Design, synthesis and molecular docking of salicylic acid derivatives containing metronidazole as a new class of antimicrobial agents

Article abstract of DOI:10.1016/j.bmc.2015.07.075

A series of novel salicylic acid derivatives containing metronidazole as Staphylococcus aureus Tyrosyl-tRNA synthetase (TyrRS) inhibitors have been synthesized and evaluated their biology activities as potential antibacterial agents. Among these compounds

Full text of DOI:10.1016/j.bmc.2015.07.075

Accepted Manuscript
Design, synthesis and molecular docking of salicylic acid derivatives containing
metronidazole as a new class of antimicrobial agents
Zhi-Hua Guo, Yong Yin, Cong Wang, Peng-Fei Wang, Xing-Tao Zhang,
Zhong-Chang Wang, Hai-Liang Zhu
PII:
S0968-0896(15)00659-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.07.075
BMC 12504
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 June 2015
30 July 2015
31 July 2015
Please cite this article as: Guo, Z-H., Yin, Y., Wang, C., Wang, P-F., Zhang, X-T., Wang, Z-C., Zhu, H-L., Design,
synthesis and molecular docking of salicylic acid derivatives containing metronidazole as a new class of
antimicrobial agents, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.07.075
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Design, synthesis and molecular docking of salicylic acid derivatives containing
metronidazole as a new class of antimicrobial agents
Zhi-Hua Guoa, b, Yong Yinb, Cong Wanga, Peng-Fei Wangb, Xing-Tao Zhanga,
Zhong-Chang Wangb*, Hai-Liang Zhub*
aCollege of Biology and Food Engineering, SuZhou University, Suzhou, 234000,
People’s Republic of China.
bState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093，People’s Republic of China.
* Corresponding author. Tel.: +86-25-83592572; Fax: +86-25-83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
Abstract
A series of novel salicylic acid derivatives containing metronidazole as S. aureus
Tyrosyl-tRNA synthetase (TyrRS) inhibitors have been synthesized and evaluated their
biology activities as potential antibacterial agents. Among these compounds,
compound 5r exhibited the most potent antibacterial activity against Gram-positive (S.
aureus ATCC 6538 and B. subtilis ATCC 6633) and Gram-negative(E. coli ATCC 35218
and P. aeruginosa ATCC 13525) with MICs of 0.39 ~ 1.57 μg/mL and showed the most
potent S. aureus Tyrosyl-tRNA synthetase inhibitory with 2.3 μM. Docking simulation
was performed to insert compound 5r into the crystal structure of S. aureus
Tyrosyl-tRNA synthetase active site to determine the probable binding model. These
results suggested that compound 5r may be a promising antibacterial agent.
Keywords:
Salicylic acid derivatives
S. aureus TyrRS
Antibacterial activity
Molecular docking
1

1. Introduction
Bacterial infections are a serious threat to human health because of emerging
resistance to existing antibiotics, so new antibacterial candidates will come from is an
open problem.1 In the past few decades the discovery of new antibacterial agents has
proven very important challenging for the research community.2 In order to resolve this
serious puzzle, the crucial task of looking for new types of antibacterial agents should be
achieved.3 So, much of the research are put into the design of new efficiency
antibacterial agents against the challenge of the drug resistance.
New antibacterial agents which can work through different kinds of targets in key
structures of the bacterial cells to overcome the problem of acquired resistance.
Aminoacyl-tRNA synthetases (AARSs) is an enzyme which can recognize specific
amino acids and ligate these amino acids to their cognate tRNAmolecules during protein
synthesis.4 When 1 of the 20 AARSs present in the cell is inhibited, the corresponding
tRNA is not translated into protein, which leads to failure of protein synthesis and
inhibition of cell growth.5 Because of these reasons, Aminoacyl-tRNA synthetases are
essential for protein synthesis and cell viability. These enzymes are found in all living
organisms, but there are great sequence differences between eukaryotes and
prokaryotes.6 These properties suggest that small molecule inhibitors of AARSs could
be promising drug candidates leading to high selectivity and broad-spectrum
antibacterial agents.4 So, inhibitors of AARSs have been used as medicine or rigorously
tested in clinical trials for therapeutic applications in microbial infections, such as, the
natural product mupirocin, an inhibitor of Isoleucyl-tRNA synthetase (IleRS), is
approved as a topical treatment for bacterial skin infections.7 Tyrosyl-tRNA synthetase
(TyrRs) is one of the 20 Aminoacyl-tRNA synthetases and plays an important role in
protein synthesis.8
Nitroimidazole derivatives have exhibited broad variety biological activities,
particularly antibacterial activity.9 5-Nitroimidazole based drugs have been extensively
applied to cure the infections induced by bacteria and to kill pathogenic protozoan
2

parasites in human body.10-11 So, nitroimidazole derivatives have attracted considerable
attention of medicinal chemists as they can resist bio-reduction to generate
electrophilic substances which can destroy proteins and acids.10 In the past decades, the
toxicity and metabolism of nitroimidazole have been characterized, especially
metronidazole.12 In the past two years, we have already synthesized several compounds
containing 5-nitroimidazole and evaluated for their antibacterial activities.6,13-16 In view
the above-mentioned findings, we continue to our previous study and designed,
synthetized a novel series which have 5-nitroimidazole in skeleton as Tyrosyl-tRNA
synthetase inhibitors, and which was evaluated for bacterial activity against
Gram-positive (S. aureus ATCC 6538 and B. subtilis ATCC 6633) and Gram-negative (E.
coli ATCC 35218 and P. aeruginosa ATCC 13525), and toxicity in human cells. At the
beginning of the study, the molecular docking was performed to looking for antibacterial
target protein to determine who work with the designed compounds. The molecular
docking of all the synthesized compounds and target proteins, such as FabH (PDB code:
1HNJ), DNA gyrase (PDB code: 3G75), Tyrosyl-tRNA synthetase (PDB code: 1JIJ) and
Isoleucyl-tRNA synthetase (PDB code: 1JZQ), were performed by using the Discovery
Studio 3.5. The results were presented in Fig. 1, it has been observed that the
CDOCKER_INTERACTION_ENERGY between the designed compounds and protein
1JIJ is the lowest. The lower value of the CDOCKER_INTERACTION_ENERGY
represented the potent binding affinity. This indicated that the designed compounds may
have good inhibitory activity against Tyrosyl-tRNA synthetase.17-18 So we evaluated
their antibacterial activities, and Tyrosyl-tRNA synthetase inhibitory activities.
3

Fig. 1. The CDOCKER_INTERACTION_ENERGY (kcal/mol) obtained from the
docking study of all synthesized compounds by the CDOCKER protocol (Discovery
Studio 3.5, Accelrys, Co. Ltd).
2. Results and discussion
2.1 Chemistry
A series of novel salicylic acid derivatives were synthesized by the routs outlined in
Scheme 1 and the structures of the desired compounds are shown in Table 1.19-21 The
Met-OTs(2-(2-methyl-5-nitro-1H-imidazol-1-yl)-ethyl-4-methyl-benzenesulfonate,
compound 1 was synthesized by the method in our previous paper.21 Firstly, compounds
3a-3w were prepared by the reaction of methyl salicylate with different substituted
benzyl bromide in acetone by adding potassium carbonate at 60 ℃. Secondly, 3a-3w
were hydrolyzed using NaOH solution in EtOH which was heated at 60 ℃ for 6 h.
After completion of the reaction, the solvent was evaporated, then 5M hydrochloric
acid was slowly added to give a pH of 1~3 to get compounds 4a-4w. Lastly,
compounds 4a-4w and Met-OTs were dissolved in DMF and refluxed to get the desired
compounds 5a-5w. The refined compounds 5a-5w were finally obtained by subsequent
purification with chromatography. All synthesized compounds were given satisfactory
analytical and spectroscopic data which were in full accordance with their depicted
4

structures. Additionally, the structure of compound 5j were determined by single crystal
x-ray diffraction analysis in Table 2 and gave perspective views of compound 5j with
the atomic labeling system in Fig. 2. .
O N
2
O
N
O
N
S
a
O
O
N
N
S
HO
NO
O
2
Cl
O
O
O
b
OH
c
Br
O
O
R
O
O
OH
R
R
N
O
O
O
N
O
N
d
OH
N
S
O
O
NO
2
O
O
NO
2
R
R
Scheme 1. The synthetic routes of compounds 5a-5w. Reagents and conditions: (a)
Et3N, CH2Cl2, 0 ℃, 6 h; (b) K2CO3, Me2CO, 60℃, 6~8 h; (c) NaOH, EtOH, 60 ℃, 6
h, pH 1~3; (d) K2CO3, DMF, 110 ℃, 20~24 h.
Table 1. Structures of compounds 5a-5w.
Compounds
R
H
Compounds
R
2-CF3
5a
5m
3-CF3
4-CF3
2-F
3-F
5b
5c
5d
5e
5f
5n
5o
5p
5q
5r
4-F
2, 6-F
3, 4-F
3, 4-Cl
2-Cl
3-Cl
5

4-Cl
4-Br
2, 5-Cl
3-F, 4-Cl
2-F, 3-Cl
3,4,5-F
5g
5h
5i
5s
5t
4-CH3
5u
5v
5w
3, 5-CH3
4-CH(CH3)2
4-OCH3
5j
2, 3, 4, 5, 6-F
5k
5l
Table 2. Crystal structure data for compound 5j.
Compound
Formula
Mr
5j
C2H23N3O5
409.43
Crystal system
triclinic
Space group
a / Ǻ
P -1
7.2644(8)
11.6040(12)
b / Ǻ
c / Ǻ
α/°
12.4151(15)
88.272(4)
β/°
86.610(4)
83.315(2)
γ/°
Volume /Å3
Z
1037.1(2)
2
Dc/(g/cm3)
1.311
μ/mm−1
0.094
F(000)
Crystal size /mm3
T /K
432
0.30×0.26×0.24
273(2)
θRange /°
2.44/27.58
Index range (h,k,l)
-8/9, -14/13, -16/16
Reflections collected /unique
10652/4684[R(int)]=0.0315
6

Data/restraints/parameters
4684/0/274
1.065
Goodness-of-fit on F2
R1, wR2 [I >2σ(I)] A
0.0545/0.1281
A
R1, wR2
0.0930/0.1404
0.204/-0.289
3
(Δρ)max, (Δρ)min/(e/Ǻ )
7

Fig. 2. Crystal structure diagram of compound 5j.
2.2 Biological activity
2.2.1 Antibacterial activity
The antibacterial efficacy of these compounds were evaluated against Gram-positive
(S. aureus ATCC 6538 and B. subtilis ATCC 6633) and Gram-negative (E. coli ATCC
35218 and P. aeruginosa ATCC 13525) bacteria by the MH media dilution method. The
MIC (minimum inhibitory concentration) values of these compounds were summarized
in Table 3. Penicillin G and Chloramphenicol were also screened under identical
conditions for comparison.
Table 3. Antibacterial activities of synthetic compounds.
MIC a (μg/mL)
Compounds
Gram-positive
B. subtilis S. aureus
ATCC 6633 ATCC 6538 ATCC 35218 ATCC 13525
Gram-negative
E. coli P. aeruginosa
5a
5b
5c
5d
5e
5f
12.5
25
12.5
12.5
12.5
12.5
6.25
6.25
25
50
50
50
25
50
50
12.5
6.25
6.25
12.5
25
25
6.25
12.5
25
12.5
12.5
12.5
5g
6.25
8

5h
6.25
3.13
1.57
3.13
0.78
50
6.25
3.13
3.13
3.13
1.57
100
50
12.5
12.5
6.25
6.25
6.25
100
100
50
12.5
12.5
12.5
6.25
6.25
100
100
50
5i
5j
5k
5l
5m
5n
100
25
5o
25
5p
25
25
50
50
5q
50
25
100
3.13
3.13
6.25
12.5
100
100
6.25
6.25
100
1.57
6.25
6.25
6.25
50
5r
0.78
0.78
3.13
1.57
25
0.39
1.57
3.13
3.13
25
5s
5t
5u
5v
5w
50
50
100
6.25
0.78
Penicillin G
0.78
1.57
0.78
1.57
Chloramphenicol
a Values are the average of three independent experiments run in triplicate.
The results were shown in Table 3, the MICs differed greatly ranging from 0.39 ~ 100
μg/mL. Overall, most of the compounds exhibited better bacterial activities against
Gram-positive bacteria strains than Gram-negative bacteria strains. Among them,
compound 5r displayed the most potent antibacterial activity, which had MIC values of
0.78, 0.39, 3.13 and 1.57 µg/mL against B. subtilis, S. aureus, P. aeruginosa and E. coli,
respectively, and was comparable to the antibiotic Penicillin G (corresponding MICs:
0.78, 0.78, 6.25 and 6.25 μg/mL) and chloramphenicol (corresponding MICs: 1.57, 1.57,
3.13 and 0.78 μg/mL).
The modification of substituents such as methoxyl, halogen, methyl was performed to
explore the structure-activity relationships of these compounds. Compounds(5g, 5f, 5e,
5r, 5s and 5w) with chlorine groups on benzyl ring exhibited antibacterial activities in
the order of di-substituted > mono-substituted. Compounds 5r and 5s, which introduced
9

two chlorine atoms at 3, 4-position or 2, 5-positon in benzene ring, showed that
compounds with substitutes at 3, 4-position displayed better antibacterial activity than
substitutes at 2, 5-postion. Compared to 5r and 5s, compounds 5u and 5t showed more
weak antibacterial activity. The MIC values of compound 5r and 5s (0.39 ~ 3.13 μg/mL
and 0.78 ~ 6.25 μg/mL), 5u and 5t (1.57 ~ 6.25 μg/mL and 3.13 ~ 6.25 μg/mL) suggested
that compounds with the same group of the benzene ring had more potent antibacterial
activity than compounds with different groups. Interestingly, compounds (5d, 5p, 5v and
5w) with fluoro groups on benzyl ring exhibited antibacterial activities in the order of
mono-substituted > di-substituted > tri-substituted > multi-substituted, we concluded
that the more electron withdrawing groups could reduce the electron density of the
benzene ring and make it difficult to form π-π bonds with amino acids containing
aromatic groups.22 This performance is particularly prominent in trifluoromethyl group,
5m and 5n showed worst antibacterial activity. Among compounds 5k, 5i, 5d, 5g, 5h, 5o,
compounds with para substituted on phenyl ring exhibited antibacterial activities in
order of electron-donating groups (-CH3, -OCH3 and –CH(CH3)2)
>
electron-withdrawing groups (-CF3, -F, -Cl and -Br). Among these compounds only one
halogen atom at the 4-position of the phenyl ring, mostly, the strength order is Br > Cl >
F.
Next, a comparison of the composition on the benzyl ring was displayed as follows:
for compounds 5b-5d, 5e-5g, and 5m-5o, the potency order was found as para > ortho >
meta in substituent groups on benzyl ring. This indicated that the position of the
substituent had an influence on the antibacterial activity.
2.2.2 S. aureus tyrosyl-tRNA inhibitory activity
In order to further study the mechanism by which the synthesized compounds induce
antibacterial activity, the S. aureus Tyrosyl-tRNA inhibitory potency of the compounds
were examined and summarized in Table 4. As we can see from Table 4, most of the
tested compounds displayed potent Tyrosyl-tRNA inhibiting activity. Among them,
compounds 5r, displaying the most potent Tyrosyl- tRNA inhibitory activity with IC50
10

of 2.3 μM. Compounds 5j, 5s and 5u also displayed good inhibitory with IC50 of 3.0 μM,
2.7 μM and 2.9 μM. The results of S. aureus Tyrosyl-tRNA inhibitory activity of
synthesized compounds (Table 4) were causally related to their antibacterial activity
(Table 3). This indicated that antibacterial activity of the synthesized compounds would
derived from the inhibition of Tyrosyl-tRNA enzymatic activities.
Table 4. Inhibitory effects of the synthetic compounds against S. aureus Tyrosyl-tRNA.
a
S.aureus tyrosyl-tRNA
IC50 (μM)
Hemolysis LC50
Macrophage
Compounds
(μM)
CC50b(μM)
5a
5b
5c
5d
5e
5f
5.6 0.12
6.7 0.03
6.3 0.45
5.7 0.08
4.7 0.21
5.0 0.12
4.8 0.13
4.9 0.17
4.7 0.09
3.0 0.12
3.3 0.20
2.9 0.26
7.8 0.34
8.3 0.45
6.3 0.25
6.9 0.33
6.0 0.15
2.3 0.10
2.7 0.05
3.2 0.12
2.9 0.17
6.7 0.23
8.0 0.45
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
231.2 1.13
154.7 2.32
123.6 2.33
156.9 1.78
213.3 1.22
187.7 1.78
167.5 1.98
198.5 1.74
139.8 1.28
251.7 0.76
271.8 0.56
238.7 0.98
233.0 1.69
250.6 2.64
241.7 2.46
276.8 1.76
256.4 1.24
187.7 1.15
193.4 1.27
179.7 1.78
190.5 2.61
267.4 2.30
236.4 1.80
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
a Lytic concentration 30%.
11

b
Minimum cytotoxic concentration required to cause a microscopically detectable
alteration of normal cell morphology.
2.2.3 Cytotoxicity test
In order to find potent antibiotics, it is important to measure cytotoxicity. Good
antibacterial agents not only have effective antibacterial activities, but also have no
damage to mammalian cells that lead to high hemolytic and cytotoxic activity dangerous
to the host organism. Generally, red blood cell is extremely fragile, hemolytic activity is
used to measure the mammalian cell toxicity. Moreover, compounds were evaluated for
their toxicity against human macrophage with the median cytotoxic concentration (CC50)
data by the MTT method.
So the hemolysis and cytotoxicity assays were both tested. The pharmacological
results are showed in Table 4. As shown Table 4 tested compounds displayed low
hemolytic activities and almost have no cytotoxicity activities in vitro against human
macrophage. The cytotoxicity assay determined the selectivity of our compounds for
bacteria over mammalian cells.
2.2.4 Drug-likeness analysis
Drug-like properties were calculated and the results were summarized in Table 5.
Drug-like properties consist of molecular weight (MW), octanol–water partitioning
coefficient (AlogP) based on Ghose and Crippen’s method,23 the number of hydrogen
bond acceptors (HBA), the number of hydrogen bond donors (HBD) and molecular
fractional polar surface area (MFPSA). All the data were calculated using the
Discovery Studio molecular simulation package.24 As shown in Table 5, all the desired
compounds had AlogP value less than 5, MW less than 480 showed favorable values
for the involved molecular parameters that is suitable for the Lipinski’s Rule of Five.25
Table 5. Molecular parameters of synthetic compounds.
Molecular parameters
Compounds
MW
381.382
AlogPa
3.273
HBAb HBDc MFPSAa
5a
4
0
0.267
12

5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
399.372
399.372
399.372
415.827
415.827
415.827
460.278
395.409
409.435
423.462
411.408
449.38
3.479
3.479
3.479
3.938
3.938
3.938
4.022
3.76
4.246
4.468
3.257
4.216
4.216
4.216
3.684
3.684
4.602
4.602
4.143
4.143
3.89
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.261
0.261
0.261
0.251
0.251
0.251
0.245
0.254
0.242
0.234
0.268
0.241
0.241
0.241
0.255
0.255
0.237
0.237
0.246
0.246
0.25
449.38
449.38
417.363
417.363
450.272
450.272
433.817
433.817
435.353
471.334
5t
5u
5v
5w
4.301
0.24
a Calculated by Discovery Studio 3.5.
b Counted as the sum of Ns and Os.
c Counted as the sum of NHs and OHs.
2.2.5 Molecular docking
In order to study the interaction between binding model and bacterial activity, a
docking study was performed to fit compound 5r into the active center of the S. aureus
Tyrosyl-tRNA (PDB:1JIJ). All docking runs were applied CDOCKER_DOCK protocol
using Discovery Studio3.5. The binding model of compound 5r with Tyrosyl-tRNA
protein are presented in Fig. 3. . In the binding model, compound 5r was well bound to
the Tyrosyl-tRNA protein with Lys84, Arg88, Tyr170 and Gln174, the four amino acids
located in the binding pocket of the protein played important action in the combination
with compound 5r. As we can see from Fig. 3. (A), Arg88 and Lys84 were respectively
formed two charge interactions with oxygen atom of nitro group (O⋯H: 2.5 Å and O⋯H:
2.4 Å), which enhanced the combination activity of compound 5r. Oxygen atom of nitro
group formed two hydrogen bonds with the backbone NH of Arg88 (N-O⋯H: 2.5 Å,
13

102.73°), Lys84 (N-O⋯H: 2.2 Å, 143.88°), respectively. And carboxyl oxygen formed
two hydrogen bonds with Gln174 (C=O⋯H: 2.3 Å, 132.65°) and Tyr170 (C=O⋯H: 2.0
Å, 161.78°). The receptor surface model shown in Fig. 3. (C), which indicated that the
compound 5r was embedded into the Tyrosyl-tRNA protein active pocket. Docking
results agreed with the antibacterial assay data, suggested that compound 5r was a
potential inhibitor of Tyrosyl-tRNA protein.
14

Fig. 3. Molecular docking modeling of compound 5r with S.aureus Tyrosyl tRNA
synthetase: or clarity, only interacting residues are displayed.
(A) 2D model of the interaction between compound 5r and S. aureus Tyrosyl-tRNA
synthetase. Blue dashed arrow represent the H-bond interactions with amino acids; pink
dashed arrow represent the charge interactions.
(B) 3D model of the interaction between compound 5r and S. aureus Tyrosyl-tRNA
synthetase.
(C) The enzyme is shown as surface; while 5r docked structures are shown as sticks.
3. Conclusion
In summary, a series of novel salicylic acid derivatives containing metronidazole had
been designed and synthesized. These compounds were evaluated for their antibacterial
activity against Gram-positive (S. aureus and B. subtilis) and Gram-negative (E. coli and
P. aeruginosa). Out of these compounds, compound 5r showed the most potent
inhibitory activity against the tested bacteria strains with MICs of 0.39 ~ 3.13 μg/mL and
Tyrosyl-tRNA inhibitory activity with IC50 value of 2.3 μM. Molecular docking was
performed to position compound 5r into S. aureus Tyrosyl- tRNA active site to further
determine the probable binding models and the result showed that compound 5r was a
potential Tyrosyl-tRNA inhibitor. This work might be useful for the design and synthesis
of Tyrosyl-tRNA inhibitors with strong activities.
15

4. Experiments
4.1 Materials and measurements
All chemicals and reagents used in current study were analytical reagent or chemical
pure. All reactions were routinely checked by thin-layer chromatography (TLC) on the
glass-backed silica gel sheets (silica GF 254) and visualized using UV illumination.
Flash column chromatography was performed on silica gel (200-300 mesh) eluting with
ethyl acetate and petroleum ether. Melting points (uncorrected) were determined on a
1
X-4 MP apparatus (Taike Corp, Beijing, China). H NMR spectra were recorded on a
DPX 400 model spectrometer and reported in ppm using residual solvent as the internal
standard (CDCl3 at 7.24 ppm, DMSO-d6 at 2.50 ppm). ESI-MS spectra were recorded on
a Mariner System 5304 Mass Spectrometer. Elemental analyses were performed on a
CHN-O-Rapid instrument and were within 0.4% of the theoretical values. All the
starting materials were commercially available unless otherwise indicated.
4.2 General method of synthesis of salicylic acid derivatives containing
metronidazole
4.2.1 General synthetic procedure of Met-OTs (2-(2-methyl-5-nitro-1H-imidazol-
1-yl) ethyl-4-methylbenzenesulfonate)( 1 )
All compounds described herein were synthesized by the following pathway depicted
in Scheme1. The key compound Met-OTs (2-(2-methyl-5-nitro-1H-imidazol-
1-yl) ethyl-4-methylbenzenesulfonate), compound 1 has been synthesized from the
metronidazole using the proper excess of tosyl chloride in the presence of
trimethylamine in dichloromethane. Metronidazole (3.14 g, 20 mmol) and Et3N (3.3 mL,
24 mmol) in CH2Cl2 (10 mL), and 4-methyl-benzenesufonyl chloride (3.83 g, 20.1
mmol) in CH2Cl2 (10 mL) was added. The reaction mixture was stirred at 0 ℃ for 6 h,
then the mixture was evaporated under reduced pressure to afford a residue. The
16

residue was washed by petroleum ether for three times to remove remaining
4-methyl-benzenesufonyl chloride. The product was dried to get a slight-yellow crystal
of compound 1 (Met-OTs).
4.2.2 General synthetic procedure of methyl salicylate derivatives(3a-3w)
Compounds 3a-3w were synthesized by the following procedure. Methyl salicylate
(4 mmol), 2a-2w (4 m mol) and K2CO3 (8 mmol) were added in acetone. The reaction
was stirred at 60 ℃ for 6 ~ 8 h to gain 3a-3w.
4.2.3 General synthetic procedure of salicylic acid derivatives(4a-4w)
3a-3w were hydrolyzed using 20% NaOH in EtOH which was stirred at 60 ℃ for 6
h. After reaction, the pH value of the solution was adjusted to 1 ~ 3 using 5M
hydrochloric acid. The mixture was extracted with CH2Cl2 for three times. The organic
layer was combined and dried with anhydrous Na2SO4 for 0.5 h to get 4a-4w.
4.2.4 General synthetic procedure of salicylic acid derivatives containing
metronidazole (5a-5w)
Met-OTs (2 mmol), 4a-4w (2 mmol) and K2CO3 (4 mmol) were added in DMF (20
mL). The reaction was stirred at 110 ℃ for 20 ~ 24 h. The reaction mixture was
poured into water and extracted with ethyl acetate (3×50 mL). The organic layer was
combined and dried with anhydrous Na2SO4 for 0.5 h. Column chromatography was
performed using silica gel (200 ~ 300 mesh) eluting with ethyl acetate and petroleum
ether to obtain the desired compounds (5a-5w).
4.2.4.1. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-(benzyloxy)benzoate(5a).
Yellow solid, yield: 63.23%, mp: 78-80 ℃. 1H NMR(400 MHz, CDCl3) δ：2.43(s, 3H,
CH3), 4.68(s, 4H, CH2), 5.19(s, 2H, CH2), 7.02-7.07(m, 2H, ArH), 7.36-7.40(m, 1H,
ArH), 7.42(s, 1H, ArH), 7.44(d, J=5.80 Hz, 2H, ArH), 7.47-7.48(m, 1H, ArH),
7.50-7.52(m, 1H, ArH), 7.73-7.75(m, 1H, ArH), 7.93(s, 1H, ArH). MS (ESI): 381.38
17

(C20H20N3O5, [M+H]+). Anal.Calcd for C20H19N3O5: C, 62.99; H, 5.02; N, 11.02%.
Found: C, 63.12; H, 5.01; N, 11.04%.
4.2.4.2. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((2-fluorobenzyl)-oxy)benz
oate(5b).
Yellow solid, yield: 58.34%, mp: 89-91 ℃. 1H NMR(400 MHz, CDCl3) δ：2.46(s, 3H,
CH3), 4.69(s, 4H, CH2), 5.25(s, 2H, CH2), 7.03-7.14(m, 3H, ArH), 7.21(t, J=8.00 Hz, 1H,
ArH), 7.32-7.38(m, 1H, ArH), 7.51-7.55(m, 1H, ArH), 7.59(t, J= 7.18 Hz, 1H, ArH),
7.73-7.75(m, 1H, ArH), 7.95 (s, 1H, ArH). MS (ESI): 399.37 (C20H19FN3O5, [M+H]+).
Anal.Calcd for C20H18FN3O5: C, 60.15; H, 4.54; N, 10.52%. Found: C, 60.11; H, 4.55;
N, 10.53%.
4.2.4.3. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((3-fluorobenzyl)-oxy)benz
oate(5c).
Yellow solid, yield: 59.07%, mp: 88-89 ℃. 1H NMR(400 MHz, CDCl3) δ：2.47(s, 3H,
CH3), 4.72(s, 4H, CH2), 5.19(s, 2H, CH2), 7.02-7.08(m, 3H, ArH), 7.23(t, J=6.34 Hz, 2H,
ArH), 7.39(m, 1H, ArH), 7.50-7.54(m, 1H, ArH), 7.76(d, J=7.72 Hz, 1H, ArH), 7.94(s,
1H, ArH). MS (ESI): 399.37 (C20H19FN3O5, [M+H]+). Anal.Calcd for C20H18FN3O5: C,
60.15; H, 4.54; N, 10.52%. Found: C, 60.05; H, 4.52; N, 10.53%
4.2.4.4. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((4-fluorobenzyl)-oxy)benz
oate(5d).
Yellow solid, yield: 62.18%, mp: 90-92 ℃. 1H NMR(400 MHz, CDCl3) δ：2.46(s, 3H,
CH3), 4.70(s, 4H, CH2), 5.14(s, 2H, CH2), 7.01-7.07(m, 2H, ArH), 7.35(d, J=8.16 Hz,
2H, ArH), 7.49-7.56(m, 3H, ArH), 7.73(d, J=7.72 Hz, 1H, ArH), 7.95(s, 1H, ArH). MS
(ESI): 399.37(C20H19FN3O5, [M+H]+). Anal.Calcd for C20H18FN3O5: C, 60.15; H, 4.54;
N, 10.52%. Found: C, 60.05; H, 4.55; N, 10.54%.
4.2.4.5. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((2-chlorobenzyl)-oxy)benz
18

oate(5e).
Yellow solid, yield: 68%, mp: 99-101 ℃. 1H NMR(400 MHz, CDCl3) δ：2.46(s, 3H,
CH3), 4.69(s, 4H, CH2), 5.25(s, 2H, CH2), 7.02-7.08(m, 2H, ArH), 7.29(t, J=6.92 Hz, 1H,
ArH), 7.34(t, J=8.00 Hz, 1H, ArH), 7.41(d, J=7.46 Hz, 1H, ArH), 7.52(t, J=7.88 Hz, 1H,
ArH), 7.68(d, J=7.52 Hz, 1H, ArH), 7.75(d, J=8.00 Hz, 1H, ArH), 7.94(s, 1H, ArH). MS
(ESI): 415.83 (C20H19ClN3O5, [M+H]+). Anal.Calcd for C20H18ClN3O5: C, 57.77; H,
4.36; N, 10.11%. Found: C, 57.79; H, 4.34; N, 10.18%.
4.2.4.6. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((3-chlorobenzyl)-oxy)benz
oate(5f).
Yellow solid, yield: 76.27%, mp: 90-91 ℃. 1H NMR(400 MHz, CDCl3) δ：2.46(s, 3H,
CH3), 4.72(s, 4H, CH2), 5.16(s, 2H, CH2), 7.01-7.08(m, 2H, ArH), 7.33(d, J=14.68 Hz,
3H, ArH), 7.52(t, J=9.08 Hz, 2H, ArH), 7.76(d, J=5.60 Hz, 1H, ArH), 7.93(s, 1H, ArH).
MS (ESI): 415.83 (C20H19ClN3O5, [M+H]+). Anal.Calcd for C20H18ClN3O5: C, 57.77;
H, 4.36; N, 10.11%. Found: C, 57.79; H, 4.32; N, 10.09%.
4.2.4.7. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((4-chlorobenzyl)-oxy)benz
oate(5g).
Yellow solid, yield: 76.87%, mp: 91-93 ℃. 1H NMR(400 MHz, CDCl3) δ：2.48(s, 3H,
CH3), 4.72(s, 4H, CH2), 5.15(s, 2H, CH2), 7.00(d, J=8.36 Hz, 1H, ArH), 7.04-7.08(m,
1H, ArH), 7.19-7.21(m, 1H, ArH), 7.31(s, 1H, ArH), 7.44(t, J=7.84 Hz, 1H, ArH),
7.50-7.54(m, 2H, ArH), 7.74-7.76(m, 1H, ArH), 7.96(s, 1H, ArH). MS (ESI): 415.83
(C20H19ClN3O5, [M+H]+). Anal.Calcd for C20H18ClN3O5: C, 57.77; H, 4.36; N, 10.11%.
Found: C, 57.80; H, 4.41; N, 10.16%.
4.2.4.8. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((4-bromobenzyl)-oxy)benz
oate(5h).
Yellow solid, yield: 74.26%, mp: 109-110 ℃. 1H NMR(400 MHz, CDCl3) δ：2.46(s,
19

3H, CH3), 4.70(s, 4H, CH2), 5.1(s, 2H, CH2), 7.01-7.06(m, 2H, ArH), 7.35(d, J=8.16 Hz,
2H, ArH), 7.49-7.56(m, 3H, ArH), 7.73(d, J=7.72 Hz, 1H, ArH), 7.95(s, 1H, ArH). MS
(ESI): 460.28 (C20H19BrN3O5, [M+H]+). Anal.Calcd for C20H18BrN3O5: C, 52.19; H,
3.94; N, 9.13%. Found: C, 52.25; H, 3.99; N, 9.21%.
4.2.4.9. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((4-methylbenzyl)-oxy)benz
oate(5i).
Yellow crystal, yield: 77.16%, mp: 98-99 ℃. 1H NMR(400 MHz, CDCl3) δ：2.40(s,
3H, CH3), 2.43(s, 3H, CH3), 4.67(s, 4H, CH2), 5.14(s, 2H, CH2), 7.00-7.06(m, 2H,
ArH), 7.22(d, J=7.8 Hz, 2H, ArH), 7.33(d, J=7.84 Hz, 2H, ArH), 7.50(t, J=7.88 Hz, 1H,
ArH), 7.73(d, J=7.68 Hz, 1H, ArH), 7.93(s, 1H, ArH). MS (ESI): 395.41 (C21H22N3O5,
[M+H]+). Anal.Calcd for C20H21N3O5: C, 63.92; H, 5.35; N, 10.63%. Found: C, 63.79;
H, 5.33; N, 10.63%.
4.2.4.10. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((3, 5-dimethylbenzyl)-ox
y)benzoate(5j).
Yellow crystal, yield: 76.18%, mp: 96-97 ℃. 1H NMR(400 MHz, CDCl3) δ：2.36(s,
6H, CH3), 2.44(s, 3H, CH3), 4.68-4.69(m, 4H, CH2), 5.12(s, 2H, CH2), 6.99-7.07(m, 5H,
ArH), 7.47-7.52(m, 1H, ArH), 7.72-7.74(m, 1H, ArH), 7.94(s, 1H, ArH). MS (ESI):
409.16 (C22H24N3O5, [M+H]+). Anal.Calcd for C20 C22H23N3O5: C, 64.54; H, 5.66; N,
10.26%. Found: C, 64.70; H, 5.68; N, 10.29%.
4.2.4.11. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((4-isopropylbenzyl)-oxy)
benzoate(5k).
Yellow solid, yield: 67.54%, mp: 80-81 ℃. 1H NMR(400 MHz, CDCl3) δ：1.28-1.31
(d, J=6.92 Hz, 6H, CH3), 2.43(s, 3H, CH3), 2.93-2.99(m, 1H, CH), 4.67(s, 4H, CH2),
5.15(s, 2H, CH2), 7.02(t, J=7.50 Hz, 1H, ArH), 7.07(d, J=8.40 Hz, 1H, ArH), 7.29(t,
J=7.90 Hz, 1H, ArH), 7.38(d, J=7.92 Hz, 2H, ArH), 7.50(t, J=7.84 Hz, 1H, ArH), 7.72
(d, J=7.64 Hz, 1H, ArH), 7.94(s, 1H, ArH). MS (ESI): 423.46 (C23H26N3O5, [M+H]+).
Anal.Calcd for C23H25N3O5: C, 65.24; H, 5.95; N, 9.92%. Found: C, 65.09; H, 5.93; N,
20

9.89%.
4.2.4.12. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((4-methoxybenzyl)-oxy)
benzoate(5l).
Yellow solid, yield: 63.94%, mp: 85-86 ℃. 1H NMR(400 MHz, CDCl3) δ：2.40(s, 3H,
CH3), 2.47(s, 3H, CH3), 4.67(s, 4H, CH2), 5.14(s, 2H, CH2), 7.00-7.06(m, 2H, ArH),
7.22(d, J=7.8 Hz, 2H, ArH), 7.33(d, J=7.84 Hz, 2H, ArH), 7.50(t, J=7.88 Hz, 1H, ArH),
7.73(d, J=7.68 Hz, 1H, ArH), 7.93(s, 1H, ArH). MS (ESI): 411.41 (C21H22N3O6,
[M+H]+). Anal.Calcd for C21H21N3O6: C, 61.31; H, 5.14; N, 10.21%. Found: C, 61.48;
H, 5.15; N, 10.23%.
4.2.4.13. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((2-(trifluoromethyl)-benz
yl)-oxy)benzoate(5m).
Yellow solid, yield: 58.48%, mp: 92-93 ℃. 1H NMR(400 MHz, CDCl3) δ：2.48(s, 3H,
CH3), 4.72(s, 4H, CH2), 5.39(s, 2H, CH2), 7.04-7.07(m, 2H, ArH), 7.45-7.55(m, 2H,
ArH), 7.64(t, J=7.61 Hz, 1H, ArH), 7.73(d, J=7.84 Hz, 1H, ArH), 7.77(d, J=7.68 Hz, 1H,
ArH), 7.93(d, J=7.88 Hz, 1H, ArH), 7.96(s, 1H, ArH). MS (ESI): 449.38
(C21H19F3N3O5, [M+H]+). Anal.Calcd for C21H18F3N3O5: C, 56.13; H, 4.04; N, 9.35%.
Found: C, 56.26; H, 4.03; N, 9.37%.
4.2.4.14. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((3-(trifluoromethyl)-benz
yl)-oxy)benzoate(5n).
Yellow solid, yield: 68.31%, mp: 78-80 ℃. 1H NMR(400 MHz,CDCl3) δ：2.46(s, 3H,
CH3), 4.71(s, 4H, CH2), 5.24(s, 2H, CH2),7.04-7.09(m, 2H, ArH), 7.53(t, J=9 Hz, 1H,
ArH), 7.57(d, J=7.84 Hz, 1H, ArH), 7.64(d, J=7.64 Hz, 1H, ArH),7.68(d, J= 7.52 Hz,
1H, ArH), 7.78(m, 2H, ArH), 7.91(s, 1H, ArH). MS (ESI): 449.38 (C21H19F3N3O5,
[M+H]+). Anal.Calcd for C21H18F3N3O5: C, 56.13; H, 4.04; N, 9.35%. Found: C, 56.28;
H, 4.05; N, 9.39%.
4.2.4.15. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((4-(trifluoromethyl)-benz
yl)-oxy)benzoate(5o).
Yellow crystal, yield: 73.03%, mp:148-149 ℃. 1H NMR(400 MHz, CDCl3) δ：2.47(s,
21

3H, CH3), 4.71(s, 4H, CH2), 5.24(s, 2H, CH2), 7.01-7.08(m, 2H, ArH)，7.50-7.54(m, 1H,
ArH), 7.61(d, J=8.08 Hz, 2H, ArH), 7.69(d, J=7.52 Hz, 2H, ArH), 7.74-7.76(m, 1H,
ArH), 7.94(s, 1H, ArH). MS (ESI): 449.38 (C21H19F3N3O5, [M+H]+). Anal.Calcd for
C21H18F3N3O5: C, 56.13; H, 4.04; N, 9.35%. Found: C, 56.23; H, 4.03; N, 9.41%.
4.2.4.16. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((2, 6-difluorobenzyl)-oxy)
benzoate(5p).
Yellow solid, yield: 69.06%, mp: 113-114 ℃. 1H NMR(400 MHz,CDCl3) δ：2.44(s,
3H, CH3), 4.62(s, 4H, CH2), 5.22(s, 2H, CH2), 6.94-6.98(t, J=7.84 Hz, 2H, ArH), 7.07(t,
J=7.52 Hz, 2H, ArH), 7.20(d, J=8.36 Hz, 1H, ArH), 7.34-7.41(m, 1H, ArH),
7.53-7.57(m, 1H, ArH), 7.69(t, J=7.72 Hz, 2H, ArH), 7.98(s, 1H, ArH). MS (ESI):
417.36(C20H18F2N3O5, [M+H]+). Anal.Calcd for C20H17F2N3O5: C, 57.56; H, 4.11; N,
10.07%. Found: C, 57.63; H, 4.18; N, 10.17%
4.2.4.17. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((3, 4-difluorobenzyl)-oxy)
benzoate(5q).
Yellow solid, yield: 71.85%, mp: 105-106 ℃. 1H NMR(400 MHz, CDCl3) δ：2.48(s,
3H, CH3), 4.70-4.73(m, 4H, CH2), 5.13(s, 2H, CH2), 7.00-7.06(m, 1H, ArH),
7.19-7.23(m, 1H, ArH), 7.31-7.37(m, 1H, ArH), 7.52-7.54(m, 1H, ArH), 7.73-7.76(m,
1H, ArH), 7.95(s, 1H, ArH). MS (ESI): 417.36(C20H18F2N3O5, [M+H]+). Anal.Calcd
for C20H17F2N3O5: C, 57.56; H, 4.11; N, 10.07%. Found: C, 57.61; H, 4.09; N, 10.08%.
4.2.4.18. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((3, 4-dichlorobenzyl)-oxy)
benzoate(5r).
1
Yellow solid, yield: 77.27%, mp: 143-144 ℃. H NMR(DMSO-d6,400 MHz) δ：
2.40(s, 3H, CH3), 4.62(t, J=4.78 Hz, 2H, CH2), 4.69(t, J=4.82 Hz, 2H, CH2), 5.20(s, 2H,
CH2), 7.06(t, J=7.52 Hz, 1H, ArH), 7.21(d, J=8.4 Hz, 1H, ArH), 7.40-7.42(m, 1H, ArH),
7.54-7.59(m, 1H, ArH), 7.61-7.63(m, 1H, ArH), 7.65(d, J=8.28 Hz, 1H, ArH), 7.71(s,
1H, ArH), 7.98(s, 1H, ArH). MS (ESI): 450.27 (C20H18Cl2N3O5,[M+H]+). Anal.Calcd
for C20H17Cl2N3O5: C, 53.35; H, 3.81; N, 9.33%. Found: C, 53.29; H, 3.85; N, 9.39%.
4.2.4.19. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((2, 6-dichlorobenzyl)-oxy)
22

benzoate(5s).
Yellow solid, yield: 76.16%, mp: 109-110 ℃. 1H NMR(400 MHz, CDCl3) δ：2.47(s,
3H, CH3),4.76(s, 4H, CH2), 5.20(s, 2H, CH2), 7.08(t, J=7.58 Hz, 2H, ArH), 7.27-7.29(m,
1H, ArH), 7.36(d, J=8.48 Hz, 1H, ArH), 7.54-7.58(m, 1H, ArH), 7.77-7.82(m, 2H, ArH),
7.94(s, 1H, ArH). MS (ESI): 450.27(C20H18Cl2N3O5, [M+H]+). Anal.Calcd for
C20H17Cl2N3O5: C, 53.35; H, 3.81; N, 9.33%. Found: C, 53.30; H, 3.88; N, 9.41%.
4.2.4.20. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((4-chloro-3-fluorobenzyl)
-oxy)benzoate(5t).
Yellow solid, yield:74.28%, mp: 121-122 ℃. 1H NMR(400 MHz, CDCl3) δ：2.48(s,
3H, CH3), 4.72(s, 4H, CH2), 5.15(s, 2H, CH2), 6.99-7.01(d, J=8.36 Hz, 1H, ArH),
7.04-7.08(m, 1H, ArH), 7.19-7.21(m, 1H, ArH), 7.31(s, 1H, ArH), 7.43(t, J=7.84 Hz, 1H,
ArH), 7.50-7.54(m, 1H, ArH), 7.74-7.76(m, 1H, ArH), 7.96(s, 1H, ArH). MS (ESI):
433.82 (C20H18ClFN3O5, [M+H]+). Anal.Calcd for C20H17ClFN3O5: C, 55.37; H, 3.95;
N, 9.69%. Found: C, 55.42; H, 4.00; N, 9.76%.
4.2.4.21. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((3-chloro-2-fluorobenzyl)
-oxy)benzoate(5u).
Yellow solid, yield: 58.15%, mp: 113-114 ℃. 1H NMR(400 MHz, CDCl3) δ：2.51(s,
3H, CH3), 4.69-4.74(s, 4H, CH2), 5.26(s, 2H, CH2), 7.06-7.10(m, 2H, ArH),
7.15-7.19(m, 1H, ArH), 7.40-7.44(m, 1H, ArH), 7.51-7.58(m, 2H, ArH), 7.74-7.76(m,
1H, ArH), 7.97(s, 1H, ArH). MS (ESI): 433.82 (C20H18ClFN3O5, [M+H]+). Anal.Calcd
for C20H17ClFN3O5: C, 55.37; H, 3.95; N, 9.69%. Found: C, 55.42; H, 3.99; N, 9.73%.
4.2.4.22. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((3, 4, 5-trifluorobenzyl)-
oxy)benzoate(5v).
Yellow solid, yield: 57.74%, mp: 148-149 ℃. 1H NMR(400 MHz, CDCl3) δ：2.52(s,
3H, CH3), 4.72-4.78(m, 4H, CH2), 5.12(s, 2H, CH2), 6.99(d, J=8.32 Hz, 1H, ArH),
7.06-7.10(m, 1H, ArH), 7.16(t, J=7.3 Hz, 2H, ArH), 7.52-7.56(m, 1H, ArH),
7.75-7.78(m, 1H, ArH), 7.97(s, 1H, ArH). MS (ESI): 435.35 (C20H17F3N3O5, [M+H]+).
Anal.Calcd for C20H16F3N3O5: C, 55.18; H, 3.70; N, 9.65%. Found: C, 55.08; H, 3.78;
23

N, 9.73%.
4.2.4.23. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-2-((perfluorophenyl)-metho
xy)benzoate(5w).
Yellow solid, yield: 57.79%, mp: 166-167 ℃. 1H NMR(400 MHz, CDCl3) δ：2.47(s,
3H, CH3), 4.64-4.70(m, 4H, CH2), 5.25(s, 2H, CH2), 7.08-7.18(m, 2H, ArH), 7.55(t,
J=7.82 Hz, 1H, ArH), 7.70(d, J=7.68 Hz, 1H, ArH), 7.98(s, 1H, ArH). MS (ESI): 471.33
(C20H15F5N3O5, [M+H]+). Anal.Calcd for C20H14F5N3O5: C, 50.96; H, 2.99; N, 8.92%.
Found: C, 50.88; H, 2.91; N, 8.99%.
4.3. Crystal structure determination
Compound 5j of X-ray single-crystal diffraction data was collected on a Bruker
D-8 venture diffractometer equipped with graphite monochromated Mo Kα (λ= 0.71073
Å) radiation by the ω scan mode. Structure solution, refinement and data output were
carried out with the SHELXTL-NT program package.26 All the non-hydrogen atoms
were refined anisotropically. All the hydrogen atoms were placed in calculated positions
and were assigned fixed isotropic thermal parameters at 1.2 times the equivalent
isotropic U of the atoms to which they are attached and allowed to ride on their
respective parent atoms. The contributions of these hydrogen atoms were included in the
structure-factors calculations. The crystal data, data collection and refinement
parameters for the compound 5j were listed in Table 2.
4.4. In Vitro Antimicrobial Activity Experiments
The minimum inhibitory concentration (MIC) was tested against two Gram-positive
bacterial strains (B. subtilis ATCC 6633 and S. aureus ATCC 6538) and two
Gram-negative bacterial strains (E. coli ATCC 35218 and P. aeruginosa ATCC
13525).27 The MIC values were determined using the broth microdilution method
according to the standards Institute (CLSI) as previously described.28 Synthesized
compounds were dissolved in 50% water in DMSO to prepare a stock solution that had
a concentration of 0.5 mg/mL. Serial 2-fold dilutions were prepared from the stock
solution with MH media and poured into 96-well plates. The tested bacteria were
24

grown in MH media at 37 ℃ for 24 h and were justed to the turbidity of the 0.5
McFarland standard. These bacterial suspensions were added to 96-well plates to
obtain the compound concentrations of 25, 12.5, 6.25, 3.13, 1.57, 0.79 μg/mL and even
lower concentrations. An inoculated micro-dilution trays were incubated in ambient air
at 37 ℃ for 18 h. The MIC values were recorded as the lowest concentration of
compounds showing no growth of bacteria.29 MIC values were determined at least
twice on separate days, with the higher value used to represent the MIC value.
Penicillin G and chloramphenicol were used as standards for bacteria. The observed
MICs were presented in Table 3.
4.5. S. aureus TyrRS inhibitory activity
S. aureus TyrRS was over-expressed in E. coli BL21 and purified to near
homogeneity using standard purification procedures.30 TyrRS activity was measured by
aminoacylation using modifications to previously described methods.8 The assays were
performed at 37 ℃ in a mixture containing (final concentrations) 100 mM Tris-HCl pH
8.0, 50 mM KC1, 16 mM MgCl2, 5 mM ATP, 3 mM DTT, 4 mg/mL MRE600 tRNA
(Roche) and 10 μM L-tyrosine (0.3 mM L-[ring-3,5-3H] tyrosine (PerkineElmer,
Specific activity: 1.48-2.22 TBq/mmol), 10 μM carrier). TyrRS (0.2 nM) was
pre-incubated with a range of inhibitor concentrations 25, 12.5, 6.25 3.13, 1.56 and 0.78
μM for 10 min at room temperature followed by the addition of pre-warmed mixture at
37 ℃.” After specific intervals, the reaction was terminated by adding aliquots of the
reaction mixture into ice-cold 7% trichloroacetic acid and harvesting onto 0.45 μm
hydrophilic Durapore filters (Millipore Multiscreen 96-well plates) and counted by
liquid scintillation. The rate of reaction in the experiments was linear with respect to
protein and time with less than 50% total tRNA acylation. IC50 values corresponding to
the concentration at which half of the enzyme activity is inhibited by the compound. The
results were showed in Table 4.
25

4.6. Hemolysis test
Hemolytic activity was assayed using fresh capillary human blood. Erythrocytes were
collected by centrifuging the blood three times in chilled phosphate buffered saline (PBS
at 4 °C) at 1000 g for10 min. The final pellet was resuspended in PBS to give a 2% w/v
solution. Using a microtiter plate, 100 mL of the erythrocyte solution was added to
dextran, PLL, stearyl-PLL or stearyl-PLL+ LDL(1–1000 μg/mL) in a volume of 100 mL.
Samples were then incubated for 3 h and the microtiter plate was then centrifuged at
1000 g for 10 min and the supernatants (100 μL) transferred into a new microtiter plate.
Hemoglobin release was determined spectrophotometrically using a microtiter plate
reader (absorbance at 550 nm). Results were expressed as the amount of released
hemoglobin induced by the compounds as a percentage of the total.
4.7. Cytotoxicity test
The cytotoxic activity in vitro was measured against mammalian cells, human
macrophage using the MTT assay. The cell was grown in DMEM medium
supplemented with 10% PBS and 1× antimycotic and antibacterial solution (sigma USA)
at 37 °C, in humidified atmosphere having 5% CO2. 100 μL of the confluent fibroblast
stock suspension was dispensed in 96-well tissue culture plate. The original medium
from the wells was replaced with 100 μL serum free DMEM when the cells reached 90%
confluence after 5 h incubation in a CO2 incubator. Various concentrations of the
compounds were added to the growing cells and incubated for 24 h. The absorbance was
measured at a wavelength of 570 nm on an ELISA microplate reader. Three replicate
wells were used for each concentration and each assay was measured three times, after
which the average of IC50 was calculated. The cytotoxicity of each compound was
expressed as the concentration of compound that reduced cell viability to 50% (IC50).
The results were summarized in Table 4.
4.8. Docking simulations
26

The crystal structure of S. aureus Tyrosyl-tRNA synthetase (PDB code: 1JIJ) was
obtained from the Protein Data Bank (http://www.rcsb.org). Molecular docking of
compound 5r into the three-dimensional X-ray structure of Tyrosyl-tRNA synthetase
was carried out using CDOCKER_DOCK protocol of Discovery Studio 3.5.
Acknowledgment
This work was supported by Science Research Project of Education Department of
Anhui Province (KJ2013B286).
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Figure Captions
Table 1. Structures of compounds 5a-5w.
Table 2. Crystal structure data for compound 5j.
Table 3. Antibacterial activities of synthetic compounds.
Table 4. Inhibitory effects of the synthetic compounds against S.aureus tyrosyl-tRNA.
Table 5. Molecular parameters of synthetic compounds.
Fig. 1. The CDOCKER_INTERACTION_ENERGY (kcal/mol) obtained from the
docking study of all synthesized compounds by the CDOCKER protocol (Discovery
Studio 3.5, Accelrys, Co. Ltd).
Fig. 2. Crystal structure diagram of compound 5j.
Fig. 3. Molecular docking modeling of compound 5r with S. aureus Tyrosyl tRNA
synthetase: or clarity, only interacting residues are displayed.
Scheme 1. The synthetic routes of compounds 5a-5w. Reagents and conditions:
29