Synthesis and Biological Evaluation of New Quinoxaline Derivatives as Antioxidant and Anti-Inflammatory Agents

Article abstract of DOI:10.1111/j.1747-0285.2011.01076.x

We report the synthesis, anti-inflammatory, and antioxidant activities of novel quinoxaline and quinoxaline 1,4-di-N-oxide derivatives. Microwave-assisted methods have been used to optimize reaction times and to improve yields. The tested compounds presented important scavenging activities and promising in vitro inhibition of soybean lipoxygenase (LOX). Two of the best LOX inhibitors (compounds 7b and 8f) were evaluated as invivo anti-inflammatory agents using the carrageenin-induced edema model. One of them (compound 7b) showed important in vivo anti-inflammatory effect (41%) similar to that of indomethacin (47%) used as the reference drug. Synthesis and Biological Evaluation of New Quinoxaline Derivatives as Antioxidant and Anti-Inflammatory Agents Asuncion Burguete, Eleni Pontiki, Dimitra Hadjipavlou-Litina*, Saioa Ancizu, Raquel Villar, Beatriz Solano, Elsa Moreno, Enrique Torres, Silvia Perez, Ignacio Aldana and Antonio Monge The synthesis, anti-inflammatory and antioxidant activities of new quinoxaline derivatives are reported. The new compounds exhibit important scavenging activities and promising values of in vitro inhibition of soybean LOX, One of them shows strong in vivo anti-inflammatory effect similar to that of indomethacin used as the reference drug.

Full text of DOI:10.1111/j.1747-0285.2011.01076.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01076.x
Chem Biol Drug Des 2011; 77: 255–267
Research Article
Synthesis and Biological Evaluation of New
Quinoxaline Derivatives as Antioxidant and
Anti-Inflammatory Agents
Asuncion Burguete1,2, Eleni Pontiki1,
´
type of pro-inflammatory mediator, that are involved in processes
Dimitra Hadjipavlou-Litina1,*, Saioa
such as fever, asthma (1), or cardiovascular disease (2). Aberrant AA
metabolism is also related to carcinogenesis. In that respect, recent
studies revealed increased LOX expression levels in a wide range of
cancers including pancreatic, bladder, and breast cancer (3–5).
Ancizu2, Raquel Villar2, Beatriz Solano2,
2
2
2
´
Elsa Moreno , Enrique Torres , Silvia Perez ,
Ignacio Aldana2 and Antonio Monge2
1Department of Pharmaceutical Chemistry, School of Pharmacy,
Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
2Unidad de Investigaciꢀn y Desarrollo de Medicamentos, Centro de
Investigaciꢀn en Farmacobiologꢁa Aplicada (CIFA), University of
Navarra, c ⁄ Irunlarrea s ⁄ n, 31080 Pamplona, Spain
*Corresponding author: Dimitra Hadjipavlou-Litina,
hadjipav@pharm.auth.gr
On the other hand, during the inflammation process, phagocytic leuko-
cytes (e.g. neutrophils, monocytes, macrophages, eosinophils) produce
reactive oxygen species (ROS), such as superoxide radical anion,
hydrogen peroxide, and hydroxyl radical. The presence of high levels
of ROS induce and heighten certain pathological conditions such as
carcinogenesis, atherosclerosis, and neurodegenerative diseases (6–
8) and are well known to be involved in the induction and prolonga-
tion of the inflammatory process (9,10). The involvement of ROS in
inflammation is confirmed by a number of commercially available
non-steroidal anti-inflammatory drugs (NSAIDs) (acetaminophen, sali-
cylates, indomethacin (IMA), and nimesulide) that have been shown
to possess radical scavenging properties (11–14).
We report the synthesis, anti-inflammatory, and
antioxidant activities of novel quinoxaline and
quinoxaline 1,4-di-N-oxide derivatives. Microwave-
assisted methods have been used to optimize
reaction times and to improve yields. The tested
compounds presented important scavenging activi-
ties and promising in vitro inhibition of soybean
lipoxygenase (LOX). Two of the best LOX inhibitors
(compounds 7b and 8f) were evaluated as in vivo
anti-inflammatory agents using the carrageenin-
induced edema model. One of them (compound 7b)
showed important in vivo anti-inflammatory effect
(41%) similar to that of indomethacin (47%) used
as the reference drug.
Taking into account the above-mentioned relationship between ROS
and inflammation as well as the importance of inhibition of LOX to
combat inflammatory and carcinogenic processes, the development
of new compounds having both anti-inflammatory and antioxidant
activities and being LOX inhibitors constitutes an interesting
approach in the obtention of new drugs for cancer prevention,
treatment of chronic inflammation and other related pathological
conditions.
Key words: anti-inflammatory, antioxidant, quinoxaline, quinoxaline
1,4-di-N-oxide
Quinoxaline 1,4-di-N-oxide derivatives are a class of compounds
having a great interest in medicinal chemistry as they display a
broad range of biological properties such as antibacterial, antican-
cer, or antiparasitic (15–17). We demonstrated (18) that quinoxaline
1,4-di-N-oxide derivatives also show very interesting antioxidant
and anti-inflammatory properties, some of them displaying an
in vivo anti-inflammatory effect higher than the reference drug,
IMA, and promising in vitro inhibition values of LOX (<1 lM).
Abbreviations: AA, arachidonic acid; AAPH, 2,2¢-azobis(2-amidino-
propane) dihydrochloride; ABTS, 2,2¢-azinobis-(3-ethylbenzothiazoline-6-
sulfonic acid); BFX, benzofuroxane; CPE, carrageenin-induced paw
edema; DMSO, dimethyl sulfoxide; DNA, deoxyribonucleic acid; DPPH,
1,1-diphenyl-2-picrylhydrazyl; IMA, indomethacin; LOX, lipoxygenase;
NADH, nicotinamide adenine dinucleotide; NBT, nitroblue tetrazolium;
NDGA, nordihydroguaeretic acid; NSAIDs, non-steroidal anti-inflamma-
tory drugs; PMS, phenazine methosulfate; ROS, reactive oxygen species;
UV, ultraviolet.
Based on these results and with the aim of obtaining new com-
pounds with improved activities, we now describe the synthesis,
antioxidant, and in vivo anti-inflammatory activity and LOX inhibition
of a wide number of new quinoxaline and quinoxaline 1,4-di-
N-oxide derivatives. Several structural modifications have been
carried out (Figure 1) to determine the structural requirements for
this kind of compounds to act as anti-inflammatory and antioxidant
agents.
Received 8 December 2009, revised 30 September 2010 and accepted
for publication 17 December 2010
Arachidonic acid (AA) metabolism, mediated by the lipoxygenase
(LOX) enzyme family, leads to the generation of leukotrienes, a
255

Burguete et al.
O–
N+
O
H
N
O
O–
N+
N
O
O
R7
R7
O
N+
N+
O
O–
O–
O
Series 1 (1a)
Series 3 (3a, 3b)
PREVIOUS LEADS
O–
N+
O
O
R7
H
N
O
O–
N+
N
O–
N+
O
N+
R4'
OH
R7
R6
O
O
R7
R6
O–
N+
Series 2
R3'
N+
O–
O–
(2a, 2b, 2c)
O
Series 3 (R3'=R4'=OCH3) 3c, 3e, 3f
Series 1
1b, 1e, 1f
Series 10 (R3'=H; R4'=OH)
10a
O–
N+
O
O–
N+
O
H
O
O
O
R7
R6
N
N
O
O
R7
R6
O
O
O
R7
R6
N
N
N
N
R7
R6
O
N+
OH
N+
O–
O–
O
O
Series 2
2e, 2f
Series 4
4a, 4b, 4c, 4d, 4e, 4f
Series 8
8c, 8f
Series 6
6a, 6b, 6c, 6d, 6e, 6f
O–
N+
O
R7
R6
O
NH2
S
O
N+
O
NH2
O–
N
N
O
N
R7
R6
N
N
O
O
N
Series 5
R7
R6
N
O
5a, 5b, 5d, 5e, 5g
O
O
N
Series 7
7a, 7b, 7c, 7d, 7e
Series 9
9a, 9b, 9c, 9d
R6/R7: H/H (a); H/F(b); H/OCH3 (c); H/Cl (d); H/CH3 (e); CH3/CH3 (f); F/F (g)
Figure 1: Chemical structure modifications of the previous lead compounds.
Alugramꢀ SIL G ⁄ UV254 (Layer: 0.2 mm) (Postfach 101352; Mache-
rey-Nagel GmbH & Co. KG, Dꢀren, Germany) was used for Thin
Layer Chromatography and Silica gel 60 (0.040–0.063 mm) for Col-
umn Flash Chromatography (Merck, Darmstadt, Germany).
Experimental Section
Chemistry
Microwave-assisted synthesis was carried out in a Discover S-
Class microwave system apparatus (CEM Corporation). The 1H
NMR spectra were recorded on a Bruker 400 UltrashieldTM (Bru-
ker BioSpin GmbH, Rheinstetten, Germany), using TMS as the
internal standard and with CDCl3 and DMSO-d6 as the solvents;
the chemical shifts are reported in ppm (d), and the coupling
constant (J) values are given in Hertz (Hz). Signal multiplicities
are represented by s (singlet), bs (broad singlet), d (doublet), dd
(double doublet), t (triplet), and m (multiplet). The IR spectra
were performed on Thermo Nicolet FT-IR Nexus Euro (Madison,
WI, USA) using KBr pellets; the frequencies are expressed in
per cm. Elemental microanalyses were obtained on an Elemental
Analyzer LECO CHN-900 (Leco Corporation. ST. Joseph, MI
49085, USA) from vacuum-dried samples. The analytical results
for C, H, and N were within €0.4 of the theoretical values, indi-
cating a purity of >95%.
All reagents and solvents were purchased from commercial sources:
E. Merck (Darmstadt, Germany), Scharlau (F.E.R.O.S.A., Barcelona,
Spain), Panreac Quꢁmica S.A. (Montcada i Reixac, Barcelona, Spain),
Sigma-Aldrich Quꢁmica, S.A., (Alcobendas, Madrid, Spain), Acros
Organics (Janssen Pharmaceuticalaan 3a, 2440 Geel, Belgiꢂ) and
Lancaster (Bischheim, Strasbourg, France).
General method for the microwave-assisted
synthesis of 1,4-dioxy-quinoxaline-2-
carbaldehyde derivatives (compounds IV)
In a microwave reaction flask, 3 mmol of the appropriate compound
III, 4.5 mmol of SeO2, and 20 mL of acetonitrile were mixed. The
reaction was carried out at 200 W for 5 min. The solvent was
evaporated, an extraction with chloroform and water was per-
256
Chem Biol Drug Des 2011; 77: 255–267

Synthesis, Antioxidant and Anti-inflammatory Activities of New Quinoxaline Derivatives
formed, and the residue purified by column chromatography using
toluene:dioxane (3:2). The compounds obtained have been previ-
ously described (20,36).
General method for the microwave-assisted
synthesis of 3-(3-methyl-1,4-dioxy-quinoxalin-2-
yl)-1-(3,4,5-trimethoxy-phenyl)-propenone
derivatives (series 4) and of 3-(1,4-dioxy-
quinoxalin-2-yl)-1-(4-methoxy-phenyl)-
propenone derivatives (series 5)
General method for the microwave-assisted
synthesis of 1-(substituted-phenyl)-2-(triphenyl-
phosphonylidene)-ethanone derivatives
(compounds VI)
Five millimoles of triphenylphosphine, 5 mmol of the corresponding
halide V, and 3 mL of xylene were mixed in a microwave reac-
tion flask. The program was run for 1 min and 30 seconds at
110 W. The residue was dissolved in 10 mL of methanol, and
8 mmol of NaH in water was added. The mixture was extracted
with ethyl ether (3 · 25 mL), the organic phase dried with anhy-
drous Na2SO4, and the solvent removed under vacuum to yield
compounds VI. Spectroscopic data for compounds VI are listed
below.
In
a microwave reaction flask, 0.25 mmol of aldehyde IV,
0.375 mmol of the corresponding ylide VI, and 1 mL of methanol
were mixed. The reaction was carried out at 25 W for 5 min. The
solid obtained was filtered and purified by flash column chromatog-
raphy using toluene:dioxane (3:2) as the eluent.
(2E)-3-(3-Methyl-1,4-dioxy-quinoxalin-2-yl)-1-(3,4,5-trimethoxy-phenyl)-
propenone (4a). The derivative 4a was obtained as yellow solid
(27%). IR (KBr) 1651 (C = O), 1317 (N-O), 1120 (C–O–C); 1H-NMR
(CDCl3) d: 2.95 (s, 3H, CH3), 3.98 (s, 3H, p-OCH3), 4.00 (s, 6H, m-
OCH3), 7.40 (s, 2H, H20 +H60 ), 7.93-7.81 (m, 3H, H6 + H7 + Hb), 8.73-
8.62 (m, 2H, H5 + H8), 9.20 (d, 1H, Ha, Jab = 15.3 Hz); Analysis cal-
culated for C21H20N2O6 (396.40): C, 63.60; H, 5.09; N, 7.07. Found:
C, 63.73; H, 4.99; N, 6.90.
1-(3,4,5-Trimethoxy-phenyl)-2-(triphenyl-phosphonylidene)-ethanone
(VI-a). The compound VI-a was obtained as a white solid (42%).
IR (KBr) 1664 (C = O), 1584-1463 (C–C ar), 1122 (C–O–C); 1NMR
(CDCl3) d: 3.88 (s, 3H, p-OCH3), 3.92 (s, 6H, m-OCH3), 4.35–4.40 (d,
1H, = CH, JH-P = 19.1 Hz), 7.26 (s, 2H, H2 + H6), 7.48–7.53 (m, 6H,
H03H03+H05), 7.57–7.61 (m, 3H, H40 ), 7.71–7.76 (m, 6H, H20 +H06);
Analysis calculated for C29H27O4P (470.51): C, 74.04; H, 5.74. Found:
C, 73.82; H, 5.70.
(2E)-3-(7-Fluoro-3-methyl-1,4-dioxy-quinoxalin-2-yl)-1-(3,4,5-trimethoxy-
phenyl)-propenone (4b). The compound 4b was obtained as yel-
low solid (24%). IR (KBr) 1650 (C = O), 1319 (N-O), 1120 (C–O–C);
1H-NMR (CDCl3) d: 2.93 (s, 3H, CH3), 3.99 (s, 3H, p-OCH3), 4.00 (s,
6H, m-OCH3), 7.40 (s, 2H, H02+H60 ), 7.61–7.65 (ddd, 1H, H6,
J65 = 9.6 Hz, J6F = 7.4 Hz, J68 = 2.5 Hz), 7.82–7.86 (d, 1H, Hb,
Jba = 15.2 Hz), 8.31–8.34 (dd, 1H, H8, J8F
= 8.7 Hz, J86 = 2.6 Hz),
1-(4-Methoxy-phenyl)-2-(triphenyl-phosphonylidene)-ethanone (VI-b).
8.70–8.74 (dd, 1H, H5, J56 = 9.5 Hz, J5F = 5.1 Hz), 9.20–9.23 (d, 1H,
Ha, Jab = 15.3 Hz); Analysis calculated for C21H19FN2O6 (414.38): C,
60.87; H, 4.62; N, 6.76. Found: C, 60.62; H, 4.76; N, 6.51.
The compound VI-b was obtained as a white solid (37%). IR (KBr)
1
1649 (C = O), 1598-1482 (C–C ar), 1184 (C–O–C); NMR (DMSO-d6)
d: 3.77 (s, 3H, OCH3), 4.37–4.44 (d, 1H, = CH, JH–P = 24.9 Hz),
6.88–6.90 (d, 2H, H3 + H5, J32 ⁄ 56 = 8.8 Hz), 7.54–7.59 (m, 6H,
H03+H05), 7.64–7.70 (m, 6H, H20 +H40 +H60 ), 7.81–7.83 (d, 2H, H2 + H6,
J23 ⁄ 65 = 8.7 Hz); Analysis calculated for C27H23O2P (410.46): C,
79.01; H, 5.65. Found: C, 78.65; H, 5.60.
(2E)-3-(7-Methoxy-3-methyl-1,4-dioxy-quinoxalin-2-yl)-1-(3,4,5-trimeth-
oxy-phenyl)-propenone (4c). The derivative 4c was obtained as
yellow solid (37%). IR (KBr) 1657 (C = O), 1320 (N-O), 1133 (C–O–
1
C); H-NMR (CDCl3) d: 2.90 (s, 3H, CH3), 3.97 (s, 3H, p-OCH3), 3.99
(s, 6H, m-OCH3), 4.05 (s, 3H, quinox-OCH3), 7.39 (s, 2H, H02+H60 ),
7.44–7.47 (dd, 1H, H6, J65 = 9.4 Hz, J68 = 2.6 Hz), 7.84–7.87 (d, 1H,
Hb, Jba = 15.2 Hz), 7.94 (d, 1H, H8, J86 = 2.7 Hz), 8.55–8.58 (d, 1H,
H5, J56 = 9.5 Hz), 9.13–9.17 (d, 1H, Ha, Jab = 15.3 Hz); Analysis cal-
culated for C22H22N2O7 (426.43): C, 61.97; H, 5.20; N, 6.57. Found:
C, 61.63; H, 5.66; N, 6.14.
Synthesis of 1-(3-methyl-quinoxalin-2-yl)-
ethanone derivatives (compound VIII)
These compounds were obtained following the procedure described
in the literature (23). Spectroscopic data for one of these deriva-
tives (VIIIa, R6 ⁄ R7 = H ⁄ H) are described below as a reference
compound.
(2E)-3-(7-Chloro-3-methyl-1,4-dioxy-quinoxalin-2-yl)-1-(3-hydroxy-4,5-
dimethoxy-phenyl)-propenone (4d). The derivative 4d was
obtained as orange solid (30%). IR (KBr) 1654 (C = O), 1321 (N-O),
1-(3-Methyl-quinoxalin-2-yl)-ethanone (VIIIa). A 218 mg portion of
2-acetyl-3-methylquinoxaline 1,4-di-N-oxide (1 mmol) was dissolved
in 20 mL of methanol. The solution was heated at 70 ꢁC, and
1.04 g of Na2S2O4 (6 mmol) in 10 mL of water was added. The mix-
ture was stirred for 10 min, and the solvent removed under reduced
pressure. The solid obtained (50% yield) was filtered and washed
with water. IR (KBr) 1694 (C = O); 1H-NMR (DMSO-d6) d: 2.76 (s,
3H, 3-CH3), 2.84 (s, 3H, CO-CH3), 7.86–7.90 (ddd, 1H, H6,
J65 = 8.3 Hz, J67 = 6.9 Hz, J68 = 1.5 Hz), 7.93–7.97 (ddd, 1H, H7,
J78 = 8.4 Hz, J76 = 6.9 Hz, J75 = 1.5 Hz), 8.05–8.07 (dd, 1H, H5,
J56 = 8.3 Hz, J57 = 1.1 Hz), 8.14–8.16 (dd, 1H, H8, J87 = 8.3 Hz,
J86 = 1.1 Hz); Analysis calculated for C11H10N2O (186.22): C, 70.95;
H, 5.41; N, 15.04. Found: C, 70.63; H, 5.33; N, 15.08.
1
1135 (C–O–C); H-NMR (CDCl3) d: 2.93 (s, 3H, CH3), 3.99 (s, 3H, p-
OCH3), 4.00 (s, 6H, m-OCH3), 7.39 (s, 2H, H02+H60 ), 7.81–7.84 (dd,
1H, H6, J65 = 9.1 Hz, J68 = 2.2 Hz), 7.81–7.85 (d, 1H, Hb,
Jba = 15.2 Hz), 8.63–8.65 (d, 1H, H5, J56 = 9.1 Hz), 8.66–8.67 (d,
1H, H8, J86 = 2.2 Hz), 8.19–8.23 (d, 1H, Ha, Jab = 15.3 Hz); Analysis
calculated for C21H19ClN2O6 (430.89): C, 58.54; H, 4.45; N, 6.50.
Found: C, 58.58; H, 4.41; N, 6.32.
(2E)-3-(3,7-Dimethyl-1,4-dioxy-quinoxalin-2-yl)-1-(3,4,5-trimethoxy-phe-
nyl)-propenone (4e). The compound 4e was obtained as yellow
solid (33%). IR (KBr) 1654 (C = O), 1322 (N-O), 1126 (C–O–C); 1H-
NMR (CDCl3) d: 2.66 (s, 3H, 7-CH3), 2.94 (s, 3H, 3-CH3), 3.99 (s, 3H,
Chem Biol Drug Des 2011; 77: 255–267
257

Burguete et al.
p-OCH3), 4.00 (s, 6H, m-OCH3), 7.41 (s, 2H, H02+H60 ), 7.71–7.73 (dd,
1H, H6, J65 = 8.7 Hz, J68 = 1.6 Hz), 7.84–7.88 (d, 1H, Hb,
Jba = 15.3 Hz), 8.45 (s, 1H, H8), 8.56–8.59 (d, 1H, H5, J56 = 8.8 Hz),
9.14–9.18 (d, 1H, Hb, Jba = 15.3 Hz); Analysis calculated for
C22H22N2O6 (410.42): C, 64.39; H, 5.37; N, 6.83. Found: C, 64.19; H,
5.40; N, 6.64.
(KBr) 1662 (C = O), 1374 (N-O), 1176 (C–O–C); H-NMR (DMSO-d6)
1
d: 3.90 (s, 3H, OCH3), 7.14–7.16 (d, 2H, H02+H60 ,
J
2¢3¢ ⁄ J6¢5¢ = 8.7 Hz), 8.01–8.05 (d, 1H, Hb, Jba = 16.0 Hz), 8.22–8.24
(d, 2H, H03+H05, J3¢2¢ ⁄ J5¢6¢ = 8.6 Hz), 8.51–8.63 (m, 3H, Ha+H5 + H8),
9.59 (s, 1H, H3); Analysis calculated for C18H12N2O4F2 (358.30): C,
60.34; H, 3.38; N, 7.82. Found: C, 60.07; H, 3.35; N, 7.65.
(2E)-1-(3-Hydroxy-4,5-dimethoxy-phenyl)-3-(3,6,7-trimethyl-1,4-dioxy-
quinoxalin-2-yl)-propenone (4f). The compound 4f was obtained as
yellow solid (36%). IR (KBr) 1654 (C = O), 1321 (N-O), 1128 (C–O–
C); 1H-NMR (CDCl3) d: 2.55 (s, 6H, 6,7-CH3), 2.93 (s, 3H, 3-CH3),
3.98 (s, 3H, p-OCH3), 3.99 (s, 6H, m-OCH3), 7.40 (s, 2H, H02+H60 ),
7.83–7.87 (d, 1H, Hb, Jba = 15.3 Hz), 8.39 (s, 1H, H5), 8.42 (s, 1H,
H8) 9.13–9.17 (d, 1H, Ha, Jab = 15.3 Hz); Analysis calculated for
C23H24N2O6 (424.45): C, 65.08; H, 5.70; N, 6.60. Found: C, 65.16; H,
5.57; N, 6.46.
General method for the synthesis of (2E)-1-(3-
methyl-quinoxalin-2-yl)-3-(3,4,5-trimethoxy-
phenyl)-propenone derivatives (series 6)
The synthesis of compounds 6 was carried out as shown in
Scheme 2. The starting reagents used (VII and VIII) were obtained
by means of previously described methods (23,24). To a solution of
1-(3-methyl-quinoxalin-2-yl)-ethanone derivative VIII (1 mmol) and
3,4,5-trimethoxy-benzaldehyde (1 mmol) in methanol, a solution of
3% NaOH in methanol (1 mL) was added. After 24 h, the reaction
mixture was filtered, and the solid washed with water.
(2E)-
3-(1,4-Dioxy-quinoxalin-2-yl)-1-(4-methoxy-phenyl)-propenone
(5a). The derivative 5a was obtained as yellow solid (46%). IR
1
(KBr) 1663 (C = O), 1374 (N-O), 1174 (C–O–C); H-NMR (DMSO-d6)
(2E)-1-(3-Methyl-quinoxalin-2-yl)-3-(3,4,5-trimethoxy-phenyl)-propenone
(6a). The derivative 6a was obtained as yellow solid (56%). IR
(KBr) 1666 (C = O), 1133 (C–O–C); H-NMR (CDCl3) d: 3.01 (s, 3H,
CH3), 3.94 (s, 3H, p-OCH3), 3.95 (s, 6H, m-OCH3), 6.94 (s, 2H,
H02+H06), 7.75–7.79 (d, 1H, Ha, Jab = 16.0 Hz), 7.81–7.90 (m, 2H,
H6 + H7), 7.84–7.88 (d, 1H, Hb, Jba = 15.9 Hz), 8.09–8.11 (d, 1H, H5,
J56 = 8.3 Hz), 8.20–8.22 (d, 1H, H8, J87 = 8.3 Hz); Analysis calcu-
lated for C21H20N2O4 (364.40): C, 69.23; H, 5.49; N, 7.69. Found: C,
69.46; H, 5.53; N, 7.45.
d: 3.90 (s, 3H, OCH3), 7.12–7.15 (d, 2H, H02+H60 ,
1
J
2¢3¢ ⁄ J6¢5¢ = 8.8 Hz), 7.98–8.01 (m, 2H, H6 + H7), 8.05–8.09 (d, 1H,
Hb, Jba = 15.9 Hz), 8.22–8.24 (d, 2H, H03+H50 , J3¢2¢ ⁄ J5¢6¢ = 8.7 Hz),
8.47–8.54 (m, 2H, H5 + H8), 8.58–8.62 (d, 1H, Ha, Jab = 16.0 Hz),
9.53 (s, 1H, H3); Analysis calculated for C18H14N2O4 (322.32): C,
67.08; H, 4.35; N, 8.70. Found: C, 67.14; H, 4.43; N, 8.71.
(2E)-3-(7-Fluoro-1,4-dioxy-quinoxalin-2-yl)-1-(4-methoxy-phenyl)-prope-
none (5b). The compound 5b was obtained as yellow solid (32%).
1
IR (KBr) 1661 (C = O), 1372 (N-O), 1177 (C–O–C); H-NMR (DMSO-d6)
(2E)-1-(7-Fluoro-3-methyl-quinoxalin-2-yl)-3-(3,4,5-trimethoxy-phenyl)-
propenone (6b). The compound 6b was obtained as yellow solid
d: 3.89 (s, 3H, OCH3), 7.13–7.15 (d, 2H, H02+H60 , J2¢3¢ ⁄ J6¢5¢ = 8.8 Hz),
7.90–7.95 (m, 1H, H6), 8.02–8.06 (d, 1H, Hb, Jba = 16.0 Hz), 8.22–8.24
(m, 3H, H8 + H03+H50 ), 8.58–8.62 (m, 2H, Ha+H5), 9.59 (s, 1H, H3); Anal-
ysis calculated for C18H13FN2O4 (340.31): C, 63.53; H, 3.82; N, 8.24.
Found: C, 63.29; H, 3.72; N, 8.02.
1
(59%). IR (KBr) 1670 (C = O), 1128 (C–O–C); H-NMR (CDCl3) d: 2.98
(s, 3H, CH3), 3.94 (s, 3H, p-OCH3), 3.95 (s, 6H, m-OCH3), 6.93 (s, 2H,
H20 +H60 ), 7.63–7.68 (ddd, 1H, H6, J65 = 9.3 Hz, J6F = 8.1 Hz,
J68 = 2.8 Hz), 7.74–7.78 (d, 1H, Ha, Jab = 16.0 Hz), 7.79–7.83 (d,
1H, Hb, Jba = 16.0 Hz), 7.82–7.85 (dd, 1H, H8, J8F = 8.8 Hz,
J86 = 2.8 Hz), 8.09–8.12 (dd, 1H, H5, J56 = 9.3 Hz, J5F = 5.6 Hz);
Analysis calculated for C21H19N2O4F (382.39): C, 65.97; H, 4.97; N,
7.33. Found: C, 66.13; H, 5.04; N, 7.11.
(2E)-3-(7-Chloro-1,4-dioxy-quinoxalin-2-yl)-1-(4-methoxy-phenyl)-propenone
(5d). The derivative 5d was obtained as yellow solid (71%). IR
1
(KBr) 1661 (C = O), 1374 (N-O), 1177 (C–O–C); H-NMR (DMSO-d6)
d: 3.89 (s, 3H, OCH3), 7.13–7.15 (d, 2H, H02+H60 ,
J
2¢3¢ ⁄ J6¢5¢ = 8.8 Hz), 8.02–8.06 (m, 2H, H6 + Hb), 8.21–8.23 (d, 2H,
(2E)-1-(7-Methoxy-3-methyl-quinoxalin-2-yl)-3-(3,4,5-trimethoxy-phenyl)-
propenone (6c). The derivative 6c was obtained as yellow solid
(31%). IR (KBr) 1670 (C = O), 1126 (C–O–C); H-NMR (CDCl3) d: 2.97
(s, 3H, CH3), 3.94 (s, 3H, p-OCH3), 3.95 (s, 6H, m-OCH3), 4.02 (s, 3H,
quinox-OCH3), 6.93 (s, 2H, H02+H60 ), 7.47–7.48 (d, 1H, H8,
J86 = 2.6 Hz), 7.51–7.54 (dd, 1H, H6, J65 = 9.2 Hz, J68 = 2.8 Hz),
7.73–7.77 (d, 1H, Ha, Jab = 16.0 Hz), 7.80–7.84 (d, 1H, Hb,
Jba = 16.0 Hz), 7.99–8.02 (d, 1H, H5, J56 = 9.2 Hz); Analysis calcu-
lated for C22H22N2O5 (394.43): C, 67.00; H, 5.58; N, 7.11. Found: C,
67.08; H, 5.61; N, 7.05.
H03+H05, J3¢2¢ ⁄ J5¢6¢ = 8.3 Hz), 8.47–8.63 (m, 3H, Ha+H5 + H8), 9.58 (s,
1H, H3); Analysis calculated for C18H13ClN2O4 (356.77): C, 60.59; H,
3.65; N, 7.85. Found: C, 60.23; H, 3.52; N, 7.64.
1
(2E)-1-(4-Methoxy-phenyl)-3-(7-methyl-1,4-dioxy-quinoxalin-2-yl)-propenone
(5e). The compound 5e was obtained as yellow solid (39%). IR
1
(KBr) 1657 (C = O), 1373 (N-O), 1171 (C–O–C); H-NMR (DMSO-d6)
d: 2.60 (s, 3H, CH3), 3.90 (s, 3H, OCH3), 7.13–7.15 (d, 2H, H02+H60 ,
J
2¢3¢ ⁄ J6¢5¢ = 8.9 Hz), 7.82–7.84 (dd, 1H, H6, J65 = 8.8 Hz,
J68 = 1.7 Hz), 8.04–8.08 (d, 1H, Hb, Jba = 16.0 Hz), 8.22–8.24 (d,
2H, H03+H05, J3¢2¢ ⁄ J5¢6¢ = 8.9 Hz), 8.30 (s, 1H, H8), 8.41–8.43 (d, 1H,
H5, J56 = 8.8 Hz), 8.56–8.60 (d, 1H, Ha, Jab = 16.0 Hz), 9.50 (s, 1H,
H3); Analysis calculated for C19H16N2O4 (336.35): C, 67.86; H, 4.76;
N, 8.33. Found: C, 67.68; H, 4.51; N, 8.55.
(2E)-1-(7-Chloro-3-methyl-quinoxalin-2-yl)-3-(3,4,5-trimethoxy-phenyl)-
propenone (6d). The derivative 6d was obtained as yellow solid
(31%). IR (KBr) 1670 (C = O), 1128 (C–O–C); H-NMR (CDCl3) d: 2.99
(s, 3H, CH3), 3.94 (s, 3H, p-OCH3), 3.96 (s, 6H, m-OCH3), 6.93 (s, 2H,
H02+H06), 7.74–7.78 (d, 1H, Ha, Jab = 16.0 Hz), 7.79–7.83 (d, 1H, Hb,
Jba = 16.0 Hz), 7.79–7.82 (dd, 1H, H6, J65 = 8.9 Hz, J68 = 2.3 Hz),
8.03–8.05 (d, 1H, H5, J56 = 9.0 Hz), 8.21–8.22 (d, 1H, H8,
1
(2E)-3-(6,7-Difluoro-1,4-dioxy-quinoxalin-2-yl)-1-(4-methoxy-phenyl)-propenone
(5g). The compound 5g was obtained as yellow solid (14%). IR
258
Chem Biol Drug Des 2011; 77: 255–267

Synthesis, Antioxidant and Anti-inflammatory Activities of New Quinoxaline Derivatives
J86 = 2.2 Hz); Analysis calculated for C21H19N2O4Cl (398.85): C,
63.24; H, 4.77; N, 7.03. Found: C, 62.92; H, 4.89; N, 6.73.
1H-NMR (DMSO-d6) d: 2.79 (s, 3H, CH3); 3.74 (s, 3H, p-OCH3); 3.88
(s, 6H, m-OCH3); 3.96 (s, 3H, 7-OCH3); 6.94 (s, 2H, NH2); 7.48 (s, 2H,
H02+H06); 7.51–7.54 (m, 2H, H6 + H8); 7.63 (s, 1H, ar-CH); 7.97–8.00
(d, 1H, H5, J56 = 9.9 Hz); Analysis calculated for C23H23N5O4
(433.47): C, 63.74; H, 5.31; N, 16.17. Found: C, 63.35; H, 5.39; N,
15.99.
(2E)-1-(3,7-Dimethyl-quinoxalin-2-yl)-3-(3,4,5-trimethoxy-phenyl)-prope-
none (6e). The compound 6e was obtained as yellow solid (49%).
1
IR (KBr) 1670 (C = O), 1126 (C–O–C); H-NMR (CDCl3) d: 2.64 (s, 3H,
7-CH3), 3.00 (s, 3H, 3-CH3), 3.94 (s, 3H, p-OCH3), 3.95 (s, 6H, m-
OCH3), 6.93 (s, 2H, H20 +H60 ), 7.69–7.72 (dd, 1H, H6, J65 = 8.6 Hz,
J68 = 1.8 Hz), 7.74–7.78 (d, 1H, Ha, Jab = 16.0 Hz), 7.85–7.89 (d,
1H, Hb, Jba = 16.0 Hz), 7.98 (s, 1H, H8), 7.99–8.01 (d, 1H, H5,
J56 = 8.8 Hz); Analysis calculated for C22H22N2O4 (378.43): C, 69.84;
H, 5.82; N, 7.41. Found: C, 69.92; H, 5.79; N, 7.48.
4-(7-Chloro-3-methyl-quinoxalin-2-yl)-6-(3,4,5-trimethoxy-phenyl)-pyr-
imidin-2-ylamine (7d). The derivative 7d was obtained as beige
solid (10%). IR (KBr) 3447-3308 (N-H), 1630 (C = N), 1132 (C–O–C);
1H-NMR (DMSO-d6) d: 2.84 (s, 3H, CH3); 3.74 (s, 3H, p-OCH3); 3.88
(s, 6H, m-OCH3); 6.99 (s, 2H, NH2); 7.47 (s, 2H, H02+H60 ); 7.65 (s, 1H,
ar-CH); 7.91–7.94 (dd, 1H, H6, J65 = 8.9 Hz; J68 = 2.4 Hz); 8.12–8.14
(d, 1H, H5, J56 = 9.0 Hz); 8.26–8.27 (d, 1H, H8, J86 = 2.3 Hz); Analy-
sis calculated for C22H20ClN5O3 (437.89): C, 60.34; H, 4.57; N,
16.00. Found: C, 60.22; H, 4.72; N, 15.66.
(2E)-3-(3,4,5-Trimethoxy-phenyl)-1-(3,6,7-trimethyl-quinoxalin-2-yl)-
propenone (6f). The compound 6f was obtained as yellow solid
1
(38%). IR (KBr) 1671 (C = O), 1127 (C–O–C); H-NMR (CDCl3) d: 2.55
(s, 3H, 6 ⁄ 7-CH3), 2.56 (s, 3H, 6 ⁄ 7-CH3), 3.01 (s, 3H, 3-CH3), 3.94 (s,
3H, p-OCH3), 3.96 (s, 6H, m-OCH3), 6.94 (s, 2H, H02+H60 ), 7.75–7.79
(d, 1H, Ha, Jab = 16.0 Hz), 7.89 (s, 1H, H8), 7.89–7.93 (d, 1H, Hb,
Jba = 16.0 Hz), 7.96 (s, 1H, H5); Analysis calculated for C23H24N2O4
(392.46): C, 70.41; H, 6.12; N, 7.14. Found: C, 70.34; H, 6.12; N, 7.00.
4-(3,7-Dimethyl-quinoxalin-2-yl)-6-(3,4,5-trimethoxy-phenyl)-pyrimidin-
2-ylamine (7e). The derivative 7e was obtained as beige solid
(13%). IR (KBr) 3458-3360 (N-H), 1616 (C = N), 1129 (C–O–C); 1H-
NMR (DMSO-d6) d: 2.58 (s, 3H, 7-CH3); 2.81 (s, 3H, 3-CH3); 3.74 (s,
3H, p-OCH3); 3.88 (s, 6H, m-OCH3); 6.96 (s, 2H, NH2); 7.47 (s, 2H,
H02+H06); 7.63 (s, 1H, ar-CH); 7.72–7.74 (d, 1H, H6, J65 = 8.6 Hz);
7.93 (s, 1H, H8); 7.97–7.99 (d, 1H, H5, J56 = 8.6 Hz); Analysis calcu-
lated for C23H23N5O3 (417.47): C, 66.19; H, 5.52; N, 16.79. Found: C,
65.93; H, 5.61; N, 16.66.
General method for the synthesis of 4-(3-
methyl-quinoxalin-2-yl)-6-(3,4,5-trimethoxy-
phenyl)-pyrimidin-2-ylamine derivatives (series
7)
To a solution of the corresponding compound of series 6 (0.5 mmol)
and guanidine hydrochloride (1 mmol) in isopropanol, 0.56 mL of a
solution 10% KOH in isopropanol was added. The reaction mixture
was refluxing over 24 h. The solvent was removed under reduced
pressure, and the crude residue was purified by flash chromatogra-
phy using hexane:ethyl acetate (50:50) to yield compounds 7.
General method for the synthesis of 2-methyl-3-
[5-(3,4,5-trimethoxy-phenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-quinoxaline derivatives (series 8)
and of 2-methoxy-4-[5-(3-methyl-1,4-dioxy-
quinoxalin-2-yl)-3,4-dihydro-2H-pyrazol-3-yl]-
phenol derivatives (series 10)
4-(3-Methyl-quinoxalin-2-yl)-6-(3,4,5-trimethoxy-phenyl)-pyrimidin-2-yl-
amine (7a). The derivative 7a was obtained as beige solid (15%).
IR (KBr) 3432–3346 (N-H), 1620 (C = N), 1126 (C–O–C); 1H-NMR
(DMSO-d6) d: 2.84 (s, 3H, CH3); 3.75 (s, 3H, p-OCH3); 3.88 (s, 6H, m-
OCH3); 6.96 (s, 2H, NH2); 7.48 (s, 2H, H02+H60 ); 7.65 (s, 1H, ar-CH);
7.84–7.92 (m, 2H, H6 + H7); 8.08–8.11 (dd, 1H, H8, J87 = 8.2 Hz;
J86 = 1.2 Hz); 8.15–8.17 (d, 1H, H5, J56 = 8.3 Hz); Analysis calcu-
lated for C22H21N5O3 (403.44): C, 65.51; H, 5.21; N, 17.37. Found: C,
65.59; H, 5.45; N, 17.48.
Two millimoles of hydrazine hydrate 98% was added to a solution
of 0.5 mmol of the appropriate compound of series 6 in absolute
ethanol. After 24 h, the dissolvent was removed under reduced
pressure, and the solid obtained (compound of series 8) was fil-
tered and washed with ethyl ether. In the same way, 2 mmol of
hydrazine hydrate 98% was added to a solution of 1 mmol of the
corresponding series 2 derivative, and the reaction mixture was stir-
red for 24 h. After removing the solvent under reduced pressure,
the residue was precipitated with ethyl acetate, and the solid
obtained (compounds of series 10) was filtered.
4-(7-Fluoro-3-methyl-quinoxalin-2-yl)-6-(3,4,5-trimethoxy-phenyl)-pyrimidin-
2-ylamine (7b). The compound 7b was obtained as beige solid
(15%). IR (KBr) 3430-3308 (N-H), 1628 (C = N), 1133 (C–O–C); 1H-
NMR (DMSO-d6) d: 2.83 (s, 3H, CH3); 3.74 (s, 3H, p-OCH3); 3.88 (s,
6H, m-OCH3); 6.98 (s, 2H, NH2); 7.47 (s, 2H, H02+H60 ); 7.65 (s, 1H,
ar-CH); 7.81–7.86 (ddd, 1H, H6, J65 = 9.1 Hz; J6F = 8.9 Hz;
J68 = 2.8 Hz); 7.94–7.97 (dd, 1H, H8, J8F = 9.4 Hz; J86 = 2.8 Hz);
8.16–8.20 (dd, 1H, H5, J56 = 9.2 Hz; J5F = 5.8 Hz); Analysis calcu-
lated for C22H20FN5O3 (421.43): C, 62.71; H, 4.75; N, 16.63. Found:
C, 62.30; H, 5.15; N, 16.40.
6-Methoxy-2-methyl-3-[5-(3,4,5-trimethoxy-phenyl)-4,5-dihydro-1H-pyr-
azol-3-yl]-quinoxaline (8c). The derivative 8c was obtained as yel-
low solid (74%). IR (KBr) 3206 (N-H), 1592 (C = N), 1127 (C–O–C);
1H-NMR (DMSO-d6) d: 2.92 (s, 3H, CH3); 3.10–3.17 (dd, 1H, HA,
upfield H of CH2, JAB = 16.7 Hz; JAX = 10.6 Hz); 3.65 (s, 3H, p-
OCH3); 3.68–3.74 (dd, 1H, HB, downfield H of CH2, JBA = 16.8 Hz;
JBX = 11.3 Hz); 3.78 (s, 6H, m-OCH3); 3.92 (s, 3H, q-OCH3); 4.89–
4.95 (ddd, 1H, HX, CH, JXA = 11.0 Hz; JXB = 11.1 Hz; JX-NH = 2.1 Hz);
6.72 (s, 2H, H02+H60 ); 7.28–7.29 (d, 1H, H8, J86 = 2.8 Hz); 7.37–7.40
(dd, 1H, H6, J65 = 9.1 Hz; J68 = 2.8 Hz); 7.85–7.87 (d, 1H, H5,
J56 = 9.1 Hz); 8.53 (d, 1H, NH, JNH-X = 2.1 Hz); Analysis calculated
for C22H24N4O4 (408.46): C, 64.71; H, 5.88; N, 13.73. Found: C,
64.26; H, 5.72; N, 13.59.
4-(7-Methoxy-3-methyl-quinoxalin-2-yl)-6-(3,4,5-trimethoxy-phenyl)-
pyrimidin-2-ylamine (7c). The derivative 7c was obtained as beige
solid (9%). IR (KBr) 3458–3212 (N-H), 1620 (C = N), 1127 (C–O–C);
Chem Biol Drug Des 2011; 77: 255–267
259

Burguete et al.
2,6,7-Trimethyl-3-[5-(3,4,5-trimethoxy-phenyl)-4,5-dihydro-1H-pyrazol-
3-yl]-quinoxaline (8f). The derivative 8f was obtained as yellow
solid (74%). IR (KBr) 3205 (N-H), 1589 (C = N), 1128 (C–O–C); 1H-
NMR (DMSO-d6) d: 2.43-2.44 (2s, 6H, 6-CH3 + 7-CH3); 2.92 (s, 3H,
3-CH3); 3.07-3.14 (dd, 1H, HA, upfield H of CH2, JAB = 16.5 Hz;
JAX = 11.1 Hz); 3.65 (s, 3H, p-OCH3); 3.69-3.73 (m, 1H, HB, down-
field H of CH2); 3.78 (s, 6H, m-OCH3); 4.87–4.92 (t, 1H, HX, CH,
JXA = 10.6 Hz; JXB = 10.6 Hz); 6.73 (s, 2H, H02+H60 ); 7.71–7.73 (2s,
2H, H5 + H8); 8.42 (s, 1H, NH); Analysis calculated for C23H26N4O3
(406.49): C, 67.98; H, 6.40; N, 13.79. Found: C, 67.65; H, 6.92; N,
13.63.
J
XA = 6.1 Hz; JXB = 12.4 Hz); 6.36 (s, 2H, NH2); 6.74 (s, 2H, H02+H60 );
7.32–7.35 (d, 2H, HC, JCD = 9.0 Hz); 7.63–7.69 (m, 2H, H6 + H8); 7.75–
7.79 (d, 2H, HD, JDC = 9.1 Hz); 8.06–8.10 (d, 1H, H5, J56 = 10.0 Hz;
J5F = 5.8 Hz); Analysis calculated for C27H26FN5O5S (551.60): C,
58.80; H, 4.72; N, 12.70. Found: C, 58.56; H, 4.76; N, 12.32.
4-[3-(7-Methoxy-3-methyl-quinoxalin-2-yl)-5-(3,4,5-trimethoxy-phenyl)-
4,5-dihydro-pyrazol-1-yl]-benzenesulfonamide (9c). The derivative
9c was obtained as yellow solid (9.5%). IR (KBr) 3313–3229 (N-H),
1593 (C = N), 1326 (as O = S = O), 1155 (sym O = S = O), 1122 (C–
1
O–C); H-NMR (DMSO-d6) d: 3.10 (s, 3H, CH3); 3.44–3.50 (dd, 1H,
HA, upfield H of CH2, JAB = 17.8 Hz; JAX = 5.5 Hz); 3.62 (s, 3H, p-
OCH3); 3.70 (s, 6H, m-OCH3); 3.92 (s, 3H, q-OCH3); 4.10–4.18 (dd,
1H, HB, downfield H of CH2, JBA = 18.2 Hz; JBX = 12.3 Hz); 5.61–
5.65 (dd, 1H, HX, CH, JXA = 5.6 Hz; JXB = 12.4 Hz); 6.90 (s, 2H,
H02+H06); 7.13 (s, 2H, NH2); 7.22–7.24 (d, 2H, HC, JCD = 8.6 Hz);
7.32–7.33 (d, 1H, H8, J86 = 2.5 Hz); 7.43–7.46 (dd, 1H, H6,
J65 = 8.8 Hz; J68 = 2.4 Hz); 7.68–7.71 (d, 2H, HD, JDC = 8.7 Hz);
7.91–7.93 (d, 1H, H5, J56 = 9.1 Hz); Analysis calculated for
C28H29N5O6S (563.64): C, 59.68; H, 5.15; N, 12.43. Found: C, 59.20;
H, 4.96; N, 12.07.
2-Methoxy-4-[5-(3-methyl-1,4-dioxy-quinoxalin-2-yl)-3,4-dihydro-2H-
pyrazol-3-yl]-phenol (10a). The derivative 10a was obtained as yel-
low solid (35%). IR (KBr) 3537-3317 (N-H, O-H), 1606 ( = N), 1331
(N-O), 1273 (C–O); 1H-NMR (DMSO-d6) d: 2.60 (s, 3H, CH3); 3.16-
3.22 (m, 1H, HA, upfield H of CH2); 3.46–3.53 (dd, 1H, HB, downfield
H of CH2, JBA = 16.2 Hz; JBX = 11.2 Hz); 3.79 (s, 3H, OCH3); 4.88–
4.93 (m, 1H, HX, CH); 6.74–6.77 (dd, 1H, H06, J6¢5¢ = 8.0 Hz;
J6¢2¢ = 1.4 Hz); 6.84–6.86 (m, 1H, H05); 7.06 (s, 1H, H20 ); 7.91–7.98
(m, 2H, H6 + H7); 8.11 (s, 1H, NH); 8.46–8.51 (m, 2H, H5 + H8); 8.92
(s, 1H, OH); Analysis calculated for C19H18N4O4 (366.38): C, 62.30;
H, 4.92; N, 15.30. Found: C, 62.24; H, 5.18; N, 14.90.
4-[3-(7-Chloro-3-methyl-quinoxalin-2-yl)-5-(3,4,5-trimethoxy-phenyl)-
4,5-dihydro-pyrazol-1-yl]-benzenesulfonamide (9d). The derivative
9d was obtained as yellow solid (12%). IR (KBr) 3302-3225 (N-H),
1594 (C = N), 1341 (as O = S = O), 1157 (sym O = S = O), 1130 (C–
General method for the synthesis of 4-[3-(3-
methyl-quinoxalin-2-yl)-5-(3,4,5-trimethoxy-
phenyl)-4,5-dihydro-pyrazol-1-yl]-
benzenesulfonamide derivatives (series 9)
The corresponding compound of series 6 (0.55 mmol), 4-hydrazino-
benzene-1-sulfonamide hydrochloride 97% (0.55 mmol), and 10 mL
of ethanol 99% was mixed in a microwave reaction flask. The pro-
gram (50 W, 5¢) was carried out four times. The solid obtained was
filtered and purified by flash column chromatography using ethyl
acetate to yield compounds of series 9.
1
O–C); H-NMR (DMSO-d6) d: 3.14 (s, 3H, CH3); 3.40–3.46 (dd, 1H,
HA, upfield H of CH2, JAB = 17.8 Hz; JAX = 6.0 Hz); 3.62 (s, 3H, p-
OCH3); 3.70 (s, 6H, m-OCH3); 4.09–4.17 (dd, 1H, HB, downfield H of
CH2, JBA = 17.9 Hz; JBX = 12.4 Hz); 5.63–5.67 (dd, 1H, HX, CH,
J
XA = 5.8 Hz; JXB = 12.2 Hz); 6.61 (s, 2H, H02+H60 ); 7.13 (s, 2H,
NH2); 7.24–7.26 (d, 2H, HC, JCD = 8.9 Hz); 7.69–7.71 (d, 2H, HD,
JDC = 9.0 Hz); 7.81–7.84 (dd, 1H, H6, J65 = 8.9 Hz; J68 = 2.3 Hz);
8.03–8.04 (d, 1H, H8, J86 = 2.5 Hz); 8.03–8.05 (d, 1H, H5,
J56 = 8.7 Hz); Analysis calculated for C27H26ClN5O5S (568.06): C,
57.09; H, 4.58; N, 12.33. Found: C, 56.68; H, 4.62; N, 11.97.
4-[3-(3-Methyl-quinoxalin-2-yl)-5-(3,4,5-trimethoxy-phenyl)-4,5-dihy-
dro-pyrazol-1-yl]-benzenesulfonamide (9a). The derivative 9a was
obtained as yellow solid (14%). IR (KBr) 3337-3248 (N-H), 1594
(C = N), 1341 (as O = S = O), 1158 (sym O = S = O), 1129 (C–O–C);
1H-NMR (acetone-d6) d: 3.20 (s, 3H, CH3); 3.63-3.57 (dd, 1H, HA,
upfield H of CH2, JAB = 18.1 Hz; JAX = 6.2 Hz); 3.71 (s, 3H, p-OCH3);
3.78 (s, 6H, m-OCH3); 4.22–4.30 (dd, 1H, HB, downfield H of CH2,
JBA = 18.1 Hz; JBX = 12.4 Hz); 5.63–5.68 (dd, 1H, HX, CH,
Biological experiments
Experiments in vitro
In the in vitro assays, each experiment was performed at least in
triplicate, and the standard deviation of absorbance was less than
10% of the mean.
J
XA = 6.2 Hz; JXB = 12.4 Hz); 6.36 (s, 2H, NH2); 6.75 (s, 2H,
H02+H06); 7.31–7.34 (d, 2H, HC, JCD = 9.0 Hz); 7.75–7.83 (d, 2H, HD,
JDC = 9.0 Hz); 7.79–7.83 (m, 2H, H6 + H7); 7.99–8.03 (m, 2H,
H5 + H8); Analysis calculated for C27H27N5O5S (533.61): C, 60.79; H,
5.07; N, 13.13. Found: C, 60.68; H, 5.22; N, 12.70.
Determination of the reducing activity of the stable radical 1,1-
diphenyl-picrylhydrazyl (33). To a solution of DPPH in absolute eth-
anol, an equal volume of the compounds dissolved in DMSO was
added. An ethanol solution was used as control. The concentrations
of the solutions of the compounds were 0.05, 0.1, and 0.2 mM.
After 20 and 60 min at room temperature, the absorbance was
recorded at 517 nm.
4-[3-(7-Fluoro-3-methyl-quinoxalin-2-yl)-5-(3,4,5-trimethoxy-phenyl)-4,5-
dihydro-pyrazol-1-yl]-benzenesulfonamide (9b). The derivative 9b
was obtained as yellow solid (29%). IR (KBr) 3306-3243 (N-H), 1594
(C = N), 1330 (as O = S = O), 1160 (sym O = S = O), 1130 (C–O–C);
1H-NMR (acetone-d6) d: 3.19 (s, 3H, CH3); 3.55–3.61 (dd, 1H, HA,
upfield H of CH2, JAB = 18.1 Hz; JAX = 6.2 Hz); 3.71 (s, 3H, p-OCH3);
3.78 (s, 6H, m-OCH3); 4.21–4.29 (dd, 1H, HB, downfield H of CH2,
JBA = 18.1 Hz; JBX = 12.5 Hz); 5.65–5.70 (dd, 1H, HX, CH,
ˇ
ˇ
+
ABTS. -decolorization assay in ethanolic solution for antioxidant
activity (26). ABTS is dissolved in water to a 7 mM concentration.
ˇ
ˇ
+
ABTS radical cation (ABTS. ) produced by reacting ABTS stock solu-
tion with 2.45 mM potassium persulfate (final concentration) and
allowing the mixture to stand in the dark at room temperature for
260
Chem Biol Drug Des 2011; 77: 255–267

Synthesis, Antioxidant and Anti-inflammatory Activities of New Quinoxaline Derivatives
ˇ
+
ˇ
12–16 h before use. For the present study, the ABTS. solution was
laboratory, were housed under standard conditions and received a
diet of commercial food pellets and water ad libitum during the
maintenance, but they were entirely fasted during the experiment
period. Our studies were in accordance with the recognized guide-
lines on animal experimentation.
diluted with ethanol to an absorbance of 0.70 (60.02) at 734 nm
and equilibrated at 30 ꢁC. Stock solutions of the tested compounds
in DMSO were diluted, so that after introduction of a 10-mL aliquot
of each dilution into the assay, they produced between 20% and
80% inhibition of the blank absorbance. After addition of 1.0 mL of
ˇ
+
ˇ
diluted ABTS. solution (A734 nm) to 10 lL of antioxidant com-
The tested compounds, 0.01 mmol ⁄ kg body weight, were suspended
in water, with few drops of Tween 80 and ground in a mortar
before use and were given intraperitoneally simultaneously with the
carrageenin injection. The rats were euthanized 3.5 h after carra-
geenin injection. The difference between the weight of the injected
and uninjected paws was calculated for each animal. The change
in paw weight was compared with that in control animals (treated
with water) and expressed as a percent inhibition of the edema
CPE % values. IMA in 0.01 mmol ⁄ kg presented 47% inhibition of
the edema. Values CPE % are the mean from two different experi-
ments with a standard error of the mean <10%.
pounds or Trolox standards (final concentration 0.1 mM) in ethanol,
the absorbance reading was taken at room temperature exactly
1 min after the initial mixing.
Non-enzymatic assay of superoxide radicals – measurement of
superoxide radical scavenging activity (33). The superoxide produc-
ing system was set up by mixing PMS, NADH, and air. The produc-
tion of superoxide was estimated by the NBT method. The reaction
mixture containing compounds, 3 lM PMS, 78 lM NADH, and
25 lM NBT in 19 lM phosphate buffer pH 7.4 was incubated for
2 min at room temperature, and the absorption measured at
560 nm against a blank containing PMS. The tested compounds
were preincubated for 2 min before adding NADH.
Results and Discussion
Competition of the tested compounds with DMSO for hydroxyl radi-
cals (33). The hydroxyl radicals generated by the Fe3+ ⁄ ascorbic
acid system were detected according to Nash, by the determination
of formaldehyde produced from the oxidation of DMSO. The reac-
tion mixture contained EDTA (0.1 mM), Fe3+ (167 lM), DMSO
(33 mM) in phosphate buffer (50 mM, pH 7.4), the tested compounds
(concentration 0.1 mM), and ascorbic acid (10 mM). After 30 min of
Synthesis
The new compounds of series 1, 2, and 3 were synthesized follow-
ing the previous described procedures (18). The starting reagents,
5-substituted or 5,6-disubstituted benzofuroxanes (BFX) I, were
obtained as described in previously reported methods (19).
The classic Claisen-Schmidt condensation, used to synthesize com-
pounds with a,b-unsaturated ketone system (such as compounds in
series 1), did not react in the same expected way to obtain com-
pounds in series 4 and 5. Thus, a Wittig reaction was proposed to
obtain the desired compounds (Scheme 1). Usually, these reactions,
which consist in a condensation between an aldehyde and an ylide
to afford the corresponding a,b-unsaturated ketone system deriva-
tive, are carried out in a dichloromethane reflux for 5 or 6 h (20).
After a deep study of the reaction conditions, we optimized a
microwave-assisted method for the synthesis of these two series
concluding that the use of the microwave-improved conversion
ratios and decreased reaction times. As shown in Scheme 1, both
aldehyde and ylide had to be previously synthesized. Aldehydes IV
were obtained by two different methods: one of them included
deprotection of an acetal group in acid medium; the other involved
a methyl oxidation using SeO2 as the oxidizing agent (21). The sec-
ond method was found to be the most appropriate for the synthesis
of these aldehydes as a huge volume of solvent was needed in the
first one to do the extraction. In addition, a microwave-assisted
method was developed by our group to carry out this reaction. The
starting compounds II and III were synthesized by the classic Beirut
reaction (22) between the appropriate BFX and the corresponding
carbonilic derivative using pyrrolidine or morpholine as the catalyst.
Obtention of ylide VI required a reaction between triphenylphos-
phine and the corresponding alkyl halide V and a subsequent treat-
ment with a weak base (20). In the case of series 5, the halide
was commercially available, and the formation of the ylide was car-
ried out by using a microwave-assisted method. The synthesis of
ylide in series 4 was performed by a conventional route as the
microwave did not work as expected with this reaction. The
required halide was previously synthesized as reported (20) and
ꢁ
incubation (37 C), the reaction was stopped with CCl3COOH (17%
w ⁄ v).
Inhibition of linoleic acid lipid peroxidation (31). Ten microliters of
the 16 mM linoleic acid dispersion was added to the UV cuvette
containing 0.93 mL of 0.05 M phosphate buffer, pH 7.4 prethermo-
stated at 37 ꢁC. The oxidation reaction was initiated at 37 ꢁC
under air by the addition of 50 lL of 40 mM AAPH solution. Oxida-
tion was carried out in the presence of aliquots in different concen-
trations. In the assay without antioxidant, lipid oxidation was
measured in the presence of the same level of DMSO. The rate of
oxidation at 37 ꢁC was monitored by recording the increase in
absorption at 234 nm caused by conjugated diene hydroperoxides.
Soybean lipoxygenase inhibition study in vitro. In vitro study was
evaluated as reported previously (33). The tested compounds dis-
solved in DMSO were incubated at room temperature in different
concentrations with sodium linoleate (0.1 mM) and 0.2 mL of
enzyme solution (1 ⁄ 9 · 10)4 w ⁄ v in saline). The conversion of
sodium linoleate to 13-hydroperoxylinoleic acid at 234 nm was
recorded and compared with the appropriate standard inhibitor. IC50
values were determined.
Experiments in vivo
Inhibition of the carrageenin-induced edema. Edema was induced
in the right hind paw of Fisher 344 rats (150–200 g) by the intra-
dermal injection of 0.1 mL 2% carrageenin in water. Both sexes
were used. Pregnant females were excluded. Each group was com-
posed of 6–15 animals. The animals, which have been bred in our
Chem Biol Drug Des 2011; 77: 255–267
261

Burguete et al.
O–
N+
O
R7
R6
O
N+ R3
c
O–
a
O–
N+
O
O–
R7
R6
N+
R6
H
II
O
N+ R3
N
R5
O–
d
O–
N+
I
b
(MW)
R7
R6
O–
N+
O
IV
R3'
R4'
R7
R6
h
N+ R3
O–
N+ R3
O–
i
R5'
(MW)
III
R3=CH3; R3', R4', R5'=OCH3
R3=H; R3', R5'=H; R4'=OCH3
Series 4
Series 5
O
O
(Ph)3P
R3'
R4'
f
Br
R3'
R4'
+
(Ph)3P
g
(MW)
R5'
R5'
V-a (Series 4)
V-b (Series 5)
VI-a (Series 4)
VI-b (Series 5)
e
O
O
O
O
Scheme 1: Synthesis of series 4 and 5. (a) 4,4-dimethoxy-butan-2-one (series 4) or 3,3-dimethoxy-propionaldehyde (series 5), pyrrolidine;
(b) butan-2-one (series 4) or propionaldehyde (series 5), morpholine (c) HCl ⁄ MeOH, DCM extraction; (d) SeO2 ⁄ acetonitrile, 5 min, 200 W
(MW); (e) Br2 ⁄ CH3COOH; (f) toluene, NaH ⁄ H2O; (g) xylene, 1.5 min, 110 W (MW), NaH ⁄ H2O; (h) DCM reflux, 5 ⁄ 6 h; (i) MeOH, 5 min, 25 W
(MW).
used without purification to obtain the ylide, which could be the
reason of the failure of the microwave-assisted synthesis.
Antioxidant activity
The estimation of the antioxidant potential of the synthesized com-
pounds was assessed by several different assays to study a wider
spectrum of scavenging properties. The results obtained were
compared to well-known antioxidants agents such as nordihydro-
guaeretic acid (NDGA), trolox, and caffeic acid. Most of the experi-
mental procedures required a spectrophotometric measurement and
a certain reaction time to obtain reproducible results.
Synthesis of series 6 was carried out by a Claisen-Schmidt conden-
sation between the corresponding 6,7-substituted 2-acetyl-3-methyl
quinoxaline VIII and 3,4,5-trimethoxy-benzaldehyde using 3%
sodium hydroxide in methanol (Scheme 2). Starting compounds VIII
were obtained by reduction of the N-oxide groups (23) of com-
pounds VII (24) with Na2S2O4. While the reaction to obtain series
1 was performed at low temperature (18), it was possible to carry
out the condensation at room temperature to obtain compounds of
series 6, resulting in much better yields.
DPPH interaction
One such method that is currently popular is based upon the use of
1,1-diphenyl-2-picrylhydrazyl (DPPH). DPPH is a stable free radical
with a spare electron, which is delocalized over the whole mole-
cule. The delocalization causes deep violet color with kmax around
517 nm. When an ethanolic solution of DPPH is mixed with that of
a compound that can donate a hydrogen atom, then this gives rise
to the reduced form with the loss of the characteristic color. This
interaction indicates radical scavenging ability in an iron-free sys-
tem. Our compounds were examined for their DPPH interaction at
0.05, 0.1, and 0.2 mM after 20 and 60 min (Table 1).
Cyclization of the a,b-unsaturated ketone system in compounds of
series 6 yielded compounds of series 7, 8, and 9 (Scheme 2). For-
mation of a six-membered ring (series 7) was carried out in an
isopropanol reflux in the presence of guanidine and KOH as the
base. Reaction between compounds of series 6 and hydrazine gave
compounds of series 8, and in the same way compounds of series
2 reacted with hydrazine to afford compounds of series 10. The
conventional method to synthesize 3,5-substituted 1-(4-sulfamylphe-
nyl) pyrazolines (compounds 9) consists of an 8-h ethanol reflux
(25). We optimized a microwave-assisted method for the prepara-
tion of series 9, and we managed to reduce the volume of sol-
Compounds of series 2, 3, 8, and 10 (2e, 2f, 3c, 3e, 3f, 8c, 8f,
and 10a) showed the best DPPH interaction percentage values;
some of them displaying similar values (70–72%) to that of the ref-
erence compound NDGA (83%) at the same concentration. These
vent, the reaction times and, as
secondary reactions.
a consequence, possible
262
Chem Biol Drug Des 2011; 77: 255–267

Synthesis, Antioxidant and Anti-inflammatory Activities of New Quinoxaline Derivatives
O–
N+
O
O
O–
N+
R7
R6
R7
R6
N
N
R6
R5
b
a
O
N+
N
O–
I
VIII
VII
c
O
O
O
R7
R6
N
N
O
Series 6
f
d
e
O
NH2
S
O
NH2
N
H
O
O
N
N
N
N
N
O
O
R7
R6
N
N
R7
R6
N
N
R7
R6
N
N
O
O
O
O
O
Series 9
Series 7
Series 8
Scheme 2: Synthesis of series 6, 7, 8, and 9. (a) Pentane-2,4-dione, triethylamine (b) Na2S2O4, methanol, 70 ꢁC; (c) 3,4,5-trimethoxy-
benzaldehyde, 3% NaOH ⁄ methanol, r.t; (d) guanidine hydrochloride, 10% KOH ⁄ isopropanol reflux, 24 h; (e) NH2NH2, ethanol, r.t.; (f) 4-hydrazi-
nobenzene-1-sulfonamide hydrochloride 97%, ethanol, 20 min, 50 W (MW).
compounds have either a phenolic group or a free amino pyrazoline
ring in their structure, so they are able to donate a hydrogen atom.
The most interesting derivatives were those with the pyrazoline
moiety (compounds 3c, 3e, 3f, 8c, 8f, and 10a) and among
them, those with N-oxide groups in the quinoxaline ring (3c, 3e,
3f, and 10a) exhibited significantly increased activity compared to
their reduced analogs (8c and 8f). Thus, the presence of the
N-oxide groups in these compounds might increase the hydrogen
donation ability of the amino group making easier the release of
this atom and so increasing their scavenging activity. The presence
of both NH and OH groups in the same molecule (compound 10a)
seemed to strengthen its antioxidant character.
In the same way as DPPH interaction, the best activities in the
ABTS assay (Table 1) were shown by compounds of series 2, 3, 8,
and 10 (2e, 2f, 3c, 3e, 3f, 8c, 8f, and 10a) with a phenolic
group and ⁄ or a free amino pyrazoline ring in the structure. Never-
theless, these activities seemed to have the opposite trend of that
showed in the DPPH assay, as the best derivatives were those with
a phenol group (compounds 2e and 2f) followed by compounds of
series 8 (8c and 8f) and series 3 (3c, 3e, and 3f). Surprisingly,
compounds of series 6 (6a–f) showed quite good values of ABTSÆ+
scavenging ability as well as compound 7b that displayed an activ-
ity similar to that of compounds of series 3.
For compounds 2e, 2f, 3c, 3e, and 3f, the interaction values were
found to be time- and concentration-dependent.
Superoxide radical anion (·O2)) and hydroxyl
radical (·OH) scavenging activity
The ability of the compounds to scavenge superoxide radical
anion and hydroxyl radical was also evaluated. Superoxide anion
is considered to be the 'primary' ROS that initiates the generation
of other ROS such as hydrogen peroxide and hydroxyl radicals
(27). These toxic oxygen species are involved in the oxidative
damage to DNA, proteins, and other macromolecules leading to
the development of several degenerative diseases associated with
aging (28).
ABTS assay
Another existing method to evaluate antioxidant activity is the
ABTS radical cation decolorization assay (26). The pre-formed radi-
cal monocation of 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulfonic
ˇ
ˇ
+
acid) (ABTS. ) is generated by oxidation of ABTS with potassium
persulfate and reduced in the presence of hydrogen-donating
antioxidants. The decolorization of the blue ⁄ green solution (due to
the ABTS. color) after the treatment with the compound is indica-
tive of its scavenging ability.
ˇ
ˇ
+
Generation of non-enzymatic superoxide anion radicals was carried
out by mixing phenazine methosulfate (PMS), nicotinamide adenine
Chem Biol Drug Des 2011; 77: 255–267
263

Burguete et al.
Table 1: Interaction percentage with DPPH (DPPH %) at 0.05, 0.1, and 0.2 mM; antioxidant determination using the ABTS cation radical-
percentage inhibition (ABTS %); superoxide radical scavenging activity (PMS %); competition percentage with DMSO for hydroxyl radical (OH
%); inhibition of linoleic acid lipid peroxidation induced by AAPH (AAPH)
DPPH %
0.05 mM
20 min
ABTS %
PMS %
OH %
AAPH
0.1 mM
20 min
0.2 mM
20 min
AAPH %
at 0.1 mM
IC50
(mM)
Compound
60 min
60 min
60 min
0.1 mM
0.01 mM
0.1 mM
0.1 mM
1b
1e
NR
6.5
2.4
11.4
15.9
58.5
44.1
24.9
NR
7.4
3.2
0.7
NR
0.3
0.6
3.7
3.3
NR
NR
6.0
2.0
7.0
5.0
8.0
3.0
1.7
6.3
NR
0.6
0.4
11.7
13.4
5.1
NR
NR
NR
49.2
68
NR
0.8
NR
12.0
18.8
65.9
54.9
41.3
NR
6.9
3.9
NR
NR
NR
1.1
4.5
1.8
NR
NR
4.0
4.0
3.0
6.0
3.0
3.0
7.3
8.9
NR
1.7
2.9
22.5
23.5
4.4
NR
NR
NR
60.6
72
1.4
2.5
NR
28.7
32.2
58.5
40.6
53.3
7.5
NR
3.9
2.4
0.6
2.9
0.8
2.1
NR
NR
NR
1.0
2.0
3.0
7.0
NR
6.0
4.3
10.7
NR
NR
NR
19.2
24.6
6.7
NR
NR
NR
62.7
81
2.3
4.3
NR
37.7
41.4
70.1
55.5
62.5
11.9
3.3
6.0
NR
2.9
1.6
3.0
NR
2.5
4.0
NR
1.0
2.0
NR
2.0
NR
1.0
0.3
14.6
0.6
NR
1.3
37.3
42.9
8.0
NR
NR
NR
72.4
83
0.4
NR
NR
39.7
50.8
65.5
55.7
89.6
2.8
NR
NR
NR
1.0
NR
4.4
NR
NR
NR
NR
NR
1.0
2.0
3.0
5.0
5.0
5.5
13.0
NR
NR
NR
26.4
35.8
10.8
NR
NR
NR
83.3
–
1.7
NR
NR
50.1
62.9
78.7
66.8
94.0
NR
NR
NR
NR
3.6
2.4
NR
1.1
NR
0.1
NR
NR
2.0
4.0
5.0
3.0
5.0
3.9
18.8
NR
0.9
2.2
45.0
52.5
6.4
NR
NR
1.0
87.7
–
19.2
2.1
8.8
92.3
84.3
48.3
40.2
50.5
NR
NR
11.1
NR
9.1
NR
20.0
–
–
NR
–
–
–
–
–
–
–
–
–
–
–
50.0
–
–
–
81.1
51.4
52.8
52.8
97.2
81.1
–
–
–
–
–
–
–
–
–
–
–
50.0
–
–
75.0
NR
NR
62.5
6.3
NR
NR
NR
NR
25.0
NR
NR
NR
57.1
50.0
96.4
NR
NR
50.0
79.0
100
100
100
100
78.9
NR
50.0
NR
31.3
NR
NR
NR
NR
NR
NR
NR
68.8
–
99.7
92.5
91.5
NR
65.3
53.0
35.3
24.3
28.2
59.0
29.0
43.8
NR
0.091
0.100
–
–
–
1f
2e
2f
3c
95.9
94.4
96.0
99.6
94.5
96.3
97.8
87.6
94.6
98.5
98.4
96.6
95.2
97.6
96.9
91.0
87.0
84.0
85.0
85.0
92.0
NR
0.084
3e
–
–
–
–
–
–
–
–
–
–
–
–
–
–
3f
4a
4b
4c
NR
NR
3.6
4d
4e
13.0
18.2
11.2
6.8
10.1
22.6
9.0
50.5
100
45.0
21.5
100
45.4
97.1
92.3
62.0
73.4
74.5
2.9
75.9
99.8
71.9
8.5
78.9
90.2
–
63.0
–
95.8
4f
5a
12.1
NR
10.0
NR
5b
5d
5e
5g
6.5
6a
6b
39.3
81.0
80.6
79.7
72.0
55.2
18.1
40.6
NR
NR
4.3
74.2
76.1
2.6
NR
10.1
5.8
71.6
–
88
0.010
–
–
<0.010
–
6c
6d
6e
6f
7a
0.053
0.073
0.094
0.078
0.089
–
0.078
0.023
0.072
–
0.077
0.074
–
–
–
7b
7c
NR
77.7
91.8
NR
7d
7e
8c
NR
8f
9a
82.6
92.9
97.4
92.6
87.0
98.3
–
88.2
–
–
9b
9c
9d
10a
NDGA
Trolox
Caffeic acid
Ascorbic acid
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
45
–
–
NR, no response; NDGA, nordihydroguaiaretic acid; –, not tested.
Each experiment was performed at least in triplicate, and the standard deviation of absorbance was <10% of the mean; significant differences from control
values.
*p < 0.05.
dinucleotide (NADH), and air-oxygen. The production of superoxide
was estimated by the nitroblue tetrazolium (NBT) method (29). As
shown in the Table 1, compounds with an a,b-unsaturated ketone
system (1b, 2e, 4f, 5a, 5b, 5g and 6a–f) presented the best
superoxide scavenging activities (higher than that of caffeic acid
used as the reference compound) being the reduced derivatives (6a–
f) the most interesting structures with interaction values between 79
and 100%. This evidence led us to affirm that the olefinic moiety
might play an important role in the activity of these compounds by
trapping the superoxide radical. The presence of the N-oxide groups
in the molecules greatly decreases their scavenging ability.
The competition of compounds with dimethyl sulfoxide (DMSO) for
.OH radicals, generated by the Fe3+ ⁄ ascorbic acid system and
ˇ
ˇ
264
Chem Biol Drug Des 2011; 77: 255–267

Synthesis, Antioxidant and Anti-inflammatory Activities of New Quinoxaline Derivatives
expressed as the inhibition of formaldehyde production, was used
Table 2: Inhibition percentage of induced carrageenin rat paw
edema (CPE %) at 0.01 mmol ⁄ kg; in vitro inhibition of soybean lip-
oxygenase (LOX) inhitibion % at 0.1 mM ⁄ IC50 (mM)
for the evaluation of their hydroxyl radical scavenging activity. Most
of the tested compounds exhibited high competition percentage val-
ues at 0.1 mM (Table 1), similar to that of Trolox used as the refer-
ence compound, which indicates that these derivatives are good
hydroxyl radical scavengers.
LOX
Inhibition %
at 0.1 mM
Compound
CPE %a
IC50 (mM)
Clog Pb
1b
1e
1f
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
57.8
35.4
36.7
12.2
26.0
38.4
51.0
50.4
30.0
No
26.6
40.3
40.4
18.7
4.0
0.090
–
–
–
–
)0.01
0.20
0.64
0.08
0.53
)0.33
)0.09
0.35
0.17
0.46
0.43
1.03
0.67
1.12
0.29
0.58
1.15
0.79
0.71
2.49
2.60
2.82
3.17
3.37
2.93
3.00
3.14
3.19
3.71
3.50
2.53
3.08
3.90
4.07
4.29
4.64
)0.71
–
Inhibition of linoleic acid lipid peroxidation
Lipid peroxidation in the cell membrane has been proposed to be
a major mechanism for several pathological events including can-
cer, Parkinson's disease, and aging (30). The ability of the com-
pounds to inhibit lipid peroxidation was measured (31). Production
of conjugated diene hydroperoxide by oxidation of linoleic acid in
an aqueous dispersion was monitored at 234 nm. 2,2¢-Azobis(2-
amidinopropane) dihydrochloride (AAPH) was used as a free radical
initiator.
2e
2f
3c
–
3e
3f
0.095
0.100
–
–
–
–
–
–
–
–
–
–
–
0.140
0.200
0.320
0.350
0.088
0.410
0.050
0.023
–
0.089
0.036
–
0.042
–
0.037
–
0.043
0.092
0.600
–
4a
4b
4c
4d
4e
4f
The results (Table 1) showed that compounds 6b and 6e were
the best inhibitors of lipid peroxidation displaying IC50 values of
0.01 and <0.01 mM respectively. Compounds of series 7 (7a, 7b,
7d, and 7e) and 9 (9b and 9d), having a free amino group in
their structure, also presented good inhibition activities, especially
those with a hydrogen atom (7a and 9a) in position R7 of the
quinoxaline ring. Replacement of the hydrogen by a fluoro atom
(7b and 9b) resulted in a decrease in the activity, although flu-
oro derivatives showed also interesting inhibition values of lipid
peroxidation.
5a
5b
5d
5e
5g
6a
6b
6c
19.7
2.4
5.0
23
NR
31.0
29.0
NR
75.0
NR
77.0
71.7
21.6
55.4
66.8
37.9
68.2
3.0
6d
6e
6f
7a
7b
7c
–
Inhibition of soybean lipoxygenase
41.3*
–
–
–
–
28.2*
–
–
–
–
The assay for LOX activity was carried out according to the UV
absorbance-based enzyme assay (32,33) using soybean LOX. While
one may not extrapolate the quantitative results of this assay to
the inhibition of mammalian 5-LOX, it has been shown that inhibi-
tion of plant LOX activity by NSAIDs is qualitatively similar to their
inhibition of the rat mast cell LOX and may be used as a simple
qualitative screen for such activity.
7d
7e
8c
8f
9a
9b
9c
78.0
NR
52.8
59.4
–
9d
10a
Caffeic acid
IMA
In general, compounds that displayed good activities as inhibitors
of lipid peroxidation also presented good values of inhibition of
LOX (Table 2). Thus, the best IC50 values were shown by compounds
of series 7 and 9 (7b > 7e > 9b > 8f > 9d > 7a). From series
8, only compound 8f presented significant activity. Nevertheless,
while the best inhibitors of lipid peroxidation were those com-
pounds without any substitution in the quinoxaline ring, the most
interesting activities of LOX inhibition were obtained by fluoro- and
methyl-substituted derivatives.
–
–
47*
–
–
NR, no response under the experimental conditions; –, not tested; each
experiment was performed at least in triplicate, and the standard deviation
of absorbance was <10% of the mean; significant differences from control
values are on the level of *p < 0.05; IMA, indomethacin.
aStatistical studies for the in vivo results were done with student's t-test,
*p < 0.01.
bhttp://www.biobyte.com. Biobyte Corp., C-QSAR Database 201 West 4th
Str., Suite 204, Claremont CA, California 91711, USA.
In vivo anti-inflammatory activity
Two compounds, 7b (the most potent LOX inhibitor) and 8f (the
most potent from series 8), were tested as in vivo anti-inflamma-
tory agents. In acute toxicity experiments, the in vivo examined
compounds 7b and 8f did not present toxic effects in doses up to
0.2 mmol ⁄ kg body weight. Ulcerogenicity was not found. Acute
inflammation is attributable to the release of chemical mediators,
which cause edema as a result of extravasations of fluid and
proteins from the local microvasculature and accumulation of poly-
morphonuclear leukocytes at the inflammatory site. The in vivo
anti-inflammarory effects of the tested quinoxaline derivatives were
assessed by using the carrageenin-induced rat paw edema (CPE)
model and are presented in Table 2 as percentage of weight
increase at the right hind paw. The induced edema is a non-specific
inflammation highly sensitive to non-steroidal anti-inflammatory
Chem Biol Drug Des 2011; 77: 255–267
265

Burguete et al.
agents (NSAIDs). Thus, it has been accepted as a useful tool for
studying new anti-inflammatory agents (34). It reliably predicts the
anti-inflammatory potency of the NSAIDs and detects during the
second phase that are anti-inflammatory agents as a result of inhi-
bition of prostaglandin amplification (35). Compound 7b showed
41.3% percentage of protection, while the reference drug IMA
induced 47% protection at an equivalent dose. Compound 8f was
less potent (28.2%). No role for lipophilicity was found within these
two coompounds. Both derivatives presented similar Clog p values
(Table 2).
promoter genotype, dietary arachidonic acid, and atherosclerosis.
N Engl J Med;350:29–37.
3. Hennig R., Ding X., Tong W., Schneider M.B., Standop J., Friess
H., Bꢀchler M.W., Pour P.M., Adrian T.E. (2002) 5-lipoxygenase
and leukotriene B4 receptor are expressed in human pancreatic
cancers but not in pancreatic ducts in normal tissue. Am J
Pathol;161:421–428.
4. Yoshimura R., Matsuyama M., Tsuchida K., Kawahito Y., Sano
H., Nakatani T. (2003) Expression of lipoxygenase in human
bladder carcinoma and growth inhibition by its inhibitors. J Urol;
170:1994–1999.
5. Jiang W.G., Douglas-Jones A., Mansel R.E. (2003) Levels of
expression of lipoxygenases and cyclooxygenase-2 in human
breast cancer. Prostaglandins Leukot Essent Fatty Acids;69:275–
281.
6. Franco R., Schoneveld O., Georgakilas A.G., Panayiotidis M.I.
(2008) Oxidative stress, DNA methylation and carcinogenesis.
Cancer Lett;266:6–11.
7. Dhalla N.S., Temsah R.M., Netticadan T. (2000) Role of oxidative
stress in cardiovascular diseases. J Hypertens;18:655–673.
8. Chen L., Liu L., Yin J., Luo Y., Huang S. (2009) Hydrogen perox-
ide-induced neuronal apoptosis is associated with inhibition of
protein phosphatase 2A and 5, leading to activation of MAPK
pathway. Int J Biochem Cell Biol;41:1284–1295.
9. Halliwell B., Hoult J.R., Blake D.R. (1988) Oxidants, inflamma-
tion, and anti-inflammatory drugs. FASEB J;2:2867–2873.
10. Weiss S.J. (1989) Tissue destruction by neutrophils. N Engl J
Med;320:365–376.
11. Udassin R., Ariel I., Haskel Y., Kitrossky N., Chevion M. (1991)
Salicylate as an in vivo free-radical trap: studies on ischemic
insult to the rat intestine. Free Radic Biol Med;10:1–6.
12. Tsujimoto Y., Saitoh K., Kashima M., Shiozawa A., Kozuka M.,
Hashizume H., Kimura K., Yamazaki M., Fujii A. (1998) Effect of
nonsteroidal anti-inflammatory drugs on lipid peroxidation by
hydroxyl radical. Gen Pharmacol;31:405–408.
13. Maharaj H., Maharaj D.S., Daya S. (2006) Acetylsalicylic acid
and acetaminophen protect against oxidative neurotoxicity.
Metab Brain Dis;21:189–199.
Conclusions
Several new quinoxaline and quinoxaline 1,4-di-N-oxide derivatives
have been synthesized with the aim of studying their antioxidant
and anti-inflammatory activities. We have optimized the synthesis
of some of these derivatives by using microwave-assisted methods
that greatly improved reaction times and conversion ratios.
In terms of biological activity, the most interesting derivatives were
those with the pyrazoline moiety (series 3, 8, and 10), and among
them those with N-oxide groups in the quinoxaline ring (series 3
and 10) exhibited significantly increased reducing activity compared
to their reduced analogs (series 8). Compounds with an a,b-unsatu-
rated ketone system (1b, 2e, 4f, 5a, 5b, 5g, and 6a–f) pre-
sented the best superoxide scavenging activities being the reduced
derivatives (6a–f) the most interesting structures with interaction
values between 79 and 100%. This evidence led us to affirm that
the olefinic moiety might play an important role in the activity of
these compounds by trapping the superoxide radical. The derivatives
are good hydroxyl radical scavengers. Compounds 6b and 6e were
the best inhibitors of lipid peroxidation displaying IC50 values of
0.01 and <0.01 mM respectively. In general, compounds that dis-
played good activities as inhibitors of lipid peroxidation also pre-
sented good values of inhibition of LOX. Compound 7b showed
significant protection against carragenin-induced paw edema.
14. Facino R.M., Carini M., Aldini G., Saibene L., Morelli R. (1995)
Differential inhibition of superoxide, hydroxyl and peroxyl radi-
cals by Nimesulide and its main metabolite 4-hydroxynimesulide.
Arzneim Forsch Drug Res;45:1102–1109.
15. Vicente E., Villar R., Burguete A., Solano B., Perez-Silanes S.,
Aldana I., Maddry J.A., Lenaerts A.J., Franzblau S.G., Cho S.,
Monge A., Goldman R.C. (2008) Efficacy of quinoxaline-2-carbox-
ylate 1,4-di-N-oxide derivatives in experimental tuberculosis.
Antimicrob Agents Chemother;52:3321–3326.
16. Zarranz B., Jaso A., Aldana I., Monge A. (2004) Synthesis and
anticancer activity evaluation of new 2-alkylcarbonyl and 2-ben-
zoyl-3-trifluoromethyl-quinoxaline 1,4-di-N-oxide derivatives. Bio-
org Med Chem;12:3711–3721.
Acknowledgments
This work has been carried out thanks to the financial support of
the FIS Project PI080817. The authors are grateful to Drs C. Hansch
and A. Leo and to Biobyte Corp. for the free access to the C-QSAR
program. Asunciꢃn Burguete was awarded a PhD fellowship
supported by the 'Gobierno de Navarra'. Eleni Pontiki is grateful to
the 'Foundation for Education and European Culture' for financial
support during PostDoc research.
References
17. Burguete A., Estevez Y., Castillo D., Gonzalez G., Villar R., Solan-
o B., Vicente E., Pꢄrez S., Aldana I., Monge A., Sauvain M.,
Deharo E. (2008) Anti-leishmanial and structure-activity relation-
ship of ring substituted 3-phenyl-1-(1,4-di-N-oxide quinoxalin-
2-yl)-2-propen-1-one derivatives. Mem Inst Oswaldo Cruz;103:
778–780.
1. Claesson H.E., Dahlen S.E. (1999) Asthma and leukotrienes: anti-
leukotrienes as novel anti-asthmatic drugs. J Intern Med;245:
205–227.
2. Dwyer J.H., Allayee H., Dwyer K.M., Fan J., Wu H., Mar R.,
Lusis A.J., Mehrabian M. (2004) Arachidonate 5-Lipoxygenase
266
Chem Biol Drug Des 2011; 77: 255–267

Synthesis, Antioxidant and Anti-inflammatory Activities of New Quinoxaline Derivatives
18. Burguete A., Pontiki E., Hadjipavlou-Litina D., Villar R., Vicente
radical cation decolorization assay. Free Radic Biol Med;26:
1231–1237.
27. Valko M., Leibfritz D., Moncol J., Cronin M.T.D., Mazur M.,
Telser J. (2007) Free radicals and antioxidants in normal physio-
logical functions and human disease. Int J Biochem Cell Biol;
39:44–84.
28. Ames B.N., Shigenaga M.K., Hagen T.M. (1993) Oxidants, anti-
oxidants, and the degenerative diseases of aging. Proc Natl
Acad Sci USA;90:7915–7922.
E., Solano B., Ancizu S., Pꢄrez-Silanes S., Aldana I., Monge A.
(2007) Synthesis and anti-inflammatory ⁄ antioxidant activities of
some new ring substituted 3-phenyl-1-(1,4-di-N-oxide quinoxalin-
2-yl)-2-propen-1-one derivatives and of their 4,5-dihydro-(1H)-pyr-
azole analogues. Bioorg Med Chem Lett;17:6439–6443.
19. Monge A., Palop J.A., De Cerain A.L., Senador V., Martinez
Crespo F.J., Sainz Y., Narro S., Garcia E., Demiguel C., Gonzalez
M., Hamilton E., Barker A.J., Clarke E.D., Greenhow D.T. (1995)
Hypoxia-selective agents derived from quinoxaline 1,4-di-N-oxi-
des. J Med Chem;38:1786–1792.
29. Pontiki E., Hadjipavlou-Litina D. (2006) Antioxidant and anti-
inflammatory activity of aryl-acetic and hydroxamic acids as
novel lipoxygenase inhibitors. Med Chem;2:251–264.
30. Halliwell B., Gutteridge J.M.C. (1984) Oxygen-toxicity, oxygen
radicals, transition-metals and disease. Biochem J;219:1–14.
31. Liegeois C., Lermusieau G., Collin S. (2000) Measuring antioxidant
efficiency of wort, malt, and hops against the 2,2¢-azobis(2-amidi-
nopropane) dihydrochloride-induce oxidation of an aqueous disper-
sion of linoleic acid. J Agric Food Chem;48:1129–1134.
32. Taraporewala I.B., Kauffman J.M. (1990) Synthesis and structure-
activity-relationships of anti-inflammatory 9,10-dihydro-9-oxo-2-
acridine-alkanoic acids and 4-(2-carboxyphenyl)aminobenzenealka-
noic acids. J Pharm Sci;79:173–178.
33. Pontiki E., Hadjipavlou-Litina D. (2007) Synthesis and pharmaco-
chemical evaluation of novel aryl-acetic acid inhibitors of lipoxy-
genase, antioxidants, and anti-inflammatory agents. Bioorg Med
Chem;15:5819–5827.
34. Winter C.A., Garattini S., Dukes M.N.G. (1965) Non-Steroidal
Anti-Inflammatory Drugs. Amsterdam: Excepta Medica.
35. Kuroda T., Suzuki F., Tamura T., Ohmori K., Hosoe H. (1992)
A novel synthesis and potent antiinflammatory activity of
4-hydroxy-2(1h)-oxo-1-phenyl-1,8-naphthyridine-3-carboxamides. J
Med Chem;35:1130–1136.
20. Monge A., Gil M.J., Gastelurrutia M.A., Pascual M. (1982) Sinte-
sis y actividad bactericida de 1,4-dioxidos de 2-acil o aroilqui-
noxalin etilenos y derivados pirazolicos. An Real Acad Farm;48:
533–542.
21. Johnston J.D. (1968) U.S. Patent 3371090.
22. Haddadin M.J., Issidorides C.H. (1976) Application of benzofura-
zan oxide to synthesis of heteroaromatic N-oxides. Hetero-
cycles;4:767–816.
23. Solano B., Junnotula V., Marin A., Villar R., Burguete A., Vicente
E., Perez-Silanes S., Aldana I., Monge A., Dutta S., Sarkar U.,
Gates K.S. (2007) Synthesis and biological evaluation of new
2-arylcarbonyl-3-trifluoromethylquinoxaline 1,4-di-N-oxide deriva-
tives and their reduced analogues. J Med Chem;50:5485–5492.
24. Jaso A., Zarranz B., Aldana I., Monge A. (2003) Synthesis of
new 2-acetyl and 2-benzoyl quinoxaline 1,4-di-N-oxide deriva-
tives as anti-Mycobacterium tuberculosis agents. Eur J Med
Chem;38:791–800.
25. Reddy M.V., Billa V.K., Pallela V.R., Mallireddigari M.R., Boomi-
nathan R., Gabriel J.L., Reddy E.P. (2008) Design, synthesis, and
biological evaluation of 1-(4-sulfamylphenyl) 3-trifluoromethyl-
5-indolyl pyrazolines as cyclooxygenase-2 (COX-2) and lipoxygen-
ase (LOX) inhibitors. Bioorg Med Chem;16:3907–3916.
36. Kim H.K., Miller L.F., Bambury R.E., Ritter H.W. (1977) Nitrones.
7. alpha.-Quinoxalinyl-N-substituted nitrone 1,4-dioxides. J Med
Chem;20:557–560.
26. Re R., Pellegrini N., Proteggente A., Pannala A., Yang M., Rice-
Evans C. (1999) Antioxidant activity applying an improved ABTS
Chem Biol Drug Des 2011; 77: 255–267
267