Synthesis and in vitro evaluation of new ethyl and methyl quinoxaline-7-carboxylate 1,4-di-N-oxide against Entamoeba histolytica

Article abstract of DOI:10.1016/j.bmc.2013.05.036

In our search for new antiamoebic agents, a new series of ethyl and methyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives have been synthesized using the Beirut reaction. All compounds were characterized by spectroscopic techniques and elemental analysis. Antiamoebic activity was evaluated in vitro against Entamoeba histolytica strain HM1:IMSS by the microdilution method, and the structure-activity relationship was analyzed. We found that eleven quinoxaline derivatives showed greater activity than metronidazole and nitazoxanide with IC₅₀ values in the range 1.99-0.35 μM. Compounds T-001 and T-016 shows IC₅₀ values of 1.41 and 1.47 μM, respectively, with a value of selectivity index >60.

Full text of DOI:10.1016/j.bmc.2013.05.036

Bioorganic & Medicinal Chemistry 21 (2013) 4550–4558
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro evaluation of new ethyl and methyl
quinoxaline-7-carboxylate 1,4-di-N-oxide against Entamoeba
histolytica
Blanca Estela Duque-Montaño a, Lilia Citlalli Gómez-Caro b, Mario Sanchez-Sanchez b, Antonio Monge c,
Efrén Hernández-Baltazar a, Gildardo Rivera b, , Oscar Torres-Angeles a,
⇑
⇑
a Laboratorio de Microbiología, Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, 62209 Cuernavaca, Mexico
b Centro de Biotecnología Genómica, Instituto Politécnico Nacional, Blvd. del Maestro, s/n, Esq. Elías Piña, 88710 Reynosa, Mexico
c Unidad de Investigación y Desarrollo de Medicamentos, Centro de Investigación en Farmacobiologia Aplicada, Universidad de Navarra, 31008 Pamplona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
In our search for new antiamoebic agents, a new series of ethyl and methyl quinoxaline-7-carboxylate
1,4-di-N-oxide derivatives have been synthesized using the Beirut reaction. All compounds were charac-
terized by spectroscopic techniques and elemental analysis. Antiamoebic activity was evaluated in vitro
against Entamoeba histolytica strain HM1:IMSS by the microdilution method, and the structure–activity
relationship was analyzed. We found that eleven quinoxaline derivatives showed greater activity than
Received 8 March 2013
Revised 10 May 2013
Accepted 18 May 2013
Available online 1 June 2013
metronidazole and nitazoxanide with IC50 values in the range 1.99–0.35 lM. Compounds T-001 and T-
Keywords:
016 shows IC50 values of 1.41 and 1.47
l
M, respectively, with a value of selectivity index >60.
Antiparasitic agents
Antiamoebic activity
Cytotoxicity
Ó 2013 Elsevier Ltd. All rights reserved.
Quinoxaline 1,4-di-N-oxide
1. Introduction
properties have been known for a century.15 They are considered
isomeric compounds of quinozaline, phtalazine and cinnoline,
and isosteric compounds of pyrazinamide, isoniazide and quino-
line (Fig. 2).16
Currently, the synthesis and development of various quinoxa-
lines and their derivatives (Fig. 3) focus on different biological tar-
gets. Some of these compounds are antitumoural agents, which act
as DNA intercalators, and inhibitors of enzymes involved in nucleic
acid biosynthesis; for example, dyhidrofolate reductase (DHFR)
and human thymidylate synthase (TS).17,18 Quinoxaline derivatives
has demonstrated anti-inflammatory and analgesic activity by act-
ing on prostaglandin E2 inhibition (PGE2) and nitric acid produc-
tion, respectively.19 Another series of quinoxaline derivatives
have antiviral activity, inhibiting viral replication of herpes
Amoebiasis is a parasitic infection caused by the microaerophilic
protozoa Entamoeba histolytica.1,2 This disease is considered a pub-
lic health problem in endemic countries where it frequently affects
children, young adults, and immunocompromised patients.3,4
Worldwide, amoebiasis is the second leading cause of death among
parasitic diseases.5 According to the World Health Organization
(WHO), this disease causes about 110,000 deaths, and more than
500 million people are infected annually.6 In Mexico it is considered
an endemic disease. Antiamoebic drugs, such as metronidazole
with a 5-nitroimidazole group, and nitazoxanide with a 5-nitro-
thiazole group (Fig. 1), among others, have been used to treat infec-
tion. However, these drugs have several gastrointestinal side
effects.7 Furthermore, in the last two decades, the literature has re-
ported that metronidazole has mutagenic and carcinogenic proper-
ties in murine models8–10 and protozoan resistance against
conventional drugs.11–14 Based on these considerations, it is impor-
tant to search for new antiamoebic compounds for use as potential
antiparasitic agents with greater potency, efficacy, and safety for
patients.
-
O
O
N
+
O
O
O
S
N
-
+
O
N
N
N
H
N
O
On the other hand, quinoxalines are heterocyclic compounds
that contain both benzene and pyrazine rings and whose biological
HO
Metronidazole
Figure 1. Chemical structure of antiamoebic drugs.
Nitazoxanide
⇑
Corresponding authors. Tel./fax: +52 777 3297089.
E-mail address: tao_ff@uaem.mx (O. Torres-Angeles).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.036

B. E. Duque-Montaño et al. / Bioorg. Med. Chem. 21 (2013) 4550–4558
4551
O
O
NH2
N
N
N
H
NH2
N
N
Pyrazinamide
Isoniazide
Quinoline
N
N
N
N
N
N
N
N
Phtalazine
Figure 2. Structure of N-heterocyclic isomeric and isosteric compounds of the ring of quinoxaline.
Quinoxaline
Cinolinne
Quinozaline
-
-
-
O
N
O
N
O
O
N
+
+
+
Cl
Cl
S
Cl
Cl
N
H
N
O
N
O
NH2
N
O
+
-
+
-
+
-
Antibacterial
Antitumoural
Antiparasitic
N
N
H
N
N
N
N
N
O
Cl
O
Antifungal
Antiparasitic
R
N
N
N
S
N
N
N
N
Antiviral
Figure 3. Structure of quinoxaline derivatives with biological activity.
Antiparasitic
simplex type 1, cytomegalovirus, and varicella-zoster virus.20 Par-
ticularly, quinoxaline 1,4-di-N-oxide derivatives are versatile com-
pounds that have been studied as antitumoural, antibacterial, and
antiparasitic agents.15,21–26 Interestingly, its activity is significantly
affected by substituents on quinoxaline nucleus. It has been ob-
served that the presence of different group at 7-position on quin-
oxaline reduces or enhance biological activity. However, Jaso
et al., suggest that activity principally depends on the substituents
in the carboxylate group in position 2.27
In the last century, quinoxaline 1,4-dioxide derivatives were
found to exert a therapeutic effect against amoebiasis in dogs, rats,
and cats models, but were also toxic.28 Recently, Budakoti et al.,
obtained quinoxaline derivatives with better antiamoebic activity
and less toxicity than metronidazole. Interestingly, modification
of the compounds from chalcones, to pyrazolines, and further to
quinoxalines results in an increase in antiamoebic activity.29 Fol-
lowing with the use of quinoxaline as a scaffold in drug antiamoe-
in new derivatives containing both carbonyl and carboxylate group
at 2- and 7-position on the quinoxaline nucleus, respectively,
which could change solubility properties and also to correlate the
structure with biological activity. Additionally, we include an ethyl
carboxylate at 7-position to show potential steric and metabolic ef-
fects by esterase enzyme on our compounds. Therefore, our pres-
ent work focuses on the in vitro evaluation of the effect of a new
series of ethyl and methyl quinoxaline-7-carboxylate 1,4-di-N-
oxide derivatives on Entamoeba histolytica.
2. Material and methods
2.1. Synthetic procedure
The Beirut reaction was the general procedure used for synthe-
sis of quinoxaline1,4-di-N-oxide derivatives, as described in meth-
ods A–C.30
bic design, in
a first approximation, we have initiated an
exploration on quinoxalines 1,4-di-N-oxide with a new methyl car-
boxylate group at 7-position on quinoxaline nucleus that has not
been reported, in an effort to analysis how a carboxylate group acts
2.1.1. Method A
Compound synthesis was achieved by the reaction of the corre-
sponding diketone derivative (10.6 mmol) with the appropriate

4552
B. E. Duque-Montaño et al. / Bioorg. Med. Chem. 21 (2013) 4550–4558
benzofuroxane (2.4 mmol) in dry chloroform (35 mL).31,32 Triethyl-
amine (TEA) was added (1 mL), and the reaction mixture was stir-
red at room temperature for 3–7 days. After evaporation to dryness
at low pressure, a crude solid or brown oil was obtained. This was
then precipitated and washed by adding diethyl ether, affording
the target compound. The residue was purified by column chroma-
tography on silica gel, when necessary using dichlorometh-
ane:methanol (95:5).
2.2.2. Culture conditions of Entamoeba histolytica
Entamoeba histolytica trophozoite strain HM1:IMSS in log-phase
were axenically grown in BI-S-33 medium38 supplemented with
20% (vol/vol) calf serum (Microlab, S.A de C.V). The number of tro-
phozoites per mL was estimated by trypan blue exclusion (In Vitro,
S.A de C.V) in a hematocytometer to confirm viability. The tropho-
zoite suspension used was diluted to 105 trophozoites per mL by
adding fresh medium and 100 lL of this suspension was added
to the test and control wells in the plate.
2.1.2. Method B
2.2.3. Antiamoebic assay
Compound synthesis was achieved with a variation of the Bei-
rut reaction. The corresponding diketone (10 mmol) was added
to a solution of the appropriate benzofuroxane (5 mmol) dissolved
in a minimum amount of methanol (20–35 mL) in the presence of
calcium chloride (0.1 mmol) and ethanolamine (1 mmol) as cata-
lysts.33,34 The reaction was stirred at room temperature for 6–
24 h. The solvent was removed under vacuum; the quinoxaline
1,4-di-N-oxide derivative was isolated by addition of 50 mL of
water followed by extraction with dichloromethane (5 Â 40 mL).
The organic layer was dried (Na2SO4), filtered, and evaporated to
dryness, and further purified by column chromatography on silica
gel when necessary using dichloromethane:methanol (95:5). The
mixture was then precipitated and washed with cold diethyl ether
in order to obtain a yellow solid.
All ethyl and methyl quinoxaline-7-carboxylate 1,4-di-N-oxide
derivatives were screened in vitro for antiamoebic activity against
Entamoeba histolytica by the microdilution method.39 A 100
lL
suspension with Entamoeba histolytica trophozoites was added to
each test and control well, and afterwards the plates were incu-
bated at 37 °C for 48 h.
2.2.4. Evaluation of antiamoebic activity
After incubation, trophozoite growth on the plate was checked
with an inverted microscope (ZEISS Allegra™ 2IR). After that, 10 lL
of WST-1 reagent (Millipore, S.A de C.V) was added to each test and
control well and plates were incubated again at 37 °C for 4 h. The
optical density of the resulting solution in each well was deter-
mined at 490 nm with a microplate reader (Biorad Ultramark).
The percentage (%) of growth inhibition of trophozoites was deter-
mined from the optical densities of control and test wells. From the
2.1.3. Method C
Acetoacetate (2 mmol) and potassium carbonate (1 mmol) were
added to a solution of 1 mmol of the appropriate benzofuroxane in
50 mL of acetone.26,35 The suspension was stirred at room temper-
ature for 2–24 h. The quinoxaline 1,4-di-N-oxide derivatives were
isolated by the addition of 50 mL of water followed by extraction
with dichloromethane (5 Â 40 mL). The organic layer was then
dried (Na2SO4), filtered and evaporated to dryness. The residue
was purified by column chromatography on silica gel when neces-
sary using dichloromethane:methanol (95:5).
% of inhibition, the half maximal inhibitory concentration (IC50
)
was obtained by linear regression between the concentration of
compounds used and for the % of inhibition. The assay was per-
formed in duplicate, and mean and standard deviation (SD) were
determined. The reference used to select compounds with anti-
amoebic activity was based on an IC50 <10
2.3. Cytotoxicity evaluation
2.3.1. Cell culture
lM.
According to previous reports,36 we have observed that 7-
substituted quinoxaline 1,4-di-N-oxide derivatives were prevailing
over the 6-isomer. In practice, the workup and purification permit-
ted the isolation of the 7-isomer and reagents. All quinoxaline 1,4-
di-N-oxide derivatives were characterized by infrared (IR) and nu-
clear magnetic resonance (NMR) spectroscopy. NMR was recorded
on a Bruker Ultrashield 400 MHz device, using Tetramethylsilane
(TMS) as the internal standard, and dimethyl sulfoxide-d6
(DMSO-d6) as a solvent. The chemical shifts are reported in ppm
(), and coupling constant (J) values are given in hertz (Hz). Signal
multiplicities are represented by s (singlet), d (doublet), t (triplet),
q (quartet) and m (multiplet). IR spectroscopy was performed on a
Perkin–Elmer 1600 FTIR in KBr Pellets. The frequencies are ex-
pressed in cmÀ1. Alugram1 SIL G/UV254 (layer: 0.2 mm) was used
for thin layer chromatography (TLC).
Green monkey kidney cell line (VERO) was cultured in minimal
essential medium MEM (In Vitro, S.A de C.V) with nonessential
aminoacid 0.1 mM (In Vitro, S.A de C.V) and 1 mM pyruvate (In Vi-
tro, S.A de C.V) and 10% fetal bovine serum (Microlab, S.A de C.V),
at 37 °C in 5% CO2 humidified incubator. The viability and number
of cell per milliliter were determined with trypan blue (In Vitro, S.A
de C.V) in a hematocytometer. Prepared a cell suspension at den-
sity of 105 cell/mL and 100
each of the wells of the plate.
lL of this suspension were added to
2.3.2. Cell proliferation assay (WST-1)
The cell proliferation assay is based on the cleavage of the tet-
razolium salt WST-1 to formazan by cellular mitochondrial dehy-
drogenases. Expansion in the number of viable cells results in an
increase in the overall activity of mitochondrial dehydrogenases
in viable cells only.40
2.2. In vitro evaluation
2.2.1. Stock solutions
2.3.3. Cytotoxic assay
Exponentially growing cells were plated at 104 cells per well
into 96-well plates (Costar, Corning) and incubated for 24 h before
the addition of compounds to achieve the maximum confluence of
the cells. Stock solutions were prepared and dissolved in 0.1% (v/v)
dimethyl sulfoxide (DMSO, Sigma–Aldrich) and further diluted
Stock solutions of new ethyl and methyl quinoxaline-7-carbox-
ylate 1,4-di-N-oxide derivatives were prepared by dissolving in
0.1% (vol/vol) dimethyl sulfoxide (DMSO, Sigma–Aldrich) at a level
at which no inhibition of trophozoites occurs.37 The solution was
further diluted to 1 mL by adding freshly prepared culture medium
to reach a concentration of 1 mg/mL. Two fold serial dilutions were
made in each well of a 96-well microtiter plate (Corning Costar) in
with fresh complete medium to achieve 0.1–100 lM concentra-
tion. Cells were treated with different concentrations of test com-
pounds for 48 h at 37 °C in 5% CO2 humidified incubator with
untreated control sample. The toxicity effect was determined using
100 lL of culture medium. Each test included metronidazole and
nitazoxanide as reference amoebicidal drugs, growth control wells
(culture medium with trophozoites only), and a blank well (culture
medium only).
10
lL WST-1 (Millipore, Inc.) and incubated for 4 h at 37 °C. The
metabolized WST-1 product was quantified by measuring the

B. E. Duque-Montaño et al. / Bioorg. Med. Chem. 21 (2013) 4550–4558
4553
Table 1
absorbance at 490 nm on a Microplate reader (Biorad Ultramark)
Antiamoebic activity of new quinoxaline 1,4-di-N-oxide derivatives
with a reference wavelength of 630 nm. All assays were performed
in quadruplicate and repeated twice. The results were determined
the mean and standard deviation, and statistical analysis of the re-
sults was assessed by analysis of variance (ANOVA). The reference
used to select compounds to determine toxicity profile was based
on an IC50 6reference amoebicidal drugs (metronidazole and nita-
zoxanide) in antiamoebic activity assay.
O
O
O
N+
R7
R2
O
N+
O
R3
2.3.4. Selectivity index (SI)
Compound
R2
R3
R7
IC50
(l
M)
SD
Selectivity index (SI) was calculated and defined as: Toxicity
IC50 value for the VERO cells/IC50 values for the Entamoeba histoly-
tica under study; where toxicity IC50 is defined as the concentra-
tion of compound that kills 50% of the kidney epithelial cells
(VERO) and protozoal IC50 is the concentration that kills 50% of
amoeba.
T-001
T-002
T-003
T-004
T-005
T-006
T-007
T-008
T-009
T-010
T-011
T-012
T-013
T-014
T-015
T-016
T-017
T-018
T-019
T-020
T-021
T-022
T-023
T-024
T-025
MTZ
CH3
OCH3
CH3
CH3
CH3
CH3
CH3
CH3
C6H5
CF3
CF3
CF3
CF3
CF3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
—
1.41
4.17
1.99
0.012
0.007
0.022
NDa
OCH2CH3
OC(CH3)3
C6H5
NHC6H5
OCH2CH3
CH3
CH(CH3)2
C6H5
C4H3S
C10H7
OCH2CH3
OCH3
OCH2CH3
NH2
C6H5
NDa
3.43
4.14
12.25
1.53
1.88
6.07
0.35
5.29
1.91
1.88
4.18
1.47
1.93
NDa
NDa
1.98
18.07
2.5
0.017
0.020
0.026
0.000
0.024
0.001
0.026
0.004
0.021
0.007
0.093
0.004
0.024
NDa
3. Results and discussion
In this work, 25 new quinoxaline-7-carboxylate 1,4-di-N-oxide
derivatives were synthesized as shown in Figure 4, following the
methods previously described. The synthesized compounds were
divided in two series of 12 and 13 compounds by substitution at
the 7-position on the quinoxaline ring: ethyl and methyl quinoxa-
line-7-carboxylate 1,4-di-N-oxide.
The next step was an in vitro evaluation of all new ethyl and
methyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives to
determine their antiamoebic activity. These results are shown in
Table 1.
Quinoxaline 1,4-di-N-oxide derivatives have been studied as
interesting compounds in organic and medicinal chemistry; from
their molecular structure, a series of new compounds with biolog-
ical activity3,11,15,21,22,27,41 have been designed, synthesized, and
evaluated. In the present work, we report in vitro screening of
new ethyl and methyl quinoxaline-7-carboxylate 1,4-di-N-oxide
derivatives on Entamoeba histolytica strain HM1:IMSS. The new
ethyl and methyl quinoxaline-7-carboxylate 1,4-di-N-oxide
CH3CH2OCOCH2
CH3
CH3
CH3
CH3
CH3
CH3
CF3
CF3
CF3
CF3
CHF2
C6H5
—
NHC6H5
OCH2C6H5
CH3
CH2CH3
C4H3S
NDa
0.050
0.038
0.001
NDa
0.002
0.017
0.020
0.024
C10H7
NDa
3.81
7.13
4.5
C4H3O
OCH2CH3
—
—
NTZ
—
—
3.9
MTZ: metronidazole.
NTZ: nitazoxanide.
IC50: half maximal inhibitory concentration.
SD: standard deviation.
M: micromolar.
ND: not determined by solubility problems.
l
a
derivatives showed an IC50 in the range of 18.07–0.35 lM. Struc-
ture–activity relationship (SAR) analysis of new methyl quinoxa-
line-7-carboxylate 1,4-di-N-oxide derivatives showed that
incorporation of aliphatic substitutions with one carbon atom at
the 2- and 3-position on the quinoxaline 1,4-di-N-oxide ring (T-
001) had better activity than both metronidazole and nitazoxanide
drugs. However, incorporation of an electron-donating (OCH3)
group at 2-position (T-002) decreased biological activity, although
potency values were similar to the reference drugs. Subsequently,
incorporation of more carbon atoms in the lineal chain (OCH2CH3)
at 2-position (T-003) enhanced amoebicidal potency. These results
show that a steric effect is very important in biological activity.
Although, addition of branched aliphatic chain [OC(CH3)3]
a
(T-004) showed solubility problems in antiamoebic assays.
On the other hand, incorporation of an aromatic group, phenyl
and phenylamide at 2-position in compounds T-005 and T-006,
respectively, showed an activity similar to that of both reference
drugs. Surprisingly, when we considered incorporation of an
-
O
O
R
+
O
N
O
O
N
C
A
-
-
O
O
N
O
O
N
B
O
O
+
+
R7
R2
R7
O
O
R2
CF3/CHF2
O
-
O
N
O
+
N
O
R3
R7
N
O
+
-
+
-
O
R2
N
O
R3
+
-
Figure 4. Synthesis of quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives.

4554
B. E. Duque-Montaño et al. / Bioorg. Med. Chem. 21 (2013) 4550–4558
Table 3
aromatic group at 3-position on the quinoxaline 1,4-di-N-oxide
ring (T-007), this produced a drastically low biological activity
Antiamoebic activity of benzofuroxane derivatives precursors
(IC50 = 12.25 l
M). In previous research,42 incorporation of an elec-
-
O
tron-withdrawing group enhanced antiprotozoal activity; there-
fore we decided to incorporate a trifluoromethyl group at 3-
position (T-008) on the quinoxaline nucleus, but results showed
an unmodifiable activity in an analogue compound (T-001) with
the methyl group at 2-position. Addition of a branched aliphatic
chain at 2-position (T-009) on 3-trifluoromethyl quinoxaline 1,4-
di-N-oxide compounds did not enhance biological activity, and
the addition of an aromatic group, such as phenyl and napthyl, in
compound T-010 and T-012, respectively, produced drastically
low activity. Interestingly, the incorporation of a thiophene ring
(T-011) enhanced biological activity 11-fold with IC50 values of
R
+
N
O
N
Compound
R
IC50
aND
221.25
74.48
11.98
4.5
(
l
M)
SD
A
B
C
D
MTZ
NTZ
HOOC
CH3CH2OOC
CH3OOC
aND
0.009
0.046
0.025
0.020
0.024
H
—
—
3.9
0.35 lM. Even though phenyl and thiophene rings are considered
bioisosteric, we suggest that this biological activity may be due
MTZ: metronidazole.
NTZ: nitazoxanide.
IC50: half maximal inhibitory concentration.
SD: standard deviation.
to the polarity of the sulfur atom. Finally, addition of an ester ali-
phatic group at 3-position (T-013, IC50 = 1.91 lM) maintained bio-
logical activity below that of both reference drugs.
l
M: micromolar.
a
ND: not determined by solubility problems.
According to the rules of drug design, if an enhanced number of
carbon atoms (less than 8 or 9) are placed in an aliphatic chain,
there is enhanced biological activity. For analysis of the steric ef-
fect, we incorporate an ethyl ester group at 7-position on the quin-
oxaline 1,4-di-N-oxide ring. Additionally, incorporation of ethyl
ester could show a potential metabolic effect of esterase enzyme
on our compounds. Compounds T-014 and T-017 showed en-
hanced activity in comparison to their counterparts T-002 and T-
005, respectively, but, compounds T-015, T-020, and T-022 showed
decreased antiamoebic activity, and compounds T-018, T-019 and
T-023 showed solubility problems. Interestingly, an amide free
group (T-016) at 2-position produced better activity than both ref-
erence drugs. These results show an important steric effect at 7-
posiiton on the quinoxaline nucleus in antiamoebic activity, and
nule or low metabolic effect.
Later, quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives (T-
001, T-003, T-008, T-009, T-011, T-014, T-016, T-017, T-020 and
T-022) which showed the better antiamoebic results than refer-
ences drugs were assessed for toxicity profile (Table 2).
Data in table 2 show that the tested compounds T-001, T-003,
T-014, T-016 and T-017 have a value of IC50 >100 lM on VERO
a value of IC50 <15 lM, similar to nitazoxanide drug. Therefore
selectivity index (SI) is a good parameter to determinate that the
toxic activity of quinoxaline 1,4-di-N-oxide derivatives is selective
on Entamoeba histolytica. Compounds T-001, and T-016 showed SI
values three fold that metronidazole. T-003, T-014 and T-017 show
a value of SI >50, which is better than both reference drugs. Com-
pounds T-008 (SI = 2.14), T-009 (SI = 3.70), T-011 (SI = 16.74), T-
020 (SI = 3.63) and T-022 (SI = 2.83) showed less SI value than met-
ronidazole (SI >22.22) and similar to nitazoxanide (SI = 2.75).
In summary, we showed that the quinoxaline-7-carboxylate
1,4-di-N-oxide derivatives T-001, T-003, T-014, T-016, and T-017
have better antiamoebic activity and SI value than the reference
drugs, and that these could be considered lead compounds for
designing potential antiamoebic agents43,44
.
Finally, on the other hand, several studies have shown that the
benzofuroxane ring has various biological activities as anti-Trypan-
osoma cruzi and -Mycobacterium tuberculosis agents, among oth-
ers.45–47 Additionally, is very common in medicinal chemistry to
evaluate the starting compounds or intermediates to discard or
confirm their potential activity, therefore we evaluated three ben-
zofuroxane derivative precursors as possible antiamoebic agents.
These results are shown in Table 3.
cells. However, metronidazole has a same value of IC50 on VERO
cells. Compounds T-008, T-009, T-011, T-020 and T-022 showed
Benzofuroxane derivative precursors B–D showed IC50 values in
the range of 221.25–11.98 lM. The incorporation of an ethyl and
Table 2
methyl ester group at 5-position on the benzofuroxane ring de-
creases antiamoebic activity, while benzofuroxane without the
addition of substituent groups at the 5-position showed better
activity against this protozoal parasite. These results lead us to
propose the development of quinoxaline derivatives without a
substituent in position 7, to determinate if the behavior of
antiamoebic activity in both benzofuroxane and quinoxaline
derivatives is identical or different.
Toxicity profile in vitro on kidney epithelial cells (VERO) of quinoxaline-7-carbox-
ilate-1,4-di-N-oxide derivatives most active against Entamoeba histolytica strain
Compound
Cytotoxic activity
M)
SI
IC50
(
l
SD
T-001
T-003
T-008
T-009
T-011
T-014
T-016
T-017
T-020
T-022
MTZ
>100
>100
3.28
6.97
5.86
>100
>100
>100
13.08
6.74
—
—
2.12
1.65
1.97
—
—
—
4.96
3.68
—
>70.92
>50.25
2.14
3.70
16.74
>53.19
>68.02
>51.81
6.60
2.69
>22.22
2.75
4. Conclusion
We examined the biological activities of 21 new ethyl and
methyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives syn-
thesized by the Beirut reaction. Five quinoxaline-7-carboxylate
1,4-di-N-oxide derivatives showed better antiamoebic activity
and SI value than the reference drugs, metronidazole and nitazox-
anide, therefore these should be considered lead compounds for
the development of new antiamoebic agents. The potency of the
new compounds was two to eleven times greater than the refer-
ence compounds. Further optimization and stability in physiologi-
cal conditions of this series are in progress in our research group.
>100
10.74
NTZ
5.66
HM1: IMSS.
MTZ: metronidazole.
NTZ: nitazoxanide.
SI: selectivity index.
IC50: half maximal inhibitory concentration.
SD: standard deviation.
lM: micromolar.

B. E. Duque-Montaño et al. / Bioorg. Med. Chem. 21 (2013) 4550–4558
4555
5.6. T-006
5. Experimental data
5.1. T-001
Methyl
2-phenylamide-3-methylquinoxaline-7-carboxylate
1,4-di-N-oxide. This compound was obtained in 32.5% yield from
methyl benzofuroxane-5-carboxylate and 3-oxo-N-phenylbutana-
mide using method B. IR (KBr): 3077 (N–H), 2949 (ArC–H), 1728
Methyl 2-acetyl-3-methyl-quinoxaline-7-carboxylate 1,4-di-N-
oxide. This compound was obtained in 32.6% yield from methyl
benzofuroxane-5-carboxylate and 2,4-pentanedione using method
and 1682 (C@O), 1328 (N-oxide) cmÀ1 1H NMR (400 MHz,
.
B. IR (KBr): 2955 (ArC–H), 1726 (C@O), 1331 (N-oxide) cmÀ1 1H
.
DMSO-d6) ppm: 2.52 (s, 3H, CH3), 3.99 (s, 3H, CH3OOC), 7.20 (t,
J = 7.38 Hz, 1H, H4, NHC6H5), 7.42 (t, J = 7.78 Hz, 2H, H3 and H5,
NHC6H5), 7.67 (d, J = 7.92 Hz, H2 and H6, NHC6H5), 8.41 (d,
J = 8.84 Hz, H5), 8.62 (d, J = 8.93 Hz, 1H, H6), 9.0 (s, 1H, H8),
11.01 (s, 1H, NH). Calculated analysis for C18H15N3O5: C, 61.19;
H, 4.28; N, 11.89. Found: C, 61.05; H, 4.12; N, 11.46.
NMR (400 MHz, DMSO-d6) ppm: 2.38 (s, 3H, CH3), 2.66 (s, 3H,
COCH3), 3.98 (s, 3H, CH3OOC), 8.37 (d, J = 8.95 Hz, 1H, H5), 8.54
(d, J = 8.90 Hz, 1H, H6), 8.94 (s, 1H, H8). Calculated analysis for
C13H12N2O5: C, 56.52; H, 4.38; N, 10.14. Found: C, 56.35; H, 4.13;
N, 10.05.
5.7. T-007
5.2. T-002
Ethyl methyl-3-phehylquinoxaline-2,7-dicarboxylate 1,4-di-N-
oxide. This compound was obtained in 17% yield from methyl ben-
zofuroxane-5-carboxylate and ethyl benzoylacetate using method
Dimethyl 3-methylquinoxaline-2,7-dicarboxylate 1,4-di-N-
oxide. This compound was obtained in 12% yield from methyl ben-
zofuroxane-5-carboxylate and methyl acetoacetate using method
C. IR (KBr): 1715.30 and 1726.88 (C@O), 1327.65 (N-oxide) cmÀ1
.
C. IR (KBr): 1715.30 (C@O), 1333.57 (N-oxide) cmÀ1 1H NMR
.
1H NMR (400 MHz, DMSO-d6) ppm: 0.96 (t, 3H, COOCH2CH3),
4.16 (s, 3H, CH3OOC), 4.45 (q, J1 = 6.67 Hz, J2 = 6.40 Hz, 2H,
COOCH2CH3), 7.57 (s, 5H, C6H5), 8.44 (d, J = 8.59 Hz, 1H, H5), 8.61
(t, J = 7.98 Hz, 1H, H6), 8.95 (d, J = 8.99 Hz, 1H, H8). Calculated anal-
ysis for C19H16N2O6: C, 61.96; H, 4.38; N, 7.61. Found: C, 61.82; H,
4.15; N, 7.53.
(400 MHz, DMSO-d6) ppm: 2.44 (s, 3H, CH3), 3.97 (s, 3H, COOCH3),
4.03 (s, 3H, CH3OOC), 8.38 (d, J = 8.93 Hz, 1H, H5), 8.56 (d,
J = 8.89 Hz, 1H, H6), 8.85 (s, 1H, H8). Calculated analysis for
13H12N2O6: C, 53.43; H, 4.14; N, 9.59. Found: C, 53.29; H, 3.97;
N, 9.45.
C
5.3. T-003
5.8. T-008
Ethyl methyl-3-methylquinoxaline-2,7-dicarboxylate 1,4-di-N-
Methyl
2-acetyl-3-trifluoromethylquinoxaline-7-carboxylate
oxide. This compound was obtained in 17% yield from methyl ben-
zofuroxane-5-carboxylate and ethyl acetoacetate using method C.
1,4-di-N-oxide. This compound was obtained in 17.7% yield from
methyl benzofuroxane-5-carboxylate and 1,1,1-trifluoro-2,4-pen-
tanedione using method A. IR (KBr): 2962 (ArC–H), 1732 (C@O),
IR (KBr): 1719.20 and 1741.12 (C@O), 1328.02 (N-oxide) cmÀ1
.
1H NMR (400 MHz, DMSO-d6) ppm: 1.36 (t, 3H, COOCH2CH3),
2.45 (s, 3H, CH3), 3.97 (s, 3H, CH3OOC), 4.5 (q, J1 = 7.10 Hz,
J2 = 7.12 Hz, and J3 = 7.10 Hz, 2H, COOCH2CH3), 8.39 (d,
J = 8.98 Hz, 1H, H5), 8.56 (d, J = 8.99 Hz, 1H, H6), 8.85 (s, 1H, H8).
Calculated analysis for C14H14N2O6: C, 54.90; H, 4.61; N, 9.15.
Found: C, 54.56; H, 4.38; N, 9.35.
1337 (N-oxide), 1236 and 1173 (Ar–CF3) cmÀ1
.
1H NMR
(400 MHz, DMSO-d6) ppm: 2.62 (s, 3H, COCH3), 3.99 (s, 3H,
CH3OOC), 8.51 (d, J = 8.95, Hz, 1H, H5), 8.58 (d, J = 8.94, 1H, H6),
8.92 (s, 1H, H8). Calculated analysis for C13H9F3N2O5: C, 47.28; H,
2.75; N, 8.48. Found: C, 47.01; H, 2.56; N 8.45.
5.9. T-009
5.4. T-004
Methyl 2-isobutyryl-3-trifluoromethylquinoxaline-7-carboxyl-
ate 1,4-di-N-oxide. This compound was obtained in 23.9% yield
from methyl benzofuroxane-5-carboxylate and 1,1,1-trifluoro-
methyl-5-methyl-2,4-hexanedione using method A. IR (KBr):
2923 (ArC–H), 1730 (C@O), 1356 (N-oxide), 1293 and 1187 (Ar-
Tert-butyl methyl-3-methylquinoxaline-2,7-dicarboxylate 1,4-
di-N-oxide. This compound was obtained in 7% yield from methyl
benzofuroxane-5-carboxylate and tert-butyl acetoacetate using
method C. IR (KBr): 1719.78 and 1738.16 (C@O), 1324.32 (N-oxide)
cmÀ1
.
1H NMR (400 MHz, DMSO-d6) ppm: 1.60 (s, 9H,
CF3) cmÀ1 1H NMR (400 MHz, DMSO-d6) ppm: 1.27 (s, 6H,
.
COOC(CH3)3), 2.45 (s, 3H, CH3), 3.96 (s, 3H, CH3OOC), 8.36 (t,
J = 9.39 Hz, 1H, H5), 8.49–8.55 (m, 1H, H6), 8.87 (s, 1H, H8). Calcu-
lated analysis for C16H18N2O6: C, 57.48; H, 5.43; N, 8.38. Found: C,
57.31; H, 5.12; N, 8.21.
CH(CH3)2), 3.17 (q, J1 = 14.09 Hz, J2 = 7.05 Hz, 1H, CH(CH3)2), 4.07
(s, 3H, CH3OOC), 8.57 (d, J = 8.98 Hz, 1H, H5), 8.64 (d, J = 8.95 Hz,
1H, H6), 9.27 (s, 1H, H8). Calculated analysis for C15H13F3N2O5: C,
50.29; H, 3.66; N, 7.82. Found: C, 49.89; H, 3.47; N, 7.35.
5.10. T-010
5.5. T-005
Methyl 2-benzoyl-3-trifluoromethylquinoxaline-7-carboxylate
1,4-di-N-oxide. This compound was obtained in 10.9% yield from
methyl benzofuroxane-5-carboxylate and 4,4,4-trifluoro-1-phe-
nyl-1,3-butanedione using method A. IR (KBr): 2957 (ArC–H),
1732 and 1689 (C@O), 1337 (N-oxide), 1255 and 1164 (Ar–CF3)
Methyl 2-benzoyl-3-methylquinoxaline-7-carboxylate 1,4-di-
N-oxide. This compound was obtained in 10.9% yield from methyl
benzofuroxane-5-carboxylate
using method B. IR (KBr): 2949 (ArC-H), 1726 and 1674 (C@O),
1328 (N-oxide) cmÀ1 1H NMR (400 MHz, DMSO-d6) ppm: 2.54
and
1-phenyl-1,3-butanedione
.
cmÀ1 1H NMR (400 MHz, DMSO-d6) ppm: 4.01 (s, 3H, CH3OOC),
.
(s, 3H, CH3), 4.04 (s, 3H, CH3OOC), 7.56 (t, J = 7.75 Hz, 2H, H3
and H5, C6H5), 7.72 (t, J = 8.08 Hz, 1H, H4, C6H5), 7.90–7.93 (m,
2H, H2 and H6, C6H5), 8.52 (d, J = 9.0 Hz, 1H, H5), 8.76 (d,
J = 9.01 Hz, 1H, H6), 9.22 (s, 1H, H8). Calculated analysis for
7.61 (t, J = 7.09 Hz, 2H, H3 and H5, C6H5), 7.79 (t, J = 7.24 Hz, 1H,
H4, C6H5), 8.15 (d, J = 7.82 Hz, 2H, H2 and H6, C6H5), 8.52–8.54
(m, 2H, H5 and H6), 8.97 (s, 1H, H8). Calculated analysis for
C18H11F3N2O5: C, 55.11; H, 2.83; N, 7.14. Found: C, 54.98; H,
C18H11F3N2O5: C, 55.11; H, 2.83; N, 7.14. Found: C, 54.89; H,
2.63; N, 6.85.
2.75; N, 7.58.

4556
B. E. Duque-Montaño et al. / Bioorg. Med. Chem. 21 (2013) 4550–4558
5.11. T-011
Methyl
2H, CH3CH2OOC), 4.5 (q, J1 = 7.08 Hz, J2 = 7.07 Hz, 2H, COOCH2CH3),
8.39 (d, J = 8.99 Hz, 1H, H5), 8.56 (d, J = 8.98 Hz, 1H, H6), 8.90 (s,
1H, H8). Calculated analysis for C15H16N2O6: C, 56.25; H, 5.04; N,
8.75. Found: C, 56.09; H, 4.96; N, 8.56.
2-(thiophene-2-carbonyl)-3-trifluoromethylquinoxa-
line-7-carboxylate 1,4-di-N-oxide. This compound was obtained
in 5.6% yield from methyl benzofuroxane-5-carboxylate and
4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione using method A. IR
(KBr): 2955 (ArC–H), 1732 and 1664 (C@O), 1360 (N-oxide), 1264
5.16. T-016
and 1162 (Ar–CF3) cmÀ1
.
1H NMR (400 MHz, DMSO-d6) ppm:
Ethyl 2-amide-3-methylquinoxaline-7-carboxylate 1,4-di-N-
oxide. This compound was obtained in 11.7% yield from ethyl ben-
zofuroxane-5-carboxylate and acetoacetamide using method B. IR
(KBr): 3300 (NH), 2990 (ArC–H), 1692 (C@O), 1321 (N-oxide)
cmÀ1. 1H NMR (400 MHz, DMSO-d6) ppm: 1.38 (t, 3H, CH3CH2OOC),
2.48 (s, 3H, CH3), 4.4 (q, J1 = 7.33 Hz, J2 = 14.57 Hz, 2H,
CH3CH2OOC), 8.25 (s, 2H, NH2), 8.38–8.40 (m, 1H, H5), 8.56–8.61
(m, 1H, H6), 8.93 (s, 1H, H8). Calculated analysis for C13H13N3O5:
C, 53.61; H, 4.50; N, 14.43. Found: C, 53.21; H, 4.35; N, 14.16.
4.09 (s, CH3OOC), 7.22 (t, J = 4.41 Hz, 1H, H4, C4H3S), 7.63 (d,
J = 3.77 Hz, H5, C4H3S), 7.9 (d, J = 4.85 Hz, H3, C4H3S), 8.58 (d,
J = 8.86 Hz, 1H, H5), 8.67 (d, J = 8.89 Hz, 1H, H6), 9.31 (s, 1H, H8).
Calculated analysis for C16H9F3N2O5S: C, 48.25; H, 2.28; N, 7.03.
Found: C, 48.01; H, 1.96; N, 6.76.
5.12. T-012
Methyl 2-(naphthyl-2-carbonyl)-3-trifluoromethylquinoxaline-
7-carboxylate 1,4-di-N-oxide. This compound was obtained in
11.2% yield from methyl benzofuroxane-5-carboxylate and 4,4,4-
trifluoromethyl-1-(2-naphthyl)-1,3-butanedione using method A.
IR (KBr): 2962 (ArC–H), 1729 and 1685 (C@O), 1349 (N-oxide),
5.17. T-017
Ethyl 2-benzoyl-3-methylquinoxaline-7-carboxylate 1,4-di-N-
oxide. This compound was obtained in 12.3% yield from ethyl ben-
zofuroxane-5-carboxylate and 1-phenyl-1,3-butanedione using
method B. IR (KBr): 2979 (ArC–H), 1720 and 1684 (C@O), 1331
1287 and 1174 (Ar–CF3) cmÀ1 1H NMR (400 MHz, DMSO-d6)
.
ppm: 4.43–4.50 (m, 3H, CH3OOC), 7.65 (t, J = 7.50 Hz, 1H, H3,
10H7), 7.75 (t, J = 7.85 Hz, 1H, H6, C10H7), 8.01 (d, J = 8.12 Hz, 1H,
C
(N-oxide) cmÀ1 1H NMR (400 MHz, DMSO-d6) ppm: 1.41 (t,
.
H7, C10H7), 8.07 (d, J = 8.14 Hz, 1H, H5, C10H7), 8.14 (s, 2H, H2
and H4, C10H7), 8.52–8.55 (m, 1H, H8, C10H7), 8.56 (d, J = 8.87 Hz,
1H, H5), 8.88 (s, 1H, H6), 9.02 (s, 1H, H8). Calculated analysis for
J = 7.11 Hz, 3H, CH3CH2OOC), 2.32 (s, 3H, CH3), 4.46 (q,
J1 = 7.10 Hz, J2 = 7.13 Hz, 2H, CH3CH2OOC), 7.6 (t, J = 7.8 Hz, 2H,
H3 and H5, C6H5), 7.79 (t, J = 7.43 Hz, 1H, H4, C6H5), 8.1 (d,
J = 7.34 Hz, 2H, H2 and H6, C6H5), 8.38 (d, J = 8.9 Hz, 1H, H5), 8.5
(d, J = 8.94 Hz, 1H, H6), 8.99 (s, 1H, H8). Calculated analysis for
C22H13F3N2O5: C, 59.74; H, 2.96; N, 6.33. Found: C, 59.48; H,
2.57; N, 6.05.
C19H16N2O5: C, 64.77; H, 4.58; N, 7.95. Found: C, 64.58; H, 4.32;
5.13. T-013
N, 7.67.
Ethyl
methyl-3-(2-ethoxy-2-oxoethyl)quinoxaline-2,7-dicar-
5.18. T-018
boxylate 1,4-di-N-oxide. This compound was obtained in 6% yield
from methyl benzofuroxane-5-carboxylate and diethyl 3-oxoglu-
tarate using method C. IR (KBr): 1716.11 and 1737.12 (C@O),
Ethyl 2-phenylamide-3-methylquinoxaline-7-carboxylate 1,4-
di-N-oxide. This compound was obtained in 22.4% yield from ethyl
benzofuroxane-5-carboxylate and 3-oxo-N-phenylbutanamide
using method B. IR (KBr): 2981 (ArC–H), 1714 and 1661 (C@O),
1326.59 (N-oxide) cmÀ1 1H NMR (400 MHz, DMSO-d6) ppm: 1.17
.
(s, 3H, COCH2CH3), 1.32 (s, 3H, COOCH2CH3), 3.95 (s, 2H,
CH2COOCH2CH3), 3.97 (s, 3H, CH3OOC), 4.12 (q, J1 = 6.25 Hz,
J2 = 13.27 Hz, 2H, COOCH2CH3), 4.50 (q, J1 = 6.50 Hz, J2 = 13.43 Hz,
2H, CH2COOCH2CH3), 8.43 (d, J = 8.26 Hz, 1H, H5), 8.57 (d,
J = 8.78 Hz, 1H, H6), 8.89 (s, 1H, H8). Calculated analysis for
1369 (N-oxide) cmÀ1 1H NMR (400 MHz, DMSO-d6) ppm: 1.36
.
(m, 3H, CH3CH2OOC), 2.51 (s, 1H, CH3), 4.39–4.47 (q, J1 = 7.10 Hz,
J2 = 7.22 Hz, 2H, CH3CH2O), 7.20 (t, J = 7.34 Hz, 1H, H4-NHC6H5),
7.41 (t, J = 7.16 Hz, 2H, H3 and H5, NHC6H5), 7.65 (d, J = 7.84 Hz,
H2 and H6, NHC6H5), 8.39 (d, J = 8.98 Hz, 1H, H5), 8.59 (d,
J = 8.97 Hz, 1H, H6), 8.96 (s, 1H, H8), 11.10 (s, 1H, NH). Calculated
analysis for C19H17N3O5: C, 62.12; H, 4.66; N, 11.44. Found: C.
61.98; H, 4.49; N, 11.23.
C16H16N2O8: C, 52.75; H, 4.43; N, 7.69. Found: C, 52.41; H, 4.25;
N, 7.46.
5.14. T-014
Ethyl methyl-3-methyl-quinoxaline-2,7-dicarboxilate 1,4-di-N-
5.19. T-019
oxide. This compound was obtained in 10% yield from ethyl ben-
zofuroxane-5-carboxylate and methyl acetoacetate using method
Benzyl ethyl-3-methylquinoxaline-2,7-dicarboxylate 1,4-di-N-
oxide. This compound was obtained in 3% yield from ethyl ben-
zofuroxane-5-carboxylate and benzyl acetoacetate using method
C. IR (KBr): 1715.30 and 1726.88 (C@O), 1327.65 (N-oxide) cmÀ1
.
1H NMR (400 MHz, DMSO-d6) ppm: 1.35 (s, 3H, CH3CH2OOC),
2.44 (s, 3H, CH3), 4.03 (s, 3H, COOCH3), 4.48 (q, J1 = 7.08 Hz,
J2 = 7.07 Hz, 2H, CH3CH2OOC), 8.39 (d, J = 8.99 Hz, 1H, H5), 8.56
(d, J = 8.99 Hz, 1H, H6), 8.84 (s, 1H, H8). Calculated analysis for
C. IR (KBr): 1715.30 and 1726.88 (C@O), 1327.65 (N-oxide) cmÀ1
.
1H NMR (400 MHz, DMSO-d6) ppm: 1.35 (t, 3H, CH3CH2OOC),
2.51 (s, 3H, CH3), 4.35 (q, J1 = 7.08 Hz, J2 = 7.15 Hz, 2H,
CH3CH2OOC), 5.47 (s, 2H: COOCH2C6H5), 7.39–7.46 (m, 3H, C6H5),
7.53 (d, J = 7.69 Hz, 2H, C6H5), 7.70 (d, J = 8.64 Hz,1H, H5), 8.03,
(d, J = 8.63 Hz, 1H, H6), 8.35 (s, 1H, H8). Calculated analysis for
C14H14N2O6: C, 54.90; H, 4.61; N, 9.15. Found: C, 54.76; H, 4.27;
N, 9.13
5.15. T-015
C20H18N2O6: C, 62.82; H, 4.74; N, 7.33. Found: C, 62.95; H, 4.71;
N, 7.42.
Diethyl 3-methylquinoxaline-2,7-dicarboxylate 1,4-di-N-oxide.
This compound was obtained in 13% yield from ethyl benzofurox-
ane-5-carboxylate and ethyl acetoacetate using method C. IR (KBr):
5.21. T-020
1715.30 and 1740.12 (C@O), 1333.57 (N-oxide) cmÀ1
.
1H NMR
Ethyl 2-acetyl-3-trifluoromethylquinoxaline-7-carboxylate 1,4-
(400 MHz, DMSO-d6) ppm: 1.34–1.40 (m, 6H, CH3CH2OOC and
COOCH2CH3), 2.45 (s, 3H, CH3), 4.4 (q, J1 = 7.10 Hz, J2 = 7.09 HZ,
di-N-oxide. This compound was obtained in 11.9% yield from ethyl
benzofuroxane-5-carboxylate and 1,1,1-trifluoro-2,4-pentanedi-

B. E. Duque-Montaño et al. / Bioorg. Med. Chem. 21 (2013) 4550–4558
4557
one using method A. IR (KBr): 2978 (ArC–H), 1746 (C@O), 1355 (N-
oxide), 1271 and 1158 (Ar–CF3) cmÀ1 1H NMR (400 MHz, DMSO-
5.25. T-025
.
d6) ppm: 1.40 (t, J = 7.11 Hz, 3H, CH3CH2OOC), 2.62 (s, 3H, COCH3),
4.45 (q, J1 = 7.12 Hz, J2 = 7.13 Hz, 2H, CH3CH2OOC), 8.52 (d,
J = 8.95 Hz, 1H, H5), 8.58 (d, J = 8.96 Hz, 1H, H6), 8.92 (s, 1H, H8).
Calculated analysis for C14H11 F3N2O5: C, 48.85; H, 3.22; N, 8.14.
Found: C, 48.58; H, 3.15; N, 8.23.
Diethyl 3-phenylquinoxaline-2,7-dicarboxylate 1,4-di-N-oxide.
This compound was obtained in 10% yield from ethyl benzofurox-
ane-5-carboxylate and ethyl benzoylacetate using method C. IR
(KBr): 1715.30 and 1726.88 (C@O), 1327.65 (N-oxide) cmÀ1 1H
.
NMR (400 MHz, DMSO-d6) ppm: 1.10 (s, 3H, CH3CH2OOC), 1.25
(t, 3H, COOCH2CH3), 4.18 (q, J1 = 6.60 Hz, J2 = 6.50 Hz, 2H,
COOCH2CH3), 4.22 (q, J1 = 6.60 Hz, J2 = 6.50 Hz, 2H, CH3CH2OOC),
7.56 (s, 5H, C6H5), 8.49–8.51 (m, 1H, H5), 8.60–8.63 (m, 1H, H6),
8.93 (s, 1H, H8). Calculated analysis for C20H18N2O6: C, 62.82; H,
4.74; N, 7.33. Found: C, 62.71; H, 4.52; N, 7.13.
5.22. T-021
Ethyl 2-propionyl-3-trifluoromethylquinoxaline-7-carboxylate
1,4-di-N-oxide. This compound was obtained in 5.15% yield from
ethyl benzofuroxane-5-carboxylate and 1,1,1-trifluoro-2,4-hex-
anedione using method A. IR (KBr): 2981 (ArC–H), 1728 (C@O),
Acknowledgments
1355 (N-oxide), 1258 and 1169 (Ar–CF3) cmÀ1
.
1H NMR
(400 MHz, DMSO-d6) ppm: 1.35 (t, 3H, COCH2CH3), 1.39 (t, 3H,
CH3CH2OOC), 4.45 (q, J1 = 6.99 Hz, J2 = 14.10 Hz, 2H, COCH2CH3),
4.5 (q, J1 = 7.05 Hz, J2 = 14.17 Hz, 2H, CH3CH2OOC), 8.50 (d,
J = 8.96 Hz, 1H, H5), 8.56 (d, J = 8.94 Hz, 1H, H6), 8.88 (s, 1H, H8).
Calculated analysis for C15H13F3N2O5: C, 50.29; H, 3.66; N, 7.82.
Found: C, 50.12; H, 3.36; N, 7.56.
Blanca Estela Duque Montaño is the recipient of a scholarship
(No. 208856) from CONACyT México. The authors are thankful to
Dra. Mireya de la Garza for her donation of the Entamoeba histoly-
tica strain HM1:IMSS. We wish to express our gratitude to CONA-
CYT (CB-2010-01, clave 0157358) and the Instituto Politecnico
Nacional (SIP-IPN, Mexico) for their financial support. Gildardo
Rivera holds a scholarship from the Comision de Operacion y
Fomento de Actividades Academicas (COFAA-IPN). All participants
declare no conflict of interest.
5.23. T-022
Ethyl 2-(thiophene-2-carbonyl)-3-trifluoromethylquinoxaline-
7-carboxylate 1,4-di-N-oxide. This compound was obtained in
23.2% yield from ethyl benzofuroxane-5-carboxylate and 4,4,4-tri-
fluoro-1-(2-thienyl)-1,3-butanedione using method A. IR (KBr):
2987 (ArC–H), 1726 and 1665 (C@O), 1336 (N-oxide), 1286 and
References and notes
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1161 (Ar–CF3) cmÀ1 1H NMR (400 MHz, DMSO-d6) ppm: 1.41 (t,
.
J = 7.1 Hz, 3H, CH3CH2OOC), 4.47 (q, J1 = 7.07 Hz, J2 = 7.12 Hz, 2H,
CH3CH2OOC), 7.32 (d, J = 4.7 Hz, 1H, H4, C4H3S), 8.24 (d,
J = 4.59 Hz, H5, C4H3S), 8.3 (d, J = 4.8 Hz, H3, C4H3S), 8.51 (d,
J = 8.95 Hz, 1H, H5), 8.56 (d, J = 8.96 Hz, 1H, H6), 8.96 (s, 1H, H8).
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5.24. T-024
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