Synthesis andanti-mycobacterial evaluation of some pyrazine-2-carboxylic acid hydrazide derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.04.025

A series of pyrazine-2-carboxylic acid hydrazide derivatives were synthesized and screened for their activity against Mycobacterium tuberculosis. The results show that pyrazine-2-carboxylic acid hydrazi-deehydrazone derivatives 3ael were less active than pyrazinamide. In contrast,the N ⁴-ethyl-N¹-pyr-azinoyl-thiosemicarbazide 4 showed the highest activity against M. tuberculosis H₃₇Rv (IC₉₀ 16.87 mg/mL). Details of the structureeactivity and structureecytotoxicity relationships are discussed.

Full text of DOI:10.1016/j.ejmech.2010.04.025

European Journal of Medicinal Chemistry 45 (2010) 3384e3388
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anti-mycobacterial evaluation of some pyrazine-2-carboxylic
acid hydrazide derivatives
,
b
Mohamed Abdel-Aziz a , Hamdy M. Abdel-Rahman
*
a Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, 61519-Minia, Egypt
b Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Assiut University, 71526-Assiut, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrazine-2-carboxylic acid hydrazide derivatives were synthesized and screened for their
activity against Mycobacterium tuberculosis. The results show that pyrazine-2-carboxylic acid hydrazi-
deehydrazone derivatives 3ael were less active than pyrazinamide. In contrast, the N4-ethyl-N1-pyr-
Received 18 July 2009
Received in revised form
14 April 2010
Accepted 21 April 2010
Available online 28 April 2010
azinoyl-thiosemicarbazide 4 showed the highest activity against M. tuberculosis H37Rv (IC90 ¼ 16.87
mg/
mL). Details of the structureeactivity and structureecytotoxicity relationships are discussed.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Tuberculosis
Pyrazinamide
Hydrazones
Thiosemicarbazides
1. Introduction
efficacious in vivo than would be predicted by its in vitro potency
with vaguely defined mechanism of action [3,6,7]. On the other
hand, the hydrazoneehydrazide derivatives [8,9] as well as the N4-
alkyl thiosemicarbazides [10] of the anti-mycobacterial drug
isoniazid were reported to improve its activity. Therefore, guided
by the above mentioned data as well as the high activity of pyr-
azinamide against MDR-TB, we would like to report the synthesis,
anti-mycobacterial screening as well as the cytotoxicity studies of
a new series of pyrazine-2-carboxylic acid hydrazide derivatives
namely hydrazones and N4-alkyl thiosemicarbazides.
The treatment of tuberculosis (TB) infections has become an
important and challenging problem due to the emergence of
multiple-drug-resistant organisms [1]. Different factors are
responsible for the resurgence of TB, such as people infected with
HIV virus, immigration, war, famine, homelessness, the lack of new
drugs and multi-drug-resistant tuberculosis resulted from incon-
sistent or partial treatment. According to WHO global report, in
2006 there were an estimated 9.2 million worldwide new cases of
TB with 0.5 million cases of multidrug-resistant TB (MDR-TB) and
estimated 1.5 million deaths from TB in HIV-negative people and
0.2 million among people infected with HIV [2]. The recommended
therapy for TB consists of an initial phase of treatment with four
drugs e isoniazid, rifampicin, pyrazinamide and ethambutol, taken
daily for two months, followed by a continuation phase of treat-
ment with isoniazid and rifampicin for another four months [3,4].
Pyrazinamide is one of the frontline agents that played a significant
role in shortening the duration of treatment of MDR-TB. It is
considered to be a prodrug that requires activation by pyr-
azinamidase to pyrazinoic acid, which is believed to be the active
form [5]. The SAR of pyrazinamide derivatives is still very limited
because the drug is active only under acidic conditions and more
2. Results and discussion
2.1. Chemistry
The synthesis of the target compounds is outlined in Schemes 1
and 2. The core intermediate pyrazine-2-carboxylic acid hydrazide
2 was prepared from pyrazine-2-carboxylic acid 1 by esterification
followed by hydrazinolysis to give 2 in a good yield.
The hydrazones 3ael were prepared in one step by the
condensation of the hydrazide 2 with the appropriate aryl alde-
hydes or ketones in ethanol according to the procedure described in
literature [11]. On the other hand the thiosemicarbazide 4 was
prepared by reaction of 2 with ethyl isothiocyanate.
The commercially non-available 2-chloroquinoline-3-carbox-
aldehyde derivatives 6aeb were prepared by heating a mixture of
the acetanilide derivatives 5aeb with phosphorus oxychloride in
* Corresponding author. Tel./fax: þ20 86 2369075.
E-mail address: abulnil@hotmail.com (M. Abdel-Aziz).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.04.025

M. Abdel-Aziz, H.M. Abdel-Rahman / European Journal of Medicinal Chemistry 45 (2010) 3384e3388
3385
O
O
R1
N
N
COOH
N
N
NH2
N
N
N
N
N
(a)
(b)
H
H
R
3a-j
1
2
3a; R = H, R1 = 4-Cl
3b; R = H, R1 = 2-OH
3c; R = H, R1 = 3-OH
3d; R = H, R1 = 4-OH
3e; R = H, R1 = 3-NH2
3f; R = H, R1 = 4-OCH3
(c)
O
H
H
N
3g; R = H, R1 = 3-OCH3, 4-OH
3h; R = CH3, R1 = H
N
N
N
N
H
3i; R = CH3, R1 = 4-OCH3
3j; R = CH3, R1 = 4-Br
S
4
Scheme 1. Reagents and conditions; a) (i) Et3N, ClCOOEt, MeOH, (ii) NH2NH2$H2O b) Aldehyde or Ketone, EtOH, reflux c) EtNCS, EtOH, reflux.
DMF (Scheme 2). The structure of the prepared compounds was
fully characterized on the basis of melting points, IR, 1H NMR, mass
spectra and elemental analysis.
Confronted by these results of the hydrazones, we pursued
another venue of structural modification of the pyrazine-2-
carboxylic acid hydrazide to N4-ethyl-N1-pyrazinoyl-thio-
semicarbazide 4 as a representative example of the N4-alkyl-N1-
pyrazinoyl thiosemicarbazides. We selected to synthesize 4 with
the hope that it would possess high anti-mycobacterial activity
based on a literature survey showing that the N4-alkyl-N1-iso-
nicotinoyl thiosemicarbazides showed higher activity than their
N4-aryl analogs in the SAR of isoniazid derivatives [10]. Further-
more, some N4-aryl-N1-pyrazinoyl thiosemicarbazides were
already reported to have low anti-mycobacterial activity [14,15].
As anticipated, compound 4 was the most active in this series
2.2. Biological assay
All of the prepared compounds (hydrazones 3ael and thio-
semicarbazide 4) are initially subjected to the TAACF’s primary
screen against Mycobacterium tuberculosis strain H37Rv (ATCC
27294) in BACTEC 12B medium using a broth microdilution assay,
the microplate Alamar Blue Assay (MABA) [12] in the dos-
eeresponse assay. The assay returns IC90, and IC50 values for all
compounds where IC stands for ‘inhibitory concentration’ e i.e the
concentration where a compound inhibits the TB strain by 90% or
50%, respectively. As shown in Table 1, all the prepared hydrazones
3ael as well as the parent hydrazide 2 were inactive with IC90, and
with IC90, and IC50 values of 16.87
respectively. This is a quite good activity compared with pyr-
azinamide having MIC90 value ranging from 6 to 50 g/mL,
specifically; MIC90 at pH 5.5 is 50 g/mL [5].
mg/mL and 11.37 mg/mL,
m
m
The high anti-mycobacterial activity of 4 can be attributed to
one or more of the following hypotheses: (1) the thiosemicarbazide
molecule masks the acid group of pyrazinoic acid, and allowing it to
pass through the bacterial cell membrane. (2) A synergistic anti-
mycobacterial effect of both the thiosemicarbazide molecule (thi-
osemicarbazides were reported to exhibit anti-mycobacterial
activity) with pyrazinoic acid produced from its hydrolysis (3) or
that 4 can act as bacterial pyrazinamidase activator.
IC50 values more than 100 mg/mL.
A rationale for the design of the anti-mycobacterial drug isoni-
azid hydrazones is the increased lipophilicity of prepared
compounds resulting in a beneficial influence on their activity.
These hydrazones undergo hydrolysis to their parent hydrazide
(isoniazid) which is responsible for their activity [8]. However this
strategy unexpectedly was non effective for the prepared pyrazine-
2-carboxylic acid hydrazide-hydrazones as none of them exhibited
improved or similar anti-mycobacterial activity to pyrazinamide.
These unforeseen results were not only in disagreement with
The later hypothesis is based on Zhang et al. [16] discovery that
M. tuberculosis pyrazinamidase is a monomeric Fe2þ⁄Mn2þ protein
and can be inhibited by an excess of other metals competing for the
a
previous report showing good anti-mycobacterial activity
same binding-site such as Zn2þ
.
(without exact MICs or detailed SARs) of some pyrazine-2-
carboxylic acid hydrazide-hydrazones [13] but also against a reality
that all the prepared hydrazones 3ael exhibited increased lip-
ophilicity compared to pyrazinamide (see ClogP values, Table 1). A
reason of the results obtained may be attributed to that the parent
hydrazide 2 is less active than pyrazinamide itself.
2.3. Cytotoxicity assay
Cytotoxicity of all the prepared compounds is done in parallel
with the TB dose response assay. Compounds are screened by serial
Cl
N
H
N
O
Cl
N
N
N
N
(a)
(b)
N
X
O
H
O
X
H
X
H
3k-l
6a-b
5a-b
5a, 6a and 3k, X = CH3
5b, 6b and 3l, X = OCH3
Scheme 2. Reagents and conditions; a) POCl3, DMF b) EtOH, reflux.

3386
M. Abdel-Aziz, H.M. Abdel-Rahman / European Journal of Medicinal Chemistry 45 (2010) 3384e3388
Table 1
manner over a period of 10 min under stream of nitrogen gas, the
mixture was continuously stirred for an additional 30 min. Meth-
anol (0.32 g, 10.0 mmol) was added over a period of 15 min, the
mixture was stirred for additional 12 h at room temperature. The
solvent was evaporated under reduced pressure and the obtained
crude oily product was dissolved in 30 mL methanol, hydrazine
monohydrate (0.25 g, 5.0 mmol) was added and the mixture was
refluxed for 6 h, the mixture was cooled to room temperature and
the obtained precipitate was recrystallized from ethanol to give 2 as
a yellowish white crystals (0.11 g; 80% yield), m.p. 169e170 ꢂC. IR
(KBr) nmax (cmꢁ1): 3435 (NH2), 1636 (C]O), 1582 (C]N). 1H NMR
The in-vitro anti-mycobacterial activity of test compounds.
Compd. # TAACFa ID IC50
(
m
g/mL) IC90
(
m
g/mL) CC50 Vero cells CLogPc
g/mL)b
(m
2
417781
417771
417774
417772
417773
417775
417779
417769
417770
417778
417777
417776
417780
415101
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
11.37
e
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
16.87
>30
>30
11.43
>30
>30
>30
>30
>30
>30
>30
>30
5.37
29.75
e
ꢁ1.16
1.72
1.54
0.91
0.91
0.31
1.16
0.71
1.90
2.84
1.55
1.31
ꢁ1.71
ꢁ0.68
3a
3b
3c
3d
3e
3f
3g
3i
3j
3k
3l
4
PZA
(300 MHz, DMSO-d6)
d (ppm): 4.66 (s, 2H, NH2), 8.72 (dd, 1H,
J ¼ 2.50 Hz, J ¼ 1.40 Hz, H5 of pyrazine), 8.85 (d, 1H, J ¼ 2.50 Hz, H6
of pyrazine), 9.16 (d, 1H, J ¼ 1.40 Hz, H3 of pyrazine), 10.18 (s, 1H,
NH). MS: m/z (%) 138 [Mþ] (100), 124 (5), 105 (5).
6e50d
a
TAACF: Tuberculosis Antimicrobial Acquisition and Coordinating Facility.
Measured by Cell Titer-GIo.
CLogP calculated using ChemDraw Ultra 9.0.
Data from Ref. [5].
3.1.2. General procedure for preparation of hydrazones 3ael
To a stirred solution of 2 (0.138 g, 1.0 mmol) in 25 mL absolute
ethanol, various substituted aldehydes or ketones (1.0 mmol) were
added, the mixture was refluxed for 12 h, then cooled to room
temperature and the obtained precipitate was recrystallized from
ethanol to give 3ael in a good yield.
b
c
d
dilution to assess toxicity to a VERO cell line. The VERO cell cyto-
toxicity assay returns a CC50 value. As shown in Table 1, all the
tested compounds are showed CC50 values more than 30
mg/mL
3.1.2.1. 4-Chlorobenzylidene pyrazine-2-carboxylic acid hydrazide
(3a) [13]. Yellowish white crystals in (0.213 g; 81.9% yield), m.p.
240e242 ꢂC. IR (KBr) nmax (cmꢁ1): 3450 (NH), 1651 (C]O), 1564
(C]N), 1534 (NeH), 1326, 1168, and 1137 (pyrazine). 1H NMR
except 3b, 3l, 4 with values 11.43, 5.37 and 29.75 g/mL, respec-
m
tively. Although compound 4 exhibited the best anti-mycobacterial
activity (Table 1), its cytotoxicity is relatively high thus having
narrow selectivity index (1.76). The selectivity index is defined as
the ratio of the measured CC50 (mammalian cell toxicity) to the IC90
(H37Rv M. tuberculosis) described above. According to TAACF rules, if
the SI value is ꢀ10, then the compound may be considered for
further screening. Therefore, compound 4 was not evaluated
further although pyrazinamide itself is considered a narrow
selectivity index drug.
Nevertheless, more information on the structureeactivity rela-
tionships of these compounds and more potent anti-mycobacterial
agents can be achieved by the preparation of N1-pyrazinolyl-N4-
alkyl thiosemicarbazides and their cyclization products.
(300 MHz, DMSO-d6)
d
(ppm): 7.42 (dd, 2H, J ¼ 8.45 Hz, AreH), 7.81
(dd, 2H, J ¼ 8.45 Hz, AreH), 8.55 (s, 1H, CH), 8.75 (dd, 1H,
J ¼ 2.50 Hz, J ¼ 1.40 Hz, H5 of pyrazine), 8.85 (d, 1H, J ¼ 2.50 Hz, H6
of pyrazine), 9.36 (d, 1H, J ¼ 1.40 Hz, H3 of pyrazine), 10.60 (s, 1H,
NH). MS: m/z (%) 260 [Mþ] (100), 262 (24), 264 (32), 141 (4). 140 (4).
3.1.2.2. 2-Hydroxybenzylidene pyrazine-2-carboxylic acid hydrazide
(3b). Yellow crystals in (0.201 g; 83% yield), m.p. 210e211 ꢂC. IR
(KBr) nmax (cmꢁ1): 3415 (OH), 3380 (NH), 1685 (C]O), 1570 (C]
N), 1512 (NeH), 1355, 1309, and 1118 (pyrazine). 1H NMR
(300 MHz, DMSO-d6)
d
(ppm): 6.93 (t, 2H, J ¼ 8.50 Hz, AreH), 7.33
(t, 1H, J ¼ 5.76 Hz, AreH), 7.51 (d, 1H, J ¼ 9.06 Hz, AreH), 8.80 (s,
1H, CH), 8.85 (dd, 1H, J ¼ 2.50 Hz, J ¼ 1.50 Hz, H5 of pyrazine), 8.95
(d, 1H, J ¼ 2.50 Hz, H6 of pyrazine), 9.31 (d, 1H, J ¼ 1.50 Hz, H3 of
3. Experimental
3.1. Chemistry
pyrazine). 13C NMR (75 MHz, DMSO-d6)
d (ppm): 117.71 (CH),
119.75 (CH), 120.58 (CH), 130.81 (CH), 132.72 (CH), 144.26 (CH),
145.07 (CH), 145.16 (C), 148.78 (CH), 151.18 (C), 158.35 (C), 160.17
(CO). MS: m/z (%) 242 [Mþ] (100), 141 (4), 122 (5). Anal. Calcd. for
C12H10N4O2: C, 59.50; H, 4.16; N, 23.13. Found: C, 59.45; H, 4.19; N,
22.79.
Reactions were monitored by TLC analysis using Merck 9385
pre-coated aluminum plate silica gel (Kieselgel 60) with F254 indi-
cator thin layer plates. Melting points were determined on Stuart
electrothermal melting point apparatus and were uncorrected.
IR spectra were recorded as KBr disks on a Brukar Vector 22 IR
spectrophotometer. 1H NMR spectra were obtained on a 300 MHz
AL spectrophotometer and on 60 MHz Varian EM-360L NMR
spectrophotometer using TMS as internal reference. Chemical shifts
3.1.2.3. 3-Hydroxybenzylidene pyrazine-2-carboxylic acid hydrazide
(3c). White powder in (0.206 g; 85.1% yield), m.p. 264e265 ꢂC. IR
(KBr) nmax (cmꢁ1): 3450 (OH), 3380 (NH), 1661 (C]O), 1592 (C]N),
1509 (NeH), 1356, 1310, and 1160 (pyrazine). 1H NMR (300 MHz,
(d) values are given in parts per million (ppm) relative to CDCl3
(7.29) or DMSO-d6 (2.5) and coupling constants (J) in Hertz. Split-
ting patterns are designated as follows: s, singlet; d, doublet; t,
triplet; q, quartet; dd, doublet of doublet; m, multiplet.
Accurate masses were obtained on Micromass LCT mass spec-
trometer, which was recorded in the positive ion mode with leucine
enkephalin as an internal lock mass standard. Elemental analysis
was performed on Vario EL III, Elemental analyzer, GMBH. D-63452
HANAU.
DMSO-d6)
d
(ppm): 5.32 (s, 1H, OH), 6.63 (d, 1H, J ¼ 2.00 Hz, AreH),
6.80 (d, 1H, J ¼ 2.00 Hz, AreH), 7.05 (s, 1H, AreH), 7.13 (d, 1H,
J ¼ 3.60 Hz, AreH), 8.50 (s, 1H, CH), 8.82 (dd, 1H, J ¼ 2.50 Hz,
J ¼ 1.40 Hz, H5 of pyrazine), 8.92 (d, 1H, J ¼ 2.50 Hz, H6 of pyrazine),
9.30 (d, 1H, J ¼ 1.40 Hz, H3 of pyrazine), 12.18 (s, 1H, NH). 13C NMR
(75 MHz, DMSO-d6)
d (ppm): 114.04 (CH), 118.94 (CH), 120.17 (CH),
130.99 (CH), 136.39 (C), 144.19 (CH), 144.99 (CH), 145.53 (CH),
148.65 (CH), 150.74 (C), 158.42 (C), 160.16 (CO). MS: m/z (%) 242
[Mþ] (100), 141 (6), 122 (5). Anal. Calcd. for C12H10N4O2: C, 59.50; H,
4.16; N, 23.13. Found: C, 59.34; H, 4.34; N, 23.48.
3.1.1. Synthesis of pyrazine-2-carboxylic acid hydrazide (2)
To a stirred solution of 1 (0.124 g, 1.0 mmol) in 30 mL dry
chloroform at ꢁ10 ꢂC, triethylamine (0.10 g, 1.0 mmol) was added,
followed by ethyl chloroformate (0.108 g, 1.0 mmol) in a drop wise
3.1.2.4. 4-Hydroxybenzylidene pyrazine-2-carboxylic acid hydrazide
(3d). Yellowish white crystals in (0.206 g; 85.1% yield), m.

M. Abdel-Aziz, H.M. Abdel-Rahman / European Journal of Medicinal Chemistry 45 (2010) 3384e3388
3387
p. > 300 ꢂC. IR (KBr) nmax (cmꢁ1): 3455 (OH), 3365 (NH), 1663 (C]
O) 1593 (C]N), 1518 (NeH), 1352, 1295, and 1143 (pyrazine). 1H
3.1.2.9. [1-(4-Methoxyphenyl)ethylidene]pyrazine-2-carboxylic acid
hydrazide (3i). White powder in (0.222 g, 82.2% yield), m.p.
179e180 ꢂC. IR (KBr) nmax (cmꢁ1): 3425 (NH), 1666 (C]O), 1633
(C]C), 1520 (NeH), 1388, 1305, and 1147 (pyrazine). 1H NMR
NMR (300 MHz, DMSO-d6)
d
(ppm): 6.85 (dd, 2H, J ¼ 6.00 Hz,
AreH), 7.58 (dd, 2H, J ¼ 6.00 Hz, AreH), 8.55 (s, 1H, AreH), 8.81 (dd,
1H, J ¼ 2.40 Hz, J ¼ 1.40 Hz, H5 of pyrazine), 8.92 (d, 1H, J ¼ 2.40 Hz,
H6 of pyrazine), 9.27 (d, 1H, J ¼ 1.40 Hz, H3 of pyrazine), 9.95 (s, 1H,
(300 MHz, CDCl3) d (ppm): 2.40 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 6.93
(dd, 2H, J ¼ 8.45 Hz, AreH), 7.88 (dd, 2H, J ¼ 8.45 Hz, AreH), 8.55
(dd, 1H, J ¼ 2.50 Hz, J ¼ 1.40 Hz, H5 of pyrazine), 8.82 (d, 1H,
J ¼ 2.50 Hz, H6 of pyrazine), 9.50 (d, 1H, J ¼ 1.40 Hz, H3 of pyrazine),
OH), 12.10 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6)
d (ppm): 116.99
(CH), 126.21 (C), 130.17 (CH), 144.13 (CH), 144.89 (CH), 145.67 (CH),
148.49 (CH), 150.94 (C), 159.85 (C), 160.37 (CO). MS: m/z (%) 242
[Mþ] (100), 141 (6), 122 (7). Anal. Calcd. for C12H10N4O2: C, 59.50; H,
4.16; N, 23.13. Found: C, 59.49; H, 4.17; N, 22.85.
10.65 (s, 1H, NH). 13C NMR (75 MHz, CDCl3)
d (ppm): 13.19 (CH3),
57.02 (OCH3) 114.71 (CH), 129.09 (CH), 130.77 (C), 131.28 (C), 142.78
(CH), 143.29 (CH), 144.60 (CH), 148.35 (C), 158.89 (C), 161.48 (CO).
MS: m/z (%) 270 [Mþ] (100), 150 (8), 141 (4). Anal. Calcd. for
C14H14N4O2$H2O: C, 58.32; H, 5.59; N, 19.43. Found: C, 58.27; H,
5.54; N, 19.37.
3.1.2.5. 3-Aminobenzylidene pyrazine-2-carboxylic acid hydrazide
(3e). Yellow crystals in (0.198 g; 82.1% yield), m.p. 253e254 ꢂC. IR
(KBr) nmax (cmꢁ1): 3435 (NH2), 3260 (NH), 1679 (C]O), 1609 (C]
N), 1511 (NeH), 1366, 1315, and 1140 (pyrazine). 1H NMR (300 MHz,
3.1.2.10. [1-(4-Bromophenyl)ethylidene]pyrazine-2-carboxylic acid
hydrazide (3j). White crystals in (0.255 g, 80% yield), m.p.
198e199 ꢂC. IR (KBr) nmax (cmꢁ1): 3310 (NH),1689 (C]O),1597 (C]
N),1506 (NeH), 1387, 1298, and 1142 (pyrazine). 1H NMR (300 MHz,
DMSO-d6)
d
(ppm): 5.30 (s, 2H, NH2), 6.65 (d,1H, J ¼ 8.01 Hz, AreH),
6.81 (d, 1H, J ¼ 7.50 Hz, AreH), 7.01 (s, 1H, AreH), 7.07e7.12 (m, 1H,
AreH), 8.50 (s, 1H, CH), 8.82 (dd, 1H, J ¼ 2.50 Hz, J ¼ 1.40 Hz, H5 of
pyrazine), 8.93 (d, 1H, J ¼ 2.50 Hz, H6 of pyrazine), 9.30 (d, 1H,
J ¼ 1.40 Hz, H3 of pyrazine), 12.16 (s, 1H, NH). 13C NMR (75 MHz,
CDCl3)
d
(ppm): 2.42 (s, 3H, CH3), 7.58 (dd, 2H, J ¼ 8.45 Hz, AreH),
7.80 (dd, 2H, J ¼ 8.45 Hz, AreH), 8.55 (dd, 1H, J ¼ 2.50 Hz,
J ¼ 1.50 Hz, H5 of pyrazine), 8.80 (d, 1H, J ¼ 2.50 Hz, H6 of pyrazine),
9.52 (d, 1H, J ¼ 1.50 Hz, H3 of pyrazine), 10.70 (s, 1H, NH). 13C NMR
DMSO-d6)
d (ppm): 112.72 (CH), 117.06 (CH), 117.49 (CH), 130.32
(CH), 135.61 (CH), 144.16 (CH), 144.96 (CH), 145.59 (C), 148.59 (CH),
149.89 (C), 151.51 (C), 160.04 (CO). MS: m/z (%) 241 [Mþ] (100), 141
(12), 121 (13). Anal. Calcd. for C12H11N5O: C, 59.74; H, 4.60; N, 29.03.
Found: C, 59.50; H, 4.57; N, 28.75.
(75 MHz, CDCl3)
d (ppm): 13.18 (CH3), 126.58 (CH), 129.41 (CH),
138.34 (C), 141.15 (CH), 141.92 (CH), 145.81 (CH), 145.90 (C), 153.03
(C), 156.57 (C), 159.46 (CO). MS: m/z (%) 319 [Mþ] (100), 321 (8), 323
(6), 198 (5), 141 (4). Anal. Calcd. for C13H11BrN4O: C, 48.92; H, 3.47;
N, 17.55. Found: C, 48.55; H, 3.81; N, 17.56.
3.1.2.6. 4-Methoxybenzylidene pyrazine-2-carboxylic acid hydrazide
(3f). Yellow crystals in (0.21 g; 82% yield), m.p. 225e226 ꢂC. IR
(KBr) nmax (cmꢁ1): 3450 (NH), 1667 (C]O), 1596 (C]N), 1497
(NeH), 1392, 1290, and 1140 (pyrazine). 1H NMR (300 MHz, CDCl3)
3.1.2.11. (2-Chloro-6-methylquinolin-3-ylmethylene)pyrazine-2-
carboxylic acid hydrazide (3k). Yellowish white crystals in (0.27 g,
83% yield), m.p. 230e231 ꢂC. IR (KBr) nmax (cmꢁ1): 3445 (NH), 1666
(C]O), 1593 (C]N), 1514 (NeH), 1382, 1262, and 1143 (pyrazine).
d
(ppm): 3.85 (s, 3H, OCH3), 7.04 (dd, 2H, J ¼ 8.76 Hz, AreH), 7.68
(dd, 2H, J ¼ 8.76 Hz, AreH), 8.58 (s, 1H, CH), 8.80 (dd, 1H,
J ¼ 2.50 Hz, J ¼ 1.50 Hz, H5 of pyrazine), 8.95 (d, 1H, J ¼ 2.50 Hz, H6
of pyrazine), 9.28 (d, 1H, J ¼ 1.50 Hz, H3 of pyrazine), 12.20 (s, 1H,
1H NMR (300 MHz, DMSO-d6)
d (ppm): 2.50 (s, 3H, CH3), 7.73 (d, 1H,
J ¼ 1.92 Hz, AreH), 7.89 (d, 1H, J ¼ 8.49 Hz, AreH), 8.00 (s, 1H,
AreCH), 8.83 (s, 1H, CH), 8.87 (dd, 1H, J ¼ 2.50 Hz, J ¼ 1.40 Hz, H5 of
pyrazine), 8.94 (d, 1H, J ¼ 2.50 Hz, H6 of pyrazine), 9.17 (s, 1H,
AreCH), 9.33 (d, 1H, J ¼ 1.40 Hz, H3 of pyrazine), 12.82 (s. 1H, NH).
MS: m/z (%) 325 [Mþ] (100), 327 (20), 329 (40), 205 (3), 141 (4).
Anal. Calcd. for C16H12ClN5O: C, 58.99; H, 3.71; N, 21.50. Found: C,
58.86; H, 3.70; N, 21.39.
NH). 13C NMR (75 MHz, CDCl3)
d (ppm): 53.69 (OCH3) 111.90 (CH),
124.28 (C),126.25 (CH),140.42 (CH),141.57 (CH),142.10 (CH),144.89
(CH), 147.10 (C), 156.45 (C), 158.28 (CO). MS: m/z (%) 256 [Mþ] (100),
141 (5), 136 (7). Anal. Calcd. for C13H12N4O2: C, 60.93; H, 4.72; N,
21.86. Found: C, 60.92; H, 4.71; N, 21.33.
3.1.2.7. 4-Hydroxy-3-methoxybenzylidene
pyrazine-2-carboxylic
acid hydrazide (3g) [17]. Yellow crystals in (0.228 g, 83.8% yield), m.
p. 249e250 ꢂC. IR (KBr) nmax (cmꢁ1): 3460 (OH), 3310 (NH), 1671
(C]O), 1595 (C]N), 1499 (NeH), 1385, 1309, and 1144 (pyrazine).
3.1.2.12. (2-Chloro-6-methoxyquinolin-3-ylmethylene) pyrazine-2-
carboxylic acid hydrazide (3l). Yellow crystals in (0.273 g, 80%
yield), m.p. 260e261 ꢂC. IR (KBr) nmax (cmꢁ1): 3275 (NH), 1681 (C]
O), 1591 (C]N), 1507 (NeH), 1382, 1299, and 1143 (pyrazine). 1H
1H NMR (60 MHz, DMSO-d6)
d (ppm): 4.20 (s, 3H, OCH3), 7.30e7.75
(m, 2H, AreH), 8.00 (s, 1H, AreH), 9.30 (s, 1H, CH), 9.70 (d, 2H,
J ¼ 8.06 Hz, CH of pyrazine), 10.20 (s, 1H, CH of pyrazine), 11.20 (s,
NMR (300 MHz, DMSO-d6) d (ppm): 3.92 (s, 3H, OCH3), 7.52 (d, 1H,
J ¼ 2.73 Hz, AreH), 7.70 (s, 1H, AreH), 7.89 (d, 1H, J ¼ 7.30 Hz,
AreH), 8.85 (s, 1H, CH), 8.93 (dd, 1H, J ¼ 2.40 Hz, J ¼ 1.40 Hz, H5 of
pyrazine), 8.97 (d, 1H, J ¼ 2.40 Hz, H6 of pyrazine), 9.18 (s, 1H,
AreCH), 9.35 (d, 1H, J ¼ 1.40 Hz, H3 of pyrazine), 12.80 (s, 1H, NH).
MS: m/z (%) 341 [Mþ] (100), 343 (19), 345 (41), 221 (3), 141 (4). Anal.
Calcd. for C16H12ClN5O2: C, 56.23; H, 3.54; N, 20.49. Found: C, 56.22;
H, 3.58; N, 20.10.
1H, NH). 13C NMR (75 MHz, DMSO-d6)
d (ppm): 57.54 (OCH3), 110.41
(CH), 116.71 (CH), 123.62 (CH), 126.62 (C), 144.16 (CH), 144.89 (CH),
145.71 (CH), 148.51 (CH), 148.90 (C), 150.11 (C), 151.14 (C), 159.86
(CO). MS: m/z (%) 272 [Mþ] (100), 152 (5), 141 (4).
3.1.2.8. 1-(Phenylethylidene) pyrazine-2-carboxylic acid hydrazide
(3h). White crystals in (0.195 g, 81.2% yield), m.p. 212e213 ꢂC. IR
(KBr) nmax (cmꢁ1): 3226 (NH), 3045-3004 (AreCH), 2960e2830
(AlipheCH), 1599 (C]N), 1567 (Ar C]C), 1375, 1312, and 1140
3.1.3. Synthesis of N-ethyl-N0-(pyrazine-2-carbonyl)
thiosemicarbazide (4)
(pyrazine). 1H NMR (300 MHz, CDCl3)
d
(ppm): 2.40 (s, 3H, CH3),
To a stirred solution of 2 (0.138 g, 1.0 mmol) in 25 mL absolute
ethanol, ethyl isothiocyanate (1.0 mmol) was added, the mixture
was refluxed for 12 h, then cooled to room temperature and the
obtained precipitate was recrystallized from ethanol to give 4 as
white crystals in (0.185 g; 82.2% yield), m.p. 218e219 ꢂC. IR (KBr)
nmax (cmꢁ1): 3450 and 3215 (NH), 1680 (C]O), 1542 (C]S), 1385,
7.00e7.00 (m, 5H, AreH), 7.88e7.94 (m, 2H, CH of pyrazine), 9.50
(d, 1H, J ¼ 1.4 Hz, CH of pyrazine), 10.60 (s, 1H, NH). 13C NMR
(75 MHz, CDCl3)
d (ppm): 13.10 (CH3), 124.88 (CH), 126.52 (CH),
128.00 (CH), 135.57 (C), 140.68 (CH), 142.00 (C), 142.42 (CH), 145.80
(CH), 152.23 (C), 156.37 (CO). MS: m/z (%) 241 (100) [Mþ], 242 (15),
141 (12). 121 (13) Anal. Calcd. for C13H12N4O: C, 64.99; H, 5.03; N,
23.32. Found: C, 64.86; H, 5.10; N, 23.30.
1316, and 1144 (pyrazine). 1H NMR (300 MHz, CDCl3)
(t, 3H, J ¼ 7.10 Hz, CH3), 3.43 (q, 2H, J ¼ 6.87 Hz and 6.03 Hz, CH2),
d (ppm): 1.05

3388
M. Abdel-Aziz, H.M. Abdel-Rahman / European Journal of Medicinal Chemistry 45 (2010) 3384e3388
8.05 (t, 1H, J ¼ 5.49 Hz, NH), 8.75 (dd, 1H, J ¼ 2.50 Hz, J ¼ 1.40 Hz, H5
of pyrazine), 8.90 (d, 1H, J ¼ 2.50 Hz, H6 of pyrazine), 9.20 (d, 1H,
J ¼ 1.40 Hz, H3 of pyrazine), 9.38 (s, 1H, NH) 10.70 (s, 1H, NH). 13C
Promega’s Cell Titer Glo Luminescent Cell Viability Assay, a homo-
geneous method of determining the number of viable cells in
culture based on quantitation of the ATP present. Cytotoxicity is
determined from the doseeresponse curve as the CC50 using
a curve-fitting program. Ultimately, the CC50 is divided by the IC90
to calculate an SI (Selectivity Index) value.
NMR (75 MHz, CDCl3)
d (ppm): 14.54 (CH3), 38.77 (CH2), 141.68
(CH), 142.50 (CH), 142.78 (C), 146.08 (CH), 160.10 (CO), 179.40 (C]
S). MS: m/z (%) 225 [Mþ] (100), 141 (4), 105 (10). Anal. Calcd. for
C8H11N5OS: C, 42.65; H, 4.92; N, 31.09. Found: C, 42.82; H, 4.92; N,
31.03.
Acknowledgements
3.1.4. Synthesis of the 2-chloro-6-(substituted)quinoline-3-
carboxaldehyde (6aeb) [18]
Anti-mycobacterial data were provided by the Tuberculosis
Antimicrobial Acquisition and coordinating Facility (TAACF)
through a research and development contract with the US National
Institute of Allergy and Infectious Diseases.
A mixture of acetanilide derivative 5aeb (1.0 mmol) and phos-
phorus oxychloride (7.0 mmol) in dimethyl formamide (2.5 mmol)
was heated at 75 ꢂC for 10 h, cooled to room temperature then
poured on 250 mL ice-water and the formed precipitate was
collected by filteration. The obtained crude product was recrystal-
lized from ethyl acetate to give title compounds 6aeb.
References
3.1.4.1. 2-Chloro-6-methoxyquinoline-3-carboxaldehyde 6a. Yellowish
white crystals in (0.125 g; 56.6% yield), m.p. 146e147 ꢂC. 1H NMR
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(300 MHz, CDCl3)
d
(ppm): 3.94 (s, 3H, OCH3), 7.18 (d, 1H, J ¼ 2.8 Hz,
H-5), 7.46 (dd, 1H, J ¼ 9.0 Hz and 2.8 Hz, H-7), 7.90 (d, 1H, J ¼ 9.0 Hz,
H-8), 8.56 (s, 1H, H-4), 10.50 (s, 1H, CHO).
3.1.4.2. 2-Chloro-6-methylquinoline-3-carboxaldehyde 6b. Yellowish
white crystals in (0.145 g; 70.7% yield), m.p. 124e125 ꢂC. 1H NMR
(300 MHz, CDCl3) d d (ppm): 2.54
(ppm): 1H NMR (300 MHz, CDCl3)
(s, 3H, CH3), 7.53 (dd, 1H, J ¼ 8.9 Hz and 2.9 Hz, H-7), 7.64 (d, 1H,
J ¼ 2.9 Hz, H-5), 7.99 (d,1H, J ¼ 8.9 Hz, H-8), 8.72 (s,1H, H-4),10.52 (s,
1H, CHO).
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3.2.1. Determination of a 90% inhibitory concentration (IC90
)
The initial screening is conducted against M. tuberculosis H37Rv
(ATCC 27294) in BACTEC 12B medium using the Microplate Alamar
Blue Assay (MABA) [17]. Compounds are tested in ten 2-fold dilu-
tions, typically from 100 mg/mL to 0.19 mg/mL. The IC90 is defined as
the concentration effecting a reduction in fluorescence of 90%
relative to controls. This value is determined from the dose-
response curve using a curve-fitting program.
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[17] T.V. Gortinskaya, K.M. Murav’eva, M.N. Shchukina, Zh. Obshch. Khim. 25
(1955) 2313e2317 [Through Chemical Abst. 50:48790].
[18] O. Meth-Cohn, B. Narine, B. Tarnowski, Tetrahedron Lett. 20 (1979)
3111e3114.
3.2.2. Determination of mammalian cell cytotoxicity (CC50
The VERO cell cytotoxicity assay is done in parallel with the TB
Dose Response assay. After 72 h exposure, viability is assessed using
)