Docking study, in vitro anticancer screening and radiosensitizing evaluation of some new fluorine-containing quinoline and pyrimidoquinoline derivatives bearing a sulfonamide moiety

Article abstract of DOI:10.1007/s00044-010-9332-3

The present work reports the synthesis of 20 novel fluorine-containing quinoline and pyrimido[4,5-b]quinoline derivatives bearing a sulfonamide moiety. The new synthesized compounds were designed in compliance with the general pharmacophoric requirements for carbonic anhydrase (CA) inhibiting anticancer drugs, as this may play a role in their anticancer activity. All the newly synthesized compounds were evaluated for their in vitro anticancer activity against human breast cancer cell line (MCF7). Compounds 11 and 12 exhibited better activities than the reference drug doxorubicin (IC50 = 71.8 μM) with IC50 values of 52.6 μM and 67.3 μM, respectively. On the other hand, compounds 6, 10, and 13 showed IC50 values (71.8 μM, 69.8 μM, and 70.8 μM, respectively) comparable to that of the reference drug doxorubicin. In addition, docking of the synthesized compounds into human carbonic anhydrase isozyme II (hCA II) active site was performed in order to predict the affinity and the orientation of these compounds at the isozyme active site. Also, the most active compounds, 11 and 12, were selected and evaluated for their ability to enhance the cell killing effect of γ-radiation. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-010-9332-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:388–400
DOI 10.1007/s00044-010-9332-3
ORIGINAL RESEARCH
Docking study, in vitro anticancer screening and radiosensitizing
evaluation of some new fluorine-containing quinoline
and pyrimidoquinoline derivatives bearing a sulfonamide moiety
•
Mostafa M. Ghorab Fatma A. Ragab
•
•
Helmy I. Heiba Reem K. Arafa
•
Ebaa M. El-Hossary
Received: 4 November 2009 / Accepted: 16 February 2010 / Published online: 13 March 2010
Ó Springer Science+Business Media, LLC 2011
Abstract The present work reports the synthesis of 20
novel fluorine-containing quinoline and pyrimido[4,5-
b]quinoline derivatives bearing a sulfonamide moiety. The
new synthesized compounds were designed in compliance
with the general pharmacophoric requirements for carbonic
anhydrase (CA) inhibiting anticancer drugs, as this may
play a role in their anticancer activity. All the newly syn-
thesized compounds were evaluated for their in vitro
anticancer activity against human breast cancer cell line
(MCF7). Compounds 11 and 12 exhibited better activities
than the reference drug doxorubicin (IC50 = 71.8 lM)
with IC50 values of 52.6 lM and 67.3 lM, respectively. On
the other hand, compounds 6, 10, and 13 showed IC50
values (71.8 lM, 69.8 lM, and 70.8 lM, respectively)
comparable to that of the reference drug doxorubicin. In
addition, docking of the synthesized compounds into
human carbonic anhydrase isozyme II (hCA II) active site
was performed in order to predict the affinity and the ori-
entation of these compounds at the isozyme active site.
Also, the most active compounds, 11 and 12, were selected
and evaluated for their ability to enhance the cell killing
effect of c-radiation.
Introduction
Sulfonamides posses many types of biological activities
and many of them are widely used in therapy as antibac-
terial (Drews, 2000), hypoglycemic (Boyd, 1988), diuretic
(Supuran and Scozzafava, 2000; Maren, 1976), anti-car-
bonic anhydrase (Supuran and Scozzafava, 2000; Supuran
and Scozzafava, 2001), and antithyroid (Thornber, 1979)
agents. Recently, a host of structurally novel sulfonamide
derivatives have been reported to show substantial antitu-
mor activity in vitro and/or in vivo (Abbate et al., 2004;
Ghorab et al., 2006; Ismail et al., 2006; Rostom, 2006;
Supuran et al., 2004).
It has been known that aryl/heteroaryl sulfonamides may
act as antitumor agents through a variety of mechanisms
such as cell cycle perturbation in the G1 phase, disruption
of microtubule assembly, angiogenesis inhibition, and
functional suppression of the transcriptional activator NF-Y,
and the most prominent mechanism is the inhibition of
carbonic anhydrase (CA) isozymes (Casini et al., 2002).
In addition, quinoline and fused quinoline derivatives
are known to possess several biological activities including
anticancer activity (Gopal et al., 2003; Kim et al., 2005;
Zhao et al., 2005). Also, several reduced quinoline deriv-
atives have shown significant anticancer activity (Liou
et al., 2008).
Keywords Quinoline ꢀ Fluorine ꢀ Sulfonamide ꢀ
Carbonic anhydrase II ꢀ Anticancer ꢀ c-Radiation
Also, the special properties of the fluorine atom, such as
strong electronegativity, small size, and the low polariz-
ability of the C–F bond, can have considerable impact on
the behavior of a molecule in a biological environment
M. M. Ghorab (&) ꢀ H. I. Heiba ꢀ E. M. El-Hossary
Medicinal, Aromatic and Poisonous Plants Research Center
(MAPPRC), College of pharmacy, King Saud University,
Riyadh, Saudi Arabia
´
´
(Begue and Delpon, 2006). The incorporation of fluorine
into a drug allows simultaneous modulation of electronic,
lipophilic, and steric parameters, all of which can critically
influence both the pharmacodynamic and pharmacokinetic
properties of drugs. Bioisosteric substitution for hydrogen
e-mail: mmsghorab@yahoo.com
F. A. Ragab ꢀ R. K. Arafa
Faculty of Pharmacy, Department of Pharmaceutical Chemistry,
Cairo University, Cairo, Egypt
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Med Chem Res (2011) 20:388–400
389
Scaffold
by fluorine is, therefore, an important strategy for incor-
poration of a group capable of reinforcing drug–receptor
interactions (electronic modulation), aiding translocation
across lipid bilayers or absorption (lipophilic modulation)
and inducing conformational change/blocking metabolism
(steric parameters) (Ismail, 2002).
O
O
S
Hydrophobic
moiety
H2N
N
F
O
H2N
CN
In the light of these facts, and as a continuation of our
previous reported work (Ghorab et al., 2007; Ghorab et al.,
2009), we report here the synthesis of some new fluorine-
containing quinoline and pyrimido[4,5-b]quinoline deriv-
atives (6–25) having a free sulfonamide moiety, where
their design complies with the general pharmacophore of
the sulfonamide CA inhibitors. The anticancer screening
was done against a human breast cancer cell line (MCF7).
We also aimed to evaluate the ability of these compounds
to enhance the cell killing effect of c-radiation.
Sulfonamide
Hydrophilic link
Scaffold
O
O
S
Hydrophobic
moiety
F
N
N
H2N
O
O
NH
Sulfonamide
Results and discussion
Hydrophilic link
A general pharmacophore (Fig. 1) for the compounds act-
ing as carbonic anhydrase inhibitors has been reported by
Thiry et al. (2006), from the analysis of the CA active site
and from the structure of inhibitors described in the liter-
ature (Supuran et al., 2003).
Fig. 2 Representative examples of the synthesized compounds
complying with the general pharmacophore of sulfonamide com-
pounds acting as CA inhibitors
This pharmacophore includes the structural elements
that are required to be present in the compounds in order to
act as CA inhibitors. This includes the presence of a sul-
fonamide moiety which coordinates with the zinc ion of the
active site of the CA and the sulfonamide is attached to a
scaffold, which is usually a benzene ring. The side chain
might posses a hydrophilic link 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.
Synthesis
Enaminone 3 was obtained from condensation of 5,5-
dimethyl-1,3-cyclohexandione 1 with sulfanilamide 2.
Treatment of enaminone 3 with 2-(4-fluorobenzylid-
ene)malononitrile 4 in ethanol containing a catalytic
amount of triethylamine, as a base catalyst, yielded the
corresponding hexahydroquinoline derivative 6, via the
formation of the intermediate Michael type product 5,
followed by intramolecular cyclization (Scheme 1) (Gho-
rab et al., 2007).
Figure 2 includes representative examples of the
synthesized compounds, showing compliance to the above-
mentioned pharmacophore, and the compounds were syn-
thesized according to Schemes 1, 2, 3, 4, and 5.
The pyrimido[4,5-b]quinoline derivative 7 was obtained
by refluxing compound 6 in formic acid. When compound
6 was refluxed in acetic anhydride for 5 h and 10 h, the
monoacetyl derivative 8 and the diacetyl derivative 9 were
obtained, respectively. While refluxing compound 6 in
acetic anhydride for 15 h yielded the fused pyrimido[4,5-
b]quinoline system 10. Also, the pyrimido[4,5-b]quinoline
derivative 11 was obtained by the reaction of compound 6
with formamide (Scheme 2).
Pro
Val
Leu
Phe
Thr
N
Hydrophobic
moiety
Glu
O
S
Treatment of compound 6 with triethylorthoformate in
the presence of acetic anhydride yielded the quinoline
derivative 12. Reaction of compound 6 with chloroacetyl
chloride at room temperature afforded the quinoline
derivative 13. While refluxing compound 6 with chloro-
acetyl chloride in dimethyl formamide for 16 h, yielded
the 2-chloromethyl-pyrimido[4,5-b]quinoline derivative
14. Fusion of compound 6 with ethyl cyanoacetate
Scaffold
H
Hydrophilic
link
O
Glu
Zn
His
His
His
Gln
Asn
Fig. 1 Structural elements of CA inhibitors in the CA enzymatic
active site
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390
Med Chem Res (2011) 20:388–400
Scheme 1 Adopted synthetic
pathway of compound 6
O
O
NH2
SO2NH2
Ethanol
O
N
H
SO2NH2
(3)
(1)
(2)
NC
CN
EtOH / TEA
Ar
(4)
O
Ar
O
Ar
CN
CN
NH2
C N
NH
N
SO2NH2
SO2NH2
(6)
(5)
Ar= 4-Fluorophenyl
O
Ar
N
Scheme 2 Adopted synthetic
pathways of compounds 7–11
CN
O
Ar
O
Ar
N
O
N
CN
N(COCH3)2
NH
O
N
N
H
SO2NH2
(9)
(10)
(8)
SO2NH2
SO2NH2
5 h
10 h
15 h
O
Ar
N
O
O
Ar
NH2
N
NH
Ac2O
N
N
N
HCONH2
HCOOH
(6)
SO2NH2
SO2NH2
(7)
(11)
Ar= 4-Fluorophenyl
yielded the corresponding pyrimido[4,5-b]quinoline
derivative 15. The formation of compound 15 was
assumed to proceed via elimination of 1 mol of ethanol
followed by intramolecular cyclization. On the other
hand, the thioureido derivative 16 was obtained by the
reaction of compound 6 with ethyl isothiocyanate in
dimethyl formamide containing a catalytic amount of
triethylamine (Scheme 3).
Fusion of compound 6 with succinic anhydride yielded
the 2,5-dioxopyrrolidinyl derivative 17. Stirring of com-
pound 6 in concentrated H2SO4 caused partial hydrolysis of
the cyano group yielding the carboxamide derivative 18.
Additionally, the imidazolyl derivative 19 was obtained by
the treatment of compound 6 with ethylene diamine in the
presence of carbon disulfide. The reaction proceeded via
intramolecular cyclization through the elimination of 1 mol
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Med Chem Res (2011) 20:388–400
391
O
Ar
N
O
N
Scheme 3 Adopted synthetic
pathways of compounds 12–16
NH
O
Ar
N
O
N
O
Ar
N
Cl
CN
O
NH
CN
Cl
N
H
SO2NH2
(14)
O
SO2NH2
SO2NH2
NC
Stirring
at R.T.
6 h
O
(13)
reflux
16 h
(15)
Fusion
O
O
Ar
O
Ar
N
DMF
Cl
CN
S
CN
N
Cl
N
N
H
N
O
HC(OCH2CH3)3
N
C S
H
(6)
DMF/TEA
Ar= 4-Fluorophenyl
SO2NH2
SO2NH2
(16)
(12)
O
Ar
N
Scheme 4 Adopted synthetic
pathways of compounds 17–20
O
Ar
N
CONH2
NH2
N
H
N
NH2
SO2NH2
SO2NH2
(19)
(18)
Stirring at
R. T. for 6 h
NH2
Conc. H2SO4
O
Ar
CS2
H2N
O
Ar
CN
O
O
CN
O
O
O
O
O
N
N
O
H
N
N
O
(6)
O
Fusion
Fusion
SO2NH2
Ar= 4-Fluorophenyl
SO2NH2
(17)
(20)
In vitro anticancer screening
of ammonia. Also, the quinoline derivative 20 was obtained
by the reaction of compound 6 with ethyl acetoacetate
(Scheme 4).
The newly synthesized compounds were evaluated for their
in vitro cytotoxic activity against human breast cancer cell
line, MCF7.
Treatment of compound 7 with phosphorus pentasulfide
in pyridine afforded the 4-thioxo-pyrimido[4,5-b]quinoline
derivative 21. While, the 4-chloro-pyrimido[4,5-b]quino-
line derivative 22 was obtained by refluxing compound 7 in
thionyl chloride for 2 h. Compound 23 was obtained by the
treatment of compound 7 with ethyl bromoacetate in dry
acetone in the presence of anhydrous potassium carbonate.
The 4-hydrazinyl derivative 24 and the 4-isothiocyanato
derivative 25 were obtained by the reaction of the 4-chloro-
pyrimido[4,5-b]quinoline derivative 22 with hydrazine
hydrate and ammonium thiocyanate, respectively
(Scheme 5).
Doxorubicin, which is one of the most effective anti-
cancer agents, was used as a reference drug in this study.
The relationship 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 syn-
thesized compounds, where some compounds exhibited
significant activity compared to the reference drug.
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Med Chem Res (2011) 20:388–400
Scheme 5 Adopted synthetic
pathways of compounds 21–25
O
Ar
N
Cl
N
O
Ar
N
NCS
N
O
Ar
N
NHNH2
N
N
N
N
NH2NH2.H2O
NH4SCN
SO2NH2
SO2NH2
SO2NH2
(25)
(22)
(24)
O
Ar
O
N
O
Ar
N
S
SOCl2
O
N
NH
O
O
N
N
Br
P2S5
O
(7)
Ar= 4-Fluorophenyl
SO2NH2
SO2NH2
(21)
(23)
Table 1 In vitro anticancer screening of the synthesized compounds against human breast cell line (MCF7)
Compound
Compound concentration (lM)
10 25
Surviving fraction (means SE)a
IC50 (lM)
50
100
Doxorubicin
0.721 0.02
0.855 0.01
0.934 0.03
0.882 0.01
0.987 0.01
0.768 0.01
0.795 0.03
0.904 0.06
0.885 0.02
0.984 0.02
0.976 0.01
0.924 0.01
0.933 0.02
0.906 0.9
0.873 0.03
0.919 0.01
0.990 0.01
0.909 0.05
0.969 0.02
0.959 0.01
0.922 0.01
0.546 0.02
0.461 0.01
0.427 0.01
0.499 0.01
0.598 0.01
0.455 0.01
0.494 0.03
0.311 0.01
0.477 0.05
0.450 0.06
0.784 0.05
0.662 0.02
0.642 0.04
0.678 0.01
0.409 0.06
0.423 0.01
0.760 0.01
0.686 0.06
0.635 0.02
0.686 0.01
0.478 0.04
0.487 0.01
0.494 0.03
0.482 0.03
0.447 0.02
0.483 0.03
0.498 0.02
0.425 0.03
0.381 0.01
0.401 0.01
0.430 0.03
0.768 0.01
0.600 0.02
0.609 0.01
0.488 0.02
0.493 0.01
0.581 0.02
0.663 0.06
0.608 0.06
0.457 0.02
0.572 0.02
0.518 0.02
0.534 0.03
71.8
71.8
77.1
85.9
79.7
69.8
52.6
67.3
70.8
[100
[100
[100
94.1
75.2
87
6
0.562 0.05
0.757 0.01
0.763 0.03
0.733 0.05
0.654 0.07
0.448 0.05
0.620 0.04
0.700 0.05
0.770 0.03
0.864 0.01
0.852 0.02
0.885 0.02
0.711 0.01
0.679 0.01
0.905 0.01
0.901 0.02
0.785 0.02
0.898 0.01
0.652 0.01
0.755 0.1
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
a
[100
[100
85.6
[100
81.2
85.6
Each value is the mean of three values standard error
From the results in Table 1, it was found that the py-
rimido[4,5-b]quinoline derivative 11 (IC50 = 52.6 lM)
and the quinoline derivative 12 (IC50 = 67.3 lM) were the
most potent compounds in this screening, and exhibited
higher cytotoxic activities when compared with the reference
drug doxorubicin (IC50 = 71.8 lM). Compounds 6, 10,
and 13 are nearly as active as doxorubicin, or slightly
higher in activity. While compounds 7–9, 17–19, 22, 24,
and 25 showed slightly lower IC50 values than that of the
reference drug, ranging from 75.2 to 94.1 lM.
123

1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
Med Chem Res (2011) 20:388–400
Radiosensitizing activity
393
Compound 11
Compound 11 + 8 Gy
The rationale for combining chemotherapy and radiother-
apy is based mainly on two ideas, one being spatial
cooperation, which is effective if chemotherapy is suffi-
ciently 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.
0.795
0.51
0.448
0.347
0.381
0.222
0.311
0.163
The other idea is the enhancement of 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
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 (Nishimura, 2004).
0
20
40
60
80
100
Concentration (µM)
Fig. 3 Survival curve for MCF7 cell line for compound 11 alone and
in combination with c-irradiation (8 Gy)
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
1
Compound 12
Consequently, the ability of the most two active com-
pounds, compounds 11 and 12, to enhance the cell killing
effect of c-irradiation was studied. From the results
obtained in Table 1, compound 11 showed an in vitro
cytotoxic activity with IC50 value of 52.6 lM, when the
cells were subjected to different concentrations of the
compound alone. While when the cells were subjected to
the same concentrations of compound 11, and irradiated
with a single dose of c-radiation at a dose level of 8 Gy, as
shown in Table 2, the IC50 value was synergistically
decreased to 28.5 lM (Fig. 3).
0.904
Compound 12 + 8 Gy
0.677
0.62
0.447
0.428
0.401
0.199
60
0.156
0
20
40
80
100
Concentration (µM)
Similarly, compound 12 showed IC50 value of 67.3 lM
when used alone, as shown in Table 1. The IC50 value was
decreased to 36 lM, when the cells were treated with
compound 12 in combination with c-radiation (Fig. 4).
From these results, we can conclude that using a com-
bination of compound 11 or 12 and ionizing radiation
synergistically enhanced growth inhibition on breast cancer
cells, compared with each agent alone.
Fig. 4 Survival curve for MCF7 cell line for compound 12 alone and
in combination with c-irradiation (8 Gy)
and this was clearly showed by Abbate et al. (2004), who
stated that the potent anticancer sulfonamide drug (E7070)
(Fig. 5), currently undergoing clinical development for the
treatment of several types of cancer, acts as a strong car-
bonic anhydrase inhibitor, and this may contribute at least
in part, to its in vivo efficacy.
Docking studies
Previous literature shows that carbonic anhydrase inhibi-
tion is one of the anticancer mechanisms of sulfonamides,
The X-ray crystal structure of the adduct of human
carbonic anhydrase II (hCA II) with E7070 revealed
Table 2 In vitro anticancer screening of compounds 11 and 12 against human breast cell line (MCF7) in combination with c-radiation
Compd. no.
Control
Irradiated (8 Gy)
Compound concentration (lM) ? irradiation (8 Gy)
IC50 (lM)
10
25
50
100
Surviving fraction (means SE)a
11
12
a
1.000
1.000
0.927 0.02*
0.927 0.02*
0.510 0.04*
0.677 0.02*
0.347 0.01*
0.428 0.01*
0.163 0.01*
0.199 0.01*
0.222 0.01*
0.156 0.01*
28.5
36
Each value is the mean of three values standard error
* Significant difference from control group at P \ 0.001
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Med Chem Res (2011) 20:388–400
Hydrophobic part
of active site
Hydrophilic part
of active site
Cl
R
S
N
O
H
N
H
NH
O
HN
O S O
Thr 199
2
Zn
O
H
His 119
His 94
His 96
SO2NH2
O
O
Fig. 5 E7070, a sulfonamide compound in advanced clinical trials as
anticancer agent
Tetrahedral adduct
(sulfonamide)
Glu 106
similar interactions between the inhibitor and the active site
as those reported by Supuran et al. These interactions are
found to be common for the sulfonamide compounds which
are CA inhibitors and include: (i) binding of the com-
pounds to the Zn(II) ion by the sulfonamide moiety in a
tetrahedral geometry which is a stable geometry for the
metal ion; (ii) the nitrogen atom of the sulfonamide is
coordinated to the Zn(II) ion of the enzyme; (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. 6) (Supuran and Scozzafava, 2007;
Pastorekova et al., 2004).
Fig. 6 CA inhibition mechanism by sulfonamides
might possibly act as CA inhibitors, and this may contribute
at least in part, to their in vitro anticancer activity.
Conclusion
We report here the synthesis of new fluorine-containing
quinolines and pyrimidoquinolines bearing a free sulfon-
amide moiety. Additionally, it was clearly observed that
some of the synthesized compounds exhibited significant
anticancer activities. Since it was reported that compounds
bearing a free sulfonamide group may exhibit potent car-
bonic anhydrase inhibition activity, which is considered to
be an interesting target for the design of anticancer agents,
docking study was performed which showed significantly
similar binding interactions to hCA II compared to that of
E7070. From the results obtained from the anticancer
screening and the docking studies, it may be suggested that
the anticancer activity may be in part due to the carbonic
anhydrase inhibition. Moreover, the most two active
compounds showed interesting radiosensitizing activity,
when evaluated for their in vitro cytotoxic activity in
combination with c-irradiation.
Since the synthesized compounds are sulfonamide
derivatives and their design complies with the general
pharmacophore of sulfonamide CA inhibitors, it was
interesting to perform docking studies on the synthesized
compounds to hCA II and to compare their docking
interactions with the previously reported interactions of
E7070.
In order to validate our docking procedure, E7070 was
docked into the active site of hCA II. The docking results
clearly show that indeed, the compound exhibits similar
interactions as those previously reported in the literature
and stated above (Fig. 7).
Docking studies of all the newly synthesized compounds
was performed, and it was found that they exhibit similar
interactions to that previously reported for E7070 and
stated above. Figure 8 shows the interaction map of the
pyrimido[4,5-b]quinoline derivative 11 docked pose with
nearby binding site amino acids of hCA II.
Experimental
Finally, it can be concluded from our docking study that
the synthesized compounds exhibit similar conformations
and binding interactions with hCA II similar to those pre-
viously reported for othersulfonamide compounds that act as
CA inhibitors. This suggests that the synthesized compounds
Synthesis
Melting points are uncorrected and were determined on a
Stuart melting point apparatus (Stuart Scientific, Redhill,
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Med Chem Res (2011) 20:388–400
395
Fig. 7 Interaction map of
E7070 with the active site of
hCA II showing similar
interactions as those previously
reported
Fig. 8 Interaction map of
compound 11 with the active
site of hCA II showing similar
interactions as those previously
reported
4-(5,5-Dimethyl-3-oxocyclohex-1-enylamino)
benzenesulfonamide (3)
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,
Munich, Germany), in DMSO-d6 as a solvent, using tet-
ramethylsilane (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 Alumi-
num sheets Silica gel Merck 60 F254 and were visualized
by UV lamp (Merck, Darmstadt, Germany).
Prepared according to the previously reported procedure
(Supuran et al., 2003).
4-(2-Amino-3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-
oxo-5,6,7,8-tetrahydroquinolin-1(4H)-
yl)benzenesulfonamide (6)
A mixture of 4-(5,5-dimethyl-3-oxocyclohex-1-enylamino)
benzenesulfonamide 3 (2.94 g, 0.01 mol) and 2-(4-fluo-
robenzylidene)malononitrile 4 (1.72 g, 0.01 mol) in etha-
nol (20 ml) containing three drops of triethylamine was
refluxed for 6 h. The reaction mixture was filtered while
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Med Chem Res (2011) 20:388–400
hot and the solid obtained was recrystallized from dioxane
to give compound 6: Yield, 86%; m.p. 282–284°C; IR,
cm-1: 3467, 3364, 3258 (2NH2), 3068 (CHarom.), 2954,
2882 (CHaliph.), 2176 (C:N), 1652 (C=O), 1325, 1153
recrystallized from ethanol to give compound 9: Yield,
89%; m.p. 178–180°C; IR, cm-1: 3451, 3250, 3207 (NH,
NH2), 3060 (CHarom.), 2959, 2876 (CHaliph.), 2214 (C:N),
1727, 1655 (3C=O), 1371, 1163 (SO2). 1H NMR (DMSO-d6)
d: 0.7, 0.9 [2s, 6H, 2CH3], 1.8–2.2 [m, 4H, 2CH2], 2.4 [s,
6H, 2COCH3], 4.8 [s, 1H, CH], 7.0–8.0 [m, 10H, Ar–H ?
SO2NH2]. Anal. Calcd. For C28H27FN4O5S: C, 61.08; H,
4.94; N, 10.18. Found: C, 61.25; H, 5.12; N, 10.39.
1
(SO2). H NMR (DMSO-d6) d: 0.7, 0.9 [2s, 6H, 2CH3],
1.7–2.3 [m, 4H, 2CH2], 4.4 [s, 1H, CH], 5.5 [s, 2H, NH2,
exchangeable with D2O], 7.1–8.0 [m, 10H, Ar–H ?
SO2NH2, exchangeable with D2O]. MS, m/z (%): 466 [M?]
(39.87), 371 (100). Anal. Calcd. For C24H23FN4O3S: C,
61.79; H, 4.97; N, 12.01. Found: C, 61.91; H, 4.73; N,
11.80.
4-(5-(4-Fluorophenyl)-2,8,8-trimethyl-4,6-dioxo-
3,4,6,7,8,9-hexahydropyrimido[4,5-b]quinolin-10(5H)-
yl)benzenesulfonamide (10)
4-(5-(4-Fluorophenyl)-8,8-dimethyl-4,6-dioxo-3,4,6,7,8,9-
hexahydropyrimido[4,5-b]quinolin-10(5H)-
yl)benzenesulfonamide (7)
A solution of compound 6 (0.466 g, 0.001 mol) in acetic
anhydride (20 ml) was refluxed for 15 h, the reaction
mixture was then concentrated, the solid separated was
recrystallized from ethanol to give compound 10: Yield,
75%; m.p. 208–210°C; IR, cm-1: 3471, 3300, 3205 (NH,
NH2), 3072 (CHarom.), 2959, 2876 (CHaliph.), 1720, 1656
(2C=O), 1591 (C=N), 1367, 1165 (SO2). 1H NMR
(DMSO-d6) d: 0.7, 0.8 [2s, 6H, 2CH3], 1.8–2.2 [m, 4H,
2CH2], 2.4 [s, 3H, CH3], 4.9 [s, 1H, CH], 7.0–8.0 [m,
11H, Ar–H ? NH ? SO2NH2]. MS, m/z (%):508 [M?]
(9.66), 456 (100). Anal. Calcd. For C26H25FN4O4S: C,
61.40; H, 4.95; N, 11.02. Found: C, 61.12; H, 4.81; N,
11.30.
A solution of compound 6 (0.466 g, 0.001 mol) in formic
acid (30 ml) was refluxed for 5 h, the reaction mixture was
cooled and then poured onto cold water, the obtained solid
was recrystallized from dioxane to give compound 7:
Yield, 79%; m.p. 168–170°C; IR, cm-1: 3390, 3300, 3262
(NH, NH2), 3082 (CHarom.), 2959, 2883 (CHaliph.), 1714,
1647 (2 C=O), 1602 (C=N), 1375, 1172 (SO2). MS, m/
z (%): 495 [M ? 1] (0.16), 101 (100). Anal. Calcd. For
C25H23FN4O4S: C, 60.72; H, 4.69; N, 11.33. Found: C,
60.82; H, 4.84; N, 11.49.
4-(4-Amino-5-(4-fluorophenyl)-8,8-dimethyl-6-oxo-6,7,8,9-
tetrahydropyrimido[4,5-b]quinolin-10(5H)-
yl)benzenesulfonamide (11)
N-(3-Cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-
sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-
yl)acetamide (8)
A solution of compound 6 (0.466 g, 0.001 mol) in form-
amide (30 ml) was refluxed for 5 h, the reaction mixture
was cooled and then poured onto cold water, the obtained
solid was recrystallized from dioxane to give compound
11: Yield, 45%; m.p. 140–142°C; IR, cm-1: 3451, 3310,
3257 (NH2), 3068 (CHarom.), 2952, 2871 (CHaliph.), 1640
(C=O), 1600 (C=N), 1330, 1157 (SO2). MS, m/z (%): 494
[M ? 1] (0.16), 90 (100). Anal. Calcd. For C25H24FN5O3S:
C, 60.84; H, 4.90; N, 14.19. Found: C, 61.02; H, 4.73; N,
14.43.
A solution of compound 6 (0.466 g, 0.001 mol) in acetic
anhydride (20 ml) was refluxed for 5 h, the reaction mixture
was then concentrated, the solid separated was recrystal-
lized from ethanol to give compound 8: Yield, 97%; m.p.
150–152°C; IR, cm-1: 3460, 3230, 3206 (NH, NH2), 3057
(CHarom.), 2957, 2876 (CHaliph.), 2212 (C:N), 1725, 1652
(2 C=O), 1364, 1164 (SO2). 1H NMR (DMSO-d6) d: 0.7, 0.9
[2s, 6H, CH3], 1.5 [s, 3H, COCH3], 1.8–2.2 [m, 4H, CH2],
4.8 [s, 1H, CH], 7.1–8.0 [m, 10H, Ar–H ? SO2NH2], 9.9 [s,
1H, NH]. MS, m/z (%): 510 [M ? 2] (1.24), 90 (100). Anal.
Calcd. For C26H25FN4O4S: C, 61.40; H, 4.95; N, 11.02.
Found: C, 61.68; H, 5.11; N, 10.83.
Ethyl N-3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-
(4-sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-
ylformimidate (12)
N-Acetyl-N-(3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-
oxo-1-(4-sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-
2-yl)acetamide (9)
A solution of compound 6 (0.466 g, 0.001 mol) in tri-
ethylorthoformate (30 ml) containing three drops of acetic
anhydride was refluxed for 8 h, the reaction mixture was
cooled and then poured onto cold water, the obtained
solid was recrystallized from methanol to give compound
12: Yield, 97%; m.p. 164–166°C; IR, cm-1: 3364, 3258
A solution of compound 6 (0.466 g, 0.001 mol) in acetic
anhydride (20 ml) was refluxed for 10 h, the reaction
mixture was then concentrated, the solid separated was
123

Med Chem Res (2011) 20:388–400
397
(NH2), 3065 (CHarom.), 2953, 2895 (CHaliph.), 2195
1
dioxane to give compound 15: Yield, 95%; m.p. [300°C;
IR, cm-1: 3356, 3260, 3227 (NH, NH2), 3040 (CHarom.),
2958, 2880 (CHaliph.), 2216 (C:N), 1677, 1645 (2C=O),
1620 (C=N), 1370, 1161 (SO2). MS, m/z (%): 534 [M ? 1]
(4.90), 97 (100). Anal. Calcd. For C27H24FN5O4S: C,
60.78; H, 4.53; N, 13.13. Found: C, 60.95; H, 4.30; N,
12.91.
(C:N), 1650 (C=O), 1585 (C=N), 1373, 1159 (SO2). H
NMR (DMSO-d6) d: 0.7, 0.9 [2s, 6H, 2CH3], 1.2 [t, 3H,
CH3 ethyl], 1.9–2.2 [m, 4H, 2CH2], 4.3 [q, 2H, CH2
ethyl], 4.6 [s, 1H, CH], 7.1–8.0 [m, 10H, Ar–
H ? SO2NH2], 8.7 [s, 1H, N=CH]. Anal. Calcd. For
C27H27FN4O4S: C, 62.05; H, 5.21; N, 10.72. Found: C,
61.81; H, 5.00; N, 11.01.
4-(3-Cyano-2-(3-ethylthioureido)-4-(4-fluorophenyl)-7,7-
dimethyl-5-oxo-5,6,7,8-tetrahydroquinolin-1(4H)-
yl)benzenesulfonamide (16)
2-Chloro-N-(3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-
oxo-1-(4-sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-
2-yl)acetamide (13)
A mixture of compound 6 (0.466 g, 0.001 mol) and ethyl
isothiocyanate (0.086 g, 0.001 mol) in dimethyl formam-
ide (20 ml) containing three drops of triethylamine was
refluxed for 10 h. The reaction mixture was cooled and
then poured onto cold water, and the solid obtained was
recrystallized from ethanol to give compound 16: Yield,
95%; m.p. 180–182°C; IR, cm-1: 3410, 3346, 3280 (NH,
NH2), 3095 (CHarom.), 2967, 2874 (CHaliph.), 2183 (C:N),
1646 (C=O), 1257 (C=S), 1369, 1137 (SO2). 1H NMR
(DMSO-d6) d: 0.7, 0.9 [2s, 6H, 2CH3], 1.2 [t, 3H, CH3
ethyl], 1.8–2.2 [m, 4H, 2CH2], 3.2 [q, 2H, CH2 ethyl], 4.4
[s, 1H, CH], 7.2–7.8 [m, 10H, Ar–H ? SO2NH2], 8.2, 9.0
[2s, 2H, 2NH]. Anal. Calcd. For C27H28FN5O3S2: C, 58.57;
H, 5.10; N, 12.65. Found: C, 58.32; H, 4.86; N, 12.41.
A mixture of compound 6 (0.466 g, 0.001 mol) and
chloroacetyl chloride (0.112 g, 0.001 mol) in dimethyl
formamide (20 ml) was stirred at room temperature for
6 h. The reaction mixture was poured onto cold water
and the solid obtained was recrystallized from dioxane
to give compound 13: Yield, 95%; m.p. 172–174°C; IR,
cm-1: 3375, 3304, 3252 (NH, NH2), 3096 (CHaliph.),
2960, 2884 (CHaliph.), 2213 (C:N), 1705, 1649
1
(2C=O), 1365, 1172 (SO2). H NMR (DMSO-d6) d: 0.7,
0.9 [2s, 6H, 2CH3], 1.8–2.2 [m, 4H, 2CH2], 4.2 [s, 1H,
CH], 4.7 [s, 2H, CH2Cl], 7.1–7.9 [m, 10H, Ar–
H ? SO2NH2], 10.2 [s, 1H, NH]. MS, m/z (%): 543
[M?] (14.63), 464 (100). Anal. Calcd. For C26H24
ClFN4O4S: C, 57.51; H, 4.45; N, 10.32. Found: C,
57.78; H, 4.22; N, 10.53.
4-(3-Cyano-2-(2,5-dioxopyrrolidin-1-yl)-4-(4-
fluorophenyl)-7,7-dimethyl-5-oxo-5,6,7,8-
tetrahydroquinolin-1(4H)-yl)benzenesulfonamide (17)
4-(2-(Chloromethyl)-5-(4-fluorophenyl)-8,8-dimethyl-4,6-
dioxo-3,4,6,7,8,9-hexahydropyrimido[4,5-b]quinolin-
10(5H)-yl)benzenesulfonamide (14)
A mixture of compound 6 (0.466 g, 0.001 mol) and suc-
cinic anhydride (0.15 g, 0.0015 mol) was fused together in
an oil bath at 250°C for 15 min, the fused mass was dis-
solved in dimethyl formamide and poured onto cold water,
the solid obtained was recrystallized from ethanol to give
A mixture of compound 6 (0.466 g, 0.001 mol) and
chloroacetyl chloride (0.112 g, 0.001 mol) in dimethyl
formamide (20 ml) was refluxed for 16 h. The reaction
mixture was cooled and then poured onto cold water
and the solid obtained was recrystallized from dioxane
to give compound 14: Yield, 84%; m.p. [300°C; IR,
cm-1: 3472, 3407, 3255 (NH, NH2), 3064 (CHarom.),
2940, 2879 (CHaliph.), 1672, 1649 (2C=O), 1595 (C=N),
1381, 1146 (SO2). MS, m/z (%): 542 [M - 1] (4.31),
232 (100). Anal. Calcd. For C26H24ClFN4O4S: C,
57.51; H, 4.45; N, 10.32. Found: C, 57.34; H, 4.61; N,
10.50.
compound 17: Yield, 46%; m.p. 196–198°C; IR, cm-1
:
3362, 3257 (NH2), 3072 (CHarom.), 2959, 2892 (CHaliph.),
1
2214 (C:N), 1734, 1658 (3C=O), 1368, 1163 (SO2). H
NMR (DMSO-d6) d: 0.7, 0.9 [2s, 6H, 2CH3], 1.9–2.2 [m,
4H, 2CH2], 4.8 [s, 1H, CH], 5.2 [t, 4H, 2CH2 pyrrolidine],
7.0–8.0 [m, 10H, Ar–H ? SO2NH2]. Anal. Calcd. For
C28H25FN4O5S: C, 61.30; H, 4.59; N, 10.21. Found: C,
61.57; H, 4.39; N, 10.33.
2-Amino-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-
sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinoline-3-
carboxamide (18)
4-(2-(Cyanomethyl)-5-(4-fluorophenyl)-8,8-dimethyl-4,6-
dioxo-3,4,6,7,8,9-hexahydropyrimido[4,5-b]quinolin-
10(5H)-yl)benzenesulfonamide (15)
A solution of compound 6 (0.466 g, 0.001 mol) in con-
centrated H2SO4 (10 ml) was stirred for 6 h at room tem-
perature, then the reaction mixture was poured onto cold
water. The obtained solid was recrystallized from ethanol
A mixture of compound 6 (0.466 g, 0.001 mol) and ethyl
cyanoacetate (10 ml) was refluxed together for 5 h. The
formed solid mass was collected and recrystallized from
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Med Chem Res (2011) 20:388–400
to give 18: Yield, 75%; m.p. 210–212°C; IR, cm-1: 3400,
3310, 3251 (NH2), 3090 (CHarom.), 2962, 2885 (CHaliph.),
(CHarom.), 2960, 2875 (CHaliph.), 1663 (C=O), 1626 (C=N),
1256 (C=S), 1374, 1164 (SO2). MS, m/z (%): 510 [M?]
(0.25), 100 (100). Anal. Calcd. For C25H23FN4O3S2: C,
58.81; H, 4.54; N, 10.97. Found: C, 59.04; H, 4.38; N,
10.71.
1
1685, 1642 (2C=O), 1376, 1165 (SO2). H NMR (DMSO-
d6) d: 0.7, 0.9 [2s, 6H, 2CH3], 1.8–2.2 [m, 4H, 2CH2], 4.6
[s, 1H, CH], 4.8 [s, 2H, NH2], 6.3 [s, 2H, CONH2], 7.0–8.0
[m, 10H, Ar–H ? SO2NH2]. Anal. Calcd. For
C24H25FN4O4S: C, 59.49; H, 5.20; N, 11.56. Found: C,
59.65; H, 5.00; N, 11.31.
4-(4-Chloro-5-(4-fluorophenyl)-8,8-dimethyl-6-oxo-
6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-10(5H)-
yl)benzenesulfonamide (22)
4-(2-Amino-3-(4,5-dihydro-1H-imidazol-2-yl)-4-(4-
fluorophenyl)-7,7-dimethyl-5-oxo-5,6,7,8-
tetrahydroquinolin-1(4H)-yl)benzenesulfonamide (19)
A solution of compound 7 (0.494 g, 0.001 mol) in thionyl
chloride (10 ml) was refluxed for 2 h, the thionyl chloride
was then removed by distillation, the obtained solid was
washed twice with benzene and recrystallized from diox-
ane to give compound 22: Yield, 96%; m.p. 226–228°C;
IR, cm-1: 3346, 3260 (NH2), 3084 (CHarom.), 2981, 2883
(CHaliph.), 1671 (C=O), 1600, 1570 (C=N), 1340, 1162
(SO2). MS, m/z (%): 513 [M?] (4.77), 100 (100). Anal.
Calcd. For C25H22ClFN4O3S: C, 58.53; H, 4.32; N, 10.92.
Found: C, 58.71; H, 4.10; N, 11.16.
A mixture of compound 6 (0.466 g, 0.001 mol) and eth-
ylene diamine (7 ml) was refluxed in carbon disulfide
(7 ml) for 6 h. The reaction mixture was cooled and then
poured onto cold water. The solid obtained was recrystal-
lized from dioxane to give compound 19: Yield, 35%; m.p.
188–190°C; IR, cm-1: 3401, 3333, 3217 (NH, NH2), 3047
(CHarom.), 2926, 2862 (CHaliph.), 1643 (C=O), 1579 (C=N),
1345, 1164 (SO2). MS, m/z (%): 508 [M - 1] (1.23), 90
(100). Anal. Calcd. For C26H28FN5O3S: C, 61.28; H, 5.54;
N, 13.74. Found: C, 61.03; H, 5.74; N, 13.90.
Ethyl 2-(5-(4-fluorophenyl)-8,8-dimethyl-4,6-dioxo-10-
(4-sulfamoylphenyl)-6,7,8,9-tetrahydropyrimido[4,5-
b]quinolin-3(4H,5H,10H)-yl)acetate (23)
N-(3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-
sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl)-3-
oxobutanamide (20)
A mixture of compound 7 (0.494 g, 0.001 mol), ethyl
bromoacetate (0.167 g, 0.001 mol) and anhydrous potas-
sium carbonate (1.5 g) was refluxed in dry acetone for
24 h. The reaction mixture was filtered while hot, and the
filtrate was cooled then poured onto cold water. The solid
obtained was recrystallized from ethanol to give compound
23: Yield, 94%; m.p. 90–92°C; IR, cm-1: 3367, 3288
(NH2), 3090 (CHarom.), 2965, 2883 (CHaliph.), 1736, 1643
A mixture of compound 6 (0.466 g, 0.001 mol) and ethyl
acetoacetate (10 ml) was refluxed together for 5 h. The
formed solid mass was collected and recrystallized from
dioxane to give compound 20: Yield, 76%; m.p.
158–160°C; IR, cm-1: 3410, 3321, 3260 (NH, NH2), 3072
(CHarom.), 2959, 2872 (CHaliph.), 2182 (C:N), 1725, 1709,
1
(C=O), 1600 (C=N), 1364, 1168 (SO2). H NMR (DMSO-
1
1646 (3C=O), 1372, 1165 (SO2). H NMR (DMSO-d6) d:
d6) d: 0.9, 1.0 [2s, 6H, 2CH3], 1.3 [t, 3H, CH3 ethyl],
1.9–2.2 [m, 4H, 2CH2], 4.1 [q, 2H, CH2 ethyl], 4.2 [s, 1H,
CH], 4.6 [s, 2H, CH2CO], 7.1–7.9 [m, 10H, Ar–
H ? SO2NH2], 8.0 [s, 1H, N=CH]. Anal. Calcd. For
C29H29FN4O6S: C, 59.99; H, 5.03; N, 9.65. Found: C,
60.23; H, 4.79; N, 9.90.
0.7, 0.9 [2s, 6H, 2CH3], 1.9–2.1 [m, 4H, 2CH2], 2.3 [s, 3H,
COCH3], 4.5 [s, 1H, CH], 4.8 [s, 1H, COCH2], 7.1–7.9 [m,
10H, Ar–H ? SO2NH2], 10.1 [s, 1H, NH]. MS, m/z (%):
552 [M ? 2] (0.80), 78 (100). Anal. Calcd. For
C28H27FN4O5S: C, 61.08; H, 4.94; N, 10.18. Found: C,
61.29; H, 5.13; N, 9.92.
4-(5-(4-Fluorophenyl)-4-hydrazinyl-8,8-dimethyl-6-oxo-
6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-10(5H)-
yl)benzenesulfonamide (24)
4-(5-(4-Fluorophenyl)-8,8-dimethyl-6-oxo-4-thioxo-
3,4,6,7,8,9-hexahydropyrimido[4,5-b]quinolin-10(5H)-
yl)benzenesulfonamide (21)
A mixture of compound 22 (0.513 g, 0.001 mol) and
hydrazine hydrate (0.1 mol) was refluxed in ethanol for
5 h. The reaction mixture was cooled and poured onto cold
water. The solid obtained was recrystallized from dioxane
to give compound 24: Yield, 72%; m.p. 150–152°C; IR,
cm-1: 3360, 3325, 3260 (NH, NH2), 3080 (CHarom.), 2967,
2872 (CHaliph.), 1653 (C=O), 1596 (C=N), 1335, 1160
(SO2). MS, m/z (%): 511 [M ? 2] (26.47), 120 (100). Anal.
A mixture of compound 7 (0.494 g, 0.001 mol) and
phosphorus pentasulfide (0.22 g, 0.001 mol) in pyridine
(20 ml) was refluxed for 8 h, the reaction mixture was
cooled and then poured onto cold water, then acidified with
diluted HCl. The solid obtained was recrystallized from
ethanol to give compound 21: Yield, 55%; m.p.
190–192°C; IR, cm-1: 3390, 3329, 3258 (NH, NH2), 3075
123

Med Chem Res (2011) 20:388–400
399
Calcd. For C25H25FN6O3S: C, 59.04; H, 4.95; N, 16.52.
Found: C, 59.29; H, 4.78; N, 16.73.
11 and the quinoline derivative 12, were selected to be
evaluated again for their in vitro anticancer activity alone
and in combination with c-radiation. This study was con-
ducted 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 2 Gy/min. Irradiation
was performed in the National Cancer Institute, Cairo
University, using Gamma cell-40 (60Co) source.
4-(5-(4-Fluorophenyl)-4-isothiocyanato-8,8-dimethyl-6-
oxo-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-10(5H)-
yl)benzenesulfonamide (25)
A mixture of compound 22 (0.513 g, 0.001 mol) and
ammonium thiocyanate (0.076 g, 0.001 mol) was refluxed
in dry acetone for 1 h. The reaction mixture was cooled and
poured onto cold water. The solid obtained was recrystal-
lized from ethanol to give compound 25: Yield, 54%; m.p.
198–200°C; IR, cm-1: 3341, 3262 (NH2), 3083 (CHarom.),
2977, 2873 (CHaliph.), 2019 (N=C=S), 1669 (C=O), 1612
(C=N), 1341, 1163 (SO2). MS, m/z (%): 536 [M ? 1] (0.25),
87 (100). Anal. Calcd. For C26H22FN5O3S2: C, 58.30; H,
4.14; N, 13.08. Found: C, 58.51; H, 4.32; N, 12.80.
The surviving fractions were expressed as means
standard error. The results were analyzed using 1-way
ANOVA test and given in Table 2.
Docking studies
All molecular modeling calculations and docking studies
were performed using ‘‘Molecular Operating Environment
(MOE) version 2007.09’’.
In vitro anticancer screening
The ligand was drawn on ChemDraw and imported in
MOE. The structure was subjected to energy minimization
using MMFF94x forcefield and the partial charges were
computed using the same forcefield.
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. (1990). The in
vitro anticancer screening was done by the pharmacology
unit at the National Cancer Institute, Cairo University.
Cells were plated in 96-multiwell plate (104 cells/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 dimethyl sulfoxide. Different concentra-
tions of the compound under test (10, 25, 50, and 100 lM)
were added to the cell monolayer. Triplicate wells were
prepared for each individual concentration. 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) SRB
dissolved in 1% acetic acid. Excess unbound dye was
removed by four washes 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 breast tumor cell line after the spec-
ified time. The molar concentration required for 50% inhi-
bition of cell viability (IC50) was calculated and compared
to the reference drug doxorubicin (CAS, 25316-40-9). The
surviving fractions were expressed as means standard
error and the results are given in Table 1.
The X-ray crystallographic structure of hCA II com-
plexed with N-(2,3,4,5,6-pentafluoro-benzyl)-4-sulfomyl-
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 Amber99 forcefield.
Docking calculations were done using Alpha triangle
placement method and poses were prioritized by affinity
dG scoring method.
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