Novel 4-(4-substituted-thiazol-2-ylamino)-N-(pyridin-2-yl)-benzene- sulfonamides as cytotoxic and radiosensitizing agents

Article abstract of DOI:10.1007/s12272-012-0106-y

A series of novel 4-(4-substituted-thiazol-2-ylamino)-N-(pyridin-2-yl) benzene-sulfonamides were synthesized and screened for their cytotoxic activity against human breast cancer cell line (MCF-7). Compounds 6, 7, 9, 10, 11, and 14 displayed significant a

Full text of DOI:10.1007/s12272-012-0106-y

Arch Pharm Res Vol 35, No 1, 59-68, 2012
DOI 10.1007/s12272-012-0106-y
Novel 4-(4-substituted-thiazol-2-ylamino)-N-(pyridin-2-yl)-benzene-
sulfonamides as Cytotoxic and Radiosensitizing Agents
Mostafa M. Ghorab1, Fatma A. Ragab2, Helmy I. Heiba3, Hebaallah M. Agha3, and Yassin M. Nissan2
1Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud University, Saudi
2
3
Arabia, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt, and Department
of Drug Radiation Research, National Center for Radiation Research and Technology, Atomic Energy Authority, Cairo,
Egypt
(Received June 24, 2011/Revised July 30, 2011/Accepted August 2, 2011)
A series of novel 4-(4-substituted-thiazol-2-ylamino)-N-(pyridin-2-yl) benzene-sulfonamides
were synthesized and screened for their cytotoxic activity against human breast cancer cell
line (MCF-7). Compounds
when compared to doxorubicin, which was used as a reference drug. The synergistic effect of
Gamma radiation for the most active derivatives , and 11 was also studied and their IC50
values markedly decreased to 11.9 M, 11.7 M, and 11.6 M, respectively.
6, 7, 9, 10, 11, and 14 displayed significant activity against MCF-7
7, 9
µ
µ
µ
Key words: Sulfonamides, Thiazoles, Cytotoxic, Radiosensetizing activities
al., 2002). A series of sulfonamide derivatives I
bearing a thiazole moiety showed a significant tumor
INTRODUCTION
Sulfonamide derivatives have been shown to display growth delay in mouse tumor xenograft models and
extensive in vitro and/or in vivo antitumor activity some examples were reported to have > 100-fold
(Supuran and Scozzafava, 2000; Casini et al., 2002a; selectivity for CDK4, as well as other examples that
Abbate et al., 2004; Ghorab et al., 2006, 2009, 2010a, were equipotent against CDK1, CDK2, and CDK4
2010b, 2010c, 2011; Ismail et al., 2006; Al-Said et al., (Fischer and Lane, 2000). Recently, N4 sulfapyridine
2010). Sulfonamide antitumor activity arises through derivatives II bearing a 2-substituted thiazolidine-4-
a wide range of different mechanisms, such as cell one moiety were synthesized and showed more
cycle arrest in the G1 phase (Fukuoka et al., 2001) cytotoxic activity against human breast cancer cell
and inhibition of carbonic anhydrase (Supuran et al., line (MCF-7) and HeLa cell lines than 5-flurouracil
2001), histone deacetylases (HDACs) (Payne et al., and doxorubicin (Kamel et al., 2010) (Fig. 1).
2008), methionine aminopeptidases (MetAPs) (Kawai
Thus, with the goal of identifying new cytotoxic
et al., 2006), matrix metalloproteinase (MMPs) (Casini compounds, we designed and synthesized novel 4-(4-
et al., 2002b), nicotinamide adenine dinucleotide substituted-thiazol-2-ylamino)-N-(pyridin-2-yl) ben-
(NADH) oxidase (Villar et al., 2004), cyclin-dependent zene-sulfonamide derivatives for use as potential
kinase (CDK) (Huang et al., 2006), binding to
Tubulin, and disruption of microtubule assembly substitutions at position 4 of the thiazole ring on their
cytotoxic activity. The synergistic effect of
(Kenneth et al., 2006). -radiation
β
-
cytotoxic agents and explored the effect of different
γ
Indisulam (E7070) is an example of an anticancer with the most potent synthesized derivatives was also
agent that contains sulfonamide moiety (Kesteren et evaluated.
MATERIALS AND METHODS
Correspondence to: Mostafa M. Ghorab, Medicinal, Aromatic
and Poisonous Plants Research Center (MAPPRC), College of
Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
Chemistry
Melting points were uncorrected and were deter-
mined using a Stuart melting point apparatus (Stuart
Tel: 966-53-429-2860, Fax: 966-1-467-0560
E-mail: mmsghorab@yahoo.com
59

60
M. M. Ghorab et al.
benzene and crystallized from dioxane to give 4: Yield
95%, m.p. 198-200oC, IR (KBr, cm−1): 3192 (NH), 3040
(CH arom.), 1616 (C=N), 1390, 1165 (SO2), 684 (C-Cl).
1H-NMR (DMSO-d6,
δ ppm): 6.6 (s, 1H, CH, thiazole),
6.8-8.0 (m, 9H, Ar-H + NH, D2O exchangeable), 10.9
(s, 1H, SO2NH, D2O exchangeable). MS (m/z): 366 (M+,
3.46%), 78 (100%). Anal. Calcd. for C14H11ClN4O2S2
(366): C, 45.90; H, 3.00; N, 15.30. Found: C, 45.63; H,
3.36; N, 15.5.
Fig. 1. Sulfonamide derivatives bearing the thiazole moiety
as cytotoxic agents
4-(4-Mercaptothiazol-2-ylamino)-N-(pyridin-2-yl))
benzenesulfonamide (5)
A mixture of compound
3 (3.48 g, 0.01 mol) and phos-
Scientific). Elemental analyses (C, H, N) were per- phorus pentasulfide (2.2 g, 0.01 mol) in pyridine (20
formed on Perkin-Elmer 2400 analyser (Perkin-Elmer) mL) was refluxed for 8 h, the reaction mixture was
at the Microanalytical Laboratories of the Faculty of cooled and then poured onto ice-cold water and acidi-
Science, Cairo University. All compounds were within fied with dilute hydrochloric acid. The solid obtained
± 0.4% of the theoretical values. Infrared spectra (KBr) was crystallized from ethanol to give
5: Yield 47%,
were determined using a Shimadzu IR-110 spectro- m.p. 250-251oC, IR (KBr, cm−1): 3445, 3233 (NH), 3056
photometer (Shimadzu). 1H-NMR spectra were carried (CH arom.), 2553 (SH), 1590 (C=N), 1388, 1138 (SO2).
out using BRUCKER proton NMR-Avance 300 (300 1H-NMR (DMSO-d6,
δ ppm): 6.86 (s, 1H, CH, thiazole),
MHz, Bruker) in DMSO-d6 as a solvent, using tetra- 6.88-8.0 (m, 9H, Ar-H + NH, D2O exchangeable), 10.9
methylsilane (TMS) as internal standard. Mass spectra (s, 1H, SO2NH, D2O exchangeable), 13.8 (s, 1H, SH,
were recorded on a JEOL JMS AX-500 spectrometer D2O exchangeable). MS (m/z): 364 (M+, 1.60%), 63
(JEOL JMS), in electronic impact (EI). All reactions (100%). Anal. Calcd. for C14H12N4O2S3 (364): C, 46.15;
were monitored by thin layer chromatograph (TLC) H, 3.29; N, 15.38. Found: C, 46.48; H, 2.99; N, 15.64.
using precoated Aluminium sheets Silica gel Merck 60
F254. Ethyl acetate-cyclohexane (2.5:7.5 mL) mixture 4-(4-(Methylthio) thiazol-2-ylamino)-N-(pyridin-
was used as the eluting solvent and TLC sheets were 2-yl) benzenesulfonamide (6)
visualized using a UV lamp (Merck).
A mixture of compound 5 (3.64 g, 0.01 mol) and methyl
iodide (1.52 mL, 1.42 g, 0.01 mol) in methanol (20 mL)
was left at ambient temperature for 48 h. The sample
was trhen heated under reflux for 6 h. The solvent
4-(4-Hydroxythiazol-2-ylamino)-N-(pyridin-2-yl)
benzenesulfonamide (3)
A solution of compound
2 (3.25 g, 0.01 mol) and Am- was removed by evaporation and the oily residue was
monium thiocyanate (0.76 g, 0.01 mol) in ethanol (20 triturated with diethyl ether. The crude solid was col-
mL) was refluxed for 1 h. The reaction mixture was lected, washed with cold ethanol and finally crystalliz-
filtered while hot and the obtained solid was cry- ed from propanol to give 6
: Yield 55%, m.p. 194-196oC,
stallized from dioxane to give 3
: Yield 85%, m.p. 240- IR (KBr, cm−1): 3440, 3239 (NH), 3057 (CH arom.),
242oC, IR (KBr, cm−1): 3448 (OH), 3366, 3191 (NH), 2928, 2860 (CH aliph), 1540 (C=N), 1390, 1140 (SO2). 1H-
1
3047 (CH arom.), 1602 (C=N), 1397, 1137 (SO2). H- NMR (DMSO-d6,
δ
ppm): 1.2 (s, 3H, CH3), 6.8 (s, 1H, CH,
NMR (DMSO-d6, δ ppm): 6.84 (s, 1H, CH, thiazole), thiazole), 6.9-8.2 (m, 9H, Ar-H + NH, D2O exchangea-
6.86-8.0 (m, 9H, Ar-H + NH, D2O exchangeable), 10.9 ble), 10.8 (s, 1H, SO2NH, D2O exchangeable). Anal.
(s, 1H, SO2NH, D2O exchangeable), 11.7 (s, 1H, OH, Calcd. for C15H14N4O2S3 (378): C, 47.61; H, 3.70; N,
D2O exchangeable). MS (m/z): 348 (M+, 1.7%), 184 14.81. Found: C, 47.34; H, 4.06; N, 14.64.
(100%). Anal. Calcd. for C14H12N4O3S2 (348): C, 48.27;
H, 3.44; N, 16.09. Found: C, 48.59; H, 3.26; N, 16.32.
4-(4-Hydrazinylthiazol-2-ylamino)-N-(pyridin-2-yl)
benzenesulfonamide (7)
4-(4-Chlorothiazol-2-ylamino)-N-(pyridin-2-yl)ben-
zenesulfonamide (4)
Method A: A mixture of compound
mol) and hydrazine hydrate (0.01 mol) was refluxed in
6 (3.78 g, 0.01
A solution of compound
3 (3.48 g, 0.01 mol) in thionyl ethanol (20 mL) for 24 h. and H2S was detected using
chloride (10 mL) was refluxed for 2 h, thionyl chloride lead acetate paper. The reaction mixture was cooled
was then removed by distillation under reduced pres- and poured onto ice-cold water. The solid obtained
sure and the residual solid was washed twice with was crystallized from dioxane to give
7 with a 20%

Sulfonamides as Antitumor and Radiosensitizer
yield.
61
Found: C, 52.91; H, 2.78; N, 14.69.
Method B: A mixture of compound
4 (3.66 g, 0.01
mol) and hydrazine hydrate (0.01 mol) was refluxed in 4-(4-(2-Phenylhydrazinyl)thiazol-2-ylamino)-N-
ethanol (20 mL) for 5 h. The reaction mixture was (pyridin-2-yl)benzenesulfon-amide (10), 4-(4-(2-
cooled and poured onto ice-cold water and acidified (2,4-Dinitrophenyl)hydrazinyl)thiazol-2-ylamino)-
with dilute hydrochloric acid. The solid obtained was N-(pyridin-2-yl) benzenesulfonamide (11)
collected by filtration and crystallized from dioxane to
A mixture of compound 4 (3.66 g, 0.01 mol) and
give
7
at a yield higher than 58%, m.p. 210-212oC, IR phenylhydrazine (0.9 mL, 1.08 g, 0.01 mol) or 2,4-dinitro-
(KBr, cm−1): 3280, 3215, 3113 (3NH, NH2), 3098 (CH phenylhydrazine (1.98 g, 0.01 mol) in ethanol (20 mL)
arom.), 1600 (C=N), 1389, 1138 (SO2). 1H-NMR (DMSO- was refluxed for 5 h. The reaction mixture was cooled
d6,
δ
ppm): 2.4 (s, 2H, NH2, D2O exchangeable), 4.7 (s, and poured onto cold water. The solid obtained was
11 respectively.
1H, NHNH2, D2O exchangeable), 6.7 (s, 1H, CH, thia- crystallized from dioxane to give 10
,
zole), 6.9-8.0 (m, 9H, Ar-H + NH, D2O exchangeable),
Microanalytical and spectral data of 10: Yield 72%,
10.2 (s, 1H, SO2NH, D2O exchangeable). Anal. Calcd. m.p.130-132oC, IR (KBr, cm−1): 3364, 3313 (NH), 3057
1
for C14H14N6O2S2 (362): C, 46.40; H, 3.86; N, 23.20. (CH arom.), 1582 (C=N), 1389, 1143 (SO2). H-NMR
Found: C, 46.67; H, 4.10; N, 23.38.
(DMSO-d6,
δ ppm): 3.5 (s, 2H, NHNH, D2O exchangea-
ble), 6.8 (s, 1H, CH, thiazole), 6.9-8.0 (m, 14H, Ar-H +
4-(5-Amino-7-(4-chlorophenyl)-6-cyano-7H-pyrano NH, D2O exchangeable), 10.1 (s, 1H, SO2NH, D2O ex-
[2,3-d]thiazol-2-ylamino)-N-(pyridin-2-yl) benzene- changeable). MS (m/z): 438 (M+, 0.74%), 61 (100%).
sulfonamide (8)
Anal. Calcd. for C20H18N6O2S2 (438): C, 54.79; H, 4.10;
A mixture of compound
3
(3.48g, 0.01 mol) and 2-(4- N 19.17,. Found: C, 54.55; H, 4.39; N, 18.98.
chlorobenzylidene) malononitrile (1.88 g, 0.01 mol) in
Microanalytical and spectral data of 11: Yield 73%,
ethanol (20 mL) containing a catalytic amount of tri- m.p. 168-170oC, IR (KBr, cm−1): 3421, 3347 (NH), 3068
ethylamine (TEA), was refluxed for 5 h. The reaction (CH arom.), 1593 (C=N), 1520, 1387 (C-NO2), 1335,
1
mixture was cooled, poured onto ice-cold water and 1135 (SO2). H-NMR (DMSO-d6,
δ ppm): 3.7 (s, 2H,
acidified with dilute hydrochloric acid. The obtained NHNH, D2O exchangeable), 6.8 (s, 1H, CH, thiazole),
solid was crystallized from dioxane to give compound 6.9-8.2 (m, 12H, Ar-H + NH, D2O exchangeable), 9.7
8
: Yield 86%, m.p. 270-272oC, IR (KBr, cm−1): 3250, (s, 1H, SO2NH, D2O exchangeable). MS (m/z): 528
3191, 3113 (NH, NH2), 3046 (CH arom.), 2939, 2825 (M+, 1.71%), 61 (100%). Anal. Calcd. for C20H16N8O6S2
(CH aliph.), 2219 (CN), 1603 (C=N), 1391, 1138 (SO2), (528): C, 45.45; H, 3.03; N, 21.21. Found: C, 45.80; H,
1
780 (C-Cl). H-NMR (DMSO-d6,
δ ppm): 3.8 (s, 2H, 3.33; N, 21.53.
NH2, D2O exchangeable), 4.1 (s, 1H, CH, pyran), 6.6-
8.0 (m, 13H, Ar-H + NH, D2O exchangeable), 8.9 (s, 4-[4-(Isothiocyanato) thiazol-2-ylamino)-N-(pyri-
1H, SO2NH, D2O exchangeable). MS (m/z): 536 (M+, din-2-yl)benzenesulfonamide (12)
1.00%), 168 (100%). Anal. Calcd. for C24H17ClN6O3S2 A mixture of compound
4 (3.66 g, 0.01 mol) and am-
(536): C, 53.73; H, 3.17; N, 15.67. Found: C, 53.39; H, monium thiocyanate (0.76 g, 0.01 mol) in dry acetone
3.51; N, 15.45.
(20 mL) was refluxed for 1 h. The reaction mixture
was cooled and poured onto ice-cold water. The solid
4-[3H-5-(4-Chlorophenyl)-thiazolo[4,5-b]-pyrano
obtained was crystallized from ethanol to give 12:
[2,3-d]pyrimidin-4-one]-N-(pyridin-2-yl) benzene- Yield 69%, m.p. 215-217oC, IR (KBr, cm−1): 3392, 3128
sulfonamide (9)
A solution of compound
(NH), 3049 (CH arom.), 2051 (N=C=S), 1587 (C=N),
8 (5.36 g, 0.01 mol) in formic 1390, 1129 (SO2). H-NMR (DMSO-d6, δ ppm): 6.8 (s,
1
acid (20 mL) was refluxed for 5 h, the reaction mix- 1H, CH, thiazole), 6.9-8.0 (m, 9H, Ar-H + NH, D2O ex-
ture was cooled and then poured onto ice-cold water. changeable), 10.8 (s, 1H, SO2NH, D2O exchangeable).
The obtained solid was crystallized from ethanol to MS (m/z): 389 (M+, 0.9%), 64 (100%). Anal. Calcd. for
give 9
: Yield 67%, m.p. > 290oC, IR (KBr, cm−1): 3273, C15H11N5O2S3 (389): C, 46.27; H, 2.82; N, 17.99. Found:
3120 (NH), 3046 (CH arom.), 2936, 2822 (CH aliph.), C, 45.98; H, 3.04; N, 17.62.
1713 (C=O), 1589 (C=N), 1371, 1138 (SO2), 778 (C-Cl).
1H-NMR (DMSO-d6,
δ
ppm): 4.1 (s, 1H, CH, pyran), 6.8- 4-(4-(4-Chlorophenylamino)thiazol-2-ylamino)-N-
8.0 (m, 15H, Ar-H + CH, pyrimidine + 2NH, D2O ex- (pyridin-2-yl)benzenesulfon-amide (13)
changeable), 10.9 (s, 1H, SO2NH, D2O exchangeable). A mixture of compound
4 (3.66 g, 0.01 mol) and 4-
MS (m/z): 564 (M+, 6.19%), 157 (100%). Anal. Calcd. chloroaniline (1.27 g, 0.01 mol) in dimethylformamide
for C25H17ClN6O4S2 (564): C, 53.19; H, 3.01; N, 14.89. (20 mL) was refluxed for 4 h. The reaction mixture

62
M. M. Ghorab et al.
was cooled and poured onto cold water. The solid
7,
9, and 11, in molar concentrations of 10, 25, 50, 100
obtained was crystallized from ethanol to give 13
:
µ
M. After 2 h, Cells were subjected to a single dose of
Yield 64%, m.p. > 290oC, IR (KBr, cm−1): 3386, 3345 ã-radiation at a dose level of 8 Gy with a dose rate of
(NH), 3076 (CH arom.), 1602 (C=N), 1397, 1130 (SO2), 2 Gy/min. After 48 h, the surviving fractions were
620 (C-Cl). MS (m/z): 457 (M+, 0.27%), 54 (100%). Anal. measured using an ELISA reader.
Calcd. for C20H16ClN5O2S2 (457): C, 52.51; H, 3.50; N,
15.31. Found: C, 52.19; H, 3.68; N, 15.54.
The cytotoxicity of the control group, the radiated
group, the compound treated groups (10, 25, 50, and
100
µ
M) and the irradiated compound groups (10, 25,
4-[4-(2-Aminobenzoic acid)thiazol-2-ylamino]-N- 50, and 100
µM) was measured using the SRB assay
(pyridin-2-yl)benzenesulfon-amide (14)
A mixture of compound (3.66 g, 0.01 mol) and an-
on the human breast cancer cell line (MCF-7).
4
thranilic acid (1.37 g, 0.01 mol) in n-butanol (20 mL)
was refluxed for 5 h. The reaction mixture was cooled
Molecular docking study
All molecular modeling studies were carried out on
and poured onto cold water. The solid obtained was an Intel Pentium 1.6 GHz processor, 512 MB memory
crystallized from ethanol to give 14: Yield 69%, m.p. > with Windows XP operating system using Molecular
290oC, IR (KBr, cm−1): 3424 (OH), 3347, 3286 (NH), Operating Environment (MOE, 10.2008) software. All
3069 (CH arom.), 1681 (C=O), 1574 (C=N), 1390, 1142 the minimizations were performed with MOE until a
(SO2). 1H-NMR (DMSO-d6 ppm): 6.5 (s, 1H, CH, thia- RMSD gradient of 0.05 kcal mol-1Ao-1 with MMFF94X
, δ
zole), 6.8-7.9 (m, 14 H, Ar-H + 2NH, D2O exchangea- forcefield and the partial charges were automatically
ble), 10.9 (s, 1H, SO2NH, D2O exchangeable), 11.8 (s, calculated.
1H, OH, D2O exchangeable). MS (m/z): 467 (M+, 2.7%),
The X-ray crystallographic structure of human
184 (100%). Anal. Calcd. for C21H17N5O4S2 (467): C, CDK2 complexes with the sulfone inhibitor (PDB ID:
53.96; H, 3.64; N, 14.98. Found: C, 54.27; H, 3.45; N, 1FVV) was obtained from the protein data bank. The
14.61.
enzyme was prepared for docking studies where: (i)
Ligand molecule was removed from the enzyme active
site. (ii) Hydrogen atoms were added to the structure
In vitro cytotoxic screening
The cytotoxic activity of the newly synthesized com- with their standard geometry. (iii) MOE Alpha Site
pounds was evaluated against a human breast cancer Finder was used to search for the active sites in the
cell line (MCF-7) in vitro using the Sulfo-Rhodamine- enzyme structure and dummy atoms were created
Bstain (SRB) assay as described previously (Skehan et from the obtained alpha spheres. (iv) The obtained
al., 1990). The human tumor cell line (MCF-7) was pro- model was then used to predict the interaction between
vided by the National Cancer Institute, Cairo Uni- he ligand and enzymes at the active site
versity, Egypt. The relationship between surviving
fraction and drug concentration (µM) was plotted
RESULTS AND DISCUSSION
using the Microsoft Office Excel 2003 program to
obtain the best fit survival curve for the breast tumor
cell line after a specified time. The molar concentra-
Chemistry
2-Chloro-N-(4-(N-pyridin-2-ylsulfamoyl) phenyl) ace-
tion required for 50% inhibition of cell viability (IC50) tamide
was calculated by solving the third order polynomial with chloroacetyl chloride (Finkelstein, 1944). Re-
equation using software that was written on the fluxing compound with ammonium thiocyanate in
ethanol efficiently yielded 4-hydroxythiazole derivat-
ive via intramolecular cyclization. Compound was
obtained by refluxing with SOCl2 (Scheme 1). When
was refluxed with phosphorus pentasulfide in pyri-
was pro-
was obtained by
with methyl iodide in methanol.
with hydrazine hydrate in ethanol for
line (MCF-7). Irradiation was performed at the Nat- 24 h afforded the 4-hydrazino derivative with a 20%
ional Center for Radiation Research and Technology, yield. A higher yield of 45% was obtained by refluxing
Atomic Energy Authority, using a Gamma cell-40 the 4-chloro derivative with hydrazine hydrate in
137Cs) source.
ethanol for 5 h. Reaction of with 2-(4-chlorobenzyli-
Cells were incubated with the selected compounds; dene) malononitrile in ethanol containing a catalytic
2 was synthesized via reaction of sulfapyridine
1
2
‘Matlab R2008a’ program.
3
4
3
Radiosensitizing evaluation
The most promising compounds based on the in
3
vitro cytotoxic screening (
subsequent experiments to assess the synergistic duced. The thio methyl derivative
effects of combined treatment with -radiation on in refluxing compound
vitro cytotoxicity against a human breast cancer cell Treatment of
7, 9, and 11) were used in dine, the 4-mercaptothiazole derivative 5
6
γ
5
6
7
4
(
3

Sulfonamides as Antitumor and Radiosensitizer
63
Scheme 1. Synthetic pathway used to prepare the starting materials 3, 4
Scheme 2. Synthetic pathway used to prepare compounds 5-9
amount of triethylamine (TEA), yielded the corres- zole derivative
ponding pyrano [2,3-d] thiazole derivative
4
with phenylhydrazine, 2,4 dinitro-
8
, via the phenyl hydrazine, ammonium thiocynate, 4-chloroani-
formation of an intermediate followed by intramole- line and anthranilic acid, respectively (Scheme 3).
cular cyclization. Thiazolo [4,5-b]-pyrano[2,3-d] pyri-
midine derivative
formic acid. This reaction proceeded via condensation
followed by cyclization (Scheme 2). Compounds 10 11
12
9 was obtained by refluxing 8 in
In vitro cytotoxic screening
In the in vitro cytotoxic screening of the synthesized
compounds against human breast cancer cell line
,
,
,
13, and 14 were obtained by reacting 4-chlorothia- (MCF-7), the thiazolo [4,5-b]-pyrano [2,3-d] pyrimidine

64
M. M. Ghorab et al.
Scheme 3. Synthetic pathway used to prepare compounds 7, 10-14
derivative
MCF-7 cells with an IC50 value of 12.0
significantly better than doxorubicin, the reference tion is effective if CT is sufficiently active to eradicate
drug (IC50 = 26.1 M). 4-hydrazinyl thiazole derivati- subclinical metastases and if the primary local tumor
ves 10, and 11 also showed high activity with IC50 is effectively treated by RT. In this regard, no interac-
values of 21.7, 22.8, 20.0 M, respectively, while the tion between RT and CT is required. In regards to the
methylthio derivative , and the anthranilic acid former concept, radiation the initial radiation damage
derivative 14 showed less activity with IC50 values of can be directly enhanced by incorporating drugs into
9
was shown to be highly cytotoxic against mainly based on two concepts; spatial cooperation,
µM, which was and enhancement of radiation effects. Spatial coopera-
µ
7,
µ
6
23.2 and 25.8
µ
M, respectively (Table I).
DNA, inhibiting cellular repair, accumulating cells in
a radiosensitive phase or eliminating radioresistant
phase cells, and eliminating hypoxic cells (Nishimura,
Radiosensitizing evaluation
It has been reported that combining chemotherapy 2004).
(CT) with radiotherapy (RT) is effective for treatment
Thus, the effect of combined treatment with the
of cancer. The rationale for combining CT and RT is most active compounds (7, 9, and 11) and γ-radiation

Sulfonamides as Antitumor and Radiosensitizer
65
Table I. In vitro anticancer screening of the synthesized compounds against the human breast cancer cell line (MCF-7)
Compound concentration ( M)
25 ( M) 50 ( M)
Surviving fraction (mean ± S.E.M.)a
µ
IC 50 (µM)
Compd. No.
10 (µM)
µ
µ
100 (µM)
3rd order
Doxorubicin
0.7210 ± 0.02
0.9294 ± 0.11
0.9032 ± 0.08
0.8624 ± 0.09
0.7797 ± 0.04
0.8047 ± 0.02
0.8502 ± 0.10
0.4921 ± 0.03
0.7347 ± 0.05
0.6666 ± 0.10
0.8940 ± 0.06
0.7831 ± 0.24
0.7510 ± 0.07
0.5460 ± 0.02
0.6825 ± 0.02
0.5566 ± 0.03
0.5843 ± 0.08
0.4775 ± 0.08
0.4019 ± 0.04
0.6243 ± 0.03
0.2722 ± 0.06
0.4925 ± 0.06
0.4294 ± 0.03
0.6655 ± 0.07
0.6426 ± 0.08
0.5662 ± 0.07
0.4610 ± 0.01
0.2583 ± 0.04
0.2540 ± 0.07
0.3262 ± 0.07
0.1452 ± 0.01
0.1122 ± 0.02
0.3514 ± 0.03
0.1659 ± 0.03
0.2606 ± 0.04
0.2076 ± 0.04
0.3155 ± 0.09
0.4252 ± 0.03
0.2576 ± 0.02
0.4940 ± 0.03
0.3387 ± 0.01
0.3272 ± 0.03
0.1474 ± 0.02
0.1448 ± 0.01
0.1521 ± 0.02
0.2735 ± 0.01
0.2160 ± 0.05
0.1716 ± 0.02
0.1975 ± 0.03
0.1533 ± 0.03
0.1397 ± 0.01
0.1972 ± 0.06
26.1
34.5
31.6
32.9
23.2
21.7
35.5
12.0
22.8
20.0
45.9
35.0
25.8
3
4
5
6
7
8
9
10
11
12
13
14
aEach value is the mean of three experiments ± S.E.M.
Table II. In vitro anticancer screening of compounds
breast cancer cell line (MCF-7)
7
,
9, and 11 in combination with γ-radiation against the human
Compound concentration (
µ
M) + Irradiation (8 Gy)
Compd. Control
Irradiated
IC50 (µM)
10
25
50
100
No.
(cells only) control (8 Gy)
Surviving fraction (Means ± S.E.M.)a
7
9
11
1.000
1.000
1.000
0.927 ± 0.02b
0.927 ± 0.02b
0.927 ± 0.02b
0.5256 ± 0.16b
0.4902 ± 0.18b
0.4286 ± 0.06b
0.1543 ± 0.04b
0.2846 ± 0.04b
0.3089 ± 0.05b
0.1020 ± 0.02b
0.2468 ± 0.03b
0.1081 ± 0.03b
0.2270 ± 0.07b
0.2742 ± 0.04b
0.1503 ± 0.03b
11.9
11.7
11.6
aEach value is the mean of three values ± S.E.M.; bSignificant difference from control group at
p
< 0.001
on cytotoxicity was evaluated. The IC50 values of the tors as antitumor agents. The CDK2/cyclin interface
tested compounds were synergistically decreased to area is 3252 Å2 in Cyclin E and 2839 Å2 in Cyclin A
11.9, 11.7, and 11.6
on these results, we can conclude that the combina-
tion (treatment) of compounds or 11 with the examined to understand the binding mode to the active
ionizing radiation synergistically enhanced inhibition site of CDK2 and to identify the key amino acids in
µ
M, respectively (Table II). Based (Jeffrey et al., 1995; Honda et al., 2005).
Two classes of synthetic inhibitors of CDK2 were
7, 9
of breast cancer.
this site. Thiazolidinone inhibitors bind to CDK2
through two hydrogen bonds with Glu 81 and Leu 83
and the sulfonate group of these inhibitors interacts
Molecular docking study
Cyclin-dependant kinase 2 enzyme (CDK2) is one of with Asp 86 and leu 10 of the backbone (Richardson et
the most important enzymes in the protein kinases al., 2007). In contrast, aminopyrimidine inhibitors were
family. It is responsible for G1/S phase in the cell cycle shown to interact with Asp 86 and Ile 10 (Lucking et
(Lucking et al., 2007), where it acts by transferring a al., 2007).
phosphoryl group from a donor to an acceptor thus
Since our compounds were designed to contain amino-
activating a cyclin protein and regulating cell division pyridines and sulfone groups, we evaluated their
(Lucking et al., 2007). CDK2 interacts with Cyclin A docking on the active site of CDK2 and determined
and E to drive the cell from the G1 phase to S phase their interactions with amino acids on the active site
(Jeffrey et al., 1995). CDK2 is a monomer composed of of this enzyme in order to better understand their
298 amino acids and consists of
-sheet (Morgan).
Understanding the binding mode of cyclin A and E selected for this purpose. The file contains the CDK2
to CDK2 was of great help in designing CDK2 inhibi- enzyme co-crystallized with a sulfone ligand (Davis et
α
-helix elements and biological activity as cytotoxic agents.
a
β
A protein data bank file with the code 1FVV was

66
M. M. Ghorab et al.
al., 2001). All docking procedures were achieved using mechanism as compound 10. However, compound
MOE (Molecular Operating Enviroment) software showed a poor energy score (S) = 10.7351 despite its
10.2008 provided by chemical computing group, Canada. IC50 value = 12.0 M, suggesting that this compound
Docking on the active site of CDK2 was performed for functions as a cytotoxic agent through another mecha-
compounds 10 11, and 14, which showed good nism.
cytotoxic activity. Based on the above results, compounds
9
µ
6,
7,
9,
,
6, 7, 9, 10,
The docking protocol was verified by redocking of
the co-crystallized ligand in the vicinity of the active
site of the enzyme with energy score (S) = 18.1741
Kcal/mol and root mean standard deviation (RMSD) =
0.7723 (Fig. 2 and 3). The sulfone ligand interacts
with the active site of CDK2 through four interactions:
the sulfonate group interacts with Lys 89 through a
hydrogen bond of 3.13 Å and Asp 86 through a hydro-
gen bond of 3.22 Å, the carbonyl group of pyrrolone
interacts with Leu 83 through a hydrogen bond of 2.96
Å and the NH group of pyrrolone interacts with Glu
81 through a hydrogen bond of 1.98 Å. All the docked
compounds bind to either Lys 89 or Asp 86 through
SO2 by one hydrogen bond. In addition, compound 11
binds to Asp 86 through NH (thiazole) and compound
14 binds to Leu 83 through NH as illustrated in Table
III.
Compound 10 showed the best energy score (S) =
21.8927 Kcal/mol with two hydrogen bond interactions
with Asp 86 through the NH group of its thiazole ring
and with Lys 89 through its SO2 group. This result
suggests that compound 10 acts as a cytotoxic agent
by inhibiting CDK2. This finding is supported by its
Fig. 3. Validation of the docking protocol on the active site
of CDK2
IC50 value = 22.8
bicin as illustrated in Fig. 4 and 5. Compounds
µ
M, which was better than Doxoru-
6,7,11
Table III. Binding scores and interaction of the amino
acids on the active site of CDK2 with the docked com-
pounds
and 14 also act as cytotoxic agents through the same
Length of
H bonds
(Å)
Compound
No.
S
Amino
Groups
interacted
Kcal/mol acids
Asp 86
Lys 89
Leu 83 C=O pyrrole
Glu 81 NH pyrrole
O=S=O
O=S=O
3.72
3.13
2.96
1.98
Co-crystallized
Ligand
18.1741
6
7
15.9589 Lys 89
O=S=O
3.16
Asp 86
Glu 51
O=S=O
NH-NH2
2.81
1.58
14.7205
10.7351
21.8927
17.5159
Lys 89
O=S=O
2.59
2.85
9
10
11
Gln 131 N (pyridine)
Lys 89 O=S=O
Asp 86 NH (thiazole)
Lys 89 O=S=O
Asp 86 NH (thiazole)
2.77
1.72
2.65
2.29
Lys 89
16.3041 Leu 83
Lys 33
O=S=O
NH
COO
2.74
1.68
2.79
14
Fig. 2. Interactions between the ligand and the active site of
CDK2

Sulfonamides as Antitumor and Radiosensitizer
67
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