Synthesis, molecular modeling and biological evaluation of PSB as targeted antibiotics

Article abstract of DOI:10.1016/j.bmc.2010.02.052

We described here the design, synthesis, molecular modeling, and biological evaluation of a series of peptide and Schiff bases (PSB) small molecules, inhibitors of Escherichia coli β-Ketoacyl-acyl carrier protein synthase III (ecKAS III). The initial lead compound was reported by us previously, we continued to carry out structure-activity relationship studies and optimize the lead structure to potent inhibitors in this research. The results demonstrated that both N-(2-(3,5-dichloro-2-hydroxybenzylideneamino)propyl)-2-hydroxybenzamide (1f) and 2-hydroxy-N-(2-(2-hydroxy-5-iodobenzylideneamino)propyl)-4-methylbenzamide (3e) posses good ecKAS III inhibitory activity and well binding affinities by bonding Gly152/Gly209 of ecKAS III and fit into the mouth of the substrate tunnel, and can be as potential antibiotics agent, displaying minimal inhibitory concentration values in the range 0.20-3.13 μg/mL and 0.39-3.13 μg/mL against various bacteria.

Full text of DOI:10.1016/j.bmc.2010.02.052

Bioorganic & Medicinal Chemistry 18 (2010) 2447–2455
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, molecular modeling and biological evaluation of PSB
as targeted antibiotics
**
*
*
Kui Cheng, Qing-Zhong Zheng, Jin Hou, Yang Zhou, Chang-Hong Liu , 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:
We described here the design, synthesis, molecular modeling, and biological evaluation of a series of pep-
tide and Schiff bases (PSB) small molecules, inhibitors of Escherichia coli b-Ketoacyl-acyl carrier protein
synthase III (ecKAS III). The initial lead compound was reported by us previously, we continued to carry
out structure–activity relationship studies and optimize the lead structure to potent inhibitors in this
research. The results demonstrated that both N-(2-(3,5-dichloro-2-hydroxybenzylideneamino)propyl)-
2-hydroxybenzamide (1f) and 2-hydroxy-N-(2-(2-hydroxy-5-iodobenzylideneamino)propyl)-4-methylb-
enzamide (3e) posses good ecKAS III inhibitory activity and well binding affinities by bonding Gly152/
Gly209 of ecKAS III and fit into the mouth of the substrate tunnel, and can be as potential antibiotics
Received 9 January 2010
Revised 23 February 2010
Accepted 24 February 2010
Available online 2 March 2010
Keywords:
Peptide & Schiff base (PSB)
ecKAS III
Antibacterial activity
Molecular modeling
agent, displaying minimal inhibitory concentration values in the range 0.20–3.13
3.13 g/mL against various bacteria.
lg/mL and 0.39–
l
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Pioneering reports demonstrated that platencin,15 thiolactomy-
cin16 and its analogues compounds17 had low toxicity and some of
which were highly active against KAS III. In particular, Schiff base
compounds were reported as a potent inhibitor of Helicobacter py-
lori KAS I,18 Plasmodium falciparum KAS III19 and ecKAS III.20 In the
previous research, we performed that a kind of peptide and Schiff
bases (PSB) small molecules with electron-withdrawing groups on
B-ring and appropriate length of aliphatic chain (n = 2 or 3) were
important factors responsible for the inhibitory activities against
ecKAS III.21
Multi-drug-resistant Gram-positive and Gram-negative patho-
gens have become a serious problem in hospitals and the commu-
nity.1 Particularly alarming is the emergence of methicillin-
resistant Staphylococcus aureus (MRSA) and Staphylococcus epide-
rmidis (MRSE) and vancomycin-resistant Enteroccocus faecium
(VRE).2,3 Drug discovery efforts have been significantly intensified
in the past years to search for more effective antibacterial agents
with a broader spectrum of activity and especially activity against
resistant pathogens to fight infectious diseases.4–6
In this study, our main objective is development of potential
inhibitors by newly synthesized PSB as targeted antibiotics agents,
based on molecular modeling and investigation of SAR between
new inhibitors and ecKAS III. Firstly, we used structure-based de-
sign method to synthesize the PSB by introducing substitute group
on A-ring and using 1,2-propanediamine as connect aliphatic chain
(Scheme 1) and test their inhibitory activity against ecKAS III. And
following, top 10 PSB compounds which posses good ecKAS III
inhibitory activity (low IC50) 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 S.
aureus ATCC 6538), expecting to exploit potent antibacterial agent
with broad-spectrum antibiotics activity. As expected, the results
demonstrated that the antimicrobial compound, N-(2-(3,5-di-
chloro-2-hydroxybenzylideneamino)propyl)-2-hydroxybenzamide
(1f) and 2-hydroxy-N-(2-(2-hydroxy-5-iodobenzylideneamino)
propyl)-4-methylbenzamide (3e), can be as an inhibitor of ecKAS
III and as potential antibiotics agent, displaying MIC values in the
Bacterial type II fatty-acid synthesis (FASII) is an attractive tar-
get for antibacterial drug discovery.7–9 The initiation condensing
enzyme, Escherichia coli b-Ketoacyl-acyl carrier protein synthase
III (ecKAS III), and elongation condensing enzymes, KAS I/II, are
essential components of fatty-acid biosynthesis10,11 and are highly
conserved among key pathogens.12 Notably, KAS III, regulates the
fatty-acid biosynthesis rate via an initiation pathway and its sub-
strate specificity is a key factor in membrane fatty-acid composi-
tion and this protein represents
a promising target for the
antimicrobial drugs design.13 Moreover, the three-dimensional
structure of the protein is highly conserved in various bacteria,
and its inhibitors may thus act as potent antibiotics with broad-
spectrum activity.14
* Corresponding authors. Tel.: +86 25 8359 2672; fax: +86 25 8359 2572.
** Co-corresponding author.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.052

2448
K. Cheng et al. / Bioorg. Med. Chem. 18 (2010) 2447–2455
O
O
OH
98% H2SO4
-H2O
+
CH3CH2OH
O
OH
OH
O
O
DCC, THF, rt, 18h
OH
O
HO
+
R
R
OH
OH
NH2
R = 5-Bror 4-Me
H2N
R2
R2
110 oC, 5h
O
II O
O
B
R3
H
OH
N
NH2
N
R3
IV
N
H
OH
A
A
III
R1
H
MeOH, 50 oC, 3h
R1
H
OH
OH
I
1a-1h, 2a-2h, 3a-3h
1: R1 = H
2: R1 = 5-Br
3: R1 = 4-Me
O
II
O
B
N
H
OH
N
III
R1
H
MeOH, 50 oC, 3h
IV
OH
H
OH
I
1i - 3i
1a
R1 = R2 = R3 = H
1b R1 = R3 = H, R2 = F
2a R1 = 5-Br, R2 = R3 = H
2b R1 = 5-Br, R3 = H, R2 = F
2c R1 = 5-Br, R3 = H, R2 = Cl
3a
3b
3c
R1 = 4-Me, R2 = R3 = H
R1 = 4-Me, R3 = H, R2 = F
R1 = 4-Me, R3 = H, R2 = Cl
1c R1 = R3 = H, R2 = Cl
1d
2d
2e R1 = 5-Br, R3 = H, R2 = I
R1 = R3 = H, R2 = Br
R1 = 5-Br, R3 = H, R2 = Br
3d R1 = 4-Me, R3 = H, R2 = Br
1e R1 = R3 = H, R2 = I
3e R1 = 4-Me, R3 = H, R2 = I
1f
1g
R1 = H, R2 = R3 = Cl
R1 = H, R2 = R3 = Br
2f
R1 = 5-Br, R2 = R3 = Cl
3f
R1 = 4-Me, R2 = R3 = Cl
2g R1 = 5-Br, R2 = R3 = Br
2h R1 = 5-Br, R2 = R3 = I
3g R1 = 4-Me, R2 = R3 = Br
1h R1 = H, R2 = R3 = I
1i
3h
3i
R1 = 4-Me, R2 = R3 = I
R1 = 4-Me
1i
R1 = H
R1 = 5-Br
Scheme 1. The general procedure of the synthesized compounds. ‘I’, ‘II’, ‘III’, ‘IV’ represent the atom number bonding to the amino acid active site.
range 0.20–3.13 lg/mL and 0.39–3.13 lg/mL against various bac-
Table 1
Crystal and experimental data for complexes 1f
teria. This is much better than or compare to the result reported
previously.21 Computational docking simulation study for the top
10 inhibitors docked into the active sites of ecKAS III also have
been carried out.
Compound
1f
Empirical formula
Mr
C17H16Br2N2O3
456.12
T (K)
298(2)
Radiation (Mo K
a
), k (Å)
0.71073
Needle/yellow
0.31 ꢀ 0.20 ꢀ 0.12
Monoclinic
P21/c
14.860(3)
16.964(3)
7.066(1)
90.00
95.71(3)
90.00
1772.4(6)
4
1.709
4.591
904
0.944
0.0608, 0.1213
0.0993, 0.1490
2. Result and discussion
2.1. Chemistry
Crystal shape/color
Crystal size (mm3)
Crystal system
Space group
a (Å)
In this study, 27 PSB were subjected by reacting salicylamide, 5-
bromo salicylamide, 4-methyl salicylamide primary amines with
salicylaldehyde or its derivatives. The general method for prepar-
ing the compounds is outlined in Scheme 1. Salicylamide type’s
primary amines, which were not commercially available, were syn-
thesized using modified procedures of Kido et al.22 The crude prod-
ucts were purified using silica gel column chromatography,
preparative TLC and recrystallization. All the compounds gave sat-
isfactory chemical analyses ( 0.4%). 1H NMR, ESI-MS spectra and
elemental analyses were consistent with the assigned structures.
Compound 1f 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-
b (Å)
c (Å)
a
(°)
b (°)
c
(°)
V (Å3)
Z
Dcalcd (g/cmꢁ3
)
l
(mmꢁ1
)
F(0 0 0)
Goodness-of-fit on F2
R1, wR [I ꢂ 2
r
(I)]a
a
R1, wR (all data)
w(F2o ꢁ F2c )2/
R r
w(F2o)2]1/2, w1 = [ 2(Fo)2 + (0.1009
a
R1
=
R
||Fo| ꢁ |Fc||/
R
|Fo|, wR2 = [
R
(F2o þ 2Fc2)/3)2]ꢁ1, w2 = [
r
2(Fo)2 + (0.0962(F2o þ 2Fc2)/3)2]ꢁ1
.

K. Cheng et al. / Bioorg. Med. Chem. 18 (2010) 2447–2455
2449
a
b
Figure 1. Molecular structure of compound 1f with atoms and hydrogen bonds labeling (1a) and
pꢃ ꢃ ꢃp interaction between the two benzyl rings (1b).
tem. In the structure of compound 1f, there are intramolecular O–
Hꢃ ꢃ ꢃO (O1–H1Bꢃ ꢃ ꢃO2, 2.506(3) Å, 146.6°), O–Hꢃ ꢃ ꢃN hydrogen bond
(O3–H3Bꢃ ꢃ ꢃN2: 2.558(7) Å, 136.9°) and an intermolecular N–
Hꢃ ꢃ ꢃO hydrogen bond (N1–H1Aꢃ ꢃ ꢃO3i: 2.909(3) Å, 149.9° (1a), sym-
metry codes: i, x, ꢁy + 1/2, z + 1/2) in the structure. The dihedral
angle between the two benzene rings is 34.1°. The bond length
of N1–C7 (1.344(8) Å) indicates a single bond and bond length of
N2@C10 (1.284(8) Å) exhibits a double bond. The two benzyl rings
Table 2
have strong
p p interaction with distance of 3.566(6) Å (1b),
ꢃ ꢃ ꢃ
Inhibition constant (IC50) against ecKAS III
making the structure stable in the crystal configuration.
Compound
IC50
g/mL)
Compound
IC50
g/mL)
Compound
IC50
g/mL)
(
l
(
l
(l
2.2. Inhibitory activities of PSB against ecKAS III
1a
1b
1c
1d
1e
1f
1g
1h
1i
8.14
2a
2b
2c
2d
2e
2f
2g
2h
2i
>50
22.12
>50
16.71
9.32
8.75
>50
3a
3b
3c
3d
3e
3f
3g
3h
3i
7.88
5.66
8.64
6.33
0.45
1.75
5.37
12.11
5.46
6.03
0.78
0.31
3.13
2.56
19.75
All the synthesized compounds (1a–1i, 2a–2i and 3a–3i) were
tested for inhibitory activity against ecKAS III. The inhibition con-
stant (IC50) of the compounds are presented in Table 2 and Figure
2 (IC50 more than 50 lg/mL not demonstrated in Fig. 2). It was ob-
served that many compounds have been found to show fairly good
>50
10.94
14.95
4.68
inhibitory activity displaying IC50 values between 0.31 and
22.12
lg/mL. Inspection of the chemical structure of the
IC50
20
15
10
5
0
1a-- -- --1e-- -- -- 1i
2a-- -- --2e-- -- -- 2i
Compounds
3a-- -- --3e-- -- -- 3i --
Figure 2. Inhibitory activities of the compounds. IC50 more than 50
lg/mL not demonstrated.

2450
K. Cheng et al. / Bioorg. Med. Chem. 18 (2010) 2447–2455
compounds (Scheme 1) suggested that it could be divided into two
subunits: A- and B-rings.
in the improving of their inhibitory activity (1c–1h). Introduced the
bromo substitution group on A-ring (2a), the inhibitory activity was
The initial lead compound was reported by us previously. Here
we used 1,2-propanediamine as connect aliphatic chain for the
appropriate length of aliphatic chain (n = 2 or 3) showed better
inhibitory activities.21 Subsequently SAR studies were performed
by modification of the parent compound to determine how the sub-
stituents of the subunits affected the inhibitory activities. Replace-
remarkably decreasing (IC50 >50 lg/mL) compared to 1a. When
modified the substituents on B-ring with one or two halogen atoms
(2b–2i), the inhibitory activity had a few improving, but the best
compounds 2e and 2f still turned out to be less potent than 1a. For
more extensive evidences, we attempted further modification of
thesubstituent on A-ringby a methyl group, and theresults are sum-
marized in Table 2 and Figure 2. As shown, 3a had an increase in the
IC50 valuecomparedto 1a. Halogensubstituentsat theB-ringalsore-
ment of H atom (1a IC50 8.14 lg/mL) at R2-position by one halogen
atom or R2- and R3-position of B-ring by two halogen atoms resulted
Table 3
Antimicrobial activity and Docking parameters of the top 10 compounds
Top 10 compounds
Minimum inhibitory concentrations (
l
g/mL)
Docking parameters
Gb (kcal/mol) Hydrogen bond
Escherichia coli
Pseudomonas aeruginosa
Bacillus subtilis
Staphylococcus aureus
D
1f
3e
1e
3f
0.39
0.20
1.56
3.13
ꢁ9.21
N
O
N
O
N
III–Hꢃ ꢃ ꢃO/Gly209
IV–Hꢃ ꢃ ꢃO/Gly152
III–Hꢃ ꢃ ꢃO/Gly152
IV–Hꢃ ꢃ ꢃO/Gly209
III–Hꢃ ꢃ ꢃO/Gly152
0.39
6.25
3.13
3.13
3.13
25.00
1.56
0.78
1.56
1.56
12.5
6.25
ꢁ9.77
ꢁ9.41
ꢁ8.17
OIV–Hꢃ ꢃ ꢃO/Gly209
OI–Hꢃ ꢃ ꢃN/Gly152
O
O
IV–Hꢃ ꢃ ꢃO/Gly209
IV–Hꢃ ꢃ ꢃO/Gly152
1h
1g
>50.00
25.00
6.25
0.78
12.50
12.50
12.50
6.25
ꢁ7.05
ꢁ7.13
OI–Hꢃ ꢃ ꢃO/Gly152
OIIꢃ ꢃ ꢃH–N/Arg36
OI–Hꢃ ꢃ ꢃO/Gly209
OIIꢃ ꢃ ꢃN–H/Asn247
OIV–Hꢃ ꢃ ꢃO/Gly152
OI–Hꢃ ꢃ ꢃO/Gly209
OI–Hꢃ ꢃ ꢃO/Asn247
3i
12.5
>50.00
6.25
>50.00
ꢁ8.39
3g
1c
3b
0.78
12.5
6.25
3.13
12.50
3.13
>50.00
3.13
3.13
>50.00
3.13
>50.00
>50.00
12.50
1.56
ꢁ7.93
ꢁ7.12
ꢁ6.73
Kanamycin
1.56
Figure 3. Compounds 1f, 3e and 1e bound into ecKAS III receptor site via hydrophilic binding. (a) 1f is in grey, 3e is in red and 1e is in blue. (b) eg. 1e is bonding into the
mouth of the substrate tunnel. (c) 1f bonding into esKAS III via NIII–Hꢃ ꢃ ꢃO/Gly209 (2.20 Å, 158.5°) and OIV–Hꢃ ꢃ ꢃO/Gly152 (1.91 Å, 163.9°). (d) 3e bonding into esKAS III via NIII
–
Hꢃ ꢃ ꢃO/Gly152 (1.99 Å, 136.9°) and OIV–Hꢃ ꢃ ꢃO/Gly209 (1.99 Å, 139.3°). (e) 1e bonding into esKAS III via NIII–Hꢃ ꢃ ꢃO/Gly152 (2.23 Å, 136.8°) and OIV–Hꢃ ꢃ ꢃO/Gly209 (1.99 Å,
151.3°).

K. Cheng et al. / Bioorg. Med. Chem. 18 (2010) 2447–2455
2451
sulted in increased IC50 values (3b–3f, 3i). This suggested that com-
pounds with electron-withdrawing groups on R1 and R2 and none of
the electron-withdrawing groups on A-ring showed better inhibi-
tory activities. The introduction of naphthyl substitute of phenyl
antibiotic kanamycin with corresponding MIC of 3.13, 3.13, 1.56
and 1.56 g/mL. Compounds 1e and 3f showed similar moderate
l
antibacterial activity with MIC of 0.78–25.00
lg/mL against all
the tested bacterial strains.
group shows improvement in potency (2i IC50 10.95
4.68 g/mL) compared to 2a and 3a. Among them, two chloro in B-
ring at R2- and R3-positions (1f) displayed the most potent activity
with IC50 0.31 g/mL, which had a little improvement compared to
the best compound reported previously. So is 3e (IC50 0.45 lg/mL)
with methyl group at A-ring and iodo group at R2 position.
l
g/mL, 3i IC50
From the binding model, we can see compounds 1f, 3e and 1e
(Fig. 3a) are bound into ecKAS III receptor site (Fig. 3b) via hydro-
philic binding by hydrogen bond between NIII–Hꢃ ꢃ ꢃO/Gly209 and
OIV–Hꢃ ꢃ ꢃO/Gly152 (Fig. 3c), NIII–Hꢃ ꢃ ꢃO/Gly152 and OIV–Hꢃ ꢃ ꢃO/
Gly209 (Fig. 3d), NIII–Hꢃ ꢃ ꢃO/Gly152 and OIV–Hꢃ ꢃ ꢃO/Gly209
(Fig. 3e). Gly152/Gly209 are very important residues in ecKAS
III,26,20 effecting to these amino acid resides may greatly influence,
inhibit or even stop its catalytic activity. Compounds 1f, 3e and 1e
bond two Gly active sites (Gly152 and Gly209) of ecKAS III and fit
into the mouth of the substrate tunnel. This fact made 1f and 3e
have excellent inhibitory activity than other compounds and can
be acted 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 triplicates experiment re-
sults) against E. coli, P. aeruginosa, B. subtilis and S. aureus proved
this point in practice. The binding energy indicates that 1f is some-
what smaller than 2d,26 but the antibacterial activity has a few
improvements against the tested bacterial strains.
l
l
2.3. Molecular docking study
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 substrate in an X-
ray structure of ecKAS III complexed with CoA or inhibitor.23 To-
wards optimization of the aforementioned compounds of the
promising antimicrobial activities, the docking program AutoDock
4.024 was used to evaluate the binding free energies as potential
inhibitors into the target ecKAS III macromolecule.
2.3.1. Validation of the accuracy and performance of AutoDock
According to the method of validation cited in literature,25
where if the RMSD (root mean square deviation) of the best docked
conformation is ꢄ2.0 Å from the experimental one, the used scor-
ing function is successful. The obtained success rates of AutoDock
(Morris et al.)24 are highly excellent as illustrated in Table 3 for
the native ligand, which was docked into its KAS III (PDB code:
1HNJ).
3. Conclusion
A series of novel PSB derivatives were prepared and tested for
their inhibitory activity against ecKAS III, and the Autodock inves-
tigation also carried out by docking them into the active site of
ecKAS III. Both the theory and practice demonstrate that both
N -(2-(3,5-dichloro-2-hydroxybenzylideneamino)propyl)-2-hydro
xybenzamide (1f) and 2-hydroxy-N-(2-(2-hydroxy-5-iodobenzy-
lideneamino)propyl)-4-methylbenzamide (3e), can be as an inhib-
itor of ecKAS III and as a potential antibiotics agent, displaying
2.3.2. AutoDock-binding affinities of the synthesized and
designed compounds into KAS III
The binding affinity was evaluated by the binding free energies
(DGb, kcal/mol) and hydrogen bonding. The compounds which re-
MIC values in the range 0.20–3.13
against various bacteria.
lg/mL and 0.39–3.13 lg/mL
vealed 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 3. These com-
pounds include N-(2-(3,5-dichloro-2-hydroxybenzylideneamino)-
propyl)-2-hydroxybenzamide (1f), 2-hydroxy-N-(2-(2-hydroxy-5-
iodobenzylideneamino)propyl)-4-methylbenzamide (3e), 2-hydro-
xy-N-(2-(2-hydroxy-5-iodobenzylideneamino)propyl)benzamide (1e),
and N-(2-(3,5-dichloro-2-hydroxybenzylideneamino)propyl)-2-hy-
droxy-4-methylbenzamide (3f). Many of these derivatives exhib-
ited one or two hydrogen bonds between OI, OII, OIV or NIII of PSB
and different amino acids of the target ecKAS III including
Gly152, Gly209, Arg36 and Asn247 as cited in Table 3. The molec-
ular docking study revealed that the majority of the compounds
docked into the ecKAS III (PDB code: 1HNJ) exhibited hydrogen
bonds via NIII–H, OIV–Hꢃ ꢃ ꢃO/Gly152 or Gly209 as illustrated for
compounds 1f, 3e and 1e in Figure 3.
4. Experimental
4.1. Materials
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
parts per million (d). ESI-MS spectra were recorded on a Mariner
System 5304 Mass spectrometer. Elemental analyses were per-
formed on a CHN-O-Rapid instrument and were within 0.4% of
the theoretical values. Melting points were measured on a Boetius
micro melting point apparatus.
The global results are listed in Table 3, and we selected the top
10 compounds (1f, 3e, 1e, 3f, 1h, 1g, 3i, 3g, 1c and 3b) which have
better inhibitory activity to test their antibacterial activities
against Gram-negative bacterial strains (E. coli ATCC 35218 and
P. aeruginosa ATCC 13525) and two Gram-positive bacterial strains
(B. subtilis ATCC 6633 and S. aureus ATCC 6538), expecting to ex-
ploit potent antibacterial agent with broad-spectrum antibiotics
activity. Out of the top 10 compounds, the results demonstrate that
the antimicrobial compounds, N-(2-(3,5-dichloro-2-hydroxyben-
zylideneamino)propyl)-2-hydroxybenzamide (1f) and 2-hydroxy-
N-(2-(2-hydroxy-5-iodobenzylideneamino)propyl)-4-methylben-
zamide (3e), can be as inhibitor of ecKAS III and a potential antibi-
4.2. General method of synthesis PSB
Ethyl salicylate: 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
evaporation and the residue was washed with water. The organic
layer was again treated with water and concentrated sodium bicar-
bonate solution to an alkaline reaction, and then again with water.
The product was dried over anhydrous MgSO4, collected by filtra-
tion, and distilled (yield: 55.0%).
Phenyl 5-bromo-2-hydroxybenzoate: 5-Bromo salicylic acid
4.32 g (20 mmol), phenol 3.72 g (40 mmol), dicyclohexylcarbodi-
imide 0.08 g and THF 10 mL were mixed and stirred at room
otics agent, displaying MIC of 0.39, 0.20, 1.56, 3.13
3.13, 1.56, 1.56 g/mL against E. coli, P. aeruginosa, B. subtilis and S.
aureus, respectively, which was similar to the broad spectrum
lg/mL and 0.39,
l

2452
K. Cheng et al. / Bioorg. Med. Chem. 18 (2010) 2447–2455
temperature for 18 h. The mixture was subsequently concentrated
under reduced pressure giving the crude product as a yellow oil.
The crude product was purified by means of flash chromatography
on silica and 25% EtOAc/hexanes as eluent to obtain as a yellow oil
(57%).
1H); 6.91 (d, J = 5.3 Hz, 1H); 6.98 (d, J = 5.2 Hz, 1H); 7.22 (m, 1H);
7.27 (m, 1H); 7.38 (m, 2H); 8.31 (s, 1H); 12.10 (s, 1H); 13.11 (s,
1H). ESI-MS: 425.0 (C17H18IN2O3 C17H18IN2O3+, [M+H]+). Anal.
þ
Calcd for C17H17IN2O3: C, 48.13; H, 4.05; N, 6.60. Found: C, 48.15;
H, 4.01; N, 6.55.
Phenyl 4-methyl-2-hydroxybenzoate: The same as phenyl 5-bro-
mo-2-hydroxybenzoate preparation. Only use 4-methyl salicylic
acid 3.04 g (20 mmol) instead of 5-bromo salicylic acid, obtained
a colorless oil (60%).
Salicylamide types primary amines: Equimolar quantities
(10 mmol) of 1,2-propanediamine, ethyl salicylate, or phenyl 5-
bromo-2-hydroxybenzoate, or phenyl 4-methyl-2-hydroxybenzo-
ate and isopropanol (20 mL) 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:
62.0%).
Equimolar quantities (0.5 mmol) of salicylamide type’s primary
amines 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. 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)pro-
pyl)-2-hydroxybenzamide (1f). Yellow crystal, yield 90%, mp:
125–126 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.37 (d, J = 6.4 Hz,
3H); 3.34 (m, 1H); 3.82 (m, 2H); 6.64 (s, 1H); 6.79 (m, 1H); 6.97
(d, J = 8.2 Hz, 1H); 7.12 (s, 1H); 7.29 (m, 1H); 7.40 (m, 2H); 8.27
(s, 1H); 12.06 (s, 1H); 14.22 (s, 1H). ESI-MS: 367.0
þ
(C17H17Cl2N2O3
,
[M+H]+). Anal. Calcd for C17H16Cl2N2O3: C,
55.60; H, 4.39; N, 7.63. Found: C, 55.60; H, 4.37; N, 7.62.
4.2.1.7. (E)-N-(2-(3,5-Dibromo-2-hydroxybenzylideneamino)pro-
pyl)-2-hydroxybenzamide (1g). Yellow powder, yield 89%, mp:
163–165 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.37 (d, J = 6.3 Hz,
3H); 3.32 (m, 1H); 3.84 (m, 2H); 6.56 (s, 1H); 6.80 (m, 1H); 6.97
(d, J = 7.8 Hz, 1H); 7.31 (m, 2H); 7.38 (m, 1H); 7.70 (s, 1H); 8.24
(s, 1H); 12.05 (s, 1H); 14.38 (s, 1H). ESI-MS: 456.9
þ
(C17H17Br2N2O3
,
[M+H]+). Anal. Calcd for C17H16Br2N2O3: C,
44.76; H, 3.54; N, 6.14. Found: C, 44.70; H, 5.56; N, 6.12.
4.2.1. General data of 1a–1i
4.2.1.8. (E)-2-Hydroxy-N-(2-(2-hydroxy-3,5-diiodobenzylidenea-
mino)propyl)benzamide (1h). Yellow powder, yield 84%, mp:
155–156 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.36 (d, J = 6.3 Hz,
3H); 3.31 (m, 1H); 3.82 (m, 2H); 6.55 (s, 1H); 6.81 (m, 1H); 6.96
(d, J = 7.4 Hz, 1H); 7.33 (m, 2H); 7.38 (m, 1H); 7.71 (s, 1H); 8.25
(s, 1H); 12.04 (s, 1H); 14.41 (s, 1H). ESI-MS: 550.9
(C17H17I2N2O3þ, [M+H]+). Anal. Calcd for C17H16I2N2O3: C, 37.12;
H, 2.93; N, 5.09. Found: C, 37.11; H, 2.92; N, 5.12.
4.2.1.1. (E)-2-Hydroxy-N-(2-(2-hydroxybenzylideneamino)pro-
pyl)benzamide (1a). Yellow powder, yield 91%, mp: 39–41 °C, 1H
NMR (300 MHz, CDCl3, d ppm): 1.35 (d, J = 6.6 Hz, 3H); 3.39 (m,
1H); 3.66 (m, 1H); 3.91 (m, 1H); 6.53 (s, 1H); 6.78 (m, 1H); 6.89
(m, 1H); 6.97 (m, 2H); 7.22 (m, 2H); 7.35 (m, 2H); 8.37 (s, 1H);
12.97 (s, 2H). ESI-MS: 299.1 (C17H19N2O3þ, [M+H]+). Anal. Calcd
for C17H18N2O3: C, 68.44; H, 6.08; N, 9.39. Found: C, 68.45; H,
6.04; N, 9.35.
4.2.1.9. (E)-2-Hydroxy-N-(2-((2-hydroxynaphthalen-1-yl)meth-
yleneamino)propyl)benzamide (1i). Yellow powder, yield 81%,
mp: 171–173 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.46 (d,
J = 6.4 Hz, 3H); 3.36 (m, 1H); 3.93 (m, 2H); 6.71 (m, 1H); 6.81 (d,
J = 9.3 Hz, 1H); 6.96 (d, J = 8.4 Hz, 1H); 7.23 (m, 1H); 7.39 (m,
4H); 7.54 (m, 2H); 7.78 (d, J = 8.4 Hz, 1H); 8.83 (s, 1H); 12.24 (s,
1H); 14.52 (s, 1H). ESI-MS: 349.1 (C21H22N2O3þ, [M+H]+). Anal.
Calcd for C21H20N2O3: C, 72.40; H, 5.79; N, 8.04. Found: C, 72.32;
H, 5.88; N, 5.69.
4.2.1.2. (E)-N-(2-(5-Fluoro-2-hydroxybenzylideneamino)propyl)-
2-hydroxybenzamide (1b). Yellow powder, yield 87%, mp: 45–
47 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.36 (d, J = 6.4 Hz, 3H);
3.39 (m, 1H); 3.71 (m, 1H); 3.84 (m, 1H); 6.48 (s, 1H); 6.80 (d,
J = 6.8 Hz, 1H); 6.94 (m, 2H); 7.21 (m, 2H); 7.39 (m, 1H); 8.31 (s,
1H); 12.61 (s, 2H). ESI-MS: 317.1 (C17H18FN2O3þ, [M+H]+). Anal.
Calcd for C17H17FN2O3: C, 64.55; H, 5.42; N, 8.86. Found: C, 64.45;
H, 5.40; N, 8.75.
4.2.1.3. (E)-N-(2-(5-Chloro-2-hydroxybenzylideneamino)pro-
pyl)-2-hydroxybenzamide (1c). Yellow powder, yield 87%, mp:
41–44 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.37 (d, J = 6.3 Hz,
3H); 3.38 (m, 1H); 3.69 (m, 1H); 3.82 (m, 1H); 6.47 (s, 1H); 6.80
(m, 1H); 6.91 (d, J = 8.8 Hz, 1H); 6.98 (d, J = 8.3 Hz, 1H); 7.22 (m,
2H); 7.38 (m, 1H); 8.30 (s, 1H); 12.16 (s, 1H); 13.05 (s, 1H). ESI-
MS: 333.1 (C17H18ClN2O3þ, [M+H]+). Anal. Calcd for C17H17ClN2O3:
C, 61.36; H, 5.15; N, 8.42. Found: C, 61.35; H, 5.11; N, 8.45.
4.2.2. General data of 2a–2i
4.2.2.1. (E)-5-Bromo-2-hydroxy-N-(2-(2-hydroxybenzylidenea-
mino)propyl)benzamide (2a). Yellow powder, yield 86%, mp:
59–61 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.38 (d, J = 6.4 Hz,
3H); 3.41 (m, 1H); 3.71 (m, 1H); 3.89 (m, 1H); 6.53 (s, 1H); 6.91
(m, 2H); 6.99 (d, J = 5.4 Hz, 1H); 7.25 (m, 1H); 7.35 (m, 1H); 7.42
(m, 1H); 7.46 (dd, J = 2.3 Hz, J = 8.9 Hz, 1H); 8.39 (s, 1H); 12.56 (s,
2H). ESI-MS: 377.0 (C17H18BrN2
,
[M+H]+). Anal. Calcd for
þ
3
C17H17BrN2O3: C, 54.13; H, 4.54; N, 7.43. Found: C, 53.10; H,
5.56; N, 7.45.
4.2.1.4. (E)-N-(2-(5-Bromo-2-hydroxybenzylideneamino)pro-
pyl)-2-hydroxybenzamide (1d). Yellow powder, yield 85%, mp:
75–77 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.35 (d, J = 6.9 Hz,
3H); 3.40 (m, 1H); 3.69 (m, 1H); 3.84 (m, 1H); 6.53 (s, 1H); 6.79
(m, 1H); 6.90 (d, J = 5.3 Hz, 1H); 6.97 (d, J = 5.2 Hz, 1H); 7.20 (m,
1H); 7.27 (m, 2H); 7.38 (m, 1H); 8.30 (s, 1H); 12.11 (s, 1H);
4.2.2.2. (E )-5-Bromo-N-(2-(5-fluoro-2-hydroxybenzylideneami-
no)propyl)-2-hydroxybenzamide (2b). Yellow powder, yield
91%, mp: 130–133 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.36 (d,
J = 6.3 Hz, 3H); 3.42 (m, 1H); 3.71 (m, 1H); 3.85 (m, 1H); 6.45 (s,
1H); 6.89 (m, 2H); 7.20 (s, 1H); 7.27 (m, 1H); 7.37 (s, 1H); 7.45
13.15 (s, 1H). ESI-MS: 377.0 (C17H18BrN2 þ, [M+H]+). Anal. Calcd
3
for C17H17BrN2O3: C, 54.13; H, 4.45; N, 7.42. Found: C, 54.35; H,
4.41; N, 7.45.
(d, J = 8.9 Hz, 1H); 8.30 (s, 1H); 12.52 (s, 2H). ESI-MS: 395.0
þ
(C17H17BrFN2O3
,
[M+H]+). Anal. Calcd for C17H16BrFN2O3: C,
51.66; H, 4.08; N, 7.09. Found: C, 51.55; H, 4.96; N, 7.15.
4.2.1.5. (E)-2-Hydroxy-N-(2-(2-hydroxy-5-iodobenzylideneami-
no)propyl)benzamide (1e). Yellow powder, yield 79%, mp: 97–
98 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.34 (d, J = 6.8 Hz, 3H);
3.41 (m, 1H); 3.71 (m, 1H); 3.83 (m, 1H); 6.52 (s, 1H); 6.77 (m,
4.2.2.3. (E)-5-Bromo-N-(2-(5-chloro-2-hydroxybenzylideneami-
no)propyl)-2-hydroxybenzamide (2c). Yellow powder, yield

K. Cheng et al. / Bioorg. Med. Chem. 18 (2010) 2447–2455
2453
89%, mp: 130–133 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.35 (d,
J = 6.3 Hz, 3H); 3.43 (m, 1H); 3.72 (m, 1H); 3.85 (m, 1H); 6.46 (s,
1H); 6.88 (m, 2H); 7.21 (s, 1H); 7.28 (m, 1H); 7.37 (s, 1H); 7.46
(d, J = 8.7 Hz, 1H); 8.31 (s, 1H); 12.53 (s, 2H). ESI-MS: 413.0
(C17H17BrClN2O3þ, [M+H]+). Anal. Calcd for C17H16BrClN2O3: C,
49.60; H, 3.93; N, 6.80. Found: C, 49.56; H, 3.96; N, 6.78.
38–41 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.35 (d, J = 6.5 Hz,
3H); 2.29 (s, 3H); 3.35 (m, 1H); 3.68 (m, 1H); 3.87 (m, 1H); 6.51
(s, 1H); 6.59 (d, J = 8.1 Hz, 1H); 6.77 (s, 1H); 6.87 (m, 1H); 6.96
(d, J = 8.4 Hz, 1H); 7.13 (d, J = 8.4 Hz, 1H); 7.21 (m, 1H); 7.32 (m,
þ
1H); 8.36 (s, 1H); 12.51 (s, 2H). ESI-MS: 313.4 (C18H21N2O3
,
[M+H]+). Anal. Calcd for C18H20N2O3: C, 69.21; H, 6.45; N, 8.97.
Found: C, 69.10; H, 6.46; N, 8.95.
4.2.2.4. (E)-5-Bromo-N-(2-(5-bromo-2-hydroxybenzylideneami-
no)propyl)-2-hydroxybenzamide (2d). Yellow powder, yield 86%,
mp: 120–121 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.35 (d,
J = 6.4 Hz, 3H); 3.41 (m, 1H); 3.69 (m, 1H); 3.85 (m, 1H); 6.43 (s,
1H); 6.87 (m, 2H); 7.36 (m, 3H); 7.45 (m, 1H); 8.30 (s, 1H); 12.73
(s, 2H). ESI-MS: 456.9 (C17H17Br2N2O3þ, [M+H]+). Anal. Calcd for
C17H16Br2N2O3: C, 44.76; H, 3.54; N, 6.14. Found: C, 44.66; H,
3.56; N, 6.08.
4.2.3.2. (E)-N-(2-(5-Fluoro-2-hydroxybenzylideneamino)propyl)-
2-hydroxy-4-methylbenzamide (3b). Yellow powder, yield 81%,
d ppm): 1.35 (d,
J = 6.6 Hz, 3H); 2.29 (s, 3H); 3.36 (m, 1H); 3.70 (m, 1H); 3.83 (m,
1H); 6.59 (m, 2H); 6.77 (s, 1H); 6.90 (d, J = 8.5 Hz, 1H); 7.14 (d,
mp: 61–62 °C, 1H NMR (300 MHz, CDCl3,
J = 8.1 Hz, 1H); 7.19 (m, 1H); 7.25 (m, 1H); 8.28 (s, 1H); 12.19 (s,
þ
2H). ESI-MS: 331.3 (C18H20FN2O3
,
[M+H]+). Anal. Calcd for
C18H19FN2O3: C, 65.44; H, 5.80; N, 8.48. Found: C, 65.26; H, 5.75;
N, 8.45.
4.2.2.5. (E)-5-Bromo-2-hydroxy-N-(2-(2-hydroxy-5-iodobenzyli-
deneamino)propyl)benzamide (2e). Yellow powder, yield 79%,
mp: 90–92 °C, 1H NMR (300 MHz, CDCl3,
d
ppm): 1.34 (d,
4.2.3.3. (E)-N-(2-(5-Chloro-2-hydroxybenzylideneamino)propyl)-
2-hydroxy-4-methylbenzamide (3c). Yellow powder, yield 81%,
J = 6.3 Hz, 3H); 3.42 (m, 1H); 3.68 (m, 1H); 3.84 (m, 1H); 6.44 (s,
1H); 6.88 (m, 2H); 7.38 (m, 3H); 7.46 (m, 1H); 8.31 (s, 1H); 12.74
(s, 2H). ESI-MS: 502.9 (C17H17BrIN2O3þ, [M+H]+). Anal. Calcd for
C17H16BrIN2O3: C, 40.58; H, 3.21; N, 5.57. Found: C, 40.61; H,
3.16; N, 5.53.
mp: 54–56 °C, 1H NMR (300 MHz, CDCl3,
d ppm): 1.36 (d,
J = 6.5 Hz, 3H); 2.29 (s, 3H); 3.35 (m, 1H); 3.69 (m, 1H); 3.82 (m,
1H); 6.56 (s, 1H); 6.60 (d, J = 8.2 Hz, 1H); 6.78 (s, 1H); 6.89 (d,
J = 8.5 Hz, 1H); 7.14 (d, J = 8.2 Hz, 1H); 7.18 (m, 1H); 7.24 (m,
1H); 8.29 (s, 1H); 12.13 (s, 1H); 13.10 (s, 1H). ESI-MS: 349.1
(C18H20ClN2O3þ, [M+H]+). Anal. Calcd for C18H19ClN2O3: C, 62.34;
H, 5.52; N, 8.08. Found: C, 62.31; H, 5.55; N, 8.07.
4.2.2.6. (E)-5-Bromo-N-(2-(3,5-dichloro-2-hydroxybenzylidenea-
mino)propyl)-2-hydroxybenzamide (2f). Yellow powder, yield
88%, mp: 161–163 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.38 (d,
J = 6.2 Hz, 3H); 3.41 (m, 1H); 3.81 (m, 2H); 6.53 (s, 1H); 6.88 (d,
J = 8.8, 1H); 7.14 (d, J = 2.2, 1H); 7.43 (m, 3H); 8.28 (s, 1H); 11.95
(s, 1H); 14.13 (s, 1H). ESI-MS: 446.9 (C17H16BrCl2N2O3þ, [M+H]+).
Anal. Calcd for C17H15BrCl2N2O3: C, 45.77; H, 3.39; N, 6.28. Found:
C, 45.68; H, 3.37; N, 6.19.
4.2.3.4. (E)-N-(2-(5-Bromo-2-hydroxybenzylideneamino)propyl)-
2-hydroxy-4-methylbenzamide (3d). Yellow powder, yield 89%,
mp: 69–70 °C, 1H NMR (300 MHz, CDCl3,
d ppm): 1.35 (d,
J = 6.4 Hz, 3H); 2.30 (s, 3H); 3.37 (m, 1H); 3.68 (m, 1H); 3.83 (m,
1H); 6.48 (s, 1H); 6.60 (d, J = 8.5 Hz, 1H); 6.78 (s, 1H); 6.90 (d,
J = 8.9 Hz, 1H); 7.13 (d, J = 7.9 Hz, 1H); 7.19 (m, 1H); 7.27 (m,
1H); 8.29 (s, 1H); 12.13 (s, 1H); 13.12 (s, 1H). ESI-MS: 391.1
(C18H20BrN2O3þ, [M+H]+). Anal. Calcd for C18H19BrN2O3: C, 55.26;
H, 4.89; N, 7.16. Found: C, 55.31; H, 4.84; N, 7.13.
4.2.2.7. (E)-5-Bromo-N-(2-(3,5-dibromo-2-hydroxybenzylidenea-
mino)propyl)-2-hydroxybenzamide (2g). Yellow powder, yield
94%, mp: 188–190 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.37 (d,
J = 6.3 Hz, 3H); 3.38 (m, 1H); 3.82 (m, 2H); 6.54 (s, 1H); 6.87 (d,
J = 8.9, 1H); 7.32 (s, 1H); 7.44 (m, 2H); 7.70 (m, 1H); 8.25 (s, 1H);
4.2.3.5. (E)-2-Hydroxy-N-(2-(2-hydroxy-5-iodobenzylideneami-
no)propyl)-4-methylbenzamide (3e). Yellow powder, yield 81%,
mp: 121–123 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.34 (d,
J = 6.3 Hz, 3H); 2.30 (s, 3H); 3.35 (m, 1H); 3.69 (m, 1H); 3.84 (m,
1H); 6.48 (s, 1H); 6.61 (d, J = 8.3 Hz, 1H); 6.78 (s, 1H); 6.91 (d,
J = 8.8 Hz, 1H); 7.14 (d, J = 7.8 Hz, 1H); 7.20 (m, 1H); 7.28 (m,
1H); 8.29 (s, 1H); 12.14 (s, 1H); 13.22 (s, 1H). ESI-MS: 439.1
(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.
þ
11.97 (s, 1H); 14.32 (s, 1H). ESI-MS: 534.8 (C17H16Br3N2O3
,
[M+H]+). Anal. Calcd for C17H15Br3N2O3: C, 38.16; H, 2.83; N,
5.24. Found: C, 38.12; H, 2.79; N, 5.22.
4.2.2.8. (E )-5-Bromo-2-hydroxy-N-(2-(2-hydroxy-3,5-diiodoben-
zylideneamino)propyl)benzamide (2h). Yellow powder, yield
91%, mp: 171–173 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.36 (d,
J = 6.2 Hz, 3H); 3.39 (m, 1H); 3.81 (m, 2H); 6.54 (s, 1H); 6.88 (d,
J = 8.8, 1H); 7.33 (s, 1H); 7.45 (m, 2H); 7.71 (m, 1H); 8.26 (s, 1H);
þ
11.93 (s, 1H); 14.33 (s, 1H). ESI-MS: 628.8 (C17H16BrI2N2O3
,
4.2.3.6. (E)-N-(2-(3,5-Dichloro-2-hydroxybenzylideneamino)pro-
pyl)-2-hydroxy-4-methylbenzamide (3f). Yellow powder, yield
85%, mp: 118–120 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.51 (d,
J = 6.1 Hz, 3H); 2.21 (s, 3H); 4.61 (m, 3H); 6.17 (s, 1H); 7.68 (m,
[M+H]+). Anal. Calcd for C17H15BrI2N2O3: C, 32.47; H, 2.40; N,
4.45. Found: C, 32.48; H, 2.39; N, 4.36.
4.2.2.9. (E)-5-Bromo-2-hydroxy-N-(2-((2-hydroxynaphthalen-1-
yl)methyleneamino)propyl)benzamide (2i). Yellow powder,
yield 95%, mp: 88–90 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.46
(d, J = 6.4 Hz, 3H); 3.32 (m, 1H); 3.93 (m, 2H); 6.67 (d, J = 9.1,
1H); 6.81 (d, J = 9.0, 1H); 7.19 (m, 1H); 7.41 (m, 4H); 7.72 (d,
3H); 7.80 (m, 1H); 7.88 (m, 1H); 8.34 (s, 1H); 12.25 (m, 2H). ESI-
[M+H]+). Anal. Calcd for
þ
MS: 381.1 (C18H19Cl2N2O3
,
C18H18Cl2N2O3: C, 56.71; H, 4.76; N, 7.35. Found: C, 56.62; H,
4.75; N, 7.40.
J = 8.2, 1H); 7.78 (s, 1H); 8.48 (s, 1H); 8.71 (s, 1H); 14.51 (s, 2H).
4.2.3.7. (E)-N-(2-(3,5-Dibromo-2-hydroxybenzylideneamino)pro-
pyl)-2-hydroxy-4-methylbenzamide (3g). Yellow powder, yield
85%, mp: 171–172 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.52 (d,
J = 6.2 Hz, 3H); 2.22 (s, 3H); 4.61 (m, 3H); 6.18 (s, 1H); 7.66 (m,
2H); 7.81 (s, 1H); 7.88 (m, 1H); 7.98 (s, 1H); 8.34 (s, 1H); 12.25
(m, 2H). ESI-MS: 470.9 (C18H19Br2N2O3þ, [M+H]+). Anal. Calcd for
C18H18Br2N2O3: C, 45.98; H, 3.86; N, 5.96. Found: C, 46.01; H,
3.73; N, 5.92.
[M+H]+). Anal. Calcd for
þ
ESI-MS: 429.0 (C21H20BrN2O3
,
C21H19BrN2O3: C, 59.03; H, 4.48; N, 6.56. Found: C, 58.98; H,
4.49; N, 6.61.
4.2.3. General data of 3a–3i
4.2.3.1. (E )-2-Hydroxy-N-(2-(2-hydroxybenzylideneamino)pro-
pyl)-4-methylbenzamide (3a). Yellow powder, yield 81%, mp:

2454
K. Cheng et al. / Bioorg. Med. Chem. 18 (2010) 2447–2455
4.2.3.8. (E)-2-Hydroxy-N-(2-(2-hydroxy-3,5-diiodobenzylidenea-
mino)propyl)-4-methylbenzamide (3h). Yellow powder, yield
93%, mp: 157–158 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.50 (d,
J = 6.1 Hz, 3H); 2.23 (s, 3H); 4.58 (m, 3H); 6.17 (s, 1H); 7.68 (m, 2H);
7.80 (s, 1H); 7.86 (m, 1H); 7.99 (s, 1H); 8.34 (s, 1H); 12.25 (m, 2H).
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, 3.23; N, 4.92.
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
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 cu-
bic 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 target site re-
gion where the native ligand was embedded. Then automated
docking studies were carried out to evaluate the binding free en-
ergy 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, default values
were used with 20 independent docking runs for each docking
case. The AutoDock performs the task of the docking, where the li-
gand 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 auto-
matically accepted as the next step in docking.
4.2.3.9. (E)-2-Hydroxy-N-(2-((2-hydroxynaphthalen-1-yl)meth-
yleneamino)propyl)-4-methylbenzamide (3i). Yellow powder,
yield 95%, mp: 66–67 °C, 1H NMR (300 MHz, CDCl3, d ppm): 1.42
(d, J = 6.3 Hz, 3H); 2.23 (s, 3H); 3.31 (m, 1H); 3.92 (m, 2H); 6.49 (d,
J = 8.0 Hz, 1H); 6.74 (m, 2H); 7.16 (m, 1H); 7.35 (m, 2H); 7.49 (m,
2H); 7.71 (d, J = 8.2 Hz, 1H); 7.90 (s, 1H); 8.72 (s, 1H); 12.36 (m,
þ
2H). ESI-MS: 363.1 (C22H23N2O3
,
[M+H]+). Anal. Calcd for
C22H22N2O3: C, 72.91; H, 6.12; N, 7.73. Found: C, 72.86; H, 6.13; N,
7.66.
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 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 ecKAS III 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 assay.
4.5.1. Preparation of ligands and target ecKAS III
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/).
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
l
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 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%.
4.5.2. Molecular modeling and analysis of the docked results
The predicted binding free energy that includes the intermolec-
ular energy and torsional free energy was used as the criterion for
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.
Cluster analysis was performed on the docked results using a root
mean square (RMS) tolerance of 0.5 Å. 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 modeling, and
evaluation of the hydrogen bonds.
4.4. Antimicrobial activity
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.27 A stock solution of the synthesized compound (50
lg/
mL) in DMSO was prepared and graded quantities of the test com-
pounds were incorporated in specified quantity of sterilized liquid
medium (MH medium for antibacterial activity 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 approx-
imately 105 cfu/mL and applied to microtitration plates with seri-
ally 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 microtitration plates, 50 L of PBS (Phosphate Buffered
Saline 0.01 mol/L, pH 7.4: Na2HPO4ꢃ12H2O 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 tem-
perature for 4–5 h. The content of each well was removed, and 100
Acknowledgement
This work was supported by National Basic Research Programs
(973) of China (No. 2008CB418004 & No. 2008CB418201) and the
Jiangsu National Science Foundation (No. BK2009239) and Anhui
National Science Foundation (No. 070416274X).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.02.052.
lL of isopropanol containing 5% 1 mol/L HCl was added to extract

K. Cheng et al. / Bioorg. Med. Chem. 18 (2010) 2447–2455
2455
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