Synthesis, in-vitro anticancer screening and radiosensitizing evaluation of some new N-(quinoxalin-2-yl)benzenesulfonamide derivatives

Article abstract of DOI:10.1055/s-0031-1295496

The objective of this work is to synthesize and investigate the anticancer activity of a new series of sulfaquinoxaline derivatives by incorporating biologically active moieties (thiourethane, thiazole, imidazole, imidazopyrimidine, imidazopyrimido-pyrimidine, thienopyrimidine, benzopyrimidinone, benzothiazole, thiazole and pyridine moieties). All the newly synthesized compounds were evaluated for their in-vitro anticancer activity against human liver cell line (HEPG2). All the tested compounds showed comparable activity to that of the reference drug 5-fluorouracil (IC ₅₀=40 M), and the most potent compounds were found to be compounds 4 and 17 (IC₅₀=4.29 and 11.27 M, respectively). On the other hand, the most potent compounds 4 and 17 were evaluated as radiosensitizing agents. Georg Thieme Verlag KG Stuttgart · New York.

Full text of DOI:10.1055/s-0031-1295496

46 Original Article
Synthesis, in-vitro Anticancer Screening and
Radiosensitizing Evaluation of some New
N-(quinoxalin-2-yl)benzenesulfonamide Derivatives
Authors
M. M. Ghorab1,3, F. A. Ragab2, H. I. Heiba1, M. G. El-Gazzar1, M. G. El-Gazzar1
Affiliations
1 Department of Drug Radiation Research, National Center for Radiation Research and Technology (NCRRT), Atomic Energy
Authority, Cairo, Egypt
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
3 Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud University, Riyadh,
Saudi Arabia
Key words
Abstract
▼
compounds were evaluated for their in-vitro
anticancer activity against human liver cell line
(HEPG2). All the tested compounds showed
comparable activity to that of the reference drug
5-fluorouracil (IC50 =40μM), and the most potent
compounds were found to be compounds 4 and
17 (IC50 =4.29 and 11.27μM, respectively). On the
other hand, the most potent compounds 4 and
17 were evaluated as radiosensitizing agents.
▶
● sulfonamide
▶
● quinoxaline
The objective of this work is to synthesize and
investigate the anticancer activity of a new series
of sulfaquinoxaline derivatives by incorporat-
ing biologically active moieties (thiourethane,
thiazole, imidazole, imidazopyrimidine, imi-
dazopyrimido-pyrimidine,
benzopyrimidinone, benzothiazole, thiazole and
pyridine moieties). All the newly synthesized
▶
● anticancer
▶
● radiosensitizing
thienopyrimidine,
Introduction
▼
zole, imidazopyrimidine, imidazopyrimido-pyri-
midine, thienopyrimidine, benzopyrimidinone,
benzothiazole, thiazole and pyridine moieties) to
be evaluated as antitumor agents against liver
human tumor cell lines (HEPG2) to study their
SAR. Moreover, we also aimed to evaluate these
new compounds for their in vitro anticancer
activity in combination with γ-irradiation.
A series of structurally novel sulfonamide deriva-
tives containing a N1-substituted sulfonamide
moiety were reported to show substantial anti-
tumor activity in-vitro and/or in-vivo [1–5]. In
order to explain this antitumor activity, several
mechanisms were adopted, including carbonic
anhydrase inhibition, cell cycle arrest at G1
phase, disruption of microtubules, and angiogen-
esis inhibition. The most prominent among these
mechanisms was carbonic anhydrase inhibition
[6–10]. Quinoxaline derivatives possess a wide
range of biological activities [11–15]. Recently,
several quinoxaline derivatives exhibited antitu-
mor activity as potent and highly selective tyro-
sine kinase inhibitors [16–19]. Furthermore,
received 14.09.2011
accepted 17.10.2011
Bibliography
Materials and Methods
▼
Chemistry
DOI http://dx.doi.org/
10.1055/s-0031-1295496
Arzneimittelforschung 2012;
62: 46–52
© Georg Thieme Verlag KG
Stuttgart · New York
ISSN 0004-4172
Melting points are uncorrected and were deter-
mined on a Stuart melting point apparatus (Stu-
art Scientific, Redhill, UK). Elemental analysis (C,
H, N) were performed on Perkin-Elmer 2400 ana-
lyser (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 (Shimadzu, Koyoto,
Japan), 1H-NMR spectra were obtained on a
Bruker proton NMR-Avance 300 (300MHz)
(Bruker, Munuch, Germany), in DMSO-d6 as a
solvent, using tetramethylsilane (TMS) as inter-
nal standard. Mass spectra were run on HP Model
MS-5988 (Hewlett Packard, Palo, Alto, California,
USA). All reactions were monitored by thin layer
▶
chloroquinoxaline sulfonamide (CQS; ● Fig. 1),
Correspondence
M. G. El-Gazzar
was reported to be an efficient antitumor agent
against breast, lung, melanoma and ovarian car-
cinomas by causing cell cycle arrest at the G1
phase [20]. Based on the above informations, and
due to the above mentioned anticancer activity
of quinoxaline-containing compounds and due to
our interest in synthesizing novel sulfonamide
derivatives, the present investigation deals with
the design and synthesis of some novel sulfaqui-
noxaline derivatives by substituting the amino
group of sulfonamide by different biologically
active moieties (thiourethane, thiazole, imida-
Department of Drug Radiation
Research, National Center
for Radiation Research and
Technology (NCRRT)
Atomic Energy Authority
PO box 29
Nasr City
Cairo
Egypt
Tel.: +20/22/2749 298
Fax: +20/22/2749 298
marwagalalgazzar@yahoo.com
Ghorab MM et al. Synthesis, in-vitro Anticancer Screening… Arzneimittelforschung 2012; 62: 46–52

Original Article 47
O-Propyl 4-(N-quinoxalin-2-ylsulfamoyl)
benzenesulfonamide (4)
Cl
O
N S
O
A mixture of 1 (0.4g, 0.01mol) and propanol (3mL, 0.01mol),
was refluxed for 3h. The reaction mixture was poured into ice
water and the solid obtained was filtered and crystallized from
dioxane to give 4. Yield %: 92, m.p.: 206–208°C. IR (KBr, cm−1):
3208, 3156 (2NH), 3070 (CH arom), 2966 (CHaliph), 1200
(C=S), 1348, 1140 (SO2). 1H-NMR DMSO-d6 (ppm): 0.9 (t, 3H,
CH3), 1.8 (m, 2H, CH2), 4.4 (t, 3H, CH2), 4.6 (s,1H, NH), 6.7, 7.5 [2d,
4H, Ar-H AB system], 7.7–8.1 (m, 4H, Ar-H), 8.4 [s, 1H, SO2NH,
exchangeable with D2O], 8.7(s,1H, CH quinoxaline). Analysis
Calc. for C18H18N4O3S2: C, 53.71; H, 4.51; N, 13.92. Found: C,
53.52; H, 4.67; N, 13.65.
H
N
N
NH2
CQS
Fig. 1 Chloroquinoxaline.
chromatograph (TLC) using precoated Aluminium sheets Silica
gel Merck 60 F254 and were visualized by UV lamp (Merck,
Damstadt, Germany).
O-Butyl 4-(N-quinoxalin-2-ylsulfamoyl)
4-Isothiocyanato-N-quinoxaline-2-yl-benzene
benzenesulfonamide (5)
sulfonamide (1)
A mixture of 1 (0.4g, 0.01mol) and butanol (3mL, 0.01mol), was
refluxed for 8h, the reaction mixture was poured into ice water,
filtered and crystallized from dioxane to give 5. Yield %: 95, m.p.:
208–210°C. IR (KBr, cm−1): 3290, 3154 (2NH), 3060 (CH arom),
2956 (CHaliph), 1198 (C=S), 1342, 1140 (SO2). 1H-NMR DMSO-
d6 (ppm): 0.95 (t, 3H, CH3), 1.4, 1.7 (2m, 4H, 2CH2), 4.5(t, 3H,
CH2), 4.6 (s,1H,NH), 6.8, 7.5 [2d, 4H, Ar-H AB system], 7.8–8.2 (m,
4H,Ar-H), 8.5 (s,1H,SO2NH), 8.7(s,1H, CH quinoxaline). Analysis
Calc. for C19H20N4O3S2: C, 54.79; H, 4.74; N, 13.45. Found: C,
54.45; H, 4.57; N, 13.85.
To a suspension of sulfaquinoxaline (3g, 0.01mol) in water
(30mL), thiophosgene (1ml, 0.01mol) was added and the reac-
tion mixture was stirred for 1h, where the red colour of the thio-
phosgene was disappeared and a white precipitate was formed.
The precipitate was filtered off, washed with water and crystal-
lized from ethanol to give 1. Yield %: 86, m.p.: 210–212°C. IR
(KBr, cm−1): 3290 (NH), 3070 (CH arom.), 2106 (NCS), 1330,
1156 (SO2). MS (m/z): 342 (M+, 10.2%), 261 (27.94%), 236
(100%), 92 (71.14%).1 H-NMR DMSO-d6 (ppm): 4.0 [s, 1H, NH,
exchangeable with D2O], 7.01, 7.5 [2d, 4H, Ar-H AB system],
7.68–8.05 [m, 4H, Ar-H], 8.07[s, 1H, CH-quinoxaline]. 13C-NMR
(DMSO-d6): 124.1, 125.2, 126.9, 128.9, 134.8, 135.1, 135.6, 136.8
(NCS), 138.3, 161.9. Analysis Calc. for C15H10N4O2S2: C, 52.61; H,
2.92; N, 16.37. Found: C, 52.40; H, 2.83; N, 16.11.
4-(4-Amino-5-cyano-2-thioxothiazol-3(2H)-yl)-N-
(quinoxalin-2-yl)benzene-sulfonamide (6)
A mixture of 1 (0.4g, 0.01mol) and malononitrile (0.1g,
0.001mol) in ethanol (20mL) and a catalytic amount of TEA in
presence of sulphur (0.03g, 0.001mol), was refluxed for 5h, the
reaction mixture was poured into ice water, filtered and crystal-
lized from dioxane to give 6. Yield %: 92, m.p.: 158–160°C. IR
(KBr, cm−1): 3312, 3220, 3160 (NH, NH2), 3080 (CH arom),
2214 (CN), 1194 (C=S), 1342, 1138 (SO2). 1H-NMR DMSO-d6
(ppm): 2.2 [S, 2H, NH2, exchangeable with D2O], 4.9 (s,1H, NH),
6.7, 7.7 [2d, 4H, Ar-H AB system], 7.7–8.1 (m, 4H, Ar-H), 8.7 [s,
1H, SO2NH, exchangeable with D2O], 8.2 (s,1H, CH quinoxaline).
13C-NMR (DMSO-d6): 58.5 (CN), 112.2 (C-CN), 124.1, 125.2,
126.9, 128.9, 134.8, 135.1, 135.6, 137.1, 154.1 (C-NH2), 161.9,
187.5 (C=S). MS (m/z): 440 (M+, 0.1%), 323 (59.31%), 277 (100%),
235 (37.25%), 90 (26,75%). Analysis Calc. for C18H12N6O2S3: C,
49.08; H, 2.75; N, 19.08. Found: C, 49.44; H, 2.83; N, 19.35.
O-2-Hydroxyethyl4-(N-quinoxalin-2-yl-sulfamoyl)benzene-
sulfonamide (2)
A mixture of 1 (0.4g, 0.01mol) and ethylene glycol (0.3mL,
0.01mol), was refluxed for 8h, the solid obtained was precipi-
tated on hot, filtered and crystallized from dioxane to give 2.
Yield %: 93, m.p.: 160–162°C. IR (KBr, cm−1): 3396 (0H), 3525,
3290 (2NH), 3080 (CH arom), 2944 (CHaliph), 1210 (C=S),
1396, 1142 (SO2). 1H-NMR DMSO-d6 (ppm): 3.8, 4.5 (2t, 4H,
2CH2), 4.2 [s, 2H, 2NH, exchangeable with D2O], 6.7, 7.1 [2d, 4H,
Ar-H AB system], 7.7–8.1 (m, 4H, Ar-H), 8.7(s,1H, CH quinoxa-
line), 11.5 (s,1H, OH). Analysis Calc. for C17H16N4O4S2: C, 50.48;
H, 3.99; N, 13.85. Found: C, 50.67; H, 3.75; N, 13.63.
O-Ethyl 4-(N-quinoxalin-2-ylsulfamoyl)
4-(3-Cyano-6-imino-8-thioxo-5a,6,9,9a-
benzenesulfonamide (3)
tetrahydropyrazolo[1,5-a]pyrimido[3,4-e]pyrimidin-
7(8H)-yl)-N-(quinoxalin-2-yl)benzenesulfonamide (7)
A mixture of 1 (0.4g, 0.01mol) and pyrazolo pyrimidine o-amino
carbonitrile (0.3g, 0.01mol) in DMF (20mL) and a catalytic
amount of TEA, was refluxed for 5h, the reaction mixture was
poured into ice water, filtered and crystallized from ethanol to
give 7. Yield %: 90, m.p.: 236–238°C. IR (KBr, cm−1): 3332, 3360,
3240 (3NH), 3075(CH arom), 2222 (CN), 1210 (C=S), 1328,
A mixture of 1 (0.4g, 0.01mol) and ethanol (3mL, 0.01mol), was
refluxed for 8h, the reaction mixture was poured into ice water,
the solid obtained was filtered and crystallized from dioxane to
give 3. Yield %: 87, m.p.: 135–136°C. IR (KBr, cm−1): 3270, 3154
(2NH), 3080 (CH arom), 2986 (CHaliph), 1194 (C=S), 1378,
1114 (SO2).1H-NMR DMSO-d6 (ppm): 1.3 (t, 3H, CH3), 4.0 [s, 1H,
NH, exchangeable with D2O], 4.5(q, 2H, CH2), 6.6, 7.4 [2d, 4H,
Ar-H ABsystem], 7.7–8.1 (m, 4H, Ar-H), 8.3[s, 1H, SO2NH,
exchangeable with D2O], 8.7(s,1H, CH quinoxaline). Analysis
Calc. for C17H16N4O3S2: C, 52.56; H, 4.15; N, 14.42. Found: C,
52.35; H, 3.98; N, 14.13.
1
1148 (SO2). H-NMR DMSO-d6 (ppm): 4.4 (s, 2H, 2NH), 6.7, 7.5
[2d, 4H, Ar-H AB system], 7.6 [s, 1H, CH pyrazole], 7.7–8.1 (m,
4H, Ar-H), 8.2 [s, 1H, CH pyrimidine], 8.4 [s, 1H, SO2NH, exchange-
able with D2O], 8.7(s,1H, CH quinoxaline). 13C-NMR (DMSO-d6):
66.9 (C-CN), 111.6, 114.1 (CN), 124.1, 125.2, 126.9, 128.9, 134.8,
135.1, 135.6, 138.9, 147, 147.1, 156.1, 156.2, 161.9, 166.9, 175.2
(C=S). MS (m/z): 526 (M+,0.1%), 236 (12.56%), 184 (100%), 119
Ghorab MM et al. Synthesis, in-vitro Anticancer Screening… Arzneimittelforschung 2012; 62: 46–52

48 Original Article
(12.91%), 92 (4.79%). Analysis Calc. for C23H16N10O2S2: C, 52.26;
H, 3.05; N, 26.50. Found: C, 52.55; H, 3.33; N, 26.61.
4-(6,8-Dichloro-4-oxo-2-thioxo-1,2-dihydroquinazolin-
3(4H)-yl)-N-(quinoxalin-2-yl)benzenesulfonamide (11)
A mixture of 1 (0.4g, 0.01mol) and 3,5-dichloroanthranilic acid
(0.3g, 0.01mol) in dioxane (20mL) and a catalytic amount of
TEA, was refluxed for 5h, the reaction mixture was poured into
ice water, filtered and crystallized from ethanol to give 11.
Yield %: 92, m.p.: 250–252°C. IR (KBr, cm−1): 3350, 3240 (NH),
3072 (CH arom), 1690 (C=O), 1220 (C=S), 1310, 1160 (SO2).
1H-NMR DMSO-d6 (ppm): 4.0 [s, 2H, 2NH, exchangeable with
D2O], 6.9, 7.5[2d, 4H, Ar-H, AB system], 6.8, 7.2[2s, 2H, Ar-H],
7.7–8.07 [m, 4H, Ar-H], 8.2 [s, 1H, CH quinoxaline], 8.3 [s, 1H,
SO2NH]. MS (m/z): 529 (M+, 10.81%), 313 (46.82%), 236 (100%),
116 (28.60%), 92 (69.07%). Analysis Calc. for C22H13Cl2N5O3S2: C,
49.82; H, 2.47; N, 13.20. Found: C, 49.75; H, 2.33; N, 12.95.
4-(5-Amino-3-(4-methoxyphenyl)-2,4-
dithioxoimidazolidin-1-yl)-N-(quinoxalin-2-yl)
benzenesulfonamide (8)
A mixture of 1 (0.4g, 0.1mol) and p-Methoxy cyano thio form-
anilide (0.2g, 0.001mol) in tetrahydrofuran (20mL) and a cata-
lytic amount of TEA, was refluxed for 6h, the reaction mixture
was poured into ice water, filtered and crystallized from ethanol
to give 8. Yield %: 89, m.p.: 97–98°C. IR (KBr, cm−1): 3330, 3212,
3250 (NH, NH2), 3065(CH arom), 2995 (CH aliph), 1250 (C=S),
1292, 1155 (SO2). MS (m/z): 536 (M+, 1.76%), 256 (3.91%), 165
(100%), 150 (5.62%), 64 (58.82%). 1H-NMR DMSO-d6 (ppm): 1.7
[s, 3H, CH3], 2.2 [s, 2H, NH2, exchangeable with D2O], 6.3, 6.5 [2d,
4H, Ar-H, AB system], 6.7, 7.1 [2d, 4H, Ar-H, AB system], 7.6–8.07
[m, 4H, Ar-H], 8.1 [s, 1H, CH quinoxaline], 8.3 [s, 1H, SO2NH].
13C-NMR (DMSO-d6): 102.1 (C-NH2), 114.1, 124.1, 125.2, 126.9,
127.2, 128.9, 133.1, 133.9, 134.8, 135.1, 135.6, 143.3, 159.9
(OCH3), 161.9. 176.2 (C=S), 196.1 (C=S). Analysis Calc. for
C24H20N6O3S3: C, 53.71; H, 3.76; N, 15.66. Found: C, 53.58; H,
3.65; N, 15.49.
4-(6-Methyl-4-oxo-2-thioxo-1,2-dihydroquinazolin-3(4H)-
yl)-N-(quinoxalin-2-yl)benzenesulfonamide (12)
A mixture of 1 (0.4g, 0.01mol) and 2-amino 5-methyl benzoic
acid (0.3g ,0.01mol) in dioxane (20mL) and a catalytic amount
of TEA, was refluxed for 6h, the reaction mixture was poured
into ice water, filtered and crystallized from ethanol to give 12.
Yield %: 91, m.p.: 238–239°C. IR (KBr, cm−1): 3242, 3120 (2NH),
3036 (CH arom), 2860 (CH aliph.), 1620 (C=O), 1206 (C=S),
1206, 1186 (SO2). MS (m/z): 475 (M+,7%), 410 (100%), 266
(27.2%), 160 (42.79%), 90 (41.61%). 1H-NMR DMSO-d6 (ppm):
2.7 [s, 3H, CH3], 4.1 [s, 1H, NH, exchangeable with D2O], 6.5,
7.3[2d, 4H, Ar-H, AB system], 7.5-8.07 [m, 7H, Ar-H], 8.1[s, 1H,
CH quinoxaline], 8.3[s, 1H, SO2NH]. Analysis Calc. for
C23H17N5O3S2: C, 58.09; H, 3.60; N, 14.73. Found: C, 58.45; H,
3.55; N, 14.85.
6-Mercapto-1-phenyl-5-(4-(N-quinoxalin-2-yl-sulfamoyl)
phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo [3,4-d]
pyrimidine-4-carboxylic acid (9)
A mixture of 1 (0.4g, 0.01mol) and 4-(2-phenyl3-amino pyra-
zole)carboxylic acid (0.3g ,0.001mol) in dioxane (20mL) and a
catalytic amount of TEA, was refluxed for 6h, the reaction mix-
ture was poured into ice water, filtered and crystallized from
ethanol to give 9. Yield %: 87, m.p.: 180–182°C. IR (KBr, cm−1):
3426 (OH), 3250, 3190 (2NH), 3080 (CH arom), 2929 (CH aliph),
4-(Benzo[d]thiazol-2-yl-amino)-N-(quinoxalin-2-yl)
benzenesulfonamide (13)
1
1690 (C=O), 1280 (C=S), 1340, 1150 (SO2). H-NMR DMSO-d6
(ppm): 4.4 (s, 2H, 2NH), 6.7, 7.5 [2d, 4H, Ar-H AB system], 7.6 [s,
1H, CH pyrazole], 7.7–8.3 (m, 9H, Ar-H), 8.4 [s, 1H, SO2NH,
exchangeable with D2O], 8.9 (s,1H, CH quinoxaline), 11.0 [s, 1H,
COOH, exchangeable with D2O]. 13C-NMR (DMSO-d6): 71.3
(C-COOH), 88.1 (C-SH), 99.5, 123.1, 124.1, 125.2, 126.2, 126.9,
128.9, 129.2, 134.8, 135.1, 135.6, 136.1, 138.9, 146.7, 152.1,
161.9, 169.1 (COOH). MS (m/z): 559 (M+, 0.16%), 264 (43.26%),
111 (100%), 95 (29.79%), 75 (24.19%). Analysis Calc. for
C26H21N7O4S2: C, 55.80; H, 3.78; N, 17.52. Found: C, 55.67; H,
3.65; N, 17.33.
A mixture of 1 (0.4g, 0.01mol) and thiophenol (0.1g ,0.01mol)
in DMF (20mL) and a catalytic amount of TEA, was refluxed for
24h, the reaction mixture was poured into ice water, filtered and
crystallized from ethanol to give 13. Yield %: 94, m.p.: 190–
192°C. IR (KBr, cm−1): 3374, 3288 (2NH), 3062 (CH arom), 1230
1
(C=S), 1300, 1150 (SO2). H-NMR DMSO-d6 (ppm): 4.0 [s, 2H,
2NH, exchangeable with D2O], 6.9, 7.5 [2d, 4H, Ar-H, AB system],
7.2–8.1 [m, 9H, Ar-H], 8.2 [s, 1H, CH quinoxaline], 8.3 [s, 1H,
SO2NH]. MS (m/z): 433 (M+, 16.89%), 369 (100%), 225 (73.21%),
108 (13.02%), 90 (27.65%). Analysis Calc. for C21H15N5O2S2: C,
58.18; H, 3.49; N, 16.16. Found: C, 58.55; H, 3.67; N, 16.43.
4-Methyl-4-oxo-6-propionyl-2-thioxo-1,2-
dihydrothieno[2,3-d]pyrimidin-3(4H)-yl)-N-(quinoxalin-
2-yl)benzenesulfonamide (10)
4-(9H-Purin-8-ylamino)-N-(quinoxalin-2-yl)
benzenesulfonamide (14)
A mixture of 1 (0.4g, 0.01mol) and 3,5-(2amino-4-methyl thi-
ophen)carboxylic acid ethyl ester (0.3g ,0.001mol) in dioxane
(20mL) and a catalytic amount of TEA, was refluxed for 6h, the
reaction mixture was poured into ice water, filtered and crystal-
lized from ethanol to give 10. Yield %: 86, m.p.: 104–106°C. IR
(KBr, cm−1): 3306, 3296 (2NH), 3095 (CH arom), 2984 (CH
aliph),1674 (C=O), 1228 (C=S), 1320, 1104 (SO2). MS (m/z):
537 (M+, 14.38%), 479 (11.81%), 368 (100%), 221 (97.12%), 147
(50.16%). 1H-NMR DMSO-d6 (ppm): 1.17 [s, 3H, CH3], 2.2 [s, 3H,
CH3], 4.0 [s, 1H, NH, exchangeable with D2O], 6.9, 7.5 [2d, 4H,
Ar-H, AB system], 7.7–8.07 [m, 4H, Ar-H], 8.1 [s, 1H, CH quinoxa-
line], 8.3[s, 1H, SO2NH]. Analysis Calc. for C24H19N5O4S3: C,
53.62; H, 3.56; N, 13.03. Found: C, 53.77; H, 3.69; N, 13.25.
A mixture of 1 (0.4g, 0.01mol) and 4, 5-diaminopyrimidine
(0.2g, 0.01mol) in DMF (20mL) and a catalytic amount of TEA,
was refluxed for 8h, the reaction mixture was poured into ice
water, filtered and crystallized from ethanol to give 14. Yield %:
86, m.p.: 236–238°C. IR (KBr, cm−1): 3358, 3244 (2NH), 3070
(CH arom), 1236 (C=S), 1316, 1148 (SO2). 1H-NMR DMSO-d6
(ppm): 4.0 [s, 2H, 2NH, exchangeable with D2O], 6.9, 7.5 [2d, 4H,
Ar-H, AB system], 7.5–8.1 [m, 4H, Ar-H], 8.2 [s, 1H, CH quinoxa-
line], 8.3 [s, 1H, SO2NH], 8.9, 9.1 [2s, 2H, 2CH pyrimidine]. MS
(m/z): 418 (M+, 0.54%), 388 (1.31%), 236 (69.51%), 145 (100%),
92 (47.33%). Analysis Calc. for C19H14N8O2S: C, 54.54; H, 3.37; N,
26.78. Found: C, 54.15; H, 3.23; N, 26.65.
Ghorab MM et al. Synthesis, in-vitro Anticancer Screening… Arzneimittelforschung 2012; 62: 46–52

Original Article 49
N-(Quinoxalin-2-yl)-4-(5-thioxo-4,5-dihydro-1H-1,2,4-
triazol-3-ylamino)benzene-sulfonamide (15)
the compounds for 48h at 37°C and in atmosphere of 5% CO2.
After 48h, cells were fixed, washed and stained for 30min with
0.4% (wt/vol) with SRB dissolved in 1% acetic acid. Unbounded
dye was removed by 4 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 was plotted to get the survival
curve of each tumor cell line after the specified time. The con-
centration required for 50% inhibition of cell viability (IC50) was
calculated and compared with the reference drug 5-flurouracil
A mixture of 1 (0.4g, 0.01mol) and thiosemicarbazide (0.1g,
0.01mol) in DMF (20mL) and a catalytic amount of TEA, was
refluxed for 8h, the reaction mixture was poured into ice water,
filtered and crystallized from ethanol to give 15. Yield %: 90,
m.p.: 198–200°C. IR (KBr, cm−1): 3364, 3256, 3210, 3160 (4NH),
3066 (CH arom), 1316, 1146 (SO2), 1213 (C=S). 1H-NMR DMSO-
d6 (ppm): 4.0 [s, 2H, 2NH, exchangeable with D2O], 6.9, 7.5 [2d,
4H, Ar-H, AB system], 7.2 [s, 2H, 2NH triazole, exchangeable with
D2O], 7.6–8.1 [m, 4H, Ar-H], 8.2 [s, 1H, CH quinoxaline], 8.3 [s,
1H, SO2NH]. MS (m/z): 399 (M+, 2.90%), 236 (100%), 145
(83.28%), 110 (3.70%), 92 (67.22%). Analysis Calc. for
C16H13N7O2S2: C, 48.11; H, 3.28; N, 24.55. Found: C, 47.95; H,
3.20; N, 24.49.
▶
and the results are given in● Table 1.
Radiosensitizing activity
The most potent compounds resulted from the in vitro antican-
cer screening; compounds 4 and 17, were selected to be evalu-
ated again for their in vitro anticancer activity alone and in
combination with γ-radiation. This study was conducted to eval-
uate the ability of these compounds to enhance the cell killing
effect of γ-radiation. Cells were subjected to a single dose of
γ-radiation at a dose level of 8Gy with a dose rate of 2Gy/min.
Irradiation was performed in the National Cancer Institute, Cairo
University, using γ-cell-40 (60CO) source. The surviving fractions
were expressed as means ± standard error. The results were
4-(O-pyridin-3-yl-carbamothioate)-N-(quinoxalin-2-yl)
benzenesulfonamide (16)
A mixture of 1 (0.4g, 0.01mol) and 3-pyridinol (0.1g, 0.01mol)
in DMF (20mL) and a catalytic amount of TEA, was refluxed for
8h, the reaction mixture was poured into ice water, filtered and
crystallized from ethanol to give 16. Yield %: 95, m.p.: 258–
260°C. IR (KBr, cm−1): 3360, 3250 (2NH), 3070 (CH arom), 1232
1
▶
(C=S), 1318, 1148 (SO2). H-NMR DMSO-d6 (ppm): 4.3 [s, 2H,
analyzed using 1-way ANOVA test and are given in ● Table 2.
2NH, exchangeable with D2O], 6.7, 7.3 [2d, 4H, Ar-H, AB system],
7.6–8.1 [m, 4H, Ar-H], 8.2[s, 1H, CH quinoxaline], 8.3 [s, 1H,
SO2NH], 8.4–8.7 [m, 4H, 4CH pyridine]. MS (m/z): 437 (M+,
0.45%), 236 (100%), 156 (18.64%), 145 (48.85%), 92 (59.01%).
Analysis Calc. for C20H15N5O3S2: C, 54.91; H, 3.46; N, 16.01.
Found: C, 54.95; H, 3.55; N, 16.35.
Results and Discussion
▼
Chemistry
The isothiocyanate derivative 1 was synthesized by the reported
method [22], due to the high reactivity if isothiocyanate group,
compound 1 was employed as starting material for the synthesis
of different sulfaquinoxaline derivatives through incorporation
of different biologically active moieties, reaction of compound 1
with ethylene glycol yielded the corresponding thiourethane
derivative 2, while condensation of compound 1 with different
aliphatic alcohols such as ethanol, propanol or butanol in DMF
containing 3 drops of TEA gave compounds 3, 4 and 5, respec-
tively, cyclization occurred when 1 was reacted with malononi-
trile in the presence of sulfur to the thiazole derivative 6, the
structure of the synthesized compounds were confirmed by
microanalytical and spectral data, reaction of 1 with pyrazol-
opyrimidine o-amino carbonitrile yielded the pyrazolopyrimi-
dopyrimidine derivative 7, interaction of 1 with p-methoxy
cyanothioformanilde, intramolecular cyclization occurred to
give the imidazole derivative 8. Refluxing 1 with 4-(2-phenyl
3-amino pyrazole)carboxylic acid in DMF yielded the purine
analogue 9. Refluxing 1 with 3,5-(2-amino-4-methylthiophen)
carboxylic acid ester, 3,5-dichloroanthranilic acid or 2-amino-5-
methyl benzoic acid resulted in the formation of pyrimido deriv-
atives 10, 11 and 12, respectively. While, reaction of 1 with
thiophenol or 4,5-diaminopyrimidine yielded compounds 13
and 14, respectively. Double cyclization occurred when com-
pound 1 was refluxed with thiosemicarbazide in DMF through
elimination of H2S which was detected by lead acetate paper to
give the triazolo derivative 15. Finally, interaction of 1 with
3-pyridinol and 4-N-amino-3-hydrazino-5-thioxo-1,2,4-tria-
2-(4-Amino-5-mercapto-4H-1,2,4-triazol-3-yl)-N-(4-(N-
quinoxalin-ylsulfamoyl)phenyl)benzenesulfonamide (17)
A mixture of compound 1 (0.4g, 0.001mol) and 4-N-amino-
3-hyrazine-5-thioxo-1,2,4-triazole (0.2g, 0.001mol) in DMF
(20mL) and a catalytic amount of TEA, was refluxed for 2h, the
reaction mixture was poured into ice water, filtered and crystal-
lized from ethanol to give 17. Yield %: 92, m.p.: 192–194°C. IR
(KBr, cm−1): 3336, 3323, 3272, 3240, 3212, 3190 (4NH, NH2),
3070 (CH arom),1232 (C=S), 1324, 1114 (SO2). 1H-NMR DMSO-
d6 (ppm): 2.2 [2, 2H, NH2, exchangeable with D2O], 3.1[s, 1H,
SH], 4.0 [s, 3H, 3NH, exchangeable with D2O], 6.9, 7.5 [2d, 4H,
Ar-H, AB system], 7.6-8.1[m, 4H, Ar-H], 8.2[s, 1H, CH quinoxa-
line], 8.3 [s, 1H, SO2NH]. MS (m/z): 488 (M+, 0.1%), 313 (1.66%),
236 (16.71%), 108 (5.54%), 73 (100%). Analysis Calc. for
C217H16N10O2S3: C, 41.79; H, 3.30; N, 28.67. Found: C, 41.66; H,
3.25; N, 28,54.
In-vitro anticancer activity
The human tumor cell line (HEPG2) was available at the National
Cancer Institute, Cairo, Egypt. Irradiation was performed in the
National Cancer Institute, Cairo, Egypt using γ-cell-40 (60CO)
source. The anticancer activity of the newly synthesized com-
pounds was mesured using the Sulfo-Rhodamine-B stain (SRB)
assay by the method of Skehan et al. [21] (1990). Cells were
plated in 96-multiwell plate (104 cells/well) for 24h before treat-
ment with the compounds to allow attachment of cell to the wall
of the plate. Tested compounds were dissolved in DMSO and
diluted with saline to the appropriate volume. Different concen-
trations of the compounds under test (5, 12.5, 25 and 40μM)
were added to the cell monolayer. Triplicate wells were prepared
for each individual dose. Monolayer cells were incubated with
▶
zole gave compounds 16 and 17, respectively (● Fig. 2) .
In-vitro anticancer evaluation
The newly synthesized compounds were evaluated for their in-
vitro anticancer activity against human liver cancer cell line
Ghorab MM et al. Synthesis, in-vitro Anticancer Screening… Arzneimittelforschung 2012; 62: 46–52

50 Original Article
Table 1 In-vitro anticancer screening of the synthesized compounds against human liver cell line (HEPG2).
Compound concentration (μM)
IC 50 (μM)
Cpd. No.
5 (μM)
12.5 (μM)
Surviving fraction (mean±SE)a
0.846±0.020
25 (μM)
40 (μM)
5-FU
1
0.921±0.020
0.934±0.002
0.855±0.018
0.882±0.012
0.395±0.064
0.921±0.031
0.925±0.014
0.892±0.009
0.912±0.014
0.896±0.007
0.894±0.016
0.927±0.021
0.758±0.032
0.91±0.006
0.93±0.014
0.923±0.017
0.886±0.006
0.888±0.011
0.761±0.010
0.848±0.028
0.493±0.022
0.625 ±0.047
0.346±0.016
0.610±0.081
0.642±0.045
0.553±0.007
0.654±0.027
0.611±0.034
0.538±0.041
0.583±0.051
0.437±0.015
0.481±0.038
0.725±0.041
0.569±0.017
0.603±0.041
0.316±0.044
0.494±0.030
0.422±0.047
0.392±0.012
0.419±0.022
0.412±0.012
0.386±0.015
0.428±0.317
0.366±0.015
0.388±0.044
0.454±0.008
0.445±0.044
0.496±0.062
0.396±0.004
0.331±0.012
0.462±0.049
0.408±0.006
0.348±0.009
0.368±0.028
40
0.896±0.009
0.653±0.031
0.693±0.041
0.259±0.014
0.731±0.043
0.839±0.007
0.681±0.016
0.739±0.019
0.703±0.017
0.761±0.031
0.721±0.021
0.563±0.026
0.705±0.039
0.813±0.015
0.823±0.027
0.753±0.021
0.445±0.025
38.12
23.35
32.21
4.29
2
3
4
5
30.87
34.63
26.84
32.48
34.36
29.26
40
6
7
8
9
10
11
12
13
14
15
16
17
16.64
23.35
38.12
29.79
30.06
11.27
a Each value is the mean of 3 experiments ± standard error
Table 2 In-vitro anticancer screening of compounds 4 and 17 against human liver cell line (HEPG2) in combination with radiation.
Cpd. No.
Control
Irradiated (8Gy)
Compound concentration (μM) + irradiation (8Gy)
IC50(μM)
Surviving fraction (mean ± SE) a
1
2.5
5
12.5
25
40
4
1.000
1.000
0.927±0.02*
0.927±0.02*
0.93±0.02*
0.98±0.01*
0.47±0.03*
0.81±0.03*
0.29±0.01*
0.67±0.07*
0.16±0.02*
0.23±0.01*
0.24±0.05*
0.11±0.01*
0.21±0.06*
0.16±0.08*
2.41
6.97
17
a Each value is the mean of three values ± Standard Error
*Significant difference from control group at p<0.001
(HEPG2). 5-Flurouracil, which is one of the most effective anti-
cancer 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 liver cancer cell line
(HEPG2). The response parameter calculated was the IC50 value,
which corresponds to the concentration required for 50% inhibi-
than the reference drug. While compounds 3, 5, 6, 8–11, 14–16
showed IC50 values ranging from 30.06–40μM.
Radiosensitizing evaluation
The rationale for combining chemotherapy and radiotherapy is
based mainly on 2 ideas, one being spatial cooperation, which is
effective if chemotherapy is sufficiently active to eradicate sub-
clinical metastases and if the primary local tumor is effectively
treated by radiotherapy. In this regard, no interaction between
radiotherapy and chemotherapy is required. The other idea is
the enhancement of radiation effects. Cytotoxic agents can
enhance radiation effects by direct enhancement of the initial
radiation damage by incorporating drugs into DNA, inhibiting
cellular repair, accumulating cells in a radiosensitive phase or
eliminating radioresistant phase cells, eliminating hypoxic cells
or inhibiting the accelerated repopulation of tumor cells [23].
Consequently, the ability of the 2 most active compounds, com-
pounds 4 and 17, to enhance the cell killing effect of γ-irradiation
▶
tion of cell viability. ● Table 1 shows the in-vitro anticancer
activity of the synthesized compounds which exhibited signifi-
cant activity compared to the reference drug. From the results
▶
in ● Table 1, it was found that some of the tested compounds
were found to be equipotent while others showed lower IC50
than 5-fluorouracil (IC50 =40μM), where, the starting material 1
showed high IC50 compared to most of the tested compounds
(IC50 =38.12μM), while, substitution on the isothiocyanato
group resulted in an increase in the activity in most of the com-
pounds and the most potent was the propyl thioureido com-
pound 4 (IC50 =4.29μM), the thioureido 2 (IC50 =23.35μM),
while, the thiouredo compounds 3 and 5 containing an aliphatic
▶
chain showed
a
slightly higher activity (IC50 =32.21 and
was studied. From the results obtained in ● Table 1, compound
30.87μM, respectively). The pyrazolopyrimidopyrimidine
7
4 showed an in vitro cytotoxic activity with IC50 value of 4.29μM,
when the cells were subjected to different concentrations of the
compound alone. While, when the cells were subjected to differ-
ent concentrations of compound 4 (1, 2.5, 5, 12.5, 25, 40μM),
and irradiated with a single dose of γ-radiation at a dose level of
(IC50 =26.84μM), the benzopyrimidine 12 (IC50 =16.64μM), the
benzothiophene 13 (IC50 =23.3μM) and the triazolo derivative
17 (IC50 =11.27μM) were the most active derivatives among
those containing cyclic heterocyclic moieties as substituent on
amino group of sulfonamide and they found to be more active
▶
8Gy, as shown in ● Table 2, the IC50 value was synergistically
Ghorab MM et al. Synthesis, in-vitro Anticancer Screening… Arzneimittelforschung 2012; 62: 46–52

Original Article 51
S
N
N
S
S
H
N
SH
S
N
R
N
H
N
H
OH
O
3
CH3
O
R N
H
CH3
R N
H
O
NH2
R N
H
S
2
17
N
4
O
R N
H
16
(xvi)
(i)
(ii)
S
(iii)
(xv)
NH
N
O
CH3
R N
H
N
S
H
N
H
R
(iv)
5
15
O
(xiv)
(xiii)
NH2
CN
(v)
N
H
N
R N
S
S
NCS
N
N
H
6
S
R
N
O
N
H
N
(vi)
N
NH
1
14
(xii)
N
N R
S
(vii)
N
N
N
NC
7
N
H
H
N
(xi)
R
(viii)
(x)
(ix)
S
OCH3
13
S
O
N
R N
H3C
COOH
O
N R
S
O
S
H2N
Cl
H3C
R
N
N R
S
N
H
N
O
N R
S
8
N
Ph
HS
N
H
N
H
12
S
N
H
Cl
9
11
10
O
S
O
N
H
N
R=
N
Fig. 2 Synthetic pathways for compounds 1–17. Conditions and reagents: (i) ethylen glycol, DMF, TEA, reflux 8h; (ii) ethanol, DMF, TEA, reflux 8h; (iii)
propanol, DMF, TEA, reflux 3h; (iv) butanol, DMF, TEA, 8h; (v) malononitrile, S, DMF, TEA, reflux 5h; (vi) pyrazolo pyrimidine o-amino carbonitrile, DMF,
TEA, reflux 6h; (vii) p-methoxy cyanothio formanilide, DMF, TEA, reflux 6h; (viii) 4-(2 phenyl 3-amino pyrazole)carboxylic acid, DMF, TEA, reflux 1h; (ix)
3,5-(2amino-4-methyl thiophen)carboxylic acid ethyl ester, DMF, TEA, reflux 5h; (x) 3,5,dichloroanthranilic acid, DMF, TEA, reflux 5h; (xi) 2-amino 5-methyl
benzoic acid, DMF, TEA, reflux 6h; (xii) thiophenol, DMF, TEA, reflux 24h; (xiii) 4,5,diamino pyrimidine, DMF, TEA, reflux 8h; (xiv) thiosemicarbazide, DMF,
TEA, reflux 8h; (xv) 3-pyridinol, DMF, TEA, reflux 5h; (xvi) 4-N-amino-3-hydrazine-5-thioxo1,2,4,triazole, DMF, TEA, reflux 2h.
1.2
1
1.2
1
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
10
0
20
Concentration (uM)
50
30
40
0
10
20
Concentration (uM)
Compound 17 Compound 17 + 8Gy
30
40
50
Compound 4
Compound 4 + 8Gy
Fig. 4 Survival curve for HEPG2 cell line for compound 17 alone and in
combination with γ-irradiation (8Gy).
Fig. 3 Survival curve for HEPG2 cell line for compound 4 alone and in
combination with γ-irradiation (8Gy).
Ghorab MM et al. Synthesis, in-vitro Anticancer Screening… Arzneimittelforschung 2012; 62: 46–52

52 Original Article
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▶
decreased to 2.41μM (● Fig. 3). Similarly, compounds 17
showed IC50 value of 11.27μM when used alone, as shown
▶
in ● Table 1. The IC50 value was decreased to 6.97μM when the
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Conclusion
▼
We report here the synthesis of new sulfaquinoxaline deriva-
tives. It was clearly observed from the results of in-vitro antican-
cer screening that the synthesized compounds exhibited
significant anticancer activity on liver human tumor cell line
(HEPG2). Combining these compounds with radiation enhances
their activity, which demonstrates the importance of combina-
tion therapy for cancer patients as it allows a reduction of the
individual doses, which in turn decreases the side effects of both
drugs and radiation.
Conflict of Interest
▼
The authors declare that they have no conflict of interest with
respect to this paper.
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