Synthesis of some new thiazolopyrane and thiazolopyranopyrimidine derivatives bearing a sulfonamide moiety for evaluation as anticancer and radiosensitizing agents

Article abstract of DOI:10.1007/s00044-011-9751-9

A new series of thiazolopyrane 5a-d, 11-13 and thiazolopyranopyrimidine 6-10, 7b, 8b, and 14 derivatives bearing a sulfonamide moiety were designed and synthesized. The molecular design was performed using molecular operating environment software to predict the binding mode of the proposed compounds on hCAII. All the newly synthesized compounds were evaluated for their in vitro anticancer activity against human liver cancer cell line in which hCAII is overexpressed. Compounds 8b and 14 showed higher activities compared with doxorubicin as a positive control. The radiosensitizing ability of the promising compounds 3, 7a, 8b, 12, and 14 was studied which showed an increase in the cell killing effect of γ-radiation after combination with them. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9751-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2395–2407
DOI 10.1007/s00044-011-9751-9
ORIGINAL RESEARCH
Synthesis of some new thiazolopyrane
and thiazolopyranopyrimidine derivatives bearing a sulfonamide
moiety for evaluation as anticancer and radiosensitizing agents
•
Dalal A. Abou El Ella Mostafa M. Ghorab
•
•
Helmy I. Heiba Aiten M. Soliman
Received: 25 March 2011 / Accepted: 18 July 2011 / Published online: 9 August 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A new series of thiazolopyrane 5a–d, 11–13
and thiazolopyranopyrimidine 6–10, 7b, 8b, and 14
derivatives bearing a sulfonamide moiety were designed
and synthesized. The molecular design was performed
using molecular operating environment software to predict
the binding mode of the proposed compounds on hCAII.
All the newly synthesized compounds were evaluated for
their in vitro anticancer activity against human liver cancer
cell line in which hCAII is overexpressed. Compounds 8b
and 14 showed higher activities compared with doxorubi-
cin as a positive control. The radiosensitizing ability of the
promising compounds 3, 7a, 8b, 12, and 14 was studied
which showed an increase in the cell killing effect of
c-radiation after combination with them.
Introduction
Sulfonamides possess many biological activities, such as
antibacterial (Drews, 2000), hypoglycemic (Boyd, 1988),
diuretic (Supuran and Scozzafava, 2000; Maren, 1976),
anti-carbonic anhydrase (Supuran and Scozzafava, 2000;
Supuran and Scozzafava, 2001), and antithyroid (Thornber,
1979). Recently, sulfonamide derivatives have been
reported to show substantial antitumor activity in vitro and/
or in vivo (Abbate et al., 2004; Ghorab et al., 2006;
Al-Said et al., 2011; Rostom, 2006; Supuran et al., 2004).
E7070 (I) and E7010 (II), are examples for anticancer
sulfonamides in advanced clinical trials (Kivela et al.,
2005) (Fig. 1).
It has been known that sulfonamides may act as anti-
tumor agents through a variety of mechanisms, such as cell
cycle perturbation in the G1 phase, disruption of microtu-
bules assembly, angiogenesis inhibition, matrix metallo-
proteinase (MMP) inhibition (Kivela et al., 2005; Supuran,
2008; Supuran and Scozzafava, 2007; Supuran et al.,
2003). The most prominent mechanism was the inhibition
of carbonic anhydrase isozymes (Casini et al., 2002).
CAs are involved in pH regulation, secretion of elec-
trolytes, respiration (Pastorekova et al., 2004; Saczewski
et al., 2006), and biosynthetic reactions which require CO2/
bicarbonate as substrate, such as gluconeogenesis, lipo-
genesis, ureagenesis, and pyrimidine synthesis (Saczewski
et al., 2008). Sulfonamide CA inhibitors may reduce the
provision of bicarbonate for the synthesis of nucleotides and
other cell components such as membrane lipids leading to
tumor inhibition (Chegwidden et al., 2000). In addition,
thiazole (Lv et al., 2010), fused pyrane (Amr et al., 2006),
and fused pyrimidine derivatives (Ghorab et al., 2010) are
known to possess several biological activities including
anticancer activity. Also, it has been reported that CA II is
Keywords Thiazole Á Thiazolopyrane Á
Thiazolopyranopyrimidine Á Sulfonamide Á Anticancer Á
Radiosensitizing activities
D. A. Abou El Ella
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Ain Shams University, Cairo, Egypt
M. M. Ghorab (&)
Medicinal, Aromatic and Poisonous Plants Research Center
(MAPPRC), College of Pharmacy, King Saud University,
P.O. Box 2457, Riyadh 11451, Saudi Arabia
e-mail: mmsghorab@yahoo.com
H. I. Heiba Á A. M. Soliman
Department of Drug Radiation Research, National Center for
Radiation Research and Technology, Nasr City, Cairo, Egypt
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Med Chem Res (2012) 21:2395–2407
CA, and the sulfonamide is attached to a scaffold which is
usually a benzene ring. The side chain might posses a hydro-
philic link that is able to interact with the hydrophilic part of
the active site and a hydrophobic moiety which can interact
with the hydrophobic part of the CA active site (Fig. 2).
Synthesis
Interaction of sulfanilamide 1 with chloroacetyl chloride
furnished, 2-chloro-N-(4-sulfamoylphenyl) acetamide 2
(Donkor et al., 1998) which upon reaction with ammonium
thiocyanate yielded a strategic starting material 4-(4-oxo-4,
5-dihydrothiazol-2-ylamino) benzenesulfonamide 3 (Siav-
osh et al., 2006) (Scheme 1).
Fig. 1 Sulfonamide containing compounds (E7070 and E7010) in
advanced clinical trials as anticancer agents
Reaction of compound 3 with different arylidine mal-
ononitriles 4a–d furnished the corresponding thiazolopy-
rane derivatives 5a–d (Scheme 2).
overexpressed in certain cell lines such as human liver
cancer cell line (HEPG2) (Chegwidden and Spencer, 1995).
Therefore, the first aim in this investigation is to prepare
some new compounds having anticancer activity targeting
hCAII isozymes. The second aim is to test the influence of
the substitution of thiazolopyrane and thiazolopyranopyr-
imidine moieties on the anticancer activity and to study
their structure–activity relationship.
The structures of compounds 5a–d were confirmed by
micro-analytical and spectral data, where the IR spectra
showed the disappearance of carbonyl group of compound
3 and the appearance of bands at 2194–2220 cm-1 attrib-
1
uted to the presence of (C:N) group. In addition, the H-
NMR spectra showed two singlets one at 4.0–4.1 ppm and
the other at 12.0–12.5 ppm attributed to the presence of CH
of pyrane ring and NH2 of the orthoaminocarbonitrile
group, respectively.
Results and discussion
Treatment of compounds 5a, b with one-carbon cyc-
lizing agents, such as formamide, formic acid, acetic
anhydride, urea, thiourea, and phenyl isothiocyanate yiel-
ded the corresponding thiazolopyranopyrimidine deriva-
tives 6–10, 7b, 8b, and 14, where IR spectra showed the
absence of (C:N) band which confirms the cyclization
and the formation of thiazolopyranopyrimidines 6–10, 7b,
8b, and 14 (Scheme 3).
In the light of these facts and guided by molecular mod-
eling results, this study was carried out, hoping that the
newly synthesized compounds might exhibit significant
anticancer and radiosensitizing activities.
A general pharmacophore for the compounds to act as
carbonic anhydrase inhibitors was reported by Thiry et al.,
(2006), and it includes the presence of a sulfonamide moiety
which coordinates with the zinc ion of the active site of the
Fig. 2 Examples of the synthesized compounds showing the compliance with the general pharmacophore of sulfonamide compounds acting as
CA inhibitors
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Med Chem Res (2012) 21:2395–2407
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Scheme 1
Scheme 2
Scheme 3
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Med Chem Res (2012) 21:2395–2407
Interaction of compound 5a with formamide caused
1685 cm-1, respectively. Mass spectra of compounds 11 and
12 exhibited a base peak at m/z 92, 63 and a molecular ion
peak at m/z 552, 570, respectively.
cyclization and the corresponding thiazolopyranopyrimi-
dine derivative 6 was obtained. 1H-NMR spectrum of
compound 6 exhibited two singlets corresponding to the
NH2 group attached to the pyrimidine at 7.0 ppm and the
CH of pyrimidine at 8.3 ppm.
Fusion of compound 5a with ethylacetoacetate afforded
the oxobutanamide derivative 13. The structure of com-
pound 13 was confirmed by IR spectrum, where the
appearance of two bands at 1717, 1710 cm-1 attributed to
the presence of 2 (C=O) groups. 1H-NMR spectrum
showed two singlets at 1.6, 2.2 ppm attributed to the
presence of COCH3 and CH2 group, respectively.
Interaction of compounds 5a, b with formic acid lead to
the formation of the thiazolopyranopyrimidine derivatives
7a, b. IR spectra of compounds 7a, b exhibited a band
attributed to the presence of (C=O) at 1705 and 1714 cm-1
,
respectively. 1H-NMR spectra of compounds 7a, b exhibited
a singlet corresponding to the CH of pyrimidine at 8.3 ppm.
When compounds 5a, b were refluxed in acetic anhy-
dride, the corresponding thiazolopyranopyrimidine deriv-
atives 8a, b were isolated instead of the expected mono or
When compound 5b was reacted with phenyl isothio-
cyanate either in pyridine, or in ethanol the thiazolopyr-
anopyrimidine derivative 14 was isolated via Dimroth
rearrangement (Dean and Papadopoules, 1982), while the
formation of the other derivative was excluded on the basis
1
1
diacetyl derivatives C and D on the basis of IR and H-
NMR spectroscopy, where the IR spectra showed the
of IR and H-NMR spectroscopy (Fig. 3). IR spectrum of
compound 14 showed the disappearance of (C:N) band
and the appearance of (C=S) band at 1270 cm-1. 1H-NMR
spectrum showed multiplet at 7.0–7.9 ppm, showing
additional aromatic protons of the phenyl ring.
appearance of (C=O) band at 1696 and 1719 cm-1
,
1
respectively. In addition, H-NMR spectra of compounds
8a, b showed a singlet attributed to the CH3 group at 1.9
and 1.8 ppm, respectively (Fig. 2).
The 2- oxo/thioxo pyrimido derivatives 9 and 10 were
obtained by fusion of compound 5a with urea and/or
thiourea, respectively. The structure of compound 9 was
confirmed by IR spectrum where the appearance of a band
at 1697 cm-1 attributed to the presence of (C=O), and the
structure of compound 10 was confirmed by the appearance
In vitro anticancer screening
All the newly synthesized compounds were subjected to in
vitro anticancer screening. Doxorubicin was used as the
reference drug in this study. The relationship between sur-
viving fraction and drug concentration was plotted to obtain
the survival curve of HEPG2. The response parameter cal-
culated was IC50 value which corresponds to the concen-
tration required for 50% inhibition of cell viability. It has
been observed that the pyrimidine derivatives 7b, 8b, and 14
were found to posses higher potency with IC50 32, 30, and
31 lM, respectively, followed by the oxobutanoic acid
derivative 12 with (IC50 = 32 lM), then the pyrimidine
of (C=S) band at 1280 cm-1
.
When compound 5a was fused with succinic anhydride,
the 2,5-dioxopyrrolidine derivative 11 was obtained. On the
other hand, when compound 5a was refluxed with succinic
anhydride in ethanol, the oxobutanoic acid derivative 12 was
obtained. The IR spectra of compounds 11 and 12 showed
two carbonyl groups at 1700, 1680 cm-1 and 1710,
Fig. 3 Synthesis of
compounds 8a, b
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Med Chem Res (2012) 21:2395–2407
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Table 1 In vitro anticancer screening of the synthesized compounds against HEPG2
Cpd. No.
Compound concentration (lM)
IC50 (lM)
Surviving fraction (mean SE)a
10
25
50
100
DOX.
3
0.537 0.026
0.848 0.012
0.846 0.010
0.628 0.049
0.909 0.022
0.846 0.015
0.814 0.029
0.625 0.005
0.561 0.008
0.764 0.005
0.529 0.018
0.779 0.027
0.615 0.045
0.710 0.006
0.626 0.007
0.546 0.012
0.593 0.019
0.479 0.003
0.501 0.025
0.767 0.016
0.564 0.061
0.861 0.036
0.564 0.046
0.605 0.030
0.498 0.025
0.446 0.035
0.498 0.062
0.477 0.036
0.487 0.004
0.432 0.006
0.474 0.033
0.382 0.031
0.456 0.015
0.434 0.012
0.122 0.005
0.168 0.008
0.376 0.005
0.640 0.017
0.582 0.015
0.388 0.010
0.287 0.022
0.168 0.007
0.164 0.019
0.301 0.008
0.118 0.018
0.244 0.005
0.284 0.018
0.342 0.018
0.127 0.015
0.206 0.006
0.128 0.006
0.125 0.016
0.231 0.022
0.312 0.007
0.672 0.018
0.655 0.061
0.509 0.066
0.489 0.020
0.015 0.015
0.127 0.020
0.213 0.017
0.115 0.08
0.262 0.014
0.246 0.014
0.231 0.004
0.166 0.005
0.299 0.018
0.137 0.036
32
43
60
–
5a
5b
5c
5d
6
–
72
65
36
32
45
30
45
39
45
32
37
31
7a
7b
8a
8b
9
10
11
12
13
14
a
Each value is the mean of three experiments standard error
derivative 7a with (IC50 = 36 lM), and the starting mate-
rial, thiazole derivative 3 with (IC50 = 43 lM) compared
with that of doxorubicin (IC50 = 32 lM) (Table 1).
These preliminary biological screening of the tested com-
pounds could offer an encouraging framework in this field,
which may lead to the discovery of potent anticancer agents.
c-irradiation to study the enhancement of the cell killing
effect of c-irradiation. Table 2 showed the effect of radia-
tion alone and was not significant when compared with the
control group which contains only the cell lines. On the
other hand, the surviving fraction of the tested compounds
decreased significantly from the control after being sub-
jected to radiation which indicates the synergistic effect.
From the literature, it was reported that sulfonamides and
nitro group can act as radiosensitizers (Ghorab et al., 2010;
Sasai et al., 1994); therefore, we aimed at introducing these
two moieties with different substitutions to see their effect
on the IC50, for example compound 12 with oxobutanoic
acid derivative showed lower IC50 than that of the pyrimi-
dine derivative 7a, while the pyrimidine derivative 8b
containing 3,4-dimethoxy group showed the least IC50.
Radiosensitizing activity
The rationale for combining chemotherapy and radiother-
apy is based mainly on two ideas, one being spatial coop-
eration 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 chemo-
therapy is required. The other idea is the enhancement of
radiation effects. Cytotoxic agents can enhance radiation
effects by direct enhancement of the initial radiation dam-
age by incorporating drugs into DNA, inhibiting cellular
repair, accumulating cells in a radiosensitive phase or
eliminating radioresistant 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 (Yoshino et al., 1992). Consequently, compounds
3, 7a, 8b, 12, and 14, showed high potency in the in vitro
anticancer screening supported by molecular modeling
data, which were further screened in combination with
Docking studies
Abbate et al., (2004) showed that carbonic anhydrase inhibi-
tion is one of the anticancer mechanisms of sulfonamides, and
he stated that the potent anticancer sulfonamide lead compound
(E7070) (Fig. 4)—currently undergoingclinicaldevelopment
for the treatment of several types of cancer—also acts as a
strong CAII inhibitor, and this may contribute at least in part,
to its in vivoefficacy. The X-ray crystal structure of the adduct
of human carbonic anhydrase II (hCAII) with E7070 revealed
similar interactions between the inhibitor and the active site
(Supuran and Scozzafava, 2007; Pastorekova et al., 2004).
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Med Chem Res (2012) 21:2395–2407
Table 2 In vitro anticancer screening of compounds 3, 7a, 8b, 12, and 14 against HEPG2 in combination with radiation
Cpd. no.
Control
Irradiated (8 Gy)
Compound concentration (lM) ? irradiation (8 Gy)
IC50 (lM)
Surviving fraction (mean SE)a
10
25
50
100
3
1.000
1.000
1.000
1.000
1.000
0.927 0.02
0.927 0.02
0.927 0.02
0.927 0.02
0.927 0.02
0.53 0.08*
0.41 0.01*
0.31 0.07*
0.36 0.07*
0.40 0.01*
0.48 0.01*
0.22 0.02*
0.12 0.01*
0.23 0.01*
0.21 0.02*
0.12 0.01*
0.12 0.05*
0.10 0.01*
0.11 0.01*
0.12 0.05*
0.12 0.01*
0.14 0.06*
0.11 0.08*
0.13 0.08*
0.13 0.06*
30
18
8
7a
8b
12
14
a
15
17
Each value is the mean of three values standard error
* Significant difference from control group at P \ 0.001
These interactions were found to be common for the sulfon-
amide-bearing compounds which act as CA inhibitors and
include: (i) binding of the compounds to the Zn(II) ion by the
sulfonamidemoietyinatetrahedralgeometrywhichisastable
geometry for the metal ion, (ii) the nitrogen atom of the sul-
fonamide is coordinated to the Zn(II) ion of the enzyme, and
(iii) the amino acid Thr 199 participates in two hydrogen
bonds, one with the NH moiety and the other with one of the
oxygen atoms of the SO2NH2 (Fig. 5) (Supuran and Sco-
zzafava, 2007; Pastorekova et al., 2004). Since our
synthesized compounds are sulfonamide derivatives and their
design complies with the general pharmacophore of sulfon-
amide CA inhibitors, it was interesting to perform docking
studies on the synthesized compounds to hCAII and to com-
pare their docking interactions with the previously reported
interactions of E7070. In order to validate our docking pro-
cedure, E7070 was docked into the active site of hCAII. The
docking results clearly showed that indeed, the newly syn-
thesized compounds exhibited similar interactions as those
previously reported in the literature (Figs. 5, 6, 7, 8).
Fig. 4 Synthesis of compound 14
Fig. 5 Interaction map of
E7070 inside the active site of
hCAII showing similar
interactions as those previously
reported
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Med Chem Res (2012) 21:2395–2407
2401
Fig. 6 Interaction map of
compound 8b inside the active
site of hCAII showing similar
interactions as those previously
reported
Fig. 7 Interaction map of
compound 12 inside the active
site of hCAII showing similar
interactions as those previously
reported
Fig. 8 Interaction map of
compound 14 inside the active
site of hCAII showing similar
interactions as those previously
reported
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Med Chem Res (2012) 21:2395–2407
Conclusion
refluxed in ethanol (50 ml) containing piperidine (1 ml) for
8 h. The reaction mixture was poured into ice cold water.
The solid obtained was crystallized from dioxane to give
5a–d, respectively.
From the above results, we can conclude that the adminis-
tration of the tested compounds 3, 7a, 8b, 12 and 14 on
HEPG2 showed promising anticancer activity. While, com-
bining these compounds with radiation at the same concen-
trations enhanced their activity indicating the importance of
combination therapy for the patients with cancer to decrease
the side effects of both drugs and radiation.
4-(5-Amino-6-cyano-7-(4-nitrophenyl)-7H-
pyrano[2,3-d]thiazol-2-ylamino)benzenesulfonamide
(5a)
Yield, 67%; m.p: 178–180°C. IR (KBr, cm-1): 3354, 3237,
3077 (NH, NH2), 3067 (CH arom.), 2943, 2974 (CH
aliph.), 2194 (C:N), 1620 (C=N), 1514, 1310 (NO2),
Experimental
1
1360, 1190(SO2). H-NMR (DMSO-d6) d: 4.0 (s, 1H, CH
Synthesis
pyrane), 6.9, 7.2 (2d, 4H, AB system of sulfanilamide), 7.1,
7.6 (2d, 4H, AB system of p-nitrophenyl ring), 7.9 (s, 2H,
SO2NH2 exchangeable with D2O), 8.2 (s, 1H, NH
exchangeable with D2O), 12.0 (s, 2H, NH2 exchangeable
with D2O). MS (m/z): 470 [M?] (2.91), 63 (100), 77
(96.62), 179 (77.46). Anal. Calcd. For C19H14N6O5S2: C,
48.50; H, 3.00; N, 17.86. Found: C, 48.38; H, 2.95; N,
17.95.
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
Perkin-Elmer 2400 analyser (Perkin-Elmer, Norwalk, CT,
USA) at the microanalytical laboratories 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 (Shi-
madzu, Koyoto, Japan), 1H-NMR spectra were obtained on
a Bruker proton NMR-Avance 300 (300 MHz) (Bruker,
Munuch, Germany), in DMSO-d6 as a solvent, using tet-
ramethylsilane (TMS) as internal standard. Mass spectra
were run on JEOL JMS AX-500 mass spectrometer (JEOL
JMS, Tokyo, Japan). All reactions were monitored using
thin layer chromatograph (TLC) with precoated Aluminum
sheets Silica gel Merck 60 F254 and were visualized by
UV lamp (Merck, Damstadt, Germany).
4-(5-Amino-6-cyano-7-(3,4-dimethoxyphenyl)-7H-
pyrano[2,3-d]thiazol-2-ylamino)benzenesulfonamide
(5b)
Yield, 64%; m.p: 216–218°C. IR (KBr, cm-1): 3390, 3300,
3280 (NH, NH2), 3060 (CH arom.), 2960, 2980 (CH
1
aliph.), 2220 (C:N), 1617 (C=N), 1380, 1130(SO2). H-
NMR (DMSO-d6) d: 4.0 (s, 6H, 2 OCH3), 4.1 (s, 1H, CH
pyrane), 7.0, 7.2 (2d, 4H, AB system of sulfanilamide), 7.1
(t, 1H, CH6 of 3,4-dimethoxyphenyl), 7.5 (d, 1H, CH5 of
3,4-dimethoxyphenyl), 7.6 (s, 1H, CH2 of 3,4-dimethoxy-
phenyl), 7.9 (s, 2H, SO2NH2 exchangeable with D2O), 8.1
(s, 1H, NH exchangeable with D2O), 12.0 (s, 2H, NH2
exchangeable with D2O). MS (m/z): 485 [M?] (2.54), 209
(100), 64 (59.34), 179 (47.49). Anal. Calcd. For
C21H19N5O5S2: C, 51.95; H, 3.94; N, 14.42. Found: C,
51.93; H, 3.82; N, 14.27.
2-Chloro-N-(4-sulfamoylphenyl)acetamide (2)
m.p.: 215–217°C as reported (Donkor et al., 1998).
4-(4-Oxo-4,5-dihydrothiazol-2-
ylamino)benzenesulfonamide (3)
Compound 3 was prepared by another method.
A mixture of compound 2 (2.48 g, 0.01 mol) and
ammonium thiocyanate (0.76 g, 0.01 mol) in ethanol
(50 ml) was refluxed for 1 h. The solid obtained was
crystallized from dioxane.
4-(5-Amino-7-(2-chlorophenyl)-6-cyano-7H-
pyrano[2,3-d]thiazol-2-ylamino)benzenesulfonamide
(5c)
Yield, 65%; m.p: [300°C. IR (KBr, cm-1): 3453, 3230,
3113 (NH, NH2), 3070 (CH arom.), 2960, 2984 (CH
aliph.), 2202 (C:N), 1610 (C=N), 1340, 1170 (SO2), 750
m.p.: 244–246°C as reported (Siavosh et al., 2006).
4-(5-Amino-7-(phenyl substituted)-6-cyano-7H-
pyrano[2,3-d]thiazol-2-ylamino) benzenesulfonamides
(5a–d)
1
(C–Cl). H-NMR (DMSO-d6) d: 4.1 (s, 1H, CH pyrane),
7.0, 7.2 (2d, 4H, AB system of sulfanilamide), 7.3–7.6 (m,
4H, Ar–H of 2-chlorophenyl), 7.9 (s, 2H, SO2NH2
exchangeable with D2O), 8.0 (s, 1H, NH exchangeable
with D2O), 12.5 (s, 2H, NH2 exchangeable with D2O).
A mixture of compound 3 (2.71 g, 0.01 mol) and
2-(substituted benzylidene) malononitrile (0.01 mol) was
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Med Chem Res (2012) 21:2395–2407
2403
Anal. Calcd. for C19H14ClN5O3S2: C, 49.62; H, 3.07; N,
15.23. Found: C, 49.47; H, 3.22; N, 15.12.
aliph.), 1705 (C=O), 1625(C=N), 1329, 1158(SO2). 1H-
NMR (DMSO-d6) d: 4.0 (s, 1H, CH pyrane), 7.0, 7.2 (2d,
4H, AB system of sulfanilamide), 7.1, 7.6 (2d, 4H, AB
system of p-nitrophenyl ring), 7.9 (s, 2H, SO2NH2
exchangeable with D2O), 8.2 (s, 2H, 2NH exchangeable
with D2O), 8.3 (s, 1H, CH pyrimidine). MS (m/z): 498
[M?] (12.15), 179 (100), 89 (73.64), 250 (25.5). Anal.
Calcd. For C20H14N6O6S2: C, 48.19; H, 2.83; N, 16.86.
Found: C, 47.98; H, 2.92; N, 16.79.
4-(5-Amino-7-(3-bromophenyl)-6-cyano-7H-
pyrano[2,3-d]thiazol-2-ylamino)benzenesulfonamide
(5d)
Yield, 61%; m.p: 250–252°C. IR (KBr, cm-1): 3343, 3251,
3150 (NH, NH2), 3080 (CH arom.), 2942, 2974 (CH
aliph.), 2199 (C:N), 1613 (C=N), 1340, 1170 (SO2) .1H-
NMR (DMSO-d6) d: 4.0(s, 1H, CH pyrane), 6.9, 7.2 (2d,
4H, AB system of sulfanilamide), 7.3–7.6 (m, 4H, Ar–H of
3-bromophenyl), 7.9 (s, 2H, SO2NH2 exchangeable with
D2O), 8.2 (s, 1H, NH exchangeable with D2O), 12.0 (s, 2H,
NH2 exchangeable with D2O). Anal. Calcd. For
C19H14BrN5O3S2: C, 45.24; H, 2.80; N, 13.89. Found: C,
45.35; H, 2.71; N, 13.78.
8-Oxo, 7-(3,4-dimethoxy phenyl) 7-H,[2-(4-sulphamoyl
phenyl amino)] thiazolo[4,5-b]pyrano[2,3-d]
pyrimidine (7b)
Yield, 64%; m.p: 250–252. IR (KBr, cm-1): 3217 (br NH,
NH2), 3085 (CH arom.), 2913, 2924 (CH aliph), 1714
(C=O), 1608 (C=N), 1380, 1130 (SO2). 1H-NMR (DMSO-
d6) d: 4.0 (s, 6H, 2 OCH3), 4.1 (s, 1H, CH pyrane), 7.0, 7.2
(2d, 4H, AB system of sulfanilamide), 7.3–7.6 (m, 3H, Ar–
8-Amino, 7-(4-nitro phenyl) 7-H,[2-(4-sulphamoyl
phenyl amino)] thiazolo[4,5-b]pyrano[2,3-d]
pyrimidine (6)
H
of 3,4-dimethoxyphenyl), 7.9 (s, 2H, SO2NH2
exchangeable with D2O), 8.2 (s, 2H, 2NH exchangeable
with D2O), 8.3 (s, 1H, CH pyrimidine). MS (m/z): 513
[M?] (0.2), 194 (100), 179 (50), 136 (29.33). Anal. Calcd.
For C22H19N5O6S2: C, 51.45; H, 3.73; N, 13.64. Found: C,
51.38; H, 3.79; N, 13.66.
A mixture of compound 5a (4.7 g, 0.01 mol) and Form-
amide (30 ml) was refluxed for 3 h. The reaction mixture
was poured into ice cold water. The solid obtained was
crystallized from ethanol to give compound 6: Yield, 65%;
m.p.: 252–254°C. IR (KBr, cm-1): 3222 (br NH, NH2),
3050 (CH arom.), 2948, 2929 (CH aliph.), 1625 (C=N),
2-Methyl,8-oxo,7-(substituted phenyl) 7-H,[2-(4-
sulphamoyl phenyl amino)] thiazolo[4,5-b]pyrano[2,3-
d] pyrimidine (8a, b)
1
1510, 1350 (NO2), 1380, 1130 (SO2). H-NMR (DMSO-
d6) d: 4.0 (s, 1H, CH pyrane), 6.9, 7.2 (2d, 4H, AB system
of sulfanilamide), 7.0 (s, 2H, NH2 exchangeable with
D2O), 7.1, 7.6 (2d, 4H, AB system of p-nitrophenyl ring),
7.9 (s, 2H, SO2NH2 exchangeable with D2O), 8.2 (s, 1H,
NH exchangeable with D2O), 8.3 (s, 1H, CH pyrimidine).
MS (m/z): 499 [M?] (13.05), 194 (100), 179 (66), 108
(62.42). Anal. Calcd. For C20H15N7O5S2: C, 48.28; H,
3.04; N, 19.71. Found: C, 48.32; H, 3.10; N, 19.83.
A solution of compound 5a, b (0.01 mol) in acetic anhy-
dride (40 ml) was refluxed for 10 min, or 10 h. The reac-
tion mixture was left to evaporate at room temperature then
triturated with ethanol and filtered. The solid obtained was
crystallized from ethanol to give 8a and 8b, respectively.
8-Oxo, 7-(substituted phenyl) 7-H,[2-(4-sulphamoyl
phenyl amino)] thiazolo[4,5-b]pyrano[2,3-d]
pyrimidine (7a, b)
2-Methyl,8-oxo,7-(4-nitro phenyl) 7-H,[2-(4-
sulphamoyl phenyl amino)] thiazolo[4,5-b]pyrano[2,3-
d] pyrimidine (8a)
Yield, 75%, m.p: 185–187°C. IR(KBr, cm-1): 3460, 3351,
3310 (NH, NH2), 3077(CH arom.), 2930, 2953 (CH aliph.),
1696 (C=O), 1600 (C=N), 1377, 1158 (SO2).1H-NMR
(DMSO-d6) d: 1.9 (s, 3H, CH3), 4.0 (s, 1H, CH pyrane), 7.0,
7.2 (2d, 4H, AB system of sulfanilamide), 7.1, 7.6 (2d, 4H,
AB system of p-nitrophenyl ring), 7.9 (s, 2H, SO2NH2
exchangeable with D2O), 8.1 (s,2H, 2 NH exchangeable
with D2O). MS (m/z):512 [M?] (2.43), 78 (100), 64 (35.05),
108 (5.72). Anal. Calcd. For C21H16N6O6S2: C, 49.21; H,
3.15; N, 16.40. Found: C, 49.38; H, 3.25; N, 16.33.
A solution of compound 5a, b (0.01 mol) in Formic acid
(30 ml) was refluxed for 3 h. The reaction mixture was
poured onto ice cold water. The solid obtained was crys-
tallized from dioxane.
8-Oxo, 7-(4-nitro phenyl) 7-H,[2-(4-sulphamoyl phenyl
amino)] thiazolo[4,5-b]pyrano[2,3-d] pyrimidine (7a)
Yield, 73%; m.p: 212–214°C. IR (KBr, cm-1): 3480, 3263,
3200 (NH, NH2), 3050 (CH arom.), 2929, 2950 (CH
123

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Med Chem Res (2012) 21:2395–2407
2-Methyl,8-oxo,7-(3,4-dimethoxy phenyl) 7-H, [2-(4-
sulphamoyl phenyl amino)] thiazolo[4,5-b]pyrano[2,3-
d] pyrimidine (8b)
D2O), 8.2 (s, 2H, 2NH exchangeable with D2O). MS (m/z):
529 [M?] (3.22), 194 (100), 179 (71.15), 26 (52.53). Anal.
Calcd. For C20H15N7O5S3: C, 45.36; H, 2.85; N, 18.51.
Found: C, 45.30; H, 2.77; N, 18.65.
Yield, 66%, m.p.: 188–190°C. IR (KBr, cm-1): 3490,
3300, 3242 (NH, NH2), 3078 (CH arom.), 2945, 2910 (CH
1
aliph.), 1719 (C=O), 1628 (C=N), 1380, 1130 (SO2). H-
4-(6-Cyano-5-(2,5-dioxopyrrolidinyl)-7-(4-
nitrophenyl)-7H-pyrano[2,3-d]thiazol-2-ylamino)
benzenesulfonamide (11)
NMR (DMSO-d6) d: 1.8 (s, 3H, CH3), 4.0 (s, 6H, 2 OCH3
phenyl), 4.1 (s, 1H, CH pyrane), 7.0, 7.2 (2d, 4H, AB
system of sulfanilamide), 7.3–7.6 (m, 3H, Ar–H of 3,4-
dimethoxyphenyl), 7.9 (s, 2H, SO2NH2 exchangeable with
D2O), 8.2(s, 2H, 2NH exchangeable with D2O). MS (m/z):
527 [M?] (4.4), 100 (100), 97 (64.13), 107 (5.01). Anal.
Calcd. For: C23H21N5O6S2: C, 52.36; H, 4.01; N, 13.27.
Found: C, 52.22; H, 4.13; N, 13.12.
A mixture of compound 5a (4.7 g, 0.01 mol) was fused
with succinic anhydride (1.0 g, 0.01 mol) for 15 min. The
reaction mixture was triturated with ethanol and the solid
obtained was crystallized from dioxane to give 11: Yield,
61%; m.p: [300°C. IR (KBr, cm-1): 3480, 3290, 3200
(NH, NH2), 3065 (CH arom.), 2925, 2912 (CH aliph.),
2200 (C:N), 1700, 1680 (2C=O), 1610(C=N), 1329, 1158
(SO2). 1H-NMR (DMSO-d6) d: 1.5, 1.6 (2t, 4H, 2CH2
succinic), 4.1 (s, 1H, CH pyrane), 7.0, 7.2 (2d, 4H, AB
system of sulfanilamide), 7.1, 7.6 (2d, 4H, AB system of p-
nitrophenyl ring), 7.9 (s, 2H, SO2NH2 exchangeable with
D2O), 8.2 (s, 1H, NH exchangeable with D2O). MS (m/z):
552 [M?] (4.09), 92(100), 172 (70.67), 156 (56.26). Anal.
Calcd. For C23H16N6O7S2: C, 50.00; H, 2.92; N, 15.21.
Found: C, 50.12; H, 2.75; N, 15.38.
8-Amino, 2-oxo and 2-thioxo, 7-(4-nitro phenyl)7-H,[2-
(4-sulphamoyl phenyl amino)] thiazolo[4,5-
b]pyrano[2,3-d] pyrimidine (9, 10)
A mixture of compound 5a (4.7 g, 0.01 mol) was fused
with urea and/or thiourea (0.01 mol) for 15 min. The
reaction mixture was triturated with ethanol and the solid
obtained was crystallized from dioxane to give 9 and 10,
respectively.
4-(6-Cyano-7-(4-nitrophenyl)-2-(4-
8-Amino, 2-oxo, 7-(4-nitro phenyl) 7-H,[2-(4-
sulphamoyl phenyl amino)] thiazolo[4,5-b]pyrano[2,3-
d] pyrimidine (9)
sulfamoylphenylamino)-7H-pyrano[2,3-d]thiazol-5-
ylamino)-4-oxobutanoic acid (12)
A mixture of compound 5a (4.7 g, 0.01 mol) and succinic
anhydride (1.0 g, 0.01 mol) in ethanol (20 ml) was
refluxed for 3 h. The reaction mixture was poured into ice
cold water and filtered. The solid obtained was crystallized
from dioxane to give 12: Yield, 63%; m.p.: 201–203°C. IR
(KBr, cm-1): 3400, 3320, 3200 (NH, NH2), 3080 (CH
arom.), 2900, 2916 (CH aliph), 1710, 1685 (2C=O), 1620
Yield, 50%; m.p: [300°C. IR (KBr, cm-1): 3400, 3337,
3200 (NH, NH2), 3060 (CH arom.), 2950, 2928 (CH
1
aliph.), 1697 (C=O), 1612 (C=N), 1329, 1158 (SO2). H-
NMR (DMSO-d6) d: 4.0 (s, 1H, CH pyrane), 7.0, 7.2 (2d,
4H, AB system of sulfanilamide), 7.1, 7.6 (2d, 4H, AB
system of p-nitrophenyl ring), 7.3 (s, 2H, NH2 exchange-
able with D2O), 7.9 (s, 2H, SO2NH2 exchangeable with
D2O), 8.2 (s, 2H, 2NH exchangeable with D2O). MS (m/z):
513 [M?] (16), 515 [M? ?2] (0.22), 78 (100), 83 (20.4).
Anal. Calcd. For C20H15N7O6S2: C, 46.78; H, 2.94; N,
19.09. Found: C, 46.49; H, 2.92; N, 19.10.
1
(C=N), 1329, 1158(SO2). H-NMR (DMSO-d6) d: 1.2, 1.6
(2t, 4H, CH2–CH2), 4.0 (s, 1H, CH pyrane), 7.0, 7.2 (2d,
4H, AB system of sulfanilamide), 7.1, 7.6 (2d, 4H, AB
system of p-nitrophenyl ring), 7.9 (s, 2H, SO2NH2
exchangeable with D2O), 8.2 (s, 2H, 2NH exchangeable
with D2O), 10.5 (s, 1H, OH exchangeable with D2O). MS
(m/z): 570 [M?] (2.5), 63 (100), 92 (66.49), 172 (90.85).
Anal. Calcd. For: C23H16N6O7S2: C, 50.00; H, 2.92; N,
15.21.Found: C, 50.14; H, 2.89; N, 15.11.
8-Amino, 2-thioxo, 7-(4-nitro phenyl) 7-H, [2-(4-
sulphamoyl phenyl amino)] thiazolo[4,5-b]pyrano[2,3-
d] pyrimidine (10)
Yield, 59%; m.p. [300°C. IR (KBr, cm-1): 3314, 3320,
3220 (NH, NH2), 3080 (CH arom.), 2945, 2920 (CH
1
aliph.), 1615 (C=N), 1280 (C=S), 1329, 1158(SO2). H-
N-(6-Cyano-7-(4-nitrophenyl)-2-(4-
sulfamoylphenylamino)-7H-pyrano[2,3-d]thiazol-5-yl)-
3-oxobutanamide (13)
NMR (DMSO-d6) d: 4.0 (s, 1H, CH pyrane), 7.0, 7.2 (2d,
4H, AB system of sulfanilamide), 7.1, 7.6 (2d, 4H, AB
system of p-nitrophenyl ring), 7.3 (s, 2H, NH2 exchange-
able with D2O), 7.9 (s, 2H, SO2NH2 exchangeable with
A solution of compound 5a (4.7 g, 0.01 mol) in ethylace-
toacetate (1.3 ml, 0.01 mol) was fused for 1 h. The reac-
tion mixture was poured onto ice cold water and filtered.
123

Med Chem Res (2012) 21:2395–2407
2405
The solid obtained was crystallized from dioxane to give
13: Yield, 53%; m.p.: 121–123°C. IR (KBr, cm-1): 3443,
3362, 3237 (NH, NH2), 3050 (CH arom.) 2939, 2923 (CH
aliph), 2195 (C:N), 1717, 1710 (C=O), 1627 (C=N),
1346, 1160 (SO2). 1H-NMR (DMSO-d6) d: 1.6 (s, 3H,
CH3), 2.2 (s, 2H, CH2), 4.0 (s, 1H, CH pyrane), 7.0, 7.2
(2d, 4H, AB system of sulfanilamide), 7.1, 7.6 (2d, 4H, AB
system of p-nitrophenyl ring), 7.9 (s, 2H, SO2NH2
exchangeable with D2O), 8.2 (s, 2H, 2NH exchangeable
with D2O). MS (m/z): 558 [M?] (7.28), 129 (100), 94
(78.46), 71 (28.93). Anal. Calcd. For C23H18N6O7S2: C,
49.81; H, 3.27; N, 15.15. Found: C, 49.76; H, 3.33; N,
15.32.
screening was done by the pharmacology unit at the
National Cancer Institute, Cairo University. Cells were
plated in 96-multiwell microtiter plate (104 cells/well) for
24 h before treatment with the compound(s) to allow the
attachment of cells to the wall of the plate. Test compounds
were dissolved in DMSO and diluted with saline to the
appropriate volume.
Different concentrations of the compound under test (10,
25, 50 and 100 lM) were added to the cell monolayer.
Triplicate wells were prepared for each individual dose.
Monolayer cells were incubated with the compound(s) for
48 h at 37°C and in atmosphere of 5% CO2. After 48 h,
cells were fixed, washed, and stained for 30 min with 0.4%
(wt/vol) of SRB dissolved in 1% acetic acid. Excess
unbound dye was removed by washing four times with 1%
acetic acid and attached stain was recovered with Tris–
EDTA buffer. Color intensity was measured in an ELISA
reader. The relation between surviving fraction and drug
concentration is plotted to get the survival curve for liver
tumor cell line after the specified time. The molar con-
centration required for 50% inhibition of cell viability
(IC50) was calculated and compared with the reference
drug doxorubicin.
8-Imino, 2-thioxo,7-(3,4-dimethoxy phenyl) 7-H, 1 N
phenyl,[2-(4-sulphamoyl phenyl amino)] thiazolo[4,5-
b]pyrano[2,3-d] pyrimidine (14)
A mixture of compound 5b (4.85 g, 0.01 mol) and phenyl
isothiocyanate (1.35 g, 0.01 mol) in ethanol and/or pyri-
dine (20 ml) was refluxed for 5 h. The reaction mixture
was poured into ice cold water. The solid obtained was
crystallized from dioxane to give 14: Yield, 65%, m.p:
244–246°C. IR (KBr, cm-1): 3337, 3255, 3123 (NH, NH2),
3050 (CH arom.), 2968, 2929 (CH aliph.), 1618 (C=N),
1380, 1130 (SO2), 1270 (C=S). 1H-NMR (DMSO-d6) d: 4.0
(s, 6H, 2 OCH3), 4.1 (s, 1H, CH pyrane), 6.9 (m, 1H, CH3
of phenyl ring), 7.0, 7.2 (2d, 4H, AB system of sulfanil-
amide), 7.3–7.6 (m, 3H, Ar–H of 3,4-dimethoxyphenyl),
7.4, 7.6 (2d, 4H, AB system of phenyl), 7.9 (s, 2H, SO2NH2
exchangeable with D2O), 8.2 (s, 2H, 2NH exchangeable
with D2O), 11.6 (s, 1H, NH imino exchangeable with
D2O). MS (m/z): 620 [M?] (10), 271 (100), 198 (51.08),
181 (27.94). Anal. Calcd. For: C28H24N6O5S3: C, 54.18; H,
3.90; N, 13.54. Found: C, 54.25; H, 3.87; N, 13.48.
Three values were obtained for each concentration in the
experiment, and then by calculating the mean and standard
error of each concentration, surviving fractions were
expressed as means standard error. Plotting the curve of
surviving fraction against concentration, using Graphpad
prism 5, the IC50 can be calculated from the equation
obtained from the graph y = ax ? b, where y = 0.5, and
the results are given in Table 1.
It is observed that there is an increase in the survival
fraction value at higher concentrations, such as 100 lM in
some samples, as the cells became more resistant to the
effect of the drug at high concentration (Kavallaris et al.,
1990).
Biological testing
Radiosensitizing activity
Materials and methods
The same compounds used in the in vitro anticancer
screening: 3, 7a, 8b, 12, and 14, were selected to be
evaluated again for their in vitro anticancer activity in
combination with c-radiation. This study was conducted to
evaluate the ability of these compounds to enhance the
cell-killing effect of c-radiation. Cells were subjected to a
single dose of c-radiation at a dose level of 8 Gy with a
dose rate of 1 Gy/min. Irradiation was performed at the
National Center for Radiation Research and Technology,
Atomic Energy Authority, using Gamma cell (137Cs)
source, the surviving fraction was measured after 1 h
subjection to radiation (Geng et al., 2005). The surviving
fractions were expressed as means standard error. The
results were analyzed using 1-way ANOVA test and are
HEPG2 were available at the National Cancer Institute,
Cairo, Egypt. Irradiation was performed at the National
Center for Radiation Research and Technology [NCRRT],
Atomic Energy Authority using Gamma cell (137Cs)
source.
In vitro anticancer screening
The cytotoxic activity was measured in vitro for the newly
synthesized compounds 3, 7a, 8b, 12, and 14 using the
Sulfo-Rhodamine-B stain (SRB) assay using the method of
Skehan and Storeng (1990). The in vitro anticancer
123

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Med Chem Res (2012) 21:2395–2407
Casini A, Scozzafava A, Mastrolorenzo A, Supuran CT (2002)
Sulfonamides and sulfonylated derivatives as anticancer agents.
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Fig. 9 Survival curve for HEPG2 cell line for compound 8b alone
and in combination with c- irradiation (8 Gy)
given in Table 2. The effect of radiation on cytotoxic
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HEPG2 cell line is presented in Fig. 9.
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Docking studies
All molecular modeling calculations and docking studies
were performed using ‘‘Molecular Operating Environment
(MOE) version 2007.09’’. The ligand was drawn on
ChemDraw and imported in MOE. The structure was
subjected to energy minimization using MMFF949
forcefield, and the partial charges were computed using the
same forcefield. The X-ray crystallographic structure of
hCA II complexed with N-(2,3,4,5,6-pentaflouro-benzyl)-
4-sulfamoyl-benzamide (1g54) was obtained from the
Protein Data Bank. The enzyme was prepared for the
docking studies, where: (i) The ligand molecule was
removed from the enzyme active site. (ii) Hydrogen atoms
were added to the structure with their standard geometry.
(iii) Partial charges were computed using Amber 99 force
field. Docking calculations were done using Alpha triangle
placement method and poses were prioritized by Affinity
dG scoring method.
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