Anticancer and radio-sensitizing evaluation of some new thiazolopyrane and thiazolopyranopyrimidine derivatives bearing a sulfonamide moiety

Article abstract of DOI:10.1016/j.ejmech.2011.08.026

Recently, it has been reported that compounds bearing a sulfonamide moiety posses many types of biological activities, including anticancer activity. There are a variety of mechanisms for their anticancer activity, and the most prominent mechanism is the inhibition of carbonic anhydrase (CA) isozymes. The present work reports the synthesis of some new thiazolo[4,5-b]pyrane, thiazolo[4,5-b]pyrano[2,3-d]pyrimidine derivatives bearing a sulfonamide moiety. The design of the structures of these compounds complies with the general pharmacophoric requirements for CA inhibiting anticancer drugs. The newly synthesized compounds were evaluated for their in vitro anticancer activity against human breast cancer cell line (MCF7). Most of the screened compounds showed interesting cytotoxic activities compared to doxorubicin as a reference drug. Compounds 5, 6, 10 and 12 (IC₅₀ values 39.4 μM, 41.6 μM, 35.72 μM and 34.64 μM, respectively) exhibited higher cytotoxic activities than the reference drug doxorubicin (IC₅₀ = 71.8 μM). Additionally, the previously mentioned compounds were evaluated again for their ability to enhance the cell killing effect of γ-radiation.

Full text of DOI:10.1016/j.ejmech.2011.08.026

European Journal of Medicinal Chemistry 46 (2011) 5120e5126
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Anticancer and radio-sensitizing evaluation of some new thiazolopyrane and
thiazolopyranopyrimidine derivatives bearing a sulfonamide moiety
,
b
c
c
Mostafa M. Ghoraba , Fatma A. Ragab , Helmy I. Heiba , Reham M. El-Hazek
*
a Medicinal, Aromatic and Poisonous Plants Research Center, College of Pharmacy, King Saud University, P.O.Box 2457, Riyadh 11451, Saudi Arabia
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
c Department of Drug Radiation Research, National Centre for Radiation Research and Technology, Atomic Energy Authority, Nasr City, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Recently, it has been reported that compounds bearing a sulfonamide moiety posses many types of
biological activities, including anticancer activity. There are a variety of mechanisms for their anticancer
activity, and the most prominent mechanism is the inhibition of carbonic anhydrase (CA) isozymes. The
present work reports the synthesis of some new thiazolo[4,5-b]pyrane, thiazolo[4,5-b]pyrano[2,3-d]
pyrimidine derivatives bearing a sulfonamide moiety. The design of the structures of these compounds
complies with the general pharmacophoric requirements for CA inhibiting anticancer drugs. The newly
synthesized compounds were evaluated for their in vitro anticancer activity against human breast cancer
cell line (MCF7). Most of the screened compounds showed interesting cytotoxic activities compared to
Received 4 February 2011
Received in revised form
16 May 2011
Accepted 17 August 2011
Available online 27 August 2011
Keywords:
Thiazolopyrane
Thiazolopyranopyrimidine
Sulfonamide
Anticancer
Radio-sensitizer
doxorubicin as a reference drug. Compounds 5, 6, 10 and 12 (IC50 values 39.4
34.64 M, respectively) exhibited higher cytotoxic activities than the reference drug doxorubicin
(IC50 ¼ 71.8 M). Additionally, the previously mentioned compounds were evaluated again for their
ability to enhance the cell killing effect of eradiation.
Ó 2011 Elsevier Masson SAS. All rights reserved.
mM, 41.6 mM, 35.72 mM and
m
m
g
1. Introduction
a free sulfonamide moiety, in order to study their structure
activity relationship, hoping that the new synthesized
compounds might show significant anticancer activity. This
anticancer activity is tested against a human breast cancer cell
line (MCF7).
The rationale for combining chemotherapy and radiotherapy is
based mainly on two ideas, one being spatial cooperation, which is
effective if chemotherapy is sufficiently active to eradicate
subclinical metastases and if the primary local tumor is effectively
treated by radiotherapy. In this regard, no interaction between
radiotherapy and chemotherapy is required. The other idea is the
enhancement of radiation effects.
Cytotoxic agents can enhance radiation effects by direct
enhancement of the initial radiation damage by incorporating
drugs into DNA, inhibiting cellular repair, accumulating cells in
a radiosensitive phase or eliminating radio resistant phase cells,
eliminating hypoxic cells or inhibiting the accelerated repopulation
of tumor cells. Virtually, all chemotherapeutic agents have the
ability to sensitize cancer cells to the lethal effects of ionizing
radiation [24]. We also aimed to evaluate the most potent
compounds for their in vitro anticancer activity in combination
Sulfonamides constitute an important class of drugs, with
several types of pharmacological activities such as antibacterial [1],
hypoglycemic [2], diuretic [3,4], anti-carbonic anhydrase [3,5],
antithyroid agents [6]. Recently, a host of structurally novel
sulfonamide derivatives have been reported to show substantial
anticancer activities in vitro and/or in vivo [7e11]. It has been
known that aryl/heteroaryl sulfonamides may act as antitumor
agents through a variety of mechanisms such as cell cycle pertur-
bation in the G1 phase, disruption of microtubule, angiogenesis
inhibition and functional suppression of the transcriptional acti-
vator NF-Y. The most prominent mechanism was the inhibition of
carbonic anhydrase isozymes [12e16].
Also, it was found that thiazole and fused thiazole derivatives
are known to possess several biological activities including anti-
cancer activity [17e23]. In the light of these facts, the present
work reports the synthesis of some new thiazolo[4,5-b]pyrane
and thiazolo[4,5-b]pyrano[2,3-d]pyrimidine derivatives bearing
with
g
-radiation, to evaluate their ability to enhance the cytotoxic
-radiation.
* Corresponding author. Tel.: þ966 534292860; fax: þ966 01 4670560.
E-mail address: mmsghorab@yahoo.com (M.M. Ghorab).
activity of
g
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.026

M.M. Ghorab et al. / European Journal of Medicinal Chemistry 46 (2011) 5120e5126
5121
2. Discussion
showed a molecular ion peak m/z at 478 [Mþ] (2.65), with a base
peak at 333 (100). Additionally, the pyrimido derivative 10 was
obtained by the reaction of compound 5 with formamide, where
intramolecular cyclization occurred through elimination of one
molecule of water. IR spectrum of 10 revealed the absence of (C^N)
band, which confirms the cyclization and the formation of the
thiazolo[4,5-b]pyrano[2,3-d]pyrimidine system (Scheme 2).
Treatment of compound 5 with aromatic aldehydes yielded the
corresponding Schiff’s bases 11 and 12. The structures of synthe-
sized compounds 11 and 12 were supported from their microana-
lytical and spectral data. 1H-NMR spectrum in DMSO-d6 of 11 and
12 revealed singlets at 9.6 and 9.9 ppm for (N]CHe), respectively.
Mass spectrum of 11 showed a molecular ion peak m/z at 548 [Mþ]
(0.76), with a base peak at 77 (100). The reactivity of compound 5
toward sulphonyl chlorides in benzene containing a catalyatic
amount of pyridine was also studied. Reaction of compound 5 with
benzene sulphonylchloride and toluene sulphonylchloride afforded
the sulfonamide derivatives 13 and 14, respectively. Mass spectrum
of 13 showed a molecular ion peak m/z at 600 [Mþ] (5.91), with
a base peak at 56 (100). 1H-NMR spectrum in DMSO-d6 of 14
showed singlet at 2.3 ppm for (CH3) group (Scheme 3).
Fusion of compound 5 with urea or thiourea, yielded the 6-oxo-
and 6-thioxo-thiazolo[4,5-b]pyrano[2,3-d]pyrimidine derivatives
15 and 16, respectively. IR spectra of compounds 15 and 16 revealed
the absence of (C^N) band, and the presence of a band at
1650 cmꢀ1 for (C]O) for the 6-oxo- derivative 15, and a band at
1250 cmꢀ1 corresponding to (C]S) for 6-thioxo- derivative 16.
Mass spectrum of 16 showed a molecular ion peak m/z at 519 [Mþ]
(10.00), with a base peak at 77 (100). While refluxing compound 5
with urea in the presence of sodium ethoxide as a catalyst yielded
the ureido derivative 17. IR spectrum of compound 17 showed the
presence of bands at 2201 cmꢀ1 for (C^N) and at 1728 cmꢀ1 for
(C]O). Also, stirring of compound 5 with chloroacetyl chloride in
dimethylformamide at room temperature for 6 h, yielded the
2-chloroacetyl derivative 18. IR spectrum of compound 18 showed
the presence of (C^N) band at 2204 cmꢀ1 and a band at 1653 cmꢀ1
for (C]O). 1H-NMR spectrum in DMSO-d6 revealed singlet at
4.2 ppm corresponding to (CH2Cl) group and a singlet at 8.2 ppm
for the (NH) group (Scheme 4).
2.1. Chemistry
The newly synthesized compounds were obtained starting with
4-(4-oxo-4,5-dihydrothiazol-2ylamino)benzenesulfonamide
3,
which was prepared according to the reported procedure [25], by
refluxing the N-chloroacetyl derivative 2 with ammonium thiocy-
anate in ethanol, through intramolecular cyclization and elimina-
tion of 1 mol of ammonium chloride. Treatment of compound 3
with 2-(4-chlorobenzylidene) malononitrile 4 in ethanol contain-
ing a catalytic amount of piperidine, as a base catalyst, resulted in
intramolecular cyclization affording the 5-amino-6-cyano-thiazolo
[4,5-b]pyrane derivative 5. Structure of compound 5 was confirmed
from its microanalytical and spectral data. IR spectrum of 5
exhibited bands at 3328, 3227, 3200 cmꢀ1 for NH and NH2, and
a band at 2193 cmꢀ1for (C^N). 1H-NMR spectra in DMSO-d6
revealed a singlet at 4.8 ppm for CH and also a singlet at 7.1 ppm for
NH2 (Scheme 1).
The thiazolopyranopyrimidine derivative 6 was obtained by
refluxing compound 5 in formic acid. This reaction proceeded via
condensation followed by elimination of 2 mol of water. IR spec-
trum of compound 6 showed the absence of (C^N) band and the
presence of band at 1709 cmꢀ1 corresponding to the (C]O).When
compound 5 was refluxed in acetic anhydride for 20 min and 10 h,
the monoacetyl derivative 7 and the diacetyl derivative 8 were
obtained, respectively. Structures of compounds 7 and 8 were
confirmed from their microanalytical and spectral data. 1H-NMR
spectrum of 7 in DMSO-d6 was characterized by a singlet at
1.08 ppm (CH3).While, the 1H-NMR spectrum of compound 8
revealed two singlets at 2.4 and 2.7 ppm (2CH3). Stirring of
compound 5 in conc. H2SO4 at room temperature for 6 h caused
partial hydrolysis of the cyano group, yielding the carboxamide
derivative 9. IR spectrum of carboxamide derivative 9 showed the
absence of the (C^N) group and the presence of a band at
1680 cmꢀ1 corresponding to (C]O) group. Mass spectrum of 9
O
Cl
S
N
O
2.2. In vitro anticancer screening
NH2
O
HN
NH
Cl
NH4NCS
EtOH
Cl
The newly synthesized compounds were evaluated for their
in vitro cytotoxic activity against human breast cancer cell line,
MCF7.
DMF
SO2NH2
SO2NH2
SO2NH2
Doxorubicin, which is one of the most effective anticancer
agents, was used as the reference drug in this study. The relation-
ship between surviving fraction and drug concentration was
plotted to obtain the survival curve of breast cancer cell line
(MCF7). The response parameter calculated was the IC50 value,
which corresponds to the concentration required for 50% inhibition
of cell viability. Table 1 shows the in vitro cytotoxic activity of the
synthesized compounds. Most of the synthesized compounds
exhibited significant activity compared to the reference drug.
From the results in Table 1, it was found that compound 12 with
(2)
(3)
(1)
NC
NH
O
NC
Ar
NH2
Ar
O
CN
CN
Ar
S
N
S
N
(4)
NH
NH
IC50 value 34.64
screening, and exhibited a higher cytotoxic activity when compared
with the reference drug doxorubicin (IC50 ¼ 71.8 M), followed by
compounds 5, 6, 7, 9, 10, 11, 13, 16, 17 with IC50 values ranging from
35.79 M to 52.77 M which showed also better activity than
doxorubicin. Compounds 8, 14 of IC50 value (74.3 M, 71.74 M,
respectively) are nearly as active as doxorubicin. While compound
mM was the most potent compound in this
EtOH/Piperidine
m
SO2NH2
m
m
SO2NH2
m
m
Ar= 4-Chlorophenyl
(5)
15 of IC50 value 84.13 mM showed lower activity than that of the
Scheme 1.
reference drug.

5122
M.M. Ghorab et al. / European Journal of Medicinal Chemistry 46 (2011) 5120e5126
NC
N(COCH3)2
O
NC
NHCOCH3
O
H2NOC
Ar
NH2
O
Ar
Ar
S
N
S
N
S
N
NH
NH
NH
SO2NH2
(8)
SO2NH2
SO2NH2
(7)
(9)
HN
S
N
Conc
Ac2O H2SO4
O
N
O
10 h
H2N
Ar
N
O
Ac2O
20 min
Stirring
at R.T
6 h
Ar
N
S
N
HCOOH
HCONH2
(5)
NH
NH
Ar= 4-Chlorophenyl
SO2NH2
SO2NH2
(6)
(10)
Scheme 2.
2.3. Radio-sensitizing evaluation
3. Conclusion
The ability of the most four active compounds, 5, 6, 10 and 12, to
enhance the cell killing effect of -irradiation was studied. From the
obtained results in Table 1, compound 12 showed cytotoxic activity
with IC50 value of 34.64 M, when the cells were subjected to
different concentrations of the compound alone. While when the
cells were subjected to the same concentrations of compound 12,
We report here the synthesis of some new thiazolo[4,5-b]pyrane
and thiazolo[4,5-b]pyrano[2,3-d]pyrimidine derivatives bearing
a sulfonamide moiety. Most of these new compounds exhibited
significant anticancer activity, when compared to doxorubicin as
a reference drug. Moreover, the most four active compounds
showed the ability to sensitize cancer cells to the lethal effects of
ionizing radiation.
g
m
and irradiated with a single dose of
8 Gy, as shown in Table 2, the IC50 value was synergistically
decreased to 17.03 M (Fig. 1).
Similarly, compounds 5, 6 and 10 showed IC50 values of 39.4,
41.6 and 35.72 M, respectively, when used alone (Table 1). The
IC50 values were decreased to 26.25, 28.41 and 23.68 M when the
cells were treated with compound 5, 6 and 10 in combination
g-radiation at a dose level of
m
4. Experimental
m
4.1. Chemistry
m
Melting points are uncorrected and were determined on a Stuart
melting point apparatus (Stuart Scientific, Redhill, UK). Elemental
analysis (C, H, N) were performed on PerkineElmer 2400 analyzer
with a single dose of
and 4).
g-radiation at a dose level of 8 Gy (Fig. 2, 3
O
NC
N
O
C
H
R
NC
HN
O
S
O
R
Ar
Ar
RSO2Cl
RCHO
EtOH
(5)
S
N
S
N
PhH/Pyridine
NH
NH
Ar= 4-Chlorophenyl
SO2NH2
SO2NH2
(13)
,
R= Phenyl
(14) R= 4-Methylphenyl
(11), R= Phenyl
(12)
,
,
R= 4-Chlorophenyl
Scheme 3.

M.M. Ghorab et al. / European Journal of Medicinal Chemistry 46 (2011) 5120e5126
5123
NC
NHCONH2
O
Ar
S
N
NH
X
N
SO2NH2
(17)
NC
NHCOCH2Cl
O
H2N
Ar
NH
O
Ar
O
S
N
S
N
EtOH H2N
NH2
X
NH
NH
H2N
NH2
ClCOCH2Cl
DMF
(5)
R.T
6 hr
Fusion
SO2NH2
SO2NH2
(18)
(15), X= O
(16), X= S
Ar= 4-Chlorophenyl
Scheme 4.
(PerkineElmer, Norwalk, CT, USA) at the microanalytical laborato-
ries of the Faculty of Science, Cairo University. All compounds were
within ꢁ0.4% of the theoretical values. The IR spectra (KBr) were
measured on Shimadzu IR 110 spectrophotometer (Shimadzu,
Koyoto, Japan), 1H-NMR spectra were obtained on a Bruker proton
NMR-Avance 300 (300 MHz) (Bruker, Munich, Germany), in DMSO-
d6 as a solvent, using tetramethylsilane (TMS) as internal standard.
Mass spectra were run on HP Model MS-5988 (Hewlett Packard,
Palo, Alto, California, USA). All reactions were monitored by thin
layer chromatograph (TLC) using precoated Aluminium sheets
Silica gel Merck 60 F254 and were visualized by UV lamp (Merck,
Darmstadt, Germany).
4.1.2. 4-(5-Amino-7-(4-chlorophenyl)-6-cyano-7H- thiazolo[4,5-b]
pyrane-2-yl amino)benzenesulfonamide (5)
A mixture of 4-(4-oxo-4, 5-dihydrothiazol-2-ylamino) benze-
nesulfonamide 3 (0.27 g, 0.001 mol) and 2-(4-chlorobenzylidene)
malononitrile 4 (0.18 g, 0.001 mol) in ethanol (20 mL) containing
5 drops of piperidine was refluxed for 5 h. The reaction mixture
was cooled then poured onto cold water, the obtained solid was
crystallized from dioxane to give compound 5: yield, 77%; m.p.
200e202 ꢂC; IR, (KBr, cmꢀ1): 3328, 3227, 3200 (NH, NH2), 3058
(CH arom.), 2939, 2870 (CH aliph.), 2193 (C^N), 1626 (C]N),
1330, 1156 (SO2). 1H-NMR (DMSO-d6)
exchangeable with D2O], 4.8 [s, 1H, CH pyrane], 7.1 [s, 2H, NH2,
exchangeable with D2O], 7.3e8.0 [m, 10H, ArH SO2NH2,
exchangeable with D2O]. 13C-NMR (DMSO-d6)
: 25.6, 56.8, 115.5,
d: 4.6 [s, 1H, NH,
þ
4.1.1. 4-(4-Oxo-4,5-dihydrothiazol-2-ylamino)benzenesulfonamide
d
(3)
117.5, 128.7, 129.1, 130.4, 131.3, 132.8, 143.7, 157.2, 170.2. MS m/z
Prepared according to the reported procedure [25].
(%): 460 [Mþ] (11.95), 56 (100).Anal. Calcd. For C19H14ClN5O3S2
Table 1
In vitro anticancer screening of the synthesized compounds against human breast cell line (MCF7).
Compound
Compound Concentration (
mM)
IC50 (mM)
10
25
50
100
Surviving Fraction (Means ꢁ SE)a
Doxorubicin
5
6
7
8
9
10
11
12
13
14
15
16
17
18
0.721 ꢁ 0.02
0.848 ꢁ 0.03
0.794 ꢁ 0.01
0.801 ꢁ 0.01
0.913 ꢁ 0.01
0.815 ꢁ 0.02
0.62 ꢁ 0.03
0.93 ꢁ 0.04
0.634 ꢁ 0.02
0.697 ꢁ 0.02
0.92 ꢁ 0.05
0.9 ꢁ 0.02
0.546 ꢁ 0.02
0.461 ꢁ 0.01
0.168 ꢁ 0.01
0.204 ꢁ 0.01
0.183 ꢁ 0.01
0.234 ꢁ 0.01
0.169 ꢁ 0.01
0.186 ꢁ 0.01
0.295 ꢁ 0.01
0.257 ꢁ 0.01
0.239 ꢁ 0.01
0.2 ꢁ 0.01
0.494 ꢁ 0.03
0.214 ꢁ 0.01
0.175 ꢁ 0.01
0.252 ꢁ 0.01
0.617 ꢁ 0.03
0.210 ꢁ 0.01
0.207 ꢁ 0.01
0.324 ꢁ 0.01
0.304 ꢁ 0.01
0.361 ꢁ 0.01
0.6 ꢁ 0.03
71.8
39.4
41.6
41.91
74.3
0.360 ꢁ 0.01
0.515 ꢁ 0.03
0.447 ꢁ 0.01
0.511 ꢁ 0.02
0.499 ꢁ 0.02
0.454 ꢁ 0.02
0.206 ꢁ 0.01
0.240 ꢁ 0.01
0.492 ꢁ 0.02
0.52 ꢁ 0.02
0.56 ꢁ 0.01
0.639 ꢁ 0.04
0.717 ꢁ 0.02
0.531 ꢁ 0.01
41.8
35.79
45.22
34.64
47.46
71.74
84.13
52.77
49.80
43.75
0.265 ꢁ 0.01
0.241 ꢁ 0.01
0.309 ꢁ 0.01
0.25 ꢁ 0.01
0.654 ꢁ 0.02
0.325 ꢁ 0.02
0.228 ꢁ 0.01
0.252 ꢁ 0.01
0.814 ꢁ 0.04
0.701 ꢁ 0.04
0.702 ꢁ 0.03
a
Each value is the mean of three values ꢁ Standard Error.

5124
M.M. Ghorab et al. / European Journal of Medicinal Chemistry 46 (2011) 5120e5126
Table 2
In vitro anticancer screening of compounds 5, 6, 10 and 12 against human breast cell line (MCF7) in combination with g-radiation.
Compd. no.
Control
Irradiated (8 Gy)
Compound Concentration (
m
M) þ Irradiation (8 Gy)
IC50 (mM)
10
25
50
100
Surviving Fraction (Means ꢁ SE)a
1.000
1.000
1.000
1.000
5
6
10
12
0.927 ꢁ 0.02b
0.927 ꢁ 0.02b
0.927 ꢁ 0.02b
0.927 ꢁ 0.02b
0.706 ꢁ 0.02b
0.650 ꢁ 0.02b
0.504 ꢁ 0.02b
0.462 ꢁ 0.02b
0.203 ꢁ 0.01b
0.371 ꢁ 0.01b
0.341 ꢁ 0.02b
0.124 ꢁ 0.01b
0.062 ꢁ 0.01b
0.060 ꢁ 0.01b
0.064 ꢁ 0.01b
0.143 ꢁ 0.01b
0.097 ꢁ 0.01b
0.040 ꢁ 0.01b
0.086 ꢁ 0.01b
0.182 ꢁ 0.01b
26.25
28.41
23.68
17.03
a
Each value is the mean of three values ꢁ Standard Error.
Significant difference from control group at p < 0.001.
b
(459.93): C, 49.62; H, 3.07; N, 15.23. Found: C, 49.70; H, 3.10; N,
15.34.
4.1.5. N-acetyl-N-(7-(4-chlorophenyl)-6-cyano-2-(4-sulfamoylp-
henylamino)-7H- thiazolo[4,5-b]pyrane-5-yl)acetamide (8)
A solution of compound 5 (0.91 g, 0.002 mol) in acetic anhydride
(20 mL) was refluxed for 10 h, the reaction mixture was then
concentrated, the solid separated was crystallized from ethanol to
give compound 8: Yield, 70%; m.p. 234e236 ꢂC; IR, (KBr, cmꢀ1):
3253, 3209 (NH, NH2), 3097 (CH arom.), 2972, 2855 (CH aliph.),
2205 (C^N), 1721,1669 (2 C]O), 1590 (C]N), 1364, 1162 (SO2).
4.1.3. 4-(9-(4-Chlorophenyl)-8-oxo-7, 9-dihydrothiazolo[4,5-b]
pyrano[2,3-d]pyrimidin-2-ylamino) benzenesulfonamide (6)
A solution of compound 5 (0.91 g, 0.002 mol) in formic acid
(30 mL) was refluxed for 5 h, the reaction mixture was cooled then
poured onto cold water, the obtained solid was recrystallized from
dioxane to give compound 6: Yield, 72%; m.p. 172e174 ꢂC; IR, (KBr,
cmꢀ1): 3310, 3241, 3186 (NH, NH2), 3053 (CH arom.), 2942, 2850
(CH aliph.), 1709 (C]O), 1599 (C]N), 1370, 1155 (SO2). 1H-NMR
1H-NMR (DMSO-d6)
d: 2.4, 2.7 [2s, 6H, 2COCH3] 4.6 [s, 1H, NH,
exchangeable with D2O], 4.8 [s, 1H, CH pyrane], 7.3e8.0 [m, 10H,
AreH þ SO2NH2, exchangeable with D2O]. MS m/z (%): 543 [M-1]
(3.19), 55 (100). Anal. Calcd. For C23H18ClN5O5S2 (544.00): C,
50.78; H, 3.34; N, 12.87. Found: C, 50.65; H, 3.20; N, 12.70.
(DMSO-d6) d: 4.6 [s, 1H, NH, exchangeable with D2O], 4.8 [s, 1H, CH
pyrano], 7.1 [s, 1H, CH pyrimidine], 7.3e8.0 [m, 10H,
AreH þ SO2NH2, exchangeable with D2O], 8.1 [s,1H, NH pyrimidine,
exchangeable with D2O]. 13C-NMR (DMSO-d6)
d: 28.0, 103.3, 113.1,
4.1.6. 5-Amino-7-(4-chlorophenyl)-2-(4-sulfamoylphenylamino)-
7H- thiazolo[4,5-b]pyrane -6-carboxamide (9)
125.9, 128.1, 129.3, 130.5, 131.0, 131.9, 134.9, 143.7, 155.89, 162.2. MS
m/z (%): 488 [Mþ] (57.77), 449 (100). Anal. Calcd. For
C20H14ClN5O4S2 (487.94): C, 49.23; H, 2.89; N, 14.35. Found: C,
49.34; H, 3.10; N, 14.41.
A solution of compound 5 (0.9 g, 0.002 mol) in conc. H2SO4
(10 mL) was stirred for 6 h at room temperature, then the reaction
mixture was poured onto cold water. The obtained solid was
recrystallized from ethanol to give compound 9: Yield, 77%; m.p.
290e292 ꢂC; IR, (KBr, cmꢀ1): 3342, 3232 (NH, NH2), 3090 (CH
arom.), 2981, 2866 (CH aliph.), 1680 (C]O), 1590 (C]N), 1334, 1157
4.1.4. N-(7-(4-chlorophenyl)-6-cyano-2-(4-sulfamoylphenyl-
amino)-7H- thiazolo[4,5-b]pyrane-5-yl)acetamide (7)
A solution of compound 5 (0.91 g, 0.002 mol) in acetic anhydride
(20 mL) was refluxed for 20 min, the reaction mixture was then
concentrated, the solid separated was recrystallized from ethanol
to give compound 7: Yield, 75%; m.p. 162e164 ꢂC; IR, (KBr, cmꢀ1):
3341, 3224 (NH, NH2), 3090 (CH arom.), 2970, 2860 (CH aliph.),
2205 (C^N), 1719 (C]O), 1590 (C]N), 1364, 1163 (SO2). 1H-NMR
(SO2). 1H-NMR (DMSO-d6)
d: 3.9 [s, 1H, NH, exchangeable with
D2O], 4.5 [s, 1H, CH pyrane], 6.7 [s, 2H, NH2, exchangeable with
D2O], 7.3e8.0 [m, 10H, Ar-H þ SO2NH2, exchangeable with D2O], 8.2
[s, 2H, CONH2, exchangeable with D2O] .MS m/z (%): 478 [Mþ]
(2.65), 333 (100). Anal. Calcd. For C19H16ClN5O4S2 (477.94): C, 47.75;
H, 3.37; N, 14.65. Found: C, 47.86; H, 3.43; N, 14.75.
(DMSO-d6) d: 1.08 [s, 3H, COCH3], 4.6 [s, 1H, NH, exchangeable with
D2O], 4.8 [s, 1H, CH pyrane], 7.3e8.0 [m, 10H, AreH þ SO2NH2,
exchangeable with D2O], 8.2 [s, 1H, NHCO, exchangeable with D2O].
Anal.Calcd.For C21H16ClN5O4S2 (501.97): C, 50.25; H, 3.21; N, 13.95.
Found: C, 50.36; H, 3.33; N, 14.05.
4.1.7. 4-(8-Amino-9(4-chlorophenyl)-9H- thiazolo[4,5-b]pyrano
[2,3-d]pyrimidine-2-ylamino) benzenesulfonamide (10)
A solution of compound 5 (0.91 g, 0.002 mol) in formamide
(30 mL) was refluxed for 5 h, the reaction mixture was cooled and
Fig. 1. Survival curve for MCF7 cell line for compound 5 alone or in combination with
Fig. 2. Survival curve for MCF7 cell line for compound 6 alone or in combination with
g
- radiation (8 Gy).
g- radiation (8 Gy).

M.M. Ghorab et al. / European Journal of Medicinal Chemistry 46 (2011) 5120e5126
5125
4.1.8.2. 4-(5-(4-Chlorobenzylideneamino)-7-(4-chlorophenyl)-6-
cyano-7H- thiazolo[4,5-b]pyrane -2-ylamino)benzenesulfonamide
(12). Yield, 78%; m.p. 223e225 ꢂC; IR, (KBr, cmꢀ1): 3319, 3246, 3191
(NH, NH2), 3070 (CH arom.), 2940, 2830 (CH aliph.), 2202 (C^N),
1581 (C]N), 1331, 1153 (SO2). 1H-NMR (DMSO-d6)
d: 3.8 [s, 1H, NH,
exchangeable with D2O], 4.5 [s, 1H, CH pyrane], 7.3e8.0 [m, 14H,
AreH þ SO2NH2, exchangeable with D2O], 9.9 [s, 1H, N]CH]. 13C-
NMR (DMSO-d6) d: 31.5, 113.8, 117.3, 127.2, 129.1, 131.1, 132.5, 133.1,
134.4, 143.7, 157.7, 163.7.MS m/z (%): 582 [Mþ] (1.82), 101 (100).
Anal. Calcd. For C26H17Cl2N5O3S2 (582.48): C, 53.61; H, 2.94; N,
12.02. Found: C, 53.74; H, 3.15; N, 12.16.
4.1.9. 4-(5-(Benzylideneamino)-7-(4-chlorophenyl)-6-cyano-7H-
thiazolo[4,5-b]pyrane -2-ylamino)benzenesulfonamide(13) and 4-
(7-(4-chlorophenyl)-6-cyano-5-(4-methylbenzylideneamino)-7H-
thiazolo[4,5-b]pyrane -2-ylamino)benzenesulfonamide (14)
A mixture of compound 5 (0.91 g, 0.002 mol) and the appro-
priate sulphonyl chloride derivative (0.002 mol) in benzene con-
taining 3 drops of pyridine was refluxed for 8 h. The obtained solid
was recrystallized from ethanol to give compounds 13 and 14
respectively.
Fig. 3. Survival curve for MCF7 cell line for compound 10 alone or in combination with
- radiation (8 Gy).
g
then poured onto cold water, the obtained solid was crystallized
from dioxane to give compound 10: Yield, 75%; m.p. 250e252 ꢂC;
IR, (KBr, cmꢀ1): 3330, 3270, 3209 (NH, NH2), 3080 (CH arom.), 2989,
2870 (CH aliph.), 1619, 1595 (2 C]N), 1398, 1157 (SO2). 1H-NMR
4.1.9.1. 4-(5-(Benzylideneamino)-7-(4-chlorophenyl)-6-cyano-7H-
thiazolo[4,5-b]pyrane -2-ylamino)benzenesulfonamide (13). Yield,
80%; m.p. 278e280 ꢂC; IR, (KBr, cmꢀ1): 3325, 3248, 3220 (NH, NH2),
3046 (CH arom.), 2930, 2870 (CH aliph.), 2194 (C^N), 1634 (C]N),
(DMSO-d6) d: 3.9 [s, 1H, NH, exchangeable with D2O], 4.8 [s, 1H, CH
pyrano], 7.3e8.0 [m, 10H, Ar-H þ SO2NH2, exchangeable with D2O],
8.2 [s, 1H, CH pyrimidine], 10.2 [s, 2H, NH2, exchangeable with D2O].
MS m/z (%): 487 [Mþ] (3.50), 125 (100). Anal. Calcd. For
C20H15ClN6O3S2 (486.95): C, 49.33; H, 3.10; N, 17.26. Found: C,
49.40; H, 3.24; N, 17.35.
1325, 1156 (SO2). 1H-NMR (DMSO-d6)
d: 3.8 [s, 1H, NH, exchange-
able with D2O], 4.5 [s, 1H, CH pyrane], 7.3e8.0 [m, 15H,
AreH þ SO2NH2, exchangeable with D2O], 12.0 [s, 1H, NHSO2,
exchangeable with D2O]. MS m/z (%): 600 [Mþ] (5.91), 56 (100).
Anal. Calcd. For C25H18ClN5O5S3 (600.09): C, 50.04; H, 3.02; N, 11.67.
Found: C, 50.18; H, 3.17; N, 11.76.
4.1.8. 4-(5-(Arylideneamino)-7-(4-chlorophenyl)-6-cyano-7H-
thiazolo[4,5-b]pyrane -2-ylamino) benzenesulfonamide (11 and 12)
A mixture of compound 5 (0.91 g, 0.002 mol) and benzaldehyde
or p-chlorobenzaldehyde (0.002 mol) in 20 mL ethanol was
refluxed for 5 h. The reaction mixture was then poured onto cold
water, the obtained solid was recrystallized from dioxane to give
compounds 11and 12 respectively.
4.1.9.2. 4-(7-(4-Chlorophenyl)-6-cyano-5-(4-methylbenzylidene-
amino)-7H- thiazolo[4,5-b]pyrane -2-ylamino)benzenesulfonamide
(14). Yield, 75%; m.p. 280e282 ꢂC; IR, (KBr, cmꢀ1): 3325, 3250,
3215 (NH, NH2), 3090 (CH arom.), 2950, 2860 (CH aliph), 2203
(C^N), 1629 (C]N), 1327, 1157 (SO2). 1H-NMR (DMSO-d6)
d: 2.3 [s,
4.1.8.1. 4-(5-(Benzylideneamino)-7-(4-chlorophenyl)-6-cyano-7H-
thiazolo[4,5-b]pyrane -2-ylamino) benzenesulfonamide (11). Yield,
80%; m.p. 260e262 ꢂC; IR, (KBr, cmꢀ1): 3335, 3295, 3231 (NH, NH2),
3080 (CH arom.), 2947, 2850 (CH aliph.), 2194 (C^N), 1594 (C]N),
3H, CH3], 3.6 [s, 1H, NH, exchangeable with D2O], 4.7 [s, 1H, CH
pyrane], 7.3e8.0 [m, 14H, AreH þ SO2NH2, exchangeable with D2O],
11.8 [s, 1H, NHSO2, exchangeable with D2O]. MS m/z (%): 613 [M-1]
(11.34), 63 (100).Anal. Calcd. For C26H20ClN5O5S3 (614.12): C, 50.85;
H, 3.28; N, 11.40. Found: C, 50.63; H, 3.17; N, 11.30.
1327,1153 (SO2). 1H-NMR (DMSO-d6)
d: 3.8 [s,1H, NH, exchangeable
with D2O], 4.5 [s, 1H, CH pyrane], 7.3e8.0 [m, 15H, AreH þ SO2NH2,
exchangeable with D2O], 9.6 [s, 1H, N]CH]. MS m/z (%): 548 [Mþ]
(0.76), 77 (100). Anal. Calcd. For C26H18ClN5O3S2 (548.04): C, 56.98;
H, 3.31; N, 12.78. Found: C, 56.81; H, 3.20; N, 12.58.
4.1.10. 4-(8-Amino-9(4-chlorophenyl)-6-oxo-5,9-dihydrothiazolo
[4,5-b]pyrano[2,3-d]pyrimidine-2-yl amino) benzenesulfonamide
(15) and 4-(8-amino-9(4-chlorophenyl)-6-thioxo-5,9-
dihydrothiazolo[4,5-b]pyrano[2,3-d]pyrimidine-2-yl amino)
benzenesulfonamide (16)
A mixture of compound 5 (0.91 g, 0.002 mol) and urea or
thiourea (0.002 mol) was fused together in an oil bath at 250 ꢂC for
15 min, the fused mass was dissolved in dimethylformamide and
poured onto cold water, the solid obtained was recrystallized from
ethanol to give compounds 15 and 16 respectively:
4.1.10.1. 4-(8-Amino-9(4-chlorophenyl)-6-oxo-5,9-dihydrothiazolo
[4,5-b]pyrano[2,3-d]pyrimidine-2-yl amino) benzenesulfonamide
(15). Yield, 70%; m.p. 310e312 ꢂC; IR, (KBr, cmꢀ1): 3327, 3260, 3184
(NH, NH2), 3080 (CH arom.), 2930, 2860 (CH aliph), 1690 (C]O),
1620 (C]N), 1327, 1153 (SO2). 1H-NMR (DMSO-d6)
d: 3.6 [s, 1H, NH,
exchangeable with D2O], 4.8 [s, 1H, CH pyrane], 6.9 [s, 1H, NH
pyrimidine, exchangeable with D2O], 7.3e8.0[m, 10H,
AreH þ SO2NH2], 10.6 [s, 2H, NH2, exchangeable with D2O]. MS m/z
(%): 503 [Mþ] (4.92), 51 (100). Anal. Calcd. For C20H15ClN6O4S2
(502.95): C, 47.76; H, 3.01; N,16.71. Found: C, 47.61; H, 2.88; N,16.65.
Fig. 4. Survival curve for MCF7 cell line for compound 12 alone or in combination with
g
- radiation (8 Gy).

5126
M.M. Ghorab et al. / European Journal of Medicinal Chemistry 46 (2011) 5120e5126
4.1.10.2. 4-(8-Amino-9(4-chlorophenyl)-6-thioxo-5,9-dihy-
The relation between surviving fraction and drug concentration
was plotted to get the survival curve for breast tumor cell line after
the specified time. The molar concentration required for 50%
inhibition of cell viability (IC50) was calculated and compared to the
reference drug doxorubicin (CAS, 25316-40-9). The surviving frac-
tions were expressed as means ꢁ standard error and the results are
given in Table 1.
drothiazolo[4,5-b]pyrano[2,3-d]pyrimidine-2-yl amino) benzene-
sulfonamide (16). Yield, 76%; m.p. 300e302 ꢂC; IR, (KBr, cmꢀ1):
3315, 3270, 3174 (NH, NH2), 3080 (CH arom.), 2950, 2870 (CH
aliph.), 1596 (C]N), 1250 (C]S), 1330, 1152 (SO2). MS m/z (%): 519
[Mþ] (10.00), 77 (100). Anal. Calcd. For C20H15ClN6O3S3 (519.02): C,
46.28; H, 2.91; N, 16.19. Found: C, 46.38; H, 3.18; N, 15.96.
4.1.11. 4-(7-(4-Chlorophenyl)-6-cyano-5-ureido-7H- thiazolo[4,5-
b]pyrane -2-yl amino)benzenesulfonamide (17)
4.3. Radio-sensitizing evaluation
A mixture of compound 5 (0.91 g, 0.002 mol) and urea (0.12 g,
0.002 mol) and sodium ethoxide (0.03 g, 0.002 mol) in ethanol
(20 mL) was refluxed for 5 h. The reaction mixture was then poured
onto cold water, the obtained solid was crystallized from dioxane to
give compound 17: Yield, 80%; m.p. 240e242 ꢂC; IR, (KBr, cmꢀ1):
3338, 3294, 3250 (NH, NH2), 3095 (CH arom.), 2970, 2860 (CH
aliph.), 2201 (C^N), 1728 (C]O), 1560 (C]N), 1331, 1156 (SO2).
The most potent compounds resulted from the in vitro anti-
cancer screening, the thiazolo[4,5-b]pyrane derivatives 5 and 12
and the thiazolo[4,5-b]pyrano[2,3-d]pyrimidine derivatives 6 and
10, were selected to be reevaluated again for their in vitro anti-
cancer activity in combination with
conducted to evaluate the ability of these compounds to enhance
the cell killing effect of -radiation.
Cells were subjected to a single dose of
g-radiation. This study was
g
1H-NMR (DMSO-d6)
d: 3.8 [s, 1H, NH, exchangeable with D2O], 4.5
g-radiation at a dose
[s, 1H, CH pyrane], 6.5 [s, 1H, NHCO, exchangeable with D2O],
7.3e8.0 [m, 10H, Ar-H þ SO2NH2], 8.3 [s, 2H, CONH2, exchangeable
with D2O]. MS m/z (%): 502 [Mþ] (7.63), 55 (100). Anal. Calcd. For
C20H15ClN6O4S2 (502.95): C, 47.76; H, 3.01; N,16.71. Found: C, 47.88;
H, 3.13; N, 16.85.
level of 8 Gy with a dose rate of 2 Gy/min. Irradiation was per-
formed in the National Cancer Institute, Cairo University, using
Gamma cell-40 (60Co) source.
The surviving fractions were expressed as means ꢁ standard
error. The results were analyzed using 1-way ANOVA test and given
in Table 2.
4.1.12. 2-Chloro-N-(7-(4-chlorophenyl)-6-cyano-2-(4-sulfamoyl-
phenylamino)-7H- thiazolo[4,5-b]pyrane -5-yl)acetamide (18)
A mixture of compound 5 (0.91 g, 0.002 mol) and chloroacetyl-
chloride (0.22 g, 0.002 mol) was stirred in dimethylformamide for
6 h at room temperature, then the reaction mixture was poured
onto cold water. The obtained solid was crystallized from ethanol to
give compound 18: Yield, 68%; m.p. 164e166 ꢂC; IR, (KBr, cmꢀ1):
3339, 3244, 3194 (NH, NH2), 3089 (CH arom.), 2970, 2860 (CH
aliph.), 2204 (C^N), 1690 (C]O), 1615 (C]N), 1331, 1155 (SO2), 750
References
[1] J. Drews, Science 287 (2000) 1960e1964.
[2] A.E. Boyd 3rd, Diabetes 37 (1988) 847e850.
[3] C.T. Supuran, A. Scozzafava, Expert Opin. Ther. Pat 10 (2000) 575e600.
[4] T.H. Maren, Annu. Rev. Pharmacol. Toxicol. 16 (1976) 309e327.
[5] C.T. Supuran, A. Scozzafava, Curr. Med. Chem. Immunol. Endocr. Metabol
Agents 1 (2001) 61e97.
[6] C.W. Thornber, Chem. Soc. Rev. 8 (1979) 563e580.
[7] F. Abbate, A. Casini, T. Owa, A. Scozzafava, C.T. Supuran, Bioorg. Med. Chem.
Lett. 14 (2004) 217e223.
[8] M.M. Ghorab, E. Noaman, M.M. Ismail, H.I. Heiba, Y.A. Ammar, M.Y. Sayed,
Arzneimittelforschung 56 (2006) 405e413.
[9] M.M. Ismail, M.M. Ghorab, E. Noaman, Y.A. Ammar, H.I. Heiba, M.Y. Sayed,
Arzneimittelforschung 56 (2006) 301e308.
[10] S.A. Rostom, Bioorg. Med. Chem. 14 (2006) 6475e6485.
[11] C.T. Supuran, A. Casini, A. Mastrolorenzo, A. Scozzafava, Mini-Rev Med. Chem.
4 (2004) 625e632.
[12] A.J. Kivela, J. Kivela, J. Saarnio, S. Parkkila, World J. Gastroenterol. 11 (2005)
155e163.
(C-Cl). 1H-NMR (DMSO-d6)
d: 3.8 [s, 1H, NH, exchangeable with
D2O], 4.2 [s, 2H, CH2Cl], 4.7 [s, 1H, CH pyrane], 7.3e8.0 [m, 10H,
Ar-H þ SO2NH2], 8.2 [s, 1H, NHCO, exchangeable with D2O]. MS m/z
(%): 537 [Mþ] (0.53), 90 (100). Anal. Calcd. For C21H15Cl2N5o4S2
(536.41): C, 47.02; H, 2.82; N,13.06. Found: C, 47.26; H, 3.04; N,13.18.
4.2. In vitro anticancer screening
Human tumor breast cell line (MCF7) was used in this study. The
cytotoxic activity was measured in vitro for the newly synthesized
compounds using the Sulfo-Rhodamine B stain (SRB) assay using
the method of Skehan et al. [26]. The in vitro anticancer screening
was done by the pharmacology unit at the National Cancer Insti-
tute, Cairo University.
[13] C.T. Supuran, Nat. Rev. Drug Discov. 7 (2008) 168e181.
[14] C.T. Supuran, A. Scozzafava, Bioorg. Med. Chem. 15 (2007) 4336e4350.
[15] C.T. Supuran, A. Scozzafava, A. Casini, Med. Res. Rev. 23 (2003) 146e189.
[16] A. Casini, A. Scozzafava, A. Mastrolorenzo, C.T. Supuran, Curr. Cancer Drug
Targets 2 (2002) 55e75.
[17] A. Aliabadi, F. Shamsa, S.N. Ostad, S. Emami, A. Shafiee, J. Davoodi,
A. Foroumadi, Eur. J. Med. Chem. 45 (2010) 5384e5389.
[18] E.L. Luzina, A.V. Popov, Eur. J. Med. Chem. 44 (2009) 4944e4953.
[19] V. Zaharia, A. Ignat, N. Palibroda, B. Ngameni, V. Kuete, C.N. Fokunang,
M.L. Moungang, B.T. Ngadjui, Eur. J. Med. Chem. 45 (2010) 5080e5085.
[20] H. Boshi, S.J. Zhang, Q.F. Ge, D.W. Guo, C.M. Cai, W.X. Hu, Biorg. Med.Chem.
Lett. 20 (2010) 6555e6559.
Cells were plated in 96-multiwell plate (104cells/well) for 24 h
before treatment with the compound(s) to allow attachment of cell
to the wall of the plate. Test compounds were dissolved in dime-
thylsulfoxide. Different concentrations of the compound under test
[21] D. Havrylyuk, L. Mosula, B. Zimenkovsky, O. Vasylenko, A. Gzella, R. Lesyk, Eur.
J. Med. Chem. 45 (2010) 5012e5021.
(10, 25, 50, and 100 mM) were added to the cell monolayer. Tripli-
[22] Z.Y. Liu, Y.M. Wang, Z.R. Li, J.D. Jiang, W. Boykin, Biorg. Med. Chem. Lett. 19
(2009) 5661e5664.
[23] R. Lesyk, B. Zimenkousky, D. Atamanyuk, F. Jensen, K.K. Konononicz, A. Gzella,
Biorg. Med. Chem 14 (2006) 5230e5240.
[24] Y. Nishimura, Int. J. Clin. Oncol. 9 (2004) 414e420.
[25] M. Siavosh, S. Andreas, H. Heymo, E. Emerich, B. Thomas, S. Mathias,
M. Thomas, S.F. Josef, B.L. Thomas, J. Med. Chem. 49 (2006) 5769e5776.
[26] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, Cancer
Inst. 82 (1990) 1107e1112.
cate wells were prepared for each individual concentration.
Monolayer cells were incubated with the compound(s) for 48 h at
37 ꢂC and in atmosphere of 5% CO2. After 48 h, cells were fixed,
washed and stained for 30 min with 0.4% (wt/vol) SRB dissolved in
1% acetic acid. Excess unbound dye was removed by four washes
with 1% acetic acid and attached stain was recovered with
TriseEDTA buffer. Color intensity was measured in an ELISA reader.