Design, synthesis and biological evaluation of some novel sulfonamide derivatives as apoptosis inducers

Article abstract of DOI:10.1016/j.ejmech.2017.04.069

Several novel thiazolidinone and fused thiazolidinone derivatives bearing benzenesulfonamide moiety were synthesized and confirmed via spectral and elemental analyses. The newly synthesized compounds were evaluated for their cytotoxic activity on colorect

Full text of DOI:10.1016/j.ejmech.2017.04.069

Accepted Manuscript
Design, synthesis and biological evaluation of some novel sulfonamide derivatives as
apoptosis inducers
Khaled O. Mohamed, Yassin M. Nissan, Afaf A. El-Malah, Wafaa A. Ahmed, Dina M.
Ibrahim, Tamer M. Sakr, Mohamed A. Motaleb
PII:
S0223-5234(17)30343-4
10.1016/j.ejmech.2017.04.069
EJMECH 9420
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 March 2017
Revised Date: 12 April 2017
Accepted Date: 25 April 2017
Please cite this article as: K.O. Mohamed, Y.M. Nissan, A.A. El-Malah, W.A. Ahmed, D.M. Ibrahim,
T.M. Sakr, M.A. Motaleb, Design, synthesis and biological evaluation of some novel sulfonamide
derivatives as apoptosis inducers, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.04.069.
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ACCEPTED MANUSCRIPT
H
N
N
O
S
H NO S
2
2
3
a
IC = 0.334 µM
5
0
Caspase-3 conc. = 0.43 nmol/mL
Fas-ligand conc. = 775.2 pg/mL
9
9m
Tc-compound 3a complex conc. = 16.02±2.43 %ID/g at 120min

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Some Novel Sulfonamide
Derivatives as Apoptosis Inducers
a
b,c*
a
d
Khaled O. Mohamed , Yassin M. Nissan , Afaf A. El-Malah , Wafaa A. Ahmed , Dina
e
f**
g
M. Ibrahim , Tamer M. Sakr , Mohamed A. Motaleb
a
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University.
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University.
b
Kasr Elini St., Cairo 11562, Egypt.
c
Pharmaceutical Chemistry Department, Faculty of Pharmacy, October University for
Modern Sciences and Arts (MSA), Giza, Egypt.
d
National Cancer Institute, Cancer Biology Department, Cairo University
e
Faculty of Science, Cairo University, Cairo.
f
Radioactive Isotopes and Generator Department, Hot Labs Center, Atomic Energy
Authority, P.O. Box 13759, Cairo, Egypt.
g
Labeled Compounds Department, Hot Labs Center, Atomic Energy Authority, P.O. Box
1
3759, Cairo, Egypt.
*
Corresponding author: Y. M. Nissan, Pharmaceutical Chemistry Department, Faculty
of Pharmacy, Cairo University, Kasr Elini Street., Cairo 11562, Egypt.
Tel.: +201000848844; E-mail address: yassin.nissan@hotmail.com.
*
*Corresponding author: T. M. Sakr, Radioactive Isotopes and Generator Department,
Hot Labs Center, Atomic Energy Authority, P.O. Box 13759, Cairo, Egypt.
Tel.: +201006884013; E-mail address: Tamer_sakr78@yahoo.com.
1

ACCEPTED MANUSCRIPT
Abstract
Several novel thiazolidinone and fused thiazolidinone derivatives bearing
benzenesulfonamide moiety were synthesized and confirmed via spectral and elemental
analyses. The newly synthesized compounds were evaluated for their cytotoxic activity
on colorectal cancer cell line (Caco-2). All the synthesized compounds showed better
activity than the reference standards (Doxorubicin and 5-FU). Investigation of the
apoptotic activity of the most active compounds revealed that compounds 3a, 5a, 5c and
6
c activate both caspase-3 and Fas-ligand in Caco-2 cell line. Compound 3a was the most
active compound with caspase-3 concentration of 0.43 nmol/mL and Fas-ligand
concentration of 775.2 pg/mL in treated Caco-2 cells. Compound 3a was radiolabeled
9
9m
with
Albino mice.
organs except for its accumulation in the colon; target organ; where it showed 8.97±1.35
ID/g at 15min p.i. that elevated till 16.02±2.43 %ID/g at 120min p.i.
Tc and its biodistribution pattern was evaluated in vivo using normal Swiss
9
9m
Tc-compound 3a complex didn’t exhibit any accumulation in any body
%
Keywords: Thiazolidinone, Caspase-3, Fas-ligand, Apoptosis, Radiolabeling,
Technetium-99m
1
. Introduction
Cancer is one of the most challenging diseases to human race being one of the highest
mortality rates all over the world. Colorectal cancer comes in the third place after lung
and breast cancer invading more than a million patients every year with more than 60%
mortality rate. Men suffer more from colorectal cancer and its incidence is increased with
age in developing countries [1]. Age, Lifestyle and genetic disorders are on the top of its
risk factors, besides lack of physical activity, smoking, alcohol, obesity, diabetes,
exposure to radiation and family history [2]. The choice of proper treatment depends on
the patient health condition and the goal of treatment. Similar to all types of cancer;
surgery, radiation and chemotherapy are used to manage the condition with limitations
and side effects [3].
Aryl sulfonamide derivatives have received great attention as anticancer agents through
the past decade [4-11]. Several sulfonamide derivatives have been evaluated as anticancer
agents and proven to exhibit good activity through many mechanisms including their
action as carbonic anhydrase inhibitors [12-14], metalloprotienase inhibitors [15],
NADPH reductase inhibitors [16], histone deacetylase inhibitors [17], PI3K inhibitors
[18] and microtubules assembly disruption [19].
On the other hand, several 4-thiazolidinone was a versatile synthone for many derivatives
with a wide range of biological activities [20-30]. Among these activities 4-
thiazolidinones and its arylidene derivatives comprise a major class in the search for new
2

ACCEPTED MANUSCRIPT
anticancer agents against many types of cancer as breast cancer cell line (MCF-7) [31],
JNK stimulatingphosphatase-1 (JSP-1) [32], tumor necrosis factor (TNFa) [33],
antiapoptotic biocomplex (Bcl-XL-BH3) [34], integrin avb3 receptor [35], CDK1/Cyclin
B inhibition [36] and coloncancer (HT29) [37]. Also fused heterocylic thiazole
derivatives especially fused pyrimidine derivatives exhibited good anticancer activity [38,
3
9]. Structure activity relationship studies on several thiazolo[4,5-d]pyrimidines have
shown good anticancer activity for those derivatives with an aryl substitution on number
[40]. Moreover, thiazolo[4,5-d]pyrimidines were evaluated for their apoptotic activity
7
through caspase-3 [41].
Furthermore, one of the major pathways for apoptosis induction in cells is the Fas/Fas-
ligand (FasL) system. In a similar way to cytolytic activity of tumor necrosis factor, Fas
is a component of the cell surface that has cell-killing activity [42, 43]. Its corresponding
ligand (FasL) is a membrane protein [44]. Activation of Fas by its natural ligand (FasL)
or an antibody transmits a death signal to the cells and cause apoptosis. Despite the
widespread of Fas in both normal and cancer cells, FasL is limited to macrophages,
natural killers and T activated cell [45, 46]. After cellular activation FasL protein exists in
intracellular stores [47]. Engagement of the cell death surface receptor Fas by Fas ligand
(
FasL) results in apoptotic cell death, mediated by caspase activation [48].
Based on the above facts and as a continuation of our search for new anticancer
agents, several benzylidinethiazolidinone derivatives bearing benzenesulfonamide moiety
were synthesized, thiazolopyrimidine thioxopyrimidine derivatives and
&
thiazoloisoxazole derivatives were cyclized from their arylidine intermediates (Figure 1).
All the newly synthesized compounds were evaluated in vitro for their cytotoxic activity
against human colon cancer cell line (Caco-2 cell line). The most active compounds were
further evaluated for their apoptotic activity through increase of caspase-3 and Fas &
FasL (Fas ligand) in Caco-2 cell line cells treated with the newly synthesized compounds.
9
9m
Moreover, biodistribution for the most active compound was evaluated through
Tc-
compound complex radiolabeling to determine its in vivo pharmacokinetic behavior and
its body organs uptakes besides its elimination pathway.
H
N
N
O
S
H NO S
2
2
Ar
3

ACCEPTED MANUSCRIPT
H
H
N
N
N
N
N
N
S
S
X
H NO S
H NO S
2 2
2
2
O
NH
Ar
Ar
Figure 1: General Structures for the newly synthesized compounds
2
. Results and Discussion
2
.1. Chemistry
Benzenesulfonamide thiazolidinone derivative 2 was synthesized from 2-chloro-N-(4-
sulfamoylphenyl)acetamide 1 through cyclization with ammonium thiocyanate [50]. The
1
structure of the formed compound was verified by its elemental and spectral analyses. H-
NMR spectrum in (DMSO-d
6
) of 2 exhibited a singlet signal at δ: 3.98 ppm
corresponding to CH of thiazolidinone and a singlet signal at δ: 11.86 ppm attributed to
2
2
NH as it was exchanged upon treatment with D O. Arylidene derivatives 3 a-c were
prepared by reaction of the active methylene group of thiazolidinone with different
aromatic aldehydes in the presence of catalytic amounts of anhydrous sodium acetate.
The structures of these derivatives were confirmed via elemental and spectral analyses.
1
H-NMR spectra in (DMSO-d ) of 3 a-c exhibited singlet signals for C=CH of arylidines
6
in the range of δ: 7.87-8.01 ppm. Reaction of arylidene intermdiates 3 a-c with urea or
thiourea and KOH afforded the cyclized thiazolo[4,5-d]pyrimidines 4 a-c & 5 a-c,
1
respectively. The structures of the synthesized compounds were verified by their H-
NMR spectra which exhibited singlet signals for NH of pyrimidine in the range of δ:
3
.33-3.40 ppm while singlet signals corresponding to NH attached to thiazole moiety
were in the range of δ: 11.96-12.24 ppm. These signals were exchanged upon treatment
with D O. Finally, thiazoloisoxazole derivatives 6 a-c were prepared by reacting
2
aryiledene derivatives 3 a-c with hydroxylamine and KOH. The structures of the
1
synthesized compounds were verified by their H-NMR that showed increased aromatic
protons (Scheme 1).
2
.2. Cytotoxic assay
Cytotoxicity of the newly synthesized compounds was studied against Caco-2 tumor cell
line using the colorimetric methyl tetrazolium test (MTT) as described and modified by
Mosmann [49]. Percentage of relative viability and the half maximal inhibitory
concentration IC50 was calculated by the prism program Version 6 (Table 1).
4

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From the results, it became clear that the newly synthesized compounds have significant
effect on Caco-2 cell line for 24 hrs. Viability test by MTT Assay using treatment of
Caco-2 cell line showed that all the synthesized compounds were much more active than
the reference standards (Doxorubicin, IC = 63.30 µM and 5-Fluorouracil, IC = 31.00
5
0
50
µM) The activity of the newly synthesized compounds can be arranged in a descending
order as follows: compounds 5a, 5c and 6c were most effective on cell line viability and
caused inhibition of cell growth, with IC value of 0.333 µM followed by compounds
5
0
3
a and 4a with IC value of 0.334 µM. The activity almost drops to its half in case of
50
compound 6b IC value of 0.559 µM followed by compounds 3c, 4b and 5b with IC
50
5
0
value of 0.667 µM with almost the same activity of compound 6a with IC50 value of
.669 µM. On the other hand, compound 3b showed higher IC50 value of 1.33 µM while
0
compound 4c was the least active compound with IC50 value of 12.70 µM.
Structure Activity Relationship
On a further look on Table 1, we can categorize the newly synthesized compounds in the
light of their biological results to study the relationship between chemical structure and
biological effect on Caco-2 cell line in some points.
Firstly, it seems that thiazolothioxopyrimidine derivatives 5a-c is the best series in
activity compared to others (arylidene derivatives 3a-c, thiazolopyrimidinone derrivatives
4
a-c and thiazoloisoxazole derivatives 6a-c). This can be concluded from the IC values
50
of the compounds belong to this series. Two of the most active four compounds, 5a and
c, are members of this series with IC value of 0.333 µM. The activity tends to be lower
5
5
0
in case of thiazoloisoxazole series 6a-c followed by arylidene derivatives 3a-c and finally
the lowest activity was attributed to the thiazolopyrimidinone series 4a-c.
Secondly, the effect of para substitution on the phenyl ring was variable among different
series. In case of arylidene derivatives 3a-c, the unsubstituted derivative 3a was the most
active compound with IC50 value of 0.334 µM. This activity drops to its half in case of p-
Cl derivative 3c and getting much worse in case of p-OCH derivative 3b.
3
In case of thiazolopyrimidinone derivatives 4a-c, again the unsubstituted derivative 4a
was the most active compound with IC50 value of 0.333 µM. However in this series the
chloro derivative 4c was least active compound with relatively high IC value of 12.7
5
0
µM. This was not the case in their thioxo nalouges 5a-c, as both the unsubstituted and the
chloro derivatives 5a and 5c showed the same activity with almost double the activity of
methoxy derivative 5b. In the last series, thiazoloisoxazole derivatives 6a-c, the chloro
derivative was the most active compound with IC value of 0.333 µM followed by the
50
methoxy derivative 6b and the unsubstituted derivative 6a.
Finally, comparing the isosteric series, thiazolopyrimidinone derivatives 4a-c &
thiazolothioxopyrimidine derivatives 5a-c, lead to the assumption of that thioxo
5

ACCEPTED MANUSCRIPT
derivatives are better derivatives for cytotoxic activity despite of the same IC50 values for
the unsubstituted and methoxy analogues, the chloro derivative of thioxo series was
almost 40 times more active than its oxo analogue.
2
.3. Fas ligand Assay
In order to investigate the apoptotic behavior for the most active compounds in cytotoxic
assay 3a, 5a, 5c and 6c, Fas ligand concentration in CaCo 2 cell line was measured by
using the colorimetric human factor-Related Apoptosis Ligand (FASL) and results were
displayed in Table 2.
All the tested compounds showed significant increase in the concentration of Fas-ligand
relative to negative control and better than the reference standards (doxorubicin and 5-
FU) supporting the assumption of the apoptotic activity of the synthesized compounds.
Compound 3a was also the most active compound in this assay with Fas-ligand
concentration of 775.2 pg/mL.
2
.4. Caspase-3 Assay
In exploring the apoptotic activity of the newly synthesized compounds, the most active
compounds in cytotoxic assay 3a, 5a, 5c and 6c were subjected to caspase-3 assay. The
caspase-3 activities were measured in Caco-2 cell line treated with drugs, doxorubicin
and 5-FU as positive control. All compounds showed significant increase in the caspase-3
activity of Caco-2 cell line compared to negative control and reference drugs as shown in
Table 3.
The increment of caspase-3 concentration was significant for all compounds relative to
control and positive control drug (doxorubicin) but less than that produced by 5-FU.
Compound 3a was also the most active compound in this assay with caspasce-3
concentration of 0.43 nmol/mL.
2
2
.5. Radiolabeling and Biodidtribution
99m
.5.1. Preparation of Tc-compound 3a complex
As presented in Figure 2 (A-D), studying four different factors optimized the preparation
9
9m
of Tc-compound 3a complex; reducing agent (NaBH ) amount, compound 3a amount,
4
9
9m
pH and the reaction time. The highest radiochemical yield of
Tc-compound 3a
complex was 85.2±0.4% that was obtained using reaction condition; 100 µl
9
9m
−
4 4
TcO (~200 MBq), 15 mg of NaBH , 0.5 mg of compound 3a, pH 9 and the reaction
mixtures were kept at room temperature for 30 min.
6

ACCEPTED MANUSCRIPT
9
9m
2
.5.2. Biological pattern of Tc-compound 3a complex
9
9m
The biodistribution compartments as (average percent-injected dose per gram) of
Tc-
compound 3a complex are illustrated in Figure 3. As reflected in this figure, high uptake
was in the kidney at 15min p.i., which dropped regularly indicating that the main
9
9m
99m
clearance route of
Tc-compound 3a complex was mainly via renal pathway.
Tc-
compound 3a complex didn’t exhibit any accumulation pattern in any body organ except
for its accumulation in the colon, which was at 15min p.i. It showed 8.97±1.35 %ID/g
and elevated till 16.02±2.43 %ID/g at 120min p.i.
Conclusion
In light of the biological results, we can conclude that compounds 3a, 5a, 5c and 6c are
good cytotoxic agents compared to the reference standards (doxorubicin and 5-FU). The
cytotoxic activity of these compounds is attributed to their apoptotic activity through
activation of caspase-3 and Fas-ligand in Caco-2 cell line. Further biological
investigation should be conducted for other mechanisms of apoptosis. Moreover,
compound 3a is accumulated selectively in colon with high renal elimination rate that
may contribute to future investigation for its selectivity for colon cancer treatment.
3
. Experimental
3
.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 centre, 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
(
F ).
254
3
.1.1. 4-((4-Oxo-4,5-dihydrothiazol-2-yl)amino)benzenesulfonamide 2
A solution of 2-chloro-N-(4-sulfamoylphenyl) acetamide 1 (2.625g, 10 mmol) and
ammonium thiocyanate (0.76 gm, 10 mmol) in absolute ethanol (20 mL) was refluxed for
3
hours and the obtained precipitate during reflux was filtered, washed with water, dried
-1
and recrystallized from ethanol, yield 70 %, m.p.250- 252 ºC. IR (KBr) cm : 3323, 3246,
205 (NH), 3051 (arom. CH), 2995, 2929 (aliph. CH), 1683 (C=O), 1635, 1604 (C=C,
3
1
C=N). H NMR (DMSO-d , D O) δ: 3.98 (s, 2H, CH -S), 7.11-7.48 (m, 6H, arom.CH,
6
2
2
7

ACCEPTED MANUSCRIPT
1
3
2 2 2 2
NH SO , D O-exchangeable), 11.86 (s, 1H, NH, D O-exchangeable). C NMR (DMSO-
d6) δ: 34.42 (C5, thiazole), 119.95 (C3, benzenesulphonamide), 121.32 (C5,
benzenesulphonamide), 127.00 (C2, 6, benzenesulphonamide), 139.59 (C1, 4-
benzenesulphonamide), 150.90 (C2, thiazole), 174.14 (C4, thiazole, C=O). Anal. Calc.
for C H N O S (271). Calculated: C, 39.85; H, 3.32; N, 15.49. Found: C, 40.04; H, 3.37;
9
9
3
3 2
N, 15.72%.
3
.1.2. 4-((5-Arylidene-4-oxo-4,5-dihydrothiazol-2-yl)amino)benzenesulfonamide 3a-c
A mixture of 4-(2-imino-4-oxothiazolidin-3-yl)benzenesulfonamide 2 (2.71g, 10 mmol),
appropriate aromatic aldehyde (10 mmol) and anhydrous sodium acetate (0.82 gm, 10
mmol) in glacial acetic acid (15 mL) was refluxed for 12 hours then allowed to cool. The
solid product was collected, dried and recrystallized from acetic acid.
3
.1.2.1. 4-((5-Benzylidene-4-oxo-4,5-dihydrothiazol-2-yl)amino)benzenesulfonamide 3a
-1
Yield 57 %, m.p.265- 267 ºC. IR (KBr) cm : 3358, 3269, 3253 (NH), 3049 (arom. CH),
1
1
676 (C=O), 1620, 1604 (C=C, C=N). H NMR (DMSO-d , D O) δ: 7.29 (s, 2H,
6 2
2 2 2
NH SO , D O-exchangeable), 7.11-7.83 (m, 9H, arom.CH), 7.87 (s, 1H, C=CH), 11.96
1
3
(
s, 1H, NH, D O-exchangeable).
C
NMR (DMSO-d6) δ: 120.20 (C3,
2
benzenesulphonamide), 120.94 (C5, benzenesulphonamide), 121.46 (C4, benzene),
1
6
1
26.94 (C2, 6, benzene), 127.21 (C5, thiazole), 129.21 (C3, 5, benzene), 129.63.14 (C2,
, benzenesulphonamide), 129.91 (C1, benzene), 139.61 (C1, 4, benzenesulphonamide),
40.05 (methylenecarbon), 152.00 (C2, thiazole), 174(C4, thiazole, C=O). Anal. Calc. for
16 13 3 3 2
C H N O S (359). Calculated: C, 53.48; H, 3.62; N, 11.69. Found: C, 53.66; H, 3.66;
N, 11.85%.
3
.1.2.2.4-((5-(4-Methoxybenzylidene-4-oxo-4,5-dihydrothiazol-2-
yl)amino)benzenesulfonamide 3b
-1
Yield 65 %, m.p.278- 280 ºC. IR (KBr) cm : 3325, 3250, 3100 (NH), 3050 (arom. CH),
1
2
3
8
956 (CH aliph), 1670 (C=O), 1637, 1595 (C=C, C=N). H NMR (DMSO-d
6 2
, D O) δ:
.84 (s, 3H, OCH ),7.34 (s, 2H, NH SO , D O-exchangeable), 7.06-7.97 (m,
H,arom.CH), 8.00 (s, 1H, C=CH), 12.20 (s, 1H, NH, D O-exchangeable). C NMR
2
3
2
2
2
1
3
(
DMSO-d ) δ: 55.82 (OCH3), 115.15 (C3, 5, benzenesulphonamide), 121.96
6
(C1,benzene), 126.86 (C3,5,benzene), 128.99 (C2,6,benzene), 131.96 (C2, 6,
benzenesulphonamide), 132.63 (C1, benzenesulphonamide), 133.74, (C5, thiazole),
39.95 (C4, benzenesulphonamide), 144.77 (methylenecarbon), 160.96 (C4,
1
benzene),161.58 (C2, thiazole), 165.30 (C4, thiazole, C=O). Anal. Calc. for
8

ACCEPTED MANUSCRIPT
C
17
H
15
N
3
O
4
S
2
(359). Calculated: C, 52.44; H, 3.85; N, 10.79. Found: C, 52.67; H, 3.41;
N, 11.04%.
3
.1.2.3.4-((5-(4-Chlorobenzylidene-4-oxo-4,5-dihydrothiazol-2-
yl)amino)benzenesulfonamide 3c
-1
Yield 68 %, m.p. >300 ºC. IR (KBr) cm : 3325, 3250, 3201 (NH), 3045 (arom. CH),
1
1
670 (C=O), 1635, 1589 (C=C, C=N). H NMR (DMSO-d
6 2
, D O) δ: 7.30 (s, 2H,
NH SO , D O-exchangeable), 7.18-7.99 (m, 8H, arom.CH), 8.01 (s, 1H, C=CH), 12.47
2
2
2
1
3
(
s, 1H, NH,
D
2
O-exchangeable).
C
NMR (DMSO-d
6
)
δ: 120.59 (C3,
benzenesulphonamide), 126.57 (C5, benzenesulphonamide), 127.25 (C1, benzene),
1
1
1
28.62 (C3, 5, benzene), 129.27 (C2, 6, benzene), 131.27 (C2, 6, benzenesulphonamide),
31.71 (C1, benzenesulphonamide, C5, thiazole), 131.80 (C4, benzenesulphonamide),
34.49 (methylenecarbon), 140.18 (C4, benzene), 164.75 (C2, thiazole), 166.46 (C4,
thiazole, C=O). Anal. Calc. for C H ClN O S (393.5). Calculated: C, 48.79; H, 3.60;
16
12
3
3 2
N, 10.67. Found: C, 49.02; H, 3.69; N, 10.79%.
3
.1.3.4-((5-Oxo-7-aryl-5,6-dihydrothiazolo[4,5-d]pyrimidin-2-
yl)amino)benzenesulfonamide 4a-c
A mixture of 0.01 mol of the required chalcone 3a-c (0.01 mol), urea (0.6g, 0.01mol) and
KOH (0.56g, 0.01 mol) in 20 mL ethanol was heated under reflux for 6 hours, then
cooled and poured onto crushed ice. The obtained solid product was filtered and
recrystallized from ethanol.
3
.1.3.1.4-((5-Oxo-7-phenyl-5,6-dihydrothiazolo[4,5-d]pyrimidin-2-
yl)amino)benzenesulfonamide 4a
-1
Yield 65 %, m.p. 273-275 ºC. IR (KBr) cm : 3360, 3317, 3201 (NH), 3049 (arom. CH),
1
1
6 2
676 (C=O), 1637, 1602 (C=C, C=N). H NMR (DMSO-d , D O) δ: 3.35 (s, 1H, NH,
D O-exchangeable), 7.29 (s, 2H, NH SO , D O-exchangeable), 7.12-7.83 (m, 9H,
2
2
2
2
1
3
arom.CH), 11.96 (s, 1H, NH, D
2
O-exchangeable). C NMR (DMSO-d
6
) δ: 120.15 (C7a,
127.00 (C5,
thiazolopyrimidine),
121.47 (C3, benzenesulphonamide),
benzenesulphonamide), 127.20 (C3, 5, benzene), 129.22 (C2, 6, benzene), 129.64 (C2, 6,
benzenesulphonamide), 129.93 (C4, benzene), 139.61 (C1, benzenesulphonamide, C1,
benzene), 140.09 (C4, benzenesulphonamide), 147.47 (C7, thiazolopyrimidine), 150.79
(C2, thiazolopyrimidine), 157.65 (C3a, thiazolopyrimidine), 174.00 (C5,
thiazolopyrimidine, C=O). Anal. Calc. for C H N O S (399). Calculated: C, 51.12; H,
1
7
13
5
3 2
3
.25; N, 17.54. Found: C, 51.38; H, 3.22; N, 17.71%.
9

ACCEPTED MANUSCRIPT
3
.1.3.2.4-((5-Oxo-7-(4-methoxyphenyl)-5,6-dihydrothiazolo[4,5-d]pyrimidin-2-
yl)amino)benzenesulfonamide 4b
-1
Yield 73 %, m.p. > 300 ºC. IR (KBr) cm : 3336, 3250, 31200 (NH), 3070 (arom. CH),
1
1
668 (C=O), 1637, 1593 (C=C, C=N). H NMR (DMSO-d
6 2
, D O) δ: 3.33 (s, 1H, NH,
D O- exchangeable), 3.78(s, 3H, OCH ), 7.34 (s, 2H, NH SO , D O-exchangeable), 7.06-
2
3
2
2
2
1
3
7
5
1
2 6
.87 (m, 8H, arom.CH), 12.24 (s, 1H, NH, D O-exchangeable). C NMR (DMSO-d ) δ:
5.37 (OCH ), 114.81 (C3, 5, benzenesulphonamide), 119.80 (C7a, thiazolopyrimidine),
20.55 (C2, 6, benzenesulphonamide), 121.49 (C3, 5, benzene), 125.68 (C1,
3
benzenesulphonamide), 127.20 (C2, 6, benzene), 129.94 (C1, benzene), 131.63 (C4,
benzenesulphonamide), 140.01 (C7, thiazolopyrimidine), 144.60 (C4, benzene), 151.45
(C2, thiazolopyrimidine), 160.62 (C3a, thiazolopyrimidine), 168.50 (C5,
thiazolopyrimidine, C=O). Anal. Calc. for C18 (429). Calculated: C, 50.34; H,
15 5 4 2
H N O S
3
.49; N, 16.31. Found: C, 50.49; H,3.51; N, 16.54%.
3
.1.3.3.4-((5-Oxo-7-(4-chlorophenyl)-5,6-dihydrothiazolo[4,5-d]pyrimidin-2-
yl)amino)benzenesulfonamide 4c
-1
Yield 67 %, m.p. >300 ºC. IR (KBr) cm : 3327, 3250, 3200 (NH), 3050 (arom. CH),
1
1
674 (C=O), 1630, 1589 (C=C, C=N). H NMR (DMSO-d
6 2
, D O) δ: 3.36 (s, 1H, NH,
D O- exchangeable), 7.35 (s, 2H, NH SO , D O-exchangeable), 7.19-7.86 (m, 8H,
2
2
2
2
1
3
arom.CH), 12.20 (s, 1H, NH, D O-exchangeable). C NMR (DMSO-d ) δ: 120.45 (C7a,
2
6
thiazolopyrimidine), 121.45 (C3, 5, benzenesulphonamide), 123.44 (C1,
benzenesulphonamide), 127.22 (C2, 6, benzene), 128.52 (C1, benzene), 129.23 (C3, 5,
benzene), 131.23 (C2, 6, benzenesulphonamide), 132.04 (C4, benzene), 132.75 (C7,
thiazolopyrimidine),
134.45
(C4,
benzenesulphonamide),
140.14
(C2,
thiazolopyrimidine), 150.54 (C3a, thiazolopyrimidine), 168.00 (C5, thiazolopyrimidine,
C=O). Anal. Calc. for C H ClN O S (433.5). Calculated: C, 47.05; H, 2.76; N, 16.14.
17
12
5
3 2
Found: C, 47.36; H, 2.79; N, 16.38%.
3
.1.4.4-((7-Aryl-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-2-
yl)amino)benzenesulfonamide 5a-c
Chalcone derivatives 3a-c (0.01 mol) thiourea (0.02 mol, 1.52 g) and KOH (0.02 mol,
1
.12 g) were taken in a 100 mL round bottom flask. To the above reaction mixture
ethanol (30 mL) were added. Reaction mixture was refluxed for 6 hrs using water
condenser. It was then cooled and poured in cold water. Acidified with dil HCl, filtered
washed with water and dried. The product was recrystallization from ethanol to get the
product 5a-c.
1
0

ACCEPTED MANUSCRIPT
3
.1.4.1.4-((7-phenyl-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-2-
yl)amino)benzenesulfonamide 5a
-1
Yield 70 %, m.p. 268-270 ºC. IR (KBr) cm : 3360, 3317, 3271 (NH), 3049 (arom. CH),
1
1
637, 1602 (C=C, C=N). H NMR (DMSO-d
6 2 2
, D O) δ: 3.33 (s, 1H, NH, D O-
exchangeable),7.34 (s, 2H, NH SO , D O-exchangeable), 7.11-7.86 (m, 9H, arom.CH),
2
2
2
1
3
1
2.11 (s, 1H, NH, D
2 6
O-exchangeable). C NMR (DMSO-d ) δ: 117.16 (C7a,
thiazolopyrimidine), 120.40 (C4, benzene), 120.76 (C3, 5, benzenesulphonamide),
1
1
26.54 (C3, 5, benzene), 127.51 (C2, 6, benzene), 130.29 (C1, benzenesulphonamide),
32.51 (C1, benzene), 138.76 (C2, 6, benzenesulphonamide), 140.09 (C4,
benzenesulphonamide),
thiazolopyrimidine), 157.82 (C3a, thiazolopyrimidine), 181.01 (C5, thiazolopyrimidine,
C=S). Anal. Calc. for C17 (415). Calculated: C, 49.15; H, 3.13; N, 16.86.
Found: C, 49.37; H, 3.19; N, 17.08%.
149.36
(C7,
thiazolopyrimidine),
152.50
(C2,
13 5 2 3
H N O S
3
.1.4.2.4-((7-(4-Methoxyphenyl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-2-
yl)amino)benzenesulfonamide 5b
-1
Yield 65 %, m.p. >300 ºC. IR (KBr) cm : 3340, 3251, 3199 (NH), 3047 (arom. CH),
1
1
639, 1593 (C=C, C=N). H NMR (DMSO-d
6 2 2
, D O) δ: 3.34 (s, 1H, NH, D O-
exchangeable), 3.77 (s, 3H, OCH ), 7.34 (s, 2H, NH SO , D O-exchangeable), 7.105-
3
2
2
2
1
3
7
5
1
.87 (m, 8H, arom.CH), 12.20 (s, 1H, NH, D O-exchangeable). C NMR (DMSO-d ) δ:
2 6
5.35 (OCH ), 114.80 (C3, 5, benzenesulphonamide), 119.44 (C7a, thiazolopyrimidine),
20.11 (C3, benzene), 121.48 (C2, 6, benzene), 125.67 (C5, benzene), 127.67 (C2, 6,
3
benzenesulphonamide), 129.96 (C1, benzene), 131.62 (C1, 4, benzenesulphonamide),
1
1
39.62 (C4, benzene), 140.00 (C7, thiazolopyrimidine), 152.00 (C2, thiazolopyrimidine),
60.61 (C3a, thiazolopyrimidine), 179.45 (C5, thiazolopyrimidine, C=S). Anal. Calc. for
C H N O S (445). Calculated: C, 48.53; H, 3.37; N, 15.73. Found: C, 48.69; H, 3.43;
18
15
5
3 3
N, 15.49%.
3
.1.4.3.4-((7-(4-Chlorophenyl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-2-
yl)amino)benzenesulfonamide 5c
-1
Yield 72 %, m.p. >300 ºC. IR (KBr) cm : 3325, 3250, 3203 (NH), 3047 (arom. CH),
1
1
637, 1597 (C=C, C=N). H NMR (DMSO-d , D O) δ: 3.40 (s, 1H, NH, D O-
6 2 2
exchangeable), 7.35 (s, 2H, NH SO , D O-exchangeable), 7.18-7.86 (m, 8H, arom.CH),
2
2
2
1
3
1
2.20 (s, 1H, NH, D O-exchangeable). C NMR (DMSO-d ) δ: 120.53 (C7a,
2
6
thiazolopyrimidine),
121.48
(C3,
benzenesulphonamide),
122.00
(C5,
benzenesulphonamide), 127.24 (C2, 6, benzene), 128.52 (C1, benzenesulphonamide),
1
1
29.27 (C3, 5, benzene), 131.27 (C2, 6, benzenesulphonamide), 132.01 (C1, benzene),
34.46 (C4, benzene, C4, benzenesulphonamide), 140.15 (C7, thiazolopyrimidine),
1
1

ACCEPTED MANUSCRIPT
1
51.35 (C2, thiazolopyrimidine), 156.58 (C3a, thiazolopyrimidine), 179.15 (C5,
thiazolopyrimidine, C=S). Anal. Calc. for C H N O S (449.5). Calculated: C, 45.38;
1
7
12
5
2 3
H, 2.66; N, 15.57. Found: C, 45.52; H, 2.64; N, 15.81%.
3
.1.5. 4-((3-Arylthiazolo[4,5-c]isoxazol-5-yl)amino)benzenesulfonamide 6a-c
To a mixture of chalcone derivatives 3a-c (0.01 mol) and hydroxylamine hydrochloride
0.7 g , 0.01 mol) in ethanol (10 mL) a solution of KOH (0.56 g , 0.01 mol) in ethanol (50
(
mL) was added. The reaction mixture was refluxed for 6 h and then kept overnight at
room temperature. After cooling in ice, the reaction mixture was acidified with aqueous
HCl (10%). The resulting precipitate was washed with distilled water and dried. The
resulting crude was crystallized from ethanol to obtained products 6a-c.
3
.1.5.1. 4-((3-phenylthiazolo[4,5-c]isoxazol-5-yl)amino)benzenesulfonamide 6a
-1
Yield 73 %, m.p. >300 ºC. IR (KBr) cm : 3315, 3246, 3200 (NH), 3050 (arom. CH),
1
1
631, 1595 (C=C, C=N). H NMR (DMSO-d
6 2 2 2 2
, D O) δ: 7.35 (s, 2H, NH SO , D O-
1
3
exchangeable), 7.19-7.87 (m, 9H, arom.CH), 12.33 (s, 1H, NH, D O-exchangeable). C
2
NMR (DMSO-d
5
6
6
) δ: 120.79 (C3a, thiazoloisoxazole), 121.81 (C4, benzene), 121.99 (C3,
, benzenesulphonamide), 127.56 (C2, 6, benzene), 129.53 (C3, 5, benzene), 129.97 (C2,
, benzenesulphonamide), 130.26 (C1, benzene, C1, benzenesulphonamide), 131.24
(C6a, thiazoloisoxazole), 133.53 (C4, benzenesulphonamide), 140.44 (C3,
thiazoloisoxazole), 169.00 (C5, thiazoloisoxazole). Anal. Calc. for C H N O S (372).
1
6
12
4
3 2
Calculated: C, 51.61; H, 3.22; N, 15.05. Found: C, 51.85; H, 3.26; N, 15.23%.
3
.1.5.2. 4-((3-(4-Methoxyphenyl)thiazolo[4,5-c]isoxazol-5-yl)amino)benzenesulfonamide
6
b
-1
Yield 63 %, m.p. >300 ºC. IR (KBr) cm : 3338, 3250, 3225 (NH), 3050 (arom. CH),
1
1
6 2 3
637, 1593 (C=C, C=N). H NMR (DMSO-d , D O) δ: 3.77 (s, 3H, OCH ) 7.46 (s, 2H,
NH SO , D O-exchangeable), 7.05-7.87 (m, 8H, arom.CH), 12.22 (s, 1H, NH, D O-
2
2
2
2
1
3
exchangeable).
C
NMR (DMSO-d
6
)
δ: 55.35 (OCH
3
), 114.79 (C3, 5,
benzenesulphonamide), 119.46 (C3a, thiazoloisoxazole), 120.35 (C1, benzene), 121.48
(
(
C3, 5, benzene), 125.65 (C1, benzenesulphonamide), 127.19 (C2, 6, benzene), 129.96
C6a, thiazoloisoxazole), 131.61 (C2, 6, benzenesulphonamide), 139.99 (C4,
benzenesulphonamide), 150.81 (C4, benzene), 151.93 (C3, thiazoloisoxazole), 167.59
(
C5, thiazoloisoxazole). Anal. Calc. for C H N O S (402). Calculated: C, 50.74; H,
17 14 4 4 2
3
.48; N, 13.93. Found: C, 51.01; H, 3.54; N, 14.19%.
1
2

ACCEPTED MANUSCRIPT
3
.1.5.3. 4-((3-(4-Chlorophenyl)thiazolo[4,5-c]isoxazol-5-yl)amino)benzenesulfonamide
6
c
-1
Yield 70 %, m.p. >300 ºC. IR (KBr) cm : 3325, 3250, 3200 (NH), 3047 (arom. CH),
1
1
637, 1589 (C=C, C=N). H NMR (DMSO-d
6 2 2 2 2
, D O) δ: 7.35 (s, 2H, NH SO , D O-
1
3
exchangeable), 7.20-7.86 (m, 8H, arom.CH), 12.35 (s, 1H, NH, D O-exchangeable). C
2
NMR
benzenesulphonamide), 121.45 (C5, benzenesulphonamide), 123.45 (C2, benzene),
27.25 (C6, benzene), 128.53 (C3, 5, benzene), 129.27 (C1, benzene, C1,
benzenesulphonamide), 131.27 (C2, 6, benzenesulphonamide), 132.06 (C4, benzene),
34.48 (C6a, thiazoloisoxazole), 140.16 (C4, benzenesulphonamide), 150.55 (C3,
thiazoloisoxazole), 167.38 (C5, thiazoloisoxazole). Anal. Calc. for C H Cl N O S
3 2
(DMSO-d
6
)
δ:
120.45
(C3a,
thiazoloisoxazole),
120.57
(C3,
1
1
1
6
11
4
(406.5). Calculated: C, 47.29; H, 2.70; N, 13.79. Found: C, 47.38; H, 2.68; N, 13.98%.
3
3
.2. In vitro cytotoxicity test by MTT assay
.2.1. Cell lines
Cancer colon cell line (Caco-2) was obtained frozen in liquid nitrogen (-180 ºC) from the
American Type Culture Collection. The cell lines were maintained in the National
Cancer Institute, Cairo, Egypt, by serial sub-culturing. Cells were cultured in RPMI-1640
medium (RPMI-1640, Sigma–Aldrich, USA). The medium was supplemented with
antibiotic-free 10% fetal bovine serum (FBS, Sigma, USA), 100 U/mL penicillin and 2-
mg/mL streptomycin. The cells were sub cultivated after trypsination (Trypsin-EDTA,
Cambrex, BioScience Verviers, Belgium) once or twice per week and resuspended in
complete medium in a 1:5 split ratio. Cell lines were maintained as monolayer in T75 cell
culture flasks with filter screw caps (TPP, Trasadingen, Switzerland) at 37 °C in a
humidified 5% CO
2
incubator.
3
.2.2. Cell viability analysis by Trypan blue:
Cells on each panel were cultured in 24-well plates and incubated for 24 hr. Cells treated
with gradual concentrations ranges (100 µM to 1µM) of each drug then cells incubated
for 24hr and the viability was examined using trypan blue dye by light microscopy.
3
.2.3. Cytotoxicity analysis by MTT assay
Cytotoxicity of compounds were studied against Caco-2 tumor cell lines and using the
colorimetric methyl tetrazolium test (MTT) as described and modified by Mosmann 1983
[49]. Percentage of relative viability and the half maximal inhibitory concentration (IC50
)
was calculated by the prism program (Graph Pad prim 7)..
3
.3. Measurement of Human Factor –Related Apoptosis Ligand (FASL) activity
1
3

ACCEPTED MANUSCRIPT
The Fas/Fas-ligand (Fas-L) system has been recognized as a major pathway for the
induction of apoptosis in cells and tissues. Fas- ligand concentration in Caco-2 cell line
was measured by using the colorimetric Human Factor-Related Apoptosis Ligand
(
FASL) ELISA kit (WKEA MED SUPPLIES company , China) (Code No.WH-174)
after incubation Caco-2 cell line with drugs 3a, 5a, 5c and 6c and positive slandered
drugs (Doxorubicin & 5-Fluorouracil) at different concentrations for 24 hrs.
3
.4. Measurements of Caspase-3 enzymatic activity
Apoptosis is mediated by proteolytic enzymes, which called Caspases, which trigger cell
death; Caspase-3 is an “effector” caspase associated with the initiation of the “death
cascade” and is therefore an important marker of the cell’s entry point into the apoptotic
signaling pathway. The caspase-3 activity was measured by using colorimetric Bender
Med System Caspase 3 assay kit; Cat No: BMS2012INST after incubation Caco-2 cell
line with drugs 3a, 5a, 5c and 6c and positive slandered drugs (Doxorubicin & 5-
Fluorouracil at different concentrations for 24 hrs.
3
3
.5. Radiolabeling and Biodistribution
.5.1. Chemicals and Equipment
Reactive grade chemicals and reagents needed were bought from Sigma-Aldrich
Chemical Company and Merck Company. We chose purged deoxygenated bidistilled
water to use in radiolabeling procedure. Technetium-99m was eluted in the form of
9
9m
-
99
99m
TcO from Mo/ Tc generator, Gentech, Turkey. A good-type NaI scintillation γ-
4
Counter model scalar ratemeter SR7 (Nuclear Enterprises Ltd., USA) and Shimadzu
reversed phase-HPLC that is composed of pumps LC-9A, Rheodyne injector, UV
spectrophotometer detector (SPD-6A) operated at a wavelength of 254 nm and a
reversed-phase column (RP-18, 250 x4.6 mm, 5µm, Lichrosorb) were used.
3
.5.2. Animal model
In accordance with the Egyptian Atomic Energy Authority (EAEA), Animal studies were
executed and it was also asserted by Animal ethics committee. Animal models used in the
in vivo experiment were Normal Swiss albino mice of body mass range 25-50g and were
purchased from Helwan University, Egypt. Mice were maintained in a cabin of size that
suits groups of five in the room temperature with a 12h light/dark cycle and accessibility
to food and water.
99m
3
.5.3. Preparation of Tc-compound 3a complex
One mL DMSO was used as a solvent for Compound 3a (0.5mg) in 10 mL penicillin
vials. Exactly 15 mg of NaBH was then added to each vial. 0.1 N Sodium Hydroxide
4
9
9m
−
was used to maintain in the pH range of 7-11. Freshly prepared 100 µL
4
TcO (~200
1
4

ACCEPTED MANUSCRIPT
MBq) was then added to each of these vials. Duration of these reactions was about 30
minutes at room temperature. Different NaBH amounts (5-25 mg), compound 3a
4
amounts (0.25-4 mg), within different time periods (5-240 min) were repeatedly used in
the aforementioned procedure to reevaluate the radiochemical yields.
99m
3
.5.4. Radiochemical yield assay of Tc-compound 3a complex
Strips of ascending Whatman paper chromatography (PC) and HPLC were used to assess
9
9m
radiochemical yield and in vitro stability of
Tc-compound 3a complex. For PC, two
strips were used for each procedure, on each strip, 2 drops of the reaction product were
placed were on origin line 2 cm far from the base. To determine percentage of free
9
9m
-
TcO radio-contaminant, the developing solvent for one PC strip was acetone, where
4
9m
9
-
99m
free
TcO R was 1 whereas
Tc- compound 3a complex and reduced hydrolyzed
4
f
technetium colloid species R was zero. Another strip was developed in C H OH: H O:
f
2
9
5
9m
2
NH
4
OH mixture (2:5:1,v/v/v) to define the ratio of the hydrolyzed
contaminant, where reduced hydrolyzed technetium colloid R is zero while free TcO4
and Tc- compound 3a complex species R
Tc radio-
9
9m
-
f
9
9m
f
is 1. As the developing process was ending,
the strips were dried, trimmed into 1 cm size pieces and counted using the sodium iodide
Tl) γ-ray scintillation counter.
(
The following equation was followed to calculate the radiochemical yield percent of
9
9m
Tc- compound 3a complex:
9
9m
-
%
ꢀRadiochemicalꢀyieldꢀ = ꢀ100ꢀ −ꢀꢁ%ꢀFreeꢀ TcO ꢀ + ꢀ%ꢀcolloidꢂ
4
More affirmation was to be gained using Shimadzu HPLC as an extra mean to evaluate
the radiochemical yields. The sample mixtures (50 µL) were injected into a reversed-
phase column (Lichrospher RP18, 4 mm X 250 mm; 5 µm). The HPLC was run at a
wavelength of 254 nm using mobile phase of acetonitrile: water (15: 85, v/v) with flow
rate adjusted to 1 mL/min. Then portions of 0.5 mL were separated distinctly using a
fraction collector up to 20 mL and counted in a good-type γ-scintillation counter.
99m
3
.5.5. Biological distribution of Tc-compound 3a complex
Guidelines of both Egyptian Atomic Energy Authority and The animal ethics committee
were followed. Pharmacokinetic and distribution studies were done in Normal Albino
9
9m
mice.
Tc- compound 3a complex biodistribution was evaluated at time periods of 15,
6
0 and 120 min post injection (p. i) in normal Albino mice (n = 5 mice/time point). Mice
9
9m
were housed in groups and nourished with food and water. Tc- compound 3a complex
was injected in the mice subcutaneously. Primarily, Chloroform was used to anaesthetize
mice, then weighted and sacrificed at different time periods. All body organs and tissues
were detached, collected and washed with saline then weighted. Specimens of blood,
bone and muscles were collected and weighted then were assumed to be 7, 10 and 40 %
1
5

ACCEPTED MANUSCRIPT
of the overall body weight, respectively [51-55]. A good type γ-counter NaI(Tl) was used
to measure radioactivities of both organs and the background. The percent-injected
dose/gram organ or fluid (% ID/g) was calculated in aggregates of five.
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0. M. Bkgaranik, R. Behera. Indian J. Chemistry Section B. 26(B) (1987) 779–781.
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derivative radiolabeled with technetium-99m., Chemical Central Journal, 10(1),
(
2016) 73.
99m
5
5
5
5
2. B.M. Essa, T. M. Sakr, M. A. Khedr, F.A. El-Essawy, A. A. El Mohty.
Tc-
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9
9m
and biological evaluation of
Tc-tricabonyl oxiracetam as a selective imaging
probe for AMPA receptors., Journal of Radioanalytical and Nuclear Chemistry,
Accepted, (2016).
Figure & Table Captions
Figure 1: General Structures for the newly synthesized compounds
9
9m
Fig. 2. Factors affecting radiochemical yield of Tc-compound 3a complex.
9
9m
Fig. 3. Biodistribution pattern of Tc-compound 3a complex
Scheme 1. Synthesis of compounds 1-6
Table 1.
In vitro anticancer screening of the synthesized compounds against colon cancer cell line
(Caco-2).
Table 2.
Fas-ligand concentration in treated Caco-2 cell line.
Table 3.
Caspase-3 activity in treated Caco-2 cell line
2
0

ACCEPTED MANUSCRIPT
Table 1.
In vitro anticancer screening of the synthesized compounds against colon cancer cell line
Caco-2).
(
Compound Compound concentration (µM)
µM 10µM 25µM
Surviving fraction
0.182
IC50
µM)
(
1
50µM
100µM
3
3
3
4
4
4
5
5
5
6
6
6
a
b
c
0.348
0.281
0.285
0.371
0.477
0.519
0.304
0.444
0.433
0.442
0.412
0.319
0.133
0.466
0.113
0.285
0.473
0.112
0.234
0.299
0.338
0.304
0.317
0.219
0.663
0.325
0.422
0.416
0.086
0.229
0.257
0.156
0.223
0.186
0.339
0.295
0.229
0.202
0.552
0.525
0.068
0.485
0.153
0.227
0.167
0.073
0.135
0.169
0.276
0.456
0.228
0.197
0.343
0.254
0.334
1.33
0.179
0.314
0.237
0.362
0.603
0.223
0.344
0.456
0.335
0.401
0.248
0.597
0.310
0.667
0.334
0.667
12.70
0.333
0.667
0.333
0.669
0.559
0.333
63.30
31.00
a
b
c
a
b
c
a
b
c
Doxorubicin 0.814
-FU
0.405
5
2
1

ACCEPTED MANUSCRIPT
Table 2.
Fas-ligand concentration in treated Caco-2 cell line.
Compound
Concentration
Concentration of Fas –
ligand (pg/mL)
(µM)
Control
0
20.5
445.2
775.2
706.12
115.5
165.7
307.5
312.4
309.4
641.8
111.3
147.5
312.2
41.65
56.41
95.8
3a
5a
5c
6c
0.15
0
.3
.6
0
0.15
0
.3
.6
0
0.15
0
.3
.6
0
0.15
0
.3
.6
0
Doxorubicin
31.5
3.5
00
62
6
1
5-FU
66.2
3
1
1
5
140.1
287.7
2
2

ACCEPTED MANUSCRIPT
Table 3.
Caspase-3 activity in treated Caco-2 cell line
Compound
Concentration
Caspase-3 concentration
(nmol/mL)
(µM)
Control
0
0.193
0.37
3a
5a
5c
6c
0.6
0
.3
.15
0.6
.3
.15
0.6
.3
.15
0.6
.3
.15
31
2.3
0.38
0
0.43
0.364
0.34
0
0
0.35
0.37
0
0.366
0.404
0.407
0.409
0.366
0.251
0.26
0
0
0
Doxorubicin
6
1
00
0.272
0.8
5-FU
31
1
6
5
2
0.92
0.73
2
3

ACCEPTED MANUSCRIPT
1
00
100
9
9m
-
%
%
%
Free TcO4
Colloid impurity
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
99m
Tc-compound 3a
99m
-
%
%
%
Free TcO4
Colloid impurity
99m
Tc-compound 3a
5
10
15
20
25
7
.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
NaBH amount, mg
4
pH
(A)
(B)
1
00
100
90
80
70
60
50
40
30
20
10
0
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
9
9m
-
99m
-
%
Free TcO4
% Free TcO4
% Colloid impurity
% Colloid impurity
99m
99m
%
Tc-compound 3a
%
Tc-compound 3a
0
25
50
75
100
125
150
175
200
225
250
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Time, min
Compound 3a, mg
(
C)
(D)
9
9m
Fig. 2. Factors affecting radiochemical yield of Tc-compound 3a complex.
2
4

ACCEPTED MANUSCRIPT
26
24
22
20
18
16
14
12
10
8
6
4
2
0
15 min
0 min
20 min
6
1
9
9m
Fig. 3. Biodistribution pattern of Tc-compound 3a complex
2
5

ACCEPTED MANUSCRIPT
H
N
Cl
O
H NO S
1
2
2
a
H
N
N
O
S
H NO S
2
2
2
b
H
N
N
N
S
N
O
HN
O
N
R
X
N
S
d
c
S
HN
H NO S
2
2
NH
H NO S
2
2
H NO S
2
2
R
R
6
a-c
3
a-c
4
5
a-c, X=O
a-c, X=S
a, R= H
b, R= OCH3
c, R= Cl
Reagents and Solvents: a: NH SCN, b: ArCHO, CH COOH, CH COONa, c: KOH, urea/thiourea, d: KOH,
4
3
3
hydroxylamine HCl
Scheme 1. Synthesis of compounds 1-6
2
6

ACCEPTED MANUSCRIPT
•
•
•
•
Novel thiazolidinone and fused thiazolidinone derivatives.
In vitro cytotoxic activity on colorectal cancer cell line (Caco-2).
In vitro activation of Caspase-3 and Fas-ligand in Caco-2 cell line.
9
9m
Radiolabeling with Tc and biodistribution pattern was evaluated in vivo.