Synthesis, antibacterial activities and molecular docking studies of Schiff bases derived from N-(2/4-benzaldehyde-amino) phenyl-N′-phenyl-thiourea

Article abstract of DOI:10.1016/j.bmc.2011.06.077

A series of novel Schiff base 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 3v 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 4.3 μM. Docking simulation was performed to position compound 3v into the E. coli FabH active site to determine the probable binding conformation.

Full text of DOI:10.1016/j.bmc.2011.06.077

Bioorganic & Medicinal Chemistry 19 (2011) 5708–5715
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, antibacterial activities and molecular docking studies of Schiff
bases derived from N-(2/4-benzaldehyde-amino)phenyl-N0-phenyl-thiourea
⇑
⇑
Hong-Jia Zhang, Xuan Qin, Kai Liu, Di-Di Zhu, Xiao-Ming Wang , 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 novel Schiff base derivatives have been designed and synthesized, and their biological activ-
ities 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. Com-
pounds with potent antibacterial activities were tested for their E. coli FabH inhibitory activity. Com-
Received 20 April 2011
Revised 24 June 2011
Accepted 25 June 2011
Available online 1 July 2011
pound 3v showed the most potent antibacterial activity with MIC of 1.56–6.25
tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with IC50 of
4.3 M. Docking simulation was performed to position compound 3v into the E. coli FabH active site to
determine the probable binding conformation.
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
lg/mL against the
Keywords:
Schiff base
Antibacterial activity
Structure–activity relationship
FabH inhibitors
l
1. Introduction
while there are no significantly homologous proteins in humans.
Importantly, the residues that comprise the active site are essen-
tially invariant in various bacterial FabH molecules.10–12 These
attribute suggest that small molecule inhibitors of FabH enzymatic
activity could be potential development candidates leading to
selective, nontoxic and broad-spectrum antibacterials. Because of
this, various kinds of compounds were screened by enzymatic as-
says to generate leads that were co-crystallized with various path-
ogenic FabH proteins and subsequently optimized using structure
guided drug design methods.13–17
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 or the
expansion of bioactivity of the previous drugs is a very important
task.1 Therefore, the research has been focused toward develop-
ment of new antibacterial agents with novel target so as to surpass
the problem of acquired resistance.
A promising target is the fatty acid synthase (FAS) pathway in
bacteria. Fatty acid biosynthesis (FAB) is an essential metabolic
process for prokaryotic organisms and is required for cell viability
and growth.2 b-Ketoacyl-acyl carrier protein (ACP) synthase III,
also known as FabH or KAS III, plays an essential and regulatory
role in bacterial FAB.3,4 The enzyme initiates the fatty acid elonga-
tion cycles,5,6 and is involved in the feedback regulation of the bio-
synthetic pathway via product inhibition.7 FabH catalyzes the
condensation reaction between a CoA-attached acetyl group and
an ACP-attached malonyl group, yielding acetoacetyl-ACP as its fi-
nal product (Fig. 1). Two other condensing enzymes, FabB (KASI)
and FabF (KASII), perform the chain elongation reactions in subse-
quent cycles leading to longchain acyl ACP products,8,9,3 while
FabB and FabF are also condensing enzymes, FabH is structurally
distinct.
Some Schiff bases were reported to possess antibacterial, anti-
fungal and antitumor activities.18 Lots of researchers studied the
synthesis, characterization and structure–activity relationship
(SAR) of Schiff bases.19–22 Kim and co-workers reported the
YKAs3003, a Schiff base condensed by 4-hydroxy salicylaldehyde
and cyclohexanamine as a potent inhibitor of Escherichia coli
(E. coli) FabH with antimicrobial activity.23 Further optimization of
this compound is required to improve its antimicrobial activity.
Thiourea has been used as purification agent for the effluent of
organic and inorganic, industrial, agricultural, and mining wastes.24
This compound and its related analogues have become the focus of
interest in recent past on account of their pharmacological activities,
such as antidiabetic and antituberculosis.25 Furthermore, thiourea
has been studied for the systematic control of tuberculosis as well
as anti-inflammatory activity during corrageenin induced edema
in rats, while thiazoles and their ester possess depressant activity
with central nervous system.26 Recently, a few compounds of thio-
urea(2-amino-1,3-thiazines) have shown bactericidal, fungicidal,
herbicidal, and algeacidal activities.27 Importantly, it is reported that
sulfur-urea-based Schiff base and its complexes exhibit excellent
FabH proteins from both Gram-positive and Gram-negative
bacteria are highly conserved at the sequence and structural level
⇑
Corresponding authors. 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.2011.06.077

H.-J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 5708–5715
5709
Figure 1. FabH-catalyzed ignition reaction of fatty acid biosynthesis.
antibacterial activity.28–30 As a result, we hypothesized that Schiff
base compounds containing thiourea skeleton may display potent
antibacterial activity. In this study, we designed and synthesized a
new series of Schiff bases derived from N-(2/4-benzaldehyde-ami-
no) phenyl-N0-phenyl-thiourea and studied their antibacterial activ-
ities and E. coli FabH inhibitory activities. Docking simulations were
performed using the X-ray crystallographic structure of the FabH of
E. coli in complex with the most potent inhibitor to explore the bind-
ing modes of these compounds at the active site.
(minimum inhibitory concentrations) of the compounds against
these bacteria were presented in Table 1. Also included was the
activity of reference compound kanamycin. The results revealed
that most of the synthesized compounds exhibited significant anti-
bacterial activities.
Out of the 22 synthetic new Schiff bases, compound 3v, (E)-1-
(4-(4-(benzyloxy)benzylideneamino)phenyl)-3-phenylthiourea,
exhibited the most potent antibacterial activity with MIC of 1.56,
3.13, 6.25 and 3.13 lg/mL against E. coli, P. aeruginosa, B. subtilis
and S. aureus, respectively, which was similar to the broad-spectrum
antibiotic Kanamycin B with corresponding MIC of 1.56, 3.13, 0.39
2. Results and discussion
2.1. Chemistry
and 3.13
lg/mL and Penicillin G with corresponding MIC of 3.13,
6.25, 1.56 and 6.25
lg/mL.
As shown in Table 1, compounds 3a–3g were condensed by o-
aniline and substituted salicylaldehydes and benzaldehydes, while
compounds 3h–3v were condensed by p-aniline and substituted
salicylaldehydes and benzaldehydes. Various substituents of alde-
hyde such as hydroxyl, halogen, methyl, methoxyl and nitro group
lead to the different antibacterial activities of these Schiff bases, as
well as the position of aniline. Based on the data obtained, we
found that compounds 3h–3v with p-aniline showed better anti-
bacterial activity than those that compounds 3a–3g with o-aniline.
Besides, compounds with halogen group on salicylaldehyde and
benzaldehyde component exhibited moderate activity with MIC
In the present study, 12 benzaldehydes and 4 salicylaldehydes
were subjected to react with 2 amines to prepare the correspond-
ing Schiff bases (Scheme 1).
2.2. Biological activity
2.2.1. Antibacterial activity
All the synthesized compounds were screened for antibacterial
activity against two Gram-negative bacterial strains:Escherichia
coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) and two
Gram-positive bacterial strains: Bacillus subtilis (B. subtilis) and
Staphylococcus aureus (S. aureus) by MTT method.31 The MICs
of 3.13 to more than 100 lg/mL. Among them, compounds 3c, 3j,
3n and 3r with chloro group on aldehyde component showed
H
N
S
NH2
NH
i
H2N
H2N
H
S
N
1
2
H
S
N
NH
N
NH
R4
R3
CHO
R1
R4
R3
ii
NH2
R1
H
N
S
R2
R2
3a-3g
H
S
N
NH
NH
R4
CHO
R1
iii
R4
R3
N
H2N
R3
R1
R2
R2
3h-3v
Scheme 1. General synthesis of N-(2/4-salicylaldehyde-amino) phenyl-N0-phenyl-thiourea and N-(2/4-benzaldehyde-amino) phenyl-N0-phenyl-thiourea derivatives (3a–3v).
Reagents and conditions: (i) PhNCS, EtOH, 20 °C, 1–2 h; (ii) EtOH, 80 °C, 2.5 h or toluene, p-toluenesulfonic acid, 80 °C, 3–4 h; (iii) EtOH, 80 °C, 2.5 h or toluene, p-
toluenesulfonic acid, 80 °C, 3–4 h.

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H.-J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 5708–5715
Table 1
Structure of N-(2/4-salicylaldehyde-amino) phenyl-N0-phenyl-thiourea and N-(2/4-benzaldehyde-amino) phenyl-N0-phenyl-thiourea derivatives (3a–3v)
H
N
H
N
S
S
NH
NH
N
R4
R3
R4
R3
N
R1
R1
R2
R2
3a-3g
3h-3v
Compound
R1
R2
R3
R4
3a
3b
3c
3d
3e
3f
3g
3h
3i
OH
OH
OH
OH
H
H
Br
H
H
H
H
H
H
Br
H
H
H
H
H
Br
H
H
H
H
H
Br
Cl
Br
H
H
H
H
Br
Cl
Br
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
Br
OCH3
H
H
H
H
H
H
Cl
H
H
F
Cl
Br
NO2
CH3
OCH2Ar
OH
OH
OH
OH
F
NO2
Cl
H
H
H
H
H
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
OCH3
H
H
H
H
H
H
H
3u
3v
H
H
better antibacterial activity with MIC of 3.13–25
lg/mL against the
The results of E. coli FabH inhibitory activity of the test com-
tested bacterial strains. However, compound 3e with p-substituted
chloro group lead to the great decrease of antibacterial activity.
These results demonstrated that hydroxyl group on aldehyde is
not conducive to the antibacterial activity of synthetic Schiff bases.
This can also be seen from the result compared with 3e and 3r, 3f
and 3s, respectively. Compounds 3m and 3t with nitro group
showed weak antibacterial activity.
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.
2.3. Binding model of compound 3v and E. coli FabH
Molecular docking of compound 3v and E. coli FabH was per-
formed on the binding model based on the E. coli FabH-CoA complex
structure (1HNJ.pdb).32 The FabH active site generally contains a
catalytic triad tunnel consisting 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, interac-
tions between Cys and substrate appear to play an important role in
substrate binding. Qiu et al. have refined three-dimensional struc-
ture 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 car-
ried out to identify a plausible malonyl-binding mode.32 They found
that in one of the binding modes appeared in the lower scored
conformations, the malonyl carboxylate formed hydrogen bonds
to the backbone nitrogen of Phe304.
2.2.2. E. coli FabH inhibitory activity
The E. coli FabH inhibitory potency of the synthetic Schiff bases
with potent antibacterial activities (3b, 3h, 3j, 3k, 3n, 3p, 3r, 3s, 3u
and 3v) was examined and the results are summarized in Table 3.
Most of the tested compounds displayed potent E. coli FabH inhibi-
tory. Among them, compound 3v condensing by 4-(benzyloxy)benz-
aldehyde and 1-(4-aminophenyl)-3-phenylthiourea showed the
most potent inhibitory with IC50 of 4.3 lM. This result supported
the potent antibacterial activities of 3v. It also indicated that com-
pounds 3c, 3h, 3j and 3k which were condensed by salicylaldehyde
and substituented amines showed moderate E. coli FabH inhibitory
activity with IC50 of 9.5–39.0 lM. Among them, compound 3c re-
acted with o-amine exhibited better inhibitory activity than those
reacted with p-amine. As shown in Table 2, compound 3p with
m-substituented displayed worst inhibitory activity. Compound
3k and 3s were found to be inactive against E. coli FabH. This indi-
cated that Schiff bases with bromine-substituted on aldehyde lead
to the decrease of E. coli FabH inhibitory activity.
The binding model of compound 3v and E. coli FabH is depicted
in Figure 2. It can be seen from Figure 2 that, in the binding model
of compound 3v and E. coli FabH, there is one hydrogen bond.

H.-J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 5708–5715
5711
Table 2
3. Conclusions
Antibacterial activity of synthetic compounds
Compounds
Minimum inhibitory concentrations (
l
g/mL)
A series of novel Schiff bases reacting by N-(2/4-benzaldehyde-
amino) phenyl-N0-phenyl-thiourea and substituted benzaldehyde
and salicylaldehyde were synthesized and assayed for their antibac-
terial activities against E. coli, P. fluorescence, B. subtilis and S. aureus.
Compound 3v, (E)-1-(4-(4-(benzyloxy)benzylideneamino)phenyl)-
3-phenylthiourea showed the most potent antibacterial activity
Gram-negative Gram-positive
E. coli
P. aeruginosa
B. subtilis
S. aureus
ATCC6538
ATCC35218
ATCC13525
ATCC6633
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
>100
>100
3.13
>100
>100
>100
>100
12.5
>100
6.25
25
>100
>100
12.5
>100
50
>100
>100
3.13
>100
>100
>100
>100
6.25
>100
6.25
25
>100
>100
12.5
>100
>100
50
>100
25
>100
12.5
50
>100
>100
6.25
>100
>100
>100
>100
6.25
>100
12.5
50
>100
25
12.5
>100
50
with MIC of 1.56–6.25
exhibited the most potent E. coli FabH inhibitory activity with IC50
of 4.3 M, which was compared with the positive control Kanamy-
lg/mL against the test bacterial strains and
l
cin B and Penicillin G. Docking simulation was performed to position
compound 3v into the E. coli FabH active site to determine the prob-
able binding conformation. Analysis of the compound 3v’s binding
conformation in active site displayed the compound 3v was stabi-
lized by hydrogen bonding interactions with THR81. Antibacterial
assay results also showed that these Schiff base derivatives had
the potential to be developed for antibacterial agents against
E. coli. Particularly, compound 3v has demonstrated significant
E. coli FabH inhibitory activity as a potential antibacterial agent.
>100
>100
25
>100
25
>100
50
12.5
>100
25
3q
50
50
50
25
3r
3s
6.25
25
3.13
25
12.5
50
6.25
50
4. Experiments
3t
25
50
50
50
3u
3v
12.5
1.56
1.56
3.13
25
25
25
4.1. Materials and measurements
3.13
3.13
6.25
6.25
0.39
1.56
3.13
3.13
6.25
Kanamycin B
Penicillin G
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). All the compounds gave satisfactory chemical
analyses ( 0.4%). 1H NMR and ESI-MS spectra were consistent with
the assigned structures.
Table 3
E. coli FabH inhibitory activity of synthetic compounds
Compounds
E. coli FabH IC50
(
lM)
Hemolysis LC (mg/mL)
3c
3h
3j
3k
3n
3p
3r
3s
3u
3v
9.5
28.7
12.8
39.0
30.4
48.1
15.6
40.8
25.9
4.3
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
4.2. General procedure for synthesis of N-(2/4-amino) phenyl-
N0-phenyl-thiourea
o-Diaminobenzene or p-diaminobenzene (1.85 mmol) was dis-
solved in ethanol (4 mL) and stirred 20 °C, a solution of phenyl iso-
thiocyanate (0.22 mL) and ethanol was dropwised into the reaction
mixture when the diaminobenzene was completely dissolved. The
reaction mixture was stirred until the solids formed largely.
The products were filtrated and washed carefully with EtOH; the
resulting N-(2/4-amino) phenyl-N0-phenyl-thiourea were purified
by crystallization from EtOH in refrigerator (Scheme 1).
4.3. General procedure for synthesis of N-(2/4-salicylaldehyde-
amino) phenyl-N0-phenyl-thiourea derivatives
N-(2/4-Amino) phenyl-N0-phenyl-thiourea (2.5 mmol) was dis-
solved in ethanol (10 mL) and stirred 80 °C, salicylaldehyde
(3.0 mmol) was dropwised into the solution when the solid was
completely dissolved. The reaction mixture was stirred for 2.5 h.
The products were filtrated timely and dried by vacuum (Scheme
1).
4.3.1. (E)-1-(2-(2-hydroxybenzylideneamino)phenyl)-3-phenylt
hiourea (3a)
Figure 2. Binding model of compound 3v (colored in green) into E. coli FabH. H-
bond is shown as dotted green lines. THR81 forms hydrogen bond with the oxygen
atom of compound 3v.
Yellow crystals, yield 81%, mp: 216–218 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 6.95–6.99 (m, 2H), 7.12 (t, J = 7.40 Hz, 1H), 7.28–
7.33 (m, 4H), 7.37–7.46 (m, 4H), 7.63–7.66 (m, 2H), 8.88 (s, 1H),
9.49 (s, 1H), 9.90 (s, 1H), 12.66 (s, 1H, OH). ESI-MS: 348.1
(C20H18N3OS, [M+H]+). Anal. Calcd for C20H17N3OS: C, 69.14; H,
4.93; N, 12.09. Found: C, 69.42; H, 5.21; N, 12.15.
THR81 forms hydrogen bond with the oxygen atom of compound
3v. The result along with the data of E. coli FabH inhibitory activity
assay indicated that compound 3v would be potential inhibitors of
E. coli FabH with potent antibacterial activity.

5712
H.-J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 5708–5715
4.3.2. (E)-1-(2-(3,5-Dibromo-2-hydroxybenzylideneamino)phe
nyl)-3-phenylthiourea (3b)
1H), 8.95 (s, 1H), 9.87 (d, J = 12.06 Hz, 2H), 13.13 (s, 1H, OH). ESI-
MS: 426.0 (C20H17BrN3OS, [M+H]+). Anal. Calcd for C20H16BrN3OS:
C, 56.34; H, 3.78; N, 9.86. Found: C, 59.42; H, 3.51; N, 9.75.
Yellow crystals, yield 78%, mp: 173–174 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.13 (t, J = 7.32 Hz, 1H), 7.31 (t, J = 7.77 Hz, 2H),
7.35–7.41 (m, 2H), 7.47 (d, J = 8.04 Hz, 4H), 7.92 (dd, J1 = 12.42 Hz,
J2 = 12.24 Hz, 2H), 8.92 (s, 1H), 9.51 (s, 1H), 9.90 (s, 1H), 14.39 (s,
1H, OH). ESI-MS: 503.9 (C20H16Br2N3OS, [M+H]+). Anal. Calcd for
4.4. General procedure for synthesis of N-(2/4-benzaldehyde-
amino) phenyl-N0-phenyl-thiourea derivatives
C
20H15Br2N3OS: C, 47.55; H, 2.99; N, 8.32. Found: C, 47.73; H,
Equimolar amount of N-(2/4-amino) phenyl-N0-phenyl-thiourea
(1.63 mmol) and benzaldehyde (1.63 mmol) were dissolved in tol-
uene, and p-toluenesulfonic acid (0.175 mmol) as a catalyst and
stirred 80 °C for 3–4 h. The products were filtrated and washed
carefully with ice water and cool EtOH; the products were purified
by crystallization from EtOH in refrigerator (Scheme 1).
3.25; N, 8.61.
4.3.3. (E)-1-(2-(5-Chloro-2-hydroxybenzylideneamino)phenyl)-
3-phenylthiourea (3c)
Yellow crystals, yield 80%, mp: 190–192 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.00 (d, J = 8.79 Hz, 1H), 7.12 (t, J = 7.32 Hz, 1H),
7.28–7.34 (m, 5H), 7.42–7.46 (m, 3H), 7.69 (t, J = 4.45 Hz, 1H), 7.77
(d, J = 2.55 Hz, 1H), 8.86 (s, 1H), 9.45 (s, 1H), 9.91 (s, 1H), 12.54 (s,
1H, OH). ESI-MS: 382.1 (C20H17ClN3OS, [M+H]+). Anal. Calcd for
4.4.1. (E)-1-(2-(4-Chlorobenzylideneamino)phenyl)-3-phenyl
thiourea (3e)
Yellow crystals, yield 80%, mp: 154–155 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.18 (t, J = 7.59 Hz, 1H), 7.25 (d, J = 6.78 Hz, 1H),
7.31–7.42 (m, 4H), 7.46 (d, J = 8.04 Hz, 2H), 7.57 (d, J = 8.40 Hz, 2H),
7.80 (d, J = 8.61 Hz, 2H), 8.49 (d, J = 6.96 Hz, 1H), 8.72 (s, 1H), 9.46
(s, 1H), 10.23 (s, 1H). ESI-MS: 366.1 (C20H17ClN3S, [M+H]+). Anal.
Calcd for C20H16ClN3S: C, 65.65; H, 4.41; N, 11.48. Found: C,
65.95; H, 4.67; N, 11.66.
C20H16ClN3OS: C, 62.90; H, 4.22; N, 11.00. Found: C, 63.14; H,
4.33; N, 12.14.
4.3.4. (E)-1-(2-(5-Bromo-2-hydroxybenzylideneamino)phenyl)-
3-phenylthiourea (3d)
Yellow crystals, yield 83%, mp: 197–198 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 6.95 (d, J = 8.79 Hz, 1H), 7.12 (t, J = 7.32 Hz, 1H),
7.28–7.34 (m, 5H), 7.45 (d, J = 7.86 Hz, 2H), 7.55 (dd, J1 = 8.79 Hz,
J2 = 8.79 Hz, 1H), 7.67 (t, J = 4.57 Hz, 1H), 7.90 (d, J = 2.55 Hz, 1H),
8.85 (s, 1H), 9.45 (s, 1H), 9.90 (s, 1H), 12.58 (s, 1H, OH). ESI-MS:
426.0 (C20H17BrN3OS, [M+H]+). Anal. Calcd for C20H16BrN3OS: C,
56.34; H, 3.78; N, 9.86. Found: C, 56.61; H, 4.03; N, 10.14.
4.4.2. (E)-1-(2-(4-Bromobenzylideneamino)phenyl)-3-phenylth
iourea (3f)
Yellow crystals, yield 84%, mp: 153–155 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.17 (t, J = 7.58 Hz, 1H), 7.24–7.31 (m, 3H), 7.33–
7.41 (m, 3H), 7.46 (d, J = 8.04 Hz, 2H), 7.69–7.75 (m, 3H), 8.49 (d,
J = 8.04 Hz, 1H), 8.07 (s, 1H), 9.45 (s, 1H), 10.23 (s, 1H). ESI-MS:
410.0 (C20H17BrN3S, [M+H]+). Anal. Calcd for C20H16BrN3S: C,
58.54; H, 3.93; N, 10.24. Found: C, 58.82; H, 4.16; N, 10.53.
4.3.5. (E)-1-(4-(2-Hydroxybenzylideneamino)phenyl)-3-phenyl
thiourea (3h)
Yellow crystals, yield 76%, mp: 187–188 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 6.95–7.01 (m, 2H), 7.14 (t, J = 7.41 Hz, 1H), 7.34
(t, J = 7.88 Hz, 2H), 7.38–7.44 (m, 3H), 7.50 (d, J = 8.04 Hz, 2H), 7.64
(d, J = 7.68 Hz, 1H), 8.97 (s, 1H), 9.86 (d, J = 12.45 Hz, 2H), 13.16 (s,
1H, OH). ESI-MS: 348.1 (C20H18N3OS, [M+H]+). Anal. Calcd for
4.4.3. (E)-1-(2-(4-Methoxybenzylideneamino)phenyl)-3-phenyl
thiourea (3g)
Yellow crystals, yield 82%, mp: 156–157 °C,1H NMR (300 MHz,
DMSO-d6, d ppm): 3.87 (s, 3H, OCH3), 7.04 (d, J = 8.79 Hz, 2H),
7.15–7.32 (m, 4H), 7.39 (t, J = 7.77 Hz, 2H), 7.46 (d, J = 8.07 Hz,
2H), 7.72 (d, J = 8.58 Hz, 2H), 8.54 (d, J = 6.57 Hz, 1H), 8.62 (s,
1H), 9.47 (s, 1H), 10.23 (s, 1H). ESI-MS: 362.1 (C21H20N3OS,
[M+H]+). Anal. Calcd for C21H19N3OS: C, 69.78; H, 5.30; N, 11.63.
Found: C, 69.98; H, 5.52; N, 11.89.
C20H17N3OS: C, 69.14; H, 4.93; N, 12.09. Found: C, 69.38; H, 4.25;
N, 12.16.
4.3.6. (E)-1-(4-(3,5-Dibromo-2-hydroxybenzylideneamino)phen
yl)-3-phenylthiourea (3i)
Yellow crystals, yield 82%, mp: 286–289 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.14 (t, J = 7.41 Hz, 1H), 7.35 (t, J = 7.86 Hz, 1H),
7.45–7.51 (m, 4H), 7.64 (t, J = 8.79 Hz, 2H), 7.90 (dd, J1 = 17.37 Hz,
J2 = 17.37 Hz, 2H), 9.01 (s, 1H), 9.91 (d, J = 12.06 Hz, 2H), 14.74 (s,
1H, OH). ESI-MS: 503.9 (C20H16Br2N3OS, [M+H]+). Anal. Calcd for
4.4.4. (E)-1-(4-(2-Fluorobenzylideneamino)phenyl)-3-phenylt
hiourea (3l)
Yellow crystals, yield 79%, mp: 179–182 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.13 (t, J = 7.31 Hz, 1H), 7.31–7.35 (m, 6H), 7.48–
7.57 (m, 5H), 8.10 (t, J = 6.95 Hz, 1H), 8.80 (s, 1H), 9.84 (d,
J = 14.25 Hz, 2H). ESI-MS: 350.1 (C20H17FN3S, [M+H]+). Anal. Calcd
for C20H16FN3S: C, 68.75; H, 4.62; N, 12.03. Found: C, 68.94; H,
4.94; N, 12.27.
C20H15Br2N3OS: C, 47.55; H, 2.99; N, 8.32. Found: C, 47.69; H,
3.14; N, 8.58.
4.3.7. (E)-1-(4-(5-Chloro-2-hydroxybenzylideneamino)phenyl)-
3-phenylthiourea (3j)
Yellow crystals, yield 78%, mp: 206–209 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.00 (d, J = 8.76 Hz, 1H), 7.14 (t, J = 7.32 Hz, 1H),
7.34 (t, J = 7.86 Hz, 2H), 7.39–7.45 (m, 3H), 7.49 (d, J = 7.68 Hz, 2H),
7.60 (d, J = 8.61 Hz, 2H), 7.74 (d, J = 2.73 Hz, 1H), 8.95 (s, 1H), 9.87
(d, J = 12.24 Hz, 2H), 13.10 (s, 1H, OH). ESI-MS: 382.1
(C20H17ClN3OS, [M+H]+). Anal. Calcd for C20H16ClN3OS: C, 62.90;
H, 4.22; N, 11.00. Found: C, 63.18; H, 4.47; N, 11.25.
4.4.5. (E)-1-(4-(2-Nitrobenzylideneamino)phenyl)-3-phenylt
hiourea (3m)
Yellow crystals, yield 77%, mp: 165–166 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.13 (t, J = 7.96 Hz, 1H), 7.29–7.27 (m, 4H), 7.49
(d, J = 7.50 Hz, 2H), 7.57 (d, J = 8.79 Hz, 2H), 7.77 (t, J = 7.79 Hz, 1H),
7.88 (t, J = 7.50 Hz, 1H), 8.11 (d, J = 9.15 Hz, 1H), 8.19 (d,
J = 9.15 Hz,1H), 8.90 (s, 1H), 9.85 (d, J = 16.29 Hz, 2H). ESI-MS:
377.1 (C20H17N4O2S, [M+H]+). Anal. Calcd for C20H16N4O2S: C,
63.81; H, 4.28; N, 14.88. Found: C, 63.69; H, 4.47; N, 15.03.
4.3.8. (E)-1-(4-(5-Bromo-2-hydroxybenzylideneamino)phenyl)-
3-phenylthiourea (3k)
Yellow crystals, yield 77%, mp: 211–212 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 6.95 (d, J = 8.76 Hz, 1H), 7.14 (t, J = 7.32 Hz, 1H),
7.34 (t, J = 7.77 Hz, 2H), 7.41 (d, J = 8.79 Hz, 2H), 7.49 (d, J = 7.68 Hz,
2H), 7.52–7.56 (m, 1H), 7.60 (d, J = 8.61 Hz, 2H), 7.86 (d, J = 2.4 Hz,
4.4.6. (E)-1-(4-(2,4-Dichlorobenzylideneamino)phenyl)-3-pheny
lthiourea (3n)
Yellow crystals, yield 79%, mp: 184–185 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.14 (t, J = 7.32 Hz, 1H), 7.32–7.37 (m, 4H), 7.49

H.-J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 5708–5715
5713
(d, J = 7.86 Hz, 2H), 7.57 (d, J = 8.76 Hz, 3H), 7.79 (s, 1H), 8.17 (d,
J = 8.61 Hz, 1H), 8.86 (s, 1H), 9.85 (d, J = 16.98 Hz, 2H). ESI-MS:
400.0 (C20H16Cl2N3S, [M+H]+). Anal. Calcd for C20H15Cl2N3S: C,
60.00; H, 3.78; N, 10.50. Found: C, 60.34; H, 4.03; N, 10.68.
J = 8.25 Hz, 2H), 8.60 (s, 1H), 9.80 (d, J = 10.23 Hz, 2H). ESI-MS:
346.1 (C21H20N3S, [M+H]+). Anal. Calcd for C21H19N3S: C, 73.01;
H, 5.54; N, 12.16. Found: C, 73.32; H, 5.77; N, 12.38.
4.4.14. (E)-1-(4-(4-(Benzyloxy)benzylideneamino)phenyl)-3-
phenylthiourea (3v)
4.4.7. (E)-1-(4-(3-Bromobenzylideneamino)phenyl)-3-pheny
lthiourea (3o)
Yellow crystals, yield 79%, mp: 198–201 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 5.20 (s, 2H, OCH2), 7.14 (t, J = 10.15 Hz, 1H), 7.24
(d, J = 8.61 Hz, 2H), 7.31–7.41 (m, 6H), 7.47–7.53 (m, 7H), 7.89 (d,
J = 8.79 Hz, 2H), 8.57 (s, 1H), 9.80 (d, J = 10.23 Hz, 2H). ESI-MS:
438.2 (C27H24N3OS, [M+H]+). Anal. Calcd for C27H23N3OS: C,
74.11; H, 5.30; N, 9.60. Found: C, 74.42; H, 5.11; N, 9.78.
Yellow crystals, yield 75%, mp: 177–178 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.13 (t, J = 7.32 Hz, 1H), 7.29–7.37 (m, 4H), 7.44–
7.57 (m, 5H), 7.72 (d, J = 7.89 Hz, 1H), 7.93 (d, J = 7.86 Hz, 1H), 8.12
(s, 1H), 8.66 (s, 1H), 9.83 (d, J = 10.05 Hz, 1H). ESI-MS: 410.0
(C20H17BrN3S, [M+H]+). Anal. Calcd for C20H16BrN3S: C, 58.54; H,
3.93; N, 10.24. Found: C, 58.84; H, 4.23; N, 10.38.
4.5. Crystal structure determination
4.4.8. (E)-1-(4-(3-Methoxybenzylideneamino)phenyl)-3-phenyl
thiourea (3p)
Crystal structure determination of compound 3e was carried
Yellow crystals, yield 74%, mp: 250–253 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 2.29 (s, 3H, OCH3), 6.84 (s, 1H), 7.10–7.14 (m,
3H), 7.21 (d, J = 4.65 Hz, 1H), 7.31–7.34 (m, 2H), 7.44–7.50 (m,
5H), 7.54–7.55 (m, 1H), 8.66 (s, 1H), 9.80 (d, J = 11.87 Hz, 2H).
ESI-MS: 362.1 (C21H20N3OS, [M+H]+). Anal. Calcd for C21H19N3OS:
C, 69.78; H, 5.30; N, 11.63. Found: C, 69.92; H, 5.54; N, 11.81.
out on a Nonius CAD4 diffractometer equipped with graphite-
mono-chromated Mo Ka (k = 0.71073 Å) radiation (Fig. 3). The
structure was solved by direct methods and refined on F2 by
full-matrix least-squares methods using SHELX-97.33 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 parameter for the com-
pound 3e are listed in Table 4.
4.4.9. (E)-1-(4-(4-Fluorobenzylideneamino)phenyl)-3-phenyl
thiourea (3q)
Yellow crystals, yield 81%, mp: 191–192 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.13 (t, J = 7.32 Hz, 1H), 7.26–7.36 (m, 6H), 7.48–
7.55 (m, 4H), 7.98–8.03 (m, 2H), 8.66 (s, 1H), 9.81 (d, J = 9.69 Hz,
2H). ESI-MS: 350.1 (C20H17FN3S, [M+H]+). Anal. Calcd for
C20H16FN3S: C, 68.75; H, 4.62; N, 12.03. Found: C, 68.89; H, 4.84;
4.6. Antimicrobial activity
N, 12.21.
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 (minimum
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 tetrazoliumbromide).31 A stock solution
4.4.10. (E)-1-(4-(4-Chlorobenzylideneamino)phenyl)-3-phenylt
hiourea (3r)
Yellow crystals, yield 82%, mp: 187–188 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.13 (t, J = 7.41 Hz, 1H), 7.28–7.37 (m, 4H), 7.48–
7.61 (m, 6H), 7.96 (d, J = 8.4 Hz, 2H), 8.67 (s, 1H), 9.82 (d,
J = 10.05 Hz, 2H). ESI-MS: 366.1 (C20H17ClN3S, [M+H]+). Anal. Calcd
for C20H16ClN3S: C, 65.65; H, 4.41; N, 11.48. Found: C, 65.94; H,
4.63; N, 12.74.
of the synthesized compound (100 lg/mL) in DMSO was prepared
and graded quantities of the test compounds were incorporated 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 microorganism was pre-
pared to contain approximately 105 cfu/mL and applied to microt-
itration plates with serially diluted compounds in DMSO to be
tested and incubated at 37 °C for 24 h. After the MICs were visually
4.4.11. (E)-1-(4-(4-Bromobenzylideneamino)phenyl)-3-phenylt
hiourea (3s)
Yellow crystals, yield 76%, mp: 196–197 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.13 (t, J = 7.32 Hz, 1H), 7.28–7.37 (m, 4H), 7.49
(d, J = 7.47 Hz, 2H), 7.54 (d, J = 8.76 Hz, 2H), 7.73 (d, J = 8.43 Hz,
2H), 7.88 (d, J = 8.61 Hz, 2H), 8.66 (s, 1H), 9.82 (d, J = 9.87 Hz,
2H). ESI-MS: 410.0 (C20H17BrN3S, [M+H]+). Anal. Calcd for
determined on each of the microtitration plates, 50 lL of PBS
(phosphate buffered saline 0.01 mol/L, pH 7.4, Na2HPO4ꢀ12H2O
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 content of each
C20H16BrN3S: C, 58.54; H, 3.93; N, 10.24. Found: C, 58.34; H,
4.17; N, 10.55.
4.4.12. (E)-1-(4-(4-Nitrobenzylideneamino)phenyl)-3-phenylt
hiourea (3t)
well was removed, and 100 lL of isopropanol containing 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 1.
Yellow crystals, yield 80%, mp: 196–197 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 7.14 (t, J = 7.32 Hz, 1H), 7.32–7.40 (m, 4H), 7.49
(d, J = 7.68 Hz, 2H), 7.60 (d, J = 8.79 Hz, 2H), 8.19 (d, J = 8.94 Hz,
2H), 8.37 (d, J = 8.79 Hz, 2H), 8.86 (s, 1H), 9.87 (d, J = 11.52 Hz,
2H). ESI-MS: 377.1 (C20H17N4O2S, [M+H]+). Anal. Calcd for
4.7. E. coli FabH purification and activity assay
C20H16N4O2S: C, 63.81; H, 4.28; N, 14.88. Found: C, 63.99; H,
4.56; N, 14.75.
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). Trans-
formed cells were grown on Luria-Bertani (LB) agar plates
supplemented with kanamycin (30 mg/mL). Sodium dodecyl
4.4.13. (E)-1-(4-(4-Methylbenzylideneamino)phenyl)-3-phenylt
hiourea (3u)
Yellow crystals, yield 75%, mp: 170–171 °C, 1H NMR (300 MHz,
DMSO-d6, d ppm): 2.49 (s, 3H, CH3), 7.13 (t, J = 7.32 Hz, 1H), 7.26 (d,
J = 8.76 Hz, 2H), 7.31–7.36 (m, 4H), 7.51 (t, J = 8.96 Hz, 4H), 7.82 (d,

5714
H.-J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 5708–5715
Figure 3. Crystal structure diagrams of compound 3e. Molecule structure diagram with displacement ellipsoids being at the 30% probability level and H atoms are shown as
small spheres of arbitrary radii.
Table 4
Tris, 2 mM DTT, 10 mM MgCl2, 600 lM CoA and 0.2 lM ACPS
Crystallographical and experimental data for compound 3e
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 2 column volumes.
Compound
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
3e
C
20H16ClN3S
363.85
Monoclinic
P2(1)/n
8.4640(17)
23.169(5)
16.905(3)
90.00
100.33(3)
90.00
3261.4(11)
8
1.482
2.14–31.27
1504
30,869/8846 [Rint = 0.0287]
8846/0/451
0.370
In a final 20
0.5 mM DTT, 0.25 mM MgCl2 and 2.5 mM holo-ACP were mixed
with 1 nM FabH, and H2O was added to 15 L. After 1 min incuba-
tion, a 2 L mixture of 25 M acetyl-CoA and 0.75
Ci [3H] acetyl-
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
lL reaction, 20 mM Na2HPO4/NaH2PO4, pH 7.0,
b (Å)
c (Å)
l
a
(°)
b (°)
(°)
l
l
l
c
V (Å)
Z
l
Dcalc (g cmꢁ3
h range (°)
F(0 0 0)
)
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 concentrated DMSO
stock such that the final concentration of DMSO did not exceed 2%.
Reflections collected/unique
Data/restraints/parameters
Absorption coefficient (mmꢁ1
)
R1; wR2 [I > 2
r(I)]
0.0621/0.1427
0.1404/0.1758
1.004
R1; wR2 (all data)
GOOF
4.8. Molecular docking modeling
The crystal structures of E. coli FabH (PDB code: 1HNJ)32 was ob-
tained from the Protein Data Bank (http://www.rcsb.org).
sulfate–polyacrylamide 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 30 mg/mL of kanamycin and grown over night at 37 °C,
1 mL of which was used to inoculate 100 mL LB medium supple-
Studies were carried out on only one subunit of the enzymes.
The graphical user interface AutoDockTools (ADT) was employed
to setup the enzymes: all hydrogens were added, Gasteiger charges
were calculated and nonpolar hydrogens were merged to carbon
atoms. For macromolecules, generated pdbqt files were saved.
The 3D structures of ligand molecules were built, optimized
(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 (version 1.4.6) 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.
mented with 30
lg/mL of kanamycin. The culture was shaken for
4 h at 37 °C, and then induced with 0.5 mM isopropyl b-
D
-thioga-
lactopyranoside (IPTG). The culture was grown for 4 h, and har-
vested by centrifugation (30 min at 15,000 rpm).
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.
AutoDock 4.0 was employed for all docking calculations.34,35
The AutoDockTools program was used to generate the docking in-
put files. In all docking a grid box size of 48 ꢂ 48 ꢂ 48 points in x, y
and z directions was built in the catalytic site of the protein. A grid
spacing of 0.375 Å (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
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

H.-J. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 5708–5715
5715
13. Lee, P. J.; Bhonsle, J. B.; Gaona, H. W.; Huddler, D. P.; Heady, T. N.; Kreishman-
Deitrick, M.; Bhattacharjee, A.; McCalmont, W. F.; Gerena, L.; Lopez-Sanchez,
M.; Roncal, N. E.; Hudson, T. H.; Johnson, J. D.; Prigge, S. T.; Waters, N. C. J. Med.
Chem. 2009, 52, 952.
14. Nie, Z.; Perretta, C.; Lu, J.; Su, Y.; Margosiak, S.; Gajiwala, K. S.; Cortez, J.;
Nikulin, V.; Yager, K. M.; Appelt, K.; Chu, S. J. Med. Chem. 2005, 48, 1596.
15. Daines, R. A.; Pendrak, I.; Sham, K.; Aller, G. S. V.; Konstantinidis, A. K.;
Lonsdale, J. T.; Janson, C. A.; Qiu, X.; Brandt, M.; Khandekar, S. S.; Silverman, C.;
Head, M. S. J. Med. Chem. 2003, 46, 5.
algorithm searches. Default settings were used with an initial pop-
ulation of 50 randomly placed individuals, a maximum number of
2.5 ꢂ 106 energy evaluations, and a maximum number of 2.7 ꢂ 104
generations. A mutation rate of 0.02 and a crossover rate of 0.8
were chosen. Results differing by less than 0.5 Å 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.
16. Asheka, A.; Cho, S. J. Bioorg. Med. Chem. 2006, 14, 1474.
17. Singh, S.; Soni, L. K.; Gupta, M. K.; Prabhakar, Y. S.; Kaskhedikar, S. G. Eur. J. Med.
Chem. 2008, 43, 1071.
18. Shi, L.; Ge, H.-M.; Tan, S.-H.; Li, H.-Q.; Song, Y.-C.; Zhu, H.-L.; Tan, R.-X. Eur. J.
Med. Chem. 2007, 42, 558.
Acknowledgment
19. Huang, G. S.; Liang, Y. M.; Wu, X. L.; Liu, W. M.; Ma, Y. X. Appl. Organo-met.
Chem. 2003, 17, 706.
20. Curini, M.; Epifano, F.; Maltese, F.; Marcotullio, M. C. Tetrahedron Lett. 2002, 43,
3821.
21. Yadav, L. D. S.; Yadav, B. S.; Rai, V. K. Tetrahedron Lett. 2004, 45, 5351.
22. Zhang, L. X.; Liu, Y.; Cia, L. H.; Hu, Y. J.; Yin, J.; Hu, P. Z. Thermochim. Acta 2006,
440, 51.
23. Lee, J.-Y.; Jeong, K.-W.; Lee, J.-U.; Kang, D.-I.; Kim, Y. Bioorg. Med. Chem. 2009,
17, 1506.
24. Nakano, H.; Haroda, H.; Funaoka, T.; Akashi, K. Chem. Abstr. 1973, 78, 43086.
25. Pluempe, H.; Pulls, W. Chem. Abstr. 1971, 74, 1251154n.
26. Salman, A. K.; Neha, S.; Kishwar, S. Eur. J. Med. Chem. 2008, 43, 2272.
27. Yonova, P. A.; Stoilkova, G. M. J. Plant Growth Regul. 2004, 23, 280.
28. Wang, J. D.; Martell, A. E.; Motekaitis, R. J.; Reibenspies, J. H. Inorg. Chin. Acta
2001, 24, 194.
29. Singhb, B.; Srivastava, U. Synth. React. Inorg. Met. Org. Chem. 1989, 19, 279.
30. Iskander, M. F.; El-Sayed, L.; El-Toukhy, A. C. J. Inorg. Nucl. Chem. 1980, 42, 145.
31. Meletiadis, J.; Meis, J. F.; Mouton, J. W.; Donnelly, J. P.; Verweij, P. E. J. Clin.
Microbiol. 2000, 38, 2949.
This work was supported by Jiangsu National Science Founda-
tion (No. BK2009239) and the Fundamental Research Funds for
the Central Universities (No. 1092020804 & 1106020824).
References and notes
1. Leeb, M. Nature 2004, 431, 892.
2. Cronan, J. E., Jr.; Rock, C. O. In Escherichia coli and Salmonella typhimurium:
Cellular and Molecular Biology; Neidhardt, F. C., Ingraham, I. L., Low, K. B., et al.,
Eds.; American Society of Microbiology: Washington, DC, 1996; pp 612–638.
3. Khandekar, S. S.; Daines, R. A.; Lonsdale, J. T. Curr. Protein Pept. Sci. 2003, 4, 21.
4. Heath, R. J.; Rock, C. O. Nat. Prod. Rep. 2002, 19, 581.
5. Tsay, J. T.; Oh, W.; Larson, T. J.; Jakowski, S.; Rock, C. O. J. Biol. Chem. 1992, 267,
6807.
6. Clough, R. C.; Matthis, A. L.; Barnum, S. R.; Jaworski, J. G. J. Biol. Chem. 1992, 267,
20992.
7. Heath, R. J.; Rock, C. O. J. Biol. Chem. 1996, 271, 1833.
8. White, S. W.; Zheng, J.; Zhang, Y. M.; Rock, C. O. Annu. Rev. Biochem. 2005, 74,
791.
9. Veyron-Churlet, R.; Guerrini, O.; Mourey, L.; Daffé, M.; Zerbib, D. Mol. Microbiol.
2004, 54, 1161.
10. Christensen, C. E.; Kragelund, B. B.; von Wettstein-Knowles, P.; Henriksen, A.
Protein Sci. 2007, 16, 261.
11. Bylka, W.; Matlawska, I.; Pilewski, N. A. JANA 2004, 7, 24.
12. Huycke, M. M.; Sahm, D. F.; Gilmore, M. S. Emerg. Infect. Dis. 1998, 4, 239.
32. Qiu, X.; Janson, C. A.; Smith, W. W.; Head, M.; Lonsdale, J.; Konstantinidis, A. K.
J. Mol. Biol. 2001, 307, 341.
33. Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112.
34. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
35. Huey, R.; Morris, G. M.; Olson, A. J.; Goodsell, D. S. J. Comput. Chem. 2007, 28,
1145.