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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,