Rational Design of Dipicolylamine-Containing Carbazole Amphiphiles Combined with Zn2+as Potent Broad-Spectrum Antibacterial Agents with a Membrane-Disruptive Mechanism

Article abstract of DOI:10.1021/acs.jmedchem.1c00858

Antibiotic resistance has become one of the most urgently important problems facing healthcare providers. A novel series of dipicolylamine-containing carbazole amphiphiles with strong Zn²⁺chelating ability were synthesized, biomimicking cationic antimicrobial peptides. Effective broad-spectrum 16 combined with 12.5 μg/mL Zn²⁺was identified as the most promising antimicrobial candidate. 16 combined with 12.5 μg/mL Zn²⁺exhibited excellent antimicrobial activity against both Gram-positive and Gram-negative bacteria (MICs = 0.78-3.125 μg/mL), weak hemolytic activity, and low cytotoxicity. Time-kill kinetics and mechanism studies revealed 16 combined with 12.5 μg/mL Zn²⁺had rapid bacterial killing properties, as evidenced by disruption of the integrity of bacterial cell membranes, effectively preventing bacterial resistance development. Importantly, 16 combined with 12.5 μg/mL Zn²⁺showed excellentin vivoefficacy in a murine keratitis model caused byStaphylococcus aureusATCC29213 orPseudomonas aeruginosaATCC9027. Therefore, 16 combined with 12.5 μg/mL Zn²⁺could be a promising candidate for treating bacterial infections.

Full text of DOI:10.1021/acs.jmedchem.1c00858

pubs.acs.org/jmc
Article
Rational Design of Dipicolylamine-Containing Carbazole
Amphiphiles Combined with Zn2+ as Potent Broad-Spectrum
Antibacterial Agents with a Membrane-Disruptive Mechanism
Jiayong Liu,# Hongxia Li,# Haizhou Li, Shanfang Fang, Jinguo Shi, Yongzhi Chen, Rongcui Zhong,
Shouping Liu,* and Shuimu Lin*
Cite This: J. Med. Chem. 2021, 64, 10429−10444
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ABSTRACT: Antibiotic resistance has become one of the most
urgently important problems facing healthcare providers. A novel
series of dipicolylamine-containing carbazole amphiphiles with
strong Zn2+ chelating ability were synthesized, biomimicking
cationic antimicrobial peptides. Effective broad-spectrum 16
combined with 12.5 μg/mL Zn2+ was identified as the most
promising antimicrobial candidate. 16 combined with 12.5 μg/mL
Zn2+ exhibited excellent antimicrobial activity against both Gram-
positive and Gram-negative bacteria (MICs = 0.78−3.125 μg/mL),
weak hemolytic activity, and low cytotoxicity. Time-kill kinetics
and mechanism studies revealed 16 combined with 12.5 μg/mL
Zn2+ had rapid bacterial killing properties, as evidenced by disruption of the integrity of bacterial cell membranes, effectively
preventing bacterial resistance development. Importantly, 16 combined with 12.5 μg/mL Zn2+ showed excellent in vivo efficacy in a
murine keratitis model caused by Staphylococcus aureus ATCC29213 or Pseudomonas aeruginosa ATCC9027. Therefore, 16
combined with 12.5 μg/mL Zn2+ could be a promising candidate for treating bacterial infections.
Cationic antimicrobial peptides (CAMPs) are widely
distributed in various organisms as immune response products
and possess a variety of biological activities.13−15 The cationic
amphiphilic conformation of CAMPs plays a vital role in their
antimicrobial effects.16,17 The cationic region of CAMPs is
attracted by negatively charged bacterial cell membranes via
electrostatic interactions, and then the lipophilic/hydrophobic
region of CAMPs is inserted into the lipid bilayer of the
bacterial cell membranes to penetrate or destroy the
membrane, leading to the leakage of intracellular contents
and bacterial death.14,18−20 This membrane-targeting mode of
action is very helpful in greatly reducing the probability of
bacterial resistance.14,21,22 However, poor in vivo stability and
efficacy, poor pharmacokinetic properties, high cytotoxicity,
and expensive preparation costs are common and notable
obstacles for the use of CAMPs as antimicrobials in clinical
applications.23 Recently, research on small molecule mimetics
of CAMPs has provided a very promising solution to overcome
INTRODUCTION
■
Antibiotic resistance has become a global health challenge and
poses a serious threat to human health.1−3 It is reported that
approximately 700,000 deaths worldwide are caused by drug-
resistant bacterial infections each year, including more than
23,000 deaths in the United States and 175,000 deaths in the
European Union.4−6 Since the 1980s, the discovery and
development of new antibacterial agents have been very slow,
and the number of newly approved antibacterial agents has
been severely reduced.7,8 In addition, no new drugs have been
approved for treating Gram-negative bacterial infections in the
past three decades.7,8 Gram-negative bacteria have special
outer membranes that consist of a lipopolysaccharide (LPS)
layer and membrane phospholipids, making it very difficult for
most antibiotics to penetrate the cell membranes to show
effective antibacterial efficacy.9,10 In a global priority list of
antibiotic-resistant bacteria published by the World Health
Organization in 2017, the listed bacteria of critical priority are
all Gram-negative bacteria, including carbapenem-resistant
Acinetobacter baumannii and Pseudomonas aeruginosa, as well
as extended-spectrum β-lactamase (ESBL)-producing carbape-
nem-resistant Enterobacteriaceae.11 Therefore, in the current
serious situation, it is urgent to develop new broad-spectrum
antibacterial agents that can overcome drug-resistant bacterial
infections.12
Received: May 12, 2021
Published: July 8, 2021
https://doi.org/10.1021/acs.jmedchem.1c00858
© 2021 American Chemical Society
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these problems associated with CAMPs.24,25 Our group and
other laboratories previously discovered and optimized
flavone,26,27 xanthone,28,29 and aminoglycoside30,31-based
peptidomimetics by biomimicking the structure and function
of CAMPs. Several successful examples, such as LTX-109 and
brilacidin, have entered or finished phase 2 clinical trials.32,33
Carbazole derivatives have many pharmacological activ-
ities,34−37 including anti-inflammatory,38 antibacterial,39 anti-
fungal,40 antitumor,41 antioxidant activities,42 etc. The
commercial antihypertensive drugs carazolol and carvedilol
possess carbazole scaffolds.36,37 In our previous work, we used
4-epoxypropoxycarbazole as the starting material to design and
synthesize a series of carbazole-based antimicrobial agents by
biomimicking CAMPs.43 The lead compound (Figure 1)
antibacterial compounds, including daptomycin45−47 and
quinolones48,49 can form metal ion complexes by interacting
with metal ions (e.g., Ca2+, Zn2+, and Mg2+) to significantly
enhance their antibacterial activity. Metal ions that act as
electron acceptors show stronger electron-accepting abilities
than H+.50 The surface of bacterial cell membranes contains
anionic phospholipids, such as phosphatidylglycerol (PG) and
cardiolipin (CL).51 After the ligand and metal ions form
complexes, the positive charge density of the ligand is
enhanced.44,50 The formed complexes can strongly bind to
anionic phospholipids on bacterial cell membranes.44,52 Zn2+
works as an essential trace element in the human body and
participates in different types of life activities.53 It is estimated
that the total concentration of Zn2+ in mammalian cells is
approximately 6.54−32.69 μg/mL.54,55 It has been reported
that Zn2+ can specifically combine with dipicolylamine to form
a Zn2+-dipicolylamine (Zn-DPA) complex that binds to
pyrophosphate with high affinity.52,56 In a positively charged
metal complex, Zn2+ can effectively interact with negatively
charged membranes of both Gram-positive and Gram-negative
bacterial cells.43,57,58 As a selective membrane-targeting agent,
the Zn-DPA complex has been widely used in cancer
detection,59 the treatment of neurodegenerative diseases,60
and bacterial infections.43,57,58
Inspired by the amphiphilic structure and function of
CAMPs as well as the high affinity between the Zn-DPA
complex and pyrophosphate, we designed a new series of
amphiphilic dipicolylamine-containing carbazole derivatives
combined with Zn2+ as antibacterial agents. In this study, we
used 4-epoxypropoxycarbazole as the starting material, which is
a commercially available intermediate for the preparation of
carvedilol and carazolol,36,37 and different hydrophobic lipid
chains were introduced into the carbazole scaffold to adjust the
hydrophobicity of the carbazole compounds (Scheme 1). The
cationic group of 2,2′-dipicolylamine with a strong chelating
ability for Zn2+ was incorporated into the carbazole scaffold to
yield a series of cationic carbazole amphiphiles. Based on the
structural properties and hypothesis mentioned above, the
designed compounds combined with Zn2+ were expected to
overcome the limitation of permeation/diffusion through the
outer membrane barrier (LPS layer) of Gram-negative bacteria.
In the presence of Zn2+, the designed dipicolylamine-
containing carbazole compounds display significantly enhanced
antibacterial activity against both Gram-positive and Gram-
negative bacteria. After a series of structural optimization and
biological activity evaluations, compound 16 combined with
Zn2+ was identified as the most promising antimicrobial
candidate that displayed excellent broad-spectrum antibacterial
Figure 1. Chemical structure of the carbazole-based lead compound.
containing guanidine and isoprenyl groups exhibited excellent
in vitro and in vivo anti-Gram-positive bacterial activity, killed
bacteria rapidly by destroying the bacterial membranes, and
effectively avoided the development of bacterial resistance.43
However, these cationic carbazole-based compounds displayed
poor antibacterial activity against Gram-negative bacteria.43
The main reason may be that these synthesized carbazoles had
poor penetration/diffusion ability through the outer membrane
barrier (LPS layer) of Gram-negative bacteria, making it very
difficult for them to enter the cell membranes of Gram-
negative bacteria to exert antibacterial activity.43
Metal ion complexes in biological systems have been widely
studied due to their unique structures and properties.44 Several
Scheme 1. Design Concept for Amphiphilic Dipicolylamine-Containing Carbazole Derivatives Combined with Zn2+ as
Antibacterial Agents by Biomimicking CAMPs
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a
Scheme 2. Synthesis of Carbazole-Based Compounds 2−16
a
Reagents and conditions: (i) 1-iodoalkanes, NaOH, DMF, 60 °C, 2 h. (ii) 3, 3-dimethylallyl bromide, KI, potassium tert-butoxide, DMF, 50 °C,
0.5 h. (iii) 2,2′-dipicolylamine, MeOH, reflux, 6 h.
a
Scheme 3. Synthesis of Carbazole-Based Compounds 17−19
a
Reagents and conditions: (i) corresponding amines, MeOH, reflux, 6 h.
activity and weak hemolytic activity. The time-killing curve,
development of drug resistance, mode of action, in vitro
cytotoxicity toward mammalian cells, and in vivo efficacy in a
murine bacterial keratitis model were further studied. This new
design strategy provides a new solution for the discovery and
development of effective broad-spectrum antibacterial agents
to combat bacterial resistance.
spectrometry, as indicated by the peak at m/z 569.3 (Figure
S1, Supporting Information).
In Vitro Antibacterial and Hemolytic Activity. The
minimum inhibitory concentrations (MICs) were used to
evaluate the in vitro antibacterial activity of these synthetic
carbazole derivatives in the presence of different concen-
trations of Zn2+ (0, 6.25, and 12.5 μg/mL) against a panel of
Gram-positive and Gram-negative bacteria, including S. aureus
ATCC29213, MRSA NCTC10442, MRSA N315, E. coli
ATCC25922, P. aeruginosa ATCC9027, Acinetobacter bauman-
nii ATCC17978, and A. baumannii R2889 (Tables 1 and 2 and
Table S1). Commercially available vancomycin and cipro-
floxacin were used as positive controls. The hemolytic activity
was evaluated by HC50 values that were measured as the
concentration required to lyse 50% of rabbit red blood cells.
Evaluation of the Influence of Zn2+ on the Activity.
Zn2+, as a strong electron-acceptor, could greatly enhance the
electron absorption property of cationic moieties of carbazole
derivatives after being chelated to dipicolylamine groups,
leading to a significant increase in their interaction with LPS or
lipoteichoic acid (LTA), as well as negatively charged bacterial
cell membranes. As shown in Table 2 and Table S1,
compounds 9−16 with a cationic group of dipicolylamine
combined with Zn2+ (6.25 or 12.5 μg/mL) showed a
significant enhancement in antibacterial activity against
RESULTS AND DISCUSSIONS
■
Design and Synthesis of Carbazole-Based Deriva-
tives. In this study, we further designed and modified cationic
amphiphilic carbazole derivatives for screening and identi-
fication of broad-spectrum antimicrobial compounds to
overcome bacterial resistance. The chemical structures and
synthetic routes of a new series of dipicolylamine-containing
carbazole derivatives are shown in Schemes 2 and 3. Starting
material 1 (4-epoxypropanoxycarbazole) was reacted with 3,3-
dimethylallyl bromide or 1-iodoalkane in the presence of
K2CO3 to yield intermediate compounds 2−8. Then,
compounds 1−8 were treated with 2,2′-dipicolylamine in
methanol to give compounds 9−16. Compounds 17−19 were
prepared by the treatment of compound 8 with the
corresponding amines. All synthesized compounds were
characterized by H NMR, C NMR, and HRMS. The formation
of compound 16-Zn2+ complexes was validated by mass
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Table 1. Antibacterial and Hemolytic Activities of 12.5 μg/mL Zn2+ and Compounds 1−19
MIC (μg/mL)
S. aureus ATCC
MRSA
N315
MRSA
NCTC10442
E. coli
ATCC25922
P. aeruginosa
ATCC9027
A. baumannii
ATCC17978
A. baumannii
HC50
(μg/mL)
compd
29213
R2889
Zn2+
1
2
3
4
5
6
7
>50
>50
>50
>50
>50
>50
>50
>50
>50
25
12.5
3.125
1.56
1.56
3.125
3.125
3.125
6.25
6.25
6.25
0.78
>50
>50
>50
>50
>50
>50
>50
>50
>50
25
6.25
1.56
1.56
1.56
0.78
3.125
1.56
6.25
6.25
6.25
1.56
0.39
>50
>50
>50
>50
>50
>50
>50
>50
>50
25
12.5
3.125
1.56
1.56
6.25
1.56
1.56
6.25
6.25
6.25
1.56
0.39
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
50
12.5
6.25
>50
>50
12.5
6.25
25
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.195
>50
>50
>50
>50
>50
>50
>50
>50
>50
50
12.5
6.25
3.125
>50
>50
6.25
3.125
12.5
50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
25
6.25
3.125
25
>50
6.25
3.125
12.5
25
>200
>200
>200
>200
>200
>200
>200
NDa
>200
>200
>200
>200
>200
>200
>200
>200
8
9
10
11
12
13
14
15
16
17
18
19
>200
119.4 2.9
88.7 4.4
>200
>50
50
>50
0.049
>50
50
0.39
>50
>50
0.78
b
a
VAN
ND
ND
c
a
CIP
0.39
a
b
c
Not determined. VAN = vancomycin. CIP = ciprofloxacin.
Table 2. Antibacterial and Hemolytic Activities of Compounds 9−19 Combined with 12.5 μg/mL Zn2+
MIC (μg/mL)
compd with 12.5 μg/ S. aureus ATCC
MRSA
N315
MRSA
NCTC10442
E. coli
ATCC25922
P. aeruginosa
ATCC9027
A. baumannii
ATCC17978
A. baumannii
HC50
(μg/mL)
mL Zn2+
29213
R2889
9
6.25
3.125
0.78
1.56
1.56
3.125
1.56
1.56
6.25
6.25
3.125
0.78
0.39
6.25
1.56
0.78
0.78
0.78
0.78
1.56
0.78
6.25
6.25
3.125
1.56
12.5
6.25
1.56
0.78
1.56
6.25
0.78
1.56
6.25
6.25
6.25
1.56
0.39
>50
50
12.5
12.5
6.25
6.25
>50
50
12.5
3.125
3.125
>50
12.5
3.125
0.78
12.5
50
>50
25
6.25
3.125
25
>50
3.125
1.56
12.5
25
>50
>50
0.78
>200
>200
>200
>200
>200
>200
>200
>200
10
11
12
13
14
15
16
17
18
19
6.25
1.56
3.125
>50
3.125
1.56
25
>50
12.5
3.125
>50
>50
>50
119.4 2.9
88.7 4.4
>200
50
>50
>50
0.049
>50
50
0.39
a
b
VAN
CIP
>50
0.195
ND
ND
c
b
0.39
a
b
c
VAN = vancomycin. Not determined. CIP = ciprofloxacin.
Gram-negative bacteria, especially P. aeruginosa ATCC9027.
To investigate the effect of different hydrophobicities on
antibacterial and hemolytic activities, dipicolylamine-contain-
ing compounds 10−16 were synthesized, in which the alkyl
chain was varied (R = −CH3, −C3H7, −C5H11, −C7H15,
−C9H19, isoprenyl group, and −C4H6F3). Compound 9
without alkyl chains was also prepared. As shown in Tables
1 and 2 and Table S1, compound 9 without alkyl chains
showed poor antibacterial activity against both Gram-positive
bacteria (MICs = 25 μg/mL) and Gram-negative bacteria
short lipophilic alkyl chain (R = −CH3), displayed stronger
antibacterial activity than compound 9. The MIC values of
compound 10 against Gram-positive bacteria were in the range
of 6.25−12.5 μg/mL, which were 2- to 4-fold reduced (1.56−
6.25 μg/mL) after the addition of Zn2+ (6.25 or 12.5 μg/mL).
However, its antibacterial activity against Gram-negative
bacteria was still weak. Compounds 11−16 with increased
lipid chains exhibited excellent antibacterial activity against
Gram-positive bacteria (MICs = 0.78−6.25 μg/mL), and their
anti-Gram-positive bacterial activity could be further increased
by 1−2 times in the presence of Zn2+. Compounds 11−16
displayed very poor antibacterial activity against P. aeruginosa
(MICs >50 μg/mL). However, compounds 11−12 and 15−16
showed good activity (MICs = 3.125−12.5 μg/mL) against
(MICs ≥50 μg/mL) and very poor hemolytic activity (HC50
>
200 μg/mL). When combined with Zn2+ (6.25 or 12.5 μg/
mL), the antibacterial activity of compound 9 was increased
1−4 times. Compound 10, which was incorporated with a
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Table 3. MIC Values (μg/mL) of Compound 16 in the Presence of Various Concentrations of Zn2+
MIC (μg/mL)
concentrations of Zn2+ (μg/mL)
E. coli ATCC25922
P. aeruginosa ATCC9027
MRSA NCTC10442
S. aureus ATCC29213
50
25
1.56
1.56
1.56
1.56
3.125
6.25
6.25
6.25
6.25
3.125
3.125
3.125
3.125
12.5
50
>50
>50
>50
0.78
0.78
0.78
0.78
1.56
1.56
1.56
1.56
1.56
0.78
1.56
1.56
1.56
3.125
3.125
3.125
3.125
3.125
12.5
6.25
3.125
1.56
0.78
0.39
a
control
a
Treatment with compound 16 only.
Gram-negative bacteria (E. coli and A. baumannii). Com-
pounds 13−14 containing longer lipid chains (R = −C7H15
and −C9H19) displayed obviously significantly reduced activity
against E. coli and A. baumannii, with MIC values of ≥25 and
>50 μg/mL, respectively.
Gram-positive bacteria (MICs = 1.56−6.25 μg/mL). However,
compounds 17−19 displayed weak or poor antibacterial
activity against Gram-negative bacteria. After the addition of
6.25−12.5 μg/mL Zn2+, their antibacterial activities were not
changed (Table 2 and Table S1). The reason for this could be
that the cationic moieties of compounds 17−19 were
incapable of chelating with Zn2+. Only compound 16 showed
good antibacterial activity against the E. coli and A. baumannii
strains tested, with MICs of 3.125−6.25 μg/mL. Notably, the
antibacterial activity of compound 16 against both Gram-
positive and Gram-negative bacteria was greatly enhanced after
the addition of 6.25−12.5 μg/mL Zn2+, including P. aeruginosa
ATCC9027, with MICs of 0.78−3.125 μg/mL.
However, after chelating with Zn2+, the antibacterial activity
of compounds 9−12 and 15−16 against P. aeruginosa
ATCC9027 was significantly improved from MICs of >50
μg/mL to MICs of 3.125−12.5 μg/mL. When combined with
Zn2+ (6.25 or 12.5 μg/mL), the antibacterial activity of
compound 16 against E. coli and A. baumannii was also 2−4
times increased compared with that without Zn2+. Compound
14 with the longest lipophilic chain showed poor activity
against Gram-negative bacteria, and its MIC values remained
almost the same after the addition of Zn2+. This result
indicated that an excessively long lipophilic alkyl chain was
detrimental to the biological activity of carbazole derivatives
because an excessively long lipophilic alkyl chain would destroy
the hydrophilic and hydrophobic balance of the amphiphilic
carbazole compounds, resulting in reduced antibacterial
activity. Compound 12 containing an n-pentyl group and
compound 16 containing an isoprenyl group displayed the
most promising antibacterial activity against Gram-positive and
Gram-negative (except P. aeruginosa) bacteria (MICs = 1.56−
6.25 μg/mL), suggesting that a medium-length alkyl chain was
conducive to the balance of hydrophilicity and hydrophobicity
of carbazole derivatives, leading to a significant increase in
antibacterial activity. When combined with Zn2+ (6.25 or 12.5
μg/mL), compound 16 with an isoprenyl group showed the
best antibacterial activity against both Gram-positive (MICs =
0.78−1.56 μg/mL) and Gram-negative bacteria (MICs =
0.78−3.125 μg/mL) among all synthesized carbazole com-
pounds. Notably, compound 16 combined with Zn2+ also
displayed potent antibacterial activity against P. aeruginosa
(MIC = 3.125 μg/mL). These results indicated that the
incorporation of an isoprenyl group was conducive to the
enhancement of the antibacterial activity of dipicolylamine-
containing carbazole derivatives, whether complexed with
Zn2+. In our previous reports, the isoprenyl group was also
considered to be the best hydrophobic alkane chain for
enhancing the interaction between the modified amphiphilic
compounds and the phospholipid layer of the bacterial cell
membranes.29
Effect of Various Concentrations of Zn2+ on the
Activity of 16. The effect of different concentrations of Zn2+
on the antibacterial activity of compound 16 was explored. As
shown in Table 3, when the added concentration of Zn2+ was
more than 3.125 μg/mL, an obvious increase in antibacterial
activity was observed. When the added concentration of Zn2+
was less than 3.125 μg/mL, the MIC values remained
unchanged. These results indicated that the incorporation of
a dipicolylamine group as the cationic moiety would
significantly enhance the antibacterial activity of carbazole
compounds against both Gram-positive and Gram-negative
bacteria in the presence of Zn2+.
After preliminary structure−activity relationship study, we
found that the incorporation of dipicolylamine can form a Zn-
DPA complex in the presence of Zn2+ and then could
efficiently interact with the negatively charged bacterial
membranes of Gram-positive and Gram-negative bacteria
through electrostatic interaction. Among all the lipid chains
investigated, the introduction of isoprenyl group is more
conducive to improving the antibacterial activity. This finding
is consistent with our earlier studies.29,43 The reason may be
that the isoprenyl group can promote the insertion of the
amphiphilic carbazole derivatives into the phospholipid bilayer
of bacterial membrane more efficiently through hydrophobic
interaction. So far, this study has illustrated the strategy of
designing and synthesizing a series of carbazole-based CAMPs
mimics as broad-spectrum antibacterial agents. Their activities
are determined by several structural parameters, including
amphiphilicity, positive charge of cationic groups, and
hydrophobic lipid chains.
To study the effect of different cationic groups on the
antibacterial and hemolytic activity, compounds 16−19
containing the same hydrophobic chain of the isoprenyl
group were designed and synthesized. As shown in Table 1,
compounds 16−19 had good antibacterial activity against
In Vitro Salt Resistance Study. The stability of
compound 16 combined with 12.5 μg/mL Zn2+ in the
presence of various salt conditions was evaluated by
determining the MIC changes. Compound 16 combined
with 12.5 μg/mL Zn2+ was incubated with bacterial
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Table 4. MIC Values (μg/mL) of Compound 16 Combined with 12.5 μg/mL Zn2+ under Physiological Concentrations of
Various Salts
MIC (μg/mL)
condition
S. aureus ATCC29213
MRSA N315
MRSA NCTC10442
E. coli ATCC25922
A. baumannii ATCC17978
a
control
1.56
1.56
1.56
3.125
1.56
0.78
0.78
0.78
3.125
1.56
1.56
1.56
1.56
3.125
1.56
1.56
3.125
1.56
6.25
3.125
0.78
0.78
0.78
3.125
1.56
3220 μg/mL Na+
175.5 μg/mL K+
100 μg/mL Ca2+
24.3 μg/mL Mg2+
a
Treatment with compound 16 combined with 12.5 μg/mL Zn2+ only.
Figure 2. Bacterial killing kinetics of compound 16 combined with 12.5 μg/mL Zn2+ against S. aureus ATCC29213 (A) and E. coli ATCC25922
(B). Data are presented as means s.d. from two independent experiments.
Figure 3. Cytoplasmic membrane permeabilization of compound 16 combined with 12.5 μg/mL Zn2+ against S. aureus ATCC29213 (A) and E. coli
ATCC25922 (B) using a SYTOX-Green assay. The data are representative of two independent experiments.
suspensions (S. aureus ATCC29213, MRSA NCTC10442,
MRSA N315, E. coli ATCC25922, or A. baumannii
ATCC17978) using Mueller Hinton broth (MHB) containing
physiological concentrations of 3220 μg/mL Na+, 175.5 μg/
mL K+, 100 μg/mL Ca2+, or 24.3 μg/mL Mg2+, respectively. As
shown in Table 4, the MIC values of compound 16 combined
with 12.5 μg/mL Zn2+ under physiological concentrations of
Na+, K+, or Mg2+ were almost consistent with the original
values (MICs = 0.78−3.125 μg/mL). After the addition of 100
μg/mL Ca2+, compound 16 combined with 12.5 μg/mL Zn2+
maintained its potent antibacterial activity, with MIC values of
3.125−6.25 μg/mL. However, the MIC values of compound
16 combined with 12.5 μg/mL Zn2+ in the presence of 100
μg/mL Ca2+ were 1- to 2-fold reduced compared with their
original values. These results demonstrated that compound 16
combined with 12.5 μg/mL Zn2+ had very high salt resistance
on the antibacterial activity and could overcome the instability
issue associated with CAMPs at physiological salt concen-
trations.
Bacterial Killing Kinetic Evaluation. The bacterial killing
kinetics of the most promising compound 16 combined with
Zn2+ were evaluated against S. aureus ATCC29213 and E. coli
ATCC25922. A suspension of S. aureus ATCC29213 or E. coli
ATCC25922 was incubated with different concentrations of
compound 16 (4× and 8× MIC) combined with 12.5 μg/mL
Zn2+ at 37 °C. As shown in Figure 2, 4× MIC compound 16
combined with 12.5 μg/mL Zn2+ obtained a 3.03 log reduction
in the bacterial counts of S. aureus ATCC29213 and a 2.91 log
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Figure 4. Membrane depolarization against S. aureus ATCC29213 (A) and E. coli ATCC25922 (B) using DiSC3 (5) dye as a fluorescence probe.
The data are representative of two independent experiments.
Figure 5. Fluorescence image of S. aureus ATCC29213 or E. coli ATCC25922 cells stained with SYTO 9 and propidium iodide after treatment with
PBS or 8× MIC compound 16 combined with 12.5 μg/mL Zn2+: (A) PBS-treated S. aureus ATCC29213 cells, (B) compound 16 combined with
12.5 μg/mL Zn2+-treated S. aureus ATCC29213 cells; (C) PBS-treated E. coli ATCC25922 cells, (D) compound 16 combined with 12.5 μg/mL
Zn2+-treated E. coli ATCC25922 cells. The figures are representative of two independent experiments.
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reduction in the bacterial counts of E. coli ATCC25922 within
1 h. Additionally, 8× MIC compound 16 combined with 12.5
μg/mL Zn2+ achieved a more than 3 log reduction in the
bacterial counts of both S. aureus ATCC29213 (4.22 log) and
E. coli ATCC25922 (3.59 log) within 0.5 h. These results
indicated that compound 16 combined with Zn2+ displayed a
rapid bactericidal property. This rapid bactericidal property is
beneficial to shorten the treatment time of infectious diseases
and reduce the probability of drug resistance.
Antibacterial Mechanism Studies (Disruption of
Bacterial Cell Membrane Integrity). Because compound
16 combined with 12.5 μg/mL Zn2+ showed the best in vitro
antibacterial activity, very low hemolytic activity, rapid
bactericidal property, and high salt resistance, we used
compound 16 combined with 12.5 μg/mL Zn2+ to explore
the antibacterial mechanism. Most antimicrobial peptidomi-
metics were reported to act on bacteria via a membrane-
disruptive mechanism.24,25 To study the mode of action for
compound 16 combined with 12.5 μg/mL Zn2+ against both
Gram-positive and Gram-negative bacteria, we used various
fluorescent probes, including SYTOX Green, DiSC3 (5),
SYTO 9, propidium iodide (PI), 1-N-phenylnaphthylamine
(NPN), and BODIPY-TR-cadaverine.
Cytoplasmic Membrane Permeabilization. SYTOX
Green dye is commonly used to measure the integrity of
bacterial cell membranes. SYTOX Green cannot pass through
intact cell membranes but can enter disrupted cell membranes
and combine with intracellular nucleic acids to significantly
enhance fluorescence.61 The effect of compound 16 in the
presence of 12.5 μg/mL Zn2+ on the permeabilization of
bacterial cell membranes was evaluated by measuring the
change in fluorescence. As shown in Figure 3, when compound
16 combined with 12.5 μg/mL Zn2+ was added to SYTOX
Green-treated S. aureus ATCC29213 or E. coli ATCC25922, a
significant increase in fluorescence intensity was observed in an
obvious concentration-dependent manner. The results dem-
onstrated that compound 16 combined with 12.5 μg/mL Zn2+
killed both Gram-positive and Gram-negative bacteria by
destroying the integrity of the bacterial cell membranes in a
concentration-dependent manner.
green.63 The bacterial suspensions of S. aureus ATCC29213 or
E. coli ATCC25922 were mixed with 8× MIC compound 16
combined with 12.5 μg/mL Zn2+ or PBS (negative control).
After 2 h of incubation at 37 °C, these bacterial cells were
stained with PI and SYTO 9. As shown in Figure 5, both S.
aureus ATCC29213 and E. coli ATCC25922 cells treated with
compound 16 combined with 12.5 μg/mL Zn2+ were stained
with strong red fluorescence and negligible green fluorescence,
suggesting that the membrane integrity of both S. aureus
ATCC29213 and E. coli ATCC25922 cells had been obviously
damaged. The PBS-treated groups were stained with only
strong green fluorescence, indicating that bacterial membranes
were intact. These results demonstrated that compound 16
combined with 12.5 μg/mL Zn2+ effectively acted on both
Gram-positive and Gram-negative bacteria by destroying the
integrity of the bacterial membranes.
Outer Membrane Permeability. The hydrophobic
fluorescent probe NPN is commonly used to explore the
interactions of antimicrobial agents with the outer membranes
of Gram-negative bacteria (e.g., E. coli). NPN cannot pass
through the intact outer membranes of bacterial cells, but
when the outer membranes of bacterial cells are damaged,
NPN can freely enter the phospholipid layer and cause a
significant increase in fluorescence intensity.64 As shown in
Figure 6, after compound 16 combined with 12.5 μg/mL Zn2+
Cytoplasmic Membrane Depolarization Ability. To
further verify the membrane-targeting mechanism of com-
pound 16 combined with 12.5 μg/mL Zn2+, DiSC3 (5) was
used to evaluate its effect on the depolarization of bacterial cell
membranes. DiSC3 (5), as a membrane potential-sensitive
probe, accumulated in the phospholipid bilayer and then self-
quenched; when the membrane potential was changed, the
fluorescence intensity sharply increased.62 As shown in Figure
4, after compound 16 combined with 12.5 μg/mL Zn2+ was
added to the DiSC3 (5)-treated Gram-positive bacterium S.
aureus ATCC29213 or Gram-negative bacterium E. coli
ATCC25922, a significant increase in fluorescence intensity
was observed. These results indicated that the membrane
potential of the bacterial cell membranes was depolarized by
compound 16 combined with 12.5 μg/mL Zn2+, demonstrat-
ing that compound 16 combined with 12.5 μg/mL Zn2+ acted
on both Gram-positive and Gram-negative bacteria via a
membrane targeting mode of action.
Figure 6. Outer membrane permeabilization of compound 16
combined with 12.5 μg/mL Zn2+ against E. coli ATCC25922 using
the probe 1-N-phenylnaphthylamine. The data are representative of
two independent experiments.
was added to NPN-treated E. coli ATCC25922, a significant
increase in fluorescence intensity was observed. This result
revealed that compound 16 in the presence of 12.5 μg/mL
Zn2+ can penetrate and destroy the outer membranes of E. coli
in a concentration-dependent manner, demonstrating that
compound 16 combined with 12.5 μg/mL Zn2+ exerted an
antibacterial effect against Gram-negative bacteria through a
membrane-targeting mode of action.
LPS Binding. The binding affinity between compound 16
combined with 12.5 μg/mL Zn2+ and LPS was investigated by
determining the displacement of BODIPY-TR-cadaverine
bonded to LPS. When the probe was bonded to LPS,
quenching of the fluorescence intensity was observed, but
when the probe was replaced and redissolved in solution, a
significant increase in fluorescence was observed.65,66 As shown
Membrane Integrity Analysis. A LIVE/DEAD BacLight
bacterial viability kit L7007 composed of SYTO 9 and PI was
used to measure the membrane integrity of bacterial cells.
Bacterial cells with damaged membranes will be stained red,
while bacterial cells with intact membranes will be stained
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Figure 7. Determination of BODIPY-TR-cadaverine displacement from lipopolysaccharides (LPS) by compound 16 without Zn2+ (A) or
combined with 12.5 μg/mL Zn2+ (B). BODIPY-TR-cadaverine displacement from LPS was calculated using the formula below: % Displacement
from LPS = [(Fcompound − Fblank)/(Fmax − Fblank)]×100, where Fblank is the fluorescence intensity of BODIPY-TR-cadaverine with only LPS, and Fmax
is the fluorescence intensity of BODIPY-TR-cadaverine without LPS and compounds. Data are presented as means s.d. from two independent
experiments.
Figure 8. Bacterial resistance studies of compound 16 combined with 12.5 μg/mL Zn2+ against S. aureus ATCC29213 (A) and E. coli ATCC25922
(B). Norfloxacin and amoxicillin were selected as comparisons for S. aureus ATCC29213 and E. coli ATCC25922, respectively. The data are
representative of two independent experiments.
in Figure 7, BODIPY-TR-cadaverine displacement from LPS
by compound 16 combined with 12.5 μg/mL Zn2+ was
significantly increased compared with that of compound 16
without Zn2+. The BODIPY-TR-cadaverine displacement from
LPS by 1× MIC compound 16 combined with 12.5 μg/mL
Zn2+ was 20.78 1.03%. However, when the concentration of
compound 16 was increased to 16× MIC (combined with 12.5
μg/mL Zn2+), BODIPY-TR-cadaverine displacement from
Gram-negative bacteria, leading to the destruction of bacterial
cell membrane integrity and bacterial death.
Tendency to Develop Bacterial Resistance. The
development of antibiotic resistance has become a crucial
evaluation factor for the discovery and development of new
antibiotics.2 Compound 16 combined with 12.5 μg/mL Zn2+
was selected to evaluate drug resistance development toward S.
aureus and E. coli, using multipassage resistance selection
studies.43,67 Compound 16 combined with 12.5 μg/mL Zn2+
or norfloxacin was continuously exposed to S. aureus
ATCC29213 at subinhibitory concentrations of antimicrobials.
As shown in Figure 8A, after 19 days, the fold change of MICs
of compound 16 combined with 12.5 μg/mL Zn2+ against S.
aureus ATCC29213 remained unchanged and stable, suggest-
ing that compound 16 combined with 12.5 μg/mL Zn2+ can
effectively avoid the development of bacterial resistance. After
17 days, the fold change in the MICs of norfloxacin was
increased by 32-fold, indicating that the bacterial resistance
development induced by norfloxacin was significant. The drug
LPS was greatly enhanced (91.04
5.77%), indicating that
the interaction between LPS and compound 16 combined with
12.5 μg/mL Zn2+ was strong and concentration-dependent.
However, compound 16 without Zn2+ showed a very weak
affinity for LPS, which was in accordance with its poor anti-
Gram-negative bacterial activity. These results proved that the
addition of Zn2+ can cause a strong interaction between
compound 16 and the LPS of Gram-negative bacteria, and
then compound 16 combined with Zn2+ could pass through
the LPS layer to interact with the bacterial membranes of
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resistance of E. coli ATCC25922 to compound 16 combined
with 12.5 μg/mL Zn2+ or amoxicillin during serial passaging in
the presence of subinhibitory concentrations of antimicrobials
is displayed in Figure 8B, after 15 days, the MIC value of
amoxicillin increased to 16-fold, suggesting that the bacterial
resistance caused by amoxicillin was obvious. By contrast, after
19 days, the antibacterial potency against E. coli ATCC25922
of compound 16 combined with 12.5 μg/mL Zn2+ was
unaltered, indicating that compound 16 combined with 12.5
μg/mL Zn2+ can effectively prevent resistance development.
The avoidance of bacterial resistance was one of the
advantages of compound 16 combined with Zn2+ over
commercial antibiotics and might be associated with its
membrane-active mode of action.
fibroblasts (NCTC clone 929) was maintained at 71.58
5.01%. This result indicated that compound 16 in the presence
of 12.5 μg/mL Zn2+ had very low cytotoxicity toward
mammalian cells.
In Vivo Antibacterial Efficacy. Finally, the therapeutic
potential of compound 16 combined with Zn2+ as a broad-
spectrum antimicrobial agent was evaluated using a murine
bacterial keratitis model induced by S. aureus ATCC29213 or
P. aeruginosa ATCC9027. In the S. aureus ATCC29213-
induced corneal infection model, the mice were immunosup-
pressed by intraperitoneal injections of cyclophosphamide
(100 mg/kg) three times 5 days before infection. One day after
infection, each group of mice (n = 5) was topically treated with
0.5% compound 16 combined with 12.5 μg/mL Zn2+, 5%
vancomycin (positive control), or 5% glucose solution
(negative control) four times daily for 3 days. As shown in
Figure 10A, compared with the negative group, the count of
viable bacteria in the infected cornea for 0.5% compound 16
combined with Zn2+ and 5% vancomycin were reduced by 6.5
log (p < 0.005) and 5.4 log (p < 0.005), respectively. This
result suggested that compound 16 in the presence of 12.5 μg/
mL Zn2+ had excellent in vivo efficacy against S. aureus
ATCC29213, and its efficacy was superior to that of
vancomycin.
In Vitro Cytotoxicity Evaluation. The in vitro cytotoxicity
of compound 16 combined with 12.5 μg/mL Zn2+ toward
mammalian cells was determined using a CCK-8 assay.43 As
shown in Figure 9, even with compound 16 in the presence of
In the P. aeruginosa ATCC9027-induced keratitis model, the
mice were not immunosuppressed, and 0.3% gatifloxacin was
used as a positive control. As shown in Figure 10B, compared
with the negative group, 0.5% compound 16 combined with
12.5 μg/mL Zn2+ and 0.3% gatifloxacin reduced the count of
viable bacteria in the infected cornea by 4.46 log (p < 0.02)
and 3.03 log (p < 0.02), respectively. This result indicated that
0.5% compound 16 combined with 12.5 μg/mL Zn2+ had
better in vivo efficacy against P. aeruginosa ATCC9027 than
0.3% gatifloxacin. In vivo efficacy studies demonstrated that
compound 16 combined with Zn2+ holds great potential as an
effective broad-spectrum antimicrobial agent for the treatment
of bacterial infections caused by both Gram-positive and
Gram-negative bacteria.
Figure 9. Cytotoxicity of compound 16 combined with 12.5 μg/mL
Zn2+ toward mouse fibroblasts (NCTC clone 929).
12.5 μg/mL Zn2+ at the highest tested concentration of 25 μg/
mL (16−32× MIC), the cell survival percentage of mouse
Figure 10. In vivo antibacterial efficacy of compound 16 combined with 12.5 μg/mL Zn2+ in a murine keratitis model induced by S. aureus
ATCC29213 or P. aeruginosa ATCC9027 (n = 5). Vancomycin (5%) or gatifloxacin (0.3%) was selected as the positive control, and 5% glucose
solution was used as a negative control. (**) P < 0.005 and (***) P < 0.02 compared with the negative control. The results are given as means
s.d. for five mice.
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mixture was diluted with ethyl acetate and washed with water three
times. The organic phase was evaporated in vacuum. The resulting
residue was purified by HPLC to give 9 as a dark green solid (127.3
mg, 78%); mp 78.6−81.9 °C. 1H NMR (400 MHz, CD3OD) δ 8.31−
8.22 (m, 2H), 7.94 (d, J = 7.8 Hz, 1H), 7.58−7.51 (m, 2H), 7.45 (d, J
= 7.8 Hz, 2H), 7.37 (d, J = 8.1 Hz, 1H), 7.31−7.22 (m, 2H), 7.12−
6.95 (m, 4H), 6.58 (d, J = 7.7 Hz, 1H), 4.34−4.25 (m, 1H), 4.18 (d, J
= 4.0 Hz, 2H), 4.03−3.89 (m, 4H), 3.16−2.93 (m, 2H). 13C NMR
(100 MHz, CD3OD) δ 158.96 (2 × C), 156.35, 149.07 (2 × CH),
142.82, 140.59, 138.97 (2 × CH), 127.37, 125.52, 125.02 (2 × CH),
123.93 (2 × CH), 123.91, 123.50, 119.75, 113.57, 111.00, 104.90,
101.25, 70.41, 68.83, 61.36 (2 × CH2), 58.47. HRMS (ESI+):
calculated for C27H27N4O2 [M + H]+ 439.2134, found 439.2127.
HPLC purity: 99.6%, tR = 6.1 min.
CONCLUSIONS
■
In this work, we rationally designed and synthesized a novel
series of dipicolylamine-containing cationic carbazole amphi-
philes by biomimicking CAMPs. After performing biological
activity evaluations and structural optimization, compound 16
was identified as the most promising antimicrobial candidate.
Compound 16 combined with Zn2+ (6.25−12.5 μg/mL)
showed potent broad-spectrum antibacterial activity against
both Gram-positive and Gram-negative bacteria (MICs =
0.78−3.125 μg/mL). The addition of Zn2+ greatly improved
the antibacterial activity of compound 16 against both Gram-
positive and Gram-negative bacteria, especially P. aeruginosa
ATCC9027 (MIC from >50 to 3.125 μg/mL). The mode of
action studies revealed that compound 16 combined with Zn2+
killed both Gram-positive and Gram-negative bacteria mainly
by affecting the permeability of bacterial cell membranes or
even disrupting the integrity of bacterial cell membranes.
Compound 16 combined with 12.5 μg/mL Zn2+ had very poor
hemolytic activity (HC50 > 200 μg/mL) as well as low
cytotoxicity toward mammalian cells and could avoid the
emergence of bacterial resistance in the laboratory simulation
of drug resistance development studies. More notably,
compound 16 combined with 12.5 μg/mL Zn2+ displayed
excellent in vivo efficacy in a murine keratitis model induced by
S. aureus ATCC29213 or P. aeruginosa ATCC9027, and the in
vivo efficacy of compound 16 combined with Zn2+ was superior
to that of commercial vancomycin and gatifloxacin. This work
presents a framework for the design and synthesis of a new
generation of membrane-active antimicrobials to combat
Gram-positive and Gram-negative bacterial infections.
1-(Bis(pyridin-2-ylmethyl)amino)-3-((9-methyl-9H-carbazol-4-
yl)oxy)propan-2-ol (10). 10 was prepared from 2,2′-dipicolylamine
(0.2 mL) and 2 (65.9 mg, 0.26 mmol) according to the similar
procedure for 9 to give 10 as a dark green solid (87.2 mg, 74%); mp
1
49.2−51.8 °C. H NMR (400 MHz, CDCl3) δ 8.58 (d, J = 4.1 Hz,
2H), 8.16 (d, J = 7.7 Hz, 1H), 7.65−7.56 (m, 2H), 7.46−7.32 (m,
5H), 7.20−7.11 (m, 3H), 7.02 (d, J = 8.1 Hz, 1H), 6.68 (d, J = 8.0
Hz, 1H), 5.13 (s, 2H), 4.46−4.38 (m, 1H), 4.36−4.19 (m, 2H),
4.12−4.00 (m, 3H), 3.83 (s, 3H), 3.28−3.03 (m, 2H). 13C NMR (100
MHz, CDCl3) δ 158.72 (2 × C), 155.33, 148.76 (2 × CH), 142.55,
140.28, 137.08 (2 × CH), 126.52, 124.72, 123.48 (2 × CH), 123.07,
122.45 (2 × CH), 122.15, 119.15, 111.98, 107.87, 101.64, 100.84,
70.09, 67.96, 60.33, 58.52, 50.84, 29.36. HRMS (ESI+): calculated for
C28H29N4O2 [M + H]+ 453.2291, found 453.2281. HPLC purity:
99.1%, tR = 6.1 min.
1-(Bis(pyridin-2-ylmethyl)amino)-3-((9-propyl-9H-carbazol-4-yl)-
oxy)propan-2-ol (11). 11 was synthesized from 2,2′-dipicolylamine
(0.2 mL) and 3 (93 mg, 0.33 mmol) according to the similar
procedure for 9 to provide 11 as a dark green solid (112.7 mg, 71%);
mp 53.1−54.7 °C. 1H NMR (400 MHz, CDCl3) δ 8.56 (d, J = 4.2 Hz,
2H), 8.14 (d, J = 7.7 Hz, 1H), 7.64−7.52 (m, 2H), 7.45−7.29 (m,
5H), 7.18−7.07 (m, 3H), 7.01 (d, J = 8.2 Hz, 1H), 6.65 (d, J = 8.0
Hz, 1H), 4.43−4.36 (m, 1H), 4.35−4.16 (m, 7H), 4.01 (d, J = 14.9
Hz, 2H), 3.27−2.99 (m, 2H), 1.96−1.80 (m, 2H), 0.95 (t, J = 7.4 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 158.62 (2 × C), 155.39, 148.70
(2 × CH), 142.07, 139.79, 137.15 (2 × CH), 126.40, 124.62, 123.54
(2 × CH), 123.13, 122.49 (2 × CH), 122.19, 119.03, 112.01, 108.18,
101.97, 100.66, 70.05, 67.95, 60.30 (2 × CH2), 58.53, 44.84, 22.39,
11.84. HRMS (ESI+): calculated for C30H33N4O2 [M + H]+
481.2604, found 481.2593. HPLC purity: 98.8%, tR = 5.2 min.
1-(Bis(pyridin-2-ylmethyl)amino)-3-((9-pentyl-9H-carbazol-4-yl)-
oxy)propan-2-ol (12). 12 was synthesized from 2,2′-dipicolylamine
(0.2 mL) and 4 (42.8 mg, 0.14 mmol) according to the similar
procedure for 9 to give 12 as a dark green solid (49.3 mg, 70%); mp
EXPERIMENTAL SECTION
■
General Chemistry. All commercial chemicals and solvents were
used without further purification. NMR spectra were recorded on a
JEOL 400 MHz spectrometer. Chemical shifts and coupling constants
are expressed in ppm and Hz, respectively. HPLC was carried out on
an Agilent 1260 Infinity system by using YMC-Pack C18 column
(20× 150 mm or 20× 100 mm, 5 μm, 120 Å) and gradient elution
mode. The following mobile phases were applied: buffer A: 0.1%
HCOOH in water; buffer B: 0.1% HCOOH in methanol. All final
products were purified to more than 95% purity. High-resolution mass
(HRMS) spectra were performed with a Thermo DFS mass
spectrometer.
Synthesis of Carbazole Derivatives. The synthesis of carbazole
1
derivatives 2−6, 8, and 17−18 was described previously.43
56.2−59.1 °C. H NMR (400 MHz, CDCl3) δ 8.59−8.48 (m, 2H),
8.15 (d, J = 7.7 Hz, 1H), 7.63−7.50 (m, 2H), 7.43−7.28 (m, 5H),
7.19−7.06 (m, 3H), 7.01 (d, J = 8.1 Hz, 1H), 6.65 (d, J = 7.9 Hz,
1H), 4.73 (s, 2H), 4.43−4.35 (m, 1H), 4.33−4.16 (m, 4H), 4.10−
3.98 (m, 3H), 3.29−2.98 (m, 2H), 1.92−1.76 (m, 2H), 1.40−1.28
(m, 4H), 0.87 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
157.90 (2 × C), 155.29, 148.38 (2 × CH), 141.98, 139.70, 137.60 (2
× CH), 126.43, 124.65, 123.87 (2 × CH), 123.09, 122.77 (2 × CH),
122.15, 119.01, 111.96, 108.16, 101.98, 100.63, 69.92, 67.73, 60.00,
58.49, 50.78, 43.28, 29.45, 28.77, 22.57, 14.06. HRMS (ESI+):
calculated for C32H37N4O2 [M + H]+ 509.2917, found 509.2911.
HPLC purity: 99.1%, tR = 7.4 min.
9-(4,4,4-Trifluorobutyl)-4-(oxiran-2-ylmethoxy)-9H-carbazole
(7). NaOH (53 mg, 0.66 mmol) and 1,1,1-trifluoro-4-iodobutane
(109 μL, 0.67 mmol) were added to a solution of 1 (100 mg, 0.42
mmol) in DMF (5 mL). The mixture was stirred for 2 h at 60 °C.
Then, the mixture was diluted with ethyl acetate and extracted with
water three times. The organic phase was concentrated under reduced
pressure. The crude product was purified by silica gel chromatography
(ethyl acetate/petroleum ether = 1/4) to give 7 as a light yellow solid
(103.2 mg, 71%); mp 75.8−78.3 °C. 1H NMR (400 MHz, CDCl3) δ
8.38 (d, J = 7.3 Hz, 1H), 7.58−7.26 (m, 4H), 7.02 (d, J = 7.7 Hz,
1H), 6.69 (d, J = 7.7 Hz, 1H), 4.53−4.21 (m, 4H), 3.71−3.37 (m,
1H), 2.96 (d, J = 43.3 Hz, 2H), 2.18−2.02 (m, 4H). 13C NMR (100
MHz, CDCl3) δ 155.24, 141.78, 139.47, 126.75, 125.56, 125.22,
123.55, 122.31, 119.69, 112.35, 107.89, 101.94, 101.31, 68.91, 50.45,
44.96, 41.86, 31.64, 21.80. HRMS (ESI+): calculated for
C19H19F3NO2 [M + H]+ 350.1368, found 350.1354. HPLC purity:
99.5%, tR = 6.4 min.
1-(Bis(pyridin-2-ylmethyl)amino)-3-((9-heptyl-9H-carbazol-4-yl)-
oxy)propan-2-ol (13). 13 was synthesized from 2,2′-dipicolylamine
(0.2 mL) and 5 (42.5 mg, 0.13 mmol) according to the similar
procedure for 9 to give 13 as a dark green solid (52.2 mg, 77%); mp
1
61.1−63.5 °C. H NMR (400 MHz, CDCl3) δ 8.60−8.46 (m, 2H),
8.15 (d, J = 7.7 Hz, 1H), 7.63−7.50 (m, 2H), 7.44−7.28 (m, 5H),
7.18−7.06 (m, 3H), 7.00 (d, J = 8.1 Hz, 1H), 6.65 (d, J = 7.9 Hz,
1H), 4.43−4.35 (m, 1H), 4.34−4.16 (m, 4H), 4.09−3.98 (m, 3H),
3.89 (s, 2H), 3.27−3.00 (m, 2H), 1.90−1.75 (m, 2H), 1.42−1.22 (m,
8H), 0.85 (t, J = 6.9 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 159.10
1-((9H-Carbazol-4-yl)oxy)-3-(bis(pyridin-2-ylmethyl)amino)-
propan-2-ol (9). 2,2′-Dipicolylamine (0.2 mL) was added to a
solution of 1 (100 mg, 0.37 mmol) in MeOH (10 mL). The mixture
was stirred at 65 °C for 6 h. After the reaction was completed, the
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(2 × C), 155.45, 149.03 (2 × CH), 141.98, 139.69, 136.79 (2 × CH),
126.40, 124.60, 123.28 (2 × CH), 123.16, 122.28 (2 × CH), 122.21,
119.00, 111.99, 108.12, 101.88, 100.61, 70.12, 68.06, 60.53, 58.53,
50.83, 43.31, 31.81, 29.17, 29.09, 27.32, 22.68, 14.16. HRMS (ESI+):
calculated for C34H41N4O2 [M + H]+ 537.3230, found 537.3220.
HPLC purity: 98.4%, tR = 9.0 min.
compounds were dissolved in DMSO/H2O to prepare a 1000 μg/mL
stock solution. Then, the stock solution was diluted with Muller
Hinton broth (MHB) containing ZnSO4·H2O (final Zn2+ concen-
tration of 0, 6.25, or 12.5 μg/mL). The bacteria were inoculated on
the Mueller Hinton agar (MHA) plate at 37 °C overnight and
adjusted to approximately 106 CFU/mL. Bacterial suspensions (100
μL) were mixed with 100 μL two-fold serial dilutions of test
compounds (final compound concentrations of 50, 25, 12.5, 6.25,
3.125, 1.56, 0.78, and 0.39 μg/mL) in a 96-well plate. The plate was
incubated for 24 h at 37 °C. Then, a Biotek multi-detector microplate
reader was used to read the absorption wavelength of the mixture at
600 nm. MICs were determined as the lowest compound
concentration, which inhibited 95% of the bacterial growth. The
experiments were performed at least twice with biologically replicates.
Hemolytic Assay. The hemolysis experiment was carried out as
described in our previous reports.43 Briefly, compounds were
dissolved in DMSO to prepare 40 mg/mL stock solutions and two-
fold serially diluted with PBS (final concentration of DMSO = 0.5%)
in 96-well plates. Then, rabbit red blood cells (RBCs) were
centrifuged at 2500 rpm for 3 min, washed twice with PBS, and
suspended in PBS to make an 8% v/v RBCs suspension. The RBCs
suspension was mixed with an equal volume of two-fold serial
dilutions of compounds in 96-well plates and incubated at 37 °C for 1
h. The 96-well plate was centrifuged at 2500 rpm for 5 min. Then, the
supernatant (100 μL) was transferred into another 96-well plate. A
Biotek multi-detector microplate reader was used to read the
absorption wavelength of the mixture at 576 nm. A 2% Triton X-
100 solution treatment was used as a positive control, and 0.5%
DMSO treatment was used as a negative control. Hemolytic activity
1-(Bis(pyridin-2-ylmethyl)amino)-3-((9-nonyl-9H-carbazol-4-yl)-
oxy)propan-2-ol (14). 14 was synthesized from 2,2′-dipicolylamine
(0.2 mL) and 6 (32.8 mg, 0.09 mmol) according to the similar
procedure for 9 to give 14 as a dark green solid (33.6 mg, 66%); mp
1
66.1−67.5 °C. H NMR (400 MHz, CDCl3) δ 8.59−8.49 (m, 2H),
8.14 (d, J = 7.7 Hz, 1H), 7.63−7.47 (m, 2H), 7.46−7.29 (m, 5H),
7.18−6.98 (m, 4H), 6.65 (d, J = 7.9 Hz, 1H), 4.46−4.37 (m, 3H),
4.33−4.17 (m, 4H), 4.11−3.98 (m, 3H), 3.28−2.99 (m, 2H), 1.91−
1.76 (m, 2H), 1.41−1.21 (m, 12H), 0.86 (t, J = 6.9 Hz, 3H). 13C
NMR (100 MHz, CDCl3) δ 159.08 (2 × C), 155.45, 149.04 (2 ×
CH), 141.98, 139.70, 136.79 (2 × CH), 126.41, 124.60, 123.29 (2 ×
CH), 123.16, 122.28 (2 × CH), 122.21, 119.01, 111.99, 108.13,
101.89, 100.62, 70.13, 68.05, 60.54 (2 × CH2), 58.52, 43.30, 31.91,
29.56, 29.51, 29.34, 29.07, 27.35, 22.73, 14.21. HRMS (ESI+):
calculated for C36H45N4O2 [M + H]+ 565.3543, found 565.3535.
HPLC purity: 99.1%, tR = 5.6 min.
1-(Bis(pyridin-2-ylmethyl)amino)-3-((9-(4,4,4-trifluorobutyl)-9H-
carbazol-4-yl)oxy)propan-2-ol (15). 15 was synthesized from 2,2′-
dipicolylamine (0.2 mL) and 7 (56.3 mg, 0.16 mmol) according to
the similar procedure for 9. The product 15 was obtained as a dark
green solid (53.2 mg, 60%); mp 62.1−63.9 °C. 1H NMR (400 MHz,
CDCl3) δ 8.30 (d, J = 4.2 Hz, 2H), 8.00 (d, J = 7.7 Hz, 1H), 7.55−
7.45 (m, 4H), 7.44−7.31 (m, 3H), 7.11−7.00 (m, 4H), 6.66 (d, J =
8.0 Hz, 1H), 4.45−4.26 (m, 3H), 4.21 (d, J = 3.9 Hz, 2H), 3.98−3.85
(m, 4H), 3.14−2.88 (m, 2H), 2.23−2.02 (m, 4H). 13C NMR (100
MHz, CDCl3) δ 159.54, 156.62, 149.21 (2 × CH), 142.97, 140.70,
138.75, 129.97, 127.82 (2 × CH), 127.23, 125.86, 125.00 (2 × CH),
124.31, 123.78 (2 × CH), 123.43, 120.29, 113.24, 109.05, 102.81,
101.96, 70.59, 68.97, 61.58, 58.50, 42.37, 32.11, 31.82, 22.74. HRMS
(ESI+): calculated for C31H32F3N4O2 [M + H]+ 549.2477, found
549.2465. HPLC purity: 99.4%, tR = 5.7 min.
was calculated by the following formula: % hemolysis = [(Abssample
−
Absnegative control)/(Abspositive control − Absnegative control)]×100. The ex-
periments were performed at least twice with three duplicates for each
time.
Time-Kill Study. The time-kill experiment was performed as
described in previous reports with minor modifications.43 The in vitro
time-kill kinetics of compound 16 in the presence of 12.5 μg/mL Zn2+
were performed against S. aureus ATCC29213 and E. coli
ATCC25922. The bacteria were adjusted to approximately 106
CFU/mL. The bacterial suspension was incubated with compound
16 at different concentrations (4× and 8× MIC) in the presence of
12.5 μg/mL Zn2+ at 37 °C. Aliquots were then removed from the
mixture at different time points (0.5, 1, 2, 4, 8, and 24 h), 10-fold
serially diluted in PBS, and plated onto MHA plates. The plates were
incubated for 24 h at 37 °C, and the bacterial colonies grown on the
plates were counted. The experiments were carried out at least twice.
Drug Resistance Study. The drug resistance experiment was
performed as described in previous reports with minor modifica-
tions.43 According to the above-mentioned methods, the initial MICs
of the compounds against S. aureus ATCC29213 and E. coli
ATCC25922 were obtained. The bacteria from the wells at 0.5×
MIC concentration of compounds were then used to prepare the
bacterial suspension for the next MIC value test. The experiment was
repeated every day for 19 days.
SYTOX Green Assay. This experiment was performed as
described in previous reports with minor modifications.43 S. aureus
ATCC29213 or E. coli ATCC 25922 was incubated in MHB at 37 °C
overnight and centrifuged at 5000 rpm for 5 min. Then, the
supernatant was removed. The inoculum was washed three times with
PBS (pH = 7.2) and resuspended in PBS to prepare bacterial
suspension (OD600 = 0.2). SYTOX Green dye (0.3 μM) was added to
the bacterial suspension and cultured in the dark. The fluorescence
intensity of mixture was monitored by a Biotek multi-detector
microplate reader (emission and excitation wavelengths were 523 and
504 nm, respectively). After the fluorescence signal was stabilized,
different concentrations (2×, 4×, and 8× MIC) of compound 16 in
the presence of 12.5 μg/mL Zn2+ were added to the mixture, and the
fluorescence intensity change was monitored and recorded for about 1
h. The experiments were carried out at least twice with biologically
replicates.
1-(Bis(pyridin-2-ylmethyl)amino)-3-((9-(3-methylbut-2-en-1-yl)-
9H-carbazol-4-yl)oxy)propan-2-ol (16). 16 was prepared from 2,2′-
dipicolylamine (0.2 mL) and 8 (100 mg, 0.33 mmol) according to the
similar procedure for 9 to give 16 as a dark green solid (106.9 mg,
1
74%); mp 56.1−57.8 °C. H NMR (400 MHz, CDCl3) δ 8.58−8.54
(m, 2H), 8.16 (d, J = 7.8 Hz, 1H), 7.61−7.54 (m, 2H), 7.42−7.31 (m,
5H), 7.16−7.07 (m, 3H), 6.99 (d, J = 8.1 Hz, 1H), 6.65 (d, J = 8.0
Hz, 1H), 5.28−5.22 (m, 1H), 4.86 (d, J = 6.4 Hz, 2H), 4.43−4.35 (m,
1H), 4.34−4.16 (m, 2H), 4.13−3.96 (m, 4H), 3.27−2.98 (m, 2H),
1.94−1.89 (m, 3H), 1.71−1.67 (m, 3H). 13C NMR (100 MHz,
CDCl3) δ 158.91 (2 × C), 155.43, 148.86 (2 × CH), 141.85, 139.57,
136.93 (2 × CH), 135.08, 126.41 (2 × C), 124.62, 123.38 (2 × CH),
123.16, 122.35 (2 × CH), 120.09, 119.10, 112.17, 108.21, 101.97,
100.79, 70.13, 68.03, 60.44, 58.54, 41.35, 25.65, 18.28. HRMS (ESI
+): calculated for C32H35N4O2 [M + H]+ 507.2760, found 507.2761.
HPLC purity: 99.2%, tR = 6.6 min.
1-((9-(3-Methylbut-2-en-1-yl)-9H-carbazol-4-yl)oxy)-3-((pyridin-
2-ylmethyl)amino)propan-2-ol (19). 19 was prepared from 2-
picolylamine (0.2 mL) and 8 (50 mg, 0.16 mmol) according to the
similar procedure for 9 to provide 19 as a dark green solid (48.6 mg,
1
72%); mp 74.5−76.4 °C. H NMR (400 MHz, DMSO-d6) δ 8.46−
8.38 (m, 1H), 8.16−8.03 (m, 1H), 7.67−7.45 (m, 1H), 7.38−7.28
(m, 2H), 7.28−7.20 (m, 2H), 7.15−7.00 (m, 2H), 6.95−6.87 (m,
1H), 6.62−6.48 (m, 1H), 5.22−5.09 (m, 1H), 4.87−4.66 (m, 4H),
4.56−4.02 (m, 5H), 3.81−3.63 (m, 1H), 3.29−2.99 (m, 1H), 1.88−
1.75 (m, 3H), 1.68−1.55 (m, 3H). 13C NMR (100 MHz, CD3OD) δ
156.21, 154.93, 150.29, 143.08, 140.83, 138.67, 136.26, 127.51,
125.76, 124.50, 124.16, 124.00, 123.29, 121.06, 120.04, 113.13,
109.48, 103.51, 101.86, 70.96, 67.84, 52.87, 51.92, 41.90, 25.73, 18.21.
HRMS (ESI+): calculated for C26H30N3O2 [M + H]+ 416.2338,
found 416.2330. HPLC purity: 99.3%, tR = 5.8 min.
MIC Determinations. The MIC values were determined as
described in previous reports with minor modifications.43 Test
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DiSC3 (5) Assay. This experiment was performed as described in
previous reports with minor modifications.68 DiSC3 (5) was used to
measure the depolarization of the bacterial cell membranes of S.
aureus ATCC29213 or E. coli ATCC 25922 after the treatment of
compound 16 combined with 12.5 μg/mL Zn2+. The bacteria (S.
aureus ATCC29213 or E. coli ATCC 25922) were incubated in MHB
at 37 °C overnight and were centrifuged at 5000 rpm for 5 min. Then,
the supernatant was removed. The inoculum was washed three times
with HEPES (5 mM, pH = 7.2) and resuspended in HEPES to
prepare bacterial suspension (OD600 = 0.3). DiSC3 (5) dye (0.2 μM)
was added to the bacterial suspension. The mixture was incubated for
15 min in the dark. A Biotek multi-detector microplate reader
(emission and excitation wavelengths were 670 and 622 nm,
respectively) was used to monitor the fluorescence intensity of the
mixture. After the fluorescence signal was stabilized, different
concentrations (2×, 4×, and 8× MIC) of compound 16 in the
presence of 12.5 μg/mL Zn2+ were added to the mixture, and the
fluorescence intensity change was monitored and recorded for about 1
h. The experiments were carried out at least twice with biologically
replicates.
emission wavelength of 580 and 620 nm, respectively. The
experiments were carried out at least twice with biologically replicates.
CCK-8 Assay. This experiment was performed as described in
previous reports with minor modifications.43 The cytotoxicity of
compound 16 in the presence of 12.5 μg/mL Zn2+ toward mouse
fibroblasts (NCTC clone 929) was determined using CCK-8 assay
according to a previously reported method. Compound 16 in the
presence of 12.5 μg/mL Zn2+ was incubated with mouse fibroblasts
for 24 h. All measurements were carried out at least twice with
biological replicates.
In Vivo Efficacy. The animal studies were performed as described
in previous reports with minor modifications.43 All animal experi-
ments were approved by the Laboratory Animal Center of South
China Agricultural University and performed in accordance with the
policy of the Ministry of Health. Six to 8 week-old female C57BL6
mice (approximately 20 g weight) purchased from Vital River
Laboratory Animal Technology Co., Ltd. were used in this study. The
bacteria (S. aureus ATCC29213 or P. aeruginosa ATCC9027) were
grown on MHA plates at 37 °C for 24 h, and the cell concentration of
the bacterial suspension was adjusted with PBS to approximately 5×
107 CFU/mL for mice corneal infection.
1-N-Phenylnaphthylamine Uptake Assay. This experiment
was performed as described in previous reports with minor
modifications.69 NPN uptake assay was used to determine the
permeability of the outer membranes of E. coli ATCC25922. The
bacteria (E. coli ATCC25922) were incubated in MHB at 37 °C
overnight and were centrifuged at 5000 rpm for 5 min. Then, the
supernatant was removed. The inoculum was washed three times with
HEPES (5 mM, pH = 7.2) and resuspended in HEPES to prepare the
bacterial suspension with a cell density of approximately 1 × 108
CFU/mL. NPN (10 μM) was added to the bacterial suspension. The
fluorescence intensity of the mixture was monitored by a Biotek multi-
detector microplate reader (emission and excitation wavelengths were
420 and 350 nm, respectively). After the fluorescence signal was
stabilized, different concentrations (1×, 2×, 4×, and 8× MIC) of
compound 16 in the presence of 12.5 μg/mL Zn2+ were added to the
mixture, and the fluorescence intensity change was monitored and
recorded for about 1 h. The experiments were carried out at least
twice with biologically replicates.
LIVE/DEAD Fluorescence Staining Assay. This experiment was
performed as described in previous reports with minor modifica-
tions.43 LIVE/DEAD BacLight bacterial viability kit L7007 (Thermo-
Fisher, USA) was used to determine the membrane integrity of
bacterial cells. The bacteria (S. aureus ATCC29213 or E. coli ATCC
25922) were incubated in MHB to the logarithmic growth phase and
were centrifuged at 5000 rpm for 5 min. Then, the supernatant was
removed. The bacterial cells were washed twice with PBS (10 mM,
pH = 7.2) and resuspended in PBS to prepare the bacterial suspension
with a cell density of approximately 1 × 108 CFU/mL. The bacterial
suspension was mixed with compound 16 in the presence of 12.5 μg/
mL Zn2+ (final concentration of 8× MIC) or PBS (negative control)
and incubated for 2 h at 37 °C. Then, these bacterial cells were
stained with SYTO 9 (5 μM) and PI (30 μM) for 15 min in the dark.
Finally, the bacterial cells were prepared into smears and were
observed under a laser confocal microscope (ZEISS LSM 880). The
experiments were carried out at least twice with biologically replicates.
BODIPY-TR-Cadaverine Displacement Assay. This experiment
was performed as described in previous reports with minor
modifications.65,70 Binding affinity between compound 16 combined
with 12.5 μg/mL Zn2+ and lipopolysaccharides (LPS) was
investigated by using BODIPY-TR-cadaverine displacement assay.
BODIPY-TR-cadaverine and LPS from P. aeruginosa were obtained
from ThermoFisher and Sigma-Aldrich, respectively. BODIPY-TR-
cadaverine (final concentration of 5 μM) and LPS (final
concentration of 12.5 μg/mL) were prepared using Tris buffer (50
mM, pH 7.4) in 24-well plates. After 15 min, compound 16 (1×, 2×,
4×, 8×, and 16× MIC) in the presence of 12.5 μg/mL Zn2+ or
without Zn2+ was added. The plates were kept in the dark at room
temperature for 30 min. Finally, fluorescence intensity was recorded
by a Biotek multi-detector microplate reader, using excitation and
The murine corneal infection model induced by S. aureus
ATCC29213 were immunosuppressed using cyclophosphamide
(100 mg/kg) via intraperitoneal injection three times 5 days before
infection. In the murine corneal infection model induced by P.
aeruginosa ATCC9027, the mice did not be immunosuppressed. The
mice were anesthetized by intraperitoneal injection of 2.5% avertin
solution (500 mg/kg), and the right corneal scratches were created by
sterile needles. Fifteen microliters of the bacterial inoculum (S. aureus
ATCC29213 or P. aeruginosa ATCC9027) was dripped onto the
injured cornea. All mice were randomly divided into 6 groups, five
mice per group (n = 5), 3 groups of mice were used in the S. aureus
ATCC29213-induced murine corneal infection study, and the other 3
groups of mice were used in the P. aeruginosa ATCC9027-induced
murine corneal infection study. One day after infection, the
corresponding drugs (0.5% compound 16 containing 12.5 μg/mL
Zn2+, 5% vancomycin as the positive control toward S. aureus
ATCC29213, and 0.3% gatifloxacin as the positive control toward P.
aeruginosa ATCC9027) were given topically four times every day for 3
days. Finally, all mice were sacrificed, and the wounded corneas were
removed. The number of living bacteria on the cornea was counted by
a standard plate counting method using MHA plates. Statistical
significance was calculated by the SPSS 22.0 software. P values were
calculated by the independent t test, and P < 0.05 was considered to
be significant.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00858.
■
sı
In vitro salt resistance study; mass analysis of complex
formation of compound 16 with Zn2+; Table S1; Figure
S1; Figure S2; NMR spectra, HR-MS spectra, and
HPLC traces of synthesized carbazole derivatives (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
■
Shouping Liu − Key Laboratory of Molecular Target &
Clinical Pharmacology and the State Key Laboratory of
Respiratory Disease, School of Pharmaceutical Sciences & the
Fifth Affiliated Hospital, Guangzhou Medical University,
Guangzhou 511436, P.R. China; Email: liushouping2018@
163.com
Shuimu Lin − Key Laboratory of Molecular Target & Clinical
Pharmacology and the State Key Laboratory of Respiratory
Disease, School of Pharmaceutical Sciences & the Fifth
10441
https://doi.org/10.1021/acs.jmedchem.1c00858
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Affiliated Hospital, Guangzhou Medical University,
Guangzhou 511436, P.R. China; orcid.org/0000-0002-
0477-8173; Email: linshuimu020@163.com
phenylnaphthylamine; MIC, minimum inhibitory concentra-
tion; MHB, Mueller Hinton broth; CFU, colony-forming units;
VAN, vancomycin; CIP, ciprofloxacin; NMR, nuclear magnetic
resonance; HPLC, high-performance liquid chromatography;
HRMS, high-resolution mass spectrometry; NCTC, National
Collection of Type Cultures; ATCC, American Type Culture
Collection
Authors
Jiayong Liu − Key Laboratory of Molecular Target & Clinical
Pharmacology and the State Key Laboratory of Respiratory
Disease, School of Pharmaceutical Sciences & the Fifth
Affiliated Hospital, Guangzhou Medical University,
Guangzhou 511436, P.R. China; orcid.org/0000-0002-
1834-0877
Hongxia Li − Key Laboratory of Molecular Target & Clinical
Pharmacology and the State Key Laboratory of Respiratory
Disease, School of Pharmaceutical Sciences & the Fifth
Affiliated Hospital, Guangzhou Medical University,
Guangzhou 511436, P.R. China
Haizhou Li − Key Laboratory of Molecular Target & Clinical
Pharmacology and the State Key Laboratory of Respiratory
Disease, School of Pharmaceutical Sciences & the Fifth
Affiliated Hospital, Guangzhou Medical University,
Guangzhou 511436, P.R. China
Shanfang Fang − Key Laboratory of Molecular Target &
Clinical Pharmacology and the State Key Laboratory of
Respiratory Disease, School of Pharmaceutical Sciences & the
Fifth Affiliated Hospital, Guangzhou Medical University,
Guangzhou 511436, P.R. China
Jinguo Shi − Department of Medicinal Chemistry, School of
Pharmaceutical Science, Sun Yat-sen University, Guangzhou
510006, P. R. China
Yongzhi Chen − Key Laboratory of Molecular Target &
Clinical Pharmacology and the State Key Laboratory of
Respiratory Disease, School of Pharmaceutical Sciences & the
Fifth Affiliated Hospital, Guangzhou Medical University,
Guangzhou 511436, P.R. China
Rongcui Zhong − Key Laboratory of Molecular Target &
Clinical Pharmacology and the State Key Laboratory of
Respiratory Disease, School of Pharmaceutical Sciences & the
Fifth Affiliated Hospital, Guangzhou Medical University,
Guangzhou 511436, P.R. China
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Author Contributions
#J.L. and H.X.L. contributed equally. S.M.L. and S.P.L.
designed and coordinated the whole study. The experiments
were carried out by J.L., H.X.L., H.Z.L., S.F., J.S., Y.C., and
R.Z.; J.L., H.X.L., and S.M.L. analyzed the data. The
manuscript was written with contributions from all authors.
All authors have approved the final version of the manuscript.
Funding
This work was supported by the National Natural Science
Foundation of China (21907019 to S.M.L.) and the Talent
Fund for High-Level University Construction of Guangzhou
(B195002009029 to S.P.L. and B195002009030 to S.M.L.).
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ABBREVIATIONS
■
LPS, lipopolysaccharide; CAMPs, cationic antimicrobial
peptides; DPA, dipicolylamine; LTA, lipoteichoic acid; PI,
propidium iodide; MRSA, methicillin-resistant S. aureus; PBS,
phosphate-buffered saline; RBCs, red blood cells; NPN, 1-N-
10442
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