Bioorganic & Medicinal Chemistry 22 (2014) 2409–2415

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Synthesis, molecular docking and biological evaluation of

metronidazole derivatives containing piperazine skeleton as
potential antibacterial agents
She-Feng Wang  , Yong Yin  , Fang Qiao, Xun Wu, Shao Sha, Li Zhang ⇑, Hai-Liang Zhu ⇑
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: Metronidazole has a broad-spectrum antibacterial activity. Hereby a series of novel metronidazole deriv-
Received 22 January 2014 atives were designed and synthesized based on nitroimidazole scaffold in order to find some more potent
Revised 3 March 2014 antibacterial drugs. For these compounds which were reported for the first time, their antibacterial activ-
Accepted 4 March 2014
ities against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus were
Available online 13 March 2014
tested. These compounds showed good antibacterial activities against Gram-positive strains. Compound
4m represented the most potent antibacterial activity against S. aureus ATCC 25923 with MIC of 0.003 lg/
Keywords:
mL and it showed the most potent activity against S. aureus TyrRS with IC50 of 0.0024 lM. Molecular
Metronidazole derivatives
Antibacterial activities
docking of 4m into S. aureus tyrosyl-tRNA synthetase active site were also performed to determine the
S. aureus TyrRS probable binding mode.
Molecular docking Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction tRNA.15 Because aminoacyl-tRNA synthetases can recognize these

Infections induced by antibiotic-resistant pathogens have al-
ready led to a crisis in people’s health care. With the advent of drug
resistance, the microbes have developed several different kinds of
resistance mechanisms. One of them is to reduce drug affinity by
target-based mutation.1 Many problems still need to be solved even
though there are a lot of antimicrobial-drugs available such as keto-
conazole, miconazole, metronidazole and so on (Scheme 1).2–9 For
example, it has already caused serious menace because of the
appearance of multidrug resistant Gram-positive bacteria, in par-
ticular, methicillin-resistant Staphylococcus aureus and vancomy-
cin-resistant. In order to prevent this serious medical problem, it
is urgent to elaborate new types of antibacterial agents.10 There-
fore, much of the research effort is put into the design of high effi-
ciency antibacterial agents to combat resistant pathogens.11
Aminoacyl-tRNA synthetases (aaRSs) have been proved to be
antimicrobial targets,12 and they have been interesting targets in
antibacterial drug design.13,14 Aminoacyl-tRNAs can provide re-
quired substrates during protein synthesis. As in mRNA, the coded
genetic information is translated into amino acid sequences which
result proteins because the coded sequences complement with the
anticodon sequences which are built into a respective aminoacyl-
⇑ Corresponding authors. Tel.: +86 25 83592572; fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
These authors contributed equally to this work.
information including the coincident tRNA molecules and the ami-
no acids’ structures,16 they are essential to translate the coded
information into protein structures in nucleic acids.17 As a member
of aminoacyl-tRNA synthetase family, tyrosyl-tRNA synthetase
also plays an important role in protein synthesis. And it is a prom-
ising target for antibacterial agents as the topology of the ATP
binding site and the interaction of bacteria aaRSs are different from
those of human.18–22
Nitroimidazole derivatives have showed broad varieties of bio-
logical activities especially antimicrobial activity.23 5-Nitroimidaz-
ole based drugs have been applied to treat the infections induced
by bacteria and a range of pathogenic protozoan parasites for many
years because nitroimidazole derivatives can undergo bioreduction
to produce electrophilic substances that can damage protein and
nucleic acids.24 What’s important is the toxicology and metabolism
of nitroimidazoles have been characterized, especially metronida-
zole (Scheme 1).25,26 As one of the important nitroimidazole deriv-
atives, metronidazole has been widely used as antimicrobial
medicine. In recent years, much attention has been paid to modify
the pendant hydroxy group of metronidazole.27 Since piperazines
and its derivatives are important pharmacophores, they are effec-
tive ingredients in many marketed drugs like the merck HIV prote-
ase inhibitor Crixivan and Piperazinyl-linked ciprofloxacin dimers
which was reported as potent antibacterial agents against resistant
strains,28,29 and imidazole is found in many marketed drugs like
ketoconazole, miconazole and metronidazole and so on (Scheme 1),

http://dx.doi.org/10.1016/j.bmc.2014.03.004
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
2410 S.-F. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2409–2415
O
CH 3
Cl
Cl
N S Cl
Cl
N N
N Cl N 1
N N
O O O N
OH
Cl Cl O 2N
O O
Ketoconazole Miconazole Metronidazole
Scheme 1. Chemical structure of metronidazole.
especially ketoconazole, it also contains piperazine skeleton. This
promoted us to synthesize new derivatives of metronidazole with
piperazine skeleton for the sake of finding new efficacy antibacterial
agents. Hence, we synthesized a series of novel metronidazole
derivatives owing piperazine skeleton (4a–4o). In our preliminary
work, the molecular docking was performed to validate which anti-
bacterial target protein the designed compounds can work, the
compounds were fitted into the ATP binding site of FabH (PDB code:
1HNJ), bacterial DNA gyrase (PDB code: 3G75), bacterial thymidyl-
ate kinase (PDB code: 4HEJ) and S. aureus tyrosyl-tRNA synthetase
(PDB code: 1JIJ).30–34 The results have been plotted as a line-scatter
graph and presented in Figure 1. It showed that the interaction en-
ergy between the designed compounds and protein 1JIJ is the low-
est. It means that the designed compounds are likely to display
more potent inhibitory activity against S. aureus tyrosyl-tRNA syn-
thetase. So we studied their antibacterial activities against Bacillus
subtilis (B. subtilis) and Staphylococcus aureus (S. aureus) Escherichia
coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and S. aureus
tyrosyl-tRNA synthetase (PDB code: 1JIJ) inhibitory activities. The
results showed that the compounds showed good activity against
Gram-positive bacterial but no activity against the Gram-negative
bacterial. Docking simulation was performed using the X-ray crys-
tallographic structure of the TyrRS of S. aureus in complex with
the most potent inhibitor to explore the binding mode of the com-
pound at the active site.
2. Results and discussion
2.1. Chemistry
Compounds 4a–4o were synthesized by the routes outlined in
Scheme 2. They were first designed and synthesized from metronida-
Figure 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).
N N
O O
N a
N S
OH O
O O
O2 N O2 N
2
R
N
N
b N
HN N R N N
N S
O
O NO2
O2N
2 3a-3o 4a-4o
Scheme 2. Reagents and conditions: (a) CH2Cl2, TEA, 0 °C, 5 h; (b) K2CO3, DMF,
80 °C, 22–24 h.
zole and piperazine derivatives. The key compound MET-OTs (2-
(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-methylbenzenesulfonate,
compound 1) was synthesized by the method in our previous
paper.35 The research showed when the ratio of MET-OTs and piper-
azine derivatives was 1:1.2, the reaction temperature was 80 °C, and
the reaction time was 24 h, compounds 4a–4o were obtained with
the higher yield. The chemical structures of these metronidazole
derivatives were summarized in Table 1. All of the synthetic com-
pounds were characterized by 1H NMR, elemental analysis and mass
spectrum, and they gave satisfactory analytical and spectroscopic
data, which were in full accordance with their depicted structures.
2.2. Biological activity
2.2.1. Antibacterial activity
The synthesized compounds were tested for their antibacterial
activity against two Gram-negative bacterial strains: E. coli and P.
aeruginosa and two Gram-positive bacterial strains: B. subtilis and
S. aureus by MTT method with MH medium (Mueller-Hinton med-
ium: casein hydrolysate 17.5 g, soluble starch 1.5 g, beef extract
1000 mL). The MICs (minimum inhibitory concentrations) of the
compounds against these bacteria were reported in Table 2, the
activity of reference drugs Penicillin G and Chloramphenicol was
also included. The results revealed that those compounds showed
good activity against Gram-positive bacterial but inactive against
Gram-negative bacterial.
Most of the new compounds exhibited good inhibitory activities
against both B. Subtilis and S. Aureus. As shown in Table 2. Compounds
4l and 4m showed most potent activities with MIC values of 0.033,
 S.-F. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2409–2415 2411

Table 1 According to these data, we found that compound 4l which had

Structures of compounds 4a–4o a methoxy group on the 4-position of benzene ring showed higher
N
R antibacterial activity against B. Subtilis with MIC values of
N 0.033 lg/mL than compound 4h which had a methoxy group on
N
N the 2-position of benzene ring with MIC values of 0.272 lg/mL
NO2 and compound 4f which had a methoxy group on the 3-position
of benzene ring with MIC values of 0.805 lg/mL, the same trend
Compounds R Compounds R was also found in S. aureus. Compound 4g (MIC = 6.872 lg/mL)
F3 C with a chlorine on the 3-position of benzene ring showed worse
4a 4i inhibitory activity than compound 4d (MIC = 0.142 lg/mL) with a
chlorine on the 2-position of benzene ring. It indicated that com-
CH3
pounds with substitution at the para or ortho position exhibited
4b F 4j more potent antibacterial activities than compounds with substi-
H3 C tution at the meta position.
Cl Cl Among compounds 4a (MIC = 1.551 lg/mL), 4m (MIC = 0.011 lg/mL)
4c
Cl
4k and 4n (MIC = 0.185 lg/mL), the order of inhibitory activities
Cl
showed the potency of 4m > 4n > 4a, which indicated the activity of
Cl compounds with substitutes of benzhydryl were superior to that of
4d 4l phenyl. Compounds 4k and 4c, which introduced two chlorine atoms
H3 CO
at 2,3-position or 3,4-positon in benzene ring, displayed a dramatic
F loss in activity compared to the analogues bearing only one substitu-
4e 4m ent (4d,4g). In addition, compounds 4b (MIC > 50 lg/mL), 4e
(MIC > 50 lg/mL) and 4o (MIC > 50 lg/mL) indicated that com-
H3 CO pounds with substitution of fluorine exhibited almost no activities.
Varieties of substitutes of phenyl like trifluoromethyl, methyl and
4f 4n
halogen can lead to different antibacterial activities. For example,
Cl
the derivatives which have electron-withdrawing substituents (like
Cl
halogen and trifluoromethyl) displayed weaker activities against
4g 4o B. subtilis and S. aureus than those having electron-donating substit-
F F uents (such as methyl and methoxyl), which suggested that substi-
OCH3 tuent group can affect the inhibitory activities, and compounds
4h with electron-donating substituents exhibited better inhibitory
activities than those with electron-withdrawing substituents.

2.2.2. S. aureus TyrRS inhibitory activity

The S. aureus TyrRS inhibitory potency of the synthetic metroni-
dazole derivatives was examined and the results were summarized
Table 2
Antibacterial activities of synthetic compounds
in Table 3. Most of the tested compounds showed potent S. aureus
TyrRS inhibitory activities. Among them, compounds 4d, 4l and 4m
Compounds Minimum inhibitory concentrations (lg/mL) with better antibacterial activities also showed better inhibitory
Gram-positive Gram-negative activity toward S. aureus TyrRS. Compound 4m exhibited the most
B. subtilis S. aureus E. coli P. aeruginosa potent inhibitory activities against S. aureus TyrRS with IC50 of
4a 1.551 >50 >50 >50
0.0024 lM. Compounds 4l and 4d also displayed good inhibitory
4b >50 >50 >50 >50 with IC50 of 0.0067 lM and 0.0086 lM. Other tested compounds
4c 14.382 11.116 >50 >50 displayed moderate inhibitory activity with IC50 ranging from
4d 0.142 0.203 >50 >50 1.12 to 25.76 lM. Compound 4m with N-benzhydryl group substi-
4e >50 17.105 >50 >50
tuent showed more preferable inhibitory activity than compound
4f 0.805 0.656 >50 >50
4g 2.567 1.801 >50 >50 4a with N-phenyl group substituent. Compound 4l with p-substi-
4h 0.272 0.583 >50 >50
Table 3
4i 3.288 18.093 >50 >50
S. aureus TyrRS inhibitory activity of synthetic compounds
4j 4.312 1.609 >50 >50
4k 6.827 11.56 >50 >50 Compounds S. aureus TyrRS IC50 (lM) Hemolysis LC30a (mg/mL)
4l 0.033 0.123 >50 >50
4m 0.011 0.003 >50 >50 4a 20.47 >10
4n 0.185 0.593 >50 >50 4b 25.2 >10
4o >50 >50 >50 >50 4c 13.55 >10
Penicillin G 0.335 1.024 22.843 18.624 4d 0.0086 >10
Chloramphenicol 0.078 0.178 0.064 0.102 4e 23.37 >10
4f 7.65 >10
4g 10.52 >10
4h 1.12 >10
4i 15.28 >10
0.011 lg/mL and 0.123, 0.003 lg/mL against B. subtilis and S. aureus, 4j 12.56 >10
respectively, which were superior to the positive control Penicillin G 4k 16.2 >10
with MIC values of 0.335 and 1.024 lg/mL. 4d (MIC = 0.142 lg/mL), 4l 0.0067 >10
4n (MIC = 0.185 lg/mL) and 4h (MIC = 0.272 lg/mL) exhibited 4m 0.0024 >10
4n 1.72 >10
significant inhibitory against B. Subtilis, which were superior to the
4o 25.76 >10
positive control Penicillin G (MIC = 0.335 lg/mL) but worse than
a
Chloramphenicol (MIC = 0.078 lg/mL). Lytic concentration 30%.
2412 S.-F. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2409–2415
tuted chloro group displayed better inhibitory activity than com-
pounds 4h and 4f with o-substituted and m-substituted chloro
group. Compounds 4c and 4k with two chloro groups exhibited
worse inhibitory activity than compound 4d with single o-substi-
tuted chloro group.
The results of S. aureus TyrRS inhibitory activity of the test com-
pounds were corresponding to the structure–activity relationships
(SAR) of their antibacterial activities. This demonstrated that the
potent antibacterial activities of the synthetic compounds were
probably correlated to their TyrRS inhibitory activities.
2.2.3. Molecular docking
With the aim to better understanding of the structure–activity
relationships observed, molecular docking of the most potent
inhibitor 4m into the active site cavity of tyrosyl-tRNA synthetase
was performed on the binding model based on the tyrosyl-tRNA
synthetase complex structure (PDB ID: 1JIJ). The binding model
of compound 4m and TyrRs was described in Figure 2, and the
enzyme surface model was shown in Figure 3, which revealed that
the molecule is well filled in the active pocket.
As illustrated in Figure 2, 4m located in the substrate binding
site of tyrosyl-tRNA synthetase. The docking results revealed that
three amino acids Arg80, Gln196 and His50 located in the binding
pocket of protein played important roles in the combination with
compound 4m, which were stabilized by one p–p bond and two
hydrogen bonds. The p–p bond, of which their lengths was
4.45 Å, was formed between phene ring and His50. There is an
unoccupied orbital on the nitrogen atom, so it is easy to form a
Figure 2. Binding mode of compound 4m with TyrRS from S. aureus. For clarity,
only interacting residues were labeled. Hydrogen bonding interactions are shown in
green dotted lines. This figure was made using PyMol.
Figure 3. Binding mode of compound 4m with TyrRS. The enzyme is shown as
surface; while 4m docked structures are shown as sticks. This figure was made
using PyMol.
coordination bond with protons. As showed in Figure 2, a carboxy
of Gln196 formed ammonium salt with N heterocyclic ring, then
the oxygen of the carbonyl group formed a hydrogen bond with
the ammonium ion, and the length of H


O bond was 2.26 Å.
The other hydrogen bond was formed between the nitrogen of
amine of Arg80 and the ammonium ion with H


N bond length
of 2.49 Å. This molecular docking result, along with the biological
assay data, suggested that compound 4m was a potential inhibitor
of TyrRS.
3. Conclusion
A series of novel metronidazole derivatives have been designed
and synthesized, and their biological activities were also evaluated
as potent TryRs inhibitory. Compound 4m demonstrated the most
potent inhibitory activity (MIC = 0.011 lg/mL for B. subtilis and
MIC = 0.003 lg/mL for S. aureus), which was compared with the
positive control Penicillin G and Chloramphenicol. Docking simula-
tion was performed to position compound 4m into the S. aureus
TryRs active site to determine the potential binding model. Analy-
sis of the binding conformation of compound 4m displayed that
compound 4m was stabilized by hydrogen bonding interactions
with ASP80 and GLN196. Antibacterial assay results also showed
that these metronidazole derivatives had the potential to be devel-
oped for antibacterial agents against S. aureus. Particularly, com-
pound 4m has demonstrated significant S. aureus TryRs
inhibitory activity as a potential antibacterial agent.
4. Experiments
4.1. Materials and measurements
All chemicals and reagents used in current study were of analyt-
ical grade. TLC was performed on the glass-backed silica gel sheets
(silica GF254) and visualized in UV light (254 nm). Column chro-
matography was performed using silica gel (200–300 mesh) elut-
ing with ethyl acetate and petroleum ether. Melting points
(uncorrected) were determined on a XT4MP apparatus (Taike
Corp., Beijing, China). All the 1H NMR spectra were recorded on a
Bruker DPX 400 model Spectrometer in CDCl3 and chemical shifts
were reported in ppm (d). ESI-MS spectra were recorded on a Mar-
iner System 5304 Mass spectrometer. Elemental analyses were
performed on a CHN-O-Rapid instrument.
 S.-F. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2409–2415
4.2. General method of synthesis of metronidazole derivatives
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-methylbenzene-
sulfonate (2) was prepared according to our previous paper. Metro-
nidazole (3.14 g, 20 mmol) and Et3N (3.0 mL, 22 mmol) were
dissolved in CH2Cl2 (20 mL), and 4-methyl-benzenesufonyl chlo-
ride (3.83 g, 20.1 mmol) in CH2Cl2 (10 mL) was added. The reaction
mixture was stirred at 0 °C for 5 h followed by the addition of
30 mL ice water, and then the layer was separated and the aqueous
layer was extracted with ethyl acetate (2  30 mL). The organic
layer was combined and washed with saturated NaHCO3, and dried
with anhydrous Na2SO4 for 0.5 h. Removal of the solvent gave a
slight-yellow crystal of compound 2 (MET-OTs). Compounds
4a–4o were synthesized by the following procedure. MET-OTs
(2 mmol) and K2CO3 (2 mmol) were dissolved in DMF (20 mL),
and then one of those substituted piperazines was added. The reac-
tion was stirred at 80 °C for 22–24 h. The reaction mixture was
poured in water and extracted with ethyl acetate (3  50 mL).
The organic layer was combined and dried with anhydrous Na2SO4
for 0.5 h. After the reaction mixture was vacuum distillated, the
target product was separated by column chromatography.
4.2.1. 1-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-phenyl
piperazine (4a)
Brown, yield: 47%, mp: 108–110 °C. 1H NMR (400 MHz, CDCl3):
d 7.97 (s, 1H), 7.25–7.29 (m, 2H, ArH), 6.90–6.92 (d, J = 7.64 Hz, 3H,
ArH), 3.49–3.61 (s, 4H, –CH2CH2–), 3.09–3.13 (s, 4H, –CH2CH2–),
2.49 (s, 3H, CH3), ESI-MS: 316.28 (C16H22N5O2, [M+H]+). Anal. Calcd
for C16H21N5O2: C, 60.94; H, 6.71; N, 22.21. Found: C, 60.92; H,
6.72, N, 22.23.
4.2.2. 1-(4-Fluorophenyl)-4-(2-(2-methyl-5-nitro-1H-imidazol-
1-yl)ethyl)piperazine (4b)
Brown, yield: 51%, mp: 65–68 °C. 1H NMR (400 MHz, CDCl3) : d
7.93 (s, 1H), 6.93–6.97 (t, J = 8.34 Hz, 2H, ArH), 6.83–6.85 (m, 2H,
ArH), 4.41–4.45 (t, J = 6.26 Hz, 2H, CH2), 3.04–3.07 (t, J = 4.86 Hz,
4H, –CH2CH2–), 2.71–2.72 (t, J = 6.26 Hz, 2H, CH2), 2.63–2.65 (t,
J = 4.86 Hz, 4H, –CH2CH2–,), 2.52 (s, 3H, CH3). ESI-MS: 334.27 (C16-
H21FN5O2, [M+H]+). Anal. Calcd for C16H20FN5O2: C, 57.65; H, 6.05;
N, 21.01. Found: C, 57.62, H, 6.04; N, 21.03.
4.2.3. 1-(2,3-Dichlorophenyl)-4-(2-(2-methyl-5-nitro-1H-imidazol-
1-yl)ethyl)piperazine (4c)
Yellow, yield: 47%, mp: 90–93 °C. 1H NMR (400 MHz, CDCl3): d
7.95 (s, 1H), 7.12–719 (m, 2H, ArH), 6.89–6.91 (m, 1H, ArH),
4.61–4.64 (t, J = 5.26 Hz, 2H, CH2), 4.43–4.46 (t, J = 5.24 Hz, 2H,
CH2), 3.50–3.62 (s, 4H, –CH2CH2–), 2.92–2.97 (s, 4H, –CH2CH2–),
2.49 (s, 3H, CH3). ESI-MS: 385.15 (C16H20Cl2N5O2, [M+H]+). Anal.
Calcd for C16H19Cl2N5O2: C, 50.01; H, 4.98; N, 18.23. Found: C,
49.98; H, 4.99; N, 18.22.
4.2.4. 1-(2-Chlorophenyl)-4-(2-(2-methyl-5-nitro-1H-imidazol-
1-yl) ethyl)piperazine(4d)
Yellow, yield: 59%, mp: 120–122 °C. 1H NMR (400 MHz, CDCl3):
d 7.96 (s, 1H), 7.34–7.37 (m, 1H, ArH), 7.20–7.26 (m, 1H, ArH),
6.98–7.02 (m, 2H, ArH), 4.62–4.64 (t, J = 5.26 Hz, 2H, CH2),
4.44–4.47 (t, J = 5.26 Hz, 2H, CH2), 3.51–3.63 (s, 4H, –CH2CH2–),
2.94–2.99 (s, 4H, –CH2CH2–), 2.50 (s, 3H, CH3). ESI-MS: 350.76
(C16H21ClN5O2, [M+H]+). Anal. Calcd for C16H20ClN5O2: C, 54.94;
H, 5.76; N, 20.02. Found: C, 54.88; H, 5.75, N, 20.01.
4.2.5. 1-(2-Fluorophenyl)-4-(2-(2-methyl-5-nitro-1H-imidazol-
1-yl)ethyl)piperazine (4e)
Yellow, yield: 48%, mp: 125–127 °C. 1H NMR (400 MHz, CDCl3):
d 7.96 (s, 1H), 7.34–7.37 (m, 1H, ArH), 7.20–7.26 (m, 1H, ArH),
6.98–7.02 (m, 2H, ArH), 4.62–4.64 (t, J = 5.26 Hz, 2H, CH2),
2413
4.44–4.47 (t, J = 5.26 Hz, 2H, CH2), 3.51–3.63 (s, 4H, –CH2CH2–),
2.94–2.99 (s, 4H, –CH2CH2–), 2.50 (s, 3H, CH3). ESI-MS: 334.32
(C16H21FN5O2, [M+H]+). Anal. Calcd for C16H20ClN5O2: C, 57.65; H,
6.05; N, 20.01. Found: C, 57.60; H, 6.02; N, 20.02.
4.2.6. 1-(3-Methoxyphenyl)-4-(2-(2-methyl-5-nitro-1H-imidazol-
1-yl)ethyl)piperazine (4f)
Brown, yield: 61%, mp: 45–48 °C. 1H NMR (400 MHz, CDCl3): d
7.96 (s, 1H), 7.16–7.20 (t, J = 8.04 Hz, 1H, ArH), 6.50–6.53
(t, J = 5 Hz, 3H, ArH), 4.61–4.63 (t, J = 5.26 Hz, 2H, CH2), 4.43–4.46
(t, J = 5.28 Hz, 2H, CH2), 3.78 (s, 3H, CH3O-), 3.47–3.60 (s, 4H, –CH2-
CH2–), 3.09–3.13 (s, 4H, –CH2CH2–), 2.49 (s, 3H, CH3). ESI-MS:
346.36 (C17H24N5O3, [M+H]+). Anal. Calcd for C17H23N5O3: C,
59.12; H, 6.71; N, 20.28. Found: C: 59.11; H: 6.71; N, 20.2.
4.2.7. 1-(3-Chlorophenyl)-4-(2-(2-methyl-5-nitro-1H-imidazol-
1-yl)ethyl)piperazine (4g)
Brown, yield: 56%, mp: 66–68 °C. 1H NMR (400 MHz, CDCl3): d
7.96 (s, 1H), 7.15–7.19 (t, J = 8.12 Hz, 1H, ArH), 6.84–6.85 (d,
J = 6.48 Hz, 2H, ArH), 6.75–6.78 (m, 1H, ArH), 4.61–4.64 (t,
J = 5.28 Hz, 2H, CH2), 4.43–4.46 (t, J = 5.26 Hz, 2H, CH2), 3.47–3.59
(s, 4H, –CH2CH2–), 3.10–3.14 (s, 4H, –CH2CH2–), 2.49 (s, 3H, CH3).
ESI-MS: 350.76 (C16H21ClN5O2, [M+H]+). Anal. Calcd for C16H20-
ClN5O2: C, 54.94; H, 5.76; N, 20.02. Found: C, 54.92; H,5.75; N,
20.02.
4.2.8. 1-(2-Methoxyphenyl)-4-(2-(2-methyl-5-nitro-1H-
imidazol-1-yl)ethyl)piperazine (4h)
Yellow, yield: 52%, mp: 112–115 °C. 1H NMR (400 MHz, CDCl3):
d 7.93 (s, 1H), 6.99–7.03 (m, 1H, ArH), 6.84–6.92 (m, 3H, ArH),
4.59–4.60 (d, J = 5.28 Hz, 2H, CH2), 4.41–4.44 (t, J = 5.22 Hz, 2H,
CH2), 3.85 (s, 3H, CH3O-), 3.49–3.61 (s, 4H, –CH2CH2–), 2.93–2.99
(s, 4H, –CH2CH2–), 2.47 (s, 3H, CH3). ESI-MS: 346.36 (C17H24N5O3,
[M+H]+). Anal. Calcd for C17H23N5O3: C, 59.12; H, 6.71; N, 20.28.
Found: C, 59.12; H, 6.70; N, 20.27.
4.2.9. 1-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-(3-
(trifluoromethyl)phenyl)piperazine (4i)
Yellow, yield: 45%, mp: 98–101 °C. 1H NMR (400 MHz, CDCl3)
d:7.96 (s, 1H), 7.34–7.38 (t, J = 7.94 Hz, 1H, ArH), 7.03–7.06 (m,
3H, ArH), 4.62–4.65 (t, J = 5.26 Hz, 2H, CH2), 4.44–4.46 (t,
J = 5.28 Hz, 2H, CH2), 3.49–3.62 (s, 4H, –CH2CH2–), 3.14–3.18 (s,
4H, –CH2CH2–), 2.50 (s, 3H, CH3). ESI-MS: 384.33 (C17H21F3N5O2,
[M+H]+). Anal. Calcd for C17H20F3N5O2: C, 53.26; H, 5.26; N,
18.27. Found: C: 53.25; H, 5.27; N, 18.27.
4.2.10. 1-(2,4-Dimethylphenyl)-4-(2-(2-methyl-5-nitro-1H-imid
azol-1-yl)ethyl)piperazine (4j)
Brown, yield: 55%, mp: 130–132 °C. 1H NMR (400 MHz, CDCl3)
d: 7.97 (s, 1H), 6.96–7.00 (t, J = 9.34 Hz, 2H, ArH), 6.86–6.88 (d,
J = 8.04 Hz, 1H, ArH), 4.61–4.64 (t, J = 5.24 Hz, 2H, CH2), 4.44–4.47
(t, J = 5.24 Hz, 2H, CH2), 3.46–3.59 (s, 4H, –CH2CH2–), 2.77–2.82
(s, 4H, –CH2CH2–), 2.51 (s, 3H, CH3), 2.26 (s, 3H, CH3), 2.27 (s, 3H,
CH3). ESI-MS: 344.38 (C18H26N5O2, [M+H]+). Anal. Calcd for
C18H25N5O2: C, 62.95; H, 7.34; N, 20.39. Found: C, 62.92; H, 7.36;
N, 20.38.
4.2.11. 1-(3,4-Dichlorophenyl)-4-(2-(2-methyl-5-nitro-1H-imid
azol-1-yl)ethyl)piperazine (4k)
Yellow, yield: 61%, mp: 106–109 °C. 1H NMR (400 MHz, CDCl3):
d 7.98 (s, 1H), 7.29–7.31 (d, J = 8.6 Hz, 1H, ArH), 6.96–6.97 (d,
J = 2.76 Hz, 1H, ArH), 6.73–6.76 (m, 1H, ArH), 4.64–4.67 (t,
J = 5.26 Hz, 2H, CH2), 4.46–4.48 (t, J = 5.26 Hz, 2H, CH2), 3.50–3.62
(s, 4H, –CH2CH2–), 3.11–3.14 (s, 4H, –CH2CH2–), 2.52 (s, 3H, CH3).
ESI-MS: 385.15 (C16H20Cl2N5O2, [M+H]+). Anal. Calcd for C16H19Cl2-
N5O2: C, 50.01; H, 4.98; N, 18.23. Found: C, 50.03; H, 4.97; N, 18.24.
2414 S.-F. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2409–2415
4.2.12. 1-(4-Methoxyphenyl)-4-(2-(2-methyl-5-nitro-1H-imid
azol-1-yl)ethyl) piperazine (4l)
Yellow, yield: 46%, mp: 104–107 °C. 1H NMR (400 MHz, CDCl3):
d 7.97 (s, 1H), 6.84–6.91 (t, J = 14.56 Hz, 4H, ArH), 4.62–4.65 (t,
J = 5.26 Hz, 2H, CH2), 4.44–4.47 (t, J = 5.22 Hz, 2H, CH2), 3.77 (s,
3H, CH3O-), 3.51–3.63 (s, 4H, –CH2CH2–), 3.00–3.01 (s, 4H, CH2CH2-
), 2.50 (s, 3H, CH3). ESI-MS: 346.36 (C17H24N5O3, [M+H]+). Anal.
Calcd for C17H23N5O3: C, 59.12; H, 6.71; N, 20.28. Found: C,
59.13; H, 6.70; N, 20.27.
4.2.13. 1-Benzhydryl-4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)
ethyl)piperazine (4m)
Yellow, yield: 58%, mp: 40–43 °C. 1H NMR (400 MHz, CDCl3): d
7.93 (s, 1H), 7.37–7.39 (d, J = 7.36 Hz, 4H, ArH), 7.24–7.28 (m, 4H,
ArH), 7.15–7.18 (t, J = 7.28 Hz, 2H, ArH), 4.54–4.57 (t, J = 5.18 Hz,
2H, CH2), 4.37–4.40 (t, J = 5.18 Hz, 2H, CH2), 4.20 (s, 1H, CH), 3.30–
3.43 (s, 4H, –CH2CH2–), 2.43 (s, 3H, CH3), 2.28–2.34 (s, 4H, –CH2CH2–
). ESI-MS: 406.46 (C23H28N5O2, [M+H]+). Anal. Calcd for C23H27N5O2:
C, 72.33; H, 6.49; N, 14.54. Found: C, 72.34; H, 6.47; N, 14.55.
4.2.14. 1-((4-Chlorophenyl)(phenyl)methyl)-4-(2-(2-methyl-5-
nitro-1H-imidazol-1-yl)ethyl)piperazine (4n)
Yellow, yield: 51%, mp: 50–52 °C. 1H NMR (400 MHz, CDCl3): d
7.98 (s, 1H), 7.22–7.40 (m, 9H, ArH), 4.61–4.63 (t, J = 4 Hz, 2H,
CH2), 4.43–4.45 (t, J = 5.16 Hz, 2H, CH2), 4.25 (s, 1H, CH),
3.36–3.49 (s, 4H, –CH2CH2–), 2.49 (s, 3H, CH3), 2.32–2.38 (s, 4H, –
CH2CH2–). ESI-MS:440.86 (C23H27ClN5O2, [M+H]+). Anal. Calcd for
C23H26ClN5O2: C, 67.50; H, 5.86; N, 13.57. Found: C, 67.51; H,
5.84; N, 13.55.
4.2.15. 1-(Bis(4-fluorophenyl)methyl)-4-(2-(2-methyl-5-nitro-1H-
imidazol-1-yl)ethyl)piperazine (4o)
Brown, yield: 47%, mp: 47–50 °C. 1H NMR (400 MHz, CDCl3): d
7.90 (m, 4H, Ar), 6.97 (s, 4H, Ar), 4.35–4.38 (t, J = 6.3 Hz, 3H,
CH2), 4.17 (s, 1H, CH), 2.61–2.66 (m, 2H, CH2), 2.49 (s, 4H, –CH2-
CH2–), 2.48 (s, 3H, CH3), 2.31–2.32 (s, 4H, –CH2CH2–). ESI-MS:
442.33 (C23H26F2N5O2, [M+H]+). Anal. Calcd for C23H25F2N5O2: C,
67.30; H, 5.65; N, 13.53. Found: C, 67.31; H, 5.64; N, 13.54.
4.3. Antibacterial activity
The antibacterial activities of the synthetic compounds were
tested against two Gram-negative bacterial strains: E. coli ATCC
25922 and P. aeruginosa ATCC 27853, two Gram-positive bacterial
strains: B. subtilis ATCC 530 and S. aureus ATCC 25923, using meth-
od recommended by National Committee for Clinical Laboratory
Standards (NCCLS).36
In vitro activities of the compounds were tested in Nutrient
broth (NB) for bacteria by the two-fold serial dilution method.
Seeded broth (broth containing microbial spores) was prepared
in NB from 24 h old bacterial cultures on nutrient agar (Hi-media)
at 37 ± 1 °C. The bacterial suspension was adjusted with sterile sal-
ine to a concentration of 1  104–105 CFU/mL. The tested com-
pounds and reference drugs were prepared by two-fold serial
dilution to obtain the required concentrations of 100, 50, 25,
12.5, 6.25, 3.13 lg/mL and even lower concentration. The tubes
were incubated in BOD incubators at 37 ± 1 °C for bacteria. The
MICs were recorded by visual observations after 24 h (for bacteria)
of incubation. Penicillin G and chloramphenicol were used as stan-
dards for bacterial. The observed MICs are presented in Table 2.
4.4. S. aureus TyrRS inhibitory activity
TyrRS activity was measured by aminoacylation using modifica-
tions to previously described methods.37 The assays were
performed at 37 °C in a mixture containing (final concentrations)
100 mM Tris/Cl pH 7.9, 50 mM KC1, 16 mM MgCl2, 5 mM ATP,
3 mM DTT, 4 mg/mL E. coli MRE600 tRNA (Roche) and 10 lM
3
L-tyrosine (0.3 lM L-[ring-3,5- H] tyrosine (Perkin-Elmer, Specific
activity: 1.48–2.22 TBq/mmol), 10 lM carrier). TyrRS (0.2 nM)
was pre-incubated with a range of inhibitor concentrations for
10 min at room temperature followed by the addition of pre-
warmed mixture at 37 °C. After specific intervals, the reaction
was terminated by adding aliquots of the reaction mix into ice-cold
7% trichloroacetic acid and harvesting onto 0.45 lm hydrophilic
Durapore filters (Millipore Multiscreen 96-well plates) and
counted by liquid scintillation. The rate of reaction in the experi-
ments was linear with respect to protein and time with less than
50% total tRNA acylation. IC50s corresponding to the concentration
at which half of the enzyme activity was inhibited by the com-
pound. The results were presented in Table 3.
4.5. Docking simulations
The crystal structures of TyrRS (PDB code: 1JIJ) was obtained
from the Protein Data Bank (http://www.rcsb.org). Molecular
docking of compound 4m into the three-dimensional X-ray struc-
ture of TyrRS was carried out using CDOCKER Dock protocol of Dis-
covery Studio 3.5.
Acknowledgment
The work was financed by Natural Science Foundation of China
(No. J1103512).
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