Synthesis, antiphospholipase A2, antiprotease, antibacterial evaluation and molecular docking analysis of certain novel hydrazones

Article abstract of DOI:10.3390/molecules21121664

Some novel hydrazone derivatives 6a-o were synthesized from the key intermediate 4-Chloro-N-(2-hydrazinocarbonyl-phenyl)-benzamide 5 and characterized using IR, ¹H-NMR, ¹³C-NMR, mass spectroscopy and elemental analysis. The inhibitor

Full text of DOI:10.3390/molecules21121664

molecules
Article
Synthesis, Antiphospholipase A2, Antiprotease,
Antibacterial Evaluation and Molecular Docking
Analysis of Certain Novel Hydrazones
Nahed N. E. El-Sayed 1,2,*, Ahmed M. Alafeefy 3, Mohammed A. Bakht 4, Vijay H. Masand 5,
Ali Aldalbahi 6, Nan Chen 7, Chunhai Fan 7 and Abir Ben Bacha 8
1
Department of Chemistry, College of Science, “Girls Section”, King Saud University, P.O. Box 22452,
Riyadh 11495, Saudi Arabia
National Organization for Drug Control and Research, Giza 35521, Egypt
Department of Chemistry, Kulliyyah of Science, International Islamic University, P.O. Box 141,
2
3
25710 Kuantan, Malyasia; ahmed.alafeefy@gmail.com
Department of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam Bin Abdulaziz University,
4
P.O. Box 173, Al-kharj 11942, Saudi Arabia; bakhtpharm@gmail.com
Department of Chemistry, Vidya Bharati College, Camp, Amravati, Maharashtra 444 602, India;
5
vijaymasand@gmail.com
Chemistry Department, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia;
6
aaldalbahi@ksu.edu.sa
7
Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility,
Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China;
chennan@sinap.ac.cn (N.C.); fchh@sinap.ac.cn (C.F.)
Biochemistry Department, Science College, King Saud University, P.O. Box 22452, Riyadh 11495,
8
Saudi Arabia; aalghanouchi@ksu.edu.sa
*
Correspondence: nelsayed@ksu.edu.sa; Tel.: +966-080-55859
Academic Editor: Derek J. McPhee
Received: 21 October 2016; Accepted: 28 November 2016; Published: 2 December 2016
Abstract: Some novel hydrazone derivatives 6a
4-Chloro-N-(2-hydrazinocarbonyl-phenyl)-benzamide
–
o
were synthesized from the key intermediate
and characterized using IR, 1H-NMR,
5
13C-NMR, mass spectroscopy and elemental analysis. The inhibitory potential against two secretory
phospholipase A2 (sPLA2), three protease enzymes and eleven bacterial strains were evaluated. The
results revealed that all compounds showed preferential inhibition towards hGIIA isoform of sPLA2
rather than DrG-IB with compounds 6l and 6e being the most active. The tested compounds exhibited
excellent antiprotease activity against proteinase K and protease from Bacillus sp. with compound
6l being the most active against both enzymes. Furthermore, the maximum zones of inhibition
against bacterial growth were exhibited by compounds; 6a
6d 6f 6l 6m 6n, and 6o against Serratia; 6k against S. mutans; and compounds 6a
6n against E. feacalis. The docking simulations of hydrazones 6a with GIIA sPLA2, proteinase K
and hydrazones 6a with glutamine-fructose-6-phosphate transaminase were performed to obtain
,
6m, and 6o against P. aeruginosa; 6a
, 6b,
,
,
,
,
,
6d 6e 6m, and
,
,
–
o
–
e
information regarding the mechanism of action.
Keywords: benzo[d][1,3]oxazin-4-one; aroylhydrazides; N-acylhydrazones; hydrazones; benzylidene
hydrazides; phospholipases; proteases; antimicrobial evaluation; molecular docking
1. Introduction
Hydrazones bearing azomethine moiety can be formed by condensation of hydrazides or aroyl
hydrazides with aldehydes [
1–
4]. These compounds are most widely used as building blocks for
the synthesis of 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazoles [
5
], different mono-, di- and
Molecules 2016, 21, 1664; doi:10.3390/molecules21121664
www.mdpi.com/journal/molecules

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trisubstituted hydrazines [
6
,7], azetidin-2-ones, thiazolidin-4-ones and methyl thiazolidines [
8
,9], and
formazan derivatives [10
activities [12] and have been utilized as useful candidates for development of antimalarial [13],
antitumor [14], antiviral [15 16], antimicrobial [17], antioxidant [18], analgesic, anti-inflammatory and
,11]. Hydrazones are well known to exhibit a wide spectrum of biological
,
anti-ulcerogenic agents [19,20].
Secreted phospholipases A2 (sPLA2s) are enzymes found in mammals and animal venoms that
catalyze hydrolysis of glycerophospholipids at the sn-2 ester position to release a free fatty acid and
a lysophospholipid [21]. Mammalian phospholipases have been categorized into ten groups IB, IIA,
IIC, IID, IIE, IIF, III, V, X and XIIA [22,23]. It has been reported that sPLA2s play a key role in a good
number of bio-chemical processes, thus, on one the hand, group IB sPLA2 has been proposed to be
involved in various physiological and pathophysiological processes such as cellular proliferation, cell
migration, hormone release [24] and apoptosis in neuronal cells [25]. In addition, higher levels of this
enzyme were detected in the serum of patient with chronic renal failure [26] and acute lung injury [27
compared to healthy controls.
]
On the other hand, the GIIA sPLA2 has been correlated to inflammatory diseases, which
is due to elevated levels of this enzyme were detected in the fluids of patients suffering from
various inflammatory diseases such as rheumatoid arthritis [28], acute lung injury-acute respiratory
distress syndrome (ALI-ARDS) [29] and pancreatitis-associated adrenal injury in acute necrotizing
pancreatitis [30], as well as in various cancers [31]. Therefore, seeking potent and selective inhibitors
to this sPLA2 may be considered an effective approach to develop novel anti-inflammatory and
antitumor agents.
Proteases or proteinases enzymes are proteolytic enzymes that break down proteins into peptides
and amino acids. They are classified into different classes depending on their source and functions [32
]
such as proteinase K, protease from Bacillus and protease-esperase. Biologically, proteases are involved
in controlling many biological pathways in the life cycle of human, plants, animals, insects and
pathogens such as fungi, bacteria and viruses. Many studies reported their involvement in the
pathogenic processes of human diseases such as bleeding disorders [33], inflammation [34], cancer [35],
hypertension [36] and AIDS [37]. Therefore, proteases inhibitors’ can be considered as targets in drug
design for developing therapeutics and prevention of diseases [38].
In continuation to our previous efforts towards synthesis of novel biologically active
compounds [39
phospholipases, proteases and bacterial inhibitory activities. The docking simulations of hydrazones
6a against GIIA sPLA2, proteinase K and hydrazones 6a against glutamine-fructose-6-phosphate
–41], we report herein the synthesis of novel hydrazones 6a–o and evaluation of their
–o
–e
transaminase were performed in order to obtain information regarding the mechanism of action.
2. Results and Discussion
2.1. Chemistry
Benzoylation of anthranillic acid
presence of triethylamine afforded the corresponding amido acid
of acetic anhydride underwent intramolecular cyclization and afforded benzo[d][1,3]oxazin-4-one
derivative Nucleophilic attack by the amino group of hydrazine hydrate on the carbonyl
group of the latter benzoxazinone, and subsequent ring opening gives the key intermediate
4-Chloro-N-(2-hydrazinocarbonyl-phenyl)-benzamide . Condensation of hydrazino derivative
with a variety of aromatic aldehydes yielded the desired hydrazones 6a (Scheme 1). The structures
of these products were confirmed based on their spectral data and elemental analyses. In general,
the IR spectra of compounds 6a displayed two absorption bands at
3421–3292 cm−1, which
1
with 4-chlorobenzoyl chloride
2 in methylene chloride in the
3
which upon boiling with excess
4
.
5
5
–
o
–
o
ν
max
are attributed to the two (NH) groups, and two absorption bands at ν
max 1684–1657 cm−1 due to the
two carbonyl groups. In addition, the (C=N) groups appeared around 1598–1579 cm−1. Moreover,
the 1H-NMR spectra of these compounds indicated the disappearance of the spectral line due to NH2

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group of the starting hydrazide
5
and appearance of two broad singlet signals at
δ
12.37–11.08 ppm
corresponding to 2NH groups, singlet signals at
δ 8.84–8.30 ppm due to the characteristic azomethines’
protons (N=CH) in addition to signals attributed to the aliphatic and aromatic protons at the expected
chemical shift values. Analogously, the 13C-NMR spectra proved the presence the two C=O groups
at δC 165.61–163.86 ppm, azomethine carbons’ at 151.46–145.10 ppm and the aliphatic and aromatic
carbons at the expected chemical shift values. The mass spectra of all of the synthesized compounds
revealed the existence of the parent ion peaks. Finally, the C, H, N elemental analyses of all of the
synthesized compounds were in agreement with the proposed structures.
Cl
Cl
O
O
NH2
N
NH
OH
O
TEA, CH2Cl2
Cl
+
Ac2O
O
O
OH
O
Cl
1
2
3
4
NH2NH2
Cl
Cl
ArCHO/ AcOH cat.
O
O
NH
Ar
NH
HN NH2
HN N
O
O
6a-o
5
a, Ar = Ph
b, Ar = 4-Cl-C6H4
c, Ar = 4-OH,3-OMe-C6H3
e, Ar = 2,4-Cl-C6H3
d, Ar = 3,4,5-(OMe)3-C6H2
f, Ar = 4-OMe-C6H4
g, Ar = 4-N,N-di-Me-C6H4
i, Ar = 4-Me-C6H4
K, Ar = CH=CH-C6H5
m, Ar = 3-OH,4-OMe-C6H3
o, Ar = 3-OMe-C6H4
h, Ar = 2-OMe-C6H4
j, Ar = 4-COOH-C6H4
l, Ar = 2-COOH-C6H4
n, Ar = 4-cyclohexyl-C6H4
Scheme 1. The synthesis of Schiff bases 6a–o.
2.2. Biological Evaluation
2.2.1. Phospholipases Inhibitory Activity
The phospholipase inhibitory activity of hydrazones 6a–o have been detected against two different
classes of sPLA2s, namely human group IIA sPLA2 (hG-IIA) and dromedary group IB sPLA2 (DrG-IB).
The obtained results revealed that compounds 6c 6d 6e and 6l exhibited selective inhibition against
hGIIA rather than DrG-IB by 50.67% 4.04%, 55.67% 4.72%, 72.67% 2.51% and 74.33% 3.21%,
respectively, as shown in Figure 1 and Table S1.
Conversely, the lowest inhibitory activity against this enzyme (9%
,
,
±
±
±
±
±
2%) was displayed by
compound 6n. These results are in agreement with the data obtained in our previous work [42].
As many clinical studies revealed that the levels of sPLA2s are increased in different inflammatory
conditions such as rheumatoid arthritis [28], these active hydrazones can be proposed as potential
candidates to explore their utility for such ailment.

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Figure 1. Inhibitory activity (%) of compounds 6a–o against sPLA2 (hG-IIA and DrG-IB).
2.2.2. Proteases Inhibitory Activity
The synthesized compounds were also screened for in vitro anti-proteases activity against the
commercially available protease-esperase, proteinase K and protease obtained from Bacillus sp.
The screening results shown in Figure 2 and Table S2 revealed that the tested compounds displayed
varied degrees of proteases inhibition. The maximum inhibitory activities against proteinase K
(86
±
2.64), protease from Bacillus (74.66
±
2.88) and protease-esperase (39.33
±
3.21) were exhibited
by compound 6l.
100
90
80
70
60
50
40
30
20
10
0
Proteinase K
Protease from Bacillus sp
Esperase
6a 6b 6c 6d 6e 6f 6g 6h 6i
6j 6k
6l 6m 6n 6o
Compound
Figure 2. Antiprotease activity (%) of compounds 6a–o against different protease enzymes.
Furthermore, the next highest inhibitory activities against proteinase K were exhibited by
compounds 6e (74.66%
±
2.51%) and 6m (69%
±
4.58%), while the next highest inhibitory activities
against protease from Bacillus sp. were exhibited by compounds 6b (72.33%
(72.33% ± 6.42%).
±
2.51%) and 6g

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It is worth mentioning that most of the compounds showed good antiprotease inhibition against
proteinase K and protease from Bacillus sp., as depicted in Figure 2. It was also noticed that there was
alignment in inhibitory activity against these two enzymes, as any single compound that showed high
inhibitory potential against one of them also reacted in a similar fashion towards the other one.
As the anti-inflammatory activities of chemical compounds can be expressed by phospholipase
A2 (hGIIA) and/or through protease inhibitor potentials [42], compound 6l, which was found to
be the most active candidate against both phospholipases A2 (sPLA2) and protease enzymes under
investigation, may be proposed as promising potential anti-inflammatory agent for treatment of
ulcerative colitis.
2.2.3. Antibacterial Screening
Finally, compounds 6a–o were further examined for in vitro antibacterial activity. Preliminary
screening against eleven strains of Gram-positive and Gram-negative bacteria was performed by
adopting standard protocol [43]. The antibacterial potency was determined by measuring the inhibition
zones. All tests were performed in duplicates and means of inhibition zones were recorded in mm as
presented in Table 1.
Analysis of the screening data revealed that the inhibition activity produced by some of the
tested compounds was found to be good to excellent compared to the used reference drug tetracycline.
Among the eleven strains that would be considered more susceptible to inhibition by one or more of the
synthetic compounds were; P. aeruginosa, Serratia, S. aureus, S. mutans and E. feacalis with genus Serratia
being the most sensitive one which was inhibited by nine hydrazones. The highest inhibition towards
this pathogen was displayed by compounds 6a
,
±
6b
1.0, 13
,
6d
,
6e
±
,
6f
,
6l
,
6m
,
6n, and 6o with inhibition zones
1.5, 14.5 0.5 and 12.5 0.5,
6m
0.5, respectively. Although compounds
6l and 6n produced the highest inhibition zones against S. aureus strain, they were considered
to be less effective compared to the reference drug tetracycline. Moreover, the maximum zone of
inhibition (24.5 0.5) was exhibited by compound 6k against S. mutans. Finally, the reference drug
tetracycline was found to be completely inactive against E. feacalis, while compounds 6a
and 6n exhibited the maximum inhibition zones of 18.5 0.5, 18.0. 1.0, 18.5 0.5, 13.5
18.5
of 15.5 0.0, 12.5 0.0, 12 0.0, 14 1.0, 14
±
±
±
±
0.01, 14.5
±
±
±
respectively. The highest inhibitory activity against P. aeruginosa was exerted by compounds 6a
,
,
and 6o with inhibition zones of 17
6j
±
1.0, 18.5
±
0.5 and 18.5
±
,
±
,
6d
±
,
6e
, 6m,
±
±
±
1.5 and
±
0.5, respectively. The rest of the compounds showed moderate inhibition against all other
bacterial strains.
2.3. Molecular Docking Analysis
Based on the data obtained from different biological evaluations, compounds 6a–o were docked
against proteinase K, GIIA sPLA2, and compounds 6a–e against glutamine-fructose-6-phosphate
transaminase (GlcN6P) synthase (GlmS, L-glutamine: D-fructose-6P amidotransferase, EC 2.6.1.16) in
order to provide a conceivable rationale for the observed activities.
2.3.1. Docking Simulations for Compounds 6a–o in Active site of GIIAsPLA2
GIIAsPLA2 is a low molecular weight enzyme (14 kDa) with seven disulfide bonds with a highly
conserved Ca2+-binding loop and a catalytic dyad consisting of His47/Asp91 along with active a
hydrophobic region lined near the N-terminal helix [44].

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Table 1. Antibacterial activities of the synthesized hydrazones 6a–o.
Gram-Negative Bacteria Gram-Positive Bacteria
Comp #
E. coli
p. aeruginosa
K. Pneumoniae
Salmonella
Serratia
S. aureus
S. aureus ALA1
MRSA ATCC3
S. mutans
E. feacalis
B. subtilis
6a
6b
6c
6d
6e
0 ± 0
14.5 ± 1.5
1 ± 0
17 ± 1
12 ± 0
0 ± 0
0 ± 0
8 ± 1
12.5 ± 0.5
10.25 ± 1
6.5 ± 0
4 ± 0
15.5 ± 0
12.5 ± 0.5
1.4 ± 1
0 ± 0
8 ± 0.5
10 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
29 ± 0.4
12 ± 0
6 ± 0
12 ± 0.5
0 ± 0
18.5 ± 0.5
9.5 ± 0.5
1.3 ± 1
18 ± 1
18.5 ± 0.5
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
8.5 ± 0.5
0 ± 0
0 ± 0
0 ± 0
0 ± 0
15 ± 0.5
1.5 ± 0
0 ± 0
0 ± 0
8 ± 0.5
0 ± 0
0.6 ± 1.2
10 ± 0.0
8 ± 1
14.5 ± 0.5
10 ± 0
0 ± 0
10 ± 0
12.2 ± 1
12.5 ± 0.5
4.5 ± 0.1
8 ± 1
12 ± 0
8.5 ± 0.5
8.5 ± 0.5
0 ± 0
12.5 ± 0.1
10.5 ± 0.1
10.5 ± 0.5
12.5 ± 0.7
10.5 ± 0.5
10 ± 0
14 ± 1
14 ± 1
0 ± 0
6f
10 ± 0
0 ± 0
14 ± 0.1
10.5 ± 0.5
10 ± 0
8 ± 1
6g
6h
6i
6j
6k
6l
6m
6n
6o
TCN
0 ± 0
8.5 ± 0
0 ± 0
0 ± 0
0 ± 0
8 ± 0
0 ± 0
0 ± 0
8.5 ± 0.7
10.5 ± 1.5
19.5 ± 1.5
10.5 ± 1.5
18.5 ± 0.5
8.5 ± 0.5
14 ± 1
0 ± 0
0 ± 0
0 ± 0
8.5 ± 0.5
8 ± 1
0 ± 0
0 ± 0
8.5 ± 0.5
8.5 ± 0.5
10 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
10 ± 0
14 ± 1
10 ± 1
10 ± 0
10 ± 0
19 ± 0.1
13.5 ± 1.5
11 ± 0
14.5 ± 1.5
11 ± 0
0 ± 0
0 ± 0
0 ± 0
24.5 ± 0.5
14 ± 2
0 ± 0
0 ± 0
8 ± 1
13 ± 0.1
14.5 ± 1.5
12.5 ± 0.5
12.5 ± 0.5
12 ± 0.3
0 ± 0
0 ± 0
18.5 ± 0.5
0 ± 0
4 ± 0
8.5 ± 0.5
10 ± 0
0 ± 0
13.5 ± 1.5
18.5 ± 0.5
0 ± 0
13 ± 1
10 ± 0
17 ± 0.5
0 ± 0
15 ± 1
9 ± 0.6
27 ± 1.2
18.5 ± 0.5
17 ± 0.2
0 ± 0
0 ± 0
16 ± 0.1
31 ± 0.6
18 ± 0.3
0 ± 0
Comp # = Compound number; TCN = Tetracycline.

Molecules 2016, 21, 1664
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The docking simulations for 6a
–
o
in active site of GIIAsPLA2 are presented in Supplementary
Materials, Figure S1. Results of docking simulations for compound 6l, the most active anti-GIIAsPLA2
enzyme, depicted here as a representative in Figure 3, revealed that it interacts with hydrophobic
and polar residues of active site of GIIAsPLA2. Compound 6l has adopted “U” shape with its central
benzene ring oriented towards the polar residues viz. His6, Gly22, Asp48, and Val30. The –CONH–N–
moiety is responsible for interactions with Phe5 and His47. The –CO–NH–N= moiety bridging the two
benzene rings as a polar linker has incorporated good flexibility to the ligand, hence such a flexible
moiety is beneficial for further amendments. The –Cl and –COOH groups are oriented toward Leu2
and Val3 due to polar interactions. The –CO– group of –CONH– group is close to Gly22. Hence,
–CONH– group is important group for retention in future optimizations.
Figure 3. Docking pose for compound 6l as a representative, in the active site of GIIAsPLA2.
2.3.2. Docking Simulations for Compounds 6a–o in Active Site of Proteinase K
Proteinase K (EC 3.4.21.64, protease K, endopeptidase K, Tritirachium alkaline proteinase,
Tritirachium album serine proteinase, and Tritirachium album proteinase K) is a broad-spectrum serine
protease belonging to Peptidase family S8 with ability to digest proteins. It is used for the destruction
of proteins in cell lysates (tissue, and cell culture cells) and for the release of nucleic acids [45].
The docking simulations for hydrazones 6a–o in active site of proteinase K are presented in
Supplementary Materials, Figure S2. Results of docking simulations for compound 6l, the most
active antiproteinase K, depicted here as a representative in Figure 4, revealed that it interacts with
hydrophobic and polar residues of active site of proteinase K. Compound 6l is unable to occupy the
complete space of active site of the enzyme. It interacts with polar residues viz. Asn161, Asn162, Ser224
and His69. In addition, it has H-bond formation with H2O (at distance of 2.86 A◦), present inside the
active site of Proteinase K, due to the –COOH group present on benzene ring. Hence, the –COOH
group is beneficial. Another important interaction is arene-cation interaction between the benzene ring
possessing –Cl atom with H2O (at distance of 4.08 A◦), present inside the active site of proteinase K.
Interestingly, the compound possesses intramolecular H-bond formation (distance 2.09 A◦) between

Molecules 2016, 21, 1664
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the –NH– part of –CONH– group with –CO– part of –CONH–N– group. This H-bond is probably
responsible for the specific orientation and shape of the molecule inside the active site. The compound
6l has acquired weird “J” or “L” shape inside the active site. The benzene ring possessing –COOH
group is closer to the active site residues. This indicates that the –CONH– and –CONH–N– groups are
important for future modifications.
Figure 4. Docking pose for compound 6l as a representative, in the active site of proteinase K.
2.3.3. Docking Simulations for Compounds 6a–e in Active Site of Glutamine-Fructose-6-Phosphate
Transaminase
The molecular mechanism of multifarious reaction catalyzed by glucosamine-6-phosphate
(GlcN6P) synthase (GlmS, L-glutamine: D-fructose-6P amidotransferase, EC 2.6.1.16) enzyme comprises
both ammonia transfer (L-glutamine to Fru-6-P) and sugar isomerization (fructosamine-6-phosphate
to glucosamine-6-phosphate). This reaction is the initiation of the pathway leading to the eventual
0
production of uridine 5 -diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), a product that is present
in all class of organisms, but in fungi and bacteria it is utilized to construct macromolecules essential
for the cell wall assembly, such as a number of amino sugar-containing macromolecules, comprising
chitin and mannoproteins in fungi, and peptidoglycan and lipopolisaccharides in bacteria. In the
prokaryotic cell, the inhibition of GlcN6P synthase even for a small time is fatal. Fortunately, because
of the longer lifespan of human, the running down of amino sugar pool for a short time is not deadly.
Consequently, it has been proposed as a possible target for developing antibacterial and antifungal
agents [46–49].
It is evident that recognition of a molecule by a particular enzyme is dependent on the structural
properties of that molecule and its distribution of the molecular electrostatic potential (MEP).
Furturmore, analysis of ligand–receptor interactions for GlcN-6-P synthase revealed that ligands
having primary amido groups can form stable hydrogen bonds with the amino acids residues present
in the binding site of the fungal enzyme thus they may serve as “anchors” to lock the inhibitor in
the binding pocket of the enzyme. In addition, previous structure–activity relationship experiments

Molecules 2016, 21, 1664
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revealed that presence of active electrophilic center at a proper position is required for inactivation
of the enzyme to take place [49]. By analogy, hydrazones 6a having the same structural features
represented by two primary amido groups and electrophilic double bond may serve as inhibitors for
bacterial GlcN6P synthase. This hypothesis is examined by docking hydrazones 6a in the active site
–
o
–
e
of the bacterial enzyme (these results are presented in Supplementary Materials, Figure S3).
Docking simulations for the most active candidate 6a which inhibits collectively three bacterial
strains and exhibiting inhibition zones larger than those produced by the reference drug tetracycline
against two bacterial strains, Serratia and E. feacalis, depicted here as a representative in Figure 5,
revealed that it interacts with a good number of residues of active site of glucosamine-6-phosphate
(GlcN6P) synthase. It mainly interacts with polar and hydrophobic residues of the active site. It has
adopted weird “J” or “L” shape in the active site. The oxygen atoms of the two amide groups are
responsible for the formation of H-bonding with the polar residues Ser A401 (distance 2.61 A◦),
Gln A348 (distance 1.94 A◦) and Ser A349 (distance 1.71 A◦). Hence, the amide groups are beneficial
for future development. The arene-cation interaction of benzene ring of ligand with the polar residue
Arg A26 has strengthened the binding of ligand with the receptor. The chlorine atom on the benzene
ring of the ligand has hydrophobic interactions with the hydrophobic residues Trp A74, Ala A602 and
Val A399 that constitute the lipophilic region of the cavity. The –CO-NH-N– moiety present between
the two benzene rings as a polar linker has furnished high flexibility to the ligand, hence such a
flexible moiety is advantageous for further modifications. The benzene ring, which is acting as a bridge
between the two –CONH– groups, is oriented toward polar region of the active site, which comprises
polar residues such as Ser A401, Cys A300, Ser A303, Ser A347, Thr A352, Gln A348 and Ser A349.
Figure 5. Docking pose for compound 6a, as a representative, in the active site of glucosamine-6-
phosphate (GlcN6P) synthase.
3. Experimental Section
3.1. Chemistry
3.1.1. General
All the chemicals were purchased from various suppliers, and were used without further
purification, unless otherwise stated. Melting points were measured on a Gallenkamp melting point

Molecules 2016, 21, 1664
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apparatus (Sanyo Gallenkamp, South borough, UK) in open glass capillaries and are uncorrected.
Infrared spectra (IR) were recorded using the KBr disc technique using a Perkin Elmer FT-IR
spectrophotometer 1000 (PerkinElmer, Waltham, MA, USA). 1H- and 13C-NMR spectra were recorded
on a BRUCKER-PLUS NMR (Billerica, MA, USA) operating at 500 MHz in deuterated dimethyl
sulfoxide (DMSO-d6). Chemical shifts are referred to in terms of ppm and J-coupling constants are
given in Hz. Mass Spectra were recorded on a Shimadzu GCMS-QP 5000 instrument (Shimadzu, Tokyo,
Japan). Elemental analysis was done to evaluate the presence of C, H, and N by Perkin Elmer-series-II
and the found results were within
products were carried out at King Saud University, Riyadh, KSA.
±
0.4% of the theoretical values. The biological evaluations of the
3.1.2. Synthetic Procedures
Compounds 3 and 4 were prepared according to reported procedures [20].
4-Chloro-N-(2-(hydrazinecarbonyl)phenyl)benzamide 5
A mixture of 2-(4-chlorophenyl)-4H-benzoyl [d][1,3]oxazin-4-one 4 (2.57 g, 10 mmol) and 10 mL
of hydrazine hydrate (80%) was refluxed for 1 h. Evaporation of the excess hydrazine under reduced
pressure and washing of the remaining solid product with plenty of water afford the title compound
in pure form.
Yield (95%); shiny white powder; m.p. 175–177 ◦C. IR ( KBr)
ν
max: 3316–3128 (NH2 and 2NH),
: 12.04 (s, 1H, NHCO), 11.92 (s, 1H,
Ar-H, J = 8.4 Hz), 7.90 (d, 1H, Ar-H, J = 7.6 Hz),
Ar-H), 7.28 (t, 1H, Ar-H, J = 7.5 Hz), 4.52 (s, 2H, NH2); 13C-NMR (DMSO-d6)
: 165.27 (CO), 164.16 (CO), 138.2, 137.10, 132.43, 129.11, 128.72, 127.80, 125.445, 124.32, 120.93
(12 Ar-
); MS (ESI): 290 [M+ + 1], Anal. Calcd. For C14H12ClN3O2: C (58.04%); H (4.17%);
1
1673, 1635 (2 C=O), 1592 (C=N) cm−1; H-NMR (DMSO-d6)
δ
NHCO), 8.51 (d, 1H, Ar-H, J = 8.3 Hz), 7.95 (d, 2H, 2
7.76–7.62 (m, 3H, 3
×
×
δ
×
C
N (14.50%); Found: C (58.08%); H (4.21%); N (14.53%).
Synthesis of Compounds 6a–o
General procedures: A mixture of 4-chloro-N-(2-(hydrazinecarbonyl)phenyl)benzamide
5 (2.89 g,
10 mmol) and the appropriate aldehyde (10 mmol) was refluxed in absolute ethanol (15 mL) in
presence of few drops of glacial acetic acid as a catalyst for about 4 hour and monitored with TLC
(n-hexane:EtOAc, 80:20) until the reaction was completed as observed by appearance of a single new
spot. The resultant reaction mixture was cooled to room temperature and the solid product obtained
in each experiment was collected by filtration and recrystallized from ethanol to afford the desired
products 6a–o
N-(2-(2-benzylidenehydrazine-1-carbonyl)phenyl)-4-chlorobenzamide (6a) Yield (89%); white crystals; m.p.
◦
1
244–246 C; IR (KBr)
(DMSO-d6) : 12.14 (s, 1H, NHCO), 11.94 (s, 1H, NHCO), 8.51 (d, 1H, Ar-H, J = 8.3 Hz), 8.47 (s, 1H,
N=CH), 7.96 (d, 2H, 2 Ar-H, J = 8.4 Hz), 7.92 (d, 1H, Ar-H, J = 7.6 Hz), 7.76–7.47 (m, 8H, 8 Ar-H),
7.30 (t, 1H, Ar-H, J = 7.5 Hz); 13C-NMR (DMSO-d6)
: 165.39 (CO), 164.00 (CO), 149.53 (N=CH),
139.53, 137.42, 134.49, 133.63, 133.06, 130.86, 129.48, 129.36, 129.10, 127.75, 123.85, 121.85, 121.75, 121.31
(18 Ar-
); MS (ESI): 379 (C21H16ClN3O2, [M+ + 1]; Anal. Calcd. for C21H16ClN3O2: C (66.76%);
ν
max: 3336–3197 (2 NH), 1673, 1635 (2 C=O), 1592 (C=N) cm−1; H-NMR
δ
×
×
δ
×
C
H (4.27%); N (11.12%); Found: C (66.72%); H (4.24%); N (11.14%).
4-Chloro-N-(2-(2-(4-chlorobenzylidene)hydrazine-1-carbonyl)phenyl)benzamide (6b) Yield (95%); white
ν ;
powder; m.p. 252–256 ◦C; IR (KBr) max: 3333–3201 (2 NH), 1675, 1639 (2 C=O), 1598 (C=N) cm−1
1H-NMR (DMSO-d6)
8.45 (s, 1H, N=CH), 7.96 (d, 2H, 2
Ar-H, J = 8.4 Hz), 7.65–7.61 (m, 3H, 3
J = 7.6 Hz); 13C-NMR (DMSO-d6)
: 165.46 (CO), 163.96 (CO), 148.13 (N=CH), 139.62, 137.43, 135.32,
133.58, 133.42, 133.10, 129.67, 129.44, 129.33, 129.07, 123.76, 121.71, 121.08 (18 Ar-C); MS (ESI): 413
δ
: 12.19 (s, 1H, NHCO), 11.95 (s, 1H, NHCO), 8.52 (d, 1H, Ar-H, J = 8.3 Hz),
Ar-H, J = 8.4 Hz), 7.90 (d, 1H, Ar-H, J = 7.8 Hz), 7.77 (d, 2H,
Ar-H), 7.52 (d, 2H, 2 Ar-H, J = 8.4 Hz), 7.28 (t, 1H, Ar-H,
×
2
×
×
×
δ
×

Molecules 2016, 21, 1664
11 of 17
[M+ + 1], Anal. Calcd. for C21H15Cl2N3O2: C (61.18%); H (3.67%); N (10.19%); Found: C (61.20%);
H (3.64%); N (10.21%).
4-Chloro-N-(2-(2-(4-hydroxy-3-methoxybenzylidene)hydrazine-1-carbonyl)phenyl)benzamide (6c) Yield (85%);
white crystals; m.p. 236–238 ◦C; IR (KBr)
ν
max: 3341–3201 (2 NH and OH), 1675, 1639 (2 C=O), 1588
11.93 (s, 2H, NH and OH), 9.59 (br. s, 1H, NH), 8.46 (d, 1H, Ar-H,
J = 8.3 Hz), 8.30 (s, 1H, N=CH), 7.90 (d, 2H, 2 Ar-H, J = 8.4 Hz), 7.83 (d, 1H, Ar-H, J = 7.7 Hz),
7.58 (d, 2H, 2 Ar-H, J = 8.4 Hz), 7.54 (d, 1H, Ar-H, J = 7.7 Hz), 7.29 (d, 1H, Ar-H J = 1.0 Hz), 7.21
(t, 1H, Ar-H, J = 7.7 Hz), 7.06 (dd, 1H, Ar-H, J = 8.1, 1.0 Hz), 6.80 (d, 1H, Ar-H, J = 8.1 Hz), 3.78 (s, 3H,
OCH3); 13C-NMR (DMSO-d6)
165.13 (CO), 163.98 (CO), 150.24, 149.81, 148.55, 139.52, 137.41, 133.65,
(C=N) cm−1; 1H-NMR (DMSO-d6)
δ
×
×
δ
132.90, 129.46, 129.01, 125.84, 123.78, 123.06, 121.63, 121.31, 115.93, 109.49 (CH=N and 18
×
Ar-C), 56.04
(OCH3); MS (ESI): 425 [M+ + 1], Anal. Calcd. for C22H18ClN3O4: C (62.34%); H (4.28%); N (9.91%);
Found: C (62.32%); H (4.24%); N (9.93%).
4-Chloro-N-(2-(2-(3,4,5-trimethoxybenzylidene)hydrazine-1-carbonyl)phenyl)benzamide (6d) Yield (93%);
white crystals; m.p. 247–248 ◦C; IR (KBr)
ν
max: 3345–3208 (2 NH), 1671, 1644 (2 C=O), 1578 (C=N)
: 12.11 (s, 1H, NHCO), 11.81 (s, 1H, NHCO), 8.46 (d, 1H, Ar-H, J = 8.2
Hz), 8.37 (s, 1H, N=CH), 7.96 (d, 2H, 2 Ar-H, J = 8.4 Hz), 7.88 (d, 1H, Ar-H, J = 7.6 Hz), 7.67 (d, 2H,
Ar-H, J = 8.4 Hz), 7.63 (t, 1H, Ar-H, J = 8.2 Hz), 7.31 (t, 1H, Ar-H, J = 7.6 Hz), 7.04 (s, 2H, 2 Ar-H),
3.85 (s, 6H, 2 : 165.25 (CO), 164.12 (CO), 153.69
OCH3), 3.74 (s, 3H, OCH3); 13C-NMR (DMSO-d6)
(3 × C-OCH3), 146.48 (N=CH), 139.32, 137.39, 133.68, 129.97, 129.52, 129.47, 129.15, 123.96, 121.96,
104.96 (15
Ar-C), 60.60 (OCH3), 56.50 (OCH3), 56.46 (OCH3). MS (ESI): 469 [M+ + 1], Anal. Calcd.
cm−1; 1H-NMR (DMSO-d6)
δ
×
2
×
×
×
δ
×
for C24H22ClN3O5: C (61.61%); H (4.74%); N (8.98%); Found: C (61.32%); H (4.71%); N (9.02%).
4-Chloro-N-(2-(2-(2,4-dichlorobenzylidene)hydrazine-1-carbonyl)phenyl)benzamide (6e) Yield (93%); creamish
powder; m.p. 262–264 ◦C; IR (KBr) max: 3421–3268 (2 NH), 1680, 1656 (2 C=O), 1592 (C=N) cm−1
ν ;
1H-NMR (DMSO-d6)
Ar-H, J = 8.3 Hz), 8.04 (d, 1H, Ar-H, J = 8.5 Hz), 7.95 (d, 2H, 2
J = 7.8 Hz), 7.75 (s, 1H, CH-Ar), 7.69–7.64 (m, 3H, 3 Ar-H), 7.55 (d, 1H, Ar-H, J = 8.5 Hz), 7.31 (t, 1H,
Ar-H, J = 7.6 Hz); 13C-NMR (DMSO-d6)
: 165.31 (CO), 164.42 (CO), 148.38 (N=CH), 139.64, 137.93,
Ar-C). MS (ESI):
δ: 12.36 (s, 1H, NHCO), 11.79 (s, 1H, NHCO), 8.82 (s, 1H, N=CH), 8.47 (d, 1H,
×
Ar-H, J = 8.4 Hz), 7.91 (d, 1H, Ar-H,
×
δ
135.26, 133.45, 133.39, 133.10, 129.51, 129.49, 129.27, 129.11, 123.80, 121.71, 121.88 (18
×
447 [M+ + 1], Anal. Calcd. for C21H14Cl3N3O2: C (56.46%); H (3.16%); N (9.41%); Found: C (56.48%);
H (3.20%); N (9.42%).
4-Chloro-N-[2-(4-methoxy-benzylidene-hydrazinocarbonyl)-phenyl]-benzamide (6f) Yield (88%); white
powder; m.p. 260–262 ◦C; IR (KBr) vmax: 3374–3242 (2 NH), 1682, 1646 (2 C=O), 1589 (C=N) cm−1
;
1H-NMR (DMSO-d6)
(s, 1H, CH=N), 7.96 (d, 2H, 2
J = 8.7 Hz), 7.64 (d, 2H, 2 Ar-H, J = 8.5 Hz), 7.60 (t, 1H, Ar-H, J = 7.5 Hz), 7.27 (t, 1H, Ar-H, J = 7.5 Hz),
Ar-H, J = 8.7 Hz), 3.80 (s, 3H, OCH3); 13C-NMR (DMSO-d6)
: 165.25 (CO), 163.91
-OCH3), 149.54 (N=CH), 139.65, 137.42, 133.60, 132.96, 129.45, 129.42, 128.99, 127.01,
Ar-C), 55.75 (OCH3). MS (ESI): 409 [M+ + 1], Anal. Calcd. for
δ: 12.06 (s, 1H, NHCO), 12.03 (s, 1H, NHCO), 8.55 (d, 1H, Ar-H, J = 8.3 Hz), 8.42
×
Ar-H, J = 8.5 Hz), 7.91 (d, 1H, Ar-H, J = 7.6 Hz), 7.69 (d, 2H, 2 Ar-H,
×
×
7.02 (d, 2H, 2
(CO), 161.55 (
×
δ
C
123.69, 121.53, 121.02, 114.82 (17
C22H18ClN3O3: C (64.79%); H (4.45%); N (10.30%); Found: C (64.78%); H (4.42%); N (10.32%).
×
4-Chloro-N-(2-(2-(4-(dimethylamino)benzylidene)hydrazine-1-carbonyl)phenyl)benzamide (6g) Yield (82%);
yellow powder; m.p. 249–251 ◦C; IR (KBr)
ν
max: 3364–3212 ((2 NH), 1679, 1640 (2 C=O), 1582 (C=N)
: 12.11 (s, 1H, NHCO), 11.85 (s, 1H, NHCO), 8.56 (d, 1H, Ar-H, J = 8.2 Hz),
8.32 (s, 1H, CH=N), 7.96 (d, 2H, 2 Ar-H, J = 8.5 Hz), 7.90 (d, 1H, Ar-H, J = 7.2 Hz), 7.68 (d, 2H,
Ar-H, J = 8.5 Hz), 7.62 (t, 1H, Ar-H, J = 7.4 Hz), 7.56 (d, 2H, 2 Ar-H, J = 8.8 Hz), 7.28 (t, 1H, Ar-H,
J = 7.4 Hz), 6.77 (d, 2H, 2 Ar-H, J = 8.8 Hz), 2.99 (s, 6H, 2 : 165.31
N-CH3); 13C-NMR (DMSO-d6)
(CO), 164.13 (CO), 152.4 ( -NCH3), 148.27 (N=CH), 138.2, 137.4, 134.84, 129.53, 129.44, 129.18, 128.8,
127.7, 124.2, 123.74, 119.8, 112.3 (17 Ar-C), 45.2 (2
CH3). MS (ESI): 422 [M+ + 1], Anal. Calcd. for
C23H21ClN4O2: C (65.63%); H (5.03%); N (13.31%); Found: C (65.59%); H (5.02%); N (13.33%).
cm−1; 1H-NMR (DMSO-d6)
δ
×
2
×
×
×
×
δ
C
×
×

Molecules 2016, 21, 1664
12 of 17
4-Chloro-N-(2-(2-(2-methoxybenzylidene)hydrazine-1-carbonyl)phenyl)benzamide (6h) Yield (98%); white
powder; m.p. 236–238 ◦C; IR (KBr) max: 3383–3251 ((2 NH), 1680, 1652 (2 C=O), 1589 (C=N) cm−1
1H-NMR (DMSO-d6)
: 12.06 (s, 1H, NHCO), 11.97 (s, 1H, NHCO), 8.77 (s, 1H, N=CH), 8.47 (d, 1H,
Ar-H, J = 8.3 Hz), 7.90 (d, 2H, 2 Ar-H, J = 8.5 Hz), 7.88–7.83 (m, 2H, 2 Ar-H), 7.60 (d, 2H, 2 Ar-H,
J = 8.5 Hz), 7.58–7.20 (m, 3H, 3 Ar-H, J = 7.5 Hz), 7.05 (d, 1H, Ar-H, J = 8.4 Hz), 6.98 (t, 1H, Ar-H,
J = 7.7 Hz), 3.80 (s, 3H, O-CH3); 13C-NMR (DMSO-d6)
: 165.28 (CO), 163.96 (CO), 158.39 ( -OCH3),
145.11 (N=CH), 139.63, 137.42, 133.64, 133.05, 132.40, 129.49, 129.46, 129.09, 126.09, 123.77, 122.47,
121.58, 121.23, 120.99, 112.37 (17
Ar-C), 56.18 (OCH3). MS (ESI): 409 [M+ + 1], Anal. Calcd. for
ν
;
δ
×
×
×
×
δ
C
×
C22H18ClN3O3: C (64.79%); H (4.45%); N (10.30%); Found: C (64.82%); H (4.46%); N (10.33%).
4-Chloro-N-(2-(2-(4-methylbenzylidene)hydrazine-1-carbonyl)phenyl)benzamide (6i) Yield (91%); white
ν ;
crystals; m.p. 256–258 ◦C; IR (KBr) max: 3372-3264 ((2 NH), 1684, 1657 (2 C=O), 1579 (C=N) cm−1
1H-NMR (DMSO-d6)
(s, 1H, N=CH), 7.98 (d, 1H, Ar-H, J = 8.5 Hz), 7.91 (d, 2H, 2
Ar-H), 7.31–7.28 (m, 3H, 3
Ar-H), 2.34 (s, 3H, CH3); 13C-NMR (DMSO-d6)
163.98 (CO), 149.61 (N=CH), 140.77, 139.55, 137.42, 133.62, 133.62, 133.02, 131.78, 129.97, 129.49, 129.46,
129.06, 127.74, 123.81, 121.67, 121.24 (18
Ar-C), 21.52 (CH3). MS (ESI): 393 [M+ + 1], Anal. Calcd. for
δ
: 12.08 (s, 1H, NHCO), 11.97 (s, 1H, NHCO), 8.52 (d, 1H, Ar-H, J = 8.3 Hz), 8.43
Ar-H, J = 7.7 Hz), 8.68–7.62 (m, 5H,
(ppm): 165.31 (CO),
×
5
×
×
δ
×
C22H18ClN3O2: C (67.43%); H (4.63%); N (10.72%); Found: C (64.42%); H (4.66%); N (10.73%).
4-((2-(2-(4-chlorobenzamido)benzoyl)hydrazono)methyl)benzoic acid (6j) Yield (85%); creamish powder;
ν ;
m.p. 252–254 ◦C; IR (KBr) max: 3373–3292 (2 NH, OH), 1759, 1680, 1652 (3 C=O), 1581 (C=N) cm−1
1H-NMR (DMSO-d6)
(m, 12H, 12
Ar-H); 13C-NMR (DMSO-d6)
(N=CH), 139.63, 138.49, 137.44, 133.56, 133.13, 132.42, 130.26, 129.40, 129.30, 129.09, 127.72, 123.75,
121.75, 121.05 (18
Ar-C). MS: m/z 423 [M+ + 1], Anal. Calcd. for C22H16ClN3O4: C (62.64%); H
δ: 12.25 (s, 1H, NHCO), 11.92 (s, 1H, NHCO), 8.51 (s, 1H, N=CH), 8.04–7.25
×
δ: 167.37 (COOH), 165.58 (CONH), 163.98 (CO), 148.24
×
(3.82%); N (9.96%); Found: C (62.63%); H (3.86%); N (9.97%).
Chloro-N-(2-(2-(3-phenylallylidene)hydrazine-1-carbonyl)phenyl)benzamide (6k) Yield (93%); creamish
ν ;
powder; m.p. 248–250 ◦C; IR (KBr) max: 3391–3271 (2 NH), 1680, 1644 (2 C=O), 1594 (C=N) cm−1
1H-NMR (DMSO-d6)
(d, 1H, N=CH, J = 8.3 Hz), 7.95 (d, 2H, 2
Ar-H, J = 8.5 Hz), 7.64–7.62 (m, 3H, 3
(m, 4H, 4
136.26, 129.50, 129.45, 129.34, 129.05, 127.67, 125.84, 123.84, 121.28 (N=
MS: m/z 405 [M+ + 1], Anal. Calcd. for C23H18ClN3O2: C (68.40%); H (4.49%); N (10.40%); Found: C
δ: 12.04 (s, 1H, NHCO), 11.97 (s, 1H, NHCO), 8.51 (d, 1H, Ar-H, J = 7.8 Hz), 8.25
×
Ar-H, J = 8.5 Hz), 7.88 (d, 1H, J = 7.8 Hz, Ar-H), 7.68 (d, 2H,
Ar-H, J = 7.6 Hz), 7.41 (t, 2H, 2 Ar-H, J = 7.3 Hz), 7.36–7.08
: 165.29 (CO), 163.92 (CO), 151.46, 140.39, 139.49, 137.44,
2
×
×
×
×
Ar-H); 13C-NMR (DMSO-d6)
δ
CH-
CH=
C
H and 18
×
Ar-C).
(68.42%); H (4.51%); N (10.44%).
2-((2-(2-(4-chlorobenzamido)benzoyl)hydrazono)methyl)benzoic acid (6l) Yield (87%); white powder; m.p.
◦
251–253 C; IR (KBr)
ν
max: 3368–3278 (2 NH and OH), 1758 (CO), 1680, 1656 (2 C=O), 1579 (C=N)
: 12.37 (s, 1H, NHCO), 11.95 (s, 1H, NHCO), 8.74 (s, 1H, N=CH), 8.52 (d,
cm−1; 1H-NMR (DMSO-d6)
δ
1H, Ar-H, J = 8.3 Hz), 8.09 (d, 1H, Ar-H, J = 7.8 Hz) 7.97 (d, 2H, 2
×
Ar-H, J = 8.5 Hz), 7.95–7.62 (m, 6H,
6 × Ar-H), 7.56 (t, 1H, Ar-H, J = 7.4 Hz), 7.28 (t, 1H, Ar-H, J = 7.4 Hz); 13C-NMR (DMSO-d6) δ: 168.51
(COOH), 165.61 (CO), 164.02 (CO), 148.38 (N=CH), 139.56, 137.39, 134.82, 133.66, 133.09, 132.49, 131.29,
130.35, 129.47, 129.25, 127.23, 123.78, 121.70, 121.19 (18
for C22H16ClN3O4: C (62.64%); H (3.82%); N (9.96%); Found: C (62.67%); H (3.85%); N (9.88%).
×
Ar-C). MS (ESI): 423 [M+ + 1], Anal. Calcd.
4-Chloro-N-(2-(2-(3-hydroxy-4-methoxybenzylidene)hydrazine-1-carbonyl)phenyl)benzamide (6m) Yield
(94%); white powder; m.p. 239–241 ◦C; IR (KBr)
ν
max: 3383–3256 (2 NH), 1680, 1657 (2 C=O), 1582
: 12.04 (s, 1H, NHCO), 11.97 (s, 1H, NHCO), 8.56 (d, 1H, Ar-H,
J = 8.3 Hz), 8.34 (s, 1H, N=CH), 7.96 (d, 2H, 2 × Ar-H, J = 8.5 Hz), 7.90 (d, 1H, J = 7.7 Hz), 7.67 (d, 2H,
Ar-H, J = 8.5 Hz), 7.62 (t, 1H, Ar-H, J = 7.5 Hz), 7.31 (d, 1H, Ar-H, J = 1.6 Hz), 7.28 (t, 1H, Ar-H,
J = 7.6 Hz), 7.08 (dd, 1H, Ar-H, J = 8.3, 1.6 Hz), 6.98 (d, 1H, Ar-H, J = 8.3 Hz), 3.82 (s, 3H, O-CH3);
13C-NMR (DMSO-d6)
: 165.16 (CO), 163.93 (CO), 150.53, ( -OCH3), 149.81 (C-OH), 147.37 (N=CH),
(C=N) cm−1; 1H-NMR (DMSO-d6)
δ
2
×
δ
C

Molecules 2016, 21, 1664
13 of 17
139.58, 137.44, 133.62, 132.62, 129.49, 129.43, 129.01, 127.29, 123.77, 121.55, 121.13, 112.90, 112.29 (16 ×
Ar-C), 56.04 (OCH3). MS (ESI): 425 [M+ + 1], Anal. Calcd. for C22H18ClN3O4: C (62.34%); H (4.28%);
N 00(9.91%); Found: C (62.38%); H (4.26%); N (9.93%).
4-Chloro-N-(2-(2-(4-cyclohexylbenzylidene)hydrazine-1-carbonyl)phenyl)benzamide (6n) Yield (85%); white
crystals; m.p. 257–259 ◦C; IR (KBr) max: 3386–3260 (2 NH), 1682, 1654 (2 C=O), 1589 (C=N) cm−1
ν ;
1H-NMR (DMSO-d6)
8.36 (s, 1H, N=CH), 7.95 (d, 2H, 2
Ar-H, J = 8.3 Hz), 7.59 (t, 1H, Ar-H, J = 7. 7Hz), 7.31–7.18 (m, 5H, 5
cyclohexyl-H); 13C-NMR (DMSO-d6)
: 165.54 (CO), 163.86 (CO), 146.14 (N=CH), 139.23, 137.44, 133.55,
Ar-C), 42.85, 41.23, 35.32, 34.39,
δ
: 12.03 (s, 1H, NHCO), 11.08 (s, 1H, NHCO), 8.51 (d, 1H, Ar-H, J = 8.2 Hz),
Ar-H, J = 8.5 Hz), 7.88 (d, 1H, Ar-H, J = 7.7 Hz), 7.65 (d, 2H,
Ar-H), 3.09–1.55 (m, 11H,
×
2
×
×
δ
132.61, 129.47, 129.39, 128.84, 127.16, 126.60, 123.76, 121.72, 121.49 (18
×
33.43, 28.39 (cyclohexyl-C). MS (ESI): 461 [M+ + 1], Anal. Calcd. For C27H26ClN3O2: C (70.50%); H
(5.70%); N (9.14%); Found: C (70.51%); H (5.71%); N (9.13%).
(Z)-4-chloro-N-(2-(2-(3-hydroxybenzylidene)hydrazine-1-carbonyl)phenyl)benzamide (6o) Yield (95%); white
◦
powder; m.p. 262–264 C; IR (KBr)
ν
max: 3386–3258 (2 NH, OH), 1680, 1652 (2 C=O), 1579 (C=N)
: 12.12 (s, 1H, NHCO), 12.04 (s, 1H, NHCO), 8.84 (s, 1H, N=CH), 8.55
(d, 1H, Ar-H, J = 8.3 Hz), 7.97 (d, 2H, 2 Ar-H, J = 8.5 Hz), 7.95–7.90 (m, 2H, 2 Ar-H), 7.67 (d, 2H,
Ar-H, J = 8.5 Hz), 7.63 (t, 1H, Ar-H, J = 7.5 Hz), 7.46–7.03 (m, 4H, 4
Ar-H); 13C-NMR (DMSO-d6)
-OCH3), 145.10 (N=CH), 139.64, 137.42, 133.64, 133.05, 132.40,
Ar-C). MS (ESI): 395
cm−1; 1H-NMR (DMSO-d6)
δ
×
×
2
δ
×
×
: 165.27 (CO), 163.96 (CO), 158.40 (
C
129.50, 129.46, 129.09, 126.09, 123.77, 122.47, 121.48, 121.57, 121.23, 120.99 (17
[M+ + 1], Anal. Calcd. For C21H16ClN3O3: C (64.05%); H (4.09%); N (10.67%); Found: C (64.07%);
H (4.12%); N (10.63%).
×
3.2. Biological Evalustion
3.2.1. Inhibition of sPLA2 Activity
The test of inhibitory activity of secretory phospholipase A2 (sPLA2) was performed as described
by De Aranjo and Radvany [50]. Briefly, the substrate consisted of 3.5 mM lecithin in a mixture of
3 mM NaTDC, 100 mM NaCl, 10 mM CaCl2 and 0.055 mM red phenol as colorimetric indicator in
100 mL H2O. The pH of the reaction mixture was adjusted to 7.6 using phosphate buffer. The Human
group IIA (hG-IIA) and dromedary group IB (DrG-IIA) sPLA2 were solubilized in 10% acetonitrile at a
concentration of 0.05
µg/µL. A volume of 10 µL of these PLA2 solutions was incubated with 10 µL
containing 10 g of each compound for 20 min at room temperature. Then, 1 mL of the PLA2 substrate
µ
was added, and the kinetic of hydrolysis was followed during 5 min by reading the optical density at
558 nm. The inhibition percentage was calculated by comparison with a control experiment (absence
of compound).
3.2.2. Protease Inhibitor Assay
Three commercially available proteases; proteinase K (Sigma-Aldrich, St. Louis, MO, USA, P2308),
esperase (Novozyme, Sigma-Aldrich, P5860) and that obtained from Bacillus sp. (Sigma-Aldrich, P3111)
were evaluated for the effect of the studied compounds on their activities. Protease assays were carried
out by adopting Kunitz caseinolytic method [51] using Hammerstein casein as substrate. Respective
protease inhibitor activities were assayed under the same conditions with the addition of the inhibitor
(0.1 mg/mL) to the respective reaction mixture and pre incubation for 10 min at 37 ◦C. The assay of
the residual enzyme activity was followed by the addition of 2 mL of 1% casein and the resulting
mixture was allowed to stand for 30 min at 37 ◦C. The reaction was stopped by the addition of 2.5 mL
of 5% TCA solution. After centrifugation of the reaction mixture (12,000 rpm, 15 min), the absorbance
was measured at 280 nm. Protease inhibitor unit is defined as the amount of protease inhibitor that
inhibited one unit of respective enzyme activity. The protease inhibitor activity was expressed in

Molecules 2016, 21, 1664
14 of 17
terms of percent inhibition. Appropriate blanks for the enzyme, inhibitor, and the substrate were also
included in the assay along with the test.
3.2.3. Antibacterial Activity
Culture of Microbial Strains Preparation
Pure standard microbial isolates collected from King Khaled University Hospital were tested in
this study; including Staphylococcus aureus ATCC 25923, Methicillin resistant Staphylococus aureus
ATCC 12498, Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212 as Gram-positive
and Escherichia coli ATCC 25966, Pseudomonas aeruginosa ATCC 27853 and Serratia marcescens as
Gram-negative bacteria. Fresh cultures of each microorganism were grown on nutrient agar plates
(Oxoid, Thermo Scientific, Basingstoke, UK); of which small inoculums were suspended in 5 mL
nutrient broth for bacterial suspension preparation of 0.5 MacFarland.
Antibacterial Assay
Antimicrobial activity was assayed using well diffusion technique according to given
literature [43]. Briefly, small inoculums of each of the microbial suspension prepared were loaded on
sterile Muller Hinton agar plates surface (Oxoid) with sterile cotton swabs. Loaded plates were then
perforated equidistantly with a sterile 6 mm diameter cork borer, and 70 µL of each compound solution
(1 mg/mL) were loaded in their respective wells. DMSO and Tetracycline were used as negative
and positive controls, respectively. Plates were kept to rest for 30 min at room temperature and then
incubated at 37 ◦C for 18–24 h. Antimicrobial activity was determined by measuring the inhibition
zone. All tests were performed in duplicates and means of inhibition zones were recorded in mm.
3.3. Molecular Docking
In the present work, AutoDock 4.0 (The Scripps Research Institute, La Jolla, CA, USA) was used
for molecular docking. The software is freely available and can be used with different molecular
viewers like PMV, PyMol, etc. For docking simulations, the structures of the molecules were drawn
using ChemSketch 12.0 (Advanced Chemistry Development, Inc., Toronto, ON, Canada) freeware
and optimized using the inbuilt methodology. The optimized structures were saved in 3D-format in
.mol file format. The structures of proteins GIIA sPLA2 (pdb: 1DB4), proteinase K (pdb: 2PWB) and
glutamine-fructose-6-phosphate transaminase (1JXA) were retrieved from www.rcsb.org. The pdb
1DB4, 2PWB, and 1JXA for the proteins were selected on the basis of reasonable X-ray resolution
and sequence completion. The proteins structures were optimized before actual docking simulations.
Following parameters were set for getting better docking results: Algorithm: genetic Algorithm;
Number of runs: 10 GA runs; Number of evaluations: 250,000 evaluation/run; population size: 150.
4. Conclusions
In conclusion, fifteen novel hydrazone derivatives 6a–o were synthesized, fully characterized
and examined to evaluate their inhibitory activity against two phospholipases A2, three protease
enzymes and a panel of Gram-negative and Gram-positive bacterial strains. Among the investigated
compounds; 6e and 6l selectively exhibited sPLA2 the highest inhibitory activity against hG-IIA
isoform. It is quite interesting to report that compound 6l also displayed the highest antiprotease
inhibition against all used protease enzymes.
Moreover, the series under investigation showed varied antibacterial inhibitory activity. Thus,
while similar inhibition to that produced by the reference drug tetracycline was displayed by compound
6a against P. aeruginosa, compounds 6m and 6o exhibited higher inhibition. In addition, the most
susceptible bacterial strain to the synthesized compounds was genus Serratia, which was inhibited by
nine of them, with compound 6a being the most powerful inhibitory agent. Furthermore, the maximum
inhibitory activities were exhibited by compounds 6j, 6l, and 6n against S. aureus and by compound

Molecules 2016, 21, 1664
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6k against S. mutans. Finally, compounds 6a
,
6d
,
6e
,
6m and 6n were found to be more potent than the
used reference drug against E. feacalis. The docking simulations of compounds 6a
–o with GIIA sPLA2,
proteinase K and compounds 6a GlcN6P were carried out in order to investigate the mode of action.
–
e
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
12/1664/s1.
Acknowledgments: The authors are thankful to “The Visiting Professor Program at King Saud University”.
Author Contributions: Nahed N. E. El-Sayed and Ahmed M. Alafeefy conceived, designed the experiments,
analyzed the spectral data and wrote the manuscript; Mohammaed A. Bakht performed the experiments;
Vijay H. Masand performed the docking studies; Abir Ben Bacha performed the biological studies; and
Ali Aldalbahi, Nan Chen and Chunhai Fan helped in discussion.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).