dd, J = 247.5,
12.0 Hz),δ 161.09 (dd, J = 248.1, 11.8 Hz), 131.01 (dd, J = 9.5,
6.4 Hz), 123.26 (dd, J = 15.3, 3.8 Hz), 111.01 (dd, J = 20.9, 3.8 Hz),
103.67 (t, J = 25.6 Hz), 48.49, 46.62 (d, J = 2.7 Hz).
3.3.2. N,N-diheptylethane-1,2-diamine (4b) [46]
Method A. Yield: 55%. 1H NMR (400 MHz, CDCl3) δ 2.71 (s, 4H),
2.59 (t, J = 7.2 Hz, 4H), 1.64 (s, 2H), 1.48 (p, J = 7.1 Hz, 4H), 1.28
(dt, J = 7.7, 3.8 Hz, 16H), 0.88 (t, J = 6.7 Hz, 6H). 13C NMR
(100 MHz, CDCl3) δ 50.08, 49.62, 31.83, 30.21, 29.25, 27.35, 22.62,
14.07.
3.3.13. N,N-bis(pyridin-2-ylmethyl)ethane-1,2-diamine (4m) [50]
Method B. Yield: 80%. 1H NMR (400 MHz, CDCl3) δ 8.54 (d,
J = 4.9 Hz, 2H), 7.64 (t, J = 7.3 Hz, 2H), 7.32 (d, J = 7.2 Hz, 2H),
7.15 (t, J = 6.0 Hz, 2H), 3.94 (d, J = 4.1 Hz, 4H), 2.85 (d, J = 4.1 Hz,
4H), 2.39 (s, 2H).
3.3.3. N,N-dioctylethane-1,2-diamine (4c) [45]
Method A. Yield: 50%. 1H NMR (400 MHz, CDCl3) δ 2.72 (s, 4H),
2.64–2.52 (m, 4H), 1.54 (s, 2H), 1.51–1.42 (m, 4H), 1.28 (t, J = 6.2 Hz,
20H), 0.93–0.81 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 50.05, 49.54,
31.84, 30.17, 29.54, 29.28, 27.39, 22.66, 14.08.
3.3.14. N,N-bis(pyridin-3-ylmethyl)ethane-1,2-diamine (4n) [49]
Method B. Yield: 80%. 1H NMR (400 MHz, CDCl3) δ 8.55 (s, 2H),
8.50 (d, J = 4.5 Hz, 2H), 7.66 (d, J = 7.6 Hz, 2H), 7.25 (dd, J = 7.8,
4.9 Hz, 2H), 3.79 (s, 4H), 2.76 (s, 4H), 1.79 (s, 2H). 13C NMR
(100 MHz, CDCl3) δ 149.67, 148.49, 135.71, 135.68, 123.37, 51.23,
48.74.
3.3.4. N,N-dibenzylethane-1,2-diamine (4d) [47]
Method B. Yield: 73%. 1H NMR (400 MHz, CDCl3) δ 7.33–7.28 (m,
8H), 7.26–7.20 (m, 2H), 3.76 (s, 4H), 2.75 (s, 4H), 1.78 (s, 2H).
3.3.5. N,N-dibenzylethane-1,2-diamine (4e) [48]
Method B. Yield: 85%. 1H NMR (400 MHz, CDCl3) δ 7.19 (d,
J = 7.9 Hz, 4H), 7.12 (d, J = 7.9 Hz, 4H), 3.72 (s, 4H), 2.74 (s, 4H),
2.32 (s, 6H), 1.68 (s, 2H).
3.3.15. N,N-bis(thiophen-2-ylmethyl)ethane-1,2-diamine (4o) [51]
Method B. Yield: 82%. 1H NMR (400 MHz, CDCl3) δ 7.19 (dd,
J = 5.0, 1.3 Hz, 2H), 6.96–6.88 (m, 4H), 3.97 (s, 4H), 2.78 (s, 4H), 1.70
(s, 2H).13C NMR (100 MHz, CDCl3) δ 144.37, 126.59, 124.77, 124.29,
48.40, 48.33.
3.3.6. N,N-bis(4-chlorobenzyl)ethane-1,2-diamine (4f) [49]
Method B. Yield: 80%. 1H NMR (400 MHz, CDCl3) δ 7.28 (d,
J = 8.4 Hz, 4H), 7.24 (d, J = 8.4 Hz, 4H), 3.74 (s, 4H), 2.73 (s, 4H),
1.60 (s, 2H).
3.3.16. N,N-bis(thiophen-3-ylmethyl)ethane-1,2-diamine (4p)
Method B. Yield: 85%. 1H NMR (400 MHz, CDCl3) δ 7.27 (dd,
J = 5.0, 3.1 Hz, 2H), 7.11 (d, J = 2.0 Hz, 2H), 7.04 (d, J = 4.9 Hz, 2H),
3.79 (s, 4H), 2.76 (s, 4H), 1.61 (s, 2H). 13C NMR (100 MHz, CDCl3) δ
141.63, 127.61, 125.70, 121.47, 48.87, 48.83.
3.3.7. N,N-bis(2-fluorobenzyl)ethane-1,2-diamine (4g)
Method B. Yield: 84%. 1H NMR (400 MHz, CDCl3) δ 7.32 (td,
J = 7.6, 1.9 Hz, 2H), 7.22 (tdd, J = 7.5, 5.3, 1.9 Hz, 2H), 7.09 (td,
J = 7.5, 1.2 Hz, 2H), 7.02 (ddd, J = 9.5, 8.1, 1.2 Hz, 2H), 3.82 (s, 4H),
2.75 (s, 4H), 1.84 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 161.21 (d,
J = 245.4 Hz), 130.35 (d, J = 4.8 Hz), 128.60 (d, J = 8.2 Hz), 127.27
(d, J = 15.1 Hz), 124.02 (d, J = 3.6 Hz), 115.24 (d, J = 21.9 Hz),
48.48, 47.11 (d, J = 3.1 Hz).
3.4. General procedure for the synthesis compounds 5a-5p
A
mixture of compound 4a-4p (1.24 mmol), Compound 3
(2.6 mmol) and K2CO3 (6.19 mmol) were dissolved in CH3CN (10 mL),
and the reaction mixture was stirred at 60 °C. When the reaction was
completed (monitored by TLC), the reaction mixture was added H2O to
quench the reaction and extracted three times with CH2Cl2. The com-
bined organic phase was dried over anhydrous MgSO4, and the solvent
was then removed under vacuum. The residue was purified by flash
column flash column chromatography to give the product 5a-5p. Yield:
50–80%.
3.3.8. N,N-bis(3-fluorobenzyl)ethane-1,2-diamine (4h)
Method B. Yield: 80%. 1H NMR (400 MHz, CDCl3) δ 7.27 (td,
J = 7.7, 6.0 Hz, 2H), 7.12–7.00 (m, 4H), 6.93 (td, J = 8.5, 2.6 Hz, 2H),
3.77 (s, 4H), 2.74 (s, 4H), 1.63 (s, 2H). 13C NMR (100 MHz, CDCl3) δ
163.02 (d, J = 245.8 Hz), 143.23 (d, J = 6.9 Hz), 129.79 (d,
J = 8.2 Hz), 123.54 (d, J = 2.8 Hz), 114.82 (d, J = 21.3 Hz), 113.75
(d, J = 21.1 Hz), 53.36 (d, J = 1.8 Hz), 48.73.
3.4.1. Dimethyl 6,6′-((ethane-1,2-diylbis(hexylazanediyl))bis(methylene))
dipicolinate (5a)
3.3.9. N,N-bis(3-fluorobenzyl)ethane-1,2-diamine (4i) [49]
Method B. Yield: 85%. 1H NMR (400 MHz, CDCl3) δ 7.29–7.23 (m,
4H), 7.04–6.96 (m, 4H), 3.73 (s, 4H), 2.73 (s, 4H), 1.67 (s, 2H). 13C
NMR (100 MHz, CDCl3) δ 161.91 (d, J = 244.7 Hz), 136.14 (d,
J = 3.1 Hz), 129.61 (d, J = 7.9 Hz), 115.14 (d, J = 21.1 Hz), 53.17,
48.70.
Yield: 53%. 1H NMR (400 MHz, CDCl3) δ 7.98 (dd, J = 6.8, 2.0 Hz,
2H), 7.78–7.70 (m, 4H), 3.99 (s, 6H), 3.84 (s, 4H), 2.63 (s, 4H),
2.51–2.37 (m, 4H), 1.42 (p, J = 7.0 Hz, 4H), 1.30–1.17 (m, 12H), 0.85
(t, J = 6.8 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 165.92, 147.08,
137.15, 125.92, 123.39, 60.97, 55.10, 52.85, 52.64, 31.71, 27.22,
27.01, 22.59, 14.01.
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3.4.2. Dimethyl 6,6′-((ethane-1,2-diylbis(heptylazanediyl))bis(methylene))
dipicolinate (5b)
(d, J = 7.9 Hz), 125.79, 123.59, 115.07 (d, J = 21.3 Hz), 60.43, 58.37,
52.91, 51.89.
Yield: 51%. 1H NMR (400 MHz, CDCl3) δ 7.98 (dd, J = 6.7, 2.1 Hz,
2H), 7.75 (d, J = 6.8 Hz, 4H), 3.98 (s, 6H), 3.85 (s, 4H), 2.64 (s, 4H),
2.46 (s, 4H), 1.42 (t, J = 7.4 Hz, 4H), 1.31–1.19 (m, 16H), 0.86 (t,
J = 6.9 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 165.96, 147.09, 137.17,
125.91, 123.42, 61.03, 55.10, 52.90, 52.70, 31.84, 29.21, 27.34, 22.62,
14.08.
3.4.10. Dimethyl 6,6′-((ethane-1,2-diylbis((4-(tert-butyl)benzyl)azanediyl))
bis(methylene))dipicolinate (5j)
Yield: 78%. 1H NMR (400 MHz, CDCl3) δ 7.96 (dd, J = 7.3, 1.4 Hz,
2H), 7.77–7.66 (m, 4H), 7.27 (d, J = 8.2 Hz, 4H), 7.19 (d, J = 8.2 Hz,
4H), 3.98 (s, 6H), 3.82 (s, 4H), 3.55 (s, 4H), 2.69 (s, 4H), 1.29 (s, 18H).
13C NMR (100 MHz, CDCl3) δ 165.90, 161.36, 149.88, 147.07, 137.24,
135.83, 128.37, 125.88, 125.13, 123.44, 60.49, 58.71, 52.89, 52.02,
34.43, 31.38.
3.4.3. Dimethyl 6,6′-((ethane-1,2-diylbis(octylazanediyl))bis(methylene))
dipicolinate (5c)
Yield: 56%. 1H NMR (400 MHz, CDCl3) δ 7.97 (dd, J = 6.7, 2.0 Hz,
2H), 7.74 (q, J = 7.4, 7.0 Hz, 4H), 3.99 (s, 6H), 3.83 (s, 4H), 2.63 (s,
4H), 2.49–2.33 (m, 4H), 1.40 (d, J = 6.8 Hz, 4H), 1.29–1.19 (m, 20H),
0.87 (t, J = 6.9 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 165.94, 147.09,
137.14, 125.90, 123.39, 61.00, 55.11, 52.86, 52.69, 31.82, 29.49,
29.27, 27.37, 27.29, 22.63, 14.08.
3.4.11. Dimethyl 6,6′-((ethane-1,2-diylbis((2,6-difluorobenzyl)azanediyl))
bis(methylene))dipicolinate (5k)
Yield: 75%. 1H NMR (400 MHz, CDCl3) δ 7.95 (dd, J = 5.1, 3.7 Hz,
2H), 7.74–7.63 (m, 4H), 7.16 (tt, J = 8.3, 6.4 Hz, 2H), 6.79 (t,
J = 7.8 Hz, 4H), 3.98 (s, 6H), 3.84 (s, 4H), 3.70 (s, 4H), 2.66 (s, 4H).
13C NMR (100 MHz, CDCl3) δ 165.96, 162.03 (dd, J = 248.8, 8.4 Hz),
161.18, 146.89, 137.09, 129.15 (t, J = 10.3 Hz), 126.07, 123.48,
113.99 (t, J = 19.4 Hz), 111.12 (dd, J = 19.5, 6.4 Hz), 60.58, 52.87,
51.92, 45.79.
3.4.4. Dimethyl 6,6′-((ethane-1,2-diylbis(benzylazanediyl))bis(methylene))
dipicolinate (5d) [52]
Yield: 69%. 1H NMR (400 MHz, CDCl3) δ 7.96 (dd, J = 5.3, 3.6 Hz,
2H), 7.72–7.68 (m, 4H), 7.27–7.17 (m, 10H), 3.98 (s, 6H), 3.82 (s, 4H),
3.57 (s, 4H), 2.68 (s, 4H).
3.4.12. Dimethyl 6,6′-((ethane-1,2-diylbis((2,4-difluorobenzyl)azanediyl))
bis(methylene))dipicolinate (5l)
3.4.5. Dimethyl 6,6′-((ethane-1,2-diylbis((4-methylbenzyl)azanediyl))bis
(methylene))dipicolinate (5e)
Yield: 72%. 1H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 7.6 Hz, 2H),
7.74 (t, J = 7.7 Hz, 2H), 7.65 (d, J = 7.8 Hz, 2H), 7.30 (td, J = 8.5,
7.9, 6.0 Hz, 2H), 6.86–6.66 (m, 4H), 3.99 (s, 6H), 3.84 (s, 4H), 3.61 (s,
4H), 2.68 (s, 4H). 13C NMR (100 MHz, CDCl3) δ 165.74, 162.87 (dd,
J = 80.0, 11.9 Hz), 160.63, 160.40 (dd, J = 81.1, 11.9 Hz), 147.12,
137.25, 131.85 (dd, J = 9.5, 6.2 Hz), 125.83, 123.56, 121.41 (dd,
J = 14.6, 3.7 Hz), 110.92 (dd, J = 20.9, 3.7 Hz), 103.61 (t,
J = 25.7 Hz), 60.43, 52.83, 51.96, 51.49.
Yield: 78%. 1H NMR (400 MHz, CDCl3) δ 7.95 (t, J = 4.4 Hz, 2H),
7.70 (d, J = 4.6 Hz, 4H), 7.14 (d, J = 7.9 Hz, 4H), 7.05 (d, J = 7.8 Hz,
4H), 3.98 (s, 6H), 3.79 (s, 4H), 3.52 (s, 4H), 2.65 (s, 4H), 2.31 (s, 6H).
3.4.6. Dimethyl 6,6′-((ethane-1,2-diylbis((4-chlorobenzyl)azanediyl))bis
(methylene))dipicolinate (5f)
Yield: 66%. 1H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 7.5 Hz, 2H),
7.73 (t, J = 7.7 Hz, 2H), 7.61 (d, J = 7.7 Hz, 2H), 7.20 (q, J = 8.5 Hz,
8H), 3.98 (s, 6H), 3.80 (s, 4H), 3.52 (s, 4H), 2.63 (s, 4H).
3.4.13. Dimethyl 6,6′-((ethane-1,2-diylbis((pyridin-2-ylmethyl)azanediyl))
bis(methylene))dipicolinate (5m)
Yield: 80%. 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 2.6 Hz, 2H),
8.01–7.91 (m, 2H), 7.72 (d, J = 4.6 Hz, 4H), 7.56 (td, J = 7.7, 1.8 Hz,
2H), 7.40 (d, J = 7.8 Hz, 2H), 7.16–7.06 (m, 2H), 3.98 (s, 6H), 3.89 (s,
4H), 3.77 (s, 4H), 2.77 (s, 4H). 13C NMR (100 MHz, CDCl3) δ 165.84,
160.71, 159.34, 149.10, 147.26, 137.29, 136.34, 125.95, 123.52,
122.79, 121.99, 60.84, 60.66, 52.86, 52.48.
3.4.7. Dimethyl 6,6′-((ethane-1,2-diylbis((2-fluorobenzyl)azanediyl))bis
(methylene))dipicolinate (5g)
Yield: 68%. 1H NMR (400 MHz, CDCl3) δ 7.96 (dd, J = 5.7, 3.2 Hz,
2H), 7.74–7.68 (m, 4H), 7.32 (t, J = 7.0 Hz, 2H), 7.19 (tdd, J = 7.4,
5.1, 1.8 Hz, 2H), 7.02 (t, J = 7.4 Hz, 2H), 6.96 (t, J = 9.2 Hz, 2H), 3.98
(s, 6H), 3.84 (s, 4H), 3.65 (s, 4H), 2.70 (s, 4H). 13C NMR (100 MHz,
CDCl3) δ 165.87, 161.35 (d, J = 246.5 Hz), 160.96, 147.06, 137.28,
131.08 (d, J = 4.4 Hz), 128.72 (d, J = 8.2 Hz), 125.92, 125.53 (d,
J = 14.3 Hz), 123.84 (d, J = 3.6 Hz), 123.53, 115.28 (d, J = 22.0 Hz),
60.57, 52.88, 52.07, 51.97 (d, J = 2.0 Hz).
3.4.14. Dimethyl 6,6′-((ethane-1,2-diylbis((pyridin-3-ylmethyl)azanediyl))
bis(methylene))dipicolinate (5n)
Yield: 75%. 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 4.9 Hz, 2H),
8.01–7.91 (m, 2H), 7.72 (d, J = 4.6 Hz, 4H), 7.56 (td, J = 7.7, 1.8 Hz,
2H), 7.40 (d, J = 7.8 Hz, 2H), 7.15–7.06 (m, 2H), 3.98 (s, 6H), 3.89 (s,
4H), 3.77 (s, 4H), 2.77 (s, 4H). 13C NMR (100 MHz, CDCl3) δ 165.71,
160.27, 150.17, 148.64, 147.26, 137.43, 136.33, 134.30, 125.86,
123.72, 123.29, 60.34, 56.46, 52.91, 51.99.
3.4.8. Dimethyl 6,6′-((ethane-1,2-diylbis((3-fluorobenzyl)azanediyl))bis
(methylene))dipicolinate (5h)
Yield: 64%. 1H NMR (400 MHz, CDCl3) δ 7.98 (dd, J = 7.5, 1.1 Hz,
2H), 7.77–7.70 (m, 2H), 7.69–7.57 (m, 2H), 7.21 (td, J = 7.7, 5.8 Hz,
2H), 7.09–6.97 (m, 4H), 6.91 (td, J = 7.7, 1.6 Hz, 2H), 3.98 (s, 6H),
3.81 (s, 4H), 3.56 (s, 4H), 2.67 (s, 4H). 13C NMR (100 MHz, CDCl3) δ
165.81, 162.93 (d, J = 245.6 Hz), 160.64, 147.20, 141.81 (d,
J = 6.8 Hz), 137.39, 129.71 (d, J = 8.3 Hz), 125.82, 124.15 (d,
J = 2.7 Hz), 123.64, 115.30 (d, J = 21.4 Hz), 114.01 (d, J = 21.2 Hz),
60.51, 58.66 (d, J = 1.9 Hz), 52.90, 52.00.
3.4.15. Dimethyl
6,6′-((ethane-1,2-diylbis((thiophen-2-ylmethyl)azan-
ediyl))bis(methylene))dipicolinate (5o)
Yield: 74%. 1H NMR (400 MHz, CDCl3) δ 7.99–7.95 (m, 2H), 7.79
(dd, J = 7.9, 1.4 Hz, 2H), 7.78–7.71 (m, 2H), 7.18 (dd, J = 5.0, 1.2 Hz,
2H), 6.90 (dd, J = 5.1, 3.4 Hz, 2H), 6.86 (dd, J = 3.5, 1.2 Hz, 2H), 3.98
(s, 6H), 3.86 (s, 4H), 3.81 (s, 4H), 2.71 (s, 4H). 13C NMR (100 MHz,
CDCl3) δ 165.85, 160.90, 147.07, 142.55, 137.39, 126.50, 125.96,
125.83, 124.83, 123.59, 60.26, 53.48, 52.88, 51.71.
3.4.9. Dimethyl 6,6′-((ethane-1,2-diylbis((4-fluorobenzyl)azanediyl))bis
(methylene))dipicolinate (5i)
Yield: 62%. 1H NMR (400 MHz, CDCl3) δ 7.97 (d, J = 7.6 Hz, 2H),
7.72 (t, J = 7.7 Hz, 2H), 7.63 (d, J = 7.8 Hz, 2H), 7.21 (dd, J = 8.3,
5.4 Hz, 4H), 6.93 (t, J = 8.5 Hz, 4H), 3.98 (s, 6H), 3.80 (s, 4H), 3.52 (s,
4H), 2.64 (s, 4H). 13C NMR (100 MHz, CDCl3) δ 165.80, 161.94 (d,
J = 245.0 Hz), 160.88, 147.18, 137.30, 134.62 (d, J = 2.6 Hz), 130.18
3.4.16. Dimethyl
6,6′-((ethane-1,2-diylbis((thiophen-3-ylmethyl)azan-
ediyl))bis(methylene))dipicolinate (5p)
Yield: 69%. 1H NMR (400 MHz, CDCl3) δ 7.97 (dd, J = 7.0, 1.7 Hz,
2H), 7.76–7.66 (m, 4H), 7.23 (dd, J = 4.9, 3.0 Hz, 2H), 7.07 (dd,
J = 3.0, 1.3 Hz, 2H), 6.99 (dd, J = 5.0, 1.2 Hz, 2H), 3.98 (s, 6H), 3.83
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2H), 7.06 (d, J = 7.8 Hz, 4H), 6.92 (d, J = 7.9 Hz, 4H), 3.86 (s, 4H),
3.37 (s, 4H), 2.28 (s, 4H), 2.27 (s, 6H). HRMS (ESI) Calculated for
C32H34N4O4Na [M+Na]+: 561.2478, found 561.2477.
(s, 4H), 3.61 (s, 4H), 2.66 (s, 4H). 13C NMR (100 MHz, CDCl3) δ 165.85,
161.11, 147.12, 139.68, 137.30, 128.16, 125.85, 125.58, 123.52,
122.62, 60.50, 53.82, 52.89, 51.92.
3.5. General procedure for the synthesis compounds 6a-6p
3.5.6. 6,6′-((ethane-1,2-diylbis((4-chlorobenzyl)azanediyl))bis
(methylene))dipicolinic acid (6f)
Method A:
Method B. White solid. Yield: 88%. 1H NMR (400 MHz, CD3OD) δ
7.89 (d, J = 7.5 Hz, 2H), 7.79 (t, J = 7.7 Hz, 2H), 7.28 (d, J = 7.6 Hz,
2H), 7.15 (d, J = 8.3 Hz, 4H), 6.96 (d, J = 8.3 Hz, 4H), 3.75 (s, 4H),
3.31 (s, 4H), 2.21 (s, 4H). HRMS (ESI) Calculated for C30H27Cl2N4O4
[M−H]−: 577.1410, found 577.1414.
Compound 5 (500 μmol) was added 6 M hydrochloric acid 10 mL.
The reaction mixture was refluxed 2 h and evaporate under vacuum to
obtain product. Yield: 84–89%.
Method B:
3.5.7. 6,6′-((ethane-1,2-diylbis((2-fluorobenzyl)azanediyl))bis
(methylene))dipicolinic acid (6g)
LiOH (2 mmol) was added to the solution of Compound
5
Method B. White solid. Yield: 90%. 1H NMR (400 MHz, CD3OD) δ
7.89 (d, J = 7.4 Hz, 2H), 7.80 (t, J = 7.7 Hz, 2H), 7.26 (d, J = 7.5 Hz,
2H), 7.21–7.14 (m, 2H), 7.10 (td, J = 7.4, 1.7 Hz, 2H), 7.06–7.00 (m,
2H), 6.91 (ddd, J = 9.6, 8.2, 1.1 Hz, 2H), 3.81 (s, 4H), 3.38 (s, 4H),
2.15 (s, 4H). 13C NMR (100 MHz, CD3OD) δ 172.32, 163.06 (d,
J = 243.2 Hz), 159.18, 154.80, 139.78, 134.08 (d, J = 4.9 Hz), 130.74
(d, J = 8.1 Hz), 125.47, 125.09 (d, J = 3.5 Hz), 122.96, 122.79 (d,
J = 17.6 Hz), 116.22 (d, J = 23.6 Hz), 60.85, 49.71, 47.65. HRMS
(ESI) Calculated for C30H28F2N4O4Na [M+Na]+: 569.1976, found
569.1978.
(500 μmol) in THF and water (V: V = 3: 1). The mixture was stirred at
room temperature for 2 h. When the reaction was completed, solvent
was evaporated to obtain product. Yield: 85%-90%.
3.5.1. 6,6′-((ethane-1,2-diylbis(hexylazanediyl))bis(methylene))
dipicolinic acid (6a)
Method A. Light yellow oil. Yield: 89%. 1H NMR (400 MHz,
DMSO‑d6) δ 11.58 (s, 2H), 8.15–8.07 (m, 4H), 7.92–7.86 (m, 2H), 4.69
(s, 4H), 3.87 (s, 4H), 3.17 (t, J = 8.4 Hz, 4H), 1.89–1.69 (m, 4H),
1.31–1.20 (m, 12H), 0.85 (q, J = 6.6 Hz, 6H). 13C NMR (100 MHz,
DMSO‑d6) δ 165.29, 150.49, 147.51, 139.21, 128.20, 124.55, 55.52,
53.63, 46.73, 30.57, 25.51, 22.70, 21.80, 13.77. HRMS (ESI)
Calculated for C28H43N4O4 [M+H]+: 499.3284, found 499.3285.
3.5.8. 6,6′-((ethane-1,2-diylbis((3-fluorobenzyl)azanediyl))bis
(methylene))dipicolinic acid (6h)
Method B. White solid. Yield: 86%. 1H NMR (400 MHz, CD3OD) δ
7.90 (d, J = 7.6 Hz, 2H), 7.80 (t, J = 7.7 Hz, 2H), 7.31 (d, J = 7.6 Hz,
2H), 7.18 (q, J = 7.9 Hz, 2H), 6.87 (td, J = 8.7, 2.2 Hz, 2H), 6.77 (t,
J = 7.9 Hz, 4H), 3.80 (s, 4H), 3.34 (s, 4H), 2.25 (s, 4H). 13C NMR
(100 MHz, CD3OD) δ 172.36, 164.10 (d, J = 245.2 Hz), 159.29,
154.80, 139.71, 139.30 (d, J = 6.2 Hz), 130.86 (d, J = 8.1 Hz), 127.19
(d, J = 2.5 Hz), 125.60, 123.08, 117.75 (d, J = 20.8 Hz), 115.08 (d,
J = 21.1 Hz), 60.84, 56.96, 47.76. HRMS (ESI) Calculated for
3.5.2. 6,6′-((ethane-1,2-diylbis(heptylazanediyl))bis(methylene))
dipicolinic acid (6b)
Method A. Light yellow oil. Yield: 86%. 1H NMR (400 MHz,
DMSO‑d6) δ 11.51 (s, 2H), 8.16–8.05 (m, 4H), 7.89 (dd, J = 7.1, 1.6 Hz,
2H), 4.68 (s, 4H), 3.86 (s, 4H), 3.17 (t, J = 8.3 Hz, 4H), 1.76 (t,
J = 8.1 Hz, 4H), 1.24 (d, J = 13.2 Hz, 16H), 0.92–0.76 (m, 6H). 13C
NMR (100 MHz, DMSO‑d6) δ 165.30, 150.54, 147.52, 139.20, 128.17,
124.57, 55.53, 53.65, 46.81, 30.93, 28.07, 25.82, 22.77, 21.93, 13.87.
HRMS (ESI) Calculated for C30H47N4O4 [M+H]+: 527.3597, found
527.3598.
C
30H29F2N4O4 [M+H]+: 547.2157, found 547.2158.
3.5.9. 6,6′-((ethane-1,2-diylbis((4-fluorobenzyl)azanediyl))bis
(methylene))dipicolinic acid (6i)
Method B. White solid. Yield: 89%. 1H NMR (400 MHz, CD3OD) δ
7.89 (d, J = 7.3 Hz, 2H), 7.79 (t, J = 7.7 Hz, 2H), 7.27 (d, J = 7.4 Hz,
2H), 7.02–6.95 (m, 4H), 6.91–6.83 (m, 4H), 3.76 (s, 4H), 3.31 (s, 4H),
2.21 (s, 4H). 13C NMR (100 MHz, CD3OD) δ 172.32, 163.62 (d,
J = 244.2 Hz), 159.31, 154.85, 139.67, 133.09 (d, J = 8.1 Hz), 132.42
(d, J = 3.2 Hz), 125.56, 123.04, 115.76 (d, J = 21.4 Hz), 60.81, 56.70,
47.85. HRMS (ESI) Calculated for C30H29F2N4O4 [M+H]+: 547.2157,
found 547.2156.
3.5.3. 6,6′-((ethane-1,2-diylbis(octylazanediyl))bis(methylene))dipicolinic
acid (6c)
Method A. Light yellow oil. Yield: 84%. 1H NMR (400 MHz,
DMSO‑d6) δ 11.56 (s, 2H), 8.15–8.06 (m, 4H), 7.93–7.86 (m, 2H), 4.68
(s, 4H), 3.87 (s, 4H), 3.17 (t, J = 8.3 Hz, 4H), 1.89–1.66 (m, 4H), 1.23
(d, J = 8.6 Hz, 20H), 0.85 (t, J = 6.8 Hz, 6H). 13C NMR (100 MHz,
DMSO‑d6) δ 165.28, 150.51, 147.50, 139.21, 128.18, 124.55, 55.53,
53.63, 46.75, 31.11, 28.39, 28.37, 25.87, 22.76, 22.00, 13.91. HRMS
(ESI) Calculated for
577.3732.
C
32H50N4O4Na [M+Na]+
:
577.3730, found
3.5.10. 6,6′-((ethane-1,2-diylbis((4-(tert-butyl)benzyl)azanediyl))bis
(methylene)) dipicolinic acid (6j)
Method B. White solid. Yield: 87%. 1H NMR (400 MHz, CD3OD) δ
7.90 (dd, J = 7.7, 1.0 Hz, 2H), 7.78 (t, J = 7.7 Hz, 2H), 7.26 (dd,
J = 7.7, 1.0 Hz, 2H), 7.21–7.15 (m, 4H), 6.96–6.79 (m, 4H), 3.77 (s,
4H), 3.29 (s, 4H), 2.19 (s, 4H), 1.18 (s, 18H). 13C NMR (100 MHz,
CD3OD) δ 172.36, 159.47, 154.79, 151.31, 139.64, 133.14, 131.20,
125.97, 125.46, 122.94, 61.00, 56.92, 47.55, 35.30, 31.80. HRMS (ESI)
Calculated for C38H45N4O4 [M−H]−: 621.3441, found 621.3443.
3.5.4. 6,6′-((ethane-1,2-diylbis(benzylazanediyl))bis(methylene))
dipicolinic acid (6d) [52]
Method B. White solid. Yield: 89%. 1H NMR (400 MHz, CD3OD) δ
7.89 (d, J = 7.4 Hz, 2H), 7.79 (t, J = 7.7 Hz, 2H), 7.27 (d, J = 7.6 Hz,
2H), 7.18–7.09 (m, 6H), 6.99–6.91 (m, 4H), 3.78 (s, 4H), 3.32 (s, 4H),
2.21 (s, 4H). 13C NMR (100 MHz, CD3OD) δ 170.91, 157.83, 153.33,
138.31, 133.68, 132.87, 131.46, 127.82, 124.21, 121.65, 59.35, 55.32,
46.46. HRMS (ESI) Calculated for C30H29N4O4 [M−H]−: 509.2189,
found 509.2186.
3.5.11. 6,6′-((ethane-1,2-diylbis((2,6-difluorobenzyl)azanediyl))bis
(methylene)) dipicolinic acid (6k)
Method A. White solid. Yield: 88%. 1H NMR (400 MHz, DMSO‑d6) δ
8.01–7.89 (m, 4H), 7.64 (dd, J = 7.6, 1.3 Hz, 2H), 7.43 (tt, J = 8.4,
6.6 Hz, 2H), 7.05 (t, J = 8.2 Hz, 4H), 4.44 (s, 4H), 4.24 (s, 4H), 3.51 (s,
4H). 13C NMR (100 MHz, DMSO‑d6) δ 165.30, 161.37 (dd, J = 249.0,
7.1 Hz), 154.35, 146.91, 138.65, 132.00 (t, J = 10.0 Hz), 126.55,
3.5.5. 6,6′-((ethane-1,2-diylbis((4-methylbenzyl)azanediyl))bis
(methylene))dipicolinic acid (6e)
Method B. White solid. Yield: 86%. 1H NMR (400 MHz, CD3OD) δ
7.99 (d, J = 7.6 Hz, 2H), 7.88 (t, J = 7.7 Hz, 2H), 7.36 (d, J = 7.6 Hz,
9

D.-Y. Cui, et al.
BioorganicChemistry101(2020)103965
123.72, 111.73 (d, J = 23.5 Hz), 108.76 (d, J = 18.3 Hz), 56.64, 49.73,
45.26. HRMS (ESI) Calculated for C30H25F4N4O4 [M−H]−: 581.1812,
found 581.1811.
3.6.2. Antibacterial activity
Antimicrobial activity was assessed using Broth Microdilution
Method, according to the national clinical laboratory standards com-
mittee (NCCLS). Bacteria are cultured in Mueller-Hinton Broth (MHB)
until it reached 105 CFU/mL. The test was carried out in a 96-well plate.
The tested compounds was dissolved in DMSO and the solution was
diluted with MHB medium Further, the MEM diluted to 32 μg/mL by
MHB contained 16/32/64 μg/mL test compounds. The final volume of
each of the 96 well plate is 200 μL, in this solution. The concentration
from 0.3125 to 16 μg/mL of MEM can obtain by gradient dilution.
Besides setting blank control group (only use the medium), positive
control group was set in each experiment (only bacteria solution). The
plate was incubated at 37 °C for 24 h. Results by visual observation, the
lowest drug concentration tube was free of bacterial growth, which was
the MIC of the drug tested.
3.5.12. 6,6′-((ethane-1,2-diylbis((2,4-difluorobenzyl)azanediyl))bis
(methylene)) dipicolinic acid (6l)
Method B. White solid. Yield: 89%. 1H NMR (400 MHz, CD3OD) δ
7.90 (d, J = 7.6 Hz, 2H), 7.81 (t, J = 7.7 Hz, 2H), 7.28 (d, J = 7.6 Hz,
2H), 7.16 (td, J = 8.3, 6.5 Hz, 2H), 6.80 (dtd, J = 18.5, 9.4, 8.9,
2.5 Hz, 4H), 3.79 (s, 4H), 3.37 (s, 4H), 2.15 (s, 4H). 13C NMR
(100 MHz, CD3OD) δ 172.28, 164.72 (dd, J = 86.4, 12.1 Hz), 162.27
(dd, J = 84.9, 11.9 Hz), 159.03, 154.81, 139.81, 135.03 (dd, J = 9.4,
6.5 Hz), 125.51, 123.01, 119.04 (dd, J = 18.0, 3.7 Hz), 112.12 (dd,
J = 21.5, 3.2 Hz), 104.39 (dd, J = 28.1, 25.3 Hz), 60.71, 49.31, 47.77.
HRMS (ESI) Calculated for C30H26F4N4O4Na [M+Na]+: 605.1788,
found 605.1789.
3.6.3. Metal suppression experiments
3.5.13. 6,6′-((ethane-1,2-diylbis((pyridin-2-ylmethyl)azanediyl))bis
(methylene)) dipicolinic acid (6m)
The experiments were had done according to the above procedure
(minimum inhibitory concentration (MIC) of the combined drug). It is
well known that Zn2+ ions play an important role in MBLs. Therefore,
we performed Zn2+ metal ion inhibition experiments in vitro to test
whether the compounds evaluated in this experiment can still restore
the sensitivity of bacteria to MEM in the presence of Zn2+ ions. The
experimental operation was the same except that an equimolar amount
of ZnCl2 was added. Eventually, the results were measured at the OD600
nm of the microplate reader.
Method A. Yellowish white solid. Yield: 85%. 1H NMR
(400 MHz,D2O) δ 8.38 (d, J = 5.2 Hz, 2H), 8.21 (t, J = 7.7 Hz, 2H),
8.02–7.94 (m, 4H), 7.78 (d, J = 7.9 Hz, 2H), 7.70–7.62 (m, 2H), 7.53
(d, J = 5.5 Hz, 2H), 4.33 (s, 4H), 4.18 (s, 4H), 3.32 (s, 4H). 13C NMR
(100 MHz, D2O) δ 166.12, 153.12, 149.76, 146.02, 145.49, 143.52,
141.51, 128.07, 127.47, 126.52, 125.45, 56.36, 55.78, 50.70. HRMS
(ESI) Calculated for C28H29N6O4 [M+H]+: 513.2250, found 513.2253.
3.5.14. 6,6′-((ethane-1,2-diylbis((pyridin-3-ylmethyl)azanediyl))bis
(methylene)) dipicolinic acid (6n)
3.6.4. Haemolytic activity
Fresh sterile sheep blood (provided by the China Nanjing Bian Zhen
biology Technology Co., Ltd, excellent grade GR), was suspended with
1 × PBS and centrifuged (1500 r. p. m,10 min) to remove excess im-
purities. The treated red blood cells were diluted and tested with the
resulting 5% erythrocyte suspension. DMSO or water as the solvent
(final concentration of no more than 0.5%) in each experiment were
haemolytic determination to prevent appearance of false positive re-
sults. The Triton-X detergent at 0.5% was used as the positive control.
The 5% erythrocyte suspension was placed in a 96-well plate of 150 μL/
well. The drug was diluted in a sterile EP tube using 1 × PBS, 50 μL/
well was added and each sample was tested in parallel using three
duplicate wells under the same concentration. After incubation at 37 °C
for 1 h, the plates were placed in a 4 °C centrifuge (3500 r. p. m, 5 min).
100 μL of the supernatant in each hole was transferred to a new 96-well
plate. Eventually, the results were measured at 540 nm on the micro-
plate reader. Percentage of haemolysis was determined as (A − A0)/
(Atotal − A0) × 100, and A was the test well, A0 was the negative
control, and Atotal was the positive.
Method B. Yellowish white solid. Yield: 86%. 1H NMR (400 MHz,
CD3OD) δ 8.32 (dd, J = 4.9, 1.3 Hz, 2H), 8.19 (d, J = 1.5 Hz, 2H), 7.91
(d, J = 7.6 Hz, 2H), 7.81 (t, J = 7.7 Hz, 2H), 7.54 (d, J = 7.8 Hz, 2H),
7.31 (d, J = 7.7 Hz, 2H), 7.28 (dd, J = 7.8, 5.0 Hz, 2H), 3.79 (s, 4H),
3.41 (s, 4H), 2.28 (s, 4H). 13C NMR (100 MHz, CD3OD) δ 172.33,
158.94, 154.82, 151.24, 149.08, 140.11, 139.80, 132.85, 125.70,
125.06, 123.19, 60.52, 54.62, 48.11. HRMS (ESI) Calculated for
C
28H29N6O4 [M+H]+: 513.2250, found 513.2252.
3.5.15. 6,6′-((ethane-1,2-diylbis((thiophen-2-ylmethyl)azanediyl))bis
(methylene)) dipicolinic acid (6o)
Method B. Yellowish white solid. Yield: 88%. 1H NMR (400 MHz,
CD3OD) δ 7.90 (dd, J = 7.6, 1.0 Hz, 2H), 7.82 (t, J = 7.7 Hz, 2H), 7.30
(dd, J = 7.7, 1.0 Hz, 2H), 7.18 (dd, J = 5.1, 1.1 Hz, 2H), 6.85 (dd,
J = 5.1, 3.4 Hz, 2H), 6.67 (dd, J = 3.4, 1.1 Hz, 2H), 3.83 (s, 4H), 3.53
(s, 4H), 2.25 (s, 4H). 13C NMR (100 MHz, CD3OD) δ 172.30, 159.12,
154.67, 139.82, 137.77, 129.35, 127.81, 126.23, 125.49, 123.02,
60.88, 50.74, 47.35. HRMS (ESI) Calculated for C26H27N4O4S2
[M + H]+: 523.1474, found 523.1475.
3.6.5. Cytotoxicity of the hela cells
HeLa cells were used in this experiment and maintained in complete
DMEM media (Biological Industries, Israel) supplemented with 10%
FBS (BI). The cells were dealt with Trypsin-EDTA solution and sus-
pended with culture medium for 5 × 103 cells/well. Then, 100 μL/well
was added to the 96-well plate. Overnight for its adherent. Then discard
the supernatant, 200 μL of each hole to join media double-dilution
drugs to be tested. The negative control group was treated only with
DMEM medium. After 24 h, Cell Counting Kit-8 10 μL/well was added
and incubated in a 37 °C incubator for 4 h. The results were measured at
450 nm on the microplate reader. The inhibition was calculated, and its
IC50 was returned.
3.5.16. 6,6′-((ethane-1,2-diylbis((thiophen-3-ylmethyl)azanediyl))bis
(methylene)) dipicolinic acid (6p)
Method B. Yellowish white solid. Yield: 89%. 1H NMR (400 MHz,
CD3OD) δ 7.88 (dd, J = 7.7, 1.0 Hz, 2H), 7.79 (t, J = 7.6 Hz, 2H), 7.26
(dd, J = 7.7, 1.0 Hz, 2H), 7.20 (dd, J = 4.9, 2.9 Hz, 2H), 6.94 (dd,
J = 3.0, 1.2 Hz, 2H), 6.79 (dd, J = 4.9, 1.3 Hz, 2H), 3.80 (s, 4H), 3.34
(s, 4H), 2.20 (s, 4H). 13C NMR (100 MHz, CD3OD) δ 172.31, 159.36,
154.71, 139.65, 137.16, 130.50, 126.22, 125.39, 122.89, 61.05, 51.15,
47.74. HRMS (ESI) Calculated for C26H27N4O4S2 [M+H]+: 523.1474,
found 523.1474
3.6. Biological activity
3.6.6. Fluorescence microscopy
HeLa cells with good growth were tested. The cells were homo-
geneously cultured in 24-well plates, and the cells were incubated for
24 h and then the test compound was added at the different con-
centration. According to the mild destruction of the cell membrane by
Triton-X, use the PI to detection the dead cells. The reason is when the
3.6.1. Inhibition assays on NDM-1
The expression and purification of NDM-1 enzyme and the inhibi-
tion testing was performed as previously described to determine the
IC50 of each inhibitor [39].
10

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BioorganicChemistry101(2020)103965
cells treated with the Triton-X, PI can be easily combined with the DNA
in the nucleus. The PI-DNA complex will release red fluorescence when
the excitation and emission wavelengths are 535 nm and 615 nm re-
spectively. However, in the living cells, the non-fluorescence of Calcium
- AM is converted to green fluorescence of calcium after the esterase of
the cells is hydrolysed by hydroxymethyl ester. There is no such process
for dead cells. Cells treated with 0.1% Triton-X was the positive control.
After 24 h, the solution with 1 × PBS and stained with 2 μM Calcein-
AM (Sigma-Aldrich) or 4.5 μM propidium iodide (PI, Aladdin) was
added and incubated for 15 min in a sterile environment. In the end, the
solution contains Calcein-AM: PI = 1: 1. At last, 1 × PBS was used to
wash the plate with the dead cells in the collection and were added to
the corresponding wells. Images were captured with 20 × objective in
Nikon 901 fluorescence microscope.
References
[1] T.P. Van Boeckel, S. Gandra, A. Ashok, Q. Caudron, B.T. Grenfell, S.A. Levin,
R. Laxminarayan, Global antibiotic consumption 2000 to 2010: an analysis of na-
tional pharmaceutical sales data, Lancet Infect. Dis. 14 (8) (2014) 742–750.
[2] J.M.A. Blair, M.A. Webber, A.J. Baylay, D.O. Ogbolu, L.J.V. Piddock, Molecular
mechanisms of antibiotic resistance, Nat. Rev. Microbiol. 13 (1) (2015) 42–51.
[3] S.M. Drawz, R.A. Bonomo, Three decades of beta-lactamase inhibitors, Clin.
Microbiol. Rev. 23 (1) (2010) 160–201.
[4] G. Cornaglia, H. Giamarellou, G.M. Rossolini, Metallo-β-lactamases: a last frontier
for β-lactams? Lancet Infect. Dis. 11 (5) (2011) 381–393.
[5] Y.-H. Yan, G. Li, G.-B. Li, Principles and current strategies targeting metallo-β-
lactamase mediated antibacterial resistance, Med. Res. Rev. (2020), https://doi.
org/10.1002/med.21665.
[6] R. del Campo, J.C. Galán, C. Tenorio, P. Ruiz-Garbajosa, M. Zarazaga, C. Torres,
F. Baquero, New aac (6′)-I genes in Enterococcus hirae and Enterococcus durans:
effect on β-lactam/aminoglycoside synergy, J. Antimicrob. Chemother. 55 (6)
(2005) 1053–1055.
[7] L.J. González, D.M. Moreno, R.A. Bonomo, A.J. Vila, Host-specific enzyme-substrate
interactions in SPM-1 metallo-β-lactamase are modulated by second sphere re-
sidues, PLoS Pathog. 10 (1) (2014) e1003817.
3.6.7. Bactericidal kinetics
[8] J. Chiou, S. Wan, K.-F. Chan, P.-K. So, 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-β-
lactamase (NDM-1), Chem. Commun. 51(46) (2015) 9543–9546.
The strains (P. mirabilis containing blaNDM-1) were grown overnight
(incubated at 37 °C with aeration at 225 r.p.m). The strains were wa-
shed and diluted 1:100000 in MHB and incubated at 37 °C with aeration
at 225 r.p.m for 2 h (early exponential phase). Bacteria were then
treated with the MEM and testing compounds at 8 × MIC (the in-
hibitors and MEM undergo Table 2 described MIC) in culture tubes at
37 °C intervals 20 μL of aliquots from the suspension was subjected to
10 fold serial dilution in 1 × PBS. 10 μL of the dilution was plated on
solid agar plates and incubated at 37 °C for 16–18 h. The bacterial
colonies were counted and results represented in log10 (CFU/mL) use
the origin 8.0. And photos the bacteria control for 24 h.
[9] G. Garau, I. García-Sáez, C. Bebrone, C. Anne, P. Mercuri, M. Galleni, J.-M. Frère,
O. Dideberg, Update of the standard numbering scheme for class B β-lactamases,
Antimicrob. Agents Chemother. 48 (7) (2004) 2347–2349.
[10] J.M. Rolain, P. Parola, G. Cornaglia, New Delhi metallo-beta-lactamase (NDM-1):
towards a new pandemia? Clin. Microbiol. Infect. 16 (12) (2010) 1699–1701.
[11] S.N. Seiffert, J. Marschall, V. Perreten, A. Carattoli, H. Furrer, A. Endimiani,
Emergence of Klebsiella pneumoniae co-producing NDM-1, OXA-48, CTX-M-15,
CMY-16, QnrA and ArmA in Switzerland, Int. J. Antimicrob. Agents 44 (3) (2014)
260–262.
[12] P. Ball, The clinical development and launch of amoxicillin/clavulanate for the
treatment of a range of community-acquired infections, Int. J. Antimicrob. Agents
30 (2007) 113–117.
[13] K. Bush, P.A. Bradford, Interplay between β-lactamases and new β-lactamase in-
hibitors, Nat. Rev. Microbiol. 17 (5) (2019) 295–306.
4. Conclusions
[14] P. Wang, J. Cheng, C.-C. Liu, K. Tang, F. Xu, Z. Yu, B. Yu, J. Chang, The develop-
ment of new small-molecule inhibitors targeting bacterial metallo-β-lactamases,
Curr. Top. Med. Chem. 18 (10) (2018) 834–843.
A series of H2dedpa derivatives was prepared. Compound 6j ex-
[15] J.-D. Docquier, S. Mangani, An update on β-lactamase inhibitor discovery and de-
velopment, Drug Resist. Updates 36 (2018) 13–29.
hibited
(IC50 = 0.06
red blood cells (HC50 > 100 μg/mL). However, compounds 6k and 6n
exhibited good enzyme inhibition activity (IC50 = 0.11 0.01 and
0.46 0.06 μM, respectively) and were nonhemolytic even at
the
highest
inhibitory
activity
against
NDM-1
0.01 μM), but this compound also showed toxicity to
[16] R.P. McGeary, D.T. Tan, G. Schenk, Progress toward inhibitors of metallo-β-lacta-
mases, Future Med. Chem. 9 (7) (2017) 673–691.
[17] C. Chen, 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-β-lacta-
mase inhibitors for targeting carbapenem-resistant enterobacteriaceae, ACS Infect.
Dis. (2020), https://doi.org/10.1021/acsinfecdis.9b00385.
1280 μg/mL. In vitro, compounds 6k and 6n could recover the activity
of MEM toward resistant bacterial strains containing MBLs. It is worth
noting that 6k and 6n improved the activity of meropenem against E.
coli producing NDM-4 by nearly 5333 times and the MICs of mer-
openem were 0.03 μg/mL. Compounds 6k and 6n were shown to in-
hibit MBL by the chelating zinc ion of NDM-1. Compounds 6k and 6n
are promising agents for treatment of antibiotic-resistant bacterial
strains containing MBLs and for further investigation as antibiotic ad-
juvant co-therapies.
[18] C. Chen, K.-W. Yang, L.-Y. Wu, J.-Q. Li, L.-Y. Sun, Disulfiram as a potent metallo-β-
lactamase inhibitor with dual functional mechanisms, Chem. Commun. 56 (18)
(2020) 2755–2758.
[19] G.-B. Li, J. Brem, R. Lesniak, M.I. Abboud, C.T. Lohans, I.J. Clifton, S.-Y. Yang, J.-
C. Jiménez-Castellanos, M.B. Avison, J. Spencer, M.A. McDonough, C.J. Schofield,
Crystallographic analyses of isoquinoline complexes reveal a new mode of metallo-
β-lactamase inhibition, Chem. Commun. 53 (43) (2017) 5806–5809.
[20] Z. Meng, M.-L. Tang, L. Yu, Y. Liang, J. Han, C. Zhang, F. Hu, J.-M. Yu, X. Sun,
Novel mercapto propionamide derivatives with potent New Delhi metallo-β-lacta-
mase-1 inhibitory activity and low toxicity, ACS Infect. Dis. 5 (6) (2019) 903–916.
[21] Y.-L. Wang, S. Liu, Z.-J. Yu, Y. Lei, M.-Y. Huang, Y.-H. Yan, Q. Ma, Y. Zheng,
H. Deng, Y. Sun, C. Wu, Y. Yu, Q. Chen, Z. Wang, Y. Wu, G.-B. Li, Structure-based
development of (1-(3′-mercaptopropanamido)methyl)boronic acid derived broad-
spectrum, dual-action inhibitors of metallo- and serine-β-lactamases, J. Med. Chem.
62 (15) (2019) 7160–7184.
Declaration of Competing Interest
[22] S. Leiris, A. Coelho, J. Castandet, M. Bayet, C. Lozano, J. Bougnon, J. Bousquet,
M. Everett, M. Lemonnier, N. Sprynski, M. Zalacain, T.D. Pallin, M.C. Cramp,
N. Jennings, G. Raphy, M.W. Jones, R. Pattipati, B. Shankar, R. Sivasubrahmanyam,
A.K. Soodhagani, R.R. Juventhala, N. Pottabathini, S. Pothukanuri, M. Benvenuti,
C. Pozzi, S. Mangani, F. De Luca, G. Cerboni, J.-D. Docquier, D.T. Davies, SAR
studies leading to the identification of a novel series of metallo-β-lactamase in-
hibitors for the treatment of carbapenem-resistant enterobacteriaceae infections
that display efficacy in an animal infection model, ACS Infect. Dis. 5 (1) (2019)
131–140.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
[23] Y. Ishii, M. Eto, Y. Mano, K. Tateda, K. Yamaguchi, In vitro potentiation of carba-
penems with ME1071, a novel metallo-β-lactamase inhibitor, against metallo-β-
lactamase- producing pseudomonas aeruginosa clinical isolates, Antimicrob. Agents
Chemother. 54 (9) (2010) 3625–3629.
This research was financially supported by the National Natural
Science Foundation of China (Nos. 21702190, 81872913) and China
Postdoctoral Science Foundation (2018M640685, 001803014,
2019T120642).
[24] A.M. Somboro, D. Tiwari, L.A. Bester, R. Parboosing, L. Chonco, H.G. Kruger,
P.I. Arvidsson, T. Govender, T. Naicker, S.Y. Essack, NOTA: a potent metallo-β-
lactamase inhibitor, J. Antimicrob. Chemother. 70 (5) (2015) 1594–1596.
[25] A.M. King, S.A. Reid-Yu, W. 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 (7506) (2014) 503–506.
[26] A. Bergstrom, A. Katko, Z. Adkins, J. Hill, Z. Cheng, M. Burnett, H. Yang, M. Aitha,
M.R. Mehaffey, J.S. Brodbelt, K.H.M.E. Tehrani, N.I. Martin, R.A. Bonomo,
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103965.
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D.-Y. Cui, et al.
BioorganicChemistry101(2020)103965
R.C. Page, D.L. Tierney, W. Fast, G.D. Wright, M.W. Crowder, Probing the inter-
H2depda: an acyclic adjuvant potentiates meropenem activity in vitro against
metallo-β-lactamase-producing enterobacterales, Eur. J. Med. Chem. 167 (2019)
367–376.
action of Aspergillomarasmine A with metallo-β-lactamases NDM-1, VIM-2, and
IMP-7, ACS Infect. Dis. 4 (2) (2018) 135–145.
[27] A.Y. Chen, P.W. Thomas, A.C. Stewart, A. Bergstrom, Z. Cheng, C. Miller,
C.R. Bethel, S.H. Marshall, 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 (17) (2017)
7267–7283.
[40] B. Shen, Y. Yu, H. Chen, X. Cao, X. Lao, Y. Fang, Y. Shi, J. Chen, H. Zheng, Inhibitor
discovery of full-length New Delhi metallo-β-lactamase-1 (NDM-1), Plos One 8
(2013) e62955.
[41] J. Hoque, M.M. Konai, S. Gonuguntla, G.B. Manjunath, S. Samaddar, V. Yarlagadda,
J. Haldar, Membrane active small molecules show selective broad spectrum anti-
bacterial activity with no detectable resistance and eradicate biofilms, J. Med.
Chem. 58 (14) (2015) 5486–5500.
[28] A. Proschak, J. Kramer, E. Proschak, T.A. Wichelhaus, Bacterial zincophore [S, S]-
ethylenediamine-N, N′-disuccinic acid is an effective inhibitor of MBLs, J.
Antimicrob. Chemother. 73 (2) (2018) 425–430.
[42] J. Hoque, M.M. Konai, S. Samaddar, S. Gonuguntala, G.B. Manjunath, C. Ghosh,
J. Haldar, Selective and broad spectrum amphiphilic small molecules to combat
bacterial resistance and eradicate biofilms, Chem. Commun. 51 (71) (2015)
13670–13673.
[29] N. Valeria Marina, C.-A. Miriam, T. Leonardo, L. Joanna Izabela, C. Guido,
Chelation therapy for metal intoxication: comments from a thermodynamic view-
point, Mini-Rev. Med. Chem. 13 (11) (2013) 1541–1549.
[30] G.J. Kontoghiorghes, K. Pattichis, K. Neocleous, A. Kolnagou, The design and de-
velopment of deferiprone (L1) and other iron chelators for clinical use: targeting
methods and application prospects, Curr. Med. Chem. 11 (16) (2004) 2161–2183.
[31] R.A. Kessler, X. Li, K. Schwartz, H. Huang, M.A. Mealy, M. Levy, Two-year ob-
servational study of deferiprone in superficial siderosis, CNS Neurosci. Ther. 24 (3)
(2018) 187–192.
[43] D.T. King, L.J. Worrall, R. Gruninger, N.C.J. Strynadka, New Delhi metallo-β-lac-
tamase: structural insights into β-lactam recognition and inhibition, J. Am. Chem.
Soc. 134 (28) (2012) 11362–11365.
[44] L. Cheng, Z. Jiang, J. Dong, B. Cai, Y. Yang, X. Li, C. Chen, Monolayers of novel
Gemini amphiphiles with phthalimide headgroups at the air/water interface: pH
and alkyl chain length effects, J. Colloid Interface Sci. 401 (2013) 97–106.
[45] Z. Jiang, X. Li, G. Yang, L. Cheng, B. Cai, Y. Yang, J. Dong, pH-Responsive surface
activity and solubilization with novel pyrrolidone-based Gemini surfactants,
Langmuir 28 (18) (2012) 7174–7181.
[32] M. Riha, P. Haskova, J. Martin, T. Filipsky, K. Vanova, J. Vavrova, M. Holeckova,
P. Homola, L. Vitek, V. Palicka, T. Simunek, P. Mladenka, Protective effects of D-
penicillamine on catecholamine-induced myocardial injury, Oxid. Med. Cell.
Longev. 2016 (2016) 5213532.
[46] P.N. Coneski, M.H. Schoenfisch, Competitive formation of N-diazeniumdiolates and
N-nitrosamines via anaerobic reactions of polyamines with nitric oxide, Org. Lett.
11 (23) (2009) 5462–5465.
[33] E. Boros, C.L. Ferreira, J.F. Cawthray, E.W. Price, B.O. Patrick, D.W. Wester,
M.J. Adam, C. Orvig, Acyclic chelate with ideal properties for 68Ga PET imaging
agent elaboration, J. Am. Chem. Soc. 132 (44) (2010) 15726–15733.
[34] W. Chu, Y. Yang, J. Cai, H. Kong, M. Bai, X. Fu, S. Qin, E. Zhang, Synthesis and
bioactivities of new membrane-active agents with aromatic linker: high selectivity
and broad-spectrum antibacterial activity, ACS Infect. Dis. 5 (9) (2019) 1535–1545.
[35] W. Chu, Y. Yang, S. Qin, J. Cai, M. Bai, H. Kong, E. Zhang, Low-toxicity amphiphilic
molecules linked by an aromatic nucleus show broad-spectrum antibacterial ac-
tivity and low drug resistance, Chem. Commun. 55 (30) (2019) 4307–4310.
[36] W.-C. Chu, P.-Y. Bai, Z.-Q. Yang, D.-Y. Cui, Y.-G. Hua, Y. Yang, Q.-Q. Yang,
E. Zhang, S. Qin, Synthesis and antibacterial evaluation of novel cationic chalcone
derivatives possessing broad spectrum antibacterial activity, Eur. J. Med. Chem.
143 (2018) 905–921.
[47] A. Mohamadi, L.W. Miller, Efficient route to pre-organized and linear poly-
aminopolycarboxylates: Cy-TTHA, Cy-DTPA and mono/di- reactive, tert-butyl
protected TTHA/Cy-TTHA, Tetrahedron Lett. 58 (15) (2017) 1441–1444.
[48] Y.-Q. Huang, Y. Zhao, P. Wang, T.-A. Okamura, B.N. Laforteza, Y. Lu, W.-Y. Sun, J.-
Q. Yu, One-pot synthesis of imidazolinium salts via the ring opening of tetra-
hydrofuran, Dalton Trans. 46 (37) (2017) 12430–12433.
[49] S.M. Kishk, K.J. McLean, S. Sood, M.A. Helal, M.S. Gomaa, I. Salama, S.M. Mostafa,
L.P.S. de Carvalho, A.W. Munro, C. Simons, Synthesis and biological evaluation of
novel cYY analogues targeting Mycobacterium tuberculosis CYP121A1, Bioorg.
Med. Chem. 27 (8) (2019) 1546–1561.
[50] Y. Yang, W. Zhong, B. Nie, J. Chen, Z. Wei, X. Liu, Synergetic oxidation of ethyl-
benzene to acetophenone catalyzed by manganese(II) complexes bearing pendant
iodophenyl groups, J. Organomet. Chem. 853 (2017) 136–142.
[37] M.-M. Wang, W.-C. Chu, Y. Yang, Q.-Q. Yang, S.-S. Qin, E. Zhang, Dithiocarbamates:
efficient metallo-β-lactamase inhibitors with good antibacterial activity when
combined with meropenem, Bioorg. Med. Chem. Lett. 28 (21) (2018) 3436–3440.
[38] E. Zhang, M.-M. Wang, S.-C. Huang, S.-M. Xu, D.-Y. Cui, Y.-L. Bo, P.-Y. Bai, Y.-
G. Hua, C.-L. Xiao, S. Qin, N.O.T.A. Analogue, a First dithiocarbamate inhibitor of
metallo-β-lactamases, Bioorg. Med. Chem. Lett. 28 (2) (2018) 214–221.
[39] X.-F. Shi, M.-M. Wang, S.-C. Huang, J.-X. Han, W.-C. Chu, C. Xiao, E. Zhang, S. Qin,
[51] V. Sharma, M.S.Y. Khan, Synthesis of novel tetrahydroimidazole derivatives and
studies for their biological properties, Eur. J. Med. Chem. 36 (7) (2001) 651–658.
[52] E. Boros, C.L. Ferreira, B.O. Patrick, M.J. Adam, C. Orvig, New Ga derivatives of the
H2dedpa scaffold with improved clearance and persistent heart uptake, Nucl. Med.
Biol. 38 (8) (2011) 1165–1174.
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