Microwave-assisted solvent-free synthesis and in vitro antibacterial screening of quinoxalines and pyrido[2, 3b]pyrazines

Article abstract of DOI:10.3390/molecules17055164

We report herein the microwave assisted synthesis, without solvents and catalysts, of 6-substituted quinoxalines and 7-substituted pyrido[2,3b] pyrazines. The compounds were obtained in good yields and short reaction times using the mentioned procedure and two new structures are reported. A complete ¹H-and ¹³C-NMR assignment was performed using 1D and 2D-NMR. Additionally, an in vitro screening was performed on Gram-positive and Gram-negative bacteria using amoxicillin as positive reference. Compounds bearing a pyridyl group tended to have higher antibacterial activity, but the best activity against Bacillus subtilis and Proteus mirabilis was observed with quinoxaline derivatives.

Full text of DOI:10.3390/molecules17055164

Molecules 2012, 17, 5164-5176; doi:10.3390/molecules17055164
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Microwave-assisted Solvent-free Synthesis and in Vitro
Antibacterial Screening of Quinoxalines and
Pyrido[2, 3b]pyrazines
J. Jesús Morales-Castellanos 1, Karina Ramírez-Hernández 1, Nancy S. Gómez-Flores 1,
Oscar R. Rodas-Suárez 2 and Javier Peralta-Cruz 1,*
1
Department of Organic Chemistry, Escuela Nacional de Ciencias Biológicas del Instituto
Politécnico Nacional, Prol. Carpio y Plan de Ayala, México 11340 D. F., Mexico;
E-Mails: jesus_qorg@hotmail.com (J.J.M.-C.); chemycal_girl@hotmail.com (K.R.-H.);
gomeznancy11@hotmail.com (N.S.G.-F.)
2
Department of Microbiology, Escuela Nacional de Ciencias Biológicas del Instituto Politécnico
Nacional, Prol. Carpio y Plan de Ayala, México 11340 D. F., Mexico; E-Mail: rodas07@gmail.com
* Author to whom correspondence should be addressed; E-Mail: jperalta@ipn.mx;
Tel.: +52-5729-6000 (ext. 62457); Fax: +52-5729-6000 (ext. 62414).
Received: 15 March 2012; in revised form: 22 April 2012 / Accepted: 23 April 2012 /
Published: 4 May 2012
Abstract: We report herein the microwave assisted synthesis, without solvents and
catalysts, of 6-substituted quinoxalines and 7-substituted pyrido[2,3b]pyrazines. The
compounds were obtained in good yields and short reaction times using the mentioned
1
13
procedure and two new structures are reported. A complete H- and C-NMR assignment
was performed using 1D and 2D-NMR. Additionally, an in vitro screening was performed
on Gram-positive and Gram-negative bacteria using amoxicillin as positive reference.
Compounds bearing a pyridyl group tended to have higher antibacterial activity, but
the best activity against Bacillus subtilis and Proteus mirabilis was observed with
quinoxaline derivatives.
Keywords: 6-substituted quinoxalines; pyrido[2,3b] pyrazines; antimicrobial activity

Molecules 2012, 17
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1. Introduction
The most recurrent diseases in North and South American countries are those caused by
Escherichia coli and Bacillus subtilis [1,2]. The antibiotics amoxicillin, norfloxacilin and ciprofloxacin
have been the therapeutic agents used to prevent their consequences [3–7], but these drugs have
undesired secondary effects and increased chronic toxicity and microbes have developed resistance to
most of them [8]. Efforts to prepare antibacterial agents from quinoxaline have resulted in compounds
used against a wide variety of disorders, including cancer, diabetes, diabetic retinopathy, rheumatoid
arthritis, hemangioma and Kaposi's sarcoma, which are related to vasculogenesis and angiogenesis [9].
The quinoxaline ring moiety is part of the chemical structure of various antibiotics such as
echinomycine, levomycine and actinoleutine [10,11] that are known to inhibit growth of Gram-positive
bacteria. The mode of action for quinoxalines is based on MAO inhibitory activity, because the docked
positions of these compounds reveal interactions with many residues that have an effect on the
inhibition of the enzyme [12].
Some quinoxaline derivatives have been synthesized using microwave irradiation [13] but there is
no systematic information about their spectroscopic attributes. Synthetic approaches by traditional
methodology involves catalysts and solvents [14–19], however, recent synthetic methods have focused
on techniques involving alternative activation modes. In this sense, microwave-assisted synthesis has
shown efficiency and versatility as an easy and convenient methodology for condensation
reactions [20–22]. It’s known that microwave conditions avoid the polymerization reactions due to the
controlled hot spots formed during the heating by molecular friction of dipolar molecules [23].
2. Results and Discussion
2.1. Synthesis and Spectroscopic Analysis
Synthesis of in good yields of quinoxalines 3a–i and pyrido[2,3b]pyrazines 5a–f (Schemes 1 and 2)
was performed after short reaction times under microwave irradiation. Reactions that involved at least
one liquid reagent showed shorter reaction times than those involving two solid reagents (Table 1).
Purification of the final crude products with a mixture of hexane-ethyl acetate gave solid
pure compounds.
Scheme 1. Synthesis of 6-sustituted quinoxalines 3a–i.
8
H2N
R1
R1
O
O
mw (400Watts)
R1
R1
N
N
8a
2
3
+
7
H2N
R2
6
4a
R2
5
3a-3i
1a-1c
2a-2c
1a: R1 = Ph, 1b: R1 = Me, 1c: R1 = Pyr, 2a: R2 = H, 2b: R2 = Cl, 2c: R2 = Br.

Molecules 2012, 17
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Scheme 2. Synthesis of pyrido[2,3b]pyrazines 5a–f.
8
O
R2
R2
R1
NH2
R1
8a
N
N
R2
R2
7
2
3
mw (400Watts)
+
6
O
N
NH2
4a
N
4a-4b
1a-1c
5a-5f
1a: R2 = Ph, 1b: R2 = Me, 1c: R2 = pyr, 4a: R1 = H, 4b: R1 = Br.
Table 1. Reaction times and yields in the synthesis of compounds 3a–i and 5a–f.
Compound
Reaction time (s)
180 b
60 b
Yield (%)
90
3a: R1 = Ph; R2 = H
3b: R1 = Ph; R2 = Cl
3c: R1 = Ph; R2 = Br
3d: R1 = Me; R2 = H
3e: R1 = Me; R2 = Cl
3f: R1 = Me; R2 = Br
3g: R1 = Pyr; R2 = H
3h: R1 = Pyr; R2 = Cl
3i: R1 = Pyr; R2 = Br
5a: R1 = H; R2 = Ph
5b: R1 = H; R2 = Me
5c: R1 = H; R2 = Pyr
5d: R1 = Br; R2 = Ph
5e: R1 = Br; R2 = Me
5f: R1 = Br; R2 = pyr
85
88
87
85
84
84
86
80
70
60
80
85
88
75
90 b
60 a
30 a
40 a
180 b
60 b
120 b
180 b
180 b
300 b
60 b
150 b
300 b
a 2,3-Butanedione is a liquid reagent, diaminopyridines are solid reagents;
b Diaminopyridines and 2,3-dicarbonyl compounds are solid reagents.
Infrared spectra for quinoxalines 3a–i and pyrido[2,3b]pyrazines 5a–6 showed the characteristic
C=N stretching band centered at 1,600 cm−1. A weak absorption at 3,050 cm−1 was attributed to the
=C-H stretching of the aromatic ring. Compounds bearing alkyl groups exhibited a signal at 3,050 cm−1
due to C-H stretching absorptions. Bromine derivatives 3c, 3f, 3i, 5d, 5e and 5f showed the typical
1
absorption for C=C-Br stretching at 700 cm−1. The H-NMR analysis of the three coupled aromatic
protons H-5, H-7 and H-8 in quinoxalines 3b, 3c, 3e, 3f, 3h and 3i were identified as an AMX system,
showing a double signal for proton H-5 (J = 6 Hz), a double of double signal for proton H-7
13
(J = 9 Hz, J = 6 Hz) and a double signal for H-8 (J = 9 Hz). C-NMR showed the quinoxaline
quaternary carbons C-2 and C-3 of compounds 3a–i ranging from 149–155 ppm.
Quinoxaline carbons of 4a and 8a were observed at 135–142 ppm. 6-Substituted quinoxalines 3b,
3c, 3e, 3f, 3h and 3i showed quaternary halogenated carbons around 120 and 132 ppm that correlate
with the aromatic protons H-5, H-7 and H-8 in HMBC. Non-planar conformation of vicinal 2–3
biphenyls and 2–3 bipyridyls in compounds 3h, 3i, 5a, 5c, 5d and 5f produced atropoisomeric
structures that displayed two 13C signals for C-1′, C-2′, C-3′, C-4′ and C-5′ carbons.
X-ray diffraction of compound 5d (Figure 1) shows the orthogonal conformation of both aromatic
benzene rings located on carbons 2 and 3, which is one of the causes for the non-magnetic equivalence

Molecules 2012, 17
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1
13
of benzene carbons. The same behavior was observed for compounds 5a, 5c and 5f. The H and C
assignments were made by means of 1D spectra (13C, APT and DEPT) and confirmed by 2D spectral
1
13
analysis (HSQC, HMBC and NOESY). The H and C-NMR attributes for these molecules are not
reported in current literature; compounds 3i and 5f are new molecules.
Figure 1. X-Ray diffraction structure of pyrido[2,3b] pyrazine 5d.
2.2. Antimicrobial Test
Moderate antimicrobial activity (60–100% of amoxicillin) against some Gram-negative and
Gram-positive bacteria was observed for quinoxalines 3f, 3g, 3h and 3i which have a pyridine group in
their structure (Table 2). This level of activity could be a consequence of the lipophilic character
provided by the pyridine groups. Pyrido[2,3b]pyrazines 5c, 5d, 5e and 5f also showed moderate
activity, but primarily against Gram-negative bacteria (Table 2). Recent literature have reported the
antifungal properties of pyrido[2,3b]pyrazines-N-oxides and pyrido[2,3b]pyrazinolones [24].
Table 2. Antimicrobial activity of compounds 3a–i and 5a–f (200 µM).
Zone of inhibition (mm)
E.
S.
S.
E.
S.
P.
B.
P.
S.
Compound
coli aureus typhi faecalis typhimurium aeruginosa subtilis mirabillis flexneri
3a
3b
3c
3d
3e
3f
14
11
16
14
12
13
12
14
14
13
11
7
13
12
11
15
15
14
13
17
10
14
16
10
9
9
8
9
15
11
10
8
8
9
10
10
8
11
10
8
10
7
12
15
10
13
14
15
16
12
11
15
11
9
8
11
10
9
9
11
10
15
16
16
14
12
10
9
11
18
10
17
8
9
8
9
18
17
19
16
12
15
3g
3h
3i
14
15
11
16
12
14
10
9
11
17
15
13
15
5a
5b
15
14
9
7

Molecules 2012, 17
5168
Table 2. Cont.
Zone of inhibition (mm)
E.
S.
S.
E.
S.
P.
B.
P.
S.
Compound
coli aureus typhi faecalis typhimurium aeruginosa subtilis mirabillis flexneri
5c
5d
5e
5f
12
14
15
16
8
11
14
15
14
19
15
11
14
11
18
15
14
17
11
23
12
11
17
14
21
10
14
12
11
19
11
9
14
15
12
12
25
14
15
11
25
16
15
19
Amoxicillin 22
Large inhibition zones and a broad spectrum of activities were observed for all the tested
organisms except P. aeruginosa, which was sensitive to only six of the fifteen compounds screened.
The small zones of inhibition exhibited by P. aeruginosa may be explained in terms of the gross cell
wall of the organism that contains porins and efflux pumps, called ABC transporters, which pump out
some antibiotics before they are able to act. It may also be due to the protective biofilms formed by
this organism [25].
The results in Table 3 shows that the MIC values of quinoxalines and pyrido[2,3b]pyrazines varied
between 0.025 µM/mL and 0.1 µM/mL for all bacterial strains. These results indicated that compounds
3g, 3h, 5e, 5f had the highest activity while 5a, 5b, 5c and 5d competed favorably. The best zone
of inhibition was 19 mm by compound 3h against P. mirabilis, which was the same activity
as amoxicillin.
Table 3. Minimum inhibitory concentration of compounds 3a–i and 5a–f.
Minimum Inhibitory Concentration (µM/mL)
E.
S.
aureus
>0.2
0.1
S.
E.
faecalis
0.1
S.
typhimurium
>0.2
P.
aeruginosa
>0.2
>0.2
>0.2
0.1
B.
subtilis
0.05
0.1
P.
mirabillis
>0.2
S.
flexneri
>0.2
0.1
Comp.
coli
typhi
0.05
0.05
0.1
3a
3b
3c
3d
3e
3f
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
>0.2
0.1
0.1
>0.2
0.1
>0.2
>0.2
>0.2
>0.2
>0.2
0.1
>0.2
0.05
>0.2
0.1
>0.2
0.1
0.05
0.05
0.05
0.05
0.025
0.1
>0.2
0.1
>0.2
>0.2
0.1
>0.2
>0.2
0.05
>0.2
>0.2
>0.2
>0.2
0.1
>0.2
0.1
>0.2
0.05
0.05
0.1
0.025
>0.2
0.025
0.025
0.025
0.05
0.05
0.05
0.05
0.1
3g
3h
3i
0.05
0.05
0.05
0.05
0.1
0.025
>0.2
0.025
0.1
5a
5b
5c
5d
5e
5f
0.05
0.025
0.1
0.025
0.1
0.05
0.1
0.1
0.1
0.05
0.1
0.05
0.05
0.05
0.05
0.1
>0.2
0.05
0.05
0.05
0.05
0.1
0.05
>0.2
0.1
>0.2
0.05
0.05
0.05
0.05
0.1
>0.2
0.05
0.1
0.025
0.1
0.025
0.1
0.1
0.05
>0.2
0.05
0.1

Molecules 2012, 17
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3. Experimental
3.1. General
Purity of the compounds was checked by gas chromatography on a Finnigan Trace GC Ultra Polaris
Q Chromatograph. Infrared spectra were recorded in CH2Cl2 solution with a Perkin-Elmer FT-IR 2000
1
13
spectrometer. Samples for H- and C-NMR were dissolved in CDCl3 and spectra recorded on a
Varian VNMR System 500 and in a Varian Mercury 300 with TMS as internal standard. Chemical
shifts were recorded as δ (ppm). High resolution mass spectra were obtained in a Jeol GCMate II TSS
2000 by electronic impact at 70 eV.
3.2. Synthesis
A mixture of the appropriate α-diketone 1a–c (1.1 mmol) and 4-substituted o-phenylenediamine
2a–c or 5-substituted 2–3 diaminopyridines 4a–b (1.0 mmol) were mixed in a glass microwave
reaction tube. The mixture was placed inside the microwave oven and irradiated at 400 Watts for short
periods of time (see Table 1). The reaction was monitored every minute by thin layer chromatography
(aluminum silica gel plates F254, hexane-ethyl acetate 7:3), and developed with UV light until the
reaction was completed. The final time was taken when only traces of o-phenylendiamine or
2–3 diaminopyridine were observed by TLC. No solvents or catalysts were used in the synthesis,
and any water formed in the reaction was quickly eliminated by evaporation due to the high
microwave temperature, eliminating the need to use dehydrating agents. Quinoxalines 3a–i and
pyrido[2,3b]pyrazines 5a–f were obtained as amorphous crystals. Subsequent crystallization from 7:3
ethyl acetate-hexane gave crystalline or semisolid products in yields ranging from 60–90%. All
compound structures were identified from their spectral and analytical data.
3.3. Chemical Data and Analysis
3'
2'
4'
1
N
5'
4a
4a
6'
2
3
1'
6
N
4
5
1
2,3-Diphenylquinoxaline (3a): mp: 128–129 °C; I.R. (CH2Cl2): 3058, 1345, 697 cm−1; H-NMR:
8.18 (1H, d, J = 5.5 Hz, H5′), 8.17 (1H, d, J = 3.0 Hz, H5), 7.77 (1H, d, J = 3.0 Hz, H6), 7.76 (1H, d,
13
J = 5.5 Hz, H6′), 7.51 (4H, m, H2′), 7.34 (6H, m, H3′, H4′); C-NMR: 155.43 (C2, C3), 141.20 (C4a),
139.05 (C1′), 129.90 (C5, C5′), 129.80 (C2′), 129.17 (C4′), 128.76 (C6, C6′), 128.22 (C3′); HR MS (m/z)
282.1157 (calc.), 282.1156 (exp.).

Molecules 2012, 17
5170
4
N
5
8
Cl
6
4a
8a
3
2
2'
1'
3'
7
N
1
4'
6-Chloro-2,3-diphenylquinoxaline (3b): mp: 117–119 °C; I.R. (CH2Cl2): 3060, 1467, 1342, 697 cm−1;
1H-NMR: 8.15 (1H, d, J = 2.5 Hz, H5), 8.08 (1H, d, J = 9.0 Hz, H8), 7.68 (1H, dd, J = 2.5, 9.0 Hz, H7),
7.50 (4H, d, J = 9.0 Hz, H2′), 7.34 (6H, m, H3′, H4′); 13C-NMR: 154.51 (C3), 153.84 (C2), 141.70 (C6),
139.93 (C8a), 138.93, 138.81 (C1′), 135.88 (C4a), 130.66 (C7), 130.20 (C8), 130.08, 130.09 (C2′),
129.36, 128.30 (C3′), 128.58 (C5), 128.30 (C4); HR MS (m/z) 316.0767 (calc.), 316.0767 (exp.).
4
N
5
Br
4a
8a
6
7
3
2
2'
1'
3'
4'
N
8
1
6-Bromo-2,3-diphenylquinoxaline (3c): mp: 117–119 °C; I.R. (CH2Cl2): 3059, 1343, 697 cm−1;
1H-NMR: 8.35 (1H, d, J = 1.5 Hz, H5), 8.01 (1H, d, J = 9.0 Hz, H8), 7.80 (1H, dd, J = 1.5, 9.0 Hz, H7),
7.52 (4H, d, J = 7.1 Hz, H2′), 7.34 (6H, m, H3′, H4′); 13C-NMR: 154.02 (C2), 153.54 (C3), 141.58 (C8a),
139.77 (C4a), 138.58; 138.58 (C1′), 132.28 (C7), 131.10 (C5), 130.35 (C8), 129.72; 128.96 (C2′), 128.96;
128.89 (C4′), 128.16; 128.14 (C3′), 123.65 (C6); HR MS (m/z) 360.0262 (calc.), 360.0262 (exp.).
1
8
9
N
2
3
8a
4a
7
6
N
4
5
1
2,3-Dimethylquinoxaline (3d): mp: 102–104 °C; I.R. (CH2Cl2): 2995, 1436, 760 cm−1; H-NMR: 7.80
13
(2H, dd, J = 2.0, 6.0 Hz, H5, H8), 7.47 (2H, dd, J = 2.0, 6.0 Hz, H6, H7), 2.51 (6H, s, H9); C-NMR:
153.48 (C2, C3), 141.08 (C4a, C8a), 128.59 (C5, C8), 128.36 (C6, C7), 23.19 (C9); HR MS (m/z) 158.0844
(calc.), 158.0845 (exp.).
4
N
5
10
9
Cl
4a
8a
3
2
6
7
N
1
8

Molecules 2012, 17
5171
6-Chloro-2,3-dimethylquinoxaline (3e): mp: 98–100 °C; I.R. (CH2Cl2): 3022, 1482, 830 cm−1;
1H-NMR: 7.93 (1H, d, J = 2.5 Hz, H5), 7.87 (1H, d, J = 9.4 Hz, H8), 7.57 (1H, dd, J = 2.5, 9.4 Hz, H7),
2.69 (6H, s, H9, H10); 13C-NMR: 150.03 (C2), 149.23 (C3), 136.83 (C8a), 135 (C4a), 129.86 (C6), 125.01
(C5), 122.81 (C8), 18.64 (C10), 18.59 (C9). HR MS (m/z) 192.0453 (calc.), 192.0454 (exp.).
4
N
5
10
Br
4a
8a
3
2
6
7
N
1
9
8
6-Bromo-2,3-dimethylquinoxaline (3f): mp: 93–94 °C; I.R. (CH2Cl2): 3019, 1479, 827 cm−1;
1H-NMR: 7.98 (1H, d, J = 2.0 Hz, H5), 7.68 (1H, d, J = 9.0 Hz, H8), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H7),
13
2.58 (3H, s, H9), 2.56 (3H, s, H10); C-NMR: 154.24 (C3), 153.66 (C2), 141.31 (C8a), 139.43 (C4a),
131.86 (C7), 130.32 (C5), 129.39 (C8), 122.00 (C6), 22.88 (C9, C10), HR MS (m/z) 235.9949 (calc.),
235.9941 (exp.).
4'
3'
N
1
N
5'
5
2
3
4a
4a
6'
6
1'
2'
N
4
N
2,3-Dipyridin-2-yl-quinoxaline (3g). mp: 188–191 °C; I.R. (CH2Cl2): 2900, 1587, 1351, 744 cm−1;
1H-NMR: 8.35 (2H, ddd, J = 5.0, 2.0, 1.0 Hz, H5′), 8.19 (1H, dd, J = 8.0, 3.0 Hz, H5), 7.94 (2H, ddd,
J = 8.0, 2.0, 1.0 Hz, H3′), 7.80 (2H, m, H2′), 7.78 (1H, m, H6), 7.20 (2H, ddd, J = 10.0, 4.0, 1.0 Hz, H4′);
13C-NMR: 157.27 (C1′), 152.32 (C2, 3), 148.46 (C5′), 141.07 (C4a), 136.59 (C2′), 130.43 (C6), 129.29
(C5), 124.20 (C3′), 122.95 (C4′); HR MS (m/z) 284.1061 (calc.), 284.1062 (exp.).
5'
4'
3'
N
1
N
8
7
8a
4a
2
1'
2'
6
3
Cl
N
4
5
N
6-Chloro-2,3-dipyridin-2-yl-quinoxaline (3h): mp: 362–364 °C; I.R. (CHCl3): 3059, 1587, 1079,
744 cm−1; 1H-NMR: 8.32 (2H, m, H5′), 8.16 (1H, d, J = 2.0 Hz, H5), 8.09 (1H, d, J = 9.0 Hz, H8), 7.91
(2H, ddd, J = 8.0, 2.0, 1.0 Hz, H2′), 7.72 (1H, dd, J = 9.0, 2.0 Hz, H7), 7.20 (2H, ddd, J = 5.0, 2.0,
13
1.0 Hz, H3′), 7.77 (2H, m, H4′); C-NMR: 156.87, 156.80 (C1′), 153.04 (C3), 152.40 (C2), 148.35,
148.32 (C5′), 141.18 (C4a), 139.43 (C8a) 136.55, 136.52 (C4′), 136.09 (C6), 131.31 (C7), 130.40 (C8),
124.09, 124.03 (C2′), 122.97, 123.05 (C3′); HR MS (m/z) 318.0671 (calc.), 318.0672 (exp.).

Molecules 2012, 17
5172
5'
2'
4'
3'
N
1
N
8
7
8a
4a
2
1'
6
3
Br
N
4
5
N
6-Bromo-2,3-dipyridin-2-yl-quinoxaline (3i): mp: 95–96 °C; I.R. (CH2Cl2): 3055, 1586, 1077,
745 cm−1; 1H-NMR: 8.41 (2H, dd, 5,5 Hz, H5′), 8.37 (1H, d, J = 1.0 Hz), 8.04 (3H, m, H8, 4′), 7.89 (3H,
13
m, H5, ), 7.34 (2H, dd, J = 11, 2 Hz, H3′); C-NMR: 155.81 155.73 (C1′), 151.68 (C3), 151.11 (C2),
2′
147.41, 147.37 (C5′), 141.49 (C4a) 137.76, 137.72 (C4′), 134.29 (C5), 131.50 (C7), 130.54 (C8), 124.81
(C6), 124.69, 124.66 (C2′), 123.66, 123.60 (C3′). HR MS (m/z) 362.0169 (calc.), 362.0167 (exp.).
4'
1
N
8
2
3
3'
8a
4a
1'
7
6
2'
N
5
N
4
2,3-Diphenylpyrido[2,3b]pyrazine (5a): mp: 107–109 °C; I.R. (CH2Cl2): 3058, 1433, 802, 697, cm−1;
1H-NMR: 9.14 (1H, dd, J = 2.0, 5.0 Hz, H6), 8.49 (1H, dd, J = 2.0, 8.0 Hz, H8), 7.68 (1H, dd, J = 5.0,
13
8.0 Hz, H7), 7.60 (2H, m, H2′), 7.25 (8H, m, H3′, 4′); C-NMR: 156.50 (C3), 154.91 (C2), 154.07 (C6),
150.50 (C4a), 138.04 (C8), 135.14 (C8a), 130.25, 129.81 (C2′), 130.12, 129.25 (C1′), 129.42, 129.01
(C4′), 128.40, 128.14 (C3′), 125.21 (C7); HR MS (m/z) 283.1109 (calc.), 283.1109 (exp.).
1
N
8
9
2
3
8a
4a
7
6
N
N
10
5
4
6b
2,3-Dimethylpyrido[2,3b]pyrazine (5b): mp: 258–260 °C; I.R. (CH2Cl2): 2994, 1397, 799 cm−1;
1H-NMR:  9.05 (1H, dd, J = 2.0, 4.5 Hz, H6), 8.33 (1H, dd, J = 2.0, 8.5 Hz, H8), 7.62 (1H, dd,
13
J = 4.0, 8.5 Hz, H7), 2.81 (3H, s, H9), 2.77 (3H, s, H10); C-NMR 157.22 (C2), 154.91 (C3), 152.49
(C6), 150.12 (C4a), 137.20 (C7), 135.77 (C8a), 124.20 (C8), 23.40 (C9), 22.94 (C10). HR MS (m/z)
159.0796 (calc.), 159.0796 (exp.).

Molecules 2012, 17
5173
4'
3'
N
1
N
8
2
3
8a
4a
7
1'
2'
6
N
5
N
4
N
2,3-Dipyridin-2-yl-pyrido[2,3-b]pyrazine (5c): mp: 418 °C (dec.); I.R. (CH2Cl2): 3054, 1587,
793 cm−1; 1H-NMR:  9.8 (1H, dd, J = 2.0, 4.0 Hz, H6), 8.53 (1H, dd, J = 2.0, 8.0 Hz, H7), 8.35 (1H, d,
J = 6.0 Hz, H5′a), 8.28 (1H, d, J = 8.0 Hz, H4′a), 8.23 (1H, d, J = 6.0 Hz, H5′b), 7.94 (1H, d, J = 8.0 Hz, H4’b),
7.82 (1H, dd, J = 2.0, 20.0 Hz H2′a), 7.82 (1H, ddd, J = 2.0, 15.0, 17.0 Hz, H2′b), 7.22 (1H, m, H3′);
13C-NMR (75 MHz, CDCl3): 157.03 (C1′a), 156.47 (C1′b), 155.17 (C3), 154.55 (C6), 153.89 (C2), 149.62
(C4a), 148.55 (C5′a), 148.09 (C5′b), 138.25 (C7), 136.84 (C2′a), 136.65 (C2′b), 136.23 (C8a),
125.65 (C8), 124.66 (C4′a), 124.08 (C4′b), 123.46 (C3′a), 123.17 (C3′b). HR MS (m/z) 285.1012 (calc.),
285.1014 (exp.).
4'
1
N
8
Br
2
3
3'
8a
4a
7
1'
2'
6
N
5
N
4
7-Bromo-2,3-diphenylpyrido[2,3b]pyrazine (5d): mp: 102–104 °C; I.R. (CH2Cl2): 3055, 1584, 1078,
791 cm−1; 1H RMN: 9.14 (1H, d, J = 2.5 Hz, H6), 8.66 (1H, d, J = 2.5 Hz, H8), 7.61 (1H, d, J = 8.0 Hz,
H2′a), 7.54 (1H, d, J = 8.0 Hz H2′b), 7.39 (2H, m, H4′), 7.36 (1H, d, J = 8.0 Hz, H3′a), 7.33 (1H, d,
13
J = 8.0 Hz, H3′b); C-NMR (75 MHz, CDCl3): 156.45 (C3), 155.42 (C2), 155.04 (C6), 148.19 (C4a),
139.33 (C8), 138.04 (C1′a), 137.74 (C1′b), 136.35 (C8a), 130.13 (C2′a), 129.84 (C2′b), 129.62 (C4′a),
129.54 (C4′b), 128.39 (C3′a), 128.18 (C3′b), 120.86 (C7). HR MS (m/z) 361.0216 (calc.), 361.0215 (exp).
8
9
N
Br
1
2
3
8a
4a
7
6
N
5
N
4
10
7-Bromopyrido[2,3b]pyrazine (5e): mp: 320 °C (dec.); I.R. (CH2Cl2): 3036, 1392, 903 cm−1;
1H-RMN: 9.04 (1H, d, J = 1.0 Hz, H6), 8.49 (1H, d, J = 1.0 Hz, H8), 2.79 (3H, s, H9), 2.77 (3H, s, H10).
13C-NMR: 157.63 (C3), 156.05 (C2), 153.63 (C6), 148.59 (C2), 138.79 (C8), 136.19 (C8a), 119.98 (C7),
23.50 (C9), 23.05 (C10). HR MS (m/z) 236.9901 (calc.), 236.9902 (exp.).

Molecules 2012, 17
5174
4'
3'
N
1
N
8
Br
2
3
8a
4a
7
1'
2'
6
N
5
N
4
N
7-Bromo-2,3-dipyridin-2-yl-pyrido[2,3b]pyrazine (5f): mp: 91–93 °C; I.R. (CH2Cl2): 2900, 1584,
1
1390, 744 cm−1; H-NMR: 9.16 (1H, d, J = 2.5 Hz, H6), 8.69 (1H, d, J = 2.5 Hz, H8), 8.32 (1H, ddd,
J = 1.0, 2.0, 5.0 Hz, H4′a), 8.24 (2H, ddd, J = 1.0, 2.0, 7.0 Hz, H5′), 7.95 (1H, ddd, J = 1.0, 2.0, 7.0 Hz,
H4′b), 7.82 (1H, ddd, J = 2.0, 15.0, 17.0 Hz, H2′a), 7.82 (1H, dd, J = 2.0, 15.0 Hz H2′b), 7.22 (1H, ddd,
13
J = 2.0, 15.0, 17.0 Hz, H3′a), 7.22 (1H, ddd, J = 1.0, 2.0, 5.0 Hz, H3′b); C-NMR: 156.52 (C1′a), 156.2
(C1′b), 155.51 (C6), 155.30 (C3), 154.55 (C2), 148.42 (C5′a), 148.06 (C5′b), 147.91 (C8a), 139.46 (C8),
136.79 (C2′a), 136.63 (C2′b), 136.34 (C4a), 124.51 (C4′a), 124.01 (C4′b), 123.5 (C3′a), 123.30 (C3′b),
121.40 (C7). HR MS (m/z) 363.0126 (calc.), 363.0120 (exp.).
3.4. Antimicrobial Activity
Antimicrobial sensitivity was assessed by the Kirby-Bauer disk diffusion assay [26] using
Mueller-Hinton agar plates inoculated with the test organisms seeded in Mueller Hinton medium.
Whatman discs (6 mm) were placed on the agar after infusion with a 50, 75, 100, or 200 μM
concentration of the test compounds dissolved in DMSO. Amoxicillin was used as a positive reference
and DMSO a negative control. The Gram-positive microorganisms tested were Staphylococcus aureus
(ATCC 6538), Bacillus subtilis (ATCC 3465) and Enterococcus faecalis (ATCC 29212), and the
Gram-negative microorganisms were Escherichia coli (ATCC 8739), Salmonella typhimurium (ATCC
15674), Salmonella thypi (ATCC 6539), Pseudomonas aeruginosa (ATCC 9027), Proteus mirabilis
(ATCC 2756) and Shigella flexneri (ATCC 12022). Zones of inhibition were measured by the Bauer
protocol [26] after incubating for 24 h at 37 °C.
The minimum inhibitory concentrations (MIC) were evaluated by the micro plate method [27].
10 µL of each microbial culture, adjusted to 0.5 in the Mc. Farland Nephelometer, were placed in a
96 micro well plate containing 180 µL of Todd-Hewitt Broth medium. Later, 10 µL of the compounds
3a–i and 5a–f at 100, 50, 25 and 12.5 µM concentrations were added to each well. The plate was
incubated at 37 °C for 24 h and absorbances measured at 620 nm. Values of MIC50 were calculated by
following the CLSI (formerly NCCLS) guidelines [27].
4. Conclusions
Solvent-free and microwave-assisted synthesis provides an easy, fast and eco-friendly methodology
for the preparation of some 6-substituted quinoxalines and pyrido[2,3b]pyrazines derivatives from
α-dicarbonyl derivatives with substituted o-phenylenediamines. The antimicrobial Kirby- Bauer test
and MIC assay showed moderate activity for some of the prepared quinoxalines 3a–i and analog [2,3b]
pyridopyrazines 5a–f against a wide group of Gram-positive and Gram-negative bacteria.

Molecules 2012, 17
5175
Acknowledgments
JJMC thanks the scholarship from Nacional Council for Science and Technology (CONACYT).
JPC Thanks to CONACYT (agreement 90833) and grants provided from Secretaría de Investigación y
Posgrado del IPN (SIP) 20101028 and 20110656.
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Sample Availability: Samples of the compounds 3a–6f are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).