Biological and quantitative-SAR evaluations, and docking studies of (E)-N′-benzylidenebenzohydrazide analogues as potential antibacterial agents

Article abstract of DOI:10.17179/excli2016-388

A series of 15 (E)-N′-benzylidenebenzohydrazide analogues were evaluated for their antimicrobial activities against eleven pathogenic and food-borne microbes, namely, S. aureus (G⁺), L. monocytogenes (G⁺), B. subtilis (G⁺), K. pneumonia (G^-), C. sakazakii (G^-), C. freundii (G^-), S. enterica (G^-), S. enteritidis (G^-), E. coli (G^-), Y. pestis (G^-), and P. aeruginosa (G^-). Most of the compounds exhibited selective activity against some Gramnegative bacterial strains. Of the compounds tested (3a-o), 3b and 3g were most active against C. freundii (MIC = ~19 μg mL^-1). Whereas, compounds 3d, 3i, 3k and 3n exhibited MIC values ranging from 37.5 to 75 μg mL^-1 against C. freundii, and compounds 3e, 3l and 3n had MIC values of ~75 μg mL^-1 against K. pneumonia. Quantitative structure-antibacterial activity relationships were studied using physicochemical parameters and a good correlation was found between calculated octanol-water partition coefficients (clogP; a lipophilic parameter) and antibacterial activities. In silico screening was also performed by docking high (3b and 3g) and low (3n) activity compounds on the active site of E. coli FabH receptor, which is an important therapeutic target. The findings of these in silico screening studies provide a theoretical basis for the design and synthesis of novel benzylidenebenzohydrazide analogues that inhibit bacterial FabH.

Full text of DOI:10.17179/excli2016-388

EXCLI Journal 2016;15:350-361 – ISSN 1611-2156
Received: May 16, 2016, accepted: June 09, 2016, published: June 17, 2016
Original article:
BIOLOGICAL AND QUANTITATIVE-SAR EVALUATIONS, AND
DOCKING STUDIES OF (E)-N-BENZYLIDENEBENZOHYDRAZIDE
ANALOGUES AS POTENTIAL ANTIBACTERIAL AGENTS
Mohammad Sayed Alam1,2, Sefat Jebin2, M. Mostafizur Rahman2, Md. Latiful Bari3,
Dong-Ung Lee1,*
1
Division of Bioscience, Dongguk University, Gyeongju 780-714, Republic of Korea
Department of Chemistry, Jagannath University, Dhaka 1100, Bangladesh
Food analysis and Research laboratory, Centre for Advance Research in Sciences,
2
3
University of Dhaka, Dhaka-1000, Bangladesh
* Corresponding author: Dong-Ung Lee, Division of Bioscience, Dongguk University, Korea;
Tel: +82 54 770 2224; Fax: +82 54 742 9833; E-mail: dulee@dongguk.ac.kr
http://dx.doi.org/10.17179/excli2016-388
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/).
ABSTRACT
A series of 15 (E)-N-benzylidenebenzohydrazide analogues were evaluated for their antimicrobial activities
against eleven pathogenic and food-borne microbes, namely, S. aureus (G+), L. monocytogenes (G+), B. subtilis
(G+), K. pneumonia (G), C. sakazakii (G), C. freundii (G), S. enterica (G), S. enteritidis (G), E. coli (G), Y.
pestis (G), and P. aeruginosa (G). Most of the compounds exhibited selective activity against some Gram-
negative bacterial strains. Of the compounds tested (3a-o), 3b and 3g were most active against C. freundii (MIC
= ~19 µg mL-1). Whereas, compounds 3d, 3i, 3k and 3n exhibited MIC values ranging from 37.5 to 75 g mL1
against C. freundii, and compounds 3e, 3l and 3n had MIC values of ~75 g mL1 against K. pneumonia. Quanti-
tative structure-antibacterial activity relationships were studied using physicochemical parameters and a good
correlation was found between calculated octanol-water partition coefficients (clogP; a lipophilic parameter) and
antibacterial activities. In silico screening was also performed by docking high (3b and 3g) and low (3n) activity
compounds on the active site of E. coli FabH receptor, which is an important therapeutic target. The findings of
these in silico screening studies provide a theoretical basis for the design and synthesis of novel benzylideneben-
zohydrazide analogues that inhibit bacterial FabH.
Keywords: Hydrazone Schiff base, antimicrobial activity, physicochemical properties, docking study
INTRODUCTION
terial cell growth. A hydrazone Schiff base
analogue of INH (Figure 1A) has been re-
ported to have antimycobacterial activity
against INH resistant M. tuberculosis strains
and to exhibit lower toxicity than INH (Sah
and Peoples, 1954; Bavin et al., 1954) and
nifuroxazide – a commercial hydrazide-
hydrazone Schiff base antibiotic used to treat
colitis and diarrhea. Another analogue of ni-
furoxazide was found to show antimicrobial
activity comparable to that of ceftriaxone
Isoniazid (isonicotinylhydrazide; INH)
(Figure 1A) is a front-line antimycobacterial
that is usually used to treat tuberculosis. INH
exerts its activity via a bacterial catalase-
peroxidase (KatG) (Suarez et al., 2009) by
forming isonicotinic acyl-NADH complex,
which interacts firmly to enoyl-acyl carrier
protein reductase (InhA), and thus, inhibits
the synthesis of mycolic acid, and mycobac-
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EXCLI Journal 2016;15:350-361 – ISSN 1611-2156
Received: May 16, 2016, accepted: June 09, 2016, published: June 17, 2016
(Rollas et al., 2002). Currently, bacterial re- ingly, these structural motifs are also found
sistance to existing drugs, such as, β-lactam in many bioactive compounds.
antibiotics, macrolides, quinolones, and van-
Hydrazone
Schiff
bases
possess
comycin, is considered one of the most im- -CONHN=CH- moiety and they are widely
portant health concerns (Cassell and exploited to syntheses of various biologically
Mekalanos, 2001), and thus, sustained re- active molecules. In addition, they have
search efforts are being made to identify new shown wide-ranging bioactivities. For exam-
drugs with wide therapeutic windows, broad ple, antimicrobial (Vijesh et al., 2013), anti-
spectrum activities, and novel modes of ac- cancer (Dandawate et al., 2012), anti-
tion. A literature survey revealed that syn- mycobacterial (Mahajan et al., 2011), anti-
thetics and natural products containing the convulsant (Kulandasamy et al., 2009), anti-
hydrazone Schiff base or Schiff base motif inflammatory (Rajitha et al., 2011), analgesic
exert their antibiotic activities by inhibiting (Kümmerle et al., 2009), anti-hypertensive
fatty acid biosynthesis (FAB) (Cheng et al., (Leal et al., 2012), anti-platelet (Mashayekhi
2009; Shi et al., 2010; Song et al., 2014a). In et al., 2013), and anti-protozoal activities
prokaryotic organisms, FAB is crucially re- (Carvalho et al., 2012). Hydrazones are also
quired for cell viability and growth (Song et used as herbicides, insecticides, nemato-
al., 2014b). β-Ketoacyl-acyl carrier protein cides, rodenticides, plant growth regulators,
(ACP) synthase III (FabH or KAS III) plays and housefly sterilants (Akelah et al., 1993;
an important role in bacterial FAB Mohan et al., 1988). Hydrazone Schiff bases
(Khandekar et al., 2003) and is considered an are versatile polydentate chelating agents and
essential functional enzyme in the bacterial form a variety of complexes with various
FAB system. Bacterial FabH has no homo- transition and inner transition metals with
log in man, and thus, is an attractive target different bioactivities and practical applica-
for the design of new antibiotics (Lee et al., tions (Gad et al., 2000; Zhong et al., 2007;
2009 and 2012). The structures of several re- Fan et al., 2010; Juliano et al., 2010). These
ported antibacterial FabH inhibitors that pos- wide-ranging biochemical activities and uses
sess a Schiff base or hydrazone Schiff base of hydrazone Schiff bases and their com-
motif are shown in Figure 1B, and interest- plexes have attracted considerable research
attention.
Figure 1: Structures of (A) key molecules and (B) some reported potential FabH inhibitors possessing
a hydrazone Schiff base or Schiff base motif
351

EXCLI Journal 2016;15:350-361 – ISSN 1611-2156
Received: May 16, 2016, accepted: June 09, 2016, published: June 17, 2016
During our continued studies (Alam et al., conformations, and binding affinities of lig-
2013, 2014, 2015) on novel bioactive com- ands at active sites of target receptors (Alam
pounds, we evaluated the antimicrobial ac- and Lee, 2016). For these reasons, this tech-
tivities of a series of (E)-N'-benzylidene- nique is helpful for identifying therapeutic
benzohydrazide analogues (3a-o) exhibiting lead compounds (Shoichet et al., 2002).
structural similarities with INH and nifurox-
azide. In a previous study, we described the
synthesis and cytotoxic and antioxidant ac-
tivities of a series of fifteen (E)-N'-
MATERIAL AND METHODS
Chemistry
The (E)-N'-Benzylidenebenzohydrazide
analogues (3a-o) examined in the present
study were prepared as we previously de-
scribed (Alam and Lee, 2016) from their cor-
responding benzohydrazides (2) as presented
in Figure 2. The mixture of methyl esters of
benzoic acid (13.6 g, 0.1 M) or salicylic acid
(15.2 g, 0.1 M) and hydrazine hydrate
(12.5 g, 0.25 M) in 100 mL ethanol (roughly)
were refluxed for 3 h. TLC method was used
to observe the reaction progress. The ob-
tained solid products were separated and pu-
rified by recrystallization using aqueous eth-
anol. The structures of the synthesized com-
pounds were characterized by comparing the
previously reported physical and spectral da-
ta. The benzohydrazides (2, 1 mM) so ob-
tained were then refluxed with suitably sub-
stituted benzaldehydes (1 mM) in ethanol for
1.5-2.5 h. The reaction progress was moni-
tored by TLC. At room temperature, the re-
action mixtures give solid crude products,
and they were filtered and crystallized using
ethanol to afford the pure compounds (3a-o)
(81-96 % yield).
benzylidenebenzohydrazide
analogues
(Alam and Lee, 2016). Here, we report the in
vitro antibacterial activities of these fifteen
(E)-N'-benzylidenebenzohydrazide
ana-
logues (3a-o) against 11 pathogenic and
food-borne bacterial strains, that is, three
Gram-positive (Staphylococcus aureus, Lis-
teria monocytogenes, and Bacillus subtilis),
and eight Gram-negative bacterial strains
(Klebsiella pneumoniae, Cronobacter sa-
kazakii, Citrobacter freundii, Salmonella en-
terica, Salmonella enteritidis, E. coli, Yer-
sinia pestis, and Pseudomonas aeruginosa).
In addition, physicochemical characteristics
of the synthesized compounds were used to
access quantitative structure-antibacterial ac-
tivity relationships (Q-SAR). Finally, in sili-
co screening was performed using docking
simulations using the X-ray crystallography
determined structure of E. coli FabH to in-
vestigate the binding affinities and interac-
tion modes of benzylidene-hydrazone ana-
logues at its active site. Computer-assisted
drug design (CADD) was used is a useful
tool for conducting docking simulations, and
provides important information regarding the
natures of interactions, favorable bioactive
Figure 2: The synthesis of novel (E)-N'-benzylidenebenzohydrazide analogues 3a-o
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EXCLI Journal 2016;15:350-361 – ISSN 1611-2156
Received: May 16, 2016, accepted: June 09, 2016, published: June 17, 2016
Antibacterial screening
defined as the lowest concentration of the
A previously reported filter paper disc tested compound (in DMSO) that inhibited
diffusion method (Alam and Lee, 2010) was bacterial growth.
used to determine the in vitro antibacterial
effects of 3a-o against eleven bacterial Computational analysis
strains. Nutrient agar (NA) media (Difco La-
The molecular geometries of the (E)-N'-
boratories, Lawrence, KS), a bacterial grow- benzylidenebenzohydrazide analogues (3a-o)
ing medium was inoculated with liquid cul- were examined using standard bond length
tures (0.2 mL) of the microorganisms. Discs and angles using the ChemBio3D ultra Ver.
socked with test samples (3a-o) were kept on 14 molecular modeling program (Cam-
pre-treated agar Petri dishes and incubated bridgeSoft Corporation, Cambridge, MA
aerobically at 37 oC (24 h). DMSO and na- 02140, USA). Physicochemical properties
lidixic acid were used as negative and posi- were calculated using Molinspiration
tive controls, respectively. Bactericidal activ- Cheminformatics Software (Molinspiration
ity was defined as inhibitory zones diameters Cheminformatics, SK 90026 Slovensky
in mm. Evaluations were performed in tripli- Grob, SR). The method used for calculating
cate.
clogP values was developed by Molinspira-
Minimal inhibitory concentrations (MIC, tion (miLogP2.2-2005) derived from the
group contributions of more than twelve
thousand drug-like compounds, and the theo-
retical logP and experimental logP values
were used as a training set for fitting correc-
in g/mL) against Klebsiella pneumonia
(JCM 1662) and Citrobacter freundii (JCM
1657) were determined using the serial dilu-
tion technique (Alam and Lee, 2010) using
nutrient broth medium (DIFCO). MIC was
Table 1: In vitro antimicrobial profiles of (E)-N'-benzylidenebenzohydrazide analogues (3a-o) as de-
termined by growth inhibition zones
Comp.
Gram-positive
Gram-negative
S. a.
L. m.
B. s.
K. p.
C. s.
C. f.
S. e.
S. t.
E. c.
Y. p.
P.a.
3a
3b
3c
3d
3e
3f
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
13±0.5
-
30±1.5
-
14±0.5
-
14±0.5 14±0.5 25±1.0
14±0.5
11±1.0 10±1.0 10±1.0 17±1.0 8±0.5 10±1.0
10±1.0 8±1.0 10±0.5 12±1.0
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
13±0.5
-
-
9±1.0
-
-
-
-
-
-
-
-
-
-
15±0.5
-
-
-
-
-
-
-
-
-
-
-
-
3g
3h
3i
-
13±0.5
30±1.0
-
-
-
13±0.5
11±1.0
-
-
-
-
-
-
-
-
-
-
-
-
-
-
23±0.5 12±1.0
15±1.0
-
3j
-
-
-
22±0.5
9±0.5
-
8±1.0
-
11±1.0
8±0.5
-
-
3k
3l
-
14±0.5
17±0.5
-
-
-
7±0.5
7±0.5
-
3m
3n
3o
NA
-
-
-
-
-
-
-
16±0.5
-
19±1.5
-
12±0.5
-
-
-
24±1.0 8.0±0.5 20±1.0 21±1.0 24±1.0 29±1.5 10±1.0 18±1.0 24±1.0
23±1.0
Inhibitory activities are expressed as diameters (in mm) of observed inhibitory zones. (-), Not active.
The activities presented are the means  SDs (three experiments). S. a., Staphylococcus aureus (JMC
2151); L. m, Listeria monocytogenes (ATCC 43256); B. s., Bacillus subtilis (IFO 13719); K. p.,
Klebsiella pneumoniae (JCM 1662); C. s., Cronobacter sakazakii (CARS 2012-J-F); C. f., Citrobacter
freundii (JCM 1657); S. e., Salmonella enterica (ATCC 10708); S. t., Salmonella enteritidis (ATCC
13076); E. c., E. coli (CARS 2011-016); Y. p., Yersinia pestis (CARS 2013-027); P.a., Pseudomonas
aeruginosa (PA01)
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EXCLI Journal 2016;15:350-361 – ISSN 1611-2156
Received: May 16, 2016, accepted: June 09, 2016, published: June 17, 2016
tion factors. The molecular polar surface are bacterial strains except S. enterica, but its ac-
as (PSAs) were obtained as sums of frag- tivity was relatively weak. A half of the test-
ment contributions (Ertl et al. 2000). Molec- ed compounds inhibited G(-)-bacteria K.
ular lipophilicity potentials (MLPs) and pneumonia, C. freundii, and E. coli, but only
PSAs maps were analyzed using Molinspira- 3e inhibited G(+)-bacteria. Of the com-
tion Galaxy 3D Structure Generator (ver. pounds tested, 3b and 3g were most effective
2013.02 beta).
against C. freundii, and had activities similar
to nalidixic acid (the positive standard).
Compounds bearing an OH group at the R4-
Docking studies
The molecular geometries of compounds position (para-position) in the benzylidene
3c, 3i and 3j were constructed using standard phenyl ring (e.g., 3b, 3g > 3d > 3i > 3k) bet-
bond lengths and angles as mentioned above, ter inhibited C. freundii. Whereas, com-
and energy minimized using the Hartree- pounds with additional OH substitution at
Fock method at 6-31G basis set with R- the meta-position (e.g. 3f) or ortho-positions
Closed-Shell wave function in the Chem- (e.g. 3j) were devoid of activity.
Bio3D Ultra Ver. 14.0 software (GAMESS
The minimal inhibitory concentrations
Interface). The docking studies were per- (MICs) of selected compounds were deter-
formed using crystal structure of E. coli mined against K. pneumonia and C. freundii;
FabH-CoA complex retrieved from the Pro- results are summarized in Table 2. Com-
tein Data Bank (PDB code: 1HNJ). To pre- pounds 3b and 3g had the lowest MIC values
pare the receptor for docking studies, co-
crystallized ligand and water molecules were
removed, while polar hydrogen atoms and
Kollman-united charges were included to the
FabH receptor molecule. The necessary pdb
and pdbqt files of ligands and E. coli FabH
receptor were prepared using AutoDock 4.2
software (Morris et al., 2009). The study was
carried out using the usual docking protocol
applied for AutoDock Vina in PyRx 0.8
software (Trott and Olson, 2010) where free
rotation was allowed through single bonds of
ligands. The docking results were analyzed
using Discovery Studio 4.0 (Accelrys, Inc.
San Diego, CA 92121, USA) and binding
scores were calculated using iGEMDOCK
software (Yang and Chen, 2004).
(18.75 g/mL), followed by 3d, 3i, and 3k
(37.5 g/mL) against C. freundii, whereas
compounds 3e, 3l, and 3n had similar MIC
values (75 g/mL) against K. pneumonia.
Above results indicate the presence of a po-
lar hydroxyl group at the R4-position (para-
position) favors activity, but that meta posi-
tioned OH groups reduce activity.
Table 2: MIC of some (E)-N'-benzylidene-
benzohydrazide analogues derivatives against
selected bacterial strains
Comp.
MIC, g/mL
K. pneumonia C. freundii
3b
3d
3e
3g
3i
-
18.75
37.5
-
-
75
-
RESULTS AND DISCUSSION
18.75
37.5
37.5
-
Antibacterial activities
-
Compounds 3a-o were evaluated for their
in vitro antibacterial activities against three
Gram-positive bacteria, S. aureus, L. mono-
cytogenes, and B. subtilis, and eight Gram-
negative bacteria K. pneumonia, C. sa-
kazakii, C. freundii, S. enterica, S. enter-
itidis, E. coli, Y. pestis, and P. aeruginosa by
disc diffusion. As presented in Table 1,
compound 3e inhibited the growths of all
3k
3l
75
75
3n
75
NA
12.5
6.25
(-), Not measured; MIC, Minimum inhibitory con-
centration; NA, Nalidixic acid.
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EXCLI Journal 2016;15:350-361 – ISSN 1611-2156
Received: May 16, 2016, accepted: June 09, 2016, published: June 17, 2016
Quantitative-SAR study
partition coefficient (logP) of a molecule de-
To explain quantitative structure- pends on its hydrophobicity and polarity,
antibacterial activity relationships (Q-SAR) which facilitate transit across cellular mem-
of the fifteen (E)-N'-benzylidenebenzo- branes. Therefore, we examined the correla-
hydrazide analogues (3a-o), physicochemical tion between the inhibitory effects of 3a-o
calculations were conducted using Chem- against K. pneumonia, C. freundii, and E.
Bio3D Pro 12 molecular modeling (Cam- coli and their clogP values. The correlation
bridgeSoft Corporation, Cambridge, MA coefficients (r2) between clogP values and
02140, USA) and Molinspiration Chem- the inhibitory potencies of active molecules
informatics software (Molinspiration Chem- against K. pneumonia, C. freundii, and E.
informatics, SK 90026 Slovensky Grob, SR). coli were; 0.86 (n = 8; 3b, 3d-3g, 3k, 3l and
The physicochemical properties of mole- 3n), 0.75 (n = 8; 3b, 3d,3e, 3g, 3i, 3k, 3l, and
cules, such as, their lipophilicities and polar 3n), and 0.71 (n = 9; 3b, 3d-3g, 3i, 3k, 3l,
surface areas (PSAs) play important roles in and 3n), respectively. Although only a rela-
determining biological responses, and are tively small number of compounds were ex-
commonly used to study the structure- amined, significant correlations were ob-
activity relationships of bioactive molecules served, whereby antibacterial activity in-
in medicinal chemistry (Alam et al., 2013; creased with clogP against K. pneumonia but
Desai et al., 2014). These parameters are decreased with clogP against C. freundii and
now well-accepted major experimental and E. coli (Figure 3). However, the above corre-
theoretical tools for drug design and discov- lations should be treated with caution be-
ery. The physiochemical parameters of all cause the clogP values of 3a, 3c, 3h, 3m, and
synthesized compounds are listed in Table 3.
3o fell within the medium range for active
In the present study, a considerable num- compounds, but they were in fact inactive.
ber of compounds were found to be active Therefore, we compared maps of lipophilici-
against K. pneumonia, C. freundii, and E. ty potential (MLP) and PSA of two selected
coli. It is well-known that the octanol-water active and inactive compounds (3b and 3g
Table 3: Molinspiration calculations of the molecular properties of (E)-N'-benzylidenebenzohydrazide
analogues (3a-o)
Comp. MW (g/mol) clogPa
TPSAb
OH-NH interactc O-N interactd nrotbe volume
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
224.263
240.262
254.289
256.261
284.315
256.261
270.288
284.315
300.314
272.26
256.261
256.261
258.708
267.332
252.273
3.099
2.619
3.155
2.536
3.14
41.462
61.69
50.696
81.918
59.93
81.918
70.924
59.93
80.158
102.146
81.918
81.918
41.462
44.7
1
2
1
3
1
3
2
1
2
4
3
3
1
1
1
3
4
4
5
5
5
5
5
6
6
5
5
3
4
4
3
3
4
3
5
3
4
5
5
3
3
3
3
4
4
210.098
218.116
235.644
226.134
261.19
226.134
243.662
261.19
269.208
234.152
226.134
226.134
223.634
256.004
229.082
2.13
2.438
2.745
2.454
2.452
2.56
3.528
3.777
3.201
2.841
58.533
aCalculated octanol/water partition coefficients; bMolecular polar surface areas; cNumbers of hydrogen-
bond donors; dNumbers of hydrogen-bond acceptors; eNumbers of rotatable bonds
355

EXCLI Journal 2016;15:350-361 – ISSN 1611-2156
Received: May 16, 2016, accepted: June 09, 2016, published: June 17, 2016
vs. 3a and 3c). It was found that lipophilicity and low (3n) activity with the active site of
potentials and the polar surface areas of these E. coli FabH receptor was performed. The
two pairs of compounds differed (Figure 4). crystal structure of E. coli FabH-CoA com-
These results suggest that the distributions of plex (Qiu et al., 2001) was retrieved from the
more hydrophilic and polar areas at the para- Protein Data Bank (PDB ID: 1HNJ) and
position of benzylidene phenyl ring im- compounds 3b, 3g, and 3n were docked at its
portantly determine activity against specific active site using a standard docking protocol.
bacterial strains.
In silico docking results are presented in Ta-
ble 4.
Molecular docking studies
-Ketoacyl-(acyl-carrier-protein)
syn-
thase III (FabH), a key bacterial enzyme
plays a crucial regulatory role in bacterial
fatty acid synthesis (FAS) by initiating fatty
acid elongation cycles and providing feed-
back regarding the regulation of FAS, which
is essentially required for prokaryotic cell
metabolism, viability, and growth. However,
the bacterial FAS pathway differs considera-
bly from in man, and bacterial FabH proteins
are highly conserved at the sequence and
structural level and show no significant ho-
mology with any human protein. In addition,
the active site residues of FabH receptor are
common for Gram-positive and -negative
bacterial strains (Nie et al., 2005). These fea-
tures make FabH protein a potential thera-
peutic target for the design of novel and
broad-spectrum antimicrobial agents as se-
lective and nontoxic FabH inhibitors. To
predict the binding affinities of hydrazone
Schiff base analogues to FabH, molecular
docking of (E)-N-benzylidenebenzohydra-
zide analogues having high (3b and 3g)
Figure 4: Molecular lipophilicity potentials (left)
and polar surface areas (right) of 3a (A), 3b (B),
3c (C), and 3g (D) showing areas of lipophilicity
(blue), intermediate lipophilicity (pink), hydro-
philicity (yellow), intermediate hydrophilicity
(green), nonpolar (gray/white), and polar (red)
areas.
20
20
18
16
14
12
10
35
30
25
20
15
10
5
C
R² = 0.71
A
B
R² = 0.86
n = 8
R² = 0.75
n =8
n =9
15
10
5
0
0
1.5
2.5
3.5
cLogP
4.5
2
2.5
3
3.5
4
2
2.5
3
3.5
4
cLogP
cLogP
Figure 3: Correlations between inhibition zones (mm) and calculated octanol-water partition coeffi-
cients (clogP) of active (E)-N'-benzylidenebenzohydrazide analogues against (A) K. pneumonia (3b,
3d-g, 3k, 3l and 3n), (B) C. freundii (3b, 3d, 3e, 3g, 3i, 3k, 3l and 3n), and (C) E. coli (3b, 3d-g, 3i, 3k,
3l and 3n).
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EXCLI Journal 2016;15:350-361 – ISSN 1611-2156
Received: May 16, 2016, accepted: June 09, 2016, published: June 17, 2016
Table 4: Docking energies and molecular interactions of the ligand molecules (3b, 3g, and 3n) with E.
coli FabH receptor
Entry
Binding
affinity
(kcal/mol)
Energy
contribution
AverConPair H-bonding
Hydrophobic
interactions residues
residues
(distance,
Å)
vdW
H-bond
3b
-80.36
-84.33
-72.22
-71.29
-9.07
-12.41
-
25.72
26.20
23.70
-
Ala111, Cys112, Leu142,
Ile156, Phe157, Leu189,
Thr190, Leu191, Leu205,
Met207,Val212, Phe213,
Ala246, Ile250, Gly305
Leu142, Ile156, Phe157,
Leu205, Val212,
Phe213, Asn247,
Ala246, Ile250, Ser276,
Gly305, Gly307
3g
-71.92
-72.22
Leu189
(2.96)
3n
-
Ala109, Ala110, Leu142,
Thr145, Thr190, Leu191,
Leu205, Ser276, Thr309
These studies revealed that all three tween the benzylidene-phenyl ring and
compounds bind to the same active site of
FabH receptor as endogenous malonyl-CoA
ligand and other reported hydrazone and
Schiff base FabH inhibitors (Song et al.,
2014a; Zhou et al., 2013; Shi et al., 2010).
The binding modes and the different types of
interactions found for compound 3b (most
active) and 3n (least active) were compared
and are shown in Figures 5 and 6, respective-
ly. To measure the affinity of ligands for the
receptor, we analyzed binding scores for
compounds 3b, 3g, and 3n by iGEMDOCK
software (Yang and Chen, 2004); results are
shown in Table 4. According to docking
scores, the high activity compounds 3b and
3g had higher binding affinities with FabH
receptor than 3n (the binding energies of 3b,
3g and 3n were -80.36, -84.33, and -72.22
kcal/mol, respectively). Regarding total
binding energies, van der Waal contributions
for 3b and 3g were -71.29 and 71.02
kcal/mol, respectively, and H-bond contribu-
tions were -9.07 and 12.41 kcal/mol, respec-
tively. While for 3b only van der Waal inter-
actions (-72.22 kcal/mol) contributed to total
binding energy. In the binding model of 3b
and E. coli FabH receptor, two -sulfur in-
teractions were observed between the benzo-
yl-phenyl ring and Met207 (3.95 Å) and be-
Cys112 (4.88 Å), together with -alkyl inter-
actions with Ala111(4.22 Å), Leu142 (4.81
Å), Leu189 (5.07 Å), Leu191 (5.36 Å),
Leu205 (5.19 Å), Val212(5.47 Å), and
Ala246 (3.59 Å). However, in the binding
model of 3g with E. coli FabH receptor, a H-
bond between the carbonyl group of Leu189
(CHO: 2.96 Å) and methoxy proton, and
-sulfur and -sigma interactions of Met207
(3.83 Å) and Ala246 (2.59 Å), respectively
were observed including -alky interactions
with Ala111 (3.84 Å), Cys112 (5.32 Å),
Leu142 (5.07 Å), Leu189 (5.08 Å), and
Leu191 (5.48 Å). Whereas in the binding
model of 3n with E. coli FabH receptor, no
H-bond, -sulfur, or -sigma interactions
were observed, although -alkyl interactions
with Ala109 (4.65 Å), Ala111 (4.45 Å),
Leu189 (4.43 Å), Leu191 (5.34 Å), Leu205
(5.27 Å), and Gly306 (4.43) involving ben-
zoyl- and benzylidene-phenyl rings were ob-
served. In addition, the following van der
Waal’s interactions were also observed: be-
tween 3b and Cys112, Ile156, Phe157,
Thr190, Met207, Phe213, Ile250, and
Gly305; between 3g and Ile156, Phe157,
Leu205, Val212, Phe213, Asn247, Ala246,
Ile250, Ser276, Gly305, and Gly307; and be-
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EXCLI Journal 2016;15:350-361 – ISSN 1611-2156
Received: May 16, 2016, accepted: June 09, 2016, published: June 17, 2016
tween 3n and Ala110, Leu142, Thr145, tightly in the active site of FabH receptor due
Thr190, Ser276, and Thr309. These results
indicate compounds 3b, 3g, and 3n bind to
the same active site of FabH receptor. How-
ever, the most active compounds 3b (Figure
5) and 3g (figure not shown) attached more
to additional H-bond, -sulfur, -sigma, and
hydrophobic interactions, which resulted in
higher binding affinities than compound 3n
(Figure 6).
Figure 5: (A) E. coli FabH receptor (PDB ID: 1HNJ). (B) HF/6-31G optimized geometry of ligand 3b.
(C) Docked ligand-receptor complex (the circle shows the ligand binding site) (D) Binding model of 3b
with E. coli FabH; -alkyl and -sulfur interactions are shown by blue and yellow broken lines, respec-
tively. (E) 2D view of interacting essential amino acid residues at the ligand binding site; The residues
involved in electrostatic and covalent interactions are shown by purple circles and amino acids in-
volved in van der Waals interactions are shown by green circles. (F) Showing hydrogen bond donor
(pink) and acceptor (green) surfaces. (G) Showing hydrophobic interactions (brown) surfaces.
Figure 6: (A) E. coli FabH receptor (PDB ID: 1HNJ). (B) HF/6-31G optimized geometry of ligand 3n.
(C) Docked ligand-receptor complex (the circle indicates the ligand binding site) (D) Binding model of
compound 3n with E. coli FabH; H-bond and -alkyl interactions are shown by red and blue broken
lines, respectively. (E) 2D view of interacting essential amino acid residues at the ligand binding site;
The residues involved in electrostatic and covalent interactions are shown by purple circles and amino
acids involved in van der Waals interactions are shown by green circles. (F) Showing hydrogen bond
donor (pink) and acceptor (green) surfaces. (G) Showing hydrophobic interactions (brown) surfaces.
358

EXCLI Journal 2016;15:350-361 – ISSN 1611-2156
Received: May 16, 2016, accepted: June 09, 2016, published: June 17, 2016
Alam MS, Nam YJ, Lee DU. Synthesis and evalua-
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CONCLUSION
The present study reports the antibacteri-
al activities of a series of 15 (E)-N'-
benzylidenebenzohydrazide
analogues
Alam MS, Lee DU, Bari ML. Antibacterial and cyto-
toxic activities of Schiff base analogues of 4-
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against eleven pathogenic and food-borne
bacterial strains, of these eleven, three were
Gram-positive, i.e., S. aureus, L. monocyto-
genes, and B. subtilis, and eight were Gram-
negative, i.e., K. pneumoniae, C. sakazakii,
C. freundii, S. enterica, S. enteritidis, E. coli,
Y. pestis, and P. aeruginosa. The compounds
subjected to antibacterial assay, 3b and 3g,
which are containing one OH group at the
para-position of the benzylidene phenyl ring,
showed lowest MIC values against C. freun-
dii. Physicochemical calculations indicated
that the antibacterial activities of (E)-N'-
benzylidenebenzohydrazide analogues (3b,
3d-3g, 3i, 3k, 3l, and 3n) against K. pneu-
monia, C. freundii, and E. coli correlated
well with calculated logP values. In silico
screening using molecular docking studies
were performed to predict the binding affini-
ties and modes of interaction of the most ac-
tive (3b and 3g) and least active (3n) com-
pounds to the active site of E. coli FabH re-
ceptor. Compounds 3b and 3g were found to
bind effectively to the active site of E. coli
FabH receptor with high affinity, suggesting
their potentials as FabH inhibitors. We be-
lieve the above findings can be used to facili-
tate the design and synthesis of novel ben-
zylidenehydrazone analogues as potential an-
tibacterial as FabH inhibitors.
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