Design, synthesis and anticancer evaluation of novel tetrahydroquinoline derivatives containing sulfonamide moiety

Article abstract of DOI:10.1016/j.ejmech.2009.05.017

Sulfonamides posses many types of biological activities and have recently been reported to show substantial antitumor activity in vitro and/or in vivo. There are a variety of mechanisms for the anticancer activity and the most prominent of these is throug

Full text of DOI:10.1016/j.ejmech.2009.05.017

European Journal of Medicinal Chemistry 44 (2009) 4211–4217
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and anticancer evaluation of novel tetrahydroquinoline
derivatives containing sulfonamide moiety
,
b
c
Mostafa M. Ghorab a , Fatma A. Ragab , Mostafa M. Hamed
*
a Department of Drug Radiation Research, National Centre for Radiation Research and Technology, Atomic Energy Authority, P.O. Box 29, 11371 Nasr City, Cairo, Egypt
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, New Cairo City, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 April 2009
Received in revised form 19 May 2009
Accepted 20 May 2009
Available online 24 May 2009
Sulfonamides posses many types of biological activities and have recently been reported to show
substantial antitumor activity in vitro and/or in vivo. There are a variety of mechanisms for the anticancer
activity and the most prominent of these is through the inhibition of carbonic anhydrase isozymes. The
present work reports the synthesis of some novel quinoline and pyrimido[4,5-b]quinoline derivatives
bearing a substituted or unsubstituted sulfonamide moiety. The design of the structures of these
compounds complies with the general pharmacophore of the sulfonamide compounds that act as
carbonic anhydrase (CA) inhibitors as this may play a role in their anticancer activity. All the newly
synthesized compounds were evaluated for their in vitro anticancer activity against breast cancer cell
line (MCF7). Most of the screened compounds showed interesting cytotoxic activities compared to
a reference drug.
Keywords:
Quinoline
Sulfonamide
Anticancer
Carbonic anhydrase
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
CO2/bicarbonate as substrate such as gluconeogenesis, lipogenesis,
ureagenesis, and pyrimidine synthesis [19]. The mechanism of
Sulfonamides posses many types of biological activities and
representatives of this class of pharmacological agents are widely
used in clinic as antibacterial [1], hypoglycemic [2], diuretic [3,4],
anti-carbonic anhydrase [3,5] and antithyroid [6]. Recently, a host
of structurally novel sulfonamide derivatives have been reported to
show substantial antitumor activity in vitro and/or in vivo [7–11].
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. Moreover, following an extensive
evaluation, numerous sulfonamides were found to act as carbonic
anhydrase (CA) inhibitors [12–15]. The most prominent mechanism
was the inhibition of carbonic anhydrase isozymes [16].
tumor inhibition by these sulfonamide CA inhibitors was suggested
by Chegwidden and Spencer [20], that these compounds may
reduce the provision of bicarbonate for the synthesis of nucleotides
and other cell components such as membrane lipids.
In addition, quinoline and fused quinoline derivatives are
known to possess several biological activities including anticancer
activity [21–23]. Also, several reduced quinoline derivatives have
shown significant anticancer activity [24]. In the light of these facts,
and as a continuation of reported work [25,26], we report here the
synthesis of a novel series of quinoline and pyrimidoquinoline
derivatives having a substituted or unsubstituted sulfonamide
moiety where their design complies with the general pharmaco-
phore of the sulfonamide CA inhibitors. We also aim to test the
influence of the substitution of the sulfonamide moiety on the
anticancer activity and to study their structure–activity relation-
ship. The anticancer activity is tested against a breast cancer cell
line (MCF7).
In brief, the a-CA is a family of metalloenzymes involved in the
catalysis of an important physiological reaction: the hydration of
CO2 to bicarbonate and a proton (CO2 þ H2O 4 HCOꢀ3 þ Hþ). At least
13 enzymatically active isoforms have been discovered in higher
vertebrates [12–15]. CAs are involved in pH regulation, secretion of
electrolytes, respiration [17,18], biosynthetic reactions which require
2. Results and discussion
A general pharmacophore (Fig. 1) for the compounds acting as
carbonic anhydrase inhibitors has been reported by Thiry et al. [27]
from the analysis of the CA active site and from the structure of
inhibitors described in the literature [15].
* Corresponding author. Tel.: þ20 (02) 2747413; fax: þ20 (02) 2749298.
E-mail address: mmsghorab@yahoo.com (M.M. Ghorab).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.05.017

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M.M. Ghorab et al. / European Journal of Medicinal Chemistry 44 (2009) 4211–4217
acetate giving an intermediate which upon intramolecular cycli-
zation and finally oxidation yielded compound 1. Compound 2 was
prepared in a similar way as compound 1 but 5,5-dimethylcyclo-
hexane-1,3-dione was used instead of cyclohexanone (Scheme 2).
Compounds 1 and 2 were confirmed from their microanalytical and
spectral data. IR spectra showed bands at 3403, 3322 cmꢀ1 and 3324,
3216 cmꢀ1 (NH2), (C^N) at 2211 and 2179 cmꢀ1 for compounds 1 and
2, respectively. 1H NMR spectrum of 1 in DMSO-d6 revealed signals at
1.7 ppm (CH3) and 6.3 ppm (NH2). While, the 1H NMR spectrum of 2
revealed signals at 1.08 ppm (CH3) and 6.8 ppm (NH2).
Treatment of compounds 1 and 2 with triethylorthoformate in
the presence of acetic anhydride yielded compounds 3 (Scheme 3)
and 11 (Scheme 4), respectively. The formation of compounds 3 and
11 was supported from their microanalytical and spectral data.
Their IR spectra showed the absence of the bands corresponding to
the (NH2) and the presence of bands at 2218 and 2204 cmꢀ1 cor-
responding to the cyano group. Also, the 1H NMR spectrum of the
compounds in DMSO-d6 showed the presence of a 3-proton triplet
at 1.3 ppm and a 2-proton quartet at 4.3 ppm for the ethoxy group.
When compound 3 was treated with hydrazine hydrate at room
temperature, compound 1 was obtained instead of the expected
imino–amino derivative 10. This can explained on the basis of
formation of an intermediate, which upon elimination of ethoxy-
methylene-hydrazone [28] furnished compound 1. This was
confirmed by IR, mixed melting point and similar Rf value on the TLC.
When compound 3 or 11 was stirred with sulfanilamide or
sulfamethoxazole at room temperature in ethanol, a nucleophilic
substitution reaction took place yielding compounds 4, 5 (Scheme 3)
or 12, 13 (Scheme 4), respectively. The compounds were confirmed
by the presence of the (C^N) band in the IR spectrum in range of
Fig. 1. Structural elements of CA inhibitors in the CA enzymatic active site.
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 sulfonamide 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.
Fig. 2 includes representative examples of the synthesized
compounds showing compliance to the above-mentioned phar-
macophore and the compounds were synthesized according to
Schemes 1–4.
2202–2223 cmꢀ1 In addition, the 1H NMR spectrum of the
.
compounds confirmed that the reaction took place by the presence
of the aromatic protons.
2.1. Chemistry
The reaction of compound 1 or 2 with the sulfonamide iso-
thiocyanate derivatives in dimethylformamide, intramolecular
cyclization took place giving the pyrimido[4,5-b]quinoline deriva-
tives 6–9 (Scheme 3) and 16–19 (Scheme 4), respectively. Structures
of compounds 6–9 and 16–19 were supported by elemental analysis
and spectral data. The IR spectrum of the compounds showed the
absence of the (C^N) bands and the presence of (C]S) bands in the
range of 1254–1285 cmꢀ1. In addition, 1H NMR spectra of these
compounds revealed the presence of multiplets at the range of 6.8–
7.9 ppm which could be assigned to the aromatic protons.
Refluxing compound 12 or 13 in pyridine caused intramolecular
cyclization and furnished the pyrimido[4,5-b]quinoline derivative
14 or 15, respectively (Scheme 4). The structure of compounds 14
and 15 was confirmed by the absence of the (C^N) bands in the IR
spectrum. In addition, 1H NMR spectrum of these compounds
revealed the presence of singlet of imino (NH) and multiplets of the
aromatic protons.
Reaction of cyclohexanone with acetaldehyde and malononitrile
in the presence of ammonium acetate yielded the corresponding
tetrahydroquinoline derivative 1 (Scheme 1). The reaction proceeds
first when the acetaldehyde reacts with malononitrile to give an
a,b-unsaturated nitrile intermediate, which reacts with the
carbonyl compound cyclohexanone in the presence of ammonium
2.2. In vitro anticancer screening
The newly synthesized compounds were evaluated for their in
vitro cytotoxic activity against the breast cancer cell line, MCF7.
Doxorubicin which is one of the most effective anticancer agents
was used as the 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 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 where the compounds exhibited significant activity
compared to the reference drug Doxorubicin.
Fig. 2. Representative examples of the synthesized compounds showing compliance to
the general pharmacophore of sulfonamide compounds acting as carbonic anhydrase
inhibitors.

M.M. Ghorab et al. / European Journal of Medicinal Chemistry 44 (2009) 4211–4217
4213
Scheme 1.
From these results, it can be seen that although the variation in
biological activity between the compounds was not very high, yet
the following points can still be concluded.
Concerning the tetrahydroquinoline derivatives, it was found
from the results that the compounds having a substituted sulfon-
amide – with methyl isoxazolyl ring – 5 and 13 were more potent
than the unsubstituted ones 4 and 12. Compound 13 was the most
potent in this group.
While for the fused tetrahydroquinoline derivatives 6–9 and
16–19, it was found that the unsubstituted sulfonamide derivatives
6 and 16 were more potent than the substituted ones 7–9 and 17–19.
The order of activity for the substituted derivatives was as follows:
the guanido derivatives 7 and 17 having the highest potency fol-
lowed by the diazine derivatives 9 and 19 and finally the isoxazole
derivatives 8 and 18. Compound 6 was the most potent in this group.
Docking of the synthesized compounds was performed and it
was found that the compounds with an unsubstituted sulfonamide
moiety exhibit similar interactions to those previously reported for
E7070 and stated above. Fig. 6 shows an example of the interaction
map of compound 12 with an unsubstituted sulfonamide moiety
docked pose with nearby binding site amino acids of hCA II.
While, it has been seen from the docking of the substituted
sulfonamide derivatives that they did not fit properly in the active
site of the enzyme and accordingly do not form the similar inter-
actions that have been reported in the literature or obtained from
the X-ray crystallography of the compounds used as carbonic
anhydrase inhibitors. Fig. 7 shows an example of the docking of
compound 5 having a substituted sulfonamide moiety.
3. Conclusions
2.3. Docking studies
We report here the synthesis of new quinoline and pyr-
imidoquinoline derivatives containing a substituted and unsub-
stituted sulfonamide moiety. In addition, it was clearly observed that
the compounds with either a substituted or unsubstituted sulfon-
amide moiety exhibit significant anticancer activity. The docking of
the compounds showed that the compounds with an unsubstituted
sulfonamide moiety may act also as CA inhibitors and this may at
least in part contribute to their anticancer activity. While, the
compounds with a substituted sulfonamide moiety would not seem
to have CA inhibitory activity based on computer modeling and
docking studies and this may suggest that the anticancer activity of
these compounds is mainly due to another mechanism.
Previous literature shows that carbonic anhydrase inhibition is
one of the anticancer mechanisms of sulfonamides, and this was
clearly showed by Abbate et al. [7], who stated that the potent
anticancer sulfonamide drug (E7070) (Fig. 3) – currently under-
going clinical development for the treatment of several types of
cancer – also acts as a strong carbonic anhydrase inhibitor, and this
may contribute at least in part, to its in vivo efficacy.
The X-ray crystal structure of the adduct of human carbonic
anhydrase II (hCA II) with E7070 revealed similar interactions
between the inhibitor and the active site as those reported by
Supuran et al. [14,17]. These interactions are found to be common for
the sulfonamide compounds which are CA inhibitors and include:
(i) binding of the compounds 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. 4) [14,17].
Since our 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. 5).
4. Experimental
4.1. Chemistry
Melting points are uncorrected and were determined on Buchi
melting point apparatus (B-540). Elemental analyses (C, H, N) were
performed on Perkin–Elmer 2400 analyzer (Perkin–Elmer, Norwalk,
CT, USA) at Microanalytical Laboratories of the Faculty of Science,
Cairo University. All compounds were within ꢁ0.4% of the theoretical
values. The IR spectra were measured on Nicolet 380 FT-IR
spectrometer. 1H NMR spectra were obtained on a Bruker proton
NMR-Avance 300 (300, MHz), in DMSO-d6 as a solvent, using tetra-
methylsilane (TMS) as internal standard. Splitting patterns were
designated as follows: s: singlet; d: doublet; t: triplet; m: multiplet.
Mass spectra (EI) were run on HP Model MS-5988 (Hewlett Packard).
Scheme 2.

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M.M. Ghorab et al. / European Journal of Medicinal Chemistry 44 (2009) 4211–4217
Scheme 3.
4.1.1. 2-Amino-4-methyl-5,6,7,8-tetrahydroquinoline-3-
carbonitrile (1) and 2-amino-4,7,7-trimethyl-5-oxo-5,6,7,8-
tetrahydroquinoline-3-carbonitrile (2)
2962, 2879 (CH aliph.), 2179 (CN), 1676 (C]O). 1H NMR (DMSO-d6)
: 1.00, 1.02 [2s, 6H, 2CH3], 1.08 [s, 3H, CH3], 2.21–2.49 [m, 4H, 2CH2
cyclo], 6.82 [s, 2H, NH2 exchangeable with D2O]. MS, m/z (%): 229
[Mþ] (7.5), 217 (100). Anal. Calcd. for C13H15N3O: C, 68.10; H, 6.59; N,
18.33. Found: C, 68.39; H, 6.22; N, 18.55.
d
A mixture of cyclohexanone (0.98 g, 0.01 mol) or 5,5-dime-
thylcyclo-hexane-1,3-dione (1.4 g, 0.01 mol) with acetaldehyde
(0.44 g, 0.01 mol), malononitrile (0.06 g, 0.01 mol), and ammonium
acetate (1.15 g, 0.015 mol) in ethanol (20 ml) was refluxed for 1 h.
The reaction mixture was then stirred for 3 h at room temperature
and the obtained solid was recrystallized from dioxane to give 1
and 2, respectively. 1: Yield, 45%; m.p. 278–280 ꢂC; IR, cmꢀ1: 3403,
3322 (NH2), 2932, 2872 (CH aliph.), 2211 (C^N). 1H NMR (DMSO-
4.1.2. Ethyl N-3-cyano-4-methyl-5,6,7,8-tetrahydroquinolin-2-
ylformimidate (3), ethyl N-3-cyano-4,7,7-trimethyl-5-oxo-5,6,7,8-
tetrahydroquinolin-2-ylformimidate (11)
A solution of 1 (1.87 g, 0.01 mol) or 2 (0.46 g, 0.002 mol) and
triethylorthoformate (10 ml) containing 3 drops of acetic anhydride
was refluxed for 8 h. The reaction mixture was cooled and then
poured onto ice water. The obtained solid was recrystallized from
ethanol to give 3 and 11, respectively. 3: Yield, 68%; m.p. 98–100 ꢂC;
d6) d: 1.71 [s, 3H, CH3], 2.23–2.61 [m, 8H, 4CH2], 6.37 [s, 2H, NH2,
exchangeable with D2O]. MS, m/z (%): 187 [Mþ] (100). Anal. Calcd.
for C11H13N3: C, 70.56; H, 7.00; N, 22.44. Found: C, 70.30; H, 6.78; N,
22.70. 2: Yield, 44%; m.p. 188–190 ꢂC; IR, cmꢀ1: 3324, 3216 (NH2),
IR, cmꢀ1: 2940 (CH aliph.), 2218 (C^N). 1H NMR (DMSO-d6)
d: 1.36
Scheme 4.

M.M. Ghorab et al. / European Journal of Medicinal Chemistry 44 (2009) 4211–4217
4215
Table 1
In vitro cytotoxic activity of the synthesized compounds.
a
b
Compound
IC50
(
m
g/ml)
IC50
NA
(mM)
4
NA
5
6
7
8
1.95
0.95
1.28
2.62
1.48
2.62
1.9
1.5
2.6
4
5.17
NA
4.33
2.37
2.89
5.44
3.09
6.37
3.86
3.65
5.28
9.03
13.43
NA
9
12
13
14
15
16
17
18
19
Dox.
9.19
0.7
17.64
1.2
NA: Compounds having IC50 value > 10 mg/ml.
a
Mean of three results obtained from three experiments.
IC50 value: concentration causing 50% inhibition of cell viability.
b
[t, 3H, CH3 ethyl], 1.78 [s, 3H, CH3], 2.23–2.79 [m, 8H, 4CH2 cyclo],
4.36 [q, 2H, CH2 ethyl], 8.44 [s, 1H, N]CH]. MS, m/z (%): 243 [Mþ]
(1.78), 187 (100). Anal. Calcd. for C14H17N3O: C, 69.11; H, 7.04; N,
17.27. Found: C, 68.83; H, 7.42; N, 17.50. 11: Yield, 80%; m.p. 95–
97 ꢂC; IR, cmꢀ1: 2962, 2873 (CH aliph.), 2204 (CN), 1678 (C]O). 1H
Fig. 4. CA inhibition mechanism by sulfonamides.
NMR (DMSO-d6) d: 1.02, 1.03 [2s, 6H, 2CH3], 1.17 [s, 3H, CH3], 1.30 [t,
3H, CH3 ethyl], 2.26–2.45 [m, 4H, 2CH2 cyclo], 4.31 [q, 2H, CH2
ethyl], 8.46 [s, 1H, N]CH]. MS, m/z (%): 285 [Mþ] (1.05), 174 (100).
Anal. Calcd. for C16H19N3O2: C, 67.35; H, 6.71; N, 14.73. Found: C,
67.11; H, 6.94; N, 15.02.
C, 58.52; H, 5.18; N,18.96. Found: C, 58.20; H, 5.05; N,18.74. 5: Yield,
46%; m.p. 217–219 ꢂC; IR, cmꢀ1: 3310, 3150 (NH), 3100 (CH arom.),
2935, 2857 (CH aliph.), 2223 (C^N), 1379, 1184 (SO2). MS, m/z (%):
450 [Mþ] (1.7), 187 (100). Anal. Calcd. for C22H22N6O3S: C, 58.65; H,
4.92; N, 18.65. Found: C, 58.90; H, 5.20; N, 18.91. 12: Yield, 50%; m.p.
201–203 ꢂC; IR, cmꢀ1: 3334, 3222, 3200 (NH, NH2), 3083 (CH
arom.), 2967, 2874 (CH aliph.), 2202 (CN), 1669 (C]O), 1376, 1157
4.1.3. N0-(3-cyano-4-methyl-5,6,7,8-tetrahydroquinolin-2-yl)-N-
(4-sulfamoylphenyl)-formimidamide (4), N0-(3-cyano-4-methyl-
5,6,7,8-tetrahydroquinolin-2-yl)-N-(4-(N-(5-methylisoxazol-3-
yl)sulfamoyl)phenyl) formimidamide (5), N0-(3-cyano-4,7,7-
trimethyl -5-oxo-5,6,7,8-tetrahydroquinolin-2-yl)-N-(4-
(SO2). 1H NMR (DMSO-d6)
d: 1.03 [s, 6H, 2CH3], 1.17 [s, 3H, CH3],
2.25–2.49 [m, 4H, 2CH2 cyclo], 7.26–8.23 [m, 4H, Ar–H], 8.85 [s, 1H,
N]CH], 10.58 [s, 1H, NH], 11.12 [s, 2H, SO2NH2]. MS, m/z (%): 411
[Mþ] (1.5), 149 (100). Anal. Calcd. for C20H21N5O3S: C, 58.38; H, 5.14;
N, 17.02. Found: C, 58.09; H, 5.41; N, 16.84. 13: Yield, 45%; m.p. 130–
132 ꢂC; IR, cmꢀ1: 3325, 3220 (NH), 3099 (CH arom.), 2959, 2875 (CH
aliph.), 2205 (CN), 1658 (C]O), 1370, 1161 (SO2). MS, m/z (%): 493
[Mþ] (1.28), 161 (100). Anal. Calcd. for C24H24N6O4S: C, 58.52; H,
4.91; N, 17.06. Found: C, 58.28; H, 5.20; N, 17.30.
sulfamoylphenyl)formimidamide (12) and N0-(3-cyano-4,7,7-
trimethyl-5-oxo-5,6,7,8-tetrahydroquinolin-2-yl)-N-(4-(N-(5-
methylisoxazol-3-yl)sulfamoyl)phenyl)formimidamide (13)
A mixture of 3 (0.243 g, 0.001 mol) or 11 (0.285 g, 0.001 mol)
and sulfanilamide or sulfamethoxazole (0.001 mol) was stirred
overnight at room temperature in ethanol (20 ml). The obtained
solid was recrystallized from dioxane to give 4, 5 or 12, 13,
respectively. 4: Yield, 43%; m.p. 224–226 ꢂC; IR, cmꢀ1: 3311, 3207,
3134 (NH, NH2), 3085 (CH arom.), 2943, 2862 (CH aliph.), 2222
(C^N), 1369, 1147 (SO2). 1H NMR (DMSO-d6)
d: 1.77 [s, 3H, CH3],
2.22–2.79 [m, 8H, 4CH2 cyclo], 6.13 [s, 1H, N]CH], 7.44, 7.80 [2d,
4H, Ar–H, AB system], 9.53 [s, 1H, NH], 11.46 [s, 2H, SO2NH2]. MS,
m/z (%): 369 [Mþ] (13.4), 187 (100). Anal. Calcd. for C18H19N5O2S:
Fig. 3. Compound E7070, a sulfonamide compound in advanced clinical trials as
Fig. 5. Interaction map of E7070 with the active site of hCA II showing similar inter-
anticancer agent.
actions as those previously reported.

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M.M. Ghorab et al. / European Journal of Medicinal Chemistry 44 (2009) 4211–4217
C18H19N5O2S2: C, 53.85; H, 4.77; N, 17.44. Found: C, 53.50; H, 4.54;
N, 17.10. 7: Yield, 61%; m.p. 213–215 ꢂC; IR, cmꢀ1: 3350, 3138 (NH,
NH2), 3077 (CH arom.), 2927, 2852 (CH aliph.), 1384, 1182 (SO2),
1277 (C]S). MS, m/z (%): 443 [Mþ] (1.5), 148 (100). Anal. Calcd. for
C19H21N7O2S2: C, 51.45; H, 4.77; N, 22.11. Found: C, 51.63; H, 4.50; N,
22.40. 8: Yield, 51%; m.p. 198–200 ꢂC; IR, cmꢀ1: 3147 (NH), 3050
(CH arom.), 2931, 2862 (CH aliph.), 1383, 1142 (SO2), 1260 (C]S). 1H
NMR (DMSO-d6) d: 1.70 [s, 3H, CH3], 2.10 [s, 3H, CH3 isoxazole],
2.21–2.89 [m, 8H, 4CH2], 6.26 [s, 1H, CH isoxazole], 6.98–7.94 [m,
4H, Ar–H], 12.41, 12.83 [2s, 2H, 2NH], 15.18 [s, 1H, SO2NH]. MS, m/z
(%): 482 [Mþ] (2.0), 187 (100). Anal. Calcd. for C22H22N6O3S2: C,
54.75; H, 4.60; N, 17.41. Found: C, 54.50; H, 4.41; N, 17.72. 9: Yield,
71%; m.p. 240–242 ꢂC; IR, cmꢀ1: 3403, 3320, 3140 (NH), 3090
(CH arom.), 2932, 2852 (CH aliph.), 1389, 1153 (SO2), 1256 (C]S).
MS, m/z (%): 480 [Mþ] (7), 55 (100). Anal. Calcd. for C22H21N7O2S2:
C, 55.10; H, 4.41; N, 20.44. Found: C, 55.46; H, 4.74; N, 20.12. 16:
Yield, 75%; m.p. 132–134 ꢂC; IR, cmꢀ1: 3340, 3199 (NH, NH2), 3080
(CH arom.), 2956, 2850 (CH aliph.), 1651 (C]O), 1383, 1154 (SO2),
1256 (C]S). 1H NMR (DMSO-d6)
d: 0.96 [2s, 6H, 2CH3], 1.01 [s, 3H,
CH3], 2.21–2.49 [m, 4H, 2CH2 cyclo], 7.21–7.95 [m, 6H, Ar–
H þ SO2NH2], 10.32 [s, 1H, NH], 13.82 [s, 1H, NH]. MS, m/z (%): 443
[Mþ] (3.11), 148 (100). Anal. Calcd. for C20H21N5O3S2: C, 54.16; H,
4.77; N, 15.79. Found: C, 54.43; H, 5.02; N, 15.97. 17: Yield, 47%; m.p.
164–166 ꢂC; IR, cmꢀ1: 3426, 3336, 3200 (NH, NH2), 2955, 2850 (CH
aliph.), 1661 (C]O), 1384, 1134 (SO2), 1254 (C]S). MS, m/z (%): 485
[Mþ] (4), 107 (100). Anal. Calcd. for C21H23N7O3S2: C, 51.94; H, 4.77;
N, 20.19. Found: C, 52.23; H, 4.36; N, 20.50. 18: Yield, 59%; m.p.
160–162 ꢂC; IR, cmꢀ1: 3380, 3343 (NH, NH2), 3055 (CH arom.),
2956, 2840 (CH aliph.), 1658 (C]O), 1389, 1161 (SO2), 1266 (C]S).
Fig. 6. Interaction map of compound 12 with the active site of hCA II showing similar
interactions as those previously reported.
4.1.4. 4-(4-Imino-5-methyl-2-thioxo-1,2,6,7,8,9-
hexahydropyrimido[4,5-b]quinolin-3 (4H)-yl)-N-(substituted or
unsubstituted)benzenesulfonamides (6–9) and 4-(4-imino-5,8,8-
trimethyl-6-oxo-2-thioxo-1,2,6,7,8,9-hexahydropyrimido[4,5-b]-
quinolin-3 (4H)-yl)-N-(substituted or
1H NMR (DMSO-d6)
d: 0.98, 0.99 [2s, 6H, 2CH3], 1.01 [s, 3H, CH3],
2.11 [s, 3H, CH3 isoxazole], 2.21–2.82 [m, 8H, 4CH2], 6.23 [s, 1H, CH
isoxazole], 6.89–7.79 [m, 5H, Ar–H þ NH], 9.82 [1s, 1H, 1NH], 11.36
[s, 1H, SO2NH]. MS, m/z (%): 524 [Mþ] (46), 168 (100). Anal. Calcd.
for C24H24N6O4S2: C, 54.95; H, 4.61; N, 16.02. Found: C, 54.69; H,
4.89; N, 16.38. 19: Yield, 71%; m.p. 117–119 ꢂC; IR, cmꢀ1: 3361, 3300
(NH), 2930, 2830 (CH aliph.), 1648 (C]O), 1388, 1155 (SO2), 1254
(C]S). MS, m/z (%): 521 [Mþ] (2.5), 82 (100). Anal. Calcd. for
C24H23N7O3S2: C, 55.26; H, 4.44; N, 18.80. Found: C, 55.54; H, 4.09;
N, 18.53.
unsubstituted)benzenesulfonamides (16–19)
A mixture of 1 (0.374 g, 0.002 mol) or 2 (0.345 g, 0.0015 mol)
and the corresponding sulfonamide isothiocyanate derivative
(0.0015 mol) in dimethylformamide (20 ml) containing 3 drops of
triethylamine was refluxed for 10 h. The reaction mixture was
cooled and then poured onto ice water. The obtained solid was
recrystallized from ethanol to give 6–9 and 16–19, respectively. 6:
Yield, 75%; m.p. 227–229 ꢂC; IR, cmꢀ1: 3388, 3324, 3152 (NH, NH2),
3050 (CH arom.), 2918, 2849 (CH aliph.), 1332, 1150 (SO2), 1285
4.1.5. 4-(4-Imino-5,8,8-trimethyl-6-oxo-6,7,8,9-
tetrahydropyrimido[4,5-b]quinolin-3-(4H)-yl)benzenesulfonamide
(14) and 4-(4-imino-5,8,8-trimethyl-6-oxo-6,7,8,9-
(C]S). 1H NMR (DMSO-d6)
d: 1.79 [s, 3H, CH3], 2.22–2.98 [m, 8H,
4CH2 cyclo], 7.03–7.82 [m, 6H, Ar–H þ SO2NH2], 9.75, 12.43 [2s, 2H,
2NH]. MS, m/z (%): 401 [Mþ] (63.5), 55 (100). Anal. Calcd. for
tetrahydropyrimido-[4,5-b]quinolin-3 (4H)-yl)-N-(5-
methylisoxazol-3-yl)benzenesulfonamide (15)
Method (A). A mixture of compound 11 (0.285 g, 0.001 mol),
sulfanilamide or sulfamethoxazole (0.0012 mol) in pyridine (20 ml)
was refluxed for 12 h. The reaction mixture was cooled and poured
onto ice water. The obtained solid was recrystallized from dioxane
to give 14 and 15, respectively.
Method (B). Compound 12 or 13 (0.001 mol) in pyridine (20 ml)
was refluxed for 10 h. The reaction mixture was then poured onto
ice water acidified with dil. HCl. The product was filtered and
recrystallized from dioxane to give 14 and 15, respectively (m.p. and
m.m.p.).
14: Yield, 55%; m.p. 179–181 ꢂC; IR, cmꢀ1: 3313, 3281, 3125 (NH,
NH2), 3050 (CH arom.), 2958, 2860 (CH aliph.), 1668 (C]O), 1383,
1153 (SO2). MS, m/z (%): 412 [Mþ] (4), 149 (100). Anal. Calcd. for
C20H21N5O3S: C, 58.38; H, 5.14; N, 17.02. Found: C, 58.69; H, 5.40; N,
17.39. 15: Yield, 61%; m.p. 212–214 ꢂC; IR, cmꢀ1: 3247, 3184 (NH),
3054 (CH arom.), 2962, 2870 (CH aliph.), 1671 (C]O), 1369, 1183
(SO2). 1H NMR (DMSO-d6)
d: 1.02, 1.03 [2s, 6H, 2CH3], 1.2 [s, 3H,
CH3], 2.07–2.49 [m, 7H, 2CH2 cyclo þ CH3 isoxazole], 6.11 [s, 1H, CH
isoxazole], 7.61–7.98 [m, 4H, Ar–H], 8.24 [s, 1H, CH pyrimidine],
10.32 [s, 1H, NH], 11.25 [s, 1H, SO2NH]. MS, m/z (%):492 [Mþ] (0.5),
Fig. 7. Interaction map of compound 5 with the active site of hCA II showing different
interactions than those previously reported.

M.M. Ghorab et al. / European Journal of Medicinal Chemistry 44 (2009) 4211–4217
4217
161 (100). Anal. Calcd. for C24H24N6O4S: C, 58.52; H, 4.91; N, 17.06.
Found: C, 58.75; H, 5.15; N, 17.33.
Docking calculations were done using Alpha triangle placement
method and poses were prioritized by Affinity dG scoring method.
4.2. In vitro anticancer screening
References
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synthesized compounds using the Sulfo-Rhodamine-B stain (SRB)
assay using the method of Skehan et al. [29]. The in vitro anticancer
screening was done by the pharmacology unit at the National
Cancer Institute, Cairo University.
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