European Journal of Medicinal Chemistry 66 (2013) 372e379

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European Journal of Medicinal Chemistry

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Short communication

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 a, *, Sumit Bansal a, Sandeep Lohan a, Vikarm Modak b,
Anil Chaudhary c, Amit Tiwari d
a
Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, Manipal 576104, Karnataka, India
b
Department of Pharmaceutical Chemistry, Maharashtra Institute of Pharmacy, Pune 411038, India
c
Mahakal Institute of Pharmaceutical Studies, Ujjain 456664, India
d
Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal 576104, India

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

Article history: The novel 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamide derivatives were designed and synthe-
Received 9 December 2012 sized for selective carbonic anhydrase IX (CA IX) inhibitory activity with anticancer potential. In the CA
Received in revised form inhibition assay, 3f was found to be the most potent and selective inhibitor of CA IX with inhibitory
31 May 2013
constant (KI) value of 2.2 nM. Among the synthesized compounds, 3f showed IC50 values of 5.03 mg/ml
Accepted 2 June 2013
Available online 12 June 2013
(cisplatin: 6.56 mg/ml), 5.81 mg/ml (cisplatin: 5.85 mg/ml), and 23.93 mg/ml (cisplatin: 2.75 mg/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
Keywords:
Benzenesulfonamide
acridine orange-ethidium bromide staining studies of COLO-205 cells. In the Dalton’s lymphoma ascites
Thiazolidin-4-one (DLA) solid tumor model 3f decreased tumor volume by 64.83% (cisplatin: 71.62%), while increase in
Carbonic anhydrase IX mean body weight was found to be only 4.09% (cisplatin: 3.47%).
Anticancer Ó 2013 Elsevier Masson SAS. All rights reserved.
Cell lines
Daltion’s lymphoma ascites

1. Introduction in several tumor tissues [9]. The CAs, specifically isozymes CA IX

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
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.
* Corresponding author. Tel.: þ91 8894861073; fax: þ91 820 2571998.
E-mail address: sharadpune_2009@yahoo.com (S.K. Suthar).
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 tu-
mors 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 (HCO
þ þ
3 ) and a proton (H ) [14] and then trap H extracellu-
larly to lower down the pH [12,15]. Thus, targeting the tumor-
associated isozymes with specific inhibitors is a promising strat-
egy in the cancer therapy [16,17].
Thiazolidin-4-ones have been reported to possess anticancer
[18], antibacterial [19], antifungal [20], anticonvulsant [21], anti-
tuberculosis [22], and anti-human immunodeficiency virus type 1
(HIV1) [23] activities. Gududuru et al. reported that thiazolidin-4-
one analogs might act as inducers of apoptosis and selectively kill

0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.003
 S.K. Suthar et al. / European Journal of Medicinal Chemistry 66 (2013) 372e379 373

reaction was monitored on pre-coated Merck TLC plates using

Fig. 1. Pharmacophoric similarity of acetazolamide (potent carbonic anhydrase in-
hibitor) and 2-aryl-4-oxo-thiazolidine amide (potent cytotoxic agent) with newly
synthesized compound 3f.
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 mM 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 re-
action of sulfanilamide (1) with various aldehydes (2aej) and thi-
oglycolic acid in presence of dicyclohexylcarbodiimide (DCC) to
yield a new series of benzenesulfonamides, containing thiazolidin-
4-one moiety (3aej) (Scheme 1) [26]. In the initial step amine re-
acts with aldehyde to form imine, followed by attack of sulfur
nucleophile (sulfur atom of thioglycolic acid) and subsequent
intramolecular cyclization with elimination of water yields 4-(4-
oxo-2-arylthiazolidin-3-yl)benzenesulfonamides. Progress of the
solvent system acetone:chloroform (6:4). The synthesized com-
pounds were purified by column chromatography using silica gel
100e200 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 NeH, C]O, O]S]O, C]C, Ce
N, and CeS 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 character-
istic to 4-C]O group. In all the synthesized compounds O]S]O
bonds of sulfonamide group appeared between 1333.60e1338.77
(asym.) and 1156.20e1159.45 (sym.) cm
1. Bands corresponding to
C]C, CeN, and CeS groups were also observed in the FT-IR spectra.
The NMR spectra of all newly synthesized compounds showed
singlet peak for two NeH protons of sulfonamide group between
12.042 and 12.051 ppm. Further, presence of two methylene pro-
tons (eCH2e, 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 and
4.112 ppm (J1 ¼ 15.2e16 Hz and J2 ¼ 0.8 or 1.2 Hz). The 5-Hb proton
appeared between 3.848 and 3.940 ppm as a doublet with J value
15.6e16.4 Hz. The 2-H proton of thiazolidin-4-one ring was
observed as a singlet between 6.631 and 6.677 ppm. All the aro-
matic protons appeared between 7.252 and 7.836 ppm. The mo-
lecular ion peak (Mþ) corresponding to molecular weight was also
observed in GCeMS spectra of all the compounds.
2.2. Biological screening
2.2.1. CA enzyme inhibition studies
All the synthesized compounds (3aej) 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 as reference drug. The results of CA inhibition assay
presented in Tables 1 and 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 3bej showed
moderate to weak CA I inhibitory activity with KI in the range of
736.5e1646.6 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
Table 1
Carbonic anhydrase inhibitory activity of test compounds and reference inhibitor.

Compound KI (nM)  SD

CA I CA II CA IX

3a 112.3  2.2 56.7  2.6 21.2  0.34

3b 1036.7  5.5 35.4  1.5 44.4  0.62
3c 1646.6  6.6 45.2  1.9 53.1  0.21
3d 1239.2  6.9 33.1  2.3 37.4  0.15
3e 736.5  7.5 42.6  3.8 7.7  0.14
3f 838.3  4.4 135.5  3.5 2.2  0.25
3g 742.3  6.3 38.7  2.6 4.7  0.04
3h 936.4  3.3 31.8  2.1 9.2  0.92
3i 1247.9  5.9 121.6  3.9 19.4  0.61
3j 1112.5  6.7 28.4  2.1 13.5  0.52
Acetazolamide 252.2  5.2 12.5  1.1 25.2  1.6
Scheme 1. Synthesis of 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamides (3aej).
374 S.K. Suthar et al. / European Journal of Medicinal Chemistry 66 (2013) 372e379

Table 2 205, MDA-MB-231, and DU-145 cell lines, respectively. Results

Selectivity ratios of test compounds and reference CA inhibitor for the inhibition of (Table 3) indicate that halogen substitutions on aryl ring were
tumor-associated (CA IX) over the cytosolic (CA I and II) isozymes.
fruitful and resulted into increased activity. Chloro substituted
Compound Selectivity ratio compounds were more active than bromo substituted ones, while
CA I/CA IX CA II/CA IX fluoro substituted derivatives were least active. Incorporation of
halogen atom at m-position in the derivatives conferred highest
3a 5.3 2.7
3b 23.3 0.79 activity [3f (3-Cl) > 3i (3-Br) > 3c (3-F)]. The activity pattern among
3c 31.0 0.85 the synthesized compounds was followed as: m-substitution > p-
3d 33.1 0.89 substitution > o-substitution. From the above study it can be
3e 95.7 5.5
inferred that: (i) m-substitution was the most favorable for activity,
3f 381.0 61.6
3g 157.9 8.23 (ii) Substitution of chloro group imparted maximum activity, (iii)
3h 101.8 3.45 All the compounds were moderately potent or potent and no wide
3i 109.5 6.3 range of activity variation was seen; this can be attributed to the
3j 82.4 2.1 presence of common pharmacophores sulfonamide and thiazoli-
Acetazolamide 10 0.48
din-4-one in all the compounds.

2.2.3. Hoechst 33258 DNA staining to study nuclear morphological

activity with KI values between 2.2 nM and 53.1 nM, while acet- changes [29]
azolamide exhibited KI of 25.2 nM. All the benzenesulfonamide DNA-binding dye Hoechst 33258 was used to study nuclear
derivatives with halogen groups (3bej) showed greater CA IX morphological changes associated with cell death triggered by 3f in
inhibitory potential than that of compound 3a not possessing the COLO-205 cells. This dye is a fluorescent stain used to label DNA and
halogen group. The compound 3f bearing chloro group at m-posi- hence, it is commonly used to visualize nuclei. Hoechst 33258 is
tion of phenyl ring emerged as the most potent inhibitor of CA IX excited by ultraviolet light at 340e380 nm and emits blueecyan
with KI value of 2.2 nM. Among all the halogenated derivatives, fluorescence at emission maximum of 465 nm. This dye binds to the
fluoro substituted ones were found to be least potent inhibitors of minor groove of double-stranded DNA, preferably to the adenine
CA IX, (iv) A very important aspect of designing CA inhibitors is and thymine rich sequences. The apoptotic cells are more perme-
their selectivity toward CA isoforms. It is evident from Table 2 that able to Hoechst 33258 than living cells, were identified based on
newly synthesized benzenesulfonamide derivatives showed the nuclear condensation, and DNA fragmentation events of
excellent selectivity ratios for inhibiting the tumor-associated iso- Hoechst 33258 stained cells in fluorescent microscope. The results
form CA IX over the cytosolic CA I and CA II. Indeed, compound 3f showed that test compound 3f potentially induced the nuclear
exhibited the selectivity ratios of 381 and 61.6 for inhibiting the condensation and fragmentation process in COLO-205 cells, which
tumor-associated isoform CA IX over CA I and CA II, respectively. is the one of the characteristics of apoptotic cells (Fig. 2). On the
Acetazolamide exhibited the selectivity ratios of only 10 and 0.48 contrary, no morphological changes were seen in the control cells.
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 2.2.4. Acridine orange (AO)eethidium bromide (EB) cell
CA II were between 0.79 and 0.89; indicating that these compounds morphological analysis [30]
were better inhibitors of CA II than that of CA IX. Acridine orangeeethidium bromide (AOeEB) staining is used to
study cell shrinkage, membrane blabbing, membrane-bound
2.2.2. In vitro cytotoxicity studies (MTT assay) [28] apoptotic bodies, and nuclear fragmentation of apoptotic cells. AO
All the synthesized compounds were screened for their cyto- is a cell permeable and nucleic acid selective fluorescent dye. It
toxic potential against COLO-205 (human colon adenocarcinoma exhibits excitation maximum at 502 nm, emission maximum at
cell line), MDA-MB-231 (human breast carcinoma cell line), and 525 nm (green), and when bound to DNA, it emits green fluores-
DU-145 (human prostate carcinoma cell line) cell lines. In 48 h MTT cence. EB is an also fluorescent dye used to detect nucleic acids. It
assay results (Table 3), 3f emerged as a lead compound with IC50 of emits orangeered fluorescence and has excitation maxima of 300e
5.03 mg/ml (cisplatin: 6.56 mg/ml), 5.81 mg/ml (cisplatin: 5.85 mg/ 360 nm and an emission maximum of 590 nm. Both living and dead
ml), and 23.93 mg/ml (cisplatin: 2.75 mg/ml) against COLO-205, cells take up AO and appear green if AO is intercalated into double
MDA-MB-231, and DU-145 cell lines, respectively. While 3g stranded DNA and red if AO is intercalated into single stranded
showed IC50 values of 15.37, 7.16, and 29.28 mg/ml against COLO- RNA. While EB is taken up by dead cells only and it fluoresces or-
angeered 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 orangeered. While cells treated with 3f at
Table 3
IC50 of test compounds toward various cell lines by MTT assay method, after 48 h.
IC50 showed obvious apoptotic characters (chromatin condensation
or fragmentation) and appeared intense orangeered, as dead cells
Compound IC50 mg/ml  SD
have ruptured membrane which allowed EB to enter into the cells.
COLO-205 MDA-MB-231 DU-145 Also due to the AO uptake, control cells appeared green while 3f
3a 30.59  0.81 23.87  0.50 39.24  0.34 treated cells appeared green to intense green as apoptotic cells have
3b 34.69  0.10 25.86  0.57 38.60  0.62 much more permeable membranes. Hence, results of our study
3c 29.84  0.74 21.36  0.89 39.15  0.21 provide a morphological proof that 3f was able to induce apoptosis
3d 31.36  0.10 22.69  0.42 37.07  0.15
as a large number of apoptotic cells were observed in AOeB dual
3e 19.51  0.15 13.57  0.11 33.27  0.14
3f 5.03  0.74 5.81  0.34 23.93  0.25 staining of COLO-205 cells.
3g 15.37  0.46 7.16  0.96 29.28  0.04
3h 27.47  0.23 20.63  0.02 35.02  0.92 2.2.5. DLA-induced solid tumor studies in mice [31e34]
3i 16.92  0.12 12.39  0.83 30.14  0.61 The overexpression of CA-IX is associated with hypoxic condi-
3j 23.05  0.90 17.48  0.13 35.09  0.52
Cisplatin 6.56  0.24 5.85  0.09 2.75  0.08
tion of solid tumors. Therefore, we decided to screen 3f against DLA
induced solid tumor model in mice.
 S.K. Suthar et al. / European Journal of Medicinal Chemistry 66 (2013) 372e379 375

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).

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 (3aej) were docked into the
active site of CA IX using Autodock 4.2 software. Virtual screening
results showed estimated free energy of binding of compounds
between
6.84 and
7.15 kcal/mol. Further analysis of results in-
dicates that Asn-62, His-64, Gln-67, Gln-92, and Thr-199 amino acid
residues were commonly involved in hydrogen bonding with li-
gands. The docking results of the compounds are represented in
Table 2 of Supplementary data. Analysis of docked structure of 3f
into CA IX active site (Figs. 4 and 5) shows that both sulfonamide
and thiazolidin-4-one moieties have been well accommodated into

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 orangee
red. Cells treated with 3f take up both AO and EB and appear intense green and intense orangeered, 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. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
376 S.K. Suthar et al. / European Journal of Medicinal Chemistry 66 (2013) 372e379
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.
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/Zn2þ, 2.3  A). The carbonyl ox-
ygen of 3f also interacts with hydroxyl group of Thr-199. The sul-
fonamide 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 eSO2NH2 group binds with
hydroxyl oxygen of Asn-62 (NeH/HeO, 2.3  A), while oxygen
atoms of sulfonamide group form hydrogen bonds with eNH2 of
Asn-62 (S]O/HeN, 2.7  A) and nitrogen of His-62 (3.0  A). In-
teractions 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.
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.
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 pharma-
cophore 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 com-
pound 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 AOeEB 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 incorpora-
tion 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 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 Spec-
trochem, Himedia, SD fine chemicals limited, Aldrich, and Sigmae
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 sol-
vent system used was acetone:chloroform (6:4). IR spectra were
 S.K. Suthar et al. / European Journal of Medicinal Chemistry 66 (2013) 372e379
recorded on a Shimadzu FTIR-8310 (Shimadzu, Japan) using po-
tassium bromide discs. 1H NMR spectra were recorded on a Bruker
400 MHz spectrophotometer (Bruker, USA) and chemical shifts are
presented in parts per million (d). Tetramethylsilane was used as
internal standard in 1H NMR. Mass spectra were recorded on GCe
MS QP 5050, (Shimadzu, Japan). Elemental analyses were per-
formed on 2400 CHN analyzer (PerkinElmer, USA).
4.2. Synthesis of 4-(4-oxo-2-arylthiazolidin-3-yl)
benzenesulfonamides (3aej) [26]
General method: A mixture of amine (1.0 mmol) (1) and alde-
hyde (1.2 mmol) (2aej) 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
5e6 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 eth-
ylacetate. The organic layer was then washed with 5% aqueous
sodium hydrogen carbonate and sodium chloride solutions. Finally,
organic layer was dried over anhydrous sodium sulfate and evap-
orated 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: 165e166  C. IR (KBr, cm
1): 3321.20 (NeH),
1698.10 (C]O), 1333.60, 1156.20 (S]O). 1H NMR (DMSO-d6,
d ppm): 12.050 (s, 2H, SO2NH2), 7.252e7.808 (m, 9H, AreH), 6.631
(s, 1H, 2-H of thiazolidinone ring), 4.033e4.072 (d, J ¼ 15.6 Hz, 1H,
5-Ha), 3.884e3.924 (d, J ¼ 16 Hz, 1H, 5-Hb). GCeMS (m/z): 334 (Mþ).
4.2.2. 4-(2-(2-Fluorophenyl)-4-oxothiazolidin-3-yl)
benzenesulfonamide (3b)
Yield: 82%. Mp: 167e168  C. IR (KBr, cm
1): 3318.78 (NeH),
1701.08 (C]O), 1336.42, 1158.60 (S]O). 1H NMR (DMSO-d6,
d ppm): 12.044 (s, 2H, SO2NH2), 7.287e7.820 (m, 8H, AreH), 6.660
(s, 1H, 2-H of thiazolidinone ring), 4.036e4.076 (d, J ¼ 16 Hz, 1H, 5-
Ha), 3.900e3.940 (d, J ¼ 15.6 Hz, 1H, 5-Hb). GCeMS (m/z): 352 (Mþ).
4.2.3. 4-(2-(3-Fluorophenyl)-4-oxothiazolidin-3-yl)
benzenesulfonamide (3c)
Yield: 65%. Mp: 104e105  C. IR (KBr, cm
1): 3320.19 (NeH),
1690.11 (C]O), 1338.62, 1159.20 (S]O). 1H NMR (DMSO-d6,
d ppm): 12.047 (s, 2H, SO2NH2), 7.280e7.819 (m, 8H, AreH), 6.657
(s, 1H, 2-H of thiazolidinone ring), 4.037e4.075 (d, J ¼ 15.2 Hz, 1H,
5-Ha), 3.894e3.933 (d, J ¼ 15.6 Hz, 1H, 5-Hb). GCeMS (m/z):
352 (Mþ).
4.2.4. 4-(2-(4-Fluorophenyl)-4-oxothiazolidin-3-yl)
benzenesulfonamide (3d)
Yield: 86%. Mp: 180e181  C. IR (KBr, cm
1): 3325.39 (NeH),
1689.70 (C]O), 1338.64, 1159.26 (S]O). 1H NMR (DMSO-d6,
d ppm): 12.042 (s, 2H, SO2NH2), 7.283e7.824 (m, 8H, AreH), 6.654
(s, 1H, 2-H of thiazolidinone ring), 4.034e4.076 (dd, J ¼ 1.2, 16 Hz,
1H, 5-Ha), 3.891e3.932 (d, J ¼ 16.4 Hz, 1H, 5-Hb). GCeMS (m/z):
352 (Mþ).
4.2.5. 4-(2-(2-Chlorophenyl)-4-oxothiazolidin-3-yl)
benzenesulfonamide (3e)
Yield: 80%. Mp: 204e205  C. IR (KBr, cm
1): 3322.17 (NeH),
1692.26 (C]O), 1338.77, 1159.39 (S]O). 1H NMR (DMSO-d6,
d ppm): 12.046 (s, 2H, SO2NH2), 7.306e7.818 (m, 8H, AreH), 6.663
(s, 1H, 2-H of thiazolidinone ring), 4.038e4.079 (dd, J ¼ 1.2, 15.6 Hz,
377
1H, 5-Ha), 3.875e3.915 (d, J ¼ 16 Hz, 1H, 5-Hb). GCeMS (m/z):
368 (Mþ).
4.2.6. 4-(2-(3-Chlorophenyl)-4-oxothiazolidin-3-yl)
benzenesulfonamide (3f)
Yield: 63%. Mp: 155e156  C. IR (KBr, cm
1): 3320.84 (NeH),
1694.92 (C]O), 1337.10, 1158.62 (S]O). 1H NMR (DMSO-d6, d ppm):
12.044 (s, 2H, SO2NH2), 7.312e7.829 (m, 8H, AreH), 6.659 (s, 1H, 2-
H of thiazolidinone ring), 4.035e4.075 (d, J ¼ 16 Hz, 1H, 5-Ha),
3.852e3.891 (d, J ¼ 15.6 Hz, 1H, 5-Hb). GCeMS (m/z): 368 (Mþ).
4.2.7. 4-(2-(4-Chlorophenyl)-4-oxothiazolidin-3-yl)
benzenesulfonamide (3g)
Yield: 83%. Mp: 174e175  C. IR (KBr, cm
1): 3324.40 (NeH),
1698.18 (C]O), 1337.72, 1159.26 (S]O). 1H NMR (DMSO-d6, d ppm):
12.045 (s, 2H, SO2NH2), 7.329e7.832 (m, 8H, AreH), 6.656 (s, 1H, 2-
H of thiazolidinone ring), 4.040e4.078 (d, J ¼ 16 Hz, 1H, 5-Ha),
3.892e3.932 (d, J ¼ 16 Hz, 1H, 5-Hb). GCeMS (m/z): 368 (Mþ).
4.2.8. 4-(2-(2-Bromophenyl)-4-oxothiazolidin-3-yl)
benzenesulfonamide (3h)
Yield: 69%. Mp: 158e159  C. IR (KBr, cm
1): 3323.00 (NeH),
1702.26 (C]O), 1336.80, 1157.89 (S]O). 1H NMR (DMSO-d6,
d ppm): 12.047 (s, 2H, SO2NH2), 7.315e7.819 (m, 8H, AreH), 6.677
(s, 1H, 2-H of thiazolidinone ring), 4.073e4.112 (d, J ¼ 15.6 Hz, 1H, 5-
Ha), 3.888e3.927 (d, J ¼ 15.6 Hz, 1H, 5-Hb). GCeMS (m/z): 413 (Mþ).
4.2.9. 4-(2-(3-Bromophenyl)-4-oxothiazolidin-3-yl)
benzenesulfonamide (3i)
Yield: 55%. Mp: 116e117  C. IR (KBr, cm
1): 3322.95 (NeH),
1695.63 (C]O), 1338.13, 1159.45 (S]O). 1H NMR (DMSO-d6,
d ppm): 12.051 (s, 2H, SO2NH2), 7.317e7.828 (m, 8H, AreH), 6.674
(s, 1H, 2-H of thiazolidinone ring), 4.071e4.112 (dd, J ¼ 0.8, 16 Hz,
1H, 5-Ha), 3.886e3.925 (d, J ¼ 15.6 Hz, 1H, 5-Hb). GCeMS (m/z): 413
(Mþ).
4.2.10. 4-(2-(4-Bromophenyl)-4-oxothiazolidin-3-yl)
benzenesulfonamide (3j)
Yield: 74%. Mp: 186e187  C. IR (KBr, cm
1): 3323.46 (NeH),
1716.70 (C]O), 1334.78, 1157.33 (S]O). 1H NMR (DMSO-d6, d ppm):
12.049 (s, 2H, SO2NH2), 7.326e7.836 (m, 8H, AreH), 6.660 (s, 1H, 2-
H of thiazolidinone ring), 4.067e4.109 (dd, J ¼ 1.2, 16 Hz, 1H, 5-Ha),
3.848e3.887 (d, J ¼ 15.6 Hz, 1H, 5-Hb). GCeMS (m/z): 413 (Mþ).
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 catalyzed CO2 hy-
dration 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 main-
taining constant the ionic strength. The rate of the CA-catalyzed
CO2 hydration reaction was monitored for a period of 10e100 s.
The CO2 concentrations ranged from 1.7 to 17 mM for the deter-
mination of the kinetic parameters and inhibition constants. Stock
solutions of inhibitors (10 mM) were prepared in distillededeion-
ized water with 10e20% (v/v) DMSO and dilutions up to 0.1 nM
were made thereafter with distillededeionized 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
378 S.K. Suthar et al. / European Journal of Medicinal Chemistry 66 (2013) 372e379
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 mg/ml
streptomycin. Cells were incubated at 37  C under 5% CO2 humid-
ified atmosphere. Dalton’s lymphoma ascites (DLA) cells were
maintained and propagated by serial intraperitoneal trans-
plantation 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 ml of media) and incubated for 24 h to obtain
70e80% confluency. The compounds to be screened were dissolved
into 50 ml DMSO and were further diluted with media so as to get
DMSO concentration below 0.25%. The cells were then treated
with varying concentration (250, 125, 62.5, 31.25, 15.62, and
7.81 mg/ml) of test compounds (100 ml/well). After 48 h, media
was discarded and 100 ml 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 ml 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 70e80% 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% formalde-
hyde 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 340e380 nm
with emission of 465 nm.
4.3.2.4. Acridine orangeeethidium staining [30]. Cell death analysis
was performed by AOeEB 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 AOeEB 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.
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% carbox-
ymethyl cellulose (CMC).
4.3.3.4. DLA-induced solid tumor studies [31e34]. 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 ¼ 4/3pr21  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 con-
trol animal on day 30th e 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 ¼ [(animal weight on day 30th/animal weight on day
0)
1]  100 [31e34].
4.3.3.5. 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.
4.4. 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 anhy-
drase 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 A (x, y, and z) with grid point spacing of 0.375 
A. The
Lamarckian genetic algorithm (LGA) was used to search con-
formers with lowest binding energy. The results of docking studies
are expressed as estimated free energy of binding in kcal/mol
(Supplementary data: Table 2).
Acknowledgments
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.
 S.K. Suthar et al. / European Journal of Medicinal Chemistry 66 (2013) 372e379
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.06.003.
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