Synthesis and in vitro anticancer evaluation of some novel hexahydroquinoline derivatives having a benzenesulfonamide moiety

Article abstract of DOI:10.1016/j.ejmech.2010.11.002

Inhibition of carbonic anhydrase isozymes has been found to have a role in the treatment of cancer. Several sulfonamide compounds bearing an aromatic or a heteroaromatic ring were found to posses potent carbonic anhydrase inhibitory activity and so can be

Full text of DOI:10.1016/j.ejmech.2010.11.002

European Journal of Medicinal Chemistry 46 (2011) 201e207
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro anticancer evaluation of some novel hexahydroquinoline
derivatives having a benzenesulfonamide moiety
,
b
c
Mansour S. Al-Said a, Mostafa M. Ghorab a , Mohammed S. Al-Dosari , Mostafa M. Hamed
*
a Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
b Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy & 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:
Inhibition of carbonic anhydrase isozymes has been found to have a role in the treatment of cancer.
Several sulfonamide compounds bearing an aromatic or a heteroaromatic ring were found to posses
potent carbonic anhydrase inhibitory activity and so can be used in the treatment of several types of
cancer. In this paper, we present the synthesis of some novel quinoline 7e13, 21e26, 28 and pyr-
imidoquinoline 14e18, 20, 27 derivatives having a sulfonamide moiety. All the newly synthesized
compounds were evaluated for their in vitro anticancer activity. Several compounds showed interesting
cytotoxic activities when compared with the used reference drug. In addition, docking of the synthesized
compounds into carbonic anhydrase isozyme II (CA II) active site was performed in order to give
a suggestion about the proposed mechanism of action.
Received 27 December 2009
Received in revised form
31 October 2010
Accepted 1 November 2010
Available online 10 November 2010
Keywords:
Sulfonamide
Quinoline
Pyrimidoquinoline
Anticancer
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
have an important role in cancer treatment through reducing the
provision of bicarbonate for the synthesis of nucleotides and other
Sulfonamide compounds have shown to posses several types of
biological activities [1e4] including anticancer activity [5e10]. The
anticancer activity of sulfonamides was found to take place 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 carbonic anhydrase inhibition [11e14] which is reported to be
the most prominent mechanism [15].
Carbonic anhydrases (CAs) are zinc metalloenzymes that cata-
lyze the reversible hydration of carbon dioxide to give bicarbonate
and a proton (CO2 þ H2O 4 HCOꢀ3 þ Hþ). CAs are involved in pH
regulation, secretion of electrolytes, respiration [16,17], biosyn-
thetic reactions which require CO2/bicarbonate as substrate such as
gluconeogenesis, lipogenesis, ureagenesis, and pyrimidines
synthesis [18]. The catalytic domain of CAs contains an active site
Zn2þ. This metal cation is a strong Lewis acid that binds to and
activates a substrate H2O molecule to catalyzes the reversible
hydration reaction of carbon dioxide. The hydration of CO2 does not
proceed at an appreciable rate under physiological conditions in
the absence of CA enzymes [19]. CA inhibition has been found to
cell components such as membrane lipids [20]. Actazolamide I and
E7070 II are examples of carbonic anhydrase inhibitors.
Furthermore, among the compounds that have shown signifi-
cant anticancer activity are the quinolines and reduced quinoline
derivatives [21e24]. Also, combination of several quinoline deriv-
atives with sulfonamide moiety has been reported to have signifi-
cant anticancer activity [7,25,26]. In the light of these facts, and in
a hope to obtain some novel compounds with significant anticancer
activity, this work reports the synthesis of a novel series of quin-
oline and pyrimidoquinoline derivatives bearing a sulfonamide
moiety and the testing of these compounds for their in vitro anti-
cancer activity.
2. Results and discussion
2.1. Chemistry
During the course of our continued interest in the development
of new general synthetic routes for the synthesis of biologically
active quinolines and pyrimidoquinolines we decided to synthesize
a new series of pyrimidoquinoline derivatives 14e18, 20 and 27
starting from enaminone 3 [27]. The designed target compounds
are depicted in Schemes 1e5. Enaminone 3 was obtained via
reaction of 1,3-cyclohexandione 1 with sulfanilamide 2. Treatment
* Corresponding author. Tel.: þ966 534292860; fax: þ966 (01) 4670560.
E-mail address: mmsghorab@yahoo.com (M.M. Ghorab).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.002

202
M.S. Al-Said et al. / European Journal of Medicinal Chemistry 46 (2011) 201e207
Scheme 3.
furnish pyrimidoquinoline derivative 20, rather than the quinoline
derivative 19, based on the elemental analysis and IR spectrum,
which showed the absence of C^N band and presence of bands at
1697, 1652 cmꢀ1 (2C]O). 1H NMR spectrum of compound 20 (in
DMSO-d6) revealed signals at 3.5 ppm corresponding to CH2Cl
group and 10.9 ppm due to NH group. When compound 6 was
stirred with concentrated H2SO4 at room temperature [28] for 2 h,
afforded carboxamide derivative 21, while refluxing of compound 6
with H2SO4 caused complete hydrolysis to afford the corresponding
quinoline derivative 22. The formation of amide 21 was confirmed
by IR spectrum, which showed the disappearance of the cyano
C^N group and exhibited a carbonyl stretch at 1697 cmꢀ1 along
with characteristic (NH2) and carboxamido (CONH2) bands at 3364,
3272 cmꢀ1. IR spectrum of compound 22 exhibited the absence of
C^N band and presence of OH band at 3478 cmꢀ1 (Scheme 3).
Condensation of compound 6 with aromatic aldehydes in glacial
acetic acid afforded Schiff’s bases 23e25. The structure of
compounds 23e25 was confirmed by elemental analysis, IR spectra
which showed the presence of C^N group at 2210e2215 cmꢀ1. This
work was extended to cover the reactivity of compound 6 toward
carbonyl compounds. Thus, condensation of 6 with ethyl acetoa-
cetate under condition of fusion gave a product which was
formulated as quinoline derivative 26. The IR spectrum of 26
exhibited bands at 1719, 1680, 1641 cmꢀ1 (3C]O). 1H NMR spec-
trum of compound 26 (in DMSO-d6) exhibited signals at 2.2 ppm
due to COCH3 group (Scheme 4).
Scheme 1.
of enaminone 3 with 2,4-dichlorobenzylidenemalononitrile 4 in
ethanol containing triethylamine as catalyst resulted in cycloaddi-
tion affording quinoline-o-aminocarbonitrile
formation of the intermediate Michael type product 5, followed by
intramolecular cyclization (Scheme 1).
The reaction of compound 6 with acid chloride derivatives in
pyridine was carried out in a trial for obtaining pyrimidoquinoline
derivatives 14e18, but instead the quinoline derivatives 7e13 were
obtained. While the reaction of compound 6 with acid chloride
derivatives only affected cyclization to furnish pyrimidoquinoline
derivatives 14e18, based on the elemental analyses, IR, 1H NMR and
mass spectral data. The structure of compounds 7e13 was proved
by elemental analyses, IR, 1H NMR and mass spectral data. The IR
spectra of compounds 7e13 revealed the presence of C^N band.
The IR spectra of compounds 14e18 exhibited the absence of C^N
band (Scheme 2).
6 [27], via the
Also, interaction of compound 6 with chloroacetyl chloride in
dimethylformamide at room temperature effected cyclization to
Treatment of compound 6 with triethyl orthoformate yielded
compound 28. The formation of compound 28 was supported by
microanalytical and spectral data, its IR spectrum showed the
presence of band at 2200 cmꢀ1 corresponding to the cyano group.
Also 1H NMR spectrum of compound 28 (in DMSO-d6) exhibited the
presence of a 3 protons triplet at 1.1 ppm and a 2 protons quartet at
4.19 ppm for the ethyl group. Finally, when compound 28 was
treated with hydrazine hydrate at room temperature, the starting
material 6 was obtained instead of the expected N-amino derivative
29. This can explained on the basis of formation of an intermediate
30, which upon elimination of ethoxymethylene-hydrazone [29]
furnished compound 6. This was confirmed by IR, mixed melting
point and similar Rf value on the TLC (Scheme 5).
2.2. Biological testing
Doxorubicin, the reference drug used in this study is one of the
most effective antitumor agents used to produce regressions in
acute leukemias, Hodgkin’s disease and other lymphomas. Doxo-
rubicin, which has a significant anticancer activity, was chosen to
be our reference drug in this phase so as to just compare its activity
with the newly synthesized compounds in order to put a hand on
Scheme 2.

M.S. Al-Said et al. / European Journal of Medicinal Chemistry 46 (2011) 201e207
203
Scheme 4.
the most active compounds and to help use in optimizing the
synthesis of the second phase compounds. The relationship
between surviving ratio and drug concentration was plotted to
obtain the survival curve of Ehrlich Ascites Carcinoma (EAC) cells.
The response parameter calculated was IC50 value, which corre-
sponds to the compound concentration causing 50% mortality in
net cells.
All the synthesized compounds 6e18, 20e28 were evaluated for
their in vitro cytotoxic activity. Compounds 6, 7, 9,17 and 18 showed
significant activity compared to the reference drug doxorubicin
(CAS-23214-92-8) (Table 1).
activity such as compound 9 having benzamide moiety at 2-posi-
tion and cyano group at the 3-position (IC50 value ¼ 17 M), the
pyrimidoquinoline 18 carrying 4-bromophenyl at the 2-position
(IC50 value ¼ 37 M), compound 7 containing butanamide at the
2-position (IC50 value ¼ 45 M), the pyrimidoquinoline 17 having
p-tolyl at the 2-position (IC50 value ¼ 46 M) and compound 6
bearing a free amino group at the 2-position with cyano group at
3-position (IC50 value ¼ 51 M). These compounds showed cyto-
toxic activity which was even higher than that of reference drug
M) with the benzamide quinoline
m
m
m
m
m
doxorubicin (IC50 value ¼ 70
m
derivative 9 being the most active compound.
The parent target molecule, containing both sulfonamide and
quinoline or sulfonamide and pyrimidoquinoline nucleus, was
synthesized with several substitutions in the 2-position, in order to
study the structure activity relationship of the synthesized
compounds 6e18, 20e28. From the obtained results (Table 1), it
was found that several compounds showed significant cytotoxic
On the other hand, compounds 8, 12, 13, 15 and 26 exhibited
a moderate activity but less active than the reference drug
Table 1
In vitro cytotoxic activity of the synthesized compounds 6e18 and 20e28.
b
Compd. No.
Non viable cells (%)a
Concentration ( g/mL)
100 50
Doxorubicin 100
68 ꢁ 1.9 32 ꢁ 1.6 19 ꢁ 1.3
IC50
IC50
M)
(m
g/mL)
(
m
m
25
10
38
25
25
75
10
70
51
45
135
17
e
6
7
8
9
95 ꢁ 1.5 65 ꢁ 1.6 50 ꢁ 2.1 40 ꢁ 1.8
60 ꢁ 1.9 55 ꢁ 2.2 50 ꢁ 2.6 20 ꢁ 1.2
60 ꢁ 2.1 40 ꢁ 2.4 40 ꢁ 2.0 20 ꢁ 1.1
75 ꢁ 1.8 70 ꢁ 1.9 60 ꢁ 1.8 50 ꢁ 1.3
10
11
12
13
14
15
16
17
18
20
21
22
23
24
25
26
27
28
40 ꢁ 3.1 40 ꢁ 2.1 30 ꢁ 1.5 20 ꢁ 2.1 >100c
45 ꢁ 1.7 40 ꢁ 2.4 30 ꢁ 1.7 30 ꢁ 1.9 >100c
e
70 ꢁ 2.2 50 ꢁ 1.9 40 ꢁ 2.4 40 ꢁ 1.7
50
60
78
88
e
95 ꢁ 2.1 95 ꢁ 1.6 20 ꢁ 2.8
0
60 ꢁ 3.3 40 ꢁ 1.2 40 ꢁ 2.1 30 ꢁ 2.2 >100c
60 ꢁ 4.1 52 ꢁ 1.6 44 ꢁ 1.8 27 ꢁ 1.8
56
>100c
28
101
e
60 ꢁ 1.8 10 ꢁ 2.1
0
0
60 ꢁ 1.6 55 ꢁ 3.2 55 ꢁ 1.7 30 ꢁ 1.4
46
37
e
100
60 ꢁ 1.8 50 ꢁ 1.5 20 ꢁ 2.3
25
40 ꢁ 2.1 35 ꢁ 1.7 30 ꢁ 2.1 30 ꢁ 1.6 >100c
45 ꢁ 3.1 40 ꢁ 1.4 30 ꢁ 1.9 30 ꢁ 2.1 >100c
e
60 ꢁ 2.2 10 ꢁ 1.9
0
0
>100c
e
40 ꢁ 2.3 30 ꢁ 2.0 30 ꢁ 2.2 10 ꢁ 1.9 >100c
20 ꢁ 3.1 20 ꢁ 2.2 20 ꢁ 3.1 10 ꢁ 1.4 >100c
20 ꢁ 4.2 20 ꢁ 1.7 20 ꢁ 2.6 10 ꢁ 1.5 >100c
e
e
e
80 ꢁ 1.5 60 ꢁ 1.4 44 ꢁ 1.7 40 ꢁ 2.1
64
112
e
60 ꢁ 2.1 40 ꢁ 1.8 40 ꢁ 1.4 30 ꢁ 2.3 >100c
40 ꢁ 1.6 35 ꢁ 2.3 30 ꢁ 1.9 30 ꢁ 1.7 >100c
e
a
Mean of non-viable percentage of three repeated experiments ꢁ SD.
b
IC50 value: corresponds to compound concentration causing 50% mortality in
net cells.
c
Scheme 5.
Compounds with IC50 > 100 mg/mL are considered to be inactive.

204
M.S. Al-Said et al. / European Journal of Medicinal Chemistry 46 (2011) 201e207
doxorubicin. Finally, compounds 10, 11, 14, 16, 20e25, 27 and 28
showed no activity.
2.3. Molecular modeling
Previous literature showed that carbonic anhydrase inhibition is
one of the anticancer mechanisms of sulfonamides, and this was
clearly reported by Abbate et al. [5]. So we have performed
a molecular modeling study to investigate the possible binding
conformationforthisgroupofcompoundstothecarbonicanhydrase
binding site, which may give a suggestion about their proposed
mechanism of action. The most active compounds 9 and 18 were
docked in the (PDB entry: 1g54) using MOE 2007.09 (MOE) [30].
In order to validate our docking procedure, we docked the
compound “N-[(2,3,4,5,6-pentaflourophenyl)methyl]-4-sulfamoyl-
benzamide” which is the ligand already co-crystallized with the
protein. The docking results showed that the best scored confor-
mation exhibits a very similar fashion as the crystallized confor-
mation where the essential interactions, between the sulfonamide
moiety and the zinc ion in the active site, are maintained with also
the benzenesulfonamide moieties bound to hCA II overlap each
other completely while the tail adopts a slightly different confor-
mation (Fig. 1).
We then docked the most active compounds, 9 and 18, and in
both cases, it was found that the sulfonamide moieties interact with
the zinc ion in the active site like the co-crystallized ligand and also
the benzenesulfonamide moieties significantly overlap each other
with the tails adopting a slightly different conformation (Fig. 2).
Finally, it can be seen from our docking study that our synthe-
sized compounds exhibit similar conformations and binding
interactions with hCA II similar to the co-crystallized ligand. This
Fig. 2. Docking poses of compound 9 "red", compound 18 "green" and the ligand (N-
Pentafluorophenylmethyl-4-sulfamoyl-benzamide) "blue" docked into the CAII active
site (PDB code 1g54) (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.).
suggests that our compounds might possess significant CA inhibi-
tory activity, and this may contribute at least in part, to their anti-
cancer activity.
3. Conclusion
The objective of the present study was to synthesize and inves-
tigate the anticancer activity of new quinoline and pyrimidoquino-
line derivatives containing a free sulfonamide moiety. Several
compounds have been shown to exhibit significant anticancer
activity such as the quinoline derivatives having benzamide 9, free
amino group 6, butanamide 7, the pyrimidoquinoline derivatives
having 4-bromophenyl 18 and p-tolyl 17. Docking of the compounds
in the carbonic anhydrase active site may give a suggestion that the
synthesized compounds may act as carbonic anhydrase inhibitors
and this may contribute in part to their anticancer activity.
4. Experimental
4.1. Chemistry
Melting points (ꢂC, uncorrected) were determined in open capil-
laries on a Gallenkemp melting point apparatus (Sanyo Gallenkemp,
Southborough, UK). Precoated silica gel plates (silica gel 0.25 mm,
60 G F 254; Merck, Germany) were used for thin layer chromatog-
raphy, dichloromethane/methanol (9.5:0.5 mL) mixture was used as
a developing solvent system and the spots were visualized by ultra-
violet light and/or iodine. Infrared spectra were recorded in KBr discs
using IR-470 Shimadzu Spectrometer (Shimadzu, Tokyo, Japan). 1H
NMR spectra (in DMSO-d6) were recorded on Bruker Ac-300 Ultra
Shield NMR Spectrometer (Bruker, Flawil, Switzerland,
d ppm) at
300 MHz, using TMS as internal standard. Electron impact Mass
Spectra were recorded on a, Shimadzu Gc-Ms-Qp 5000 instrument
(Shimadzu, Tokyo, Japan). Elemental analyses were performed on
Carlo Erba 1108 Elemental Analyzer (Heraeus, Hanau, Germany). All
compounds were within ꢁ0.4% of the theoretical values.
Fig. 1. Ligand (N-Pentafluorophenylmethyl-4-sulfamoyl-benzamide) co-crystallized
with CAII (PDB code 1g54) "green" and its best scoring redocked conformer "red" in
CAII active site (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.).

M.S. Al-Said et al. / European Journal of Medicinal Chemistry 46 (2011) 201e207
205
4.1.1. 4-(3-Oxocyclohex-1-enylamino)benzenesulfonamide (3) and
4-(2-amino-3-cyano-4-(2,4-dichlorophenyl)-5-oxo-5,6,7,8-tetrahydro
quinolin-1(4H)-yl)-benzenesulfonamide (6)
Ar-H þ SO2NH2], 12.5 [s, 1H, NH]. 13C NMR (DMSO-d6)
d
: 21.4, 28.7,
35.6, 37.1, 60.2, 112.6, 117.0, 117.6, 118.7, 124.6, 125.8, 127.1, 127.8,
128.6, 129.5, 130.1, 130.7, 131.6, 133.1, 137.5, 141.3, 143.7, 145.2, 152.7,
153.6, 165.8, 197.4. Anal. Calcd. for C31H24Cl2N4O4S: C, 60.10; H,
3.90; N, 9.04. Found: C, 60.43; H, 3.76; N, 9.37.
Prepared according to the previously reported procedure [27].
4.1.2. N-(3-Cyano-4-(2,4-dichlorophenyl)-5-oxo-1-(4-
sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl)
substitutedamides (7e13)
A mixture of 6 (4.89 g, 0.01 mol) and acid chloride derivatives
(0.01 mol) in pyridine (20 mL) was refluxed for 5 h. The reaction
mixture was cooled and poured onto cold water, then acidified by
dilute HCl. The solid obtained was crystallized from the appropriate
solvent to give 7e13, respectively.
4.1.8. N-(3-Cyano-4-(2,4-dichlorophenyl)-5-oxo-1-(4-sulfamoyl-
phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl)-4-nitrobenzamide (12)
Cryst. Solvent, Dioxane; Yield, 79%: m.p. 220e222 ꢂC; IR (KBr,
cmꢀ1): 3459, 3390, 3320 (NH, NH2), 3071 (CH arom.), 2948, 2920
(CH aliph.), 2181 (C^N), 1741, 1647 (2C]O), 1371, 1189 (SO2), 724
(CeCl). 1H NMR (DMSO-d6)
d: 1.9e2.4 [m, 6H, 3CH2], 5.1 [s, 1H, CH],
7.2e8.0 [m, 13H, Ar-H þ SO2NH2], 8.9 [s, 1H, NH]. Anal. Calcd. for
C29H21Cl2N5O6S: C, 54.55; H, 3.32; N,10.97. Found: C, 54.83; H, 3.61;
N, 11.32.
4.1.3. 3-Chloro-N-(3-cyano-4-(2,4-dichlorophenyl)-5-oxo-1-(4-
sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl)butanamide
(7)
4.1.9. N-(3-Cyano-4-(2,4-dichlorophenyl)-5-oxo-1-(4-sulfamoyl-
phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl)-3,4,5-trimethoxy-
benzamide (13)
Cryst. Solvent, Dioxane; Yield, 79%: m.p. >330 ꢂC; IR (KBr,
cmꢀ1): 3450, 3422-3380 (NH, NH2), 3100 (CH arom.), 2929, 2880
(CH aliph.), 2188 (C^N), 1680, 1627 (2C]O), 1369, 1188 (SO2), 755
Cryst. Solvent, Ethanol; Yield, 76%: m.p. 201e203 ꢂC; IR (KBr,
cmꢀ1): 3410, 3356, 3180 (NH, NH2), 3089 (CH arom.), 2950, 2860
(CH aliph.), 2182 (C^N), 1680, 1647 (2C]O), 1372, 1193 (SO2), 708
(CeCl). 1H NMR (DMSO-d6)
d: 1.4e2.6 [m, 6H, 3CH2, cyclo], 1.85
[d, 3H, CH3, J ¼ 7.8 Hz], 2.3 [m, 1H, CHeCl], 2.6 [d, 2H, CH2CO,
J ¼ 7.4 Hz], 4.9 [s,1H, CH, pyridine], 7.2e8.0 [m, 9H, Ar-H þ SO2NH2],
9.5 [s, 1H, NH]. Anal. Calcd. for C26H23Cl3N4O4S: C, 52.58; H, 3.90; N,
9.43. Found: C, 52.20; H, 3.60; N, 9.70.
(CeCl). 1H NMR (DMSO-d6)
d: 1.8e2.4 [m, 6H, 3CH2], 3.7, 3.8, 3.9 [3s,
9H, 3OCH3], 5.2 [s, 1H, CH], 7.3e8.0 [m, 11H, Ar-H þ SO2NH2], 8.7
[s, 1H, NH]. Anal. Calcd. for C32H28Cl2N4O7S: C, 56.23; H, 4.13; N,
8.20. Found: C, 56.50; H, 4.37; N, 8.46.
4.1.4. N-(3-Cyano-4-(2,4-dichlorophenyl)-5-oxo-1-(4-sulfamoyl-
phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl)cyclopropane-
carboxamide (8)
4.1.10. 4-(5-(2,4-Dichlorophenyl)-4,6-dioxo-2-substituted-3,4,6,7,8,
9-hexahydropyrimido[4,5-b]quinolin-10(5H)-yl)benzenesulfonamides
(14e18)
To a solution of 6 (4.89 g, 0.01 mol), in acid chloride derivatives
(10 mL) was refluxed for 4 h. The reaction mixture was evaporated
under vacuum and the obtained product was crystallized from the
appropriate solvent to give 14e18, respectively.
Cryst. Solvent, Ethanol; Yield, 77%: m.p. 241e243 ꢂC; IR (KBr,
cmꢀ1): 3435, 3390, 3280 (NH, NH2), 3093 (CH arom.), 2949, 2836
(CH aliph.), 2210 (C^N), 1740, 1658 (2C]O), 1370, 1170 (SO2), 730
(CeCl). 1H NMR (DMSO-d6)
d: 0.3e0.9 [m, 4H, 2CH2, cyclopropyl],
1.3 [m, 1H, CH cyclopropyl], 1.6e2.6 [m, 6H, 3CH2, cyclo], 5.2 [s, 1H,
CH pyridine], 7.3e8.0 [m, 9H, Ar-H þ SO2NH2],10.1 [s,1H, NH]. Anal.
Calcd. for C26H22Cl2N4O4S: C, 56.02; H, 3.98; N, 10.05. Found: C,
55.76; H, 3.71; N, 10.35.
4.1.11. 4-(2-(2-Chloropropyl)-5-(2,4-dichlorophenyl)-4,6-dioxo-3,4,
6,7,8,9-hexahydropyrimido[4,5-b]quinolin-10(5H)-yl)benzene-
sulfonamide (14)
4.1.5. N-(3-Cyano-4-(2,4-dichlorophenyl)-5-oxo-1-(4-sulfamoyl-
phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl)benzamide (9)
Cryst. Solvent, Dioxane; Yield, 68%: m.p. >330 ꢂC; IR (KBr,
cmꢀ1): 3435, 3390, 3360 (NH, NH2), 3090 (CH arom.), 2927, 2883
(CH aliph.), 1734, 1654 (2C]O), 1378, 1188 (SO2), 746 (CeCl). 1H
Cryst. Solvent, Ethanol; Yield, 87%: m.p. 173e175 ꢂC; IR (KBr,
cmꢀ1): 3390, 3310, 3260 (NH, NH2), 3068 (CH arom.), 2950, 2860
(CH aliph.), 2210 (C^N), 1382, 1170 (SO2), 750 (CeCl). 1H NMR
NMR (DMSO-d6)
d
: 1.4 [d, 3H, CH3, J ¼ 7.4 Hz], 1.6e2.6 [m, 6H, 3CH2
cyclo], 3.5 [m, 1H, CHeCl], 4.3 [s, 1H, CH pyridine], 4.6 [d, 2H, CH2,
J ¼ 7.1 Hz], 7.4e8.0 [m, 9H, Ar-H þ SO2NH2], 8.4 [s, 1H, NH]. Anal.
Calcd. for C26H23Cl3N4O4S: C, 55.58; H, 3.90; N, 9.43. Found: C,
55.22; H, 3.57; N, 9.12.
(DMSO-d6) d: 1.9e2.6 [m, 6H, 3CH2 cyclo], 4.9 [s, 1H, CH], 7.1e7.9
[m, 14H, Ar-H þ SO2NH2], 9.8 [s, 1H, NH]. Anal. Calcd. for
C29H22Cl2N4O4S: C, 58.69; H, 3.74; N, 9.44. Found: C, 58.33; H, 3.48;
N, 9.20.
4.1.12. 4-(2-Cyclopropyl-5-(2,4-dichlorophenyl)-4,6-dioxo-3,4,6,7,
8,9-hexahydropyrimido[4,5-b]quinolin-10(5H)-yl)benzene-
sulfonamide (15)
4.1.6. N-(3-Cyano-4-(2,4-dichlorophenyl)-5-oxo-1-(4-sulfamoyl-
phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl)-4-methylbenzamide (10)
Cryst. Solvent, Dioxane; Yield, 89%: m.p. 125e127 ꢂC; IR (KBr,
cmꢀ1): 3410, 3366, 3290 (NH, NH2), 3067 (CH arom.), 2950, 2860
(CH aliph.), 2184 (C^N), 1691, 1648 (2C]O), 1371, 1170 (SO2), 747
Cryst. Solvent, Dioxane; Yield, 77%: m.p. 174e176 ꢂC; IR (KBr,
cmꢀ1): 3422, 3390, 3320 (NH, NH2), 3094 (CH arom.), 2926, 2860
(CH aliph.), 1714, 1655 (2C]O), 1371, 1186 (SO2), 715 (CeCl). 1H
(CeCl). 1H NMR (DMSO-d6)
d
: 1.6e2.6 [m, 6H, 3CH2 cyclo], 2.4 [s,1H,
NMR (DMSO-d6) d: 0.4e0.5 [m, 4H, 2CH2 cyclopropyl], 1.6e2.6 [m,
CH3], 5.0 [s, 1H, CH], 7.0e8.1 [m, 13H, Ar-H þ SO2NH2], 12.8 [s, 1H,
NH]. Anal. Calcd. for C30H24Cl2N4O4S: C, 59.31; H, 3.98; N, 9.22.
Found: C, 59.63; H, 3.64; N, 9.46.
6H, 3CH2], 4.1 [s, 1H, CH], 5.4e5.6 [m, 1H, CH cyclopropyl], 7.3e8.0
[m, 10H, Ar-H þ SO2NH2], 12.5 [s, 1H, NH]. Anal. Calcd. for
C26H22Cl2N4O4S: C, 56.02; H, 3.98; N, 10.05. Found: C, 56.36; H,
3.67; N, 9.78.
4.1.7. N-(3-Cyano-4-(2,4-dichlorophenyl)-5-oxo-1-(4-sulfamoyl-
phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl)cinnamamide (11)
Cryst. Solvent, Ethanol; Yield, 81%: m.p. 180e182 ꢂC; IR (KBr,
cmꢀ1): 3399, 3374, 3300 (NH, NH2), 3062 (CH arom.), 2923, 2870
(CH aliph.), 2183 (C^N), 1700, 1629 (2C]O), 1370, 1185 (SO2), 765
4.1.13. 4-(5-(2,4-Dichlorophenyl)-4,6-dioxo-2-phenyl-3,4,6,7,8,9-
hexahydropyrimido[4,5-b]quinolin-10(5H)-yl)benzenesulfonamide
(16)
Cryst. Solvent, Dioxane; Yield, 81%: m.p. >330 ꢂC; IR (KBr,
cmꢀ1): 3447, 3370, 3330 (NH, NH2), 3064 (CH arom.), 2945, 2880
(CH aliph.), 1690, 1646 (2C]O), 1370, 1170 (SO2), 706 (CeCl).
(CeCl). 1H NMR (DMSO-d6)
d: 1.6e2.4 [m, 6H, 3CH2], 5.1 [s, 1H,
CH], 6.5, 6.6 [2d, 2H, CH]CH, J ¼ 7.5, 7.6 Hz], 7.2e8.1 [m, 14H,

206
M.S. Al-Said et al. / European Journal of Medicinal Chemistry 46 (2011) 201e207
1H NMR (DMSO-d6)
[m, 14H, Ar-H þ SO2NH2], 12.4 [s, 1H, NH]. Anal. Calcd. for
C29H22Cl2N4O4S: C, 58.69; H, 3.74; N, 9.44. Found: C, 58.46; H, 3.48;
N, 9.76.
d
: 1.6e2.4 [m, 6H, 3CH2], 4.3 [s,1H, CH], 7.4e8.0
4.1.19. 4-(3-Cyano-4-(2,4-dichlorophenyl)-5-oxo-2-substituted-
5,6,7,8-tetrahydroquinolin-1(4H)-yl)benzenesulfonamides (23e25)
A mixture of 6 (4.89 g, 0.01 mol) and aromatic aldehydes
(0.01 mol) in acetic acid (20 mL) was refluxed for 3 h. The reaction
mixture was poured onto ice water and the obtained solid was
crystallized from the appropriate solvent to give 23e25,
respectively.
4.1.14. 4-(5-(2,4-Dichlorophenyl)-4,6-dioxo-2-p-tolyl-3,4,6,7,8,9-
hexahydropyrimido[4,5-b]quinolin-10(5H)-yl)benzenesulfonamide
(17)
Cryst. Solvent, Ethanol; Yield, 79%: m.p. >330 ꢂC; IR (KBr, cmꢀ1):
3447, 3370, 3280 (NH, NH2), 3068 (CH arom.), 2934, 2883 (CH
aliph.), 1653, 1635 (2C]O), 1399, 1174 (SO2), 755 (CeCl). 1H NMR
4.1.20. 4-(3-Cyano-4-(2,4-dichlorophenyl)-2-(4-(dimethylamino)
benzylideneamino)-5-oxo-5,6,7,8-tetrahydroquinolin-1(4H)-yl)
benzenesulfonamide (23)
(DMSO-d6)
d
: 1.2e2.4 [m, 6H, 3CH2], 2.45 [s, 3H, CH3], 4.3 [s, 1H,
Cryst. Solvent, Ethanol; Yield, 65%: m.p. >330 ꢂC; IR (KBr, cmꢀ1):
3380, 3310 (NH2), 2940, 2836 (CH aliph.), 2210 (C^N), 1651 (C]O),
CH], 7.2e8.0 [m, 13H, Ar-H þ SO2NH2], 8.3 [s, 1H, NH]. Anal. Calcd.
for C30H24Cl2N4O4S: C, 59.31; H, 3.98; N, 9.22. Found: C, 59.66; H,
3.71; N, 9.55.
1612 (C]N), 1389, 1168 (SO2), 753 (CeCl). 1H NMR (DMSO-d6)
d:
1.6e2.4 [m, 6H, 3CH2 cyclo], 3.0 [s, 6H, N(CH3)2] 7.0e8.0 [m, 13H,
Ar-H þ SO2NH2], 8.2 [s, 1H, N]CH]. MS m/z (%): 620 [Mþ] (2.4), 63
(100). Anal. Calcd. for C31H27Cl2N5O3S: C, 60.00; H, 4.39; N, 11.29.
Found: C, 60.33; H, 4.71; N, 11.57.
4.1.15. 4-(2-(4-Bromophenyl)-5-(2,4-dichlorophenyl)-4,6-dioxo-
3,4,6,7,8,9-hexahydropyrimido[4,5-b]quinolin-10(5H)-yl)benzene-
sulfonamide (18)
Cryst. Solvent, Ethanol; Yield, 76%: m.p. 104e106 ꢂC; IR (KBr,
cmꢀ1): 3446, 3390, 3340 (NH, NH2), 3068 (CH arom.), 1724, 1635
(2C]O), 1569 (C]N), 1381, 1165 (SO2), 730 (CeCl). 1H NMR (DMSO-
4.1.21. 4-(3-Cyano-4-(2,4-dichlorophenyl)-2-((E)-3-(4-(dimethyl-
amino)phenyl)allylideneamino)-5-oxo-5,6,7,8-tetrahydroquinolin-1
(4H)-yl)benzenesulfonamide (24)
d6)
d: 1.2e2.4 [m, 6H, 3CH2], 4.3 [s, 1H, CH], 7.1e8.0 [m, 13H, Ar-
Cryst. Solvent, Dioxane; Yield, 62%: m.p. >330 ꢂC; IR (KBr,
cmꢀ1): 3446, 3360 (NH2), 3094 (CH arom.), 2929, 2886 (CH aliph.),
2215 (C^N), 1654 (C]O), 1617 (C]N), 1384, 1180 (SO2), 740 (CeCl).
H þ SO2NH2], 13.2 [s, 1H, NH]. Anal. Calcd. for C29H21Cl2BrN4O4S: C,
51.80; H, 3.15; N, 8.33. Found: C, 51.98; H, 3.50; N, 8.70.
1H NMR (DMSO-d6)
d: 1.6e2.4 [m, 6H, 3CH2 cyclo], 1.7 [d, 2H, CH]
4.1.16. 4-(2-(Chloromethyl)-5-(2,4-dichlorophenyl)-4,6-dioxo-3,4,
6,7,8,9-hexahydropyrimido[4,5-b]quinolin-10(5H)-yl)benzene-
sulfonamide (20)
CH-Ph, J ¼ 7.2 Hz], 3.0 [s, 6H, N(CH3)2], 5.3 [s, 1H, CH pyridine],
7.3e8.1 [m, 13H, Ar-H þ SO2NH2], 9.8 [d, 1H, N]CH, J ¼ 7.6 Hz]. MS
m/z (%): 646 [Mþ] (2.6), 47(100). Anal. Calcd. for C35H33Cl2N5O3S: C,
62.31; H, 4.93; N, 10.38. Found: C, 62.68; H, 4.66; N, 10.73.
A mixture of 6 (4.89 g, 0.01 mol) and chloroacetyl chloride
(1.13 g, 0.01 mol) in DMF (20 mL) was stirred at room temperature
for 2 h. The reaction mixture was poured onto ice water and the
obtained solid was crystallized from ethanol to give 20. Yield, 91%:
m.p. 324e326 ꢂC; IR (KBr, cmꢀ1): 3364, 3272, 3240 (NH, NH2), 3104
(CH arom.), 2958, 2929 (CH aliph.), 1697, 1652 (2C]O),1627 (C]N),
4.1.22. 4-(3-Cyano-4-(2,4-dichlorophenyl)-2-(2,4-dinitrobenzyl-
ideneamino)-5-oxo-5,6,7,8-tetrahydroquinolin-1(4H)-yl)benzene-
sulfonamide (25)
Cryst. Solvent, Dioxane; Yield, 74%: m.p. 317e319 ꢂC; IR (KBr,
cmꢀ1): 3364, 3272 (NH2), 3106 (CH arom.), 2965, 2865 (CH aliph.),
2210 (C^N), 1651 (C]O), 1626 (C]N), 1372, 1185 (SO2), 796 (CeCl).
1372, 1185 (SO2), 796 (CeCl). 1H NMR (DMSO-d6)
d: 1.6e2.4 [m, 6H,
3CH2], 5.2 [s, 1H, CH], 5.5 [s, 2H, CH2eCl], 7.3e8.0 [m, 9H, Ar-
H þ SO2NH2], 10.9 [s,1H, NH]. 13C NMR (DMSO-d6)
d
: 21.7, 28.2, 36.1,
1H NMR (DMSO-d6).
d: 1.6e2.4 [m, 6H, 3CH2], 5.1 [s, 1H, CH], 7.0e7.9
36.2, 41.2, 99.9, 115.1, 116.3, 127.4, 128.8, 129.7, 129.9, 130.5, 133.8,
134.7, 139.3, 139.5, 145.1, 155.6, 162.7, 168.1, 194.6. Anal. Calcd. for
C24H19Cl3N4O4S: C, 50.94; H, 3.38; N, 9.90. Found: C, 50.61; H, 3.71;
N, 9.63.
[m, 12H, Ar-H þ SO2NH2], 10.4 [s, 1H, N]CH]. MS m/z (%): 667 [Mþ]
(3.2), 45 (100). Anal. Calcd. for C29H20Cl2N6O7S: C, 52.18; H, 3.02; N,
12.59. Found: C, 52.53; H, 3.29; N, 12.84.
4.1.23. N-(3-Cyano-4-(2,4-dichlorophenyl)-5-oxo-1-(4-sulfamoyl-
phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl)-3-oxo-butanamide
(26)
4.1.17. 2-Amino-4-(2,4-dichlorophenyl)-5-oxo-1-(4-sulfamoyl-
phenyl)-1,4,5,6,7,8-hexahydroquinoline-3-carboxamide (21)
A solution of 6 (4.89 g, 0.01 mol), in conc. H2SO4 (20 mL) was
stirred at room temperature for 2 h. The obtained solid was crys-
tallized from ethanol to give 21.Yield, 88%: m.p. 207e209 ꢂC; IR
(KBr, cmꢀ1): 3420, 3380, 3255 (NH2), 3091 (CH arom.), 2953, 2860
(CH aliph.), 1683, 1629 (2C]O), 1373, 1164 (SO2), 713 (CeCl). 1H
A solution of 6 (4.89 g, 0.01 mol), in ethyl acetoacetate (10 mL)
was refluxed for 5 h. The reaction mixture was removed under
vacuum and the obtained solid was crystallized from ethanol to
give 26. Yield, 79%: m.p. 113e115 ꢂC; IR (KBr, cmꢀ1): 3421, 3380,
3360 (NH, NH2), 3086 (CH arom.), 2929, 2870 (CH aliph.), 2164
(C^N), 1719, 1680, 1641 (3C]O), 1373, 1170 (SO2), 778 (CeCl) 1H
NMR (DMSO-d6) d: 1.6e2.4 [m, 6H, 3CH2], 4.8 [s, 1H, CH], 6.2 [s, 2H,
NH2], 6.9 [s, 2H, CONH2], 7.0e8.0 [m, 9H, Ar-H þ SO2NH2]. Anal.
Calcd. for C22H20Cl2N4O4S: C, 52.08; H, 3.97; N, 11.04. Found: C,
52.28; H, 3.67; N, 10.86.
NMR (DMSO-d6) d: 1.6e2.4 [m, 6H, 3CH2], 2.2 [s, 3H, COCH3], 3.3 [s,
2H, CH2CO], 4.4 [s, 1H, CH], 7.0e7.6 [m, 9H, Ar-H þ SO2NH2], 8.4 [s,
1H, NH]. Anal. Calcd. for C26H22Cl2N4O5S: C, 54.46; H, 3.87; N, 9.77.
Found: C, 54.81; H, 3.56; N, 9.44.
4.1.18. 2-Amino-4-(2,4-dichlorophenyl)-5-oxo-1-(4-sulfamoyl-
phenyl)-1,4,5,6,7,8-hexahydroquinoline-3-carboxylic acid (22)
A solution of 6 (4.89 g, 0.01 mol), in conc. H2SO4 (10 mL) was
refluxed for 1 h. The reaction mixture was cooled and poured onto
ice water and the obtained solid was crystallized from dioxane to
give 22. Yield, 69%: m.p. >330 ꢂC; IR (KBr, cmꢀ1): 3478 (OH), 3390,
3260 (NH2), 3100 (CH arom.), 2950, 2929 (CH aliph.), 1719, 1654
(2C]O), 1313, 1189 (SO2), 669 (CeCl). MS m/z (%): 508 [Mþ] (3.2),
125 (100). Anal. Calcd. for C22H19Cl2N3O5S: C, 51.98; H, 3.77; N, 8.27.
Found: C, 51.60; H, 3.45; N, 8.56.
4.1.24. 4-(5-(2,4-Dichlorophenyl)-2-methyl-6-oxo-4-thioxo-3,4,6,
7,8,9-hexahydropyrimido[4,5-b]quinolin-10(5H)-yl)benzene-
sulfonamide (27)
A mixture of 6 (4.89 g, 0.01 mol), and thioacetamide (0.75 g,
0.01 mol) in trifluoroacetic acid (10 mL) was refluxed for 5 h. The
reaction mixture was cooled and poured onto ice water. The
obtained solid was crystallized from dioxane to give 27. Yield, 80%:
m.p. 141e143 ꢂC; IR (KBr, cmꢀ1): 3370, 3251, 3190 (NH, NH2), 3090
(CH arom.), 2951, 2860 (CH aliph.), 1624 (C]O), 1286 (C]S), 1370,

M.S. Al-Said et al. / European Journal of Medicinal Chemistry 46 (2011) 201e207
207
1164 (SO2), 713 (CeCl). MS m/z (%): 547 [Mþ] (3.6), 62 (100). Anal.
Calcd. for C24H20Cl2N4O3S2: C, 52.65; H, 3.68; N, 10.23. Found: C,
52.29; H, 3.41; N, 10.56.
The carbonic anhydrase structure was downloaded from the
PDB data bank (http://www.rcsb.org/ e PDB code: 1g54). The
protein was prepared for the docking where: (i) Hydrogen atoms
were added to the protein with MOE and minimized keeping all the
heavy atoms fixed until an RMSD gradient of 0.05 kcal molꢀ1 Åꢀ1
was reached (ii) Partial charges were computed using Amber99
forcefield.
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.
4.1.25. Ethyl N-(3-cyano-4-(2,4-dichlorophenyl)-5-oxo-1-(4-sulfamoyl-
phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl)formimidate (28)
A solution of 6 (4.89 g, 0.01 mol), in triethyl orthoformate
(10 mL) was refluxed for 4 h. The reaction mixture was removed
under vacuum and the obtained solid was crystallized from ethanol
to give 28. Yield, 88%: m.p. 117e119 ꢂC IR (KBr, cmꢀ1): 3365, 3290
(NH2), 3067 (CH arom.), 2978, 2880 (CH aliph.), 2200 (C^N), 1643
(C]O), 1372, 1188 (SO2), 732 (CeCl). 1H NMR (DMSO-d6)
d
: 1.1 [t,
Docking calculations were done using Alpha triangle placement
method and poses were prioritized by London dG scoring method.
3H, CH3 ethyl]. 1.6e2.4 [m, 6H, 3CH2], 4.3 [q, 2H, CH2 ethyl], 5.2 [s,
1H, CH], 7.4e8.0 [m, 9H, Ar-H þ SO2NH2], 8.8 [s, 1H, N]CH]. Anal.
Calcd. for C25H22Cl2N4O4S: C, 55.05; H, 4.07; N, 10.27. Found: C,
55.42; H, 4.37; N, 10.58.
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4.2.1. Animals, chemicals and facilities
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Swiss albino mice weighing 25e30 g (the holding company for
biological products and vaccines, VACSERA, Cairo, Egypt). The
animals were housed at a constant temperature (24 ꢁ 2 ꢂC) with
alternating 12 h light and dark cycles and fed standard laboratory
food (Milad CO., Cairo, Egypt) and water ad libitum. All chemicals
and reagents were of the highest grade commercially available.
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and Technology (NCRRT), Atomic Energy Authority (AEA), Cairo,
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lines set by the world health organization, Geneva, Switzerland and
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4.2.2. Anticancer activity
Ehrlich Ascites Carcinoma cells (EAC) were obtained by needle
aspiration of ascitic fluid from preinoculated mice under aseptic
conditions. Tumor cell suspension (2.5 ꢃ 106 per mL) was prepared
in RPMI-1640 media. Tested compounds were prepared with
various dilutions by dissolving: 100, 50, 25 and 10
compounds in DMSO (1 mL).
mg of the tested
In a set of sterile test tubes 0.8 mL RPMI-1640 media containing
(glutamine, fetal calf serum as nutrient, streptomycin and peni-
cillin), 0.1 mL of each of the tested compounds (corresponding to
100, 50, 25, 10 mg) were mixed then 0.1 mL of tumor cell suspension
(2.5 ꢃ 105) was added. The test tubes were incubated at 37 ꢂC for
2 h. Trypan blue exclusion test was carried out to calculate the
percentage of non-viable cells after 2 h of incubation [31]. Trypan
blue dye was prepared as follows; A stoke solution was prepared by
dissolving 1 g of the dye in distilled water (100 mL). The working
solution was prepared by diluting 1 mL of the stock solution with
9 mL of distilled water. The stain was used for staining the dead EAC
cells. The results of in vitro cytotoxic activity experiments are pre-
sented in Table 1.
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4.3. Molecular modeling
All molecular modeling calculations and docking studies were
performed using “Molecular Operating Environment (MOE) version
2007.09”.