Synthesis and antimycobacterial evaluation of N'-(E)- heteroaromaticpyrazine-2-carbohydrazide derivatives

Article abstract of DOI:10.2174/157340611795564303

A series of nine N'-(E)-heteroaromatic-pyrazine-2-carbohydrazide derivatives (5a-f and 6a-c) have been synthesized and evaluated against M. tuberculosis ATCC 27294 using the micro plate Alamar Blue assay (MABA), being the activities expressed as the minim

Full text of DOI:10.2174/157340611795564303

Medicinal Chemistry, 2011, 7, 245-249
245
Synthesis and Antimycobacterial Evaluation of N´-(E)-heteroaromatic-
pyrazine-2-carbohydrazide Derivatives
C. H. S. Limaa,b, M. G. M. O. Henriquesa, A. L. P. Candéaa, M. C. S. Lourençoc, F. A. F. M. Bezerrac, M. L.
Ferreiraa,b, C. R. Kaiserb and M. V. N. de Souzaa,b*
ꢀ
aFundação Oswaldo Cruz, Instituto de Tecnologia em Fármacos-Far Manguinhos, Fundação Oswaldo Cruz, 21041-
250, Rio de Janeiro, RJ, Brazil
bPós-Graduação em Química da Universidade Federal do Rio de Janeiro, Instituto de Química, CP 68563, 21945-970-
Rio de Janeiro – RJ
cFundação Oswaldo Cruz, Instituto de Pesquisas Clínicas Evandro Chagas, Departamento de Bacteriologia, Av. Brasil,
4365, Manguinhos, Rio de Janeiro, Brazil
Abstract: A series of nine N´-(E)-heteroaromatic-pyrazine-2-carbohydrazide derivatives (5a-f and 6a-c) have been syn-
thesized and evaluated against M. tuberculosis ATCC 27294 using the micro plate Alamar Blue assay (MABA), being the
activities expressed as the minimum inhibitory concentration (MIC) in μg/ml. Compounds 5a and 5f exhibited potent ac-
tivities (3.12 and 50μg/mL, respectively) when compared to the first line drug pyrazinamide (MIC>100 μg/mL). After-
wards, these compounds were evaluated for their cell viabilities in non-infected and infected macrophages with Myco-
baterium bovis Bacillus Calmette-Guerin (BCG) and 5f was not cytotoxic to host cells in the effective concentration to in-
hibit the growth of M. tuberculosis.
Keywords: Pyrazine, tuberculosis, drugs.
1. INTRODUCTION
hydrazide derivatives, including some derivatives that
showed better in vitro antitubercular activities than PZA
(Fig. 1) [6,7]. Hence, in our continuous research program in
TB drug discovery, we propose the synthesis and antimyco-
Tuberculosis is a disease caused by Mycobacterium tu-
berculosis that infects one-third of global population, leading
to almost two million deaths each year. [1,2]. The main
problem in tuberculosis treatment is the emergence of resis-
tant strains that can be classified in two types: multidrug-
resistant tuberculosis (MDR-TB, resistant to at least isoni-
azid or rifampicin) and extensively drug-resistant TB (XDR-
TB, is MDR-TB that is also resistant to three or more sec-
ond-line drugs). World Health Organization (WHO) esti-
mated that in 2008 there were 440.000 cases of MDR-TB
and 58 countries reported at least one case of XDR-TB until
March 2010 [2].
bacterial
evaluation
of
nine
heteroaromatic
2-
pyrazinecarbohydrazide derivatives 5a-f and 6a-c, also de-
signed by molecular hybridization (Scheme 1).
The criteria used to select the five-membered heterocyc-
lic nucleus was based on isosteric replacements: i) substitu-
tion of oxygen atom of furane ring (3a) by sulfur (3d) or
nitrogen (3e); ii) substitution of –CH= by –N= in pyrrole
ring (3e) to furnish an imidazole ring (3f). The six-
membered heterocyclic nucleus (4a-c) was also chosen with
the aim to analyze the influence of these structural modifica-
tions on the biological activity of this series.
Considering the high impact of resistant strains in tuber-
culosis treatment, there is an urgent requirement of new
drugs to treat it efficiently. A strategy commonly used in
drug discovery is to synthesize analogs of drugs in current
use to improve biological activity or pharmacokinetic pa-
rameters. In this context, pyrazinamide (PZA) could be con-
sidered a good start point to the development of new drugs
against TB, due to its excellent sterilizing effect on semi-
dormant tubercle bacilli [3]. This characteristic is responsible
to reduce the time of treatment from twelve to six months
[4,5]. Considering that, we have reported in our previous
works two series of mono and disubstituted pyrazinecarbo-
R2
R1
R3
O
O
N
N
N
N
N
N
R4
NH2
H
R5
Pyrazinamide
MIC >100 ꢀg/ml
R2= Cl; R1=R3 =R4 = R5 = H MIC = 50 ꢀg/ml
R2= CN; R1=R3 =R4 = R5 = H MIC = 50 ꢀg/ml
R1= NO2; R2=R3 =R4 = R5 = H MIC = 50 ꢀg/ml
R2= NO2; R1=R3 =R4 = R5 = H MIC = 100 ꢀg/ml
R2= F;
R1=R3 =R4 = R5 = H MIC = 100 ꢀg/ml
*Address correspondence to this author at the Fundação Oswaldo Cruz,
Instituto de Tecnologia em Fármacos-Far Manguinhos, Fundação Oswaldo
Cruz, 21041-250, Rio de Janeiro, RJ, Brazil; Tel: +552139772404; Fax:
+552125602518; E-mail: marcos_souza@far.fiocruz.br
Fig. (1). Pyrazinamide and some pyrazinecarbohydrazide deriva-
tives previously synthesized by our group with their respective
antimycobacterial activities.
1573-4064/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

246 Medicinal Chemistry, 2011, Vol. 7, No. 3
Lima et al.
2. MATERIALS AND METHODS
2.1. General Procedures
2.1.3.3. N’-[(E)-((5-nitrothiophen-2-yl)methylene)pyrazine-
2-carbohydrazide (5c)
Yield: 62%; mp: 263-265oC
Melting points were determined on a Buchi apparatus
and are uncorrected. Infrared spectra were recorded on a
Thermo Nicolet Nexus 670 spectrometer as potassium bro-
mide 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 (1H) and 100.0 MHz (13C) and Bruker Avance
500 spectrometer operating at 500.00 MHz (1H) and 125.0
MHz (13C), in deuterated dimethylsulfoxide. Chemical shifts
are reported in ppm (ꢀ) relative to tetramethylsilane and J-
coupling in Hertz (Hz). Proton and carbon spectra were typi-
cally 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 nin-
hidrine (0.2% p/v in ethanol).
1H NMR (500 MHz, DMSO-d6) ꢀ: 12.76(1H; s; NH);
9.27(1H; s; H3); 8.95(1H; s; H6); 8.84(1H; s; N=CH);
8.80(1H; s; H5); 8.13 (1H; d; J=4.0 Hz; H - nitrothiophenyl);
7.58 (1H; d; J= 4.0 Hz; H - nitrothiophenyl) ppm;
13C NMR (125MHz, DMSO-d6) ꢀ: 159.9; 151.1; 148.1;
146.3; 144.3; 144.2; 143.4; 142.9; 130.4; 130.0 ppm;
ESI/MS: [M-H]: 276
IR: (KBr pellets, cm-1): 3483 (NH); 1682 (CO).
2.1.3.4.
N’-[(E)-(thiophen-2-ylmethylene)pyrazine-2-
carbohydrazide (5d)
Yield: 50 %; mp: 185-186oC; (187°C) [9]
2.1.3.5.
N’-[(E)-((1H-pyrrol-2-yl)methylene)pyrazine-2-
carbohydrazide (5e)
Yield: 50 %; mp: 176-178oC; (179°C) [9]
2.1.2. Synthesis of pyrazine-2-carbohydrazide 2
2.1.3.6. N’-(E) -((1H-imidazol-2-yl)methylene)pyrazine-2-
carbohydrazide (5f)
Pyrazine-2-carboxihydrazide 2 was prepared by addition
of N2H4.H2O (25%, 20 equiv., 144 mmol) in 36 mL ethano-
lic solution of methyl pyrazine-2-carboxylate 1 (1.0g, 7.2
mmol) and the mixture was refluxed for 2 hours. The solvent
was removed under reduced pressure and the residue was
purified by washing cold Et2O (30mL) to afford the com-
pound 2 as solid. Yield: (0.81g, 80%), mp:158°C (158-
159°C)[8].
Yield: 58%; mp: 217-219oC
1H NMR (500 MHz, DMSO-d6) ꢀ: 12.88(1H; s; NH-
imidazolyl); 12.42(1H; s; NH - pyrazine) 9.27(1H; s; H3);
8.93(1H; s; H6); 8.79(1H; s; H5); 8.56(1H; s; N=CH); 7.24-
7.10(2H; m; H- - imidazolyl) ppm;
13C NMR (125 MHz, DMSO-d6) ꢀ: 159.4; 152.7; 149.3;
147.8; 144.5; 143.3; 137.8; 127.8; 127.0 ppm;
2.1.3. General Procedures of Synthesis of N’-[(E)-
(heteroaromatic)]-pyrazine-2-carbohydrazide 5a-f and 6a-c
ESI/MS: [M-H]: 231
IR: (KBr pellets, cm-1): 3468 (NH); 1666 (CO)
N’-(E)-(heteroaromatic)-pyrazine-2-carbohydrazide 4a-f
and 6a-c were prepared by reaction between pyrazine-2-
carbohydrazide (1.0 equiv.) and the appropriate heteroaro-
matic aldehyde (1.2 equiv.) in a mixture of ethanol and water
(1:1, 10 ml). The reaction mixture was stirred for 4-16 hours
at room temperature. After that, the excess of solvent was
concentrated under reduced pressure and the residue was
purified by washing with cold Et2O (3 X 10 ml), leading the
pure derivatives 5a-f and 6a-c as solids in 50-86% yields,
except for 5d and 6c, which were recrystallized in ethanol.
2.1.3.7.
N’-[(E)-(pyridine-2-ylmethylene)pyrazine-2-
carbohydrazide (6a)
Yield: 60%; mp: 203-204oC
1H NMR (500 MHz, DMSO-d6) ꢀ: 12.60(1H; s; NH);
9.28(1H; d; J= 1.5 Hz; H3); 8.94(1H; d; J= 2.4 Hz; H6);
8.80(1H; dd; J= 1.5 and 2.4 Hz; H5); 8.69(1H; s; N=CH);
8.63 (1H; d; J= 4.5 Hz; H1’); 8.01 (1H; d; J= 7.5 Hz H4’);
7.90 (1H; ddd; J= 1.0, 7.5 and 8.0Hz; H3’ ); 7.43 (1H; dd; J=
4.5 and 7.5 Hz; H2’ ) ppm;
2.1.3.1. N’-[(E)-((5-nitrofuran-2-yl)methylene)pyrazine-2-
carbohydrazide (5a)
13C NMR (125MHz, DMSO-d6) ꢀ: 158.9; 147.6; 145.0;
144.0; 143.2; 142.6; 127.0; 1123.0; 114.1; 109.4ppm;
Yield: 58%; mp: 217-219oC
1H NMR (500 MHz, DMSO-d6) ꢀ: 12.77(1H; s; NH);
9.29(1H; s; H3); 8.95(1H; s; H6); 8.81(1H; s; H5); 8.61(1H; s;
N=CH); 7.80 (1H; d; J=3.4 Hz; H- nitrofuranyl); 7.30 (1H;
d; J=3.4 Hz; H- nitrofuranyl) ppm;
13C NMR (125 MHz, DMSO-d6) ꢀ: 159.4; 153.1; 151.5;
149.3; 147.8; 144.5; 144.1; 143.3; 114.6; 112.3 ppm;
ESI/MS: [M-H]: 226
IR: (KBr pellets, cm-1): 3418 (NH); 1706 (CO)
2.1.3.8.
N’-[(E)-(4-(pyridin-2-yl)benzylidene)pyrazine-2-
carbohydrazide (6b)
Yield: 86%; mp: 254-256oC
ESI/MS: [M-H]: 260
IR: (KBr pellets, cm-1): 3415 (NH); 1675 (CO)
1H NMR (400 MHz, DMSO-d6) ꢀ: 12.35(1H; s; NH);
9.28(1H; d; J= 1.3 Hz; H3); 8.94(1H; d; J= 2.2 Hz; H6);
8.80(1H; dd; J= 1.3 and 2.2 Hz; H5); 8.71(1H; s; N=CH);
8.70 (1H; d; J=7.7 Hz; H- pyridinyl); 8.21 (2H; d; J=8.4 Hz;
H- benzylidene); 8.03 (1H; d; J=8.0 Hz; H- pyridinyl); 7.91
(1H; ddd; J= 1.7; 7.7 and 8.0 Hz; H- pyridinyl); 7.85 (2H; d;
2.1.3.2.
N’-[(E)-((furan-2-ylmethylene)pyrazine-2-
carbohydrazide (5b)
Yield: 50 %; mp: 190-192oC; (192°C)[9]

Synthesis and Antimycobacterial Evaluation
Medicinal Chemistry, 2011, Vol. 7, No. 3 247
R1
R1
O
X
X
N
N
N
CHO
Y
N
H
Y
3a-f
5a-f
EtOH:H2O, r.t.
O
N
CO2CH3
NH2NH2.H20
EtOH, reflux
N
NHNH2
N
OHC
X
N
1
2
R1
R1
4a-c
O
EtOH:H2O, r.t.
N
N
N
N
H
X
6a-c
Entry
X
Y
R1
3a
3b
3c
3d
3e
3f
O
O
CH
CH
CH
CH
CH
N
NO2
H
S
NO2
H
S
NH
NH
N
H
H
4a
4b
4c
---
---
CH
CH
---
Pyridin-2-yl
---
---
Scheme 1. Synthetic routes for the preparation of the N´-(E)-heteroaromatic-pyrazine-2-carbohydrazide derivatives (5a-f and 6a-c).
J= 8.4 Hz; H- benzylidene); 7.39 (1H; ddd; J= 1.7; 7.7 and
8.0 Hz; H- pyridinyl) ppm;
13C NMR (100 MHz, DMSO-d6) ꢀ: 159.5; 155.1; 149.3;
147.8; 144.6; 144.1; 143.3; 140.2; 137.3; 134.7; 127.6;
126.9; 122.9; 120.5 ppm;
Adherent cells were infected or not with BCG (2.5 X 106
UFC/well/100μL) cultured in the presence of medium alone,
tween 20 (3%) (live and dead controls, respectively) or dif-
ferent concentrations of compounds (1.0, 10.0 and
100μg/mL) in a triplicate assay. After 48 h, stock MTT solu-
tion (5 mg/mL of saline; 20 mL/well) was added to the cul-
ture and 4 h later, the plate was centrifugate for 2 minutes at
2800 rpm, supernatant was discharged and Dimethyl
sulfoxide (DMSO) (100 μL/well) was added for formazan
crystals solubilization and the absorbance was read at 540
nm in a plate reader (Biorad – 450).
ESI/MS: [M-H]: 302
IR: (KBr pellets, cm-1): 3420 (NH); 1778 (CO)
2.1.3.9.
N’-[(E)-phenylmethylidene]-2-
pyrazinecarbohydrazide (6c)
Yield: 50%; mp: 224°C; (228-230°C) [10]
2.2. General Procedures for Biological Tests
2.2.1. Cell Viability Assay
2.1.2. Antimycobacterial Activity
Briefly, 200μL 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 evapora-
tion of the medium in the test wells during incubation. The
96 plates received 100μL of the Middlebrook 7H9 broth con-
taining the mycobacterial cells (Difco laboratories, Detroit,
MI, USA) and a serial dilution of the compounds (5a-f and
6a-c) was made directly on the plate. The final drug concen-
trations tests were 0.01-100 μg/mL. Plates were covered and
sealed with parafilm and incubated at 37ºC for five days.
After this time, 25μL of a freshly prepared 1:1 mixture of
Alamar Blue (Accumed International, Westlake, Ohio) rea-
gent and 10% tween 80 was added to plate and incubated for
24h. A blue color in the well was interpreted as no bacterial
growth, and a pink color was scored as growth. The MIC
The cellular viability for a macrophage cell line J774
(ATCC TIB-67™) was determined by Mosmans´s MTT (3-
(4,5- dimethylthylthiazol-2yl)-2,5-dimethyl tetrazolium
bromide; Merck) microcultured tetrazolium assay. We
evaluated non infected or infected macrophages with Myco-
baterium bovis Bacillus Calmette-Guerin (BCG) in the pres-
ence and absence of test compounds (5a-f and 6a-c). The
cells were plated in flat bottom 96 well plates (2.5 X 106
cells/well/100μL) cultured for 24 h in a controlled atmos-
phere (CO2 5% at 37 °C), and non-adherent cells were
washed by gentle flushing with RPMI 1640 supplemented
with fetal bovine serum (10%) and gentamicin (25μg/mL).

248 Medicinal Chemistry, 2011, Vol. 7, No. 3
Lima et al.
Table 2. Antimycobacterial Activities and clogP Measurements
of Pyrazinamide and heteroaromatic-pyrazine-2-
carbohydrazides Derivatives (5a-f and 6a-c)
(minimal inhibition concentration) was defined as the lowest
drug concentration, which prevented a color change from
bleu to pink.
3. RESULTS AND DISCUSSION
3.1. Chemistry
MIC a
Entry
cLogPb
(ꢀg/mL)
The synthetic route for preparation of N´-(E)-
heteroaromatic-pyrazine-2-carbohydrazide derivatives (5a-f
and 6a-c) is summarized in Scheme 1. Firstly, the pyrazine-
2-carbohydrazide was synthesized by the reaction of methyl
pyrazine-2-carboxylate with hydrazine hydrate under reflux.
After that, compounds 5a-f and 6a-c were obtained from
reactions of pyrazine-2-carbohydrazide and heteroaromatic
aldehydes, as described in the general procedure (Scheme 1
and Table 1). [6-11]
5a
5b
5c
5d
5e
5f
3.12
>100
>100
>100
>100
50
0.71
0.889
1.613
1.531
0.786
-0.274
0.461
2.281
1.631
-0.711
1
In general, H NMR spectra of the compounds 5a-f and
6a
6b
6c
>100
>100
>100
>100
6a-c showed the signals of the respective protons of the syn-
thesized compounds, which were verified on the basis of
their chemicals shifts, multiplicities and coupling constants.
These spectra showed two characteristic signals about N=CH
proton at 8.56-8.71 ppm and CONH protons at 12.35-12.77
ppm. The 13C NMR spectra showed the C=O and C=N sig-
nals at 158.4-159.9 and 145.0-147.8 ppm, respectively.
PZA
a Minimum inhibitory concentration.
b Calculated using www.molinspiration.com
Table 1. Yields and Melting Points of N´-(E)-heteroaromatic-
methodology is nontoxic, uses a thermally-stable reagent and
shows good correlation with proportional and BACTEC
radiometric methods [14, 15].
pyrazine-2-Carbohydrazide Derivatives (5a-f and 6a-
c)
This table showed that the compounds 5a and 5f exhib-
ited antimycobacterial activities of 3.12 and 50μg/mL, re-
spectively. Therefore, these compounds were selected for
cytotoxicity evaluation by Mosmans´s assay.
Entry
Yield (%)
m.p. (°C)
5a
5b
5c
5d
5e
5f
58
50
62
50
50
58
60
86
50
217-219
190-192[9]
263-265
3.2. Cell Viability Assay
The cellular viability in the presence and absence of test
compound (5a-f and 6a-c) was determined by Mosmans´s
MTT (3-(4.5-demethylthylthiazol-2-yl)-2.5-dimethyl tetra-
zolium bromide; Merck) microcultured tetrazolium assay
[16, 17]. The results were represented as percentage cell vi-
ability (Table 3).
209-210 [9]
176-179 [9]
217-219
6a
6b
6c
203-204
This table shows that the compound 5f did not kill more
than 5% of the host cells in the minimum concentration
tested. Hence, this compound was selected to be tested on
macrophages infected with Mycobaterium bovis Bacillus
Calmette- Guerin (BCG) (Table 4).
254-256
204 [10]
3.2. Antimycobacterial Activity
The purpose of this test is to evaluate the action of these
compounds against macrophages that show their metabolism
changed after infection. Then, the derivative 5f was not cyto-
The antimycobacterial activities of 5a-f and 6a-c were
assessed against M. tuberculosis ATCC 27294[12] using the
micro plate Alamar Blue assay (MABA)[13] (Table 4). This
Table 3. Data of the Cellular Viability for a Macrophage Cell Line J774 (ATCC TIB-67™) by Mosmans´s Assay
% Cell Viability/Dose (ꢀg/ml)
Entry
3.12
50
100
5a
5f
88
84
96
76
95
93
100
100
Pyrazinamide
100

Synthesis and Antimycobacterial Evaluation
Medicinal Chemistry, 2011, Vol. 7, No. 3 249
Table 4. Data of the Cellular Viability for a Macrophage Cell Line J774 Infected (ATCC TIB-67™) with BCG by Mosmans´s Assay
% Cell Viability/Dose (ꢀg/ml)
Entry
3.12
50
100
5f
100
100
100
Pyrazinamide
100
92
87
[8]
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Z
4. CONCLUSION
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The synthesis of nine heteroaromatic pyrazine-2-
carbohydrazide derivatives (5a-f and 6a-c), including five
new compounds (5a, 5c, 5f, 6a and 6b), was performed in
good yields (50-86%). All these compounds were evaluated
against M. tuberculosis and compounds 5a and 5f (3.12 and
50ꢀg/mL) exhibited antitubercular activities better than
pyrazinamide (>100ꢀg/mL) in MABA assay. These results
suggest that 5a and 5f could be acting through a different
mechanism of action of that proposed to PZA. However,
only the derivative 5f was not cytotoxic in non infected or
infected macrophages with Mycobaterium bovis Bacillus
Calmette-Guerin (BCG). Therefore, this compound could be
considered a good start point to find new lead compounds in
the fight against tuberculosis.
[10]
[11]
[12]
[13]
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Received: August 20, 2010
Revised: January 06, 2011
Accepted: March 24, 2011