New 3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide derivatives as anti-Mycobacterium tuberculosis agents

Article abstract of DOI:10.1016/j.bmc.2010.02.024

Mycobacterium tuberculosis (M.Tb) is a bacillus capable of causing a chronic and fatal condition in humans known as tuberculosis (TB). It is estimated that there are 8 million new cases of TB per year and 3.1 million infected people die annually. Thirty-six new amide quinoxaline 1,4-di-N-oxide derivatives have been synthesized and evaluated as potential anti-tubercular agents, obtaining biological values similar to the reference compound, Rifampin (RIF).

Full text of DOI:10.1016/j.bmc.2010.02.024

Bioorganic & Medicinal Chemistry 18 (2010) 2713–2719
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New 3-methylquinoxaline-2-carboxamide 1,4-di-N-oxide derivatives as
anti-Mycobacterium tuberculosis agents
Saioa Ancizu, Elsa Moreno, Beatriz Solano, Raquel Villar, Asunción Burguete, Enrique Torres,
*
Silvia Pérez-Silanes , Ignacio Aldana, Antonio Monge
Unidad de Investigación y Desarrollo de Medicamentos, Centro de Investigación en Farmacobiología Aplicada (CIFA), University of Navarra, C/Irunlarrea s/n, 31008 Pamplona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Mycobacterium tuberculosis (M.Tb) is a bacillus capable of causing a chronic and fatal condition in humans
known as tuberculosis (TB). It is estimated that there are 8 million new cases of TB per year and 3.1 mil-
lion infected people die annually. Thirty-six new amide quinoxaline 1,4-di-N-oxide derivatives have been
synthesized and evaluated as potential anti-tubercular agents, obtaining biological values similar to the
reference compound, Rifampin (RIF).
Received 23 September 2009
Revised 9 February 2010
Accepted 12 February 2010
Available online 20 February 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Anti-tuberculosis agents
Quinoxaline 1,4-di-N-oxide derivatives
Mycobacterium tuberculosis (M.Tb.)
Rifampin
1. Introduction
anti-parasitic, etc.) with application in many different therapeutic
areas.6–8 As a result of the anti-tuberculosis research project, our
Tuberculosis (TB) is a respiratory disease mainly caused by the
bacillus Mycobacterium tuberculosis (M.Tb.). It is responsible for 8
million new cases and 3.1 million deaths per year, mostly occur-
ring in developing countries, although there are over 400,000
new cases annually in industrialized countries. As infection with
human immunodeficiency virus (HIV) becomes more prevalent,
tuberculosis is becoming a serious problem in developed countries
as well,1 as can be observed in Figure 1. Of particular concern is the
development of drug-resistant forms of the disease, Multidrug-
Resistant Tuberculosis (MDR-TB) and Extensively Drug-Resistant
Tuberculosis (XDR-TB). These forms of the disease are more often
fatal and are difficult and expensive to treat. It has been estimated
that up to 50 million people are infected with drug-resistant forms
of TB.2,3
group has published several papers in which synthesis and biolog-
ical activity assessments of a large number of quinoxaline and
quinoxaline 1,4-di-N-oxide derivatives have been described,9–17
identifying some structural requirements for optimal activity.13–19
A few years ago, a group of thirty-one 3-methylquinoxaline-2-
carboxamide 1,4-di-N-oxide derivatives were prepared and tested
against M.Tb., obtaining some interesting results.13 Three of these
compounds presented enough selectivity and good results in mac-
rophage assay so as to merit continuation of their study. With the
aim of improving the previous results, new amide quinoxaline 1,4-
di-N-oxide derivatives have been synthesized and evaluated as po-
tential anti-tubercular agents, obtaining excellent results as can be
observed in this report.
Due to the problems related to MDR-TB and XDR-TB, it is neces-
sary to develop new, potent, fast-acting anti-tuberculosis drugs
with low-toxicity profiles that can be given in conjunction with
drugs used to treat HIV infections.4,5 New drugs and improved
delivery methods will be integral parts of a strategy to fully control
future outbreaks of TB, particularly MDR-TB, which has severely
challenged the limited number of effective treatment options.3
Quinoxaline and quinoxaline 1,4-di-N-oxide derivatives display
excellent biological activities (antiviral, anticancer, antibacterial,
2. Results and discussion
Thirty-six new 1,4-di-N-oxide-3-methylquinoxaline-2-carbox-
ylic acid aryl amide derivatives were prepared through the syn-
thetic route as follows:
First, the starting benzofuroxanes (BFX) were obtained by previ-
ously described methods.20,12 Next, the initial b-acetoacetamide
derivatives (1–4) were synthesized through acetoacetylation of
corresponding aryl amines by diketene, using methanol as the sol-
vent as shown in Scheme 1.21
Finally, the new 1,4-di-N-oxide-quinoxaline derivatives were
prepared using a variation of the Beirut reaction, where the
* Corresponding author. Tel.: +34 948 425653; fax: +34 948 425652.
E-mail address: sperez@unav.es (S. Pérez-Silanes).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.024

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S. Ancizu et al. / Bioorg. Med. Chem. 18 (2010) 2713–2719
Figure 1. Estimated number of new TB cases, by country, 2007. [Source, Global Tuberculosis Control WHO REPORT 2009].
neously cytotoxicity (IC50) in VERO cells is evaluated. Next, the
MIC and IC50 values are formed into a ratio termed Selectivity In-
dex (SI). If the SI level is 10 or greater, a compound is considered
active at the second level.
O
O
O
N
H
n
O
n= 1, 2
H2N
n
MeOH
+
W= H, OCH3
W
W
H2C
(1-4)
As can be observed in Table 1, compounds 10 and 27 were iden-
tified as the most interesting derivatives, with great anti-tubercu-
lar activity and low values of cytotoxicity, as can be observed by
their SI values. Derivatives 9, 13, 28, 29, 36 and 38 have also shown
very good results. Some structure–activity relationships could be
established.
Scheme 1. Synthetic scheme of initial b-acetoacetamide derivatives (1–4).
different BFX react with the corresponding b-acetoacetamide in the
presence of calcium chloride and ethanolamine as catalysts
(Scheme 2).22
Quinoxaline derivatives were un-substituted or substituted in
positions 6 and 7 by chloro, fluoro or trifluoromethyl moiety as
electron-withdrawing groups and by methyl or methoxy moiety
as electron-releasing groups. When the new quinoxalines were
prepared from monosubstituted-BFX, mixtures of positional
isomers were obtained. Generally, it could be observed that the
7-substituted isomer prevailed over 6-substituted isomer.23
Table 1 summarizes the biological values of the synthesized
compounds. The primary screening level determines the activity
of the compounds against M.Tb. in H37Rv strain. Samples showing
a percentage of TB growth inhibition greater than or equal to 90%
are considered active and therefore, move on to the secondary
screening. Active compounds are tested in order to determine the
actual minimum inhibitory concentration (MIC), and simulta-
Looking at the values for compounds 9–13, 20–22, 27–31 and
36–40, it can be said that, in general, the introduction of an elec-
tron-withdrawing substituent in the quinoxaline ring results in
an increment in the anti-tubercular activity of the derivatives. On
the contrary, the insertion of an electro-releasing moiety results
in a reduction of this activity, as can be observed by the results ob-
tained for the compounds 6–8, 15–17, 24–26 and 33–35. In short, it
can be concluded that the insertion of an electron-withdrawing
moiety in the quinoxaline ring is an essential requirement in order
to improve the anti-tubercular activity, as established in previous
works reported by the group.13
To further explore the SAR of these compounds, a methoxy
group was substituted in the para position of the benzene ring. This
derivatization led to an increase of the cytotoxicity of the
O-
O
O-
O
O
N+
N+
O
R7
R6
R7
R6
CaCl2
MeOH
N
H
n
N
H
n
+
N+
W
N
W
O-
HO
NH2
(1-4)
BFX
(5-40)
n= 1, 2
W= H, OCH3
R6, R7= H, Cl, F, OCH3, CH3, CF3
Scheme 2. Synthetic route of 1,4-di-N-oxide-3-methylquinoxaline-2-carboxylic acid aryl amide derivatives (5–40).

S. Ancizu et al. / Bioorg. Med. Chem. 18 (2010) 2713–2719
2715
Table 1
Biological values of compounds (5–40) at secondary screening
Compd
R7
R6
n
W
MIC (
l
g/mL) H37Rva
IC50
(l
g/mL) VEROb
SIc (IC50/MIC)
5
6
7
8
H
H
H
CH3
H
H
Cl
H
F
H
H
H
CH3
H
H
Cl
H
F
H
H
H
CH3
H
H
Cl
H
F
H
H
H
CH3
H
H
Cl
H
F
H
1
H
H
H
H
H
H
H
H
2.606
4.207
>100
12.346
0.433
<0.2
1.522
1.727
0.406
2.847
8.903
>100.00
12.007
NT
>100
>100
NT
>38.372
>23.77
NT
NT
>230.94
>500
>65.70
10.64
>246.30
>10.537
>3.3696
NT
NT
NT
NT
>19.893
21.734
>30.03
>32.81
>11.60
NT
CH3
CH3
OCH3
Cl
Cl
F
F
CF3
H
CH3
CH3
OCH3
Cl
Cl
F
F
CF3
H
CH3
CH3
OCH3
Cl
Cl
F
F
CF3
H
CH3
CH3
OCH3
Cl
Cl
F
F
CF3
—
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
—
NT
9
>100
>100
>100
18.378
>100
>30
>30
NT
NT
NT
NT
>30
16.605
>30
>100
>100
NT
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
RIF
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
—
NT
1.508
0.764
0.999
3.048
8.621
16.359
13.657
<0.2
<0.195
0.504
0.517
1.153
3.464
8.852
>100.00
16.866
<0.195
0.454
<0.195
2.058
1.427
0.015–0.125
NT
NT
>500
>100
>30
>100
>30
>30
>30
>30
NT
>153.84
>198.41
>58.027
>26.019
>8.6605
>3.389
NT
NT
NT
>30
>30
>30
>30
>30
>100
>153.84
>66.079
>153.84
>14.577
>21.023
>800
—
NT, Non tested.
In the first screening, all of the compounds were evaluated against M.Tb. in H37Rv strain. All of them were active at this level. In the secondary screening, the actual minimum
inhibitory concentration (MIC) in H37Rv strain and cytotoxicity (IC50) in VERO cells were determined. Finally, the SI was calculated and the compounds containing SI
level P 10 were considered active at the second level. All of these active compounds are highlighted in green in the table, with the most active ones being marked in dark
green.
a
Actual minimum inhibitory concentration in M.Tb. H37Rv strain.
Cytotoxicity in VERO cells.
Selectivity index.
b
c
compounds, as can be observed by comparing the following deriv-
atives 5 versus 14, 6 versus 15, 11 versus 20, 13 versus 22, 24 ver-
sus 33, 23 versus 32, 27 versus 36 and 29 versus 38. With the aim
of improving these results, current studies are ongoing to modify
not only the substituent on the benzene ring but also the aromatic
ring itself.
Finally, looking at the values of the compounds 10 versus 28
and 13 versus 31, it can be observed that lengthening the aliphatic
chain can also result in a greater value of cytotoxicity. Taking into
account these results and previous ones obtained by the group,13
the most suitable linker is established as a single methylene group
between the carboxamide and the benzene ring.
inhibition percentage greater than or equal to 90%. These com-
pounds were considered active at first level and moved on to the
secondary screening, where the actual minimum inhibitory con-
centration (MIC) in H37Rv strain and cytotoxicity (IC50) in VERO
cells were determined. In this case, twenty three of the twenty-
six evaluated derivatives showed an SI greater than 10, being con-
sidered to be active at the second level.
Taking into account the SI values shown in Table 1, it can be said
that these values are similar to the reference compound, Rifampin
(RIF). In conclusion, the potency, selectivity, and low cytotoxicity of
these compounds make them valid leads for synthesizing new
compounds that possess better activity.
3. Conclusions
4. Experimental section
4.1. General remarks
Thirty-six new 1,4-di-N-oxide-3-methylquinoxaline-2-carbox-
ylic acid aryl amide derivatives were synthesized using a variation
of the Beirut reaction. Thirty-four of these compounds were evalu-
ated against M.Tb. in H37Rv strain; all of them showed a TB growth
All of the synthesized compounds were chemically character-
ized by thin layer chromatography (TLC), infrared (IR), proton

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S. Ancizu et al. / Bioorg. Med. Chem. 18 (2010) 2713–2719
nuclear magnetic resonance (1H NMR) and elemental microanaly-
ses (CHN).
phase was dried with sodium sulphate and filtered. The solvent
was removed in vacuo and precipitated with cold diethyl ether in
order to obtain a yellow solid. The solid was purified by column
chromatography, if necessary.
Alugram SIL G/UV254 (Layer: 0.2 mm) (Macherey-Nagel GmbH
& Co. KG., Düren, Germany) was used for TLC, and Silica gel 60
(0.040–0.063 mm, Merck) was used for Flash Column Chromatog-
raphy. The 1H MNR spectra were recorded on a Bruker 400 Ultra-
shield instrument (400 MHz), using TMS as internal standard and
with DMSO-d6 or CDCl3 as solvents; the chemical shifts are re-
ported in ppm (d) and coupling constant (J) values are given in
Hertz (Hz). Signal multiplicities are represented by: s (singlet), bs
(broad singlet), d (doublet), dd (double doublet), ddd (double dou-
ble doblet), t (triplet), q (quadruplet) and m (multiplet). The IR
spectra were recorded on a Nicolet Nexus FTIR (Thermo, Madison,
USA) in KBr pellets. Elemental microanalyses were obtained on a
CHN-900 Elemental Analyzer (Leco, Tres Cantos, Spain) from vac-
uum-dried samples. The analytical results for C, H and N were
within 0.4 of the theoretical values. Chemicals were purchased
from Panreac Química S.A. (Barcelona, Spain), Sigma-Aldrich Quí-
mica, S.A. (Alcobendas, Spain), Acros Organics (Janssen Pharmaceu-
ticalaan, Geel, Belgium) and Lancaster (Bischheim-Strasbourg,
France).
4.2.2.1. 1,4-Di-N-oxide-3-methylquinoxaline-2-carboxylic acid
2-benzylamide (5). Yield: 47%. IR (KBr): 3218 (m, NH); 1677 (s,
CO); 1324 (s, N+Oꢀ). 1H MNR (400 MHz, DMSO-d6) d ppm: 9.36
(t, 1H, JNH–CH2 = 5.8 Hz, NH); 8.52–8.49 (m, 2H, H5+H8); 8.02–7.95
(m, 2H, H6+H7); 7.46–7.28 (m, 5H, Ph); 4.57 (d, 2H, CH2); 2.43 (s,
3H, CH3). Anal. Calcd for C17H15N3O3: C, 66.02; H, 4.85; N, 13.59.
Found: C, 66.02; H, 4.96; N, 13.69.
4.2.2.2. 3,7-Dimethyl-1,4-di-N-oxidequinoxaline-2-carboxylic
acid 2-benzylamide (6). Yield: 1.3%. IR (KBr): 3222 (m, NH); 1661
(s, CO); 1328 (s, N+Oꢀ). 1H MNR (400 MHz, DMSO-d6) d ppm: 9.37
(t, 1H, JNH–CH2 = 5.9 Hz, NH); 8.39 (d, 1H, JH5–H6 = 8.8 Hz, H5); 8.31
(d, 1H, JH8–H6 = 1.5 Hz, H8); 7.83 (dd, 1H, H6); 7.46–7.28 (m, 5H,
Ph); 4.56 (d, 2H, CH2); 2.59 (s, 3H, CH3–C3); 2.41 (s, 3H, CH3–C7).
Anal. Calcd for C18H17N3O3: C, 66.87; H, 5.26; N, 13.00. Found: C,
66.79; H, 5.26; N, 12.91.
4.2. General procedure of synthesis
4.2.2.3. 1,4-Di-N-oxide-3,6,7-trimethylquinoxaline-2-carboxylic
acid 2-benzylamide (7). Yield: 21%. IR (KBr): 3199 (m, NH); 1679
(s, CO); 1328 (s, N+Oꢀ). 1H MNR (400 MHz, CDCl3) d ppm: 8.94 (t,
1H, JNH–CH2 = 5.6 Hz, NH); 8.05 (s, 1H, H5); 7.98 (s, 1H, H8); 7.49–
7.34 (m, 5H, Ph); 4.67 (d, 2H, CH2); 2.65 (s, 3H, CH3–C3); 2.51 (s,
3H, CH3–C7); 2.48 (s, 3H, CH3–C6). Anal. Calcd for C19H19N3O3: C,
67.66; H, 5.64; N, 12.46. Found: C, 67.89; H, 5.50; N, 12.45.
4.2.1. Synthesis of b-acetoacetamide derivatives (1–4)
The corresponding aryl amines (20.0 mmol) were diluted in
methanol (20.0 mL) in N2 atmosphere and cooled in ice bath until
0 °C. Next, diketene (22.0 mmol) was added dropwise and the reac-
tion was stirred for 2–3 h. The obtained residue was precipitated
with cold diethyl ether and filtered in order to obtain a red–brown
solid. The compound was used without further purification.
4.2.2.4. 1,4-Di-N-oxide-7-methoxy-3-methylquinoxaline-2-car-
boxylic acid 2-benzylamide (8). Yield: 8%. IR (KBr): 3276 (m,
NH); 1655 (s, CO); 1323 (s, N+Oꢀ). 1H MNR (400 MHz, DMSO-d6)
4.2.1.1. N-Benzyl-3-oxobutanamide (1). Yield 25%. IR (KBr): 3318
(m, NH); 1617 (s, CO); 1540 (s, CO). 1H MNR (400 MHz, CDCl3) d
ppm: 9.52 (br s, 1H, NH); 7.33–7.21 (m, 5H, Ph); 4.56–4.35 (m,
4H, CH2–NH–CO–CH2–CO); 1.89 (s, 3H, CH3).
d
ppm: 9.37 (t, 1H,
JNH–CH2 = 5.9 Hz, NH); 8.42 (d, 1H,
JH5–H6 = 9.5 Hz, H5); 7.81 (d, 1H, JH8–H6 = 2.5 Hz, H8); 7.60 (dd, 1H,
H6); 7.47–7.28 (m, 5H, Ph); 4.56 (d, 2H, CH2); 4.00 (s, 3H, OCH3);
2.40 (s, 3H, CH3). Anal. Calcd for C18H17N3O4: C, 63.53; H, 5.00;
N, 12.35. Found: C, 63.92; H, 5.05; N, 12.48.
4.2.1.2. N-(4-Methoxybenzyl)-3-oxobutanamide (2). Yield 77%.
IR (KBr): 3317 (s, NH); 1619 (s, CO); 1539 (s, CO). 1H MNR
(400 MHz, DMSO-d6) d ppm: 8.46 (t, 1H, JNH–CH2 = 5.0 Hz, NH);
7.20 (d, 2H, JH2–H3 = 8.6 Hz, H2+H6); 6.89 (d, 2H, H3+H5); 4.22 (d,
2H, CH2–NH); 3.73 (s, 2H, CO–CH2–CO); 3.35 (s, 3H, OCH3); 2.15
(s, 3H, CH3).
4.2.2.5. 7-Chloro-1,4-di-N-oxide-3-methylquinoxaline-2-car-
boxylic acid 2-benzylamide (9). Yield: 35%. IR (KBr): 3235 (m,
NH); 1669 (s, CO); 1321 (s, N+Oꢀ). 1H MNR (400 MHz, DMSO-d6)
d ppm: 8.55–8.52 (m, 2H, H5+H8); 8.49 (t, 1H, JNH–CH2 = 5.7 Hz,
NH); 7.81 (dd, 1H, JH6–H5 = 9.2 Hz, JH6–H8 = 2.2 Hz, H6); 7.45–7.32
(m, 5H, Ph); 4.74 (d, 2H, CH2); 2.79 (s, 3H, CH3). Anal. Calcd for
C17H14ClN3O3: C, 59.38; H, 4.07; N: 12.23. Found: C, 59.06; H,
4.19; N, 12.13.
4.2.1.3. 3-Oxo-N-phenethylbutanamide (3). Yield 46%. IR (KBr):
3257 (s, NH); 1714 (s, CO); 1644 (s, CO). 1H MNR (400 MHz,
DMSO-d6) d ppm: 8.13 (t, 1H, JNH–CH2 = 5.2 Hz, NH); 7.34–7.14 (m,
5H, Ph); 3.33–3.30 (m, 2H, CH2–NH); 3.27 (s, 2H, CO–CH2–CO);
2.72 (t, 2H, JCH2–Ph = 7.2 Hz, CH2–Ph); 2.10 (s, 3H, CH3).
4.2.2.6. 6,7-Dichloro-1,4-di-N-oxide-3-methylquinoxaline-2-
carboxylic acid 2-benzylamide (10). Yield: 61%. IR (KBr): 3274
(m, NH); 1649 (s, CO); 1328 (s, N+Oꢀ). 1H MNR (400 MHz, DMSO-
d6) d ppm: 8.72 (s, 1H, H5); 8.68 (s, 1H, H8); 8.41 (t, 1H, JNH–
CH2 = 5.8 Hz, NH); 7.44–7.32 (m, 5H, Ph); 4.74 (d, 2H, CH2); 2.79
(s, 3H, CH3). Anal. Calcd for C18H15Cl2N3O4: C, 54.11; H, 3.44; N,
11.14. Found: C, 54.08; H, 3.38; N, 10.87.
4.2.1.4. N-(4-Methoxyphenethyl)-3-oxobutanamide (4). Yield
56%. IR (KBr): 3263 (m, NH); 1719 (m, CO); 1641 (s, CO). 1H MNR
(400 MHz, DMSO-d6) d ppm: 8.10 (t, 1H, JNH–CH2 = 5.3 Hz, NH);
7.13 (d, 2H, JH2–H3 = 8.4 Hz, H2+H6); 6.85 (d, 2H, H3+H5); 3.72 (s,
3H, OCH3); 3.32–3.18 (m, 4H, CH2–NH–CO–CH2–CO); 2.65 (t, 2H,
JCH2–Ph = 7.3 Hz, CH2–Ph); 2.10 (s, 3H, CH3).
4.2.2.7. 1,4-Di-N-oxide-7-fluoro-3-methylquinoxaline-2-car-
boxylic acid 2-benzylamide (11). Yield: 45%. IR (KBr): 3300 (m,
NH); 1671 (s, CO); 1323 (s, N+Oꢀ). 1H MNR (400 MHz, DMSO-d6)
d ppm: 8.61 (dd, 1H, JH5–H6 = 9.5 Hz, JH5–F = 5.0 Hz, H5); 8.52 (d,
4.2.2. Synthesis of 1,4-di-N-oxide-3-methyl-quinoxaline-2-car-
boxylic acid aryl amide derivatives (5–40)
The corresponding BFX (1.0 mmol) and the corresponding
b-acetoacetamide (1.2 mmol) were dissolved in the minimum
amount of methanol. Next, calcium chloride (0.1 mmol) and etha-
nolamine (5 drops) were added as catalyst, as described by Stumm
and Niclas.22 The reaction was stirred at room temperature from 1
to 5 h, filtered, and washed with cold ethanol. The solid was dis-
solved in dichloromethane and quenched with water. The organic
1H,
JNH–CH2 = 5.7 Hz, NH); 8.19 (dd, 1H, JH8–F = 8.5 Hz,
JH8–H6 = 2.7 Hz, H8); 7.62 (ddd, 1H, JH6–F = 7.3 Hz, H6); 7.46–7.28
(m, 5H, Ph); 4.74 (d, 2H, CH2); 2.77 (s, 3H, CH3). Anal. Calcd for
C17H14FN3O3: C, 62.37; H, 4.28; N, 12.84. Found: C, 62.28; H,
4.32; N, 12.96.

S. Ancizu et al. / Bioorg. Med. Chem. 18 (2010) 2713–2719
2717
4.2.2.8. 6,7-Difluoro-1,4-di-N-oxide-3-methylquinoxaline-2-
carboxylic acid 2-benzylamide (12). Yield: 38%. IR (KBr): 3292
(m, NH); 1670 (s, CO); 1529 (s, N+Oꢀ). 1H MNR (400 MHz, DMSO-
d6): 9.38 (t, 1H, JNH–CH2 = 6.0 Hz, NH); 8.59–8.48 (m, 2H, H5+H8);
7.46–7.26 (m, 5H, Ph); 4.56 (d, 2H, CH2); 2.42 (s, 3H, CH3). Anal.
Calcd for C17H13F2N3O3: C, 59.13; H, 3.77; N, 12.17. Found: C,
59.36; H, 3.62; N, 11.90.
4.2.2.15. 6,7-Dichloro-1,4-di-N-oxide-3-methylquinoxaline-2-
carboxylic acid 2-(4-methoxybenzyl)-amide (19). Yield, 18%. IR
(KBr), 3199 (m, NH); 1643 (f, CO); 1323 (f, N+O). 1H MNR
(400 MHz, DMSO-d6) d ppm, 9.30 (t, 1H, JNH–CH2 = 5.8 Hz, NH);
0
0
0
0
8.67 (s, 2H, H5+H8); 7.35 (d, 2H, JH2 –H3 = 8.5 Hz, H2 +H6 ); 6.94 (d,
0
0
1H, H3 +H5 ), 4.48 (d, 2H, CH2), 3.75 (s, 3H, OCH3), 2.40 (s, 3H,
CH3). Anal. Calcd for C18H15Cl2N3O4, C, 57.84; H, 4.31; N, 11.24.
Found: C, 57.92; H, 4.32; N, 11.22.
4.2.2.9. 1,4-Di-N-oxide-3-methyl-7-trifluoromethylquinoxa-
line-2-carboxylic acid 2-benzylamide (13). Yield: 28%. IR (KBr):
3294 (m, NH); 1670 (s, CO); 1323 (s, N+Oꢀ). 1H MNR (400 MHz,
DMSO-d6) d ppm: 8.92 (d, 1H, JH8–H6 = 1.7 Hz, H8); 8.70 (d, 1H,
4.2.2.16. 1,4-Di-N-oxide-7-fluoro-3-methylquinoxaline-2-car-
boxylic acid 2-(4-methoxybenzyl)-amide (20). Yield, 31%. IR
(KBr), 3213 (m, NH); 1677 (f, CO); 1323 (f, N+O). 1H MNR
(400 MHz, DMSO-d6) d ppm, 9.28 (t, 1H, JNH–CH2 = 5.9 Hz, NH);
8.57 (dd, 1H, JH5–H6 = 9.5 Hz, JH5–F = 5.2 Hz, H5); 8.24 (dd, 1H,
JH8–F = 8.8, JH8–H6 = 2.8 Hz, H8); 7.92 (ddd, 1H, JH8–F = 8.0 Hz, H6);
J
H5–H6 = 9.0 Hz, H5); 8.38 (br s, 1H, NH); 8.03 (dd, 1H, H6); 7.45–
7.32 (m, 5H, Ph); 4.75 (d, 2H, CH2); 2.82 (s, 3H, CH3). Anal. Calcd
for C18H14F3N3O3: C, 57.29; H, 3.71; N, 11.14. Found: C, 57.25; H,
3.62; N, 11.15.
0
0
0
0
0
0
7.36 (d, 2H, JH2 –H3 = 8.7 Hz, H2 +H6 ); 6.94 (d, 2H, H3 +H5 ); 4.49
(d, 2H, CH2); 3.76 (s, 3H, OCH3); 2.40 (s, 3H, CH3). Anal. Calcd for
C18H16FN3O4, C, 60.50; H, 4.51; N, 11.76. Found: C, 60.74; H,
4.51; N, 11.73.
4.2.2.10. 1,4-Di-N-oxide-3-methylquinoxaline-2-carboxylic acid
2-(4-methoxybenzyl)-amide (14). Yield: 82%. IR (KBr): 3174 (m,
NH); 1674 (f, CO); 1329 (f, N+Oꢀ). 1H MNR (400 MHz, DMSO-d6)
d ppm: 9.26 (t, 1H, JNH–CH2 = 5.7 Hz, NH); 8.52–8.50 (m, 2H,
4.2.2.17. 6,7-Difluoro-1,4-di-N-oxide-3-methylquinoxaline-2-
carboxylic acid 2-(4-methoxybenzyl)-amide (21). Yield, 71%. IR
(KBr), 3199 (m, NH); 1645 (f, CO); 1333 (f, N+O). 1H MNR
(400 MHz, DMSO-d6) d ppm, 9.29 (t, 1H, JNH–CH2 = 5.8 Hz, NH);
0
0
H8+H5); 8.02–7.95 (m, 2H, H6+H7); 7.37 (d, 2H, JH2 –H3 = 8.7 Hz,
0
0
0
0
H2 +H6 ); 6.95 (d, 2H, H3 +H5 ); 4.49 (d, 2H, CH2); 3.76 (s, 3H,
OCH3); 2.42 (s, 3H, CH3). Anal. Calcd for C18H17N3O4: C, 63.71; H,
5.05; N, 12.38. Found: C, 63.43; H, 5.11; N, 12.03.
0
0
0
0
8.56–8.50 (m, 2H, H5+H8); 7.36 (d, 2H, JH2 –H3 = 8.5 Hz, H2 +H6 );
0
0
6.94 (d, 2H, H3 +H5 ); 4.48 (d, 2H, CH2); 3.76 (s, 3H, OCH3); 2.41
(s, 3H, CH3). Anal. Calcd for C18H15F2N3O4, C, 57.60; H, 4.03; N,
11.20. Found: C, 57.33; H, 3.91; N, 11.14.
4.2.2.11. 3,7-Dimethyl-1,4-di-N-oxidequinoxaline-2-carboxylic
acid 2-(4-methoxybenzyl)-amide (15). Yield: 71%. IR (KBr): 3206
(m, NH); 1674 (f, CO); 1326 (f, N+O). 1H MNR (400 MHz, DMSO-d6)
d
ppm: 9.27 (t, 1H,
J
NH–CH2 = 5.9 Hz, NH); 8.39 (d, 1H,
4.2.2.18. 1,4-Di-N-oxide-3-methyl-7-trifluoromethylquinoxa-
line-2-carboxylic acid 2-(4-methoxybenzyl)-amide (22). Yield,
78%. IR (KBr), 9.88%. IR (KBr), 3276 (m, NH); 1648 (f, CO); 1133
(f, N+O). 1H MNR (400 MHz, DMSO-d6) d ppm, 9.29 (t, 1H,
J
H5–H6 = 8.8 Hz, H5); 8.30 (s, 1H, H8); 7.82 (dd, 1H, JH6–H8 = 1.7 Hz,
0
0
0
0
0
0
H6); 7.36 (d, 2H, JH2 –H3 = 8.5 Hz, H2 +H6 ), 6.94 (d, 2H, H3 +H5 ),
4.48 (d, 2H, CH2), 3.76 (s, 3H, OCH3), 2.59 (s, 3H, CH3–C7), 2.39 (s,
3H, CH3–C3). Anal. Calcd for C19H19N3O4: C, 64.58; H, 5.42; N,
11.89. Found: C, 64.83; H, 5.61; N, 11.50.
JNH–CH2 = 6.1 Hz, NH); 8.75 (s, 1H, H5); 8.70–8.69 (m, 1H, H8);
0
0
0
0
8.32–8.26 (m, 1H, H6); 7.36 (d, 2H, JH2 –H3 = 8.5 Hz, H2 +H6 ); 6.95
0
0
(d, 2H, H3 +H5 ); 4.49 (d, 2H, CH2); 3.76 (s, 3H, OCH3); 2.44 (s, 3H,
CH3). Anal. Calcd for C19H16F3N3O4, C, 56.02; H, 3.93; N, 10.32.
Found: C, 56.39; H, 3.85; N, 10.44.
4.2.2.12. 1,4-Di-N-oxide-3,6,7-trimethylquinoxaline-2-carbox-
ylic acid 2-(4-methoxybenzyl)-amide (16). Yield, 64%. IR (KBr),
3210 (m, NH); 1677 (f, CO); 1332 (f, N+O). 1H RMN (400 MHz,
4.2.2.19. 1,4-Di-N-oxide-3-methylquinoxaline-2-carboxylic acid
2-phenylethylamide (23). Yield, 55%. IR (KBr), 3213 (m, NH);
1668 (s, CO); 1334 (s, N+Oꢀ). 1H MNR (400 MHz, DMSO-d6) d
ppm, 8.91 (t, 1H, JNH–CH2 = 5.6 Hz, NH); 8.50–8.45 (m, 2H, H5+H8);
8.02–7.95 (m, 2H, H6+H7); 7.35–7.22 (m, 5H, Ph); 3.62–3.57 (m,
2H, NH–CH2); 2.87 (t, 2H, JCH2–Ph = 7.1 Hz, CH2–Ph), 2.25 (s, 3H,
CH3). Anal. Calcd for C18H17N3O3, C, 66.87; H, 5.26; N, 13.00. Found:
C, 66.50; H, 5.30; N, 12.79.
DMSO-d6) d ppm, 9.28 (t, 1H,
H5); 8.25 (s, 1H, H8); 7.37 (d, 2H, JH2 –H3 = 8.5 Hz, H2 +H6 ); 6.94
JNH–CH2 = 5.7 Hz, NH); 8.27 (s, 1H,
0
0
0
0
0
0
(d, 2H, H3 +H5 ); 4.48 (d, 2H, CH2); 3.76 (s, 3H, OCH3); 2.50 (s, 6H,
CH3–C6+CH3–C7); 2.39 (s, 3H, CH3–C3). Anal. Calcd for C20H21N3O4,
C, 65.38; H, 5.76; N, 11.44. Found: C, 65.09; H, 5.62; N, 11.63.
4.2.2.13. 1,4-Di-N-oxide-7-methoxy-3-methylquinoxaline-2-
carboxylic acid 2-(4-methoxybenzyl)-amide (17). Yield, 79%. IR
(KBr), 3283 (m, NH); 1652 (f, CO); 1329 (f, N+O). 1H MNR
(400 MHz, DMSO-d6) d ppm, 9.28 (t, 1H, JNH–CH2 = 5.7 Hz, NH);
8.40 (d, 1H, JH5–H6 = 9.5 Hz, H5); 7.79 (d, 1H, JH8–H6 = 2.6 Hz, H8);
4.2.2.20. 3,7-Dimethyl-1,4-di-N-oxidequinoxaline-2-carboxylic
acid 2-phenylethylamide (24). Yield, 67%. IR (KBr), 3203 (m, NH);
1666 (s, CO); 1326 (s, N+Oꢀ). 1H MNR (400 MHz, DMSO-d6) d ppm,
8.91 (t, 1H, JNH–CH2 = 5.7 Hz, NH); 8.38 (d, 1H, JH5–H6 = 8.8 Hz, H5);
8.23 (s, 1H, H8); 7.82 (d, 1H, H6); 7.35–7.21 (m, 5H, Ph); 3.62–
3.57 (m, 2H, NH–CH2); 2.87 (t, 2H, JCH2–Ph = 7.1 Hz, CH2–Ph); 2.59
(s, 3H, CH3–C7); 2.23 (s, 3H, CH3–C3). Anal. Calcd for C19H19N3O3,
C, 67.65; H, 5.64; N, 12.46. Found: C, 67.28; H, 5.87; N, 12.16.
0
0
0
0
7.59 (dd, 1H, H6); 7.37 (d, 2H, JH2 –H3 = 8.5 Hz, H2 +H6 ); 6.94 (d,
0
0
2H, H3 +H5 ); 4.48 (d, 2H, CH2), 3.99 (s, 3H, OCH3–C7), 3.75 (s, 3H,
0
OCH3–C4 ), 2.38 (s, 3H, CH3). Anal. Calcd for C19H19N3O5, C, 61.78;
H, 5.18; N, 11.38. Found: 61.58; H, 5.21; N, 11.32.
4.2.2.14. 7-Chloro-1,4-di-N-oxide-3-methylquinoxaline-2-car-
boxylic acid 2-(4-methoxybenzyl)-amide (18). Yield, 30%. IR
(KBr), 3270 (m, NH); 1654 (f, CO); 1330 (f, N+O). 1H MNR
(400 MHz, DMSO-d6), 9.27 (t, 1H, JNH–CH2 = 5.6 Hz, NH); 8.53–8.47
(m, 2H, H5+H8); 8.03 (dd, 1H, JH6–H5 = 9.2 Hz, JH6–H8 = 2.3 Hz, H6);
4.2.2.21. 1,4-Di-N-oxide-3,6,7-trimethylquinoxaline-2-carbox-
ylic acid 2-phenylethylamide (25). Yield, 61%. IR (KBr), 3203 (m,
NH); 1663 (s, CO); 1328 (s, N+Oꢀ). 1H RMN (400 MHz, DMSO-d6) d
ppm, 8.93 (t, 1H, JNH–CH2 = 5.6 Hz, NH); 8.26 (s, 1H, H5), 8.23 (s, 1H,
H8); 7.36–7.22 (m, 5H, Ph); 3.61–3.56 (m, 2H, NH–CH2); 2.87 (t, 2H,
JCH2–Ph = 7.1 Hz, CH2–Ph); 2.50 (s, 6H, CH3–C6+CH3–C7); 2.22 (s, 3H,
CH3–C3). Anal. Calcd for C20H21N3O3, C, 68.38; H, 5.98; N, 11.97.
Found: C, 68.47; H, 6.08; N, 11.75.
0
0
0
0
0
0
7.36 (d, 2H, JH2 –H3 = 8.7 Hz, H2 +H6 ); 6.94 (d, 2H, H3 +H5 ); 4.48
(d, 2H, CH2); 3.76 (s, 3H, OCH3); 2.40 (s, 3H, CH3). Anal. Calcd for
C18H16ClN3O4, C, 57.84; H, 4.31; N, 11.24. Found: C, 57.92; H,
4.32; N, 11.22.

2718
S. Ancizu et al. / Bioorg. Med. Chem. 18 (2010) 2713–2719
4.2.2.22. 1,4-Di-N-oxide-7-methoxy-3-methylquinoxaline-2-car-
boxylic acid 2-phenylethylamide (26). Yield, 36%. IR (KBr), 3180
(m, NH); 1665 (s, CO); 1329 (s, N+Oꢀ). 1H MNR (400 MHz, DMSO-
Anal. Calcd for C19H19N3O4, C, 64.59; H, 5.38; N, 11.90. Found: C,
64.25; H, 5.24; N, 11.81.
d6)
d
ppm, 8.93 (t, 1H,
J
NH–CH2 = 5.5 Hz, NH); 8.40 (d, 1H,
4.2.2.29. 3,7-Dimethyl-1,4-di-N-oxidequinoxaline-2-carboxylic
acid 2-(4-methoxyphenyl)-ethylamide (33). Yield, 19.11%. IR
(KBr), 3209 (m, NH); 1666 (s, CO); 1329 (s, N+Oꢀ). 1H MNR
(400 MHz, DMSO-d6) d ppm, 8.88 (t, 1H, JNH–CH2 = 5.5 Hz, NH);
8.38 (d, 1H, JH5–H6 = 8.9 Hz, H5); 8.26 (s, 1H, H8); 7.81 (d, 1H, H6);
JH5–H6 = 9.5 Hz, H5); 7.77 (d, 1H, JH8–H6 = 2.5 Hz, H8); 7.60 (dd, 1H,
H6); 7.36–7.23 (m, 5H, Ph); 3.99 (s, 3H, OCH3); 3.61–3.56 (m, 2H,
NH–CH2); 2.87 (t, 2H, JCH2–Ph = 7.02 Hz, CH2–Ph); 2.22 (s, 3H,
CH3). Anal. Calcd for C19H19N3O4, C, 64.59; H, 5.38; N, 11.90. Found:
C, 64.46; H, 5.44; N, 11.66.
0
0
0
0
0
0
7.22 (d, 2H, JH2 –H3 = 8.2 Hz, H2 +H6 ); 6.89 (d, 2H, H3 +H5 ), 3.72
(s, 3H, OCH3); 3.56–3.52 (m, 2H, NH–CH2); 2.80 (t, 2H,
JCH2–Ph = 7.0 Hz, CH2–Ph); 2.58 (s, 3H, CH3–C7); 2.25 (s, 3H, CH3–
C3). Anal. Calcd for C20H21N3O4, C, 65.40; H, 5.72; N, 11.44. Found:
C, 65.62; H, 5.66; N, 11.58.
4.2.2.23. 7-Chloro-1,4-di-N-oxide-3-methylquinoxaline-2-car-
boxylic acid 2-phenylethylamide (27). Yield, 44%. IR (KBr), 3224
(m, NH); 1667 (s, CO); 1324 (s, N+Oꢀ). 1H MNR (400 MHz, DMSO-
d6)
d ppm, 8.91 (t, 1H, JNH–CH2 = 5.2 Hz, NH); 8.49 (d, 1H,
J
H5–H6 = 9.2 Hz, H5); 8.45 (d, 1H, JH8–H6 = 2.3 Hz, H8); 8.03 (dd, 1H,
4.2.2.30. 1,4-Di-N-oxide-3,6,7-trimethylquinoxaline-2-carbox-
ylic acid 2-(4-methoxyphenyl)-ethylamide (34). Yield, 52.94%.
IR (KBr), 3203 (m, NH); 1665 (s, CO); 1326 (s, N+Oꢀ). 1H MNR
(400 MHz, DMSO-d6) d ppm, 8.90 (t, 1H, JNH–CH2 = 5.6 Hz, NH);
H6); 7.36–7.29 (m, 5H, Ph); 3.62–3.57 (m, 2H, NH–CH2); 2.87 (t,
JCH2–Ph = 7.1 Hz, 2H, CH2–Ph); 2.23 (s, 3H, CH3). Anal. Calcd for
C18H16ClN3O3, C, 60.42; H, 4.47; N, 11.75. Found: C, 60.14; H,
4.50; N, 11. 58.
0
8.26 (s, 1H, H5); 8.22 (s, 1H, H8); 7.22 (d, 2H, JH2 –H3 = 8.5 Hz,
0
0
0
0
H2 +H6 ); 6.88 (d, 2H, H3 +H5 ); 3.73 (s, 1H, OCH3); 3.56–3.51 (m,
2H, NH–CH2); 2.79 (t, 2H, JCH2–Ph = 7.1 Hz, CH2–Ph); 2.46 (s, 6H,
CH3–C7+CH3–C6); 2.25 (s, 3H, CH3–C3). Anal. Calcd for C21H23N3O4,
C, 66.14; H, 6.04; N, 11.02. Found: C, 66.02; H, 6.09; N, 10.99.
4.2.2.24. 6,7-Dichloro-1,4-di-N-oxide-3-methylquinoxaline-2-
carboxylic acid 2-phenylethylamide (28). Yield, 38%. IR (KBr),
3231 (m, NH); 1666 (s, CO); 1322 (s, N+Oꢀ). 1H MNR (400 MHz,
DMSO-d6) d ppm, 88.93 (t, 1H, JNH–CH2 = 5.6 Hz, NH); 8.66 (s, 1H,
H5); 8.64 (s, 1H, H8); 7.34–7.22 (m, 5H, Ph); 3.62–3.57 (m, 2H,
NH–CH2); 2.87 (t, 2H, JCH2–Ph = 7.0 Hz, CH2–Ph); 2.24 (s, 3H, CH3–
C3). Anal. Calcd for C18H15Cl2N3O3, C, 55.10; H, 3.82; N, 10.71.
Found: C, 54.70; H, 3.92; N, 10.44.
4.2.2.31. 1,4-Di-N-oxide-7-methoxy-3-methylquinoxaline-2-
carboxylic acid 2-(4-methoxyphenyl)-ethylamide (35). Yield,
20.97%. IR (KBr), 3221 (m, NH); 1666 (s, CO); 1325 (s, N+Oꢀ). 1H
MNR (400 MHz, DMSO-d6) d ppm, 8.89 (t, 1H, JNH–CH2 = 5.5 Hz,
NH); 8.40 (d, 1H, JH5–H6 = 9.5 Hz, H5); 7.76 (d, 1H, JH8–H6 = 2.7 Hz,
0
0
0
0
4.2.2.25. 1,4-Di-N-oxide-7-fluoro-3-methylquinoxaline-2-car-
boxylic acid 2-phenylethylamide (29). Yield, 77%. IR (KBr), 3231
(m, NH); 1669 (s, CO); 1328 (s, N+Oꢀ). 1H MNR (400 MHz, DMSO-
d6) d ppm, 8.92 (t, 1H, JNH–CH2 = 5.7 Hz, NH); 8.56 (dd, 1H,
H8); 7.59 (dd, 1H, H6); 7.22 (d, 2H, JH2 –H3 = 8.6 Hz, H2 +H6 ); 6.88
0
0
0
(d, 2H, H3 +H5 ); 3.98 (s, 3H, OCH3–C7); 3.72 (s, 3H, OCH3–C4 );
3.57–3.52 (m, 2H, NH–CH2); 2.80 (t, 2H, JCH2–Ph = 7.1 Hz, CH2–Ph);
2.22 (s, 3H, CH3). Anal. Calcd for C20H21N3O5, C, 62.66; H, 5.48; N,
10.97. Found: C, 62.37; H, 5.66; N, 10.66.
JH5–H6 = 9.5 Hz, JH5–F = 5.2 Hz, H5); 8.20 (dd, 1H, JH8–F = 8.8 Hz,
H8–H6 = 2.5 Hz, H8); 7.91 (ddd, 1H, JH6–F = 8.0 Hz, H6); 7.36–7.23
J
(m, 5H, Ph); 3.62–3.57 (m, 2H, NH–CH2); 2.87 (t, 2H,
JCH2–Ph = 7.0 Hz, CH2–Ph); 2.23 (s, 3H, CH3). Anal. Calcd for
C18H16FN3O3, C, 63.34; H, 4.69; N, 12.32. Found: C, 63.02; H,
4.80; N, 12.32.
4.2.2.32. 7-Chloro-1,4-di-N-oxide-3-methylquinoxaline-2-car-
boxylic acid 2-(4-methoxyphenyl)-ethylamide (36). Yield,
48.00%. IR (KBr), 3287 (m, NH); 1655 (s, CO); 1328 (s, N+Oꢀ). 1H
MNR (400 MHz, DMSO-d6) d ppm, 8.87 (s, 1H, NH); 8.49 (d,1H,
JH5–H6 = 9.3 Hz, H5); 8.45 (d, 1H, JH8–H6 = 2.1 Hz, H8); 8.02 (dd, 1H,
0
0
0
0
0
0
4.2.2.26. 6,7-Difluoro-1,4-di-N-oxide-3-methylquinoxaline-2-
carboxylic acid 2-phenylethylamide (30). Yield, 39%. IR (KBr),
3240 (m, NH); 1668 (s, CO); 1332 (s, N+Oꢀ). 1H MNR (400 MHz,
DMSO-d6) d ppm, 8.93 (t, 1H, JNH–CH2 = 5.6 Hz, NH); 8.53–8.47 (m,
2H, H6+H8); 7.36–7.20 (m, 5H, Ph); 3.61–3.56 (m, 2H, NH–CH2);
2.87 (t, 2H, JCH2–Ph = 7.1 Hz, CH2–Ph); 2.24 (s, 3H, CH3). Anal. Calcd
for C18H15F2N303, C, 60.17; H, 4.18; N, 11.70. Found: C, 60.32; H,
4.34; N, 11.87.
H6); 7.22 (d, 2H, JH2 –H3 = 8.5 Hz, H2 +H6 ); 6.88 (d, 2H, H3 +H5 );
3.73 (s, 3H, OCH3); 3.57–3.52 (m, 2H, NH–CH2); 2.80 (t, 2H,
JCH2–Ph = 7.0 Hz, CH2–Ph); 2.26 (s, 3H, CH3). Anal. Calcd for
C19H18ClN3O4, C, 58.84; H, 4.65; N, 10.84. Found: C, 58.62; H,
4.72; N, 10.40.
4.2.2.33. 6,7-Dichloro-1,4-di-N-oxide-3-methylquinoxaline-2-
carboxylic acid 2-(4-methoxyphenyl)-ethylamide (37). Yield,
28.40%. IR (KBr), 3230 (m, NH); 1667 (s, CO); 1324 (s, N+Oꢀ). 1H
MNR (400 MHz, DMSO-d6) d ppm, 8.90 (t, 1H, JNH–CH2 = 5.6 Hz,
4.2.2.27. 1,4-Di-N-oxide-3-methyl-7-trifluoromethylquinoxa-
line-2-carboxylic acid 2-phenylethylamide (31). Yield, 9%. IR
(KBr), 3243 (m, NH); 1669 (s, CO); 1323 (s, N+Oꢀ). 1H MNR
(400 MHz, DMSO-d6) d ppm, 8.93 (t, 1H, JNH–CH2 = 5.4 Hz, NH);
8.74 (s, 1H, H8); 8.66 (d, 1H, JH5–H6 = 9.2 Hz, H5); 8.27 (d, 1H, H6);
7.33–7.31 (m, 5H, Ph); 3.58–3.63 (m, 2H, NH–CH2); 2.88 (t,
JCH2–Ph = 7.0 Hz, 2H, CH2–Ph); 2.27 (s, 3H, CH3). Anal. Calcd for
C19H16F3N3O3, C, 58.31; H, 4.09; N, 10.74. Found: C, 58.10; H,
4.025; N, 10.76.
0
0
NH); 8.66 (s, 1H, H5); 8.64 (s, 1H, H8); 7.22 (d, 2H, JH2 –H3 = 8.5 Hz,
0
0
0
0
H2 +H6 ); 6.88 (d, 2H, H3 +H5 ); 3.72 (s, 1H, OCH3); 3.57–3.52 (m, 2H,
NH–CH2); 2.79 (t, 2H, JCH2–Ph = 6.9 Hz, CH2–Ph); 2.26 (s, 3H, CH3).
Anal. Calcd for C19H17Cl2N3O4, C, 54.03; H, 4.03; N, 9.95. Found:
C, 53.66; H, 3.92; N, 9.65.
4.2.2.34. 1,4-Di-N-oxide-7-fluoro-3-methylquinoxaline-2-car-
boxylic acid 2-(4-methoxyphenyl)-ethylamide (38). Yield,
18.76%. IR (KBr), 3223 (m, NH); 1668 (s, CO); 1330 (s, N+Oꢀ). 1H
MNR (400 MHz, DMSO-d6) d ppm, 8.88 (t, 1H, JNH–CH2 = 5.6 Hz,
NH); 8.56 (dd, 1H JH5–H6 = 9.4 Hz, JH5–F = 5.2 Hz, H5); 8.20 (dd, 1H,
JH8–F = 8.8 Hz, JH8–H6 = 2.8 Hz, H8); 7.94–7.81 (m, 1H, H6); 7.22 (d,
2H, JH2 –H3 = 8.6 Hz, H2 +H6 ); 6.88 (d, 2H, H3 +H5 ); 3.72 (s, 3H,
OCH3); 3.57–3.52 (m, 2H, NH–CH2); 2.80 (t, 2H, JCH2–Ph = 7.1 Hz,
CH2–Ph); 2.28 (s, 3H, CH3). Anal. Calcd for C19H18FN3O4, C, 61.46;
H, 4.85; N, 11.32. Found: C, 61.10; H, 4.78; N, 11.54.
4.2.2.28. 1,4-Di-N-oxide-3-methylquinoxaline-2-carboxylic acid
2-(4-methoxyphenyl)-ethylamide (32). Yield, 10,90%. IR (KBr),
3212 (m, NH); 1670 (s, CO); 1328 (s, N+Oꢀ). 1H MNR (400 MHz,
DMSO-d6) d ppm, 8.88 (t, 1H, JNH–CH2 = 5.5 Hz, NH); 8.48–8.46 (m,
0
0
0
0
0
0
0
0
2H, H5+H8); 8.00–7.98 (m, 2H, H6+H7); 7.22 (d, 2H, JH2 –H3 = 8.6 Hz,
0
0
0
0
H2 +H6 ); 6.88 (d, 2H, H3 +H5 ), 3.72 (s, 3H, OCH3); 3.57–3.52 (m, 2H,
NH–CH2); 2.80 (t, 2H, JCH2–Ph = 7.1 Hz, CH2–Ph); 2.28 (s, 3H, CH3).

S. Ancizu et al. / Bioorg. Med. Chem. 18 (2010) 2713–2719
2719
4.2.2.35. 6,7-Difluoro-1,4-di-N-oxide-3-methylquinoxaline-2-
carboxylic acid 2-(4-methoxyphenyl)-ethylamide (39). Yield,
12.75%. IR (KBr), 3242 (m, NH); 1669 (s, CO); 1334 (s, N+Oꢀ). 1H
MNR (400 MHz, DMSO-d6) d ppm, 8.90 (t, 1H, JNH–CH2 = 5.5 Hz,
Assay. RIF was used as the reference compound (RIF IC50
>100 g/mL).
l
4.3.4. Determination of selectivity index (SI)
0
0
NH); 8.48–8.46 (m, 2H, H5+H8); 7.22 (d, 2H, JH2 –H3 = 8.4 Hz,
The selectivity index (SI) is defined as the ratio of the measured
IC50 in VERO cells to the MIC for M.Tb. H37Rv. In general, require-
ments for moving a compound into in vivo testing include,
MIC 6 6.25 lg/mL and SI P 10 (occasionally lower). RIF was used
as the reference compound (RIF SI >800).
0
0
0
0
H2 +H6 ); 6.88 (d, 2H, H3 +H5 ); 3.76 (s, 3H, OCH3); 3.57–3.52 (m,
2H, NH–CH2); 2.79 (t, 2H, JCH2–CH2 = 7.0 Hz, CH2–CH2–Ph); 2.26 (s,
3H, CH3). Anal. Calcd for C19H17F2N3O4, C, 58.61; H, 4.37; N,
10.80. Found: C, 58.86; H, 4.435; N, 10.55.
4.2.2.36. 1,4-Di-N-oxide-3-methyl-7-trifluoromethylquinoxaline-
2-carboxylic acid 2-(4-methoxyphenyl)-ethylamide (40). Yield,
6.79%. IR (KBr), 3243 (m, NH); 1669 (s, CO); 1324 (s, N+Oꢀ). 1H
MNR (400 MHz, DMSO-d6) d ppm, 8.90 (t, 1H, JNH–CH2 = 5.7 Hz,
NH); 8.74 (s, 1H, H5); 8.66 (d, 1H, JH8–H6 = 1.8 Hz, H8); 8.27 (dd,
Acknowledgments
This work has been carried out with the financial support of the
RIDIMEDCHAG-CYTED and PIUNA project from University of Nava-
rra. We also wish to express our gratitude to the Tuberculosis Anti-
microbial Acquisition & Coordinating Facility (TAACF) for the
evaluation of the anti-tuberculosis activity through research and
development contracts. S.A. is indebted to the Navarra Government
for a grant. E.M. is indebted to the La Rioja Government for a grant.
0
0
0
0
1H, JH6–H5 = 9.1 Hz, H6); 7.22 (d, 2H, JH2 –H3 = 8.6 Hz, H2 +H6 );
0
0
6.88 (d, 2H, H3 +H5 ); 3.71 (s, 3H, OCH3); 3.58–3.53 (m, 2H, NH–
CH2), 2.80 (t, 2H, JCH2–Ph = 7.0 Hz, CH2–Ph); 2.22 (s, 3H, CH3). Anal.
Calcd for C20H18F3N3O4, C, 57.01; H, 4.28; N, 9.98. Found: C,
57.33; H, 4.22; N, 9.88.
4.3. General procedure of anti-tuberculosis activity
References and notes
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Hakkou, A.; Bouakka, M.; Ellis, T. Molecules 2002, 7, 641.
In vitro evaluation of the anti-tuberculosis activity was carried
out at the GWL Hansen’s Disease Center within the Tuberculosis
Antimicrobial Acquisition & Coordinating Facility (TAACF) screen-
ing program for the discovery of novel drugs for the treatment of
tuberculosis. 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 experi-
mental compounds can be tested for their capability to inhibit
the growth of virulent M.Tb.24
4.3.1. Determination of growth inhibition percentage via MABA
The initial screen is conducted against M.Tb. H37Rv (ATCC
27294) in BACTEC 12B medium using the Microplate Alamar Blue
Assay (MABA).25 Compounds exhibiting fluorescence were tested
in the BACTEC 460-radiometric system. Compounds effecting
9. Ortega, M. A.; Sainz, Y.; Montoya, M. E.; López de Ceráin, A.; Monge, A.
Pharmazie 1999, 54, 24.
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A. Arzneim.-Forsch. 2002, 52, 113.
<90% inhibition in the primary screen (MIC >6.25
not further evaluated.
lg/mL) were
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Lenaerts, A. J.; Franzblau, S. G.; Cho, S. H.; Monge, A.; Goldman, R. C. J.
Antimicrob. Chemother. 2008, 62, 547.
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Maddry, J. A.; Lenaerts, A. J.; Franzblau, S. G.; Cho, S. H.; Monge, A.; Goldman, R.
C. Antimicrob. Agents Chemother. 2008, 52, 3321.
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Villar, R.; Aldana, I.; Monge, A. Bioorg. Med. Chem. 2009, 17, 385.
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Aldana, I.; Monge, A. Molecules 2008, 13, 86.
4.3.2. Determination of minimum inhibitory concentration
(MIC) via MABA
Compounds demonstrating at least 90% inhibition in the pri-
mary screen were re-tested against M.Tb. H37Rv at lower concen-
trations in order to determine the actual minimum inhibitory
concentration (MIC) in the MABA. The MIC was defined as the low-
est concentration effecting a reduction in fluorescence of 90% rela-
tive to controls. Rifampicin was used as the reference compound
(RIF 0.015–0.125 lg/mL).
20. González, M.; Cerecetto, H. In Topics in Heterocyclic Chemistry; Khan, M. T. H.,
Ed.; Bioactive Heterocycles IV. Benzofuroxan and Furoxan Chemistry and
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4.3.3. Determination of cytotoxicity in VERO cells
Compounds are screened by serial dilution to assess toxicity to
a VERO cell line at concentrations less than or equal to 6.25 lg/ml
or 10 times the MIC for M.Tb. H37Rv if sample solubility in culture
medium permits. 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 Non-radioactive Cell Proliferation
24. TAACF, http://www.taacf.org/Process-text.htm#assays.
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