Synthesis and antitubercular evaluation of N-Arylpyrazine and N,Na-Alkyl-diylpyrazine-2-carboxamide derivatives

Article abstract of DOI:10.1002/jhet.921

Two series of pyrazinamide (PZA) derivatives have been synthesized and evaluated for their in vitro antibacterial activity against Mycobacterium tuberculosis H37Rv. Some compounds exhibited minimum inhibitory concentration activity of 50-100 μg/mL, greater than the first line antituberculosis drug PZA in Alamar Blue assay (>100 μg/mL). The obtained activities can be considered promising results, which characterizes these compounds as good start points to development of new antitubercular agents.

Full text of DOI:10.1002/jhet.921

November 2012
Synthesis and Antitubercular Evaluation of N‐Arylpyrazine and
N,N′‐Alkyl‐diylpyrazine‐2‐carboxamide Derivatives
1317
Marcelle de Lima Ferreira Bispo,a Raoni Schroeder Borges Gonçalves,a
Camilo Henrique da Silva Lima,a Laura Nogueira de Faria Cardoso,a Maria
b
a
*
Cristina Silva Lourenço, and Marcus Vinícius Nora de Souza
aFioCruz‐Fundação Oswaldo Cruz, Instituto de Tecnologia em Fármacos‐Far Manguinhos,
Manguinhos, Rio de Janeiro, Brazil
bInstituto de Pesquisas Clínica Evandro Chagas – IPEC, Manguinhos, Rio de Janeiro, Brazil
*
E-mail: marcos_souza@far.fiocruz.br
Received November 25, 2010
DOI 10.1002/jhet.921
View this article online at wileyonlinelibrary.com.
Two series of pyrazinamide (PZA) derivatives have been synthesized and evaluated for their in vitro
antibacterial activity against Mycobacterium tuberculosis H37Rv. Some compounds exhibited minimum
inhibitory concentration activity of 50–100 μg/mL, greater than the first line antituberculosis drug PZA in
Alamar Blue assay (>100 μg/mL). The obtained activities can be considered promising results, which
characterizes these compounds as good start points to development of new antitubercular agents.
J. Heterocyclic Chem., 49, 1317 (2012).
INTRODUCTION
acts in acid medium, where it is hydrolyzed by the myco-
bacterial enzyme pyrazinemidase to pyrazinoic acid, which
is considered the active metabolite [8], [9].
Tuberculosis (TB) is a serious infectious disease caused
by the Mycobacterium tuberculosis. According to the
World Health Organization (WHO), one‐third of popula-
tion is infected with TB and nearly 2.0 million people die
yearly [1]. The standard treatment against this disease is
an association of four drugs, isoniazid, rifampicin, ethambutol,
and pyrazinamide (PZA), which are called the first‐line drugs
[2]. In spite this regiment cure about 95% of TB cases, the
rates of nonadherence is higher because of the occurrence of
several side effects, such as hepatotoxicity, which can be
serious and fatal [3], [4].
Among the first‐line drugs, PZA has an important steril-
izing activity, which is essential to a successful treatment
[5]. PZA is considered to be a paradox drug because when
used against M. tuberculosis, it shows activity in vivo, but
not in vitro due to the pH of the usual culture medium used
to M. tuberculosis growth be almost neutral [6], [7]. As a
consequence of this, PZA is considered a prodrug that only
In previous studies of our research program for the de-
velopment of new antiTB agents, we indentified a series
of thiophen‐2‐acetamide derivatives with moderated anti-
mycobacterial activity (Fig. 1) [10–14]. Considering these
results exhibited by this series, and the importance of
PZA in TB treatment, the aim of the present work is the de-
sign, synthesis, and antimycobacterial evaluation of new
derivatives, based on the molecular hybridization of our
previous analogs with PZA (Fig. 1). The design concept
of these compounds explores the maintenance of the
scaffold aryl‐amide (A) and alkyl‐diamide (B) of our
thiophen‐2‐acetamide derivatives and the replacement of
the tiophene by the pyrazine nucleus (C). This modification
aims to investigate the influence of the heterocyclic
nucleus bounded to the amide function in the biological
activity of these series.
© 2012 HeteroCorporation

1318
M. L. F. Bispo, R. S. B. Gonçalves, C. H. S. Lima, L. N. F. Cardoso, M. C. S. Lourenço,
and M. V. N. de Souza
Vol 49
Figure 1. Molecular planning of the two series of 2‐pyrazinecarboxamide derivatives.
RESULTS AND DISCUSSION
with different diamines, in THF, under reflux (Scheme 1
and Table 2). In general, 1H‐NMR spectra of the compounds
5 and 6a–f showed two characteristic signals corresponding
to CONH protons at 9.06–11.6 ppm and the 13C‐NMR spectra
Chemistry. The synthesis of N‐arylpyrazine‐2‐carboxamide
derivatives 3a–q is summarized in Scheme 1 and Table 1.
First, 2‐pyrazinecarbonyl chloride 2 was prepared by reacting
pyrazinecarboxylic acid 1 with an excess of thionyl chloride
in dichloromethane with catalytic amounts of N,N‐
dimethylformamide, under nitrogen atmosphere. After that,
the crude compound 2 reacted with different substituted
anilines in tetrahydrofuran (THF), leading to the desired
products in 47–70% yields. The products were characterized
showed the C O signals at 178.0–162.0 ppm.
Antimycobacterial activity. The antimycobacterial activities
of the derivatives 3a–q, 5 and 6a–f were assessed against
M. tuberculosis ATCC 27294 [21] using the micro plate
Alamar Blue assay [22] (Table 3). This methodology is
nontoxic, uses a thermally stable reagent and shows good
correlation with proportional and BACTEC radiometric
methods [23], [24].
1
by H‐NMR spectra showing a characteristic large singlet
These results showed that the activities of compounds 5
and 6a–f could not be determined, as these compounds
were insoluble in the medium culture during the assay.
However, compounds 3c, 3m, 3o, and 3q exhibited
antimycobacterial activities between 50 and 100 μg/mL.
Considering that the activity of PZA in Alamar Blue assay
is >100 μg/mL, the obtained activities can be considered
at 9.25–10.78 ppm, relative to CONH. In addition, the
13C‐NMR spectra showed the C O signals at 170.0–161.0 ppm.
The synthesis of the N,N′‐alkyl‐diylpyrazine‐2‐carboxamide
derivatives 5 and 6a–f is summarized in Scheme 2 and Table 2.
Methylic ester 4 was obtained from the reaction of 2‐pyrazine-
carboxylic acid 1 with SOCl2 in MeOH. Besides, compounds 5
and 6a–f were prepared by the reaction of methylic ester 4
Scheme 1. Reagents and conditions: (a) SOCl2, DMF, CH2Cl2, r.t., 1 h and (b) THF, 0°–r.t., 1–3 h, 47–70%.
Journal of Heterocyclic Chemistry
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November 2012
Synthesis and Antitubercular Evaluation of N-Arylpyrazine and N,N0-Alkyl-diylpyrazine-
1319
2-carboxamide Derivatives
Table 1
Melting points and yields of N‐arylpyrazine‐2‐carboxamide derivatives 3a–q.
Substituted
Compound
R1
R2
R3
Yield (%)
m.p (°C)
125 [15]
143–145 [15]
183 [15]
154–155 [16]
200 [17]
117 [18]
115 [18]
146 [19]
150 [19]
160 [19]
222 [19]
120 [16]
100–102 [16]
150 [16]
115 [20]
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
H
H
H
H
H
OCH3
H
H
NO2
H
H
CF3
H
H
CH3
H
H
Cl
H
H
H
H
OCH3
H
H
NO2
H
H
CF3
H
H
CH3
H
H
H
Cl
F
Br
H
H
OCH3
H
H
NO2
H
H
CF3
H
H
52%
60%
54%
50%
48%
50%
58%
62%
64%
50%
70%
47%
52%
55%
50%
55%
56%
147 [19]
147 [19]
H
CH3
Scheme 2. Reagents and conditions: (a) SOCl2, MeOH, 0ꢀC–r.t., 1 h and (b) THF, 80ꢀC, 12–18 h.
promising results, which characterizes these compounds as
good start points to development of new antitubercular agents.
The comparison of the active compounds 3c, 3m, 3o, and
3q with the analogous thiophene series (Fig. 2) [10–14] shows
that in derivative 3m, the introduction of the pyrazine nucleus
leads to an improvement of the antimycobacterial activity.
While the derivatives 3c, 3o, and 3q displayed the same activ-
ities (100 μg/mL) (Fig. 2).
antimycobacterial activities were assayed against M. tuber-
culosis. It was found that the compounds 3c, 3m, 3o, and
3q exhibited better activities than PZA in Alamar Blue as-
say, displaying a minimum inhibitory concentration range
of 50–100 μg/mL. These results suggest that they may be
selectively targeted to M. tuberculosis growth, also consid-
ering that they were not cytotoxic to host cells at the same
concentration. The comparison between thiophen‐2‐acet-
amide and 2‐pyrazinecarboxamide series suggests that the
introduction of the pyrazine nucleus could be an important
feature to the improvement of the biological activity of the
earliest series. More information about structure–activity
relationship and their in vivo antibacterial activity test are
in progress in our laboratory.
CONCLUSION
In the present work, we described the synthesis of two
different series of 2‐pyrazinecarboxamides. The com-
pounds were obtained in good yields (47–97%) and their
Journal of Heterocyclic Chemistry
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M. L. F. Bispo, R. S. B. Gonçalves, C. H. S. Lima, L. N. F. Cardoso, M. C. S. Lourenço,
and M. V. N. de Souza
Vol 49
Table 2
Yields and melting points of the diamino‐pyrazinecarboxamide
derivatives (5 and 6a–f).
Entry
N
Yield (%)
m.p. (°C)
5
–
2
3
4
6
96
88
80
83
82
97
83
120–121
145–147
223–225
169–173
140–142
136–138
137–138
6a
6b
6c
6d
6e
6f
8
10
EXPERIMENTAL
General procedures. Melting points were determined on a
Buchi apparatus and are uncorrected. Infrared spectra were
recorded on a Thermo Nicolet Nexus 670 spectrometer as
potassium bromide pellets and frequencies are expressed in
cm−1. Mass spectra (ESI assay in solution of ammonium
chloride) were recorded on Micromass ZQ Waters mass
spectrometer. NMR spectra were recorded on a Bruker Avance
400 operating at 400.00 MHz (H1) and 100.0 MHz (13C) and
Bruker Avance 500 spectrometer operating at 500.00 MHz (1H) and
125.0 MHz (13C), in deuterated dimethylsulfoxide or acetic acid.
Chemical shifts are reported in ppm (δ) relative to tetramethylsilane
and J‐coupling in Hertz (Hz). Proton and carbon spectra were
typically obtained at room temperature. For TLC plates coated with
silica gel were run in chloroform/methanol mixture and spots were
developed in ultraviolet and solution of ninhidrine (0.2% p/v in
ethanol).
Figure 2. Thiophen-2-acetamide derivatives previously synthesized by
our group and its respective antimycobacterial activities.
1 h, the excess thionyl chloride and the other volatiles were
removed under reduced pressure to leave the acyl chloride,
which was used without purification in the next stage.
General procedure for the synthesis of N‐arylpyrazine‐2‐
carboxamide derivatives (3a–q). A solution of 2 (1.21 mmol), as
prepared above, and the appropriate substituted aniline (1.45 mmol)
in tetrahydrofuran (10 mL) was stirred at ambient temperature, for
periods between 1 and 4 h depending on the rate of reaction as
monitored by thin‐layer chromatography. The reaction mixtures
were then cooled and the solvent was removed under reduced
pressure. Saturated aqueous sodium hydrogen carbonate solution
was added to the residue, and the resulting mixture was
exhaustively extracted with ethyl acetate. The combined organic
fractions were dried over anhydrous sodium sulfate and
concentrated under reduced pressure. The resulting solids were
purified by successive washings with cold ethanol and diethyl ether,
and finally recrystallized from ethanol, to give the products 3a–q as
a solid in 47–70% yields.
Synthesis of 2‐Pyrazinecarbonyl chloride (2). 2‐Pyrazinecarbonyl
chloride was prepared by reacting pyrazinecarboxylic acid
(1.6 mmol) with a three‐fold molar excess of thionyl chloride
in dichloromethane solution at ambient temperature with
continuous stirring under a dinitrogen atmosphere in the
presence of 0.1 equivalents of N,N‐dimethylformamide. After
Synthesis of methyl 2‐pyrazinecarboxylate (4). The methyl
2‐pyrazinecarboxylate 4 was prepared by slow addition (during
15 min) of SOCl2 (0.48 mole) in MeOH (300 mL) under
stirring at 0°C, followed by the introduction of the compound 1
(15 g, 0.12 mole). The temperature of the reaction mixture was
then, increased from 0°C to the room temperature and after 1 h,
the excess of SOCl2 was removed under reduced pressure. The
crude product was neutralized with saturated aqueous solution of
NaHCO3 (100 mL) and extracted with CH2Cl2 (3 × 30 mL). The
combined organic phases were dried over NaSO4 and
concentrated under reduced pressure to afford only the desired
product 2 as a white solid. This compound was used as such in
the synthesis of 2‐pyrazinehidrazide without further purification.
Yield: 80%; mp: 59–60°C (62°C) [23–29].
General procedures for the synthesis of N,N′‐bis‐
pyrazinecarboxamide derivatives (5 and 6a–f). The synthesis of N,
N′‐bis‐2‐pyrazinecarboxamide derivatives 5 and 6a–f were prepared
by reaction between the appropriate diamines (1.44 mmol) and
methyl 2‐pyrazinecarboxylate (2.88 mmol) in tetrahydrofuran
(30 mL). After 12–18 h of stirring, at 80°C, the resulting mixture
was concentrated under reduced pressure and the residue was
purified by washing with cold ethyl ether (3 × 20 mL), leading the
pure derivatives 5 and 6a–f as a solid in 80–97% yields.
Table 3
Antimycobacterial activities of PZA, N‐arylpyrazine‐2‐carboxamide
(3a–q) and N,N′‐alkyl‐diylpyrazine‐2‐carboxamide (5 and 6a–f) derivatives.
Entry
MIC (μg/mL)a
Entry
MIC (μg/mL)a
3a
3b
3c
3d
3e
3f
3g
3h
3i
>100
>100
50
3m
3n
3o
3p
3q
5
6a
6b
6c
6d
6e
6f
50
>100
100
>100
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
N.D.b
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
>100
3j
3k
3l
Pyrazinamide
Pyrazinamide
aMinimum inhibitory concentration.
bN.D. = not determined.
Journal of Heterocyclic Chemistry
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November 2012
Synthesis and Antitubercular Evaluation of N-Arylpyrazine and N,N0-Alkyl-diylpyrazine-
1321
2-carboxamide Derivatives
N,N′‐1,2‐cyclohexanediyl‐bis‐2‐pyrazinecarboxamide (5). This
N,N′‐1,10‐decanediyl‐bis‐2‐pyrazinecarboxamide (6f). This
compound was obtained as pallet yellow, IR (cm−1; KBr): 3299
compound was obtained as pallet yellow, IR (cm−1; KBr): 3340
1
1
(NH); 1659 (C O). H‐NMR [500.00 MHz (FIDRES 0.15 Hz),
(NH); 1656 (C O). H‐NMR [500.00 MHz (FIDRES 0.15 Hz),
Acetic acid‐d6] δ: 11.41 (2H; s; NH); 9.15 (2H; ls; H3); 8.77 (2H;
d; J = 2.0 Hz; H6); 8.66 (2H; ls; H5); 4.26 (2H; t; J = 9.2 Hz; H1′
and H6′); 2.13 (2H; d; J = 12.4 Hz; H2′ or H5′); 1.86–1.49 (6H; m;
H2′ or H5′; H3′ and H4′) ppm; 13C‐NMR (125.0 MHz, Acetic acid‐
d6) δ: 178.3; 164.9; 147.6; 145.9; 144.9; 143.9; 54.5; 32.6; 25.6
ppm. LC/MS: m/z [M‐H]: 325. Anal. Calcd. for C16H18N6O2·H2O:
C, 55.80; H, 5.85; N, 24.40. Found: C, 55.72; H, 5.83; N, 24.47.
N,N′‐1,2‐ethanediyl‐bis‐2‐pyrazinecarboxamide (6a). This
compound was obtained as pallet yellow, IR (cm−1; KBr):
3340 (NH); 1661 (C O). 1H‐NMR [500.00 MHz (FIDRES
0.15 Hz), Acetic acid‐d6] δ: 9.34 (2H; d; J = 1.5 Hz; H3);
8.88 (2H; d; J = 2.5 Hz; H6); 8.75 (2H; dd; J = 1.5 and 2.5
Hz; H5); 3.87 (2H; t; J = 5.5 Hz; CH2); 3.41 (2H; t; J = 5.5
Hz; CH2) ppm; 13C‐NMR (125.0 MHz, Acetic acid‐d6) δ:
165.9; 147.7; 145.9; 144.9; 144.0; 40.7; 38.1 ppm. LC/MS:
m/z [M‐H]: 271. Anal. Calcd. for C12H12N6O2: C, 52.94;
H, 4.44; N, 30.87. Found: C, 53.02; H, 4.47; N, 30.92
Acetic acid‐d6] δ: 11.59 (2H; s; NH); 9.35 (2H; s; H3); 8.87
(2H; s; H6); 8.70 (2H; s; H5); 3.50 (4H; s; NCH2(CH2)8CH2N);
2.04 (4H; s; NCH2CH2(CH2)6CH2CH2N); 1.37 (12H; s;
NCH2CH2(CH2)6CH2CH2N) ppm. 13C‐NMR (125.0 MHz,
Acetic acid‐d6) δ: 164.7; 147.5; 146.2; 144.8; 144.1; 40.6; 30.4;
30.1; 27.7 ppm. LC/MS: m/z [M‐H]: 383. Anal. Calcd. for
C20H28N6O2.H2O: C, 59.68; H, 7.51; N, 20.88. Found: C,
59.71; H, 7.48; N, 20.85.
General procedures for biological tests. Briefly, 200 μL of
sterile deionized water was added to all outer‐perimeter wells of
sterile 96 well plates (falcon, 3072: Becton Dickinson, Lincoln
Park, NJ) to minimize evaporation of the medium in the test
wells during incubation. The 96 plates received 100 μL of the
Middlebrook 7H9 broth (Difco laboratories, Detroit, MI) and a
serial dilution of the compounds 3a–q, 5, and 6a–f was made
directly on the plate. The final drug concentrations tests were
0.01–100 μg/mL. Plates were covered and sealed with parafilm
and incubated at 37°C for five days. After this time, 25 μL of a
freshly prepared 1:1 mixture of Alamar Blue (Accumed
International, Westlake, OH) reagent and 10% tween 80 was
added to plate and incubated for 24 h. A blue color in the well
was interpreted as no bacterial growth, and a pink color was
scored as growth. The MIC was defined as the lowest drug
concentration, which prevented a color change from blue to pink.
N,N′‐1,3‐propanediyl‐bis‐2‐pyrazinecarboxamide (6b). This
compound was obtained as pallet yellow, IR (cm−1; KBr): 3512
1
(NH); 1658 (C O). H‐NMR [500.00 MHz (FIDRES 0.15 Hz),
Acetic acid‐d6] δ: 9.19 (2H; ls; H3); 9.09 (2H; ls; NH); 8.87 (2H; ls;
H6); 8.73 (2H, ls, H5) 3.39 (4H; m; NCH2); 1.66–1.63 (2H; m;
NCH2CH2CH2N) ppm; 13C‐NMR (125.0 MHz, Acetic acid‐d6)
δ: 162.8; 147.4; 144.8; 143.2; 39.1; 36.7; 29.0 ppm. LC/MS:
m/z [M‐H]: 285. Anal. Calcd. for C13H14N6O2·2H2O: C, 48.44;
H, 5.63; N, 26.07. Found: C, 53.02; H, 4.47; N, 30.92.
N,N′‐1,4‐butanediyl‐bis‐2‐pyrazinecarboxamide (6c). This
compound was obtained as pallet yellow, IR (cm−1; KBr):
3325 (NH); 1667 (C O). 1H‐NMR [400.00 MHz (FIDRES
0.12 Hz), Acetic acid‐d6] δ: 11.49 (1H; s; NH); 9.34 (1H; s;
H3); 8.70 (1H; s; H5); 8.66 (1H; s; H6); 3.54 (2H; t; J = 4.0
Hz; NCH2(CH2)2CH2N); 2.05 (2H; ls; NCH2(CH2)2CH2N)
ppm. 13C‐NMR (100.0 MHz Acetic acid‐d6) δ: 164.9; 147.6;
146.2; 144.8; 144.1; 40.2; 27.5 ppm. LC/MS: m/z [M‐H]: 299.
Anal. Calcd. for C14H16N6O2: C, 55.99; H, 5.37; N, 27.98.
Found: C, 55.73; H, 5.32; N, 27.90.
REFERENCES AND NOTES
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2009/pdf/chapter1.pdf (date of access: 10‐24‐2010).
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[4] De Souza, M. V. N. Recent Pat Antiinfect Drug Discov 2006,
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[9] Vergara, F. M. F.; Lima, C. H. S.; Henriques, M. G. M. O.;
Candéa, A. L. P.; Lourenço, M. C. S.; Ferreira, M. L.; Kaiser, C. R.;
De Souza, M. V. N. Eur J Med Chem 2009, 44, 4954.
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Lett Drug Des Discov 2008, 5, 221.
N,N′‐1,6‐hexanediyl‐bis‐2‐pyrazinecarboxamide (6d). This
compound was obtained as pallet yellow, IR (cm−1; KBr): 3341
1
(NH); 1653 (C O). H‐NMR [500.00 MHz (FIDRES 0.12 Hz),
DMSO‐d6] δ: 9.16 (2H; d; J = 1.5 Hz; H3); 8.94 (2H; t; J = 6.0
Hz; NH); 8.86 (2H; d; J = 2.5 Hz; H6); 8.72 (2H; dd; J = 2.5 e
1.5 Hz; H5); 6.59 (4H; q; J = 7.0 Hz; NCH2(CH2)4CH2N); 1.54
(4H; t; J = 7.0 Hz; NCH2CH2(CH2)2CH2CH2N); 1.33–1.30
(4H; m; NCH2CH2(CH2)2CH2CH2N) ppm. 13C‐NMR (125.0
MHz DMSO‐d6) δ: 162.6; 147.2; 144.8; 143.3; 143.2; 38.6;
28.9; 26.0 ppm. LC/MS: m/z [M‐H]: 327. Anal. Calcd. for
C16H20N6O2: C, 58.52; H, 6.14; N, 25.59. Found: C, 58.49; H,
6.18; N, 25.65.
N,N′‐1,8‐octanediyl‐bis‐2‐pyrazinecarboxamide (6e). This
compound was obtained as pallet yellow, IR (cm−1; KBr):
1
3337 (NH); 1657 (C O). H‐NMR [500.00 MHz (FIDRES 0.15
[12] De Souza, M. V. N.; Lourenço, M. C. S.; Peralta, M. A.;
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M. L.; Da Silva, E. T. Phosphorus Sulfur Silicon Relat Elem 2008, 183, 2990.
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S. R.; Smith, V. K. Jr; Williams, J. H. J Am Chem Soc 1952, 74, 3617.
Hz), Acetic acid‐d6] δ: 11.57 (1H; s; NH); 9.35 (2H; ls; H2); 8.86
(2H; d; J = 2 Hz; H6); 8.70 (2H; ls; H5); 3.50 (4H; t; J = 5.6 Hz;
NCH2(CH2)6CH2N); 1.66 (4H; t; J = 5.6Hz; NCH2CH2(CH2)
4CH2CH2N); 1.38 (8H; ls; NCH2CH2(CH2)4CH2CH2N) ppm.
13C‐NMR (125.0 MHz, Acetic acid‐d6) δ: 164.7; 147.5; 146.2;
144.8; 144.1; 40.6; 30.1; 30.0 ppm. LC/MS: m/z [M‐H]: 355.
Anal. Calcd. for C18H24N6O2: C, 60.66; H, 6.79; N, 23.58. Found:
C, 60.80; H, 6.83; N, 23.62.
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet