Synthesis, molecular modeling and biological evaluation of β-ketoacyl-acyl carrier protein synthase III (FabH) as novel antibacterial agents

Article abstract of DOI:10.1016/j.bmc.2011.06.021

A series of novel cinnamic acid secnidazole ester derivatives have been designed and synthesized, and their biological activities were also evaluated as potential inhibitors of FabH. These compounds were assayed for antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus. Compounds with potent antibacterial activities were tested for their E. coli FabH inhibitory activity. Compound 3n showed the most potent antibacterial activity with MIC of 1.56-6.25 μg/mL against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with IC₅₀ of 2.5 μM. Docking simulation was performed to position compound 3n into the E. coli FabH active site to determine the probable binding conformation.

Full text of DOI:10.1016/j.bmc.2011.06.021

Bioorganic & Medicinal Chemistry 19 (2011) 4513–4519
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, molecular modeling and biological evaluation of b-ketoacyl-acyl
carrier protein synthase III (FabH) as novel antibacterial agents
⇑
Hong-Jia Zhang, Di-Di Zhu, Zi-Lin Li, Juan Sun, Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR 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 novel cinnamic acid secnidazole ester derivatives have been designed and synthesized, and
their biological activities were also evaluated as potential inhibitors of FabH. These compounds were
assayed for antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and
Staphylococcus aureus. Compounds with potent antibacterial activities were tested for their E. coli
FabH inhibitory activity. Compound 3n showed the most potent antibacterial activity with MIC of
Received 20 April 2011
Revised 5 June 2011
Accepted 8 June 2011
Available online 6 July 2011
1.56–6.25
activity with IC50 of 2.5
E. coli FabH active site to determine the probable binding conformation.
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
l
g/mL against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory
Keywords:
Cinnamic acid
Secnidazole
Antibacterial activity
FabH inhibitors
lM. Docking simulation was performed to position compound 3n into the
1. Introduction
used as an effective molecular target for the development of new
antimicrobial agents.
Although several classes of antibacterial agents are presently
available, resistance in most of the pathogenic bacteria to these
drugs constantly emerges. In order to prevent this serious medical
problem, the elaboration of new types of antibacterial agents is a
very important task.1 Recent 10 years, different targets in key areas
of the bacterial cell cycle have been studied that would be a new
weapon against the problem of acquired resistance. One of the
most attractive biochemical pathways to be used as the target
for new antibacterial agents is the fatty acid biosynthesis (FAS).
This pathway has been demonstrated to be essential for bacteria
cell survival, and differs considerably from human FAS pathway.2,3
While in humans fatty acid synthesis occurs in a homodimeric
multifunctional enzyme,4 in bacteria the pathway is composed of
various discrete enzymes and each one can be considered a puta-
tive molecular target. Those features make the type II FAS pathway
a potential target for new antimicrobial agents.
A key enzyme in this pathway is the b-ketoacyl-acyl carrier pro-
tein synthase III (FabH), which is the enzyme responsible for the
first reaction in the pathway and plays an important regulatory
role.5 FabH has also been demonstrated to be essential for organis-
mal survival and is present in a large number of important human
pathogens.6 Further more, some chemical compounds had shown
to inhibit FabH from diverse microorganisms, including multi-drug
resistant strains.7,8 These facts support the idea that FabH can be
Nitroimidazoles have been extensively used as antimicrobial
chemotherapeutics and as antiangiogenic hypoxic cell radiosensi-
tizers.9 Nitroimidazole derivatives have attracted considerable
attention as they can undergo bioreduction to yield electrophilic
substances which can damage protein and nucleic acids.10
Importantly, the toxicology and metabolism of nitroimidazoles,
particularly secnidazole, have been characterized. Secnidazole
(a,2-dimethyl-5-nitro-1H-imidazole-1-ethanol) is used as an anti-
protozoal, antiamebic, and antibacterial drug.11 It is an antimicro-
bic agent which is structurally related to the commonly used
5-nitromidazoles metronidazole and tinidazole. Secnidazole is
particularly effective in the treatment of amebiasis, giardiasis,
trichomoniasis, and bacterial vaginosis12 as it is rapidly and com-
pletely absorbed after oral administration and has a longer termi-
nal elimination half-life (17–29 h) than commonly used drugs in
this class.13 In these cases, the treatment with secnidazole is short-
er and significantly more effective than the treatment using other
imidazole drugs and the adverse effects are not very drastic.14 As a
result, excellent reviews have been published on the activity and
pharmacokinetics of secnidazole and their determination in phar-
maceutical is of great importance.
As apart of our research for novel antibacterial agents, we de-
signed and synthesized a series of secnidazole analogs based on
cinnamic acid scaffold. We chose cinnamoyl moiety as it was found
in a variety of biologically active substances.15,16 Antibacterial
activities of various cinnamic acid derivatives were also explored
by many research groups.17 Particularly, cinnamic acid ester
derivatives showed the potential antibacterial activity.18 However,
⇑
Corresponding author. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.021

4514
H.-J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 4513–4519
to our knowledge, few reports have been dedicated to the synthe-
sis and FabH inhibitory activity of cinnamic acid secnidazole ester
derivatives. In continuation to extend our research on the activity
of cinnamic acid nitroimidazole ester derivatives, we report in the
present work the synthesis and structure–activity relationships of
a series of secnidazole derivatives.19 Herein, we described the syn-
thesis and the SAR of the novel series of secnidazole derivatives,
and the biological activity evaluation indicated that some of these
compounds are potent inhibitors of Escherichia coli FabH. Docking
simulations were also performed using the X-ray crystallographic
structure of the E. coli FabH with an inhibitor to explore the bind-
ing models of these compounds at the active site.
2. Results and discussion
2.1. Chemistry
The synthetic route for the novel cinnamic acid secnidazole es-
ter derivatives 3a–t is outlined in Scheme 1. These compounds
were synthesized from the start cinnamic acids (1) and secnidazole
CHO
COOH
i
HOOC
COOH
COOH
O2N
R
R
O2N
1
O
N
N
ii
O
N
R
R
N
HO
3a-3t
1
2
Scheme 1. General synthesis of cinnamic acid secnidazole ester derivatives (3a–t). Reagents and conditions: (i) piperidine, pyridine, 80–90 °C, 24 h; (ii) DCC, DMAP, 50–60 °C,
24 h.
Table 1
Structure of cinnamic acid secnidazole ester derivatives (3a–t)
O2N
O
N
N
O
R3
R1
R2
Compound
R1
R2
R3
3a
3b
3c
3d
3e
3f
H
F
Cl
Br
OCH3
H
H
H
H
H
H
F
H
H
H
H
H
H
3g
3h
3i
H
H
H
Br
OCH3
H
H
H
F
3j
H
H
Cl
3k
3l
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
Br
CH3
OCH3
Ph
OBn
N(CH3)2
Cl
3m
3n
3o
3p
3q
O2N
O2N
O
O
N
N
N
N
O
O
3r
3t
3s
O2N
O
N
N
O
s

H.-J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 4513–4519
4515
(2). Compound (1) was prepared according the modified procedure
of Davis et al.20 Aromatic aldehydes and malonic acid were dis-
solved in a mixture of pyridine and piperidine and refluxed for
24 h, and cinnamic acids were obtained with yields of 75–85%.
Then, cinnamic acids (1), secnidazole (2) and K2CO3 were taken
in DMF and refluxed to obtain the desired compounds (3a–t)
(Table 1). All of the synthetic compounds gave satisfactory
analytical and spectroscopic data, which were in full accordance
with their depicted structures.
positive control Kanamycin. However, a methyl or methoxy group
substituent (3l and 3m) led to a great loss of activity. Meanwhile, a
significant loss of activity was observed when the chlorine group
substituent was removed from the para position, compared with
3c, which had one single chlorine group substituent at the ortho
position. When the benzene ring was replaced by naphthalene or
thiophene ring, a significant loss of activity was observed. Based
on the data obtained from compounds with bromine group substi-
tuent (3d, 3g, 3k), we found that the potency order of antibacterial
activity was para > meta > ortho.
2.2. Biological activity
2.2.2. E. coli FabH inhibitory activity
2.2.1. Antibacterial activity
The E. coli FabH inhibitory potency of the synthetic ester deriv-
atives with potent antibacterial activities (3d, 3f, 3g, 3j, 3k, 3p–r)
was examined and the results are summarized in Table 3. Most
of the tested compounds displayed potent E. coli FabH inhibitory.
Among them, compound 3n condensing by (E)-3-(biphenyl-4-
yl)acrylic acid and secnidazole showed the most potent inhibitory
All the synthesized compounds were screened for antibacterial
activity against two Gram-negative bacterial strains: E. coli and
Pseudomonas aeruginosa and two Gram-positive bacterial strains:
Bacillus subtilis and Staphylococcus aureus by MTT method. The
MICs (minimum inhibitory concentrations) of the compounds
against these bacteria were presented in Table 2. Also included
was the activity of reference compound Kanamycin. The results re-
vealed that most of the synthesized compounds exhibited signifi-
cant antibacterial activities.
Out of the 20 synthetic new ester derivatives, compound 3n,
(E)-1-(2-methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-(biphe-
nyl-4-yl)acrylate, exhibited the most potent antibacterial activity
with MIC of 1.56, 3.13, 6.25, and 1.56 lg/mL against E. coli, P. aeru-
ginosa, B. subtilis and S. aureus, respectively, which was similar to
with IC50 of 2.5
activities of 3n. Compound 3o which with similar structure to 3n
exhibited good inhibitory with MIC of 6.7 M. Other tested com-
pounds displayed moderate inhibitory activity with MIC ranging
from 8.2 to 48.9 M. Compound 3k with p-substituted bromine
lM. This result supported the potent antibacterial
l
l
group on cinnamic acid component showed better E. coli FabH
inhibitory activity than compounds 3d and 3g with o-substituted
and m-substituted bromine group. Compound 3q with both
o-substituted and p-substituted chloro groups showed more
preferable inhibitory activity than compound 3j with single
p-substituted chloro group. In addition, the inhibitory activity of
compound 3r condensing by (E)-3-(naphthalen-1-yl)acrylic acid
and secnidazole was obviously decreased.
The results of E. coli FabH inhibitory activity of the test com-
pounds were corresponding to the structure relationships (SAR)
of their antibacterial activities. This demonstrated that the potent
antibacterial activities of the synthetic compounds were probably
correlated to their FabH inhibitory activities.
the broad-spectrum antibiotic Kanamycin with corresponding
MIC of 3.13, 3.13, 1.56, and 1.56 lg/mL. Structure–activity relation-
ships in these cinnamic acid secnidazole ester derivatives demon-
strated that compounds with substitution at the meta (3f–h) or
para (3i–p) position showed more potent activities than those with
substitution at the ortho position (3b–e). A comparison of the para
position substitution on benzene ring demonstrated that a para
halogen group (3i–k) may have more slightly improved antibacte-
rial activity and the potency order is F < Cl < Br, and other para sub-
stituents prepared (3n–q) had outstanding effect compared with
3a, especially compound 3n showed the best biological activity
2.3. Molecular docking study of synthetic compounds and
with MIC of 1.56
lg/mL against E. coli, which was superior to the
binding model of compound 3n and E. coli FabH
The binding affinity was evaluated by binding energy and inhib-
itory constant. Molecular docking of all the synthesized com-
pounds and E. coli FabH was performed on the binding model
based on the E. coli FabH-CoA complex structure (1HNJ.pdb).21
The FabH active site generally contains a catalytic triad tunnel con-
sisting of Cys-His-Asn, which is conserved in various bacteria. This
catalytic triad plays an important role in the regulation of chain
elongation and substrate binding. Since the alkyl chain of CoA is
broken by Cys of the catalytic triad of FabH, interactions between
Cys and substrate appear to play an important role in substrate
Table 2
Antibacterial activity of synthetic compounds
Compounds
Minimum inhibitory concentrations (
Gram-negative Gram-positive
P. aeruginosa
ATCC35218 ATCC13525
lg/mL)
E. coli
B. subtilis
ATCC 6633
S. aureus
ATCC 6538
3a
3b
3c
>100
>100
50
>100
>100
25
>100
>100
50
>100
>100
50
3d
25
25
50
25
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
>100
3.13
12.5
>100
50
12.5
6.25
100
>100
1.56
3.13
6.25
3.13
25
>100
3.13
12.5
>100
50
25
6.25
50
>100
3.13
6.25
6.25
6.25
50
>100
6.25
25
>100
50
12.5
3.13
>100
50
6.25
6.25
12.5
6.25
25
>100
6.25
12.5
>100
100
25
3.13
>100
50
1.56
3.13
6.25
12.5
50
Table 3
E. coli FabH inhibitory activity of synthetic compounds
Compounds
E. coli FabH IC50
(lM)
Hemolysis LC (mg/mL)
3d
3f
3g
3j
3k
3n
3o
3p
40.1
8.2
27.5
34.3
15.6
2.5
6.7
19.8
9.4
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
3q
3r
48.9
28.5
43.4
3s
3t
50
50
3.13
50
50
3.13
50
>100
1.56
100
50
1.56
Secnidazole
Cinnamic acid
Kanamycin

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H.-J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 4513–4519
binding. Qiu et al. have refined three-dimensional structure of
E. coli FabH in the presence and absence of malonyl-CoA by X-ray
spectroscopy. Since malonyl moiety is degraded by E. coli FabH,
molecular docking studies for FabH and malonyl-CoA was carried
out to identify a plausible malonyl-binding model.21 They found
that in one of the binding modes appeared in the lower scored con-
formations, the malonyl carboxylate formed hydrogen bonds to the
backbone nitrogen of Phe 304.
The binding affinity of the synthesized compounds and E. coli
FabH is depicted in Table 4. As shown in Table 4, the results of
binding energy and inhibitory constant have the same trend. Struc-
ture–activity relationships in compounds 3a–t demonstrated that
compounds with different substituent group exhibit different
parameter. Among them, compound 3n with p-substituted phenyl
group showed minimum data, which suggested that this com-
pound was mostly easy to combine with E. coli FabH. The selected
pose of compound 3n had an estimated free energy of binding of
ꢀ20.2 kcal/mol and inhibitory constant of 1.56 fm. The estimated
free energy of other compounds (3a–m, 3o–t) are ranging from
ꢀ19.1 to ꢀ15.78 kcal/mol and inhibitory constants are ranging
from 10.01 fm to 2.73 pm, respectively. This molecular docking re-
sult, along with the biological assay data, suggesting that com-
pound 3n is a potential inhibitor of E. coli FabH.
Fig. 1 (continued)
Compound 3n which displayed the most potent E. coli FabH
inhibitory activity was selected for further molecular docking
study. The binding model of compound 3n and E. coli FabH is de-
picted in Figure 1. It can be seen from Figure 1 that, in the binding
model of compound 3n and E. coli FabH, there is one hydrogen
bond. Asn247 froms hydrogen bond with the oxygen atom of the
nitro group of compound 3n. The result along with the data of
E. coli FabH inhibitory activity assay indicated that compound 3n
would be potential inhibitors of E. coli FabH with potent antibacte-
rial activity.
Table 4
Docking parameters of synthetic compounds
Compounds
Binding energy
D
Gb (kcal/mol)
Inhibitory constant
3a
3b
3c
3d
3e
3f
3g
3h
3i
ꢀ16.2
1.32 pm
ꢀ16.39
ꢀ17.99
ꢀ16.75
ꢀ16.03
ꢀ18.38
ꢀ17.28
ꢀ15.78
ꢀ17.43
ꢀ18.0
974.95 fm
65.24 fm
528.96 fm
1.79 pm
33.61 fm
214.08 fm
2.73 pm
168.56 fm
63.96 fm
79.87 fm
895.78 fm
609.79 fm
1.56 fm
10.01 fm
111.2 fm
26.14 fm
443.44 fm
525.58 fm
555.12 fm
3. Conclusions
A series of novel ester derivatives reacting by cinnamic acid and
secnidazole were synthesized and assayed for their antibacterial
activities against E. coli, P. aeruginosa, B. subtilis and S. aureus. Com-
pound 3n, (E)-1-(2-methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl
3-(biphenyl-4-yl)acrylate showed the most potent antibacterial
3j
3k
3l
ꢀ17.87
ꢀ16.44
ꢀ16.66
ꢀ20.2
3m
3n
3o
3p
3q
3r
3s
3t
ꢀ19.1
activity with MIC of 1.56–6.25
strains and exhibited the most potent E. coli FabH inhibitory activ-
ity with IC50 of 2.5 M, which was compared with the positive con-
lg/mL against the test bacterial
ꢀ17.67
ꢀ18.53
ꢀ16.85
ꢀ16.75
ꢀ16.72
l
trol Kanamycin. Docking simulation was performed to position
compound 3n into the E. coli FabH active site to determine the
probable binding conformation. Analysis of the compound 3n’s
binding conformation in active site displayed the compound 3n
was stabilized by hydrogen bonding interactions with Asn247.
Antibacterial assay results also showed that these cinnamic acid
secnidazole ester derivatives had the potential to be developed
for antibacterial agents against E. coli. Particularly, compound 3n
has demonstrated significant E. coli FabH inhibitory activity as a
potential antibacterial agent.
4. Experiments
4.1. Materials and measurements
All chemicals and reagents used in current study were analyti-
cal grade. All the 1H NMR spectra were recorded on a Bruker DPX
300 model Spectrometer in DMSO–d6 and chemical shifts were re-
ported in ppm (d). ESI-MS spectra were recorded on a Mariner Sys-
tem 5304 Mass spectrometer. Elemental analyses were performed
on a CHN-O-Rapid instrument. TLC was performed on the glass-
backed silica gel sheets (silica gel 60 Å GF254) and visualized in
UV light (254 nm).
Figure 1. (A) Binding model of compound 3n (colored in green) into E. coli FabH. H-
bond is shown as dotted green lines. Asn247 forms hydrogen bond with the oxygen
atom of the nitro group of compound 3n. (B) 3D model of the interaction between
compound 3n and the colchicine binding site. The protein is represented by
molecular surface. Compound 3n is depicted by sticks and balls.

H.-J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 4513–4519
4517
4.2. General procedure for synthesis of cinnamic acids
1H), 3.84 (s, 3H), 4.03–4.17 (m, 2H), 6.75 (d, J = 15.36 Hz, 1H), 6.92–
6.96 (m, 2H), 7.02 (s, 1H), 7.10 (d, J = 7.68 Hz, 1H), 7.28–7.34 (m,
1H), 7.66 (d, J = 15.36 Hz, 1H). ESI-MS: 346.1 (C17H20N3O5,
[M+H]+). Anal. Calcd for C17H19N3O5: C, 59.12; H, 5.55; N, 12.17.
Found: C, 58.81; H, 5.75; N, 11.95.
A mixture of aromatic aldehydes (3.2 mmol), malonic acid
(3.87 mmol), piperidine (0.387 mmol) was dissolved in pyridine
and stirred on 80–90 °C for 24 h. The pyridine was removed at
the vacuum. The reaction mixture was poured in water and
washed with HCl, the precipitate was filtered and washed with
hexane about three times, and dried under vacuum to afford the
cinnamic acids (Scheme 1)
4.3.6. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(3-fluorophenyl)acrylate (3f)
White powder, yield 66.2%, mp: 133–136 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.41 (d, J = 7.09 Hz, 3H), 2.42 (s, 3H), 4.13–4.18 (m,
1H), 4.34–4.36 (m, 1H), 4.61–4.66 (m, 1H), 6.56 (d, J = 15.36 Hz,
1H), 7.24–7.26 (m, 1H), 7.39 (d, J = 8.24 Hz, 1H), 7.56 (t,
J = 6.93 Hz, 2H), 7.71 (s, 1H), 7.97 (s, 1H). ESI-MS: 334.1
(C16H17FN3O4, [M+H]+). Anal. Calcd for C16H16FN3O4: C, 57.65; H,
4.84; N, 12.61. Found: C, 58.05; H, 4.62; N, 12.34.
4.3. General procedure for synthesis of cinnamic acid
secnidazole ester derivatives
Equimolar amount of secnidazole (1.0 mmol) and cinnamic
acid (1.0 mmol) were dissolved in dichloromethane, DCC
(1.5 mmol) and DMAP (0.5 mmol) as catalyst and stirred at 50–
60 °C for 24 h. The reaction mixture was extracted with ethyl ace-
tate and saturated sodium bicarbonate, respectively. Then, the or-
ganic layer was collected and crystallized to get the product
(Scheme 1).
4.3.7. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(3-bromophenyl)acrylate (3g)
Light yellow powder, yield 67.2%, mp: 102–104 °C, 1H NMR
(300 MHz, CDCl3, d ppm): 1.43 (d, J = 6.57 Hz, 3H), 2.54 (s, 3H),
4.09–4.16 (m, 1H), 4.32–4.40 (m, 1H), 4.63–4.69 (m, 1H), 6.27
(d, J = 15.90 Hz, 1H), 7.26 (t, J = 7.86 Hz, 1H), 7.41 (d,
J = 7.68 Hz, 1H), 7.51 (t, J = 7.97 Hz, 2H), 7.64 (s, 1H), 7.94 (s,
1H). ESI-MS: 394.0 (C16H17BrN3O4, [M+H]+). Anal. Calcd for
4.3.1. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl
cinnamate (3a)
Light grey powder, yield 60.9%, mp: 136–139 °C, 1H NMR
(300 MHz, CDCl3, d ppm): 1.42 (d, J = 4.62 Hz, 3H), 2.49 (s, 3H),
3.76–3.78 (m, 1H), 4.10–4.14 (m, 2H), 6.75 (d, J = 9.24 Hz, 1H),
7.02 (s, 1H), 7.37–7.39 (m, 3H), 7.48–7.50 (m, 2H), 7.67 (s,
1H). ESI-MS: 316.1 (C16H18N3O4, [M+H]+). Anal. Calcd for
C16H16BrN3O4: C, 48.75; H, 4.09; N, 10.66. Found: C, 48.89; H,
3.98; N, 11.12.
4.3.8. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(3-methoxyphenyl)acrylate (3h)
C16H17N3O4: C, 60.94; H, 5.43; N, 13.33. Found: C, 60.67; H,
5.56; N, 13.45.
White powder, yield 72.9%, mp: 148–151 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.42 (d, J = 6.66 Hz, 3H), 2.40 (s, 3H), 3.82 (s, 3H),
4.04 (d, J = 7.50 Hz, 2H), 4.12–4.16 (m, 1H), 6.73 (d, J = 15.39 Hz,
1H), 6.91–6.94 (m, 2H), 7.00 (s, 1H), 7.09 (d, J = 7.50 Hz, 1H),
7.26–7.33 (m, 1H), 7.64 (d, J = 15.36 Hz, 1H). ESI-MS: 346.1
(C17H20N3O5, [M+H]+). Anal. Calcd for C17H19N3O5: C, 59.12; H,
5.55; N, 12.17. Found: C, 59.33; H, 4.98; N, 12.52.
4.3.2. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(2-fluorophenyl)acrylate (3b)
White powder, yield 65.6%, mp: 106–107 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.42 (d, J = 4.62 Hz, 3H), 2.54 (s, 3H), 4.29–4.37 (m,
1H), 4.64–4.71 (m, 1H), 5.38–5.43 (m, 1H), 6.39 (d, J = 16.08 Hz,
1H), 7.08–7.19 (m, 2H), 7.34–7.41 (m, 1H), 7.49 (t, J = 7.50 Hz,
1H), 7.71 (d, J = 16.26 Hz, 1H), 7.93 (s, 1H). ESI-MS: 334.1
(C16H17FN3O4, [M+H]+). Anal. Calcd for C16H16FN3O4: C, 57.65; H,
4.84; N, 12.61. Found: C, 57.67; H, 4.87; N, 12.77.
4.3.9. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(4-fluorophenyl)acrylate (3i)
White powder, yield 66.7%, mp: 145–147 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.39 (d, J = 8.69 Hz, 3H), 2.04 (s, 3H), 3.48 (s, 1H),
3.71–3.81 (m, 1H), 4.08–4.15 (m, 1H), 6.66 (d, J = 15.36 Hz, 1H),
6.95 (s, 1H), 7.07 (t, J = 8.68 Hz, 2H), 7.45–7.49 (m, 2H), 7.64 (d,
J = 15.18 Hz, 1H). ESI-MS: 334.1 (C16H17FN3O4, [M+H]+). Anal. Calcd
for C16H16FN3O4: C, 57.65; H, 4.84; N, 12.61. Found: C, 57.23; H,
5.01; N, 12.49.
4.3.3. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(2-chlorophenyl)acrylate (3c)
White powder, yield 61.1%, mp: 125–127 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.44 (d, J = 6.42 Hz, 3H), 2.55 (s, 3H), 4.30–4.38 (m,
1H), 4.64–4.70 (m, 1H), 5.38–5.43 (m, 1H), 6.27 (d, J = 16.08 Hz,
1H), 7.25–7.35 (m, 2H), 7.40–7.43 (m, 1H), 7.55–7.58 (m, 1H),
7.93 (s, 1H), 8.00 (d, J = 16.08 Hz, 1H). ESI-MS: 350.1
(C16H17ClN3O4, [M+H]+). Anal. Calcd for C16H16ClN3O4: C, 54.94;
H, 4.61; N, 12.01. Found: C, 54.69; H, 4.81; N, 12.45.
4.3.10. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(4-chlorophenyl)acrylate (3j)
White powder, yield 56.1%, mp: 217–219 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.43 (d, J = 8.23 Hz, 3H), 1.92 (s, 3H), 3.36 (s, 1H),
3.71–3.78 (m, 1H), 4.08–4.15 (m, 1H), 6.66 (d, J = 15.36 Hz, 1H),
6.84 (s, 1H), 7.35 (d, J = 8.58 Hz, 2H), 7.41 (d, J = 8.61 Hz, 2H),
7.62 (d, J = 15.39 Hz, 1H). ESI-MS: 350.1 (C16H17ClN3O4, [M+H]+).
Anal. Calcd for C16H16ClN3O4: C, 54.94; H, 4.61; N, 12.01. Found:
C, 54.89; H, 4.81; N, 12.04.
4.3.4. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(2-bromophenyl)acrylate (3d)
White powder, yield 57.0%, mp: 112–115 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.44 (d, J = 6.39 Hz, 3H), 2.55 (s, 3H), 4.09 (d,
J = 8.40 Hz, 1H), 4.30–4.38 (m, 1H), 4.64–4.70 (m, 1H), 5.36–5.44
(m, 1H), 6.23 (d, J = 15.99 Hz, 1H), 7.21–7.26 (m, 1H), 7.33 (t,
J = 7.13 Hz, 1H), 7.54–7.57 (m, 1H), 7.59–7.62 (m, 1H), 7.93 (s,
1H). ESI-MS: 394.0 (C16H17BrN3O4, [M+H]+). Anal. Calcd for
C16H16BrN3O4: C, 48.75; H, 4.09; N, 10.66. Found: C, 48.61; H,
4.51; N, 11.15.
4.3.11. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(4-bromophenyl)acrylate (3k)
White powder, yield 62.1%, mp: 220–222 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.40 (d, J = 7.98 Hz, 3H), 2.05 (s, 3H), 3.74–3.76 (m,
1H), 4.08–4.13 (m, 2H), 6.54 (d, J = 15.00 Hz, 1H), 6.80 (s, 1H), 7.01–
7.06 (m, 1H), 7.23–7.26 (m, 2H), 7.62 (d, J = 5.13 Hz, 1H), 7.80 (d,
J = 15.00 Hz, 1H). ESI-MS: 394.0 (C16H17BrN3O4, [M+H]+). Anal.
Calcd for C16H16BrN3O4: C, 48.75; H, 4.09; N, 10.66. Found: C,
48.87; H, 4.31; N, 10.49.
4.3.5. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(2-methoxyphenyl)acrylate (3e)
White powder, yield 59.2%, mp: 158–162 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.59 (d, J = 7.21 Hz, 3H), 1.98 (s, 3H), 3.76–3.80 (m,

4518
H.-J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 4513–4519
4.3.12. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
p-tolylacrylate (3l)
J = 7.50 Hz, 2H), 8.19 (d, J = 7.50 Hz, 1H), 8.51 (d, J = 15.18 Hz,
1H). ESI-MS: 366.1 (C20H20N3O4, [M+H]+). Anal. Calcd for
White powder, yield 62.9%, mp: 194–195 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.42 (d, J = 8.12 Hz, 3H), 2.03 (s, 3H), 2.37 (s, 3H),
3.44–3.51 (m, 1H), 3.70–3.76 (m, 1H), 4.07–4.1 (m, 1H) 6.70 (d,
J = 15.36 Hz, 1H), 7.08 (s, 1H), 7.18 (d, J = 7.89 Hz, 2H), 7.38 (d,
J=8.22 Hz, 2H), 7.65 (d, J = 15.36 Hz, 1H). ESI-MS: 330.1
(C17H20N3O4, [M+H]+). Anal. Calcd for C17H19N3O4: C, 62.00; H,
5.81; N, 12.76. Found: C, 61.87; H, 5.36; N, 12.49.
C20H19N3O4: C, 65.74; H, 5.24; N, 11.50. Found: C, 65.53; H, 5.37;
N, 11.85.
4.3.19. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(naphthalen-2-yl)acrylate (3s)
White powder, yield 63.4%, mp: 204–206 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.46 (d, J=9.04 Hz, 3H), 2.35 (s, 3H), 3.43–3.49 (m,
1H), 3.71–3.80 (m, 1H), 4.00–4.15 (m, 1H), 6.69 (s, 1H), 6.74 (s,
1H), 6.80–6.82 (m, 2H), 7.26 (s, 1H), 7.34 (d, J = 8.58 Hz, 2H), 7.51
(d, J = 8.40 Hz, 2H), 7.60 (d, J = 15.36 Hz, 1H). ESI-MS: 366.1
(C20H20N3O4, [M+H]+). Anal. Calcd for C20H19N3O4: C, 65.74; H,
5.24; N, 11.50. Found: C, 65.68%; H, 5.10; N, 11.97.
4.3.13. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(4-methoxyphenyl)acrylate (3m)
White powder, yield 67.8%, mp: 146–148 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.41 (d, J = 6.79 Hz, 3H), 2.33 (s, 3H), 3.48 (s, 3H),
3.72–3.74 (m, 1H), 4.09–4.13 (m, 2H), 6.54 (d, J = 15.00 Hz, 1H),
6.80–6.91 (m, 1H), 7.01–7.06 (m, 1H), 7.23–7.26 (m, 2H), 7.35 (d,
J = 5.13 Hz, 1H), 7.80 (d, J = 15.00 Hz, 1H). ESI-MS: 346.1
(C17H20N3O5, [M+H]+). Anal. Calcd for C17H19N3O5: C, 59.12; H,
5.55; N, 12.17. Found: C, 59.31; H, 5.30; N, 11.79.
4.3.20. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(thiophen-2-yl)acrylate (3t)
Light grey powder, yield 72.9%, mp: 164–168 °C, 1H NMR
(300 MHz, CDCl3, d ppm): 1.40 (d, J = 6.79 Hz, 3H), 2.39 (s, 3H),
3.77–3.80 (m, 2H), 4.12–4.20 (m, 1H), 6.86 (d, J = 15.36 Hz, 1H),
6.96 (s, 1H), 7.50–7.53 (m, 1H), 7.60–7.63 (m, 1H), 7.81–7.87 (m,
1H), 7.91 (s, 1H). ESI-MS: 322.1 (C14H16N3O4S, [M+H]+). Anal. Calcd
for C14H15N3O4S: C, 52.33; H, 4.70; N, 13.08. Found: C, 51.96; H,
4.89; N, 12.83.
4.3.14. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(biphenyl-4-yl)acrylate (3n)
White powder, yield 58.7%, mp: 221–223 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.42 (d, J = 7.99 Hz, 3H), 2.09 (s, 3H), 3.06 (s, 1H),
3.76–3.78 (m, 1H), 4.10–4.17 (m, 1H), 6.79 (d, J = 15.36 Hz, 1H),
7.03 (s, 1H), 7.35–7.39 (m, 1H), 7.46 (t, J = 7.41 Hz, 2H), 7.55–
7.64 (m, 6H), 7.72 (d, J = 15.36 Hz, 1H). ESI-MS: 392.1
(C22H22N3O4, [M+H]+). Anal. Calcd for C22H21N3O4: C, 67.51; H,
5.41; N, 10.74. Found: C, 67.31; H, 5.20; N, 10.99.
4.4. Antimicrobial activity
The antibacterial activity of the synthesized compounds was
tested against B. subtilis, E. coli, P. aeruginosa and S. aureus using
MH medium (Mueller–Hinton medium: casein hydrolysate
17.5 g, soluble starch 1.5 g, beef extract 1000 mL). The MICs (min-
imum inhibitory concentrations) of the test compounds were
determined by a colorimetric method using the dye MTT (3-(4,5-
dimethylth-iazol-2-yl)-2,5-diphenyl trtrazoliumbromide). A stock
4.3.15. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(4-(benzyloxy)phenyl)acrylate (3o)
White powder, yield 53.36%, mp: 185–188 °C, 1H NMR
(300 MHz, CDCl3, d ppm): 1.40 (d, J = 8.05 Hz, 3H), 2.35 (s, 3H),
2.52 (s, 1H), 3.74–3.76 (m, 1H), 4.06–4.14 (m, 1H), 5.10 (s, 2H),
6.62 (d, J = 15.36 Hz, 1H), 6.91–7.00 (m, 2H), 7.13–7.15 (m, 1H),
7.31–7.45 (m, 7H), 7.64 (d, J = 15.36 Hz, 1H). ESI-MS: 422.2
(C23H24N3O5, [M+H]+). Anal. Calcd for C23H23N3O5: C, 65.55; H,
5.50; N, 9.97. Found: C, 65.36; H, 5.78; N, 9.65.
solution of the synthesized compound (100 lg/mL) in DMSO was
prepared and graded quantities of the test compounds were incor-
porated in specified quantity of sterilized liquid MH medium. A
specified quantity of the medium containing the compound was
poured into microtitration plates. Suspension of the microorgan-
ism was prepared to contain approximately 105 cfu/mL and ap-
plied to microtitration plates with serially diluted compounds in
DMSO to be tested and incubated at 37 °C for 24 h. After the MICs
were visually determined on each of the microtitration plates,
4.3.16. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(4-(dimethylamino)phenyl)acrylate (3p)
Light grey powder, yield 57.1%, mp: 122–123 °C, 1H NMR
(300 MHz, CDCl3, d ppm): 1.40 (d, J = 3.84 Hz, 3H), 2.53 (s, 3H),
3.02 (s, 6H), 4.32–4.36 (m, 1H), 4.62–4.65 (m, 1H), 5.36 (t,
J = 3.71 Hz, 1H), 6.04 (d, J = 9.42 Hz, 1H), 6.65 (d, J = 5.22 Hz, 2H),
7.37 (d, J = 5.22 Hz, 2H), 7.52 (d, J = 9.42 Hz, 1H), 7.92 (s, 1H). ESI-
MS: 359.1 (C18H23N4O4, [M+H]+). Anal. Calcd for C18H22N4O4: C,
60.32; H, 6.19; N, 15.63. Found: C, 60.54; H, 6.10; N, 15.39.
50 lL of PBS (phosphate buffered saline 0.01 mol/L, pH 7.4, Na2H-
Å
PO4 12H2O 2.9 g, KH2PO4 0.2 g, NaCl 8.0 g, KCl 0.2 g, distilled water
1000 mL) containing 2 mg of MTT/mL was added to each well.
Incubation was continued at room temperature for 4–5 h. The con-
tent of each well was removed, and 100 lL of isopropanol contain-
ing 5% 1 mol/L HCl was added to extract the dye. After 12 h of
incubation at room temperature, the optical density (OD) was
measured with a microplate reader at 550 nm. The observed MICs
are presented in Table 2.
4.3.17. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(2,4-dichlorophenyl)acrylate (3q)
White powder, yield 56.9%, mp: 165–169 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.43 (d, J = 8.44 Hz, 3H), 2.56 (s, 3H), 3.47–3.51 (m,
1H), 3.73–3.75 (m, 1H), 4.05–4.12 (m, 1H), 6.72 (d, J = 9.24 Hz, 1H),
6.96 (s, 1H), 7.23–7.26 (m, 1H), 7.40–7.46 (m, 2H), 7.94 (d,
J = 9.24 Hz, 1H). ESI-MS: 384.0 (C16H16Cl2N3O4, [M+H]+). Anal.
Calcd for C16H15Cl2N3O4: C, 50.02; H, 3.94; N, 10.94. Found: C,
50.43; H, 4.10; N, 10.77.
4.5. E. coli FabH purification and activity assay
Full-length E. coli acyl carrier protein (ACP), acyl carrier protein
synthase (ACPS), and b-ketoacyl-ACP synthase III (FabH) were indi-
vidually cloned into pET expression vectors with an N-terminal
His-tag (ACP, ACPS in pET19; FabH in pET28).
All proteins were expressed in E. coli strain BL21 (DE3).
Transformed cells were grown on Luria–Bertani (LB) agar plates
supplemented with Kanamycin (30 lg/mL). Sodium dodecyl sul-
fatepolyacrylamide gel electrophoresis (SDS–PAGE) analysis was
used to screen colonies for overexpression of proteins. One such
positive colony was used to inoculate 10 mL of LB medium with
4.3.18. (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 3-
(naphthalen-1-yl)acrylate (3r)
White powder, yield 68.7%, mp: 176–179 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.42 (d, J = 7.21 Hz, 3H), 2.12 (s, 3H), 3.72–3.81 (m,
2H), 4.11–4.20 (m, 1H), 6.83 (d, J = 15.18 Hz, 1H), 7.19 (s, 1H), 7.42–
7.52 (m, 1H), 7.52–7.58 (m, 2H), 7.67 (d, J = 7.29 Hz, 1H), 7.87 (d,
30 lg/mL of Kanamycin and grown over night at 37 °C, 1 mL of

H.-J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 4513–4519
4519
which was used to inoculate 100 mL LB medium supplemented
with 30 g/mL of Kanamycin. The culture was shaken for 4 h at
37 °C, and then induced with 0.5 mM isopropyl b- -thiogalactopy-
AutoDock 4.0 was employed for all docking calculations.22,23
The AutoDockTools program was used to generate the docking in-
put files. In all docking a grid box size of 48 ꢁ 48 ꢁ 48 points in x, y,
l
D
ranoside (IPTG). The culture was grown for 4 h, and harvested by
and z directions was built in the catalytic site of the protein. A grid
0
centrifugation (30 min at 15000 rpm).
spacing of 0.375 ÅA (approximately one forth of the length of car-
bon–carbon covalent bond) and a distances-dependent function
of the dielectric constant were used for the calculation of the ener-
getic map. Ten runs were generated by using Lamarckian genetic
algorithm searches. Default settings were used with an initial pop-
ulation of 50 randomly placed individuals, a maximum number of
Harvested cells containing His-tagged ACP, ACPS, and FabHs
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 superna-
tant was applied to a Ni-NTA agarose column, washed, and eluted
using a 5–500 mM imidazole gradient over 20 column volumes.
Eluted protein was dialyzed against 20 mM Tris, pH 7.6, 1 mM
DTT, and 100 mM NaCl. Purified FabHs were concentrated up to
2 mg/mL and stored at ꢀ80 °C in 20 mM Tris, pH 7.6, 100 mM NaCl,
1 mM DTT, and 20% glycerol for enzymatic assays.
2.5 ꢁ 106 energy evaluations, and
a maximum number of
2.7 ꢁ 104 generations. A mutation rate of 0.02 and a crossover rate
0
of 0.8 were chosen. Results differing by less than 0.5 ÅA in positional
root–mean–square deviation (RMSD) were clustered together and
the results of the most favorable free energy of binding were se-
lected as the resultant complex structures.
Purified ACP contains the apo-form that needs to be converted
into the holo-form. The conversion reaction is catalyzed by ACP
synthase (ACPS). In the final volume of 50 mL, 50 mg ACP, 50 mM
Tris, 2 mM DTT, 10 mM MgCl2, 600
l
M CoA, and 0.2
l
M ACPS
Acknowledgments
was incubated for 1 h at 37 °C. The pH of the reaction was then ad-
justed to approximately 7.0 using 1 M potassium phosphate. Holo-
ACP was purified by fractionation of the reaction mixture by Source
Q-15 ion exchange chromatography using a 0–500 mM NaCl gradi-
ent over two column volumes.
This work was supported by Jiangsu National Science Founda-
tion (No. BK2009239) and the Fundamental Research Funds for
the Central Universities (Nos. 1092020804 & 1106020824).
In a final 20
0.5 mM DTT, 0.25 mM MgCl2, and 2.5
with 1 nM FabH, and H2O was added to 15
tion, a 2 L mixture of 25 M acetyl-CoA and 0.75
CoA was added for FabH reaction for 25 min. The reaction was
stopped by adding 20 L of ice-cold 50% TCA, incubating for
5 min on ice, and centrifuging to pellet the protein. The pellet
l
L reaction, 20 mM Na2HPO4/NaH2PO4, pH 7.0,
M holo-ACP were mixed
L. After 1 min incuba-
Ci [3H] acetyl-
References and notes
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was washed with 10% ice-cold TCA and resuspended with 5 lL
of 0.5 M NaOH. The incorporation of the 3H signal in the final
product was read by liquid scintillation. When determining the
inhibition constant (IC50), inhibitors were added from a concen-
trated DMSO stock such that the final concentration of DMSO
did not exceed 2%.
4.6. Molecular docking modeling
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to carbon atoms. For macromolecules, generated pdbqt files were
saved.
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(PM3) level, and saved in mol2 format with the aid of the molecu-
lar modeling program Spartan (Wavefunction Inc.). These partial
charges of Mol2 files were further modified by using the ADT pack-
age (version1.4.6) so that the charges of the nonpolar hydrogens
atoms assigned to the atom to which the hydrogen is attached.
The resulting files were saved as pdbqt files.
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