New quinoxaline 1, 4-di-N-oxides: Anticancer and hypoxia-selective therapeutic agents

Article abstract of DOI:10.1016/j.ejmech.2010.02.052

A new series of quinoxaline 1,4-di-N-oxides was synthesized and evaluated for antitumor and hypoxic-selective cytotoxic activities. Antitumor activity against liver carcinoma (Hepg2) and brain tumor (U251) human cell lines were evaluated, among the tested compounds, 5b and 9b exhibited potential cytotoxic effect against Hepg2 with IC50 values of 0.77 and 0.50 μg/mL respectively, whereas, all the tested compounds lack antitumor activity against U251 human cell line. Moreover, compound 4 was the most potent hypoxia selective-cytotoxin on EAC cell line; IC50 2.5 μg/mL, potency 22 μg/mL, and was approximately 5.4-times more selective cytotoxin (HCR > 40) than 3-amino-2- quinoxalinecarbonitrile1,4-dioxide (standard, HCR > 7.4). Compounds 8b and 9b were more selective than the standard.

Full text of DOI:10.1016/j.ejmech.2010.02.052

European Journal of Medicinal Chemistry 45 (2010) 2733e2738
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New quinoxaline 1, 4-di-N-oxides: Anticancer and hypoxia-selective
therapeutic agents
,
b
c
a
d
Magda M.F. Ismail a , Kamelia M. Amin , Eman Noaman , Dalia H. Soliman , Yousry A. Ammar
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
c Department of Radiation Biology, Natural Center for Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt
d Department of Chemistry, Faculty of Science, Al-Azhar University, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of quinoxaline 1,4-di-N-oxides was synthesized and evaluated for antitumor and hypoxic-
selective cytotoxic activities. Antitumor activity against liver carcinoma (Hepg2) and brain tumor (U251)
human cell lines were evaluated, among the tested compounds, 5b and 9b exhibited potential cytotoxic
Received 19 December 2009
Received in revised form
6 February 2010
Accepted 21 February 2010
Available online 1 March 2010
effect against Hepg2 with IC50 values of 0.77 and 0.50
compounds lack antitumor activity against U251 human cell line. Moreover, compound 4 was the most
potent hypoxia selective-cytotoxin on EAC cell line; IC50 2.5 g/mL, potency 22 g/mL, and was approxi-
mg/mL respectively, whereas, all the tested
m
m
mately 5.4-times more selective cytotoxin (HCR > 40) than 3-amino-2-quinoxalinecarbonitrile1,4-dioxide
(standard, HCR > 7.4). Compounds 8b and 9b were more selective than the standard.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Quinoxaline 1,4-di-N-oxide
Antitumor activity
Hypoxia-selective cytotoxicity
1. Introduction
2. Results and discussion
Some quinoxaline 1,4-di-N-oxide derivatives have been
synthesized and evaluated as direct antitumor against MCF7
(breast), NCI-H460 (lung) and SF268 (CNS) human cell lines [1]. In
addition, antitumor agents can be made selective for tumors by
virtue of high activity under hypoxic conditions [2]. The lead
compound in this regard is tirapazamine, which acts as bio-
reductive cytotoxic agent [3]. Extensive studies have been carried
out on 3-amino-2-quinoxalinecarbonitrile 1,4-dioxide, indicated
that, it is more susceptible to reductive activation than tirapaz-
amine [4] and causes redox-activated DNA damage analogous
to it [5].
Recently, we reported on the antitumor and hypoxia-selective
cytotoxicity of certain quinoxaline 1,4-di-N-oxides [6]. As a continu-
ation to our previous efforts aiming to enhance the potency and
selective cytotoxicity, we designed a new series having an electron-
withdrawing (Cl) or an electron-donating (OCH3) group at C7 and
diverse substituents at position 2 and 3 of the quinoxaline 1,4-di-N-
oxides nucleus.
2.1. Chemistry
Scheme 1 outlines the synthetic pathway used to obtain
compounds 2e6. 3-Amino-2-quinoxalinecarbonitrile 1,4-dioxide, 2
[4] was prepared by reaction of 5(6)-chlorobenzofuroxane, 1a with
malononitrile in presence of triethylamine [7]. Methylation of 2
using dimethylsulphate in dioxane afforded the 3-methylamino
derivative 3, while acetylation of 2 with acetic anhydride furnished
3-diacetylaminoquinoxaline derivative, 4. Additionally, the reaction
of 5(6)-methoxybenzofuroxane 1b with prepared styrenes yielded
the 2-aryliminomethylquinoxaline derivatives 5a,b which were
further reduced by lithium borohydride to give 2-arylaminomethyl-
7-methoxy-3-phenylquinoxaline-4-oxides 6a,b.
Nitrosation [8] of the acid hydrazides 7a,b [9] with sodium nitrite
in acetic acid afforded the acid azides 8a,b. The latter underwent
Curtius rearrangement with various aromatic amines in N,N-dime-
thylformamide (DMF) to yield the urea derivatives 9aee while
urethans or carbamates 10a-e were produced by boiling 8a,b with
different alcohols (Scheme 2).
2.2. Antitumor screening
The antitumor screening is divided into two main parts. The first
part is a preliminary screening of all the synthesized compounds
against two human cell lines: liver carcinoma (Hepg2) and brain
* Corresponding author. Tel.: þ20 2 22713531.
E-mail address: magdafathi_111@hotmail.com (M.M.F. Ismail).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.052

2734
M.M.F. Ismail et al. / European Journal of Medicinal Chemistry 45 (2010) 2733e2738
Scheme 1.
tumor (U251). Some compounds precipitated in the culture medium
during the experiment and were rejected from any further investi-
gation. The second part is a study on their hypoxia-selective cyto-
toxicity against Ehrlich ascites carcinoma (EAC) cell line.
2.2.1. Activity against liver carcinoma (Hepg2) and brain tumor
(U251) human cell lines
Antitumor screening was performed at the National Cancer
Institute, Cancer Biology Department, Cairo, Egypt. Cytotoxicityof the
Scheme 2.

M.M.F. Ismail et al. / European Journal of Medicinal Chemistry 45 (2010) 2733e2738
2735
Table 2
selected compounds 3, 4, 5b, 6b, 8b, 9b,e, 10c,e was tested on two
human cell lines: liver carcinoma (Hepg2) and brain tumor (U251)
using the method of Skehan and Storeng [10]. The growth inhibitory
action is summarized in Table 1. The results showed that compounds
9b and 5b were highly potent against the liver carcinoma cell line
Data of IC50's, potencies and hypoxia-selectivity ratios of the tested quinoxalines on
Ehrlich Ascites Carcinoma (EAC).
Compd. no.
IC50 air (
m
g/mL)
IC50 hypoxia
g/mL)
Potencyb
(mg/mL)
>100c
>100
22
>100
>100
>100
90
>100
>100
>100
HCRc
(
m
Standard (2)a
3
4
5b
6b
8a
8b
9b
10c
10e
>100d
>100
>100
>100
>100
>100
>100
100
13.5
>100
2.5
>7.4
>1
>40
>1.4
>1
>1
>8
w10
>1
>1
showing IC50 values 0.50 and 0.77
of the other compounds was 8b>3 > 6b > 4 > 9e ¼ 10e showing IC50
values 3.61, 4.75, 4.95, 5.69 and 6.00 g/mL respectively. Both 9b and
mg/mL respectively. Activity order
m
70
9e were active against liver carcinoma cell line, showing that the urea
moiety is important for the activity [11], yet the introduction of an
electron-withdrawing moiety (Cl) at position 7 of the quinoxaline
>100
>100
12.5
10
>100
>100
favored the antitumor activity (9b, IC50 ¼ 0.5
mg/mL) rather than the
g/mL) .However, in case of
>100
>100
7-methoxyl function (9e, IC50 ¼ 6.0
m
carbamate derivatives, 7-methoxy-2-alkyl carbamate 10e elicited
higher cytotoxicity than 7-chloro derivative 10c which lacks the
cytotoxic activity. The reduction of the 2-aryliminomethylquinoxa-
9e could not be accurately determined because at doses higher than 25
m
g/mL this
compound precipitated in the culture medium.
a
Standard, 3-aimo-2-quinoxalinecarbonitrile 1,4-dioxide.
Potency, concentration that gives 1% cell survival in hypoxia.
HCR (hypoxic cytotoxicity ratio), IC50 in air / IC50 in hypoxia.
>100, Potency or IC50 of a tested compound was more than 100 mg/mL.
b
line (5b, IC50 ¼ 0.77
resulted in a decrease in the activity to the moderate range (6b,
IC50 ¼ 4.95 g/mL). All the tested compounds showed low activity
towards the brain tumor cell line (IC50 values > 10 g/mL).
mg/mL) to the corresponding quinoxaline 4-oxide
c
d
m
m
2.2.2. Activities against Ehrlich ascites carcinoma (EAC) cell line
[12]: hypoxia-selective cytotoxicity screening
These biological studies were performed at the National Center for
Radiation Research and Technology (NCRRT), Cairo, Egypt.
group for example 8b showed higher selectivity than the
7-chloro derivative 8a with HCR values >8 and >1, respectively
and even the IC50 value of 8b was 12.5
mg/mL while 8a was
A
>100 g/mL. Moreover, the urea derivative 9b (HCR w 10)
m
preliminary screening was performed against EAC. In-vitro study was
carried out both in air and under hypoxic conditions following the
reported methods [4,13]. Three response parameters were calculated,
IC50, potency and hypoxic cytotoxicity ratio (HCR), (Table 2). Where
IC50 value corresponds to the compound's concentration causing 50%
mortality in net cell numbers, the potency is the dose which gives 1%
cell survival in hypoxia, while HCR is the ratio between doses in air
and in hypoxia giving the same cellsurvival(calculated at IC50) [14,15]
The 3-diacetylamino-7-chloro-2-quinoxaline 1,4-dioxide 4
showed the best in-vitro hypoxia-selective results of this series
showed higher hypoxia-selectivity than the standard. Dos-
eeresponse curves for 4; 8b and 9b are presented in Figs. 1e3.
3. Conclusion
We successfully conclude that 3-diacetylamino-7-chloro-qui-
noxaline-2-carbonitrile 1,4-dioxide (4) is the most promising
compound acting both as an antitumor and as a hypoxia-selective
agent. It was found that the cytotoxicity and the potency were
greatly improved. Compounds 9b and 5b were the most toxic against
liver carcinoma (Hepg2). 7-Cl (electron-withdrawing gp) is preferred
in 3-ureido/3-substituted-amino derivatives whereas, 7-OCH3
(electron-donaing gp) is important for cytotoxicity in carbamate and
acid azide derivatives. Reduction of quinoxaline 1,4-dioxide to qui-
noxaline 4-oxide reduces the activity. Compounds 4, 8b and 9b have
well in-vitro profile and they might have useful in-vivo properties
which will be investigated in future work.
with IC50 2.5 mg/mL, potency 22 mg/mL and HCR >40. It
demonstrated better cytotoxicity, potency and 5.4-times more
selective than the standard (HCR > 7.4). The results were in
favor of adding the electron-withdrawing moieties (2COCH3) to
the 3-amino group, however, its counterpart 3 having an elec-
tron-donating group (CH3) showed insignificant HCR. The acid
azide derivative seemed to be more active and even more
selective when the 7 position was substituted with methoxyl
Table 1
Growth inhibitory action (IC50
U251 human cell lines.
mg/mL) of the tested compounds against Hepg2 and
Compound no.
Hepg2b
U251c
Standard (TPZ)a
3
4
5b
6b
8b
9b
9e
10c
10e
56.6
4.75
5.69
0.77
4.95
3.61
0.50
6.00
>10
6.00
71.2
>10
>10
>10
>10
>10
>10
>10
>10
>10
This screening was performed under aerobic conditions. The IC50 of the other
compounds could not be accurately determined because they precipitated in the
culture medium.
a
Standard, tirapazamine (TPZ) tested against Hepg2 and U251 human cell lines
under aerobic conditions.
b
Hepg2, liver carcinoma human cell line.
U251, brain tumor human cell line.
c
Fig. 1. Doseeresponse curve in air and hypoxia of 4 against EAC.

2736
M.M.F. Ismail et al. / European Journal of Medicinal Chemistry 45 (2010) 2733e2738
(25 mL), was stirred and heated at 90 ꢁC for 4 h. After cooling a brown
solid was obtained, filtered and recrystallized (Table 3). IR (KBr, cmꢂ1
3350 (NH), 2219 (CN), 1620 (C]N), 1553 (NO). 1HNMR
: 3.53 (s, 3H,
)
d
NHCH3), 7.65 (d,1H, H6, J ¼ 9 Hz), 8.25 (d,1H, H5, J ¼ 9 Hz), 8.39 (s,1H,
H8), 8.63 (bs, 1H, NHeD2O exchangeable); MS: m/z (%) 252 (M þ 2,
10.51), 250 (Mþ, 32.98); 204 (16.48),124 (100). Anal. Calc. for CHN: C,
(47.92); H, (2.82); N, (22.36). Found: C, (47.63); H, (3.19); N, (22.15).
4.1.2. 3-Diacetylamino-7-chloro-2-quinoxalinecarbonitrile
1,4-dioxide (4)
A mixture of 2 (2.36 g, 0.01 mol) and acetic anhydride (30 mL)
was heated until complete dissolution (2 h.). Then, the reaction
mixture was cooled, the product filtered and recrystallized (Table 3).
IR (KBr, cmꢂ1): 2216 (CN), 1700e1691 (2C]O), 1604 (C]N),
1364e1567 (NO). 1HNMR
d: 2.18 (s, 6H, 2COCH3), 7.62 (d, 1H, H6,
J ¼ 8.9 Hz), 8.06 (d, 1H, H5, J ¼ 8.9 Hz), 8.16 (s, 1H, H8). MS: m/z (%)
320 (Mþ,1.78), 220 (100). Anal. Calc. for CHN: C, (48.67); H, (2.83); N,
(17.47). Found: C, (48.82); H, (2.95); N, (17.83).
Fig. 2. Doseeresponse curve in air and hypoxia of 8b against EAC.
4.1.3. 2[(Arylimino)methyl]-7-methoxy-3-phenylquinoxaline
1,4-dioxides (5a,b)
4. Experimental
Equimolar amounts of 1b (0.1 mol) and 2-[N (4-methyl (ethoxy)
phenyl) iminomethyl] styrene (0.01 mol) in benzene (50 mL) was
heated for 3 h, the residue was allowed to settle overnight. The
obtained residue was extracted with chloroform and crystallized
(Table 3). IR (KBr, cmꢂ1) 5a: 1610 (C]N), 1328e1303 (NO). MS: m/z
(%): 385 (Mþ, 1.3), 352 (18.9), 55 (100). Anal. Calc. for CHN: C,
(71.68); H, (4.97); N, (10.90). Found: C, (71.35); H, (4.67); N, (10.85).
4.1. Synthesis
Melting points were determined on electrothermal 9100 digital
melting point apparatus and were uncorrected. 1HNMR spectra
were recorded in DMSO-d6 on Varian Gemini 200 (200 MHz) using
tetramethylsilane (TMS) as an internal standard (chemical shift in
IR (KBr, cmꢂ1) 5b: 1615 (C]N), 1338e1505 (NO). 1HNMR 5b:
d 1.27
d
ppm). The IR spectra were performed on a Perckin-Elmer 1600
(t, 3H, CH2eCH3, J ¼ 7.0 Hz); 3.98 (s, 3H, OCH3), 4.00 (q, 2H,
CH2eCH3, J ¼ 7.0 Hz); 6.84 (m, 4H, AreH); 7.43 (m, 5H, PheH); 7.63
(d, 1H, H6, J ¼ 9.4 Hz); 7.86 (s, 1H, H8); 8.48 (d, 1H, H5, J ¼ 9.4 Hz);
8.73 (s, 1H, CH]N). Anal. Calc. for CHN: C, (69.39); H, (5.10); N,
(10.12). Found: C, (69.15); H, (5.24); N, (10.30).
FTIR in KBr pellets. Elemental microanalysis (C, H, N) were per-
formed on a Perkin-Elmer 2400 analyzer from vacuum-dried
samples. All compounds were within ꢀ0.4% of the theoretical
values. The mass spectra were recorded on a Hewlett-Packard 5988-
A instrument at 70 eV.
Two intermediates, 2-[N-(4-ethoxy (methyl) phenyl) imino-
methyl] styrenes were prepared according to the reported method
[16,17] and used as reagents. M.F.: C16H15N, C17H17NO, M.P. 110e111,
105e106 ꢁC. Yield: 97, 95%.
4.1.4. 2-[(Arylamino)-methyl]-7-methoxye3-phenylquinoxaline
4-oxides (6a,b)
A mixture of 5a or 5b (0.01 mol) and lithium borohydride
(0.02 mol) in ethanol (20 mL) was stirred for 2 h, decomposed on
ice-water, filleted, washed, and recrystallized (Table 3). IR (KBr,
4.1.1. 7-Chloro-3-methylamino-2-quinoxalinecarbonitrile
1,4-dioxide (3)
cmꢂ1) 6a: 3396 (NH), 1622 (C]N), 1356, 1516 (NO). 1HNMR 6a
d:
2.09 (s, 3H, CH3), 3.94 (s, 3H, OCH3), 4.10 (d, 2H, CH2NH); 5.60 (t, 1H,
NHeD2O exchangeable), 6.39(d, 2H, AB system, J ¼ 9 Hz), 6.80
A mixture of 2 (2.36 g, 0.01 mol), dimethylsulphate (1.4 g,
0.015 mol) and sodium bicarbonate (1.0 g, 0.011 mol) in dry dioxane
Table 3
Physical properties and molecular formulae of the new quinoxalines.
Compd R1
No.
R2
Solvent
Mp. (ꢁC) Yield Formula
%
3
4
5a
5b
6a
6b
8a
8b
9a
9b
9c
9d
9e
e
e
Dioxane
CH3OH
EtOH
170e172 50
179e180 80
198e200 56
205e207 60
162e163 45
158e160 55
99e100 75
110e111 68
258e260 50
200e201 77
246e248 87
248e250 60
250e252 72
220e222 80
260e263 93
C10H7ClN4O2
e
e
C13H9ClN4O4
C23H19N3O3
C24H21N3O4
C23H21N3O2
C24H23N3O3
C10H6ClN5O3
C11H9N5O4
C16H12Cl2N4O3
C17H15ClN4O4
C18H17ClN4O3
C18H18N4O4
C18H18N4O5
C11H10ClN3O4
C12H12ClN3O4
C13H14ClN3O4
C13H15N3O5
C14H17N3O5
e
C6H4CH3-4
e
C6H4OC2H5-4 EtOH
C6H4CH3-4 EtOH
C6H4OC2H5-4 EtOH
e
e
Cl
OCH3
Cl
Cl
Cl
OCH3 4-CH3
OCH3 4-OCH3
Cl
Cl
Cl
e
H2O
H2O
e
3-Cl
4-OCH3
2,6-(CH3)2
EtOH-DMF
EtOH-DMF
EtOH-DMF
EtOH-DMF
EtOH-DMF
CH3OH
10a
10b
10c
10d
10e
CH3
C2H5
CH(CH3)2
EtOH
(CH3)2CHOH 258e259 80
EtOH 238e240 90
(CH3)2CHOH 218e220 88
OCH3 C2H5
OCH3 CH(CH3)2
Fig. 3. Doseeresponse curve in air and hypoxia of 9b against EAC.

M.M.F. Ismail et al. / European Journal of Medicinal Chemistry 45 (2010) 2733e2738
2737
(d, 4H, AB system, J ¼ 9 Hz), 7.58 (m, 6H, PheH þ H6), 7.77 (s, 1H,
H8), 8.04 (d, 1H, H5). Anal. Calc. for CHN: C, (74.38); H, (5.70); N,
(11.31). Found: C, (74.80); H, (5.65); N, (10.99).IR (KBr, cmꢂ1) 6b:
J ¼ 9 Hz), 7.73 (d, 1H, H5, J ¼ 9 Hz), 8.01 (s, 1H, H8). Anal. Calc. for
CHN: C, (48.42); H, (4.06); N, (14.12). Found: C, (48.65); H, (4.09); N,
(13.92). 1HNMR 10c:
d 1.02 (d , 6H, CH(CH3)2); 2.45 (s, 3H, CH3); 4.81
(m,1H, CH (CH3)2), 7.09 (bs,1H, NHeD2O exchangeable), 7.34 (d,1H,
H6, J ¼ 9.3 Hz), 7.83 (d, 1H, H5, J ¼ 9.3 Hz),), 8.17 (s, 1H, H8); Anal.
Calc. for CHN: C, (50.09); H, (4.53); N, (13.48). Found: C, (50.21); H,
3396 (NH), 1622 (C]N), 1349, 1515 (NO). 1HNMR
d: 1.20 (t, 3H,
CH2CH3, J ¼ 7.0 Hz), 3.83 (q, 2H, CH2CH3, J ¼ 7.0 Hz), 4.96 (s, 3H,
OCH3), 4.09 (d, 2H, CH2NH), 5.52 (t, 1H, NHeD2O exchangeable),
6.40 (d, 2H, AB system, J ¼ 8.8 Hz), 6.62 (d, 2H, AB system,
J ¼ 8.8 Hz), 7.56 (m, 6H, PheH þ H6), 7.78 (s,1H, H8), 8.06 (d,1H, H5).
MS: m/z (%): 401 (Mþ, 100), 385 (38.75). Anal. Calc. for CHN: C,
(71.81); H, (5.78); N, (10.47). Found: C, (72.12); H, (5.82); N, (10.63).
(4.52); N, (13.65). 1HNMR 10d:
d
1.23 (t, 3H, CH2eCH3, J ¼ 7.5 Hz) ;
2.42 (s, 3H, CH3), 3.92 (s, 3H, OCH3); 4.13 (q, 2H, CH2eCH3,
J ¼ 7.5 Hz), 7.30 (d, 1H, H6, J ¼ 9.3 Hz),), 7.37 (s, 1H, H8), 8.26 (d, 1H,
H5, J ¼ 9.3 Hz),), 10.11 (bs, 1H, NHeD2O exchangeable). Anal. Calc.
for CHN: C, (53.25); H, (5.16); N, (14.33). Found: C, (55.64); H, (5.51);
4.1.5. 2-Azidocarbonyl-7-chloro (methoxy)-3-
N, (13.99). 1HNMR 10e:
d 1.20 (d, 6H, CH(CH3)2), 2.40 (s, 3H, CH3);
methylquinoxaline1,4-dioxide (8a,b)
3.90 (s, 3H, OCH3); 4.90 (m, 1H, CH (CH3)2), 7.30 (d, 1H, H6,
J ¼ 9 Hz),), 7.37 (s, 1H, H8), 8.20 (d, 1H, H5, J ¼ 9 Hz),), 10.10 (bs, 1H,
NHeD2O exchangeable). Anal. Calc. for CHN: C, (54.72); H, (5.58); N,
(13.68). Found: C, (54.73); H, (5.99); N, (13.42).
As suspension of 7a or 7b [9] (0.01 mol) in acetic acid (10 mL)
was stirred and cooled for 10 min. Then, a solution of Na NO2
(0.1 mol) in water (10 ml) was added drop wise, stirring was
continued for 15 min at 0 ꢁC. The precipitated solid was filtered and
washed with water and dried (Table 3). MS: m/z (%) 8a: 281 (M þ 2,
26.5), 279 (Mþ, 82.3), 213 (64.7), 183 (94.1) 54 (100). Anal. Calc. for
CHN: C, (42.95); H, (2.16); N, (25.05). Found: C, (42.66); H, (2.25); N,
(25.31). IR (KBr, cmꢂ1) 8b: 2158 (N3), 1689 (CO), 1602 (C]N),
1340e1567 (NO). MS: m/z (%): 275 (Mþ, 0.16); 235 (100). Anal. Calc.
for CHN: C, (48.01); H, (3.30); N, (25.45). Found: C, (47.82); H,
(3.22); N, (25.34).
4.2. Antitumor screening
4.2.1. Activity against Hepg2 and U251
Cells were plated in 96- multiwell plate (104 cells/well) for 24 h
before treatment with the compounds to allow attachment of cell
to the wall of the plate. Different concentrations in micrograms per
milliliter [18] (0.1, 2.5, 5 and 10) of the compounds under test were
solubilized in dimethylsulfoxide (DMSO) and were added to the cell
monolayer of the two human cell lines. Monolayer cells were
incubated with the compounds for 48 h at 37 ꢁC and in atmosphere
of 5% CO2. After 48 h, cells were fixed, washed and stained with
sulforhodamine B stain. The colour intensity was measured in an
ELISA reader [10].
4.1.6. 2-(3Arylureido)-7-chloro (methoxy)-3-methyl quinoxaline
1,4-dioxides (9aee)
A mixture of equimolar amounts of 8a or 8b and the appropriate
amine was refluxed in DMF (50 mL) for 3 h; the obtained product was
filtered and crystallized (Table 3). IR (KBr cmꢂ1) 9aee: 3365e3139
(2NH), 1685e1661 (C]O), 1630e1595 (C]N), 1561-1320 (NO). MS:
m/z (%) 9a: 380 (M þ 2, 0.16), 378 (Mþ, 0.5), 362 (8.1), 220 (9.2), 56
(100). Anal. Calc. for CHN: C, (50.68); H, (3.19); N, (14.78). Found: C,
4.2.2. Activity against (EAC) experimental cell line
Animals, chemicals and facilities: Female swiss albino mice
weighing 25e30 g obtained from (the holding company of biolo-
gical products and vaccines, VACSERA, Cairo, Egypt) were housed at
a constant temperature (24 ꢀ 2 ꢁC) with alternating 12 h light and
dark cycles and fed standard laboratory food (Milad Co., Cairo
Egypt) and water adlibitum. All chemicals and reagents were from
SigmaeAldrich and MerckeGermany.
(50.98); H, (3.31); N, (14.97). 1HNMR 9b:
d 2.44 (s, 3H, CH3); 3.80
(s, 3H, OCH3); 6.98 (d, 2H, AB system, J ¼ 9 Hz), 7.31 (d, 2H, AB system,
J ¼ 9 Hz), 7.91 (d,1H, H6, J ¼ 9 Hz), 8.20 (d,1H, H5, J ¼ 9 Hz), 8.32 (s,1H,
H8), 7.09 (s, 1H, NHeD2O exchangeable), 10.72 (s, 1H, NHeD2O
exchangeable). Anal. Calc. for CHN: C, (54.48); H, (4.03); N, (14.95).
Found: C, (54.07); H, (4.20); N, (14.76). 1HNMR 9c:
d 2.21 (s, 3H, CH3),
2.26 (s, 3H, CH3); 2.41 (s, 3H, CH3-quinox); 6.87 (bs, 1H, NHeD2O
exchangeable), 7.11 (bs, 1H, NHeD2O exchangeable); 7.31e8.21
(m, 6H, Ar-H, quinox-H); Anal. Calc. for CHN: C, (57.99); H, (4.60); N,
4.2.2.1. Aerobic and hypoxic cytotoxicity. Cells: Ehrlich Ascites
carcinoma cells EAC were obtained by needle aspiration of ascitic
fluid from pre-inoculated mice, under aseptic conditions. Tumor
cell suspension (2.5 ꢃ106 per ml) was prepared.
(15.03). Found: C, (57.87); H, (4.74); N, (14.92). 1HNMR 9d:
d 2.24
(s, 3H, CH3), 2.42 (s, 3H, CH3-quinox), 3.96 (s, 3H, OCH3), 6.45 (d, 2H,
AB system J ¼ 9 Hz), 6.73 (d, 2H, AB system J ¼ 9 Hz), 7.18e7.87
(m, 3H, quinox-H); 7.52,10.69 (bs,1H, NHeD2O exchangeable); Anal.
Calc. for CHN: C, (61.01); H, (5.12); N, (15.81). Found: C, (60.76); H,
Suspension cultures: cell suspensions of EAC (2.5 ꢃ106 per ml)
were prepared in sterile growth medium RPMI-164 (Sigma-
eAldrich, Germany), supplemented with 10% V/V fetal bovine
serum (FBS) (SigmaeAldrich, Germany) and penicillin /strepto-
(5.06); N, (15.93). 1HNMR 9e :
d 2.42 (s, 3H, CH3); 3.61(s, 3H, aryl-
OCH3), 3.84 (s, 3H, quinox.-OCH3), 6.51 (d, 2H, AB system J ¼ 9 Hz),
6.54 (d, 2H, AB system J ¼ 9 Hz), 6.78 (bs, 2H, 2NHeD2O exchange-
able); 6.78 (d, 1H, H6, J ¼ 9.3 Hz),) ; 7.92 (s, 1H, H8); 8.08 (d, 1H, H5,
J ¼ 9.3 Hz),). Anal. Calc. for CHN: C, (58.38); H, (4.90); N, (15.13).
Found: C, (58.77); H, (5.25); N, (14.82).
mycin (100 U/100
different concentrations (1e100
m
g/ml). 0.1 ml of compounds solution containing
mg) were added to 0.9 ml of the
prepared culture media. Two sets of sterile tubes for aerobic and
anaerobic assays were prepared. Suspension cultures were intro-
duced into the two sets of sterile tubes. For the hypoxic condition
evacuated tubes were used. These tubes were tightly closed with
rubber caps which are perforated with two needles of (19 Gauss), to
provide gas (Nitrogen gas) inlet and outlet. The tubes were incu-
bated at 37 ꢁC in air or under hypoxia (N2 gas) with pressure of
(5 barrometer). Supplementation of N2 gas continued for 2 h,
(allover the experimental time).
Preparation of tested compounds: drug solutions, with various
dilutions were freshly prepared in pure dimethyl-sulfoxide
(DMSO). Drug solutions (0.1 ml) were added to each tube in the two
sets of aerobic and anaerobic tubes corresponding to (100, 50, 25,
4.1.7. 2-Alkoxycarbonylamino-7-chloro/methoxy-3-methyl-
quinoxalin1,4-dioxides (10aee)
A solution of compounds 8a or 8b (0.01 mol) was refluxed with
different alcohols (60 mL) for 3 h, the separated crystalline prod-
ucts were recrystallized (Table 3). IR (KBr, cmꢂ1) 10aee: 3336e3147
(NH), 1733e1723 (C]O), 1630e1590 (C]N), 1320e1350 (NO). MS:
m/z (%) 10a: 285 (M þ 2, 0.04), 283 Mþ (0.10), 267 (74.0), 251 (10.4),
218 (100), 214 (37.8). Anal. Calc. for CHN: C, (46.58); H, (3.55); N,
(14.82). Found: C, (46.94); H, (3.93); N, (14.40). 1HNMR 10b:
d 1.02
(t, 3H, CH2eCH3, J ¼ 7.5 Hz); 2.44 (s, 3H, CH3); 4.13 (q, 2H, CH2CH3,
10, 5, 1
mg/ml). This condition was applied for at least three times
J ¼ 7.5 Hz); 7.08 (bs, 1H, NHeD2O exchangeable); 7.31 (d, 1H, H6,
for each tested compound. Control group without any treatment

2738
M.M.F. Ismail et al. / European Journal of Medicinal Chemistry 45 (2010) 2733e2738
[2] A. Monge, J.A. Palop, A. Pinol, F.J. Martinez Crespo, S. Narro, M. Gonzalez,
Y. Sainz, A. Lopez de Cerain, J. Heterocycl. Chem. 31 (1994) 1135.
[3] T. Fuchs, G. Chowdhury, C.L. Barnes, K.S. Gates, J. Org. Chem. 66 (2001) 107.
[4] A. Monge, J.A. Palop, A. Lopez de Cerain, V. Senador, F.J. Martinez Crespo,
Y. Sainz, S. Narro, E. Garcia, C. DeMiguel, M. Gonzalez, E. Hamilton, A.J. Barker,
E.D. Clarke, D.T. Greenhow, J. Med. Chem. 38 (1995) 1786.
[5] G. Chowdhury, D. Kotandeniya, J.S. Daniels, C.L. Barnes, K.S. Gates, Chem. Res.
Toxicol. 17 (2004) 1399.
[6] K.M. Amin, M.M.F. Ismail, E. Noaman, D.H. Soliman, Y.A. Ammar, Bioorg. Med.
Chem. 14 (2006) 6917.
[7] M.J. Haddadin, C.H. Issidorides., Heterocycles (Rev.) 4 (1976) 767.
[8] M.M. F Ismail, S.M. Nofal, M.K. Ibrahim, H.S.A. El-Zahaby, Y.A. Ammar, Afinidad
63 (521) (2006) 57.
[9] D. Walter, H. Herbert, S. Elmar, Ger. Offen. 2 (002) (29 Jul. 1971) 712 (Cl. C 07d)
Appl. 22 Jan.1970; 16 pp., C.A.1971, 75, 98590 pp.
[10] P. Skehan, R. Storeng, et al., J. Natl. Cancer Inst. 82 (1990) 1107.
[11] C. Sanmartin, M. Echeverria, B. Mendivil, L. Cordeu, E. Cubedo, J. Garcia-
Foncillas, M. Font, J.A. Palop, Bioorg. Med. Chem. 13 (6) (2005) 2031.
[12] J.Y. Lin, M.J. Hou, Y.C. Chen, Toxicology 16 (1978) 120.
[13] J.P. Dirlam, J.W. McFarland, J. Org. Chem. 42 (8) (1977) 360.
[14] E. Hatzigrigoriou, M.V. Papadopoulou, D. Shields, W.D. Bloomer, Oncol. Res. 5
(1) (1993) 29.
(positive control) and group treated with 0.1 ml. of DMSO (negative
control) were performed parallel to compounds tested in each
experiment.
Determination of total cell counts and viable cell numbers: after 2 h
of cells incubation with different compounds, the cells were centri-
fuged. Cell numbers were determined with a haemocytometer
(Sigma catalogue) after staining with Trypan blue (Trypan blue
exclusion test) [19]. Trypan blue is one of several stains recom-
mended for use in dye exclusion procedures for viable cell counting.
This method is based on the principle that live cells do not take up
certain dyes, whereas dead cells do. The percent of control cell
survival for the treated suspension cultures were calculated,
% cell viability ¼ total viable cells (unstained)/total cells ꢃ 100.
Supplementary data
[15] M.P. Hay, F.B. Pruijn, S.A. Gamage, H.D. Liyanage, M.S. Kovacs, A.V. Patterson,
W.R. Wilson, J.M. Brow, W.A. Denny, J. Med. Chem. 47 (2) (2004) 475.
[16] H.N. Borah, P. Devi, J.N. Bauah, Tetrahedron 40 (1984) 1617.
[17] P. Devi, J.S. Sandhu, G. Thyagarajan, J. Chem. Soc. Chem. Comm. (1970) 710.
[18] M.P. Lo Russo, R. Parchment, L. Demchik, J. Knight, L. Polin, J. Dzubow,
C. Behrens, B. Harrison, G. Trainor, T.H. Corbett, Invest. New Drugs 16 (4)
(1998) 287.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2010.02.052.
References
[19] D.J. Brusick, Cytogenic Assays. Human Press Inc., Clifton, NJ, 1984, 265.
[1] B. Zarranz, A. Jaso, I. Aldana, A. Monge, Bioorg. Med. Chem. 12 (2004) 3711.