Substituted (E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-carboxylates as dual inhibitors of 15-lipoxygenase & carbonic anhydrase II: synthesis, biochemical evaluation and docking studies

Article abstract of DOI:10.1016/j.bbrc.2016.11.028

15-Lipoxygenase (15-LOX) plays a major role in many inflammatory lung diseases including chronic obstructive pulmonary disease (COPD), asthma and chronic bronchitis. Over-expression of 15-LOX is related with some specific carcinomas including pancreatic, gastric and brain tumor. Similarly among different isozymes of carbonic anhydrase (CA), CA II is expressed in pancreatic, gastric carcinomas as well as in brain tumors. Therefore, novel potent inhibitors of both 15-LOX and CA II are required to explore the role of these enzymes further and to enable the drug discovery efforts. For this purpose, a series of benzyledinyl-hydrazinyl substituted thiazole derivatives were designed, synthesized and characterized by FTIR, ¹H, & ¹³C NMR spectroscopy. The derivatives were then evaluated for their potential to inhibit 15-LOX and bovine carbonic anhydrase II (bCA II). Most of these compounds showed excellent inhibitory potential for 15-LOX with an IC₅₀ of 0.12?±?0.002 to 0.69?±?0.5?μM and showed moderate inhibition potency for bCA II with compound 5h (IC₅₀?=?1.26?±?0.24?μM) being the most active. The most potent compound 5a that emerged as a dual inhibitor of both enzymes, exhibiting 24 times greater selectivity for 15-LOX over bCA II. Compound 5a exhibited dual potent inhibitory activity against both 15-LOX and bCA II enzymes having IC₅₀ values of 0.12?±?0.002 and 2.93?±?0.22?μM, respectively. Molecular docking studies of potent as well as dual inhibitors were also carried out to provide an insight into the binding site interactions.

Full text of DOI:10.1016/j.bbrc.2016.11.028

Accepted Manuscript
Substituted (E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-carboxylates as dual
inhibitors of 15-lipoxygenase & carbonic anhydrase II: Synthesis, biological evaluation
and docking studies
Aamer Saeed, Shafi Ullah Khan, Parvez Ali Mahesar, Pervaiz Ali Channar, Ghulam
Shabir, Jamshed Iqbal
PII:
S0006-291X(16)31883-6
10.1016/j.bbrc.2016.11.028
YBBRC 36730
DOI:
Reference:
To appear in: Biochemical and Biophysical Research Communications
Received Date: 1 November 2016
Accepted Date: 6 November 2016
Please cite this article as: A. Saeed, S.U. Khan, P.A. Mahesar, P.A. Channar, G. Shabir, J. Iqbal,
Substituted (E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-carboxylates as dual inhibitors of 15-
lipoxygenase & carbonic anhydrase II: Synthesis, biological evaluation and docking studies, Biochemical
and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.11.028.
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Graphical Abstract
Substituted (E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-carboxylates as dual
inhibitors of 15-Lipoxygenase & Carbonic Anhydrase II: Synthesis, Biological Evaluation
and Docking Studies
Aamer Saeed, Shafi Ullah Khan, Parvez Ali Mahesar, Pervaiz Ali Channar, Ghulam Shabir,
Jamshed Iqbal*
Putative binding mode of most potent compound in the active pocket of 15-lipoxygenase

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Substituted (E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-carboxylates as dual
inhibitors of 15-Lipoxygenase and Carbonic Anhydrase II: Synthesis, biological evaluation
and docking studies
Aamer Saeeda*, Shafi Ullah Khanb, Parvez Ali Mahesara, Pervaiz Ali Channara, Ghulam Shabira,
Jamshed Iqbalb*
aDepartment of Chemistry, Quaid-i-Azam University-45320, Islamabad, Pakistan.
bCentre for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabad-22060, Pakistan
Abstract
15-Lipoxygenase (15-LOX) plays a major role in many inflammatory lung diseases including
chronic obstructive pulmonary disease (COPD), asthma and chronic bronchitis. Over-expression
of 15-LOX is related with some specific carcinomas including pancreatic, gastric and brain
tumor. Similarly among different isozymes of carbonic anhydrase (CA), CA II is expressed in
pancreatic, gastric carcinomas as well as in brain tumors. Therefore, novel potent inhibitors of
both 15-LOX and CA II are required to explore the role of these enzymes further and to enable
the drug discovery efforts. For this purpose, a series of benzyledinyl-hydrazinyl substituted
1
thiazole derivatives were designed, synthesized and characterized by FTIR, H, & 13C NMR
spectroscopy. The derivatives were then evaluated for their potential to inhibit 15-LOX and
bovine carbonic anhydrase II (bCA II). Most of these compounds showed excellent inhibitory
potential for 15-LOX with an IC50 of 0.12±0.002 to 0.69±0.5 µM and showed moderate
inhibition potency for bCA II with compound 5h (IC50=1.26±0.24 µM) being the most active.
The most potent compound 5a that emerged as a dual inhibitor of both enzymes, exhibiting 24
times greater selectivity for 15-LOX over bCA II. Compound 5a exhibited dual potent inhibitory
activity against both 15-LOX and bCA II enzymes having IC50 values of 0.12±0.002 and
2.93±0.22 µM, respectively. Molecular docking studies of potent as well as dual inhibitors were
also carried out to provide an insight into the binding site interactions.
Keywords: Thiazoles, 15-Lipoxygenase, Bovine carbonic anhydrase II, Docking studies.
Correspondence: Prof. Dr. Jamshed Iqbal (Tel.: +92-992-383591-6; Fax: +92-992-383441);
drjamshed@ciit.net.pk; aamersaeed@yahoo.com (Aamer Saeed)

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1. Introduction
Heterocyclic moieties, such as pyrazoles, piperazines, thiazolidine-4-ones, indoles, pyridines,
etc., always have significant role due to their therapeutic effects [1,2]. The heterocyclic
compounds have wide-range applications comprising the treatment of allergies, bacterial and
HIV infections, hypertension, ligands for estrogen receptors and as antibiotics [3,4].
Additionally, these compounds have an inhibitory effects against various enzymes such as
carbonic anhydrase isoforms [5] fructose 1,6-bisphosphatase [6], sphingosine kinase [7] and also
have antitumor activity [8]. Nitrogen and sulfur containing small-ring heterocyclic compounds
have been explored due to their synthetic nature and therapeutic applications. Among the broad
range of heterocyclic compounds explored in drug discovery, thiazoles have been recognized to
play a crucial role in medicinal chemistry [8]. Thiazoles are common heterocyclic core in natural
products and synthetic scaffolds. Among different natural products, the substances containing the
thiazole functionality are becoming attractive targets for medicine and pharmaceutical industries
due to their broad spectrum of biological activities [8]. For many years, the clinician has treated
patients with combination of compounds having different pharmacotherapeutic activities. It is
also known that balanced modulation of many targets can show a higher therapeutic effect and
lesser side effect as compared to the action of a selective ligand. By comparison to a combination
of drugs, there are several advantages linked to compounds acting on more than one target as
dual inhibitors [9].
Lipoxygenases (LOXs) are non-heme iron containing proteins that catalyze the incorporation of
dioxygen into 1,4-cis-pentadiene containing polyunsaturated fatty acids (e.g., arachidonic acid
and linoleic acid) to generate the hydroperxide derivatives. On the basis of regioselectivity,
LOXs can be divided into four isoforms i.e., 5, 8, 12 and 15-LOX. Each isoform oxidizes the
polyunsaturated fatty acids at respective position i.e., 5, 8, 12 and 15 resulting in the production
of corresponding hydroperoxyeicosatetraenoic acid (HPETE), which is further reduced to
hydroxyeicosatetraenoic acid (HETE) by the action of glutathione reductase [10,11]. These
metabolites act as signaling molecules and perform various functions of physiological
significance like cell growth, monocyte binding and modulation of different inflammatory
diseases [12]. Among mammals, human 15-LOX is an essential isoform that is found to be
present in eosinophils, reticulocytes, macrophages and airway epithelia cell [11]. It has unfolded

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itself as an important target in therapeutic interventions as it has been associated with the
development of chronic obstructive pulmonary disease and certain cancers [13]. Moreover it has
also manifested itself as an indispensible biological player in numerous diseases such as diabetes,
atherogenesis, Alzhiemer’s disease, stroke and several types of cancer including breast, colon,
pancreas and prostate cancer [10, 14] etc. Thus inhibitors of LOX, that have earned much
recognition as potential agents for the treatment of inflammatory diseases, are now expanding
their spheres as prospective agents for the treatment of various other diseases including different
types of cancers [15]. Few previously reported inhibitors of 15-LOX are given below.
S
F
N N
O
O
S
N
H
O
6,11-dihydrothiochromeno[4,3-b]indole
((5-(naphthalen-1-yl)-1,3,4-oxadiazol-2-yl)thio)ethynyl 4-
fluorobenzoate
O
O S
O
Ph
R1
CN
H
N
N
N
S
R2
Ph
X
7-isocyano-2,3-diphenyl-1,2,3,8a-tetrahydroindolizin-1-yl4-
methylbenzenesulfonate
N-aryl-4-aryl-1,3-thiazole-2-amines
Another enzyme, carbonic anhydrases (CAs, EC 4.2.1.1) constitute a family of metalloenzymes
that are ubiquitously distributed in nature and are responsible for catalyzing the reversible
interconversion between carbon dioxide and bicarbonate. They are encoded by six different gene
families and thus divided into six different classes i.e. α, β, γ δ, ε and η. Amongst them, α class
is medically most important and requires zinc for its activity. In mammals, at least 16 isozymes
of α class have been identified that vary in their tissue distribution and subcellular localization.
Some of them exist in cytosolic forms (CA I-III, VII and XIII) whereas others are found to be
membrane bound (CA IV, IX, XII, XIV and XV). Two of them are mitochondrial i.e. CA VA
and VB, while VI is a secreted form [16, 17]. They are involved in a variety of biosynthetic
reactions like gluconeogenesis, lipogenesis and ureagenesis as well as in numerous physiological
processes including respiration, secretion of electrolytes and pH regulation [18, 19]. In this
regard, their key function is to maintain acid base balance, which is of particular importance for

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the normal growth of cell. Any aberration in this balance could result in the development of
tumor. Numerous studies have indicated the presence of different isoforms of CAs (CA II, IX
and XII) in many tumors. For example, CA II is expressed in pancreatic and gastric carcinomas
as well as in brain tumors of malignant type, although this isoform has also been implicated in
many other diseases like epilepsy, edema, glaucoma and altitude sickness [19, 20]. Interestingly
various studies have demonstrated that manipulation of CA inhibitors such as ethoxzolamide and
methazolamide significantly inhibited the development of cancer cells. Similarly acetazolamide
an effective inhibitor of CAs that significantly hampered the invasion capacity in renal cancer
cell lines [21]. Hence CAs also comprises an attractive target for novel approaches while
designing the anticancer therapies.
Keeping in view the importance of thiazole moieties and our targeted enzymes, the novel
substituted (E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-carboxylates derivatives were
designed and synthesized. These compounds were evaluated for inhibitory potential against 15-
LOX and bCA II and docking studies of the potent as well as dual inhibitors were performed to
investigate the binding site interactions.
2. Materials and methods
2.1. General procedure for synthesis
All commercial products were purchased from Sigma-Aldrich. Melting points were determined
in open capillary tubes on a Stuart melting point apparatus. The IR spectra were run in the single
1
13
beam Nicolet IR 100 (Fourier-Transform). The HNMR and CNMR spectra were recorded in
DMSO-d6 using NMR Bruker DPX 400 spectrophotometer operating at 300 MHz.
Tetramethylsilane was used as internal standard with the deuterium signal of the solvent as the
lock and chemical shifts were recorded in ppm. All crude products were isolated as solid and
purified by recrystallization (Details are provided in supporting information).
2.2. Biochemical Assays
2.2.1. 15-Lipoxygenase Inhibition Assay
Lipoxygenase inhibition studies were performed by doing slight modifications in previously
reported spectrophotometric method [22]. Briefly, 10 µL of 15-LOX (Soybean source, 42.5 U)
was mixed with 20 µL compound (0.1 mM in 1% DMSO) in 145 µL of KH2PO4 buffer (100

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mM, pH 8.0). The reaction mixture was incubated at 25 °C for 10 min and absorbance was
measured at 234 nm by using micro plate reader (FLUOstar Omega (BMG Labtech, Offenburg,
Germany). Then the reaction was started by the addition of 25 µL of linoleic acid as substrate,
followed by incubation for 6 min and then change in absorbance was measured. Quercetin was
used as a positive control. All the experiments were performed in triplicates. The compounds
exhibiting >50% inhibition were subjected to further evaluation of IC50 values by non-linear
regression analysis software PRISM 5 (GraphPad, San Diego, California, USA).
2.2.2. Carbonic anhydrase II Inhibition Assay
Carbonic anhydrase (bCA II) was purchased from Sigma-Aldrich, USA and the inhibition
activity was determined according to the already reported methods [23, 24]. This method
involved the spectrophotometric determination of p-nitrophenol that was produced from a CA
catalyzed hydrolysis of p-nitrophenyl acetate substrate. Shortly total reaction volume of 100 µL
containing 60 µL of tris-sulfate buffer (50 mM, pH 7.6, 0.1 mM ZnCl2), 10 µL test compound
(0.5 mM) and 10 µL bCA II (50 U) was run by incubating at 25 °C for 10 min. Then absorbance
was measured at 348 nm using a 96 well plate reader. After that 20 µL of substrate i.e., p-
nitrophenyl acetate (6 mM), was added to each well to initiate the reaction and incubated further
for 30 min at 25 °C, followed by absorbance at 348 nm. The reported results were mean of three
independent experiments (± SEM) and expressed as percent inhibition. IC50 values of selected
compounds exhibiting > 50% inhibitory activity were calculated after suitable dilutions with the
help of non-linear regression analysis program of GraphPad Prism 5 Software Inc., San Diego,
California, USA.
2.2.3. Molecular Docking
Molecular docking studies of the most potent as well as dual inhibitors of CA II and 15-
Lipoxygenase enzymes were performed using AutoDock 4.2 to predict the binding mode inside
the active site of target enzymes. Crystal structures of both target enzymes were downloaded
from RCSB protein data bank. PDB ID: 1V9E for docking of bCA II [25] while, PDB ID: 1IK3
for soybean lipoxygenase docking [26] were retrieved as target enzyme structures. The 3D
structures of ligands were generated using ACD/ChemSketch [27]. Gasteiger charges were added
to both ligand and enzymes structure using ANTECHAMBER [28] and energy minimization of

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ligands were done through 100 steepest descents and 100 conjugate gradient steps using a step
size of 0.02. Prior to docking, preparation of both targets were carried out with DockPrep utility
of Chimera [29]. AutoDock Tool (ADT) was used for generation of appropriate charged files of
both targets as well as ligands [30]. AutoGrid was used for the calculation of affinity and map,
allowing ligands to be flexible. Both enzymes were held rigid during molecular docking studies.
Grid box having dimensions of 60 x 60 x 60 in xyz direction within 0.375 Å was kept center at
the active site of both receptors enzyme and the size of grid box was big enough to allow free
movement of ligands within active site of enzyme. Lamarckian Genetic Algorithm (LGA) was
used as searching parameter for docking of ligands in AutoDock4. After successful completion
of molecular docking steps, Discovery Studio Visualizer 4.1 was used for analyzing the
predicted and putative binding interaction of potent and dual compounds [31].
3. Results and discussion
3.1. General procedure for the synthesis of benzyledinyl-hydrazinyl substituted thiazoles
(3a-j)
Suitably substituted aldehydes (1a-e) or ketones (1e-j) dissolved in ethanol were condensed with
thiosemicarbazide (2) leading to the formation of thiosemicarbazones (3a-j) as intermediates.
Ethanolic solutions of these intermediates were then refluxed with α-haloketones (4) to afford the
benzyledinyl-hydrazinyl substituted thiazoles (5a-j) in good to excellent (71-79%) yields.

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Scheme 1. Synthesis of substituted (E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-
carboxylates
3.2. In vitro 15-Lipoxygenase and carbonic anhydrase II inhibition studies and structure
activity relationship (SAR)
All the synthesized compounds were evaluated for their potential to inhibit 15-LOX and CA II.
Three out of ten compounds i.e. 5a, 5b and 5e showed the phenomenon of dual inhibition.
Among 15-LOX inhibitors, six derivatives exhibited remarkable inhibitory potential with IC50 in
the range of 0.12±0.002 to 0.69±0.02 µM. The highest inhibition was revealed by the parent
compound i.e., 5a exhibiting an IC50 value (0.12±0.002 µM) that is ~ 132 fold more as compared
to reference compound quercetin (IC50=15.8±0.6 µM). Introduction of substitution at different
positions of benzene ring was well tolerated. Among substituted derivatives, most potent
inhibitory potential was shown by the compound 5b, followed by the compounds 5j, 5i and 5c.

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The reason behind the remarkable inhibition potency of 5b (IC50=0.21±0.03 µM) might be the
presence of electron withdrawing group i.e., NO2 at meta position. Likewise, substitution of
benzene ring with electron donating groups (methoxy and hydroxyl) in compounds 5i and 5j
resulted in the retention of considerable inhibitory potential with an IC50 value of 0.36±0.04 and
0.32±0.07 µM, respectively.
In case of bCA II inhibition, the compound 5h appeared to be the most potent and selective
inhibitor with an IC50 value of 1.26±0.14 µM. This potency of 5h could be attributed to the
presence of two halogen atoms i.e., F and Cl at ortho positions, which activate the thiourea
nitrogen by exerting negative inductive effect. A general overview of results helps us to deduce
that the presence of electron donating groups resulted in higher IC50 values than that of electron
withdrawing group. It can be exemplified by comparing the compounds 5b and 5e sharing the
same chemical structure except the presence of benzyloxy group (electron donating group) in 5e.
The inclusion of this electron donating group in 5e resulted in enhanced IC50 value (2.26±0.13
µM) which is almost 4 fold higher than 5b. Although methyl group is electron donating yet
rendered the compound 5c inactive that could be ascribed to the presence of steric hindrance.
Furthermore intermediate IC50 values were also shown by the compounds 5g and 5a i.e.,
1.89±0.14 and 2.93±0.22 µM, respectively. An interesting observation could be made from this
results that presence of pi electrons in derivatives exhibited high inhibitory activities against both
target enzymes i.e., compound 5f have no pi electron, therefore, didn’t show any inhibitory
activity towards any enzyme. High affinity and specificity of these derivatives toward either
target is briefly discussed in molecular docking portion.
Table 1. 15-Lipoxygenase and carbonic anhydrase II inhibition in presence of the synthesized
compounds
Code
15-LOX
CA II
IC50 ±SEM (µM)
0.12±0.002
0.21±0.06
0.53±0.19
2.93±0.22
8.55±0.17
-
4.39±0.10
2.26±0.13
-
5a
5b
5c
5d
5e
5f
-
0.69±0.01
-
-
-
1.89±0.14
1.26±0.14
5g
5h

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0.36±0.04
0.32±0.07
15.8±0.61
-
-
-
-
5i
5j
Quercetin
Acetazolamide
0.96 ± 0.18
IC50 = Concentration at which 50% of the enzyme activity is inhibited. 15-LOX and CA II
activities were performed at the final concentration of 0.1 mM and 0.5 mM, respectively.
“-” the symbol indicated that the compounds showed less than 50% inhibition when tested at 0.1
mM and 0.5 mM against 15-LOX and CA II, respectively.
3.3. Molecular docking studies against 15-LOX and CA II
Molecular docking studies were performed to predict the binding interactions of compound 5a
(most potent dual inhibitor of 15-LOX and bCA II). Three-dimensional predicted binding
interaction of compound 5a in active site of 15-LOX is illustrated in Figure 1. Amino acid
residue of 15-LOX surrounding the ligand mainly consist of Val372, Ser510, His513, Gln514,
Ile572, Phe576, Gln716, Gly720, Asp766 and Ile770. Critical analysis of docking results
predicted three key interactions i.e., two hydrogen bonds and one pi-pi T-shaped interactions
were formed with Ser510, His513 and Phe576 amino acid residues. Nitrogen in thiazole moiety
of ligand was involved in making one hydrogen bond with Ser510, whereas oxygen adjacent to
carbonyl group formed the second hydrogen bond with residue of His518 amino acid. In addition
to the above mentioned hydrogen bond, thiazole moiety also formed pi-pi T-shaped interactions
with residue of Phe576 amino acid in the active site of target enzyme.
Figure 1. Putative binding mode of 5a (most active and dual inhibitor, gold colored) in the active
site pocket of 15-lipoxygenase (amino acid residue in ice blue color)

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Molecular docking studies of 5h (most active bCA II inhibitor and 5a as dual inhibitor (bCA II
and 15-LOX) were also carried out to investigate putative interactions in the active site of
enzymes. Putative binding modes of 5h and 5a are shown in Figure 2, which presented broad
network of different binding interactions with side chains of several amino acids. Active site of
bCA II mainly consisted of amino acid residue such as Gln91, His63, Gln91, His93, His118,
Val120, Leu196, Thr197 and Thr198. Docking studies of compounds 5h and 5a revealed that
key interactions were noticed with His63, His93 and Thr197 amino acid residues. The carbonyl
group of both ligands formed hydrogen bonds with amino acid Thr197. Thiazole moiety of the
ligand was involved in pi-pi interaction with His93 amino acid residue. Nitrogen of hydrazine
moiety in compound 5h was making hydrogen bonding with His63 amino acid, However no
such bonding interactions was observed in hydrazine moiety of compound 5a and any amino
acid residue. It might be due to absence of strong electron withdrawing moiety like flurobenzene
adjacent to hydrazine moiety in compound 5h as shown in Figure 2. High inhibitory activity and
affinity of compound 5a inside the active site of 15-LOX was due to many strong hydrophobic
interaction (two hydrogen bond and two pi-pi interactions) as compared to few binding
interactions (one hydrogen and one pi-pi interaction) inside the active site of bCA II as depicted
in Figure 1 and 2. Potential dual inhibition of 5a derivatives was due to the presence of same
hydrophobic amino acid residue interacting with compounds inside the active site of both target
enzymes.
Figure 2. Putative binding mode of 5a (at left side dual potent inhibitor of bCA II and 15-LOX,
light black colored ), 5h (at right side most active inhibitor of bCA II, violet colored) in the
active site pocket of bCA II (ice blue colored)

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Acknowledgements
J. Iqbal is thankful to the Organization for the Prohibition of Chemical Weapons (OPCW), The
Hague, The Netherlands and Higher Education Commission of Pakistan for the financial support
through Project No. 20-3733/NRPU/R&D/ 14/520 for the financial support."
Conflict of interest
The authors confirm that this article content has no conflict of interest.
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