Design, synthesis and biological evaluation of novel pyridine-thiazolidinone derivatives as anticancer agents: Targeting human carbonic anhydrase IX

Article abstract of DOI:10.1016/j.ejmech.2017.12.049

In order to obtain novel Human carbonic anhydrase IX (CAIX) inhibitors, a series of pyridine-thiazolidinone derivatives was synthesized and characterized by various spectroscopic techniques. The binding affinity of the compounds was measured by fluorescence binding studies and enzyme inhibition activity using esterase assay of CAIX. It was observed that compound 8 and 11 significantly inhibit the CAIX activity with the IC₅₀ value, 1.61 μM and 1.84 μM, respectively. The binding-affinity of compound 8 and 11 for CAIX was significantly high with their K_D values 11.21 μM and 2.32 μM, respectively. Docking studies revealed that compound 8 and 11 efficiently binds in the active site cavity of CA IX by forming sufficient numbers of H-bonds and van der Waals interactions with active side residues. All the compounds were further screened in vitro for anticancer activity and found that compound 8 and 11 exhibit considerable anticancer activity against MCF-7 and HepG-2 cell lines. All these findings suggest that compound 8 and 11 may be further exploited as a novel pharmacophore model for the development of anticancer agents.

Full text of DOI:10.1016/j.ejmech.2017.12.049

Accepted Manuscript
Design, synthesis and biological evaluation of novel pyridine-thiazolidinone
derivatives as anticancer agents: Targeting human carbonic anhydrase IX
Fawad Ansari, Danish Idrees, Md Imtaiyaz Hassan, Kamal Ahmad, Fernando
Avecilla, Amir Azam
PII:
S0223-5234(17)31081-4
10.1016/j.ejmech.2017.12.049
EJMECH 10027
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 August 2017
Revised Date: 12 December 2017
Accepted Date: 13 December 2017
Please cite this article as: F. Ansari, D. Idrees, M.I. Hassan, K. Ahmad, F. Avecilla, A. Azam, Design,
synthesis and biological evaluation of novel pyridine-thiazolidinone derivatives as anticancer agents:
Targeting human carbonic anhydrase IX, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2017.12.049.
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ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of novel pyridine-thiazolidinone derivatives as anticancer
agents: Targeting human carbonic anhydrase IX
Fawad Ansari1, Danish Idrees2, Md. Imtaiyaz Hassan2, Kamal Ahmad2, Fernando Avecilla3, Amir Azam1,*
1Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, 110 025, New Delhi, India
2Centre for Interdisciplinary Research in Basic Science, Jamia Nagar, 110 025, New Delhi, India
3Grupo Xenomar, Centro de Investigacións Científicas Avanzadas (CICA), Departamento de Química,
Facultade de Ciencias, Universidade da Coruña, Campus de A Coruña, 15071 A Coruña, Spain*
Corresponding author. Tel.: +91 11 2698 1717/3253;
fax: +91 11 2698 0229/1232. E-mail address: amir_sumbul@yahoo.co.in (A. Azam)

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of novel pyridine-
thiazolidinone derivatives as anticancer agents: Targeting
human carbonic anhydrase IX
Mohammad Fawad Ansari1, Danish Idrees2, Md. Imtaiyaz Hassan2, Kamal Ahmad2, Fernando
Avecilla3, Amir Azam1,*
1Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, 110 025, New Delhi, India
2Centre for Interdisciplinary Research in Basic Science, Jamia Nagar, 110 025, New Delhi,
India
3Grupo Xenomar, Centro de Investigacións Científicas Avanzadas (CICA), Departamento de
Química, Facultade de Ciencias, Universidade da Coruña, Campus de A Coruña, 15071 A
Coruña, Spain
*To whom correspondence should be addressed: tel.: +91 11 2698 1717/3254; fax: +91 11
26980229; E-mail: amir_sumbul@yahoo.co.in
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Abstract
In order to obtain novel Human carbonic anhydrase IX (CAIX) inhibitors, a series of pyridine-
thiazolidinone derivatives was synthesised and characterized by various spectroscopic
techniques. The binding affinity of the compounds was measured by fluorescence binding
studies and enzyme inhibition activity using esterase assay of CAIX. It was observed that
compound 8 and 11 significantly inhibit the CAIX activity with the IC50 value, 1.61 µM and 1.84
µM, respectively. The binding-affinity of compound 8 and 11 for CAIX was significantly high
with their KD values 11.21 µM and 2.32 µM, respectively. Docking studies revealed that
compound 8 and 11 efficiently binds in the active site cavity of CA IX by forming sufficient
numbers of H-bonds and van der Waals interactions with active side residues. All the compounds
were further screened in vitro for anticancer activity and found that compound 8 and 11 exhibit
considerable anticancer activity against MCF-7 and HepG-2 cell lines. All these findings suggest
that compound 8 and 11 may be further exploited as a novel pharmacophore model for the
development of anticancer agents.
Key words: Pyridine, Thiazolidinone, Anticancer activity, Carbonic anhydrase.
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1. Introduction
Cancer is considered as the second major cause of the death after cardiovascular
disorders worldwide [1]. The inability to to distinguish between normal cell and cancerous cell
has been the main drawback of the present chemotherapeutic system [2]. In past two decades,
studies based on screening of the compounds for inhibition of tumour growth have been shifted
towards the identification of novel therapeutic targets [3]. Therefore, development of safe and
effective chemotherapeutic agents has been the major challenge to the medicinal chemists.
Currently, in the field of medicinal chemistry, five membered heterocyclic compounds
received the special concern, especially compounds having two hetero atom showed a diverse
range of biological activities [4-13]. Thiazolidinone is considered as indispensable anchor for
development of new therapeutic agents because this five membered magic moiety possesses all
types of biological activities [14-20]. The successful introduction of ralitoline as a potent
anticonvulsant, etozoline as an antihypertensive, pioglitazone as a hypoglycemic agent and
thiazolidomycin as antifungal agent proved the diverse range of biological activities associated
with thiazolidinone moiety (Figure 1) [21].
A variety of thiazolidinone based derivatives having different substituents around the
core nucleus are being considered as potential anticancer agents (Figure 2) [22-30]. Antitumor
activity of these compounds may be due to their affinity toward various biological targets, such
as JNK-stimulating phosphatase-1 (JSP-1) [31], phosphatase of a regenerating liver (PRL-3) [32-
33], non-membrane protein tyrosine phosphatase (SHP-2) [34], tumour necrosis factor TNFα
[35], antiapoptotic biocomplex Bcl-XL-BH3 [36], integrin avb3 [37], etc.
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Furthermore, pyridine nucleus a well-studied six-membered heterocyclic moiety also
displayed various biological activities, and is found in a variety of drugs such as isoniazid,
ethionamide, amrinone, bupicomide, sulphapyridine, etc. [38]. It is reported in the literature that
molecular hybridization of two or more biologically active pharmacophore into a single chemical
structure showed significant synergistic effects [39]. The combination of thiazolidinones with
other heterocyclic rings showed wide range of biological activities [40].
Recently, thiazolo- linked pyrimidine and thiazolidinone-oxoindoline base compounds
have been used as a potent CA inhibitor [41-42]. Hence, thiazolidinone coupled pyridine
molecules would be a promising scaffold for the development of potent CAIX inhibitors toward
the anticancer therapy. CAIX is an attractive target for cancer treatment because of its limited
expression in the normal tissues and predominant expression in varieties of tumour cells [43].
CAIX is a hypoxia-inducible protein that regulates cellular pH to encourage cancer cell survival
[44]. Moreover, it contributes to cancer progression by stimulating cancer cell migration,
adhesion and invasion [45]. Nowadays CAIX has been identified as an important biomarker of
hypoxia and its over-expression is often associated with a poor responsiveness to the classical
radio and chemotherapies [46].
The present study reports the synthesis, characterization, CAIX inhibition activity of
pyridine-thiazolidinone derivatives and their screening against breast and liver cancer cell lines
along with normal cell line.
2. Results and Discussion
2.1. Chemistry
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The synthetic route for the synthesis of the desired pyridine-thiazolidinone derivatives are shown
in the Scheme 1. 1-Phenyl-3-(pyridine-4-ylmethyl)thiourea 1 were synthesized by reacting
isothiocyanate with 4-picolylamine in toluene. 1-Phenyl-3-(pyridine-4-ylmethyl)thiourea 1 in
presence of anhydrous sodium acetate and monochloroacetic acid undergo cyclization to form
2(phenylimino)-3-(pyridine-4-ylmethyl)thiazolidin-4-one 2, which on further reaction with
different aldehyde derivatives in presence of piperidine, ethanol as solvent gave the final
1
products 3-20. The structure of synthesised compounds was confirmed by IR, H NMR, 13C
NMR and mass spectrometry. Elemental analysis justified the purity of the compounds.
2.1.1. Single crystal structure
2.1.2. Single crystal structures of 2, 3, 6 and 12
The compounds crystallize in centrosymmetric space groups, 2 in a triclinic system, 3, 6 and 12
in monoclinic system (Table 1). The asymmetric unit only contain one molecule of thiazolidin-
4-one derivatives in all cases. The different benzyl groups bonded to C(1) are almost flat with
thiazolindin-4-one group [C(1), C(2), C(3), S(1), N(1), C(16), C(17), C(18), C(19), C(20), C(21),
C(22), mean deviation from plane, 0.0927(10) Å in 3, 0.1358(10) Å in 6 and 0.1488(10) Å in 12,
which form dihedral angles with the phenyl groups of 76.18(3)º in 2, 65.62(3)º in 3, 57.61(3)º in
6 and 62.57(3)º in 12 and with the pyridine groups of 45.95(3)º in 2, 61.52(4)º in 3, 74.38(4)º in
6 and 59.42(5)º in 12 (Figure 3-6). (Bond lengths and angles are summarized in Table SI 1
Supplementary data).
X-ray structure analysis of 2, 3, 6 and 12 shows two enantiomers of the compounds in the
racemic crystals, the chirality being due to a twist of the phenyl and pyridine units. This
enantiomerization occurs by simultaneously rotating about the two single bonds, N(1)-C(10) and
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N(2)-C(4). The compounds present a mirror plane which contains the point of inversion (Figures
7-10) [47].
Depending on the substituents in the thiazolidinone rings, crystal structures adopt different
crystal packing (Figure 11). The crystal packing of the structures don´t present π-π stacking
interactions between the rings either hydrogen bonds as occur in other structures which contain
thiazolindinone rings with other substituents [48].
2.2. Esterase assay
The inhibition activity of all synthesized compounds 1-20 was tested against human
CAIX and CAII using the esterase activity, with 4-nitrophenyl acetate (4-NPA) as substrate. The
inhibition data of target compounds are shown in Table 2. The inhibitor acetazolamide (AZM)
was used as a standard to investigate the inhibitory activity of these compounds.
2.2.1. Structure Activity Relationship
Structure-activity relationship of pyridine-thiazolidinone derivatives 1-20 demonstrated
that compounds having unsubstituted phenyl ring 3 and alkyl group substitution on phenyl ring;
4 and 10 showed less inhibitory activity against CAIX having IC50 = 50.92 µM, 57.31 µM and
63.11 µM respectively. Alkoxy substituted compounds; 6, 13 and 14 also showed less activity
against CAIX with IC50 = 40.41 µM, 60.92 µM and 38.40 µM respectively. Compound 12 and
15 having disubstituted methoxy group displayed good inhibitory activity against CAIX (IC50 =
20.92 µM and 16.68 µM). Compound with chloro substituted; 7 and 17 were found to show
lesser activity against CA IX with IC50 = 40.18 µM and 43.52 µM respectively.
Compound with nitro substitution on benzene ring 8 showed excellent inhibitory activity
against CAIX with IC50 = 1.61 µM. SAR revealed that the compouds with hydroxyl groups had
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excellent impact on CAIX inhibition activity. Among the hydroxyl group containing compound
5, 11 and 16, compound 11 having di-hydroxyl group exhibited excellent inhibitory activity
against CA IX (IC50 = 1.84 µM). Further it was found that compounds having heterocyclic
substitution 18 (IC50 = 6.64 µM) and compound 19 (IC50 = 10.04 µM) displayed good binding
inhibition aginst CAIX. On the basis of SAR studies it can be concluded that the compounds
containing polar moieties 8 and 11 on phenyl ring were favourable for excellent inhibitory
activity against CAIX. In fact the inhibiton of CAIX by a paricular compound not depends only
on the nature of substituents but entire molecular skeleton is responsible for its activity.
To check the selectivity of compounds 8 and 11 against CAIX, these two compounds
were tested against human CAII using the esterase activity (Table 2). The compounds 8 and 11
have shown selectivity for CAIX over CAII approximately 9 times and 14 times respectively.
2.3. Fluorescence binding study
The fluorescence emission spectra CAIX with increasing concentrations of compounds 8
and 11 in Tris at 25 °C are shown in Figure 12 and 13. The emission maxima peak of CAIX at
341 nm (λexc=280 nm) is drastically reduced on increasing concentrations (1–100 µM) of
compounds, indicating a significant binding. The values fluorescence intensity at 341 nm was
plotted as a function of [ligand] to calculate the binding affinity of compounds against CAIX
(Table 2). The binding affinity of compound 8 for CAIX is 11.21 µM (Figure 12) and for
compound 11 is 2.32 µM (Figure 13). Results clearly indicate that compounds 8 and 11
exhibited excellent affinity for CAIX.
2.4. Cytotoxicity assay
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The cytotoxicity and cell proliferation activity of synthesized compounds 1-20 were
evaluated by MTT assay. Human embryonic kidney normal cell line (HEK-293) was used for the
cytotoxicity. Compounds 2, 5, 8, 10, 11, 12, 16, 18 and 19 were less toxic upto 100 µM on HEK-
293 cells. The IC50 values of these compounds ranging from 99.5 µM to 249.6 µM as compared
to standard drug doxorubicin 148.4 µM (Table 2).
The anticancer activity of these compounds against MCF-7 and HepG-2 with dose
dependent concentration were studied and found cells viability reduced significantly,
doxorubicin taken as standard in the experiment. In case of breast cancer cell line (MCF-7),
compounds 5, 8 and 11 showed good activity with IC50 value 18.9±2.19 µM, 13.0±2.28 µM and
12.4±1.39 µM while compounds 18 and 19 were moderately active with IC50 value 21.1±2.68
µM and 28.7±1.90 µM, respectively. Compound 9, 10 and 12 having IC50 value 76.3±1.56 µM,
53.3±2.05 µM and 80.7±1.96 µM showed week activity against this cancer cell line.
Concerning activity against HepG-2, the synthesized pyridine-thiazolidinone hybrid
showed good to moderate growth inhibitory activity. Compounds 5, 8, 11 and 16 were the most
potent against HepG-2 as their IC50 values were 11.8±1.95 µM, 18.9±1.34 µM, 16.2±1.34 µM
and 17.6±2.12 µM. Compounds 10, 12, 18 and 19 having IC50 value in the range of 23.1±1.90
µM - 32.3±1.48 µM were the moderately active against this cell line. Furthermore, compounds 9
and 15 showed fair antiproliferative activity with IC50 value 52.6±1.45 µM and 35.8±1.45 µM.
Selective index (SI) values were calculated for the effectiveness of synthesized
compounds 1-20 against cancerous cells. High SI value (>3) of compounds gives a selective
inhibition towards cancer cells. While the compounds with SI value <3 is considered to be toxic
for normal cells [49]. Compounds 2, 5, 8, 10, 11, 12, 16, 18 and 19 showed good selectivity
against MCF-7, HepG-2 cell lines with the SI values more than 3 (Table 2). Interestingly, if we
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compared and correlate the binding affinity and esterase activity of compounds with that of
cytotoxicity activity, it was found that compound 8 and 11 which shows significant binding with
CAIX, also inhibited the enzyme activity to the most. Consistant to the binding and activity
results, these two compounds 8 and 11 also inhibited the cell viability of cancerous cells more
pronouncly than other compounds. All these observations clearly suggested that the results of
binding, enzyme activity and cell cytotoxicity are in good agreement with each other.
2.5. Molecular docking
Auto dock 4.2 was used to determine the orientation of inhibitors bound in the active site
of the CAIX and the conformation with the highest binding energy value for each molecule was
chosen for further analysis and results of these studies are given in Table 3. The binding mode of
CAIX inhibitors are visualized by PyMOL [50]. The binding site CAIX has been used to
elucidate the interactions as reported earlier [51].
The compounds 8 and 11 bind into the active site of CAIX with minimum binding energy
(∆G) −8.95 kcal/mol (Ki = 7.01µM) and −8.01 kcal/mol (Ki = 10.21µM), respectively as
compared to AZM (∆G) −6.43 kcal/mol (Ki = 14.04µM) [52] (Table 3). The nitrobenzylidine
ring in compound 8 formed six H-bond interaction with His94, His96, Thr199 , Thr200 and one
H-bond formed between thiazolidin-4-one and Thr200 (Figure 14A). Active site residues Gln67,
Thr125, Phe128, Leu135 and Pro138 are involved in hydrophobic interaction and Tyr7 and
Trp97 are stabilized phenylimino and nitrobenzylidine ring respectively of compound 8 with π–π
interaction (Figure 14B).
At the active site of CAIX, compound 11 shows 7 H-bond interaction with Asn62, Gln92,
Thr199, Thr199, whereas residues His64, Gln67, His119 and Val121 are involved in
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hydrophobic interaction (Figure 14C and D). The 3, 4-dihydroxybenzylidine of compound 11 is
stabilized with π–π interaction of His96. However, molecular docking result shows a quite
different orientation of compounds 8 and 11 at the active site of CAIX due to substituent
changing at 5 position of thiazolidin-4-one. These results supports our binding and enzyme
activity results, that these compound 8 and 11 offers sufficient number of interactions to CAIX,
resultantly formed a stable complex. Thus these results are also in close agreement with the
results of binding, enzyme activity and cell viability studies.
3. Conclusion
A series of pyridine-thiazolidinone derivatives was synthesized and evaluated for their
anticancer activity. CAIX inhibition and fluorescence binding studies have shown that
compounds 8 and 11 were most effective inhibitors with a significant binding affinity and
exhibits the highest selectivity towards MCF-7 and HepG-2 cell lines. Molecular docking studies
showed that compounds 8 and 11 strongly bind to the active site residues of CAIX. In summary,
compounds 8 and 11 potentially inhibits CAIX and may be further exploited to fight hypoxia-
induced tumours in future studies.
4. Experimental
All chemicals were purchased from Merck and Aldrich Chemical Company (USA).
Precoated aluminum sheets (Silica gel 60 F254, Merck Germany) were used for thin-layer
chromatography (TLC) and spots were visualized under UV light. The melting points were
recorded on Veego instrument with model specifications REC-22038 A2 and are uncorrected.
Elemental analyses were performed on Elementar Vario analyzer and the results are within
±0.4% of the theoretical values. IR spectra were acquired at Bruker FT-IR spectrophotometer. 1H
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and 13C NMR were recorded on a Bruker Spectrospin DPX 300 MHz and Bruker Spectrospin
DPX 75 MHz spectrometer, respectively using CDCl3 and DMSO-d6 as a solvent and
trimethylsilane (TMS) as the internal standard. Splitting patterns are designated as follows; s,
singlet; d, doublet; t, triplet; m, multiplet. Chemical shift values are given in ppm. Mass spectra
were recorded by ESI-MS (AB-Sciex 2000, Applied Biosystem).
4.1. Chemistry
4.1.1. General procedure for the synthesis of 1-phenyl-3-(pyridine-4-ylmethyl)thiourea (1)
On treating phenylisothiocyanate (59.17 mmol, 8g) with 4-picolinylamine (59.17 mmol,
6.39g) in toluene white precipitates were obtained which were collected by filtration, washed
with toluene, and dried to afford 1-phenyl-3-(pyridine-4-ylmethyl)thiourea (1).
4.1.1.1. 1-Phenyl-3-(pyridine-4-ylmethyl)thiourea (1) Yield: 90%; m.p.: 168˚C; Anal. Calc. (%)
for C13H13N3S: C 64.17, H 5.39, N 17.27, S 13.18; found: C 64.12, H 5.19, N 17.47, S 13.42;
FT-IR νmax (cm-1): 3260 (NH), 3098 (C-H, Ar-H), 1235 (C=S); 1H NMR (CDCl3) δ (ppm): 4.92
(d, 2H, J=5.7 Hz, CH2), 6.39 (s, 1H, N-H), 7.18-7.30 (m, 4H, Ar-H), 7.35 (d, 1H, J=6.9 Hz, Ar-
H), 7.47 (t, 2H, J=7.2 Hz, Ar-H), 8.16 (s, 1H, N-H), 8.54 (d, 2H, J=7.5 Hz, Ar-H); 13C NMR
(CDCl3) δ (ppm): 47.89, 76.59, 77.02, 77.44, 122.08, 125.64, 127.88, 130.42, 135.74, 146.65,
150.06, 181.88; ESI-MS: m/z = 244.0 (M++1).
4.1.2. General procedure of synthesis of 2(phenylimino)-3-(pyridine-4-ylmethyl)thiazolidin-
4-one (2)
1-phenyl-3-(pyridine-4-ylmethyl)thiourea (53.42 mmol, 13.0g) was dissolved in ethanol and then
anhydrous sodium acetate (107.28 mmol, 8.8g) and chloroacetic acid (66.98 mmol, 6.33g) were
added in the reaction mixture. The suspension was heated under reflux for 12 h and then the
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solvent was evaporated. Water was added and the aqueous layer was extracted with ethyl acetate.
The organic layer was washed with saturated sodium bicarbonate and brine. The combined
organic extracts were dried over Na2SO4 and concentrated in vacuo, consequently yellow solid
product 2(phenylimino)-3-(pyridine-4-ylmethyl)thiazolidin-4-one 2 was obtained.
4.1.2.1. 2-(Phenylimino)-3-(pyridine-4-ylmethyl)thiazolidin-4-one (2) Yield: 87%; m.p.: 122˚C;
Anal. Calc. (%) for C15H13N3OS: C 63.58, H 4.62, N 14.83, S 11.32; found: C 63.38, H 4.69, N
14.65, S 11.58; FT-IR νmax (cm-1): 1719 (-N-C=O), 1640 (C=N), 809 (C-S-C), 1371 (tert. N); 1H
NMR (CDCl3) δ (ppm): 3.87 (s, 2H, S-CH2-C=O), 5.02 (s, 2H, CH2), 6.93 (d, 2H, J=7.5 Hz, Ar-
H), 7.17 (t, 1H, J=7.2 Hz, Ar-H), 7.36 (t, 4H, J=6.0 Hz, Ar-H), 8.59 (d, 2H, J=5.7 Hz, Ar-H); 13C
NMR (CDCl3) δ (ppm): 32.69, 45.16, 76.61, 77.04, 77.46, 120.87, 123.30, 124.87, 129.31,
144.38, 147.47, 150.10, 153.47, 171.42; ESI-MS: m/z = 284.1 (M++1).
4.1.3. General procedure of synthesis of 2(phenylimino)-3-(pyridine-4-ylmethyl)thiazolidin-
4-one hybrids (3-20)
A mixture of 3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-thiazolidin-4-one (2.00 mmol, 0.56g),
different aldehydes (2.00 mmol, 0.21-.33g), hexahydropyridine (2.30 mmol, 0.19g), and ethanol
(35 mL) were heated under reflux for 11-12 h. The reaction mixture was cooled to room
temperature and the precipitates were obtained which were filtered, washed and recrystallized
from ethanol to furnished final products 3-20.
4.1.3.1. 5-Benzylidene-2-phenylimino-3-pyridine-4-ylmethyl-thiazolidin-4-one (3) Yield: 84%;
m.p.: 162˚C; Anal. Calc. (%) for C22H17N3OS: C 71.14, H 4.61, N 11.31, S 8.63; found: C 71.38,
H 4.33, N 11.10, S 8.31; FT-IR νmax (cm-1): 1706 (-N-C=O), 1640 (C=N), 760 (C-S-C), 1375
(tert. N), 1583 (C=C); 1H NMR (CDCl3) δ (ppm): 5.17 (s, 2H, CH2), 6.98 (d, 2H, J=7.5 Hz, Ar-
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H), 7.22 (d, 2H, J=7.2 Hz, Ar-H), 7.38-7.43 (m, 8H, Ar-H), 7.79 (s, 1H, CH=C), 8.60 (s, 2H, Ar-
13
H); C NMR (CDCl3) δ (ppm): 45.19, 121.07, 123.21, 125.11, 129.08, 129.44, 130.02, 130.08,
131.77, 133.53, 140.11, 144.53, 147.66, 149.71, 150.14, 166.52; ESI-MS: m/z = 372.1 (M++1).
4.1.3.2. 5-(4-Methylbenzylidene)-2-(phenylimino)-3-(pyridine-4-ylmethyl)thiazolidin-4-one (4)
Yield: 82%; m.p.: 165˚C; Anal. Calc. (%) for C23H19N3OS: C 71.66, H 4.97, N 10.90, S 8.32;
found: C 71.91, H 4.61, N 11.15, S 8.21; FT-IR νmax (cm-1): 1703 (-N-C=O), 1653 (C=N), 764
(C-S-C), 1381 (tert. N), 1578 (C=C); 1H NMR (CDCl3) δ (ppm): 2.36 (s, 3H, CH3), 5.16 (s, 2H,
CH2), 6.98 (d, 2H, J=7.5 Hz, Ar-H), 7.22 (d, 1H, J=7.5 Hz, Ar-H), 7.33-7.39 (m, 6H, Ar-H),
7.77 (s, 1H, CH=C), 8.59 (d, 2H, J=4.5 Hz, Ar-H); 13C NMR (CDCl3) δ (ppm): 21.25, 45.14,
76.64, 77.06, 77.49, 119.85, 121.10, 123.20, 125.04, 129.41, 129.83, 130.08, 130.79, 131.89,
140.67, 144.62, 147.74, 149.87, 150.12, 166.62; ESI-MS: m/z = 386.1 (M++1).
4.2. Crystal structure determination
X-ray data were collected on a Bruker Kappa Apex CCD diffractometer at low
temperature for 2, 3, 6 and 12, by the
φ
-
ω
scan method. Reflections were measured from a
. A total of 40101 for
hemisphere of data collected from frames, each of them covering 0.3º in
ω
2, 47051 for 3, 55044 for 6, and 69246 for 12, reflections measured were corrected for Lorentz
and polarization effects and for absorption by multi-scan methods based on symmetry-equivalent
and repeated reflections. Of the total, 3876 for 2, 4193 for 3, 4374 for 6 and 5584 for 12,
independent reflections exceeded the significance level ( F /
σ F ) > 4.0. After data collection,
in each case a multi-scan absorption correction (SADABS) [53] was applied, and the structure
was solved by direct methods and refined by full matrix least-squares on F2 data using SHELX
suite of programs [54]. Hydrogen atoms were located in difference Fourier map and left to refine
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freely. Refinements were done with allowance for thermal anisotropy of all non-hydrogen atoms.
A final difference Fourier map showed no residual density outside: 0.503 and -0.205 e.Å–3 for 2,
0.346 and -0.293 for 3 e.Å–3, 0.344 and -0.279 e.Å–3 for 6 and 0.469 and -0.425 e.Å–3 for 12. A
2
2
weighting scheme w = 1/[σ2(Fo ) + (0.040400 P)2 + 0.296300 P] for 2, w = 1/[σ2(Fo ) +
2
(0.041100 P)2 + 0.905000 P] for 3, w = 1/[σ2(Fo ) + (0.054100 P)2 + 2.207800 P] for 6 and w =
2
1/[σ2(Fo ) + (0.063300 P)2 + 0.570100 P] for 12, where P = (|Fo|2 + 2|Fc|2)/3, were used in the
latter stages of refinement. Further details of the crystal structure determination are given in
Table 1. CCDC 1564721-1564724 contain the supplementary crystallographic data for the
structures reported in this paper. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223 336 033; or e-mail:
deposit@ccdc.cam.ac.uk. Supplementary data associated with this article can be found, in the
online version, at doi: $$$$$.
4.3. Biological Evaluation
4.3.1. Esterase assay
CAs inhibition was measured by following the change in absorbance at 400 nm of p-
nitrophenyl acetate (4-NPA) to p-nitrophenol (a yellow colour compound). This method is based
on spectrophotometric determination of p-nitrophenol which is formed as CA catalysed
hydrolysis 4-NPA. Esterase assay was performed in buffer containing 50 mM Tris pH 8.0, 100
mM NaCl, and 1mM EDTA. Acetazolamide (AZM) and test compounds (1-20) were in 100%
DMSO. The concentration of CAIX was used 1 µM and 4-NPA was used in 0.5 mM
concentration. Test compounds concentration was used in 0.01 to 100 µM in a final volume of
100µl. Stock solutions and dilutions of inhibitors were prepared in DMSO such that there was a
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constant 1% DMSO in the assay. The reaction was initiated by addition of 4-NPA and reading
°
was taken after 3 min incubation of test compounds at 25 C. The absorbance of each well was
measured with a Spectra Max Plus384 microplate spectrophotometer (Molecular Devices,
Sunnyvale, CA). IC50 values for each test compounds were calculated by using Graph Pad Prism
(Version 5.0) graphing software.
4.3.2. Fluorescence Measurements
Fluorescence measurements were performed on Jasco-spectrofluorimeter (Model FP-
6200) using 5 mm quartz cuvette. The protein concentration 0.5 µM was used for CAIX.
Working solutions of compounds were made in 50mM Tris buffer, pH 8.0. and used in the
concentration 1-100 µM. Protein was excited at 280 nm and the emission spectra were recorded
between 300 - 400 nm at 25 °C using slits with a 5 nm band pass for excitation and emission,
respectively. Final spectrum was collected after subtracting intensity of the buffer from each
sample spectrum. Fluorescence-quenching studies were carried out and the results have also been
used to estimate the binding affinity by Stern–Volmer equations with slight modification.
log(Fo –F)/F = logKa + nlog[Q]
(1)
Where Fo is the fluorescence intensity of protein and F is the fluorescence intensity of ligand, Ka
is the binding constant and n is the number of binding sites, Q represents quenching constant. For
the ligand–protein complex, the values for Ka and n can be derived from the intercept and slope.
4.3.3. Cell Culture
Human breast cancer cells (MCF-7), human liver cancer cells (HepG-2) and human
embryonic kidney cells (HEK-293) were obtained from National Centre for Cell Science, Pune,
India. The cells were cultured in Roswell Park Memorial Institute (RPMI) (Sigma Aldrich) with
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10% foetal bovine serum (Gibco-life technologies), 1% penicillin-streptomycin-Neomycin in a
humidified 5% CO2 atmosphere at 37 °C. Cells were routinely cultured and maintained not more
than 35 passages.
4.3.4. Cytotoxicity assay
The cell cytotoxicity was detected by standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide) assay [55-56]. Approximately, 5× 103 cells/well were seeded into
the 96-well plate media containing fetal bovine serum (10%) and 1 % penicillin–streptomycin
°
and then incubated in a humidified 5 % CO2 atmosphere at 37 C overnight. After 24 h
incubation, media were removed, and reduce serum media were added for serum starvation. The
next day, media were replaced with 200 µL of fresh complete medium containing different
concentrations of compounds and doxorubicin ranging from 2.5 µM to 80 µM for cancer cell
lines and from 5 µM to 200 µM for HEK 293. Doxorubicin was included as standard reference
drug (positive control). After 48 h, the supernatants were removed, and cell were washed with
PBS and 20 µL of MTT solution (5 mg/mL in PBS) was added to each well, and the cells were
incubated at 37 °C in a humidified chamber for 4 h. After 4 h, the supernatant was removed from
each well and the coloured formazan crystal produced from MTT was dissolved in 100 µL of
DMSO. The absorbance was measured at 570 nm by using enzyme linked immune absorbent
assay reader (BioRad). The IC50 values of the tested compound were estimated using the best fit
regression curve method in Excel. All experiments were repeated at least three times.
4.3.5. Calculation of the selectivity index
The selectivity index corresponded to the IC50 value determined for activity of
synthesized compounds and reference compound on HEK-293 cells divided by the IC50
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determined for cancer cells (MCF-7 and HepG-2). The selectivity index values greater than three
was considered significant [55, 57].
4.3.6. Preparation of Ligands and Protein Molecule
The structures of ligands were drawn by using the software ChemBioDraw Office 12.0
(licensed @ Cambridge’s soft). The PDB file of ligand was then generated and converted into
PDBQT file by process like detect root, chose the torsion and set the number of torsion by using
ADT [58]. Crystal structure of CAIX was taken from the Protein Data Bank (www.rcsb.org)
[59]. Then, the hydrogen atoms having polar nature were added, the residue structures having
lower occupancy were deleted, and the side chains that were incomplete were then replaced by
using Auto Dock Tools (ADT) version 1.5.6 from the Scripps Research Institute. Further, to each
atom having Gasteiger charges were added and the non-polar hydrogen atoms were merged to
the protein structure. After that the structures constructed were saved in PDBQT file format, for
further analysis in ADT [59].
4.3.7. Molecular docking
Molecular docking stimulation using the ligand molecules with CAIX (PDB ID: 3IAI)
was conducted using Autodock 4.2 docking suite by employing Lamarckian genetic algorithm
[60]. The ligands were set to explore and flexible to rotate most probable binding poses, while
receptor was kept rigid. The grid maps representing the center of active site pocket for the ligand
were calculated with Autogrid. The dimensions of the grid for CAIX was 85 × 85 × 85 grid
points with a spacing of 0.435 Å between the grid points but center on the ligand for receptor
(64.496, 61.661 and -8.547 coordinates). In the present docking study was performed by each
run with population of 150 individuals, rate of gene mutation 0.02, cross-over rate 0.8 and the
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remaining parameters were set as default [51, 53]. Ten poses docking conformations were
generated and the best docked conformation was selected based on the Autodock binding energy
(Kcal/mol), for further analysis. Finally, the conformations with the most favourable free binding
energy were selected for analyzing the interactions between the target receptor and ligands by
PyMOL [61-62].
Acknowledgement
One of the authors MFA is thankful to University Grant Commission, India for providing BSR
meritorious fellowship.
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References
[1] Jemal, A., Bray, F., Center, M.M., Ferlay, J., Ward, E. and Forman, D., 2011. Global cancer
statistics. CA Cancer J. Clin., 61, 69-90.
[2] Zwick, E., Bange, J. and Ullrich, A., 2001. Receptor tyrosine kinase signalling as a target for
cancer intervention strategies. Endocr. Relat. Cancer, 8, 161-173.
[3] Grever, M.R., Schepartz, S.A. and Chabner, B.A., 1992, December. The National Cancer
Institute: cancer drug discovery and development program. In Seminars in oncology (Vol. 19,
No. 6, 622-638). Independence Square West Curtis Center, STE 300, Philadelphia, PA 19106-
3399: WB Saunders CO.
[4] Romagnoli, R., Baraldi, P.G., Prencipe, F., Oliva, P., Baraldi, S., Salvador, M.K., Lopez-
Cara, L.C., Brancale, A., Ferla, S., Hamel, E. and Ronca, R., 2017. Synthesis and Biological
Evaluation of 2-Methyl-4, 5-Disubstituted Oxazoles as a Novel Class of Highly Potent
Antitubulin Agents. Sci. Rep., 7.
[5] Dewangan, D., Kumar, T., Alexander, A., Nagori, K. and Tripathi, D.K., 2011. Pyrazole:
Their Chemistry and Pharmacological Potentials: A Review. Curr Pharma Res, 1, 369.
[6] Li, Y.S., Hu, D.K., Zhao, D.S., Liu, X.Y., Jin, H.W., Song, G.P., Cui, Z.N. and Zhang, L.H.,
2017. Design, synthesis and biological evaluation of 2, 4-disubstituted oxazole derivatives as
potential PDE4 inhibitors. Bioorg. Med. Chem., 25, 1852-1859.
[7] Ansari, M.F., Siddiqui, S.M., Agarwal, S.M., Vikramdeo, K.S., Mondal, N. and Azam, A.,
2015. Metronidazole hydrazone conjugates: design, synthesis, antiamoebic and molecular
docking studies. Bioorg. Med. Chem. Lett., 25, 3545-3549.
[8] Jin, R.Y., Sun, X.H., Liu, Y.F., Long, W., Chen, B., Shen, S.Q. and Ma, H.X., 2016.
Synthesis, crystal structure, biological activity and theoretical calculations of novel isoxazole
derivatives. Spectrochim. Acta A Mol. Biomol. Spectrosc., 152, 226-232.
[9] Tomi, I.H., Tomma, J.H., Al-Daraji, A.H. and Al-Dujaili, A.H., 2015. Synthesis,
characterization and comparative study the microbial activity of some heterocyclic compounds
containing oxazole and benzothiazole moieties. J.Saudi Chem. Soc., 19, 392-398.
[10] Rouf, A. and Tanyeli, C., 2015. Bioactive thiazole and benzothiazole derivatives. Eur. J.
Med. Chem., 97, 911-927.
[11] Su, Q., Ioannidis, S., Chuaqui, C., Almeida, L., Alimzhanov, M., Bebernitz, G., Bell, K.,
Block, M., Howard, T., Huang, S. and Huszar, D., 2013. Discovery of 1-Methyl-1 H-imidazole
Derivatives as Potent Jak2 Inhibitors. J. Med. Chem., 57, 144-158.
19

ACCEPTED MANUSCRIPT
[12] Lan, R., Liu, Q., Fan, P., Lin, S., Fernando, S.R., McCallion, D., Pertwee, R. and
Makriyannis, A., 1999. Structure-activity relationships of pyrazole derivatives as cannabinoid
receptor antagonists. J. Med. Chem., 42, 769-776.
[13] Nakamura, T., Sato, M., Kakinuma, H., Miyata, N., Taniguchi, K., Bando, K., Koda, A. and
Kameo, K., 2003. Pyrazole and isoxazole derivatives as new, potent, and selective 20-hydroxy-5,
8, 11, 14-eicosatetraenoic acid synthase inhibitors. J. Med. Chem., 46, 5416-5427.
[14] Patel, N.B. and Shaikh, F.M., 2010. Synthesis and antimicrobial activity of new 4-
thiazolidinone derivatives containing 2-amino-6-methoxybenzothiazole. Saudi Pharm. J., 18,
129-136.
[15] Hu, J., Wang, Y., Wei, X., Wu, X., Chen, G., Cao, G., Shen, X., Zhang, X., Tang, Q., Liang,
G. and Li, X., 2013. Synthesis and biological evaluation of novel thiazolidinone derivatives as
potential anti-inflammatory agents. Eur. J. Med. Chem., 64, 292-301.
[16] Omar, K., Geronikaki, A., Zoumpoulakis, P., Camoutsis, C., Soković, M., Ćirić, A. and
Glamočlija, J., 2010. Novel 4-thiazolidinone derivatives as potential antifungal and antibacterial
drugs. Bioorg. Med. Chem., 18, 426-432.
[17] Ruiz, F.A.R., García-Sánchez, R.N., Estupiñan, S.V., Gómez-Barrio, A., Amado, D.F.T.,
Pérez-Solórzano, B.M., Nogal-Ruiz, J.J., Martínez-Fernández, A.R. and Kouznetsov, V.V.,
2011. Synthesis and antimalarial activity of new heterocyclic hybrids based on chloroquine and
thiazolidinone scaffolds. Bioorg. Med. Chem., 19, 4562-4573.
[18] Shiradkar, M.R., Ghodake, M., Bothara, K.G., Bhandari, S.V., Nikalje, A., Akula, K.C.,
Desai, N.C. and Burange, P.J., 2007. Synthesis and anticonvulsant activity of clubbed
thiazolidinone–barbituric acid and thiazolidinone–triazole derivatives. Arkivoc, 14, 58-74.
[19] Ravichandran, V., Kumar, B.P., Sankar, S. and Agrawal, R.K., 2009. Predicting anti-HIV
activity of 1, 3, 4-thiazolidinone derivatives: 3D-QSAR approach. Eur. J. Med. Chem., 44, 1180-
1187.
[20] Ottanà, R., Maccari, R., Giglio, M., Del Corso, A., Cappiello, M., Mura, U., Cosconati, S.,
Marinelli, L., Novellino, E., Sartini, S. and La Motta, C., 2011. Identification of 5-arylidene-4-
thiazolidinone derivatives endowed with dual activity as aldose reductase inhibitors and
antioxidant agents for the treatment of diabetic complications. Eur. J. Med. Chem., 46, 2797-
2806.
[21] Jain, A.K., Vaidya, A., Ravichandran, V., Kashaw, S.K. and Agrawal, R.K., 2012. Recent
developments and biological activities of thiazolidinone derivatives: a review. Bioorg. Med.
Chem., 20, 3378-3395.
[22] Havrylyuk, D., Mosula, L., Zimenkovsky, B., Vasylenko, O., Gzella, A. and Lesyk, R.,
2010. Synthesis and anticancer activity evaluation of 4-thiazolidinones containing benzothiazole
moiety. Eur. J. Med. Chem., 45, 5012-5021.
20

ACCEPTED MANUSCRIPT
[23] Wang, S., Zhao, Y., Zhang, G., Lv, Y., Zhang, N. and Gong, P., 2011. Design, synthesis and
biological evaluation of novel 4-thiazolidinones containing indolin-2-one moiety as potential
antitumor agent. Eur. J. Med. Chem., 46, 3509-3518.
[24] Romagnoli, R., Baraldi, P.G., Salvador, M.K., Camacho, M.E., Balzarini, J., Bermejo, J.
and Estévez, F., 2013. Anticancer activity of novel hybrid molecules containing 5-benzylidene
thiazolidine-2, 4-dione. Eur. J. Med. Chem., 63, 544-557.
[25] Havrylyuk, D., Zimenkovsky, B., Vasylenko, O., Day, C.W., Smee, D.F., Grellier, P. and
Lesyk, R., 2013. Synthesis and biological activity evaluation of 5-pyrazoline substituted 4-
thiazolidinones. Eur. J. Med. Chem., 66, 228-237.
[26] Havrylyuk, D., Zimenkovsky, B., Vasylenko, O., Gzella, A. and Lesyk, R., 2012. Synthesis
of new 4-thiazolidinone-, pyrazoline-, and isatin-based conjugates with promising antitumor
activity. J. Med. Chem., 55, 8630-8641.
[27] Wu, J., Yu, L., Yang, F., Li, J., Wang, P., Zhou, W., Qin, L., Li, Y., Luo, J., Yi, Z. and Liu,
M., 2014. Optimization of 2-(3-(arylalkyl amino carbonyl) phenyl)-3-(2-methoxyphenyl)-4-
thiazolidinone derivatives as potent antitumor growth and metastasis agents. Eur. J. Med.
Chem., 80, 340-351.
[28] Kumar, K.S.S., Hanumappa, A., Vetrivel, M., Hegde, M., Girish, Y.R., Byregowda, T.R.,
Rao, S., Raghavan, S.C. and Rangappa, K.S., 2015. Antiproliferative and tumor inhibitory
studies of 2, 3 disubstituted 4-thiazolidinone derivatives. Bioorg. Med. Chem. Lett., 25, 3616-
3620.
[29] Senkiv, J., Finiuk, N., Kaminskyy, D., Havrylyuk, D., Wojtyra, M., Kril, I., Gzella, A.,
Stoika, R. and Lesyk, R., 2016. 5-Ene-4-thiazolidinones induce apoptosis in mammalian
leukemia cells. Eur. J. Med. Chem., 117, 33-46.
[30] Appalanaidu, K., Kotcherlakota, R., Dadmal, T.L., Bollu, V.S., Kumbhare, R.M. and Patra,
C.R., 2016. Synthesis and biological evaluation of novel 2-imino-4-thiazolidinone derivatives as
potent anti-cancer agents. Bioorg. Med. Chem. Lett., 26, 5361-5368.
[31] Cutshall, N.S., O’Day, C. and Prezhdo, M., 2005. Rhodanine derivatives as inhibitors of
JSP-1. Bioorg. Med. Chem. Lett., 15, 3374-3379.
[32] Ahn, J.H., Kim, S.J., Park, W.S., Cho, S.Y., Du Ha, J., Kim, S.S., Kang, S.K., Jeong, D.G.,
Jung, S.K., Lee, S.H. and Kim, H.M., 2006. Synthesis and biological evaluation of rhodanine
derivatives as PRL-3 inhibitors. Bioorg. Med. Chem. Lett., 16, 2996-2999.
[33] Park, H., Jung, S.K., Jeong, D.G., Ryu, S.E. and Kim, S.J., 2008. Discovery of novel PRL-3
inhibitors based on the structure-based virtual screening. Bioorg. Med. Chem. Lett., 18, 2250-
2255.
21

ACCEPTED MANUSCRIPT
[34] Geronikaki, A., Eleftheriou, P., Vicini, P., Alam, I., Dixit, A. and Saxena, A.K., 2008. 2-
Thiazolylimino/heteroarylimino-5-arylidene-4-thiazolidinones as new agents with SHP-2
inhibitory action. J. Med. Chem., 51, 5221-5228.
[35] Carter, P.H., Scherle, P.A., Muckelbauer, J.A., Voss, M.E., Liu, R.Q., Thompson, L.A.,
Tebben, A.J., Solomon, K.A., Lo, Y.C., Li, Z. and Strzemienski, P., 2001. Photochemically
enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding
of TNF-α. Proc. Natl. Acad. Sci., 98, 11879-11884.
[36] Degterev, A., Lugovskoy, A., Cardone, M., Mulley, B., Wagner, G., Mitchison, T. and
Yuan, J., 2001. Identification of small-molecule inhibitors of interaction between the BH3
domain and Bcl-xL. Nat. Cell Biol., 3, 173-182.
[37] Dayam, R., Aiello, F., Deng, J., Wu, Y., Garofalo, A., Chen, X. and Neamati, N., 2006.
Discovery of small molecule integrin αvβ3 antagonists as novel anticancer agents. J. Med.
Chem., 49, 4526-4534.
[38] Jemmezi, F., Kether, F.B.H., Amri, I., Bassem, J. and eddine Khiari, J., Synthesis and
biological activity of novel benzothiazole pyridine derivatives. IOSR JAC., 7, 62-66.
[39] Nepali, K., Sharma, S., Sharma, M., Bedi, P.M.S. and Dhar, K.L., 2014. Rational
approaches, design strategies, structure activity relationship and mechanistic insights for
anticancer hybrids. Eur. J. Med. Chem., 77, 422-487.
[40] Jain, A.K., Vaidya, A., Ravichandran, V., Kashaw, S.K. and Agrawal, R.K., 2012. Recent
developments and biological activities of thiazolidinone derivatives: A review. Bioorg. Med.
Chem., 20, 3378-3395.
[41] El Ella, D.A.A., Ghorab, M.M., Heiba, H.I. and Soliman, A.M., 2012. Synthesis of some
new thiazolopyrane and thiazolopyranopyrimidine derivatives bearing a sulfonamide moiety for
evaluation as anticancer and radiosensitizing agents. Med. Chem. Res., 21, 2395–2407.
[42] Eldehna, W.M., Abo-Ashour, M.F., Nocentini, A., Gratteri, P., Eissa, I.H., Fares, M.,
Ismael, O.E., Ghabbour, H.A., Elaasser, M.M., Abdel-Aziz, H.A. and Supuran, C.T., 2017.
Novel
4/3-((4-oxo-5-(2-oxoindolin-3-ylidene)
thiazolidin-2-ylidene)
amino)
benzenesulfonamides: Synthesis, carbonic anhydrase inhibitory activity, anticancer activity and
molecular modelling studies. Eur. J. Med. Chem., 139, 250-262.
[43] Narges, K., Tafreshi, Mark C., Lloyd, Marilyn M., Bui, Robert J., Gillies, and David L.
Morse, 2014. Carbonic Anhydrase IX as an Imaging and Therapeutic Target for Tumors and
Metastases Subcell. Biochem. 75, 221–254.
[44] Sedlakova, O., Svastova, E., Takacova, M., Kopacek, J., Pastorek, J. and Pastorekova, S.,
2013. Carbonic anhydrase IX, a hypoxia-induced catalytic component of the pH regulating
machinery in tumors. Front Physiol, 4, 400-414.
22

ACCEPTED MANUSCRIPT
[45] Kato, Y., Ozawa, S., Miyamoto, C., Maehata, Y., Suzuki, A., Maeda, T. and Baba, Y., 2013.
Acidic extracellular microenvironment and cancer. Cancer Cell Int., 13, 89-96.
[46] Patard, J., Fergelot, P., Karakiewicz, P.I., Klatte, T., Trinh, Q.D. and Rioux-Leclercq, N.,
2008. CAIX and VHL status define two types of clear cell renal cell carcinoma: low CAIX
expression and absence of VHL gene mutation are associated with tumour aggressiveness and
poor survival. Int. J. Cancer, 123, 395-400.
[47] Côrte-Real, L., Robalo, M.P., Marques, F., Nogueira, G., Avecilla, F., Silva, T.J., Santos,
F.C., Tomaz, A.I., Garcia, M.H. and Valente, A., 2015. The key role of coligands in novel
ruthenium (II)-cyclopentadienyl bipyridine derivatives: ranging from non-cytotoxic to highly
cytotoxic compounds. J. Inorg. Biochem. 150, 148-159.
[48] Akkurt, M., Çelik, Í., Demir, H., Özkırımlı, S. and Büyükgüngör, O., 2011. N-[2-(4-
Bromophenyl)-5-methyl-4-oxo-1, 3-thiazolidin-3-yl] pyridine-3-carboxamide. Acta Crystallogr.
Sect. E, 67,o914-o915.
[49] Syarifah, M.S., Nurhanan, M.Y., Haffiz, J.M., Ilham, A.M., Getha, K., Asiah, O., Norhayati,
I., Sahira, H.L. and Suryani, S.A., 2011. Potential anticancer compound from Cerbera
odollam. J. Trop. For. Sci., 89-96.
[50] Alexandre, J., Bleuzen, P., Bonneterre, J., Sutherland, W., Misset, J.L., Guastalla, J.P.,
Viens, P., Faivre, S., Chahine, A., Spielman, M. and Bensmaine, A., 2000. Factors predicting for
efficacy and safety of docetaxel in a compassionate-use cohort of 825 heavily pretreated
advanced breast cancer patients. J. Clin. Oncol., 18, 562-562.
[51] Badisa, R.B., Ayuk-Takem, L.T., Ikediobi, C.O. and Walker, E.H., 2006. Selective
anticancer activity of pure licamichauxiioic-B acid in cultured cell lines. Pharm. Biol., 44, 141-
145.
[52] Amresh, P., Kumar, K., Islam, A., Hassan, I. and Ahmad, F., 2013. Receptor chemoprint
derived pharmacophore model for development of CAIX inhibitors. J. Carcinog. Mutagen S, 8,
17-20.
[53]. Kumari, S., Idrees, D., Mishra, C.B., Prakash, A., Ahmad, F., Hassan, M.I. and Tiwari, M.,
2016. Design and synthesis of a novel class of carbonic anhydrase-IX inhibitor 1-(3-(phenyl/4-
fluorophenyl)-7-imino-3H-[1, 2, 3] triazolo [4, 5d] pyrimidin 6 (7H) yl) urea. J. Mol. Graph.
Model., 64, 101-109.
[54]. Fuhrmann, J., Rurainski, A., Lenhof, H.P. and Neumann, D., 2010. A new Lamarckian
genetic algorithm for flexible ligand receptor docking. J. Comput. Chem., 31, 1911-1918.
[55] G. M. Sheldrick, SADABS, version 2.10, University of Göttingen, Germany, (2004)
[56] G. M. Sheldrick, SHELX, Acta Crystallogr., Sect. A, 64 (2008) 112–122.
23

ACCEPTED MANUSCRIPT
[57] Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays. J. Immunol. Methods, 65(1-2), 55-63.
[58] Alterio, V., Hilvo, M., Di Fiore, A., Supuran, C.T., Pan, P., Parkkila, S., Scaloni, A.,
Pastorek, J., Pastorekova, S., Pedone, C. and Scozzafava, A., 2009. Crystal structure of the
catalytic domain of the tumor-associated human carbonic anhydrase IX. Proc. Natl Acad.
Sci., 106, 16233-16238.
[59] Ahmad, K., Roouf Bhat, A., and Athar, F., 2017. Pharmacokinetic Evaluation of
Callistemon viminalis Derived Natural Compounds as Targeted Inhibitors Against δ-Opioid
Receptor and Farnesyl Transferase. Lett. Drug Des. Discov., 14, 488-499.
[60] Morris, G.M., Goodsell, D.S., Halliday, R.S., Huey, R., Hart, W.E., Belew, R.K. and Olson,
A.J., 1998. Automated docking using a Lamarckian genetic algorithm and an empirical binding
free energy function. J. Comput. Chem., 19,1639-1662.
[61] Ansari, M.F., Siddiqui, S.M., Ahmad, K., Avecilla, F., Dharavath, S., Gourinath, S. and
Azam, A., 2016. Synthesis, antiamoebic and molecular docking studies of furan-thiazolidinone
hybrids. Eur. J. Med. Chem., 124, 393-406.
[62] Zaidi, S.L., Agarwal, S.M., Chavalitshewinkoon-Petmitr, P., Suksangpleng, T., Ahmad, K.,
Avecilla, F. and Azam, A., 2016. Thienopyrimidine sulphonamide hybrids: design, synthesis,
antiprotozoal activity and molecular docking studies. RSC Adv., 6, 90371-90383.
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Caption
Figure 1: Structure of some Thiazolidinone based drugs which are currently used for the treatment of
various disease.
Figure 2: Structure of some thiazolidinone derivatives showing anticancer activity.
Figure 3: ORTEP plot for the compound of 2(Phenylimino)-3-(pyridine-4-ylmethyl) thiazolidin-
4-one (2). All the non-hydrogen atoms are presented by their 50% probability
ellipsoids. Hydrogen atoms are omitted for clarity.
Figure 4: ORTEP plot for the compound of 5-Benzylidene-2-phenylimino-3-pyridine-4-
ylmethyl-thiazolidin-4-one (3). All the non-hydrogen atoms are presented by their
50% probability ellipsoids. Hydrogen atoms are omitted for clarity.
Figure 5: ORTEP plot for the compound of 5-(4-Methoxybenzylidene)-2-(phenylimino)-3-
(pyridine-4-ylmethyl)thiazolidin-4-one (6). All the non-hygrogen atoms are presented
by their 50% probability ellipsoids. Hydrogen atoms are omitted for clarity.
Figure 6: ORTEP plot for the compound of -(3,4-Dimethoxy-benzylidene)-2-(phenylimino)-3-
(pyridine-4-ylmethyl)thiazolidine-4-one (12). All the non-hygrogen atoms are
presented by their 50% probability ellipsoids. Hydrogen atoms are omitted for clarity.
Figure 7: Enantiomers present in the compound 2(Phenylimino)-3-(pyridine-4-ylmethyl)
thiazolidin-4-one (2). Enantiomerization occurs by simultaneously rotating about the
two single bonds, N(1)-C(10) and N(2)-C(4) (Figure 3). Drawings were done with
mercury 2.3 program with balls and sticks.
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Figure 8: Enantiomers present in the compound 5-Benzylidene-2-phenylimino-3-pyridine-4-
ylmethyl-thiazolidin-4-one (3). Enantiomerization occurs by simultaneously rotating
about the two single bonds, N(1)-C(10) and N(2)-C(4) (Figure 4). Drawings were
done with mercury 2.3 program with balls and sticks.
Figure 9: Enantiomers present in the compound 5-(4-Methoxybenzylidene)-2-(phenylimino)-3-
(pyridine-4-ylmethyl)thiazolidin-4-one
(6).
Enantiomerization
occurs
by
simultaneously rotating about the two single bonds, N(1)-C(10) and N(2)-C(4) (Figure
5). Drawings were done with mercury 2.3 program with balls and sticks.
Figure 10: Enantiomers present in the compound 5-(3,4-Dimethoxy-benzylidene)-2-
(phenylimino)-3-(pyridine-4-ylmethyl)thiazolidine-4-one (12). Enantiomerization
occurs by simultaneously rotating about the two single bonds, N(1)-C(10) and N(2)-
C(4) (Figure 6). Drawings were done with mercury 2.3 program with balls and sticks.
Figure 11: View along edge of the crystal packing of compounds, 2, 3, 6 and 12.
Figure 12: Fluorescence binding studies: (A). Fluorescence emission spectra of CAIX titrated
with compound 8, Quenching in fluorescence intensity after the addition of compound
indicating binding. (B). Modified Stern-Volmer plot (log (Fo-F)/F versus log [L]).
Figure 13: Fluorescence binding studies: (A). Fluorescence spectra of CAIX were titrated with
compound 11 gradually adding compounds with increasing concentration,
fluorescence intensity was quenched indicating binding. (B). Plot between log (Fo-
F)/F versus log [L].
Figure 14: Molecular docking studies: (A, C) Cartoon view of compound 8 and 11 docked
with CAIX. (B D,) Shows the active site residues interact with compound 8 and 11.
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Residues of CAIX are shown with ball and stick and compounds are shown with stick
model. Hydrogen bonds are shown as broken lines (black).
Tables Legends:
Table 1: Crystal Data and Structure Refinement for 2(Phenylimino)-3-(pyridine-4-
ylmethyl)thiazolidin-4-one (2), 5-Benzylidene-2-phenylimino-3-pyridine-4-ylmethyl-
thiazolidin-4-one (3), 5-(4-Methoxybenzylidene)-2-(phenylimino)-3-(pyridine-4-
ylmethyl)thiazolidin-4-one (6) and 5-(3,4-Dimethoxy-benzylidene)-2-(phenylimino)-
3-(pyridine-4-ylmethyl)thiazolidine-4-one (12).
Table 2: Cytotoxic activity of the synthesized pyridine-thiazolidinone derivatives against HEK-
293, MCF-7 and HepG2 cell lines, selective index, binding affinity on CA IX and
fluorescence binding study
Table 3: Binding energy and specific interaction of CA IX with compound
Scheme:
Scheme 1: Synthesis of pyridine-thiazolidinone derivatives (3-20)
27

ACCEPTED MANUSCRIPT
H
N
O
O
S
O
H
N
N
O
O
N
O
S
S
S
O
N
O
HN
Cl
N
O
O
COOH
Figure 1: Structure of some Thiazolidinone based drugs which are currently used for the treatment of
various disease.
O
S
O
S
N
H
N
N
N
N
O
H
S
Br
N
IC50 = 11.9 µM (Reh)
IC50 = 3.4 ± 2 µM (B16F10)
O
O
CF
3
N
O
O
O
O
S
F
S
N
N
O
N
N
H
Br
H
IC50 = 0.025 ± 0.008 (HT-29)
IC50 = 0.72 ± 0.04 (L1210)
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