Synthesis, antitubercular and antimicrobial potential of some new thiazole substituted thiosemicarbazide derivatives

Article abstract of DOI:10.1007/s00044-017-1955-1

The increase in antibiotic resistance due to multiple factors has warranted the need for search of new compounds which are active against multidrug resistant pathogens. In this context a small focused library of thiosemicarbazide derivatives of 2-arylthiazole-4-carbaldehyde, 4-methyl-2-arylthiazole-5-carbaldehyde and 1-(4-methyl-2-arylthiazol-5-yl) ethanone, (5a–l) has been synthesized. The title compounds were screened for inhibitory activity against Mycobacterium tuberculosis H37Ra (ATCC 25177) and Mycobacterium bovis Bacille Calmette Guerin (ATCC 35743) strains. The synthesized compounds, 5a–l were further assayed for their cytotoxic activity against the two human cancer cell lines, HeLa and human colon carcinoma 116 cell lines and showed no significant cytotoxic activity against these two cell lines at the maximum concentration evaluated. Further, the synthesized compounds were found to have potential antibacterial activity against Gram-negative bacteria, Escherichia coli, Pseudomonas flurescence and Gram-positive bacteria, Staphylococcus aureus, Bacillus subtilis. Most of the synthesized compounds showed moderate activity against fungal strain Candida albicans. This study provides valuable directions to our ongoing endeavor of rationally designing more potent antimycobacterial agent.

Full text of DOI:10.1007/s00044-017-1955-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1955-1
ORIGINAL RESEARCH
Synthesis, antitubercular and antimicrobial potential of some new
thiazole substituted thiosemicarbazide derivatives
Yogita K. Abhale1 Abhijit Shinde2 Keshav K. Deshmukh1 Laxman Nawale3
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Dhiman Sarkar3 Pravin C. Mhaske2
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Received: 5 February 2017 / Accepted: 7 June 2017
© Springer Science+Business Media, LLC 2017
Abstract The increase in antibiotic resistance due to mul-
tiple factors has warranted the need for search of new
compounds which are active against multidrug resistant
pathogens. In this context a small focused library of thio-
semicarbazide derivatives of 2-arylthiazole-4-carbaldehyde,
4-methyl-2-arylthiazole-5-carbaldehyde and 1-(4-methyl-2-
arylthiazol-5-yl) ethanone, (5a–l) has been synthesized. The
title compounds were screened for inhibitory activity
against Mycobacterium tuberculosis H37Ra (ATCC 25177)
and Mycobacterium bovis Bacille Calmette Guerin (ATCC
35743) strains. The synthesized compounds, 5a–l were
further assayed for their cytotoxic activity against the two
human cancer cell lines, HeLa and human colon carcinoma
116 cell lines and showed no significant cytotoxic activity
against these two cell lines at the maximum concentration
evaluated. Further, the synthesized compounds were found
to have potential antibacterial activity against Gram-
negative bacteria, Escherichia coli, Pseudomonas flures-
cence and Gram-positive bacteria, Staphylococcus aureus,
Bacillus subtilis. Most of the synthesized compounds
showed moderate activity against fungal strain Candida
albicans. This study provides valuable directions to our
ongoing endeavor of rationally designing more potent
antimycobacterial agent.
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Keywords Thiosemicarbazide Thiazole Antitubercular
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activity Antimicrobial activity Cytotoxicity
Introduction
Tuberculosis (TB), an infection caused by Mycobacterium
tuberculosis (MTB) is a huge menace to the human race. To
combat with microbial pathogens, different kinds of anti-
microbial agents have been developed substantially. How-
ever, the emergence of multidrug-resistant bacterial strains,
the restricted spectrum of action, toxicity, extensive side
effects and drug interactions, create a serious challenge to
the scientific community and the need for an effective
therapy has led to a search for new therapeutic options
(Kunin and Ellis 2000; Cannon et al. 2009; Achkar and
Fries 2010; Kathiravan et al. 2012).
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1955-1) contains supplementary material,
which is available to authorized users.
* Pravin C. Mhaske
The simplest hydrazine derivative of thiocarbamic acid
called thiosemicarbazide has received considerable attention
because of their variable bonding modes, structural diversity,
promising biological activities, and ion sensing ability
(Casas et al. 2000; Kumari et al. 2012). During the past few
decades, interest has been rapidly growing in gaining insight
into the properties and transformations of thiosemicarbazides
and their derivatives due to their appreciable pharmacolo-
gical activities viz. antitubercular activity (Patel et al. 2014;
Tan et al. 2012; Kucukguzel et al. 2006; Sriram et al. 2006;
mhaskepc18@gmail.com
1
Post-Graduate Department of Chemistry and Research Centre,
S. N. Arts, D. J. M. Commerce and B. N. S. Science College,
Savitribai Phule Pune University, College Road, Sangamner,
District Ahmednagar 422 605, India
2
Post Graduate Department of Chemistry, S. P. Mandali’s Sir
Parashurambhau College, Savitribai Phule Pune University, Tilak
Road, Pune 411 030, India
3
CombiChemBio Resource Centre, CSIR-National Chemical
Laboratory, Pune 411 008, India

Med Chem Res
Fig. 1 Antibacterial and
antitubercular agents containing
thiosemicarbazone moiety
Moglea et al. 2016), anti-inflammatory activity (Salgın-
Goksen et al. 2007), antibacterial activity (Alagarsamy et al.
2012; Rane et al. 2014; Sheikhy et al. 2012; Umadevi et al.
2012; Plech et al. 2011; Chornous et al. 2014; Pitucha et al.
2016), anticancer activity (Arora et al. 2014; Perkoviisć
et al. 2012; Malki et al. 2015; Zhang et al. 2011), cytotoxic
and antiproliferative activity (Mavrova et al. 2014), anti-
protozoal activity (Leite et al. 2006; Haraguchi et al. 2011),
etc. Use of these compounds in organic synthesis has
become a classical strategy for the synthesis of several
heterocycles. Thiosemicarbazide is a useful structural moi-
ety and optimization of this structure can result in ground
breaking discovery of new class of therapeutic agents.
Similarly, Thiazole and its derivatives are also important
pharmacophore in medicinal chemistry that could provide a
rich spectrum of biological activities (Abhale et al. 2015,
2016, 2017; Johnson et al. 2012; Gaikwad et al. 2012;
Tomasic et al. 2015; Urbanek et al. 2013; Inamdar et al.
2013; Lee et al. 2012; Malik et al. 2013; Navale et al. 2013;
Mishra et al. 2015; Siddiqui and Ahsan 2010; Yusufzai
et al. 2017) (Fig. 1).
internal tetramethylsilane standard and are given in δ units.
Infrared spectra were recorded on Shimadzu Fourier transform
infrared spectroscopy (KBr)-408 in KBr. The LC–MS spectra
were recorded on a Shimadzu 2010 LC–MS. Column chro-
matography was performed on silica gel (100–200 mesh)
supplied by Acme Chemical Co. The chemicals and solvents
used were laboratory grade and were purified as per literature
methods.
General procedure for synthesis of ethyl 2-phenylthiazole-
4-carboxylate (2a)
The mixture of ethyl 3-bromo-2-oxopropanoate (40 mmol)
and benzothiomide, 1a (40 mmol) in ethanol (30 mL) was
refluxed for 3 h. After completion of reaction (TLC) half of
the solvent was distilled off and reaction mass was cooled to
room temperature. The product was filtered, washed with
water and recrystallized from ethanol.
General procedure for synthesis of 2-(2-phenylthiazol-4-yl)
methanol (3a)
Considering the importance of thiosemicarbazide and
thiazole derivatives, it was thought worthwhile to synthesize
thiazole substituted thiosemicarbazide derivatives. In the
present work the synthesis and antimycobacterial screening
of thiosemicarbazide derivatives of 2-arylthiazole-4-carbal-
dehyde, 4-methyl-2-arylthiazole-5-carbaldehyde, and 1-(4-
methyl-2-arylthiazol-5-yl) ethanone is reported.
To a cold solution of lithium aluminum hydride (20 mmol)
in diethyl ether (20 mL) 2-phenylthiazole-4-carboxylate, 2a
(10 mmol) in diethyl ether (20 mL) was added dropwise
over a period of 30 min and the reaction mixture was further
stirred for 1 h at 0 °C. After completion of the reaction
(TLC), the reaction mixture was quenched by saturated
solution of sodium sulphate. The reaction mixture was fil-
tered and the aqueous layer was extracted with diethyl ether
(2 × 30 mL), the combined organic layer was washed with
water, brine, and dried over sodium sulphate. 2-(2-phe-
nylthiazol-4-yl) methanol was obtained by removing the
solvent by distillation.
Materials and methods
Chemistry
All the reactions were monitored by thin layer chromatography
(TLC). TLC was performed on Merck 60 F-254 silica gel
plates. Melting points were determined in capillary tubes in
silicon oil bath using a Veego melting point apparatus and are
General procedure for synthesis of 2-phenylthiazole-4-
carbaldehyde (4a)
1
uncorrected. H (300 MHz) NMR and 13C (75 MHz) NMR
To a solution of 2 (2-phenylthiazol-4-yl) methanol, 3a
(14 mmol) in DMSO (30 mL), iodoxy benzoic acid (IBX,
18 mmol) was added and reaction mixture was stirred at
0–20 °C. The progress of reaction was monitored on TLC.
spectra were recorded on Varian mercury XL-300 and Bruker
at either 400 MHz (1H NMR) and 100 MHz (13C NMR),
spectrometer instruments. Chemical shifts are reported from

Med Chem Res
After completion of reaction, reaction mixture was filtered.
Water (90 mL) was added in the filtrate and the filtrate was
extracted with diethyl ether (3 × 30 mL). The organic layer
was washed with water, brine and dried over sodium sul-
phate. Product was obtained by distillation of solvent.
1-((2-p-tolylthiazol-4-yl)methylene)thiosemicarbazide (5d)
Yield 65 %, m.p. 230 °C (dec.); IR (KBr): 3470, 3430,
3260, 3165, 3012, 2970, 1688, 1646, 1600, 1540, 1500,
1
1368, 1320,1284, 1122, 1001, 839, 799 cm−1; H NMR
(500 MHz, DMSO-d6) δ: 2.42, (s, 3H, Ar–CH3) 7.35 (d, J
= 6.8 Hz, 2H, H-3′, H-5′), 7.84 (d, J = 6.8 Hz, 2H, H-2′, H-
6′), 8.00 (s, 1H, Thiazole H-5), 8.22 (s, 1H, NH), 8.32 (s,
2H, NH2), 11.58 (s, 1H, N = CH); 13C NMR (125 MHz,
DMSO-d6) δ: 21.2 (Ar–CH3), 119.0 (Thiazole C-5), 127.0
(C-2′, -6′), 130.1 (C-3′, -5′), 130.2 (C-4′), 137.1 (C = N),
140.4 (C-1′), 152.6 (Thiazole C-4), 167.8 (Thiazole C-2),
179.1 (C = S); LC–MS m/z: 277.1 (M + H)+.
General procedure for synthesis of -1-((2-phenylthiazol-4-
yl) methylene) thiosemicarbazide (5a)
The mixture of 2-phenylthiazole-4-carbaldehyde, 4a (2 mmol)
and thiosemicarbazide (2.5 mmol) in ethanol (10 mL) was
refluxed for 2 h. After completion of reaction (TLC) solvent
was cooled to room temperature. The product was filtered,
washed with water and recrystallized from ethanol.
General procedure for synthesis of Ethyl 4-methyl-2-
phenylthizole-5-carboxylate (2e)
1-((2-phenylthiazol-4-yl)methylene)thiosemicarbazide (5a)
Yield 70 %, m.p. 230–232 °C; IR (KBr): 3470, 3430, 3259,
3150, 3018, 2978, 1687, 1645, 1597, 1546, 1491, 1369,
The mixture of 2-bromo-3-oxoethylbutanoate (36.4 mmol)
and benzothioamide (36.4 mmol) in 25 mL ethanol was
refluxed for 8 h. After completion of reaction (TLC), half of
the solvent was distilled-off and reaction mass cooled to
room temperature. The product was filtered, washed with
water and recrystallized from ethanol. Yield: 4.0 g, (80 %),
m.p. 90–92 °C. The compounds 2f–h were synthesized by
same method.
1
1318, 1284, 1111, 841 cm−1; H NMR (DMSO-d6, 500
MHz) δ: 7.46–7.49 (m, 3H, H-3′, H-4′, H-5′), 7.82–7.84 (m,
2H, H-2′,H-6′), 7.87 (s, 1H, Thiazole H-5), 8.10 (s, 1H,
NH), 8.25 (s, 1H, NH), 8.26 (s, 1H, NH), 11.45 (s, 1H,
N = CH); 13C NMR (DMSO-d6,125 MHz) δ: 118.5 (Thia-
zole C-5), 126.5 (C-4′), 129.3 (C-3′,-5′), 130.5 (C-2′,-6′),
132.5 (C-1′), 136.4 (C = N), 153.4 (Thiazole C-4), 167.7
(Thiazole C-2), 178.5 (C = S); LC–MS m/z: 263.0 (M +
H)+.
General procedure for synthesis of (4-Methyl-2-
phenylthiazol-5-yl) methanol (3e)
1-((2-(4-chlorophenyl)thiazol-4-yl)methylene)thiosemicar-
bazide (5b) Yield 68 %, m.p. 240 °C (dec.); IR (KBr):
3468, 3430, 3261, 3168, 3010, 2976, 1685, 1645, 1597,
To a cold solution of lithium aluminum hydride (20 mmol)
in dry diethyl ether (25 mL), ethyl 4-methyl-2-phenylthia-
zole-5-carboxylate, 2e (10 mmol) in diethyl ether (25 mL)
was added dropwise over a period of 30 min and the reac-
tion mixture was further stirred for 1 h at 0 °C. After com-
pletion of the reaction (TLC), the reaction mixture was
quenched by saturated solution of sodium sulphate. The
reaction mixture was filtered and the aqueous layer was
extracted with diethyl ether (2 × 30 mL), the combined
organic layer was washed with water, brine and dried over
sodium sulphate furnished (4-Methyl-2-phenylthiazol-5-yl)
methanol.
1542, 1502, 1368, 1320, 1284, 1122, 1001, 839, 801 cm−1
;
1H NMR (500 MHz, DMSO-d6) δ: 7.50 (d, J = 8 Hz, 2H,
H-3′, H-5′), 7.92 (d, J = 8 Hz, 2H, H-2′, H-6′), 7.95 (s, 1H,
Thiazole H-5), 8.30 (s, 1H, NH), 8.35 (s, 2H, NH2), 11.46
(s, 1H, N = CH); 13C NMR (125 MHz, DMSO-d6) δ: 119.0
(Thiazole C-5), 128.8 (C-2′, -6′), 129.9 (C-3′, -5′), 131.9 (C-
4′), 135.7 (C-1′), 136.8 (C = N), 152.3 (Thiazole C-4),
165.5 (Thiazole C-2), 178.0 (C = S); LC–MS m/z: 297.0
(M + H)+, m/z: 299.0 (M + H + 2)+.
1-((2-(4-fluorophenyl)thiazol-4-yl)methylene)thiosemicar-
bazide (5c) Yield 75 %, m.p. 230 °C (dec.); IR (KBr):
3472, 3433, 3264, 3165, 3010, 2978, 1687, 1645, 1597,
General procedure for synthesis of 4-Methyl-2-
phenylthiazole-5-carbaldehyde (4e)
1542, 1502, 1368, 1320, 1284, 1122, 1001, 840, 800 cm−1
;
1H NMR (500 MHz, DMSO-d6,) δ: 7.35 (t, J = 6.8 Hz, 2H,
H-3′, H-5′), 8.00 (m, 2H, H-2′, H-6′), 8.2 (s, 1H, Thiazole
H-5), 8.33 (s, 1H, NH), 8.34 (s, 2H, NH2), 11.58 (s, 1H, N
= CH); 13C NMR (125 MHz, DMSO-d6,) δ: 116.8 (C-3′,
-5′), 119.5 (Thiazole C-5), 129.4 (C-2′, -6′), 129.9 (C-1′),
136.0 (C = N), 153.6 (Thiazole C-4), 163.5 (C-4′),
166.8 (Thiazole C-2), 178.8 (C = S); LC–MS m/z: 281.0
(M + H)+.
To a solution of (4-methyl-2-phenylthiazole-5-yl) methanol,
3e (10 mmol) in DMSO (30 mL), IBX (11 mmol) was added
and the reaction mixture was stirred at 0 to 20 °C. The
progress of reaction was monitored on TLC. After com-
pletion of the reaction (20 min), reaction mixture was fil-
tered and washed with DMSO (5 mL). Water (90 mL) was
added in the filtrate and the filtrate was extracted with
diethyl ether (3 × 30 mL). The organic layer was washed

Med Chem Res
with water, brine and dried over sodium sulphate. The
solvent was distilled to afford white solid 4e in 95% yield.
1-((4-methyl-2-p-tolylthiazol-5-yl)methylene)thiosemicar-
bazide (5h) Yield 72 %, m.p. 240 °C (dec.); IR (KBr):
3466, 3430, 3261, 3163, 3009, 2974, 1687, 1645, 1597,
General procedure for synthesis of 1-((4-methyl-2-
1545, 1500, 1368, 1320,1284, 1124, 1001, 841, 801 cm−1
;
phenylthiazol-5-yl) methylene) thio-semicarbazide (5e)
1H NMR (500 MHz, DMSO-d6) δ: 2.45, (s, 3H, Thiazole-
CH3), 2.56 (s, 3H, Ar-CH3), 7.35 (d, J = 6.8 Hz, 2H, H-3′,
H-5′), 7.60 (s, 1H, NH), 7.84 (d, J = 6.8 Hz, 2H, H-2′, H-
6′), 8.25 (s, 1H, NH), 8.35 (s, 1H, NH), 11.48 (s, 1H, N =
CH); 13C NMR (125 MHz, DMSO-d6) δ: 16.0 (Thiazole-
CH3), 21.5 (Ar–CH3), 126.8 (C-2′, -6′), 127.5 (C = N),
130.5 (C-3′, -5′), 130.6 (C-4′), 135.5 (Thiazole C-5), 141.2
(C-1′), 155.1 (Thiazole C-4), 167.1 (Thiazole C-2), 178.5
(C = S); LC–MS, m/z: 291.07 (M + H)+.
The mixture of 4-methyl-2-phenylthiazole-5-carbaldehyde,
4e (2 mmol) and thiosemicarbazide (2.5 mmol) in ethanol
(10 mL) was refluxed for 2 h. After completion of reaction
(TLC) solvent was cooled to room temperature. The product
was filtered, washed with water and recrystallized from
ethanol.
1-((4-methyl-2-phenylthiazol-5-yl)methylene)thiosemicar-
bazide (5e) Yield 66 %, m.p. 230 °C. IR (KBr): 3474,
3432, 3261, 3158, 3018, 2978, 1688, 1647, 1597, 1546,
General procedure for synthesis of 1-(4-methyl-2-
phenylthiazol-5-yl) ethanone (4i)
1491, 1369, 1318, 1284, 1111, 841 cm−1
;
1H NMR
(DMSO-d6, 500 MHz) δ: 2.46 (s, 3H, Thiazole-CH3),
7.50–7.52 (m, 3H, H-3′, H-4′, H-5′), 7.60 (s, 1H, NH),
7.90–7.92 (m, 2H, H-2′, H-6′), 8.28 (s, 1H, NH), 8.35 (s,
2H, NH2), 11.45 (s, 1H, N = CH); 13C NMR (DMSO-
d6,125 MHz) δ: 15.5 (Thiazole-CH3), 128.0 (C-3′, -5′),
129.5 (C = N), 130.9 (C-4′), 132.5 (C-2′, -6′), 132.7 (C-1′),
135.4 (Thiazole C-5), 154.9 (Thiazole C-4), 166.5 (Thiazole
C-2), 177.5 (C = S); LC–MS m/z: 277.1 (M + H)+.
The mixture of thiobenzamide (6.62 mmol) and 3-bromo-
pentane-2,4-dione, (6.62 mmol) was refluxed in ethanol.
After completion of reaction (TLC), solvent was removed
under reduced pressure and the residue was dissolved in
ethyl acetate. Organic layer was washed with sodium
bicarbonate and then water. Organic layer was dried over
sodium sulphate and distilled under vacuum. The product
obtained was purified by column chromatography using
hexane:ethyl acetate (9:1) as eluent.
1-((2-(4-chlorophenyl)-4-methylthiazol-5-yl)methylene)
thiosemicarbazide (5f) Yield 65 %, m.p. 240 °C (dec.); IR
(KBr): 3465, 3428, 3260, 3166, 3010, 2976, 1687, 1646,
1598, 1546, 1502, 1368, 1320, 1284, 1124, 1001, 840, 801
General procedure for synthesis of 1-(1-(4-methyl-2-
phenylthiazol-5-yl) ethylidene) thio-semicarbazide (5i)
cm−1 1H NMR (DMSO-d6, 500 MHz) δ: 2.46 (s, 3H,
;
The mixture of 1-(4-methyl-2-phenylthiazol-5-yl) ethanone,
4i (2 mmol) and thiosemicarbazide (2.5 mmol) in ethanol
(10 mL) was refluxed for 2 h. After completion of reaction
(TLC) solvent was cooled to room temperature. The product
was filtered, washed with water and recrystallized from
ethanol.
Thiazole-CH3), 7.57 (d, J = 8 Hz, 2H, H-3′, H-5′), 8.60 (s,
1H, NH), 7.90 (d, J = 8 Hz, 2H, H-2′, H-6′), 8.28 (s, 1H,
NH), 8.33 (s, 2H, NH2), 11.48 (s, 1H, N = CH); 13C NMR
(DMSO-d6, 125 MHz) δ: 16.0 (Thiazole-CH3), 128.3 (C-2′,
-6′), 128.8 (C = N), 129.9 (C-3′, -5′), 131.9 (C-4′), 135.7
(Thiazole C-5), 135.8 (C-1′), 155.3 (Thiazole C-4), 165.5
(Thiazole C-2), 178.0 (C = S); LC–MS m/z: 311.0 (M +
H)+, m/z: 313.0 (M + H + 2)+.
1-(1-(4-methyl-2-phenylthiazol-5-yl)ethylidene)thiosemi-
carbazide (5i) Yield 66 %, m.p. 230 °C (dec.); IR (KBr):
3470, 3425, 3255, 3145, 3018, 2972, 1679, 1640, 1595,
1-((2-(4-fluorophenyl)-4-methylthiazol-5-yl)methylene)
thiosemicarbazide (5g) Yield 75 %, m.p. 230 °C (dec.);IR
(KBr): 3468, 3434, 3265, 3160, 3012, 2975, 1688, 1649,
1600, 1549, 1500, 1368, 1320,1284, 1124, 1001, 839,
1546, 1491,1311, 1279, 1111, 840 cm−1
;
1H NMR
(DMSO-d6, 500 MHz) δ: 2.38 (s, 3H, Thiazole-CH3), 2.60
(s, 3H, N = CCH3), 7.38–7.42 (m, 4H, H-3′, H-4′, H-5′,
NH), 7.80–7.82 (m, 2H, H-2′, H-6′), 8.45 (s, 1H, NH),
10.51 (s, 1H, NH).13C NMR (DMSO-d6,125 MHz) δ: 18.4
(Thiazole-CH3), 31.1 (N = C–CH3), 128.3 (C-3′, -5′), 129.5
(C-4′), 129.6 (C-2′, -6′), 130.5 (Thiazole C-5), 132.2 (C =
N), 135.8 (C-1′), 157.9 (Thiazole C-4), 167.5 (Thiazole C-
2), 180.5 (C = S); LC–MS m/z: 291.08 (M + H).+
800 cm−1 1H NMR (500 MHz, DMSO-d6) δ: 2.43, (s,
:
3H, Thiazole-CH3), 7.40 (t, J = 7.6 Hz, 2H, H-3′, H-5′),
7.60 (s, 1H, NH), 7.94–7.96 (m, 2H, H-2′, H-6′), 8.28
(s, 1H, NH), 8.35 (s, 1H, NH), 11.46 (s, 1H, N = CH); 13C
NMR (125 MHz, DMSO-d6) δ: 16.6 (Thiazole-CH3), 115.8
(C-3′, -5′), 129.3 (C = N), 129.6 (C-2′, -6′), 130.5 (C-1′),
135.4 (Thiazole C-5), 155.8 (Thiazole C-4), 162.2 (C-4′),
166.9 (Thiazole C-2), 178.5 (C = S); LC–MS m/z: 295.1
(M + H)+.
1-(1-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)ethylidene)
thiosemicarbazide (5j) Yield 65 %, m.p. 240 °C (dec.); IR

Med Chem Res
(KBr): 3468, 3430, 3266, 3167, 3012,2975, 1689, 1644,
1596, 1544, 1498, 1365, 1321,1284, 1124, 1001, 838, 800
inoculation for experiments, 50 µL of glycerol stock was
pre-inoculated in the corresponding medium to obtain
metabolically active mycobacteria. For each experiment,
cultures were grown to log phase [optical density at 595 nm
(O.D.595) = 1] under aerobic conditions at 37 °C and 150 r.
p.m. As mycobacteria grow in visibly aggregated clumps in
the culture medium, cultures were sonicated for 2 min using
a water-bath sonicator (Ultrasonic, Freeport, IL) to obtain
viable dispersed cells. This step was introduced to repro-
ducibly inoculate mycobacterial bacilli in fresh medium for
carrying out the experiments.
cm−1 1H NMR (500 MHz, DMSO-d6) δ: 2.35 (s, 3H,
;
Thiazole-CH3), 2.62 (s, 3H, N = CCH3), 7.41 (s, 1H, NH),
7.50 (d, J = 7.6 Hz, 2H, H-3′, H-5′), 7.85 (d, J = 7.6 Hz, 2H,
H-2′, H-6′), 8.40 (s, 1H, NH), 10.53 (s, 1H, NH); 13C NMR
(DMSO-d6, 125 MHz) δ: 20.0 (Thiazole-CH3), 31.5 (N =
C–CH3), 126.0 (C-2′, -6′), 129.5 (C-3′, -5′), 130.3 (Thiazole
C-5), 132.0 (C = N), 132.8 (C-4′), 143.9 (C-1′), 152.3
(Thiazole C-4), 165.2 (Thiazole C-2), 178.6 (C = S);
LC–MS m/z: 325.03 (M + H)+, m/z: 327.2 (M + H + 2).+
Primary screening
1-(1-(2-(4-fluorophenyl)-4-methylthiazol-5-yl)ethylidene)thio-
semicarbazide (5k) Yield 70 %, m.p. 203 °C (dec.); IR
(KBr): 3462, 3433, 3265, 3015, 2976, 1685, 1642, 1599,
1540, 1496, 1365, 1321,1284, 998, 841, 802 cm−1; 1H NMR
(500 MHz, DMSO-d6) δ: 2.34 (s, 3H, Thiazole-CH3), 2.58, (s,
3H, N = CCH3), 7.32 (t, J = 7.6 Hz, 2H, H-3′, H-5′), 7.42 (s,
1H, NH), 8.84–8.86 (m, 2H, H-2′, H-6′), 8.36 (s, 1H, NH),
10.56 (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6) δ: 20.0
(Thiazole-CH3), 31.4 (N = C–CH3), 116.8 (C-3′, -5′), 130.0
(C-2′, -6′), 133.0 (C = N), 135.8 (Thiazole C-5), 142.6 (C-1′),
154.6 (Thiazole C-4), 163.0 (C-4′), 167.2 (Thiazole C-2),
178.7 (C = S); LC–MS m/z: 309.06 (M + H).+
Activity against dormant stage MTB was determined
through the XTT reduction menadione assay (XRMA),
reading absorbance at 470 nm as per the protocol described
by Singh et al. A compound solution (2.5 μL) was added in
a total volume of 250 μL of M. pheli medium consisting of
the MTB and BCG; sealed with plate sealers and allowed to
incubate for 12 days at 37 °C. The XRMA was then carried
out to estimate viable cells present in different wells of the
assay plate. To all wells, 200 μM XTT was added and
incubated at 37 °C for another 20 min. It was followed by
the addition of 60 μM of menadione and incubated at 37 °C
for 40 min. The optical density was measured using a
microplate reader (SpectraMaxPlus 384 plate reader,
Molecular Devices Inc.) at 470 nm filter against a blank
prepared from a well free of cells. Absorbance obtained
from the cells treated with 1% DMSO alone was considered
as 100% cell growth. The nitrate reductase (NR) assay was
performed to estimate inhibition of M. bovis BCG by the
compounds. In the NR assay, 80 μL of culture from an
incubated 96-well-plate was taken into another 96-well-
plate, then 80 μL of 1% sulfanilic acid in 20% of conc. HCl
was added, incubated for 10 min. at room temperature and
then 80 μL of 0.1% N-(1-naphthyl)ethylenediaminedihy-
drochloride solution in distilled water was added. Finally,
absorbance for the NR assay was measured at 540 nm.
The % Inhibition in the presence of test material is cal-
culated by using formula, % inhibition = (Average of
Control−Average of Compound)/(Average of Control
−Average of Blank) × 100). Where, Control is culture
medium with cells and DMSO and blank is culture medium
without cells. For all samples, each compound concentra-
tion was tested in triplicates in a single experiment and the
quantitative value was expressed as the mean standard
deviation (S.D.).
1-(1-(4-methyl-2-p-tolylthiazol-5-yl)ethylidene)thiosemi-
carbazide (5l) Yield 75 %, m.p. 230 °C (dec.); IR (KBr):
3460, 3430, 3260, 3011, 2975, 1686, 1640, 1600, 1544,
1
1499, 1366, 1322, 1285, 1000, 841, 800 cm−1; H NMR
(500 MHz, DMSO- d6) δ: 2.15 (s, 3H, Ar-CH3), 2.40 (s, 3H,
Thiazole-CH3), 2.66 (s, 3H, N = CCH3), 7.42 (s, 1H, NH),
7.62 (d, J = 6.8 Hz, 2H, H-3′, H-5′), 8.05 (d, J = 6.8 Hz,
2H, H-2′, H-6′), 8.35 (s, 1H, NH), 10.58 (s, 1H, NH); 13C
NMR (125 MHz, DMSO-d6) δ: 17.7 (Thiazole-CH3), 21.4
(Ar–CH3), 31.2 (N = C–CH3), 126.4 (C-2′, -6′), 128.5 (C-
3′, -5′), 129.9 (C-4′), 130.3 (C = N), 136.0 (Thiazole C-5),
142.2 (C-1′), 154.0 (Thiazole C-4), 167.4 (Thiazole C-2),
178.3 (C = S); LC–MS m/z: 305.1 (M + H).+
Pharmacology
Antitubercular activity
Standard cultures of M. tuberculosis H37Ra (ATCC 25177)
(MTB) and M. bovis Bacille Calmette Guerin (BCG)
(ATCC 35743) were procured from the American Type
Culture Collection (Manassas, VA). MTB and M. bovis
BCG were grown in a defined Mycobacterium phlei med-
ium and in 50 mM sodium nitrate-containing Dubos med-
ium (Difco, Detroit, MI), respectively. These were
maintained as glycerol stocks at −70 °C. Prior to
Antibacterial activity
All bacterial cultures were first grown in Luria Burtony
media at 37 °C at 180 r.p.m. Once the culture reaches one

Med Chem Res
O.D., it is used for anti-bacterial assay. Bacterial strains
E. coli (NCIM 2576), P. flurescence (NCIM 2059) as
Gram-negative and S. aureus (NCIM 2602), B. subtilis
(NCIM 2162) as Gram-positive were obtained from NCIM
(NCL, Pune) and were grown in Luria Burtony medium
from Hi Media, India. The assay was performed in 96-well-
plates after 8 and 12 h for Gram negative and Gram positive
bacteria, respectively (Ciapetti et al. 1993). 0.1% of one O.
D. culture at 620 nm was used for screening inoculated
culture was added into each well of 96-well-plate containing
the compounds to be tested. Optical density for each plate
was measured at 620 nm after 8 h for Gram-negative bac-
teria and after 12 h for Gram-positive bacteria.
culture plates and was incubated at 37 °C in an atmosphere
of 5% CO2 and 95% relative humidity in a CO2 incubator.
After 24 h, synthesized compounds at varying concentra-
tions of 3, 10, and 30 µg/mL were added to the wells con-
taining cells. Rifampicin was used as positive control.
Suitable controls with an equivalent concentration of
dimethyl sulphoxide (DMSO) (Sigma-Aldrich) were also
included. The plates were further incubated for 48 h. At the
end of the incubation period, the solution containing the
unattached cells was discarded and the wells were washed
three times with 1 mL of PBS followed by addition of 10 µL
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) (5 mg/mL in PBS) to adherent cells in growth
medium. After 4 h at 37 °C for MTT cleavage, the formazan
product was solubilised by addition of 100 µL of 0.04 N
HCl in isopropanol. Absorbance was measured on a Spec-
traMax® PLUS 384 plate reader (Molecular Devices, Sun-
nyvale, CA) at a wavelength of 570 nm. Percentage
cytotoxicity was calculated using the formula: % cytotoxi-
city = (average of control−average of compound)/(average
of control−average of blank) × 100). Each concentration
was tested in triplicate in a single experiment and the
quantitative value was expressed as the mean S.D.
Antifungal activity
The in vitro antifungal activity of all the synthesized com-
pounds was done by the disk diffusion method (Alley et al.
1988; Singh et al. 2015) against the C. albicans (NCIM
3100) obtained from National Chemical Laboratory Pune,
India. All cultures were maintained at 4 °C over nutrient
agar slants throughout the experiment. The cultures incu-
bated overnight at 37 °C in nutrient broth before using for
antifungal activity. Five hundred microliters of overnight
old fungal suspension were spread over the nutrient agar
plates using a sterile cotton swab in order to get a uniform
microbial growth. The synthesized compounds were dis-
solved in DMSO. Under aseptic conditions, empty sterilized
disks (Whatman no. 5, 6 mm diameter) were impregnated
with concentration 100 µg/disk of respective synthesized
compounds and placed on the agar surface. Paper disk
moistened with aqueous DMSO was placed on seeded
petriplates as a vehicle control. A standard disk containing
Fluconazole (25 µg/disk) was used as positive control. The
plates were left for 30 min. at room temperature to allow the
diffusion of synthesized compounds and then incubated at
37 °C for 24 h. The antifungal activity was evaluated by
measuring the zone of inhibition against the test micro-
organism. All experiments were carried in triplicates.
Results and discussion
Chemistry
A general route for the synthesis of title compounds 5a–l is
depicted in Scheme 1.
All the compounds, 5a–l were characterized by spec-
troscopic analysis and were studied for their anti-
tuberculosis and antimicrobial activity. Their toxicity was
also studied against two human cancer cell lines.
Anti-tubercular activity
The antitubercular activity for each synthesized compound
was determined by measuring inhibition of growth against
M. tuberculosis H37Ra (MTB, ATCC 25177) and M. bovis
(BCG, ATCC 35743) in liquid medium. In a preliminary
screening, the antimycobacterial activity of these com-
pounds was assessed at concentrations of 30, 10, 3 μg/mL
using first-line antitubercular drug Rifampicin as reference
standard. In vitro activity studies against MTB and M. bovis
BCG were performed using the XRMA (Singh et al. 2011;
Khan et al. 2008) and NR assays (Khan et al. 2008; Sarkar
and Sarkar 2012), respectively. The results of antitubercular
activity are summarized in Table 1.
Cytotoxic activity
Cell lines were maintained under standard cell culture
conditions at 37 °C and 5% CO2 in a humidified environ-
ment. The cytotoxic effect of synthesized compounds was
checked on cancer cell lines using the concentrations 30, 10,
3 μg/mL to determine the growth inhibition. In short, log-
phase cells were harvested using trypsin (0.05% trypsin and
0.02% ethylene diamine tetra-acetic acid in PBS) from tis-
sue culture flasks and the suspension was diluted with
appropriate culture medium to obtain a cell density of 105
cells/mL was determined by haemocytometry. An aliquot of
100 µL of each suspension was seeded in 96-well cell
Analysis of the anti-tubercular activity results provides
some lead molecules with good anti-tubercular activity. The
results revealed that, in case of thiosemicarbazide

Med Chem Res
Scheme 1 Synthesis of Target
Compounds 5a–l
Table 1 Antitubercular activity
data of compounds 5a–l
Comp.
M. tuberculosis H37Raa
M. bovis BCGa
30 µg/mL
30 µg/mL
10 µg/mL
3 µg/mL
10 µg/mL
3 µg/mL
5a
61.50
45.22
27.50
66.10
22.59
62.61
68.69
67.23
61.10
96.50
41.10
41.60
99.50
52.40
43.05
9.20
43.70
48.49
5.90
96.52
88.30
85.50
91.27
77.3
94.39
87.70
72.99
79.56
75.7
94.00
86.00
23.86
57.76
73.0
5b
5c
5d
55.30
23.66
56.94
65.08
64.28
11.70
73.50
36.20
35.20
98.00
6.40
5e
23.72
48.24
59.98
59.13
5.74
5f
92.2
90.5
89.6
5g
77.8
79.3
65.8
5h
90.5
90.1
87.2
5i
96.38
86.90
82.91
78.87
99.00
94.15
77.12
81.66
73.68
96.80
60.85
69.14
65.05
61.94
94.60
5j
50.70
15.20
24.50
96.40
5k
5l
Rifampicin
Bold entries are active compounds (that shown higher % inhibition)
a
% inhibition
derivatives of 2-arylthiazole-4-carbaldehyde the compounds
5a (R = H), 5b (R = 4-Cl), 5c (R = 4-F) and 5d (R = 4-
CH3) showed good activity against M. bovis at 30 µg/mL
concentration whereas in compounds 5a (R = H) and 5b
(R = 4-Cl) anti-tubercular activity is retained after the
concentration of compounds was decreased by 10 times

Med Chem Res
Table 2 Antibacterial screening of synthesized compounds 5a–l (% inhibition)
Comp. E. coli P. flurescence S. aureus
30 µg/mL 10 µg/mL 3 µg/mL 30 µg/mL 10 µg/mL 3 µg/mL 30 µg/mL 10 µg/mL 3 µg/mL 30 µg/mL 10 µg/mL 3 µg/mL
B. subtilis
5a
5b
5c
5d
5e
5f
65.68
62.02
55.13
97.70
35.32
80.26
61.08
92.23
90.40
40.78
91.13
30.01
58.49
55.70
45.79
93.25
25.38
70.15
18.23
80.24
83.17
28.40
89.17
28.28
95.25
49.41
47.58
37.80
90.19
16.05
66.33
1.01
64.55
65.32
53.39
97.73
31.09
89.42
64.38
93.98
92.77
40.73
91.24
32.49
96.60
54.68
60.45
49.85
92.10
21.22
70.92
16.65
80.37
81.19
24.57
84.43
29.19
92.00
41.74
44.93
35.12
86.78
13.49
60.66
6.63
76.56
61.24
60.62
97.15
31.54
82.15
66.40
92.87
95.27
39.73
90.87
36.29
95.60
52.08
59.80
47.31
92.20
23.85
70.79
20.16
89.03
84.31
30.95
89.52
25.98
93.80
48.46
48.85
38.85
87.19
17.93
67.49
2.06
66.38
67.17
53.60
99.27
35.92
87.21
62.66
91.94
94.68
38.63
92.51
36.47
98.50
59.11
58.08
42.73
94.25
21.91
77.68
16.56
88.68
82.69
26.61
88.20
21.79
95.00
46.19
41.08
38.56
89.97
11.21
69.61
5.16
5g
5h
5i
74.68
72.03
11.16
72.13
17.19
92.25
71.83
73.55
18.43
75.62
14.27
92.15
79.33
77.77
19.99
80.69
15.64
91.15
73.75
78.78
13.40
76.44
12.49
90.50
5j
5k
5l
AMP 97.00
Bold entries are active compounds (that shown higher % inhibition)
anti-tubercular activity against M. bovis BCG, whereas
compounds 5f (R = 4-Cl) and 5h (R = 4-CH3) reported
good activity and it retained at 3 µg/mL concentration. From
the SAR study, it is noteworthy to mention that as phenyl
ring of 4-methyl-2-arylthiazole-5-carbaldehyde is substituted
by 4-Cl or 4-CH3 group, thiosemicarbazide derivative
showed good anti-tubercular activity against M. bovis.
Also, in case of thiosemicarbazide derivatives of 1-(4-
methyl-2-arylthiazol-5-yl) ethanone, compound 5j (R = 4-
Cl) presented good anti-tubercular activity against both
tested strains, whereas compounds 5i (R = H) and 5k (R =
4-F) showed good activity against M. bovis at 30 µg/mL
concentration. The compounds 5i, 5k, and 5l showed
moderate activity against M. tuberculosis H37Ra. From the
SAR study, it is noteworthy to mention that thiosemi-
carbazide derivative of unsubstituted 2-phenylthiazole-4-
carbaldehyde showed excellent activity against M. bovis.
When phenyl ring is substituted by 4-Cl, activity decreases
for M. bovis but it shows good activity against M. tuber-
culosis H37Ra.
Table 3 Minimum inhibitory concentration (MIC90) in µg/mL of the
synthesized compounds 5a–l
Comp.
E. coli
P. flurescence
S. aureus
B. subtilis
5a
>30
>30
>30
2.5
>30
>30
>30
7.9
>30
>30
>30
8.6
>30
>30
>30
2.9
5b
5c
5d
5e
>30
>30
>30
26.7
27.4
>30
25.9
>30
2.0
>30
>30
>30
20.7
21.9
>30
25.4
>30
2.0
>30
>30
>30
22.3
17.6
>30
29.4
>30
0.125
>30
>30
>30
24.7
19.4
>30
22.5
>30
0.5
5f
5g
5h
5i
5j
5k
5l
Ampicillin
(3 µg/mL). All the compounds showed moderate
activity against M. tuberculosis H37Ra. From the pre-
liminary structure activity relationship (SAR) study it is
noteworthy to mention that thiosemicarbazide of unsub-
stituted 2-phenylthiazole-4-carbaldehyde, 5a showed
excellent activity against M. bovis, whereas replacement of
hydrogen atom of phenyl ring by substituent groups like 4-
Cl, 4-F, or 4-CH3 significantly decrease the anti-tubercular
activity.
Similarly, thiosemicarbazide derivatives of 4-methyl-2-
arylthiazole-5-carbaldehyde 5e (R = H), 5f (R = 4-Cl), 5g
(R = 4-F) and 5h (R = 4-CH3) showed moderate activity
against M. tuberculosis H37Ra at 30 µg/mL concentration.
Compounds 5e (R = H) and 5g (R = 4-F) showed moderate
Antimicrobial activity
The antibacterial activity (Singh et al. 2011; Khan et al.
2008) of synthesized compounds was against the standard
Gram-negative bacteria, E.coli (NCIM 2576), P. flurescence
(NCIM 2059) and Gram-positive bacteria, S. aureus (NCIM
2602), B. subtilis (NCIM 2162); while the antifungal
activity Khan and Sarkar (2008); Khan et al. 2008; Sarkar
and Sarkar 2012; Mosmann 1983) was against C. albicans
(NCIM 3100). Ampicillin served as positive control for
antibacterial activity. The in vitro preliminary screening
values (% inhibition) and MIC90 against microorganisms

Med Chem Res
tested are summarized in Tables 2 and 3, respectively.
Fluconazole served as positive control for antifungal
activity and in vitro preliminary screening values (zone of
inhibition) are summarized in Table 4.
Among the thiosemicarbazide derivatives of 1-(4-
methyl-2-arylthiazol-5-yl) ethanone, the compounds 5i (R
= H) and 5k (R = 4-F) reported good activity at 30 µg/mL
concentration, against all the four species. The compounds
5j (R = 4-Cl) and 5 l (R = 4-CH3) were found less active
against all tested strains. In general, compound with phenyl
group or 4-fluorophenyl at 2-position of thiazole moiety
showed enhanced activity.
Careful analysis of the MICs in Table 3 provides com-
pound 5d, 5h, 5i, and 5k as lead molecules with good
antibacterial activity.
The result of antifungal activity (Table 4) revealed that
most of the synthesized compounds showed moderate
activity against C. albicans.
Most of the synthesized compounds exhibited moderate
to good antibacterial activity. The compounds 5a, 5b, and
5c showed moderate activity against all tested strains.
Compound 5d (R = 4-CH3) showed good antibacterial
activity against all tested strains. It is also noticed that the
activity retained at 3 µg/mL concentration. Thus, it is con-
cluded that in case of thiosemicarbazide derivatives of 2-
arylthiazole-4-carbaldehyde, compounds with R = 4-CH3
group show good antibacterial activity whereas compounds
with unsubstituted phenyl ring and substitution with 4-Cl or
4-F phenyl group showed moderate activity.
Similarly, in case of thiosemicarbazide derivatives of 4-
methyl-2-arylthiazole-5-carbaldehyde, the compounds 5f
(R = 4-Cl) and 5h (R = 4-CH3) showed good antibacterial
activity at 30 µg/mL concentration against all tested strains.
Compounds 5e (R = 4-H) and 5g (R = 4-F) showed mod-
erate antibacterial activity against all tested strains. Thus, it
is concluded that compounds with R = 4-Cl or 4-CH3 group
showed good antibacterial activity.
Cytotoxic activity
The synthesized compounds 5a–l were further assayed for
their cytotoxic activity against the two different human
cancer cell lines, cervix adenocarcinoma HeLa and color-
ectal carcinoma human colon carcinoma (HCT) 116 using
MTT assay (Mosmann 1983; Ciapetti et al. 1993) with 48 h
exposure time of the tested compounds. Rifampicin was
Table 4 Antifungal screening
of synthesized compounds 5a–l
Comp.
C. albicans
Comp.
C. albicans
Comp.
C. albicans
(zone of inhibition in mm)
5a
8
7
5e
5f
10
10
7
5i
10
8
5b
5j
5k
5l
5c
8
5g
5h
8
5d
9
12
9
Fluconazole
17
Fluconazole (25 µg/disk) were used as reference; synthesized compounds (100 µg/disk)
Table 5 Cytotoxic activity of
compounds 5a–l
Comp.
HeLa
HCT 116
30 µg/mL
10 µg/mL
3 µg/mL
30 µg/mL
10 µg/mL
3 µg/mL
5a
60.96
51.81
10.76
51.66
39.79
–
20.23
42.87
–
–
35.64
–
24.05
nd
9.54
nd
–
nd
5b
5c
16.71
36.84
nd
14.49
32.04
nd
6.41
7.39
nd
5d
48.21
45.41
–
40.70
15.98
–
5e
5f
nd
nd
nd
5g
15.24
82.87
40.10
38.51
39.52
44.22
25.50
45.91
35.84
5.67
16.37
–
nd
nd
nd
5h
nd
nd
nd
5i
15.98
–
14.19
14.35
26.49
46.70
22.15
–
–
5j
34.57
36.10
33.72
20.10
–
–
5k
–
–
–
5l
25.48
15.80
27.77
16.50
3.17
10.12
Rifampicin
(-) = inactive; nd, not derermined

Med Chem Res
5-yl)H imidazo [1,2-a]pyridine.
229–233
J
Heterocyclic Chem 53:
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MTT cytotoxicity studies most active compounds are leads
as antimicrobials owing to no significant cell toxicity
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Conclusions
In conclusion, three series of thiosemicarbazide derivatives
of 2-arylthiazole-4-carbaldehyde, 4-methyl-2-arylthiazole-
5-carbaldehyde and 1-(4-methyl-2-arylthiazol-5-yl) etha-
none were synthesized. The antimycobacterial activity stu-
dies were undertaken to evaluate the effects of substituents/
group on the antitubercular and antimicrobial activities.
From the SAR, in case of thiosemicarbazide derivatives of
2-arylthiazole-4-carbaldehyde, compounds with un-
substituted phenyl ring showed excellent antitubercular
activity whereas, substituents on phenyl ring significantly
decrease the antitubercular activity. However, 4-CH3 sub-
stituted phenyl ring at 2-position of thiazole moiety is
required for antibacterial activity. In case of thiosemi-
carbazide derivatives of 4-methyl-2-arylthiazole-5-carbal-
dehyde, 4-Cl and 4-CH3 substituted phenyl ring at 2-
position of thiazole moiety is required for anti-tubercular, as
well as antibacterial activities. However in case of thiose-
micarbazide derivatives of 1-(4-methyl-2-arylthiazol-5-yl)
ethanone, it is concluded that, compound with phenyl group
or 4-fluorophenyl at 2-position of thiazole moiety showed
enhanced activity. Most of the thiosemicarbazide deriva-
tives showed moderate to good antimycobacterial activity.
Acknowledgements Y.K.A. expresses her gratefulness to CSIR
[File No. 08/590(0001)/2012-EMR-I] for the financial support. P.C.M.
would like to thank University Grant Commission (UGC No. 42-355/
2013(SR), New Delhi, India for the financial assistance and CSIR-
NCL, Pune for supporting biological activity. Central Analysis facility,
Savitribai Phule Pune University, Pune is also acknowledged for
spectral analysis.
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Conflict of interest The authors declare that they have no competing
interests.
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