New quinoxaline 1,4-di-N-oxides. Part 1: Hypoxia-selective cytotoxins and anticancer agents derived from quinoxaline 1,4-di-N-oxides

Article abstract of DOI:10.1016/j.bmc.2006.06.038

Hypoxic cells which are common feature of solid tumors are resistant to both anticancer drugs and radiation therapy. Thus, the identification of drugs with the selective toxicity toward hypoxic cells is an important target in anticancer chemotherapy. Tira

Full text of DOI:10.1016/j.bmc.2006.06.038

Bioorganic & Medicinal Chemistry 14 (2006) 6917–6923
New quinoxaline 1,4-di-N-oxides. Part 1: Hypoxia-selective
cytotoxins and anticancer agents derived
from quinoxaline 1,4-di-N-oxides
Kamelia M. Amin,a Magda M. F. Ismail,b,* Eman Noaman,c
Dalia H. Solimanb and Yousry A. Ammard
aPharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
bPharmaceutical Chemistry Department, Faculty of Pharmacy (Girls), Al-Azhar University, Nasr City, Cairo, Egypt
cRadiation Biology Department, Natural Center for Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt
dChemistry Department, Faculty of Science, Al-Azhar University, Nasr City, Cairo, Egypt
Received 29 April 2006; revised 15 June 2006; accepted 19 June 2006
Available online 14 July 2006
Abstract—Hypoxic cells which are common feature of solid tumors are resistant to both anticancer drugs and radiation therapy.
Thus, the identification of drugs with the selective toxicity toward hypoxic cells is an important target in anticancer chemotherapy.
Tirapazamine has been shown to be an efficient and selective cytotoxin after bioreductive activation in hypoxic cells which is thought
to be due to the presence of the 1,4-di-N-oxide. A new series of quinoxaline 1,4-di-N-oxides and fused quinoxaline di-N-oxides were
synthesized and evaluated for hypoxic–cytotoxic activity on EAC cell line. Compound 10a was the most potent cytotoxin IC50
0.9 lg/mL, potency 75 lg/mL, and was approximately 15 times more selective cytotoxin (HCR > 111) than 3-aminoquinoxaline-
2-carbonitrile which has been used as a standard (HCR > 7.5). Compounds 4 and 3a,b were more selective than the standard. In
addition, antitumor activity against Hepg2 (liver) and U251 (brain) human cell lines was evaluated, compounds 9c and 8a were
the most active against Hepg2 with IC50 values 1.9 and 2.9 lg/mL, respectively, however, all the tested compounds were nontoxic
against U251 cell line.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
3-aminoquinoxaline-2-carbonitrile 1,4-dioxide, its elec-
trochemical measurements indicating that, it is more
susceptible to reductive activation than tirapazamine6
and causes redox-activated DNA damage analogous to
it.7 Herein, we report the preparation and cytotoxicities
of several new quinoxaline 1,4-dioxides and fused quin-
oxalines, with the objective of determining the influence
of different substituents in positions 2 and 3 of the qui-
noxaline ring on the biological activity.
Quinoxaline 1,4-di-N-oxide derivatives seem to have
very interesting anticancer activity.1 Hypoxic cells in sol-
id tumors are an important target for cancer chemother-
apy.2 It has been proposed that these hypoxic cells play
a negative role in the success of some antitumor thera-
pies because of their resistance to radiotherapy and con-
ventional chemotherapeutic agents.3 Lin et al.4
introduced the concept of bioreductive alkylation, that
is the hypoxic cells in solid tumors could transform some
drugs into cytotoxic species capable of alkylating DNA.
Tirapazamine5 is the first drug to be introduced into the
clinic purely as bioreductive cytotoxic agent. In the last
few years, extensive studies have been carried out on
2. Results and discussion
2.1. Chemistry
Scheme 1 shows the synthetic pathways to prepare the
target compounds 3–7. The key substrates 3-methylqui-
noxaline 1,4-dioxides 2a–c1 were obtained by the
well-known Biuret reaction8 between 5-chloro/meth-
oxybenzo[c][1,2,5]oxadiazol 1-oxides (benzofuroxanes,
1a,b)9 and the appropriate b–diketone, acetyl acetone
Keywords: Quinoxaline 1,4-di-N-oxide; Fused quinoxaline di-N-oxide;
Antitumor activity.
*
Corresponding author. Tel.: +2022713531; fax: +2024052968;
e-mail: magda_f_ismail@yahoo.com
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.038

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K. M. Amin et al. / Bioorg. Med. Chem. 14 (2006) 6917–6923
Scheme 1.
or benzoyl acetone. In principle, isomeric product mix-
tures can be formed from unsymmetrical 5(6)-substitut-
ed benzofuroxanes10 although in practice, the workup
and crystallization afford only one isomer probably the
7-isomer,11 while the other one is discarded.
derivatives 8a–f as well as 3-amino-2-carboxamide
derivatives 9a–g.
3-Arylquinoxaline 1,4-dioxides were obtained through
reaction of benzofuroxanes with enamines derived from
morpholine.15 The instability of the prepared enamines,
which in fact resulted in very low yields of the products,
was a limiting factor to their use. In this work the bub-
bling of ammonia gas in a warm alcoholic solution of
benzofuroxane 1a and substituted acetophenones for
3 h followed by cooling, surprisingly, furnished yellow
Reaction of 2-benzoyl-3-methylquinoxaline 1,4-dioxides
2a,c with dimethylformamide–dimethylacetal (DMF–
DMA) in xylene gave the enamine derivatives 3a,b. On
the other hand, when the 2-acetyl-3-methyl-7-methoxy-
quinoxaline 1,4-dioxide 2b was reacted with the same
reagent in xylene,12 the enaminone derivative 4 was ob-
tained in good yield (Table 1). Compound 2b was em-
ployed as key intermediate in the synthesis of the two
chalcone derivatives 5a,b. Thus, when equimolar
amounts of 2b and 4-substituted benzaldehyde were
reacted in the presence of 10% sodium hydroxide, the
chalcones were obtained as yellow crystalline products.
The chalcone 5a was then used as a precursor for the
preparation of pyrazole 6 and thiopyrimidine 7 deriva-
tives through its cyclocondensation with hydrazine hy-
drate13 and thiourea, respectively.
crystalline 6-chloro-2-substituted-phenylquinoxaline
1,4-dioxides 10a,b in high yields (Table 1).
Moreover, reaction of 1a,b with diketone, for example,
indandione or dimidone in a warm ethanolic solution
for 4 h in presence of ammonia gas, provided high yields
of the indeno[1,2-b]quinoxalines 11a,b and phenazines
12a,b, respectively. All the prepared compounds were
proved in light of their microanalysis and spectral data
1
including IR, H NMR and mass spectrum.
2.2. Antitumor screening
Scheme 2 shows the synthesis of compounds 8a–f and
12a,b. Treatment of benzofuroxanes 1a,b with prepared
intermediates14 such as acetoacetanilides, benzoylace-
tanilides or cyanoacetanilides in ethanolic potassium
carbonate produced 3-methyl/phenyl-2-carboxamide
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. Potential cytotoxic-

K. M. Amin et al. / Bioorg. Med. Chem. 14 (2006) 6917–6923
Table 1. Physical properties and molecular formula of the synthesized compounds
6919
Compound
R1
R2
Ar
X
Mp (ꢁC)
Yield (%)
Formula
C19H16ClN3O3
3a
3b
Cl
—
—
—
—
—
—
—
Cl
CHO
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Cl
CH3
—
—
—
—
223–224
210–212
239–240
227–228
215–216
169–170
152–154
248–250
220–222
232–234
240–241
258–260
280–281
250–252
220–222
243–245
217–219
187–190
217–218
210–212
169–170
180–182
275–276
244–245
300–301
290–292
83
65
89
98
97
56
60
85
90
87
67
80
89
60
88
80
77
83
79
77
60
78
80
62
78
58
OCH3
—
—
C20H19N3O4
C15H17N3O4
4
—
—
—
—
5ab
5bb
6c
C19H15ClN2O4
C20H16N2O5
—
—
—
—
—
—
C19H15ClN4O3
C20H15ClN4O3S
C18H17N3O5
7
8a
—
—
—
OCH3
OCH3
OCH3
OCH3
OCH3
Cl
CH3
CH3
CH3
Ph
C6H4–4-OCH3
C6H3–2,4-(CH3)2
C6H3–2,6-(CH3)2
C6H4–4-F
C6H3–2,6-(CH3)2
C6H3–2,6-(CH3)2
C6H4–4-Cl
C6H4–4-F
C6H4–4-OCH3
C6H4–3-OC2H5
C6H4–4-OC2H5
–CH2–C6H5
C6H4–4-F
—
8b
C19H19N3O4
C19H19N3O4
8c
8da
8ea
8fa
9a
C22H16FN3O4
C24H21N3O4
Ph
Ph
C23H18ClN3O3
C16H13ClN4O4
C16H13FN4O4
C17H16N4O5
OCH3
OCH3
OCH3
OCH3
OCH3
Cl
NH2
NH2
NH2
NH2
NH2
NH2
NH2
—
9b
9c
9d
9e
C18H18N4O5
C18H18N4O5
9f
9g
C16H13ClN4O3
C15H10ClFN4O3
C14H8Cl2N2O2
C15H11ClN2O2
C15H7ClN2O3
C16H10N2O4
Cl
—
10a
10b
11a
11b
12a
12b
—
Cl
—
—
—
—
OCH3
Cl
OCH3
—
—
—
—
C14H13ClN2O3
C15H16N2O4
—
—
All compounds are recrystallized from ethanol except.
a The solvent is DMF/EtOH.
b The solvent is MeOH.
c The solvent is dioxane.
Scheme 2.

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K. M. Amin et al. / Bioorg. Med. Chem. 14 (2006) 6917–6923
Table 3. Data of IC50, potency, and hypoxic selectivity of the tested
quinoxaline derivatives
ity of the selected compounds 4, 6, 8a,d, 9b,c,f, 10a, 11a,
and 12b was tested using the method of Skehan and
Storeng.16 Two human cell lines: liver carcinoma
(Hepg2) and brain tumor (U251) were incubated with
five concentrations 0–10 lg/mL16,17 for each compound.
The growth inhibitory action of the selected compounds
is summarized in Table 2. The results showed that
compounds 9c and 8a were the most cytotoxic of all
the tested compounds with IC50 1.91 and 2.91 lg/mL,
respectively. Compound 9b and the fused quinoxaline
11a showed moderate activity with IC50 6.97 and
4.55 lg/mL, respectively. The rest of the tested com-
pounds were nontoxic against the liver carcinoma
(Hepg2), all the tested compounds were also nontoxic
against the brain tumor. In open chain structures as
the carboxamide derivatives substituted with a methoxyl
group in the 7-position and different substituents in the
3-position as 9c and 8a,d, it was observed that as the
lipophilicity of the substituent in position 3 increased
the toxicity decreased in the order 9c > 8a > 8d with
IC50 values 1.91, 2.91, and >10 lg/mL, respectively.
Activity of 9c over 9b (IC50 = 1.91 and 6.97 lg/mL) sug-
gests that an electron-donating group in position 4 of
the 2-aryl carboxamide moiety imparts higher cytotoxic-
ity than an electron-withdrawing group, 9c showed cyto-
toxicity approximately threefold that of 9b. The
combination of a lipophilic group in the 3-position
and an electron-withdrawing group in the 4-phenylcarb-
oxamide seems to abolish the activity completely and
this is demonstrated in the 2-(4-fluoro-phenyl)-carbox-
amide-3-phenylquinoxaline 1,4-dioxide 8d. In order to
assess the activity of quinoxaline 1,4-dioxide bearing a
heterocyclic ring at position 2, 2-pyrazolyl quinoxaline
6 was tested and the result revealed that it was inactive.
On the other hand, the tricyclic derivative 12b was also
inactive and this shows that rigidification is not favor-
able for the antitumor activity. However, the tetracyclic
11a was active with an IC50 4.55 lg/mL.
Compound
IC50 hypoxiaa
Potencyb
HCRc
(lg/mL)
(lg/mL)
Reference (B)
13.5
11.5
8.5
10
>100
100
90
>7.5
>8.7
3a
3b
4
9.41
100
>10
’1
6
>100
90
50
>100
>100d
>100
100
7
>1.1
>2
4.34
8e
8f
25
20
9b
9g
10a
10b
11a
>100
>100
75
3.25
>1.6
60
0.9
20
>111
>5
>100
>100
30
>3.3
* Other compounds could not be accurately determined because at
dose higher than 25 lg/mL, they precipitated in the culture medium.
a Concentration in (lg/mL) causing 50% mortality in cell numbers.
b Concentration that gives 1% cell survival in hypoxia.
c Hypoxic cytotoxicity ratio (dose in air/dose in hypoxia giving the
same cell survival, IC50).
d Potency of tested compound was more than 100 lg/mL.
Further in vitro study was carried out both in oxic and
hypoxic conditions, for the active compounds, following
the reported method.6,11,19,20 Three response parameters
were calculated, IC50, potency, and hypoxic cytotoxicity
ratio (HCR) (Table 3). 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 cell survival
(calculated at IC50).21,22 Compound 10a was the most
potent cytotoxin, IC50 0.9 lg/mL, potency 75 lg/mL,
and was approximately 15 times more hypoxia-selective
cytotoxin (HCR > 111) than 3-aminoquinoxaline-2-car-
bonitrile (B) which has been used as a standard
(HCR > 7.5). Moreover, it is approximately 22 times
more selective than its counterpart 10b (HCR > 5)
which may be due to the electron-withdrawing effect of
4-Cl group on the 3-phenyl moiety of 10a. Compounds
2.2.2. Activities against Ehrlich ascites carcinoma (EAC)
cell line: Hypoxia-selective cytotoxicity screening. These
biological studies were performed at the National Cen-
ter for Radiation Research and Technology (NCRRT),
Cairo, Egypt. A preliminary screening on compounds
3a,b, 4, 6, 7, 8a,d–f, 9b,c,f,g, 10a,b, 11a, and 12b was per-
formed against EAC cells.18 The tumor cell suspensions
were incubated with different concentrations in micro-
grams per mililiter16,17 for each compound in air (oxia).
Table 2. Growth inhibitory action (IC50) of the tested compounds
against Hepg2 and U251 human cell lines
Compound
Hepg2
U251
4
6
>10
>10
2.91
>10
6.97
1.91
>10
>10
4..55
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
8a
8d
9b
9c
9f
10a
11a
12b
Figure 1.

K. M. Amin et al. / Bioorg. Med. Chem. 14 (2006) 6917–6923
6921
of the solvent, the product was crystallized (Table 1).
IR (KBr, cmꢀ1): 1654 (CO) 1638–1595 (C@N), 1320–
1
1350 (NO). H NMR: d 2.3 (s, 3H, CH3-quinox.); 2.8,
3.0 (2s, 6H, N (CH3)2); 3.9 (s, 3H, OCH3); 5.2 (d, 1H,
NCH@CH); 7.4 (d, 2H, COCH@CH + H6); 7.7 (s, 1H,
H8); 8.3 (d, 1H, H5).
3.1.3.
7-Methoxy-3-methyl-2-[3(4-substituted-phenyl)-
propenoyl]-quinoxaline 1,4-di-N-oxides (5a,b). A mixture
of 2b (2.48 g, 0.01 mol) and 4-chlorobenzaldehyde or
terphthaldehyde (0.01 mol) in methanol (60 mL) was
stirred, while a few drops of sodium hydroxide (10%
in methanol) was added. Bright yellow crystals separat-
ed within 15 min. The mixture was stirred for an addi-
tional 30 min, filtered, washed with water, and
crystallized (Table 1). IR (KBr, cmꢀ1) 5a: weak band
at 1678 (CO–CH@CH–), 1620 (C@N), 1320–1350
Figure 2.
1
(NO). H NMR 5a: d 2.3 (s, 3H, CH3-quinox.); 4.0, (s,
3H, OCH3); 5.4 (d, 1H, COCH@CH); 7.3 (d, 1H, CO–
CH@CH); 7.4, 7.7 (2d, 4H, Ar-H, AB system
J = 8.4); 7.6 (d, 1H, H6); 7.8 (s, 1H, H8); 8.4 (d, 1H,
H5). IR (KBr, cmꢀ1) 5b: 1770 (HCO); 1681 (CO). MS
(m/z, %) 5b: 364 (M+, 4.0); 332 [M+-32 (2O), 23.5]; 173
(100).
3a,b and 4 were more selective (HCR > 8.7, 9.4, and
>10, respectively) than the standard (B). Poor selectivity
observed by compounds 6 and 7 is thought to be due to
the heterocyclic moiety (pyrazole/pyrimidine) at 2-posi-
tion. Dose–response curves for 10a and 4 are presented
in Figures 1 and 2.
3.1.4. 3-[5-(4-Chloro-phenyl)-1H-pyrazol-3-yl]-6-meth-
oxy-2-methyl-quinoxaline-1,4- di-N-oxide (6). A solution
of hydrazine hydrate 98% (0.01 mol) in ethanol (10 mL)
was added to a boiling solution of 5a (3.79 g, 0.01 mol)
in ethanol (30 mL), the mixture was refluxed for 7 h.
After the solvent removal, the product was crystallized
(Table 1). IR (KBr, cmꢀ1): 3321 (NH), 1616 (C@N),
1320–1350 (NO). MS (m/z, %): 382 (M+, 9.2); 359
(6.1), 357 (18.5), 148 (100).
3. Experimental
3.1. Synthesis
Melting points were determined on electrothermal 9100
digital melting point apparatus and are uncorrected. H
1
NMR spectra were recorded in DMSO-d6 on Varian
Gemini 200 (200 MHz) using tetramethylsilane (TMS)
as an internal standard (chemical shift in d parts per mil-
lion). The IR spectra were performed on a Perkin-Elmer
1600 FTIR in KBr pellets. Elemental microanalysis (C,
H, and N) was performed on a Perkin-Elmer 2400 ana-
lyzer 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. Chemicals were purchased from E. Merck
(Darmstadt, Germany), Sigma–Aldrich (Germany); sol-
vents used were of the highest grade. Compound B was
prepared according to the reported methods.6 MF:
C9H6N4O2, mp 240–242 ꢁC; yield: 93%.
3.1.5. 2-[6-(4-Chloro-phenyl)-2-thioxo-1H-pyrimidin-2yl]-
7-methoxy-3-methyl-quinoxaline 1,4-di-N-oxide (7). A
mixture of (3.79 g, 0.01 mol), thiourea (0.67 g,
0.01 mol), and sodium ethoxide in ethanol (50 mL)
was refluxed for 10 h. The product was washed with
water and crystallized (Table 1). IR (KBr, cmꢀ1): 3445
(NH), 1598 (C@N), 1320–1350 (NO). MS (m/z, %):
426 (M+, 29.1); 428 (M+2, 14.2); 355 (100).
3.1.6. 7-Substituted-3-methyl/phenyl-2-[N-(substituted-
phenyl)-amino-carbonyl]-quinoxaline 1,4-di-N-oxides (8a–f).
A warm solution of 5(6)-substituted benzofuroxane and
acetoacetanilides or benzoylacetanilides (0.01 mol) in etha-
nol (50 mL) was stirred at room temperature in the pres-
ence of catalytic amount of potassium carbonate. The
yellow products precipitated in a period of 2 h and were
crystallized (Table 1). IR (KBr, cmꢀ1) 8a–f: 3242–3261
(NH), 1656–1665 (CO), 1630–1590 (C@N), 1320–1350
(NO). 1H NMR 8a: d 2.4 (s, 3H, CH3); 3.8 (s, 3H,
OCH3); 4.0 (s, 3H, OCH3); 6.9, 7.5 (2d, 4H, Ar-H, AB sys-
tem J = 9 Hz); 7.6 (d, 1H, H6); 7.8 (s, 1H, H8); 8.4 (d, 1H,
H5); 11.0 (s, 1H, NH–D2O exchangeable). MS (m/z, %) 8a:
3.1.1. 2-Benzoyl-3-(2-dimethylamino-vinyl)-7-substituted-
quinoxaline 1,4-di-N-oxides (3a,b). A mixture of 2a or 2c
(0.01 mol) and DMF–DMA (0.015 mol) in xylene
(30 mL) was refluxed for 6 h. The solvent was removed
and the red precipitate was crystallized (Table 1). IR
(KBr, cmꢀ1) 3a,b 1674–1678 (CO), 1630–1590 (C@N),
1320–1350 (NO). 1H NMR 3a: d 2.49 (s, 6H, N
(CH3)2), 4.3 (d, 1H, NCH@CH); 7.5–7.9 (m, 5H, Ph-
H); 8.0 (d, 1H, H6); 8.2 (s, 1H, H8); 8.3 (d, 1H, H5);
9.4 (d, 1H, CH@CH).
1
355 (M+, 65.4); 338 [M+ꢀ17(OH), 100], 322 (55.1). H
3.1.2. 7-Methoxy-3-methyl-2-(3-dimethylamino-2-prope-
noyl)-quinoxaline 1,4-dioxide (4). A mixture of 2b
(2.48 g, 0.01 mol) and DMF–DMA (1.78 g, 0.015 mol)
in xylene (30 mL) was refluxed for 8 h. After removal
NMR 8b: d 2.2, 2.3 (2s, 6H, 2CH3); 2.4, (s, 3H, CH3-qui-
nox.); 4.0 (s, 3H, OCH3); 6.9–7.1, 7.5 (2m, 3H, Ar-H); 7.5
(d, 1H, H6); 7.8 (s, 1H, H8); 8.4 (d, 1H, H5); 10.2 (s, 1H,
NH–D2O exchangeable). 1H NMR 8c: d 2.34 (s, 6H,

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K. M. Amin et al. / Bioorg. Med. Chem. 14 (2006) 6917–6923
2CH3); 2.4 (s, 3H, CH3-quinox.); 3.9 (s, 3H, OCH3); 7.0–
7.4 (m, 3H, Ar-H); 7.45 (d, 1H, H6); 7.74 (s, 1H, H8); 8.3
(d, 1H, H5); 10.3 (s, 1H, NH–D2O exchangeable). MS
(m/z, %) 8c: 353 (M+, 6.2), 337 [M+ꢀ16 (O), 12.5], 320
[M+ꢀ33 (2O+H), 100].1H NMR 8d: d 3.8 (s, 3H,
OCH3); 7.05 (d, 2H, AB system, J = 9 Hz); 7.55 (d, 1H,
H6); 7.7 (d, 2H, AB system, J = 9 Hz); 7.83 (s, 1H, H8);
8.3 (d, 1H, H5); 10.2 (s, 1H, NH). MS (m/z, %) 8d: 405
CH3); 7.3, 7.9–8.5 (2m, 6H, 4Ar-H + H7 + H8); 8.9 (s,
1H, H5); 9.2 (s, 1H, H3).
3.1.9. 8-Substituted-11-oxo-indeno [1,2-b] quinoxaline-
5,10-dioxides (11a,b). A mixture of 5(6)-substituted ben-
zofuroxane 1a,b (0.01 mol) and indanedione (0.01 mol)
in ethanol (70 mL) was stirred and heated at 60 ꢁC,
while ammonia gas was bubbled into the solution for
4 h after cooling and evaporating the solvent in a rotary
evaporator the products were crystallized (Table 1). IR
(KBr, cmꢀ1) 11a,b: 1712–1715 (CO), 1638–1590 (C@N),
1
(M+, 19.3); 373 [M+ꢀ32 (2O), 28.4]; 109 (100). H NMR
8e: d 1.8 (s, 6H, 2CH3); 4.0 (s, 3H, OCH3); 7.0, 7.5 (2m,
8H, Ar-H); 7.6 (d, 1H, H6); 7.9 (s, 1H, H8); 8.4 (d, 1H,
H5); 10.1 (s, 1H, NH). MS (m/z, %) 8f: 419 (M+, 9.57);
403 [M+ꢀ16 (O), 23.2]; 386 [M+ꢀ33 (2O+H), 100].
1
1320–1350 (NO). H NMR 11a: 7.25–8.3 (m, 6H, Ar-
H), 8.85 (s, 1H, H9). MS (m/z, %) 11a: 298 (M+, 75.3);
300 (M+2, 30.4); 282 (M+ꢀ16 (O), 100). 1H NMR
11b: 3.75 (s, 3H, OCH3), 7.16–8.2 (m, 6H, Ar-H), 8.6
(s, 1H, H9).
3.1.7. 3-Amino-7-substituted-2-[N-(substituted-phenyl)-
amino-carbonyl]-quinoxaline 1,4-di-N-oxides (9a–e,g)
and 3-amino-7-chloro-2-benzyl-amino-carbonyl quinoxa-
line 1,4-di-N-oxides (9f). A warm solution of 5(6)-substi-
tuted benzofuroxane 1a,b (0.01 mol) and the
appropriate cyanoacetanilide (0.01 mol) in ethanol
(50 mL) was stirred at room temperature in the presence
of catalytic amount of potassium carbonate. The yellow
products precipitated in a period of 2 h and were crystal-
lized (Table 1). IR (KBr, cmꢀ1) 9a–g: 3400–3251 (NH2,
NH), 1661–1655 (CO), 1617–1593 (C@N), 1320–1350
(NO). MS (m/z, %) 9a: 360 (M+, 8.48); 328 [M+ꢀ32
(2O), 1.67], 127 (100). 1H NMR 9b: d 3.9 (s, 3H,
OCH3); 7.1–8.7 (m, 7H, Ar-H), 10.1, 12.4 (2s, 3H,
NH2, NH–D2O exchangeable). MS (m/z, %) 9b: 344
3.1.10. 3,3-Dimethyl-1-oxo-8-substituted-3,4-dihydro-2H-
phenazine-5,10-di-N-oxides (12a,b). The same procedure
as 11a,b. IR (KBr, cmꢀ1) 12a,b: 1650 (CO), 1611
1
(C@N), 1320–1350 (NO). H NMR 12b: d 1.5 (s, 6H,
2CH3); 1.7 (s, 2H, CH2), 3.6 (s, 3H, OCH3); 5.4 (s, 2H,
CH2–CO); 7.8 (d, 1H, H6); 8.4 (s, 1H, H8); 8.6 (d, 1H, H5).
3.2. Antitumor screening
3.2.1. Activity against Hepg2 and U251. Cells were plat-
ed 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 of
the compounds under test (0.1, 2.5, 5, and 10 lg/mL)
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 sulforhoda-
mine B stain. The color intensity was measured in an
ELISA reader.16
1
(M+, 6.6), 312 [M+ꢀ32 (2O), 100]. H NMR 9c: d 3.70
(s, 3H, OCH3); 3.77 (s, 3H, OCH3); 6.8, 7.3 (2d, 4H,
AB system, J = 9 Hz); 7.4 (d, 1H, H6); 7.8 (s, 1H, H8);
8.4 (d, 1H, H5); 8.8 (br s, 3H, NH2, NH–D2O exchange-
able). MS (m/z, %) 9c: 356 (M+, 7.5), 340 [M+ꢀ16 (O),
1
11.5], 124 (100). H NMR 9d: d 1.2 (t, 3H, CH2CH3);
3.9 (s, 3H, OCH3), 4.0 (q, 2H, CH2CH3), 6.6–7.2 (m,
4H, Ar-H), 6.7 (d, 1H, H6), 7.2 (s, 1H, H8), 8.00 (d,
1H, H5), 10.2, 12.1 (2s, 3H, NH2, NH–D2O exchange-
1
able). H NMR 9e: d 1.2 (t, 3H, CH2CH3); 3.8 (s, 1H,
OCH3); 3.9 (q, 2H, CH2CH3); 6.8, 7.2 (2d, 4H, Ar-H,
AB system J = 9 Hz); 7.8 (d, 1H, H6); 8.2 (d, 1H, H5);
8.4 (s, 1H, H8) 10.2, 12.4 (br s, 3H, NH2, NH–D2O
exchangeable); 1H NMR 9f: d 4.6, 5.4 (2d, 2H,
N–CH2); 7.2–7.4 (m, 6H, 5Ph-H + H6); 7.7 (s, 1H,
H8); 7.8 (d, 1H, H5); 8.6 (t, 1H, NH–D2O exchangeable),
9.3 (br s, 2H, NH2–D2O exchangeable). MS (m/z, %) 9f:
344 (M+, 30.3), 312 [M+ꢀ32 (2O), 5.4], 92 (100). MS (m/
z, %) 9g: 348 (M+, 34.2); 178 (65.7), 111 (100).
3.2.2. Activity against EAC experimental cell line. Animals,
chemicals, and facilities: Female Swiss albino mice weigh-
ing25–30 g obtainedfrom (the holdingcompany ofbiolog-
ical 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 lab-
oratory food (Milad Co., Cairo, Egypt) and water ad libi-
tum. All chemicals and reagentswere from Sigma–Aldrich,
Germany and Merck, Germany.
3.1.8. 6-Chloro-2-(4-chloro-phenyl)-quinoxaline 1,4-di-N-
oxide (10a) and 6-chloro-2-p-tolyl-quinoxaline-1,4-di-N-
oxide (10b). A mixture of 5(6)-chloro-benzofuroxane 1a
(1.79 g, 0.01 mol) and 4-methyl or chloro acetophenone
(0.01 mol) in ethanol (50 mL) was stirred at 60 ꢁC, while
ammonia gas was bubbled into the solution for 3 h stir-
ring continued for another 4 h, after which the reaction
mixture was cooled overnight, after solvent removal the
3.2.3. Aerobic and hypoxic cytotoxicity
3.2.3.1. 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 · 106/mL) was prepared.
3.2.3.2. Suspension cultures. Cell suspensions were
prepared in sterile growth medium RPMI-164 (Sigma–
Aldrich, Germany), supplemented with 10% v/v fetal
bovine serum (FBS) (Sigma–Aldrich, Germany) and
penicillin–streptomycin 100 U/100 lg/mL. To 0.9 mL
of the prepared suspension culture different concentra-
tions (1–100 lg/mL) of the prepared compounds were
added to the media (0.1 mL). Suspension cultures were
products were crystallized (Table 1). IR (KBr, cmꢀ1
)
10a,b: 3079 (CH-aromatic) 2854 (CH-aliphatic); 1608
1
(C@N), 1320–1350 (NO). H NMR 10a: 7.55–8.6 (m,
6H, 4 Ar-H + H7 + H8), 8.94 (s, 1H, H5), 9.34 (s, 1H,
H3). MS (m/z, %) 10a: 308 (M+ 2, 51.5); 306 (M+,
1
78.6); 290 (Mꢀ16 (O), 100). H NMR 10b: 2.4 (s, 3H,

K. M. Amin et al. / Bioorg. Med. Chem. 14 (2006) 6917–6923
6923
introduced into two sets of sterile tubes for aerobic and
anaerobic assays. For the hypoxic condition evacuated
tubes were used. These vacated tubes were tightly closed
with rubber caps which were perforated with two nee-
dles of (19 G), to provide gas (N2 gas) inlet and outlet.
The tubes were incubated at 37 ꢁC under oxic (O2 air)
or hypoxic (N2 gas) with pressure of (5 barometer). Sup-
plementation of N2 gas continued for 2 h.
found, in the online version, at doi:10.1016/
j.bmc.2006.06.038.
References and notes
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pure (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, 10, 5, and 1 lg/mL). This
condition was applied for at least three times for each
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pan blue (Trypan blue exclusion test).23 Trypan blue is one
of several stains recommendedfor use in dyeexclusion pro-
cedures 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 was calculated.
% cell viability ¼ total viable cells ðunstainedÞ=total cells
ꢁ100:
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4. Conclusion
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In our attempt to synthesize and evaluate the anticancer
activity and hypoxia-selectivity of quinoxaline 1,4-diox-
ide derivatives, we were overwhelmed by the results of
10a. It was 15 times more selective than the standard
suggesting that it may act by the same mechanism. Com-
pounds 4 and 3a,b elicited more selectivity than the stan-
dard. Moreover, compounds 9c and 8a were the most
potent antitumor agents against Hepg2 (liver) human
cell line. Our future studies would be concerned with
the in vivo examination of these compounds.
21. Hatzigrigoriou, E.; Papadopoulou, M. V.; Shields, D.;
Bloomer, W. D. Oncol. Res. 1993, 5, 29.
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Kovacs, M. S.; Patterson, A. V.; Wilson, W. R.; Brow, J.
M.; Denny, W. A. J. Med. Chem. 2004, 47, 475.
23. Brusick, D. J.. Cytogenic Assays; Human Press Inc.:
Clifton, NJ, 1984, p 256.
Supplementary data
Supplementary data associated with this article includ-
ing calculation details and predicted activities can be