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

Article abstract of DOI:10.1016/j.ejmech.2017.07.073

Herein we report the synthesis of two series of novel 4/3-((4-oxo-5-(2-oxoindolin-3-ylidene)thiazolidin-2-ylidene)amino)benzenesulfonamides (4a-m and 7a-g). All the newly prepared sulfonamides were in vitro investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, II, IV and IX, using a stopped-flow CO₂ hydrase assay. In particular, hCA isoforms II and IX (tumor-associated) were more susceptible to inhibition by the synthesized derivatives, with K_Is in the range of 2.6–598.2 nM for hCA II, and of 16.1–321 nM for hCA IX. All compounds (4a-m and 7a-g) were evaluated for their anti-proliferative activity against breast cancer MCF-7 and colorectal cancer Caco-2 cell lines. Compound 4c was found to be the most potent derivative against MCF-7 (IC₅₀ = 3.96 ± 0.21 μM), while 4j was the most active member against Caco-2 cells (IC₅₀ = 5.87 ± 0.37 μM). Compound 4c induced the intrinsic apoptotic mitochondrial pathway in MCF-7 cells; evidenced by the enhanced expression of the pro-apoptotic protein Bax and the reduced expression of the anti-apoptotic protein Bcl-2, and the up-regulated active caspase-9 and caspase-3 levels.

Full text of DOI:10.1016/j.ejmech.2017.07.073

Accepted Manuscript
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
Wagdy M. Eldehna, Mahmoud F. Abo-Ashour, Alessio Nocentini, Paola Gratteri,
Ibrahim H. Eissa, Mohamed Fares, Omnia E. Ismael, Hazem A. Ghabbour, Mahmoud
M. Elaasser, Hatem A. Abdel-Aziz, Claudiu T. Supuran
PII:
S0223-5234(17)30595-0
10.1016/j.ejmech.2017.07.073
EJMECH 9636
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 June 2017
Revised Date: 22 July 2017
Accepted Date: 29 July 2017
Please cite this article as: W.M. Eldehna, M.F. Abo-Ashour, A. Nocentini, P. Gratteri, I.H. Eissa, M.
Fares, O.E. Ismael, H.A. Ghabbour, M.M. Elaasser, H.A. Abdel-Aziz, C.T. Supuran, 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, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.073.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract
Novel 4/3-((4-oxo-5-(2-oxoindolin-3-ylidene)thiazolidin-2-ylidene)amino)
benzenesulfonamides: Synthesis, carbonic anhydrase inhibitory activity,
anticancer activity and molecular docking studies
Two different series of novel isatin-based benzenesulfonamide were synthesized and evaluated
for their inhibitory activity against a panel of carbonic anhydrase isoforms, hCA I, II, IV and IX.
Also, they were evaluated for their anti-proliferative activity against breast cancer MCF-7 and
colorectal cancer Caco-2 cell lines.

ACCEPTED MANUSCRIPT
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
Wagdy M. Eldehnaa,*, Mahmoud F. Abo-Ashourb, Alessio Nocentinic,d, Paola Gratterid,
Ibrahim H. Eissae, Mohamed Faresb,f, Omnia E. Ismaelg, Hazem A. Ghabbourh,i, Mahmoud
M. Elaasserj, Hatem A. Abdel-Azizk, Claudiu T. Supuranc,*
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University,
Kafrelsheikh, Egypt.
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University,
Badr City, Cairo, P.O. Box 11829, Egypt
cDepartment of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University
of Florence, Polo Scientifico, Via U. Schiff 6, 50019 Sesto Fiorentino (Firenze), Italy.
dDepartment of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences,
Laboratory of Molecular Modeling Cheminformatics & QSAR, University of Florence, Polo
Scientifico, Via U. Schiff 6, 50019 Sesto Fiorentino (Firenze), Italy.
eDepartment of Pharmaceutical Organic Chemistry, College of Pharmacy, Al-Azhar University,
Cairo 11884, Egypt.
fSchool of Chemistry, University of Wollongong, Wollongong 2522, New South Wales, Australia.
gDepartment of Biochemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, Cairo,
Egypt.
hDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box
2457, Riyadh 11451, Saudi Arabia.
iDepartment of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura
35516, Egypt.
jThe Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt.
kDepartment of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622, Egypt.
Abstract
Herein we report the synthesis of two series of novel 4/3-((4-oxo-5-(2-oxoindolin-3-
ylidene)thiazolidin-2-ylidene)amino)benzenesulfonamides (4a-m and 7a-g). All the newly
prepared sulfonamides were in vitro investigated as inhibitors of the metalloenzyme carbonic
anhydrase (CA, EC 4.2.1.1) isoforms hCA I, II, IV and IX, using a stopped-flow CO2
hydrase assay. In particular, hCA isoforms II and IX (tumor-associated) were more
susceptible to inhibition by the synthesized derivatives, with KIs in the range of 2.6–598.2
nM for hCA II, and of 16.1–321 nM for hCA IX. All compounds (4a-m and 7a-g) were
evaluated for their anti-proliferative activity against breast cancer MCF-7 and colorectal
1

ACCEPTED MANUSCRIPT
cancer Caco-2 cell lines. Compound 4c was found to be the most potent derivative against
MCF-7 (IC50 = 3.96 ± 0.21 µM), while 4j was the most active member against Caco-2 cells
(IC50 = 5.87 ± 0.37 µM). Compound 4c induced the intrinsic apoptotic mitochondrial
pathway in MCF-7 cells; evidenced by the enhanced expression of the pro-apoptotic protein
Bax and the reduced expression of the anti-apoptotic protein Bcl-2, and the up-regulated
active caspase-9 and caspase-3 levels.
____________
Keywords: Carbonic anhydrase inhibitors; Isatin; Benzenesulfonamide; Anticancer;
Apoptosis.
Corresponding authors. Tel.: +2-1068837640; e-mail: wagdy2000@gmail.com (WME);
Tel./fax:+39-055-4573729; e-mail: claudiu.supuran@unifi.it (CTS)
2

ACCEPTED MANUSCRIPT
1-Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1), the prevalent metalloenzymes in all life
kingdoms, catalyze the efficient interconversion between CO2 and bicarbonate [1-3]. In
humans, the CA-catalyzed reaction encompasses three simple chemical entities, CO2,
−
HCO3 , and H+ essential in a host of physiological and pathological processes, such as
electrolyte secretion, bone resorption, calcification, respiration, pH and CO2 homeostasis,
biosynthetic reactions (as gluconeogenesis and lipogenesis), tumorigenicity and others [1-
3]. Seven distinct genetic enzymatic families have been identified so far: the α-, β-, γ-, δ-,
ζ-. η- and θ-Cas [1-3]. The sixteen human (h) isoforms of CA, which all belong to the α-
class, displayed different patterns of location and tissue distribution, thus cytosolic (I, II,
III, VII, and XIII), membrane-bound (IV, IX, XII, and XIV), secreted (VI) and
mitochondrial (VA and VB) forms have been described in mammals [1-3].
Such isozymes represent valuable biological targets for the design of CA inhibitors
(CAIs) with many biomedical applications [2-4]. Noteworthy, many sulfonamides-CAIs
are clinically used for decades to treat several diseases (e.g., glaucoma, edema, epilepsy).
The ubiquitous hCAI and CAII, which are involved in many physiological processes,
were found to be off-target in the treatment of several pathologies [1-3], since they are
the main responsible of most side effects of non-selective inhibitors. The membrane-
associated isoform hCA IV is a drug target for retinitis pigmentosa and stroke, in addition
to glaucoma, together with hCA II [5]. The transmembrane hCA IX is overexpressed in
many solid tumors associated with the hypoxic phenotype, where it is pivotal in the
growth of solid tumors, tuning the pH to support an extracellular hypoxic
microenvironment suited for hypoxic tumor cell survival and proliferation, thus emerged
as an innovative target for the development of anticancer agents [6,7].
Encouraged by these findings, the goal of developing isoform-selective hCAIs has
been nurtured over the latter years with several studies [1,4]. The most exploited method
to overcome the typical lack in selectivity of sulfonamide-like CAIs against the different
human isozymes turned out to be the “tail approach” which consists in modulating the
functionality (tail) appended to the aromatic/heterocyclic ring present in the scaffold of
the CAIs, in order to differently interact with the middle and the rim parts of the active
3

ACCEPTED MANUSCRIPT
site cavity, which is the most variable region among the sixteen isoforms mentioned
above [8-10].
Over the last few years, isatin (1H-indole-2,3-dione) stood out as a promising tail
scaffold to design compounds with interesting inhibitory activity profiles towards
different carbonic anhydrase isoforms. Numerous studies have developed diverse isatin-
based derivatives as potent CA inhibitors, compounds I-IV (Fig. 1) [11-15]. Moreover,
isatin, as a privileged scaffold, is endowed with excellent anticancer profile [16, 17] and
represents an important pharmacophore in two clinically approved anticancer drugs;
Sunitinib V (Sutent®) and Nintedanib VI (Ofev®) (Fig. 2) [18, 19]. Consequently, design
and synthesis of various effective isatin-based anticancer agents have attracted
considerable attention in the current medical era [20-25]. Several research groups
explored the anti-proliferative activity of many isatin-thiazolidine/thiazolidinone analogs,
such as compounds VII-IX (Fig. 2) [26-30].
Figure 1. Structures of isatin-based carbonic anhydrase inhibitors I-IV and the target
sulfonamides 4a-m and 7a-g.
Taking the above into account, herein we report two novel series of 4/3-((4-oxo-5-(2-
oxoindolin-3-ylidene)thiazolidin-2-ylidene)amino)benzenesulfonamides (4a-m and 7a-g)
4

ACCEPTED MANUSCRIPT
(Fig. 1), with the prime aim of combining a strong hCAIX inhibition together with the
intrinsic anti-tumor activity of the isatin scaffold. Indeed, all the newly synthesized
sulfonamides were evaluated in vitro for their inhibitory activity against a panel of hCA I,
II, IV and IX isoforms, using stopped-flow CO2 hydrase assay. Furthermore, compounds
4a-m and 7a-g were evaluated for their anti-proliferative activity against breast cancer
MCF-7 and colorectal cancer Caco-2 cell lines. Compound 4c was further investigated for
its apoptosis induction potential in MCF-7 cells, to gain mechanistic insights into the anti-
proliferative activity of the newly prepared sulfonamides.
Figure 2. Structures of isatin-based approved anticancer drugs (V& VI) and some reported
isatin-based derivatives with potent anti-proliferative activity (VII - IX).
2. Results and Discussion
2.1. Chemistry
The synthesis of the new sulfonamides in this study is outlined in Schemes 1 and 2. In
scheme 1, synthesis of the target derivatives was started by acetylation of sulfanilamides
1a,b with chloroacetyl chloride in dry DMF to produce derivatives 2a, b, which
subsequently cyclized in absolute ethanol with ammonium thiocyanate to furnish the key
5

ACCEPTED MANUSCRIPT
intermediates 3a, b. The later condensed with different isatins in acetic acid in the presence
of sodium acetate to afford the target sulfonamides 4a-m (Scheme 1).
Scheme 1. Synthesis of target sulfonamides 4a-m; Reagents and conditions: (i) Dry DMF /
ClCOCH2Cl / r.t. 2 h, (ii) NH4SCN / Absolute ethyl alcohol / reflux 4 h, (iii) Glacial acetic
acid / Sodium acetate / reflux 3 h.
Scheme 2 depicts the synthesis protocol followed to get sulfonamides 7a-g. It started by
N alkylation of different isatins via methyl bromide 5a or benzyl bromide 5b to afford
derivatives 6a-f, which subsequently treated at reflux temperature with the key
intermediates 3a, b in glacial acetic acid in the presence of sodium acetate to furnish the
target compounds 7a-g (Scheme 2).
X
H
7
a
b
c
d
e
f
p/m
p
p
p
p
p
p
m
R
H
H
H
C6H5
C6H5
C6H5
C6H5
O
O
X
S
O
O
NH2
Cl
Br
H
Cl
Br
H
O
O
NH
Br
R
X
X
ii
5a, b
N
S
O
O
i
N
N
N
R
H
R
6a-f
7a-g
g
Scheme 2. Synthesis of sulfonamides 7a-g; Reagents and conditions: (i) DMF, K2CO3,
reflux 3 h, (ii) Glacial acetic acid / Sodium acetate / reflux 3 h.
6

ACCEPTED MANUSCRIPT
Postulated structures of the newly synthesized sulfonamides 4a-m and 7a-g were in full
agreement with their spectral and elemental analyses data.
2.2. Biological Evaluation
2.2.1. Carbonic anhydrase inhibition
The newly prepared sulfonamides 3a-b, 4a-m and 7a-g were evaluated for their ability
to inhibit the physiologically relevant hCA isoforms, hCA I, II (cytosolic) and hCA IV
(transmembrane) as well as the tumor-associated hCA IX (transmembrane), by a stopped-
flow CO2 hydrase assays [31], in comparison to the clinically used acetazolamide (AAZ) as
standard CAI. The following structure–activity relationship (SAR) can be compiled from
the inhibition data reported in Table 1:
(i) The cytosolic isoform hCA I was moderately inhibited by most of the sulfonamides with
inhibition constants (KIs) ranging in the high nanomolar - low micromolar range, in detail,
between 513.7 and 9269.7 nM, except for the p-substituted precursor 3a which arose as the
best hCA I inhibitor with a KI of 91.9 nM.
(ii) The physiologically dominant isoform hCA II was very potently inhibited by most of
the p-substituted derivatives 3a, 4a-h, 7a-7f (KI values ranging between 1.8 and 69.6 nM,
Table 1), apart from the N-unsubstituted 5-Br and 5,7-(CH3)2 isatin-bearing derivatives 4d
and 4h, which exhibited a slightly reduced inhibitory efficacy (KI values of 210.5 and 466.0
nM). The incorporation of a methyl group on the isatin core (7a-c) did not substantially
interfere with the derivatives hCA II inhibitory potency, whereas an analogue benzylic
substitution (7d-f) led to a three-fold to twenty-fold efficacy enhancement likened to the N-
unsubstituted compounds (4a-4g).
Conversely, it is noteworthy that all the m-substituted derivatives (4i-m) were found to
possess a generally ten-fold diminished hCA II inhibition efficacy (KIs ranging between
176.4 and 598.2 nM) in comparison to their p-substituted analogues. Likewise, the meta-
substituted precursor 3b (KI of 73.2 nM) was forty times less potent than 3a (KI of 1.8 nM).
On the other hand, the N-benzyl moiety furnished the 3-substituted 7g with a comparable
inhibitory potency likened to the 4-substituted N-benzyl analogues (7d-f).
7

ACCEPTED MANUSCRIPT
Table 1. Inhibition data of human CA isoforms hCA I, II, IV and IX with sulfonamides 3a-b,
4a-m and 7a-g reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by
a stopped flow CO2 hydrase assay31
KI (nM)*
Comp. p/m
X
R
hCA I
630.8
513.7
2290.4
3611.4
2881.6
4243.7
2883.7
7602.0
2662.6
728.5
718.7
3424.4
884.2
4001.7
923.9
872.7
5630.7
881.1
9269.7
856.4
91.9
hCA II
23.0
7.1
hCA IV
>10000
4290.5
3256.1
2986.4
>10000
2864.4
3682.3
4751.0
1972.7
395.5
hCA IX
47.0
26.6
62.4
66.6
23.1
72.1
34.1
100.1
16.1
62.0
15.9
24.7
48.0
146.3
123.3
38.9
117.5
90.6
321.1
169.8
4.5
p
H
-
4a
4b
4c
4d
4e
4f
p
5-F
-
p
5-Cl
5-Br
5-OCH3
5-CH3
5-NO2
5,7-(CH3)2
H
-
69.6
210.5
24.3
57.4
35.7
466.0
273.8
598.2
416.0
176.4
506.6
43.5
38.4
17.8
8.3
p
-
p
-
p
-
p
-
4g
4h
4i
p
-
m
m
m
m
m
p
-
5-Cl
5-Br
5-OCH3
5-NO2
H
-
4j
-
4k
4l
235.0
-
2526.6
441.9
-
4m
7a
7b
7c
7d
7e
7f
H
4712.2
3040.7
2634.0
3299.6
4305.8
2897.3
2244.6
348.3
p
5-Cl
5-Br
H
H
p
H
p
C6H5
C6H5
C6H5
C6H5
-
p
5-Cl
5-Br
H
2.6
p
59.8
18.2
1.8
m
p
7g
3a
3b
AAZ
m
-
-
741.4
250
73.2
12
101.9
17.7
25
-
-
74
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of ±
5-10 % of the reported values).
8

ACCEPTED MANUSCRIPT
(iii) The data reported in Table 1 ascribed to the most reported sulfonamides poor efficacy
in inhibiting the trans-membrane isoform hCA IV. Indeed, the p-substituted derivatives 4a-
h, 7a-f were shown to inhibit such isozyme in the micromolar range (KIs ranging between
2634.0 and 4751.0 nM), except 4a and 4e which did not inhibit hCA IV up to 10000 nM. It
is worth highlighting that the 3-substituted 4i-m showed a generally improved inhibitory
profile against hCA IV, in particular derivatives 4j, 4k and 4m (KIs of 395.5, 235.0 and
441.9 nM), which were endowed with electro-withdrawing substituents at the 5- position of
the isatin scaffold (respectively 5-Cl, 5-Br, 5-NO2). The precursors 3a and 3b, devoid of
the isatin core appended at the oxothiazolidin-2-ylidene moiety, arose among the best
inhibitors against such trans-membrane isozyme, again with the m-substitution preferred to
the p-substituted one (KIs of 348.3 vs. 101.9 nM).
(iv) The target tumor-associated isoform hCA IX was effectively inhibited by all the isatin-
bearing sulfonamides herein reported. Furthermore, the inhibition profiles were found to be
rather flat, since the measures KIs ranged between 15.9 and 169.8 nM, aside from
derivative 7f whose efficacy raised at slightly higher concentration (KI of 321.1 nM). It is
worth stressing the comparable inhibitory effectiveness of the N-unsubstituted 3- and 4-
pendant bearing derivatives (although slightly heightened for derivatives 4i-4m; KIs
ranging between 16.1 and 62.0 nM), unlike arose for the cytosolic hCA II.
The incorporation of a methyl or benzyl group on the isatin N atom elicit a worsening
of effectiveness against hCA IX both for the p-substituted derivatives 7a-7f (except 7c, 5-
Br; KI of 38.9 nM) and the m-pendant bearing 7g (KI 169.8 nM). Finally, the isatin-devoid
precursor 3a and 3b inhibited hCA IX in the low nanomolar range with KIs of 4.5 and 17.7
nM.
(v) The inhibitory profiles reported in Table 1 undeniably ascribed to the m-substituted
amminobenzenesulfonamide 4i-4m a rather selective hCA IX/II inhibitory efficacy. Indeed,
it is satisfying to note that such compounds displayed a selectivity ratio hCA IX/hCA II
which spanned from 7.1 and 26.2. Considering that isoform hCA IX is a validated target
for the diagnosis and treatment of cancers, discovery of selective inhibitors represents a
promising step to unveil a more effective cancer therapy devoid of the classical side effects
owing to hCA I/II inhibition.
9

ACCEPTED MANUSCRIPT
2.2.2. In vitro anti-proliferative activity against MCF-7 breast cancer cell line
Anti-proliferative activity of the newly synthesized sulfonamides (4a-m and 7a-g) was
evaluated against breast cancer MCF-7 and colorectal cancer Caco-2 cell lines.
Doxorubicin was included in the experiment as a reference drug. The results were
expressed as IC50 values, and listed in Table 2.
Table 2. In vitro anti-proliferative activity of sulfonamides 4a-m and 7a-g against breast
MCF-7 and colorectal Caco-2 cancer cell lines.
IC50 (µM)a
Comp.
MCF-7
NAb
Caco-2
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
7a
7b
7c
7d
7e
7f
NAb
20.50 ± 2.25
3.96 ± 0.21
11.3 ± 0.77
125.0 ± 10.53
NAb
59.90 ± 4.01
20.30 ± 1.35
42.30 ± 2.57
98.30 ± 6.28
174.0 ± 11.09
13.80 ± 1.16
146 ± 9.44
12.10 ± 1.05
5.87 ± 0.37
10.80 ± 0.81
8.39 ± 0.55
20.62 ± 1.64
163 ± 14.74
42.30 ± 4.02
13.20 ± 1.16
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
51.9 ± 4.74
NAb
196 ± 9.75
NAb
124 ± 6.21
NAb
NAb
13.70 ± 1.24
57.80 ± 3.10
103 ± 8.25
7g
Dox.
2.37 ± 0.09
3.83 ± 0.19
a IC50 values are the mean ± S.D. of three separate experiments.
b NA: Compounds having IC50 value >200 µM.
As shown in table 2, the Caco-2 cell line is more susceptible to the synthesized
sulfonamides than MCF-7 cancer cell line. Investigations of the anti-proliferative activity
against MCF-7 indicated that compounds 4c and 4d, only, displayed good activity with
IC50 values of 3.96±0.21 and 11.3±0.77 µM, respectively. While compounds 4b and 7a
10

ACCEPTED MANUSCRIPT
were moderately active with IC50 = 20.50±2.25 and 51.9±4.74 µM, respectively. Also,
compounds 4e, 7c and 7e elicited weak activity with IC50 values ranging from 124±6.21 to
196 ± 9.75 µM.
Concerning activity against Caco-2 cells, the synthesized sulfonamides showed
excellent to modest growth inhibitory activity. Compound 4j (IC50 = 5.87±0.37 µM) was
found to be the most potent derivative overall the tested compounds against Caco-2 as it
was 1.5 times less active than doxorubicin (IC50 = 3.83±0.19). Besides, compounds 4g, 4i,
4k, 4l, 7c and 7e displayed good activity with IC50 values of 13.80±1.16, 12.10±1.05,
10.8±0.81, 8.39±0.55, 13.20±1.16 and 13.70±1.24 µM, respectively. Moreover, compounds
4b-d, 4m, 7b and 7f elicited moderate anti-proliferative activity (IC50: 20.30±1.35 -
59.90±4.01 µM). Furthermore, compounds 4e, 4f, 4h, 7a and 7g showed fair anti-
proliferative against Caco-2 with IC50 values ranging from 98.30±6.28 to 174 ± 11.09 µM.
2.2.3. Induction of apoptosis in breast cancer MCF-7 cells
2.2.3.1. Effects on mitochondrial apoptosis pathway (Bcl-2 family) proteins
The Bcl-2 family, the best characterized protein family involved in the regulation of
apoptosis, is consisting of anti-apoptotic and pro-apoptotic members [32]. The anti-
apoptotic members, such as Bcl-2, attenuate apoptosis either by preventing the release of
mitochondrial apoptogenic factors like cytochrome c into the cytoplasm or by sequestering
pro-forms of the caspases. On the other hand, pro-apoptotic members of Bcl-2 family, such
as Bax, trigger the release of caspases [32]. Thence, the Bcl-2 family of proteins acts as a
crucial life–death decision point within the common pathway of apoptosis.
Herein, the study was further substantiated to investigate the ability of compound 4c to
provoke apoptosis in MCF-7 cells. Treatment of MCF-7 cells with compound 4c
significantly increased the expression levels of the pro-apoptotic molecule Bax by 8.5 folds
compared to control (Table 3). In contrast, exposure of MCF-7 cells to compound 4c
resulted in significant decrease in the protein expression levels of the anti-apoptotic protein
Bcl-2 by about 45 % compared to control (Table 3). Analyzing the results revealed that
sulfonamide 4c boosted the Bax/Bcl-2 ratio fifteen folds in comparison to the control,
11

ACCEPTED MANUSCRIPT
where the bax/bcl-2 ratio serving to as a decisive value to determine cell susceptibility to
apoptosis [33].
Table 3. Effect of compound 4c on the expression levels of Bax, Bcl-2, active caspases-3 and
-9, cytochrome C and p53 levels in MCF-7 breast cancer cells treated with the compound at
its IC50 concentration.
Comp.
Bax
ng/ml
1.64
Bcl-2
ng/ml
0.46
Bax/Bcl-2
ratio
3.56
Caspase-3
ng/ml
0.049
Caspase-9
ng/ml
1.64
Cyt-c
ng/ml
0.234
p53
ng/ml
172
Control
14.03*** 0.26*
0.195*
14.03***
0.395*
860.8**
53.96
4c
Data are mean ± SD of three separate experiments. * Significantly different from control (1%
**
***
DMSO) at P <0.05.
Significantly different from control at P <0.01.
Significantly
different from control at P <0.001.
2.2.3.2. Effects on the levels of active caspase-3 and caspase-9
Caspases, cysteine-containing aspartic acid-specific proteases, provide pivotal links in
cell regulatory networks controlling cell death [34]. Caspase-3 is the key executioner
protease which activated by upstream initiator caspases as caspase-9 [34]. Therefore, the
elevated Bax/Bcl-2 ratios aroused the investigation of the protein expression levels of
active caspases-3 and caspases-9. Treatment of MCF-7 cells with compound 4c produced a
significant elevation in the level of active caspase-3 and caspases-9 by about 4 and 8.5
folds, respectively, compared to control (Table 3).
2.2.3.3. Effect on the level of Cytochrome C
As a result of Bax/Bcl2 increments, mitochondrial cytochrome C is released into the
cytosol where it potentiates a cascade of caspases that finally triggers the executioner
caspase; caspase 3 [35]. Thus, we measured the level of cytochrome C to assure the
adoption of the intrinsic pathway. Results revealed that cytochrome C level increased by
1.7 folds, compared to control, upon treatment of MCF-7 cells with the IC50 of sulfonamide
4c (Table 3).
2.2.3.4. Effect on the level of p53
The p53 tumor suppressor gene is critically involved in cell cycle regulation, DNA repair,
and programmed cell death [36]. Finally, in this study we analyzed p53 expression in breast
12

ACCEPTED MANUSCRIPT
MCF-7 cancer cells after treatment with 4c. Results confirmed that 4c has strong ability to
enhance tumor suppressor p53 expression by 5 folds compared to control (Table 3).
In conclusion, the ability of compound 4c to down regulate Bcl-2 level while boosting
Bax, caspase-3, caspase-9, cytochrome C and p53 levels confirms its effectiveness as
apoptosis inducer through, at least in part, inducing the mitochondrial pathway of
apoptosis.
2.3. Molecular modeling studies
The inhibitory profiles of the derivatives in Table 1 were further investigated carrying out
docking and Molecular Mechanics Generalized Born Surface Area (MM-GBSA) calculation
to predict their binding mode and free energy as inhibitors of the hCA II (PDB 5LJT) [9] and
IX (PDB 5FL4) [37a] isoforms.
As it could be foreseen, all docking solutions oriented the benzenesulfonamide moieties
deeply into the active site region of both the isozymes. Indeed, it is worth highlighting that all
the reported derivatives were found to inhibit both hCA II and IX in the nanomolar range. In
detail, the sulfonamide established two hydrogen bonds with the Thr199 residue, its
negatively charged nitrogen atom coordinated the zinc ion and the phenyl ring was involved
in several hydrophobic contacts (V121, H94 and L198).
Despite the different size of the hydrophobic pocket in hCA II and hCA IX isoforms,
mainly due to the Phe131/Val131 mutation, the tails of the p-substituted derivatives 3a, 4a-
4h and 7a-7f located comparably within the two isozyme binding sites (Fig.3a and 4a). In
detail, the heterocyclic tails were found to lie in the hydrophobic areas defined by Phe131,
Ile91, Val121, Gln92 and Val131, Leu91, Val121 in hCA II and hCA IX, respectively. The
C=O moiety of the oxothiazolidin-2-ylidene was involved in a three-centre H-bond involving
the HE/Gln92 and HD/Asn67 (hCA II) or HE/Gln71 (hCA IX) hydrogen atoms as donor
groups. The isatin scaffolds was stabilized by edge-to-face and π-alkyl hydrophobic
interactions occurring with Phe131 (V131 in hCA IX) and Ile91. X-substitution on the isatin
benzene ring did not substantially affect ligands positioning within the hydrophobic pockets,
although they differently interacted with the residues nearby (Fig.3b and 4b). It is worth
noting that the contacts involving the N-methyl and N-benzyl groups of derivatives 7a-7f
within the aforementioned hydrophobic regions contributed in increasing the inhibitory
13

ACCEPTED MANUSCRIPT
efficacy against the cytosolic isozyme, whereas they had no, or at least little, influence on the
inhibitory efficacy of the hCA IX.
(a)
(b)
(c)
Figure 3. (a) Simulated binding modes of compounds 4a (4-substituted, grey) and 4i (3-
substituted, aquamarine) within hCA II active site; (b) 4-substituted and (c) 3-substituted
benzenesulfonamides docked within hCA II active site.
Conversely, docking solutions found for the 3- substituted derivatives 3a, 4i-4m and 7g
identified two cavities, which differ depending on the enzyme isoform considered, within
which the N-pendant on the amminobenzensulfonamide moieties locate (Fig.3a and 4a). In
hCA IX, the pocket defined by Val131, Leu91, Gln67, Ser69 and Gln92 is roomy enough to
accommodate the N-pendant, though rotated by 90° (Fig. 4a). Again, the endocyclic carbonyl
of the oxothiazolidin-2-ylidene acts as H-bond acceptor to HE/Gln67 which, in turn, is
involved in weak C-H…π H-bonds with Val121 and Leu91 (Fig. 4a). The isatin benzene ring
additional formed π-alkyl hydrophobic interactions with Ser69.
In hCA II, the N-pendant of the amminobenzensulfonamide was oriented towards Asn62
and His64, and was engaged in several H-bonds (Fig.3a). The imine nitrogen act as H-bond
acceptor from HG/Thr200, the carbonyl of the oxothiazolidin-2-ylidene was part of
bifurcated H-bonds with donor being HE/Gln92 and HD/Asn67, whereas the C=O of the
isatin accepts the HE/His64. The isatin benzene ring formed additional π- alkyl hydrophobic
contacts with Leu60. The introduction of a benzyl group on the nitrogen atom of the isatin
provided the 3-substituted compound 7g with an inhibitory efficacy against hCA II
comparable to the one observed for the 4-substituted N-benzyl derivatives 7d-7f. Again, the
14

ACCEPTED MANUSCRIPT
type and nature of the X-substitution at the isatin benzene ring did not substantially affect
ligands positioning within both the isozymes binding pockets.
(b)
(c)
(a)
Figure 4. (a) Simulated binding modes of compounds 4a (4-substituted, grey) and 4i (3-
substituted, aquamarine) within hCA IX active site; (b) 4-substituted and (c) 3-substituted
benzenesulfonamides docked within hCA IX active site.
Analysing the results obtained from docking simulations, it is possible to assume that
switching the substituents from the para to the meta position caused the increase in the steric
hindrance that, together with a torsional strain, prevent the heterocyclic tails of the 3-
substituted derivatives to lie within the hCA II hydrophobic cavity, thus worsening the
inhibitory efficacy of the compounds against such isoform.
Basing on the enzyme-inhibitor complexes obtained from molecular docking, ∆G binding
energy were calculated using the Prime MM-GBSA approach [37b], either or not defining a
4Å shell from the ligands within which hCA II and hCA IX residues were relaxed during the
computations (Table 4).
For all derivatives, but 7d-f in the case of hCA II, the ranking of the ligands based on the
calculated MM-GBSA ∆Gbinding values substantially agreed with ranking based on
experimental inhibition data. As far as it concerns the hCA IX isoform, the ∆G binding
values found reflected the experimental inhibition data in Table1 (Fig. 5). In the case of hCA
II, a better agreement was achieved not considering the 7d-f (featured by the N-benzyl
substitution at the isatin scaffold).
15

ACCEPTED MANUSCRIPT
Within the here considered congeneric series of sulfonamides, the MM-GBSA approach has
shown good success in rank-ordering the inhibitor potencies of the studied compounds
making it possible to prioritize derivatives for synthesis as inhibitors against hCA II and hCA
IX enzyme isoforms.
Figure 5. MMGBSA ∆G binding energy vs. pKi values. Because of the absence of the isatin
moiety, derivatives 3a and 3b, were not considered. Energy values were computed (a) not
allowing residues to relax and (b) defining an enzyme flexible region (4Å from the ligand).
16

ACCEPTED MANUSCRIPT
Table 4. Results of the MM-GBSA calculation on hCA II (5LJT) and hCA IX (5FL4)
∆Gexp (kcal/mol)
Compound
5LJT
5LJT 4Ȧ (a) 5FL4
5FL4 4Ȧ(a)
3a
3b
4a
4b
4c
4d
4e
4f
-33.965
-37.229
-48.294
-47.054
-53.238
-52.393
-52.470
-51.247
-49.431
-51.580
-49.434
-54.111
-54.057
-52.317
-52.289
-50.096
-54.387
-52.935
-58.443
-62.064
-61.063
-53.362
-42.301
-39.449
-51.442
-51.073
-58.898
-61.659
-58.807
-59.706
-58.535
-59.519
-52.147
-54.874
-56.809
-52.146
-52.928
-52.559
-63.293
-62.923
-71.069
-69.749
-69.790
-58.269
-34.196
-34.543
-36.224
-54.033
-54.442
-59.021
-56.322
-55.507
-57.587
-56.657
-59.265
-46.780
-52.226
-57.458
-53.287
-49.812
-58.783
-56.696
-60.361
-70.339
-64.349
-63.488
-56.508
-37.523
-51.102
-49.334
-56.486
-55.364
-54.195
-55.772
-53.066
-56.823
-47.805
-51.015
-51.408
-50.098
-49.969
-53.794
-59.336
-58.048
-60.630
-59.418
-58.381
-54.903
4g
4h
4i
4j
4k
4l
4m
7a
7b
7c
7d
7e
7f
7g
(a) Residues within 4Å from the ligand were treated as flexible
17

ACCEPTED MANUSCRIPT
3. Conclusion
In summary, this study reports the synthesis of two series of novel sulfonamides (4a-m
and 7a-g) incorporating substituted indolin-2-one moieties linked to benzenesulfonamide (as
zinc anchoring moieties). The newly synthesized sulfonamides were evaluated in vitro for
their inhibitory activity against hCA I, II, IV and IX. All these isoforms were inhibited by the
sulfonamides reported here in variable degrees. hCA isoforms II and IX were more
susceptible to the synthesized sulfonamides, with KIs in the range of 2.6–598.2 nM for hCA
II, and of 16.1–321 nM for hCA IX. Noteworthy, m-substituted derivatives 4i-m were found
to exhibit a selective hCA IX/II inhibitory efficacy. Moreover, all sulfonamides were
examined for their anti-proliferative activity against breast cancer MCF-7 and colorectal
cancer Caco-2 cell lines. In particular, compound 4c was the most potent derivative against
MCF-7 (IC50 = 3.96 ± 0.21), while 4j was the most active member against Caco-2 cells (IC50
= 5.87 ± 0.37). Interestingly, compound 4c provoked the intrinsic apoptotic mitochondrial
pathway in MCF-7 cells; evidenced by the enhanced expression of the pro-apoptotic protein
Bax and the reduced expression of the anti-apoptotic protein Bcl-2, and the up-regulated
active caspase-3 and caspase-9, cytochrome C and p53 levels. Finally, the inhibitory profiles
of the synthesized derivatives were further investigated carrying out docking and Molecular
Mechanics Generalized Born Surface Area (MM-GBSA) calculation to predict their binding
mode and free energy as inhibitors of the hCA II (PDB: 5LJT) and IX (PDB: 5FL4)
isoforms.
4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were measured with a Stuart melting point apparatus and were
uncorrected. Infrared spectra were recorded on Schimadzu FT-IR 8400S
spectrophotometer. The NMR spectra were recorded by Varian Mercury or Bruker
spectrophotometers at 400 MHz. 13C NMR spectra were run at 100 MHz in deuterated
dimethylsulfoxide (DMSO-d6). Chemical shifts (δH) are reported relative to TMS as
18

ACCEPTED MANUSCRIPT
internal standard. All coupling constant (J) values are given in hertz. Chemical shifts (δC
)
are reported relative to DMSO-d6 as internal standards. Elemental analyses were carried out
at the Regional Center for Microbiology and Biotechnology, Al-Azhar University, Cairo,
Egypt. Unless otherwise noted, all solvents and reagents were commercially available and
used without further purification.
4.1.2. General procedure for preparation of 2-chloro-N-(4/3-sulfamoylphenyl)acetamides
2a, b.
To a solution of sulfanilamides 1a, b (1.72 gm, 10 mmol) in dry DMF, chloroacetyl
chloride (0.8 mL, 10 mmol) was added dropwise at 0°C then stirred at room temperature for
2 h. The reaction mixture was then poured into ice-water. The obtained solid was filtered
off, washed with water several times and crystallized from dioxane to give compounds 2a,
b [38-40].
4.1.3.
General procedure for preparation of 4/3-((4-oxothiazolidin-2-ylidene)amino)
benzenesulfonamides 3a,b.
To a hot solution of derivatives 2a,b (1.24 gm, 5 mmol) in absolute ethyl alcohol,
ammonium thiocyanate (0.76 gm, 10 mmol) was added. The reaction mixture was refluxed
for 4 h then allowed to cool to room temperature. The formed precipitate was filtered off,
washed with water and recrystallized from ethanol to furnish the key intermediates 3a, b.
4.1.3.1. 4-((4-Oxothiazolidin-2-ylidene)amino)benzenesulfonamide (3a). [41]
4.1.3.2. 3-((4-Oxothiazolidin-2-ylidene)amino)benzenesulfonamide (3b). White powder
(yield 80% ), m.p. > 300 °C ; IR (KBr, ν cm-1): 3183 (NH), 1675 (C=O) and 1331, 1159
1
(SO2); H NMR (DMSO-d6, 400 MHz)
δ ppm: 4.00 (s, 2H, CH2), 7.14 (d, 1H, Ar-H, J =
8.4 Hz), 7.37 (s, 2H, NH2, D2O exchangeable), 7.54-7.56 (m, 2H, Ar-H), 7.85 (d, 1H, Ar-
H, J = 8.48 Hz), 11.39 (s, 1H, NH, D2O exchangeable); Anal. Calcd. (Found) For
C9H9N3O3S2 : C, 39.84 (40.11); H, 3.34 (3.40); N, 15.49 (15.82).
4.1.4. General procedure for synthesis of the target sulfonamides 4a-m
.
19

ACCEPTED MANUSCRIPT
The appropriate isatin derivatives was added to a hot stirred solution of the key
intermediates 3a, b (0.27 gm, 1 mmol) and sodium acetate (0.16 gm, 2 mmol) in glacial
acetic acid (15 mL). The reaction mixture was heated under reflux for 3 h. The formed
solid was filtered off while hot, washed with hot ethanol, dried and recrystallized from
DMF/ethanol to afford the target sulfonamides 4a-m.
4.1.4.1.
4-((4-Oxo-5-(-2-oxoindolin-3-ylidene)thiazolidin-2-ylidene)amino)
benzenesulfonamide (4a). Red powder (yield 72%); m.p. > 300 °C; IR (KBr, ν
cm-1): 3290,
1
3255 (NH, NH2), 1663 (C=O) and 1330, 1153 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
ppm: 6.89-7.09 (m, 2H, Ar-H), 7.20 (d, 0.5H, Ar-H, J = 8.0 Hz), 7.32-7.39 (m, 1.5H, Ar-
H), 7.52 (s, 2H, NH2, D2O exchangeable), 7.65 (d, 1H, Ar-H, J = 8.4 Hz), 7.86 (t, 1H, Ar-
H, J = 8.36 Hz), 7.96 (d, 1H, Ar-H, J = 8.32 Hz), 8.71 (d, 0.5H, Ar-H, J = 7.88 Hz), 8.80
(d, 0.25H, Ar-H, J = 7.84 Hz), 8.94 (d, 0.25H, Ar-H, J = 7.52 Hz), 10.02, 12.03 (2s, 1H,
NH isatin, D2O exchangeable), 11.15, 12.58 (2s, 1H, NH thiazolidinone, D2O
13
exchangeable); C NMR (DMSO-d6, 100 MHz)
δ ppm: 110.71, 120.60, 120.85, 121.19,
122.09, 122.23, 122.37, 124.11, 125.32, 126.77, 127.47, 127.71, 128.12, 128.43, 129.90,
131.98, 132.25, 132.37, 133.05, 133.82, 137.74, 140.63, 143.57, 143.63, 144.50, 156.61,
165.75, 168.91, 169.04, 169.33; MS, m/z [%]: 400 [M+, 2.97]; Anal. Calcd. (Found) for
C17H12N4O4S2: C, 50.99 (51.23); H, 3.02 (3.08); N, 13.99 (14.21).
4.1.4.2.
4-((5-(5-Fluoro-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene)amino)
benzenesulfonamide (4b). Red powder (yield 70%); m.p. > 300 °C; IR (KBr, ν
cm-1): 3294,
1
3271 (NH, NH2), 1693 (C=O) and 1334, 1157 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
ppm: 6.86-6.94 (m, 1H, Ar-H), 7.17-7.21 (m, 1H, Ar-H), 7.35-7.39 (m, 1H, Ar-H), 7.52 (s,
2H, NH2, D2O exchangeable), 7.65 (d, 1H, Ar-H, J = 8.4 Hz), 7.86-7.99 (m, 2H, Ar-H),
8.48 (d, 0.5H, Ar-H, J = 10.28 Hz), 8.58 (d, 0.25H, Ar-H, J = 10.04 Hz), 8.72 (d, 0.25H,
Ar-H, J = 9.4 Hz), 10.11, 11.99 (2s, 1H, NH isatin, D2O exchangeable), 11.16, 12.74 (2s,
1H, NH thiazolidinone, D2O exchangeable); 13C NMR (DMSO-d6, 100 MHz)
δ ppm:
111.38, 111.45, 114.53, 114.80, 115.14, 118.13, 118.37, 121.29, 121.40, 122.08, 123.46,
126.80, 127.48, 127.72, 129.87, 134.80, 135.68, 137.59, 139.89, 139.98, 140.05, 140.88,
144.58, 156.31, 156.74, 159.07, 165.78, 168.86, 169.01, 169.28; MS, m/z [%]: 418 [M+,
20

ACCEPTED MANUSCRIPT
4.8]; Anal. Calcd. (Found) for C17H11FN4O4S2: C, 48.80 (48.97); H, 2.65 (2.63); N, 13.39
(13.60).
4.1.4.3.
4-((5-(5-Chloro-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene)amino)
benzenesulfonamide (4c). Red powder (yield 62%); m.p. > 300 °C; IR (KBr,
ν
cm-1): 3295,
1
3249 (NH, NH2), 1690 (C=O) and 1330, 1153 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
ppm: 6.88-6.95 (m, 1H, Ar-H), 7.20 (d, 1H, Ar-H, J = 8.2 Hz), 7.35-7.39 (m, 1H , Ar-H),
7.52 (s, 2H, NH2, D2O exchangeable), 7.65 (d,1H, Ar-H , J = 8.4 Hz), 7.86-7.99 (m, 1H,
Ar-H), 7.95-7.99 (m, 1H, Ar-H), 8.72 (s, 0.25H, Ar-H), 8.81 (s, 0.5H, Ar-H), 8.96 (s, 0.25
H, Ar-H), 10.12, 11.97 (2s, 1H, NH isatin, D2O exchangeable), 11.26, 12.71 (2s, 1H, NH
thiazolidinone, D2O exchangeable); Anal. Calcd. (Found) for C17H11ClN4O4S2: C, 46.95
(47.32); H, 2.55 (2.59); N, 12.88 (13.04).
4.1.4.4.
((5-(5-Bromo-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene)amino)
benzenesulfonamide (4d). Red powder (yield 75%); m.p. > 300 °C; IR (KBr, ν
cm-1): 3332,
1
3251 (NH, NH2), 1693 (C=O) and 1334, 1157 (SO2); H NMR (DMSO-d6, 400 MHz)
δ
ppm: 6.83-6.90 (m, 1H, Ar-H), 7.20 (d, 1H, Ar-H, J = 7.36 Hz), 7.35-7.39 (m, 1H, Ar-H),
7.47-7.52 (m, 3H, 1H Ar-H and 2H of NH2, D2O exchangeable), 7.65 (d, 0.5H, Ar-H, J =
8.0 Hz), 7.86-7.99 (m, 1.5H, Ar-H), 8.85 (s, 0.25H, Ar-H), 8.94 (s, 0.5H, Ar-H), 9.09 (s,
0.25H, Ar-H), 10.12, 12.30 (2s, 1H, NH1isatin, D2O exchangeable), 11.25, 12.62 (2s, 1H,
13
NH thiazolidinone, D2O exchangeable); C NMR (DMSO-d6, 100 MHz)
δ ppm: 112.52,
113.89, 113.96, 121.30, 122.09, 122.35, 122.54, 123.91, 126.82, 127.46, 127.72, 129.85,
130.12, 130.46, 133.96, 134.23, 134.33, 135.12, 135.91, 137.57, 140.73, 140.84, 142.60,
142.68, 144.58, 156.24, 165.74, 168.50, 168.63, 168.92; Anal. Calcd. (Found) for
C17H11BrN4O4S2: C, 42.60 (42.76); H, 2.31 (2.24); N, 11.69 (11.87).
4.1.4.5.
4-((5-(5-Methoxy-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene)amino)
benzenesulfonamide (4e). Red powder (yield 55% ), m.p. > 300 °C; IR (KBr,
ν
cm-1):
1
3321, 3248 (NH, NH2), 1708 (C=O) and 1330, 1157 (SO2); H NMR (DMSO-d6, 400
MHz)
δ ppm: 3.71 (s, 3H, OCH3), 6.79-7.82 (m, 7H, 4 Ar-H and 2H, NH2, D2O
exchangeable), 8.39-8.63 (m, 2H, Ar-H), 10.89 (s, 1H, NH isatin, D2O exchangeable),
21

ACCEPTED MANUSCRIPT
12.29 (2s, H, NH thiazolidinone, D2O exchangeable); MS, m/z [%]: 430 [M+, 8.18]; Anal.
Calcd. (Found) for C18H14N4O5S2: C, 50.23 (50.60); H, 3.28 (3.37); N, 13.02 (13.34).
4.1.4.6.
4-((5-(5-Methyl-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene)amino)
benzenesulfonamide (4f). Red powder (yield 73% ), m.p. > 300 °C ; IR (KBr,
ν
cm-1):
1
3321, 3294 (NH, NH2), 1693 (C=O) and 1330, 1157 (SO2); H (DMSO-d6, 400 MHz)
δ
ppm: 2.48 (s, 3H, CH3), 6.75-6.82 (m, 1H, Ar-H), 7.12-7.19 (m, 1H, Ar-H), 7.31-7.35 (m,
1H, Ar-H), 7.49 (s, 2H, NH2, D2O exchangeable), 7.62-7.64 (m,1H, Ar-H), 7.82-7.96 (m,
2H, Ar-H), 8.54 (s, 0.5H, Ar-H), 8.62 (s, 0.25H, Ar-H), 8.75 (s, 0.25H, Ar-H), 9.97, 11.86
(2s, 1H, NH isatin , D2O exchangeable), 11.00, 12.59 (2s, 1H, NH thiazolidinone, D2O
exchangeable); 13C NMR (DMSO-d6, 100 MHz)
δ ppm: 21.34, 21.44, 110.38, 120.63,
120.71, 121.13, 122.07, 124.36, 126.77, 127.44, 127.67, 128.53, 128.83, 129.92, 130.91,
130.99, 132.38, 132.67, 133.36, 137.75, 140.60, 141.34, 141.41, 141.47, 144.52, 156.66,
165.76, 168.92, 169.06, 169.45, 169.92; MS, m/z [%]: 414 [M+, 6.69]; Anal. Calcd.
(Found) for C18H14N4O4S2: C, 52.16 (52.32); H, 3.40 (3.46); N, 13.52 (13.75).
4.1.4.7.
4-((5-(5-Nitro-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene)amino)
cm-1):
3352, 3263 (NH, NH2), 1693 (C=O), 1527, 1362 (NO2) and 1342, 1157 (SO2); H NMR
(DMSO-d6, 400 MHz) ppm: 7.00-7.07 (m, 1H, Ar-H), 7.18 (d, 1H, Ar-H, J = 8.4 Hz),
benzenesulfonamide (4g). Red powder (yield 62% ), m.p. > 300 °C; IR (KBr,
ν
1
δ
7.33-7.36 (m, 1H, Ar-H), 7.48 (s, 2H, NH2, D2O exchangeable), 7.64-7.66 (m, 1H, Ar-H),
7.84-7.98 (m, 1.5H, Ar-H), 8.18-8.23 (m, 0.5H, Ar-H), 9.53 (s, 0.5H, Ar-H), 9.63 (s,
0.25H, Ar-H), 9.75 (s, 0.25H, Ar-H), 10.22 (s, 1H, NH isatin, D2O exchangeable), 11.73 (t,
1H, NH thiazolidinone, D2O exchangeable, J = 8.8 Hz); 13C NMR (DMSO-d6, 100 MHz)
δ
ppm: 110.80, 110.86, 120.47, 120.57, 120.72, 121.42, 121.74, 122.06, 122.96, 123.23,
123.46, 123.59, 126.86, 127.42, 127.59, 127.71, 127.84, 129.82, 137.43, 137.82, 140.83,
140.90, 142.46, 142.55, 144.63, 148.61, 148.65, 155.71, 165.60, 169.17, 169.62; Anal.
Calcd. (Found) for C17H11N5O6S2: C, 45.84 (46.13); H, 2.49 (2.53); N, 15.72 (15.98).
4.1.4.8.
4-((5-(5,7-Dimethyl-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene)amino)
benzenesulfonamide (4h). Red powder (yield 70% ), m.p. > 300 °C; IR (KBr,
ν
cm-1):
1
3332, 3224 (NH, NH2), 1685 (C=O) and 1323, 1153 (SO2); H NMR (DMSO-d6, 400
22

ACCEPTED MANUSCRIPT
MHz)
δ
ppm: 2.14 (s, 3H, CH3), 2.26 (s, 3H, CH3), 6.97 (s, 1H, H-6 isatin), 7.48 (s, 2H,
NH2, D2O exchangeable), 7.61 (d, 2H, H-3 and H-5 of -C6H4-SO2NH2, J = 8.4 Hz), 7.93 (d,
2H, H-2 and H-6 of -C6H4-SO2NH2, J = 8.4 Hz), 8.4 (s, 1H, H-4 isatin) , 9.94 (s, 1H, NH
isatin , D2O exchangeable), 11.03 (s, 1H, NH thiazolidinone, D2O exchangeable); 13C NMR
(DMSO-d6, 100 MHz)
δ ppm: 16.76, 21.24, 119.50, 120.47, 124.80, 126.03, 126.76,
129.93, 130.75, 133.15, 133.89, 137.78, 139.94, 144.50, 156.73, 165.76, 169.52; MS, m/z
[%]: 428 [M+, 3.99]; Anal. Calcd. (Found) for C19H16N4O4S2: C, 53.26 (53.52); H, 3.76
(3.80); N, 13.08 (13.29).
4.1.4.9.
3-((4-Oxo-5-(2-oxoindolin-3-ylidene)thiazolidin-2-ylidene)amino)
benzenesulfonamide (4i). Red powder (yield 75% ); m.p. > 300 °C; IR (KBr,
ν
cm-1):
1
3307, 3217 (NH, NH2), 1688 (C=O) and 1334, 1156 (SO2); H NMR (DMSO-d6, 400
MHz)
δ ppm: 6.91 (m, 1H, Ar-H), 7.04 (q, 1H, Ar-H, J = 7.76 Hz), 7.26 (d, 0.5H, Ar-H, J
= 7.32 Hz), 7.33 (q, 1.5H, Ar-H, J = 8.08 Hz), 7.45,7.50 (s, 2H, NH2, D2O exchangeable),
7.59-7.68 (m, 2H, Ar-H), 7.98 (s br, 1H, Ar-H), 8.33 (s, 0.5H, Ar-H), 8.81 (d, 0.25H, Ar-H,
J = 7.84 Hz), 8.95 (d, 0.25H, Ar-H, J = 7.88 Hz), 11.15, 12.55 (2s, 1H, NH isatin, D2O
exchangeable), 11.89, 12.68 (2s, 1H, NH thiazolidinone, D2O exchangeable); MS, m/z [%]:
400 [M+, 3.83]; Anal. Calcd. (Found) for C17H12N4O4S2: C, 50.99 (51.27); H, 3.02 (3.09);
N, 13.99 (14.23).
4.1.4.10.
3-((5-(5-Chloro-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene)amino)
benzenesulfonamide (4j). Red powder (yield 72% ), m.p. > 300 °C; IR (KBr,
ν
cm-1):
1
3317, 3275 (NH, NH2), 1685 (C=O) and 1330, 1157 (SO2); H NMR (DMSO-d6, 400
MHz)
δ ppm: 6.88-6.95 (m, 1H, Ar-H,), 7.23 (d, 0.5H, Ar-H, J = 7.2 Hz), 7.35-7.43 (m,
1.5H, Ar-H), 7.48 (s, 2H, NH2, D2O exchangeable), 7.57-7.66 (m, 2H, Ar-H), 7.95 (d,
0.5H, Ar-H, J = 6.8 Hz), 8.29 (s, 0.5H, Ar-H), 8.82 (s, 0.5H, Ar-H), 8.96 (s, 0.5H, Ar-H),
10.82, 11.94 (2s, 1H, NH isatin , D2O exchangeable), 11.24, 12.76 (2s, 1H, NH
thiazolidinone, D2O exchangeable); 13C NMR (DMSO-d6, 100 MHz)
δ ppm: 112.06, 118.2,
118.92, 121.85, 122.08, 122.08, 122.34, 122.99, 123.33, 124.03, 124.35, 124.90, 125.16,
126.15, 127.55, 127.70, 130.58, 130.66, 131.46, 131.55, 135.40, 138.04, 138.90, 142.29,
142.33, 143.37, 145.55, 145.78, 146.91, 168.62, 169.08; Anal. Calcd. (Found) for
C17H11ClN4O4S2: C, 46.95 (47.18); H, 2.55 (2.57); N, 12.88 (13.07).
23

ACCEPTED MANUSCRIPT
4.1.4.11.
3-((5-(5-Bromo-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene)amino)
benzenesulfonamide (4k). Red powder (yield 72% ), m.p. > 300 °C; IR (KBr,
ν
cm-1):
3282, 3209 (NH, NH2), 1678 (C=O) and 1330, 1157 (SO2); H NMR (DMSO-d6, 400
MHz) ppm: 6.83-6.89 (m, 1H, Ar-H), 7.23 (s br, 0.5H, Ar-H), 7.43-7.51 (m, 3.5H, 1.5Ar-
1
δ
H and 2H of NH2, D2O exchangeable), 7.57-7.65 (m, 2H, Ar-H), 7.95 (s, 0.5H, Ar-H), 8.29
(s, 0.5H, Ar-H), 8.95 (s, 0.5H, Ar-H), 9.09 (s, 0.5H, Ar-H), 11.10, 11.94 (2s, 1H, NH isatin
, D2O exchangeable), 11.24, 12.76 (2s, 1H, NH thiazolidinone, D2O exchangeable); 13C
NMR (DMSO-d6, 100 MHz)
δ ppm: 112.53 (2 C), 113.90, 113.93, 118.26, 118.93, 121.85,
122.32, 122.55, 122.98, 123.90, 124.34, 125.02, 126.20, 127.55, 130.33, 130.46, 130.58,
130.66, 134.23, 134.23, 134.32, 135.24, 138.01, 138.90, 142.63, 142.67, 143.37, 145.54,
145.78, 168.49, 168.94, 174.94, 180.76; Anal. Calcd. (Found) for C17H11BrN4O4S2: C,
42.60 (42.56); H, 2.31 (2.27); N, 11.69 (11.88).
4.1.4.12.
benzenesulfonamide (4l). Red powder (yield 70% ), m.p. > 300 °C; IR (KBr,
3275 (NH, NH2), 1678 (C=O) and 1330, 1157 (SO2); 1H NMR (DMSO-d6, 400 MHz)
3-((5-(5-Methoxy-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene)amino)
ν
cm-1): 3309,
δ
ppm: 3.73, 3.75 (s, 3H, OCH3), 6.78-6.85 (m, 1H, Ar-H, J = 8.4 Hz), 6.92-6.97 (m, 1H, Ar-
H), 7.24 (d, 0.5H, Ar-H, J = 6.8 Hz), 7.42,7.47 (2s, 2H, NH2, D2O exchangeable), 7.57-
7.65 (m, 2.5H, Ar-H), 7.94 (d, 0.5H, Ar-H, J = 6 Hz), 8.32 (s, 0.5H, Ar-H), 8.50 (s, 0.5H,
Ar-H), 8.65 (s, 0.5H, Ar-H), 10.92 (s, 1H, NH isatin , D2O exchangeable), 11.87, 12.70 (2s,
1H, NH thiazolidinone, D2O exchangeable); 13C NMR (DMSO-d6, 100 MHz)
δ ppm:
55.93, 110.98, 114.41, 117.77, 118.20, 118.90, 121.22, 121.47, 122.25, 122.84, 124.23,
124.97, 125.78, 126.91, 130.52, 130.64, 136.23, 137.46, 139.00, 145.55, 145.77, 154.98,
155.01, 168.84, 168.29, 175.19, 180.83; MS, m/z [%]: 430 [M+, 1.6]; Anal. Calcd. (Found)
for C18H14N4O5S2: C, 50.23 (50.49); H, 3.28 (3.34); N, 13.02 (13.16).
4.1.4.13.
3-((5-(5-Nitro-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene)amino)
benzenesulfonamide (4m). Red powder (yield 55% ), m.p. > 300 °C; IR (KBr,
ν
cm-1):
1
3286, 3244 (NH, NH2), 1693 (C=O) and 1330, 1153 (SO2); H NMR (DSMO-d6)
δ
ppm:
7.04 (q, 1H, Ar-H, J = 8.8 Hz), 7.25 (d, 1H, Ar-H, J = 8 Hz), 7.43, 7.48 (2s, 2H, NH2, D2O
exchangeable), 7.58-7.66 (m, 2H, Ar-H), 7.95 (s, 1H, Ar-H), 8.22-8.29 (m,1H,Ar-H), ),
9.68 (s, 0.5H, Ar-H), 9.84 (s, 0.5H, Ar-H), 11.77, 12.03 (2s, 1H, NH isatin, D2O
24

ACCEPTED MANUSCRIPT
exchangeable), 11.79, 12.95 (2s, 1H, NH thiazolidinone, D2O exchangeable); 13C NMR
(DMSO-d6, 100 MHz)
δ ppm: 110.84, 118.35, 120.56, 120.78, 123.07, 123.65, 124.42,
127.89, 127.98, 130.59, 130.71, 142.59, 142.62, 145.56, 145.81, 148.74, 148.80, 169.23,
169.71, 180.66; Anal. Calcd. (Found) for C17H11N5O6S2: C, 45.84 (46.12); H, 2.49 (3.47);
N, 15.72 (15.94).
4.1.5. N-Methyyl/Benzyl isatin 6a-f.
Compounds 6a-f were prepared according to the literature procedure [11].
4.1.6. General procedure for synthesis of targeted compounds 7a-g.
To a hot stirred solution of the key intermediates 3a, b (0.27 gm, 1 mmol) and sodium
acetate (0.16 gm, 2 mmol) in acetic acid (15 mL), N-substituted isatins 6a-f were added and
this mixture was heated under reflux for 3 h. The formed solid was filtered while hot, dried
and recrystallized from DMF/ethanol to obtain the target compounds 7a-g.
4.1.6.1.
4-((5-(1-Methyl-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene)amino)
benzenesulfonamide (7a). Red powder (yield 68% ), m.p. > 300 °C; IR (KBr,
ν
cm-1):
1
3266, 3227 (NH, NH2), 1675 (C=O) and 1335, 1151 (SO2); H NMR (DMSO-d6, 400
MHz)
δ ppm: 3.29 (s, 3H, N-CH3), 7.02 (t, 1H, H-5 isatin, J = 7.76 Hz), 7.13 (d, 1H, H-7
isatin, J = 7.8 Hz), 7.43 (t, 1H, H-6 isatin, J = 7.68 Hz), 7.51 (s, 2H, NH2, D2O
exchangeable), 7.65 (d, 2H, H-3 and H-5 of -C6H4-SO2NH2, J = 8.44 Hz), 7.96 (d, 2H, H-2
and H-6 of -C6H4-SO2NH2, J = 8.44 Hz), 8.77 (d, 1H, H-4 isatin, J = 7.84 Hz), 10.08 (s,
1H, NH thiazolidinone, D2O exchangeable); MS, m/z [%]: 414 [M+, 3.06]; Anal. Calcd.
(Found) for C18H14N4O4S2: C, 52.16 (52.43); H, 3.40 (3.45); N, 13.52 (13.81).
4.1.6.2.
4-((5-(5-Chloro-1-methyl-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-
ylidene)amino) benzenesulfonamide (7b). Red powder (yield 75% ), m.p. > 300 °C; IR
1
(KBr,
(DMSO-d6, 400 MHz)
ν
cm-1): 3275, 3224 (NH, NH2), 1678 (C=O) and 1327, 1153 (SO2); H NMR
δ
ppm: 3.25 (s, 3H, N-CH3), 7.02-7.08 (m, 1H, Ar-H), 7.17 (d, 1H,
Ar-H, J = 7.6 Hz), 7.32 (d, 1H, Ar-H, J = 8 Hz), 7.48 (s, 2H, NH2, D2O exchangeable)
7.56-7.65 (m, 1H, Ar-H), 7.83-7.96 (m, 2H, Ar-H), 8.87 (s, 0.5H, Ar-H), 8.95 (s, 0.25H,
Ar-H), 9.09 (s, 0.25H, Ar-H), 10.15, 12.05 (2s, 1H, NH thiazolidinone, D2O
25

ACCEPTED MANUSCRIPT
13
exchangeable); C NMR (DMSO-d6, 100 MHz)
δ ppm: 26.93, 111.42, 114.51, 121.31,
121.56, 126.80, 127.44, 127.69, 129.82, 129.86, 133.83, 136.66, 137.49, 143.52, 144.59,
155.88, 165.60, 167.08; Anal. Calcd. (Found) for C18H13ClN4O4S2: C, 48.16 (48.40); H,
2.92 (2.89); N, 12.48 (12.69).
4.1.6.3.
4-((5-(5-Bromo-1-methyl-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-
ylidene)amino) benzenesulfonamide (7c). Red powder (yield 59% ), m.p. > 300 °C; IR
1
(KBr,
(DMSO-d6, 400 MHz)
ν
cm-1): 3275, 3221 (NH, NH2), 1678 (C=O) and 1330, 1153 (SO2); H NMR
δ
ppm: 3.25 (s, 3H, N-CH3), 7.12 (d, 1H, H-7 isatin, J = 8.44 Hz),
7.46 (dd, 1H, H-6 isatin, J = 2.08- 8.4 Hz), 7.52 (s, 2H, NH2, D2O exchangeable), 7.66 (d,
2H, H-3 and H-5 of -C6H4-SO2NH2, J = 8.48 Hz), 7.97 (d, 2H, H-2 and H-6 of -C6H4-
SO2NH2, J = 8.48 Hz), 8.75 (s, 1H, H-4 isatin), 10.19 (s, 1H, NH thiazolidinone, D2O
exchangeable); Anal. Calcd. (Found) for C18H13BrN4O4S2: C, 43.82 (43.97); H, 2.66 (2.71);
N, 11.36 (11.58).
4.1.6.4.
4-((5-(1-Benzyl-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene)amino)
benzenesulfonamide (7d). Red powder (yield 75% ), m.p. > 300 °C; IR (KBr,
ν
cm-1):
1
3352, 3244 (NH, NH2), 1674 (C=O) and 1396, 1153 (SO2); H NMR (DMSO-d6, 400
MHz)
δ ppm: 5.05 (s, 2H, benzylic CH2), 7.03-7.07 (m, 1H, Ar-H), 7.24-7.35 (m, 7H, Ar-
H), 7.48 (s, 2H, NH2, D2O exchangeable), 7.63 (d, 2H, Ar-H, J = 8.8 Hz), 7.94 (d, 2H, Ar-
H, J = 8.4 Hz), 8.77 (s, 0.5H, Ar-H), 8.80 (s, 0.5H, Ar-H), 10.09 (s, 1H, NH thiazolidinone,
D2O exchangeable); 13C NMR (DMSO-d6, 100 MHz)
δ ppm: 43.47, 110.08, 120.15,
122.83, 122.95, 123.45, 126.76, 127.59, 128.01, 129.19, 129.86, 131.86, 135.80, 136.51,
137.68, 143.46, 144.54, 156.09, 165.59, 167.71; MS, m/z [%]: 490 [M+, 1.68]; Anal. Calcd.
(Found) for C24H18N4O4S2: C, 58.76 (58.98); H, 3.70 (3.79); N, 11.42 (11.71).
4.1.6.5.
4-((5-(1-Benzyl-5-chloro-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-
ylidene)amino) benzenesulfonamide (7e).Red powder (yield 65% ), m.p. > 300 °C; IR
1
(KBr,
(DMSO-d6, 400 MHz)
ν
cm-1): 3269, 3230 (NH, NH2), 1683 (C=O) and 1344, 1156 (SO2); H NMR
δ
ppm: 4.96, 5.05 (2s, 2H, benzylic CH2), 7.05 (t, 1H, Ar-H, J = 8.8
Hz), 7.20-7.32 (m, 7H, Ar-H), 7.37 (s, 2H, NH2, D2O exchangeable), 7.42-7.45 (m, 1H, Ar-
H), 7.83-7.89 (m, 1H, Ar-H), 7.94 (d, 1H, Ar-H, J = 8.8 Hz), ), 8.91 (s, 0.5H, Ar-H), 9.06
26

ACCEPTED MANUSCRIPT
13
(s, 0.5H, Ar-H), 12.07, 12.96 (2s, 1H, NH thiazolidinone, D2O exchangeable); C NMR
(DMSO-d6, 100 MHz) ppm: 43.54, 111.50, 114.75, 121.33, 121.37, 127.02, 127.49,
δ
127.58, 127.70, 127.75, 128.05, 129.17, 129.20, 131.28, 136.12, 141.45, 142.18, 155.73,
165.63, 167.30; Anal. Calcd. (Found) for C24H17ClN4O4S2: C, 54.91 (55.23); H, 3.26
(3.30); N, 10.67 (10.92).
4.1.6.6.
4-((5-(1-Benzyl-5-bromo-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-
ylidene)amino) benzenesulfonamide (7f). Red powder (yield 66% ), m.p. > 300 °C; IR
1
(KBr,
(DMSO-d6, 400 MHz)
ν
cm-1): 3271, 3232 (NH, NH2), 1693 (C=O) and 1384, 1157 (SO2); H NMR
δ
ppm: 5.05 (s, 2H,benzylic CH2), 7.02 (d, 1H, H-7 isatin, J = 8.4
Hz), 7.24-7.34 (m, 5H, phenyl ring), 7.48 (s, 2H, NH2, D2O exchangeable), 7.52 (d, 1H, H-
6 isatin, J = 8.4 Hz), 7.63 (d, 2H, H-3 and H-5 of -C6H4-SO2NH2, J = 8.4 Hz), 7.94 (d, 2H,
H-2 and H-6 of -C6H4-SO2NH2, J = 8.4 Hz), 8.96 (s, 1H, H-7 isatin), 10.20 (s, 1H, NH
thiazolidinone, D2O exchangeable); 13C NMR (DMSO-d6, 100 MHz)
δ ppm: 43.54, 112.00,
114.75, 121.33, 121.87, 126.83, 127.55, 128.08, 129.22, 129.84, 130.13, 133.80, 136.20,
137.53, 137.69, 142.45, 144.63, 155.73, 165.63, 167.36; Anal. Calcd. (Found) for
C24H17BrN4O4S2: C, 50.62 (50.86); H, 3.01 (3.07); N, 9.84 (10.01).
4.1.6.7.
3-((5-(1-Benzyl-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene)amino)
benzenesulfonamide (7g). Red powder (yield 70% ), m.p. > 300 °C; IR (KBr,
ν
cm-1):
1
3282, 3201 (NH, NH2), 1678 (C=O) and 1311, 1149 (SO2); H NMR (DMSO-d6, 400
MHz)
δ ppm: 4.96, 5.05 (2s, 2H,benzylic CH2), 7.03 (t, 1H, Ar-H, J = 7.04 Hz), 7.09
(q,1H, Ar-H, J = 7.8 Hz), 7.25-7.38 (m, 5H, Ar-H), 7.46-7.50 (m, 3H, 1H Ar-H and 2H,
NH2, D2O exchangeable), 7.59-7.68 (m, 2H, Ar-H), 7.95 (d, 1H, Ar-H, J = 7.36 Hz), 8.31
(s, 1H, Ar-H), 8.89 (d, 0.5H, Ar-H, J = 7.8 Hz), 9.03 (d, 0.5H, Ar-H, J = 7.8 Hz), 12.36,
13
12.41 (2s, 1H, NH thiazolidinone, D2O exchangeable); C NMR (DMSO-d6, 100 MHz)
δ
ppm: 43.52, 43.68, 110.00, 118.47, 119.00, 120.15, 120.44, 122.27, 122.67, 123.04, 123.84,
124.56, 125.02, 127.62, 127.75, 128.00, 128.22, 128.37, 129.20, 130.46, 130.64, 131.97,
136.44, 136.53, 143.48, 145.49, 145.78, 167.55, 168.18; Anal. Calcd. (Found) for
C24H18N4O4S2: C, 58.76 (58.94); H, 3.70 (3.67); N, 11.42 (11.59).
27

ACCEPTED MANUSCRIPT
4.2. Biological Evaluation
4.2.1. CA inhibitory assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA
catalysed CO2 hydration activity, as reported earlier [31]. The inhibition constants were
obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff
equation as reported earlier [42], and represent the mean from at least three different
determinations. The four tested CA isofoms were recombinant ones obtained in-house as
reported earlier [43].
4.2.2. Anti-proliferative activity against MCF-7
MCF-7 cells (human breast cancer cell line), were obtained from VACSERA Tissue
Culture Unit. The cells were propagated in DMEM supplemented with 10% heat-
inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer and 50µg/ml gentamycin.
All cells were maintained at 37ºC in a humidified atmosphere with 5% CO2 and were
subcultured two times a week. Cytotoxicity was determined following a reported procedure
[44]. The relation between surviving cells and drug concentration is plotted to get the
survival curve of each tumor cell line after treatment with the specified compound. The
50% inhibitory concentration (IC50) was estimated from graphic plots of the dose response
curve for each conc. using Graphpad Prism software (San Diego, CA. USA). The data
presented are the mean of at least three separate experiments.
4.2.3. ELISA immunoassay
The levels of the apoptotic markers Bax, caspase-3, caspase-9, cytochrome C and p53 as
well as the anti-apoptotic marker Bcl-2 were assessed using ELISA colorimetric kits per the
manufacturer’s instructions, as reported earlier [20, 45].
4.2.4. Statistical analysis
Data are presented as the mean ±S.D. Comparisons were carried out using one-way
analysis of variance followed by Tukey–Kramer’s test for post hoc analysis. Statistical
significance was considered to be P<0.05. All statistical analyses were performed using
28