Design and synthesis of 2-styryl of 5-Nitroimidazole derivatives and antimicrobial activities as FabH inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.02.004

A series of 2-Styryl-5-Nitroimidazole derivatives (25-48) have been synthesized and their biological activities were also evaluated against two Gram-negative bacterial strains: Escherichia coli and Pseudomonas aeruginosa and two Gram-positive bacterial strains: Bacillus subtilis and Bacillus thuringiensis as potential FabH inhibitors. All the compounds were structurally determined by ¹H NMR, MS, and elemental analysis. E. coli β-ketoacyl-acyl carrier protein synthase III inhibitory assay and docking simulation indicated that compound 33 with IC₅₀ of 9.0-36.4 μg/mL and compound 47 with IC₅₀ of 6.3-34.3 μg/mL against bacterial strains were most potent inhibitors of E. coli FabH. And more, compounds 33 and 47 which possessed a broad-spectrum of antibacterial activities didn't exhibit any toxicity towards macrophage.

Full text of DOI:10.1016/j.ejmech.2014.02.004

European Journal of Medicinal Chemistry 76 (2014) 387e396
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of 2-styryl of 5-Nitroimidazole derivatives and
antimicrobial activities as FabH inhibitors
Yong-Tao Duan 1, Zhong-Chang Wang 1, Ya-Li Sang, Xiang-Xiang Tao, Shashikant B. Teraiya,
*
*
Peng-Fei Wang, Qing Wen, Xiao-Jing Zhou, Liang Ding, Yong-Hua Yang , 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:
A series of 2-Styryl-5-Nitroimidazole derivatives (25e48) have been synthesized and their biological
activities were also evaluated against two Gram-negative bacterial strains: Escherichia coli and Pseudo-
monas aeruginosa and two Gram-positive bacterial strains: Bacillus subtilis and Bacillus thuringiensis as
potential FabH inhibitors. All the compounds were structurally determined by 1H NMR, MS,
Received 21 April 2013
Received in revised form
7 January 2014
Accepted 5 February 2014
Available online 15 February 2014
and elemental analysis. E. coli
simulation indicated that compound 33 with IC50 of 9.0e36.4
e34.3 g/mL against bacterial strains were most potent inhibitors of E. coli FabH. And more, compounds
b-ketoacyl-acyl carrier protein synthase III inhibitory assay and docking
m
g/mL and compound 47 with IC50 of 6.3
m
Keywords:
33 and 47 which possessed a broad-spectrum of antibacterial activities didn’t exhibit any toxicity to-
wards macrophage.
5-Nitroimidazole
FabH inhibitors
Antibacterial activities
Cytotoxicity
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
role and ubiquitous structure of FabH have guaranteed that FabH
inhibitors are potent antibiotics with broad-spectrum activity.
Infections due to bacteria such as Escherichia coli have been
major cause of human morbidity and mortality [1,2]. Although the
innovative research for antibiotics has improved mankind’s health
status by confining life threatening infections, the emergence and
spread of bacterial resistance represents a severe global problem
[3,4], and resistance against causal organisms has been reported
and development of agents targeted at these organisms pose a
challenge presently [5,6]. Therefore, the development of new types
of antibacterial agents is a very important task.
In recent years, much of the research effort is oriented to the
design of new antibacterial agents with high efficiency [7e12]. One
of the most attractive biochemical pathways to be used as the
target for new antibacterial agents is the fatty acid biosynthesis
(FAB). This pathway has been demonstrated to be essential for
bacteria cell survival [13,14]. Fatty acid biosynthesis (FAB) is an
essential metabolic process for prokaryotic organisms and is
Importantly, the residues that comprise the active site are essen-
tially invariant in various bacterial FabH molecules [18]. And more,
our previous research also suggests that FabH can be used as an
effective molecular target for the development of new antibacterial
agents.
Presently, infection is primarily treated by instituting antimi-
crobials therapy. Antimicrobial drugs such as metronidazole, sec-
nidazole kill microbials in host tissue and organs [19]. Particularly
metronidazole is the most preferred treatment choice as 90% of
patients respond to the therapy and has been widely used as an
antimicrobial medicine [20]. However, resistances to metronida-
zole in many pathogenic bacteria as well as several side effects are
also documented [21]. Therefore it is desirable to search for
metronidazole derivatives or new lead compounds.
Our earlier investigations have provided evidence of the anti-
bacterial properties of nitroimidazole derivatives, which support
the idea that FabH can be used as an effective molecular target for
nitroimidazole derivatives [8,22,23]. Some metronidazole de-
rivatives have been predicted as notable radiosensitizers, anti-
protozoal and antibacterial or antiepileptic agents [24]. However, to
our knowledge, few reports have been dedicated to the synthesis
and FabH inhibitory activity of compounds of modifications in the
2-position of the imidazole ring. Prior studies suggest that the 2-
position modified 5-nitroimidazole drugs can overcome metroni-
dazole resistance [25,26], and they raised the possibility that
required for cell viability and growth [15]. b-ketoacyl-acyl carrier
protein (ACP) synthase III, also known as FabH or KAS III, plays an
essential and regulatory role in bacterial FAB [16,17]. In spite of the
denil necessity of FabB and FabF for Gram-positive bacteria, the key
* Corresponding authors.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
These two authors equally contributed to this paper.
1
http://dx.doi.org/10.1016/j.ejmech.2014.02.004
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

388
Y.-T. Duan et al. / European Journal of Medicinal Chemistry 76 (2014) 387e396
Table 1
metronidazole derivatives substituted in the 2-methyl group may
be promising for developing new, potent, and safe drugs. Now it is
clear that hydroxy and nitro group of metronidazole play a key role
in the metabolic activation [27]. Thus, the structure modification at
the pendant methyl group (2-position) of metronidazole has
received our attention and then we designed and synthesized a
library of substituted 2-styryl 5-nitroimidazole derivatives.
Herein, we describe the synthesis and structureeactivity rela-
tionship (SAR) of a series of 2-styryl 5-nitroimidazole derivatives,
and present their in vitro antibacterial activity against Escherichia
coli (E. coli), Bacillus thuringiensis (B. thuringiensis), Bacillus subtilis
(B. subtilis) and Pseudomonas aeruginosa (P. aeruginosa). Docking
simulations are performed using the X-ray crystallographic struc-
ture of the FabH in complex with an inhibitor to explore the binding
modes of these compounds at the active site. And more, to test the
toxicity of compounds 33 and 47 against human macrophages, a
research of cytotoxicity has been made.
Structures of compounds 25e48.
Compound Ar
Compound Ar
25
26
27
28
37
38
39
40
2. Results and discussion
29
30
41
42
2.1. Chemistry
Twenty four 2-styryl 5-nitroimidazole derivatives were syn-
thesized by the reaction of metronidazole with different
substituted benzaldehyde in DMSO by adding rapidly a stirred so-
lution of sodium methoxide in methanol at room temperature
(Scheme 1). The refined compounds were finally obtained by sub-
sequent purification with recrystallization. The chemical structures
of these metronidazole derivatives were summarized in Table 1.
These compounds gave satisfactory elementary analyses (ꢀ0.4%).
1H NMR and ESI MS spectra data was consistent with the assigned
structures. Among these compounds 28, 30, 33, 35, 37, 40e45 and
47 were reported for the first time.
31
43
32
33
44
45
Furthermore, the crystal structure of compounds 35 and 37 was
determined by single crystal X-ray diffraction analysis in Figs. 1 and
2, and their crystal data, data collection and refinement parameters
for the compound 35 and 37 were listed in Table 2.
2.2. Biological activity
2.2.1. Antimicrobial activity
34
35
36
46
47
48
All the synthesized compounds (25e48) were screened for their
antibacterial activities against two Gram-negative bacterial strains:
E. coli and P. aeruginosa and two Gram-positive bacterial strains:
B. subtilis and B. thuringiensis by MTT method. The IC50 of the
compounds against these bacteria were presented in Table 3. Also
included was the activity of reference compound Kanamycin and
Penicillamine under identical conditions for comparison. The re-
sults revealed that most of the synthesized compounds exhibited
significant antibacterial activities.
For most of the studied compounds, various substituents such as
halogen, methyl, methoxyl and nitro group led to distinct
antibacterial activities which always show more potent inhibitory
activity against E. coli than compounds 25 and 46 with a bare ar-
omatic ring (phenyl ring and naphthalene ring respectively), also
we can see that the antibacterial activities were inferior than pos-
itive control Kanamycin and Penicillamine. However, compounds
33 and 47, whose IC50 value were 36.4 and 34.3
superior activity to the positive control Kanamycin with corre-
sponding IC50 of 47.3 g/mL and Penicillamine with corresponding
IC50 of 42.3 g/mL. Addition of larger side chains at the phenyl
mg/mL, displayed
a Reagents and conditions: Sodium methoxide, DMSO, methanol, room temperature;
m
Scheme 1.
m

Y.-T. Duan et al. / European Journal of Medicinal Chemistry 76 (2014) 387e396
389
Table 2
Crystallographical and experimental data for compounds 35 and 37.
Compound
35
37
Empirical formula
Formula weight
Crystal system
Space group
C13H12BrN3O3
338.21
Monoclinic
P c
7.6657 (8)
8.7493 (9)
10.4323 (11)
83.093 (3)
73.977 (3)
84.711(3)
666.35(12)
8
C13H12FN3O3
277.12
Monoclinic
P c
7.5690 (5)
7.9036 (5)
10.8599 (7)
87.613 (2)
82.578 (2)
85.173(2)
641.63(7)
11
ꢀ
a (A)
ꢀ
b (A)
ꢀ
c (A)
a
b
g
(ꢁ)
(ꢁ)
(ꢁ)
ꢀ
V (A)
Z
Dcalcd/g cmꢂ3
h range (ꢁ)
2.451
2.04e25.86
456
1.766
3.11e27.52
341
F (000)
Reflections collected
Data/restraints/parameters
Absorption coefficient (mmꢂ1
5720 (Rint ¼ 0.0305)
2562/0/182
12.083
0.0406/0.1027
0.0580/0.1112
1.021
6790 (Rint ¼ 0.0198)
2863/0/182
0.198
0.0435/0.1179
0.0550/0.1281
1.033
Fig. 1. Crystal structure diagram of compound 35. H atoms are shown as small spheres
of arbitrary radii.
)
R1; wR2 [I > 2
s(I)]
R1; wR2 (all data)
GOF
moiety, including CH2(OC2H5)2 (36) and N(CH3)2 (42), and
OCH2Ph(47), can markedly improve the activity against Escherichia
coil. For electron-withdrawing group NO2, F, and Br, the activity
gradient of substituent group on the phenyl ring in p-position is
NO2 > F > Br which accords with their ability of withdrawing
electron. However, when NO2, F, and Br moiety is in o-position, the
ability against E. coil decreases almost simultaneously. Compound
40 with two different halogen groups on the o-position manifested
the results were summarized in Table 4. As shown in Table 4, among
the tested compounds, compounds 33 and 47, displaying the most
potent E. coli inhibitory activity, showed potent inhibitory activities
with IC50 of 2.1 and 3.4
the positive control Kanamycin with IC50 of 3.1
as the most promising anti-P. aeruginosa agent with IC50 of 1.7
(Table 3), showed low P. aeruginosa FabH inhibitory activity with
IC50 of 22.5 M. In contrast, compounds 46 and 47, which exhibited
mM, respectively, which were comparable to
m
M. Compound 30,
higher antibacterial activity with IC50 value of 82 mg/mL against
mM
E. coil than compounds 37, 38 and 44 with only one halogen moiety.
On the contrary, compound 41, two same halogen groups on the
phenyl ring, performed inferior antibacterial activity. In compari-
son, we found that the derivatives which have electron-
withdrawing substituents (such as F, Cl, Br) and bulky group on
the benzene ring exhibited more potent antibacterial activities than
those have electron-donating substituents (such as CH3, OCH3). We
proposed that, for these compounds, electron-withdrawing and
bulky groups were conducive to the antibacterial activity and
compounds with electron-donating halogen groups were not
favorable for activity.
m
average P. aeruginosa inhibitory activity, showed more strong
inhibitory effect and its 50% P. aeruginosa FabH inhibition concen-
tration of 7.9 and 10.2 mM. This may imply that the potent inhibitory
Table 3
Antibacterial activities of synthetic compounds.
Compound
50% inhibitory concentrations (
Gram-negative
m
g/mL)
Gram-positive
Escherichia
coli
Pseudomonas
aeruginosa
Bacillus
thuringiensis
Bacillus
subtilis
2.2.2. E. coli FabH and P. aeruginosa FabH inhibitory activity
The E. coli FabH and P. aeruginosa FabH inhibitory potency of the
selected compound 30, 33, 36e38 and 46e47 was examined and
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
>200
12.4 ꢀ 0.44
30.2 ꢀ 0.34
32.2 ꢀ 0.23
16.8 ꢀ 0.13
48.3 ꢀ 0.89
1.7 ꢀ 0.64
49.5 ꢀ 0.13
67.4 ꢀ 0.32
19.2 ꢀ 0.42
55.1 ꢀ 0.74
32.1 ꢀ 0.34
12.5 ꢀ 0.78
25.7 ꢀ 0.29
12.1 ꢀ 0.43
13.9 ꢀ 0.55
37.3 ꢀ 0.60
23.4 ꢀ 0.71
5.9 ꢀ 0.63
29.9 ꢀ 0.66
37.8 ꢀ 0.54
28.6 ꢀ 0.41
25.3 ꢀ 0.34
36.7 ꢀ 0.40
7.2 ꢀ 0.71
7.0 ꢀ 0.22
9.3 ꢀ 0.27
5.8 ꢀ 0.42
11.9 ꢀ 0.90
9.8 ꢀ 0.24
8.6 ꢀ 0.23
9.5 ꢀ 0.25
6.3 ꢀ 0.72
49.3 ꢀ 0.43
146.4 ꢀ 0.14
113.5 ꢀ 0.51
36.6 ꢀ 0.65
32.8 ꢀ 0.67
8.8 ꢀ 0.45
89.1 ꢀ 6.09
119.3 ꢀ 4.22
114.1 ꢀ 3.05
>200
57.2 ꢀ 2.39
119.1 ꢀ 15.24
>200
36.4 ꢀ 2.33
187.8 ꢀ 9.65
115.8 ꢀ 8.44
59.2 ꢀ 0.37
>200
174.7 ꢀ 0.78
144.8 ꢀ 10.22
82.1 ꢀ 9.90
>200
14.7 ꢀ 0.23
46.1 ꢀ 0.13
15.5 ꢀ 0.54
20.4 ꢀ 0.16
43.6 ꢀ 0.75
70.3 ꢀ 0.03
6.8 ꢀ 0.13
40.7 ꢀ 0.43
26.2 ꢀ 0.10
9.0 ꢀ 0.33
19.8 ꢀ 0.43
24.4 ꢀ 0.56
17.9 ꢀ 0.47
61.5 ꢀ 0.23
2.9 ꢀ 0.13
17.8 ꢀ 0.43
2.9 ꢀ 0.17
42.6 ꢀ 0.13
19.6 ꢀ 0.17
57.1 ꢀ 0.12
17.2 ꢀ 0.45
53.1 ꢀ 0.21
27.9 ꢀ 0.13
11.8 ꢀ 0.10
3.6 ꢀ 0.50
38.1 ꢀ 0.19
39.3 ꢀ 0.45
120.9 ꢀ 0.44
74.6 ꢀ 0.32
7.6 ꢀ 0.27
88 ꢀ 5.03
>200
>200
157.2 ꢀ 8.23
12.8 ꢀ 0.40
19.8 ꢀ 0.54
14.5 ꢀ 0.17
13.1 ꢀ 0.30
6.8 ꢀ 0.13
>200
34.3 ꢀ 4.46
153.4 ꢀ 6.31
47.3 ꢀ 7.83
42.3 ꢀ 3.91
6.3 ꢀ 0.53
13.6 ꢀ 0.34
8.9 ꢀ 0.73
Kanamycin
Penicillamine
Fig. 2. Crystal structure diagram of compound 37. H atoms are shown as small spheres
of arbitrary radii.
5.8 ꢀ 0.19
6.3 ꢀ 0.43

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Y.-T. Duan et al. / European Journal of Medicinal Chemistry 76 (2014) 387e396
Table 4
E. coli FabH and P. aeruginosa FabH inhibitory activities of selected compounds.
Compound
E. coli FabH IC50
(m
M)
P. aeruginosa FabH IC50 (mM)
30
33
36
37
38
42
46
47
16.5
2.1
4.2
4.4
6.5
10.2
11.0
3.4
22.5
12.4
15.9
19.2
29.2
25.6
10.2
7.9
Kanamycin
3.1
6.7
effects of the synthetic compounds against P. aeruginosa may not
correlated to P. aeruginosa FabH, and this result complied with the
new research from different groups [28e30]. Other tested com-
pounds displayed moderate inhibitory activities with IC50 ranging
Fig. 4. Viability (determined by MTT assay) of macrophage exposed to compound 47
after 24 h.
from 4.2 to 16.5
mM against E. coli FabH. It suggested that the
modifications in the 2-methyl group of metronidazole were favor-
able for the FabH inhibitory activity. We can also conclude from our
observed activity results that the compound which showed good
docking energies against E. coli FabH and good IC50 against E. coli
FabH, should not necessarily that they show good antibacterial
activities against E. coli.
2.2.3. Cytotoxicity
As compounds 33 and 47 exhibited potent antimicrobial activity,
their detailed toxicological studies on human macrophage were
carried out. As showed in Figs. 3 and 4, the MTT assay revealed that
the viability of macrophage was inversely proportional to the
concentration of the compounds 33 and 47. Further more, after
cultured for 24 h, Morphological anomalies in macrophage exposed
to different concentration compound 33 (0.25, 160 mg/mL) showed
nothing different from morphological in control under phase
contrast microscope (Figs. 5e7). It recommended that both com-
pounds 33 and 47 did not exhibit any toxicity towards
morphological.
Fig. 5. Morphological changes in macrophage at 0.25 mg/mL of compound 33.
2.2.4. Binding model of compounds 33, 47 and E. coli FabH
Molecular docking of the synthesized compounds and E. coli
FabH was performed on the binding model based on the E. coli
FabHeCoA complex structure (1HNJ.pdb) [31]. The coordinate of
the input site sphere is 28.4494, 9.90349, 33.4428 and the radius of
the sphere is 11 A.
ꢀ
All docking runs were applied Ligand Fit Dock protocol of Dis-
covery Studio 3.1. The binding model of compounds 33, 47 and
Fig. 3. Viability (determined by MTT assay) of macrophage exposed to compound 33
after 24 h.
Fig. 6. Morphological changes in macrophage at 160 mg/mL of compound 33.

Y.-T. Duan et al. / European Journal of Medicinal Chemistry 76 (2014) 387e396
391
interactions with two nitro group, which were accordant exactly
with the previous work of FabH inhibitors. Meanwhile, the nitrogen
atom of nitro group formed a
compound 47, it also performed a nice bonding situation via three
hydrogen bonds together with two ecation interactions. This
ensured the binding affinity and results in an increased FabH
inhibitory activity. Arg219 and Arg56 formed two remarkable
pep interaction with Phe213. As for
p
p
e
cation interactions with two benzene rings respectively. Besides,
the hydrogen of Asn274, Asn274 and Arg56 were formed three
hydrogen bonds interaction with oxygen atom of nitro group of
ꢁ
ꢀ
compound 47 (angle O $ HeN ¼ 131.1 , distance ¼ 2.10 A), nitrogen
atom of pyrazol group of compound 47 (angle N $ HeN ¼ 147.9ꢁ,
ꢀ
distance ¼ 2.40 A) and oxygen atom linking two benzene rings of
ꢁ
ꢀ
compound 47 (angle N $ HeN ¼ 101.8 , distance ¼ 2.60 A) each
other. This molecular docking result, along with the biological assay
data, suggested that compounds 33 and 47 were a potential in-
hibitor of FabH. The docking calculation of all the compounds was
also depicted in Table 5. The CDocker Energy (energy of the ligande
receptor complexes) agreed with the FabH inhibitory trend for all
the synthesized compounds.
Fig. 7. Normal growth of macrophage (control).
E. coli FabH was depicted in Figs. 8 and 9. The amino acid residues
which had interaction with FabH as well as bond length were
labeled. In the binding mode, compound 33 was nicely bound to
FabH via ꢁthree hydrogen bonds with Met207 (angle O He
3. Conclusion
ꢁ
ꢀ
N ¼ 141.6 , distance ¼ 2.33 A), Asn247 (angle O HeN ¼ 124.5 ,
To conclude, a series of novel 2-styryl 5-nitroimidazole de-
rivatives 25e48 were synthesized through straightforward chem-
istry and tested for their inhibitory activities against E. coli,
P. aeruginosa, B. subtilis and B. thuringiensis. Most of them exhibited
ꢁ
ꢀ
ꢀ
distance ¼ 2.34 A and angle O $ HeN ¼ 137.4 , distance ¼ 2.30 A),
six charge interactions and one interaction. The end group of
Arg249, Arg46 and Asp150 were respectively formed six charge
pep
Fig. 8. (A) 2D model of the interaction between compound 33 and E. coli FabH. Pecation bond is displayed as orange lines. H-bonds are displayed as blue and green dashed lines. (B)
3D model of the interaction between compound 33 and E. coli FabH. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
this article.)

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Y.-T. Duan et al. / European Journal of Medicinal Chemistry 76 (2014) 387e396
Fig. 9. (A) 2D model of the interaction between compound 47 and E. coli FabH. P-cation bonds are displayed as orange lines. H-bonds are displayed as blue and green dashed lines.
(B) 3D model of the interaction between compound 47 and E. coli FabH. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
this article.)
potent antibacterial and E. coli FabH inhibitory activities. Com-
pounds 33 and 47 showed the most potent FabH inhibition activ-
might be helpful for the design and synthesis of FabH inhibitors
with stronger activities.
ities (IC50 ¼ 2.1, 3.1
34.3
g/mL for E. coli, IC50 ¼ 15.5, 14.5
IC50 ¼ 13.9, 9.8
m
M) and antimicrobial activities (IC50 ¼ 33.0,
g/mL for P. aeruginosa,
g/mL for B. thuringiensis and IC50 ¼ 9.0, 6.3 g/mL
m
m
4. Experiments
m
m
for B. subtilis), and more, did not show any toxicity towards
morphological. The docking simulation was performed to get the
probable binding models and poses. The results indicated that
compounds 33 and 47, which acted as potential FabH inhibitors, can
both bind well into the active site of FabH. The result of this work
4.1. Materials and measurements
All chemicals used were purchased from Aldrich (USA). Melting
points (uncorrected) were determined on a X-4 MP apparatus
(Taike Corp, Beijing, China). All the 1H NMR spectra were recorded
on a Bruker DPX 300 model Spectrometer in DMSO- d6 and
chemical shifts were reported in ppm (d). Elemental analyses were
Table 5
performed on a CHNeO-Rapid instrument and were within ꢀ0.4%
of the theoretical values. ESI-MS spectra were recorded on a Mar-
iner System 5304 Mass spectrometer.
The docking calculation of the synthesized compounds.
Compound
CDocker Energy
Gb (kcal/mol)
Compound
CDocker Energy
Gb (kcal/mol)
D
D
25
26
27
28
29
30
31
32
33
34
35
36
ꢂ22.34
ꢂ21.23
ꢂ15.34
ꢂ28.22
ꢂ23.12
ꢂ19.89
ꢂ13.13
ꢂ25.23
ꢂ35.54
ꢂ31.12
ꢂ23.56
ꢂ31.75
37
38
39
40
41
42
43
44
45
46
47
48
ꢂ30.01
ꢂ29.60
ꢂ18.83
ꢂ15.27
ꢂ28.65
ꢂ25.32
ꢂ21.47
ꢂ20.26
ꢂ27.76
ꢂ20.33
ꢂ33.17
ꢂ31.08
4.2. General method of synthesis of metronidazole derivatives
Reaction of metronidazole (12 mmol) with different substituted
benzaldehyde (16 mmol) in 6 mL DMSO by adding rapidly a stirred
solution of sodium methoxide (12.8 mmol) in methanol at room
temperature resulted in the formation of target compounds.
4.2.1. 2-(5-Nitro-2-styryl-1H-imidazol-1-yl)ethanol(25)
Yield 83.1%; M.p. 75e78 ꢁC. 1H NMR (DMSO-d6, 300 MHz)
d
: 8.22
(s, 1H, CH), 7.79e7.61 (m, 2H, ArH), 7.52 (d, J ¼ 7.6 Hz,1H, CH), 7.51e

Y.-T. Duan et al. / European Journal of Medicinal Chemistry 76 (2014) 387e396
393
7.40 (m, 3H, ArH), 7.30e7.19 (m, 1H, CH), 4.96 (s, 1H, OH), 4.61 (t,
4.2.9. 2-(2-(4-Nitrostyryl)-5-nitro-1H-imidazol-1-yl)ethanol(33)
Yield 73.1%. M.p. 79e83 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:
J ¼ 5.1 Hz, 2H, CH2), 3.70 (m, 2H, CH2). ESI-MS: 260.10 (C13H14N3O3,
[MþH]þ). Anal. Calcd for C13H13N3O3: C, 60.22; H, 5.05; N, 16.21.
Found: C, 57.12; H, 5.03; N, 16.15.
8.29e8.20 (m, 4H, ArH, CH), 8.05e7.87 (m, 2H, ArH), 7.61 (d,
J ¼ 15.8 Hz, 1H, CH), 5.01 (t, J ¼ 5.6 Hz, 1H, OH), 4.8 (t, J ¼ 5.0 Hz, 2H,
CH2), 3.75e3.70 (m, 2H, CH2). ESI-MS: 305.08 (C13H13N4O5,
[MþH]þ). Anal. Calcd for C13H12N4O5: C, 51.32; H, 3.98; N, 18.41.
Found: C, 50.71; H, 3.96; N, 16.37.
4.2.2. 2-(2-(4-Methoxystyryl)-5-nitro-1H-imidazol-1-yl)
ethanol(26)
Yield 74.5%; M.p. 82e85 ꢁC. 1H NMR (DMSO-d6, 300 MHz)
d: 8.12
4.2.10. 2-(2-(3-Methoxystyryl)-5-nitro-1H-imidazol-1-yl)
(s, 1H, CH), 7.71e7.11 (m, 4H, ArH), 6.93 (d, J ¼ 5.4 Hz, 2H, CH), 4.93
(s, 1H, OH), 4.55 (t, J ¼ 5.1 Hz, 2H, CH2), 3.73 (t, J ¼ 6.3 Hz, 2H, CH2),
1.13 (s, 3H, CH3). ESI-MS: 290.11 (C14H16N3O4, [MþH]þ). Anal. Calcd
for C14H15N3O4: C, 58.13; H, 5.23; N, 14.53. Found: C, 59.12; H, 5.20;
N, 15.04.
ethanol(34)
Yield 68.1%; M.p. 80e82 ꢁC. 1H NMR (DMSO-d6, 300 MHz)
d: 8.15
(s, 1H, CH), 7.81e7.43 (m, 4H, ArH), 6.93 (d, J ¼ 6.4 Hz, 2H, CH), 5.01
(s,1H, OH), 4.53 (t, J ¼ 5.6 Hz, 2H, CH2), 3.79 (m, 2H, CH2),1.18 (s, 3H,
CH3). ESI-MS: 290.11 (C14H16N3O4, [MþH]þ). Anal. Calcd for
C14H15N3O4: C, 58.13; H, 5.23; N, 14.53. Found: C, 50.17; H, 5.21; N,
4.2.3. 2-(2-(4-Bromostyryl)-5-nitro-1H-imidazol-1-yl)ethanol(27)
16.01.
Yield 77.0%; M.p. 76e79 ꢁC. 1H NMR (DMSO-d6,300 MHz)
d: 8.22
(d, J ¼ 6.8 Hz, 1H, CH), 7.97e7.78 (m, 3H, ArH), 7.54e7.39 (m, 3H,
ArH, CH), 5.01 (t, J ¼ 4.5 Hz, 1H, OH), 4.70 (t, J ¼ 6.0 Hz, 2H, CH2),
3.72e3.65 (m, 2H, CH2). ESI-MS: 238.01 (C13H13BrN3O3, [MþH]þ).
Anal. Calcd for C13H12BrN3O3: C, 46.17; H, 3.58; N, 12.43. Found: C,
44.58; H, 3.54; N, 12.07.
4.2.11. 2-(2-(3-bromostyryl)-5-nitro-1H-imidazol-1-yl)ethanol(35)
Yield 64.8%. M.p. 81e83 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d
:
8.22 (s, 1H, CH), 8.08 (s, 1H, ArH), 7.76e7.71 (m, 2H, ArH), 7.57e7.36
(m, 3H, ArH, CH), 4.99 (t, J ¼ 4.0 Hz,1H, OH), 4.65 (t, J ¼ 5.7, 2H, CH2),
3.73e3.68 (m, 2H, CH2). ESI-MS: 238.01 (C13H13BrN3O3, [MþH]þ).
Anal. Calcd for C13H12BrN3O3: C, 46.17; H, 3.58; N, 12.43. Found: C,
43.75; H, 3.55; N, 12.13.
4.2.4. 2-(2-(3-Fluorostyryl)-5-nitro-1H-imidazol-1-yl)ethanol(28)
Yield 76.4%. M.p. 75e77 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:8.21
4.2.12. 2-(2-(4-(Dimethoxymethyl)styryl)-5-nitro-1H-imidazol-1-
(s, 1H, CH), 7.79e7.69 (m, 2H, ArH), 7.57 (d, J ¼ 7.7 Hz,1H, CH), 7.51e
7.42 (m, 2H, ArH), 7.24e7.18 (m, 1H, CH), 4.99 (s, 1H, OH), 4.65 (t,
J ¼ 5.1 Hz, 2H, CH2), 3.71 (m,2H, CH2). ESI-MS: 278.09 (C13H13FN3O3,
[MþH]þ). Anal. Calcd for C13H12FN3O3: C, 56.32; H, 4.36; N, 15.16.
Found: C, 43.24; H, 4.38; N, 13.59.
yl)ethanol(36)
Yield 76.5%. M.p. 83e86 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.00 (s, 1H, CH), 7.67e7.41 (m, 4H, ArH), 6.57 (t, J ¼ 7.14 Hz 2H, CH2),
4.80 (t, J ¼ 5.6 Hz, 1H, OH), 4.40 (t, J ¼ 6.0 Hz, 2H, CH2), 3.75e3.50
(m, 6H, CH2), 1.21e1.10 (m, 6H, CH3). ESI-MS: 362.16 (C18H24N3O5,
[MþH]þ). Anal. Calcd for C18H23N3O5: C, 59.82; H, 6.41; N, 11.63.
Found: C, 54.73; H, 6.38; N, 11.58.
4.2.5. 2-(2-(4-Chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethanol(29)
Yield 68.3%. M.p. 76e78 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.23 (d, J ¼ 5.0 Hz, 1H, CH), 7.89e7.65 (m, 3H, ArH), 7.43e7.27 (m,
3H, ArH, CH), 5.04e4.95 (m, 1H, OH), 4.70e4.54 (m, 2H, CH2), 3.86e
3.65 (m, 2H, CH2). ESI-MS: 294.06 (C13H13ClN3O3, [MþH]þ). Anal.
Calcd for C13H12ClN3O3: C, 53.16; H, 4.12; N, 14.31. Found: C, 48.57;
H, 4.09; N, 15.23.
4.2.13. 2-(2-(2-Fluorostyryl)-5-nitro-1H-imidazol-1-yl)ethanol(37)
Yield 69.2%. M.p. 77e80 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.22 (d, J ¼ 4.74, 1H, CH), 7.82e7.70 (m, 3H, ArH), 7.64 (d, J ¼ 5.4 Hz,
1H, CH), 7.42e7.30 (m, 2H, ArH, CH), 5.15e4.94 (m, 1H, OH), 4.54 (t,
J ¼ 8.4 Hz, 2H, CH2), 3.77e3.70 (m, 2H, CH2). ESI-MS: 278.09
(C13H13FN3O3, [MþH]þ). Anal. Calcd for C13H12FN3O3: C, 56.32; H,
4.36; N, 15.16. Found: C, 54.20; H, 4.34; N, 14.78.
4.2.6. 2-(2-(4-Fluorostyryl)-5-nitro-1H-imidazol-1-yl)ethanol(30)
Yield 73.2%. M.p. 77e80 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:
4.2.14. 2-(2-(2-Chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethanol(38)
8.22 (d, J ¼ 4.7 Hz, 1H, CH), 7.86e7.75 (m, 3H, ArH), 7.39e7.25 (m,
3H, ArH, CH), 5.04e4.97 (m, 1H, OH), 4.64e4.49 (m, 2H, CH2), 3.73e
3.68 (m, 2H, CH2). ESI-MS: 278.09 (C13H13FN3O3, [MþH]þ). Anal.
Calcd for C13H12FN3O3: C, 56.32; H, 4.36; N, 15.16. Found: C, 47.68;
H, 4.37; N, 16.63.
Yield 71%. M.p. 74e77 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d: 8.20
(s, 1H, CH), 7.85e7.72 (m, 3H, ArH), 7.61e7.32 (m, 3H, ArH, CH), 5.00
(t, J ¼ 5.4 Hz, 1H, OH), 4.50 (t, J ¼ 6.7 Hz, 2H, CH2), 3.65 (m, 2H, CH2).
ESI-MS: 294.06 (C13H13ClN3O3, [MþH]þ). Anal. Calcd for
C13H12ClN3O3: C, 53.16; H, 4.12; N, 14.31. Found: C, 49.54; H, 4.09; N,
13.67.
4.2.7. 2-(2-(3-Chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethanol(31)
Yield 69.7%. M.p. 75e78 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:
4.2.15. 2-(2-(4-Methylstyryl)-5-nitro-1H-imidazol-1-yl)
ethanol(39)
8.22 (s, 1H, CH), 7.83e7.69 (m, 3H, ArH), 7.57e7.36 (m, 3H, ArH, CH),
5.02 (t, J ¼ 3.7 Hz, 1H, OH), 4.34 (t, J ¼ 6.0 Hz, 2H, CH2), 3.62e3.49
(m, 2H, CH2). ESI-MS: 294.06 (C13H13ClN3O3, [MþH]þ). Anal. Calcd
for C13H12ClN3O3: C, 53.16; H, 4.12; N, 14.31. Found: C, 38.69; H,
4.10; N, 14.92.
Yield 65.3%; M.p. 82e85 ꢁC. 1H NMR (DMSO-d6, 300 MHz)
d: 7.94
(s, 1H, CH), 7.81e7.62 (m, 4H, ArH), 7.44 (d, J ¼ 7.9 Hz, 2H, CH), 4.86
(s, 1H, OH), 4.35 (t, J ¼ 6.4 Hz, 2H, CH2), 3.63 (t, J ¼ 7.2 Hz, 2H, CH2),
1.22 (s, 3H, CH3). ESI-MS: 290.11 (C14H16N3O3, [MþH]þ). Anal. Calcd
for C14H15N3O3: C, 61.53; H, 5.53; N, 15.38. Found: C, 57.43; H, 5.49;
N, 16.25.
4.2.8. 2-(2-(2-Methoxystyryl)-5-nitro-1H-imidazol-1-yl)
ethanol(32)
Yield 73.2%; M.p. 79e82 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:
4.2.16. 2-(2-(2-chloro-6-fluorostyryl)-5-nitro-1H-imidazol-1-yl)
ethanol (40)
8.22 (s, 1H, CH), 7.76e7.65 (m, 1H, CH), 7.60e7.44 (m, 4H, ArH), 6.83
(d, J ¼ 6.0 Hz, 1H, CH), 5.02 (s, 1H, OH), 4.62e4.50 (m, 2H, CH2), 3.71
(t, J ¼ 6.2 Hz, 2H, CH2),1.14 (s, 3H, CH3). ESI-MS: 290.11 (C14H16N3O4,
[MþH]þ). Anal. Calcd for C14H15N3O4: C, 58.13; H, 5.23; N, 14.53.
Found: C, 47.54; H, 5.20; N, 13.91.
Yield 72.5%. M.p. 86e88 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.05 (s, 1H, CH), 7.67e7.38 (m, 4H, ArH, CH), 6.78 (d, J ¼ 5.4 Hz, 1H,
CH), 4.97 (t, J ¼ 5.6 Hz, 1H, OH), 4.35 (t, J ¼ 6.0 Hz, 2H, CH2), 3.68e
3.53 (m, 6H, CH2). ESI-MS: 314.06 (C13H14ClFN3O3, [MþH]þ). Anal.

394
Y.-T. Duan et al. / European Journal of Medicinal Chemistry 76 (2014) 387e396
Calcd for C13H13ClFN3O3: C, 49.77; H, 4.18; N, 13.39. Found: C, 48.38;
H, 4.14; N, 12.27.
4.2.24. 2-(2-(2-(Naphthalen-1-yl)vinyl)-5-nitro-1H-imidazol-1-yl)
ethanol (48)
Yield 75.7%. M.p. 86e89 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:
4.2.17. 2-(2-(2,4-Dichlorostyryl)-5-nitro-1H-imidazol-1-yl)
8.16e7.93 (m, 4H, ArH, CH), 7.76e7.43 (m, 4H, ArH), 6.63 (t,
J ¼ 4.3 Hz, 2H, CH2), 4.81 (t, J ¼ 6.9 Hz, 1H, OH), 4.30 (t, J ¼ 5.1 Hz,
2H, CH2), 3.70 (m, 2H, CH2), ESI-MS: 310.11(C17H16N3O3, [MþH]þ).
Anal. Calcd for C17H15N3O3: C, 66.01; H, 4.89; N, 13.58. Found: C,
64.29; H, 4.88; N, 12.91.
ethanol(41)
Yield 69.5%. M.p. 85e88 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.00 (s, 1H, CH), 7.65e7.43 (m, 2H, ArH), 7.37e7.20 (m, 2H, ArH, CH),
6.94 (d, J ¼ 8.5 Hz, H, CH), 4.57 (t, J ¼ 6.1 Hz, 1H, OH), 4.38 (t,
J ¼ 9.6 Hz, 2H, CH2), 3.72e3.57 (m, 2H, CH2). ESI-MS: 330.03
(C13H14Cl2N3O3, [MþH]þ). Anal. Calcd for C13H13Cl2N3O3: C, 47.29;
H, 3.97; N, 12.73. Found: C, 45.35; H, 3.95; N, 12.53.
4.3. Crystal structure determination
Crystal structure determination of compounds 35 and 37 were
4.2.18. 2-(2-(4-Dimethylaminostyryl)-5-nitro-1H-imidazol-1-yl)
carried out on Bruker SMARTAPEX CCD diffractometer at 293(2) K
ꢀ
ethanol (42)
using Mo Ka radiation (
l
¼ 0.71073A) by the
u scan mode. The
Yield 68.4%. M.p. 86e88 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:
structure was solved by direct methods and refined on F2 by full-
matrix least-squares methods using SHELX-97. 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 35 and 37 were listed in Table 2.
8.06 (s, 1H, CH), 7.83e7.70 (m, 2H, ArH), 7.00e6.73 (m, 2H, ArH),
6.97 (t, J ¼ 6.7 Hz, 2H, CH), 5.00 (t, J ¼ 6.2 Hz, 1H, OH), 4.37 (t,
J ¼ 7.4 Hz, 2H, CH2), 3.68e3.53 (m, 2H, CH2), 1.46e1.19 (m, 6H, CH3).
ESI-MS: 305.15 (C15H19N4O3, [MþH]þ). Anal. Calcd for C15H18N4O3:
C, 59.59; H, 6.00; N, 18.53. Found: C, 43.87; H, 6.03; N, 17.73.
4.2.19. 2-(2-(3-Nitrostyryl)-5-nitro-1H-imidazol-1-yl)ethanol(43)
Yield 74.1%. M.p. 81e82 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.31e7.73 (m, 3H, ArH, CH), 7.70e7.54 (m, 2H, ArH), 7.35e7.16 (m,
2H, CH), 4.85 (t, J ¼ 7.3 Hz, 1H, OH), 4.53 (t, J ¼ 6.4 Hz, 2H, CH2), 3.65
(m, 2H, CH2). ESI-MS: 305.08 (C13H13N4O5, [MþH]þ). Anal. Calcd for
4.4. Antibacterial activity
C
13H12N4O5: C, 50.98; H, 4.61; N, 18.29. Found: C, 47.87; H, 4.57; N,
17.53.
The antibacterial activity of the synthesized compounds was
tested against E. coli, B. thuringiensis, B. subtilis and P. aeruginosa
using MH medium (MuellereHinton medium: casein hydrolyzate
17.5 g, soluble starch 1.5 g, beef extract 1000 mL). Seed 104 bacte-
rials per well into 96-well plates, incubate at 37 ꢁC for 24 h. Then
4.2.20. 2-(2-(3-Bromostyryl)-5-nitro-1H-imidazol-1-yl)
ethanol(44)
Yield 68.6%. M.p. 79e82 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.22 (d, J ¼ 4.74, 1H, CH), 7.86e7.75 (m, 3H, ArH), 7.39e7.25 (m, 3H,
ArH, CH),5.04e4.97 (m, 1H, OH), 4.64e4.49 (m, 2H, CH2), 3.73e3.68
(m, 2H, CH2). ESI-MS: 238.01 (C13H13BrN3O3, [MþH]þ). Anal. Calcd
for C13H12BrN3O3: C, 46.17; H, 3.58; N, 12.43. Found: C, 38.94; H,
3.56; N, 11.75.
add 100
wells to maintain the final concentration of drug as 320, 160, 40, 10,
2.5 and 0.25 g/mL. One concentration should be triplicated. After
12 h, bacterial survival was determined by the addition of an MTT
(3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl tetrazolium bromide)
solution (25 mL of 5 mg/mL MTT in PBS). After 4 h, discard the
mL a series concentration of drug-containing medium into
m
4.2.21. 2-(2-(2-Nitrostyryl)-5-nitro-1H-imidazol-1-yl)ethanol(45)
medium and add 150 mL DMSO. The plates were voted for 10 min to
make completely dissolution. Optical absorbance was measured at
490 nm.
Yield 72.1%. M.p. 77e80 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.25 (d, J ¼ 6.73 Hz, 1H, CH), 8.03e7.94 (m, 3H, ArH, CH), 7.83e7.58
(m, 3H, ArH, CH), 4.99 (t, J ¼ 6.3 Hz, 1H, OH), 4.54 (t, J ¼ 5.4 Hz, 2H,
CH2), 3.74e3.52 (m, 2H, CH2). ESI-MS: 305.08 (C13H13N4O5,
[MþH]þ). Anal. Calcd for C13H12N4O5: C, 51.32; H, 3.98; N, 18.41.
Found: C, 49.26; H, 3.94; N, 16.54.
4.5. E. coli FabH purification and activity assay
Full-length E. coli ACP, acyl carrier protein synthase (ACPS), and
FabH were individually cloned into pET30 expression vectors with
an N-terminal His-tag.
4.2.22. 2-(2-(2-(Naphthalen-2-yl)vinyl)-5-nitro-1H-imidazol-1-yl)
ethanol (46)
All proteins were expressed in E. coli strain BL21 (DE3). Trans-
formed cells were grown on LuriaeBertani (LB) agar plates sup-
Yield 71.4%. M.p. 86e89 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:
8.05e7.92 (m, 3H, ArH, CH), 7.89e7.64 (m, 2H, ArH), 7.61 (t,
J ¼ 6.5 Hz, 2H, ArH), 7.43 (t, J ¼ 7.3 Hz, 1H, ArH), 7.09e6.87 (m, 2H,
CH), 5.02 (t, J ¼ 7.6 Hz, 1H, OH), 4.41(t, J ¼ 6.0 Hz, 2H, CH2), 3.70e
3.58 (m, 2H, CH2). ESI-MS: 310.11 (C17H16N3O3, [MþH]þ). Anal.
Calcd for C17H15N3O3: C, 66.01; H, 4.89; N, 13.58. Found: C, 57.89; H,
4.86; N, 14.25.
plemented with kanamycin (30 mg/mL). Sodium dodecyl sulfatee
polyacrylamide gel electrophoresis (SDSePAGE) analysis was used
to screen colonies for overexpression of proteins. One such positive
colony was used to inoculate 10 mL of LB medium with 30 mg/mL of
kanamycin and grown overnight at 37 ꢁC,1 mL of which was used to
inoculate 100 mL LB medium supplemented with 30 mg/mL of
kanamycin. The culture was shaken for 4 h at 37 ꢁC, and then
4.2.23. 2-(2-(4-(Benzyloxy)styryl)-5-nitro-1H-imidazol-1-yl)
induced with 0.5 mM isopropyl b-D-thiogalactopy-ranoside (IPTG).
ethanol(47)
The culture was grown for 4 h, and harvested by centrifugation
(30 min at 15,000 rpm).
Yield 72.3%. M.p. 84e87 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
d:
7.99 (d, J ¼ 5.8 Hz,1H, CH) 7.64e7.50 (m, 4H, ArH), 7.42e7.26 (m, 3H,
ArH), 7.08e6.90 (m, 4H, Ar, CH), 5.16 (t, J ¼ 5.1 Hz, 2H, CH2),4.54 (t,
J ¼ 6.7 Hz, 1H, OH), 4.39(t, J ¼ 8.3 Hz, 2H, CH2), 3.68e3.54 (m, 2H,
CH2). ESI-MS: 366.14 (C20H21N3O4, [MþH]þ). Anal. Calcd for
Harvested cells containing FabH were lysed by sonication in
20 mM Tris, pH 7.6, 5 mM imidazole, 0.5 M NaCl and centrifuged at
20,000 rpm for 30 min. The supernatant was applied to a Ni-NTA
agarose column, washed, and eluted using a 5e500 mM imid-
azole gradient over 20 column volumes. Eluted protein was dia-
lyzed against 20 mM Tris, pH 7.6, 1 mM DTT, and 100 mM NaCl.
C
20H19N3O4: C, 65.74; H, 5.24; N, 11.50. Found: C, 63.83; H, 5.23; N,
12.98.

Y.-T. Duan et al. / European Journal of Medicinal Chemistry 76 (2014) 387e396
395
Purified FabD and FabHs were concentrated up to 2 mg/mL and
References
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