Synthesis of new quinoxaline-2-carboxylate 1,4-dioxide derivatives as anti-Mycobacterium tuberculosis agents

Article abstract of DOI:10.1021/jm049952w

Twenty-nine new 6(7)-substituted quinoxaline-2-carboxylate 1,4-dioxide derivatives were synthesized and evaluated for in vitro antituberculosis activity. In general, the in vitro activity is significantly affected by substituents on the quinoxaline nucleu

Full text of DOI:10.1021/jm049952w

J. Med. Chem. 2005, 48, 2019-2025
2019
Synthesis of New Quinoxaline-2-carboxylate 1,4-Dioxide Derivatives as
Anti-Mycobacterium tuberculosis Agents
Andre´s Jaso, Bele´n Zarranz, Ignacio Aldana,* and Antonio Monge
Unidad en Investigacio´n y Desarrollo de Medicamentos, Centro de Investigacio´n en Farmacobiologı´a Aplicada (CIFA),
Universidad de Navarra, c/Irunlarrea s/n, 31080 Pamplona, Spain
Received January 16, 2004
Twenty-nine new 6(7)-substituted quinoxaline-2-carboxylate 1,4-dioxide derivatives were
synthesized and evaluated for in vitro antituberculosis activity. In general, the in vitro activity
is significantly affected by substituents on the quinoxaline nucleus. It has been observed that
the presence of a chloro, methyl, or methoxy group in position 7 of the benzene moiety reduces
the MIC and IC50 values. However, antituberculosis activity principally depends on the
substituents in the carboxylate group, improving in the following order: benzyl > ethyl >
2-methoxyethyl > allyl > tert-butyl. Fourteen compounds have been selected for macrophage
assay, and the results show that ethyl and benzyl 3-methylquinoxaline-2-carboxylate 1,4-dioxide
derivatives with the chlorine group in position 7 of the benzene moiety (compounds 10 and 26)
and the unsubstituted derivatives (compounds 11 and 27) have good antitubercular activity,
including activity in macrophages. In addition, compounds 7 and 28 (the only ones tested up
to now) are active against drug-resistant strains of M. tuberculosis H37Rv. In conclusion, the
potency, selectivity, and low cytotoxicity of these compounds make them valid leads for
synthesizing new compounds that possess better activity.
Introduction
Tuberculosis (TB), an infection of Mycobacterium
tuberculosis, still remains the leading cause of world-
wide death among infectious diseases. The statistics
indicate that 3 million people throughout the world die
annually from tuberculosis,1,2 and there are an esti-
mated 8 million new cases each year, 95% of which occur
in developing countries.3 One-third of the population is
infected with M. tuberculosis and the World Health
Organization (WHO) estimates that within the next 20
years about 30 million people will be infected with the
bacillus.4 Because of the fact that the current frontline
therapy for TB consists of administering three different
drugs (isoniazid, rifampin, and pyrazinamide) during
an extended period of time5 as well as the problems that
arise due to MDR-TB (multidrug-resistant tuberculosis),
it is necessary to develop new therapeutic agents that
have a unique mechanism of action, different from those
of the currently used antitubercular drugs, to treat drug-
resistant forms of the disease.6
The quinoxaline derivatives show very interesting
biological properties (antibacterial, antiviral, anticancer,
antifungal, antihelmintic, insecticidal).7,8 Over the past
2 decades, many mono- and di-N-oxides and 2-oxo
derivatives of this heterocyclic system have been pre-
pared and their biological activities have been reported.
Some quinoxalin-2-ones have evidenced antifungal ac-
tivity,9,10 whereas the quinoxaline 1-oxides have shown
antibacterial activity.11 Oxidation of both nitrogens of
the quinoxaline ring dramatically increases the diversity
of biological properties such as antibacterial activity,12-15
promotion of animal growth in feed additives,16-18
hypoxia-selective activity,19 etc.
Figure 1. General structure of 3-methyl-2-carbonylquinoxa-
line 1,4-dioxide.
Several papers have been published in which both
synthesis and biological activity assessments of a large
number of quinoxaline and quinoxaline 1,4-dioxide
derivatives have been described. Different 2-acetyl and
2-benzoyl-3-methylquinoxaline 1,4-dioxide20 (I, Figure
1) and N-aryl-3-methylquinoxaline-2-carboxamide 1,4-
dioxides (II, Figure 1) derivatives showed high lead
activity.21 On the other hand, we observed that the lack
of the two N-oxide groups generally led to the loss of
the antimycobacterial activity.22,23
As a result of this research and for the purpose of
obtaining new and more potent antitubercular com-
pounds that can improve the current chemotherapeutic
antituberculosis treatments, we have synthesized and
evaluated 29 new quinoxaline-2-carboxylate 1,4-dioxide
derivatives possessing methyl, ethyl, benzyl, and tert-
butyl groups in position 2 and substituents such as
methyl or 2-ethoxy-2-oxoethyl in position 3 (III, Figure
1).
Chemistry
The aforementioned compounds were prepared ac-
cording to the synthetic sequences illustrated in Figure
* To whom correspondence should be addressed. Telephone: +34
948 425653. Fax: +34 948 425652. E-mail: ialdana@unav.es.
10.1021/jm049952w CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/28/2004

2020 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Jaso et al.
Figure 2. Synthetic route for compounds 1-29.
2. The starting compound (benzofuroxane, 5-substituted
or 5,6-disubstituted benzofuroxane) was obtained by
previously described methods.23
lent M. tuberculosis. Biological tests have been per-
formed according to the previously described method.26,27
Results and Discussion
The synthesis of compounds 1-13, 15, 16, and 18-
29 was carried out by reaction of the appropriate
benzofuroxane with the corresponding â-ketoester, using
triethylamine as the catalyst. Formation of isomeric
quinoxaline 1,4-di-N-oxides was observed in the case of
monosubstituted benzofuroxanes. According to previous
reports,7 we have observed that 7-substituted quinoxa-
line 1,4-di-N-oxides prevailed over the 6-isomer or only
the 7-isomer formed in the case of a methoxy substitu-
ent. In practice, the workup and purification allow
isolation of the major isomer.24 Another synthetic route
was carried out with a molecular sieve as the catalyst
in order to obtain compounds 14 and 17.25
The results of the in vitro evaluation of antitubercu-
losis activity are reported in Tables 1-3. In general,
quinoxaline-2-carboxylate 1,4-dioxide derivatives have
been shown to possess good antimycobacterial activity
at the first level screening, with growth inhibition
values ranging from 92% to 100%. Only compounds 16-
19 (tert-butylquinoxalin-2-carboxylate 1,4-dioxide de-
rivatives) failed to show activity. In the case of com-
pound 21, the presence of 6,7-dimethyl groups showed
low activity (32% growth inhibition value).
Second level and cytotoxicity results are summarized
in Table 1. All of the compounds that were active at the
first level screening were then tested to determine the
actual MIC. MIC is defined as the lowest concentration
causing a 90% reduction in fluorescence with regard to
controls. A significant number of samples (5, 11, 12, 15,
and 26-29) showed high activity comparable to RMP
with MICs ranging from 0.05 to 0.20 µg/mL.
All of the compounds were chemically characterized
by thin-layer chromatography (TLC), melting point,
infrared (IR), and nuclear magnetic resonance (1H
NMR) spectra, as well as elemental microanalysis.
Pharmacology
Concurrent with the determination of MIC’s, com-
pounds were tested for cytotoxicity (IC50) in VERO cells,
and the selectivity index (SI ) IC50/MIC) was calculated.
The activity is significantly affected by substituents on
the quinoxaline nucleus. It has been observed that the
presence of a chloro, methyl, or methoxy group in
position 7 of the benzene moiety reduces both the MIC
and IC50 values. The results have shown that in general
antituberculosis activity depends on the substituents in
the carboxylate group, improving in the following or-
der: benzyl > ethyl > 2-methoxyethyl > allyl > tert-
butyl.
In vitro evaluation of the antituberculosis activity was
carried out at the GWL Hansen’s Disease Center within
the Tuberculosis Antimicrobial Acquisition & Coordi-
nating Facility (TAACF) screening program for the
discovery of novel drugs for the treatment of tubercu-
losis. Under the direction of the U.S. National Institute
of Allergy and Infectious Disease (NIAID), the Southern
Research Institute coordinates the overall program.
The purpose of the screening program is to provide a
resource whereby new experimental compounds can be
tested for their capability to inhibit the growth of viru-

Quinoxaline-2-carboxylate 1,4-dioxide Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2021
Table 1. Results of Second Level and Cytotoxicity Antituberculosis Assays
compd
Ra
Rb
Rc
Rd
MIC (µg/mL)a
IC50 (µg/mL)b
SI (IC50/MIC)c
1
2
CH3
CH3
OCH3
H
CH3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
3.13
1.56
>62.5
>62.5
52.58
>10
1.68
1.48
>62.5
>62.5
39.95
47
>20
>40.06
33.7
>6.41
8.4
3
H
1.56
4
H
Cl
F
1.56
5
Cl
0.20
6
F
0.39
3.8
7
CH3
CH3
OCH3
H
CH3
H
CH2CH3
6.25
>10
>40.06
6.4
8
CH2CH3
1.56
9
H
CH2CH3
6.25
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
H
CH2CH3
1.56
30.13
>50
>20
0.77
1.2
Cl
H
CH2CH3
0.20
>10
4
Cl
Cl
F
CH2CH3
<0.20
1.56
F
CH2CH3
1.2
Cl
H
CH2COOCH2CH3
CH2CH3
1.56
0.20
>6.25
>6.25
>6.25
>6.25
0.39
1.89
1.22
ND
Cl
Cl
CH3
H
CH2COOCH2CH3
CH2CH3
6.1
CH3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C(CH3)3
ND
C(CH3)3
ND
ND
Cl
H
C(CH3)3
ND
ND
Cl
Cl
Cl
CH3
Cl
CH3
H
C(CH3)3
ND
ND
Cl
CH3CH2OCH3
CH2CHdCH2
CH2CHdCH2
CH2C6H5
CH2C6H5
CH2C6H5
CH2C6H5
CH2C6H5
CH2C6H5
CH2C6H5
4.5
11.54
ND
CH3
Cl
>6.25
6.25
ND
5.8
0.93
CH3
CH3
OCH3
H
3.13
insoluble
56.05
>62.5
47
0.39
143.7
>80.1
470
H
0.78
H
0.10
Cl
H
0.10
7.6
76
Cl
Cl
F
>6.25
>6.25
ND
ND
F
ND
ND
a Actual minimum inhibitory concentration (MABA assay). MIC of rifampin: 0.015-0.125 µg/mL versus M. tuberculosis H37Rv.
b Measurement of cytotoxicity in VERO cells. c Selectivity index.
Table 2. Results of Macrophage Assay
their unsubstituted analogues, with a benzyl or ethyl
group in the carboxylate. All of the compounds tested
were active at this level and have been selected for in
vivo assays.
SDRMIC assay results are reported in Table 3.
Compounds 7 and 28 were evaluated against seven
drug-resistant strains of M. tuberculosis H37Rv. They
were active in all of the resistant strains.
compd
EC90 (µg/mL)a
EC99 (µg/mL)a
EC90/MICb
1
2
1.64
1.94
6.15
5.83
0.52
1.24
0.56
2.30
>3.15
>4.00
2.90
1.50
0.01
10
11
12
20
24
26
27
0.88
3.32
0.46
0.63
>1.56
1.13
0.15
1.14
5.99
>1.56
3.79
>2.00
0.08
The compounds that were active in Tables 2 and 3
were selected for in vivo assay.
0.0005
a The EC90 and EC99 are defined as the concentrations causing
90% and 99% reductions in residual mycobacterial growth after 7
Conclusions
days, compared to untreated controls. b Compounds with EC90
16 MIC are considered inactive.
>
Screening of the in vitro antimycobacterial activity
of these novel series, quinoxaline-2-carboxylate 1,4-
dioxides, has indicated that the ethyl and benzyl 3-
methylquinoxaline-2-carboxylate 1,4-dioxide derivatives
with a chlorine group in position 7 of the benzene moiety
(compounds 11 and 27) and the unsubstituted deriva-
tives (compounds 10 and 26) have emerged as new
compounds endowed with antitubercular activity, ex-
hibiting EC90/MIC values between 0.01 and 2.30. In
conclusion, it has been shown that the potency, selectiv-
ity, and low cytotoxicity of these compounds make them
valid leads for synthesizing new compounds that possess
better activity.
The best SI values have been obtained from unsub-
stituted compounds or from compounds having just one
substituent in position 7. The benzyl quinoxaline-2-
carboxylate 1,4-dioxide derivatives (24-27) have been
proven to be the most active, with SI values ranging
from 76 to 470.
Fourteen compounds (Tables 2 and 3) with a high SI
value were then selected for testing for in vitro efficacy
in a TB-infected macrophage model and for single-drug-
resistant minimun inhibitory concentration (SDRMIC)
assay. Macrophage assay results are reported in Table
2. On the basis of the data obtained, five compounds
stand out in the macrophage assay, e.g., 10, 26, and 27
(EC90/MIC ) 0.56, 1.50, and 0.01, respectively). They
are 7-chloroquinoxaline-2-carboxylate derivatives and
Experimental Section
General. Melting points were determined using a Mettler
FP82+FP80 apparatus (Greifense, Switzerland) and have not
been corrected. The 1H NMR spectra were recorded on a

2022 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Jaso et al.
Table 3. Results of SDRMIC Assay
compound 7
compound 28
(resistant strain MIC)/
(resistant strain MIC)/
assay
straina
MIC (µg/mL)
(H37Rv MIC)b
MIC (µg/mL)
(H37Rv MIC)
bactec
H37Rv
H37Rv
erdman
EMB-R
INH-R
RMP-R
ETA-R
TAC-R
CIP-R
0.20
0.39
0.39
0.78
1.56
0.39
0.78
0.78
0.39
0.39
6.25
e3.13
e3.13
e3.13
6.25
e3.13
6.25
e3.13
12.5
e3.13
alamar
alamar
alamar
alamar
alamar
alamar
alamar
alamar
alamar
2.00
4.00
1.00
2.00
2.00
1.00
1.00
1.00
g2.00
1.00
g2.00
1.00
g3.99
1.00
KM-R
a Strains resistant to the following drugs: EMB ) ethambutol; INH )i soniazid; RMP ) rifampin; ETA ) ethionamide; TAC )
thiacetazone; CIP ) ciprofloxacin; KM ) kanamycin. b In order for a compound to be considered active against a strain, the relationship
(resistant strain MIC)/(H37Rv MIC) should be less than 8.
Bruker AC-200E instrument (200 MHz) and Bruker 400
Ultrashield (400 MHz), using TMS as the internal standard
and with DMSO-d6 as the solvent. The chemical shifts are
reported in ppm (δ), and coupling constant (J) values are given
in hertz (Hz). Signal multiplicities are represented by s
(singlet), d (doublet), t (triplet), c (quadruplet), dd (double
doublet), and m (multiplet). The IR spectra were obtained from
a Perkin-Elmer 1600 FTIR (Norwalk, CT) in KBr pellets. The
frequencies are expressed in cm-1. Elemental microanalysis
results were obtained on an elemental analyzer (Carlo Erba
1106, Milan, Italy) from vacuum-dried samples. The analytical
results for C, H, and N were within (0.4 of the theoretical
values.
C3-CH3), 2.46 (s, 6H, 2CH3-Ar), 4.02 (s, 3H, COOCH3), 8.11
(s, 1H, H5), 8.15 (s, 1H, H8) ppm. Anal. C13H14N2O4 (C, H, N).
HPLC: ACN/propan-2-ol (50:50), tR ) 2.64 min.
Methyl 3,7-Dimethylquinoxaline-2-carboxylate 1,4-
Dioxide (2). This compound was obtained in 5% yield from
5-methylbenzofuroxane (0.36 g, 2.4 mmol) and methyl aceto-
acetate (1.23 g, 10.6 mmol) after 24 h under stirring (method
A): mp 125-126 °C; IR (KBr) ν 1743, 1330, 1285, cm-1 1H
;
NMR (200 MHz, DMSO-d6) δ 2.41 (s, 3H, C3-CH3), 2.57 (s,
3H, CH3-Ar), 4.01 (s, 3H, COOCH3), 7.82 (d, 1H, H6, J6-5
)
8.1 Hz), 8.28 (s, 1H, H8), 8.35 (d, 1H, H5, J5-6 ) 9 Hz) ppm.
Anal. C12H12N2O4‚1/4H2O (C, H, N). HPLC: ACN/propan-2-ol
(50:50), tR ) 2.64 min.
Alugram SIL G/UV254 (layer: 0.2 mm) (Macherey-Nagel
GmbH & Co. KG, Postfach 101352, D-52313 Du¨ren, Germany)
was used for thin layer chromatography, and silica gel 60
(0.040-0.063 mm) was used for column chromatography
(Merck). HPLC conditions were the following: column Nova
Pack C18 60 A 4 µm (3.9 mm × 150 mm); mobile phase,
acetonitrile/propan-2-ol (50:50) or acetonitrile/water (60:40);
flux, 1 mL/min.
Chemicals were purchased from E. Merck (Darmstadt,
Germany), Scharlau (F.E.R.O.S.A., Barcelona, Spain), Panreac
Qu´ımica S.A. (Montcada i Reixac, Barcelona, Spain), Sigma-
Aldrich Qu´ımica, S.A., (Alcobendas, Madrid), Acros Organics
(Janssen Pharmaceuticalaan 3a, 2440 Geel, Belgium), and
Lancaster (Bischheim-Strasbourg, France).
General Procedure for Compounds 1-29. Method A.
The corresponding â-ketoester (10.6 mmol) was added to a
solution of the appropriate benzofuroxane (2.4 mmol). The
mixture was allowed to stand at 0 °C. Triethylamine was
added dropwise (1 mL), and the reaction mixture was stirred
at room temperature in darkness for 1-3 days. After evapora-
tion to dryness under pressure, either a crude solid or a brown
oil was obtained. It was then precipitated and washed by
adding diethyl ether (or n-hexane), affording the target
compound. The obtained yellow precipitate was purified by
recrystallization from methanol. Flash column chromatogra-
phy on silica gel (flash chromatography) was applied when
necessary. Yields: 2-44%.
Methyl 7-Methoxy-3-methylquinoxaline-2-carboxylate
1,4-Dioxide (3). This compound was obtained in 2% yield from
5-methoxybenzofuroxane (0.40 g, 2.4 mmol) and methyl ace-
toacetate (1.23 g, 10.6 mmol) after 24 h under stirring. It was
purified by recrystallization from methanol/n-hexane (method
A): mp 146-147 °C; IR (KBr) ν 1749, 1330, 1253, 1177 cm-1
;
1H NMR (200 MHz, DMSO-d6) δ 2.37 (s, 3H, CH3), 3.95 (s,
3H, COOCH3), 3.99 (s, 3H, OCH3), 7.58 (dd, 1H, H6, J6-5
)
9.2 Hz, J6-8 ) 2.6 Hz), 7.68 (ds, 1H, H8, J8-6 ) 2.6 Hz), 8.36
(d, 1H, H5, J5-6 ) 9.2 Hz) ppm. Anal. C12H12N2O5‚1/4H2O (C,
H, N). HPLC: ACN/propan-2-ol (50:50), tR ) 2.36 min.
Methyl 3-Methylquinoxaline-2-carboxylate 1,4-Diox-
ide (4). This compound was obtained in 11% yield from
benzofuroxane (0.33 g, 2.4 mmol) and methyl acetoacetate
(1.23 g, 10.6 mmol) after 24 h under stirring. It was purified
by recrystallization from methanol/n-hexane (method A): mp
155-156 °C; IR (KBr) ν 1746, 1524, 1454, 1341, 1245 cm-1
;
1H NMR (200 MHz, DMSO-d6) δ 2.41 (s, 3H, CH3), 4.00 (s,
3H, COOCH3), 7.91-8.02 (m, 2H, H6 + H7), 8.38-8.48 (m, 2H,
H5 + H8) ppm. Anal. C11H10N2O4‚1/2H2O (C, H, N). HPLC:
ACN/propan-2-ol (50:50), tR ) 2.48 min.
Methyl 6,7-Dichloro-3-methylquinoxaline-2-carboxy-
late 1,4-Dioxide (5). This compound was obtained in 26%
yield from 5,6-dichlorobenzofuroxane (0.46 g, 2.24 mmol) and
methyl acetoacetate (2.47 g, 19.12 mmol) after 24 h under
stirring (method A): mp 201-202 °C; IR (KBr) ν 1749, 1327,
1
1260 cm-1; H NMR (200 MHz, DMSO-d6) δ 2.42 (s, 3H, C3-
CH3), 4.03 (s, 3H, COOCH3), 8.60 (s, 1H, H5), 8.64 (s, 1H, H8)
ppm. Anal. C11H8Cl2N2O4 (C, H, N). HPLC: ACN/H2O (60:40),
tR ) 3.80 min.
Method B. Molecular sieves [4Å (powder), 3 mmol] were
added to a solution of the corresponding â-ketoester (2.4 mmol)
and the appropriate benzofuroxane (2.4 mmol) in methanol
(100 mL). The solvent (methanol) was evaporated in an
evaporator at 20 °C. The molecular sieve containing the
adsorbed reagents was allowed to stand for 0.5-1 h without
drying, at 90 °C. The product was purified by means of flash
column chromatography on silica gel, using a gradient of
dichloromethane/methanol as the mobile phase. Yield: 3%.
Methyl 3,6,7-Trimethylquinoxaline-2-carboxylate 1,4-
Dioxide (1). This compound was obtained in 20% yield from
5,6-dimethylbenzofuroxane (1.00 g, 6.10 mmol) and methyl
acetoacetate (5.90 g, 50.83 mmol) after 24 h under stirring
(method A): mp 200-201 °C; IR (KBr) ν 1747, 1508, 1438,
1326, 1265 cm-1; 1H NMR (200 MHz, DMSO-d6) δ 2.40 (s, 3H,
Methyl 6,7-Difluoro-3-methylquinoxaline-2-carboxy-
late 1,4-Dioxide (6). This compound was obtained in 3% yield
from 5,6-difluorobenzofuroxane (0.40 g, 2.33 mmol) and methyl
acetoacetate (0.54 g, 4.18 mmol) after 24 h under stirring
(method A): mp 148-149 °C; IR (KBr) ν 1735, 1333, 1260,
1
1176 cm-1; H NMR (400 MHz, DMSO-d6) δ 2.42 (s, 3H, C3-
CH3), 4.02 (s, 3H, COOCH3), 8.47 (dd, 1H, H5, J5-6 ) 8.5 Hz),
8.51 (dd, 1H, H8, J8-7 ) 8.5 Hz) ppm. Anal. C11H8F2N2O4 (C,
H, N). HPLC: ACN/H2O (60:40), tR ) 2.00 min.
Ethyl 3,6,7-Trimethylquinoxaline-2-carboxylate 1,4-
Dioxide (7). This compound was obtained in 30% yield from
5,6-dimethylbenzofuroxane (0.40 g, 2.4 mmol) and ethyl ace-

Quinoxaline-2-carboxylate 1,4-dioxide Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2023
toacetate (1.38 g, 10.6 mmol) after 24 h under stirring (method
A): mp 183-184 °C; IR (KBr) ν 2988, 1748, 1518, 1328, 1255
cm-1; 1H NMR (200 MHz, DMSO-d6) δ 0.36 (t, 3H, CH2CH3, J
) 7.2 Hz), 2.41 (s, 3H, C3-CH3), 2.48 (s, 6H, 2CH3-Ar), 4.50
(c, 2H, CH2CH3, J ) 7.1 Hz), 8.15 (s, 1H, H5), 8.20 (s, 1H, H8)
ppm. Anal. C14H16N2O4 (C, H, N).
J ) 7.0 Hz), 3.93 (s, 2H, CH2COOCH2CH3), 4.11 (c, 2H,
CH2COOCH2CH3, J ) 7.1 Hz), 4.48 (c, 2H, COOCH2CH3, J )
7.0 Hz), 8.06 (dd, 1H, H6, J6-5 ) 9.2 Hz, J6-8 ) 1.6 Hz), 8.44
(s, 1H, H8), 8.47 (d, 1H, H5, J5-6 ) 9.2 Hz) ppm. Anal. C15H15-
ClN2O6 (C, H, N). HPLC: ACN/H2O (60:40), tR ) 4.76 min.
Ethyl 6,7-Dichloro-3-(2-ethoxy-2-oxoethyl)quinoxaline-
2-carboxylate 1,4-Dioxide (15). This compound was obtained
in 9% yield from 5,6-dichlorobenzofuroxane (0.50 g, 2.44 mmol)
and diethyl 3-oxoglutarate (4.33 g, 21.41 mmol) after 24 h
under stirring (method A): mp 136-137 °C; IR (KBr) ν 1736,
Ethyl 3,7-Dimethylquinoxaline-2-carboxylate 1,4-Di-
oxide (8). This compound was obtained in 5% yield from
5-methylbenzofuroxane (0.36 g, 2.4 mmol) and ethyl acetoace-
tate (1.38 g, 10.6 mmol) after 24 h under stirring. It was
purified by recrystallization from methanol/n-hexane (method
A): mp 159-160 °C; IR (KBr) ν 2980, 1740, 1525, 1331, 1244
cm-1; 1H NMR (200 MHz, DMSO-d6) δ 1.36 (t, 3H, CH2CH3, J
) 7.1 Hz), 2.42 (s, 3H, C3-CH3), 2.58 (s, 3H, CH3-Ar), 4.50
(c, 2H, CH2CH3, J ) 7.1 Hz), 7.83 (d, 1H, H6, J6-5 ) 8.8 Hz),
8.22 (s, 1H, H8), 8.36 (d, 1H, H5, J5-6 ) 8.8 Hz) ppm. Anal.
C13H14N2O4 (C, H, N). HPLC: ACN/H2O (60:40), tR ) 2.03 min.
Ethyl 7-Methoxy-3-methylquinoxaline-2-carboxylate
1,4-Dioxide (9). This compound was obtained in 11% yield
from 5-methoxybenzofuroxane (0.20 g, 1.2 mmol) and ethyl
acetoacetate (0.69 g, 5.3 mmol) after 24 h under stirring
(method A): mp 157-158 °C; IR (KBr) ν 1732, 1334, 1250
cm-1; 1H NMR (200 MHz, DMSO-d6) δ 1.30 (t, 3H, CH2CH3, J
) 7.0 Hz), 2.36 (s, 3H, CH3), 3.93 (s, 3H, OCH3), 4.45 (c, 2H,
1
1329, 1262, 1023 cm-1; H NMR (200 MHz, DMSO-d6) δ 1.18
(t, 3H, CH2COOCH2CH3, J ) 7.1 Hz), 1.32 (t, 3H, COOCH2CH3,
J ) 7.0 Hz), 3.94 (s, 2H, CH2COOCH2CH3), 4.11 (c, 2H, CH2-
COOCH2CH3, J ) 7.1 Hz), 4.49 (c, 2H, COOCH2CH3, J ) 7.0
Hz), 8.65 (s, 1H, H5), 8.67 (s, 1H, H8) ppm. Anal. C15H14Cl2N2O6
(C, H, N). HPLC: ACN/H2O (60:40), tR ) 8.32 min.
tert-Butyl 3,6,7-Trimethylquinoxaline-2-carboxylate
1,4-Dioxide (16). This compound was obtained in 3% yield
from 5,6-dimethylbenzofuroxane (0.40 g, 2.4 mmol) and tert-
butyl acetoacetate (1.67 g, 10.6 mmol) after 24 h under stirring
(method A): mp 191-192 °C; IR (KBr) ν 2932, 1742, 1458,
1330, 1154 cm-1; 1H NMR (200 MHz, DMSO-d6) δ 1.60 (s, 9H,
C(CH3)3), 2.43 (s, 3H, C3-CH3), 2.49 (s, 6H, 2CH3-Ar), 8.16
(s, 1H, H5), 8.22 (s, 1H, H8) ppm. Anal. C16H20N2O4 (C, H, N).
tert-Butyl 3-Methylquinoxaline-2-carboxylate 1,4-Di-
oxide (17). This compound was obtained in 3% yield from
benzofuroxane (1.00 g, 7.4 mmol) and tert-butyl acetoacetate
(1.17 g, 7.4 mmol) after 1 h of heating (method B). After column
cromatography, the product was washed with diethyl ether
and recrystallized from methanol: mp 128-129 °C; IR (KBr)
ν 1734, 1341, 1259, 1163 cm-1; 1H NMR (200 MHz, DMSO-d6)
δ 1.59 (s, 9H, C(CH3)3), 2.44 (s,3H, CH3), 7.94-7.99 (m, 2H,
H6 + H7), 8.43 (m, 2H, H5 + H8) ppm. Anal. C14H16N2O4‚1/4H2O
(C, H, N). HPLC: ACN/propan-2-ol (50:50), tR ) 3.06 min.
CH2CH3, J ) 7.0 Hz), 7.55 (dd, 1H, H6, J6-5 ) 9.4 Hz, J6-8
)
2.7 Hz), 7.67 (ds, 1H, H8, J8-6 ) 2.6 Hz), 8.34 (d, 1H, H5, J5-6
) 9.4 Hz) ppm. Anal. C13H14N2O5‚1/2H2O (C, H, N). HPLC:
ACN/H2O (60:40), tR ) 1.96 min.
Ethyl 3-Methylquinoxaline-2-carboxylate 1,4-Dioxide
(10). This compound was obtained in 30% yield from benzo-
furoxane (2.50 g, 18.38 mmol) and ethyl acetoacetate (9.23 g,
70.91 mmol) after 24 h under stirring (method A): mp 135-
136 °C; IR (KBr) ν 1739, 1332, 1288 cm-1; 1H NMR (200 MHz,
DMSO-d6) δ 1.36 (t, 3H, COOCH2CH3, J ) 6.9 Hz), 2.44 (s,
3H, C3-CH3), 4.50 (c, 2H, COOCH2CH3, J ) 7.0 Hz), 7.95-
8.01 (m, 2H, H6 + H7), 8.40-8.49 (m, 2H, H5 + H8) ppm. Anal.
C12H12N2O4 (C, H, N). HPLC: ACN/H2O (60:40), tR ) 2.21 min.
Ethyl 7-Chloro-3-methylquinoxaline-2-carboxylate 1,4-
Dioxide (11). This compound was obtained in 26% yield from
5-chlorobenzofuroxane (1.50 g, 8.80 mmol) and ethyl acetoace-
tate (5.14 g 39.49 mmol) after 24 h under stirring (method A):
tert-Butyl 7-Chloro-3-methylquinoxaline-2-carboxy-
late 1,4-Dioxide (18). This compound was obtained in 3%
yield from 5-chlorobenzofuroxane (0.30 g, 1.76 mmol) and tert-
butyl acetoacetate (1.36 g, 8.60 mmol) after 24 h under stirring
(method A): mp 143-144 °C; IR (KBr) ν 1736, 1329, 1278
cm-1 1H NMR (200 MHz, DMSO-d6) δ 1.60 (s, 9H, COOC-
;
(CH3)3), 2.43 (s, 3H, C3-CH3), 8.03 (dd, 1H, H6, J6-5 ) 9.1 Hz,
J6-8 ) 1.9 Hz), 8.41 (s, 1H, H8), 8.46 (d, 1H, H5, J5-6 ) 9.1 Hz)
ppm. Anal. C14H15ClN2O4 (C, H, N). HPLC: ACN/H2O (60:40),
tR ) 5.26 min.
1
mp 173-174 °C; IR (KBr) ν 1744, 1326, 1277 cm-1; H NMR
(200 MHz, DMSO-d6) δ 1.35 (t, 3H, COOCH2CH3, J ) 7.0 Hz),
2.42 (s, 3H, C3-CH3), 4.51 (c, 2H, COOCH2CH3, J ) 7.0 Hz),
8.02 (dd, 1H, H6, J6-5 ) 9.1 Hz, J6-8 ) 1.6 Hz), 8.40 (s, 1H,
H8), 8.46 (d, 1H, H5, J5-6 ) 9.1 Hz) ppm. Anal. C12H11ClN2O4
(C, H, N). HPLC: ACN/H2O (60:40), tR ) 3.23 min.
tert-Butyl 6,7-Dichloro-3-methylquinoxaline-2-carbox-
ylate 1,4-Dioxide (19). This compound was obtained in 9%
yield from 5,6-dichlorobenzofuroxane (0.20 g, 0.98 mmol) and
tert-butyl acetoacetate (0.67 g, 4.24 mmol) after 24 h under
stirring (method A): mp 174-175 °C; IR (KBr) ν 1734, 1326
Ethyl 6,7-Dichloro-3-methylquinoxaline-2-carboxylate
1,4-Dioxide (12). This compound was obtained in 25% yield
from 5,6-dichlorobenzofuroxane (0.30 g, 1.50 mmol) and
ethyl acetoacetate (0.80 g, 6.15 mmol) after 24 h under stir-
ring (method A): mp 197-198 °C; IR (KBr) ν 1748, 1326,
cm-1 1H NMR (200 MHz, DMSO-d6) δ 1.58 (s, 9H, COOC-
;
(CH3)3), 2.41 (s, 3H, C3-CH3), 8.58 (s, 1H, H5), 8.60 (s, 1H,
1264 cm-1 1H NMR (200 MHz, DMSO-d6) δ 1.44 (t, 3H,
;
H8) ppm. Anal. C14H14Cl2N2O4 (C, H, N).
2-Methoxyethyl 6,7-Dichloro-3-methylquinoxaline-2-
carboxylate 1,4-Dioxide (20). This compound was obtained
in 11% yield from 5,6-dichlorobenzofuroxane (0.30 g, 1.46
mmol) and 2-methoxyethyl acetoacetate (1.06 g, 6.62 mmol)
after 24 h under stirring (method A): mp 150-151 °C; IR (KBr)
ν 1738, 1328, 1277, 1131 cm-1; 1H NMR (400 MHz, DMSO-d6)
δ 2.44 (s, 3H, C3-CH3), 3.29 (s, 3H, COOCH2CH2OCH3), 3.66
(t, 2H, COOCH2CH2OCH3, J ) 4.5 Hz), 4.60 (t, 2H, COOCH2-
CH2OCH3, J ) 4.5 Hz), 8.61 (s, 1H, H5), 8.65 (s, 1H, H8) ppm.
Anal. C13H12Cl2N2O5 (C, H, N). HPLC: ACN/H2O (60:40), tR
) 4.09 min.
Allyl 3,6,7-Trimethylquinoxaline-2-carboxylate 1,4-
Dioxide (21). This compound was obtained in 10% yield from
5,6-dimethylbenzofuroxane (0.50 g, 3.0 mmol) and allyl aceto-
actate (1.88 g, 13.3 mmol) after 24 h under stirring (method
A): mp 139-140 °C; IR (KBr) ν 1741, 1443, 1328, 1273, 1221
cm-1; 1H NMR (200 MHz, DMSO-d6) δ 2.50 (s, 6H, 2CH3-Ar),
2.55 (s, 3H, C3-CH3), 4.97 (d, 2H, COOCH2CH, J ) 5.9 Hz),
5.38 (d, 1H, CHdCH2, Hcis, J ) 10.2 Hz), 5.52 (d, 1H, CHd
CH2, Htrans, J ) 17.1 Hz), 5.98-6.06 (m, 1H, CHdCH2, J )
6.0 Hz), 8.29 (s, 1H, H5), 8.34 (s, 1H, H8) ppm. Anal.
COOCH2CH3, J ) 7.1 Hz), 2.52 (s, 3H, C3-CH3), 4.59 (c, 2H,
COOCH2CH3, J ) 7.1 Hz), 8.69 (s, 1H, H5), 8.73 (s, 1H, H8)
ppm. Anal. C12H10Cl2N2O4 (C, H, N). HPLC: ACN/H2O (60:
40), tR ) 5.12 min.
Ethyl 6,7-Difluoro-3-methylquinoxaline-2-carboxylate
1,4-Dioxide (13). This compound was obtained in 11% yield
from 5,6-difluorobenzofuroxane (0.40 g, 2.33 mmol) and ethyl
acetoacetate (2.06 g, 15.60 mmol) after 24 h under stirring
(method A): mp 114-115 °C; IR (KBr) ν 1738, 1331, 1257,
1171 cm-1 1H NMR (400 MHz, DMSO-d6) δ 1.36 (t, 3H,
;
COOCH2CH3, J ) 7.1 Hz), 2.43 (s, 3H, C3-CH3), 4.50 (c, 2H,
COOCH2CH3, J ) 7.1 Hz), 8.47 (dd, 1H, H5, J5-6 ) 7.3 Hz),
8.51 (dd, 1H, H8, J8-7 ) 7.3 Hz) ppm. Anal. C12H10F2N2O4 (C,
H, N). HPLC: ACN/H2O (60:40), tR ) 2.77 min.
Ethyl 7-Chloro-3-(2-ethoxy-2-oxoethyl)quinoxaline-2-
carboxylate 1,4-Dioxide (14). This compound was obtained
in 3% yield from 5-chlorobenzofuroxane (0.40 g, 2.35 mmol)
and diethyl 3-oxoglutarate (2.22 g, 10.98 mmol) after 0.5 h
of heating (method B): mp 102-103 °C; IR (KBr) ν 1739,
1331, 1283 cm-1 1H NMR (200 MHz, DMSO-d6) δ 1.15 (t,
;
3H, CH2COOCH2CH3, J ) 7.0 Hz), 1.33 (t, 3H, COOCH2CH3,

2024 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Jaso et al.
C15H16N2O4‚1/4H2O (C, H, N). HPLC: ACN/propan-2-ol (50:50),
tR ) 3.05 min.
8.64 (s, 1H, H8) ppm. Anal. C17H12Cl2N2O4 (C, H, N). HPLC:
ACN/H2O (60:40), tR ) 12.79 min.
Benzyl 6,7-Difluoro-3-methylquinoxaline-2-carboxy-
late 1,4-Dioxide (29). This compound was obtained in 20%
yield from 5,6-difluorobenzofuroxane (0.40 g, 2.33 mmol) and
benzyl acetoacetate (0.55 g, 2.86 mmol) after 24 h under
stirring (method A): mp 148-149 °C; IR (KBr) ν 1687, 1323,
1249, 1074 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 2.39 (s, 3H,
C3-CH3), 5.54 (s, 2H, COOCH2C6H5), 7.39-7.45 (m, 3H, H3′
+ H4′ + H5′), 7.52 (d, 2H, H2′ + H6′, J2′-3′ ) 7.6 Hz), 8.47-8.52
(m, 2H, H5 + H8) ppm. Anal. C17H12F2N2O4 (C, H, N). HPLC:
ACN/H2O (60:40), tR ) 3.47 min.
Allyl 6,7-Dichloro-3-methylquinoxaline-2-carboxylate
1,4-Dioxide (22). This compound was obtained in 7% yield
from 5,6-dichlorobenzofuroxane (0.30 g, 1.46 mmol) and allyl
acetoacetate (0.93 g, 6.54 mmol) after 24 h under stirring
(method A): mp 141-142 °C; IR (KBr) ν 1686, 1637, 1329,
1
1241 cm-1; H NMR (200 MHz, DMSO-d6) δ 2.42 (s, 3H, C3-
CH3), 4.98 (d, 2H, COOCH2CHdCH2, J ) 5.4 Hz), 5.35 (d, 1H,
COOCH2CHdCH2, Hcis, J ) 10.5 Hz), 5.51 (d, 1H, COOCH2-
CHdCH2, Htrans, J ) 17.8 Hz), 6.01 (m, 1H, COOCH)CH2),
8.61 (s, 1H, H5), 8.63 (s, 1H, H8) ppm. Anal. C13H10Cl2N2O4
(C, H, N).
Biological Evaluation. In Vitro Evaluation of Antitu-
berculosis Activity. Primary Screen (Level 1). The pri-
mary screen is conducted at 6.25 µg/mL against Mycobacterium
tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium,
using the microplate alamar blue assay (MABA).26 Compounds
exhibiting fluorescence are tested in the BACTEC 460 radio-
metric system.28 Generally, compounds effecting <90% inhibi-
tion in the primary screen (MIC > 6.25 µg/mL) are not further
evaluated.
Determination of Minimum Inhibitory Concentration
(MIC) (Level 2). Compounds demonstrating at least 90%
inhibition in the primary screen are retested at lower concen-
trations against M. tuberculosis H37Rv in order to determine
the actual MIC in the MABA. The MIC is defined as the lowest
concentration effecting a reduction in fluorescence of 90%
relative to controls. Rifampin (RMP) was used as the reference
compound (RMP MIC ) 0.015-0.125 µg/mL).
Benzyl 3,6,7-Trimethylquinoxaline-2-carboxylate 1,4-
Dioxide (23). This compound was obtained in 21% yield from
5,6-dimethylbenzofuroxane (0.50 g, 3.0 mmol) and benzyl
acetoacetate (2.50 g, 13.3 mmol) after 24 h under stirring
(method A): mp 159-160 °C; IR (KBr) ν 3065, 2936, 1740,
1514, 1328 cm-1; 1H NMR (200 MHz, DMSO-d6) δ 2.36 (s, 3H,
C3-CH3), 2.47 (s, 6H, 2CH3-Ar), 5.52 (s, 2H, COOCH2C6H5),
7.37-7.54 (m, 5H, H2′ + H3′ + H4′ + H5′ + H6′), 8.17 (s, 1H,
H5), 8.20 (s, 1H, H8) ppm. Anal. C19H18N2O4‚1/4H2O (C, H, N).
HPLC: ACN/propan-2-ol (50:50), tR ) 3.05 min.
Benzyl 3,7-Dimethylquinoxaline-2-carboxylate 1,4-
Dioxide (24). This compound was obtained in 17% yield from
5-methylbenzofuroxane (0.36 g, 2.4 mmol) and benzyl acetoace-
tate (2.04 g, 10.6 mmol) after 24 h under stirring and purified
by flash chromatography, eluting with DCM/MeOH 97.5:2.5
(method A): mp 111-112 °C; IR (KBr) ν 1741, 1329, 1236
Determination of 50% Inhibitory Concentrations (IC50)
(Level 2). Concurrent with the determination of MIC values,
compounds are tested for cytotoxicity (IC50) in VERO cells at
concentrations less than or equal to 62.5 µg/mL or 10 times
the MIC for M. tuberculosis H37Rv. After 72 h of exposure,
viability is assessed on the basis of cellular conversion of MTT
into a formazan product using the Promega CellTiter 96
nonradioactive cell proliferation assay. The selectivity index
(SI ) IC50/MIC) was also determined; it is considered signifi-
cant when greater than 10 (RMP IC50 > 100 µg/mL, SI > 800).
Macrophage Assay. Determination of 90% and 99%
Effective Concentration (EC90 and EC99) (Level 3). Com-
pounds with MIC e 6.25 µg/mL and SI > 10 were then tested
to evaluate in vitro efficacy in a TB-infected macrophage
model.26 Compounds are tested for the killing of M. tuberculosis
Erdman (ATCC 35801) in monolayers of mouse bone marrow
macrophages (EC90 and EC99; lowest concentration effecting
a 90% and 99% reduction, respectively, in colony forming units
at 7 days compared to drugfree controls) at 4-fold concentra-
tions equivalent to 0.25, 1, 4, and 16 times the MIC. Com-
pounds with EC90 > 16 MIC are considered inactive in the
model (RMP EC90 ) 0.04-0.1 µg/mL, EC99 ) 0.5-1.5 µg/mL).
Determination of MIC against Three Strains of Single-
Drug-Resistant M. tuberculosis (SDRMIC) (Level 3). Con-
current with the testing of compounds in macrophages, MIC
values are determined in the MABA for a minimum of three
strains of drug-resistant M. tuberculosis (each strain resistant
to a single TB drug). Typically, all compounds progressing to
this stage of screening will be tested against M. tuberculosis
strains resistant to isoniazid (ATCC 35822), rifampin (ATCC
35838), and other drug-resistant strains (these latter strains
determined by the compound type) as well as the drug-
sensitive strains H37Rv and Erdman. Generally, MIC’s for SDR
strains should not be greater than 10 MIC for nonresistant
strains in order for compound evaluation to continue.
1
cm-1; H NMR (200 MHz, DMSO-d6) δ 2.40 (s, 3H, C3-CH3),
2.54 (s, 3H, CH3-Ar), 5.46 (s, 2H, COOCH2C6H5), 7.32-7.41
(m, 5H, H2′ + H3′ + H4′ + H5′ + H6′), 7.62 (d, 1H, H6), 8.29 (s,
1H, H8), 8.41 d, 1H, H5) ppm. Anal. C18H16N2O4‚1/2H2O (C, H,
N).
Benzyl 7-Methoxy-3-methylquinoxaline-2-carboxylate
1,4-Dioxide (25). This compound was obtained in 17% yield
from 5-methoxybenzofuroxane (0.20 g, 1.2 mmol) and benzyl
acetoacetate (1.02 g, 5.3 mmol) after 24 h under stirring. It
was purified by flash chromatography, eluting with DCM/
MeOH 98:2. The obtained oil was precipitated by adding cold
diethyl ether (method A): mp 129-130 °C; IR (KBr) ν 1739,
1331, 1237 cm-1; 1H NMR (200 MHz, DMSO-d6) δ 2.39 (s, 3H,
C3-CH3), 3.98 (s, 3H, OCH3), 5.46 (s, 2H, COOCH2C6H5),
7.39-7.51 (m, 5H, H2′ + H3′ + H4′ + H5′ + H6′), 7.59 (d, 1H,
H6), 8.29 (s, 1H, H8), 8.41 d, 1H, H5) ppm. Anal. C18H16N2O5
(C, H, N).
Benzyl 3-Methylquinoxaline-2-carboxylate 1,4-Dioxide
(26). This compound was obtained in 41% yield from benzo-
furoxane (0.40 g, 2.4 mmol) and benzyl acetoacetate (1.70 g,
10.6 mmol) after 24 h under stirring (method A): mp 102-
103 °C; IR (KBr) ν 3087, 1752, 1519, 1330, 1233 cm-1; 1H NMR
(200 MHz, DMSO-d6) δ 2.39 (s, 3H, CH3), 5.54 (s, 2H,
COOCH2C6H5), 7.95-8.01 (m, 2H, H6 + H7), 8.41-8.44 (m, 2H,
H5 + H8) ppm. Anal. C17H14N2O4 (C, H, N). HPLC: ACN/H2O
(60:40), tR ) 2.59 min.
Benzyl 7-Chloro-3-methylquinoxaline-2-carboxylate
1,4-Dioxide (27). This compound was obtained in 14% yield
from 5-chlorobenzofuroxane (1.50 g, 8.80 mmol) and benzyl
acetoacetate (5.56 g, 28.93 mmol) after 24 h under stirring
(method A): mp 148-149 °C; IR (KBr) ν 1749, 1322, 1287
1
cm-1; H NMR (400 MHz, DMSO-d6) δ 2.39 (s, 3H, C3-CH3),
5.54 (s, 2H, COOCH2C6H5), 7.40-7.45 (m, 3H, H3′ + H4′ + H5′),
7.52 (d, 2H, H2′ + H6′, J2′-3′ ) 8.1 Hz), 8.02 (dd, 1H, H6, J6-5
9.2 Hz, J6-8 ) 2.3 Hz), 8.42 (s, 1H, H8), 8.45 (d, 1H, H5, J5-6
)
)
Acknowledgment. Antimycobacterial data were
provided by the Tuberculosis Antimicrobial Acquisition
and Coordinating Facility (TAACF) through a research
and development contract with the U.S. National In-
stitute of Allergy and Infectious Diseases. Thanks are
addressed to Dr. Joseph A. Maddry (TAACF) and his
team for their collaboration. We also thank CYTED (Red
Iberoamericana para la Investigacio´n y Descubrimiento
de Medicamentos).
9.2 Hz) ppm. Anal. C17H13ClN2O4 (C, H, N). HPLC: ACN/H2O
(60:40), tR ) 7.02 min.
Benzyl 6,7-Dichloro-3-methylquinoxaline-2-carboxy-
late 1,4-Dioxide (28). This compound was obtained in 44%
yield from 5,6-dichlorobenzofuroxane (0.50 g, 2.44 mmol) and
benzyl acetoacetate (2.40 g, 12.49 mmol) after 24 h under
stirring (method A): mp 147-148 °C; IR (KBr) ν 1749, 1327,
1
1252 cm-1; H NMR (400 MHz, DMSO-d6) δ 2.39 (s, 3H, C3-
CH3), 5.54 (s, 2H, COOCH2C6H5), 7.39-7.45 (m, 3H, H3′ + H4′
+ H5′), 7.52 (d, 2H, H2′ + H6′, J2′-3′ ) 8.0 Hz), 8.62 (s, 1H, H5),

Quinoxaline-2-carboxylate 1,4-dioxide Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2025
(16) Hutchinson, M. J.; Young, P. Y.; Hewitt, S. A.; Faulkner, D.;
Kennedy, D. G. Development and validation of an improved
method for confirmation of the carbadox metabolite, quinoxaline-
2-carboxylic acid, in porcine liver using LC-electrospray MS-
MS according to revised EU criteria for veterinary drug residue
analysis. Analyst 2002, 127, 342-346.
Supporting Information Available: Results from el-
emental analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
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