Mechanistic investigations on substituted benzene sulphonamides as apoptosis inducing anticancer agents

Article abstract of DOI:10.1016/j.bioorg.2019.103539

In an approach to develop potent cytotoxic compounds with targeted action, a systematic methodology was employed to design and initially synthesize parent compounds A1, A8, A13 and A14 followed by synthesis of further analogs of A1 (A2-A7) and A8 (A9-A12)

Full text of DOI:10.1016/j.bioorg.2019.103539

Journal Pre-proofs
Mechanistic investigations on substituted benzene sulphonamides as apoptosis
inducing anticancer agents
Akhila Mettu, Venu Talla, Soujanya Thumma, Subhashini Naikal James
Prameela
PII:
S0045-2068(19)31166-6
DOI:
https://doi.org/10.1016/j.bioorg.2019.103539
YBIOO 103539
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
22 July 2019
27 November 2019
21 December 2019
Please cite this article as: A. Mettu, V. Talla, S. Thumma, S. Naikal James Prameela, Mechanistic investigations
on substituted benzene sulphonamides as apoptosis inducing anticancer agents, Bioorganic Chemistry (2019),
doi: https://doi.org/10.1016/j.bioorg.2019.103539
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. 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.
© 2019 Published by Elsevier Inc.

Mechanistic investigations on substituted benzene sulphonamides as apoptosis inducing
anticancer agents
Akhila Mettu,a,c Venu Talla,b Soujanya Thumma,a Subhashini Naikal James Prameela,*c
aDepartment of Pharmaceutical Chemistry, Gokaraju Rangaraju College of Pharmacy, Kukatpally,
Hyderabad, India 500049.
bDepartment of Pharmacology, National Institute of Pharmaceutical Education and Research, Balanagar,
Hyderabad, India 500037.
cDepartment of Chemistry, University College of Science, Osmania University, Hyderabad, India 500007.
Email: njsubhashini@yahoo.co.in
ABSTRACT
In an approach to develop potent cytotoxic compounds with targeted action, a systematic methodology was
employed to design and initially synthesize parent compounds A1, A8, A13 and A14 followed by synthesis
of further analogs of A1 (A2-A7) and A8 (A9-A12) with characterization by IR, NMR, mass and elemental
techniques. These compounds were evaluated for their in vitro anti-proliferative activities against DU-145,
MCF-7, HCT-15, HT-29 cell lines and apoptosis inducing potential via various mechanistic studies.
Compounds A2, A9, A10 exhibited significant cytotoxic activities compared to their parent compounds and
standard drug 5-fluorouracil. Compound A2 displayed superior cytotoxicity with IC50 values less than 1
µM in most of the tested cell lines. Further, compound A2 also induced apoptosis in DU-145 cells as
exemplified from DAPI staining, Annexin V-FITC assay, ROS generation and mitochondrial membrane
alteration studies. The above studies depict the synthesized compound A2 as potent anticancer agent with
the ability to induce apoptosis in prostate cancerous cells.
Keywords: Benzene sulphonamide; Apoptosis; ROS; Anticancer; Annexin V.
1

INTRODUCTION
Research on cancer remains a continuous strive to unveil the novel drugs possessing targeted action with
minimal side effects [1]. Generation of a tumor involves activation/deactivation of numerous pathways and
proteins involved in cell growth and regulation [2,3]. Design and development of novel drugs for cancer
therapy involves activation of proapoptotic factors and inhibition of antiapoptotic factors [4,5]. The various
modes of cancerous cell death include the activation of intrinsic and extrinsic pathways of apoptosis,
followed by release of caspase-3, release of cytochrome-c following mitochondrial stress, ROS mediated
cell death and so on [6-9].
Pyrimidine is a renowned endogenous metabolite and its analogs have various therapeutic roles
including anticancer action [10]. 5-Fluoro uracil, Tegafar are renowned pyrimidine antimetabolites for
cancer therapy [11]. Various fused pyrimidines and aryl pyrimidines are reported in literature for their
prominent anticancer activities [12-15]. Benzene sulphonamides are reported to have anticancer activity
exhibited through multiple mechanisms, the most prominent ones being carbonic anhydrase inhibition and
apoptotic induction [16,17]. Indisulam, SLC-0111 are the sulphonamide analogs possessing anticancer
activity with induction of apoptosis [18,19]. Literature reports thioureido benzene sulphonamides [20],
thiazolidone benzene sulphonamides [21], 7-chloro quinoline benzene sulphonamides [22], guanidinyl
benzene sulphonamides [23] as anticancer compounds exhibited through various mechanisms.
Owing to the crucial role of pyrimidine analogs as antimetabolites and sulphonamide moiety as an
essential pharmacophoric group in multiple anticancer drugs [19,24,25], we attempted to link substituted
pyrimidine/ tetrahydro quinazoline with benzene sulphonamide and initially synthesized a set of four
variously substituted benzene sulphonamide derivatives as parent compounds (A1, A8, A13 and A14)
employing four different schemes, and thereby evaluated for anticancer activity (Fig. 1). Subsequent
analogs were synthesized from A1 (A2-A7) and A8 (A9-A12) and mechanistically investigated for their
apoptotic potentials.
2

2. RESULTS AND DISCUSSION
2.1. Chemistry
The scaffold was designed based on the indisulam anticancer drug. Two variants of pyrimidines were
synthesized initially, one with attached aromatic groups and the other fused with cyclohexene. Then, the
sulphonamide group was attached in two different ways, one substituted on the pyrimidine ring as
sulphomoyl group (A1-A12) and other on the benzene ring as terminal sulphonamide (A13, A14). Overall,
four variants of analogs were synthesized employing two common synthetic routes (Fig. 1).
Claisen-Schmidt condensation of substituted benzaldehydes with substituted acetophenones
/cyclohexanone in 40% aq. NaOH and methanol afforded chalcones C1-C12. Reaction with cyclohexanone
yielded mixture of products generating mono and di substituted alkenes. The required compound was
separated by column chromatography. The IR spectrum showed the characteristic (C=O) peaks between
1680-1730 cm-1 and (C=C) peaks at 1600 to 1650 cm-1 along with molecular ion peaks [M+] in mass
spectrum. The chalcones were converted to 2-amino pyrimidines P1-P12 by refluxing guanidine
hydrochloride in DMF with sodium ethoxide as base. The IR spectra of compounds showed the presence
of N-H, C=C, C=N, C-N stretching around 3300-3400 cm-1, 1600-1650 cm-1, 1690-1640 cm-1, 1300-1350
3

cm-1 respectively; 1H NMR signals were observed at δ 5.0-5.5 ppm representing amino protons and δ 7.7-
8.0 ppm indicating pyrimidine proton. The characteristic mass fragmentation pattern was observed at amino
(-NH2) group. In the third step, the scheme was bifurcated into two parts, one part involved the nucleophilic
acyl substitution reaction between 2-amino pyrimidine with benzene sulphonyl chloride in pyridine to
produce benzene sulphonamides A1-A12 (Scheme 1 and 2, Table 1 and 2). The IR spectrum of these
compounds showed characteristic N-H stretching at 3300-3600 cm-1 and 1300-1360, 1100-1150 cm-1 for
1
O=S=O stretching; H-NMR depicted the peaks for aliphatic methylene protons of cyclohexene ring at δ
1.6-1.7 and δ 2.6-2.7 ppm, methoxy protons at δ 3.5-4.0 ppm, aromatic protons at δ 6.8-8.3 ppm and the
proton of sulphonamide group at δ 11.0-11.8 ppm. In another part, the amino group of pyrimidine (P2, P9)
reacted with benzoyl chloride to generate benzamide group (Q1, Q2), which was further reacted with chloro
sulphonyl chloride to obtain intermediates (R1, R2) and then refluxed with ammonium hydroxide to yield
the final products (A13, A14) possessing terminal sulphonamide group (Scheme 3 and 4). The IR spectrum
of these compounds showed absorptions bands characteristic for N-H stretching at 3500-3600 cm-1; O=S=O
stretching at 1360, 1150 cm-1; C=O stretching at 1690-1720 cm-1, 1H-NMR confirmed the presence of two
NH groups , free amino group at δ 5.5-6.0 ppm and NH group of benzamide at δ 9.5-9.8 ppm. Mass
spectrums (molecular ion peaks) and elemental analysis (limits within 0.4) further confirmed the chemical
structures of the final compounds.
4

5

2.2. Biology
2.2.1 Anticancer activity
To comprehend the effect the sulphonamide substitution, initially A1, A8 (parent compounds) with
sulphomoyl group, A13 and A14 (parent compounds) with terminal sulphonamide group were synthesized
and evaluated for anticancer screening by MTT assay. Compounds A1, A8 were moderately active and
A13, A14 were least active (Table 1,2,3). Therefore, further analogs of A1 (pyrimidinyl series) were
6

developed with various substitutions (A2-A7) and similarly for A8 (tetrahydro quinazolinyl series), further
analogs (A9-A12) were developed. A2 was the most potent compound in all the tested cell lines except
MCF-7. Its potency was comparably higher than the tested standard drugs, 5-fluoro uracil (5-FU) against
DU-145 (prostrate carcinoma), HCT-15, HT-29 (colon carcinoma) cell lines. Compound A3 was the most
potent one in MCF-7 (breast carcinoma) cell line. Compared to A1; compounds A2, A3, A4, A6 were more
active in DU-145 cell line; similarly, A2, A3 in MCF-7; A2, A3, A4 in HCT-15 cell line; A2, A4 in HT-29
cell line. Compared with A8, compounds A9, A10, A12 were more active in DU-145 cell line; A10, A12
in HCT-15 cell line; A9, A10 in HT-29 cell line. Overall, A2, A9 and A10 were comparatively more active
WRT anticancer activity. This could be due to multiple methoxy substitutions on the phenyl rings. Further,
compounds with methoxy substitutions were more active than chloro substituted ones, tri/di substitution
predominated over mono substitution (A6, A7, A11 were least active); dichloro compounds were more
active than mono chloro compounds as elucidated in SAR studies.
Table 1
Structural features of compounds A1-A7 along with their IC50 values (µM) for anticancer activity
obtained by MTT assay.
Structural features of A1-A7
R3 R4 R5 R6
Results of MTT assay (IC50, µM)
#
R1
R2
R7
OCH3 OCH3
DU-145 MCF-7 HCT-15 HT-29
A1
18.45±
1.52
16.87±
1.61
5.54±
0.59
(parent
compd)
H
H
Cl
H
H
>20
0.76±
0.11
8.56±
0.96
0.10±
0.04
0.22±
0.05
A2
A3
A4
A5
H
OCH3 OCH3 OCH3 OCH3
H
OCH3
4.29±
0.25
6.33±
0.54
3.41±
0.20
17.80±
0.23
OCH3
OCH3
H
H
H
OCH3
OCH3
H
H
H
H
Cl
Cl
OCH3 OCH3
2.08±
0.25
11.52±
4.27
0.86±
0.27
H
H
Cl
Cl
>20
>20
14.60±
0.96
OCH3 OCH3
>20
>20
13.72±
4.29
A6
A7
H
H
H
H
OCH3
OCH3
H
H
H
H
Cl
Cl
OCH3
Cl
>20
>20
>20
>20
>20
>20
>20
7

Cytotoxicity value in µM between 0-10: most active; 10-20: moderately active; >20: least active. Each
reading represents mean of three ±SD; MCF-7, breast carcinoma; HCT-15, HT-29, colon adenocarcinoma;
DU-145, prostate carcinoma.
Table 2
Structural features of compounds A8-A12 along with their IC50 values (µM) for anticancer activity
obtained by MTT assay.
Structural features of A8-A12
Results of MTT assay (IC50, µM)
#
R1
R2
R3
R4
R5
DU-145
MCF-7
HCT-15
HT-29
A8
(parent
compd)
7.34±
0.37
10.40±
0.33
11.04±
0.23
14.43±
0.66
H
H
Cl
H
H
3,4,5-
3.43±
0.28
14.84±
1.31
3.11±
0.45
A9
H
H
OCH3 OCH3 OCH3 trimethoxy
>20
>20
>20
benzylidene
3,4-
dimethoxy
benzylidene
3,4-
dichloro
benzylidene
3,4-
1.14±
0.08
1.87±
0.41
4.99±
0.60
A10
A11
A12
OCH3 OCH3
H
H
H
OCH3
H
H
OCH3
H
>20
>20
>20
5.55±
0.29
19.59±
1.51
9.74±
0.42
14.38±
0.97
OCH3
dimethoxy
benzylidene
Cytotoxicity value in µM between 0-10: most active; 10-20: moderately active; >20: least active. Each
reading represents mean of three ±SD; MCF-7, breast carcinoma; HCT-15, HT-29, colon adenocarcinoma;
DU-145, prostate carcinoma.
Table 3
Cytotoxicity (IC50) values in µM obtained by MTT assay.
DU-145
MCF-7
HCT-15 HT-29
A13
>20
>20
>20
>20
>20
>20
(parent
compd)
A14
(parent
compd)
>20
>20
1.15±0.07
5.12±1.33 1.02±0.4 4.2±1.2
5-FU
Cytotoxicity value >20 µM represents least activity. Each reading
represents mean of three ±SD; MCF-7, breast carcinoma;
8

HCT-15, HT-29, colon adenocarcinoma; DU-145, prostate
carcinoma.5-FU, 5-fluoro uracil: positive control.
\2.2.2. Morphological changes observed through DAPI staining
Nucleomorphological changes like horse shoe shaped nuclei and nuclear fragmentation were
observed upon treatment of DU-145 cells with compound A2 for 24 h and further staining with DAPI. The
effect was observed in concentration dependent manner (0.5, 1, 2, 4 µM) similar to the standard 5-FU.
Nuclear morphology of control cells was devoid of any changes. (Fig. 2). These nuclear changes indicate
the progression of apoptosis following drug treatment.
2.2.3. Apoptosis induction confirmed by Annexin V binding assay
To exploit the underlying mechanism of title compound A2, Annexin V/PI double staining assay
was performed. The percentage of cells in various stages of apoptosis were noted after treatment with A2
and the results were compared with control and standard. There was a rise in total percentage of apoptotic
9

and dead cell population from 6.70 % in control to 14.91 % at 0.5 µM and 35.46 % at 4 µM respectively in
a dose dependent manner, while standard (5-FU, 1 µM) displayed 26.78 % of apoptotic population. The
percentage of live cells declined in a dose dependent manner from 93.30% in control to 64.54 % at 4 µM,
while the standard displayed 73.22% of live cells (Fig. 3.). The increase in apoptotic population indicates
the ability of title compound A2 to induce apoptosis mediated cytotoxicity.
2.2.4. Alteration of mitochondrial membrane potential in apoptotic cells identified by JC-1 stain.
JC-1 stain is an indicator of mitochondrial membrane potential in healthy cells due to accumulation
of J-aggregates with excitation frequency at 590 nm. In apoptosis, there is a significant change in
mitochondrial potential due to the opening of mitochondrial permeability transition pore (MPTP) leading
to decoupling of respiratory chain and release of cytochrome c. This is evidenced by blue fluorescence
(540 nm) due to the formation of J-monomers upon blue light excitation (490 nm). Flow cytometric analysis
10

displayed an increase in apoptotic population (depolarized cell population) from 23.11% in control to 39.54
% at 4 µM. The alteration of mitochondrial membrane potential upon treatment with A2 was manifested
by decrease in orange fluorescence and increase in blue fluorescence in dose dependent manner (Fig. 4).
This could be due to increase in J-monomer formation upon apoptosis depicting the mitochondrial
involvement in cell arrest probably via release of cytochrome c.
2.2.5. ROS generation by DCFDA staining
ROS is a reactive molecular entity involved in many biological pathways. When the levels of ROS
increases in cancer cells, there would be an irreparable damage to DNA and proteins followed by activation
of caspase-3 with apoptosis induction. tumor cells. In the present study, production of ROS was observed
when DU-145 cells were treated with increasing concentrations (2.5, 5, 10 µM) of compound A2 followed
by DCFDA staining (Fig. 5.). The dose dependent shift in peaks towards right indicates the rise in
fluorescence intensity. Similar effect was observed with standard 5-FU at 10 µM concentration.
11

2.3. Structure Activity Relationship (SAR)
To understand the substituent effects on cytotoxicity, structure activity relationship studies were
established (Table 1,2 and 3). The highlights are summarized below.
Basic skeleton: Compounds bearing benzene sulphonamide moiety (A1-A12) were more active
than 3-sulphomoyl benzamide derivatives (A13, A14). 4,6-disubstituted pyrimidine nucleus (A1-A7) was
comparatively more active than tetrahydro quinazolinyl nucleus (A8-A12).
Methoxy substitution: Increasing the methoxy groups significantly increased the cytotoxicity of the
molecules. This was observed with compounds A2, A3, A9 and A10. Increasing the methoxy groups in the
range of 4-6 was more advantageous. This could be due to enhanced lipophilicity that allows the molecule
to enter into the cytotoxic cells easily and thereby display their inhibitory potential.
Chloro substitution: Dichloro substitution was more predominate over mono chloro substitution.
This is well exemplified with compounds A4, A5, A11 with dichloro substitution being more active than
parent analogs A1 and A8 with mono chloro substitution. Increase in chloro groups enhances the polarity
12

of the molecule and therefore could be the reason for enhanced activity observed with A4 and A5. However,
methoxy substituted compounds (A2, A3, A9, A10) were more active than chloro substituted ones.
Benzylidene substitution: Compounds bearing benzylidene group at 8th position on quinazoline ring
(A9-A12) were more active than the parent compound A8. Trimethoxy benzylidene group (A9) and
dimethoxy (A10) were more active than mono methoxy benzylidene group (A12). Further, dichloro
substitution on benzylidene decreased the activity of the molecule (A11).
CONCLUSION
Four variants of benzene sulphonamides were developed and their effect on anticancer activity was
analyzed. Preliminary anti-proliferative screening has identified compounds A1, A8 as lead parent
molecules. Further analogs of A1 and A8 were developed as 4,6-disubstituted pyrimidinyl benzene
sulphonamides (A2-A7) and other as tetrahydro quinazolinyl benzene sulphonamides (A9-A12). Most of
them have shown improvised activity profiles than parent compounds. A2 was the most potent compound
of the series and also potent than the standard drug, 5-fluorouracil with IC50 values less than 1 µM in DU-
145, HCT-15, HT-29 cell lines. The drug also induced apoptosis in DU-145 cells as evidenced by various
mechanistic studies on apoptosis. All of the above studies proved compound A2 as excellent apoptosis
inducing cytotoxic agent with potential source as lead for next generation.
4. MATERIALS AND METHODS
4.1. Chemistry
IR spectra were recorded by KBr pellet technique on Shimadzu IR affinity-1, FT-IR
spectrophotometer (Schimadzu, Germany). Nuclear magnetic resonance (1H and 13C NMR) spectra were
recorded using Bruker Avance spectrophotometer (Bruker Analytic, Karlsruhe, Germany; Bruker AG,
Fallanden, Switzerland) with chemical shifts in parts per million (ppm, δ) downfield from TMS as an
internal standard using CDCl3 or DMSO solvent. Mass spectra were recorded with 6540 Qtof - Infinity
1290 (Agilent Technologies, India). Elemental analyses were carried out on Perkin-Elmer 2400 analyser
(Waltham, MA, USA) and the results were found to be in good agreement within ±0.4 compared to their
13

theoretical valves. All the melting points were determined using BÜCHI Melting Point B-450 apparatus
(Büchi Labortechnik, Switzerland) and were uncorrected. Commercially available reagents and solvents
were procured from Sigma Aldrich/Merck and used without any purification unless stated. All the
synthesized compounds were purified on Buchi Flash Chromatography using Merck silica gel 200-400#.
Analytical thin-layer chromatography was carried out on Merck silica gel plates. All reactions were carried
out nitrogen or under air atmosphere. General procedures for Chalcone synthesis (C1-C12) and pyrimidine
synthesis (P1-P12) was performed in according to the procedures prescribed by Verma et.al.[26].
General procedure for synthesis of N-[4,6-diaryl pyrimidin-2-yl]benzene sulfonamides and N-
[2,6-diaryl (5,6,7,8-tetrahydro-quinazolin-2-yl)]-benzenesulfonamides
To an ice cooled solution of intermediates (P1-P12, 1 mmol) in pyridine, benzene sulfonyl chloride (5
mmol) was slowly added. Then the reaction mixture was stirred at 0 °C to room temperature for 24 h.
Further, dil. HCl was added and the obtained crystals were filtered, washed with water and recrystallized
from ethanol to yield final compounds A1-A12 (Scheme 1 and 2; Table 1 and 2).
N-[4-(4-chloro-phenyl)-6-(3,4-dimethoxy-phenyl)-pyrimidin-2-yl]-4-sulfamoyl-benzamide A1
Yellowish solid, yield 75%, mp 102 oC. FT-IR (KBr) cm-1: 3649 (N-H stretching), 1350, 1150 (O=S=O
1
stretching), 1616, 1454 (C=C aromatic stretching). H-NMR (300 MHz, CDCl3) δ (ppm): 3.86 (s, 3H,
OCH3), 3.94 (s, 3H, OCH3) 7.05-7.07 (d, 1H, ArH), 7.09-7.11 (d, 1H, ArH), 7.44 (s, 1H, ArH), 7.53-7.55
(d, 2H, ArH), 7.77-7.96 (m, 3H, ArH), 8.05-8.07 (d, 2H, ArH), 8.19-8.21 (d, 2H, ArH), 8.35-8.37 (s, 1H,
ArH), 11.46 (s, 1H, NH).13C-NMR (75 MHz, CDCl3): 167.7, 164.2, 160.3, 149.9, 148.6, 144.3, 134.2,
133.5, 132.4, 130.3, 129.5, 129.4, 128.9, 127.6, 122.8, 109.6, 100.2, 97.4, 56.4. ESI-MS: m/z 482 (M+H)+.
Anal. Calcd. for C24H20ClN3O4S: C, 59.81; H, 4.81; N, 8.72. Found: C, 59.85; H, 4.83; N, 8.61.
N-[4-(3,4-dimethoxy-phenyl)-6-(3,4,5-trimethoxy-phenyl)-pyrimidin-2-yl]-benzenesulfonamide A2
Yellowish solid, yield 45%, mp 220 oC. FT-IR (KBr) cm-1: 3612 (N-H stretching), 1338, 1126 (O=S=O
1
stretching), 1650, 1456 (C=C aromatic stretching). 1H-NMR (300 MHz, CDCl3) δ (ppm): H-NMR (300
MHz, CDCl3) δ (ppm): 3.79 (s, 3H, OCH3), 3.83 (s, 6H, OCH3), 3.85 (s, 3H, OCH3), 3.87 (s, 3H, OCH3),
14

7.04-7.06 (d, 1H, ArH), 7.10-7.12 (d, 1H, ArH), 7.33-7.35 (d, 1H, ArH), 7.40 (d, 1H, ArH), 7.59-7.65 (m,
2H, ArH), 7.77 (s, 1H, ArH), 7.85-7.87 (dd, 1H, ArH), 8.05-8.07 (dd, 1H, ArH) , 8.14 (s, 1H, ArH), 8.20-
8.22 (d, 1H, ArH), 11.86 (d, 1H, NH). 13C-NMR (75 MHz, CDCl3): 165.1, 163.7, 160.5, 153.5, 150.8,
149.6, 144.3, 137.7, 132.4, 131.5, 129.4, 128.7, 128.0, 122.8, 110.8, 110.2, 106.2, 104.7, 56.4, 56.0. ESI-
MS: m/z 538 (M + H+). Anal. Calcd. for C27H27N3O7S: C, 60.32; H, 5.06; N, 7.82. Found: C, 60.35; H, 5.07;
N, 7.78.
N-[4-(3,4-dimethoxy-phenyl)-6-(2,4-dimethoxy-phenyl)-pyrimidin-2-yl]-benzenesulfonamide A3
o
Colorless solid, yield 45%, mp 160 C, FT-IR (KBr) cm-1 : 3489 (N-H stretching), 1367, 1145 (O=S=O
stretching) , 1607, 1467 (C=C aromatic stretching).1H-NMR (300 MHz, CDCl3) δ (ppm): 3.78 (s, 3H,
OCH3), 3.81 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 7.11-7.13 (d, 1H, ArH), 7.15 (s, 1H,
ArH), 7.56 (s, 1H, ArH), 7.58-7.60 (d, 1H, ArH), 7.61-7.63 (d, 2H, ArH), 7.72-7.73 (m, 2H, ArH), 7.84-
7.87 (t, 1H, ArH), 8.01-8.02 (d, 1H, ArH), 8.10 (s, 1H, ArH), 8.22-8.23 (d, 1H, ArH) ), 11.66 (s, 1H, NH)
. 13C-NMR (75 MHz, CDCl3): 167.3, 163.9, 161.7, 159.4, 158.2, 154.6, 150.3, 134.2, 132.4, 131.1, 130.4,
129.4, 129.5, 128.9, 127.6, 122.8, 105.6, 100.2, 97.4, 56.1. ESI-MS: m/z 530 (M+Na+). Anal. Calcd. for
C26H25N3O6S: C, 61.53; H, 4.96; N, 8.28. Found: C, 61.51; H, 4.94; N, 8.30.
N-(4-(2,4-dichlorophenyl)-6-(2,4-dimethoxyphenyl) pyrimidin-2-yl) benzenesulfonamide A4
Colorless solid, yield 60 %, mp 112 oC, FT-IR (KBr) cm-1: 3365 (N-H stretching), 1314, 1156 (O=S=O
stretching), 1649, 1445 (C=C aromatic stretching).1H-NMR (300 MHz, CDCl3) δ (ppm): 3.83 (s, 3H,
OCH3), 3.85 (s, 3H, OCH3), 7.04-7.12 (d, 2H, ArH), 7.28-7.31 (d, 1H, ArH), 7.39 (s, 2H, ArH), 7.45-7.48
(d, 1H, ArH), 7.62-7.65 (m, 3H, ArH), 7.85-7.87 (dd, 2H, ArH), 8.14 (s, 1H, ArH), 11.58 (s, 1H, NH). 13C-
NMR (75 MHz, CDCl3): 165.4, 163.2, 156.7, 153.5, 152.1, 149.1, 136.5, 133.3, 130.8, 129.9, 129.4, 128.8,
128.3, 126.2, 122.8, 110.9, 110.7, 110.0, 106.6, 100.2, 56.1. ESI-MS: m/z 515 (M+). Anal. Calcd. for
C24H19Cl2N3O4S: C, 55.82; H, 3.71; N, 8.14. Found: C, 55.61; H, 3.94; N, 8.27.
N-[4-(2,4-dichloro-phenyl)-6-(3,4-dimethoxy-phenyl)-pyrimidin-2-yl]-benzenesulfonamide A5
Yellowish solid, yield 67%, mp 108 oC, FT-IR (KBr) cm-1: 3371 (N-H stretching), 1312, 1162 (O=S=O
1
stretching), 1656, 1459 (C=C aromatic stretching). H-NMR (300 MHz, CDCl3) δ (ppm): 3.84 (s, 3H,
15

OCH3), 3.86 (s, 3H, OCH3), 7.02-7.11 (dd, 1H, ArH), 7.28-7.48 (m, 1H, ArH), 7.56-7.65 (d, 2H, ArH),
7.77-7.89 (m, 3H, ArH), 8.02-8.05 (d, 2H, ArH), 8.19 (d, 2H, ArH), 8.38 (s, 1H, ArH), 11.92 (s, 1H, NH).
13C-NMR (75 MHz, CDCl3): 164.4, 161.2, 160.0, 157.6, 154.6, 147.3, 136.4, 134.2, 132.4, 130.4, 129.9,
129.0, 128.5, 128.2, 127.6, 126.2, 122.8, 111.5, 108.9, 100.6, 56.1. ESI-MS: m/z 516 (M+H+). Anal. Calcd.
for C24H19Cl2N3O4S: C, 61.53; H, 4.96; N, 8.28. Found: C, 61.51; H, 4.94; N, 8.30.
N-[4-(4-methoxy phenyl)-6-(4-methoxy-phenyl)-pyrimidin-2-yl]-benzenesulfonamide A6
Colorless solid, yield 72 %, mp 242 oC, FT-IR (KBr) cm-1: 3592 (N-H stretching), 1378, 1143 (O=S=O
stretching), 1650, 1489 (C=C aromatic stretching). 1H-NMR (300 MHz, CDCl3) δ (ppm): 3.83 (s, 6H,
OCH3), 7.05-7.08 (d, 4H, ArH), 7.58-7.60 (d, 3H, ArH), 8.00 (s, 1H, ArH), 8.03-8.06 (m, 2H, ArH), 8.13-
8.16 (d, 4H, ArH), 11.74 (s, 1H, NH). 13C-NMR (75 MHz, CDCl3): 166.6, 163.3, 159.3, 146.4, 131.6,
129.4, 128.3, 128.8, 128.1, 127.8, 101.2, 56.1. ESI-MS: m/z 447 (M+). Anal. Calcd. for C24H21N3O4S: C,
64.41; H, 4.73; N, 9.39. Found: C, 64.56; H, 4.65; N, 9.40.
N-(4-(3,4-dichlorophenyl)-6-(4-methoxy phenyl) pyrimidin-2-yl) benzenesulfonamide A7
Yellowish solid, yield 57 %, mp 163 oC, FT-IR (KBr) cm-1: 3584 (N-H stretching), 1369, 1139 (O=S=O
stretching), 1642, 1476 (C=C aromatic stretching). 1H-NMR (300 MHz, CDCl3) δ (ppm): 3.89 (s, 3H,
OCH3), 6.68-6.70 (d, 2H, ArH), 7.01-7.03 (d, 2H, ArH), 7.68-7.79 (m, 6H, ArH), 7.97-8.00 (d, 2H, ArH),
8.21-8.23 (d, 1H, ArH), 11.32 (s, 1H, NH). 13C-NMR (75 MHz, CDCl3): 165.7, 163.9, 160.2, 158.8,
145.1, 134.7, 132.8, 132.4, 131.6, 130.5, 129.4, 128.5, 128.3, 127.2, 127.0, 114.8, 101.5, 108.9, 55.4. ESI-
MS: m/z 487 (M+). Anal. Calcd. for C23H17Cl2N3O3S: C, 56.80; H, 3.52; N, 8.64. Found: C, 56.76; H, 3.59;
N, 8.71.
N [4-(4- chloro -phenyl)-5,6,7,8-tetrahydro-quinazolin-2-yl]-benzenesulfonamide A8
Yellowish solid, yield 65 %, mp 130 oC, FT-IR (KBr) cm-1: 3678 (N-H stretching), 1350, 1150
(O=S=O stretching), 1624, 1431 (C=C aromatic stretching). 1H-NMR (300 MHz, CDCl3) δ (ppm): 1.61-
1.82 (m, 4H, CH2), 2.42-2.55 (m, 4H, CH2), 7.05-7.15 (d, 2H, ArH), 7.38-7.51 (m, 3H, ArH), 7.81-7.89 (d,
13
2H, ArH), 8.05-8.07 (d, 2H, ArH), 11.25 (s, 1H, NH). C-NMR (75 MHz, CDCl3): 167.8, 164.7, 144.4,
16

134.2, 132.4, 131.3, 129.4, 129.0, 128.7, 127.5, 126.9, 123.3, 29.8, 28.7, 24.7, 23.9. ESI-MS: m/z 422 (M
+ Na+). Anal. Calcd. for C20H18ClN3O2S: C, 60.07; H, 4.54; N, 10.51. Found: C, 62.51; H, 4.59; N, 10.67.
N-[8-(3,4,5-timethoxy benzylidene)-2-(3,4,5-trimethoxy phenyl)-5,6,7,8-tetrahydro-quinazolin-4-yl]-
benzenesulfonamide A9
Yellow solid, yield 45%, mp 195oC, FT-IR (KBr) cm-1: 3649 (N-H stretching), 1336, 1128 (O=S=O
1
stretching), 1630, 1446 (C=C aromatic stretching). H-NMR (300 MHz, CDCl3) δ (ppm): 1.69-1.92 (m,
4H, CH2), 2.85-2.88 (m, 2H, CH2), 3.89 (s, 6H, OCH3), 3.91(s, 6H, OCH3), 3.92 (s, 6H, OCH3), 6.66 (s,
2H, ArH), 6.74 (s, 2H, ArH), 7.45-7.60 (m, 3H, ArH), 7.98 (s, 1H, =CH), 8.16-8.19 (d, 2H, ArH), 11.23 (s,
1H, NH).13C-NMR (75 MHz, CDCl3): 167.6, 163.4, 162.2, 153.2, 153.0, 139.7, 139.4, 138.8, 138.4, 132.4,
130.1, 129.4, 129.0, 127.3, 124.8, 115.8, 104.6, 103.7, 60.5, 56.1, 30.6, 27.8, 23.7. ESI-MS: m/z 634 (M +
H+). Anal. Calcd. for C33H35N3O8S: C, 62.54; H, 5.57; N, 6.63. Found: C, 62.51; H, 5.49; N,
N-[6-(3,4-dimethoxy-benzylidene)-2-(3,4-dimethoxy-phenyl)-5,6,7,8-tetrahydro-quinazolin-4-yl]-
benzenesulfonamide A10
Yellowish solid, yield 45%, mp 110 oC, FT-IR (KBr) cm-1: 3673 (N-H stretching), 1372, 1156 (O=S=O
1
stretching), 1606, 1478 (C=C aromatic stretching). H-NMR (300 MHz, CDCl3) δ (ppm): 1.57-1.59 (m,
2H, CH2), 1.78-1.80 (m, 2H, CH2), 2.67-2.74 (m, 2H, CH2), 3.79 (s, 6H, OCH3), 3.81 (s, 6H, OCH3), 7.00-
7.03 (d, 4H, ArH), 7.38-7.62 (m, 5H, ArH), 7.76 (s, 1H, =CH), 7.98-8.00 (d, 2H, ArH), 11.58 (s, 1H, NH).
13C-NMR (75 MHz, CDCl3): 166.7, 166.5, 161.9, 150.3, 149.8, 149.7, 149.1, 144.3, 139.7, 134.3, 131.4,
129.4, 127.6, 126.8, 122.5, 121.7, 121.3, 120.2, 117.6, 114.9, 109.6, 56.1, 30.3, 27.6, 24.4. ESI-MS: m/z
573 (M+). Anal. Calcd. for C31H31N3O6S: C, 64.90; H, 5.45; N, 7.32. Found: C, 64.81; H, 5.55; N, 7.45.
N-[6-(3,4-dichloro-benzylidene)-2-(3,4-dichloro-phenyl)-5,6,7,8-tetrahydro-quinazolin-4-yl]-
benzenesulfonamide A11
Colorless solid, yield 45%, mp 230 oC, FT-IR (KBr) cm-1: 3493 (N-H stretching), 1369, 1143 (O=S=O
1
stretching), 1655, 1467 (C=C aromatic stretching). H-NMR (300 MHz, CDCl3) δ (ppm): 1.59-1.60 (m,
4H, CH2), 2.63-2.72 (m, 2H, CH2), 6.60 (s, 1H, ArH), 6.76 (s, 1H, ArH), 7.36-7.59 (m, 6H, ArH), 7.72-
7.76 (m, 3H, ArH), 7.99 (s, 1H, =CH), 9.82 (s, 1H, NH). 13C-NMR (75 MHz, CDCl3): 166.3, 166.1, 163.5,
17

144.3, 134.5, 133.8, 133.6, 132.7, 132.5, 132.3, 131.1, 130.6, 130.2, 129.4, 129.0, 128.6, 127.8, 127.3,
127.1, 122.5, 30.5, 27.6, 24.8. ESI-MS: m/z 591 (M+). Anal. Calcd. for C31H31N3O6S: C, 62.10; H, 5.45;
N, 9.88. Found: C, 62.11; H, 5.43; N, 9.87.
N-[6-(4-methoxy-benzylidene)-2-(4-methoxy-phenyl)-5,6,7,8-tetrahydro-quinazolin-4-yl]-
benzenesulfonamide A12
Colorless solid, yield 45%, mp 165 oC. FT-IR (KBr) cm-1: 3567 (N-H stretching), 1310, 1162 (O=S=O
1
stretching), 1630, 1467 (C=C aromatic stretching). H-NMR (300 MHz, CDCl3) δ (ppm): 1.52-1.78 (m,
4H, CH2), 2.73-2.87 (m, 2H, CH2), 3.11 (s, 6H, OCH3), 6.38-6.41 (d, 1H, ArH), 6.56-6.64 (d, 2H, ArH),
6.78-6.81 (d, 1H, ArH), 7.01-7.13 (dd, 2H, ArH), 7.25-7.27 (d, 1H, ArH), 7.45-7.60 (m, 5H, ArH), 7.69-
7.77 (d, 1H, ArH), 8.02 (s, 1H, =CH), 11.62 (s, 1H, NH). 13C-NMR (75 MHz, CDCl3): 164.8, 164.3, 161.2,
159.6, 144.7, 135.3, 134.2, 132.7, 131.1, 130.4, 129.4, 129.0, 128.5, 128.1, 127.6, 125.4, 117.8, 114.2, 56.1,
34.6, 31.3, 29.8. ESI-MS: m/z 516. (M+H+). Anal. Calcd. for C31H31N3O6S: C, 67.82; H, 5.30; N, 8.18.
Found: C, 67.95; H, 5.42; N, 8.25.
Procedure for synthesis of N-[4-(4-chloro-phenyl)-6-(3,4-dimethoxy-phenyl)-pyrimidin-2-yl]-4-sulfamoyl-
benzamide (A13) and N-[4-(4-chloro-phenyl)-5,6,7,8-tetrahydro-quinazolin-2-yl]-4-sulfamoyl-benzamide
(A14)
To the mixture of ice cooled solution of pyrimidines (P2, P9; 1 mmol) in pyridine, triethylamine (5 mmol)
and benzoyl chloride (5 mmol) were added and the reaction was allowed to stir at room temperature for
about 24 h. To the reaction mixture, dil. HCl was added and the obtained solid was filtered, washed with
water and recrystallized from ethanol to produce Q1 and Q2. The products were confirmed by their
molecular ion (M+) peaks observed in mass spectrums [27].
A mixture of ice cooled benzamides (Q1, Q2; 1 mmol) in chloro sulfonic acid (0.33 mL, 5 mmol)
was heated on water bath for 1 h then poured onto crushed ice and the obtained solid was filtered, washed
with water and recrystallized from ethanol to produce R1 and R2. Further, the benzamido sulphonyl
chloride (R1, R2; 1mmol) was refluxed with ammonium hydroxide (2 mL) for 1 h. After cooling to room
18

temperature, the obtained solid was filtered and washed with water to obtain the final products A13 and
A14 (Scheme 3 and 4).
o
A13: Yellowish solid, yield 41%, mp 124 C. FT-IR (KBr) cm-1: 3565 (N-H stretching), 1321, 1165
(O=S=O stretching), 1604, 1458 (C=C aromatic stretching). 1H-NMR (300 MHz, CDCl3) δ (ppm): 3.85 (s,
6H, OCH3), 5.55 (s, 2H, NH2), 6.81-6.90 (d, 2H, ArH), 7.12-7.15 (d, 2H, ArH), 7.24 (s, 1H, ArH), 7.72-
7.76 (d, 2H, ArH), 7.88-7.99 (m, 3H, ArH), 8.32-8.38 (d, 2H, ArH), 9.62 (s, 1H, NH). 13C-NMR (75 MHz,
CDCl3): 166.8, 164.5, 164.3, 159.4, 151.6, 149.6, 146.8, 137.3, 135.6, 131.8, 130.2, 129.4, 128.6, 127.5,
126.1, 121.3, 110.5, 107.6, 101.7, 56.2. ESI-MS: m/z 526 (M+H)+. Anal. Calcd. for C25H21ClN4O5S: C,
59.81; H, 4.81; N, 8.72. Found: C, 59.85; H, 4.83; N, 8.61.
o
A14: Yellowish solid, yield 47 %, mp 116 C, FT-IR (KBr) cm-1: 3630 (N-H stretching), 1360, 1150
(O=S=O stretching), 1641, 1466 (C=C aromatic stretching). 1H-NMR (300 MHz, CDCl3) δ (ppm): 1.65-
1.92 (m, 4H, CH2), 2.44-2.55 (t, 2H, CH2), 2.65-2.71 (t, 2H, CH2), 5.38 (s, 2H, NH2) 7.48-7.68 (d, 2H,
ArH), 7.72-7.83 (d, 2H, ArH), 7.92-8.00 (d, 2H, ArH), 8.32-8.48 (d, 2H, ArH), 9.52 (s, 1H, NH). 13C-NMR
(75 MHz, CDCl3): 165.2, 163.8, 159.4, 146.9, 136.8, 133.3, 132.3, 130.5, 129.6, 128.6, 127.6, 127.4, 122.7,
29.6, 28.2, 24.3, 23.7. ESI-MS: m/z 463 (M + Na+). Anal. Calcd. for C21H19ClN4O3S: C, 56.95; H, 4.32;
N, 12.65. Found: C, 56.86; H, 4.23; N, 12.67.
4.2. Biology
4.2.1. Cytotoxicity assay
Cytotoxicity studies on the test compounds was carried by MTT assay. DU-145, MCF-7, HCT-15,
HT 29, cell lines were seeded at a density of 4 x 103, 3.7 × 104, 3.5 × 104 cells per well in 100 µL of
respective media and were allowed to attach overnight for 24 h. The cells were treated with test compounds
(A1-A14) at different concentrations for a period of 48 h. After the treatment, MTT (0.5 mg/ml) was added
o
and incubated at 37 C for 4 h. The insoluble purple formazan crystals were dissolved in 100 µL DMSO
and cell viability was assessed by measuring the absorbance at 570 nm in a multi detection plate reader
19

(Spectramax M4, Molecular devices, USA). The IC50 values shown are the means and standard deviation
of at least three independent experiments performed in duplicate [28].
4.2.2. DAPI staining
To evaluate the morphological changes following treatment with compound A2, DAPI staining was
performed. DU-145 cells were grown in 24-well plates at a seeding density of 5×104 cells/well for 24 h and
then treated with compound A2 at various concentrations (0.5, 1, 2, 4 µM). After 24 h, untreated cells, cells
treated with PS01 and standard 5-FU (1 µM) were washed thrice with PBS. Then, 200 µL of 0.1 % triton
X was added for permeabilization and stained with 1 µM of DAPI at 37 oC for 10 min. The stained cells
with fragmented nuclei/ condensed chromatin were observed under fluorescence microscope with
excitation at 359 nm and emission at 461 nm using DAPI filter at 200X magnification.
4.2.3. FITC- Annexin V apoptosis detection
Annexin V is labeled with a fluorescent tag, FITC, to detect and quantify the apoptotic cells
generated by treatment with compound A2. DU-145 cells (1×106) were seeded in 12-well plates and allowed
to grow for 24 h. After cell attachment, the supernatant liquid was replaced by new 10% FBS/ RPMI 1640
medium containing DMSO alone for negative control, varying concentrations of compound A2 (0.5, 1, 2,
4 µM) and standard 5-FU (1 µM). After 24 h of treatment, cells from the supernatant and adherent
monolayer cells were harvested by trypsinization, washed with PBS and centrifuged at 3000 rpm. Then
cells were processed with Annexin V- assay kit (FITC Annexin V Apoptosis Detection Kit, BD
Pharmingen, USA) according to the manufacturer’s instruction [29,30]. Further, flow cytometric analysis
was performed using a flow cytometer (BD FACS Verse USA).
4.2.4. Analysis of mitochondrial membrane potential (MMP)
Apoptosis induces changes in mitochondrial membrane permeability that allows accumulation of
JC-1 dye and thereby decrease the red/ green fluorescence intensity ratio. DU-145 cells were plated at a
seeding density of 1×106 cells/well in a 12-well plate. They were treated with DMSO as negative control
and varying concentrations of compound A2 (0.5, 1, 2, 4) and standard 5-FU (1 µM) for 24 h. After
20

o
treatment, cells were incubated with JC-1 (5 µM) for 30 min at 37 C. Finally, cells were harvested,
collected in tubes, washed with PBS and analyzed using flow cytometer [31].
4.2.5. Detection of intracellular ROS
2, 7’-dichlorofluorescindiacetate (DCFDA), is a cell permeable fluorogenic dye that measures
hydroxyl, peroxyl and other reactive oxygen species (ROS) activity within the cell. DU-145 cells were
o
seeded in 12-well plate at density of 1×106 cells/well and incubated for 24 h at 37 C. After incubation,
cells were treated with DMSO for negative control or with varying concentrations of compound A2 (2.5,
5, 10 µM) concentrations along with standard 5-FU (10 µM). After 24 h of treatment, 400µL of DCFDA
working solution was added per well containing 5µM of DCFDA and incubated for 30 min. Then, DCFDA
working solution was removed and the cells were trypsinised and centrifuged at 5000 rpm for 5 min. The
obtained pellet was rinsed with PBS and centrifuged at 3000 rpm for 3 min [32, 33]. Finally, 400µL of PBS
was added and analyzed by flow cytometer (BD FAC Verse, USA).
2.9. Statistical Analysis
All the results were expressed as mean ± SD and IC50, standard deviation and P-value were
evaluated by one way analysis of variance (ANOVA). P < 0.05 was the allowed standard deviation. All the
experiments were independently repeated thrice and the readings from the triplicates were combined and
analyzed.
Conflict of Interest:
There are no conflicts to declare.
Acknowledgements
This research was supported by Women Scientist Scheme (WOS-A), Department of Science and
Technology (DST), New Delhi, India (Grant no. SR/WOS-A/CS-1003/2014).
REFERENCES
1) F. U. Hartl, A. Bracher, M. H. Hartl, Nature 475 (2011) 324-432.
2) J. N. Moloney, T. G. Cotter, Semin. Cell Dev. Biol. 80 (2018) 50-64.
21

3) J. Paluncic, Z. Kovacevic, P. J. Jansson, D. Kalinowski, A. M. Merlot, M. L. Huang, H. C. Lok, S.
Sahni, D. J. Lane, D. R. Richardson, Biochim. Biophys. Acta. 1863 (2016) 770-784.
4) R. S. Wong, J. Exp. Clin. Cancer Res. 30 (2011) 87.
5) G. Pistritto, D. Trisciuoglio, C. Ceci, A. Garufi, G. D'Orazi, Aging (Albany NY) 8 (2016) 603–
619.
6) A. Gross, J. M. McDonnell, S. J. Korsmeyer, Genes Dev. 13 (1999) 1899–1911.
7) I. M. Ghobrial, T. E. Witzig, A. A. Adjei, CA Cancer J. Clin. 55 (2005) 178–194.
8) S. Galadari, A. Rahman, S. Pallichankandy, F. Thayyullathil, Free Radic. Biol. Med. 104 (2017)
144-164.
9) Z. Zou, H. Chang, H. Li, S. Wang, Apoptosis 22 (2017) 1321-1335.
10) X. Xu, J. Wang, Q. Yao, Bioorg. Med. Chem. Lett. 25 (2015) 241-244.
11) Y. M. El Sayed, W. Sadee, Cancer Res 43 (1983) 4039-4044.
12) X. J. Song, Y. Shao, X. Gao, Chin. Chem. Lett. 22 (2011) 1036-1038.
13) Z. Liu, Y. Wang, H. Lin, D. Zuo, L. Wang, Y. Zhao, P. Gong, Eur. J. Med. Chem. 85 (2014) 215-
227.
14) D. A. Ibrahim, N. S. Ismail, Eur. J. Med. Chem. 46 (2011) 5825-5832.
15) B. Barlaam, S. Cosulich, S. Degorce, M. Fitzek, F. Giordanetto, S. Green, T. Inghardt, L.
Hennequin, U. Hancox, C. Lambert-van der Brempt, R. Morgentin, S. Pass, P. Plé, T. Saleh, L.
Ward, Bioorg. Med. Chem. Lett. 24 (2014) 3928-3935.
16) Y. H. Kim, K. J. Shin, T. G. Lee, E. Kim, M. S. Lee, S. H. Ryu, P. G. Suh, Biochem. Pharmacol.
69 (2005) 1333-1341.
17) A. Casini, A. Scozzafava, A. Mastrolorenzo, L. T. Supuran, Curr. Cancer Drug Targets 2 (2002)
55-75.
18) D. C. Talbot, J. von Pawel, E. Cattell, S.M. Yule, C. Johnston, A.S. Zandvliet, A. D. Huitema, C.
J. Norbury, P. Ellis, L. Bosquee, M. Reck, Clin. Cancer Res. 13 (2007) 1816-1822.
19) C. T. Supuran, Expert Opin. Investig. Drugs 27 (2018) 963-970.
20) M. M. Ghorab, M. S. Al said, M. S. Al-Dossary, Y. M. Nissan, S. M. Attia, Chem. Cent. J. 10
(2016) 19.
21) K. O. Mohamed, Y. M. Nissan, A. A. El-Malah, W. A. Ahmed, D. M. Ibrahim, T. M. Sakr, Eur.
J. Med. Chem. 135 (2017) 424-433.
22) S. Mohammed, M. M. Al-Dosari, M. M. Ghorab; M.S. AlSaid, Y. M. Nissan, A. B. Ahmed, Eur.
J. Med. Chem. 69 (2013) 373-383.
23) M. G. Mostafa, M. G. El-Gazzar, M.S. Alsaid, Int. J. Mol. Sci. 15 (2014) 5582-5595.
22

24) M.V. Kachaeva, D. M. Hodyna, I.V. Semenyuta, S. G. Pilyo, V. M. Prokopenko, V. V. Kovalishyn,
L. O. Metelytsia, V. S. Brovarets, Comput. Biol. Chem. 74 (2018) 294-303.
25) K. P. Rakesh, S. M. Wang, J. Leng, L. Ravindar, A. M. Asiri, H. M. Marwani, H. L. Qin,
Anticancer Agents Med. Chem. 18 (2018) 488-505.
26) K. F. Hussain, A. Ashawa, B. L. Verma, Arabian J. Chem. 9 (1997) 86-90.
27) V. Theodorou, M. Gogou, A. Giannoussi, K. Skobridis, ARKIVOC IV (2014) 11-23.
28) Y. Priyadarshini Devi, A. Uma, M. N. Lakshmi, C. Kalyani, Int. J. Res. Appl. Nat. Soc. Sci. 2
(2014) 269-272.
29) I. Vermes, C. Haanen, H. Steffens-Nakken, C. Teutelinger, J. Immunol. Methods 184 (1995) 39–
51.
30) M. Van Engeland, L. J. W. Nieland, F. C. S. Ramaekers, B. Schutte, C. P. M. Reutelingsperger,
Cytometry 31 (1998) 1–9.
31) A. Cossarizza, M. Baccaranicontri, G. Kalashnikova, C. Franceschi, Biochem. Biophys. Res.
Comm. 197 (1993) 40-45.
32) C. Focaccetti, A. Bruno, E. Magnani, D. Bartolini, E. Principi, K. Dallaglio, E. O. Bucci, G. Finzi,
F. Sessa, D. M. Noonan, A. Albini,, PLoS One 10 (2015) e0115686.
33) D. Trachootham, W. Lu, M. A. Ogasawara, R. D. Nilsa, P. Huang, Antioxid. Redox Signal 10
(2008) 1343–1374.
23

Mechanistic investigations on substituted benzene sulphonamides as apoptosis inducing
anticancer agents
Akhila Mettu,a,c Venu Talla,b Soujanya Thumma,a Subhashini Naikal James Prameela,*c
aDepartment of Pharmaceutical Chemistry, Gokaraju Rangaraju College of Pharmacy, Kukatpally,
Hyderabad, India 500049.
bDepartment of Pharmacology, National Institute of Pharmaceutical Education and Research, Balanagar,
Hyderabad, India 500037.
cDepartment of Chemistry, University College of Science, Osmania University, Hyderabad, India 500007.
Email: njsubhashini@yahoo.co.in
Graphical Abstract
24

Mechanistic investigations on substituted benzene sulphonamides as apoptosis inducing
anticancer agents
Akhila Mettu,a,c Venu Talla,b Soujanya Thumma,a Subhashini Naikal James Prameela,*c
aDepartment of Pharmaceutical Chemistry, Gokaraju Rangaraju College of Pharmacy, Kukatpally,
Hyderabad, India 500049.
bDepartment of Pharmacology, National Institute of Pharmaceutical Education and Research, Balanagar,
Hyderabad, India 500037.
cDepartment of Chemistry, University College of Science, Osmania University, Hyderabad, India 500007.
Email: njsubhashini@yahoo.co.in
Highlights

Four variants of benzene sulphonamides A1-A14 were designed, synthesized and characterized
analytically.




Compounds were excellent anti-proliferative agents tested on four cell lines.
Apoptotic mechanisms were exemplified in DU-145 cells by various mechanistic studies.
Compounds A2 was the most potent compound of the series.
Structure activity relationships were established.
25