Detailed investigation of anticancer activity of sulfamoyl benz(sulfon)amides and 1H–pyrazol–4–yl benzamides: An experimental and computational study

Article abstract of DOI:10.1016/j.ejphar.2018.05.011

Cancer is the second leading cause of mortality worldwide. Therapeutic approach to cancer is a multi-faceted one, whereby many cellular/enzymatic pathways have been discovered as important drug targets for the treatment of cancer. A major disadvantage of most of the currently available anticancer drugs is their non-selective cytotoxicity towards cancerous as well as healthy cells. Another major hurdle in cancer therapy is the development of resistance to anticancer drugs. This necessitates the discovery of new molecules with potent and selective cytotoxic activity towards only cancerous cells, with minimum or no damage to the normal/healthy cells. Herein we report detailed investigation into the anticancer activity of sulfamoyl benz(sulfon)amides (1a-1g, 2a-2k) and 1H–pyrazol–4–yl benzamides (3a-3j) against three cancer cell lines, breast cancer cells (MCF–7), bone-marrow cancer cells (K–562) and cervical cancer cells (HeLa). For comparison, screening against healthy baby hamster kidney cells (BHK-21) was carried out. All compounds exhibited selective cytotoxicity towards cancerous cells. Cell cycle analysis was carried out using flow cytometry, followed by fluorescence microscopic analysis. DNA interaction and docking studies were also carried out.

Full text of DOI:10.1016/j.ejphar.2018.05.011

EuropeanJournalofPharmacology832(2018)11–24
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Detailed investigation of anticancer activity of sulfamoyl benz(sulfon)
amides and 1H–pyrazol–4–yl benzamides: An experimental and
computational study
Jamshed Iqbala,⁎, Syeda Abida Ejaza, Aamer Saeedb, Mariya al-Rashidac,⁎
a
Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
Department of Chemistry, Forman Christian College (A Chartered University), Lahore, Pakistan
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Cancer is the second leading cause of mortality worldwide. Therapeutic approach to cancer is a multi-faceted
one, whereby many cellular/enzymatic pathways have been discovered as important drug targets for the
treatment of cancer. A major disadvantage of most of the currently available anticancer drugs is their non-
selective cytotoxicity towards cancerous as well as healthy cells. Another major hurdle in cancer therapy is the
development of resistance to anticancer drugs. This necessitates the discovery of new molecules with potent and
selective cytotoxic activity towards only cancerous cells, with minimum or no damage to the normal/healthy
cells. Herein we report detailed investigation into the anticancer activity of sulfamoyl benz(sulfon)amides (1a-
1g, 2a-2k) and 1H–pyrazol–4–yl benzamides (3a-3j) against three cancer cell lines, breast cancer cells (MCF–7),
bone-marrow cancer cells (K–562) and cervical cancer cells (HeLa). For comparison, screening against healthy
baby hamster kidney cells (BHK-21) was carried out. All compounds exhibited selective cytotoxicity towards
cancerous cells. Cell cycle analysis was carried out using flow cytometry, followed by fluorescence microscopic
analysis. DNA interaction and docking studies were also carried out.
Breast cancer cells (MCF–7)
Bone marrow cells (K–562)
Cervical cancer cells (HeLa)
Anti-cancer activity
Flow cytometry, DNA interaction and docking
1. Introduction
Different molecules containing sulfonamide moieties exhibit diverse
biological activities including anticancer activity (Ghorab et al., 2014,
Cancer is among the most prevailing diseases of the current decade,
with one of the highest mortality rate (Danaei et al., 2005). A variety of
factors are known to contribute to cancer; many cellular/enzymatic
pathways have been discovered as important drug targets for the
treatment of cancer. However development of resistance in the can-
cerous cells to the available anticancer therapeutics is a growing con-
cern among researchers which necessitates the discovery of new potent
molecules with enhanced anticancer activity and less resistance (Dutta
and Garner, 2003; Kateb et al., 2011). Currently, a variety of potent
anticancer drugs are available, yet most of the drugs are cytotoxic not
only to the cancerous cells but to the healthy cells as well, this results in
development of severe side effects to these drugs such as weight loss,
hair loss, nausea, and loss of appetite etc. (Fortin and Bérubé, 2013;
Nepali et al., 2014a, 2014b; Nepali et al., 2014a, 2014b).
2015, 2016; Scozzafava et al., 2003). A comprehensive review by
Rakesh et al. (2017) describes in detail the anticancer activities of
sulfonyl and sulfonamide hybrid molecules, furthermore sulfonamides
are also found to have minimum adverse effects on healthy cells. Si-
milarly pyrazole derivatives also constitute a class of important biolo-
gically active molecules exhibiting various activities including antic-
ancer activity (Liu et al., 2013; Faria et al., 2017). Pyrazolo[4,3-d]
oxazole derivatives were found to possess anticancer as well as radio-
sensitizing properties (Aly and El-Gazzar, 2012).
Herein we carried out extensive studies to investigate anticancer po-
tential of sulfamoylbenz(sulfon) amides (1a-1g, 2a-2k) (al-Rashida et al.,
2015) and 1H–pyrazol–4–yl benzamides (3a-3j) (Saeed et al., 2015).
These compounds were screened for their cytotoxic potential against
three cancer cell lines, breast cancer cells (MCF–7), bone-marrow cancer
cells (K–562) and cervical cancer cells (HeLa). For comparison, screening
against healthy baby hamster kidney cells (BHK-21) was also carried out,
GI50 values were determined. The results are significant since none of the
compounds showed any significant cytotoxicity towards normal/healthy
The sulfonamide pharmacophore is known to be biologically active
exhibiting different activities such as antimicrobial (Alegaon and
Alagawadi, 2012), fungicidal (Soni et al., 2010), antiviral and anti-
neurodegenerative activity (Kouatly et al., 2009; Bhatt et. al, 2016).
⁎
Corresponding authors.
E-mail addresses: drjamshed@ciit.net.pk (J. Iqbal), abidaejaz2010@gmail.com (S.A. Ejaz), aamersaeed@yahoo.com (A. Saeed), maria_al_rashida@hotmail.com (M. al-Rashida).
https://doi.org/10.1016/j.ejphar.2018.05.011
Received 23 February 2018; Received in revised form 8 May 2018; Accepted 9 May 2018
0014-2999/©2018ElsevierB.V.Allrightsreserved.

J. Iqbal et al.
EuropeanJournalofPharmacology832(2018)11–24
R2
R2
R1
( )n
R1
( )n
n = 0, 1, 2
(1a-1g)
R1 = p-SO NH m-SO NH
R2 = H, F
Scheme 1. Synthesis of sulfamoylphenyl benzamides (1a-1g).
R2
( )n
R2
R1
( )
R1
(2a-2k)
n = 0, 1, 2
R1 = p-SO NH m-SO
R2 = H, F, Me, OEt
2
NH F, Me
Scheme 2. Synthesis of sulfamoylphenyl sulfonamides (2a-2k).
Table 1
Table 2
Purity of sulfamoylphenyl benzamides (1a-1g) and sulfa-
moylphenyl sulfonamides (2a-2k) derivatives through ele-
mental analysis.
Purity of 1H–pyrazol–4–yl benzamides (3a-3j) derivatives
through elemental analysis.
Compound
Purity of compoundsa
Compound
Purity of compoundsa
3a
3b
3c
3d
3e
3f
3g
3h
3i
≥ 99.9%
≥ 99.8%
≥ 99.6%
≥ 99.7%
≥ 99.9%
≥ 99.8%
≥ 99.9%
≥ 99.8%
≥ 99.9%
≥ 99.8%
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
≥ 99.6%
≥ 99.8%
≥ 99.9%
≥ 99.6%
≥ 99.7%
≥ 99.9%
≥ 99.9%
≥ 99.8%
≥ 99.6%
≥ 99.7%
≥ 99.9%
≥ 99.8%
≥ 99.6%
≥ 99.7%
≥ 99.9%
≥ 99.8%
≥ 99.9%
≥ 99.8%
3j
a
Purity of compounds was determined through elemental
analysis.
2g
2h
2i
2j
2k
cells, instead selective cytotoxicity against only cancerous cells was ob-
served. Cell cycle progression analysis and detection of apoptotic events
in different cancer cells, upon treatment with these compounds was in-
vestigated using flow cytometry. This was further complimented from
results of fluorescence microscopic analysis of treated cancer cells, using
two different fluorescent dyes (DAPI and PI). DNA interaction studies
were carried out using UV–visible spectroscopy indicating that the com-
pounds exhibit non-covalent groove binding to the DNA. To further ra-
tionalize binding modes, DNA docking studies were also carried out.
a
Purity of compounds was determined through elemental
analysis.
O
O
OH
Cl
O
Pyridine
Reflux
HN
SOCl2
R
R
N
Reflux, 2hr
NH2
O
N
N
(1'a-1j)
O
(3')
(2'a-2j)
N
(3a-3j)
Scheme 3. Synthesis of pyrazole benzamide derivatives (3a–3j).
12

J. Iqbal et al.
EuropeanJournalofPharmacology832(2018)11–24
Table 3
Cytotoxic potential of sulfamoylphenyl benzamides (1a-1g), sulfamoylphenyl sulfonamides (2a-2k) and standard carboplatin against breast cancer cells (MCF–7),
bone marrow lymphoblast cells (K–562), cervical cancer cells (HeLa) and normal/healthy baby hamster kidney cells (BHK–21) cell lines.
.
Code
n
R1
R2
Cytotoxic Potential (% Growth Reduction
S.E.M)
MCF–7
K–562
HeLa
BHK–21
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
2g
2h
2i
0
1
2
0
1
2
0
0
1
2
0
0
1
2
0
0
0
0
p-SO2NH2
p-SO2NH2
p-SO2NH2
p-SO2NH2
p-SO2NH2
p-SO2NH2
m-SO2NH2
p-SO2NH2
p-SO2NH2
p-SO2NH2
m-SO2NH2
p-SO2NH2
p-SO2NH2
p-SO2NH2
m-SO2NH2
F
H
H
H
F
F
F
F
CH3
CH3
CH3
CH3
F
F
F
F
58.5
50.5
64.7
72.7
61.3
66.2
72.2
61.3
84.7
54.9
59.1
62.1
57.3
60.2
63.6
62.5
63.1
61.7
90.7
2.91
2.67
3.21
3.33
2.87
1.98
3.77
2.56
3.11
1.08
2.34
2.11
1.67
1.98
2.67
2.38
1.98
1.11
3.08
50.9
80.3
80.4
49.7
70.3
65.4
64.2
78.4
72.5
76.4
64.7
59.3
78.4
78.4
86.3
60.7
74.5
76.4
82.8
2.67
3.21
2.87
2.11
3.09
1.98
2.67
2.45
1.98
3.56
2.15
1.78
2.24
2.98
3.67
1.45
2.08
1.78
2.67
70.6
69.6
68.2
56.4
89.6
70.5
60.6
65.2
63.1
56.3
54.1
34.3
79.9
77.2
56.4
73.5
59.9
69.3
85.2
1.78
1.34
1.45
2.06
3.45
3.05
2.34
2.78
1.79
1.34
1.11
2.56
2.67
3.89
2.99
3.25
1.87
2.11
2.98
9.35
19.6
9.40
14.7
5.81
12.8
19.5
14.5
16.7
11.9
12.1
17.9
17.1
11.4
10.1
13.8
17.6
9.03
18.4
0.98
2.34
0.89
1.11
1.45
1.67
2.45
1.45
1.34
1.08
1.56
2.06
2.67
1.56
1.03
1.78
1.54
1.56
2.67
OC2H5
OC2H5
H
2j
2k
CH3
CH3
Carboplatin
The cytotoxic potential was measured at the final concentration of 100 μM of tested compounds. Data represents the mean values (S.E.M) for three independent
determinations.
Fig. 1. Bar graph representation of cytotoxic potential
of sulfamoylphenyl benzamides (1a-1g), sulfamoyl-
phenyl sulfonamides (2a-2k) and standard carbo-
platin, against breast cancer cells (MCF–7), bone
marrow lymphoblast cells (K–562), cervical cancer
cells (HeLa) and normal/healthy baby hamster kidney
cells (BHK–21) cell lines. All the groups in the graph
are statistically significant (P < 0.05) using one way
ANOVA except those with a cap (^).
13

J. Iqbal et al.
EuropeanJournalofPharmacology832(2018)11–24
Table 4
(HeLa) and bone marrow cells i.e., Lymphoblast cells (K-562). The effect of
the selected derivatives was also investigated against normal cells i.e. baby
hamster kidney cells (BHK-21). All the cells were seeded in 96–well flat–-
bottom plates at a final concentration of 2.5 × 104 cells/ ml. The plates
were incubated for 24 h under conditions of 5% CO2 and temperature of
37 °C. The compounds were added to the respective wells at the final
concentration of 100 µM and the well containing only 100 µl of culture
medium without any compound was taken as blank control. Carboplatin
was used as positive control against all the cell lines, at the concentration of
100 µM. After 24 h, 10 µl of MTT reagent was added to each reaction well
and plates were incubated for another 4 h under the same conditions as
above. At the end, the enzyme reaction was stopped by adding 100 µl of
stopping reagent (50% isopropanol and 50 ml of 10% sodium dodecyl
sulfate) and plates were kept at room temperature for 30 min under agi-
tation. The absorbance was recorded at 570 nm after subtracting the
background signal (690 nm), using a 96–well microplate reader (Bio–Tek
ELx 800™). The experiment was performed in triplicate and the results
were calculated as percent growth inhibition values with the mean of three
Growth inhibitory values GI50
showing maximum inhibition against growth of cancerous cells MCF-7, K-562
and HeLa.
S.E.M (μM) for compounds 1e, 2b and 2h
Code
MCF–7
K–562
HeLa
GI50
S.E.M (μM)
1e
2b
2h
6.58
0.75
10.4
3.91
0.63
0.02
1.67
0.32
19.1
16.6
12.2
4.11
1.66
1.11
1.09
0.78
4.64
8.09
15.1
5.13
0.34
0.88
1.77
0.45
Carboplatin
GI50 denotes compound concentrations that result in a 50% decrease in the cell
number compared to non–treated controls and were derived after 24 h treat-
ment. Results from three independent experiments are presented.
2. Material and methods
2.1. Synthesis of sulfamoylphenyl (sulfon)/benzamides and pyrazole
benzamides
independent values (
SEM). Growth inhibitory (GI50) values of potent
inhibitors (≥50%) were calculated by using a non–linear regression ana-
lysis program PRISM 5.0 (GraphPad, San Diego, California, USA).
The sulfamoylphenyl benzamides (1a-1g) and sulfamoylphenyl
sulfonamides (2a-2k) were synthesized according to our already re-
ported method (al-Rashida et al., 2015). Similarly pyrazole benzamide
derivatives (3a-3j) were also synthesized according to our already re-
ported method (Saeed et al., 2015).
2.2.2. Cell cycle analysis assay
The effect of the most potent derivatives (having lower micro-molar
GI50 values) on the cell cycle of different cell lines was determined
according to the previously reported method (Saito et al., 2010). All the
cells were seeded at the final concentration of 2.5 × 104 cells/ ml,
treated with 100 µM of selected compounds and were kept at 37 °C.
After 24 h of incubation the treated cells were centrifuged at 3000 rpm
for 3 min. The supernatant was discarded and the pallet was re-
suspended in 200 µl of PBS solution having 3% FBS. After that, the cells
were treated with 5 µl of propidium iodide (250 µg/ml), 5 µl of Triton
X–100 (0.1%) and 25 µl of ribonuclease A (10 mg/ml). The reaction
mixture was kept in dark for 1 h. The 10,000 events were observed for
2.2. Anticancer assays
2.2.1. Cell Viability Assay (MTT Assay)
The cytotoxic potential of sulfamoylphenyl benzamides (1a-1g), sul-
famoylphenyl sulfonamides (2a-2k) and pyrazole benzamides (3a-3j) was
evaluated
by
using
MTT
(dimethyl–2–thiazolyl–2,
5–diphenyl–2H–tetrazolium bromide)–based cell viability assay
(Mosmann, 1983) and (Nikš, 1990). Three different cancer cell lines were
used for the study i.e., breast cancer cells (MCF-7), cervical cancer cells
Table 5
Cytotoxic potential of 1H–pyrazol–4–yl benzamides (3a-3j) and standard carboplatin against breast cancer cells (MCF–7), bone marrow lymphoblast cells (K–562),
cervical cancer cells (HeLa) and normal/healthy baby hamster kidney cells (BHK–21) cell lines.
R
.
Code
R
Cytotoxic Potential (% Growth Reduction
S.E.M)
MCF–7
K–562
HeLa
BHK–21
3a
3b
3c
3d
3e
3f
3g
3h
Phenyl
65.1
50.9
66.5
59.8
61.3
42.4
60.2
69.4
76.6
55.5
90.7
2.76
2.98
3.78
2.87
3.12
2.06
2.78
3.89
2.98
3.16
3.08
80.4
66.2
84.3
9.81
90.2
98.1
5.88
60.8
56.5
70.6
82.8
3.67
2.67
3.78
0.98
2.78
2.89
0.56
1.89
2.06
1.89
2.67
72.1
77.6
70.2
58.9
66.1
66.1
67.3
69.6
49.2
72.3
85.2
2.87
3.89
2.78
1.78
2.56
1.89
1.67
1.78
2.56
3.78
2.98
4.18
17.3
29.7
9.43
5.97
13.1
4.79
10.1
15.2
13.1
18.4
0.34
1.09
2.67
0.78
0.56
1.58
0.23
1.45
2.16
2.78
2.67
2-Methylphenyl
4-Methylphenyl
2-Chlorophenyl
4-Chlorophenyl
2-Bromophenyl
3,5-Dihydroxyphenyl
Benzyl
3i
3j
2-Furanyl
Methyl
Carboplatin
The cytotoxic potential was measured at the final concentration of 100 μM of tested compounds. Data represents the mean values (S.E.M) for three independent
determinations.
14

J. Iqbal et al.
EuropeanJournalofPharmacology832(2018)11–24
Fig. 2. Bar graph representation of cytotoxic potential
of 1H–pyrazol–4–yl benzamides (3a-3j) and standard
carboplatin, against breast cancer cells (MCF–7), bone
marrow lymphoblast cells (K–562), cervical cancer
cells (HeLa) and normal/healthy baby hamster kidney
cells (BHK–21) cell lines. All the groups in the graph
are statistically significant (P < 0.05) using one way
ANOVA except those with a cap (^).
Table 6
Growth inhibitory values GI50
showing maximum inhibition against growth of cancerous cells MCF-7, K-562
and HeLa.
were kept at room temperature for 30 min. The absorption spectra were
measured with blank correction using 96–well plates of 5.5 mm path
length and the mode of interaction of compounds was determined by
using Omega– Data Analysis Software, Program Version: 3.00 R3.
S.E.M (μM) for compounds 3b, 3 f and 3i
Code
MCF–7
K–562
HeLa
GI50
S.E.M (μM)
2.2.5. DNA docking
The binding orientation of compounds with DNA was investigated by
molecular operating environment (MOE) by Chemical Computing Group
Inc (MOE, version, 2014) and LeadIT. The compound structures were
drawn using builder tool of MOE 2014.09. Furthermore the 3D structures
of compounds were energy minimized and optimized through semi-em-
pirical method PM3 inbuilt in MOE. The X-ray crystallographic DNA
structure was obtained from Protein Data Bank (PDB IDs: 1BNA and
127D) (Drew et al., 1981). The structure of DNA was 3D protonated with
its standard geometry; followed by energy optimization tool using
MOPAC 7.0. Whole DNA structure was used to search for ideal con-
formation at default parameters with root mean squared (RMS) gradient
of 0.01 kcalmol−1. Total 100 poses per compound were generated. The
best conformation was selected and then imported to Discovery Studio
Visualizer (Visualizer, 2005) for visualization purpose.
3b
3f
3i
21.5
> 100
4.18
2.11
12.1
7.27
18.4
4.11
1.11
0.48
1.99
0.78
3.26
8.22
> 100
5.13
0.03
0.78
0.01
0.32
Carboplatin
3.91
0.45
GI50 denotes compound concentrations that result in a 50% decrease in the cell
number compared to non–treated controls and were derived after 24 h treat-
ment. Results from three independent experiments are presented.
each sample by using the BD accuri Flow Cytometer and the analysis
was done by using BD Accuri software.
2.2.3. Microscopic analysis of apoptosis
In order to support the results obtained from flow cytometry, the
fluorescence microscopic analysis of the cells after treatment with most
active compounds was carried out according to the previously reported
method (Ban et al., 2014). The confluent cells with the density of
2 × 105 cells/well were treated with 100 µM of test compound and kept
in CO2 incubator at 37 °C for 24 h. Next day, the medium was discarded
and cells were washed three times with cold PBS. Then the cells were
fixed with 4% formalin and allowed to become permeable with 0.1%
Triton X–100. After 5 min room incubation, 10 µl of DAPI (0.1 mg/ml)
or PI (0.1 mg/ml) dye was added to stain the nuclear material and the
glass slides were kept in dark for 10 min. The images were captured by
using the fluorescence microscope at excitation/emission wavelength of
350/460 nm and 493/632 for DAPI and PI, respectively.
3. Results
3.1. Chemistry
3.1.1. Synthesis of sulfamoylphenyl benzamides and sulfamoylphenyl
sulfonamides
Sulfamoylphenyl benzamides (1a-1g) were synthesized by reacting
different aminobenzenesulfonamides with benzoyl chlorides (Scheme
1), and sulfamoylphenyl sulfonamides (2a-2k) were synthesized by
reacting different aminobenzenesulfonamides with sulfonyl chlorides
(Scheme 2) as previously reported by us (al-Rashida et al., 2015).
The purity of synthesized compounds was determined through ele-
mental analysis as shown in Table 1 (al-Rashida et al., 2015).
2.2.4. DNA interaction studies
The DNA interaction studies of the most potent compounds were
performed according to the previously reported method (Sirajuddin et al.,
2014). For this purpose 100 µM of each selected compound was inter-
acted with the different concentrations of salmon sperm DNA i.e., 0, 40,
90, 140, 190, 240, 290 and 340 µM obtained from the molar absorption
coefficient of 6600 M–1 cm–1 i.e., 4.15 × 10–4 M. The same amount of
DNA was also added to their respective reference solutions. The plates
3.1.2. Synthesis of 1H–Pyrazol–4–yl benzamides derivatives
The synthetic pathway leading to the formation of 1H–pyrazol–4–yl
benzamides (3a–3j) is depicted in Scheme 3. Firstly, the aromatic car-
boxylic acids (1′a–1′j) were refluxed with thionyl chloride to prepare
respective acid halides (2′a–2′j), in the second step the obtained acid
halides were further reacted with the 4–aminophenazone (3′) to afford
15

J. Iqbal et al.
EuropeanJournalofPharmacology832(2018)11–24
Fig. 3. Cell cycle analysis using propidium iodide (PI) DNA staining and flow cytometry. Data showing cell population in G0/G1, S and G2/M phases of the cell cycle
(A) untreated MCF–7 cells, (B) carboplatin against MCF–7, (C) Compound 2b against MCF–7, (D) Compound 3i against MCF–7, (E) untreated K–562 cells, (F)
carboplatin against K–562, (G) Compound 2 h against K–562, (H) Compound 3f against K–562, (I) untreated HeLa cells, (J) Carboplatin against HeLa, (K) Compound
1e against HeLa, (L) Compound 3b against HeLa.
the desired pyrazole benzamide derivatives (3a–3j) (Saeed et al., 2015).
The purity of synthesized compounds was determined through ele-
mental analysis as shown in table (Saeed et al., 2015).
untreated cells and are presented in Table 3 and Fig. 1.
Most potent compounds were further evaluated for the determina-
tion of GI50 values against MCF–7, K–562 and HeLa cells. The antic-
ancer potency of the compounds was calculated by linear regression
analysis of the concentration–response curves obtained for each com-
pound (Table 4).
3.2. Evaluation of anticancer activity
3.2.1. Cytotoxic potential by MTT assay
Similar to the results obtained for previous compounds (1a-1g, 2a-
2k), all 1H–pyrazol–4–yl benzamides (3a-3j) also did not inhibit
growth of normal/healthy baby hamster kidney cells (BHK–21)
(Table 5). Fig. 2 shows anticancer potential of these compounds in the
form of a bar graph.
Most potent compounds were further evaluated for the determina-
tion of GI50 values against MCF–7, K–562 and HeLa cells. The antic-
ancer potency of the compounds was calculated by linear regression
analysis of the concentration–response curves obtained for each com-
pound (Table 6).
In vitro evaluation of anticancer activity of all compounds 1a-1g,
2a-2k and 3a-3j was carried out using the 3–(4,5–dimethylthiazol–
2–yl)–2,5–diphenyltetrazolium bromide (MTT) assay (Mosmann, 1983;
Nikš, 1990). The cell viability was determined by measurement of purple
formazan that was formed after metabolization of original yellow colored
tetrazolium bromide (MTT). This conversion of yellow tetrazolium bro-
mide to purple formazan is brought about by oxidoreductase enzymes
that are typically present in the cytosol of living cells, thereby providing
an estimate of number of viable (healthy (control) and cancerous) cells
present after treatment with test compounds. The screening of all the
derivatives was carried out using breast cancer cells (MCF-7), cervical
cancer cells (HeLa) and bone marrow cells i.e., lymphoblast cells (k-562),
in comparison to normal cell line i.e. baby hamster kidney cells (BHK-21).
The viability of cells was assessed after 24 h treatment with 100 µM
concentration of respective derivative. The cytotoxic potential was mea-
sured at the final concentration of 100 µM of tested compounds. The
obtained results are expressed as percentage of viability stated in
3.2.2. Cell cycle analysis and detection of apoptosis in different cell lines by
flow cytometry
In order to investigate the effect of most potent compounds on cell
cycle progression and apoptosis, flow cytometric analysis was carried
out using human breast cancer cell line (MCF–7), bone marrow lym-
phoblast cell lines (K–562) and human cervical cancer cell line (HeLa).
The results after 24 h treatment of cells are shown in Figs. 3 and 4.
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EuropeanJournalofPharmacology832(2018)11–24
Fig. 4. Apoptosis induced by the most potent compounds in different cell lines (A) Compound 2b and 3i against MCF–7 (B) Compound 2h and 3f against K–562 (C)
Compound 1e and 3b against HeLa. Data were analyzed by one way analysis of variance (ANOVA) using PRISM 5 (GraphPad).
Fig. 5. Cell apoptosis observed under fluorescence microscope using DAPI (a; left, blue), and PI (b; right, red) staining respectively. In both (a) and (b), (A) untreated
MCF–7 cells (B) carboplatin against MCF–7 (C) Compound 2b against MCF–7 (D) Compound 3i against MCF–7 (E) untreated K–562 cells (F) carboplatin against K–562 (G)
Compound 2h against K–562 (H) Compound 3f against K–562 (I) untreated HeLa cells (J) carboplatin against HeLa (K) Compound 1e against HeLa (L) Compound 3b
against HeLa. In comparison to negative control, the compounds exhibited chromatin condensation, nuclear fragmentation and apoptotic body formation.
Carboplatin was used as positive control and also treated at final con-
centration of 100 µM. Untreated cells, MCF–7, K–562 or HeLa in the
G0/G1, S and G2/M phases were considered 100% (control). The dif-
ferent cell cycle phases of the treated derivatives were analyzed and the
percentages of G0– G1, S, and G2/ M phase cells are presented gra-
phically in Figs. 3 and 4.
and carboplatin (positive control) were treated with respective cancer
cell lines at final concentration of 100 µM. After 24 h, the images re-
vealed the characteristic apoptotic changes like higher percentage of
rounded cells, condensed and fragmented nuclei unlike untreated
control. Fluorescent microscopic analysis was carried out to confirm the
nuclear fragmentation using nuclear staining dyes (DAPI, PI). The nu-
clei in the viable cells have a regular, oval shape with homogeneous
chromatin, whereas in the apoptotic cells, shrinking and fragmentation
of the nuclei could be observed (Fig. 5(a, b)).
3.2.3. Microscopic analysis of potent derivatives (using DAPI & PI)
The most potent anti-cancer compounds (2b, 3i, 2h, 3f, 1e and 3b)
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EuropeanJournalofPharmacology832(2018)11–24
Fig. 6. Absorption spectra of 40 µM of (a) 2b, 2h and 1e in absence and presence of 40 µM, 90 µM, 140 µM, 190 µM, 240 µM, 290 µM and 340 µM SS-DNA. The arrow
direction indicates increasing concentration of DNA. The inset graph is the plot of A°/ (A–A°) vs 1/[DNA] for the determination of binding constant and Gibb's free
energy of compound–DNA adduct.
3.2.4. DNA interaction studies
3.2.5. DNA docking studies
The mechanism of action of the cytotoxic compounds against
MCF–7 cells (2b, 3i), K–562 cells (2h, 3f) and HeLa cells (1e, 3b) was
further evaluated by their interaction studies with SS–DNA in
UV–visible range, using Omega FLUOstar, microplate reader. The ab-
sorption spectra of test compounds in the absence and presence of in-
creasing concentration of SS–DNA was observed as depicted in Figs. 6
and 7.
From the results of DNA interaction via UV–visible spectroscopy, it
was found that all compounds were interacting with DNA as groove
binders In order to rationalize these binding modes, DNA docking
studies were carried out according to our previously reported protocol
(al-Rashida and Ahsen, 2015). Crystal structure of DNA co-crystallized
with Hoechst (minor groove binder) was downloaded from the Protein
Data Bank (PDB ID 127D). Docking studies were carried out using
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EuropeanJournalofPharmacology832(2018)11–24
Fig. 7. Absorption spectra of 40 µM of (a) 3i, 3f and 3b in absence and presence of 40 µM, 90 µM, 140 µM, 190 µM, 240 µM, 290 µM and 340 µM SS-DNA. The arrow
direction indicates increasing concentration of DNA. The inset graph is the plot of A°/ (A–A°) vs 1/[DNA] for the determination of binding constant and Gibb's free
energy of compound–DNA adduct.
BioSolveIT's LeadIT software (LeadIT, 2014). In order to validate the
docking protocol, the original co-crystallized ligand, Hoechst was re-
docked into the same DNA, the docking protocol was able to reproduce
the experimentally observed bound conformation with rmsd of 0.99 Å.
Figs. 8 and 9 show docked conformations of compounds 2b, 2h and 1e,
all showing DNA groove binding, which is in agreement with the ex-
perimental DNA interaction studies (Fig. 8).
Similar results were obtained from docking of 1H–pyrazol–4–yl
benzamides 3i, 3f and 3b, as shown in Figs. 10 and 11.
4. Discussion
The sulfonamide moiety is a highly active pharmacophore and is
responsible for a number of biological activities. Sulfamoylphenyl
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EuropeanJournalofPharmacology832(2018)11–24
Fig. 8. Docked conformations of 2b (blue), 2h (green) and 1e (pink), indicating DNA groove binding mode.
benzamides (1a-1g) and sulfamoylphenyl sulfonamides (2a-2k) were
synthesized by reacting different aminobenzenesulfonamides with
benzoyl chlorides and sulfonyl chlorides respectively (Schemes 1 and
2), as previously reported by us al-Rashida et al. (2015). The pyrazole
benzamide derivatives (3a–3j) were synthesized via a two-step process
starting with preparation of aroyl chlorides from their respective car-
boxylic acids, and further reaction of obtained aroyl chlorides with
4–aminophenazone as reported by us Saeed et al. (2015).
than the inhibition shown by standard carboplatin (82%). Compounds
1b and 1c showed 80% inhibition, followed by compounds 2a, 2f and
2g exhibiting 78% growth inhibition. Compound 1d showed least in-
hibition of 49.7%. Against cervical cancer cells (HeLa), maximum
growth inhibition of 89% was shown by compound 1e, it is important
to note here that the inhibition shown by 1e is higher than that of
carboplatin (85%). Compounds 2f and 2g exhibited 79% and 77% in-
hibition respectively. Least inhibition was shown by compound 2e
which showed less than 50% inhibition (34%).
Most interestingly, all compounds showed no inhibition of growth
(less than 19%) against normal/healthy baby hamster kidney cells
(BHK–21). In the search of new potent anticancer agents, one of the
most desirable trait is their ability to selectively target only cancerous
cells, ideally without damaging normal or healthy cells, or (less ideally)
with minimum damage to the healthy cells. In this respect, compound
1e has emerged as the most promising candidate as a lead compound
for further development of anticancer compounds from this class of
compounds (Table 2). Compound 1e exhibited maximum inhibition of
89% against cervical cancer cells (HeLa) with minimum damage (5%
inhibition) to normal healthy cells (BHK–21). Against bone marrow
lymphoblast cells (K–562), compound 2h was most active (86% growth
inhibition), with minimum damage to healthy cells (only 10% growth
inhibition). Against breast cancer cells (MCF–7), most promising com-
pound was 2b (84% growth inhibition) with minimum damage to
healthy cells (only 16% growth inhibition) (Fig. 1).
The cytotoxic potential of all compounds was evaluated using MTT
assay. By virtue of this test, cellular metabolic activity can be assessed,
hence it provides
a means to determine cell viability. MTT
(dimethyl–2–thiazolyl–2,5–diphenyl–2H–tetrazolium bromide), is
a
yellow colored tetrazolium dye that is taken up by cells via en-
docyctosis. In living cells, the NADPH dependent mitochondrial dehy-
drogenases reduce yellow MTT to purple colored formazan, which is
determined spectrophotometrically. Since mitochondrial activity is
only present in living cells, MTT test is a reliable indicator of cell via-
bility. The viability of cells 24 h after treatment with test compounds, at
concentration of 100 µM, was assessed against three different cancer
cell lines i.e., breast cancer cells (MCF-7), cervical cancer cells (HeLa)
and bone marrow cells i.e., Lymphoblast cells (K-562). The effect
against normal cells i.e. baby hamster kidney cells (BHK-21) was also
determined. All compounds with the exception of compound 2e showed
more than 50% growth inhibition of cancerous cells (Table 1). Against
breast cancer cells (MCF-7), compound 2b showed maximum inhibition
of 84%, followed by compounds 1d and 1g exhibiting 72% growth
inhibition. Third most active compound was 1f, showing 66% inhibi-
tion. Whereas least growth inhibition was exhibited by compound 1b,
showing only 50% inhibition.
The results of cytotoxic activity for 1H–pyrazol–4–yl benzamides (3a-
3j) are given in Table 3. Against the breast cancer cells (MCF–7), max-
imum growth inhibition was exhibited by compound 3i (76%), followed
by compound 3h which showed 69% inhibition. Compounds 3a and 3c
showed growth inhibition of 65% and 66% respectively. Against bone
marrow lymphoblast cells (K–562), compound 3f showed excellent
Against bone marrow lymphoblast cells (K–562), compound 2h
showed maximum growth inhibition of 86% which is slightly higher
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Fig. 9. 2D DNA-docked conformations of compounds 2b, 2h and 1e.
growth inhibition of 98%, followed by compound 3e that showed 90%
inhibition. Compounds 3a and 3c were also found to be potent anticancer
agents exhibiting 80% and 84% growth inhibition respectively. Against
cervical cancer cells (HeLa), compound 3b exhibited maximum growth
inhibition of 77%. Compounds 3a and 3j exhibited 72% inhibition, fol-
lowed by compound 3c that showed 70% inhibition.
Similar to the results obtained for previous compounds (1a-1g, 2a-
2k), all 1H–pyrazol–4–yl benzamides (3a-3j) also did not inhibit
growth of normal/healthy baby hamster kidney cells (BHK–21). In the
quest to design new potent anticancer agents, the ability of anticancer
compounds to selectively inhibit the growth of only cancerous cells, and
not healthy cells is of utmost importance (Fig. 2). Hence compounds
from this series present attractive lead molecules for further develop-
ment as anticancer agents.
Next we performed flow cytometric analysis in order to determine
whether or not the tested compounds had the ability to affect the cell cycle
and induce apoptosis. The flow cytometric method is based on analysis of
cellular DNA content, which was stained with fluorescent DNA binding
dye, propidium iodide (PI). The selected derivatives were treated at final
concentration of 100 µM with respective cell line i.e. MCF–7, K–562 or
HeLa. The compounds 2b and 3i exhibited 58.1% and 57.6% apoptosis in
MCF–7 cell line (Figs. 3 and 4). The detailed cell cycle analysis suggests
that these compounds interfere with, and inhibit the mitotic spindling that
ultimately results in DNA damage and cell arrest in G2 phase. The G2 phase
is a period of rapid cell growth where the cell prepares itself for the next
stage, mitosis. Concerted interplay between a number of important proteins
(PLK1, CHK1, Wee1, CDC25C and CDK1) determine successful conclusion
of G2 checkpoint, these proteins are strongly influenced by DNA damage
and repair signaling (Gooijer et al., 2017).
compounds 2h and 3f were found to induce comparable apoptosis
(53.5%) with respect to positive control, carboplatin (58.1%). The cell
cycle analysis suggested that more cell death occurred in S phase as
compared to other two phases (Go and G2). DNA replication is the
hallmark event in S phase. It is crucial that during cell replication,
identical copies of DNA be made with minimum or no errors. During
this phase any damage to DNA is detected and a cascade of tightly
regulated events is started that attempts to repair the damaged DNA.
While this repair mechanism is going on, mitosis and cell replication is
put on hold. In case of failure to repair the damaged DNA, cell death
mechanisms are initiated (Branzei et al., 2005). Against human cervical
cancer cells (HeLa), the compound 3b induced maximum apoptosis
(33.4%), while compound 1e induced approximately 22.2% apoptosis.
Compound 3b was found to be the most effective with respect to S
phase apoptotic cell death. In comparison to 1e derivative, 3b pos-
sessed more apoptotic activity with more cell arrest or accumulation of
cell population in S phase (Fig.s 3 and 4). Thus the percentage of
apoptotic cells with respect to the total cell count revealed that the
compound 3b induced significantly higher pro–apoptotic activity than
1e, which correlates with the results obtained in the MTT assay (Figs. 1
and 2).
In order to support the results (DNA damage) obtained from flow
cytometry, cell nucleus morphology was investigated 24 h after treat-
ment of cancerous cells (MCF-7, K–562 and HeLa cells) with selected
test compounds at 100 µM concentration using DAPI (4′,6-diamidino-2-
phenylindole) and PI (propidium iodide) staining followed by ob-
servation using fluorescence microscopy. For comparison untreated
MCF-7, K–562 and HeLa cells were was stained and observed under
fluorescence microscope, carboplatin treated cells were used as control.
The microscopic images with DAPI staining (Fig. 5(a)) show bluish
When the effect was observed against K–562 cells, the test
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EuropeanJournalofPharmacology832(2018)11–24
Fig. 10. Docked conformations of 3i (tan), 3f (purple) and 3b (black), indicating DNA groove binding mode.
A0
εG
εG
1
intact nuclei in the control and bright fragmented nuclei in cells treated
with derivatives 2b, 3i, 2h, 3f, 1e and 3b. Similarly another dye, PI is
impermeable to nuclei of living cells because of intact nuclear mem-
brane. While in dead cells, the nuclear membrane is compromised that
allows the permeability of PI with resultant image of red nuclei in front
of black background (Fig. 5(b)). Among all the tested compounds, 2b,
3f, and 3b induced maximum apoptosis as indicated by DAPI and PI
stained images in their respective cell lines (MCF-7, K-562 and Hela
respectively), as compared to carboplatin, positive control. The results
were in correlation with the flow cytometric analysis and suggested that
there is a significant increase in DNA damage in each case. While other
derivatives exhibited comparatively less apoptotic activity.
Since our investigation strongly suggested DNA damage to con-
tribute to the observed apoptosis, we decided to carry out detailed DNA
interaction studies. Small molecules can bind to and damage DNA in
different ways, either via electrostatic interactions, or by binding in the
major or minor groove of the DNA, or by intercalating in between the
DNA base pairs. Cell death is triggered if DNA repair mechanisms fail or
are compromised. The absorption spectra of test compounds in the
absence and presence of increasing concentration of SS–DNA was ob-
served as depicted in Fig.s 6 and 7. In each case of DNA interaction
studies, with the increase in DNA concentration, a corresponding in-
crease in the intensity of absorption bands at 220 nm and 260 nm was
observed (hyperchromic effect), with no shift in the position of the
bands, which remained unaffected by increase in concentration of SS-
DNA. The hyperchromic effect indicates the non–covalent, groove
binding interaction with DNA (Sirajuddin et al., 2014). Binding con-
stants were determined using Benesi–Hildebrand's equation which is
given below (Benesi and Hildebrand, 1949).
=
+
×
A − A0
εH−G − εG
εH−G − εG
K [DNA]
Where K is the association/binding constant, Ao and A are the absor-
bance of the compound and its complex with DNA, respectively, and εG
and εH-G are the absorption coefficients of the compound and com-
pound–DNA complex, respectively. The binding constants (K) were
obtained from the intercept-to-slope ratios of Ao/(A-Ao) vs. 1/[DNA]
plots. The Gibbs free energy (ΔG) was determined from the following
equation, where R is the general gas constant (8.314 JK−1 mol−1), and
T is temperature (298 K).
ΔG = −RT ln K
The binding constant K for compounds 2b, 2h and 1e were calcu-
lated to be 8.52 × 102
M M
−1, 11.31 × 102 −1, and 19.80 × 102 M−1
respectively in the order 2b < 2h < 1e. The corresponding Gibbs free
energies (ΔG) were found to be − 17.48 kJ/mol, − 17.16 kJ/mol and
− 17.88 kJ/mol respectively. Similar results were obtained for
1H–pyrazol–4–yl benzamides 3i, 3f and 3b (Fig. 7). Upon increasing the
concentration of added DNA, an increase in the intensity of absorption
bands at 220 nm and 260 nm was observed (hyperchromic effect),
whereas the absorption bands did not shift their position, indicating
groove binding as the mechanism responsible for observed DNA inter-
action (Fig. 8). Maximum binding constant was observed for compound
3b (K = 10.15 × 102
M
−1, ΔG = −17.87 kJ/mol).
Groove binding interaction was stabilized by formation of hydrogen
bonds between the compounds and the DNA base pairs. As can be seen
from Fig. 9, compounds 2b, 2h and 1e have identical binding pattern,
where the sulfonamide NH group was making hydrogen bonds with
guanine (dg 10) and cytosine (dc11), one of the sulfonamide oxygen
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EuropeanJournalofPharmacology832(2018)11–24
Fig. 11. 2D DNA-docked conformations of compounds 3b, 3f and 3i.
was making a hydrogen bond with another guanine (dg16) (Fig. 9).
Similar results were obtained from docking of 1H–pyrazol–4–yl ben-
zamides 3i, 3f and 3b, as shown in Fig. 10. All docked conformations
indicated goove binding mode of interaction with the DNA, which is in
agreement with the experimental data. From docking studies, it was
evident that all compounds bind in the minor groove of the DNA
(Fig. 10). Hydrogen bonded interactions with the DNA base pairs were
also observed (Fig. 11). Compounds 3b and 3f had identical binding
modes, contrary to sulfamoyl benz(sulfon)amides, these two com-
pounds prefer to bind in AT rich region, as indicated by hydrogen bond
formation between the NH of amide group and carbonyl oxygen of
thymine (dt8). However compound 3i was found to bind in GC rich
region, due to the presence of furan group, which is absent in other
compounds. The NH amide group was making a hydrogen bond with
cytosine (dc11), whereas the carbonyl oxygen of amide group was
making a hydrogen bond with amino group of guanine (dg16). The
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J. Iqbal et al.
EuropeanJournalofPharmacology832(2018)11–24
furan ring oxygen was also making a hydrogen bond with amino group
of another guanine (dg10) (Fig. 11).
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Sulfamoyl benz(sulfon)amides (1a-1g, 2a-2k) and 1H–pyrazol–4–yl
benzamides (3a-3j) were evaluated for their anticancer potential against
MCF–7, K–562 and HeLa cancer cell lines. Majority of the derivatives
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most active against MCF–7, K–562 and HeLa cancer cells, respectively.
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Acknowledgments
The authors are grateful to the Organization for the Prohibition of
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