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Biochemical and Biophysical Research Communications xxx (2016) 1e6

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Biochemical and Biophysical Research Communications


journal homepage: www.elsevier.com/locate/ybbrc

Substituted (E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-
carboxylates as dual inhibitors of 15-lipoxygenase & carbonic
anhydrase II: synthesis, biochemical evaluation and docking studies
Aamer Saeed a, **, Sha Ullah Khan b, Parvez Ali Mahesar a, Pervaiz Ali Channar a,
Ghulam Shabir a, Jamshed Iqbal b, *
a
Department of Chemistry, Quaid-i-Azam University, 45320, Islamabad, Pakistan
b
Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad, 22060, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: 15-Lipoxygenase (15-LOX) plays a major role in many inammatory lung diseases including chronic
Received 1 November 2016 obstructive pulmonary disease (COPD), asthma and chronic bronchitis. Over-expression of 15-LOX is
Accepted 6 November 2016 related with some specic carcinomas including pancreatic, gastric and brain tumor. Similarly among
Available online xxx
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
Keywords:
role of these enzymes further and to enable the drug discovery efforts. For this purpose, a series of
Thiazoles
benzyledinyl-hydrazinyl substituted thiazole derivatives were designed, synthesized and characterized
15-Lipoxygenase
Bovine carbonic anhydrase II
by FTIR, 1H, & 13C NMR spectroscopy. The derivatives were then evaluated for their potential to inhibit 15-
Docking studies 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 mM and showed moderate inhibition
potency for bCA II with compound 5h (IC50 1.26 0.24 mM) 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 mM, respectively. Molecular docking
studies of potent as well as dual inhibitors were also carried out to provide an insight into the binding
site interactions.
2016 Elsevier Inc. All rights reserved.

1. Introduction antitumor activity [8]. Nitrogen and sulfur containing small-ring


heterocyclic compounds have been explored due to their syn-
Heterocyclic moieties, such as pyrazoles, piperazines, thiazoli- thetic nature and therapeutic applications. Among the broad range
dine-4-ones, indoles, pyridines, etc., have always played a signi- of heterocyclic compounds explored in drug discovery, thiazoles
cant role due to their therapeutic effects [1,2]. The heterocyclic have been recognized to play a crucial role in medicinal chemistry
compounds have wide-range applications comprising the treat- [8]. Thiazole is a common heterocyclic core found in natural
ment of allergies, bacterial and HIV infections, hypertension, li- products and synthetic scaffolds. Among different natural products,
gands for estrogen receptors and as antibiotics [3,4]. Additionally, the substances containing the thiazole functionality are becoming
these compounds have an inhibitory effects against various en- attractive targets for medicine and pharmaceutical industries due
zymes such as carbonic anhydrase isoforms [5] fructose 1,6- to their broad spectrum of biological activities [8]. For many years,
bisphosphatase [6], sphingosine kinase [7] and also have 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
* Corresponding author.
a selective ligand. By comparison to a combination of drugs, there
** Corresponding author.
E-mail addresses: aamersaeed@yahoo.com (A. Saeed), drjamshed@ciit.net.pk are several advantages linked to compounds acting on more than
(J. Iqbal). one target as dual inhibitors [9].

http://dx.doi.org/10.1016/j.bbrc.2016.11.028
0006-291X/ 2016 Elsevier Inc. All rights reserved.

Please cite this article in press as: A. Saeed, et al., Substituted (E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-carboxylates as dual
inhibitors of 15-lipoxygenase & carbonic anhydrase II: synthesis, biochemical evaluation and docking studies, Biochemical and Biophysical
Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.028
2 A. Saeed et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6

Lipoxygenases (LOXs) are non-heme iron containing proteins electrolytes and pH regulation [18,19]. In this regard, their key
that catalyze the incorporation of dioxygen into 1,4-cis-pentadiene function is to maintain acid base balance, which is of particular
containing polyunsaturated fatty acids (e.g., arachidonic acid and importance for the normal growth of cell. Any aberration in this
linoleic acid) to generate the hydroperoxide derivatives. On the balance could result in the development of tumor. Numerous
basis of regioselectivity, LOXs can be divided into four isoforms i.e., studies have indicated the presence of different isoforms of CAs (CA
5, 8, 12 and 15-LOX. Each isoform oxidizes the polyunsaturated fatty II, IX and XII) in many tumors. For example, CA II is expressed in
acids at respective position i.e., 5, 8, 12 and 15 resulting in the pancreatic and gastric carcinomas as well as in brain tumors of
production of corresponding hydroperoxyeicosatetraenoic acid malignant type, although this isoform has also been implicated in
(HPETE), which is further reduced to hydroxyeicosatetraenoic acid many other diseases like epilepsy, edema, glaucoma and altitude
(HETE) by the action of glutathione reductase [10,11]. These me- sickness [19,20]. Interestingly various studies have demonstrated
tabolites act as signaling molecules and perform various functions that manipulation of CA inhibitors such as ethoxzolamide and
of physiological signicance like cell growth, monocyte binding and methazolamide signicantly inhibited the development of cancer
modulation of different inammatory diseases [12]. Among mam- cells. Similarly acetazolamide an effective inhibitor of CAs that
mals, human 15-LOX is an essential isoform that is found to be signicantly hampered the invasion capacity in renal cancer cell
present in eosinophils, reticulocytes, macrophages and airway lines [21]. Hence CAs also comprises an attractive target for novel
epithelia cell [11]. It has unfolded itself as an important target in approaches while designing the anticancer therapies.
therapeutic interventions as it has been associated with the Keeping in view the importance of thiazole moieties and our
development of chronic obstructive pulmonary disease and certain targeted enzymes, the novel substituted (E)-2-(2-
cancers [13]. Moreover it has also manifested itself as an indis- benzylidenehydrazinyl)-4-methylthiazole-5-carboxylates de-
pensible biological player in numerous diseases such as diabetes, rivatives were designed and synthesized. These compounds were
atherogenesis, Alzhiemer's disease, stroke and several types of evaluated for inhibitory potential against 15-LOX and bCA II and
cancer including breast, colon, pancreas and prostate cancer [10,14] docking studies of the potent as well as dual inhibitors were per-
etc. Thus inhibitors of LOX, that have earned much recognition as formed to investigate the binding site interactions.
potential agents for the treatment of inammatory diseases, are
now expanding their spheres as prospective agents for the treat- 2. Materials and methods
ment of various other diseases including different types of cancers
[15]. Few previously reported inhibitors of 15-LOX are given below. 2.1. General procedure for synthesis

All commercial products were purchased from Sigma-Aldrich.

Another enzyme, carbonic anhydrases (CAs, EC 4.2.1.1) consti- Melting points were determined in open capillary tubes on a Stu-
tute a family of metalloenzymes that are ubiquitously distributed in art melting point apparatus. The IR spectra were run in the single
nature and are responsible for catalyzing the reversible intercon- beam Nicolet IR 100 (Fourier-Transform). The 1HNMR and 13CNMR
version between carbon dioxide and bicarbonate. They are encoded spectra were recorded in DMSO-d6 using NMR Bruker DPX 400
by six different gene families and thus divided into six different spectrophotometer operating at 300 MHz. Tetramethylsilane was
classes i.e. a, b, g d, and h. Amongst them, a class is medically most used as internal standard with the deuterium signal of the solvent
important and requires zinc for its activity. In mammals, at least 16 as the lock and chemical shifts were recorded in ppm. All crude
isozymes of a class have been identied that vary in their tissue products were isolated as solid and puried by recrystallization
distribution and subcellular localization. Some of them exist in (Details are provided in supporting information).
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]. 2.2. Biochemical assays
They are involved in a variety of biosynthetic reactions like gluco-
neogenesis, lipogenesis and ureagenesis as well as in numerous 2.2.1. 15-Lipoxygenase inhibition assay
physiological processes including respiration, secretion of Lipoxygenase inhibition studies were performed by doing slight
modications in previously reported spectrophotometric method

Please cite this article in press as: A. Saeed, et al., Substituted (E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-carboxylates as dual
inhibitors of 15-lipoxygenase & carbonic anhydrase II: synthesis, biochemical evaluation and docking studies, Biochemical and Biophysical
Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.028
A. Saeed et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6 3

Scheme 1. Synthesis of substituted (E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-carboxylates.

[22]. Briey, 10 mL of 15-LOX (Soybean source, 42.5 U) was mixed inhibition. IC50 values of selected compounds exhibiting >50%
with 20 mL compound (0.1 mM in 1% DMSO) in 145 mL of KH2PO4 inhibitory activity were calculated after suitable dilutions with the
buffer (100 mM, pH 8.0). The reaction mixture was incubated at help of non-linear regression analysis program of GraphPad Prism 5
25  C for 10 min and absorbance was measured at 234 nm by using Software Inc., San Diego, California, USA.
micro plate reader (FLUOstar Omega (BMG Labtech, Offenburg,
Germany). Then the reaction was started by the addition of 25 mL of
linoleic acid as substrate, followed by incubation for 6 min and then 2.2.3. Molecular docking
change in absorbance was measured. Quercetin was used as a Molecular docking studies of the most potent as well as dual
positive control. All the experiments were performed in triplicates. inhibitors of CA II and 15-Lipoxygenase enzymes were performed
The compounds exhibiting >50% inhibition were subjected to using AutoDock 4.2 to predict the binding mode inside the active
further evaluation of IC50 values by non-linear regression analysis site of target enzymes. Crystal structures of both target enzymes
software PRISM 5 (GraphPad, San Diego, California, USA). 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
2.2.2. Carbonic anhydrase II inhibition assay structures of ligands were generated using ACD/ChemSketch [27].
Carbonic anhydrase (bCA II) was purchased from Sigma-Aldrich, Gasteiger charges were added to both ligand and enzymes struc-
USA and the inhibition activity was determined according to the ture using ANTECHAMBER [28] and energy minimization of ligands
already reported methods [23,24]. This method involved the were done through 100 steepest descents and 100 conjugate
spectrophotometric determination of p-nitrophenol that was pro- gradient steps using a step size of 0.02. Prior to docking, prepara-
duced from a CA catalyzed hydrolysis of p-nitrophenyl acetate tion of both targets were carried out with DockPrep utility of
substrate. Shortly total reaction volume of 100 mL containing 60 mL Chimera [29]. AutoDock Tool (ADT) was used for generation of
of tris-sulfate buffer (50 mM, pH 7.6, 0.1 mM ZnCl2), 10 mL test appropriate charged les of both targets as well as ligands [30].
compound (0.5 mM) and 10 mL bCA II (50 U) was run by incubating AutoGrid was used for the calculation of afnity and map, allowing
at 25  C for 10 min. Then absorbance was measured at 348 nm ligands to be exible. Both enzymes were held rigid during mo-
using a 96 well plate reader. After that 20 mL of substrate i.e., p- lecular docking studies. Grid box having dimensions of 60  60 x 60
nitrophenyl acetate (6 mM), was added to each well to initiate the in xyz direction within 0.375 was kept center at the active site of
reaction and incubated further for 30 min at 25  C, followed by both receptors enzyme and the size of grid box was big enough to
absorbance at 348 nm. The reported results were mean of three allow free movement of ligands within active site of enzyme. La-
independent experiments (SEM) and expressed as percent marckian Genetic Algorithm (LGA) was used as searching

Please cite this article in press as: A. Saeed, et al., Substituted (E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-carboxylates as dual
inhibitors of 15-lipoxygenase & carbonic anhydrase II: synthesis, biochemical evaluation and docking studies, Biochemical and Biophysical
Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.028
4 A. Saeed et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6

parameter for docking of ligands in AutoDock4. After successful Table 1


completion of molecular docking steps, Discovery Studio Visualizer 15-Lipoxygenase and carbonic anhydrase II inhibition in presence of the synthesized
4.1 was used for analyzing the predicted and putative binding compounds.

interaction of potent and dual compounds [31]. Code 15-LOX bCA II

IC50 SEM (mM)


3. Results and discussion 5a 0.12 0.002 2.93 0.22
5b 0.21 0.06 8.55 0.17
3.1. General procedure for the synthesis of benzyledinyl-hydrazinyl 5c 0.53 0.19 e
substituted thiazoles (3a-j) 5d e 4.39 0.10
5e 0.69 0.01 2.26 0.13
5f e e
Suitably substituted aldehydes (1a-e) or ketones (1e-j) dissolved 5g e 1.89 0.14
in ethanol were condensed with thiosemicarbazide (2) leading to 5h e 1.26 0.14
the formation of thiosemicarbazones (3a-j) as intermediates. The 5i 0.36 0.04 e
5j 0.32 0.07 e
ethanolic solutions of the latter were reuxed with ethyl alpha-
Quercetin 15.8 0.61 e
chloro acetoacetate to afford the benzyledinyl-hydrazinyl Acetazolamide e 0.96 0.18
substituted thiazoles (5a-j) in good to excellent (71e79%) yields
IC50 Concentration at which 50% of the enzyme activity is inhibited. 15-LOX and
as shown in scheme 1. CA II activities were performed at the nal concentration of 0.1 mM and 0.5 mM,
respectively.
e the symbol indicated that the compounds showed less than 50% inhibition
3.2. In vitro 15-lipoxygenase and carbonic anhydrase II inhibition
when tested at 0.1 mM and 0.5 mM against 15-LOX and CA II, respectively.
studies and structure activity relationship (SAR)

All the synthesized compounds were evaluated for their po-


tential to inhibit 15-LOX and CA II and inhibitory activities are 1.26 0.14 mM. This potency of 5h could be attributed to the
tabulated in Table 1. Three out of ten compounds i.e. 5a, 5b and 5e presence of two halogen atoms i.e., F and Cl at ortho positions,
showed the phenomenon of dual inhibition. Among 15-LOX in- which activate the thiourea nitrogen by exerting negative inductive
hibitors, six derivatives exhibited remarkable inhibitory potential effect. A general overview of results helps us to deduce that the
with IC50 in the range of 0.12 0.002 to 0.69 0.02 mM. The highest presence of electron donating groups resulted in higher IC50 values
inhibition was revealed by the parent compound i.e., 5a exhibiting than that of electron withdrawing group. It can be exemplied by
an IC50 value (0.12 0.002 mM) that is ~132 fold more as compared comparing the compounds 5b and 5e sharing the same chemical
to reference compound quercetin (IC50 15.8 0.6 mM). Intro- structure except the presence of benzyloxy group (electron
duction of substitution at different positions of benzene ring was donating group) in 5e. The inclusion of this electron donating group
well tolerated. Among substituted derivatives, most potent inhibi- in 5e resulted in enhanced IC50 value (2.26 0.13 mM) which is
tory potential was shown by the compound 5b, followed by the almost 4 fold higher than 5b. Although methyl group is electron
compounds 5j, 5i and 5c. The reason behind the remarkable inhi- donating yet rendered the compound 5c inactive that could be
bition potency of 5b (IC50 0.21 0.03 mM) might be the presence ascribed to the presence of steric hindrance. Furthermore inter-
of electron withdrawing group i.e., NO2 at meta position. Likewise, mediate IC50 values were also shown by the compounds 5g and 5a
substitution of benzene ring with electron donating groups i.e., 1.89 0.14 and 2.93 0.22 mM, respectively. An interesting
(methoxy and hydroxyl) in compounds 5i and 5j resulted in the observation could be made from this results that presence of pi
retention of considerable inhibitory potential with an IC50 value of electrons in derivatives exhibited high inhibitory activities against
0.36 0.04 and 0.32 0.07 mM, respectively. both target enzymes i.e., compound 5f have no pi electron, there-
In case of bCA II inhibition, the compound 5h appeared to be the fore, didn't show any inhibitory activity towards any enzyme. High
most potent and selective inhibitor with an IC50 value of afnity and specicity of these derivatives toward either target is

Fig. 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). (For inter-
pretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: A. Saeed, et al., Substituted (E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-carboxylates as dual
inhibitors of 15-lipoxygenase & carbonic anhydrase II: synthesis, biochemical evaluation and docking studies, Biochemical and Biophysical
Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.028
A. Saeed et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6 5

Fig. 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). (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

briey discussed in molecular docking portion. Conict of interest

3.3. Molecular docking studies on 15-LOX and CA II The authors conrm that this article content has no conict of
interest.
Molecular docking studies were performed to predict the
binding interactions of compound 5a (most potent dual inhibitor of Acknowledgements
15-LOX and bCA II). Three-dimensional predicted binding interac-
tion of compound 5a in active site of 15-LOX is illustrated in Fig. 1. J. Iqbal is thankful to the Organization for the Prohibition of
Amino acid residue of 15-LOX surrounding the ligand mainly Chemical Weapons (OPCW), The Hague, The Netherlands and
consist of Val372, Ser510, His513, Gln514, Ile572, Phe576, Gln716, Higher Education Commission of Pakistan for the nancial support
Gly720, Asp766 and Ile770. Critical analysis of docking results through Project No. 20-3733/NRPU/R&D/14/520 for the nancial
predicted three key interactions i.e., two hydrogen bonds and one support.
pi-pi T-shaped interactions were formed with Ser510, His513 and
Phe576 amino acid residues. Nitrogen in thiazole moiety of ligand Appendix A. Supplementary data
was involved in making one hydrogen bond with Ser510, whereas
oxygen adjacent to carbonyl group formed the second hydrogen Supplementary data related to this article can be found at http://
bond with residue of His518 amino acid. In addition to the above dx.doi.org/10.1016/j.bbrc.2016.11.028.
mentioned hydrogen bond, thiazole moiety also formed pi-pi T-
shaped interactions with residue of Phe576 amino acid in the active References
site of target enzyme.
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Please cite this article in press as: A. Saeed, et al., Substituted (E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-carboxylates as dual
inhibitors of 15-lipoxygenase & carbonic anhydrase II: synthesis, biochemical evaluation and docking studies, Biochemical and Biophysical
Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.028
6 A. Saeed et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6

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Please cite this article in press as: A. Saeed, et al., Substituted (E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-carboxylates as dual
inhibitors of 15-lipoxygenase & carbonic anhydrase II: synthesis, biochemical evaluation and docking studies, Biochemical and Biophysical
Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.028