Design and synthesis of potent inhibitors of β-ketoacyl-acyl carrier protein synthase III (FabH) as potential antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2010.05.033

Twenty new Schiff bases were synthesized by reacting 5-fluoro- salicylaldehyde and primary amine as potent inhibitors of FabH. These compounds were assayed for antibacterial activity against Escherichia coli, Pseudomonas fluorescence, Bacillus subtilis an

Full text of DOI:10.1016/j.ejmech.2010.05.033

European Journal of Medicinal Chemistry 45 (2010) 4358e4364
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Design and synthesis of potent inhibitors of
b-ketoacyl-acyl carrier protein
synthase III (FabH) as potential antibacterial agents
,
a,
*
Lei Shi a, Rui-Qin Fang a, Zhen-Wei Zhu a, Ying Yang a, Kui Cheng a, Wei-Qing Zhong b , Hai-Liang Zhu
*
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty new Schiff bases were synthesized by reacting 5-fluoro-salicylaldehyde and primary amine as
potent inhibitors of FabH. These compounds were assayed for antibacterial activity against Escherichia
coli, Pseudomonas fluorescence, Bacillus subtilis and Staphylococcus aureus. Compounds with potent
antibacterial activities were tested for their E. coli FabH inhibitory activity. (E)-4-fluoro-2-((4-hydrox-
yphenethylimino)methyl)phenol (10) showed the most potent antibacterial activity with MIC of
Received 26 August 2009
Received in revised form
7 December 2009
Accepted 14 May 2010
Available online 24 May 2010
1.56e6.25
activity with IC50 of 2.7
FabH active site to determine the probable binding conformation.
Ó 2010 Elsevier Masson SAS. All rights reserved.
m
g/mL against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory
m
M. Docking simulation was performed to position compound 10 into the E. coli
Keywords:
FabH
Inhibitor
Antibacterial
Structureeactivity relationship
Schiff base
1. Introduction
inhibition [12]. FabH catalyzes the condensation reaction between
a CoA-attached acetyl group and an ACP-attached malonyl group,
yielding acetoacetyl-ACP as its final product (Fig. 1). Two other
condensing enzymes, FabB (KAS I) and FabF (KAS II), perform the
chain elongation reactions in subsequent cycles leading to long-
chain acyl ACP products [6,8], while FabB and FabF are also
condensing enzymes, FabH is structurally distinct.
FabH proteins from both Gram-positive and Gram-negative
bacteria are highly conserved at the sequence and structural level
while there are no significantly homologous proteins in humans.
Importantly, the residues that comprise the active site are essen-
tially invariant in various bacterial FabH molecules [13e15]. FabH
represents a promising target for the design of novel antimicrobial
drugs, since it regulates the fatty acid biosynthesis rate via an
initiation pathway and its substrate specificity is a key factor in
membrane fatty acid composition [16e18]. These attributes suggest
that small molecule inhibitors of FabH enzymatic activity could be
potential development candidates leading to selective, nontoxic,
and broad-spectrum antibacterials.
Although several classes of antibacterial agents are presently
available, resistance in most of the pathogenic bacteria to these
drugs constantly emerges. In order to prevent this serious medical
problem, the elaboration of new types of antibacterial agents or the
expansion of bioactivity of the previous drugs is a very important
task [1]. Therefore, in recent years, the research has been focused
toward development of new antibacterial agents, which may act
through novel target, surpassing the problem of acquired resistance.
A promising target is the fatty acid synthase (FAS) pathway in
bacteria. Fatty acid biosynthesis (FAB) is an essential metabolic
process for prokaryotic organisms and is required for cell viability
and growth [2]. Large multifunctional proteins termed type I fatty
acid synthases (FAS I) catalyze these essential reactions in
eukaryotes [3e5]. In contrast, bacteria use multiple enzymes to
accomplish the same goal and are referred to as type II, or disso-
ciated, fatty acid synthases (FAS II) [6,7].
b-Ketoacyl-acyl carrier
protein (ACP) synthase III, also known as FabH or KAS III, plays an
essential and regulatory role in bacterial FAB [8,9]. The enzyme
initiates the fatty acid elongation cycles [10,11], and is involved in
the feedback regulation of the biosynthetic pathway via product
Various kinds of compounds were screened by enzymatic assays
to generate leads that were co-crystallized with various pathogenic
FabH proteins and subsequently optimized using structure guided
drug design methods [19e23]. Y. Kim and coworkers reported the
YKAs3003, a Schiff base condensed by 4-hydroxy salicylaldehyde
and cyclohexanamine as a potent inhibitor of Escherichia coli
(E. coli) FabH with antimicrobial activity [24]. Further optimization
* Corresponding author. Tel.: þ86 25 8359 2572; fax: þ86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.033

L. Shi et al. / European Journal of Medicinal Chemistry 45 (2010) 4358e4364
4359
Fig. 1. FabH-catalyzed initiation reaction of fatty acid biosynthesis.
of this compound is required to improve its antimicrobial activity.
It was well known that some fluoro-substituted organic substances,
such as norfloxacin, ciprofloxacin, levofloxacin, linezolid, exhibited
favorable antibacterial activities because fluorin atom may play an
important role in improving the pharmacokinetics properties of the
compounds [25e28]. In this study, we designed and synthesized
a new series of Schiff bases derived from 5-fluoro-salicylaldehyde
and studied their antibacterial activities and E. coli FabH inhibitory
activities. Docking simulations were performed using the X-ray
crystallographic structure of the FabH of E. coli in complex with the
most potent inhibitor to explore the binding modes of these
compounds at the active site.
The structure of compound 2, (E)-4-fluoro-2-((cyclohexylimino)
methyl)phenol was very similar to 4-cyclohexyliminomethyl-
benzene-1,3-diol (YKAs3003) [24]. The difference between them is
that 4-substituent of YKAs3003 is eOH while the 5-substituent of
compound 2 is ꢂF. However, compound 2 showed much more
potent antibacterial activity than YKAs3003. The MIC of compound
2 against E. coli, P. fluorescence, B. Subtilis and S. aureus was 6.25,
12.5, 25 and 12.5
mg/mL, respectively while the MIC of YKAs3003
against E. coli and S. aureus was 128 and 256
mg/mL. This result
suggested that the introduction of fluoro substituent increased the
hydrophobicity of Schiff base and lead to the increase of the anti-
bacterial activity and the location of substituent may also influence
the activity. Compounds 1 and 3 with cyclopentyl and methyl-
cyclohexyl substituent replacing the cyclohexyl substituent of
compound 2 exhibited similar antibacterial activity to compound 2
2. Results and discussion
with the MIC of 6.25e25
Compounds 4 and 6 showed similar moderate antibacterial
activity with MIC of 12.5e50 g/mL against all the tested bacterial
strains. This indicated that the introduction of morpholine or
piperazine substituent cannot enhance the inhibitory activity
obviously.
mg/mL against the tested bacterial strains.
2.1. Chemistry
m
In the present study, 20 primary amines were respectively
subjected to react with 5-fluoro-salicylaldehyde to prepare the
corresponding Schiff bases (Scheme 1). All the compounds gave
satisfactory chemical analyses (ꢀ0.4%). 1H NMR and ESI-MS spectra
were consistent with the assigned structures. Compound 10 was
successfully crystallized and its structure was determined by
single-crystal X-ray diffraction analysis. Fig. 2 gives a perspective
view of this compound together with the atomic labeling system. In
the structure of compound 10, all bond lengths are within normal
ꢀ
ranges [29]. The bond length between C7 and N1 (1.284 A) conform
to the value for a carbon-nitrogen double bond, while the bond
ꢀ
length between C8 and N1 (1.463 A) conform to the value for
ꢀ
a carbon-nitrogen single bond. The bond length of C2eO1 (1.302 A)
ꢀ
is shorter than C13eO1 (1.363 A) because of the existence of the
ꢁ
ꢀ
hydrogen bond (O2eH2.N1: 2.565(3) A, 149.5 ). There is an
ꢀ
intermolecular OeH.O hydrogen bond (O1eH1.O2: 2.707(3) A,
172.1ꢁ, Symmetry codes: ꢂx þ 1/2, y þ 1/2, ꢂz þ 1/2) in the
structure. The dihedral angle between the two benzene rings is
14.22 (0.21)ꢁ.
2.2. Biological activity
2.2.1. Antibacterial activity
All the synthesized compounds were screened for antibacterial
activity against two Gram-negative bacterial strains: E. coli (E. coli)
and Pseudomonas fluorescence (P. fluorescence) and two Gram-posi-
tive bacterial strains: Bacillus Subtilis (B. Subtilis) and Staphylococcus
aureus (S. aureus) by MTT method. The MICs (minimum inhibitory
concentrations) of the compounds against these bacteria were pre-
sented in Table 1. Also included was the activity of reference
compounds kanamycin. The results revealed that most of the
synthesized compounds exhibited significant antibacterial activities.
Out of the 20 synthetic new Schiff bases, compound 10, (E)-
4-fluoro-2-((4-hydroxyphenethylimino)methyl)phenol, exhibited
the most potent antibacterial activity with MIC of 1.56, 3.13, 6.25
and 1.56
mg/mL against E. coli, P. fluorescence, B. Subtilis and
S. aureus, respectively, which was similar to the broad-spectrum
antibiotic kanamycin with corresponding MIC of 3.13, 3.13, 1.56 and
1.56
mg/mL.
Scheme 1. Syntheses of the Schiff bases derived from 5-fluoro-salicylaldehyde.

4360
L. Shi et al. / European Journal of Medicinal Chemistry 45 (2010) 4358e4364
component were not favorable for activity. Compound 20 was
condensed by 5-fluoro-salicylaldehyde and 2-aminopyridin and
showed weak antibacterial activity.
2.2.2. E. coli FabH inhibitory activity
The E. coli FabH inhibitory potency of the synthetic Schiff bases
with potent antibacterial activities (1e3, 6, 10, 13, 14, 18 and 19) was
examined and the results are summarized in Table 2. Most of the
tested compounds displayed potent E. coli FabH inhibitory. Among
them, compound 10 condensing by 5-fluoro-salicylaldehyde and
Fig. 2. Molecular structure of compound 10 with all atoms labeling. Displacement
ellipsoids are drawn at the 30% probability level and H atoms are shown as small
spheres of arbitrary radii.
tyramine showed the most potent inhibitory with IC50 of 2.7 mM.
This result supported the potent antibacterial activities of 10. It also
indicated that the introduction of fluoro substituent and tyramine
increased the inhibitory activity of Schiff bases. Compounds 1e3
with similar structure to YKAs3003 exhibited good inhibitory with
Compounds 5, 7, 8 and 9 were found to be inactive against all the
tested bacterial strains. This indicated that the introduction of long
aliphatic chain lead to the decrease of antibacterial activity.
Compounds 11e19 were condensed by 5-fluoro-salicylaldehyde
and substituted anilines. Various substituents of anilines such as
hydroxyl, halogen and saturated alkyl lead to the different antibac-
terial activities of these Schiff bases. Compounds 18 and 19 with
o-substituted fluoroand chloro group on aniline component showed
IC50 ranged from 6.8 to 18.4 mM, which indicating that hydrophobic
cyclopentyl and cyclohexyl substituent were favorable for the E. coli
FabH inhibitory activity. Compounds 18 and 19 with o-substituted
fluoro and chloro group on aniline component showed better E. coli
FabH inhibitory activity than compounds 13 and 14 with
p-substituted halogen group on aniline component. The results of
E. coli FabH inhibitory activity of the test compounds were corre-
sponding to the structure relationships (SAR) of their antibacterial
activities. This demonstrated that the potent antibacterial activities
of the synthetic compounds were probably correlated to their FabH
inhibitory activities.
significant antibacterial activity with MIC of 6.25e12.5
mg/mL
against the tested bacterial strains. Compounds 13e15 with
p-substituted halogen group on aniline component exhibited
moderate activity with MIC of 12.5e50
mg/mL. These results
demonstrated that the synthetic Schiff bases with o-substituted
halogen group on aniline component showed slightly more potent
antibacterial activity than that of p-substituted. The antibacterial
activity of these compounds enhanced slightly in the order of
substituent on aniline component: Br < Cl < F. We proposed that
hydrophobic and electron-withdrawing halogen groups on aniline
component were conducivetothe antibacterial activity. Compounds
16 and 17 with hydroxyl and nitro substituent on aniline component
were not active against all the tested bacterial strains. This indicated
that hydrophilic groups on aniline component were not conducive
to the antibacterial activity. Compounds 11 and 12 with methyl and
isopropyl substituent on aniline component showed weak antibac-
2.3. Binding model of compound 10 and E. coli FabH
Molecular docking of compound 10 and E. coli FabH was per-
formed on the binding model based on the E. coli FabHeCoA
complex structure (1HNJ.pdb) [30]. The FabH active site generally
contains a catalytic triad tunnel consisting of Cys-His-Asn, which is
conserved in various bacteria. This catalytic triad plays an impor-
tant role in the regulation of chain elongation and substrate
binding. Since the alkyl chain of CoA is broken by Cys of the cata-
lytic triad of FabH, interactions between Cys and substrate appear
to play an important role in substrate binding. Qiu, X. et al. have
refined three-dimensional structure of E. coli FabH in the presence
and absence of malonyl-CoA by X-ray spectroscopy. Since malonyl
moiety is degraded by E. coli FabH, molecular docking studies for
FabH and malonyl-CoA was carried out to identify a plausible
malonyl-binding mode [30]. They found that in one of the binding
modes appeared in the lower scored conformations, the malonyl
carboxylate formed hydrogen bonds to the backbone nitrogen of
Phe304.
terial activity with the MIC ranged from 50 to more than 100 mg/mL.
This suggested that the electron-donating groups on aniline
Table 1
Antibacterial activity of synthetic compounds.
Compounds
Minimum inhibitory concentrations (mg/mL)
Gram-negative
Gram-positive
E. coli
P. fluorescence
B. subtilis
S. aureus
The binding model of compound 10 and E. coli FabH is depicted
in Fig. 3. In the binding model, amino hydrogen of Asn247 forms
1
2
3
4
12.5
6.25
6.25
25
12.5
12.5
6.25
25
25
25
12.5
50
25
12.5
25
ꢀ
hydrogen bond with phenolic O1 of compound 10 (2.024 A,
172.165ꢁ). There is a
pep interaction between the benzyl ring of
50
compound 10 (C10, C11, C12, C13, C14, C15) and the benzyl ring of
5
6
7
8
9
10
11
12
>100
12.5
>100
>100
>100
1.56
50
>100
12.5
12.5
25
>100
25
>100
50
>100
25
>100
>100
>100
3.13
50
100
12.5
25
>100
>100
>100
6.25
>100
>100
25
>100
>100
>100
1.56
100
>100
12.5
25
Table 2
E. coli FabH inhibitory activity of synthetic compounds.
Compound
IC50 (mM)
13
14
15
1
2
3
18.4
7.4
6.8
50
50
25
50
16
17
18
19
>100
>100
6.25
6.25
50
>100
>100
6.25
12.5
100
>100
>100
12.5
12.5
>100
1.56
>100
>100
6.25
12. 5
100
6
48.5
2.7
28.6
42.5
8.6
10
13
14
18
19
20
Kanamycin
3.13
3.13
1.56
11.7

L. Shi et al. / European Journal of Medicinal Chemistry 45 (2010) 4358e4364
4361
from methanol, washed with methanol for three times, and dried in
a vacuum desiccator containing anhydrous CaCl2.
4.2.1. (E)-2-((Cyclopentylimino)methyl)-4-fluorophenol (1)
Yellow oil, yield 85%, 1H NMR (500 MHz, CDCl3,
d, ppm):
1.60e1.63 (m, 4H); 1.72e1.76 (m, 2H); 1.93e1.95 (m, 2H); 3.92e3.96
(m, 1H); 6.85e6.88 (m, 1H); 6.99e7.01 (m, 1H); 7.04 (d, J ¼ 3.0 Hz,
1H); 8.28 (s, 1H); 13.46 (s, 1H). MS (ESI): 208.13 (C12H15FNOþ,
[M þ H]þ). Anal. Calcd for C12H14FNO: C, 69.55%; H, 6.81%; N, 6.76%.
Found: C, 69.72%; H, 6.84%; N, 6.75%.
4.2.2. (E)-4-Fluoro-2-((cyclohexylimino)methyl)phenol (2)
Yellow oil, yield 84%, 1H NMR (500 MHz, CDCl3,
d, ppm):
1.41e1.80 (m, 10H); 3.38e3.40 (m, 1H); 6.88e6.91 (m, 1H); 6.98 (dd,
J ¼ 3.0, 8.5 Hz,1H); 7.06 (d, J ¼ 3.0 Hz,1H); 8.25 (s,1H); 13.47 (s, 1H).
ESI-MS: 222.12 (C13H17FNOþ, [M þ H]þ). Anal. Calcd for C13H16FNO:
C, 70.56%; H, 7.29%; N, 6.33%. Found: C, 70.47%; H, 7.82%; N, 6.32%.
4.2.3. (E)-2-((Cyclohexylmethylimino)methyl)-4-fluorophenol (3)
Yellow powder, yield 87%, mp: 36e37 ꢁC, 1H NMR (500 MHz,
CDCl3, d, ppm): 1.00e1.07 (m, 2H); 1.16e1.28 (m, 3H); 1.66e1.68 (m,
2H); 1.73e1.78 (m, 4H); 3.45 (d, J ¼ 6.3 Hz, 2H); 6.88e6.92 (m, 1H);
6.94 (d, J ¼ 3.0 Hz, 1H); 7.01 (d, J ¼ 3.0 Hz, 1H); 8.23 (s, 1H); 13.41 (s,
1H). MS (ESI): 236.13 (C14H19FNOþ, [M þ H]þ). Anal. Calcd for
C14H18FNO: C, 71.46%; H, 7.71%; N, 5.95%; Found: C, 71.35%; H,
7.74%; N, 5.93%.
Fig. 3. Binding model of compound 10 (colored in green) into E. coli FabH (colored in
blue). H-bond is shown as dotted green lines. interaction is shown as yellow
column (For interpretation of the references to colour in this figure legend, the reader
pep
is referred to the web version of this article.).
Phe 157. C1eN1eC8eC9 of compound 10 may form a hydrophobic
interaction with Ile156, Phe304 and Met207 of E. coli FabH.
4.2.4. (E)-4-Fluoro-2-((2-morpholinoethylimino)methyl)phenol (4)
Yellow oil, yield 89%, 1H NMR (500 MHz, CDCl3,
d, ppm): 2.53 (t,
3. Conclusions
J ¼ 4.6 Hz, 4H); 2.70 (t, J ¼ 6.7 Hz, 2H); 3.71 (t, J ¼ 4.7 Hz, 4H); 3.74
(t, J ¼ 6.6 Hz, 2H); 6. 90 (t, J ¼ 4.5 Hz, 1H); 6.94 (dd, J ¼ 3.0, 8.4 Hz,
1H); 7.02 (d, J ¼ 3.1 Hz, 1H); 8.30 (s, 1H); 13.10 (s, 1H). MS (ESI):
253.12 (C13H18FN2O2þ, [M þ H]þ). Anal. Calcd for C13H17FN2O2: C,
61.89%; H, 6.79%; N, 11.10%; Found: C, 62.11%; H, 6.76%; N, 11.08%.
A series of novel Schiff bases reacting by 5-fluoro-salicylalde-
hyde and substituted primary amines were synthesized and
assayed for their antibacterial activities against E. coli, P. fluores-
cence, B. Subtilis and S. aureus. Compound 10, (E)-4-fluoro-2-((4-
hydroxyphenethylimino)methyl)phenol showed the most potent
antibacterial activity with MIC of 1.56e6.25
bacterial strains and exhibited the most potent E. coli FabH inhib-
itory activity with IC50 of 2.7 M. The introduction of hydrophobic
and electron-withdrawing halogeno groups was conducive to the
antibacterial and E. coli FabH inhibitory activity. The introduction of
tyramine into the Schiff base was favorable for the activity. Docking
simulation was performed to position compound 10 into the E. coli
FabH active site to determine the probable binding conformation.
mg/mL against the test
4.2.5. (E)-4-Fluoro-2-((3-morpholinopropylimino)methyl)phenol (5)
Yellow oil, yield 89%, 1H NMR (500 MHz, CDCl3,
d,
m
ppm):1.86e1.91 (m, 2H); 2.41e2.44 (m, 6H); 3.66 (t, J ¼ 6.7 Hz, 2H);
3.71 (t, J ¼ 4.6 Hz, 4H); 6.85 (d, J ¼ 9.3 Hz, 1H); 7.34e7.38 (m, 2H);
8.28 (s,1H); 13.51 (s,1H). MS (ESI): 267.15 (C14H20FN2Oþ2 , [M þ H]þ).
Anal. Calcd for C14H19FN2O2: C, 63.14%; H, 7.19%; N, 10.52%; Found:
C, 63.28%; H, 7.15%; N, 10.49%.
4.2.6. (E)-4-Fluoro-2-((2-(piperazin-1-yl)ethylimino)methyl)
phenol (6)
4. Experimental section
Yellow oil, yield 74%, 1H NMR (500 MHz, CDCl3,
d, ppm):
2.46e2.80 (m, 10H); 3.78 (t, J ¼ 6.7 Hz, 2H); 6. 89 (t, J ¼ 4.5 Hz, 1H);
6.94 (m, 1H); 7.04 (d, J ¼ 3.2 Hz, 1H); 8.32 (s, 1H); 13.21 (s, 1H). MS
(ESI): 252.2 (C13H19FN3Oþ, [M þ H]þ). Anal. Calcd for C13H18FN3O: C,
62.13%; H, 7.22%; N, 16.72%. Found: C, 62.28%; H, 7.24%; N, 16.78%.
4.1. Chemistry
All chemicals (reagent grade) used were commercially available.
Melting points (uncorrected) were determined on a XT4 MP
apparatus (Taike Corp., Beijing, China). ESI mass spectra were
obtained on a Mariner System 5304 mass spectrometer, and 1H
NMR spectra were recorded on a Bruker PX500 or DPX300 spec-
trometer at 25 ꢁC with TMS and solvent signals allotted as internal
standards. Chemical shifts were reported in ppm (d). Elemental
analyses were performed on a CHNeO-Rapid instrument and were
within ꢀ0.4% of the theoretical values.
4.2.7. (E)-2-((1-Benzylpiperidin-4-ylimino)methyl)-4-fluorophenol
(7)
Yellow powder, yield 89%, mp: 93e95 ꢁC, 1H NMR (500 MHz,
CDCl3,
d
, ppm): 1.83e1.86 (m, 4H); 2.22 (t, J ¼ 5.6 Hz, 2H); 2.88 (d,
J ¼ 11.6 Hz, 2H); 3.30e3.32 (m, 1H); 3.53 (d, J ¼ 12.6 Hz, 2H);
6.88e6.95 (m, 2H); 7.01 (d, J ¼ 2.6 Hz, 1H); 7.26e7.35 (m, 5H); 8.32
(s, 1H); 13.25 (s, 1H). MS (ESI): 313.15 (C19H22FN2Oþ, [M þ H]þ).
Anal. Calcd for C19H21FN2O: C, 73.05%; H, 6.78%; N, 8.97%; Found: C,
73.26%; H, 6.75%; N, 9.01%.
4.2. General method of synthesis of Schiff bases
Equimolar quantities (0.5 mmol) of 5-fluoro-salicylaldehyde
and the substituted amines with one amino group were dissolved
in methanol (10 mL) and stirred at room temperature for several
hours. The precipitates were separated by filtration, recrystallized
4.2.8. (E)-2-((3-(Dimethylamino)propylimino)methyl)-4-
fluorophenol (8)
Yellow oil, yield 86%, 1H NMR (500 MHz, CDCl3,
d, ppm):
1.86e1.92 (m, 2H); 2.26 (s, 6H); 2.39 (t, J ¼ 7.0 Hz, 2H); 3.66 (d,

4362
L. Shi et al. / European Journal of Medicinal Chemistry 45 (2010) 4358e4364
J ¼ 6.7 Hz, 2H); 6.90 (dd, J ¼ 9.2, 4.6 Hz, 1H); 6.95 (dd, J ¼ 8.3, 3.1 Hz,
1H); 7.00e7.04 (m, 1H); 8.30 (s, 1H); 13.23 (s, 1H). MS (ESI): 225.15
(C12H18FN2Oþ, [M þ H]þ). Anal. Calcd for C12H17FN2O: C, 64.26%; H,
7.64%; N, 12.49%; Found: C, 64.34%; H, 7.66%; N, 12.45%.
7.19e7.23 (m, 2H); 7.29 (d, J ¼ 8.8 Hz, 2H); 8.66 (s,1H); 13.76 (s, 1H);
MS (ESI): 232.07 (C13H11FNOþ2 , [M H]þ). Anal. Calcd for
C13H10FNO2: C, 67.53%; H, 4.36%; N, 6.06%; Found: C, 67.52%; H,
4.38%; N, 6.08%.
þ
4.2.9. (E)-2-((3-(Diethylamino)propylimino)methyl)-4-
4.2.17. (E)-4-fluoro-2-((4-nitrophenylimino)methyl)phenol (17)
Yellow crystals, yield 85%, mp: >200 ꢁC, 1H NMR (500 MHz,
fluorophenol (9)
Yellow oil, yield 87%, 1H NMR (500 MHz, CDCl3,
d, ppm): 1.02 (t,
CDCl3,
d, ppm): 7.07 (d, J ¼ 8.6 Hz, 1H); 7.54e7.60 (m, 3H);
J ¼ 7.2 Hz, 6H); 1.83e1.88 (m, 2H); 2.50e2.56 (m, 6H); 3.65 (t,
J ¼ 6.8 Hz, 2H); 6.89 (dd, J ¼ 4.6, 8.9 Hz, 1H); 6.93e6.95 (m, 1H);
6.99e7.03 (m, 1H); 8.30 (s, 1H); 13.28 (s, 1H). MS (ESI): 253.14
(C14H22FN2Oþ, [M þ H]þ). Anal. Calcd for C14H21FN2O: C, 66.64%; H,
8.39%; N, 11.10%; Found: C, 66.78%; H, 8.36%; N, 11.07%.
7.85e7.87 (m, 1H); 8.34 (d, J ¼ 8.7 Hz, 2H); 8.64 (s, 1H); 13.53 (s,
1H); MS (ESI): 261.05 (C13H10FN2Oþ3 , [M þ H]þ). Anal. Calcd for
C13H9FN2O3: C, 60.00%; H, 3.49%; N, 10.77%; Found: C, 60.23%; H,
3.46%; N, 10.79%.
4.2.18. (E)-2-Fluoro-6-((2-fluorophenylimino)methyl)phenol (18)
Yellow crystals, yield 86%, mp: 111e112 ꢁC, 1H NMR (300 MHz,
4.2.10. (E)-4-Fluoro-2-((4-hydroxyphenethylimino)methyl)phenol
(10)
CDCl3, d, ppm): 7.18e7.30 (m, 7H); 8.74 (s, 1H); 13.40 (s, 1H); MS
Yellow crystals, yield 83%, mp: 161e163 ꢁC, 1H NMR (500 MHz,
(ESI): 234.07 (C13H10F2NOþ, [M þ H]þ). Anal. Calcd for C13H9F2NO:
CDCl3,
d
, ppm): 2.93 (t, J ¼ 7.1 Hz, 2H); 3.81 (t, J ¼ 7.0 Hz, 2H); 6.75
C, 66.95%; H, 3.89%; N, 6.01%; Found: C, 66.92%; H, 3.90%; N, 6.03%.
(d, J ¼ 8.6 Hz, 2H); 6.86e6.90 (m, 2H); 6.99e7.02 (m, 1H); 7.06 (d,
J ¼ 8.2 Hz, 2H); 8.12 (s, 1H); 8.90 (s, 1H); 13.48 (s, 1H). MS (ESI):
260.12 (C15H15FNO2þ, [M þ H]þ). Anal. Calcd for C15H14FNO2: C,
69.49%; H, 5.44%; N, 5.40%; Found: C, 69.63%; H, 5.46%; N, 5.37%.
4.2.19. (E)-2-((2-Chlorophenylimino)methyl)-6-fluorophenol (19)
Yellow powder, yield 86%, mp: 100e104 ꢁC, 1H NMR (300 MHz,
CDCl3,
d
, ppm): 7.19e7.28 (m, 5H); 7.33 (d, J ¼ 7.4 Hz, 1H); 7.50
(d, J ¼ 7.8 Hz, 1H); 13.45 (s, 1H); MS (ESI): 250.04 (C13H10ClFNOþ,
[M þ H]þ). Anal. Calcd for C13H9ClFNO: C, 62.54%; H, 3.63%; N,
5.61%; Found: C, 62.52%; H, 3.66%; N, 5.63%.
4.2.11. (E)-2-Fluoro-6-((p-tolylimino)methyl)phenol (11)
Yellow powder, yield 88%, mp: 128e130 ꢁC, 1H NMR (300 MHz,
CDCl3,
d
, ppm): 2.36 (s, 3H); 6.81e6.88 (m, 1H); 7.15 (d, J ¼ 1.5 Hz,
1H); 7.18e7.19 (m,1H); 7.21e7.23 (m, 4H); 8.64 (s,1H); 13.74 (s,1H);
MS (ESI): 230.09 (C14H13FNOþ, [M þ H]þ). Anal. Calcd for C14H12FNO:
C, 73.35%; H, 5.28%; N, 6.11%; Found: C, 73.38%; H, 5.26%; N, 6.12%.
4.2.20. (E)-4-Fluoro-2-((pyridin-2-ylimino)methyl)phenol (20)
Yellow crystal, yield 85%, mp: 94e95 ꢁC, 1H NMR (300 MHz,
CDCl3,
d, ppm): 6.96e7.00 (m, 1H); 7.09e7.22 (m, 4H); 7.33
(d, J ¼ 7.9 Hz, 1H); 7.79 (t, J ¼ 7.9 Hz, 1H); 8.51e8.52 (m, 1H); 9.39 (s,
1H); 13.20 (s, 1H); MS (ESI): 217.06 (C12H10FN2Oþ, [M þ H]þ). Anal.
Calcd for C12H9FN2O: C, 66.66%; H, 4.20%; N, 12.96%; Found: C,
66.82%; H, 4.18%; N, 13.01%.
4.2.12. (E)-2-Fluoro-6-((4-isopropylphenylimino)methyl)phenol (12)
Yellow crystals, yield 93%, mp: 64e65 ꢁC, 1H NMR (300 MHz,
CDCl3,
d
, ppm): 1.27 (d, J ¼ 4.0 Hz, 3H); 1.32 (d, J ¼ 4.2 Hz, 3H); 2.99
(m, 1H); 6.87e6.91 (m, 1H); 7.14 (d, J ¼ 1.5, 1H); 7.18e7.19 (m, 1H);
7.29 (d, J ¼ 7.2 Hz, 2H); 7.30 (s, 1H); 7.34 (d, J ¼ 7.0 Hz, 2H); 8.69 (s,
1H); 13.77 (s, 1H); MS (ESI): 258.12 (C16H17FNOþ, [M þ H]þ). Anal.
Calcd for C16H16FNO: C, 74.69%; H, 6.27%; N, 5.44%; Found: C,
74.65%; H, 6.28%; N, 5.42%.
4.3. Crystal structure determination
Crystal structure determination of compound 10 was carried out
on a Nonius CAD4 diffractometer equipped with graphite-mono-
ꢀ
chromated MoK
a
(l
¼ 0.71073 A) radiation. The structure was
4.2.13. (E)-2-Fluoro-6-((4-fluorophenylimino)methyl)phenol (13)
Yellow powder, yield 76%, mp: 127e129 ꢁC, 1H NMR (300 MHz,
solved by direct methods and refined on F2 by full-matrix least-
squares methods using SHELX-97 [31]. All the non-hydrogen atoms
were refined anisotropically. All the hydrogen atoms were placed in
calculated positions and were assigned fixed isotropic thermal
parameters at 1.2 times the equivalent isotropic U of the atoms to
which they are attached and allowed to ride on their respective
parent atoms. The contributions of these hydrogen atoms were
included in the structure-factors calculations. The crystal data, data
collection, and refinement parameter for the compound 10 are
listed in Table 3.
CDCl3, d, ppm): 6.83e6.90 (m, 1H); 7.09e7.22 (m, 4H); 7.27e7.30
(m, 2H); 8.61 (s, 1H); 13.40 (s, 1H); MS (ESI): 234.07 (C13H10F2NOþ,
[M þ H]þ). Anal. Calcd for C13H9F2NO: C, 66.95%; H, 3.89%; N, 6.01%;
Found: C, 66.91%; H, 3.90%; N, 6.03%.
4.2.14. (E)-2-((4-Chlorophenylimino)methyl)-6-fluorophenol (14)
Yellow powder, yield 87%, mp: 129e131 ꢁC, 1H NMR (500 MHz,
CDCl3,
d
, ppm): 6.86e6.90(m, 1H); 7.19 (d, J ¼ 7.3 Hz, 2H); 7.23 (d,
J ¼ 8.6 Hz, 2H); 7.40 (s, 1H); 7.41 (s, 1H); 8.62 (s, 1H); 13.28 (s, 1H);
MS (ESI): 251.03 (C13H10ClFNOþ, [M þ H]þ). Anal. Calcd for
C13H9ClFNO: C, 62.54%; H, 3.63%; N, 5.61%; Found: C, 62.52%; H,
3.65%; N, 5.62%.
4.4. Antibacterial activity
The antibacterial activity of the synthesized compounds was
tested against E. coli, P. fluorescence, B. subtilis and S. aureus using MH
medium (MuellereHinton medium: casein hydrolysate 17.5 g,
soluble starch 1.5 g, beef extract 1000 mL), The MICs (minimum
inhibitory concentrations) of the test compounds were determined
by a colorimetric method using the dye MTT (3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyl trtrazolium bromide). A stock solution of
4.2.15. (E)-2-((4-Bromophenylimino)methyl)-6-fluorophenol (15)
Yellow powder, yield 89%, mp: 148e149 ꢁC, 1H NMR (300 MHz,
CDCl3,
d
, ppm): 6.95e6.99 (m, 1H); 7.17 (d, J ¼ 8.6 Hz, 2H); 7.19 (d,
J ¼ 8.7 Hz, 2H); 7.54 (s, 1H); 7.57 (s, 1H); 8.62 (s, 1H); 13.27 (s, 1H);
MS (ESI): 293.99 (C13H10BrFNOþ, [M þ H]þ). Anal. Calcd for
C13H9BrFNO: C, 53.09%; H, 3.08%; N, 4.76%; Found: C, 53.06%; H,
3.10%; N, 4.72%.
the synthesized compound (100 mg/mL) in DMSO was prepared and
graded quantities of the test compounds were incorporated in
specified quantity of sterilized liquid MH medium. A specified
quantity of the medium containing the compound was poured into
microtitration plates. Suspension of the microorganism was
prepared to contain approximately 105 cfu/mL and applied to
4.2.16. (E)-2-Fluoro-6-((4-hydroxyphenylimino)methyl)phenol (16)
Yellow crystals, yield 89%, mp: 168e170 ꢁC, 1H NMR (500 MHz,
CDCl3,
d, ppm): 6.87e6.90 (m, 1H); 6.94 (d, J ¼ 8.5 Hz, 2H);

L. Shi et al. / European Journal of Medicinal Chemistry 45 (2010) 4358e4364
4363
Table 3
eluted using a 5e500 mM imidazole gradient over 20 column
volumes. Eluted protein was dialyzed against 20 mM Tris, pH 7.6,
1 mM DTT, and 100 mM NaCl. Purified FabHs were concentrated up
to 2 mg/mL and stored at ꢂ80 ꢁC in 20 mM Tris, pH 7.6, 100 mM
NaCl, 1 mM DTT, and 20% glycerol for enzymatic assays.
Purified ACP contains the apo-form that needs to be converted
into the holo-form. The conversion reaction is catalyzed by ACP
synthase (ACPS). In the final volume of 50 mL, 50 mg ACP, 50 mM
Crystallographical and experimental data for compound 10.
Compound
10
Formula
C15H14FNO2
259.27
Monoclinic
C2/c
15.979(3)
12.941(3)
15.040(3)
90
121.72(3)
90
2645.5(9)
8
1.302
0.096
1088
Mr (Formula weight)
Crystal system
Space group
ꢀ
a (A)
ꢀ
b (A)
ꢀ
Tris, 2 mM DTT, 10 mM MgCl2, 600 mM CoA, and 0.2 mM ACPS was
c (A)
(ꢁ)
(ꢁ)
(ꢁ)
incubated for 1 h at 37 ꢁC. The pH of the reaction was then adjusted
to approximately 7.0 using 1 M potassium phosphate. Holo-ACP
was purified by fractionation of the reaction mixture by Source
Q-15 ion exchange chromatography using a 0e500 mM NaCl
gradient over 25 column volumes.
a
b
g
3
ꢀ
Volume (A )
Z (formula units)
Dc/(g/cm3) (Calculated density)
a
m
(mmꢂ1
)
In a final 20
0.5 mM DTT, 0.25 mM MgCl2, and 2.5
with 1 nM FabH, and H2O was added to 15
bation, a 2 L mixture of 25 M acetyl-CoA and 0.75
CoA was added for FabH reaction for 25 min. The reaction was
stopped by adding 20 L of ice-cold 50% TCA, incubating for 5 min
on ice, and centrifuging to pellet the protein. The pellet was washed
m
L reaction, 20 mM Na2HPO4/NaH2PO4, pH 7.0,
M holo-ACP were mixed
L. After 1 min incu-
Ci [3H]acetyl-
F(000)b
m
Crystal size (mm3)
0.35 ꢃ 0.30 ꢃ 0.30
m
T/K (Data collection temperature)
293(2)
range/ꢁ
2.17/25.96
0<¼h<¼19
0<¼k<¼15
ꢂ18<¼l<¼15
2692/2599 [R(int) ¼ 0.0518]
1431
m
m
m
q
Index ranges
m
Reflections collected/unique
Observed reflections
with 10% ice-cold TCA and resuspended with 5 mL of 0.5 M NaOH.
Data/restraints/parameters
2599/0/176
1.021
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%.
Goodness-of-fit on F2
Final R indices [I ꢄ 2
R indices (all data)c
Largest diff. peak and hole (eA
s
(I)]c
R1 ¼ 0.0659,
u
R2 ¼ 0.1524
R2 ¼ 0.1868
R1 ¼ 0.1223,
u
ꢀꢂ3
)
0.305 and ꢂ0.225
a
Absorption coefficient.
4.6. Docking simulations
b
The number of electrons in a single cell.
c
R1 ¼ SkFoj ꢂ jFck/SjFoj, wR2 ¼ [Sw(Fo2 ꢂ Fc2)2/Sw(Fo2)2]1/2
.
The crystal structures of E. coli FabH (PDB code: 1HNJ) [30] was
obtained from the Protein Data Bank (http://www.rcsb.org).
Studies were carried out on only one subunit of the enzymes.
The graphical user interface AutoDockTools (ADT) was employed to
setup the enzymes: all hydrogens were added, Gasteiger charges
were calculated and nonpolar hydrogens were merged to carbon
atoms. For macromolecules, generated pdbqt files were saved.
The 3D structures of ligand molecules were built, optimized
(PM3) level, and saved in mol2 format with the aid of the molecular
modeling program Spartan (Wavefunction Inc.). These partial
charges of Mol2 files were further modified by using the ADT
package (version 1.4.6) so that the charges of the nonpolar hydro-
gens atoms assigned to the atom to which the hydrogen is attached.
The resulting files were saved as pdbqt files.
microtitration plates with serially diluted compounds in DMSO to be
tested and incubated at 37 ꢁC for 24 h. After the MICs were visually
determined on each of the microtitration plates, 50
mL of PBS
(Phosphate Buffered Saline 0.01 mol/L, pH 7.4: Na2HPO4.12H2O 2.9 g,
KH2PO4 0.2 g, NaCl 8.0 g, KCl 0.2 g, distilled water 1000 mL) con-
taining 2 mg of MTT/mL was added to each well. Incubation was
continued at room temperature for 4e5 h. The content of each well
was removed, and 100 mL 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 micro-
plate reader at 550 nm. The observed MICs were presented inTable 1.
4.5. E. coli FabH purification and activity assay
AutoDock 4.0 was employed for all docking calculations [32,33].
The AutoDockTools program was used to generate the docking
input files. In all docking a grid box size of 48 ꢃ 48 ꢃ 48 points in x,
y, and z directions was built in the catalytic site of the protein.
Full-length E. coli acyl carrier protein (ACP), acyl carrier protein
synthase (ACPS), and
b-ketoacyl-ACP synthase III (FabH) were
ꢀ
individually cloned into pET expression vectors with an N-terminal
His-tag (ACP, ACPS in pET19; FabH in pET28).
All proteins were expressed in E. coli strain BL21(DE3). Trans-
formed cells were grown on Luria-Bertani (LB) agar plates
supplemented with kanamycin (30 mg/ml). Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used
to screen colonies for overexpression of proteins. One such positive
A grid spacing of 0.375 A (approximately one forth of the length of
carbonecarbon covalent bond) and a distances-dependent function
of the dielectric constant were used for the calculation of the
energetic map. Ten runs were generated by using Lamarckian
genetic algorithm searches. Default settings were used with an
initial population of 50 randomly placed individuals, a maximum
number of 2.5 ꢃ106 energy evaluations, and a maximum number of
2.7 ꢃ 104 generations. A mutation rate of 0.02 and a crossover rate
colony was used to inoculate 10 ml of LB medium with 30
kanamycin and grown overnight at 37 ꢁC, 1 ml of which was used to
inoculate 100 ml LB medium supplemented with 30 g/ml of
kanamycin. The culture was shaken for 4 h at 37 ꢁC, and then
induced with 0.5 mM isopropyl -thiogalactopyranoside (IPTG).
mg/ml of
ꢀ
of 0.8 were chosen. Results differing by less than 0.5 A in positional
m
root-mean-square deviation (RMSD) were clustered together and
the results of the most favorable free energy of binding were
selected as the resultant complex structures.
b-D
The culture was grown for 4 h, and harvested by centrifugation
(30 min at 15 000 rpm).
Acknowledgements
Harvested cells containing His-tagged ACP, ACPS, and FabHs
were lysed by sonication in 20 mM Tris, pH 7.6, 5 mM imidazole,
0.5 M NaCl and centrifuged at 20,000 rpm for 30 min. The super-
natant was applied to a Ni-NTA agarose column, washed, and
This work was supported by the Jiangsu National Science
Foundation (No. BK2009239) and Anhui National Science Founda-
tion (No. 070416274X).

4364
L. Shi et al. / European Journal of Medicinal Chemistry 45 (2010) 4358e4364
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