Design, synthesis and antibacterial activities of vanillic acylhydrazone derivatives as potential β-ketoacyl-acyl carrier protein synthase III (FabH) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.09.009

Fatty acid biosynthesis is essential for bacterial survival. FabH, β-ketoacyl-acyl carrier protein (ACP) synthase III, is a particularly attractive target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and Gram-negative bacteria. A series of acylhydrazone derivatives were synthesized and developed as potent inhibitors of FabH. This inhibitor class demonstrates strong broad-spectrum antibacterial activity. Compounds with potent antibacterial activities were tested for their Escherichia coli FabH inhibitory activity. Especially, compound E9 showed the most potent antibacterial activity with MIC values of 0.39-1.56 μg/mL against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with IC₅₀ of 2.5 μM. Docking simulation was performed to position compound E9 into the E. coli FabH active site to determine the probable binding conformation.

Full text of DOI:10.1016/j.ejmech.2012.09.009

European Journal of Medicinal Chemistry 57 (2012) 373e382
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and antibacterial activities of vanillic acylhydrazone derivatives
as potential -ketoacyl-acyl carrier protein synthase III (FabH) inhibitors
b
*
Xiao-Liang Wang, Yan-Bin Zhang, Jian-Feng Tang, Yu-Shun Yang, Ruo-Qi Chen, Fei Zhang, Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Fatty acid biosynthesis is essential for bacterial survival. FabH,
b
-ketoacyl-acyl carrier protein (ACP)
Received 10 July 2012
Received in revised form
24 August 2012
Accepted 6 September 2012
Available online 13 September 2012
synthase III, is a particularly attractive target, since it is central to the initiation of fatty acid biosynthesis
and is highly conserved among Gram-positive and Gram-negative bacteria. A series of acylhydrazone
derivatives were synthesized and developed as potent inhibitors of FabH. This inhibitor class demon-
strates strong broad-spectrum antibacterial activity. Compounds with potent antibacterial activities were
tested for their Escherichia coli FabH inhibitory activity. Especially, compound E9 showed the most potent
antibacterial activity with MIC values of 0.39e1.56
exhibited the most potent E. coli FabH inhibitory activity with IC50 of 2.5
performed to position compound E9 into the E. coli FabH active site to determine the probable binding
conformation.
m
g/mL against the tested bacterial strains and
M. Docking simulation was
Keywords:
m
FabH inhibitor
Antibacterial
Hemolytic activity
Acylhydrazone
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
invariant in various bacterial FabH molecules [8,9]. These attributes
suggest that small molecule inhibitors of FabH enzymatic activity
Bacterial infection remains a seriousthreat to human lives because
of emerging resistance to existing antibiotics, which is an increasing
public health problem [1]. 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
[2]. To meet critical clinical needs, especially to overcome the
emerging drug resistance, the discovery of novel antibiotic chemical
scaffolds with new modes of action is crucial for saving lives. Fatty
acid biosynthesis (FAB) is an essential metabolic process for
prokaryotic organisms and is required for cell viabilityand growth [3].
Significant differences between bacterial and human fatty acid
synthesis systems exist that include the organization and structure of
enzymes and the specific roles played by fatty acids make this system
could be potential development candidates leading to selective,
nontoxic and broad-spectrum antibacterial agents.
Thus, various kinds of compounds were screened by enzymatic
assays and subsequently optimized using structure guided drug
design methods [7,10e19]. Compounds containing Schiff base
moiety or hydrozone moiety show antimicrobial activity against
various bacteria, including inhibitors of FabH, which were reported
in the previous studies of our laboratory [13e17] and Kim et al.
[18,19]. For example, compounds (11 and 18 [13]; 19 [14]; 10 [15];
2d [16]; 12 and 13 [17]; YKAs3003 [18]; 6, 6c and 6e [19]) reported
as inhibitors of Escherichia coli FabH showed the most potent
antibacterial activity and FabH inhibitory activity in each article
respectively, which are displayed in Fig. 2. As shown in Fig. 2, these
compounds share similar core structures. Obviously, there always
exists a benzene-1,3-diol or benzene-1-ol group and a hydrazone
moiety (eNHeN]CHe) or a Schiff base moiety (eC]Ne) in the
core structures. Taking into account the instability of hydrazone
moiety and Schiff base moiety, the N-acylhydrazone moiety (eCOe
NHeN]CHe) was considered as a privileged structure employed
to improve the chemical stability for the design of new FabH
inhibitors. Moreover, acylhydrazones have aroused considerable
attention due to their particular physical, chemical, and biological
activities based on their unique structures and they are known to
present a wide spectrum of biological properties, such as antimi-
crobial activity against several bacteria [20,21].
an attractive target for antibacterial drug discovery [4,5]. The
b-
ketoacyl-acyl carrier protein synthase III (FabH) is one of essential
functional enzymes in FAS II, which initiates the FAB cycle by cata-
lyzing the first condensation step between acetyl-CoA and malonyl-
ACP, playing a key regulatory role in the bacterial FAB cycle (Fig. 1)
[6]. Additionally, FabH is highly conserved among key pathogens
while there are no significantly homologous proteins in humans [7].
Importantly, the residues that comprise the active site are essentially
* 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 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.009

374
X.-L. Wang et al. / European Journal of Medicinal Chemistry 57 (2012) 373e382
structureeactivity relationships in antibacterial activity for further
optimization. Thus we studied their antimicrobial activities and
E. coli FabH inhibitory activities. Docking simulations were per-
formed using the X-ray crystallographic structure of the FabH of
E. coli in complex with the most potent inhibitor to explore the
binding model of the compound at the active site.
2. Results and discussion
2.1. Chemistry
Fig. 1. FabH accomplishes the initial condensation step in FAS II.
In this study, 30 vanillic acylhydrazone derivatives (E1eE30)
were synthesized. The synthetic route of compounds E1eE30 was
shown in Scheme 1. They were synthesized from 4-hydroxy-3-
methoxybenzoic acid (A). Esterification of the 4-hydroxy-3-
methoxybenzoic acid with methanol and concentrated sulfuric
acid afforded the corresponding ester B. The aroyl hydrazide C was
obtained by reacting B with 85% hydrazine monohydrate in
ethanol. The synthesis of compounds E1eE30 was accomplished by
reacting C with aldehyde D in ethanol [31].
All of the synthetic compounds gave satisfactory analytical and
spectroscopic data, which were in full accordance with their
depicted structures. Additionally, the structure of compound E22
was further confirmed by X-ray diffraction. Its crystal data are
presented in Table 1 and Fig. 4 gives a perspective view of this
compound together with the atomic labeling system.
Compounds containing a benzene-1-methoxy-2-ol scaffold also
have received significant attention in natural medicinal chemistry
research as this structural scaffold is found in a variety of natural
products. As shown in Fig. 3, vanillic acid (A), which is found in
many traditional Chinese medicines, such as Rhizoma Picrorhizae,
Ginseng, Propolis and BaiHao, has been reported to possess anti-
microbial activity [22]. Ferulic acid (B) is also an active ingredient of
many Chinese herbal medicines, such as Chuanxiong, Danggui and
Awei, which have been used to treat cardiovascular diseases by
Chinese physicians for thousands of years [23]. Curcumin (C), the
active ingredient in the traditional herbal remedy turmeric, has
a surprisingly wide range of beneficial properties, including anti-
oxidant [24], anti-inflammatory [25,26], antimicrobial [27] and
anti-cancer [28] activity, so curcumin research has been a hot
scientific topic of study. Eugenol (D) exhibits an excellent broad-
spectrum bactericidal activity [29]. Silymarin (E) is well known as
a kind of hepatoprotective herbal drug [30]. Hesperidin (F) is
clinically taken supplements along with vitamin C to treat diseases
like purpura and scurvy as hesperidin can promote vitamin C more
easily absorbed because of its beneficial effects on the blood
vessels.
In view of the above mentioned findings, we report in the
present work the synthesis of a series of acylhydrazone derivatives
to extend the research to achieve new potential antibacterial FabH
inhibitors by structural optimization. The benzene-1-methoxy-2-ol
moiety was introduced to enhance the molecular interactions,
which based on the results of CADD (computer assistant drug
design). With the benzene-1-methoxy-2-ol moiety fixed, we
introduced different substituents on the other benzene ring which
connected with the azomethine moiety (eC]Ne) to study the
2.2. Biological activity
2.2.1. Antibacterial activity
All the synthesized vanillic acylhydrazone derivatives E1eE30
were evaluated for their antimicrobial activities against two
Gram-negative bacterial strains: E. coli ATCC 25922 and Pseudo-
monas aeruginosa ATCC 27853 and two Gram-positive bacterial
strains: Staphylococcus aureus ATCC 6538 and Bacillus subtilis ATCC
530 by the standard two-fold serial broth dilution method [32,33].
The MICs (minimum inhibitory concentrations) of those
compounds against these bacteria were presented in Table 2. DDCP
[34] and standard antibacterial agent kanamycin B were also
screened under identical conditions for comparison. The results
revealed that most of the synthetic compounds exhibited signifi-
cant antibacterial activities.
Out of the 30 vanillic acylhydrazone derivatives, compound E9
displayed the most potent activity with MIC values of 0.39, 0.39,
Fig. 2. New potential FabH inhibitors: 11, 18, 2d, 10, 19, 12 and 13 discovered by Zhu et al.; YKAs3003, 6, 6e and 6c discovered by Kim et al.

X.-L. Wang et al. / European Journal of Medicinal Chemistry 57 (2012) 373e382
375
Fig. 3. Natural products AeF containing a benzene-1-methoxy-2-ol scaffold.
1.56 and 0.78
B. subtilis respectively, which was superior to DDCP and kanamycin
B with corresponding MIC values of 3.13, 1.56, 12.5, 25 g/mL and
0.78, 0.78, 1.56, 1.56 g/mL, respectively.
m
g/mL against E. coli, P. aeruginosa, S. aureus and
MIC values of 1e2
bacterial strains were 32 to >512
compounds in previous studies, our compound E9 showed much
more potent broad-spectrum antibacterial activity with the MIC
m
g/mL while the MIC values against other
mg/mL [19]. Compared to the
m
m
Compounds 11, 18, 2d, 10, 19, 12 and 13 in previous studies of our
laboratory displayed broad-spectrum antimicrobial activity against
all tested bacterial strains (E. coli, P. aeruginosa, S. aureus and
values of 0.39e1.56 mg/mL against the tested bacterial strains. The
results suggested further structural optimization leaded to a posi-
tive result in a significant increase of its antibacterial activity.
Subsequently structureeactivity relationships (SAR) studies
were performed to determine how the substituents on benzene
ring affected the antimicrobial activity. Compound E1 without any
substituent group on benzene ring showed moderate antimicrobial
B. subtilis) with MIC values of 0.78e25
mg/mL [13e17]. YKAs3003
displayed MIC values in the range 128e256
mg/mL against various
bacteria [18]. Besides, compounds 6 and 6c also displayed broad-
spectrum antimicrobial activity against bacterial strains (E. coli,
E. faecalis, VREF and S. aureus) with MIC values of 4e64
mg/mL; But
activity with the mean MIC values of 25, 12.5, 25 and 25
mg/mL
6e showed selective antimicrobial activity against S. aureus with
against E. coli, P. aeruginosa, S. aureus and B. subtilis, respectively.
Scheme 1. General synthesis of compounds E1eE30. Reagents and conditions: (i) methanol, concentrated sulfuric acid; reflux; (ii) NH2NH2$H2O, ethanol; 90 ꢀC; (iii) ethanol, water,
acetic acid; rt.

376
X.-L. Wang et al. / European Journal of Medicinal Chemistry 57 (2012) 373e382
Table 1
Table 2
Crystal data for compound E22.
Antimicrobial activity of the synthesized compounds.
Crystal parameters
Compound E22
16H16N2O3
Compounds
Minimum inhibitory concentrations (mg/mL)
Formula
C
Gram-negative
Gram-positive
Crystal size (mm)
Formula weight
Crystal system
0.1 ꢁ 0.1 ꢁ 0.2
E. coli
P. aeruginosa
12.5
1.56
25
6.25
S. aureus
B. subtilis
284.12
Monoclinic
90.00
99.441(7)
90.00
12.002(8)
8.592(6)
15.569(10)
1583.6(18)
E1
E2
E3
E4
E5
E6
E7
E8
25
25
3.13
50
6.25
12.5
25
25
3.13
25
6.25
12.5
12.5
6.25
25
0.78
6.25
25
25
50
50
25
>50
50
>50
50
50
>50
25
12.5
25
50
a
b
g
(ꢀ)
(ꢀ)
1.56
12.5
3.13
12.5
25
(ꢀ)
ꢀ
a (A)
b (A)
c (A)
V (A )
25
12.5
ꢀ
ꢀ
3
ꢀ
6.25
12.5
0.39
6.25
50
3.13
25
0.39
3.13
25
6.25
12.5
1.56
12.5
50
Z
q
4
E9
limits (ꢀ)
2.34 ꢂ h ꢂ 26.00
E10
E11
E12
E13
E14
E15
E16
E17
E18
E19
E20
E21
E22
E23
E24
E25
E26
E27
E28
E29
E30
DDCP
Kanamycin B
hkl limits
F (000)
ꢃ14 ꢂ h ꢂ 14, ꢃ10 ꢂ k ꢂ 10, ꢃ19 ꢂ l ꢂ 14
704
25
25
12.5
50
25
50
Data/restraints/parameters
Absorption coefficient
Reflections collected
Independent reflections
R1/wR2 [I > 2&Gs (I)]
R1/wR2 (all data)
GOF
3092/0/190
0.130
7637
3092 [Rint ¼ 0.0499]
0.2767/0.6485
0.3159/0.6810
3.002
50
25
25
25
12.5
25
>50
25
50
>50
25
50
>50
25
25
25
25
50
25
25
50
>50
25
12.5
25
50
25
25
25
25
50
>50
25
6.25
25
50
12.5
25
12.5
25
>50
1.56
1.56
>50
12.5
12.5
50
>50
25
50
25
50
Then, we evaluated the effect of introducing different substituent
group(s) on benzene ring in E1. Firstly, introducing hydroxyl
group(s) on the benzene ring in E1 to form a class of compounds
(E2eE6) increased the antimicrobial activity. E2 with the hydroxyl
group on the ortho-position of benzene ring showed MIC values of
3.13e6.25 mg/mL which was better than that of E3 (12.5e50 mg/mL)
with the hydroxyl group on the para-position of benzene ring. E4
with two hydroxyl groups on the ortho-position and para-position
of benzene ring whose MIC values (1.56e3.13
25
50
25
50
>50
25
3.13
>50
12.5
3.13
3.13
1.56
mg/mL) was superior
to that of E5 (12.5e25 g/mL) with two hydroxyl groups on the
m
para-position and meta-position of benzene ring. And the MIC
values of E6 with hydroxyl group on the para-position and
methoxyl group on the meta-position of benzene ring was in the
Thirdly, the introduction of the electron-withdrawing groups
(halogen groups or eNO2) at the position of benzene ring weak-
ened the antimicrobial effects. For example, compounds E11eE18
with halogen group(s) and E19eE21 with nitro group on benzene
range of 12.5e25
mg/mL. The results indicated that the potent
inhibitory activity order of compounds with hydroxyl group(s) on
the benzene ring was ortho
>
2,4-disubstituded
>
3,4-
ring exhibited poor activities with MIC values of both 25 to >50 mg/
disubstituded > para.
mL. However, the introduction of the electron-donating groups
(here it was eCH3 or eOCH3) at the position of benzene ring slightly
strengthened the antimicrobial effects of compounds E22eE26
Secondly, compounds E7eE10 condensed by
a series of
substituted salicylaldehyde and 4-hydroxy-3-methoxybenzo-
hydrazide showed potent antimicrobial activities with MIC values
with MIC values of 6.25e50
a meta-methyl group on benzene ring showed the best antibacte-
rial activity in the range of 6.25e12.5 g/mL.
mg/mL. Among them, E23 with
of 0.39e25
3-position and 5-position of salicylaldehyde whose MIC values
were 0.39e1.56 g/mL. Compound E7 with a chlorine atom on the
mg/mL, especially for E9 with two chlorine atoms on the
m
m
Finally, the benzene ring of E1 was replaced with naphthalene
ring, furan ring or thiophene ring respectively (E28eE30), which
resulted in an obvious decrease in potency with MIC values of 25 to
>50 mg/mL. This suggested that the benzene ring contributed to the
antibacterial activity of our compounds.
5-position of salicylaldehyde displayed lower antibacterial activity
than E9 and when the substitute happened on the same position of
benzene ring, the potency order was Cl > Br. For example, E7 (3.13e
6.25
mg/mL) > E8 (12.5e25
mg/mL); E9 (0.39e1.56 mg/mL) > E10
(3.13e12.5
mg/mL). Besides, compared to E2, E9 showed better
antibacterial activities. It referred that the 4-hydrazonomethyl-
benzene-2,4-dichloro-1-ol moiety is an important core structure
for the antibacterial activity of E9.
2.2.2. E. coli FabH inhibitory activity
In addition, we selected the top 9 compounds (E1, E2, E3, E4, E5,
E7, E8 E9 and E23) and the bottom 3 compounds (E16, E21 and E30)
of antibacterial activity according to their MIC values to examine
their E. coli FabH inhibitory activity and the results were summa-
rized in Table 3. As shown in Table 3, all the bottom compounds
showed poor E. coli FabH inhibitory activities while most of the top
compounds displayed potent E. coli FabH inhibitory activities.
Among the tested compounds, E9 showed the most potent inhib-
itory activity with IC50 of 2.5
positive control DDCP [34] with IC50 of 2.8
compounds displayed moderate inhibitory activity with IC50
ranging from 10.5 to 45.8 M while the IC50 of the bottom 3
m
M, which was comparable to the
mM. Other tested top
Fig. 4. Crystal structure diagram of compound E22.
m

X.-L. Wang et al. / European Journal of Medicinal Chemistry 57 (2012) 373e382
377
Table 3
2.2.3. Cytotoxicity test
E. coli FabH inhibitory activities, hemolytic activities and cytotoxicity of the selected
In order to find potent antibiotics, it is important to measure
cytotoxicity. One of the major hindrances to the clinical use of many
compounds with effective antibacterial activities, however, is their
ability to damage mammalian cell that leads to high hemolytic and
cytotoxic activity dangerous to the host organism. Hemolytic activity
is conventionally used as a measure of cytotoxicity and model for
mammalian cells because red blood cells are, in general, extremely
fragile. However, sometimes, compounds which show low hemolytic
activities have the severe cytotoxicity against mammalian cells. So
the hemolysis and cytotoxicity assays were both tested.
The compounds selected above were also detected for their
hemolytic activity. Then we further tested their cytotoxic activity
on a mouse embryonic fibroblast cell line (NIH-3T3) using the MTT
assay to prove the potency of the compounds [19].
The pharmacological results of these compounds were
summarized in Table 3. It can be seen from Table 3 that the selected
compounds displayed low hemolytic activities. Besides, the cyto-
toxicity assay determined the selectivity of our compounds for
bacterial over mammalian cells. What we can see from the data is
that the compounds with potent inhibitory activity were low toxic.
compounds.
Compounds
E. coli FabH
IC50 M)
Hemolysis
Cytotoxicity
IC50 M)
(
m
LC30a (mg/mL)
(m
E1
E2
E3
E4
E5
E7
E8
E9
E16
E21
E23
E30
DDCP
41.2
13.8
23.2
11.3
24.1
12.6
14.7
2.1
>100
>100
28.9
>100
2.6
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
143.1
155.3
113.4
158.6
123.7
180.4
200.6
130.5
125.6
232.3
198.4
210.5
123.7
a
Lytic concentration 30%.
compounds was all >100 mM. The results indicated that E9 had
a high binding affinity with E. coli FabH, which supported the
potent antibacterial activity of E9.
Then, an analysis between the antibacterial activity against
E. coli and the E. coli FabH inhibitory activity of the top 9 compounds
(E1, E2, E3, E4, E5, E7, E8, E9 and E23) indicated that there was
a moderate correlation between FabH inhibition and inhibition of
E. coli cellular proliferation, as evidenced in Fig. 5, with a correlation
coefficient of 1.5808, R square value 0.9146.
The results of E. coli FabH inhibitory activities of the test
compounds were corresponding to the structureeactivity rela-
tionships (SAR) of their antibacterial activities. This demonstrated
that the potent antibacterial activities of the synthetic compounds
were probably correlated to their FabH inhibitory activities.
However, P. aeruginosa was reported to have a different FabH
with E. coli. So it is curious indeed that the MIC for E9 in these two
strains is the same. Fortunately, we evaluated the antibacterial
activities of our compounds with both MIC and IC50 values
(not listed) when we did the bioassay. Although MIC values are the
same, the effect of anti-proliferation is different. The data indicated
that the antibacterial activities of our compounds against E. coli
were better than that of P. aeruginosa, including compound E9. The
IC50 value of compound E9 against E. coli and P. aeruginosa are 0.81
2.3. Binding model of E9 and E. coli FabH
To gain better understanding on the potency of the compound E9
and guide further SAR studies, the molecular docking of all the
synthetic acylhydrazone derivatives and E. coli FabH was performed
on the binding model based on the E. coli FabHeCoA complex
structure (1HNJ pdb) [35] by using the Discovery Stutio (version 3.1).
The docking calculation of all the compounds was depicted in
Table 4. As shown in Table 4, our compounds had good binding
affinity to FabH and the results of EDOCKER_ENERGY almost have
the same trend with the structureeactivity relationships of anti-
bacterial activities, which further confirmed the correlation
between the antibacterial activities and E. coli FabH inhibitory
activities of our compounds.
Among them, compound E9 with the most potent inhibitory
activity showed the minimum data, which suggested it was mostly
easy to combine with E. coli FabH. The binding model of compound
E9 with 1HNJ is depicted in Fig. 6. In the binding model of E9, E9 is
nicely bound to the 1HꢀNJ via four hydrogen bonds with ARGꢀ36 (He
and 2.14
mM, respectively. This suggested that our compounds
ꢀ
ꢀ
O/H: 1.95 A, 144.497 ), GLY 209 (HeO/H: 2.25 A, 131.92 ), MET
maybe acting as multi-kinase inhibitors, and FabH was one of their
targets. In addition, the antibacterial effects of our compounds
against P. aeruginosa are more likely to off-target due to inhibit
targets, such as FabB, FabF and other enzymes in FAS pathway or
even other pathways.
ꢀ
ꢀ
ꢀ
207 (HeO/H: 2.02 A 144.966 ) and ASN 247 (HeO/H: 1.93 A,
160.54ꢀ), which enhance the binding affinity, resulting in the
increased antimicrobial activity of this compound. The binding
Table 4
CDOCKER_ENERGY of compounds and 1HNJ.
Compounds CDOCKER ENERGY PSA Compounds CDOCKER ENERGY PSA
D
Gb (kcal/mol)
DGb (kcal/mol)
E1
E2
E3
E4
E5
E6
E7
E8
ꢃ16.13
ꢃ18.49
ꢃ16.26
ꢃ18.67
ꢃ16.31
ꢃ16.63
ꢃ18.65
ꢃ18.66
ꢃ21.33
ꢃ18.21
ꢃ15.28
ꢃ12.75
ꢃ10.35
ꢃ13.79
ꢃ10.68
70.9 E16
91.2 E17
91.2 E18
111.4 E19
111.4 E20
100.4 E21
91.2 E22
91.2 E23
91.2 E24
91.2 E25
70.9 E26
70.9 E27
70.9 E28
70.9 E29
70.9 E30
ꢃ9.38
ꢃ12.87
ꢃ11.31
ꢃ12.89
ꢃ15.54
ꢃ10.78
ꢃ15.78
ꢃ17.02
ꢃ16.70
ꢃ10.73
ꢃ16.32
ꢃ15.58
ꢃ16.22
ꢃ14.26
ꢃ10.80
70.9
70.9
70.9
116.7
116.7
116.7
70.9
70.9
70.9
80.16
80.16
70.9
E9
E10
E11
E12
E13
E14
E15
70.9
84.1
70.9
Fig. 5. Correlation between the antibacterial activity against E. coli and the E. coli FabH
inhibitory activity, r ¼ 1.5808, R2 ¼ 0.9146.

378
X.-L. Wang et al. / European Journal of Medicinal Chemistry 57 (2012) 373e382
model of E9 inferred that the 4-hydrazonomethyl-benzene-2,4-
dichloro-1-ol moiety made the ligand easier to conduct hydrogen
bond with FabH and the introduction of benzene-1-methoxy-2-ol
moiety also enhanced the binding affinity, which confirmed our
structural optimization. Therefore, high binding affinities resulted
in strong antimicrobial activity and the initial attempt to achieve
new potential antibacterial FabH inhibitors has been verified with
the expected results.
spectra were recorded on a Bruker DPX 300 model Spectrometer 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 ꢄ0.4%
of the theoretical values. TLC was run on the silica gel coated
ꢀ
aluminum sheets (Silica Gel 60 A GF254, E. Merk, Germany) and
visualized in UV light (254 nm).
However, compound E8 showed good binding affinity for FabH
with the CDOCKER_ENERGY of ꢃ18.06 kcal/mol and IC50 of 14.7
against FabH. Unfortunately, it showed low antimicrobial activity
4.2. General procedure for synthesis of the target compounds
To a stirred solution of hydrazide C (1 mmol) and compound D
(1 mmol) in ethanol (15 mL), water (1 mL) was added followed by
dropwise addition of glacial acetic acid (0.2 mL). The resulting
mixture was stirred at room temperature until the target product
precipitated from the solvent, which was collected using suction
filtration and dried, followed by recrystallization in ethanol.
against E. coli with MIC value of 12.5 mg/mL. Then molecular polar
surface area (PSA) was also calculated in Table 4, which is a very
useful parameter for prediction of drug transport properties, related
to the binding affinity and the cell permeability of FabH inhibitors.
PSA values in the range of 55e100 Å2 increase the probability of
a compound being a potent inhibitor of FabH. The results shown E8
satisfied the PSA criteria, so the interactions between E8 and E. coli
FabH maybe not critical for antimicrobial activity against E. coli.
4.2.1. (E)-N0-Benzylidene-4-hydroxy-3-methoxybenzohydrazide (E1)
White powder, yield 89%; mp: 111e112 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.85 (s, 3H), 6.86e6.88 (d, J ¼ 8.22 Hz, 1H), 7.43e7.49 (m,
5H), 7.70e7.72 (d, J¼ 6.39 Hz, 2H), 8.44(s,1H), 9.70 (s,1H),11.60(s,1H).
MS (ESI): 271.10 (C15H15N2O3, [M þ H]þ). Anal. Calcd for C15H14N2O3:C,
66.66; H, 5.22; N, 10.36%. Found: C, 66.45; H, 5.21; N, 10.33%.
3. Conclusions
In conclusion, a series of vanillic acylhydrazone derivatives was
designed, prepared and evaluated for their antibacterial activity
against E. coli ATCC 25922, P. aeruginosa ATCC 27853, B. subtilis ATCC
530 and S. aureus ATCC 25923 in order to achieve new better
potential antibacterial FabH inhibitors. Out of the 30 vanillic acyl-
hydrazone derivatives, E9 showed the most potent antibacterial
4.2.2. (E)-4-Hydroxy-N0-(2-hydroxybenzylidene)-3-
methoxybenzohydrazide (E2)
White powder, yield 89%; mp: 221e222 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.81 (s, 3H), 6.85e6.87 (d, J ¼ 7.89 Hz,1H), 7.00e7.03 (m,
2H), 7.41e7.44 (m, 1H), 7.47 (s, 1H), 7.53e7.56 (m, 2H), 8.33 (s, 1H),
9.64 (s, 1H), 9.84 (s, 1H), 11.39 (s, 1H). MS (ESI): 287.10 (C15H15N2O4,
[M þ H]þ). Anal. Calcd for C15H14N2O4: C, 62.93; H, 4.93; N, 9.79%.
Found: C, 62.75; H, 4.95; N, 9.82%.
activity with MIC values of 0.39e1.56
tested bacterial strains and exhibited the most potent E. coli
FabH inhibitory activity with IC50 of 2.5 M. Preliminary structuree
mg/mL against the
m
activity relationships and molecular modeling study provided
further insight into interactions between the enzyme and its ligands.
This study shows that E9 is a novel compound that can be potent
antimicrobial inhibitor of FabH and provides valuable information
for the design of E. coli FabH inhibitors as antibacterial agents.
4.2.3. (E)-4-Hydroxy-N0-(4-hydroxybenzylidene)-3-
methoxybenzohydrazide (E3)
White powder, yield 87%; mp: 245e246 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.82 (s, 3H), 6.82e6.87 (m, 3H), 7.41e7.44 (m, 1H), 7.47
(s,1H), 7.53e7.56 (m, 2H), 8.33 (s,1H), 9.64 (s,1H), 9.87 (s,1H),11.39
(s, 1H). MS (ESI): 287.10 (C15H15N2O4, [M þ H]þ). Anal. Calcd for
4. Experimental section
4.1. Materials and measurements
C15H14N2O4: C, 62.93; H, 4.93; N, 9.79%. Found: C, 62.80; H, 4.92; N,
9.75%.
All chemicals (reagent grade) used were purchased from Sigma
Aldrich (USA) and Sinopharm Chemical Reagent Co., Ltd (China).
Melting points (uncorrected) were determined on a XT4 MP
apparatus (Taike Corp., Beijing, China). ESI-MS spectra were recor-
ded on a Mariner System 5304 Mass spectrometer, and 1H NMR
4.2.4. (E)-N0-(2,4-Dihydroxybenzylidene)-4-hydroxy-3-
methoxybenzohydrazide (E4)
White powder, yield 85%; mp: 255e256 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.83 (s, 3H), 6.89e6.91 (m, 2H), 7.52 (s, 1H), 7.55 (s, 1H),
Fig. 6. (A) 2D molecular docking modeling of compound E9 with 1HNJ. The purple circles showed the amino acids which participated in hydrogen bonding and the green circles
showed the amino acids which participated in the Van der Waals interactions. (B) 3D model of the interaction between compound E9 and 1HNJ bonding site. The four H-bonds
(green lines) are displayed as dotted lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

X.-L. Wang et al. / European Journal of Medicinal Chemistry 57 (2012) 373e382
379
7.59e7.63 (m, 2H), 8.44 (s, 1H), 9.64 (s, 1H), 9.71 (s, 1H), 9.88 (s, 1H),
11.62 (s, 1H). MS (ESI): 303.09 (C15H15N2O5, [M þ H]þ). Anal. Calcd
for C15H14N2O5: C, 59.60; H, 4.67; N, 9.27%. Found: C, 59.80; H, 4.68;
N, 9.32%.
for C15H13FN2O3: C, 62.50; H, 4.55; N, 9.72%. Found: C, 62.68; H,
4.56; N, 9.75%.
4.2.12. (E)-N0-(3-Fluorobenzylidene)-4-hydroxy-3-
methoxybenzohydrazide (E12)
4.2.5. (E)-N0-(3,4-Dihydroxybenzylidene)-4-hydroxy-3-
methoxybenzohydrazide (E5)
White powder, yield 92%; mp: 102e103 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.82 (s, 3H), 6.85e6.87 (d, J ¼ 8.43 Hz,1H), 7.41e7.44 (m,
2H), 7.47 (s, 1H), 7.53e7.56 (m, 2H), 8.33 (s, 1H), 9.64 (s, 1H), 9.87 (s,
1H), 11.39 (s, 1H). MS (ESI): 289.09 (C15H14FN2O3, [M þ H]þ). Anal.
Calcd for C15H13FN2O3: C, 62.50; H, 4.55; N, 9.72%. Found: C, 62.70;
H, 4.57; N, 9.75%.
White powder, yield 85%; mp: 260e261 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.81 (s, 3H), 6.85 (s, 1H), 7.29e7.34 (m, 2H), 7.55 (s, 1H),
7.55e7.58 (m, 2H), 8.49 (s, 1H), 9.66 (s, 1H), 9.74 (s, 1H), 9.89 (s, 1H),
11.59 (s, 1H). MS (ESI): 303.09 (C15H15N2O5, [M þ H]þ). Anal. Calcd
for C15H14N2O5: C, 59.60; H, 4.67; N, 9.27%. Found: C, 59.77; H, 4.68;
N, 9.22%.
4.2.13. (E)-N0-(2-Fluorobenzylidene)-4-hydroxy-3-
methoxybenzohydrazide (E13)
4.2.6. (E)-4-Hydroxy-N0-(4-hydroxy-3-methoxybenzylidene)-3-
methoxybenzohydrazide (E6)
White powder, yield 90%; mp: 114e115 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.82 (s, 3H), 6.86e6.89 (d, J ¼ 8.04 Hz, 1H), 7.27e7.33
(m, 2H), 7.44e7.49 (m, 3H), 7.93 (s, 1H), 8.69 (s, 1H), 9.73 (s, 1H),
11.74 (s, 1H). MS (ESI): 289.09 (C15H14FN2O3, [M þ H]þ). Anal. Calcd
for C15H13FN2O3: C, 62.50; H, 4.55; N, 9.72%. Found: C, 62.67; H,
4.57; N, 9.76%.
White powder, yield 87%; mp: 102e103 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.80 (s, 3H), 3.82 (s, 3H), 6.81e6.84 (m, 2H), 7.33e7.36
(m, 2H), 7.39e7.40 (m, 2H), 8.46 (s, 1H), 8.48 (s, 1H), 9.57 (s, 1H),
11.47 (s, 1H). MS (ESI): 317.11 (C16H17N2O5, [M þ H]þ). Anal. Calcd
for C16H16N2O5: C, 60.75; H, 5.10; N, 8.86%. Found: C, 60.58; H, 5.10;
N, 8.83%.
4.2.14. (E)-N0-(4-Chlorobenzylidene)-4-hydroxy-3-
methoxybenzohydrazide (E14)
4.2.7. (E)-N0-(5-Chloro-2-hydroxybenzylidene)-4-hydroxy-3-
methoxybenzohydrazide (E7)
White powder, yield 93%; mp: 211e212 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.85 (s, 3H), 6.84e6.89 (d, J ¼ 6.96 Hz, 1H), 7.27e7.33 (t,
J ¼ 8.97 Hz, 2H), 7.27e7.49 (m, 2H), 7.74e7.79 (m, 2H), 8.44 (s, 1H),
9.70 (s, 1H), 11.61 (s, 1H). MS (ESI): 305.06 (C15H14ClN2O3,
[M þ H]þ). Anal. Calcd for C15H13ClN2O3: C, 59.12; H, 4.30; N, 9.19%.
Found: C, 59.08; H, 4.31; N, 9.23%.
White powder, yield 92%; mp: 210e211 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.80 (s, 3H), 6.84e6.87 (d, J ¼ 8.43 Hz,1H), 7.01e7.03 (m,
1H), 7.41e7.43 (m, 2H), 7.54e7.57 (m, 2H), 8.34 (s, 1H), 9.63 (s, 1H);
9.85 (s, 1H); 11.39 (s, 1H). MS (ESI): 321.06 (C15H14ClN2O4,
[M þ H]þ). Anal. Calcd for C15H13ClN2O4: C, 56.17; H, 4.09; N, 8.73%.
Found: C, 56.36; H, 4.12; N, 8.77%.
4.2.15. (E)-N0-(3-Chlorobenzylidene)-4-hydroxy-3-
methoxybenzohydrazide (E15)
4.2.8. (E)-N0-(5-Bromo-2-hydroxybenzylidene)-4-hydroxy-3-
methoxybenzohydrazide (E8)
White powder, yield 89%; mp: 223e224 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.85 (s, 3H), 6.87e6.90 (d, J ¼ 7.68 Hz,1H), 7.38e7.49 (m,
3H), 7.61e7.64 (m, 1H), 7.70e7.72 (m, 1H), 7.91 (s, 1H), 8.41 (s, 1H),
9.74 (s,1H),11.73 (s,1H). MS (ESI): 305.06 (C15H14ClN2O3, [M þ H]þ).
Anal. Calcd for C15H13ClN2O3: C, 59.12; H, 4.30; N, 9.19%. Found: C,
58.92; H, 4.30; N, 9.22%.
White powder, yield 91%; mp: 216e217 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.81 (s, 3H), 6.87e6.88 (d, J ¼ 7.67 Hz,1H), 7.02e7.04 (m,
1H), 7.41e7.44 (m, 2H), 7.54e7.57 (m, 2H), 8.34 (s, 1H), 9.58 (s, 1H),
9.72 (s, 1H), 11.51 (s, 1H). MS (ESI): 365.01 (C15H14BrN2O4,
[M þ H]þ). Anal. Calcd for C15H13BrN2O4: C, 49.33; H, 3.59; N, 7.67%.
Found: C, 49.10; H, 3.61; N, 7.70%.
4.2.16. (E)-N0-(2,4-Dichlorobenzylidene)-4-hydroxy-3-
methoxybenzohydrazide (E16)
4.2.9. (E)-N0-(3,5-Dichloro-2-hydroxybenzylidene)-4-hydroxy-3-
methoxybenzohydra-zide (E9)
White powder, yield 88%; mp: 128e129 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.85 (s, 3H), 6.86e6.89 (d, J ¼ 8.04 Hz, 1H), 7.44e7.54
(m, 3H), 7.72e7.73 (d, J ¼ 2.01 Hz, 1H), 8.00e8.03 (d, J ¼ 7.86 Hz,
1H), 8.78 (s, 1H), 9.76 (s, 1H), 11.89 (s, 1H). MS (ESI): 339.02
(C15H13Cl2N2O3, [M þ H]þ). Anal. Calcd for C15H12Cl2N2O3: C, 53.12;
H, 3.57; N, 8.26%. Found: C, 53.01; H, 3.56; N, 8.23%.
White powder, yield 90%; mp: 180e181 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.81 (s, 3H), 6.85e6.88 (s, 1H), 7.22e7.24 (m, 1H), 7.43e
7.47 (m, 1H), 7.51 (s, 1H), 7.54e7.57 (m, 1H), 8.34 (s, 1H), 9.58 (s, 1H),
9.72 (s, 1H), 11.51 (s, 1H). MS (ESI): 355.02 (C15H13Cl2N2O4,
[M þ H]þ). Anal. Calcd for C15H12Cl2N2O4: C, 50.72; H, 3.41; N, 7.89%.
Found: C, 50.92; H, 3.42; N, 7.91%.
4.2.17. (E)-N0-(4-Bromobenzylidene)-4-hydroxy-3-
methoxybenzohydrazide (E17)
4.2.10. (E)-N0-(3,5-Dibromo-2-hydroxybenzylidene)-4-hydroxy-3-
methoxybenzohydrazide (E10)
White powder, yield 90%; mp: 124e125 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.84 (s, 3H), 6.85e6.88 (d, J ¼ 8.04 Hz,1H), 7.43e7.48 (m,
2H), 7.66 (s, 4H), 8.41 (s, 1H), 9.72 (s, 1H), 11.67 (s, 1H). MS (ESI):
349.01 (C15H14BrN2O3, [M þ H]þ). Anal. Calcd for C15H13BrN2O3: C,
51.60; H, 3.75; N, 8.02%. Found: C, 51.81; H, 3.78; N, 8.06%.
White powder, yield 84%; mp: 204e205 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.81 (s, 3H), 6.84e6.87 (s, 1H), 7.22e7.25 (m, 1H), 7.41e
7.45 (m, 1H), 7.50 (s, 1H), 7.53e7.57 (m, 1H), 8.38 (s, 1H), 9.49 (s, 1H),
9.68 (s, 1H), 11.47 (s, 1H). MS (ESI): 442.92 (C15H13Br2N2O4,
[M þ H]þ). Anal. Calcd for C15H12Br2N2O4: C, 40.57; H, 2.72; N,
6.31%. Found: C, 40.69; H, 2.71; N, 6.29%.
4.2.18. (E)-N0-(3-Bromobenzylidene)-4-hydroxy-3-
methoxybenzohydrazide (E18)
White powder, yield 93%; mp: 113e114 ꢀC. 1H NMR
(300 MHz, DMSO-d6): 3.85 (s, 3H), 6.86e6.89 (d, J ¼ 8.25 Hz,
1H), 7.39e7.49 (m, 3H), 7.60e7.63 (d, J ¼ 8.04 Hz, 1H), 7.69e7.72
(d, J ¼ 7.5 Hz, 1H), 7.91 (s, 1H), 8.40 (s, 1H), 9.73 (s, 1H), 11.74 (s,
1H). MS (ESI): 349.0 (C15H14BrN2O3, [M þ H]þ). Anal. Calcd for
4.2.11. (E)-N0-(4-Fluorobenzylidene)-4-hydroxy-3-
methoxybenzohydrazide (E11)
White powder, yield 89%; mp: 196e197 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.81e3.83 (m, 3H), 6.84e6.87 (d, J ¼ 8.35 Hz, 1H), 7.41e
7.73 (m, 4H), 7.51e7.73 (d, J ¼ 8.4 Hz, 2H), 8.41 (s, 1H), 9.69 (s, 1H),
11.65 (s, 1H). MS (ESI): 289.09 (C15H14FN2O3, [M þ H]þ). Anal. Calcd
C
15H13BrN2O3: C, 51.60; H, 3.75; N, 8.02%. Found: C, 51.40; H,
3.76; N, 8.05%.

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X.-L. Wang et al. / European Journal of Medicinal Chemistry 57 (2012) 373e382
4.2.19. (E)-4-Hydroxy-3-methoxy-N0-(4-nitrobenzylidene)
benzohydrazide (E19)
4.2.26. (E)-4-Hydroxy-3-methoxy-N0-(3-methoxybenzylidene)
benzohydrazide (E26)
Yellow powder, yield 93%; mp: 189e190 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.86 (s, 3H), 6.88e6.91 (d, J ¼ 8.25 Hz, 1H), 7.46e7.60
(m, 2H), 7.96e8.00 (d, J ¼ 8.58 Hz, 2H), 8.29e8.32 (d, J ¼ 8.76 Hz,
2H), 8.53 (s, 1H), 9.77 (s, 1H), 11.91 (s, 1H). MS (ESI): 316.09
(C15H14N3O5, [M þ H]þ). Anal. Calcd for C15H13N3O5: C, 57.14; H,
4.16; N, 13.33%. Found: C, 57.01; H, 4.17; N, 13.35%.
White powder, yield 89%; mp: 174e175 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.81 (s, 3H), 3.85 (s, 3H), 6.86e6.89 (d, J ¼ 8.22 Hz, 1H),
6.99e7.02 (m, 1H), 7.26e7.28 (m, 2H), 7.35e7.40 (m, 2H), 7.48e7.49
(m, 1H), 8.42 (s, 1H), 9.71 (s, 1H), 11.62 (s, 1H). MS (ESI): 301.11
(C16H17N2O4, [M þ H]þ). Anal. Calcd for C16H16N2O4: C, 63.99; H,
5.37; N, 9.33%. Found: C, 63.80; H, 5.35; N, 9.36%.
4.2.27. (E)-4-Hydroxy-3-methoxy-N0-((E)-3-phenylallylidene)
benzohydrazide (E27)
4.2.20. (E)-4-Hydroxy-3-methoxy-N0-(3-nitrobenzylidene)
benzohydrazide (E20)
White powder, yield 90%; mp: 222e223 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.84 (s, 3H), 6.84e6.87 (d, J ¼ 8.22 Hz, 1H), 7.03e7.06 (d,
J ¼ 8.40 Hz, 2H), 7.32e7.46 (m, 5H), 7.61e7.63 (d, J ¼ 6.96 Hz, 2H),
8.20 (s, 1H), 9.69 (s, 1H), 11.50 (s, 1H). MS (ESI): 297.12 (C17H17N2O3,
[M þ H]þ). Anal. Calcd for C17H16N2O3: C, 68.91; H, 5.44; N, 9.45%.
Found: C, 68.79; H, 5.43; N, 9.49%.
Yellow powder, yield 85%; mp: 193e194 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.84 (s, 3H), 6.88e6.90 (d, J ¼ 8.01 Hz,1H), 7.47e7.54 (m,
2H), 7.68e7.72 (m, 2H), 7.84e7.88 (m, 1H), 8.07e8.15 (m, 1H), 8.90
(s, 1H), 9.76 (s, 1H), 11.93 (s, 1H). MS (ESI): 316.09 (C15H14N3O5,
[M þ H]þ). Anal. Calcd for C15H13N3O5: C, 57.14; H, 4.16; N, 13.33%.
Found: C, 57.29; H, 4.14; N, 13.34%.
4.2.28. (E)-4-Hydroxy-3-methoxy-N0-(naphthalen-2-ylmethylene)
benzohydrazide (E28)
4.2.21. (E)-4-Hydroxy-3-methoxy-N0-(2-nitrobenzylidene)
benzohydrazide (E21)
White powder, yield 86%; mp: 144e145 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.83 (s, 3H), 6.84e6.86 (m,1H), 7.43e7.49 (m, 5H), 7.59e
7.61 (d, J ¼ 7.4 Hz, 2H), 7.70e7.72 (m, 2H), 8.44 (s, 1H), 9.70 (s, 1H),
11.60 (s, 1H). MS (ESI): 320.12 (C19H17N2O3, [M þ H]þ). Anal. Calcd
for C19H16N2O3: C, 71.24; H, 5.03; N, 8.74%. Found: C, 71.01; H, 5.04;
N, 8.70%.
Yellow powder, yield 86%; mp: 198e199 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.85 (s, 3H), 6.87e6.90 (d, J ¼ 8.04 Hz, 1H), 7.46e7.50 (d,
J ¼ 9.27 Hz, 2H), 7.64e7.70 (t, J ¼ 5.12 Hz, 1H), 7.79e7.84 (t,
J ¼ 7.5 Hz, 1H), 8.05e8.13 (m, 2H), 8.88 (s, 1H), 9.75 (s, 1H), 11.95 (s,
1H). MS (ESI): 316.09 (C15H14N3O5, [M þ H]þ). Anal. Calcd for
C
15H13N3O5: C, 57.14; H, 4.16; N, 13.33%. Found: C, 57.01; H, 4.17; N,
13.37%.
4.2.29. (E)-N0-(Furan-2-ylmethylene)-4-hydroxy-3-
methoxybenzohydrazide (E29)
4.2.22. (E)-4-Hydroxy-3-methoxy-N0-(4-methylbenzylidene)
benzohydrazide (E22)
White powder, yield 89%; mp: 112e113 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 4.08 (s, 3H), 6.62e6.64 (m, 1H), 6.85e6.91 (m, 2H),
7.41e7.46 (t, J ¼ 8.05 Hz, 2H), 7.84 (s, 1H), 8.34 (s, 1H), 9.72 (s, 1H),
11.59 (s, 1H). MS (ESI): 260.08 (C13H13N2O4, [M þ H]þ). Anal. Calcd
for C13H12N2O4: C, 60.00; H, 4.65; N, 10.76%. Found: C, 60.11; H,
4.66; N, 10.80%.
White powder, yield 87%; mp: 124e125 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 2.34 (s, 3H); 3.85 (s, 3H), 6.85e6.88 (d, J ¼ 8.25 Hz, 1H),
7.25e7.27 (d, J ¼ 7.71 Hz, 2H), 7.42e7.47 (m, 2H), 7.59e7.62 (d,
J ¼ 7.71 Hz, 2H), 8.40 (s, 1H), 9.67 (s, 1H), 11.52 (s, 1H). MS (ESI):
285.12 (C16H17N2O3, [M þ H]þ). Anal. Calcd for C16H16N2O3: C,
67.59; H, 5.67; N, 9.85%. Found: C, 67.41 H, 5.67; N, 9.89%.
4.2.30. (E)-4-Hydroxy-3-methoxy-N0-(thiophen-2-ylmethylene)
benzohydrazide (E30)
4.2.23. (E)-4-Hydroxy-3-methoxy-N0-(3-methylbenzylidene)
benzohydrazide (E23)
White powder, yield 85%; mp: 115e116 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 3.84 (s, 3H), 6.85e6.88 (d, J ¼ 8.22 Hz, 1H), 7.13e7.15 (t,
J ¼ 4.31 Hz, 1H), 7.41e7.46 (m, 3H), 7.65e7.66 (d, J ¼ 5.13 Hz, 1H),
8.66 (s, 1H), 9.71 (s, 1H), 11.55 (s, 1H). MS (ESI): 277.06
(C13H13N2O3S, [M þ H]þ). Anal. Calcd for C13H12N2O3S: C, 56.51; H,
4.38; N, 10.14%. Found: C, 56.33; H, 4.37; N, 10.17%.
White powder, yield 87%; mp: 135e136 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 2.50 (s, 3H), 3.86 (s, 3H), 6.86e6.89 (d, J ¼ 8.34 Hz, 1H),
7.20e7.21 (d, J ¼ 8.01 Hz, 1H), 7.26e7.28 (d, J ¼ 7.76 Hz, 1H), 7.42e
7.47 (m, 2H), 7.59e7.63 (m, 2H), 8.48 (s, 1H), 9.69 (s, 1H), 11.54 (s,
1H). MS (ESI): 285.12 (C16H17N2O3, [M þ H]þ). Anal. Calcd for
C
16H16N2O3: C, 67.59; H, 5.67; N, 9.85%. Found: C, 67.77 H, 5.66; N,
9.87%.
4.3. Crystal structure determination
4.2.24. (E)-4-Hydroxy-3-methoxy-N0-(2-methylbenzylidene)
benzohydrazide (E24)
Crystal structure determination of compound E22 was carried
out on a Nonius CAD4 diffractometer equipped with graphite-
White powder, yield 89%; mp: 153e154 ꢀC. 1H NMR (300 MHz,
DMSO-d6): 2.45 (s, 3H), 3.87 (s, 3H), 6.86e6.88 (d, J ¼ 7.65 Hz, 1H),
7.23e7.25 (m, 2H), 7.40e7.46 (m, 2H), 7.57e7.60 (m, 2H), 8.46 (s,
1H), 9.68 (s, 1H), 11.51 (s, 1H). MS (ESI): 285.12 (C16H17N2O3,
[M þ H]þ). Anal. Calcd for C16H16N2O3: C, 67.59; H, 5.67; N, 9.85%.
Found: C, 67.45; H, 5.66; N, 9.87%.
monochromated MoK
a
(l
¼ 0.71073 A) radiation. The structure
ꢀ
was solved by direct methods and refined on F2 by full-matrix least
squares methods using SHELX-97 [36]. 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 E22 are
listed in Table 1.
4.2.25. (E)-4-Hydroxy-3-methoxy-N0-(4-methoxybenzylidene)
benzohydrazide (E25)
White powder, yield 85%; mp: 118e119 ꢀC. 1H NMR
(300 MHz, DMSO-d6): 3.81 (s, 3H), 3.84 (s, 3H), 6.84e6.87 (d,
J ¼ 8.25 Hz, 1H), 7.00e7.03 (m, J ¼ 8.76 Hz, 2H), 7.41e7.47 (m,
2H), 7.64e7.67 (m, J ¼ 6.75 Hz, 2H), 8.38 (s, 1H), 9.67 (s, 1H),
11.46 (s, 1H). MS (ESI): 301.11 (C16H17N2O4, [M þ H]þ). Anal.
Calcd for C16H16N2O4: C, 63.93; H, 5.37; N, 9.33%. Found: C,
63.80; H, 5.36; N, 9.36%.
4.4. Antibacterial activity
The antibacterial activities of the synthetic compounds were
tested against two Gram-negative bacterial strains: E. coli ATCC

X.-L. Wang et al. / European Journal of Medicinal Chemistry 57 (2012) 373e382
381
25922 and P. aeruginosa ATCC 27853, two Gram-positive bacterial
4.7. Cytotoxicity test
strains: B. subtilis ATCC 530 and S. aureus ATCC 25923, using
method recommended by National Committee for Clinical Labo-
ratory Standards (NCCLS).
The cytotoxic activity in vitro was measured against mouse
fibroblast NIH-3T3 cells using the MTT assay. Cells were cultured in
a 96-well plate at a density of 5 ꢁ 105 cells and different concen-
trations of compounds were respectively added to each well. The
incubation was permitted at 37 ꢀC, 5% CO2 atmosphere for 24 h
before the cytotoxicity assessments. 20 mL MTT reagent (4 mg/mL)
was added per well 4 h before the end of the incubation. Four hours
later, the plate was centrifugated at 1200 rcf for 5 min and the
In vitro activities of the compounds were tested in Nutrient
broth (NB) for bacteria by the two-fold serial dilution method.
Seeded broth (broth containing microbial spores) was prepared in
NB from 24 h old bacterial cultures on nutrient agar (Hi-media) at
37 ꢄ 1 ꢀC. The bacterial suspension was adjusted with sterile saline
to a concentration of 1 ꢁ 104e105 CFU. The tested compounds and
reference drugs were prepared by two-fold serial dilution to obtain
the required concentrations of 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 and
supernatants were removed, each well was added with 200
mL
DMSO. The absorbance was measured at a wavelength of 570 nm
(OD570 nm) on an ELISA microplate reader. Three replicate wells
were used for each concentration and each assay was measured
three times, after which the average of IC50 was calculated. The
cytotoxicity of each compound was expressed as the concentration
of compound that reduced cell viability to 50% (IC50). The results
were summarized in Table 3.
0.39
mg/mL. The tubes were incubated in BOD incubators at
37 ꢄ 1 ꢀC for bacteria. The MICs were recorded by visual observa-
tions after 24 h (for bacteria) of incubation. Kanamycin and DDCP
were used as standards for bacterial. The observed MICs are pre-
sented in Table 2.
4.5. E. coli FabH purification and activity assay
4.8. Docking simulations
Native E. coli FabH protein was overexpressed in E. coli DH10B
cells using the pET30 vector and purified to homogeneity in three
chromatographic steps (Q-Sepharose, MonoQ, and hydroxyapatite)
at 4 ꢀC. The selenomethionine-substituted protein was expressed
in E. coli BL21 (DE3) cells and purified in a similar way. Harvested
cells containing FabH 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 supernatant was applied to a Ni-NTA agarose column,
washed, and 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 ꢃ80 ꢀC in 20 mM Tris,
pH 7.6, 100 mM NaCl, 1 mM DTT, and 20% glycerol for enzymatic
assay.
Molecular docking of compounds E9 into the three-dimensional
X-ray structure of E. coli FabH (PDB code: 1HNJ) was carried out
using the Discovery Stutio (version 3.1) as implemented through
the graphical user interface DS-CDocker protocol.
The three-dimensional structures of the aforementioned
compounds were constructed using Chem 3D ultra 12.0 software
[Chemical Structure Drawing Standard; Cambridge Soft corpora-
tion, USA (2010)], then they were energetically minimized by using
MMFF94 with 5000 iterations and minimum RMS gradient of 0.10.
The crystal structures of E. coli FabH were retrieved from the RCSB
Protein Data Bank (http://www.rcsb.org/pdb/home/home.-do). All
bound water and ligands were eliminated from the protein and the
polar hydrogen was added. The whole E. coli FabH was defined as
a receptor and the site sphere was selected based on the ligand
binding location of malonyl-CoA, then the malonyl-CoA molecule
was removed and E9 was placed during the molecular docking
procedure. Types of interactions of the docked protein with ligand
were analyzed after the end of molecular docking.
In a final 20
DTT, 0.25 mMMgCl2, and 2.5
1 nM FabH, and H2O was added to 15
a 2 L mixture of 25 M acetyl-CoA, 0.5 mM NADH, and 0.5 mM
NADPH 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
m
L reaction, 20 mM Na2HPO4, pH 7.0, 0.5 mM
M holo-ACP were mixed with
L. After 1 min incubation,
m
m
m
m
m
Acknowledgment
was washed with 10% ice-cold TCA and resuspended with 5 mL of
0.5 M NaOH. The incorporation of the 3H signal in the final
product was read by liquid scintillation. When determining the
inhibition constant (IC50), inhibitors were added from a concen-
trated DMSO stock such that the final concentration of DMSO did
not exceed 2%.
The work was financed by Natural Science Foundation of China
(No. J1103512).
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