New benzenesulfonamide scaffold-based cytotoxic agents: Design, synthesis, cell viability, apoptotic activity and radioactive tracing studies

Article abstract of DOI:10.1016/j.bioorg.2020.103577

A new series of thiazolidinone (5a-g), thiazinone (9a-g) and dithiazepinone (9a-g) heterocycles bearing a benzenesulfonamide scaffold was synthesized. Cytotoxicity of these derivatives was assessed against MCF-7, HepG2, HCT-116 and A549 cancer cell lines and activity was compared to the known cytotoxic agents doxorubicin and 5-FU where the most active compounds displayed better to nearly similar IC₅₀ values to the reference compounds. For assessing selectivity, the most active derivatives against MCF-7, 5b, 5c and 5e, were also assessed against the normal breast cell line MCF-10 A where they demonstrated high selective cytotoxicity to cancerous cells over that to normal cells. Further, the effect of the most active compounds 5b-e on MCF-7 and HepG2 cell cycle phase distribution was assessed and the tested sulfonamide derivatives were found to induce accumulation of cells in the <2n phase. To further confirm apoptosis induction, caspase 8 and 9 levels in MCF-7 and HepG2 were evaluated before and after treatment with compounds 5b-e and were found to be significantly higher after exposure to the test agents. Since 5c was the most active, its effect on the cell cycle regulation was confirmed where it showed inhibition of the CDK2/cyclin E1. Finally, in vivo biodistribution study using radioiodinated-5c revealed a significant uptake and targeting ability into solid tumor in a xenograft mouse model.

Full text of DOI:10.1016/j.bioorg.2020.103577

Journal Pre-proofs
New Benzenesulfonamide Scaffold-Based Cytotoxic Agents: Design, Synthe-
sis, cell viability, apoptotic activity and radioactive tracing studies
Yassin M. Nissan, Khaled O. Mohamed, Wafaa A. Ahmed, Dina M. Ibrahim,
Marwa M. Sharaky, Tamer M. Sakr, Mohamed A. Motaleb, Ahmed Maher,
Reem K. Arafa
PII:
S0045-2068(19)31059-4
DOI:
https://doi.org/10.1016/j.bioorg.2020.103577
YBIOO 103577
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
3 July 2019
11 December 2019
9 January 2020
Please cite this article as: Y.M. Nissan, K.O. Mohamed, W.A. Ahmed, D.M. Ibrahim, M.M. Sharaky, T.M. Sakr,
M.A. Motaleb, A. Maher, R.K. Arafa, New Benzenesulfonamide Scaffold-Based Cytotoxic Agents: Design,
Synthesis, cell viability, apoptotic activity and radioactive tracing studies, Bioorganic Chemistry (2020), doi:
https://doi.org/10.1016/j.bioorg.2020.103577
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New Benzenesulfonamide Scaffold-Based Cytotoxic Agents: Design, Synthesis, cell viability,
apoptotic activity and radioactive tracing studies
Yassin M. Nissana,b, Khaled O. Mohamedc, Wafaa A. Ahmedd, Dina M. Ibrahime, Marwa M.
Sharakyd, Tamer M. Sakrb,f,*, Mohamed A. Motalebg, Ahmed Maherh, Reem K. Arafai,*
aPharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr Elini St., Cairo
11562, Egypt
bPharmaceutical Chemistry Department, Faculty of Pharmacy, October University for Modern Sciences
and Arts (MSA), Giza, Egypt
cPharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Egypt
dNational Cancer Institute, Cancer Biology Department, Cairo University, Egypt
eFaculty of Science, Cairo University, Cairo, Egypt
fRadioactive Isotopes and Generator Department, Hot Labs Center, Atomic Energy Authority, P.O. Box
13759, Cairo, Egypt
gLabeled Compounds Department, Hot Labs Center, Atomic Energy Authority, P.O. Box 13759, Cairo,
Egypt
hBiochemistry Department, Faculty of Pharmacy, October University for Modern Sciences and Arts
(MSA), Giza, Egypt
iBiomedical Sciences Program, University of Science and Technology, Zewail City of Science and
Technology, 12578, Cairo, Egypt
Corresponding authors:
Prof. Reem K. Arafa
Biomedical Sciences Program
University of Science and Technology
Zewail City of Science and Technology
Ahmed Zewail Road
October Gardens, 6th of October City, Giza, Egypt, 12578
Mobile: +2-01002074028
E-mail: rkhidr@zewailcity.edu.eg
Associate Prof. Tamer M. Sakr
Radioactive Isotopes and Generator Department
Hot Labs Center
Atomic Energy Authority, Cairo, Egypt, 13759
Mobile: +2-01006884013
E-mail: tamer_sakr78@yahoo.com
Page 1 of 27

Abstract
A new series of thiazolidinone (5a-g), thiazinone (9a-g) and dithiazepinone (9a-g)
heterocycles bearing a benzenesulfonamide scaffold was synthesized. Cytotoxicity of these
derivatives was assessed against MCF-7, HepG2, HCT-116 and A549 cancer cell lines and activity
was compared to the known cytotoxic agents doxorubicin and 5-FU where the most active
compounds displayed better to nearly similar IC50 values to the reference compounds. For
assessing selectivity, the most active derivatives against MCF-7, 5b, 5c and 5e, were also assessed
against the normal breast cell line MCF-10 A where they demonstrated high selective cytotoxicity
to cancerous cells over that to normal cells. Further, the effect of the most active compounds 5b-
e on MCF-7 and HepG2 cell cycle phase distribution was assessed and the tested sulfonamide
derivatives were found to induce accumulation of cells in the <2n phase. To further confirm
apoptosis induction, caspase 8 and 9 levels in MCF-7 and HepG2 were evaluated before and after
treatment with compounds 5b-e and were found to be significantly higher after exposure to the
test agents. Since 5c was the most active, its effect on the cell cycle regulation was confirmed
where it showed inhibition of the CDK2/cyclin E1. Finally, in vivo biodistribution study using
radioiodinated-5c revealed a significant uptake and targeting ability into solid tumor in a
xenograft mouse model.
Key words: Benzenesulfonamide scaffold-based derivatives, cytotoxicity, caspase 8 and 9 levels,
cell cycle phase distribution analysis.
Page 2 of 27

1. Introduction
Cancer is the second leading cause of death worldwide. Statistical surveys show that 14.1
million new cases and 8.2 million cancer-related deaths were reported worldwide in 2012. [1]
The choice of proper treatment depends on the patient’s health condition and the goal of
treatment. While surgery and radiation are treatment options, chemotherapy alone or in
combination with the previously mentioned methods still holds a front line in treatment
protocols [2]. Cytotoxic chemotherapeutics elicit their activity through preventing cell growth
and/or inducing cell death (apoptosis) by inhibiting microtubule function, protein function,
or DNA synthesis [3].
Those clinically used cytotoxins can be categorized as cell cycle-independent or cell cycle-
dependent where they arrest cancer cell growth at specific cell cycle phases.[4] Molecular
mechanisms underlying triggering of apoptotic pathway(s) involve activation and inactivation of
key apoptotic proteins with an intricate interplay between the apoptotic machinery members.
[5] Caspase 8, a cysteine protease which is a member of the apoptotic family proteins, is the
initiator of a proteolytic cascade via the extrinsic pathway culminating in cell death [6,7]. Caspase
8 was found to be silenced in most patient tumor samples giving cancer cells a survival advantage
[8]. Another important apoptosis linked protein is caspase 9 which is mainly induced by the
mitochondria-linked intrinsic apoptotic pathway. Both the initiator caspases 8 and 9 are
causatives of the successive activation of down-stream proteolytic executional caspases 3 and 7.
[9] Cyclin-dependent kinase-2 (CDK2) is protein kinase responsible for cell cycle regulation and
recently its overexpression is highly related to hyperproliferation [31] and in some cases
recurrence and invasion. [32] Sulfonamide scaffold bearing aromatic/heterocyclic compounds
have been frequently reported to possess cytotoxic activity. The observed anticancer activity was
attributed to various modes of action such as carbonic anhydrase inhibition, cell cycle arrest,
microtubule disruption, angiogenesis inhibition, apoptosis and autophagy induction, epigenetic
modulation among others. [10-16]
In continuation to our quest for new cytotoxic agents based on the sulfonamide motif [17,
18], we herein present the synthesis and cytotoxic assessment of a new series of thiazolidinone,
thiazinone and dithiazepinone derivatives bearing a benzenesulfonamide functionality. In
addition, caspase 8 and 9 levels, cell cycle phase distribution analysis were determined for the
most active compounds based on the apoptotic morphology of the cytotoxic agents treated cells
upon microscopic evaluation. Finally, the CDK2 activity and the pharmacokinetic behavior of the
most active compound 5c was studied with the aid of radiolabeling technique to evaluate its
targeting ability to solid tumor in tumor bearing mouse model [19-22].
2. Results and discussion
2.1. Chemistry
The synthesis routes of the target thiazolidinone (5a-g), thiazinone (9a-g) and dithiazepinone
(9a-g) benzenesulfonamide derivatives is depicted in schemes 1 and 2. First, the key intermediate
Page 3 of 27

chloroacetyl derivative 2 was synthesized from the thioureido derivative 1 through the reaction
of the nucleophilic N-1 of thiourea with chloroacetyl chloride according to the reported method
[23]. Compound 2 was further subjected to cyclization to the corresponding 1,3-thiazolidin-4-one
derivative 3 upon refluxing in ethanol in the presence of catalytic amount of anhydrous sodium
acetate [24]. Chemical structure of compound 3 was verified by elemental and spectral analyses.
IR spectrum of compound 3 showed a characteristic band for C=O group at 1734 cm-1. 1H-NMR
spectrum of compound 3 revealed a singlet at 4.17 ppm attributed to CH2 of thiazolidinone ring.
On the other hand, treatment of compound 2 with KSCN under reflux in absolute ethanol
furnished the seven member 1,3,5-dithiazepine derivative 4. Chemical structure of compound 4
was verified by elemental and spectral analyses. IR spectrum of compound 4 showed a band for
1
C=O group at 1674 cm-1. H-NMR spectrum of compound 4 revealed a singlet at 4.03 ppm
attributed to CH2 group. The arylidene derivatives 5a-g were synthesized from the thiazolidinone
derivative 3 by condensing the active methylene group with several aromatic aldehydes in glacial
acetic acid and a catalytic amount of anhydrous sodium acetate. Chemical structures of
1
compounds 5a-g were verified by elemental and spectral analyses. H-NMR spectra of
compounds 5a-g revealed the absence of the singlet band at 4.17 ppm and the increment of
aromatic proton bands confirming condensation. Applying the same procedure on the 1,3,5-
dithiazepine derivative 4, compounds 6a-g were obtained. Chemical structures of compounds 6a-
g were verified by elemental and spectral analyses. 1H-NMR spectra of compounds 6a-g revealed
the absence of the singlet band at 4.03 ppm and the increment of aromatic proton bands
confirming condensation (Scheme 1).
H
N
NH2
S
H2NO2S
1
a
H
N
H
N
H
N
O
H
N
N
b
N
c
Cl
O
S
O
S
S
N
H2NO2S
H2NO2S
S
H2NO2S
3
2
HN
4
d
d
H
a
b
c
d
e
f
, R= H
, R= F
N
O
O
H
N
N
, R= N(CH3)2
, R= Br
, R= OH
, R= OCH3
S
H2NO2S
S
S
H2NO2S
HN
6a-g
Scheme 1. Sovents and Reagents:
g
, R= Cl
R
R
5a-g
a, Chlororacetyl chloride, DMF, anhydrous Na acetate; b, Ethanol,
anhydrous Na acetate; c, Ethanol, KSCN; d, Aromatic aldehydes, Glacial Acetic Acid, anhydrous Na acetate
Page 4 of 27

Several attempts were adopted to synthesize the six membered 1,3-thiazine derivative 8 from
the thioureido derivative 1 through its reaction with 3-chloropropionyl chloride and further
cyclization in ethanol but unfortunately compound 8 could not be obtained using this pathway.
A different strategy was conducted through the reaction of 3-chloropropionyl chloride with KSCN
in acetone followed by the addition of a solution sulfanilamide 7 in acetone which successfully
furnished compound 8 in a good yield [25]. Chemical structure of compound 8 was verified by
elemental and spectral analyses. IR spectrum of compound 8 showed bands 3377-3197 cm-1 for
the NH and NH2 groups, a band for C=O group at 1689 cm-1 and two bands at 1330, 1149 cm-1 for
the SO2 group. 1H-NMR spectrum of compound 8 showed two triplets at 2.76 and 3.12 attributed
to the 2CH2 groups. The arylidene derivatives 9a-g could not be obtained by condensing the six
membered 1,3-thiazine derivative 8 with aromatic aldehydes as done before in case of the
thiazolidinone derivatives 5a-g or the 1,3,5-dithiazepine derivatives 6a-g. This lack of success in
obtaining the target compounds can be attributed to the weakness of active methylene in this
case due to the electron donating effect of the adjacent CH2 and so more drastic condition
needed to be applied. Neither increasing the reaction time to 72 hours nor the use of different
bases such as piperidine, sodium ethoxide or even sodium hydroxide, succeeded to give the
target compounds 9a-g. In order to obtain the desired arylidene derivatives 9a-g, fusion
conditions were applied between the 1,3-thiazine derivative 8 and several aromatic aldehydes in
an oil bath which finally furnished the target compounds in good yields [20]. Chemical structures
1
of compounds 9a-g were verified by elemental and spectral analyses. H-NMR spectra of
compounds 9a-g revealed the disappearance of the two triplets at 2.76, 3.17 ppm and the
appearance of new singlets in the range of 3.35-3.47 ppm corresponding to the CH2 groups
(Scheme 2).
NH2
H
N
H
N
a
N
N
O
O
b
H2NO2S
S
S
H2NO2S
H2NO2S
7
8
9a-g
a
, R= H
b
, R= F
c
d
, R= N(CH3)2
, R= Br
R
e
f
, R= OH
, R= OCH3
g
, R= Cl
Scheme 2. Sovents and Reagents:
a, 1,3-Chloropropionylchloride, KSCN,
Acetone; b, Aromatic aldehydes, Glacial Acetic Acid, anhydrous Na acetate
2.2. Cytotoxicity Screening
Cytotoxicity of the newly synthesized compounds was assessed against breast, liver, colon
and lung cancer cell lines MCF-7, HepG2, HCT-116 and A549, respectively using SRB method as
previously described by Skehan et al. [26]. The half maximal inhibitory concentration values IC50
Page 5 of 27

of the tested compounds were calculated utilizing the prism program Version 5 (Table 1) from
their respective dose response curves (Figures S22-S26 supplementary material).
While HCT-116 and A549 cell lines were responsive to all the tested compounds, MCF-7 and
HepG2 were only sensitive to the cytotoxicity of a few derivatives.
For the HCT-116 colon cancer cell line, none of the tested compounds showed equal or better
potency than the reference anticancer agent doxorubicin (IC50 = 0.058±0.02 µM). Yet, 5e and 9d
exhibited almost equipotent cytotoxicity profile when compared to 5-FU (IC50 = 8.91±0.66,
6.93±0.51 and 7.7±1.25 µM, respectively). Also, 6f and 9e displayed almost a 2-fold potency
increase relative to 5-FU (IC50 = 3.50±0.26 and 3.88±0.29 µM, respectively).
With regards to the A549 lung cancer cells, tested compounds showed excellent to moderate
cytotoxicity profile (IC50 values between 1.69±0.11 and 157.17±10.48, 157.17±10.48 µM).
Derivatives 6f and 9d were very potent where they were 5.4 and 2.7-fold more potent than 5-FU
(IC50 1.69±0.11, 3.36±0.23 and 9.27±0.69 µM, respectively). On the other hand, 5e, 5g, 6c, 9c, 9f
and 9g all demonstrated high cytotoxicity (IC50 between 11.73±0.78 and 18.21±1.25 µM).
With respect to the MCF-7 breast cancer cell line, while all compounds were poorly active to
inactive, derivatives 5b and 5c were between 4.5 and 2.7-fold more potent than both doxorubicin
and 5-FU (IC50 = 0.37±0.005, 0.54±0.002, 1.50±0.02 and 1.70±0.03 µM, respectively).
Finally, HepG2 liver cancer cell line was mostly poorly sensitive to the cytotoxicity of the
tested compounds. An exception was 5c which was very cytotoxic being 3.7 times more potent
than doxorubicin (IC50 = 0.24±0.012 and 0.90±0.12 µM, respectively). Also, 5b and 5d exhibited
high cytotoxicity (IC50 = 1.58±0.031 and 2.28±0.120 µM, respectively); while 5e was weakly active
(IC50 >100 µM).
Table 1: In vitro cytotoxicity screening against cancer cell lines (data presented are IC50 µM ± SEM).
Compd. #
HCT-116*
58.66±4.39
53.73±4.02
25.85±1.93
27.32±2.04
8.91±0.66
22.16±1.66
12.38±0.92
>100
26.97±2.02
18.85±1.41
15.73±1.17
45.35±3.39
>100
A549
MCF-7
65.00±3.21
0.37±0.005
0.54±0.002
ND
17.04±0.65
78.30±4.21
71.70±3.58
ND
HepG2
ND**
1.58±0.031
0.24±0.012
2.28±0.120
>100
5a
5b
5c
5d
5e
5f
60.47±4.03
57.78±3.85
27.11±1.80
26.30±1.75
11.73±0.78
32.61±2.17
13.25±0.91
95.71±6.28
31.69±2.19
29.70±2.05
18.21±1.25
50.27±3.47
>100
ND
ND
>100
ND
ND
ND
ND
ND
5g
4
6a
6b
6c
6d
6e
6f
ND
>100
ND
ND
ND
ND
ND
ND
3.50±0.26
10.00±0.74
>100
1.69±0.11
9.38±0.64
>100
38.79±2.34
26.49±1.86
ND
6g
8
Page 6 of 27

9a
9b
9c
9d
9e
9f
9g
Dox
5-FU
10.00±0.74
48.09±3.60
51.40±3.84
6.93±0.51
3.88±0.29
96.81±7.24
12.08±0.90
0.058±0.02
7.7±1.25
30.96±2.14
43.63±3.01
13.07 ± 0.90
3.36±0.23
82.47±5.70
17.92±1.23
15.55±1.07
0.27±0.01
ND
ND
>100
>100
ND
ND
ND
ND
ND
ND
ND
ND
1.50±0.02
1.70±0.03
ND
ND
0.90±0.12
38.50±2.12
9.27±0.69
* Results are average of triplicate experiments ± SEM
** ND = not detectable under the employed experimental conditions (> 1000 µM)
To further assess the selectivity profile of derivatives 5b, 5c and 5e, their cytotoxic potential of
against the normal breast cell line MCF-10A was evaluated (Table 2). All three test compounds
demonstrated an IC50 of >100 µM. These results reflect the highly selective cytotoxicity of these agents (SI
ranging between >5.8 to >270 fold) (dose response curves are provided in the supplementary material).
Table 2: In vitro cytotoxicity screening against normal breast cell line MCF-10 (data presented are IC50
µM).
Compd. # MCF-10A* SI**
5b
5c
5e
>100
>100
>100
>270
>185
>5.8
* Results are average of triplicate experiments
** SI (selectivity Index) = IC50 against MCF-10A/IC50 against MCF-7
2.3. Examination of cell viability under inverted microscope
All test compounds treated cells showed marked shrinkage, accumulation and observable
apoptosis after 48 h of test agents’ exposure (figures 1 and 2 for MCF-7 and HepG2 treated cells,
respectively) compared to the untreated control cells. Moreover, the new compounds 5a-d
treated cells displayed higher rates of apoptosis than the standard reference drugs, doxorubicin
and 5-FU, treated cells. To further confirm this observation, cell cycle analysis and caspases 8 and
9 cell concentrations were investigated as discussed in the following sections.
Page 7 of 27

Fig. 1: Microscopic images of MCF-7 treated cells.
Fig. 2: Microscopic images of HepG2 treated cells.
2.4. Cell cycle analysis
To determine the effect of the new compounds on MCF-7 and HepG2 cell cycle phase
distribution, cells were treated with the test agents for 24 h after which cell cycle histograms
were obtained and results were analyzed (Figures 3 and 4).
Untreated MCF-7 cancer cells displayed a cell cycle population of 48%, 44.4% and 5.9% for
the G0/G1, S and G2/M phases, respectively. Cell cycle analysis of 5b treated MCF-7 breast cancer
cells revealed the occurrence of population of 16.31% of the cells at the <2n phase, significant
accumulation of cells at G0/G1 (74.4%) and a small percent of cells at the G2/M phase (1.6%)
(Figure 3). On the other hand, 5c cellular treatment led cells to accumulate at both <2n and G0/G1
Page 8 of 27

phases (75 and 23%, respectively). Similarly, for derivative 5e, major cell populations were found
to exist at the <2n (54.5%) and G0/G1 (40%) phases.
5b treated
MCF-7 cells
(0.371 µM)
5c treated
MCF-7 cells
(0.547 µM)
5e treated
MCF-7 cells
(17.04 µM)
Page 9 of 27

Untreated
MCF-7 cells
(negative
control)
Fig. 3: Cell cycle analysis of MCF-7 5b, 5c and 5e treated cells versus untreated negative control cells.
Cell cycle analysis of untreated HepG2 cells showed that cells were distributed between the
G0/G1, S and G2/M phases in 52.2%, 35.4 % and 5.1%, respectively (Figure 4). On the other hand,
cell cycle analysis of HepG2 liver cancer cells revealed moderate <2n phase accumulation (28.1%),
very high population at G0/G1 phase (67.9%) and poor existence at the G2/M phase (1.6%) for
5b treated cells. Furthermore, 5c treated cells showed a little higher <2n phase accumulation
(41.3%), noticeable G0/G1 arrest (56.2%) and a small G2/M (1.7%) population. For derivative 5d,
it showed major accumulation of cells at both <2n (32%) and G0/G1 (65.3%) phases with a very
small population at the G2/M phase (1.5%).
5b treated
HepG2 cells
(1.58 µM)
Page 10 of 27

5c treated
HepG2 cells
(0.24 µM)
5d treated
HepG2 cells
(2.28 µM)
HepG2
untreated
cells
(negative
control)
Fig. 4: Cell cycle analysis of HepG2 5b, 5c and 5d treated cells versus untreated negative control cells.
It can be concluded from this experiment that all test compounds induced accumulation of
cells in the <2n phase and an induction of apoptosis in both MCF-7 and HepG2 treated cells. Also,
the observed significant cell cycle arrest in the G0/G1 phase by exposure to the test agents is
another indication of cell growth arrest which ultimately leads to apoptosis induction.
Page 11 of 27

2.5. Caspase 8 and 9 levels
To further exploit the apoptotic behavior of MCF-7 and HepG2 treated cells, caspase 8 and 9
levels were examined in both cell lines (Tables 3 and 4).
Caspase 8 level was measured in MCF-7 and HepG2 cancer cell lines after treatment with the
different test agents (5b,c&e for MCF-7; and 5b-d for HepG2) at their respective IC50
concentrations and results were compared with doxorubicin and 5-FU as positive controls (Table
3). Experimental results showed significant increase in caspase 8 activity in both MCF-7 and
HepG2 cancer cells treated with the test agents compared to the untreated control (p value
<0.001) and both positive control drugs doxorubicin and 5-FU.
Table 3: Caspase 8 activity (absorbance at 400 or 405 nm).
Treatment
Control
Dox
Conc. in MCF-7 cells* P value Conc. in HepG2 cells*
p value
NA
0.3±0.02
0.75±0.03
0.6±0.03
1.02±0.04
1.5±0.03
NT***
NA**
<0.01
<0.05
<0.001
<0.001
NT
0.48±0.02
0.52±0.03
0.7±0.02
0.9±0.03
1.3±0.05
1.1±0.01
NT
NS >0.05
<0.01
<0.001
<0.001
<0.001
NT
5-FU
5b
5c
5d
5e
1.2±0.02
<0.001
* Results are average of triplicate experiments ± SEM
** NA = not applicable
*** NT = not tested
Similarly, capase-9 level was measured in MCF-7 and HepG2 cancer cell lines treated with the
compounds under investigation (5b-e) at their respective IC50 values and were compared to
controls (Table 4). MCF-7 test agents-treated cells showed extremely significant increase in
caspase 9 enzymatic concentration compared to negative control and positive control
(doxorubicin and 5-FU) treated counterparts. Analogous effect was observed for HepG2 treated
cells.
Table 4: Caspase 9 activity (absorbance at 400 or 405 nm).
Treatment
Control
Dox
Conc. in MCF-7 cells*
0.38±0.02
0.48±0.01
0.4±0.02
P value
NA**
<0.01
NS >0.05
<0.05
<0.001
NT
Conc. in HepG2 cells*
0.21±0.02
0.31±0.01
0.23±0.02
0.28±0.01
0.38±0.03
0.36±0.01
NT
P value
NA
<0.01
NS >0.05
<0.001
<0.001
<0.001
NT
5-FU
5b
5c
5d
5e
0.66±0.02
0.78±0.01
NT***
0.56±0.01
<0.001
* Results are average of triplicate experiments ± SEM
** NA = not applicable
*** NT = not tested
Page 12 of 27

2.6. CDK-2 inhibition activity for 5c
Compound 5c was tested for inhibitory effect on CDK2 and showed moderate potency (IC50 =
56.97 ± 2 µM) (Figure 5). This could account for the cell cycle arrest in the G0/G1 phase [27] and
in part account for the compound’s high cytotoxicity.
150
100
50
0
0.0
0.5
1.0
1.5
2.0
2.5
Log Conc M
Fig. 5: Dose-dependent inhibitory effect of 5c on CDK2.
2.7. Radiolabeling and in vivo biodistribution of radioiodinated-5c
Since compound 5c showed the highest results in almost all conducted assays. It was selected
for radiolabeling and biodistribution studies.
2.7.1. Radiosynthesis of radioiodinated-5c
The highest radiochemical yield (RCY) of radioiodinated-5c was 89.5±2.25%. Such maximum
yield was obtained using 0.5 mg of compound 5c and 50 mg chloramine-T. Radiolabeling
reaction was done for 30 min reaction time at pH 4 at ambient temperature (27±3 °C). The in-
vitro stability of radioiodinated-5c was up to 10 h.
2.7.2. Pharmacokinetic study of radioiodinated-5c
Results of radioiodinated-5c biodistribution pattern in Swiss albino mice with right tumor
bearing thigh muscle are shown in Table 5. It was evident that there is a significant difference in
uptake of radioiodinated-5c by the tumor tissues as represented by the tumor bearing right thigh
muscle compared to non-cancerous tissues represented by the normal left thigh muscle at all-
tested time intervals. Besides, the target to non-target ratio (T/NT) was greater than 1 at all-time
intervals and reached its maximum value at 4 h post injection (T/NT= 4.66). Liver and intestine
high radioactivity levels indicate that hepatobiliary pathway is the main excretion route of
radioiodinated-5c. Low radioactivity levels of thyroid at different time intervals indicate the good
in-vivo stability of radioiodinated-5c.
Page 13 of 27

Table 5. Biodistribution of radioiodinated-5c in Swiss albino mice with right tumor bearing thigh muscle
at different time intervals post-injection. (% ID/organ ± S.D., n = 5).
Organs
15 min
60 min
2 h
4 h
Blood
47.07±3.78
5.73±0.47
16.03±1.90
0.66±0.12
8.62±0.91
1.39±0.05
3.88±0.7
28.84±3.51
4.34±0.73
26.82±2.67
1.22±0.03
12.15±1.63
4.88±0.42
1.16±0.05
0.34±0.09
8.78±0.94
1.87±0.11
11.48±1.23
27.36±3.65
31.69±3.55
4.21±0.52
28.86±3.5
1.56±0.18
14.45±1.32
5.01±0.68
2.15±0.18
0.34±0.04
3.09±0.21
2.30±0.35
8.62±0.89
29.38±3.51
16.93±2.43
6.88±0.75
32.95±4.21
1.37±0.13
24.57±3.71
4.07±0.44
2.23±0.09
0.24±0.01
2.18±0.24
1.90±0.22
8.59±0.83
40.10±5.99
Kidneys
Liver
Spleen
Intestine
Stomach
Lungs
Heart
1.02±0.03
2.79±0.17
1.34±0.22
12.82±0.99
20.17±1.86
Bone
Thyroid
Normal left thigh muscle
Tumor right thigh muscle
T/NT
1.57
2.38
3.41
4.66
3. Conclusion
Twenty-one new compounds belonging to the thiazolidinone (5a-g), thiazinone (9a-g) and
dithiazepinone (9a-g) heterocycles bearing a benzenesulfonamide scaffold were synthesized.
These derivatives were found to display promising cytotoxicity against MCF-7, HepG2, HCT-116
and A549 cancer cell lines and activity as compared to the known cytotoxic agents doxorubicin
and 5-FU. The IC50 values for compounds 5b and 5c on MCF-7 cell line were 0.37 and 0.54 µM,
respectively, while those for compounds 5b, 5c and 5d on HepG2 cell line were 1.58, 0.24 and
2.28 µM, respectively. Cytotoxicity of the 5b, 5c and 5e towards MCF-10A, normal breast cell
line, was also assessed and all three compounds were found to be selectively much more toxic
to MCF-7 breast cancer cell line and far less toxic to MCF-10A. Additionally, cell cycle analysis
showed that the most active compounds 5b-e were able to disrupt MCF-7 and HepG2 cell cycle
phase distribution inducing cellular accumulation in the <2n phase which is a hall-mark of
apoptosis. Furthermore, evaluation of caspase 8 and 9 levels in MCF-7 and HepG2 before and
after treatment with the most active compounds 5b-e demonstrated a significant increase in the
concentration of these apoptotic proteins which is a second hall-mark for programmed cell death
induction. Compound 5c has increased the level of caspase 8 by 5 folds in MCF-7 cells and almost
for folds in HepG2 cells. The same compound has also increased the level of caspase 9 by almost
two folds in HepG2 cells. Testing compound 5c showed it had some inhibitory effect on CDK2
explaining the accumulation of treated cells in the G0/G1 phase. The IC50 value for this inhibition
was 56.97 µM. Finally, pharmacokinetic study of the 125I radioiodinated-5c was performed and
biodistribution results showed a significant high accumulation of radioactivity in cancer bearing
thigh muscle compared to the normal thigh muscle (high T/NT) that reached 4.66 in 4 hours. In
Page 14 of 27

conclusion, the derivative 5c displays the qualities of a promising lead compound, which can be
adopted for further optimization.
4. Experimental
4.1. Chemistry
Melting points were determined on Electro thermal Stuart 5MP3 digital melting point
apparatus and were uncorrected. Elemental microanalyses were performed at the micro
analytical center, Al-Azhar University, Cairo, Egypt. IR spectra were recorded on a Bruker Fourier
transform (FT)-IR spectrophotometer as KBr discs. NMR spectra (in DMSO-d6) were recorded on
Bruker AC-300 Ultra Shield NMR spectrometer (Bruker, Flawil, Switzerland, δ ppm) at 400 MHz
using TMS as internal Standard and peak multiplicities are designed as follows: s, singlet; d,
doublet; t, triplet; m, multiplet. Silica gel used for column chromatography was obtained from
Fluka, 70-230 mesh thin layer chromatography was carried out on silica gel TLC plates with
fluorescence indicator (F254).
4.1.1. 2-Chloro-N-(4-sulfamoylphenyl)acetamide 2
The target compound was prepared according the reported method. Yield: 63%; m.p: 207–208 °C
(as reported). [23]
4.1.2. 4-(4-Oxo-4,5-dihydrothiazol-2-ylamino)benzenesulfonamide 3
To a solution of the choloro derivative 2 (0.01 mol, 2.48 g) in absolute ethanol (20 mL), a catalytic
amount of anhydrous sodium acetate (0.5 g) was added and the reaction mixture was refluxed
for three hours. The white precipitate formed was filtered, dried, washed with hot ethanol and
crystallized from DMF/H2O to give white powder of 3.
Yield: 68%; m.p.: 213–215 °C; IR (KBr) (cm-1): 3390, 3204 (NH, NH2), 3050-3015 (CH arom.), 1734
1
(C=O), 1344, 1163 (SO2); HNMR (400 MHz, DMSO-d6) δ: 4.17 (s, 2H, CH2), 7.47 (s, 2H, NH2SO2,
D2O exchangeable), 7.47-7.50 (d, 2H, Ar H), 7.91-7.93 (d, 2H, Ar H), 9.38 (s, 1H, NH, D2O
exchangeable); 13CNMR (100.63 MHz, DMSO-d6) δ: 34.30(1), 126.75(2), 129.72(2), 138.41(1),
144.13(1), 158.34(1), 171.97(1); Anal. Calcd. for C9H9N3O3S2 (Mwt. 271.32): C, 39.84; H, 3.34; N,
15.49; found: C, 39.91; H, 3.38; N, 15.63.
4.1.3. 4-(2-Imino-6-oxo-6,7-dihydro-1,3,5-dithiazepin-4-ylamino)benzenesulfonamide 4
To a solution of the chloro derivative 2 (0.01 mol, 2.48 g) in absolute ethanol (20 mL), KSCN (0.01
mol, 0.97 g) was added and the reaction mixture was refluxed for five hours. The canary yellow
precipitate formed was filtered, dried, washed with hot ethanol and crystallized from DMF/H2O
to give yellow powder of 4.
Yield: 75%; m.p.: 260–262 °C; IR (KBr) (cm-1): 3360, 3271 (NH, NH2), 3051-3001 (CH arom.), 1674
1
(C=O), 1296, 1151(SO2); HNMR (400 MHz, DMSO-d6) δ: 4.03 (s, 2H, CH2 ), 7.10 (s, 2H, NH2SO2,
D2O exchangeable), 7.31 (d, 2H, Ar H), 7.82 (d, 2H, Ar H), 11.51 (s, 1H, NH, D2O exchangeable),
Page 15 of 27

11.92 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) δ: 19.02(1), 120.40(2),
121.77(2), 127.26(2), 140.05(2), 152(1); Anal. Calcd. for C10H10N4O3S3 (Mwt. 330.41): C, 36.35; H,
3.05; N, 16.96; found C, 36.44; H, 3.03; N, 17.08.
4.1.4. General procedure for preparation of compounds 5a-g
To a solution of compound 3 (0.01 mol, 2.71 g) in glacial acetic acid (20 mL), catalytic amount of
anhydrous sodium acetate (0.5 g) was added. After complete dissolution, the appropriate
aromatic aldehyde (0.01 mol) was added and the reaction mixture was refluxed for 6-8 hours.
The precipitate formed was filtered, dried, washed with hot ethanol and crystallized from
DMF/H2O to give 5a-g, respectively.
4.1.4.1. 4-(5-Benzylidene-4-oxo-4,5-dihydrothiazol-2-ylamino)benzenesulfonamide 5a
Yield: 72%; m. p.:
°C; IR (KBr) (cm-1): 3313, 3246 (NH, NH ), 3050-3010 (CH arom.), 1678
< 300
2
(C=O), 1319, 1153 (SO2); Anal. Calcd. for C16H13N3O3S2 (Mwt. 359.42): C, 53.47; H, 3.65; N, 11.69;
found C, 53.54; H, 3.68; N, 11.77.
4.1.4.2. 4-(5-(4-Fluorobenzylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino)benzenesulfonamide 5b
Yield: 65%; m.p.: 279–281°C; IR (KBr) (cm-1): 3317, 3242 (NH, NH2), 3055-3010 (CH arom.), 1683
(C=O), 1338, 1157 (SO2); 1HNMR (400 MHz, DMSO-d6) δ: 7.20 (s, 2H, NH2SO2, D2O exchangeable),
7.22-7.85 (m, 8H, Ar H), 7.95 (s, 1H, CH=C), 12.42 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63
MHz, DMSO-d6) δ: 116.80(2), 120.78(1), 121.98(2), 122.82(1), 127.48(1), 127.75(2), 129.39(1),
130.35(1), 132.65(2), 140.12(1), 140.65(1), 161.82(1); Anal. Calcd. for C16H12FN3O3S2 (Mwt.
377.41): C, 50.92; H, 3.20; N, 11.13; found C, 51.03; H, 3.24; N, 11.26.
4.1.4.3.
4-(5-(4-(Bimethylamino)benzylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino)benzene-
sulfonamide 5c
Yield: 55%; m.p.: 285–287 °C; IR (KBr) (cm-1): 3356, 3238 (NH, NH2), 3122-3043 (CH arom.), 2922
(CH aliph.), 1670 (C=O), 1319, 1155 (SO2); 1HNMR (400 MHz, DMSO-d6) δ: 3.04 (s, 6H, N(CH3)2),
6.80-6.84 (m, 3H, Ar H), 7.20 (s, 2H, NH2SO2, D2O exchangeable), 7.35-7.50 (m, 3H, Ar H), 7.66-
7.96 (d, 2H, Ar H), 7.98 (s, 1H, CH=C), 12.27 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz,
DMSO-d6) δ: 40.00(2), 112.55(2), 120.20(1), 126.98(2), 129.22(2), 132.17(2), 132.96(2),
135.06(1), 144.80(1), 152.19(1), 165.57(1), 167.45(1); Anal. Calcd. for C18H18N4O3S2 (Mwt.
402.49): C, 53.71; H, 4.51; N, 13.92; found C, 53.87; H, 4.57; N, 14.08.
4.1.4.4. 4-(5-(4-Bromobenzylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino)benzenesulfonamide 5d
Yield: 55%; m.p.
°C; IR (KBr) (cm-1): 3332, 3201 (NH, NH ), 3043-3010 (CH arom.), 1670
< 300
2
(C=O), 1317, 1155 (SO2); 1HNMR (400 MHz, DMSO-d6) δ: 7.19 (d, 2H, Ar H), 7.21 (s, 2H, NH2SO2,
D2O exchangeable),7.44 (d, 2H, Ar H), 7.58 (d, 2H, Ar H), 7.90 (d, 2H, Ar H), 7.95 (s, 1H, CH=C),
12.59 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) δ: 120.90(1), 121.98(2),
Page 16 of 27

123.88(2), 127.76(2), 129.22(1), 132.00(2), 132.87(2), 140.68(2), 167.79(1), 171.83(1); Anal.
Calcd. for C16H12BrN3O3S2 (Mwt. 438.32): C, 43.84; H, 2.76; N, 9.59; found C, 43.91; H, 2.74; N,
9.67.
4.1.4.5. 4-(5-(4-Hydroxybenzylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino)benzenesulfonamide
5e
Yield: 52%; m.p.
°C; IR (KBr) (cm-1): 3446 (OH), 3346, 3255 (NH, NH ), 3041-3010 (CH
< 300
2
arom.), 1670 (C=O), 1311, 1151 (SO2); 1HNMR (400 MHz, DMSO-d6) δ: 6.87 (d, 2H, Ar H), 7.19 (d,
2H, Ar H), 7.21 (s, 2H, NH2SO2, D2O exchangeable), 7.58 (d, 2H, Ar H), 7.92 (d, 2H, Ar H), 7.95 (s,
1H, CH=C), 10.20 (s, 1H, OH, D2O exchangeable), 12.37 (s,1H,NH, D2O exchangeable); 13CNMR
(100.63 MHz, DMSO-d6) δ: 116.76(4), 122.00(2), 124.56(1), 127.74(2), 130.98(1), 132.48(2),
140.45(1), 151.50(1), 159.99(1), 168.10(1); Anal. Calcd. for C16H13N3O4S2 (Mwt. 375.42): C, 51.19;
H, 3.49; N, 11.19; found C, 51.32; H, 3.54; N, 11.26.
4.1.4.6. 4-(5-(4-Methoxybenzylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino)benzenesulfonamide
5f
Yield: 67%; m.p.
°C; IR (KBr) (cm-1): 3334, 3248 (NH, NH ), 3045-3020 (CH arom.), 2926 (CH
< 300
2
aliph.), 1678 (C=O), 1319, 1153 (SO2); Anal. Calcd. for C17H15N3O4S2 (Mwt. 389.45): C, 52.43; H,
3.88; N, 10.79; found C, 52.57; H, 3.92; N, 10.87.
4.1.4.7. 4-(5-(4-Chlorobenzylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino)benzenesulfonamide 5g
Yield: 52%; m.p.
°C; IR (KBr) (cm-1): 3325, 3250 (NH, NH ), 3090-3025 (CH arom.), 1670
< 300
2
(C=O), 1319, 1157 (SO2); Anal. Calcd. for C16H12ClN3O3S2 (Mwt. 393.87): C, 48.79; H, 3.07; N, 10.67;
found C, 48.88; H, 3.11; N, 10.82.
4.1.5. General procedure for prepration of compounds 6a-g
To a solution of compound 4 (0.01 mol, 3.30 g) in glacial acetic acid (20 mL), catalytic amount of
anhydrous sodium acetate (0.5 g) was added. After complete dissolution, the appropriate
aromatic aldehyde (0.01 mol) was added and the reaction mixture was refluxed for 6-8 hours.
The precipitate formed was filtered, dried, washed with hot ethanol and crystallized from
DMF/H2O to give 6a-g, respectively.
4.1.5.1.
4-(7-Benzylidene-2-imino-6-oxo-6,7-dihydro-1,3,5-dithiazepin-4-ylamino)-benzene-
sulfonamide 6a
Yield: 80%; m.p.
°C; IR (KBr) (cm-1): 3315-3197 (2NH, NH ), 3090-3039 (CH arom.), 1676
< 300
2
(C=O), 1319, 1155 (SO2); 1HNMR (400 MHz, DMSO-d6) δ: 7.19 (d, 2H, Ar H ) , 7.21 (d, 2H, Ar H),
7.26 (s, 2H, NH2SO2, D2O exchangeable), 7.59-7.85 (m, 5H, Ar H), 7.95 (s, 1H, CH=C), 11.93 (s, 1H,
NH, D2O exchangeable), 12.53 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) δ
ppm: 120.92(2), 121.99(2), 127.75(2), 129.76(2), 130.19(2), 130.48(2), 133.68(1), 140.63(1),
Page 17 of 27

152.50(1), 161.00(1), 166.34(1); Anal. Calcd. for C17H14N4O3S3 (Mwt. 418.51): C, 48.79; H, 3.37; N,
13.39; found C, 48.89; H, 3.40; N, 13.46.
4.1.5.2.
benzenesulfonamide 6b
Yield 70%; m.p.
(C=O), 1317, 1159 (SO2); HNMR (400 MHz, DMSO-d6) δ: 7.19 (d, 2H, Ar H), 7.21 (d, 2H, Ar H),
7.31 (s, 2H, NH2SO2, D2O exchangeable), 7.58-7.86 (m, 4H, Ar H), 7.95 (s, 1H, CH=C), 11.53 (s, 1H,
NH, D2O exchangeable), 12.49 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6)
δ: 116.79(2), 120.89(1), 121.98(2), 127.75(2), 129.37(1), 130.33(1), 132.64(2), 140.65(2),
151.19(1), 161.82(1), 164.29(1), 167.95(1); Anal. Calcd. for C17H13FN4O3S3 (Mwt. 436.50); C,
46.78; H, 3.00; N, 12.84; found C, 46.89; H, 3.02; N, 12.89.
4-(7-(4-Fluorobenzylidene)-2-imino-6-oxo-6,7-dihydro-1,3,5-dithiazepin-4-ylamino)-
°C; IR (KBr) (cm-1): 3317-3199 (2NH, NH ), 3085-3045 (CH arom.), 1681
< 300
2
1
4.1.5.3.
ylamino)benzenesulfonamide 6c
Yield: 64%; m.p.
°C; IR (KBr) (cm-1): 3300-3197 (2NH, NH ), 3080-3039 (CH arom.), 2953
4-(7-(4-(Dimethylamino)benzylidene)-2-imino-6-oxo-6,7-dihydro-1,3,5-dithiazepin-4-
< 300
2
(CH aliph.), 1680 (C=O), 1321, 1155 (SO2); 1HNMR (400 MHz, DMSO-d6) δ: 2.98 (s, 6H, N(CH3)2),
6.80 (d, 2H, Ar H), 7.20 (d, 2H, Ar H), 7.35 (s, 2H, NH2SO2, D2O exchangeable),7.55-7.85 (m, 4H,
Ar H), 7.96 (s, 1H, CH=C), 11.73 (s, 1H, NH, D2O exchangeable), 12.26 (s, 1H, NH, D2O
exchangeable); 13CNMR (100.63 MHz, DMSO-d6) δ: 39.79(2), 112.55(2), 119.68(2), 120.76(2),
122.75(2), 127.75(2), 132.16(2), 143.73(2), 152.19(1), 157.86(1), 167.48(1); Anal. Calcd. for
C19H19N5O3S3 (Mwt. 461.58): C, 49.44; H, 4.15; N, 15.17; found C, 49.53; H, 4.19; N, 15.28.
4.1.5.4.
benzenesulfonamide 6d
Yield: 58%; m.p.
4-(7-(4-Bromobenzylidene)-2-imino-6-oxo-6,7-dihydro-1,3,5-dithiazepin-4-ylamino)-
°C; IR (KBr) (cm-1): 3334-3195 (2NH, NH ), 3091-3035 (CH arom.), 1670
< 300
2
(C=O), 1317, 1155 (SO2); 1HNMR (400 MHz, DMSO-d6) δ: 7.19 (d, 2H, Ar H), 7.37 (s, 2H, NH2SO2,
D2O exchangeable), 7.46 (d, 2H, Ar H), 7.68-7.89 (m, 4H, Ar H), 7.95 (s, 1H, CH=C), 11.97 (s, 1H,
NH, D2O exchangeable), 12.61 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6)
δ: 121.93(2), 123.88(2), 127.76(2), 132.02(2), 132.76(4), 140.66(2), 152.59(1), 168.80(1),
172.00(1); Anal. Calcd. for C17H13BrN4O3S3 (Mwt. 497.41): C, 41.05; H, 2.63; N, 11.26; found C,
41.21; H, 2.60; N, 11.39.
4.1.5.5.
benzenesulfonamide 6e
Yield: 65%; m.p.
4-(7-(4-Hydroxybenzylidene)-2-imino-6-oxo-6,7-dihydro-1,3,5-dithiazepin-4-ylamino)-
°C; IR (KBr) (cm-1): 3440 (OH), 3360-3271 (2NH, NH ), 3049-3001 (CH
< 300
2
1
arom.), 1674 (C=O), 1332, 1149 (SO2); HNMR (400 MHz, DMSO-d6) δ: 6.88-7.19 (m, 4H, Ar H),
7.21 (s, 2H, NH2SO2, D2O exchangeable), 7.31 - 7.74 (m, 4H, Ar H), 7.82 (s, 1H, CH=C), 10.20 (s,
1H, OH, D2O exchangeable), 11.48 (s, 1H, NH, D2O exchangeable), 11.90 (s, 1H, NH, D2O
Page 18 of 27

exchangeable); 13CNMR (100.63 MHz, DMSO-d6) δ: 116.76(2), 120.49(2), 121.83(2), 127.52(2),
132.47(2), 140.12(4), 151.81(1), 160.12(1), 172.23(1); Anal. Calcd. for C17H14N4O4S3 (Mwt.
434.51): C, 46.99; H, 3.25; N, 12.89; found C, 47.06; H, 3.28; N, 13.02.
4.1.5.6. 4-(2-Imino-7-(4-methoxybenzylidene)-6-oxo-6,7-dihydro-1,3,5-dithiazepin-4-ylamino)-
benzenesulfonamide 6f
Yield: 58%; m.p.
°C; IR (KBr) (cm-1): 3325-3197 (2NH, NH ), 3090-3045 (CH arom.), 2926
< 300
2
(CH aliph.), 1668 (C=O), 1328, 1157 (SO2); 1HNMR (400 MHz, DMSO-d6) δ: 3.83 (s, 3H, OCH3), 7.05-
7.21 (m, 4H, Ar H), 7.36 (s, 2H, NH2SO2, D2O exchangeable),7.47-7.85 (m, 4H, Ar H), 7.95 (s, 1H,
CH=C), 11.82 (s, 1H, NH, D2O exchangeable), 12.42 (s, 1H, NH, D2O exchangeable); 13CNMR
(100.63 MHz, DMSO-d6) δ: 55.90(1), 115.36(4), 122.00(2), 127.73(2), 132.17(4), 140.55(2),
151.00(1), 162.33(1), 171.00(1); Anal. Calcd. for C18H16N4O4S3 (Mwt. 448.54): C, 48.20; H, 3.60; N,
12.49; found C, 48.28; H, 3.64; N, 12.63.
4.1.5.7.
benzenesulfonamide 6g
Yield: 63%; m.p.
4-(7-(4-Chlorobenzylidene)-2-imino-6-oxo-6,7-dihydro-1,3,5-dithiazepin-4-ylamino)-
°C; IR (KBr) (cm-1): 3325-3201 (2NH, NH ), 3090-3047 (CH arom.), 1670
< 300
2
(C=O), 1319, 1159 (SO2); 1HNMR (400 MHz, DMSO-d6) δ: 7.19 (d, 2H, Ar H ), 7.37 (s, 2H, NH2SO2,
D2O exchangeable), 7.55 (d, 2H, Ar H ), 7.68-7.89 (m, 4H, Ar H), 7.95 (s, 1H, CH=C), 11.85 (s, 1H,
NH, D2O exchangeable), 12.57 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6)
δ: 121.97(2), 127.75(2), 129.84(4), 131.85(2), 135.00(2), 140.68(2), 152.00(1), 163.50(1),
169.00(1); Anal. Calcd. for C17H13ClN4O3S3 (Mwt. 452.96): C, 45.08; H, 2.89; N, 12.37; found C,
45.15; H, 2.87; N, 12.49.
4.1.6. 4-(4-Oxo-5,6-dihydro-4H-1,3-thiazin-2-ylamino)benzenesulfonamide 8
A solution of KSCN (0.01 mol, 0.97 g) and 1,3-choloropropionyl chloride (0.01 mol, 1.26 g) in
acetone (30 mL) was refluxed for 30 minutes. The solution was left to cool then a solution of
sulfanilamide 7 (0.01 mol, 1.72 g) in acetone (20 mL) was added drop wise in 15 minutes and left
to stir for three hours in room temperature then 100 ml 1N HCl was added to the solution. The
formed yellow precipitate was dissolved in acetone/toluene 1:1 and refluxed for six hours. The
yellow precipitate formed was filtered, dried, washed with hot ethanol and crystallized from
DMF/H2O to give 8.
Yield: 70%; m.p.: 243–245 °C; IR (KBr) (cm-1): 3377-3197 (NH, NH2), 3088-3068 (CH arom.), 2947-
2926 (CH aliph.), 1689 (C=O), 1330, 1149 (SO2); 1HNMR (400 MHz, DMSO-d6) δ: 2.76 (t, 2H, CH2),
3.12 (t, 2H, CH2), 6.97-7.00 (d, 2H, Ar H), 7.28 (s, 2H, NH2SO2, D2O exchangeable), 7.75-7.77 (d,
2H, Ar H), 11.15 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) δ: 23.18(1),
33.63(1), 121.73(2), 126.71(2), 139.58(1), 151.62(1), 152.71(1), 170.82(1); Anal. Calcd. for
C10H11N3O3S2 (Mwt. 285.34): C, 42.09; H, 3.89; N, 14.73; found C, 42.16; H, 3.92; N, 14.86.
Page 19 of 27

4.1.7. General procedure for the preparation of compounds 9a-g
A mixture of compound 8 (0.01 mol, 2.85 g) and the appropriate aldehyde (0.01 mol) was fused
on oil bath for six hours. The formed mass was washed with hot ethanol, dried and crystallized
form DMF/H2O to give 9a-g, respectively.
4.1.7.1. 4-(5-benzylidene-4-oxo-5,6-dihydro-4H-1,3-thiazin-2-ylamino)benzenesulfonamide 9a
Yield: 52%; m.p.: 155–157 °C; IR (KBr) (cm-1): 3325-3244 (NH, NH2), 3101-3062 (CH arom.), 1689
1
(C=O), 1319, 1153 (SO2); HNMR (400 MHz, DMSO-d6) δ: 3.45 (s, 2H, CH2), 6.58-6.93 (m, 4H, Ar
H),7.25 (s, 2H, NH2SO2, D2O exchangeable), 7.50-7.99 (m, 5H, Ar H), 8.70 (s, 1H, CH=C), 10.44(s,
1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) δ: 25.55(1), 112.89(2), 119.04(2),
127.15(2), 128.27(4), 129.03(2), 130.49(2), 152.50(1), 172.78(1); Anal. Calcd. for C17H15N3O3S2
(Mwt. 373.45): C, 54.67; H, 4.05; N, 11.25; found C, 54.82; H, 4.09; N, 11.34.
4.1.7.2.
4-(5-(4-Fluorobenzylidene)-4-oxo-5,6-dihydro-4H-1,3-thiazin-2-ylamino)benzene-
sulfonamide 9b
Yield: 52%; m.p.: 185–187 °C; IR (KBr) (cm-1): 3336-3250 (NH, NH2), 3097-3066 (CH arom.), 1690
1
(C=O), 1328, 1155 (SO2); HNMR (400 MHz, DMSO-d6) δ: 3.47 (s, 2H, CH2), 6.58-7.00 (m, 4H, Ar
H), 7.24 (s, 2H, NH2SO2, D2O exchangeable), 7.52-8.01 (m, 4H, Ar H), 8.70 (s, 1H, CH=C), 10.50 (s,
1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) δ: 23.30(1), 112.88(2), 116.75(2),
119.05(1), 121.72(2), 127.14(2), 127.39(2), 132.90(1), 139.58(1), 152.38(1), 171.27(1), 192.16(1);
Anal. Calcd. for C17H14FN3O3S2 (Mwt. 391.44): C, 52.16; H, 3.60; N, 10.73; found C, 52.34; H, 3.59;
N, 10.90.
4.1.7.3.
4-(5-(4-(Dimethylamino)benzylidene)-4-oxo-5,6-dihydro-4H-1,3-thiazin-2-ylamino)-
benzenesulfonamide 9c
Yield: 52%; m.p.: 205–207 °C; IR (KBr) (cm-1): 3369-3196 (NH, NH2), 3088-3055 (CH arom.), 2920
(CH aliph.), 1697 (C=O), 1301, 1180 (SO2); 1HNMR (400 MHz, DMSO-d6) δ: 3.13 (s, 6H, N(CH3)2),
3.47 (s, 2H, CH2 ), 6.58-7.04 (m, 4H, Ar H), 7.24 (s, 2H, NH2SO2, D2O exchangeable), 7.44-7.87 (m,
4H, Ar H), 8.83 (s, 1H, CH=C), 10.37 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-
d6) δ: 23.20(1), 33.5(2), 112.05(2), 119.32(2), 121.73(2), 122.27(2), 127.39(2), 128.83(2),
134.83(1), 139.58(1), 152.84(1), 169.61(1); Anal. Calcd. for C19H20N4O3S2 (Mwt. 416.52): C, 54.79;
H, 4.84; N, 13.45; found C, 54.88; H, 4.89; N, 13.62.
4.1.7.4.
4-(5-(4-Bromobenzylidene)-4-oxo-5,6-dihydro-4H-1,3-thiazin-2-ylamino)benzene-
sulfonamide 9d
Yield: 55%; m.p.: 203–205 °C; IR (KBr) (cm-1): 3313-3246 (NH, NH2), 3050-3010 (CH arom.), 1678
1
(C=O), 1319, 1153 (SO2); HNMR (400 MHz, DMSO-d6) δ: 3.47 (s, 2H, CH2), 6.63-7.17 (m, 4H, Ar
H), 7.25 (s, 2H, NH2SO2, D2O exchangeable), 7.45-7.95 (m, 4H, Ar H), 8.80 (s, 1H, CH=C), 10.40 (s,
1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) δ: 23.40(1), 117.50(2), 119.24(2),
Page 20 of 27

127.53(2), 128.54(1), 132.20(2), 133.43(1), 136.47(1), 139.44(1), 143.50(1), 149.00(1), 153.11(1),
171.00(1); Anal. Calcd. for C17H14BrN3O3S2 (Mwt. 452.35): C, 45.14; H, 3.12; N, 9.29; found C,
45.28; H, 3.14; N, 9.34.
4.1.7.5.
4-(5-(4-Hydroxybenzylidene)-4-oxo-5,6-dihydro-4H-1,3-thiazin-2-ylamino)benzene-
sulfonamide 9e
Yield: 65%; m.p.: 272–274 °C; IR (KBr) (cm-1): 3447 (OH), 3334-3228 (NH, NH2), 3082-3057 (CH
arom.), 1680 (C=O), 1327, 1155 (SO2); 1HNMR (400 MHz, DMSO-d6) δ: 3.47 (s, 2H, CH2 ), 6.63-7.00
(m, 4H, Ar H),7.28 (s, 2H, NH2SO2, D2O exchangeable), 7.40-7.89 (m, 4H, Ar H), 8.81 (s, 1H, CH=C),
9.3 (s, 1H, OH, D2O exchangeable), 10.45 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz,
DMSO-d6) δ: 23.40(1), 112.89(2), 119.04(1), 121.73(2), 127.15(2), 127.39(2), 127.88(2),
128.33(1), 139.58(1), 149.00(1), 152.38(1), 172.00(1); Anal. Calcd. for C17H15N3O4S2 (Mwt.
389.45): C, 52.43; H, 3.88; N, 10.79; found C, 52.59; H, 3.86; N, 10.92.
4.1.7.6.
4-(5-(4-Methoxybenzylidene)-4-oxo-5,6-dihydro-4H-1,3-thiazin-2-ylamino)benzene-
sulfonamide 9f
Yield: 68%; m.p.: 125–127 °C; IR (KBr) (cm-1): 3336-3248 (NH, NH2), 3068-3005 (CH arom.), 2966
(CH aliph.), 1680 (C=O), 1327, 1155 (SO2); 1HNMR (400 MHz, DMSO-d6) δ: 3.35 (s, 2H, CH2 ), 3.85
(s, 3H, CH3 ), 6.58-6.92 (m, 4H, Ar H), 7.20 (s, 2H, NH2SO2, D2O exchangeable), 7.44-7.86 (m, 4H,
Ar H), 7.89 (s, 1H, CH=C), 10.34 (s, 1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6)
δ: 23.02(1), 55.47(1), 112.89(2), 114.13(2), 114.99(2), 119.04(2), 127.16(2), 127.88(2), 130.12(1),
130.50(1), 152.38(1), 172.55(1); Anal. Calcd. for C18H17N3O4S2 (Mwt. 403.48): C, 53.58; H, 4.25; N,
10.41; found C, 53.74; H, 4.28; N, 10.49.
4.1.7.7.
4-(5-(4-Chlorobenzylidene)-4-oxo-5,6-dihydro-4H-1,3-thiazin-2-ylamino)benzene-
sulfonamide 9g
Yield: 65%; m.p.: 173–175 °C; IR (KBr) (cm-1): 3345-3238 (NH, NH2), 3097-3064 (CH arom.), 1685
1
(C=O), 1323, 1155 (SO2); HNMR (400 MHz, DMSO-d6) δ: 3.61 (s, 2H, CH2), 6.58-7.03 (m, 4H, Ar
H), 7.33 (s, 2H, NH2SO2, D2O exchangeable), 7.45-7.97 (m, 4H, Ar H), 8.80 (s, 1H, CH=C), 10.40 (s,
1H, NH, D2O exchangeable); 13CNMR (100.63 MHz, DMSO-d6) δ: 23.00(1), 112.92(2), 119.04(2),
120.39(1), 127.16(2), 127.88(2), 129.02(1), 130.25(1), 130.53(1), 138.69(1), 142.00(1), 152.34(1),
172.00(1); Anal. Calcd. for C17H14ClN3O3S2 (Mwt. 407.89): C, 50.06; H, 3.46; N, 10.30; found C,
50.23; H, 3.50; N, 10.42.
4.2. In vitro cytotoxicity screening
a. Cell culture
Human cancer cells were grown in DMEM, supplemented with 10% heat inactivated FBS, 50
units/mL of penicillin and 50 µg/mL of streptomycin and maintained at 37 °C in a humidified
Page 21 of 27

atmosphere containing 5% CO2. The cells were maintained as “monolayer culture” by serial
subculturing. Normal breast cancer cells, MCF-10A, were treated similarly.
b. SRB cytotoxicity assay
Cytotoxicity was determined using SRB method as previously described by Skehan et al. [28]
Test compounds were dissolved in DMSO and serial dilutions were used for cell treatment (0.01,
0.1, 1, 10, 100, 1000 µM). Final working DMSO concentration was kept to 1% to minimize
potential cyctotoxicity therewith. Exponentially growing cells were collected using 0.25% Trypsin-
EDTA and seeded in 96-well plates at 1000-2000 cells/well in supplemented DMEM. After 24 h of
incubation in fresh culture medium and non-adherent cells were removed by suction. Then,
adherent cells were incubated for 72 h in the presence of various concentrations of the test
compounds. Following 72 h treatment, the cells were fixed with 10% trichloroacetic acid for 1 h
at 4 °C. Wells were stained for 10 min at room temperature with 0.4% SRB dissolved in 1% acetic
acid. The plates were air dried for 24 h and the dye was solubilized with Tris-HCl for 5 min on a
shaker at 1600 rpm. The optical density (OD) of each well was measured spectrophotometrically
at 564 nm with an ELISA microplate reader (ChroMate-4300, FL, USA). The following equation
was employed to cancel solvent cytotoxicity: Cytotoxicity (%) = O.D. (DMSO, corrected) - O.D.
(Sample, corrected) /O.D. (DMSO, corrected) * 100%. IC50 values were calculated according to
the equation for Boltzmann sigmoidal concentration–response curve using the nonlinear
regression fitting models (Graph Pad, Prism Version 5).
c. Microscopic imaging
After treatment of the cells with the investigated compounds, they were photographed by
inverted microscopy (×100).
4.3. Caspase 8 and 9 assays
a. Procedure
Apoptosis is controlled by proteolytic enzymes called caspases which stimulate cell death.
Caspase 8 is one of the cysteine proteases implicated in apoptosis and cytokine processing while
caspase 9 is mainly responsible for activation of caspases cascade responsible for apoptosis
execution. Caspases 8 and 9 activity was measured by using the colorimetric assay kit (BIOVISION,
K113-25 and K119-25, respectively) following the kit assay protocol. To induce apoptosis, MCF-7
and HepG2 cell lines were incubated with the different test agents (Doxorubicin, 5-FU and 5a-d)
at their respective IC50 value dose for 48 h was determined. Cells were then treated with cell lysis
buffer, lysate was centrifuged and the supernatant was transferred to fresh tubes. Lysate with
200 µg protein equivalent was diluted with 50 µl cell lysis buffer then 50 µl of reaction buffer
(containing 10 mM DTT) was added to each sample. Finally, 5 µl of the appropriate substrate
solution was added (IETD-pNA for caspase 8 and LEHD-pNA for caspase 9). After incubation at 37
° C for 2 hr. samples were read at 400 or 405 nm.
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b. Statistical analysis
Data were statistically described in terms of mean  standard deviation ( SD). Comparison
of numerical variables between the study groups was done using Mann Whitney U test for
independent samples. P values less than 0.05 were considered statistically significant. All
statistical calculations were done using the computer program SPSS (Statistical Package for the
Social Science; SPSS Inc., Chicago, IL, USA) release 15 for Microsoft Windows.
4.4. Cell cycle analysis
To analyze cell cycle phase distribution, MCF-7 and HepG2 cells were treated with the test
agents as well as by positive controls (doxorubicin and 5-FU) at their respective IC50
concentrations for 24 h at 37 °C in a 5% CO2 incubator. Cells were then stained and worked with
the Cytell™ Cell Cycle Kit (GE Healthcare Japan, Tokyo, Japan) and then analyzed using a CytellTM
cell imaging system (GE Healthcare Japan) according to the manufacturer’s procedure.
4.5. CDK2/cyclin E1 inhibition
To assess the CDK2 activity, GST-labelled CDK2/cyclin E1 (PV6295), LanthaScreen™ Eu-anti-
GST Antibody (A15116) and Kinase Tracer 236 (PV5592) from ThermoFisher Scientific were used.
The assay was done according to the LanthaScreenTM Eu Kinase Binding Assay protocol developed
by the manufacturer and Staurosporine was used as a positive control.
4.6. Radiolabeling and in vivo biodistribution of radioiodinated-5c
a. Radioiodinated-5c preparation
Radiolabeling technique was used as a tool to monitor the in vivo biodistribution of 5c.
Radiolabeling of radioiodinated-5c by 125I was performed using the electrophilic substitution
technique under oxidative conditions by chloramine-T [19-22]. Different factors that affect the
radiolabeling process were studied (5c amount, chloramine-T amount, pH, reaction time and
temperature) to reach the maximum radiochemical yield (RCY). Each factor studying
experiment was performed in triplicate and one-way ANOVA test was used to evaluate data
differences (level of significance set at P<0.05).
b. Analysis of the radiochemical yield:
Efficiency of radiolabeling and stability of radioiodinated-5c in-vitro were evaluated by
ascending paper chromatography (P.C.) and TLC to determine the percent of radioiodinated-5c
and free 125I- [29,30].
Page 23 of 27

4.6.1. Pharmacokinetic study of radioiodinated-5c
a. Induction of cancer in mice
Induction of tumor in the right lateral thigh muscle of the Swiss albino mice was done by
intramuscularly injecting 200 mL of 12.5x106 cells.mL-1 Ehrlich ascites carcinoma. Solid tumors
appeared in mice injected muscles 4-6 days after injection [19,22].
b. Biodistribution studies of the radioiodinated-5c in solid tumor bearing mice model
The animal ethics committee of Egyptian Atomic Energy Authority approved the
biodistribution studies performed. Male Swiss albino mice, with body mass 20-25 g, were
segregated in groups of five and fed up with food and water. Aliquots of 150 μL containing 6.2
MBq of radioiodinated-5c were injected intravenously in male Swiss albino mice with cancer in
right thigh muscle. Mice were anaesthetized by chloroform and dissected at 15min, 1, 2 and 4 h
post-injection. Each mouse was weighed and fresh blood, bone and muscle samples were
separated, weighed and their radioactivities were counted. Blood, bone and muscles were
calculated in percentage of 7, 10 and 40% of the total body weight, respectively [20,21]. Other
body organs and tissues were also separated, weighed and their radioactivities were counted
using NaI (Tl) crystal gamma counter. Percent-injected dose per organ (% ID/organ ± S.D.) at
each time point for a population of five mice were reported. One-way ANOVA test was used to
evaluate data differences (P<0.05).
Declaration of interest
RKA wishes to acknowledge STDF for supporting part of this research project through the grant
fund provided to her “STDF 25839”.
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Graphical abstract
Highlights

New thiazolidinone, thiazinone and dithiazepinone-benzenesulfonamides as cytotoxic
agents.



Effect of 5a-d on MCF-7 and HepG2 cell cycle phase distribution.
Caspase 8 and 9 levels determination in MCF-7 and HepG2 after test agents’ treatment.
In vivo biodistribution study of radioiodinated-5c.
Conflict of interest
RKA wishes to acknowledge STDF for supporting part of this research project through the grant
fund provided to her “STDF 25839”.
Page 27 of 27