Design and synthesis of novel 4-(4-oxo-2-arylthiazolidin-3-yl) benzenesulfonamides as selective inhibitors of carbonic anhydrase IX over I and II with potential anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2013.06.003

The novel 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamide derivatives were designed and synthesized for selective carbonic anhydrase IX (CA IX) inhibitory activity with anticancer potential. In the CA inhibition assay, 3f was found to be the most poten

Full text of DOI:10.1016/j.ejmech.2013.06.003

Accepted Manuscript
Design and synthesis of novel 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamides
as selective inhibitors of carbonic anhydrase IX over I and II with potential anticancer
activity
Sharad Kumar Suthar, Sumit Bansal, Sandeep Lohan, Vikarm Modak, Anil
Chaudhary, Amit Tiwari
PII:
S0223-5234(13)00368-1
10.1016/j.ejmech.2013.06.003
EJMECH 6228
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 December 2012
Revised Date: 31 May 2013
Accepted Date: 2 June 2013
Please cite this article as: S.K. Suthar, S. Bansal, S. Lohan, V. Modak, A. Chaudhary, A. Tiwari, Design
and synthesis of novel 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamides as selective inhibitors of
carbonic anhydrase IX over I and II with potential anticancer activity, European Journal of Medicinal
Chemistry (2013), doi: 10.1016/j.ejmech.2013.06.003.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design and synthesis of novel 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamides as
selective inhibitors of carbonic anhydrase IX over I and II with potential anticancer
activity
Sharad Kumar Suthara,*, Sumit Bansala, Sandeep Lohana, Vikarm Modakb, Anil Chaudharyc,
Amit Tiwarid
aDepartment of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences,
Manipal-576104, India
bDepartment of Pharmaceutical Chemistry, Maharashtra Institute of Pharmacy, Pune-411038,
India
cMahakal Institute of Pharmaceutical Studies, Ujjain-456664, India
dDepartment of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal-576104,
India
*Corresponding author. Tel.: +91 8894861073; fax: +91 820-2571998; E-mail address: sharadpune_2009@yahoo.com.
ABSTRACT
The novel 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamide derivatives were designed and
synthesized for selective carbonic anhydrase IX (CA IX) inhibitory activity with anticancer
potential. In the CA inhibition assay, 3f was found to be most potent and selective inhibitor of
CA IX with inhibitory constant (KI) value of 2.2 nM. Among the synthesized compounds, 3f
showed IC50 values of 5.03 µg/ml (cisplatin: 6.56 µg/ml), 5.81 µg/ml (cisplatin: 5.85 µg/ml), and
23.93 µg/ml (cisplatin: 2.75 µg/ml) against COLO-205, MDA-MB-231, and DU-145 cell lines,
respectively. At IC50, 3f caused cell shrinkage, nuclear condensation, and nuclear fragmentation
events characteristic to apoptosis in the Hoechst 33258 and acridine orange-ethidium bromide
staining studies of COLO-205 cells. In the Dalton’s lymphoma ascites (DLA) solid tumor model
3f decreased tumor volume by 64.83% (cisplatin: 71.62%), while increase in mean body weight
was found to be only 4.09% (cisplatin: 3.47%).
Key words: Benzenesulfonamide, thiazolidin-4-one, carbonic anhydrase IX, anticancer, cell
lines, Daltion’s lymphoma ascites.
Abbreviations: CA, carbonic anhydrase; HIF-1, hypoxia-inducible transcription factor-1; DLA,
Dalton’s lymphoma ascites; DCC, N,N-dicyclohexylcarbodiimide; MTT, (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; AO, acridine orange; EB, ethidium
bromide; OECD, Organisation for Economic Cooperation and Development.
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1. Introduction
Cancer is a disease of complex etiology and its chemotherapy requires combinations of drugs
which can act simultaneously on different targets [1]. Therefore, development of multifunctional
and multitargeting chemotherapeutic agents is an alternative to drug combination protocols [2,3].
Sulfonamides have been reported to possess antibacterial [4], antifungal [5], insulin releasing [6],
and carbonic anhydrase inhibitory activity [7]. Acetazolamide (Fig. 1), a sulfonamide is a potent
carbonic anhydrase (CA) inhibitor. In the year1992, Pastoreková et al. reported the involvement
of CA IX in cancer [8]. Later, CA XII was also found to be coexpressed with CA IX in several
tumor tissues [9]. The CAs, specifically isozymes CA IX and CA XII regulate pH system by
maintaining intracellular pH while acidifying the extracellular pH compatible for cancer cell
survival and proliferation [10]. The hypoxic condition in solid tumors induces expression of CA
IX [9]. Chronic hypoxia leads to an increased rate of glycolysis through stabilization of hypoxia-
inducible transcription factor-1 (HIF-1) [11] that results into elevated lactic acid production and
drop in the intracellular pH [12]. The CAs sense and maintain the acidic tumor environment; a
fundamental character of solid tumors [13] by catalyzing the reversible hydration of tumor cell-
generated CO2 into bicarbonate anion (HCO3 ) and a proton (H+) [14] and then trap H+
−
extracellularly to lower down the pH [12,15]. Thus, targeting the tumor-associated isozymes
with specific inhibitors is a promising strategy in the cancer therapy [16,17].
Thiazolidin-4-ones have been reported to possess anticancer [18], antibacterial [19], antifungal
[20], anticonvulsant [21], antituberculosis [22], and anti-human immunodeficiency virus type 1
(HIV1) [23] activities. Gududuru et al. reported that thiazolidin-4-one analogues might act as
inducers of apoptosis and selectively kill a variety of prostate cancer cell lines [24]. They used
thiazolidin-4-one moiety as a biomimetic replacement for the phosphate group and synthesized
2-aryl-4-oxo-thiazolidine amide based 2-arylthiazolidine-4-carboxylic acid amides. The lead
compound (Fig. 1) obtained in their study showed IC50 values of 12.6, 11.1, 9.3, 7.1, and 8.5 ꢀM
against DU-145, PC-3, LNCaP, PPC-1, and TSU human prostate cancer cell lines, respectively
[24]. Zhou et al. synthesized thiazolidin-4-one derivatives targeting drug-resistant lung cancer
cells [25]. These compounds selectively killed both non-small cell lung cancer cell line H460
and its paclitaxel-resistant variant H460taxR at an IC50 between 0.21 and 2.93 mM [25]. Based
on above findings, it was of interest to design and synthesize novel hybrid compounds
possessing both sulfonamide and thiazolidin-4-one pharmacophores (Fig. 1) and screen them for
selective CA IX inhibitory activity with anticancer potential.
2. Results and discussion
2.1 Chemistry
All the synthesized compounds were prepared by one pot reaction of sulfanilamide (1) with
various aldehydes (2a-j) and thioglycolic acid in presence of dicyclohexylcarbodiimide (DCC) to
yield a new series of benzenesulfonamides, containing thiazolidin-4-one moiety (3a-j) (Scheme
1) [26]. In the initial step amine reacts with aldehyde to form imine, followed by attack of sulfur
nucleophile (sulfur atom of thioglycolic acid) and subsequent intramolecular cyclization with
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elimination of water yields 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamides. Progress of the
reaction was monitored on pre-coated Merck TLC plates using solvent system acetone:
chloroform (6:4). The synthesized compounds were purified by column chromatography using
silica gel 100-200 mesh and final yield of products ranged from 55% to 86%. Formation of all
newly synthesized products was confirmed by FT-IR, NMR, and Mass spectral studies.
The FT-IR spectra of all the synthesized compounds showed characteristic absorption bands for
N-H, C=O, O=S=O, C=C, C-N, and C-S groups. The amino group present in sulfonamide moiety
was absorbed between 3318.78 and 3325.39 cm-1. The synthesis of thiazolidin-4-one moiety was
evident by presence of vibrational frequency ranged from 1689.70 cm-1 to 1716.70 cm-1
characteristic to 4-C=O group. In all the synthesized compounds O=S=O bonds of sulfonamide
group appeared between 1333.60-1338.77 (asym.) and 1156.20-1159.45 (sym.) cm-1. Bands
corresponding to C=C, C-N, and C-S groups were also observed in the FT-IR spectra. The NMR
spectra of all newly synthesized compounds showed singlet peak for two N-H protons of
sulfonamide group between 12.042 and 12.051ppm. Further, presence of two methylene protons
(-CH2-, 5-Ha and 5-Hb protons) indicates cyclization into thiazolidin-4-one ring. The 5-Ha proton
due to its proximity with carbonyl group appeared downfield to 5-Hb proton either as a double
doublet or as a doublet ranging between 4.033 to 4.112 ppm (J1= 15.2-16 Hz and J2= 0.8 or 1.2
Hz). The 5-Hb proton appeared between 3.848-3.940 ppm as a doublet with J value 15.6-16.4 Hz.
The 2-H proton of thiazolidin-4-one ring was observed as a singlet between 6.631 to 6.677 ppm.
All the aromatic protons appeared between 7.252 to 7.836 ppm. The molecular ion peak (M+)
corresponding to molecular weight was also observed in GC-MS spectra of all the compounds.
2.2. Biological screening
2.2.1. CA enzyme inhibition studies
All the synthesized compounds (3a-j) were assayed for their potential to inhibit physiologically
relevant enzymes ubiquitous CA I and II, and the tumor-associated isoform CA IX by carrying
out stopped-flow assay [27]. Clinically used CA inhibitor acetazolamide was used a reference
drug. The results of CA inhibition assay presented in Table 1 & 2 indicated that: (i) Unlike the
compound 3a, which exhibited potent inhibitory activity (KI of 112.3 nM) against the slow
cytosolic isoform CA I, the congeners 3b-j showed moderate to weak CA I inhibitory activity
with KI in the range of 736.5-1646.6 nM. It indicated that, substitution of halogen atoms in
phenyl ring of benzenesulfonamide derivatives resulted into the dramatic loss of CA I inhibition
activity, (ii) All the compounds showed a better activity profile against the CA II than that of CA
I, (iii) All the tested derivatives exhibited significant CA IX inhibitory activity with KI values
between 2.2 nM and 53.1 nM, while acetazolamide exhibited KI of 25.2 nM. All the
benzenesulfonamide derivatives with halogen groups (3b-j) showed greater CA IX inhibitory
potential than that of compound 3a not possessing the halogen group. The compound 3f bearing
chloro group at m-position of phenyl ring emerged as the most potent inhibitor of CA IX with KI
value of 2.2 nM. Among all the halogenated derivatives, fluoro substituted ones were found to
be least potent inhibitors of CA IX, (iv) A very important aspect of designing CA inhibitors is
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their selectivity toward CA isoforms. It is evident from the Table 2 that newly synthesized
benzenesulfonamide derivatives showed excellent selectivity ratios for inhibiting the tumor-
associated isoform CA IX over the cytosolic CA I and CA II. Indeed, compound 3f exhibited the
selectivity ratios of 381 and 61.6 for inhibiting the tumor-associated isoform CA IX over CA I
and CA II, respectively. Acetazolamide exhibited the selectivity ratios of only 10 and 0.48 for
the inhibition of CA IX over CA I and CA II, respectively. Though, the selectivity ratios of
compounds 3b, 3c, and 3d for CA IX over the CA II were between 0.79 and 0.89; indicating that
these compounds were better inhibitors of CA II than that of CA IX.
2.2.2. In vitro cytotoxicity studies (MTT assay) [28]
All the synthesized compounds were screened for their cytotoxic potential against COLO-205
(human colon adenocarcinoma cell line), MDA-MB-231 (human breast carcinoma cell line), and
DU-145 (human prostate carcinoma cell line) cell lines. In 48 h MTT assay results (Table 3), 3f
emerged as a lead compound with IC50 of 5.03 µg/ml (cisplatin: 6.56 µg/ml), 5.81 µg/ml
(cisplatin: 5.85 µg/ml), and 23.93 µg/ml (cisplatin: 2.75 µg/ml) against COLO-205, MDA-MB-
231, and DU-145 cell lines, respectively. While 3g showed IC50 values of 15.37, 7.16, and 29.28
µg/ml against COLO-205, MDA-MB-231, and DU-145 cell lines, respectively. Results (Table 3)
indicate that halogen substitutions on aryl ring were fruitful and resulted into increased activity.
Chloro substituted compounds were more active than bromo substituted ones, while fluoro
substituted derivatives were least active. Incorporation of halogen atom at m-position in the
derivatives conferred highest activity [3f (3-Cl) > 3i (3-Br) > 3c (3-F)]. The activity pattern
among the synthesized compounds was followed as: m-substitution > p-substitution > o-
substitution. From the above study it can be inferred that: (i) m-substitution was the most
favorable for activity, (ii) Substitution of Chloro group imparted maximum activity, (iii) All the
compounds were moderately potent or potent and no wide range of activity variation was seen;
this can be attributed to the presence of common pharmacophores sulfonamide and thiazolidin-4-
one in all the compounds.
2.2.3. Hoechst 33258 DNA staining to study nuclear morphological changes [29]
DNA-binding dye Hoechst 33258 was used to study nuclear morphological changes associated
with cell death triggered by 3f in COLO-205 cells. This dye is a fluorescent stain used to label
DNA and hence, it is commonly used to visualize nuclei. Hoechst 33258 is excited by ultraviolet
light at 340-380 nm and emits blue-cyan fluorescence at emission maximum of 465 nm. This dye
binds to the minor groove of double-stranded DNA, preferably to the adenine and thymine rich
sequences. The apoptotic cells are more permeable to Hoechst 33258 than living cells, were
identified based on the nuclear condensation, and DNA fragmentation events of Hoechst 33258
stained cells in fluorescent microscope. The results showed that test compound 3f potentially
induced the nuclear condensation and fragmentation process in COLO-205 cells, which is the
one of the characteristic of apoptotic cells (Fig. 2). On the contrary, no morphological changes
were seen in the control cells.
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2.2.4. Acridine orange (AO)-ethidium bromide (EB) cell morphological analysis [30]
Acridine orange-ethidium bromide (AO-EB) staining is used to study cell shrinkage, membrane
blabbing, membrane-bound apoptotic bodies, and nuclear fragmentation of apoptotic cells. AO is
a cell permeable and nucleic acid selective fluorescent dye. It exhibits excitation maximum at
502 nm, emission maximum at 525 nm (green), and when bound to DNA, it emits green
fluorescence. EB is an also fluorescent dye used to detect nucleic acids. It emits orange-red
fluorescence and has excitation maxima of 300-360 nm and an emission maximum of 590 nm.
Both living and dead cells take up AO and appear green if AO is intercalated into double
stranded DNA and red if AO is intercalated into single stranded RNA. While EB is taken up by
dead cells only and it fluoresces orange-red because of its intercalation into the DNA of dead
cells. Results depicted in Fig. 3 indicate that control cells did not take up EB and appeared faint
orange-red. While cells treated with 3f at IC50, showed obvious apoptotic characters (chromatin
condensation or fragmentation) and appeared intense orange-red, as dead cells have ruptured
membrane which allowed EB to enter into the cells. Also due to the AO uptake, control cells
appeared green while 3f treated cells appeared green to intense green as apoptotic cells have
much more permeable membranes. Hence, results of our study provide a morphological proof
that 3f was able to induce apoptosis as a large number of apoptotic cells were observed in AO-
EB dual staining of COLO-205 cells.
2.2.5. DLA-induced solid tumor studies in mice [31-34]
The overexpression of CA-IX is associated with hypoxic condition of solid tumors. Therefore,
we decided to screen 3f against DLA induced solid tumor model in mice.
2.2.5.1. Effect of 3f against DLA induced solid tumor volume in mice
In the DLA induced solid tumor model, maximum increase in tumor volume was observed on
day 30th in the control group. Both the reference drug cisplatin and test compound 3f
significantly reduced development of solid tumor in mice (supplementary data: Fig. 1, Table 1).
The test drug 3f at 50 mg/kg remarkably decreased tumor volume by 64.83% (P < 0.0001), while
cisplatin caused reduction in tumor volume by 71.62% (P < 0.0001).
2.2.5.2. Effect of 3f on body weight changes in DLA induced solid tumor in mice
An inoculation of DLA cells in mice significantly increased the body weight in control group
(supplementary data: Fig. 1, Table 1). Maximum increase in body weight was observed on day
30th. The test compound 3f significantly inhibited raise in body weight and in 3f treated group,
body weight was increased by only 4.09% (P < 0.0001) compared to 13.79% increase observed
in control group. While in cisplatin treated group, body weight was increased by 3.47%.
2.2.6. In silico docking studies [1]
All the designed compounds (3a-j) were docked into the active site of CA IX using Autodock
4.2 software. Virtual screening results show estimated free energy of binding of compounds
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between −6.84 and −7.15 kcal/mol. Further analysis of results indicates that Asn-62, His-64,
Gln-67, Gln-92, and Thr-199 amino acid residues were commonly involved in hydrogen bonding
with ligands. The docking results of the compounds are represented in (supplementary data:
Table 2). Analysis of docked structure of 3f into CA IX active site (Fig. 4 & 5) shows that both
sulfonamide and thiazolidin-4-one moieties have been well accommodated into binding pocket
of CA IX. Enhanced cytotoxicity of 3f may be attributed to the coordination of catalytic Zn (II)
ion by anionic oxygen of thiazolidin-4-one (C=O…Zn2+, 2.3 Å). The carbonyl oxygen of 3f also
interacts with hydroxyl group of Thr-199. The sulfonamide moiety present in the compounds has
played a crucial role in the activity as this functionality is involved in hydrogen bonding with
Asn-62, His-64, and Gln-67 residues of the target protein. In 3f, one of the hydrogens of -
SO2NH2 group binds with hydroxyl oxygen of Asn-62 (N-H…H-O, 2.3 Å), while oxygen atoms
of sulfonamide group form hydrogen bonds with -NH2 of Asn-62 (S=O…H-N, 2.7 Å) and
nitrogen of His-62 (3.0 Å). Interactions other than hydrogen bonding including hydrophobic and
van der Waals were also observed. The most common amino acid residues to participate in such
interactions were Leu-93 and Leu-198. A simplified picture of 3f docked into the active site of
CA IX is depicted in Fig. 6.
3. Conclusion
Our approach to design and synthesize single structural scaffold containing sulfonamide and
thiazolidin-4-one pharmacophores for potent anticancer activity has been met with success.
Virtual screening studies indicated that both of the pharmacophores were involved in interaction
with CA IX and were well accommodated within the active site of target protein. The
sulfonamide pharmacophore interacted with Asn-62, His-64, and Gln-67 residues of CA IX,
while thiazolidin-4-one pharmacophore interacted with target protein by means of hydrogen
bonding through Thr-199 residue of the target. Among the virtually screened compounds, 3f
exhibited the lowest estimated free energy of binding of −7.15 kcal/mol. Molecular docking
analysis showed that the most potent compound 3f was able to coordinate with catalytic Zn (II)
ion that may be attributed to higher binding affinity of 3f toward CA IX. Among the compounds
tested for CA inhibitory activity, 3f displayed the lowest KI value of 2.2 nM toward CA IX and
its selectivity ratio was found to be greater than acetazolamide with respect to inhibition of
cancer-associated CA IX over the inhibition of ubiquitous CA I and CA II. IC50 values of 3f
against COLO-205 and MDA-MB-231 cells were found to be lower than cisplatin. While
compound 3g showed IC50 value close to cisplatin against MDA-MB-231 cells. All the other
compounds also exhibited moderate potent to potent activity. The m-substitution was the most
favorable for activity and substitution of chloro group conferred potency higher than bromo and
fluoro groups. Hoechst 33258 and AO-EB staining studies showed apoptosis mediated cell death.
Potency of 3f in DLA induced solid tumor model in mice was also comparable to cisplatin.
Hence, our assumption in the designing of anticancer scaffold that incorporation of sulfonamide
moiety will cause CA IX inhibition while that of thiazolidin-4-one moiety will act as biomimetic
replacement for the phosphate group; might have played a greater role in the cytotoxicity and
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antitumor activity of 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamides, although the detail
molecular mechanism still remains to be discovered.
4. Experimental
4.1. Chemistry
All the chemicals and reagents were purchased from Spectrochem, Himedia, SD fine chemicals
limited, Aldrich, and Sigma-aldrich, India and were of analytical or reagent grades. The purity of
synthesized compounds was assessed by single spot on pre-coated silica gel plates (TLC silica
gel F254, Merck, Germany). The TLC solvent system used was acetone: chloroform (6:4). IR
spectra were recorded on a Shimadzu FTIR-8310 (Shimadzu, Japan) using potassium bromide
1
discs. H NMR spectra were recorded on a Bruker 400 MHz spectrophotometer (Bruker, USA)
and chemical shifts are presented in parts per million (δ). Tetramethylsilane was used as internal
1
standard in H NMR. Mass spectra were recorded on GC-MS QP 5050, (Shimadzu, Japan).
Elemental analyses were performed on 2400 CHN analyzer (PerkinElmer, USA).
4.2. Synthesis of 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamides (3a-j) [26]
General method: A mixture of amine (1.0 mmol) (1) and aldehyde (1.2 mmol) (2a-j) was stirred
in tetrahydrofuran under ice-cold condition. After 5 min, thioglycolic acid (2.0 mmol) was added
followed by addition of dicyclohexylcarbodiimide (1.2 mmol) at 0 °C and the reaction mixture
was stirred for another 5-6 h at room temperature (RT) to complete the reaction. The
dicyclohexylurea formed during the reaction was filtered off and the filtrate was concentrated to
dryness under reduced pressure. Distilled water was added to the residue and extracted with
ethylacetate. The organic layer was then washed with 5% aqueous sodium hydrogen carbonate
and sodium chloride solutions. Finally organic layer was dried over anhydrous sodium sulphate
and evaporated under reduced pressure to yield solid product which was further purified by using
column chromatography.
4.2.1. 4-(4-Oxo-2-phenylthiazolidin-3-yl)benzenesulfonamide(3a). Yield: 69%. Mp: 165-166 °C.
1
IR (KBr, cm−1): 3321.20 (N-H), 1698.10 (C=O), 1333.60, 1156.20 (S=O). H NMR (DMSO-d6,
δ ppm): 12.050 (s, 2H, SO2NH2), 7.252-7.808 (m, 9H, Ar-H), 6.631 (s, 1H, 2-H of thiazolidinone
ring), 4.033-4.072 (d, J = 15.6 Hz, 1H, 5-Ha), 3.884-3.924 (d, J = 16 Hz, 1H, 5-Hb). GC-MS
(m/z): 334 (M+).
4.2.2. 4-(2-(2-Fluorophenyl)-4-oxothiazolidin-3-yl)benzenesulfonamide(3b). Yield: 82%. Mp:
167-168 °C. IR (KBr, cm−1): 3318.78 (N-H), 1701.08 (C=O), 1336.42, 1158.60 (S=O). 1H NMR
(DMSO-d6, δ ppm): 12.044 (s, 2H, SO2NH2), 7.287-7.820 (m, 8H, Ar-H), 6.660 (s, 1H, 2-H of
thiazolidinone ring), 4.036-4.076 (d, J = 16 Hz, 1H, 5-Ha), 3.900-3.940 (d, J = 15.6 Hz, 1H, 5-
Hb). GC-MS (m/z): 352 (M+).
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4.2.3. 4-(2-(3-Fluorophenyl)-4-oxothiazolidin-3-yl)benzenesulfonamide(3c). Yield: 65%. Mp:
104-105 °C. IR (KBr, cm−1): 3320.19 (N-H), 1690.11 (C=O), 1338.62, 1159.20 (S=O). 1H NMR
(DMSO-d6, δ ppm): 12.047 (s, 2H, SO2NH2), 7.280-7.819 (m, 8H, Ar-H), 6.657 (s, 1H, 2-H of
thiazolidinone ring), 4.037-4.075 (d, J = 15.2 Hz, 1H, 5-Ha), 3.894-3.933 (d, J = 15.6 Hz, 1H, 5-
Hb). GC-MS (m/z): 352 (M+).
4.2.4. 4-(2-(4-Fluorophenyl)-4-oxothiazolidin-3-yl)benzenesulfonamide(3d). Yield: 86%. Mp:
180-181 °C. IR (KBr, cm−1): 3325.39 (N-H), 1689.70 (C=O), 1338.64, 1159.26 (S=O). 1H NMR
(DMSO-d6, δ ppm): 12.042 (s, 2H, SO2NH2), 7.283-7.824 (m, 8H, Ar-H), 6.654 (s, 1H, 2-H of
thiazolidinone ring), 4.034-4.076 (dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.891-3.932 (d, J = 16.4 Hz, 1H,
5-Hb). GC-MS (m/z): 352 (M+).
4.2.5. 4-(2-(2-Chlorophenyl)-4-oxothiazolidin-3-yl)benzenesulfonamide(3e). Yield: 80%. Mp:
204-205 °C. IR (KBr, cm−1): 3322.17 (N-H), 1692.26 (C=O), 1338.77, 1159.39 (S=O). 1H NMR
(DMSO-d6, δ ppm): 12.046 (s, 2H, SO2NH2), 7.306-7.818 (m, 8H, Ar-H), 6.663 (s, 1H, 2-H of
thiazolidinone ring), 4.038-4.079 (dd, J = 1.2, 15.6 Hz, 1H, 5-Ha), 3.875-3.915 (d, J = 16 Hz, 1H,
5-Hb). GC-MS (m/z): 368 (M+).
4.2.6. 4-(2-(3-Chlorophenyl)-4-oxothiazolidin-3-yl)benzenesulfonamide(3f). Yield: 63%. Mp:
155-156 °C. IR (KBr, cm−1): 3320.84 (N-H), 1694.92 (C=O), 1337.10, 1158.62 (S=O). 1H NMR
(DMSO-d6, δ ppm): 12.044 (s, 2H, SO2NH2), 7.312-7.829 (m, 8H, Ar-H), 6.659 (s, 1H, 2-H of
thiazolidinone ring), 4.035-4.075 (d, J = 16 Hz, 1H, 5-Ha), 3.852-3.891 (d, J = 15.6 Hz, 1H, 5-
Hb). GC-MS (m/z): 368 (M+).
4.2.7. 4-(2-(4-Chlorophenyl)-4-oxothiazolidin-3-yl)benzenesulfonamide(3g). Yield: 83%. Mp:
174-175 °C. IR (KBr, cm−1): 3324.40 (N-H), 1698.18 (C=O), 1337.72, 1159.26 (S=O). 1H NMR
(DMSO-d6, δ ppm): 12.045 (s, 2H, SO2NH2), 7.329-7.832 (m, 8H, Ar-H), 6.656 (s, 1H, 2-H of
thiazolidinone ring), 4.040-4.078 (d, J = 16 Hz, 1H, 5-Ha), 3.892-3.932 (d, J = 16 Hz, 1H, 5-Hb).
GC-MS (m/z): 368 (M+).
4.2.8. 4-(2-(2-Bromophenyl)-4-oxothiazolidin-3-yl)benzenesulfonamide(3h). Yield: 69%. Mp:
158-159 °C. IR (KBr, cm−1): 3323.00 (N-H), 1702.26 (C=O), 1336.80, 1157.89 (S=O). 1H NMR
(DMSO-d6, δ ppm): 12.047 (s, 2H, SO2NH2), 7.315-7.819 (m, 8H, Ar-H), 6.677 (s, 1H, 2-H of
thiazolidinone ring), 4.073-4.112 (d, J = 15.6 Hz, 1H, 5-Ha), 3.888-3.927 (d, J = 15.6 Hz, 1H, 5-
Hb). GC-MS (m/z): 413 (M+).
4.2.9. 4-(2-(3-Bromophenyl)-4-oxothiazolidin-3-yl)benzenesulfonamide(3i). Yield: 55%. Mp:
116-117 °C. IR (KBr, cm−1): 3322.95 (N-H), 1695.63 (C=O), 1338.13, 1159.45 (S=O). 1H NMR
(DMSO-d6, δ ppm): 12.051 (s, 2H, SO2NH2), 7.317-7.828 (m, 8H, Ar-H), 6.674 (s, 1H, 2-H of
thiazolidinone ring), 4.071-4.112 (dd, J = 0.8, 16 Hz, 1H, 5-Ha), 3.886-3.925 (d, J = 15.6 Hz, 1H,
5-Hb). GC-MS (m/z): 413 (M+).
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4.2.10. 4-(2-(4-Bromophenyl)-4-oxothiazolidin-3-yl)benzenesulfonamide(3j). Yield: 74%. Mp:
186-187 °C. IR (KBr, cm−1): 3323.46 (N-H), 1716.70 (C=O), 1334.78, 1157.33 (S=O). 1H NMR
(DMSO-d6, δ ppm): 12.049 (s, 2H, SO2NH2), 7.326-7.836 (m, 8H, Ar-H), 6.660 (s, 1H, 2-H of
thiazolidinone ring), 4.067-4.109 (dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.848-3.887 (d, J = 15.6 Hz, 1H,
5-Hb). GC-MS (m/z): 413 (M+).
4.3. Biological screening
4.3.1 CA enzyme inhibition assay [27]
An Applied Photophysics stopped-flow instrument was used to assay the inhibition of various
CA isozymes (CA catalysed CO2 hydration activity). Phenol red (at a concentration of 0.2 mM)
was used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH
7.5) as buffer and 0.1 M Na2SO4 for maintaining constant the ionic strength. The rate of the CA-
catalyzed CO2 hydration reaction was monitored for a period of 10-100 s. The CO2
concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and
inhibition constants. Stock solutions of inhibitors (10 mM) were prepared in distilled-deionized
water with 10-20 % (v/v) DMSO and dilutions up to 0.1 nM were made thereafter with distilled–
deionized water. Inhibitor and enzyme solutions were preincubated together for 15 min at room
temperature prior to assay, in order to allow for the formation of the enzyme-inhibitor complex.
The experiments were done in triplicate for each inhibitor concentration, and the values reported
in the paper are the mean of such results. The IC50 values were obtained by nonlinear least-
squares methods using PRISM 3 and represented the mean from at least three different
determinations. Enzyme concentrations in the assay system were: 9.2 nM for CA I, 7.6 nM for
CA II, and 12 nM for CA IX.
4.3.2. In vitro anticancer activity
4.3.2.1. Cell lines
Three human carcinoma causing cell lines viz. COLO-205, MDA-MB-231, and DU-145 were
procured from National Centre for Cell Science, Pune, India. The all three cell lines were
cultured in the RPMI-1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/l sodium
bicarbonate. The medium was supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml
streptomycin. Cells were incubated at 37 °C under 5% CO2 humidified atmosphere. Dalton’s
lymphoma ascites (DLA) cells were maintained and propagated by serial intraperitonial
transplantation in adult male Swiss albino mice.
4.3.2.2. In vitro cytotoxicity study (MTT assay) [28]
All the newly synthesized compounds were screened for in vitro cytotoxicity by MTT assay
method. In brief, exponentially growing COLO-205, MDA-MB-231, and DU-145 cells were
seeded into 96-well plates (104 cells/well in 100 µl of media) and incubated for 24 h to obtain 70-
80% confluency. The compounds to be screened were dissolved into 50 µl DMSO and were
further diluted with media so as to get DMSO concentration below 0.25%.The cells were then
9

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treated with varying concentration (250, 125, 62.5, 31.25, 15.62, and 7.81 µg/ml) of test
compounds (100 µl/well). After 48 h, media was discarded and 100 µl MTT reagents (1 mg/ml)
was added to cell culture and then incubated for 4 h at 37 °C. The control wells contained cells
with media and 0.25% DMSO. Standard drug cisplatin was used as a positive control. The viable
cells developed formazan complex which was solubilized by adding 100 µl DMSO. The plates
were then kept on micro-vibrator for 5 min. The absorbance was measured on BIOTEK EL
X800- MS microtiter plate reader at 540 nm and percentage cytotoxicity was calculated as
(control − test / control) × 100.
4.3.2.3. Hoechst 33258 staining to study nuclear morphological changes [29]
COLO-205 cells (105 cells/ml) were plated into 12-well plate. When cells achieved 70-80%
confluency, old media was discarded from wells and cells were treated with IC50 of 3f and
incubated for 48 h. After this, cells were fixed with 4% formaldehyde for 30 min at RT and then
stained with Hoechst dye for 15 min at RT. Cells were then washed thrice with PBS and
examined under a fluorescence inverted microscope at excitation of 340-380 nm with emission
of 465 nm.
4.3.2.4. Acridine orange-ethidium staining [30]
Cell death analysis was performed by AO-EB dual staining assay. COLO-205 cells (105 cells/
ml) were seeded into 12-well plate and incubated for 24 h. Cells were then treated with IC50 of 3f
for 48 h. Cell suspension was then transferred to new vial, trypsinized and centrifuged at 129 g
for 5 min. Supernatant obtained was discarded and the pellet remained behind was washed with
PBS and centrifuged again. Finally, cells were stained with 1:1 ratio of AO-EB mixture and
examined under fluorescence microscope.
4.3.3. In vivo anticancer activity
4.3.3.1. Animals
The animal study protocol was approved by the Institutional Animal Ethics Committee (IAEC)
and complied with the NIH guidelines on handling of experimental animals. Six to eight week
old Swiss albino mice weighing 29 ± 2 g were selected from an inbred colony maintained under
the controlled conditions of temperature (30 ± 3 °C), humidity (60 ± 5%), and light (13 and 11 h
of light and dark, respectively). Four animals were housed in each polypropylene cage
containing paddy husk as bedding. The mice were provided with standard mice food pellets and
water ad libitum.
4.3.3.2. Toxicological study
Safe dose was calculated as per OECD test guideline 425 [35].
4.3.3.3. Preparation of test solution of compounds for animal activity
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The compound 3f was tested at the dose of 50 mg/kg body weight. The cisplatin was given as 3.5
mg/kg, i.p. The solution of test compound 3f was prepared by suspending it in the 0.25%
carboxymethyl cellulose (CMC).
4.3.3.4. DLA-induced solid tumor studies [31-34]
Solid tumor was induced in mice by injecting Dalton’s lymphoma ascites cells (1 × 106
cells/mouse) subcutaneously into the right hind limb of male Swiss albino mice. The animals
were grouped randomly immediately after tumor inoculation as: Group A: animals treated with
vehicle, Group B: animals treated with Cisplatin (3.5 mg/kg, i.p.), and Group C: animals treated
with test compound 3f (50 mg/kg, p.o). Cisplatin (single dose, 3.5 mg/kg, i.p.) was administered
on day 3rd which served as standard drug, while test compound 3f was administered on days 3rd,
5th, 7th, 10th, 12th, and 14th of tumor inoculation. Tumor progression was checked by
measuring tumor volume and body weight at the different intervals of time. Initial diameter of
the right hind limb was recorded using vernier caliper. From the 3rd day onwards, tumor
diameter was recorded on every third day and volume was calculated using the formula: V =
2
4/3πr1 × r2, where V is volume, r1 and r2 represent the radii of the tumor at two different planes.
Percentage inhibition in tumor volume was calculated by using formula: [(Tumor volume of
control animal on day 30th – Tumor volume of treated animal on day 30th) / (Tumor volume of
control animal on day 30th)] × 100. The animals were weighed on day 30th and the percentage
increase in weight was calculated using the formula: % Increase in weight = [(animal weight on
day 30th / animal weight on day 0) − 1] × 100 [31-34].
4.4.3.5. Statistical analysis
The statistical analyses were performed by one-way ANOVA followed by Tukey’s post hoc test
using GraphPad Prism 6. The results were expressed as the mean ± SEM. Statistical significance
was set at P < 0.05 level.
4.5. Molecular docking studies [1]
The 3D structures of ligands in pdb form were prepared by using CS ChemDraw Ultra 8.0. The
3D crystal structure of the catalytic domain of the tumor-associated human carbonic anhydrase
IX was obtained from Protein Data Bank (PDB ID: 3IAI). Acetazolamide, other hetero atoms,
and water molecules were removed and hydrogens were added to the protein for correct
calculation of partial atomic charges. The AutoDock 4.2 software was used for virtual screening
studies. The grid box was set at 40, 40, and 40 Å (x, y, and z) with grid point spacing of 0.375 Å.
The Lamarckian genetic algorithm (LGA) was used to search conformers with lowest binding
energy. The results of docking studies are expressed as estimated free energy of binding in
kcal/mol (supplementary data: Table 2).
Acknowledgements
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The authors thank Indian Institute of Science, Bangalore, India for recording NMR spectra and
SAIF labs of Central Drug Research Institute, Lucknow and Panjab University, Chandigarh,
India for carrying out NMR spectra and CHN analysis.
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Scheme 1. Synthesis of 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamides (3a-j).
Fig. 1. Pharmacophoric similarity of acetazolamide (potent carbonic anhydrase inhibitor) and 2-
aryl-4-oxo-thiazolidine amide (potent cytotoxic agent) with newly synthesized compound 3f.
Fig. 2. Hoechst 33258 DNA staining for the effects of 3f on nuclear morphology of COLO-205
cells. Cells were incubated with control (c) and IC50 dose of 3f for 48 h (CC: control cells, NC:
nuclear condensation, NF: nuclear fragmentation).
Fig. 3. Apoptosis analysis of COLO-205 cells treated with control (c) and IC50 dose of 3f for 48
h. Control cells take up AO and appear green, while exclude EB and appear orange-red. Cells
treated with 3f take up both AO and EB and appear intense green and intense orange-red,
respectively (apoptotic or nonviable cells have more permeable or ruptured membranes and
stained by both AO and EB). Nuclear condensation (NC) and nuclear fragmentation (NF) are
evident in the figure.
Fig. 4. Docking of compound 3f within the CA IX active site. Amino acid residues and Zn (II)
ion (grayish sphere) interact with inhibitor is evident in figure.
Fig. 5. Orientation of 3f in the active site of CA IX.
Fig. 6. Simplified presentation of 3f docked into the active site of CA IX. Active amino acid
residues surrounding 3f and hydrogen bonds in dashed lines are depicted in figure.
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Highlights
ꢀ Novel 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamides were synthesized.
ꢀ Compounds were evaluated for carbonic anhydrase inhibitory activity.
ꢀ Cytotoxicity evaluation was performed against three cancer cell lines.
ꢀ Hoechst 33258 and AO-EB staining studies were carried out in COLO-205 cells.
ꢀ Compound 3f was evaluated in the DLA induced solid tumor model in mice.

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Table 1
Carbonic anhydrase inhibitory activity of test compounds and reference inhibitor
Compound
KI (nM) ± SD
CA II
CA I
CA IX
3a
3b
3c
3d
3e
3f
3g
3h
112.3 ± 2.2
1036.7 ± 5.5
1646.6 ± 6.6
1239.2 ± 6.9
736.5 ± 7.5
838.3 ± 4.4
742.3 ± 6.3
936.4 ± 3.3
1247.9 ± 5.9
1112.5 ± 6.7
252.2 ± 5.2
56.7 ± 2.6
35.4 ± 1.5
45.2 ± 1.9
33.1 ± 2.3
42.6 ± 3.8
135.5 ± 3.5
38.7 ± 2.6
31.8 ± 2.1
121.6 ± 3.9
28.4 ± 2.1
12.5 ± 1.1
21.2 ± 0.34
44.4 ± 0.62
53.1 ± 0.21
37.4 ± 0.15
7.7 ± 0.14
2.2 ± 0.25
4.7 ± 0.04
9.2 ± 0.92
19.4 ± 0.61
13.5 ± 0.52
25.2 ± 1.6
3i
3j
Acetazolamide

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Table 2
Selectivity ratios of test compounds and reference CA inhibitor for the inhibition of tumor-
associated (CA IX) over the cytosolic (CA I and II) isozymes
Compound
Selectivity ratio
CA I/CA IX
CA II/CA IX
2.7
3a
3b
3c
3d
3e
3f
3g
3h
5.3
23.3
31.0
33.1
95.7
381.0
157.9
101.8
109.5
82.4
10
0.79
0.85
0.89
5.5
61.6
8.23
3.45
6.3
3i
3j
2.1
0.48
Acetazolamide

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Table 3
IC50 of test compounds towards various cell lines by MTT assay method, after 48 h
Compound
IC50 µg/ml ± SD
MDA-MB-231
23.87 ± 0.50
25.86 ± 0.57
21.36 ± 0.89
22.69 ± 0.42
13.57 ± 0.11
5.81 ± 0.34
COLO-205
30.59 ± 0.81
34.69 ± 0.10
29.84 ± 0.74
31.36 ± 0.10
19.51 ± 0.15
5.03 ± 0.74
15.37 ± 0.46
27.47 ± 0.23
16.92 ± 0.12
23.05 ± 0.90
6.56 ± 0.24
DU-145
3a
3b
3c
3d
3e
3f
3g
3h
3i
39.24 ± 0.34
38.60 ± 0.62
39.15 ± 0.21
37.07 ± 0.15
33.27 ± 0.14
23.93 ± 0.25
29.28 ± 0.04
35.02 ± 0.92
30.14 ± 0.61
35.09 ± 0.52
2.75 ± 0.08
7.16 ± 0.96
20.63 ± 0.02
12.39 ± 0.83
17.48 ± 0.13
5.85 ± 0.09
3j
Cisplatin

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Design and synthesis of novel 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamides as selective inhibitors of carbonic
anhydrase IX over I and II with potential anticancer activity
Sharad Kumar Suthara,*, Sumit Bansala, Sandeep Lohana, Vikarm Modakb, Anil Chaudharyc,
Amit Tiwarid
aDepartment of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, Manipal-576104, India
bDepartment of Pharmaceutical Chemistry, Maharashtra Institute of Pharmacy, Pune-411038, India
cMahakal Institute of Pharmaceutical Studies, Ujjain-456664, India
dDepartment of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal-576104, India
*Corresponding author. Tel.: +91 8894861073; fax: +91 820-2571998; E-mail address: sharadpune_2009@yahoo.com.
2.2.5. DLA-induced solid tumor studies in mice
Table 1
Effect of 3f on tumor volume and body weight changes in DLA cells induced solid tumor in mice
Group
Control
3f
Dose (mg/kg) % reduction in tumor volume % increase in body weight
13.92 ± 1.50
50
3.5
64.83 ± 1.07a
71.62 ± 1.77a
4.15 ± 1.10a
3.46 ± 0.86a
Cisplatin
All the values are mean ± SEM of six mice. a P < 0.0001, compared to control.
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Fig. 1. Effect of 3f on tumor volume and body weight changes in DLA cells induced solid tumor in mice. All the values are mean ±
SEM of six mice. ****P < 0.0001, compared to control.
2