Synthesis and Antitubercular activity of New Thiazolidinones with Pyrazinyl and Thiazolyl Scaffolds

Article abstract of DOI:10.1002/jhet.2552

Emergence of multidrug resistant and extensively drug resistant tuberculosis has prompted to develop new molecular entities to treat the disease. A series of new 4-thiazolidinones with pyrazinyl and thiazolyl scaffolds has been synthesized, and their antitubercular activity is reported. The title 4-thiazolidinones, N-(pyrazinyl substituted thiazoloylamino)-2-aryl-4-thiazolidinones (6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j) have been first time prepared using pyrazinamide as a starting material via five successive steps. The purity and the structures of the intermediates (carboethoxythiazole, acid hydrazide, and azomethines) and title thiazolidinones (6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j) have been confirmed by TLC and spectral analyses, respectively. An antitubercular screening of the new 4-thiazolidinones has been performed on bacterial strains, Mycobacterium tuberculosis H37Ra and Mycobacterium BCG using the solutions of different concentrations of the compounds (6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j) and the screening results are presented. Compound 6a has displayed notable antitubercular activity.

Full text of DOI:10.1002/jhet.2552

Month 2015
Synthesis and Antitubercular activity of New Thiazolidinones with
Pyrazinyl and Thiazolyl scaffolds
Sambhaji T. Dhumal,a Amarsinh R. Deshmukh,a Lalit D. Khillare,a Manisha Arkile,b
b
a
*
Dhiman Sarkar, and Ramrao A. Mane
aDepartment of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, 431004, India
bCombi Chem-Bio Resource Centre, CSIR-National Chemical Laboratory, Pune, 411008, India
*
E-mail: manera2011@gmail.com
Additional Supporting Information may be found in the online version of this article.
Received June 17, 2015
DOI 10.1002/jhet.2552
Published online in 00 Month 2015 Wiley Online Library (wileyonlinelibrary.com).
Emergence of multidrug resistant and extensively drug resistant tuberculosis has prompted to develop
new molecular entities to treat the disease. A series of new 4-thiazolidinones with pyrazinyl and thiazolyl
scaffolds has been synthesized, and their antitubercular activity is reported. The title 4-thiazolidinones, N-
(pyrazinyl substituted thiazoloylamino)-2-aryl-4-thiazolidinones (6a–j) have been first time prepared using
pyrazinamide as a starting material via five successive steps. The purity and the structures of the intermedi-
ates (carboethoxythiazole, acid hydrazide, and azomethines) and title thiazolidinones (6a–j) have been con-
firmed by TLC and spectral analyses, respectively. An antitubercular screening of the new 4-thiazolidinones
has been performed on bacterial strains, Mycobacterium tuberculosis H37Ra and Mycobacterium BCG using
the solutions of different concentrations of the compounds (6a–j) and the screening results are presented.
Compound 6a has displayed notable antitubercular activity.
J. Heterocyclic Chem., 00, 00 (2015).
INTRODUCTION
of the serious threats of TB, the search and development of
new bioactive therapeutic lead as effective anti-TB drugs
against the resistant strains having fewer to no side effects
is currently gaining more importance. As such, the develop-
ment of novel molecular scaffolds targeting Mtb is an area of
increasing active investigation globally [4].
First-line antitubercular agent, PZA is a nicotinamide
analog. This in combination with rifampicin displays ster-
ilizing activity, the ability to kill the dormant non-growing
tubercle bacilli of low metabolism activity [5]. This syner-
getic effect of PZA and rifampicin against dormant sub-
population of Mtb is responsible to decrease the time of
treatment from 12 to 6months. PZA is a prodrug, and after
getting converted into pyrazinoic acid [6], acts as inhibitor of
mycobacterial fatty acid synthase-I [7]. Pyrazines possess wide
range of pharmacological activities, viz antitubercular [8],
Tuberculosis (TB) is a major global health problem and
highly contagious infectious disease, caused by Mycobacte-
rium tuberculosis (Mtb). One third of the world’s population
is currently infected with Mtb [1]. In 2012, the World Health
Organization reported 8.6 million new cases and 1.3 million
TB deaths [2]. Increasing incidence of multidrug resistant
TB (resistant to at least isoniazid and rifampicin), extensively
drug resistant TB (XDR-TB; multidrug resistant plus resis-
tant to fluroquinoline and aminoglycoside), totally drug re-
sistant TB, and coinfection with HIV remain a serious
problem [3]. Currently available TB treatment is an adminis-
tration of combination of first-line antituberculars; rifampi-
cin, isoniazid, ethambutol, and pyrazinamide (PZA) for
6months and has side effects and noncompliance. Because
© 2015 HeteroCorporation

S. T. Dhumal, A. R. Deshmukh, L. D. Khillare, M. Arkile, D. Sarkar, and R. A. Mane
antifungal [9], antitumor [10], and antibacterial [11]. The
and 10.77 ppm, corresponds to the signals because of pro-
tons of N–CH–S and NH–C¼O, The presence of three
characteristics carbon signals is observed at δ 29.31,
61.6, and 169.01 ppm in 13C NMR spectrum of (6a) owing
to the signals of carbons of S-CH2-CO, N-CH-S and C¼O
groups, respectively, confirming the presence of a 4-
thiazolidinone ring in (6a). The HRMS spectrum further
strengthen the structure assigned as 4-methyl-N-(4-oxo-2-
phenylthiazolidin-3-yl)-2-(pyrazin-2-yl)thiazole-5-carboxamide
as it displays [M+ H]+ ion peak at m/z 398.0735 for the molec-
ular formula C18H15N5O2S2 (Scheme 1).
Biological evaluation. Antitubercular activity. All the
synthesized compounds were evaluated for there in vitro
activity against all M. Tuberculosis H37Ra, M. Bovis BCG.
All compounds showed moderate activity against
Mycobacterium species (Table 1). All compounds did not
show any activity against other bacteria, which means it does
not affect human’s normal flora. Compound (6a) showed
70% inhibition at 30μg/mL concentration, which is highest
among the series. The title compound (6a) is a parent
compound not having any substituent in the benzonoid ring.
The other compounds (6b-j), most of them are having
electron donating substituents at four position, responsible for
enhancing electron availability and activation of benzonoid
ring. This is the only difference with respect to the electron
availability, causing more favor to (6a) to display better
antitubercular activity than the others.
previously mentioned clinical properties of PZA/its deriva-
tives have increased the interest to synthesize its more new
derivatives/analogues with the hope to obtain potentially
active antitubercular compounds [4].
Literature survey reveals that 4-thiazolidinone is a versatile
scaffold observed in various pharmaceutical agents and
displays broad spectrum of biological activities such as
antitubercular [12], anti-inflammatory [13], anticancer [14],
antihyperglycemic [15], antifungal [16], anticonvulsant [17],
antihistaminic [18], and anti-HIV [19]. Some of the thiazoles
are the key scaffolds/clinical drugs and have displayed
numerous biological and pharmaceutical activities such as
antitubercular [20], anti-inflammatory [21], anticancer [22], an-
tibacterial [23], antifungal [24], and antiviral [25].
Considering the therapeutic significance of pyrazines,
thiazoles, and thiazolidinones here it was thought to
synthesize some new 4-thiazolidinones having these
biodynamic rings, i.e., thiazole and pyrazine within one
molecular framework with expectation to obtain the new
leads with enhanced antitubercular activity, because of
the combined synergetic effect of the rings.
RESULTS AND DISCUSSION
Chemistry. The target compounds have been synthesized
by following multistep route, designed on the basis of
retrosynthetic analysis, starting from PZA.
In the first step, PZA (1) was refluxed with phosphorus
pentasulphide in pyridine and converted into thio PZA
(2). The freshly prepared thio PZA was then condensed
with 2-chloroethylacetoacetate in refluxed ethanol and
obtained 5-carboethoxy-4-methyl (pyrazinyl-2-yl) thiazole
(3) following Hantzsch Synthesis. A mixture of hydrazine
hydrate and carboethoxythiazole (3) was dissolved in etha-
nol, and the solution was refluxed for getting the respective
acid hydrazide (4). Condensation of aryl aldehydes and the
acid hydrazide (4) has been carried in PEG-400 at 110°C
and obtained the corresponding Schiff bases/azomethines
(5a–j). Finally, the cyclocondensation of mercaptoacetic
acid and azomethines /Schiff bases has been carried in
glycerol at 100°C and obtained the title new compounds
N-(pyrazinyl substituted thiazoloylamino)-2-aryl-4-thiazo-
lidinones, i.e., 4-methyl-N-(4-oxo-2-phenylthiazolidin-3-yl)-
2-(pyrazin-2-yl)thiazole-5-carboxamides (6a–j, Scheme 1).
All the newly synthesized compounds have been charac-
CONCLUSION
New N-(pyrazinylsubstituted thiazoloylamino)-2-aryl-4-
thiazolidinones (6a–j) have been synthesized by developing
convenient multistep synthetic route starting from readily
available PZA, an existing antitubercular drug. The
antitubercular evaluation has also been carried out of the
new thiazolidinones using their lower concentrations. All
of them have shown moderate activity against M. tuberculosis
H37Ra and M. Bovis BCG. The most active compound
among them is 6a.
EXPERIMENTAL
Methods and Materials. The chemicals used were of
laboratory grade. Melting points of all the synthesized
compounds were determined in open capillary tubes and
are uncorrected. The IR spectra (In KBr pellets) were
recorded on Brukar FTIR spectrometer.1H NMR spectra
were recorded on a Brukar DRX-300 and 300MHz NMR
spectrometer, and 13C NMR spectra were recorded on a
Brukar DRX-75 and 75MHz NMR in CDCl3/DMSO-d6
using tetramethylsilane as an internal standard and
chemical shifts are in δ (ppm). High-resolution mass
spectra (HRMS) were recorded on Agilent 6520 (Q-TOF)
1
terized using their IR, H NMR, 13C NMR, and HRMS
spectral data. The IR spectrum of compound (6a) indicates
that formation of product as it shows a characteristics
absorption peak at 1706 cmꢀ1, which corresponds to the
1
carbonyl group of cyclic amide. The H NMR spectrum
of compound (6a) displays a pair of doublet at δ 3.81 and
3.98ppm, because of the geminal coupling between two
hydrogens of S–CH2–C¼O and a sharp singlet at δ 5.88
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
New Thiazolidinones with Pyrazinyl and Thiazolyl scaffolds
Scheme 1. Synthesis of 4-methyl-N-(4-oxo-2-phenyl-thiazolidin-3-yl)-2-(pyrazin-2-yl)thiazole-5-carboxamides (6a–j).
ESI-HRMS instrument. The purity of each of the
compound was checked by thin-layer chromatography
(TLC) using silica-gel, 60 F254 aluminum sheets as an
adsorbent, and visualization was accomplished by
iodine/ultraviolet light.
Yield 72%, mp 91–92°C; IR (KBr) ν cmꢀ1: 3260, 3180,
2979, 1704, 1523, 1665, and 1254; H NMR (300MHz,
1
CHCl3-d) δ ppm = 1.40 (t, 3H, CH3), 2.82 (s, 3H, CH3),
4.38 (q, 2H, CH2), 8.62 (d, J= 12Hz, 2H), and 9.43
(s, 1H); HRMS m/z: 250.0643 [M+ H]+ for C11H11N3O2S.
Synthesis of 4-methyl-2-(pyrazin-2-yl)thiazole-5-carbo-
hydrazide (4). A mixture of ethyl-4-methyl-2-(pyrazin-2-yl)
thiazole-5-carboxylate (3) (0.01 mol) and excess of hydrazine
hydrate (0.03mol) was refluxed in ethanol. The progress of
the reaction was monitored by TLC using ethyl acetate: hex-
ane (3:7) as solvents. Then after 4h, ethanol was removed un-
der reduced pressure. The obtained residue was then poured in
ice cold water. The solid that was obtained was filtered,
washed with water and crystallized from ethanol.
Synthesis of pyrazine-2-carbothiamide (2), (prepared by
following reported method [26]). A mixture of pyrazine-2-
carboxamide (1) (0.01 mol) and phosphorous pentasulphide
(0.004 mole ) was dissolved in pyridine (8 mL) and then the
solution was refluxed for 4h. The progress of the reaction
was monitored by TLC using ethyl acetate: hexane (6:4) as
solvents. After 4h, reaction mass was poured in ice water
and kept overnight, the solid obtained was filtered and
was washed with water and the obtained crude solid was
crystallized from ethanol-DMF. (yield 59%, mp 191ꢀ193°C).
The melting point is an agreement with the reported.
Synthesis of ethyl-4-methyl-2-(pyrazin-2-yl)thiazole-5-
carboxylate (3). Pyrazine-2-carbothiamide (2) (0.01 mol)
and 2-chloroethylacetoacetate (0.02 mol) were dissolved in
ethanol (10 mL), and the solution was refluxed. The progress
of the reaction were monitored by TLC using ethyl acetate:
hexane (3:7) as solvents. After refluxing for 5h, ethanol was
removed from the reaction mass under reduced pressure.
The residue obtained was added in ice cold water, neutral-
ized by NaHCO3.The solid obtained was filtered, washed
with water and crystallized from ethanol.
Yield 74 %, mp 208–211°C; IR (KBr) ) ν cmꢀ1: 3307,
3060, 1674, 1602 and 1585; 1H NMR (300MHz,
CHCl3-d) δ ppm = 2.80 (s, 3H), 4.16 (br. s., 2H), 7.20
(br. s., 1 H), 8.50–8.77 (m, 2H), and 9.44 (s, 1H), LCMS
m/z: 235.58 [M]+ for C9H9N5OS.
General Procedure for the synthesis of N-arylidene-4-methyl-
2-(pyrazin-2-yl)thiazole-5-carbohydrazide (5a–j). 4-Methyl-2-
(pyrazin-2-yl)thiazole-5-carbohydrazide (4) (0.01mol) and
benzaldehydes (0.01 mol) were dissolved in PEG-400
(8 mL), and the solution was stirred at 110°C. The progress
of the reaction was monitored by TLC using ethyl acetate:
hexane (3:7) as solvents. After 2 h of stirring, the reaction
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

S. T. Dhumal, A. R. Deshmukh, L. D. Khillare, M. Arkile, D. Sarkar, and R. A. Mane
Table 1
Table 2
Antimycobacterial activity of compounds (6a–j).
Physical data of N-benzylidene-4-methyl-2-(pyrazin-2-yl)thiazole-5-
carbohydrazides (5a–j).
Antimycobacterial activity (% Inhibition) at 30 μg/mL
concentration the of compound
Melting
point (°C)
Entry
Compounds
R
Yielda (%)
Compound
code
M. tuberculosis
H37Ra
1
2
3
4
5
6
7
8
9
5a
5b
5c
5d
5e
5f
5 g
5 h
5i
4-H
4-CH3
4-Cl
4-OCH3
4-F
2-F
85
77
82
73
80
72
74
68
74
78
207–209
218–220
252–255
210–211
172–174
182–184
195–198
222–224
232–234
168–171
M. bovis BCG
6a
6b
6c
6d
6e
6f
6g
6h
71.16
39.24
48.94
27.64
17.96
37.14
42.12
14.82
62.67
28.65
46.72
37.83
31.72
33.54
21.48
24.16
2-Cl
3,4-OCH3
4-N(CH3)2
4-OH
10
5j
6i
6j
29.65
28.49
45.21
19.46
aIsolated yields.
Rifampicin
90 (0.03 ug/mL Conc.)
91 (0.04 ug/mL Conc.)
Table 3
Physical data of 4-methyl–N-(4-oxo-2-phenylthiazolidin-3-yl)-2-(pyr-
azine-2- yl)thiazol-5-carboxamides (6a–j).
mass was poured in cold water and the obtained crude prod-
uct wascrystallizedfrom ethanol-DMF. Thephysical data of
the compounds (5a–j) of the series are given in the Table 2.
Synthesis of N-benzylidene-4-methyl-2-(pyrazin-2-yl)-
thiazole-5-carbohydrazide (5a). IR (KBr) ν cmꢀ1: 3391,
Melting
Entry Compounds
R
Yieldsa (%) points (°C)
1
2
3
4
5
6
7
8
9
10
6a
6b
6c
6d
6e
6f
6g
6h
6i
4-H
4-CH3
4-Cl
4-OCH3
4-F
2-F
78
72
82
76
84
62
66
68
72
72
205–207
170–172
106–107
168–170
174–177
205–208
146–148
172–174
156–158
171–172
1
3157, 3048, 1668, 1574 and 1385; H NMR (300MHz,
DMSO-d6) δ ppm=2.93 (s, 3H), 7.45–7.79 (m, 5H), 7.91
(s, 1H), 8.62–8.65 (d, J=12Hz, 2H), and 9.44 (s, 2H);
LCMS m/z: 324.01 [M+H]+ for C16H13N5OS.
2-Cl
Synthesis of N′-(4-chlorobenzylidene)-4-methyl-2-(pyra-
3,4-OCH3
4-N(CH3)2
4-OH
zin-2-yl)thiazole-5-carbohydrazide (5c). IR (KBr) ν cmꢀ1
:
1
3360, 3112, 2958, 1664 and 857; H NMR (300MHz,
DMSO-d6) δ=2.51 (s, 3H, CH3), 7.25 (s, 1H, CH), 7.37–
7.38 (m, 4H, Ar-H), 8.49–9.29 (m, 3H, pyrazine-H), and
11.64 (s, 1H, Amido N-H); HRMS m/z: 358.0521 [M+H]+
for C16H12ClN5OS.
6j
aIsolated yields.
Synthesis of 4-Methyl–N-(4-oxo-2-pheylthiazolidin-3-
yl)-2-(pyrazine-2-yl)thiazol-5-carboxamide (6a). IR (KBr)
ν cmꢀ1: 3376, 2957, 2929, 2836, 1706, 1664, and 1511;
1H NMR (300MHz, DMSO-d6) δ ppm =2.50 (s, 3H, CH3),
3.81–3.98 (dd, 2H, CH2), 5.93 (s, 1H, CH), 7.35–7.53.(m,
5H, Ar-H), 8.69–9.26 (m, 3H, pyrazine-H), and 10.77 (s, 1H,
Amido N-H);13C NMR (DMSO-d6, 75MHz) δ ppm= 16.92,
29.31, 61.68, 124.94, 127.87, 128.56, 129.10, 137.83,
140.65, 144.59, 144.70, 146.59, 157.87, 159.86, 165.79, and
169.01; HRMS m/z: 398.0735 [M +H]+ for C18H15N5O2S2.
Synthesis of N-(4-methoxybenzylidene)-4-methyl-2-(py-
razin-2-yl)thiazole-5-carbohydrazide (5d). IR (KBr) νcmꢀ1
:
3312, 3112, 3048, 1662, 1648 and 1320; 1H NMR (300MHz,
DMSO-d6) δ=2.13 (s, 3H, CH3), 3.10 (s, 3H, OCH3), 6.79
(s, 1H, CH), 6.20–6.22 (dd, J=8 Hz, 2H, Ar-H), 6.93–6.95
(dd, J= 8Hz, 2H, Ar-H), 7.59 (s, 1H, NH), 7.90–8.69
(m, 3H, pyrazine-H); HRMS m/z: 354.1016 [M +H]+ for
C17H15N5O2S.
General procedure for the synthesis of 4-methyl–N-(4-oxo-
2-arylthiazolidin-3-yl)-2-(pyrazine-2-yl)thiazol-5-carboxamide
(6a–j). A mixture of N-arylidene-4-methyl-2-(pyrazin-2-
yl)thiazole-5-carbohydrazide (5a–j) (0.01mol) and merca-
ptoacetic acid (0.04mol) in glycerol (8mL) was stirred at
100°C. The progress of the reaction was monitored by
TLC using ethyl acetate: hexane (4:7) as solvents. After 2h
of stirring, the reaction mass poured in cold water, washed
with NaHCO3, and the obtained crude product was crystal-
lized from ethanol. The physical data of the compounds
(6a–j) of the series are given in the Table 3.
Synthesis of 4-Methyl-N-(4-oxo-2-(p-tolyl)thiazolidin-3-
yl)-2-(pyrazin-2-yl)thiazole-5-carboxamide (6b). IR (KBr)
ν cmꢀ1: 3389, 2968, 2923, 1707, 1665, and 1648; 1H
NMR (300 MHz, DMSO-d6) δ ppm=2.50 (s, 3H, CH3),
2.72 (s, 3H, CH3), 3.78–3.95 (dd, 2H, CH2), 5.88 (s, 1H,
CH), 7.19–7.21 (dd, J=8Hz, 2H, Ar-H), 7.37–7.40 (dd,
J=8Hz, 2H, Ar-H), 8.70–9.27 (m, 3H, pyrazine-H), and
10.74 (s, 1H, Amido N-H); 13C NMR (DMSO-d6,
75MHz) δ ppm=16.93, 20.75, 29.31, 61.55, 124.94,
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New Thiazolidinones with Pyrazinyl and Thiazolyl scaffolds
127.84, 129.08, 134.73, 138.56, 140.65, 144.71, 144.99,
146.60, 157.91, 159.84, 165.76, and 168.98; HRMS m/z:
412.0879 [M+H]+ for C19H17O2N5S2.
ppm=17.60, 29.82, 59.76, 123.85, 127.65, 128.60, 130.38,
130.49, 133.83, 134.81, 141.75, 144.05, 145.78, 146.00,
160.45, 166.50, and 170.41; HRMS m/z: 432.0332 [M+ H]+
for C18H14ClN5O2S2.
Synthesis of N-(2-(4-chlorophenyl)-4-oxothiazolidin-3-
Synthesis of N-(2-(3,4-dimethoxyphenyl)-4-oxothiazolidin-3-
yl)-4-methyl-2-(pyrazin-2-yl)thiazole-5-carboxamide (6 h). IR
yl)-4-methyl-2-(pyrazin-2-yl)thiazole-5-carboxamide (6c).IR
1
(KBr) ν cmꢀ1: 3360, 2961, 1700, 1664, 1525 and 753; H
1
(KBr) νcmꢀ1: 3362, 2964, 1715, 1668, 1654, and 1254; H
NMR (300 MHz, DMSO-d6) δ ppm=2.72 (s, 3H, CH3),
3.80–4.00 (dd, 2H, CH2), 5.93 (1H, s, CH), 7.45–7.48.
(dd, J= 8Hz, 2H, Ar-H), 7.74–7.95 (dd, J= 8Hz, 2H, Ar-H),
8.71–9.28 (m, 3H, pyrazinyl), and 10.76 (s, 1H, Amido N-
H); 13C NMR (DMSO-d6, 75MHz) δ ppm=16.98, 20.75,
60.92, 124.77, 128.56, 129.84, 133.61, 137.16, 140.67,
144.73, 144.99, 146.63, 158.03, 159.88, 165.83, and 168.84;
HRMS m/z: 432.0332 [M +H]+ for C18H14ClN5O2S2.
NMR (300 MHz, DMSO-d6) δ ppm= 2.65 (s, 3H, CH3),
3.67–3.77 (dd, 2H, CH2), 3.90 (s, 3H, OCH3), 4.10 (s, 3H,
OCH3), 5.97 (s, 1H, CH), 6.80–7.02.( m, 3H, Ar-H), 7.91–
8.63 (m, 3H, pyrazine-H), and 9.36 (s, 1H, Amido N-H); HRMS
m/z: 458.0952 [M+ H]+ for C20H19N5O4S2.
Antitubercular screening. All the chemicals such as XTT
(Sodium Salt Powder), sulfanilic acid, sodium nitrate, HCl,
NEED rifampicin, and DMSO were purchased from
Sigma-Aldrich, USA. Dubos medium was purchased
from DIFCO, USA.
Synthesis of N-(2-(4-methoxyphenyl)-4-oxothiazolidin-3-yl)-
4-methyl-2-(pyrazin-2-yl)thiazole-5-carboxamide (6d).
IR
(KBr) ν cmꢀ1: 3345, 2963, 1706, 1657, 1652, and 1527; H
NMR (300 MHz, DMSO-d6) δ ppm= 2.64 (s, 3H, CH3), 3.82
(s, 3H, OCH3), 3.80–4.00 (dd, 2H, CH2), 5.97 (s, 1H, CH),
6.88–6.92 (dd, J= 8Hz, 2H, Ar-H) 7.35–7.39 (dd, J= 8 Hz, 2H,
Ar-H), 8.52–8.64 (m, 3H, pyrazine-H), and 9.36 (s, 1H, Amido
N-H); 13C NMR (CDCl3, 125 MHz) δ ppm= 17.50, 30.14,
60.11, 62.42, 116.11, 123.92, 128.01, 129.68, 130.07, 132.50,
134.06, 147.74, 151.61, 157.91, 159.82, 160.29, 162.42,
164.40, 165.05, and 170.13; HRMS m/z: 428.0843 [M +H]+
for C19H17N5O3S2.
1
Microbial strains such as Mycobacterium tuberculosis
H37Ra (ATCC 25177) and M. bovis BCG (ATCC 35734)
were obtained from AstraZeneca, India. The stock culture
was maintained at ꢀ80°C and subcultured once in a liq-
uid medium before inoculation into an experimental
culture. Cultures were grown in Dubos media (enrich-
ment media).
Antimycobacterium testing. For antimycobacterial assay,
M. pheli medium (minimal essential medium) was used
for both M.tuberculosis H37Ra and M.bovis BCG. It
contains 0.5 g KH2PO4, 0.25g trisodium citrate, 60mg
MgSO4, 0.5 g aspargine, and 2mL glycerol in distilled
water (100mL) followed by pH adjustment to 6.6. Stock
solutions of the compounds were prepared in DMSO and
were used for further antimycobacterial testing.
Synthesis of N-(2-(4-fluorophenyl)-4-oxothiazolidin-3-
yl)-4-methyl-2-(pyrazin-2-yl)thiazole-5-carboxamide (6e). 1H
NMR (300MHz, CDCl3) δ=2.50 (s, 3H, CH3), 3.59–3.85
(dd, 2H, CH2), 5.89 (s, 1H, CH), 6.93–7.02 (dd, J=8Hz,
2H, Ar-H), 7.30–7.34 (dd, J= 8Hz, 2H, Ar-H), 8.12–8.21
(m, 3H, pyrazine-H), and 9.22 (s, 1H, Amido N-H), 13C
NMR (125MHz, CHCl3-d) δ ppm=17.45, 30.10, 63.01,
122.18, 123.89, 128.02, 128.68, 129.11, 134.03, 136.81,
147.77, 151.61, 157.91, 159.52, 160.26, 165.08, and
170.12; HRMS m/z: 416.0643 [M+H]+ for C18H14FN5O2S2.
Synthesis of N-(2-(2-fluorophenyl)-4-oxothiazolidin-3-yl)-
M. tuberculosis H37Ra (ATCC 25177) was grown to
logarithmic phase (O.D.595 ~1.0) in a defined medium
(M. pheli_medium). The stock culture was maintained
at ꢀ70°C and subcultured once in M. pheli_medium
before inoculation into experimental culture. Drugs were
solubilized in dimethyl sulfoxide (DMSO) and stored in
aliquots at ꢀ20°C. XTT sodium salt powder (Sigma)
was prepared as a 1.25 mM stock solution in sterile 1×
PBS and used immediately. Menadione (Sigma) was al-
ways freshly prepared as a 6 mM solution in DMSO be-
fore use. Compounds were screened for their inhibitory
effect on MTB by following XTT Reduction Menadione
Assay protocol published earlier [27]. Briefly, in all wells
of assay plate 200 μM XTT was added as a final concen-
tration and incubated at 37°C for 20min. Then 60 μM
Menadione was added as a final concentration and incu-
bated at 37°C for 40min. The optical density was read
on a micro plate reader (Spectramax plus384 plate
reader, Molecular Devices Inc) at 470nm filter against
a blank prepared from cell-free wells. Absorbance given
by cells treated with the vehicle alone was taken as
4-methyl-2-(pyrazin-2-yl)thiazole-5-carboxamide (6f).
IR
(KBr) νcmꢀ1: 3354, 2967, 1706, 1655, and 1641; H NMR
(300 MHz, DMSO-d6) δ ppm=2.61 (s, 3H, CH3), 3.70–3.84
(dd, 2H, CH2), 6.28 (s, 1H, CH), 7.04–7.52 ( m, 4H, Ar-H),
8.44–8.62 (m, 3H, pyrazine-H), and 9.33 (s, 1H, Amido N-H);
13C NMR (CHCl3-d, 125 MHz) δ ppm=17.60, 29.82, 59.76,
123.85, 127.65, 128.60, 130.38, 130.49, 133.83, 134.81,
141.75, 144.05, 145.78, 146.00, 160.45, 166.50, and 170.41;
HRMS m/z: 416.0643 [M + H]+ for C18H14FN5O2S2.
1
SynthesisofN-(2-(2-chlorophenyl)-4-oxothiazolidin-3-yl)-
4-methyl-2-(pyrazin-2-yl)thiazole-5-carboxamide (6g).IR
(KBr) ν cmꢀ1: 3360, 2952, 2923, 1704, 1667, 1515, and
748cmꢀ1; 1H NMR (300MHz, DMSO-d6) δ ppm=2.72 (s,
3H, CH3), 3.78–3.92 (dd, 2H, CH2), 6.50 (s, 1H, CH), 7.36–
7.56.( m, 4H, Ar-H), 7.89–8.66 (m, 3H, pyrazinyl), and 9.40
(s, 1H, Amido N-H); 13C NMR (CHCl3-d, 125MHz) δ
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

S. T. Dhumal, A. R. Deshmukh, L. D. Khillare, M. Arkile, D. Sarkar, and R. A. Mane
[9] Dolezal, M.; Cmedlova, P.; Palek, L.; Vinsova, J.; Kunes, J.;
100% cell growth. All experiments were performed in
triplicates along with rifampicin as standard (MIC 0.03
(μg/mL), and the quantitative value was expressed as
the average standard deviation.
For M. bovis BCG nitrate reduction (NR) assay was used
[28]. An additional 0.1% inoculated culture of 1 OD was
mixed into drug solution, and NR was carried out after
12days. Briefly 1:1:1 ration of culture:sulfanulic acid in
HCl:NEED in D/W was added. Optical density was mea-
sured at 540nm filter against a blank prepared from cell-
free wells.
Buchta, V.; Jampilek, J.; Kralova, K. Eur J Med Chem 2008, 43, 1105.
[10] Zhang, Y.; Wang, X-L.; Liu, W.; Yang, Y-S.; Tang, J-F.; Zhu,
H-L. Bioorg Med Chem 2012, 20, 6356.
[11] Zitko, J.; Dolezal, M.; Svobodova, M.; Vejsova, M.; Kunes, J.;
Kucera, R.; Jilek, P. Bioorg Med Chem 2011, 19, 1471.
[12] (a) Sharma, S.; Sharma, P. K.; Kumar, N.; Dudhe, R.
Biomed Pharmacother 2011, 65, 244; (b) Pathak, R.B; Chovatia, P.T;
Parekh, H. H. Bioorg Med Chem Lett 2012, 22, 5129–5133.
[13] Murphy, G. J.; Holder, J. C. Trends Pharmacol Sci 2000, 2, 469.
[14] (a) Havrylyuk, D.; Zimenkovsky, B.; Vasylenko, O.; Gzella, A.;
Lesyk, R. J Med Chem 2012, 55, 8630; (b) Havrylyuk D.; Zimenkovsky B.;
Vasylenko O.; Day C.W.; Smee D.F.; Grellier P.; Lesyk R. Eur J Med Chem
2013, 66, 228–237.
[15] (a) Bhosle, M. R.; Mali, J. R.; Pal, S.; Srivastava, A. K.; Mane, R. A.
Bioorg Med Chem Lett 2014, 24, 2651.: (b) Raza, S.; Srivastava, S. P.;
Srivastava, D. S.; Srivastava, A.K.; Haq, W.; Katti S.B.; Eur J Med Chem
2013, 63, 611–620.
[16] Jain, A. K.; Vaidya, A.; Ravichandran, V.; Kashaw, S. K.;
Agrawal, R. K. Bioorg Med Chem 2012, 03, 69.
Acknowledgments. Authors are thankful to Professor D.B. Ingle
for his invaluable discussions and guidance. The authors are very
much thankful to CDRI, Lucknow, for providing spectral
facilities. Authors are also thankful to National Chemical
Laboratory, Pune, for screening antitubercular/antibacterial
activity. One of the authors S.T.D. is grateful to U.G.C., New
Delhi, India, for financial assistance in the form of NET Senior
Research Fellowship.
[17] Archana, Srivastava, V. K.; Kumar, A. Eur J Med Chem 2002,
37, 873.
[18] (a) Diurno, M. V.; Mazzoni, O.; Piscopo, E.; Calignano, A.;
Giordano, F.; Bolognesel, A. J Med Chem 1992, 35, 2910.: (b) Arya, K.;
Rawat, D.S.; Dandia, A.; Sasai, H. J Flu Chem 2012,137, 117–122.
[19] Rawal, R. K.; Tripathi, R.; Katti, S. B.; Pannecouque, C.;
Clercq, E. D. Bioorg Med Chem 2007, 15, 1725–1731.
[20] Makam, P.; Kankanala, R.; Prakash, A.; Kannan, T. Eur J Med
Chem 2013, 69, 564.
REFERENCES AND NOTES
[1] http://www.who.int/tb/challenges/xdr/xdr_map_june08.pdf.
[2] Zitko, J.; Servusova B., Janoutova, A.; Paterova, P.;
Mandikova, J.; Garaj, V.; Vejsova, M.; Marek, J.; Dolezal, M. Bioorg
Med Chem 2015, 23, 174.
[3] Boogaard, J. D.; Kibiki, G. S.; Kisanga, E. R.; Boeree, M. J.;
Aarnoutse, R. E. Antimicrob Agents Chemother 2009, 53, 849.
[4] (a) Chauhan, K.; Sharma, M.; Trivedi, P.; Chaturvedi, V.;
Chauhan, P. M. S. Bioorg Med Chem Lett 2014, 24, 4166; (b) Altamari,
J. M.; Hockey, S. C.; Boshoff, H. I.; Sajid, A.; Henderson, L. C. Chem
Med Chem 2015, 10, 787.
[21] Khillare, L. D.; Bhosle, M.; Deshmukh, A.; Mane, R. A. Med
Chem Res 2015, 24, 1380.
[22] Romagnoli, R.; Baraldi, P. G.; Salvador, M. K.; Camacho, M. E.;
Preti, D.; Tabrizi, M. E.; Basatto, M.; Brancale, A.; Hamel, E.; Bortolozzi, R.;
Basso, G.; Viola, g. Bioorg Med Chem 2012, 20, 7083.
[23] Kalhor, M.; Salehifar, M.; Nikokar, I. Med Chem Res 2014,
23, 2947.
[24] Sarojini, B. K.; Krishna, B. G.; Darshanraj, C. G.; Bharath, B. R.;
Manjunatha, H. Eur J Med Chem 2010, 45, 3490.
[25] Stankova, I.; Chuchkov, K.; Shishkov, S.; Kostova, K.; Mukova, L.;
Galabov, A. S. Amino Acids 2009, 37, 383.
[5] Zhang, Y. Front Biosci 2004, 9, 1136.
[6] Judge, V.; Narasimhan, B.; Ahuja, M. Hygeia J D Med 2012, 4, 1.
[7] Sayahi, H.; Zimhony, O.; Jacobs, W. R.; Shekhtman, A.;
Welch, J. T. Bioorg Med Chem Lett 2011, 21, 4804.
[8] Sriram, D.; Yogeeswari, P.; Reddy, S. P. Bioorg Med Chem
Lett 2006, 16, 2113.
[26] Radwan S.M.; Bakhite, E.A. Monatshefte fur Chemie 1999,
130, 1117.
[27] Singh, U.; Akhtar, S.; Mishra, A.; Sarkar D. J Micro Methods
2011, 84, 202.
[28] Khan, A.; Sarkar, D. J Micro Methods 2008, 73, 6.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet