Synthesis, antibacterial activities and molecular docking studies of peptide and Schiff bases as targeted antibiotics

Article abstract of DOI:10.1016/j.bmc.2009.10.037

A series of peptide and Schiff bases (PSB) were synthesized by reacting salicylic acid, primary diamines with salicylaldehyde or its derivatives, and 40 of which were newly reported. The inhibitory activities against Escherichia coli β-ketoacyl-acyl carrier protein synthase III (ecKAS III) were investigated in vitro and molecular docking simulation also surveyed. Top 10 PSB compounds which posses both good inhibitory activity and well binding affinities were picked out, and their antibacterial activities against Gram-negative and Gram-positive bacterial strains were tested, expecting to exploit potent antibacterial agent with broad-spectrum antibiotics activity. The results demonstrate compound N-(3-(5-bromo-2-hydroxybenzylideneamino)propyl)-2-hydroxybenzamide (2d) can be as a potential antibiotics agent, displaying minimal inhibitory concentration values in the range of 0.39-3.13 μg/mL against various bacteria. Crown Copyright

Full text of DOI:10.1016/j.bmc.2009.10.037

Bioorganic & Medicinal Chemistry 17 (2009) 7861–7871
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, antibacterial activities and molecular docking studies of peptide
and Schiff bases as targeted antibiotics
*
*
Kui Cheng, Qing-Zhong Zheng, Yong Qian, Lei Shi, Jing Zhao , 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 peptide and Schiff bases (PSB) were synthesized by reacting salicylic acid, primary diamines
with salicylaldehyde or its derivatives, and 40 of which were newly reported. The inhibitory activities
against Escherichia coli b-ketoacyl-acyl carrier protein synthase III (ecKAS III) were investigated in vitro
and molecular docking simulation also surveyed. Top 10 PSB compounds which posses both good inhib-
itory activity and well binding affinities were picked out, and their antibacterial activities against Gram-
negative and Gram-positive bacterial strains were tested, expecting to exploit potent antibacterial agent
with broad-spectrum antibiotics activity. The results demonstrate compound N-(3-(5-bromo-2-hydrox-
ybenzylideneamino)propyl)-2-hydroxybenzamide (2d) can be as a potential antibiotics agent, displaying
Received 12 September 2009
Revised 17 October 2009
Accepted 20 October 2009
Available online 23 October 2009
Keywords:
Peptide and Schiff base (PSB)
Antibacterial activity
Autodock
minimal inhibitory concentration values in the range of 0.39–3.13 lg/mL against various bacteria.
Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
ecKAS III
1. Introduction
conserved in various bacteria, the active site contains a Cys-His-
Asn catalytic triad,10 and its inhibitors may thus act as potent anti-
biotics with broad-spectrum activity.
During the past decades, the human population affected with
life-treating infectious diseases caused by multi-drug resistant
Gram-positive and Gram-negative pathogen bacteria increased an
alarming level around the world1 and represent the second leading
cause of death.2
Type II fatty acid synthesis (FAS II) pathway has been recently
reported as a attractive targeting for their efficacy against infec-
tions caused by mutiresistant Gram-positive bacteria.3,4 Also have
people not agree with this point for there are plenty of fatty acids
available to the bacteria inside of the host.5 However, FAS II it’s
proven to be a good target for Gram-negative.6
Among the related FAS II enzymes, the condensing protein, b-
ketoacyl-acyl carrier protein synthase (KAS), is an essential target
for novel antibacterial drug design.7 In bacteria, there appear to
be three KAS enzymes, which are denoted KAS I, KAS II, and KAS
III (the Escherichia coli equivalents are FabB, FabF, and FabH,
respectively). The initial C2–C4 step of fatty acid biosynthesis is
catalyzed by KAS III; thereafter, KAS I and KAS II are involved in
chain elongation.8 Notably, KAS III, regulates the fatty acid biosyn-
thesis rate via an initiation pathway and its substrate specificity is
a key factor in membrane fatty acid composition and this protein
represents a promising target for the antimicrobial drugs design.9
Moreover, the three-dimensional structure of the protein is highly
Recent reports demonstrated that thiolactomycin11 and its ana-
logues compounds12 have low toxicity and some of which are
highly active against KAS III. Acyl derivative13 and Schiff bases
were also well document as a wide variety of biological activities
such as antimicrobacterial, antimicrobial, and antitumor agents,
etc.14–16 Recently, Schiff bases were reported as a potent inhibitor
of Plasmodium falciparum KAS III17 and ecKAS III with antimicrobial
activity against various bacteria.18 In view of this, we developed a
series of new PSB compounds and suppose them possess similar or
much better biological activities as salicylamide peptide or salen-
type Schiff base by the fact that PSB own both peptide and Schiff
base structures (Scheme 1).
In this study, our main objective is the development of
potential inhibitors by newly synthesized PSB as targeted antibi-
otics agents, based on molecular modeling and the investigation
of SAR between new inhibitors and ecKAS III. Firstly, we use
structure-based design method to synthesized the PSB and test
their inhibitory activity against ecKAS III. And following, top
ten PSB compounds which posses good ecKAS III inhibitory
activity (low IC50) and well binding affinities (binding free ener-
gies, inhibition constants, hydrogen bonding) by docking into
the active site of ecKAS III were picked out to test their antibac-
terial activities against two Gram-negative bacterial strains
(E. coli ATCC 35218 and Pseudomonas aeruginosa ATCC 13525)
and two Gram-positive bacterial strains (Bacillus subtilis ATCC
6633 and Staphylococcus aureus ATCC 6538), expecting to exploit
potent antibacterial agent with broad-spectrum antibiotics activ-
* Corresponding authors. Tel.: +86 25 8359 2672; fax: +86 25 8359 2572 (J.Z.);
tel.: +86 25 8359 2082; fax: +86 25 8359 3201 (H.-L.Z.).
E-mail addresses: jingzhao@nju.edu.cn (J. Zhao), zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.037

7862
K. Cheng et al. / Bioorg. Med. Chem. 17 (2009) 7861–7871
Scheme 1. The general procedure of the synthesized compounds. ‘1’, ‘2’, ‘3’, ‘4’, ‘5’ represent the atom number bonding to the amino acid active site.
ity. As expected, the results demonstrate that the antimicrobial
compound, N-(3-(5-bromo-2-hydroxybenzylideneamino)propyl)-
2-hydroxybenzamide (2d), can be as an inhibitor of ecKAS III
and as a potential antibiotics agent, displaying MIC values in
Table 1
Crystal and experimental data for complexes 1c
Compound
1c
Empirical formula
Mr
T (K)
C16H15ClN2O3
318.75
298(2)
the range 0.39–3.13 lg/mL against various bacteria. Which is
much better than the best compound (YKAs3003) reported by
Kim and co-workers.18 The AutoDock study for new inhibitors
docked into the active sites of ecKAS III has also been carried
out.
0
0.71073
Radiation (Mo Ka), k (AÅ)
Crystal shape/color
Crystal size (mm3)
Crystal system
Space group
a (Å)
block/yellow
0.32 ꢁ 0.20 ꢁ 0.17
monoclinic
P21/c
16.527(2)
6.401(1)
14.117(1)
90.00
2. Result and discussion
2.1. Chemistry
b (Å)
c (Å)
a
(°)
b (°)
90.49(5)
90.00
1493.4(2)
4
1.418
0.270
664
1.003
0.0487, 0.1306
0.0942, 0.1687
In the present study, 36 PSB were subjected by reacting sali-
cylamide types primary amines with salicylaldehyde or its deriv-
atives. And 8 di-peptide and di-Schiff base compounds were
synthesized through primary diamines reaction with ethyl salicy-
late or salicylaldehyde, respectively. The general method for pre-
paring the compounds is outlined in Scheme 1. Salicylamide types
primary amines (C), which were not commercially available, were
synthesized using modified procedures of Kido.19 The crude prod-
ucts were purified using silica gel column chromatography, pre-
parative TLC and recrystallization. All the compounds gave
satisfactory chemical analyses ( 0.4%). 1H NMR, ESI-MS spectra
and elemental analyses were consistent with the assigned
structures.
c
(°)
V (Å3)
Z
Dcalcd (g/cmꢂ3
)
l
(mmꢂ1
)
F(0 0 0)
Goodness of fit on F2
R1, wR [I P 2
r
(I)]a
R1, wR (all data)a
P
P
P
P
2
2
1=2
2
||Fo| ꢂ |Fc||/ |Fo|, wR2 ¼ ½ wðF2o ꢂ Fc2Þ = wðFo2Þ ꢃ
,
w1 ¼ ½r2ðFoÞ þ
a
R1
=
ð0:1009ðFo2 þ 2Fc2Þ=3Þ ꢃꢂ1, w2 ¼ ½r2ðFoÞ þ ð0:0962ðFo2 þ 2F2c Þ=3Þ ꢃꢂ1
.
2
2
2
Compound 1c was successfully crystallized and its structure
was determined by single-crystal X-ray diffraction analysis. The
crystal data are presented in Table 1, and Figure 1 gives a perspec-
tive view of this compound together with the atomic labeling sys-
tem. In the structure of compound 1c, there are an intramolecular
O–Hꢀ ꢀ ꢀO (O1–H1ꢀ ꢀ ꢀO2, 2.504(3) Å, 148.6°) and O–Hꢀ ꢀ ꢀN hydrogen
bond (O3–H3Aꢀ ꢀ ꢀN2: 2.602(3) Å, 147.3°) and an intermolecular
N–Hꢀ ꢀ ꢀO hydrogen bond (N1–H1Aꢀ ꢀ ꢀO1i: 2.974(3) Å, 137.2°, sym-
metry codes: i, x, 1 + y, z) in the structure. The dihedral angle be-
tween the two benzene rings is 34.1 (0.3)°. The bond length of
N1–C7 (1.330(4) Å) indicates a single bond and bond length of
N2@C10 (1.266(3) Å) exhibits a double bond.

K. Cheng et al. / Bioorg. Med. Chem. 17 (2009) 7861–7871
7863
Figure 1. Molecular structure of compound 1c with atoms and hydrogen bonds labeling.
2.2. Inhibitory activities of PSB against ecKAS III
substrate in an X-ray structure of ecKAS III complexed with
CoA or inhibitor.20 Towards optimization of the aforementioned
compounds of the promising antimicrobial activities, the docking
program AutoDock 4.021 was used to evaluate the binding free
energies as potential inhibitors into the target ecKAS III
macromolecule.
All the synthesized compounds (1a–1k, 2a–2k, 3a–3k and 4a–
4k) were tested for inhibitory activity against ecKAS III. The inhibi-
tion constant (IC50) of the compounds were presented in Table 2
and Figure 2 (IC50 more than 50 lg/mL not demonstrate in
Fig. 2). It was observed that many compounds have been found
to show fairly good inhibitory activity displaying IC50 values be-
tween 0.33 and 40.0 lg/mL.
2.3.1. Validation of the accuracy and performance of AutoDock
According to the method of validation cited in literature,22
where if the RMSD (root mean square deviation) of the best docked
conformation is 62.0 Å from the experimental one, the used scor-
ing function is successful. The obtained success rates of AutoDock
(Morris et al.)21 was highly excellent as illustrated in Table 2 for
the native ligand, which was docked into its KAS III (PDB code:
1HNJ).
Inspection of the chemical structure of the compounds
(Scheme 1) suggested that it could be divided into two subunits:
the length of aliphatic chain-n and the substitution on aromatic
ring-R1 and R2. Initial SAR studies were performed by modification
of the parent compound to determine how the substituents of the
subunits affected the inhibitory activities. Replacement of H atom
(1a) at R1-position by one halogen atom or R1 and R2-position by
two halogen atoms resulted in the improving of their inhibitory
activity (1c–1e and 1f–1g). Increment of the aliphatic chain from
2.3.2. AutoDock binding affinities of the synthesized and
designed compounds into KAS III
1 (1b IC50 10.35
ilar increasing of inhibitory activity, while n increased from 2 (2b)
to 5 (4b IC50 40.00 g/mL), the inhibitory activity was decrease
l
g/mL) to 2 (2b IC50 1.76
l
g/mL) also led to a sim-
The binding affinity was evaluated by the binding free energies
(DGb, kcal/mol), inhibition constants (Ki) and hydrogen bonding.
l
The compounds which revealed the highest binding affinities, that
is, lowest binding free energies, within KAS III and the hydrogen
bond interactions into the target macromolecule are represented
in Table 2. These compounds include N-(3-(5-bromo-2-hydroxyb-
enzylideneamino)propyl)-2-hydroxybenzamide (2d), (E)-2-hydro-
xy-N-(4-(2-hydroxy-5-iodobenzylideneamino)butyl)benzamide (3e),
N-(3-(5-chloro-2-hydroxybenzylideneamino)propyl)-2-hydroxybenz-
amide (2c), 2-hydroxy-N-(3-(2-hydroxy-5-iodobenzylideneami-
no)propyl)benzamide (2e) and (E)-2-hydroxy-N-(2-(2-hydroxy-5-
iodobenzylideneamino)ethyl)benzamide (1e). Many of these deriv-
atives exhibited one or two hydrogen bonds between O1, O2, O5 or
N3 of PSB and different amino acids of the target ecKAS III includ-
ing Asn 274, Asn 247, Arg 36, Arg 151, Gly 209, Trp36, Gly152, Asn
210, and Arg 249 as cited in Table 2.
(Fig. 2). For more extensive evidences, the substitution of H atom
at R1 (2b–2e, 3b–3e, 4c–4d), R1 and R2 (2f–2g, 3f–3h, 4g–4h) by
halogen atoms, also produced some increment compared to the
corresponding precursor 2a, 3a and 4a, respectively. This sug-
gested that compounds with electron-withdrawing groups on R1
and R2 and appropriate length of aliphatic chain (n = 2, 3) showed
better inhibitory activities, excepting 1b, 2h, 4b, 4e and 4f. The
introduction naphthyl (1i, 2i, 3i and 4i) substitute of phenyl group,
led to inhibitory activity increase in 2i (IC50 3.00
lg/mL), and 3i
(IC50 3.10 g/mL). Compared to PSB, the di-peptide and di-schiff
l
base compounds exhibit weak inhibitory activities.
2.3. Molecular docking study
The molecular docking study revealed that the majority of the
compounds docked into the ecKAS III (PDB code: 1HNJ) exhibited
hydrogen bonds via O2 (or O5)–NH group as illustrated for com-
pounds 2d in Figure 3.
Here, we investigated the AutoDock binding affinities of the
synthesized PSB into ecKAS III. We defined the active site of
ecKAS III, based on the center and radius of the binding

Table 2
Inhibition constant (IC50) against ecKAS III and docking simulation parameters
Compound
Inhibitory constant
Docking parameters
Hydrogen bond atom of compound
Total score
IC50
(l
g/mL)
Observed activity class
D
Gb (kcal/mol)
Ki
D
G and Ki score
Amino acids
Hydrogen bond score
1a
1b
1c
1d
1e
1f
1g
1h
1i
7.25
++
+
ꢂ7.14
ꢂ7.80
ꢂ7.5
5.70 ꢁ 10ꢂ6
1.39 ꢁ 10ꢂ6
3.18 ꢁ 10ꢂ6
1.34 ꢁ 10ꢂ5
9.51 ꢁ 10ꢂ8
4.80 ꢁ 10ꢂ7
1.98 ꢁ 10ꢂ7
3.27 ꢁ 10ꢂ7
1.96 ꢁ 10ꢂ6
2.84 ꢁ 10ꢂ7
1.08 ꢁ 10ꢂ6
8.22 ꢁ 10ꢂ6
1.40 ꢁ 10ꢂ7
+
+
+
0
+++
++
+++
++
+
O2
O1
O5
—
O2
O2
O2
—
N–H of Asn 274
N–H of Asn 247
N–H of Arg 36
—
N–H of Asn 247
N–H of Asn 247
N–H of Arg 151
—
N–H of Asn 247
N–H of Asn 274
N–H of Asn 247
N–H of Arg151
N–H of Asn 247
N–H of Gly 209
N–H of Asn 247
N–H of Asn 247
N–H of Asn 274
N–H of Asn 247
N–H of Asn 247
N–H of Gly 209
—
N–H of Trp 36
N–H of Asn 247
N-H of Asn 247
N–H of Asn 274
N–H of Gly 209
O of Gly152
+
+
+
0
+
+
+
0
+
+
+
+
++
++++
+++
10.35
2.77
6.22
1.13
2.5
+++
++
++++
+++
+++++
+
+
0
0
+
+++++
++
++++++++
++++++
+++++++++
+++
+++
+++
+++
++
ꢂ6.65
ꢂ9.58
ꢂ8.62
ꢂ9.15
ꢂ8.85
ꢂ7.79
ꢂ8.93
ꢂ8.14
ꢂ6.94
ꢂ9.35
0.54
12.72
9.75
16.51
>50.00
12.5
1.76
O5
O1
O1
O2
O2
O5
O2
O2
O5
O2
O2
O1
—
O5
O1
O2
O2
O1
O1-H
O2
—
O5
O5
O2
—
O5
—
O2
O1
O1
N3-H
O2
O2
O2
O5
O2
1j
++
++
0
1k
2a
2b
++++
+++
+++++++++
2c
2d
1.64
0.33
++++
+++++
ꢂ9.6
9.23 ꢁ 10ꢂ8
4.85 ꢁ 10ꢂ9
+++
+++
+
++
++++
++++++++++
ꢂ11.34
2e
2f
2g
2h
2i
1.50
2.75
6.31
12.95
3.00
++++
+++
++
+
+++
ꢂ9.51
ꢂ7.82
ꢂ7.82
ꢂ6.46
ꢂ8.66
1.06 ꢁ 10ꢂ7
1.86 ꢁ 10ꢂ6
1.84 ꢁ 10ꢂ6
1.84 ꢁ 10ꢂ8
4.51 ꢁ 10ꢂ7
+++
+
+
0
++
+
+
+
0
++++++++
+++++
++++
+
++
+++++++
2j
13.15
+
ꢂ9.71
7.63 ꢁ 10ꢂ8
+++
++
++++++
2k
3a
3b
3c
3d
3e
12.25
13.57
6.35
2.32
6.25
0.65
+
+
++
+++
++
ꢂ9.01
ꢂ6.78
ꢂ6.19
ꢂ9.49
ꢂ6.32
ꢂ10.15
2.49 ꢁ 10ꢂ7
1.06 ꢁ 10ꢂ5
2.91 ꢁ 10ꢂ5
1.11 ꢁ 10ꢂ7
2.33 ꢁ 10ꢂ5
3.66 ꢁ 10ꢂ8
+++
0
0
+++
0
+++
+
+
+
0
+
++
+++++
++
+++
++++++
+++
++++++++++
N–H of Asn 247
—
N–H of Asn 274
N–H of Trp 36
N–H of Asn 247
—
+++++
3f
2.95
3.11
6.25
3.10
12.59
13.04
22.16
40.00
10.00
12.53
26.55
>50.00
+++
+++
++
+++
+
+
0
0
+
ꢂ4.67
ꢂ6.58
ꢂ9.1
3.74 ꢁ 10ꢂ4
1.49 ꢁ 10ꢂ5
2.12 ꢁ 10ꢂ7
1.79 ꢁ 10ꢂ4
1.61 ꢁ 10ꢂ6
2.69 ꢁ 10ꢂ6
4.72 ꢁ 10ꢂ6
3.60 ꢁ 10ꢂ5
3.06 ꢁ 10ꢂ5
1.11 ꢁ 10ꢂ5
3.63 ꢁ 10ꢂ7
1.46 ꢁ 10ꢂ5
0
0
+++
0
+
+
+
0
0
0
+
0
+
+
+
+
+
+
+
+
+
+++
++++
+++++
++++
+++
+++
++
+
++
++
+++
+
3g
3h
3i
N–H of Asn 247
—
ꢂ5.11
ꢂ7.9
N–H of Asn 247
N–H of Asn 210
N–H of Asn 247
O of Gly 152
N–H of Arg 36
N–H of Asn 247
N–H of Arg 151
N–H of Asn 247
N–H of Arg 249
3j
3k
4a
4b
4c
4d
4e
4f
ꢂ7.6
ꢂ7.27
ꢂ6.06
ꢂ6.16
ꢂ6.76
ꢂ8.79
ꢂ6.6
+
0
0
0
++
0

K. Cheng et al. / Bioorg. Med. Chem. 17 (2009) 7861–7871
7865
A data set, shown in Table 2 and 44 compounds were selected
for future evaluation, and the inhibitory activity (IC50) and Docking
parameters (DGb, Ki and hydrogen bond) are fifty–fifty weight. The
correlation between inhibitory activities and the binding affinities
predicted by AutoDock modeling was highly good for some
compounds.
As IC50 consider, the compounds were categorized into six
activity classes and based on the observed bioactivity data distri-
bution with <1
+++++, 1–2 g/mL as second active represented by symbol
++++, 2–4 g/mL as third active represented by symbol +++, 4–
g/mL as fourth active represented by symbol ++, 8–12 g/
mL as fifth active represented by symbol +, and >12 g/mL as
last active represented by symbol 0. As binding free energies
Gb, kcal/mol) consider, the compounds were categorized into
lg/mL as very active represented by symbol
l
l
8
l
l
l
(D
four activity classes and based on the docking data distribution
with <ꢂ9 kcal/mol as very active represented by symbol +++,
ꢂ9 to ꢂ8 kcal/mol as moderately active represented by symbol
++, ꢂ8 to ꢂ7 kcal/mol as third active represented by symbol +,
and >ꢂ7 kcal/mol as less active represented by symbol 0. Inhibi-
tion constants (Ki) vary tendency is according with
DGb, so just
study one parameter is enough. Base on hydrogen bond with
active sites, the compounds were categorized into three activity
classes and the docking data distribution with 2 bonds as very
active represented by symbol ++, 1 bond as moderately active
represented by symbol +, and no bond as less active represented
by symbol 0.
The global results are list in Table 2, and we selected the top
10 compounds (2d, 3e, 2b, 1g, 2c, 1e, 2e, 2i, 3c and 1f) which
have more ‘+’ symbol in the ‘Total Score’ column to test their
antibacterial activities against Gram-negative bacterial strains
(E. coli ATCC 35218 and Pseudomonas aeruginosa ATCC 13525)
and two Gram-positive bacterial strains (B. subtilis ATCC 6633
and Staphylococcus aureus ATCC 6538) (Table 3), expecting to
exploit potent antibacterial agent with broad-spectrum antibiot-
ics activity. Out of the top 10 compounds, the results demon-
strate that the antimicrobial compound, N-(3-(5-bromo-2-hydro-
xybenzylideneamino)propyl)-2-hydroxybenzamide (2d), can be
as an inhibitor of ecKAS III and a potential antibiotics agent,
displaying MIC of 0.39, 3.13, 1.56 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 with corre-
sponding MIC of 3.13, 3.13, 1.56 and 1.56
lg/mL. Compounds
2b and 1g showed similar moderate antibacterial activity with
MIC of 1.56–25.00
strains.
lg/mL against all the tested bacterial
From the binding model, we can see compound 2d (Fig. 3) (col-
ored by element, ball and stick) is bound into ecKAS III receptor site
via hydrophilic binding by hydrogen bond between O2ꢀ ꢀ ꢀN–H of
Asn 274 and O5ꢀ ꢀ ꢀN–H of Asn 247. The ecKAS III active site gener-
ally contains a catalytic triad tunnel consisting of Cys-His-Asn,10
effecting to these amino acid resides may greatly influence, inhibit
or even stop its catalytic activity. The direct result is the fatty acid
biosynthesis can’t be smoothly proceeded, leading the energy sup-
ply to organism is not sufficient and could not forming the compo-
nent of all cell membranes, so display its antimicrobial activity.
Compound 2d bonds two Asn active site (Asn 247 and Asn 274)
of ecKAS III and fit into the mouth of the substrate tunnel
(Fig. 3). This fact made 2d had excellent inhibitory activity than
other compounds and can be act as an inhibitor of KAS III and as
a potential antibiotics agent in theory. The antimicrobial activity
(minimum inhibitory concentrations are the average values of trip-
licates experiment results) against E. coli, P. aeruginosa, B. Subtilis
and S. aureus proved this point in practice (Table 3). Compounds
2b and 1g also have moderate inhibitory activity against various
bacteria.

7866
K. Cheng et al. / Bioorg. Med. Chem. 17 (2009) 7861–7871
40
35
30
25
20
15
10
5
IC50
u
0
1a--------1f--------1k 2a--------2f--------2k 3a--------3f--------3k 4a--------4f--------4k
Compounds
Figure 2. Inhibitory activities of the compounds. IC50 more than 50 lg/mL not demonstrate.
Figure 3. Compound 2d (colored in green) is bound into ecKAS III receptor site via hydrophobic interactions and hydrophilic binding by hydrogen bond between its O2 and
H–N of Asn 274 (N–Hꢀ ꢀ ꢀO2: 2.14 Å, 138.7°), O5 and H–N of Asn 247 (N–Hꢀ ꢀ ꢀO5: 2.00 Å, 147.2°) (2d(i)), and extending into the mouth of the substrate tunnel (2d(ii)).
Table 3
Antimicrobial activity of the top 10 compounds
Top 10 compounds
Minimum inhibitory concentrations (lg/mL)
Escherichia coli
Pseudomonas aeruginosa
Bacillus subtilis
Staphylococcus aureus
2d
0.39
3.13
1.56
3.13
3e
2b
1g
2c
1e
2e
2i
3c
6.25
3.13
6.25
25.00
25.00
3.13
>50.00
>50.00
1.56
3.13
1.56
12.50
>50.00
>50.00
6.25
1.56
25.00
1.56
>50.00
>50.00
25.00
>50.00
25.00
3.13
>50.00
1.56
12.50
25.00
>50.00
>50.00
>50.00
25.00
>50.00
1.56
3.13
12.50
>50.00
3.13
1f
Kanamycin
3.13
1.56

K. Cheng et al. / Bioorg. Med. Chem. 17 (2009) 7861–7871
7867
(300 MHz, CDCl3, d ppm): 3.83 (m, 4H); 6.71 (s, 1H); 6.80 (m, 1H);
6.88 (m, 1H); 6.97 (m, 2H); 7.23 (m, 1H); 7.35 (m, 3H); 8.37 (s, 1H);
12.67 (s, 2H). ESI-MS: 285.1 (C16H17N2O3þ, [M+H]+). Anal. Calcd for
C16H16N2O3: C, 67.59; H, 5.67; N, 9.85. Found: C, 67.45; H, 5.69; N,
9.79.
3. Conclusion
In summary, a series of novel PSB derivatives were prepared and
tested for their inhibitory activity against ecKAS III, and the Auto-
dock investigation also carried out by docking them into the active
site of ecKAS III. Both the theory and practice demonstrate that N-
(3-(5-bromo-2-hydroxybenzylideneamino)propyl)-2-hydroxyben-
zamide (2d), can be as a potent inhibitor of ecKAS III, displaying MIC
4.2.1.2. (E)-N-(2-(5-Fluoro-2-hydroxybenzylideneamino)ethyl)-
2-hydroxybenzamide (1b). Yellow power, yield 90%, mp: 95–
96 °C, 1H NMR (300 MHz, CDCl3, d ppm): 3.81 (m, 4H); 6.65 (s,
1H); 6.80 (m, 1H); 6.89 (d, J = 7.1 Hz, 1H); 6.96 (m, 1H); 7.21 (s,
1H); 7.26 (m, 1H); 7.31 (m, 1H); 7.38 (m, 1H); 8.33 (s, 1H); 12.11
(s, 1H); 13.21 (s, 1H). ESI-MS: 303.1 (C16H16FN2O3þ, [M+H]+). Anal.
Calcd for C16H15FN2O3: C, 63.57; H, 5.00; N, 9.27. Found: C, 63.48;
H, 4.94; N, 9.22.
values in the range 0.39–3.13 lg/mL against various bacteria. While
a few compounds, for example 3e, is potent inhibitors of ecKAS III
but fail to inhibit S. aureus, suggesting that a correlation may exist.
Here, the preliminary structure–activity relationships and molecu-
lar modeling study just shed a light on the further insight into inter-
actions between the macromolecular enzyme and its inhibitor
ligands.
4.2.1.3. (E)-N-(2-(5-Chloro-2-hydroxybenzylideneamino)ethyl)-
2-hydroxybenzamide (1c). Yellow power, yield 82%, mp: 85–
88 °C, 1H NMR (300 MHz, CDCl3, d ppm): 3.82 (m, 4H); 6.69 (s,
1H); 6.81 (m, 1H); 6.90 (d, J = 7.1 Hz, 1H); 6.97 (d, J = 8.4 Hz, 1H);
4. Experiment
4.1. Materials
7.21 (s, 1H); 7.25 (m, 1H); 7.32 (m, 1H); 7.38 (m, 1H); 8.31 (s,
þ
1H); 12.21 (s, 1H); 13.03 (s, 1H). ESI-MS: 219.1 (C16H16ClN2O3
,
All chemicals (reagent grade) used were commercially avail-
able. All the 1H NMR and spectra were recorded on a Bruker DRX
500 or DPX 300 model Spectrometer at 25 °C with TMS and solvent
signals allotted as internal stands. Chemical shifts were reported in
ppm (d). ESI-MS spectra were recorded on a Mariner System 5304
Mass spectrometer. Elemental analyses were performed on a CHN-
O-Rapid instrument and were within 0.4% of the theoretical val-
ues. Melting points were measured on a Boetius micro melting
point apparatus.
[M+H]+). Anal. Calcd for C16H15ClN2O3: C, 60.29; H, 4.74; N, 8.79.
Found: C, 60.35; H, 4.79; N, 8.82.
4.2.1.4. (E)-N-(2-(5-Bromo-2-hydroxybenzylideneamino)ethyl)-
2-hydroxybenzamide (1d). Yellow power, yield 86%, mp: 135–
137 °C, 1H NMR (300 MHz, CDCl3, d ppm): 3.62 (m, 2H); 3.80 (m,
2H); 6.86 (m, 3H); 7.36 (m, 1H); 7.46 (d, J = 8.7 Hz, 1H); 7.66 (s,
1H); 7.82 (d, J = 7.8 Hz, 1H); 8.54 (s, 1H); 8.95 (s, 1H); 12.41 (s,
1H); 13.37 (s, 1H). ESI-MS: 363.0 (C16H16BrN2O3þ, [M+H]+). Anal.
Calcd for C16H15BrN2O3: C, 52.91; H, 4.16; N, 7.71. Found: C,
52.95; H, 4.09; N, 7.72.
4.2. General method of synthesis PSB
A: Concentrated sulfuric acid (1.0 mL) was added slowly with
vigorous agitation to a mixture of 20.0 g (144 mmol) of salicylic
acid and 63.0 mL (1.08 mol) of anhydrous ethanol. The mixture
was then reflux for 15 h. Excess ethanol was removed by evapora-
tion and the residue was washed with water. The organic layer was
again treated with water and concentrated sodium bicarbonate
solution to an alkaline reaction, and then again with water. The
product was dried over anhydrous MgSO4, collected by filtration,
and distilled (yield: 55.0%).
4.2.1.5. (E)-2-Hydroxy-N-(2-(2-hydroxy-5-iodobenzylideneami-
no)ethyl)benzamide (1e). Yellow power, yield 92%, mp: 151–
153 °C, 1H NMR (300 MHz, CDCl3, d ppm): 3.63 (m, 2H); 3.81 (m,
2H); 6.83 (m, 3H); 7.35 (m, 1H); 7.48 (d, J = 8.9 Hz, 1H); 7.64 (s,
1H); 7.81 (d, J = 8.0 Hz, 1H); 8.54 (s, 1H); 8.96 (s, 1H); 12.40 (s,
1H); 13.36 (s, 1H). ESI-MS: 410.1 (C16H16IN2O3þ, [M+H]+). Anal.
Calcd for C16H15IN2O3: C, 46.85; H, 3.69; N, 6.83. Found: C, 46.93;
H, 3.71; N, 6.82.
C: Equimolar quantities (10 mmol) of A and the primary dia-
mines (1,2- diaminoethane, 1,3-diaminopropane, 1,4-diaminobuy-
ane, 1,6-diaminohexane) were mixed and vigorously stirred at
110 °C under oil bath for 5 h to give a melicera solution. The crude
product was then purified by column chromatography in 10%
MeOH/CH2Cl2 to afford the desired compound (yield: 60.0%).
Equimolar quantities (0.5 mmol) of C and salicylaldehyde or its
derivatives, or 2-hydroxy-1-naphthaldehyde were dissolved in
methanol (10 mL) and stirred at 50 °C for 3 h to give a clear solution
(1a–1i, 2a–2i, 3a–3i and 4a–4i). The crude product was then puri-
fied by column chromatography in 25% EtOAc/hexanes to afford
the desired compound. 0.5 mmol of diamines (1,2- diaminoethane,
1,3-diaminopropane, 1,4-diaminobuyane, 1,6-diaminohexane) was
mixed with 2 N A and vigorous stirring 5 h at 110 °C under oil bath.
The crude product was then purified by column chromatography in
25% EtOAc/hexanes to afford 1i–4j. 0.5 mmol of B and 2 N salicylal-
dehyde were dissolved in methanol (10 mL) and stirred at 50 °C for
3 h to give a clear solution (1k–4k). The crude product was then
purified by column chromatography in 25% EtOAc/hexanes to af-
ford the desired compound.
4.2.1.6. (E)-N-(2-(3,5-Dichloro-2-hydroxybenzylideneamino)ethyl)-
2-hydroxybenzamide (1f). Yellow power, yield 95%, mp: 135–136 °C,
1H NMR (300 MHz, CDCl3, d ppm): 3.78 (m, 2H); 3.89 (m, 2H); 6.82 (m,
2H); 6.98 (d, J = 8.4 Hz, 1H); 7.13 (s, 1H); 7.39 (m, 3H); 8.28 (s, 1H); 12.09
(s, 1H); 14.12 (s, 1H). ESI-MS: 357.0 (C16H15Cl2N2O3þ, [M+H]+). Anal.
Calcd for C16H14Cl2N2O3: C, 54.41; H, 4.00; N, 7.39. Found: C, 54.38; H,
3.97; N, 7.42.
4.2.1.7. (E)-N-(2-(3,5-Dibromo-2-hydroxybenzylideneami-
no)ethyl)-2-hydroxybenzamide (1g). Yellow power, yield 79%,
mp: 145–147 °C, 1H NMR (300 MHz, CDCl3, d ppm): 3.79 (m, 2H);
3.90 (m, 2H); 6.70 (s, 1H); 6.83 (m, 1H); 6.98 (d, J = 8.2 Hz, 1H);
7.32 (m, 2H); 7.39 (m, 1H); 7.70 (s, 1H); 8.26 (s, 1H); 12.05 (s,
1H); 14.24 (s, 1H). ESI-MS: 441.9 (C16H15Br2N2O3þ, [M+H]+). Anal.
Calcd for C16H14Br2N2O3: C, 43.47; H, 3.19; N, 6.34. Found: C,
43.48; H, 3.17; N, 6.32.
4.2.1.8. (E)-2-Hydroxy-N-(2-(2-hydroxy-3,5-diiodobenzylide-
neamino)ethyl)benzamide (1h). Yellow power, yield 86%, mp:
123–125 °C, 1H NMR (300 MHz, CDCl3, d ppm): 3.80 (m, 2H);
3.91 (m, 2H); 6.71 (s, 1H); 6.84 (m, 1H); 6.99 (d, J = 8.3 Hz, 1H);
7.34 (m, 2H); 7.41 (m, 1H); 7.71 (s, 1H); 8.26 (s, 1H); 12.06 (s,
1H); 14.25 (s, 1H). ESI-MS: 536.9 (C16H15I2N2O3þ, [M+H]+). Anal.
4.2.1. General data of 1a–1k
4.2.1.1. (E)-2-Hydroxy-N-(2-(2-hydroxybenzylideneamino)ethyl)
benzamide (1a). Yellowpower, yield 88%, mp: 125–127 °C, 1H NMR

7868
K. Cheng et al. / Bioorg. Med. Chem. 17 (2009) 7861–7871
Calcd for C16H14I2N2O3: C, 35.85; H, 2.63; N, 5.23. Found: C, 35.92;
H, 3.67; N, 5.22.
1H); 7.28 (d, J = 8.45 Hz, 1H); 7.37 (m, 3H); 8.31 (s, 1H); 12.28 (s,
1H); 13.31 (s, 1H). ESI-MS: 425.0 (C17H18IN2O3þ, [M+H]+). Anal.
Calcd for C17H17IN2O3: C, 48.13; H, 4.04; N, 6.60. Found: C, 48.14;
H, 4.08; N, 6.63.
4.2.1.9. (E)-2-Hydroxy-N-(2-((2-hydroxynaphthalen-1-yl)meth-
yleneamino)ethyl)benzamide (1i). Yellow power, yield 84%, mp:
151–153 °C, 1H NMR (300 MHz, CDCl3, d ppm): 3.82 (m, 2H); 3.91
(m, 2H); 6.75 (m, 1H); 6.84 (d, J = 9.3 Hz, 1H); 6.98 (d, J = 8.4 Hz,
1H); 7.22 (m, 1H); 7.37 (m, 2H); 7.56 (m, 4H); 7.80 (d, J = 8.4 Hz,
1H); 8.80 (s, 1H); 12.17 (s, 1H); 14.44 (s, 1H). ESI-MS: 335.1
(C20H19N2O3þ, [M+H]+). Anal. Calcd for C20H18N2O3: C, 71.84; H,
5.43; N, 8.38. Found: C, 71.92; H, 5.47; N, 8.91.
4.2.2.6. N-(3-(3,5-Dichloro-2-hydroxybenzylideneamino)pro-
pyl)-2-hydroxybenzamide (2f). Yellow power, yield 89%, mp:
55–57 °C, 1H NMR (300 MHz, CDCl3, d ppm): 2.07 (m, 2H); 3.59
(m, 2H); 3.74 (m, 2H); 6.48 (s, 1H); 6.80 (m, 1H); 6.95 (d,
J = 8.22 Hz, 1H); 7.11 (s, 1H); 7.35 (m, 3H); 8.28 (s, 1H); 12.21 (s,
1H); 14.45 (s, 1H). ESI-MS: 425.0 (C17H17Cl2N2O3þ, [M+H]+). Anal.
Calcd for C17H16Cl2N2O3: C, 55.60; H, 4.39; N, 7.63. Found: C,
55.62; H, 4.38; N, 7.65.
4.2.1.10. N,N0-(Ethane-1,2-diyl)bis(2-hydroxybenzamide)
(1j). White power, yield 67%, mp: 111–113 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 3.94 (m, 4H); 6.71 (s, 2H); 6.80 (m, 2H); 6.98 (m,
2H); 7.20 (m, 2H); 7.29 (m, 2H); 13.19 (s, 2H). ESI-MS: 301.1
(C16H17N2O4þ, [M+H]+). Anal. Calcd for C16H16N2O4: C, 63.99; H,
5.37; N, 9.33. Found: C, 64.03; H, 5.33; N, 9.91.
4.2.2.7. N-(3-(3,5-Dibromo-2-hydroxybenzylideneamino)pro-
pyl)-2-hydroxybenzamide (2g). Yellow power, yield 88%, mp:
163–165 °C, 1H NMR (300 MHz, CDCl3, d ppm): 2.08 (m, 2H);
3.60 (m, 2H); 3.74 (m, 2H); 6.44 (s, 1H); 6.81 (m, 1H); 6.95 (d,
J = 8.25 Hz, 1H); 7.33 (m, 3H); 7.69 (s, 1H); 8.24 (s, 1H); 12.20 (s,
1H); 14.61 (s, 1H). ESI-MS: 456.9 (C17H17Br2N2O3þ, [M+H]+). Anal.
Calcd for C17H16Br2N2O3: C, 44.76; H, 3.54; N, 6.14. Found: C,
44.67; H, 3.58; N, 6.15.
4.2.1.11. N,N0-Disalicylalethylenediamine (1k). Yellow power,
yield 97%, mp: 73–75 °C, 1H NMR (300 MHz, CDCl3, d ppm): 3.91
(m, 4H); 6.86 (m, 2H); 6.95 (m, 2H); 7.21 (m, 2H); 7.31 (m, 2H);
8.36 (s, 2H); 13.21 (s, 2H). ESI-MS: 269.1 (C16H17N2O2þ, [M+H]+). Anal.
Calcd for C16H16N2O2: C, 71.62; H, 6.01; N, 10.44. Found: C, 71.52; H,
5.97; N, 10.41.
4.2.2.8. 2-Hydroxy-N-(3-(2-hydroxy-3,5-diiodobenzylideneami-
no)propyl)benzamide (2h). Yellow power, yield 86%, mp: 154–
155 °C, 1H NMR (300 MHz, CDCl3, d ppm): 2.08 (m, 2H); 3.61 (m,
2H); 3.75 (m, 2H); 6.44 (s, 1H); 6.80 (m, 1H); 6.95 (d, J = 8.40 Hz,
1H); 7.35 (m, 3H); 7.67 (s, 1H); 8.24 (s, 1H); 12.20 (s, 1H); 14.60
(s, 1H). ESI-MS: 550.9 (C17H17I2N2O3þ, [M+H]+). Anal. Calcd for
C17H16I2N2O3: C, 37.12; H, 2.93; N, 5.09. Found: C, 37.22; H, 2.95;
N, 5.11.
4.2.2. General data of 2a–2k
4.2.2.1. 2-Hydroxy-N-(3-(2-hydroxybenzylideneamino)pro-
pyl)benzamide (2a). Oil, yield 83%, 1H NMR (300 MHz, CDCl3, d
ppm): 2.05 (m, 2H); 3.58 (m, 2H); 3.69 (m, 2H); 6.52 (s, 1H);
6.77 (m, 1H); 6.89 (m, 1H); 6.96 (m, 2H); 7.30 (m, 4H); 8.64 (s,
þ
1H); 12.24 (s, 1H); 13.42 (s, 1H). ESI-MS: 299.1 (C17H19N2O3
,
[M+H]+). Anal. Calcd for C17H18N2O3: C, 68.44; H, 6.08; N, 9.39.
Found: C, 68.54; H, 6.15; N, 9.37.
4.2.2.9. 2-Hydroxy-N-(3-((2-hydroxynaphthalen-1-yl)methyle-
neamino)propyl)benzamide (2i). Yellow power, yield 77%, mp:
166–167 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.45 (m, 3H);
3.40 (m, 1H); 3.96 (m, 2H); 6.71 (m, 1H); 6.82 (d, J = 9.33 Hz,
1H); 6.96 (d, J = 8.40 Hz, 1H); 7.22 (m, 1H); 7.40 (m, 4H); 7.55 (d,
4.2.2.2. N-(3-(5-Fluoro-2-hydroxybenzylideneamino)propyl)-2-
hydroxybenzamide (2b). Yellow power, yield 90%, mp: 60–63 °C,
1H NMR (300 MHz, CDCl3, d ppm): 2.04 (m, 2H); 3.62 (m, 2H); 3.73
(m, 2H); 6.49 (s, 1H); 6.79 (m, 1H); 6.91 (d, J = 8.53 Hz, 1H); 6.99 (d,
J = 7.36 Hz, 1H); 7.25 (m, 2H); 7.42 (m, 2H); 8.35 (s, 1H); 12.28 (s,
1H); 13.28 (s, 1H). ESI-MS: 299.1 (C17H18FN2O3þ, [M+H]+). Anal.
Calcd for C17H17FN2O3: C, 64.55; H, 5.42; N, 8.86. Found: C,
64.54; H, 5.36; N, 8.91.
J = 9.12 Hz, 2H); 7.78 (d, J = 8.4 Hz, 1H); 12.24 (s, 1H); 14.61 (s,
þ
1H). ESI-MS: 349.1 (C21H21N2O3
,
[M+H]+). Anal. Calcd for
C21H20N2O3: C, 72.40; H, 5.79; N, 8.04. Found: C, 72.42; H, 5.75;
N, 8.06.
4.2.2.10. N,N0-(Propane-1,3-diyl)bis(2-hydroxybenzamide)
(2j). White power, yield 93%, mp: 181–182 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.87 (m, 2H); 3.59 (m, 4H); 6.88 (m, 2H); 6.99 (d,
J = 8.43 Hz, 2H); 7.27 (m, 2H); 7.41 (m, 2H); 8.49 (d, J = 8.07 Hz,
2H); 12.32 (s, 2H). ESI-MS: 316.1 (C17H19N2O4þ, [M+H]+). Anal.
Calcd for C17H18N2O4: C, 64.96; H, 5.77; N, 8.91. Found: C, 65.00;
H, 5.79; N, 8.88.
4.2.2.3. N-(3-(5-Chloro-2-hydroxybenzylideneamino)propyl)-2-
hydroxybenzamide (2c). Yellow power, yield 84%, mp: 98–
100 °C, 1H NMR (300 MHz, CDCl3, d ppm): 2.06 (m, 2H); 3.59 (m,
2H); 3.71 (m, 2H); 6.44 (s, 1H); 6.79 (m, 1H); 6.88 (d, J = 8.58 Hz,
1H); 6.96 (d, J = 7.86 Hz, 1H); 7.22 (m, 2H); 7.36 (m, 2H); 8.32 (s,
þ
1H); 12.28 (s, 1H); 13.28 (s, 1H). ESI-MS: 333.1 (C17H18ClN2O3
,
[M+H]+). Anal. Calcd for C17H17ClN2O3: C, 61.36; H, 5.15; N, 8.42;
Found: C, 61.44; H, 5.15; N, 8.38.
4.2.2.11. N,N0-Disalicylidenetrimethylenediamine (2k). Yellow
power, yield 70%, mp: 76–77 °C, 1H NMR (300 MHz, CDCl3, d
ppm): 2.09 (m, 2H); 3.68 (m, 4H); 6.87 (m, 2H); 6.96 (d,
J = 8.3 Hz, 2H); 7.23 (d, J = 6.0 Hz, 2H); 7.29 (m, 2H); 8.35 (s, 2H);
13.44 (s, 2H). ESI-MS: 283.1 (C17H19N2O2þ, [M+H]+). Anal. Calcd
for C17H18N2O2: C, 72.32; H, 6.43; N, 9.92. Found: C, 72.40; H,
6.45; N, 9.96.
4.2.2.4. N-(3-(5-Bromo-2-hydroxybenzylideneamino)propyl)-2-
hydroxybenzamide (2d). Yellow power, yield 91%, mp: 74–76 °C,
1H NMR (300 MHz, CDCl3, d ppm): 2.06 (m, 2H); 3.60 (m, 2H); 3.71
(m, 2H); 6.43 (s, 1H); 6.81 (m, 2H); 6.97 (d, J = 8.43 Hz, 1H); 7.30 (d,
J = 8.04 Hz, 1H); 7.38 (m, 3H); 8.30 (s, 1H); 12.27 (s, 1H); 13.29 (s,
1H). ESI-MS: 377.0 (C17H18BrN2O3þ, [M+H]+). Anal. Calcd for
C17H17BrN2O3: C, 54.13; H, 4.54; N, 7.44. Found: C, 54.14; H,
4.63; N, 7.47.
4.2.3. General data of 3a–3k
4.2.3.1. (E)-2-Hydroxy-N-(4-(2-hydroxybenzylideneami-
no)butyl)benzamide (3a). Yellow oil, yield 82%, 1H NMR
(300 MHz, CDCl3, d ppm): 1.78 (m, 4H); 3.48 (m, 2H); 3.62 (m,
4.2.2.5. 2-Hydroxy-N-(3-(2-hydroxy-5-iodobenzylideneami-
no)propyl)benzamide (2e). Yellow power, yield 79%, mp: 90–
92 °C, 1H NMR (300 MHz, CDCl3, d ppm): 2.05 (m, 2H); 3.56 (m,
2H); 3.70 (m, 2H); 6.44 (s, 1H); 6.85 (m, 2H); 6.96 (d, J = 7.86 Hz,
2H); 6.50 (s, 1H); 6.84 (m, 2H); 6.96 (m, 2H); 7.21 (m, 1H); 7.37
þ
(m, 3H); 8.34 (s, 1H); 13.23 (s, 2H). ESI-MS: 313.1 (C18H21N2O3
,
[M+H]+). Anal. Calcd for C18H20N2O3: C, 69.21; H, 6.45; N, 8.97.
Found: C, 69.32; H, 6.41; N, 8.96.

K. Cheng et al. / Bioorg. Med. Chem. 17 (2009) 7861–7871
7869
4.2.3.2. (E)-N-(4-(5-Fluoro-2-hydroxybenzylideneamino)butyl)-
2-hydroxybenzamide (3b). Yellow power, yield 89%, mp: 72–
74 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.78 (m, 4H); 3.51 (m,
2H); 3.64 (m, 2H); 6.41 (s, 1H); 6.80 (m, 1H); 6.91 (d, J = 8.6 Hz,
1H); 6.99 (m, 1H); 7.23 (m, 2H); 7.36 (m, 2H); 8.28 (s, 1H); 12.34
(s, 1H); 13.46 (s, 1H). ESI-MS: 331.1 (C18H20FN2O3þ, [M+H]+). Anal.
Calcd for C18H19FN2O3: C, 65.44; H, 5.80; N, 8.48; Found: C, 65.42;
H, 5.75; N, 8.46.
1H); 6.97 (d, J = 7.68 Hz, 1H); 7.24 (m, 1H); 7.39 (m, 3H); 7.61 (d,
J = 7.1 Hz, 1H); 7.69 (d, J = 9.3 Hz, 1H); 7.85 (d, J = 8.2 Hz, 1H);
8.74 (s, 1H); 12.35 (s, 1H); 14.55 (s, 1H). ESI-MS: 363.1
(C22H23N2O3þ, [M+H]+). Anal. Calcd for C22H22N2O3: C, 72.91; H,
6.12; N, 7.73. Found: C, 72.88; H, 6.17; N, 7.69.
4.2.3.10. N,N0-(Butane-1,4-diyl)bis(2-hydroxybenzamide)
(3j). White power, yield 90%, mp: 165–167 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.75 (m, 4H); 3.54 (m, 4H); 6.67 (s, 2H); 6.85 (m, 2H);
6.99 (d, J = 8.3 Hz, 2H); 7.39 (m, 4H); 12.28 (s, 2H). ESI-MS: 329.1
(C18H21N2O4þ, [M+H]+). Anal. Calcd for C18H21N2O4: C, 65.84; H, 6.14;
N, 8.53. Found: C, 68.73; H, 6.16; N, 8.56.
4.2.3.3. (E)-N-(4-(5-Chloro-2-hydroxybenzylideneamino)butyl)-
2-hydroxybenzamide (3c). Yellow power, yield 92%, mp: 72–
74 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.77 (m, 4H); 3.50 (m,
2H); 3.66 (m, 2H); 6.41 (s, 1H); 6.78 (m, 1H); 6.90 (d, J = 8.6 Hz,
1H); 6.96 (m, 1H); 7.21 (m, 2H); 7.35 (m, 2H); 8.28 (s, 1H); 12.33
(s, 1H); 13.46 (s, 1H). ESI-MS: 347.1 (C18H20ClN2O3þ, [M+H]+). Anal.
Calcd for C18H19ClN2O3: C, 62.34; H, 5.52; N, 8.08. Found: C, 62.32;
H, 5.55; N, 8.03.
4.2.3.11. N,N0-Disalicylidenetetramethylenediamine (3k). Yellow
power, yield 78%, mp: 131–134 °C, 1H NMR (300 MHz, CDCl3, d
ppm): 1.77 (m, 4H); 3.53 (m, 4H); 6.87 (m, 2H); 6.97 (d, J = 7.8 Hz,
2H); 7.41 (m, 4H); 8.25 (s, 2H); 12.31 (s, 2H). ESI-MS: 297.1
(C18H21N2O2þ, [M+H]+). Anal. Calcd for C18H20N2O2: C, 72.95; H, 6.80;
N, 9.45. Found: C, 72.89; H, 6.77; N, 9.47.
4.2.3.4. (E)-N-(4-(5-Bromo-2-hydroxybenzylideneamino)butyl)-
2-hydroxybenzamide (3d). Yellow power, yield 89%, mp: 75–
77 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.77 (m, 4H); 3.49 (m,
2H); 3.65 (m, 2H); 6.40 (s, 1H); 6.83 (m, 2H); 6.97 (d, J = 8.2 Hz,
1H); 7.38 (m, 4H); 8.28 (s, 1H); 12.32 (s, 1H); 13.50 (s, 1H).
ESI-MS: 391.1 (C18H20BrN2O3þ, [M+H]+). Anal. Calcd for C18H19Br-
N2O3: C, 55.26; H, 4.89; N, 7.16. Found: C, 55.31; H, 4.87; N,
7.11
4.2.4. General data of 4a–4k
4.2.4.1. (E)-2-Hydroxy-N-(6-(2-hydroxybenzylideneamino)hexyl)-
benzamide (4a). Yellow oil, yield 82%, 1H NMR (300 MHz, CDCl3, d
ppm): 1.45 (m, 4H); 1.69 (m, 4H); 3.46 (m, 2H); 3.59 (m, 2H); 6.43
(s, 1H); 6.84 (m, 2H); 6.96 (m, 2H); 7.24 (m, 1H); 7.33 (m, 1H); 7.36
þ
(m, 2H); 8.33 (s, 1H); 12.70 (s, 2H). ESI-MS: 341.1 (C20H25N2O3
,
[M+H]+). Anal. Calcd for C20H24N2O3: C, 70.56; H, 7.11; N, 8.23.
Found: C, 70.62; H, 7.07; N, 8.17.
4.2.3.5. (E)-2-Hydroxy-N-(4-(2-hydroxy-5-iodobenzylideneami-
no)butyl)benzamide (3e). Yellow power, yield 84%, mp: 79–
81 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.78 (m, 4H); 3.48 (m,
2H); 3.66 (m, 2H); 6.40 (s, 1H); 6.85 (m, 2H); 6.96 (d, J = 8.3 Hz,
1H); 7.35 (m, 4H); 8.28 (s, 1H); 12.32 (s, 1H); 13.51 (s, 1H). ESI-
MS: 339.0 (C18H20IN2O3þ, [M+H]+). Anal. Calcd for C18H19IN2O3:
C, 49.33; H, 4.37; N, 6.39. Found: C, 49.31; H, 4.33; N, 6.42.
4.2.4.2. (E)-N-(6-(5-Fluoro-2-hydroxybenzylideneamino)hexyl)-
2-hydroxybenzamide (4b).
Yellow power, yield 88%, mp:
86–89 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.42 (m, 4H); 1.69
(m, 4H); 3.45 (m, 2H); 3.59 (m, 2H); 6.41 (s, 1H); 6.84 (m, 1H);
6.88 (d, J = 8.7 Hz, 1H); 6.97 (d, J = 8.4 Hz, 1H); 7.21 (m, 2H); 7.38
(m, 2H); 8.26 (s, 1H); 12.37 (s, 1H); 13.62 (s, 1H). ESI-MS: 359.1
(C20H25N2O3þ, [M+H]+). Anal. Calcd for C20H24N2O3: C, 67.02; H,
6.47; N, 7.82. Found: C, 67.01; H, 6.43; N, 7.81.
4.2.3.6. (E)-N-(4-(3,5-Dichloro-2-hydroxybenzylideneami-
no)butyl)-2-hydroxybenzamide (3f). Yellow power, yield 85%,
mp 118–120 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.77 (m, 4H);
3.51 (m, 2H); 3.69 (m, 2H); 6.51 (s, 1H); 6.83 (m, 1H); 6.98 (d,
J = 8.0 Hz, 1H); 7.14 (s, 1H); 7.41 (m, 3H); 8.25 (s, 1H); 12.31 (s,
1H); 14.65 (s, 1H). ESI-MS: 381.1 (C18H19Cl2N2O3þ, [M+H]+). Anal.
Calcd for C18H18Cl2N2O3: C, 56.71; H, 4.76; N, 7.35. Found: C,
56.67; H, 4.73; N, 7.41.
4.2.4.3. (E)-N-(6-(5-Chloro-2-hydroxybenzylideneamino)hexyl)-
2-hydroxybenzamide (4c). Yellow power, yield 87%, mp: 90–
101 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.43 (m, 4H); 1.68 (m,
4H); 3.46 (m, 2H); 3.60 (m, 2H); 6.41 (s, 1H); 6.84 (m, 1H); 6.89
(d, J = 8.8 Hz, 1H); 6.98 (d, J = 8.0 Hz, 1H); 7.20 (m, 2H); 7.38 (m,
2H); 8.25 (s, 1H); 12.36 (s, 1H); 13.61 (s, 1H). ESI-MS: 375.1
(C20H24ClN2O3þ, [M+H]+). Anal. Calcd for C20H23ClN2O3: C, 64.08;
H, 6.18; N, 7.47. Found: C, 64.01; H, 6.16; N, 6.21.
4.2.3.7. (E)-N-(4-(3,5-Dibromo-2-hydroxybenzylideneami-
no)butyl)-2-hydroxybenzamide (3g). Yellow power, yield 91%,
mp: 110–111 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.76 (m, 4H);
3.49 (m, 2H); 3.69 (m, 2H); 6.48 (s, 1H); 6.84 (m, 1H); 6.98 (d,
J = 8.2 Hz, 1H); 7.57 (m, 3H); 7.69 (s, 1H); 8.21 (s, 1H); 12.30 (s,
1H); 14.79 (s, 1H). ESI-MS: 470.9 (C18H19Br2N2O3þ, [M+H]+). Anal.
Calcd for C18H18Br2N2O3: C, 45.98; H, 3.86; N, 5.96. Found: C,
46.02; H, 3.71; N, 5.91.
4.2.4.4. (E)-N-(6-(5-Bromo-2-hydroxybenzylideneami-
no)hexyl)-2-hydroxybenzamide (4d). Yellow power, yield 92%,
mp: 90–101 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.44 (m, 4H);
1.69 (m, 4H); 3.45 (m, 2H); 3.60 (m, 2H); 6.37 (s, 1H); 6.83 (m,
2H); 6.98 (d, J = 8.2 Hz, 1H); 7.38 (m, 4H); 8.25 (s, 1H); 12.38 (s,
1H); 13.67 (s, 1H). ESI-MS: 419.1 (C20H24BrN2O3þ, [M+H]+). Anal.
Calcd for C20H23BrN2O3: C, 57.29; H, 5.53; N, 6.68. Found: C,
57.31; H, 5.56; N, 6.73.
4.2.3.8. (E)-2-Hydroxy-N-(4-(2-hydroxy-3,5-diiodobenzylide-
neamino)butyl)benzamide (3h). Yellow power, yield 90%, mp:
130–133 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.77 (m, 4H);
3.50 (m, 2H); 3.69 (m, 2H); 6.48 (s, 1H); 6.85 (m, 1H); 6.97 (d,
J = 8.1 Hz, 1H); 7.55 (m, 3H); 7.70 (s, 1H); 8.21 (s, 1H); 12.31 (s,
1H); 14.80 (s, 1H). ESI-MS: 564.9 (C18H19I2N2O3þ, [M+H]+). Anal.
Calcd for C18H18I2N2O3: C, 38.32; H, 3.22; N, 4.97. Found: C,
38.23; H, 3.19; N, 4.96.
4.2.4.5. (E)-2-Hydroxy-N-(6-(2-hydroxy-5-iodobenzylideneami-
no)hexyl)benzamide (4e). Yellow power, yield 83%, mp: 112–
114 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.43 (m, 4H); 1.67 (m,
4H); 3.45 (m, 2H); 3.61 (m, 2H); 6.37 (s, 1H); 6.84 (m, 2H); 6.97
(d, J = 8.0 Hz, 1H); 7.40 (m, 4H); 8.26 (s, 1H); 12.38 (s, 1H); 13.66
(s, 1H). ESI-MS: 467.1 (C20H24IN2O3þ, [M+H]+). Anal. Calcd for
C20H23IN2O3: C, 51.51; H, 4.97; N, 6.01. Found: C, 51.47; H, 4.51;
N, 5.96.
4.2.3.9. (E)-2-Hydroxy-N-(4-((2-hydroxynaphthalen-1-yl)meth-
yleneamino)butyl)benzamide (3i). Yellow power, yield 95%, mp:
136–137 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.80 (m, 4H); 3.52
(m, 2H); 3.68 (m, 2H); 6.74 (s, 1H); 6.81 (m, 1H); 6.92 (d, J = 9.3 Hz,

7870
K. Cheng et al. / Bioorg. Med. Chem. 17 (2009) 7861–7871
4.2.4.6. (E)-N-(6-(3,5-Dichloro-2-hydroxybenzylideneami-
no)hexyl)-2-hydroxybenzamide (4f). Yellow power, yield 91%,
mp: 125–127 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.43 (m, 4H);
1.65 (m, 4H); 3.44 (m, 2H); 3.62 (m, 2H); 6.45 (s, 1H); 6.82 (m,
In a final 20
0.5 mM DTT, 0.25 mM MgCl2, and 2.5
with 1 nM ecKAS III, and H2O was added to 15
bation, a 2 L mixture of 25 M acetyl-CoA, 0.5 mM NADH, and
0.5 mM NADPH was added for ecKAS III reaction for 25 min. The
reaction was stopped by adding 20 L of ice-cold 50% TCA, incubat-
ing for 5 min on ice, and centrifuging to pellet the protein. The pel-
l
L reaction, 20 mM Na2HPO4/NaH2PO4, pH 7.0,
M holo-ACP were mixed
L. After 1 min incu-
l
l
l
l
1H); 6.97 (d, J = 7.8 Hz, 1H); 7.11 (m, 1H); 7.37 (m, 3H); 8.19 (s,
þ
1H); 12.33 (s, 1H); 14.76 (s, 1H). ESI-MS: 409.1 (C20H23Cl2N2O3
,
l
[M+H]+). Anal. Calcd for C20H22Cl2N2O3: C, 58.69; H, 5.42; N, 6.84.
Found: C, 58.49; H, 5.41; N, 6.88.
let 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 prod-
uct was read by liquid scintillation. When determining the inhibi-
tion constant (IC50), inhibitors were added from a concentrated
DMSO stock such that the final concentration of DMSO did not ex-
ceed 2%.
4.2.4.7. (E)-N-(6-(3,5-Dibromo-2-hydroxybenzylideneami-
no)hexyl)-2-hydroxybenzamide (4g). Yellow power, yield 87%,
mp: 130–131 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.44 (m, 4H);
1.65 (m, 4H); 3.44 (m, 2H); 3.63 (m, 2H); 6.38 (s, 1H); 6.83 (m,
1H); 6.98 (d, J = 8.0 Hz, 1H); 7.28 (m, 1H); 7.37 (m, 3H); 8.16 (s,
þ
1H); 12.35 (s, 1H); 14.91 (s, 1H). ESI-MS: 499.0 (C20H23Br2N2O3
,
4.4. Antimicrobial activity
[M+H]+). Anal. Calcd for C20H22Br2N2O3: C, 48.22; H, 4.45; N,
5.62. Found: C, 48.09; H, 4.41; N, 5.58.
The antibacterial activity of the synthesized compounds was
tested against E. coli, P. aeruginosa, B. subtilis 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 of
the test compounds were determined by a colorimetric method
using the dye MTT.23 A stock solution of the synthesized com-
4.2.4.8. (E)-2-Hydroxy-N-(6-(2-hydroxy-3,5-diiodobenzylide-
neamino)hexyl)benzamide (4h). Yellow power, yield 85%, mp:
143–145 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.43 (m, 4H);
1.66 (m, 4H); 3.44 (m, 2H); 3.65 (m, 2H); 6.39 (s, 1H); 6.85 (m,
1H); 6.97 (d, J = 7.9 Hz, 1H); 7.28 (m, 1H); 7.38 (m, 3H); 8.17 (s,
pound (50 lg/mL) in DMSO was prepared and graded quantities
þ
1H); 12.31 (s, 1H); 14.90 (s, 1H). ESI-MS: 592.9 (C20H23I2N2O3
,
of the test compounds were incorporated in specified quantity
of sterilized liquid medium (MH medium for antibacterial activ-
ity and RPMI-1640 medium for antifungal activity). A specified
quantity of the medium containing the compound was poured
into microtitration plates. Suspension of the microorganism
was prepared to contain approximately 105 cfu/mL and applied
to microtitration plates with serially diluted compounds in
DMSO to be tested and incubated at 37 °C for 24 h for bacterial.
After the MICs were visually determined on each of the microt-
[M+H]+). Anal. Calcd for C20H22I2N2O3: C, 40.56; H, 3.74; N, 4.73.
Found: C, 40.59; H, 3.71; N, 4.68.
4.2.4.9. (E)-2-Hydroxy-N-(6-((2-hydroxynaphthalen-1-yl)meth-
yleneamino)hexyl)benzamide (4i). Yellow power, yield 82%, mp:
139–141 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.49 (m, 4H); 1.77
(m, 4H); 3.47 (m, 2H); 3.62 (m, 2H); 6.79 (m, 1H); 6.89 (s, 1H); 6.95
(m, 2H); 7.23 (m, 1H); 7.42 (m, 3H); 7.61 (d, J = 7.8 Hz, 1H); 7.68 (d,
J = 9.1 Hz, 1H); 7.84 (d, J = 8.4 Hz, 1H); 8.69 (s, 1H); 12.44 (s, 1H);
14.55 (s, 1H). ESI-MS: 391.2 (C24H27N2O3þ, [M+H]+). Anal. Calcd
for C24H26N2O3: C, 73.82; H, 6.71; N, 7.17. Found: C, 73.77; H,
6.65; N, 7.21.
itration 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 well was
4.2.4.10. N,N0-(Hexane-1,6-diyl)bis(2-hydroxybenzamide)
(4j). White power, yield 86%, mp: 171–173 °C, 1H NMR (300 MHz,
CDCl3, d ppm): 1.44 (m, 4H); 1.68 (m, 4H); 3.63 (m, 4H); 6.39 (s,
2H); 6.91 (m, 2H); 7.22 (m, 2H); 7.60 (d, J = 7.3 Hz, 2H); 7.85 (m,
2H); 12.41 (s, 2H). ESI-MS: 357.2 (C20H25N2O4þ, [M+H]+). Anal.
Calcd for C20H24N2O4: C, 67.40; H, 6.79; N, 7.86. Found: C, 67.52;
H, 6.81; N, 7.88.
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 were presented
in Table 3.
4.5. Experimental protocol of docking study
4.2.4.11. N,N0-Disalicylidene-1,6-hexanediamine (4k). Yellow
power, yield 81%, mp: 133–134 °C, 1H NMR (300 MHz, CDCl3, d
ppm): 1.26 (m, 4H); 1.54 (m, 4H); 3.63 (m, 4H); 6.85 (m, 2H);
The automated docking studies were carried out using Auto-
Dock version 4.0. First, AutoGrid component of the program
pre-calculates a three-dimensional grid of interaction energies
based on the macromolecular target using the AMBER force field.
The cubic grid box of 60 Å size (x, y, z) with a spacing of 0.375 Å
and grid maps were created representing the catalytic active tar-
get site region where the native ligand was embedded. Then
automated docking studies were carried out to evaluate the bind-
ing free energy of the inhibitors within the macromolecules. The
GALS search algorithm (genetic algorithm with local search) was
chosen to search for the best conformers. The parameters were
set using the software ADT (Auto-Dock Tool Kit) on PC which is
associated with Auto-Dock 4.0. For all docking parameters, de-
fault values were used with 20 independent docking runs for each
docking case. The AutoDock performs the task of the docking,
where the ligand moves randomly in any one of six degrees of
freedom, and the energy of the new ligand ‘state’ is calculated.
If the energy of the new state is lower than that of the old state,
the new one is automatically accepted as the next step in
docking.
6.95 (m, 2H); 7.22(m, 2H); 7.85 (m, 2H); 8.72 (s, 2H); 13.61 (s,
þ
2H). ESI-MS: 345.2 (C20H25N2O2
,
[M+H]+). Anal. Calcd for
C20H24N2O2: C, 74.04; H, 7.46; N, 8.64. Found: C, 74.12; H, 7.42;
N, 7.51.
4.3. E. coli KAS III purification and activity assay
Native E. coli KAS III protein was expressed in E. coli strain
BL21(DE3) using the pET30 vector. Harvested cells containing
KAS III were lysed by sonication in 20 mM Tris, pH 7.6, 5 mM imid-
azole, 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 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 ecKAS III were concentrated
up to 2 mg/mL and stored at ꢂ80 °C in 20 mM Tris, pH 7.6, 100 mM
NaCl, 1 mM DTT, and 20% glycerol for enzymatic assay.

K. Cheng et al. / Bioorg. Med. Chem. 17 (2009) 7861–7871
7871
4.5.1. Preparation of ligands and target ecKAS III
References and notes
The compounds were studied for their binding activities into
protein ecKAS III. The three-dimensional structures of the afore-
mentioned compounds were constructed using Chem 3D ultra
11.0 software [Chemical Structure Drawing Standard; Cambridge
Soft corporation, USA (2009)], then they were energetically mini-
mized by using MOPAC with 100 iterations and minimum RMS
gradient of 0.10. The Gasteiger–Hückel charges of ligands were as-
signed. The crystal structures of protein ecKAS III (PDB code: 1hnj)
complex were retrieved from the RCSB Protein Data Bank (http://
www.rcsb.org/pdb/home/home.do). All bound waters and ligands
were eliminated from the protein and the polar hydrogens and
the Kollman-united charges were added to the proteins. The amino
acids of the ligand-target binding site were defined using data in
pdbsum (http://www.ebi.ac.uk/pdbsum/).
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4.5.2. Molecular modeling and analysis of the docked results
The predicted binding free energy that includes the intermolec-
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ranking. Furthermore, the intermolecular hydrogen bonds, whose
effect has already been counted in the binding energy, were also
investigated in order to find useful information for drug design.
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mean square (RMS) tolerance of 0.5 ÅA. Each of the clusters that
exhibited significant negative interaction energies was examined
by Accelrys, DS visualizer 2.0 [Accelrys Inc., San Diego, CA
(2007)] to determine their binding orientations, molecular model-
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The work was financed by a grant (Project 30772627) from Na-
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Supplementary data
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