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European	Journal	of	Chemistry	5	(3)	(2014)	550‐556	

	

European	Journal	of	Chemistry	
ISSN	2153‐2249	(Print)	/	ISSN	2153‐2257	(Online)		2014	Eurjchem	Publishing	‐	Printed	in	the	USA	

http://dx.doi.org/10.5155/eurjchem.5.3.550‐556.1059	

	
	

	

	

European	Journal	of	Chemistry	
Journal	homepage:	www.eurjchem.com	

	 	 	

Synthesis	and	antitubercular	evaluation	of	2‐iminothiazolidine‐4‐ones	

Ganesh	Samala,	Chunduri	Madhuri,	Jonnalagadda	Padma	Sridevi,		
Radhika	Nallangi,	Yogeeswari	Perumal,	and	Sriram	Dharmarajan	*	

Antitubercular	Drug	Discovery	Laboratory,	Department	of	Pharmacy,	Birla	Institute	of	Technology	and	Science,	Pilani,	Hyderabad	Campus,	Jawahar	Nagar,	
Hyderabad,	500078,	India	

*Corresponding	author	at:	Antitubercular	Drug	Discovery	Laboratory,	Department	of	Pharmacy,	Birla	Institute	of	Technology	and	Science,	Pilani,	Hyderabad	
Campus,	Jawahar	Nagar,	Hyderabad,	500078,	India.	
Tel.:	+91.40.66303506.	Fax:	+91.40.66303998.	E‐mail	address:	dsriram@hyderabad.bits‐pilani.ac.in	(S.	Dharmarajan).	

	
	

	 	

	 	 	
COMMUNICATION	INFORMATION	 	 ABSTRACT

	
DOI:	10.5155/eurjchem.5.3.550‐556.1059	

Received:	25	March	2014	
Received	in	revised	form:	29	April	2014	
Accepted:	30	April	2014	
Online:	30	September	2014	

KEYWORDS	

	 In	 the	 present	 manuscript,	 we	 report	 synthesis	 of	 new	 3	 and	 5	 substituted	 2‐imino
thiazolidine‐4‐ones	by	three	step	synthetic	protocols	from	3‐trifluormethyl	aniline	or	2‐amino
heterocycle.	 The	 compounds	 were	 evaluated	 for	 in	 vitro	 activities	 against	 Mycobacterium
tuberculosis	(MTB)	in	presence	and	absence	of	efflux	pump	inhibitor,	cytotoxicity	against	RAW
264.7	 cells.	 Among	 the	 thirty	 six	 compounds,	 2‐imino‐3‐(5‐nitrothiazol‐2‐yl)‐5‐(3,4,5‐
trimethoxybenzylidene)thiazolidin‐4‐one	(5g)	was	 found	to	be	the	most	active	compound	 in
vitro	with	MICs	of	3.31	µM	against	log‐phase	culture	of	MTB	and	also	non‐toxic	up	to	100	µM.
Compound	5g	 showed	minimum	 inhibitory	 concentration	 (MIC)	 of	 0.82	 µM	 against	MTB	 in
presence	of	efflux	pump	inhibitor	verapamil.	

Cytotoxicity	
Antitubercular	activity	
Thiazolidine	derivatives	
Mycobacterium	tuberculosis		
Structure	activity	relationship	
Minimum	inhibitory	concentration	

	
1.	Introduction	
	

Despite	 the	 existence	 of	 treatments	 for	 tuberculosis	 (TB),	
nine	million	 people	 are	 currently	 infected	 and	 one	 and	 a	 half	
million	 die,	 each	 year	 [1].	 The	 disease	 also	 represents	 an	
escalating	 threat	 for	 global	 health,	 with	 the	 increasing	
prevalence	of	multi	drug	resistant	(MDR)	and	extensively	drug	
resistant	(XDR)	TB	strains	[2].	Despite	this,	the	development	of	
new	drugs	with	novel	modes	of	action	for	the	treatment	of	TB	
would	likely	still	be	the	most	cost‐effective	way	of	tackling	the	
pandemic.	Specifically,	any	new	drug	should	be	able	to	shorten	
the	duration	of	treatment,	avoid	significant	drug‐drug	interact‐
tion	 with	 current	 regimens,	 to	 treat	 MDR	 as	 well	 as	 XDR‐TB	
patients	[3,4].		

Target‐based	 approaches	 are	 widely	 used	 in	 drug	
discovery;	 questions	 have	 been	 raised	 about	 the	 efficiency	 of	
this	 approach	 given	 the	 very	 high	 attrition	 rates	 that	 these	
projects	have	historically	shown	in	the	anti‐infective	field	[5,6].	
Furthermore,	 the	 state	 of	 affairs	 is	 a	 symptom	 of	 wider	
inconsistencies	 between	 results	 obtained	with	 animal	models	
of	infection	and	their	translation	to	clinical	therapeutic	value	in	
humans.	 Compounds	 identified	 in	 whole‐cell	 screens	 fulfil	 a	
double	 function:	 (i)	 they	 provide	 lead	 structures	 for	 further	
optimization	 within	 the	 drug	 development	 progression	
sequence,	and	(ii)	they	can	be	exploited	as	tools	to	identify	new	

targets.	 Notably,	 cell‐based	 hits	 already	 fulfil	 some	 important	
criteria,	 including	 permeability	 issues	 and,	 given	 the	 progres‐
sion	 criteria	 in	 the	 high‐throughput	 screen,	 higher	 activity	
against	mycobacteria	than	mammalian	cells.	Thus,	they	provide	
suitable	chemical	and	biological	starting	points.	

The	 Tuberculosis	 Antimicrobial	 Acquisition	 and	 Coordi‐
nating	 Facility	 (TAACF)	 was	 established	 by	 the	 National	
Institute	of	Allergy	and	Infectious	Diseases	(NIAID)	in	1994	to	
allow	 researchers	 access	 to	 high	 quality	 screening	 services	 in	
order	 to	 encourage	 antituberculosis	 drug	 discovery	 research.	
Recently	TAACF	reported	their	anti‐TB	high‐throughput	results	
of	 large	 libraries	 of	 drug	 like	 small	 molecules	 [7‐9],	 among	
them	 one	 of	 the	 molecule	 SID:	 24823007	 5‐(furan‐2‐yl	
methylene)‐2‐imino‐3‐(thiazol‐2‐yl)thiazolidin‐4‐one)	 showed	
good	 activity	 against	Mycobacterium	 tuberculosis	 H37Rv	with	
minimum	 inhibitory	 concentration	of	<	0.2	µM	and	selectivity	
index	of	>	115	(Figure	1).		

We	 have	 taken	 SID:	 24823007	 as	 the	 starting	 point	 to	
design	more	 analogues	by	 keeping	 2‐imino	 thiazolidine‐4‐one	
nucleus	intact	and	modify	3rd	and	5th	position	with	various	aryl	
and	 heteroaryl	 moiety	 to	 study	 the	 structure	 activity	
relationship	(SAR)	of	the	lead	compound.		
	



Samala	et	al.	/	European	Journal	of	Chemistry	5	(3)	(2014)	550‐556	 551	
 

 
	

Figure	1.	Antitubercular	lead	compound.	
	
2.	Experimental	
	
2.1.	Instrumentation	
	

All	 the	 reagents	 obtained	 from	 commercial	 sources	 were	
used	without	further	purification.	All	reactions	were	run	under	
inert	 atmosphere	of	 nitrogen	or	 argon.	All	 the	 reactions	were	
monitored	by	thin	layer	chromatography	(TLC)	on	silica	gel	40	
F254	(Merck,	Darmstadt,	Germany)	coated	on	aluminium	plates.	
1H	and	13C	NMR	spectra	were	recorded	on	a	Bruker	AM‐300	or	
400	 NMR	 spectrometer,	 Bruker	 BioSpin	 Corp.,	 Germany.	
Chemical	 shifts	 are	 in	parts	per	million	 (ppm).	 In	 the	Nuclear	
Magnetic	resonance	spectra	(1H	NMR	and	13C	NMR),	the	signals	
of	 the	respective	protons	of	 the	prepared	2‐iminothiazolidine‐
4‐one	 derivatives	were	 verified	on	 the	basis	 of	 their	 chemical	
shifts,	multiplicities,	and	coupling	constants.	Temperatures	are	
reported	 in	 degrees	 Celsius	 and	 are	 uncorrected.	 Compounds	
were	analysed	for	C,	H,	N	and	analytical	results	obtained	were	
within	 ±0.4%	of	 the	 calculated	 values	 for	 the	 formula	 shown.	
Molecular	weights	of	the	synthesised	compounds	were	checked	
by	(Shimadzu,	LC/MS‐2020)	ESI‐MS	method.	
	
2.2.	General	procedure	for	synthesis	of	N‐substituted	
chloroacetamides	(2a‐c)	
	

Compound	 1	 (1.0	 equiv.)	 and	 chloroacetyl	 chloride	 (1.0	
equiv.)	 were	 taken	 in	 benzene	 and	 the	 reaction	mixture	 was	
refluxed	for	6	h.	after	completion	of	the	reaction	monitored	by	
TLC,	the	reaction	mixture	was	diluted	with	EtOAc	and	washed	
with	 sat	NaHCO3,	H2O	 and	Brine.	 The	 combined	 organic	 layer	
was	dried	over	anhy	Na2SO4,	evaporated	to	get	compound	2.	

2‐Chloro‐N‐(3‐(trifluoromethyl)phenyl)acetamide	 (2a):	 3‐
(Trifluoromethyl)aniline	 (3.0	 g,	18.63	mmol)	 and	 chloroacetyl	
chloride	(1.48	mL,	18.63	mmol)	were	taken	in	benzene	(25	mL)	
and	 the	 reaction	 mixture	 was	 refluxed	 for	 6	 h.	 The	 reaction	
mixture	was	diluted	with	EtOAc	and	washed	with	sat.	NaHCO3	
(3	 ×	 30	 mL),	 H2O	 (2	 ×	 30	 mL)	 and	 Brine	 (2	 ×	 20	 mL).	 The	
combined	 organic	 layer	 was	 dried	 over	 anhydrous	 Na2SO4,	
evaporated	under	vacuum	to	get	solid	product,	the	solids	were	
washed	 with	 hexanes	 to	 get	 2‐chloro‐N‐(3‐(trifluoromethyl)	
phenyl)acetamide.	Color:	Off‐white	solid.	Yield:	3.60	g,	81%.	1H	
NMR	(400	MHz,	CDCl3,	δ,	ppm):	8.21	(s,	1H,	Ar‐H),	7.77	(s,	1H,	
NH),	7.54	(d,	J	=	8.4	Hz,	1H,	Ar‐H),	7.49	(d,	J	=	7.6	Hz,	1H,	Ar‐H),	
7.36	(t,	J	=	7.6	Hz,	1H,	Ar‐H),	4.23	(s,	2H,	CH2).	
MS	(EI,	m/z	(%)):	238	[M+H]+.	

2‐Chloro‐N‐(5‐nitrothiazol‐2‐yl)acetamide	 (2b):	 5‐Nitro	
thiazol‐2‐amine	(3.0	g,	20.80	mmol)	and	chloroacetyl	chloride	
(1.64	mL,	20.80	mmol)	were	taken	in	benzene	(25	mL)	and	the	
reaction	mixture	was	refluxed	for	6	h.	The	reaction	mixture	was	
diluted	with	EtOAc	and	washed	with	sat.	NaHCO3	(3	×	30	mL),	
H2O	(2	×	30	mL)	and	Brine	(2	×	20	mL).	The	combined	organic	
layer	 was	 dried	 over	 anhydrous	 Na2SO4,	 evaporated	 under	
vacuum	 to	 get	 solid	 product,	 the	 solids	 were	 washed	 with	
hexanes	 to	 get	 2‐chloro‐N‐(5‐nitrothiazol‐2‐yl)acetamide.	
Color:	 Off‐white	 solid.	 Yield:	 3.40	 g,	 74%.	 1H	NMR	 (400	MHz,	
DMSO‐d6,	δ,	ppm):	9.43	(s,	1H,	NH),	8.64	(s,	1H,	Ar‐H),	4.24	(s,	
2H,	CH2).	MS	(EI,	m/z	(%)):	222	[M+H]+.	

2‐Chloro‐N‐(6‐nitrobenzo[d]thiazol‐2‐yl)acetamide	 (2c):	 6‐
Nitrobenzo[d]thiazol‐2‐amine	 (3.0	 g,	 15.38	 mmol)	 and	
chloroacetyl	 chloride	 (1.21	 mL,	 15.38	 mmol)	 were	 taken	 in	

benzene	(25	mL)	and	the	reaction	mixture	was	refluxed	for	6	h.	
The	reaction	mixture	was	diluted	with	EtOAc	and	washed	with	
sat.	NaHCO3	 (3	×	30	mL),	H2O	 (2	×	30	mL)	and	Brine	 (2	×	20	
mL).	 The	 combined	 organic	 layer	 was	 dried	 over	 anhydrous	
Na2SO4,	 evaporated	 under	 vacuum	 to	 get	 solid	 product,	 the	
solids	 were	 washed	 with	 hexanes	 to	 get	 2‐chloro‐N‐(6‐nitro	
benzo[d]thiazol‐2‐yl)acetamide.	 Color:	 Off‐white	 solid.	 Yield:	
2.97	g,	71%.	1H	NMR	(400	MHz,	DMSO‐d6,	δ,	ppm):	10.03	(s,	1H,	
NH),	8.49	(s,	1H,	Ar‐H),	8.32	(d,	J	=	8.4	Hz,	1H,	Ar‐H),	8.22	(d,	J	=	
8.0	 Hz,	 1H,	 Ar‐H),	 4.26	 (s,	 2H,	 CH2).	 MS	 (EI,	m/z	 (%)):	 272	
[M+H]+.	
	
2.3.	General	procedure	for	synthesis	of	3‐substituted‐2‐
iminothiazolidine‐4‐one	(3a‐c)	
	

Compound	 2a‐c	 (1.0	 equiv)	 and	 KSCN	 (1.6	 equiv)	 were	
taken	in	dry	acetone	and	refluxed	for	2	h.	The	reaction	mixture	
was	concentrated	and	obtained	solid	was	washed	with	H2O	and	
dried	in	vacuum	oven	to	get	compound	3a‐c.		

2‐Imino‐3‐(3‐(trifluoromethyl)phenyl)thiazolidin‐4‐one	(3a):	
2‐Chloro‐N‐(3‐(trifluoromethyl)phenyl)acetamide	 (3.60	 g,	
15.18	mmoles)	and	KSCN	(2.36	g,	24.30	mmoles)	were	taken	in	
dry	 acetone	 and	 refluxed	 for	 2	 h.	 The	 reaction	 mixture	 was	
concentrated	 and	 obtained	 solid	 was	 washed	 with	 H2O	 and	
dried	 in	 vacuum	 oven	 to	 get	 compound	 2‐imino‐3‐(3‐
(trifluoromethyl)phenyl)thiazolidin‐4‐one.	 Yield:	 3.30	 g,	 83%.	
Color:	Off‐white	solid.	 1H	NMR	(400	MHz,	CDCl3,	δ,	ppm):	9.12	
(s,	1H,	NH),	7.78	(s,	1H,	Ar‐H),	7.64	(d,	J	=	8.0	Hz,	1H,	Ar‐H),	7.54	
(d,	 J	=	7.6	Hz,	1H,	Ar‐H),	7.45	(t,	 J	=	7.6	Hz,	1H,	Ar‐H),	4.32	(s,	
2H,	CH2).	MS	(EI,	m/z	(%)):	261	[M+H]+.	

2‐Imino‐3‐(5‐nitrothiazol‐2‐yl)thiazolidin‐4‐one	 (3b):	 2‐
Chloro‐N‐(5‐nitrothiazol‐2‐yl)acetamide	 (3.40	 g,	 15.38	
mmoles)	 and	KSCN	 (2.40	g,	24.61	mmoles)	were	 taken	 in	dry	
acetone	 and	 refluxed	 for	 2	 h.	 The	 reaction	 mixture	 was	
concentrated	 and	 obtained	 solid	 was	 washed	 with	 H2O	 and	
dried	 in	 vacuum	 oven	 to	 get	 compound	 2‐imino‐3‐(5‐
nitrothiazol‐2‐yl)thiazolidin‐4‐one.	 Yield:	 3.15	 g,	 84%.	 Color:	
Off‐white	solid.	 1H	NMR	(400	MHz,	DMSO‐d6,	δ,	ppm):	9.97	(s,	
1H,	NH),	8.81	(s,	1H,	Ar‐H),	4.27	(s,	2H,	CH2).	
MS	(EI,	m/z	(%)):	245	[M+H]+.	

2‐Imino‐3‐(6‐nitrobenzo[d]thiazol‐2‐yl)thiazolidin‐4‐one	
(3c):	 2‐Chloro‐N‐(6‐nitrobenzo[d]thiazol‐2‐yl)acetamide	 (2.97	
g,	10.95	mmoles)	and	KSCN	(1.71	g,	17.53	mmoles)	were	taken	
in	dry	acetone	and	refluxed	 for	2	h.	The	reaction	mixture	was	
concentrated	 and	 obtained	 solid	 was	 washed	 with	 H2O	 and	
dried	 in	 vacuum	 oven	 to	 get	 compound	 2‐imino‐3‐(6‐
nitrobenzo[d]thiazol‐2‐yl)thiazolidin‐4‐one.	Yield:	2.34	g,	72%.	
Color:	Off‐white	 solid.	 	 1H	NMR	 (400	MHz,	DMSO‐d6,	 δ,	 ppm):	
9.72	(s,	1H,	NH),	8.53	(s,	1H,	Ar‐H),	8.34	(d,	J	=	8.4	Hz,	1H,	Ar‐H),	
8.17	 (d,	 J	 =	 8.0	 Hz,	 1H,	 Ar‐H),	 4.20	 (s,	 2H,	 CH2).	 MS	 (EI,	m/z	
(%)):	295	[M+H]+.	
	
2.4.	General	procedure	for	synthesis	of	compounds	4a‐l,	5a‐l	
and	6a‐l	
	

2‐Imino‐3‐(3‐(trifluoromethyl)phenyl)thiazolidin‐4‐one	
(3a)	(0.25	g,	0.96	mmol),	NaOAc	(0.15	g,	1.92	mmoles)	and	2‐
hydroxybenzaldehyde	(0.14	g,	1.15	mmol)	were	taken	in	acetic	
acid	 (2.0	 mL)	 and	 heated	 at	 100	 °C	 for	 3	 hours,	 the	 solids	
formed	in	the	reaction	mixture	were	filtered	and	washed	with	
water,	 little	 amount	 of	 ethanol	 and	 hexanes	 to	 afford	 title	
compound	(0.38	g,	90%)	as	an	off‐white	solid	(4a).		

5‐(2‐Hydroxybenzylidene)‐2‐imino‐3‐(3‐(trifluoromethyl)	
phenyl)thiazolidin‐4‐one	 (4a):	 Color:	 Brown.	 Yield:	 90%.	 1H	
NMR	(400	MHz,	DMSO‐d6,	δ,	ppm):	11.51	(s,	1H,	OH),	10.02	(s,	
1H,	NH),	8.30	(s,	1H,	Ar‐H),	7.81	(d,	J	=	8.0	Hz,	1H,	Ar‐H),	7.78‐
7.60	(m,	4H,	Ar‐H	+	CH),	7.54‐7.36	(m,	3H,	Ar‐H).	13C	NMR	(100	
MHz,	DMSO‐d6,	δ,	ppm):	169.0,	153.4,	152.3,	144.9,	136.5,	133.2,	
131.5,	 128.4,	 127.3,	 127.0,	 126.4,	 125.7,	 124.4,	 123.6,	 122.4,	
121.3,	 119.4.	 	 MS	 (EI,	m/z	 (%)):	 365	 [M+H]+.	 Anal.	 calcd.	 for	



552	 Samala	et	al.	/	European	Journal	of	Chemistry	5	(3)	(2014)	550‐556	
	
C17H11F3N2O2S:	 C,	 56.04;	 H,	 3.04;	 N,	 7.69.	 Found:	 C,	 56.12;	 H,	
3.10;	N,	7.72%.	

5‐(4‐Hydroxybenzylidene)‐2‐imino‐3‐(3‐(trifluoromethyl)	
phenyl)thiazolidin‐4‐one	 (4b):	 Color:	 White.	 Yield:	 88%.	 1H	
NMR	 (400	MHz,	DMSO‐d6,	 δ,	 ppm):	 12.91	 (s,	 1H,	OH),	 9.42	 (s,	
1H,	NH),	8.34	(s,	1H,	Ar‐H),	7.99	(d,	J	=	7.6Hz,	2H,	Ar‐H),	7.72‐
7.63	(m,	4H,	Ar‐H	+	CH),	7.27	(d,	J	=	8.0	Hz,	2H,	Ar‐H).	13C	NMR	
(100	MHz,	DMSO‐d6,	δ,	ppm):	166.8,	160.6,	152.4,	149.4,	134.5,	
133.4,	 130.5	 (2C),	 129.8,	 129.1,	 127.4,	 126.3,	 125.4,	 124.7,	
123.4	(2C),	119.7.	MS	(EI,	m/z	(%)):	365	[M+H]+.	Anal.	calcd.	for	
C17H11F3N2O2S:	 C,	 56.04;	 H,	 3.04;	 N,	 7.69.	 Found:	 C,	 56.14;	 H,	
3.10;	N,	7.77%.	

2‐Imino‐5‐(4‐methoxybenzylidene)‐3‐(3‐(trifluoromethyl)	
phenyl)thiazolidin‐4‐one	 (4c):	 Color:	 Brown.	 Yield:	 79%.	 1H	
NMR	 (300	MHz,	 δ,	 ppm):	12.40	 (s,	1H,	NH),	8.78‐7.20	 (m,	3H,	
Ar‐H),	 7.92‐7.77	 (m,	 4H,	 Ar‐H	+	 CH),	 7.69‐7.54	 (m,	 2H,	 Ar‐H),	
4.05	 (s,	 3H,	CH3).	 13C	NMR	 (75	MHz,	DMSO‐d6,	 δ,	 ppm):	163.5,	
156.4,	 149.4,	 144.5,	 136.4,	 135.3,	 133.4	 (2C),	 132.6,	 130.5,	
129.5,	127.6,	126.0,	125.4,	124.6,	123.3	(2C),	120.6.	MS	(EI,	m/z	
(%)):	 379	 [M+H]+.	 Anal	 calcd	 for	 C18H13F3N2O2S:	 C,	 57.14;	 H,	
3.46;	N,	7.40.	Found:	C,	57.21;	H,	3.48;	N,	7.43%.	

5‐(4‐(Benzyloxy)benzylidene)‐2‐imino‐3‐(3‐(trifluoromethyl)	
phenyl)thiazolidin‐4‐one	 (4d):	 Color:	 Brown.	 Yield:	 93%.	 1H	
NMR	(300	MHz,	CDCl3,	δ,	ppm):	12.45	(s,	1H,	NH),	8.56	(s,	1H,	
Ar‐H),	8.12	 (d,	 J	=	7.6	Hz,	2H,	Ar‐H),	7.99‐7.72	 (m,	6H,	Ar‐H	+	
CH),	 7.63‐7.51	 (m,	 5H,	 Ar‐H),	 4.99	 (s,	 2H,	 CH2).	 13C	 NMR	 (75	
MHz,	 CDCl3,	 δ,	 ppm):	 167.3,	 161.1,	 154.3,	 148.7,	 139.5,	 138.4,	
137.4,	 136.3(2C),	 134.5,	 133.2(2C),	 131.4,	 130.4(2C),	 129.6,	
128.4,	127.4,	126.3(2C),	125.2,	123.4,	120.5,	69.3.	MS	(EI,	m/z	
(%)):	 455	 [M+H]+.	 Anal	 calcd	 for	 C24H17F3N2O2S:	 C,	 63.43;	 H,	
3.77;	N,	6.16.	Found:	C,	63.52;	H,	3.87;	N,	6.25%.	

2‐Imino‐5‐(4‐methylbenzylidene)‐3‐(3‐(trifluoromethyl)	
phenyl)thiazolidin‐4‐one	(4e):	Color:	White.	Yield:	81%.	1H	NMR	
(300	MHz,	DMSO‐d6,	δ,	ppm):	12.24	(s,	1H,	NH),	8.34	(s,	1H,	Ar‐
H),	7.89	(d,	J	=	7.6	Hz,	2H,	Ar‐H),	7.81‐7.72	(m,	6H,	Ar‐H	+	CH),	
2.52	 (s,	 3H,	 CH3).	 13C	 NMR	 (75	 MHz,	 CDCl3,	 δ,	 ppm):	 164.3,	
159.4,	 155.3,	 152.4,	 141.3,	 139.5,	 138.4,	 136.4,	 135.4	 (2C),	
133.2,	130.4	(2C),	127.4,	126.4,	125.3,	122.4,	22.5.	MS	(EI,	m/z	
(%)):	 363	 [M+H]+.	 Anal	 calcd	 for	 C18H13F3N2OS:	 C,	 59.66;	 H,	
3.62;	N,	7.73.	Found:	C,	59.73;	H,	3.67;	N,	7.87%.	

5‐(4‐(Dimethylamino)benzylidene)‐2‐imino‐3‐(3‐(trifluoro	
methyl)phenyl)thiazolidin‐4‐one	(4f):	Color:	Brown.	Yield:	84%.	
1H	NMR	(400	MHz,	CDCl3,	δ,	ppm):	9.99	(s,	1H,	NH),	8.28	(s,	1H,	
Ar‐H),	7.97	 (d,	 J	=	7.6	Hz,	2H,	Ar‐H),	7.82‐7.74	 (m,	2H,	Ar‐H	+	
CH),	7.63‐7.45	(m,	4H,	Ar‐H),	3.15	(s,	6H,	2CH3).	13C	NMR	(100	
MHz,	 CDCl3,	 δ,	 ppm):	 164.4,	 154.4,	 152.6,	 143.4,	 136.4,	 134.5,	
133.6	(2C),	132.9,	132.1,	131.3	(2C),	127.5,	126.4,	124.6,	123.4,	
120.4,	43.2	(2C).	MS	(EI,	m/z	(%)):	392	[M+H]+.	Anal	calcd	for	
C19H16F3N3OS:	 C,	 58.30;	 H,	 4.12;	 N,	 10.74.	 Found:	 C,	 58.41;	 H,	
4.18;	N,	10.83%.	

2‐Imino‐3‐(3‐(trifluoromethyl)phenyl)‐5‐(3,4,5‐trimethoxy	
benzylidene)thiazolidin‐4‐one	(4g):	Color:	White.	Yield:	89%.	1H	
NMR	(400	MHz,	CDCl3,	δ,	ppm):	12.60	(s,	1H,	NH),	8.49	(s,	1H,	
Ar‐H),	8.10‐8.01	(m,	2H,	Ar‐H),	7.99	(s,	1H,	CH),	7.63	(d,	J	=	7.2	
Hz,	 1H,	 Ar‐H),	 7.49	 (s,	 2H,	 Ar‐H),	 4.12	 (s,	 9H,	 3CH3).	 13C	NMR	
(100	MHz,	CDCl3,	δ,	ppm):	168.3,	162.4(2C),	161.1,	152.4,	149.6,	
142.3,	 138.5,	 137.4,	 135.9,	 133.0,	 131.4,	 128.4,	 126.2,	 124.2,	
123.4	(2C),	63.4,	61.2	(2C).	MS	(EI,	m/z	(%)):	439	[M+H]+.	Anal.	
calcd.	 for	 C20H17F3N2O4S:	 C,	 54.79;	 H,	 3.91;	 N,	 6.39.	 Found:	 C,	
54.82;	H,	3.98;	N,	6.36%.	

5‐(2‐Fluorobenzylidene)‐2‐imino‐3‐(3‐(trifluoromethyl)	
phenyl)thiazolidin‐4‐one	 (4h):	 Color:	 Light	 Yellow.	 Yield:	 80%.	
1H	NMR	(400	MHz,	DMSO‐d6,	δ,	ppm):	12.42	(s,	1H,	NH),	7.99	(s,	
1H,	Ar‐H),	7.92	(s,	1H,	CH),	7.81‐7.74	(m,	2H,	Ar‐H),	7.63	(d,	J	=	
6.8	Hz,	1H,	Ar‐H),	7.58‐7.49	(m,	4H,	Ar‐H).	13C	NMR	(100	MHz,	
DMSO‐d6,	 δ,	 ppm):	 167.2,	 162.2,	 154.3,	 149.6,	 135.3,	 134.6,	
133.2,	 129.2,	 126.3,	 126.0,	 125.2,	 123.7,	 123.2,	 122.6,	 121.4,	
120.3,	 119.9.	 MS	 (EI,	 m/z	 (%)):367	 [M+H]+.	 Anal.	 calcd.	 for	
C17H10F4N2OS:	 C,	 55.74;	 H,	 2.75;	 N,	 7.65.	 Found:	 C,	 55.81;	 H,	
2.81;	N,	7.74%.	

5‐(4‐Chlorobenzylidene)‐2‐imino‐3‐(3‐(trifluoromethyl)	
phenyl)thiazolidin‐4‐one	 (4i):	 Color:	 Light	 Yellow.	 Yield:	 79%.	
1H	NMR	(400	MHz,	DMSO‐d6,	δ,	ppm):	12.88	(s,	1H,	NH),	8.21	(s,	
1H,	Ar‐H),	7.81	(d,	J	=	7.2	Hz,	2H,	Ar‐H),	7.77	(d,	J	=	6.8	Hz,	1H,	
Ar‐H),	7.72‐7.68	(m,	3H,	Ar‐H	+	CH),	7.35	(d,	J	=	8.0	Hz,	2H,	Ar‐
H).	13C	NMR	(100	MHz,	DMSO‐d6,	δ,	ppm):	167.2,	163.3,	155.2,	
148.9,	 136.8,	 136.2,	 135.3(2C),	 133.8,	 130.4,	 129.4,	 127.2,	
126.4,	124.7(2C),	123.1,	120.6.	 	MS	(EI,	m/z	(%)):	383	[M+H]+.	
Anal.	 calcd.	 for	 C17H10ClF3N2OS:	 C,	 53.34;	 H,	 2.63;	 N,	 7.32.	
Found:	C,	53.37;	H,	2.72;	N,	7.41%.	

2‐Imino‐5‐(3‐nitrobenzylidene)‐3‐(3‐(trifluoromethyl)	
phenyl)thiazolidin‐4‐one	 (4j):	 Color:	 Yellow.	 Yield:	 76%.	 1H	
NMR	(400	MHz,	DMSO‐d6):	12.94	(s,	1H,	NH),	8.34	(s,	1H,	Ar‐H),	
8.21	(s,	1H,	Ar‐H),	7.98	(d,	J	=	7.6	Hz,	1H,	Ar‐H),	7.81‐7.74	(m,	
3H,	 Ar‐H	 +	 CH),	 7.63‐7.54	 (m,	 3H,	 Ar‐H).	 13C	NMR	 (100	MHz,	
DMSO‐d6,	 δ,	 ppm):	 164.6,	 162.2,	 151.2,	 146.4,	 137.8,	 136.3,	
134.2,	 133.8,	 132.6,	 131.6,	 130.4,	 128.2,	 126.7,	 125.1,	 124.2,	
123.2,	 121.5.	 MS	 (EI,	m/z	 (%)):	 394	 [M+H]+.	 Anal.	 calcd.	 for	
C17H10F3N3O3S:	C,	 51.91;	H,	2.56;	N,	10.68.	Found:	C,	51.94;	H,	
2.62;	N,	10.73%.	

2‐Imino‐5‐((5‐nitrofuran‐2‐yl)methylene)‐3‐(3‐(trifluoro	
methyl)phenyl)thiazolidin‐4‐one	(4k):	Color:	Yellow.	Yield:	92%.	
1H	NMR	(400	MHz,	CDCl3,	δ,	ppm):	12.63	(s,	1H,	NH),	8.31	(d,	J	=	
7.2	Hz,	1H,	Ar‐H),	8.21	(s,	1H,	Ar‐H),	7.92	(d,	J	=	7.2	Hz,	1H,	Ar‐
H),	7.81‐7.72	(m,	2H,	Ar‐H	+	CH),	7.58	(d,	J	=	7.2	Hz,	1H,	Ar‐H),	
7.54	(t,	J	=	7.2	Hz,	1H,	Ar‐H).	13C	NMR	(100	MHz,	CDCl3,	δ,	ppm):	
168.1,	 160.2,	 153.2,	 152.1,	 149.3,	 136.3,	 135.3,	 134.9,	 133.9,	
131.4,	127.2,	126.4,	124.2,	122.1,	120.5.	MS	(EI,	m/z	(%)):	384	
[M+H]+.	 Anal.	 calcd.	 for	 C15H8F3N3O4S:	 C,	 47.00;	 H,	 2.10;	 N,	
10.96.	Found:	C,	47.11;	H,	2.13;	N,	10.98%.	

2‐Imino‐5‐((5‐nitrothiophen‐2‐yl)methylene)‐3‐(3‐(trifluoro	
methyl)phenyl)thiazolidin‐4‐one	(4l):	Color:	Light	Yellow.	Yield:	
72%.	1H	NMR	(400	MHz,	CDCl3,	δ,	ppm):	12.51	(s,	1H,	NH),	8.29	
(d,	 J	=	7.2	Hz,	1H,	Ar‐H),	8.19	(s,	1H,	Ar‐H),	7.89	(d,	J	=	7.2	Hz,	
1H,	Ar‐H),	7.79‐7.69	(m,	2H,	Ar‐H+	CH),	7.63	(d,	J	=	7.2	Hz,	1H,	
Ar‐H),	7.58	(t,	J	=	7.2	Hz,	1H,	Ar‐H).	13C	NMR	(100	MHz,	CDCl3,	δ,	
ppm):	 166.9,	 161.1,	 152.3,	 150.3,	 148.2,	 135.5,	 134.1,	 133.4,	
132.9,	 130.2,	 128.4,	 126.2,	 122.3,	 120.3,	 118.8.	 MS	 (EI,	 m/z	
(%)):	 400	 [M+H]+.	 Anal.	 calcd.	 for	 C15H8F3N3O3S2:	 C,	 45.11;	 H,	
2.02;	N,	10.52.	Found:	C,	45.19;	H,	2.10;	N,	10.59%.	

5‐(2‐Hydroxybenzylidene)‐2‐imino‐3‐(5‐nitrothiazol‐2‐yl)	
thiazolidin‐4‐one	(5a):	Color:	White.	Yield:	88%.	 1H	NMR	(400	
MHz,	DMSO‐d6,	δ,	ppm):	12.42	(s,	1H,	OH),	9.70	(s,	1H,	NH),	8.31	
(s,	 1H,	 Ar‐H),	 7.92	 (d,	 J	=	 7.2	 Hz,	 1H,	 Ar‐H),	 7.81	 (s,	 1H,	 CH),	
7.72‐7.69	(m,	2H,	Ar‐H),	7.63	(t,	J	=	7.6	Hz,	1H,	Ar‐H).	13C	NMR	
(100	MHz,	DMSO‐d6,	δ,	ppm):	172.6,	163.4,	156.4,	149.4,	137.4,	
131.4,	 128.5,	 126.4,	 125.2,	 123.8,	 121.4,	 120.4,	 119.3.	MS	 (EI,	
m/z	(%)):	349	[M+H]+.	Anal.	calcd.	for	C13H8N4O4S2:	C,	44.82;	H,	
2.31;	N,	16.08.	Found:	C,	44.92;	H,	2.41;	N,	16.12%.	

5‐(4‐Hydroxybenzylidene)‐2‐imino‐3‐(5‐nitrothiazol‐2‐yl)	
thiazolidin‐4‐one	(5b):	Color:	White.	Yield:	79%.	1H	NMR	(400	
MHz,	DMSO‐d6,	δ,	ppm):	12.78	(s,	1H,	OH),	9.21	(s,	1H,	NH),	8.31	
(s,	1H,	Ar‐H),	7.92	(s,	1H,	CH),	7.63	(d,	J	=	7.6	Hz,	2H,	Ar‐H),	7.32	
(d,	J	=	8.0	Hz,	2H,	Ar‐H).	13C	NMR	(100	MHz,	DMSO‐d6,	δ,	ppm):	
169.9,	 163.6,	 154.9,	 148.5,	 134.5,	 132.4,	 129.5	 (2C),	 127.8,	
124.9,	123.1	 (2C),	 121.5.	MS	 (EI,	m/z	 (%)):	 349	 [M+H]+.	Anal.	
calcd.	 for	 C13H8N4O4S2:	 C,	 44.82;	 H,	 2.31;	 N,	 16.08.	 Found:	 C,	
44.94;	H,	2.34;	N,	16.18%.	

2‐Imino‐5‐(4‐methoxybenzylidene)‐3‐(5‐nitrothiazol‐2‐yl)	
thiazolidin‐4‐one	(5c):	Color:	Light	Yellow.	Yield:	94%.	1H	NMR	
(300	MHz,	DMSO‐d6,	δ,	ppm):	13.12	(s,	1H,	NH),	8.31	(s,	1H,	Ar‐
H),	7.69	(s,	1H,	CH),	7.65	(d,	J	=	7.5	Hz,	2H,	Ar‐H),	7.29	(d,	J	=	7.2	
Hz,	2H,	Ar‐H),	3.99	(s,	3H,	CH3).	13C	NMR	(75	MHz,	DMSO‐d6,	δ,	
ppm):	 171.9,	 166.6,	 156.2,	 150.5,	 141.5,	 136.2,	 132.5	 (2C),	
128.3,	 126.3,	 122.4	 (2C),	 120.6,	 60.3.	 MS	 (EI,	m/z	 (%)):	 363	
[M+H]+.	Anal.	calcd.	for	C14H10N4O4S2:	C,	46.40;	H,	2.78;	N,	15.46.	
Found:	C,	46.51;	H,	2.81;	N,	15.52%.	

5‐(4‐(Benzyloxy)benzylidene)‐2‐imino‐3‐(5‐nitrothiazol‐2‐yl)	
thiazolidin‐4‐one	(5d):	Color:	Brown.	Yield:	93%.	1H	NMR	(300	
MHz,	CDCl3,	δ,	ppm):	13.12	(s,	1H,	NH),	8.53	(s,	1H,	Ar‐H),	7.81	
(s,	1H,	CH),	7.72	(d,	J	=	7.2	Hz,	2H,	Ar‐H),	7.67‐7.54	(m,	7H,	Ar‐



Samala	et	al.	/	European	Journal	of	Chemistry	5	(3)	(2014)	550‐556	 553	
 

H),	5.22	(s,	2H,	CH2).	 13C	NMR	(75	MHz,	CDCl3,	δ,	ppm):	169.6,	
162.6,	 154.7,	 144.9,	 139.6,	 136.6,	 134.2,	 133.2	 (2C),	 132.5,	
132.0	(2C),	130.5,	128.2	(2C),	126.2,	124.8	(2C),	120.6,	70.2.	MS	
(EI,	 m/z	 (%)):	 439	 [M+H]+.	 Anal.	 calcd.	 for	 C20H14N4O4S2:	 C,	
54.78;	H,	3.22;	N,	12.78.	Found:	C,	54.85;	H,	3.26;	N,	12.81%.	

2‐Imino‐5‐(4‐methylbenzylidene)‐3‐(5‐nitrothiazol‐2‐yl)	
thiazolidin‐4‐one	(5e):	Color:	Light	Yellow.	Yield:	89%.	1H	NMR	
(400	MHz,	DMSO‐d6,	δ,	ppm):	12.54	(s,	1H,	NH),	8.41	(s,	1H,	Ar‐
H),	7.71	(d,	J	=	7.2	Hz,	2H,	Ar‐H),	7.67	(s,	1H,	CH),	7.58	(d,	J	=	7.2	
Hz,	 2H,	 Ar‐H),	 2.49	 (s,	 3H,	 CH3).	 13C	NMR	 (100	MHz,	 CDCl3,	 δ,	
ppm):	 172.1,	 166.4,	 152.5,	 149.4,	 138.4,	 136.3,	 135.2	 (2C),	
134.2,	 131.4	 (2C),	 126.2,	 122.1,	 22.3.	 MS	 (EI,	m/z	 (%)):	 347	
[M+H]+.	Anal.	calcd.	for	C14H10N4O3S2:	C,	48.54;	H,	2.91;	N,	16.17.	
Found:	C,	48.61;	H,	2.93;	N,	16.21%.	

5‐(4‐(Dimethylamino)benzylidene)‐2‐imino‐3‐(5‐nitrothia	
zol‐2‐yl)thiazolidin‐4‐one	 (5f):	 Color:	 Yellow.	 Yield:	 76%.	 1H	
NMR	 (400	MHz,	DMSO‐d6,	 δ,	 ppm):	12.87	 (s,	 1H,	NH),	8.35	 (s,	
1H,	Ar‐H),	7.72	(s,	1H,	CH),	7.63	(d,	J	=	7.2	Hz,	2H,	Ar‐H),	7.54	(d,	
J	=	 8.0	Hz,	 2H,	 Ar‐H),	 3.15	 (s,	 6H,	 2CH3).	 13C	NMR	 (100	MHz,	
DMSO‐d6,	 δ,	 ppm):	 169.9,	 164.1,	 153.3,	 149.4,	 141.4,	 136.4,	
133.2	 (2C),	 130.9,	 128.8,	 126.3	 (2C),	 125.5,	 44.4	 (2C).	MS	 (EI,	
m/z	 (%)):	376	 [M+H]+.	Anal.	 calcd.	 for	C15H13N5O3S2:	C,	47.99;	
H,	3.49;	N,	18.65.	Found:	C,	47.97;	H,	3.58;	N,	18.71%.	

2‐Imino‐3‐(5‐nitrothiazol‐2‐yl)‐5‐(3,4,5‐trimethoxybenzylide	
ne)thiazolidin‐4‐one	 (5g):	 Color:	 Yellow.	 Yield:	 88%.	 1H	 NMR	
(400	MHz,	DMSO‐d6,	δ,	ppm):	13.23	(s,	1H,	NH),	8.45	(s,	1H,	Ar‐
H),	7.72	 (s,	1H,	CH),	7.11	 (s,	2H,	Ar‐H),	3.90	 (s,	9H,	3CH3).	 13C	
NMR	 (100	 MHz,	 DMSO‐d6,	 δ,	 ppm):	 171.7,	 166.2,	 152.1	 (2C),	
148.2,	146.3,	142.3,	137.2,	135.9,	132.6,	130.3,	126.4	(2C),	61.3,	
60.6	 (2C).	 MS	 (EI,	 m/z	 (%)):	 423	 [M+H]+.	 Anal.	 calcd.	 for	
C16H14N4O6S2:	 C,	 45.49;	 H,	 3.34;	 N,	 13.26.	 Found:	 C,	 45.52;	 H,	
3.41;	N,	13.32%.	

5‐(2‐Fluorobenzylidene)‐2‐imino‐3‐(5‐nitrothiazol‐2‐yl)	
thiazolidin‐4‐one	(5h):	Color:	Light	Yellow.	Yield:	78%.	1H	NMR	
(400	MHz,	DMSO‐d6,	δ,	ppm):	11.91	(s,	1H,	NH),	8.31	(s,	1H,	Ar‐
H),	8.18	(s,	1H,	CH),	7.94	(d,	J	=	7.6	Hz,	1H,	Ar‐H),	7.81‐7.63	(m,	
3H,	Ar‐H).	13C	NMR	(100	MHz,	DMSO‐d6,	δ,	ppm):	168.3,	163.5,	
156.1,	 146.2,	 134.8,	 134.2,	 133.5,	 132.9,	 128.4,	 126.9,	 124.4,	
120.3,	 119.4.	 MS	 (EI,	m/z	 (%)):	 350	 [M+H]+.	 Anal.	 calcd.	 for	
C13H7FN4O3S2:	 C,	 44.57;	 H,	 2.01;	 N,	 15.99.	 Found:	 C,	 44.61;	 H,	
1.98;	N,	16.01%.	

5‐(4‐Chlorobenzylidene)‐2‐imino‐3‐(5‐nitrothiazol‐2‐yl)	
thiazolidin‐4‐one	(5i):	Color:	Brown.	Yield:	81%.	1H	NMR	(400	
MHz,	DMSO‐d6,	 δ,	 ppm):	 13.02	 (s,	 1H,	NH),	 8.41	 (s,	 1H,	Ar‐H),	
7.81	(s,	1H,	CH),	7.72	(d,	J	=	8.0	Hz,	2H,	Ar‐H),	7.56	(d,	J	=	8.0	Hz,	
2H,	Ar‐H).	13C	NMR	(100	MHz,	DMSO‐d6,	δ,	ppm):	166.9,	160.2,	
152.7,	146.8,	137.4,	134.8,	133.3(2C),	132.3,	128.4(2C),	126.2,	
121.5.	 MS	 (EI,	 m/z	 (%)):	 367	 [M+H]+.	 Anal.	 calcd.	 for	
C13H7ClN4O3S2:	C,	 42.57;	H,	 1.92;	N,	 15.27.	 Found:	 C,	 42.63;	H,	
1.99;	N,	15.32%.	

2‐Imino‐5‐(3‐nitrobenzylidene)‐3‐(5‐nitrothiazol‐2‐yl)	
thiazolidin‐4‐one	(5j):	Color:	Yellow.	Yield:	88%.	1H	NMR	(400	
MHz,	DMSO‐d6,	δ,	ppm):	δ	13.11	(s,	1H,	NH),	8.32	(s,	1H,	Ar‐H),	
8.27	(s,	1H,	CH),	7.94‐7.76	(m,	3H,	Ar‐H),	7.62	(t,	J	=	7.6	Hz,	1H,	
Ar‐H).	 13C	 NMR	 (100	 MHz,	 DMSO‐d6,	 δ,	 ppm):	 170.1,	 163.2,	
152.2,	 147.3,	 138.1,	 136.2,	 135.0,	 133.3,	 132.4,	 127.3,	 125.9,	
123.3,	 121.6.	 MS	 (EI,	m/z	 (%)):	 378	 [M+H]+.	 Anal.	 calcd.	 for	
C13H7N5O5S2:	 C,	 41.38;	 H,	 1.87;	 N,	 18.56.	 Found:	 C,	 41.41;	 H,	
1.96;	N,	18.61%.	

2‐Imino‐5‐((5‐nitrofuran‐2‐yl)methylene)‐3‐(5‐nitrothiazol‐
2‐yl)thiazolidin‐4‐one	 (5k):	Color:	Brown.	Yield:	69%.	 1H	NMR	
(400	MHz,	CDCl3,	δ,	ppm):	13.21	(s,	1H,	NH),	8.49	(s,	1H,	Ar‐H),	
8.39	(d,	J	=	6.8	Hz,	1H,	Ar‐H),	8.21	(d,	J	=	7.2	Hz,	1H,	Ar‐H),	7.99	
(s,	 1H,	 CH).	 13C	 NMR	 (100	MHz,	 CDCl3,	 δ,	 ppm):	 172.1,	 163.3,	
152.8,	150.8,	143.2,	142.4,	136.3,	133.9,	126.8,	124.2,	123.6.	MS	
(EI,	 m/z	 (%)):	 368	 [M+H]+.	 Anal.	 calcd.	 for	 C11H5N5O6S2:	 C,	
35.97;	H,	1.37;	N,	19.07.	Found:	C,	36.02;	H,	1.39;	N,	19.19%.	

2‐Imino‐3‐(5‐nitrothiazol‐2‐yl)‐5‐((5‐nitrothiophen‐2‐yl)	
methylene)thiazolidin‐4‐one	 (5l):	Color:	Brown.	Yield:	76%.	 1H	
NMR	(400	MHz,	CDCl3,	δ,	ppm):	13.14	(s,	1H,	NH),	8.53	(s,	1H,	
Ar‐H),	8.34	(d,	J	=	6.8	Hz,	1H,	Ar‐H),	8.17	(d,	J	=	7.2	Hz,	1H,	Ar‐

H),	7.82	(s,	1H,	CH).	13C	NMR	(100	MHz,	CDCl3,	δ,	ppm):	169.4,	
161.3,	 151.4,	 150.3,	 142.8,	 141.3,	 135.2,	 132.7,	 125.9,	 123.9,	
122.4.	 MS	 (EI,	 m/z	 (%)):	 384	 [M+H]+.	 Anal	 calcd	 for	
C11H5N5O5S3:	 C,	 34.46;	 H,	 1.31;	 N,	 18.27.	 Found:	 C,	 34.52;	 H,	
1.32;	N,	18.29%.	 	

5‐(2‐Hydroxybenzylidene)‐2‐imino‐3‐(6‐nitrobenzo[d]	
thiazol‐2‐yl)thiazolidin‐4‐one	 (6a):	 Color:	 Light	 Yellow.	 Yield:	
83%.	1H	NMR	(400	MHz,	CDCl3,	δ,	ppm):	12.60	(s,	1H,	OH),	9.49	
(s,	1H,	NH),	8.31	(d,	J	=	7.2	Hz,	1H,	Ar‐H),	8.12	(s,	1H,	CH),	8.01	
(d,	J	=	7.2	Hz,	1H,	Ar‐H),	7.81‐7.74	(m,	4H,	Ar‐H),	7.63	(d,	J	=	8.0	
Hz,	 1H,	 Ar‐H).	 13C	 NMR	 (100	 MHz,	 DMSO‐d6,	 δ,	 ppm):	 169.6,	
161.1,	 155.3,	 148.8,	 138.0,	 136.1,	 132.2,	 130.5,	 128.1,	 127.4,	
126.3,	 125.8,	 125.0,	 124.0,	 122.1,	 120.6,	 120.0.	 MS	 (EI,	 m/z	
(%)):	 399	 [M+H]+.	 Anal.	 calcd.	 for	 C17H10N4O4S2:	 C,	 51.25;	 H,	
2.53;	N,	14.06.	Found:	C,	51.30;	H,	2.65;	N,	14.16%.	

5‐(4‐Hydroxybenzylidene)‐2‐imino‐3‐(6‐nitrobenzo[d]	
thiazol‐2‐yl)thiazolidin‐4‐one	 (6b):	 Color:	 Light	 Yellow.	 Yield:	
81%.	1H	NMR	(400	MHz,	DMSO‐d6,	δ,	ppm):	13.05	(s,	1H,	OH),	
9.36	(s,	1H,	NH),	8.21	(d,	J	=	7.2	Hz,	1H,	Ar‐H),	8.18	(s,	1H,	Ar‐H),	
8.01	(d,	J	=	6.8	Hz,	1H,	Ar‐H),	7.92	(s,	1H,	CH),	7.77	(d,	J	=	7.6	Hz,	
2H,	 Ar‐H),	 7.54	 (d,	 J	=	 8.0	 Hz,	 2H,	 Ar‐H).	 13C	 NMR	 (100	MHz,	
DMSO‐d6,	 δ,	 ppm):	 166.4,	 162.6,	 157.6,	 153.7,	 148.4,	 143.4,	
137.5,	133.4	(2C),	130.6,	129.3,	127.2,	125.6,	124.4	(2C),	122.4,	
120.6.	 MS	 (EI,	 m/z	 (%)):	 399	 [M+H]+.	 Anal.	 calcd.	 for	
C17H10N4O4S2:	 C,	 51.25;	 H,	 2.53;	 N,	 14.06.	 Found:	 C,	 51.29;	 H,	
2.62;	N,	14.12%.	

2‐Imino‐5‐(4‐methoxybenzylidene)‐3‐(6‐nitrobenzo[d]	
thiazol‐2‐yl)thiazolidin‐4‐one	(6c):	Color:	Brown.	Yield:	84%.	1H	
NMR	 (400	MHz,	DMSO‐d6,	 δ,	 ppm):	12.98	 (s,	 1H,	NH),	8.47	 (s,	
1H,	Ar‐H),	8.39	(d,	J	=	6.8	Hz,	1H,	Ar‐H),	8.29	(d,	J	=	7.2	Hz,	1H,	
Ar‐H),	7.87	(d,	J	=	7.6	Hz,	2H,	Ar‐H),	7.83	(s,	1H,	CH),	7.72	(d,	J	=	
7.2	Hz,	2H,	Ar‐H),	3.94	(s,	3H,	CH3).		13C	NMR	(100	MHz,	DMSO‐
d6,	 δ,	 ppm):	 165.3,	 160.6,	 156.5,	 152.4,	 144.3,	 142.4,	 139.3,	
132.9	(2C),	130.4,	128.6,	126.9,	126.0,	123.9	(2C),	122.4,	120.3,	
60.1.	 MS	 (EI,	 m/z	 (%)):	 413	 [M+H]+.	 Anal.	 calcd.	 for	
C18H12N4O4S2:	 C,	 52.42;	 H,	 2.93;	 N,	 13.58.	 Found:	 C,	 52.49;	 H,	
3.00;	N,	13.67%.	

5‐(4‐(Benzyloxy)benzylidene)‐2‐imino‐3‐(6‐nitrobenzo[d]	
thiazol‐2‐yl)thiazolidin‐4‐one	 (6d):	 Color:	 Brown.	 Yield:	 91%.	
1H	NMR	 (400	MHz,	CDCl3,	 δ,	 ppm):	 12.42	 (s,	 1H,	NH),	8.61	 (s,	
1H,	Ar‐H),	8.32	(d,	J	=	7.2	Hz,	1H,	Ar‐H),	8.10	(d,	J	=	7.2	Hz,	1H,	
Ar‐H),	7.99	 (d,	 J	=	7.2	Hz,	2H,	Ar‐H),	7.87‐7.69	 (m,	6H,	Ar‐H	+	
CH),	 7.63‐7.58	 (m,	 2H,	 Ar‐H),	 5.15	 (s,	 2H,	 CH2).	 13C	 NMR	 (75	
MHz,	 CDCl3,	 δ,	 ppm):	 168.7,	 163.0,	 155.4,	 145.4,	 141.6,	 139.2,	
138.3,	136.4,	135.3,	134.2,	133.8(2C),	132.6,	131.8	(2C),	131.2,	
130.7,	 127.4	 (2C),	 125.9,	 125.2	 (2C),	 121.5,	 71.1.	MS	 (EI,	m/z	
(%)):	 489	 [M+H]+.	 Anal.	 calcd.	 for	 C24H16N4O4S2:	 C,	 59.00;	 H,	
3.30;	N,	11.47.	Found:	C,	59.12;	H,	3.34;	N,	11.52%.	

2‐Imino‐5‐(4‐methylbenzylidene)‐3‐(6‐nitrobenzo[d]thiazol‐
2‐yl)thiazolidin‐4‐one	 (6e):	 Color:	Yellow.	Yield:	88%.	 1H	NMR	
(400	MHz,	CDCl3,	δ,	ppm):	12.33	(s,	1H,	NH),	8.32	(s,	1H,	Ar‐H),	
8.21	(d,	J	=	7.2	Hz,	1H,	Ar‐H),	8.01	(d,	J	=	7.6	Hz,	1H,	Ar‐H),	7.72	
(s,	1H,	CH),	7.63	(d,	J	=	7.2	Hz,	2H,	Ar‐H),	7.56	(d,	J	=	7.2	Hz,	2H,	
Ar‐H),	 2.43	 (s,	 3H,	 CH3).	 13C	 NMR	 (100	 MHz,	 CDCl3,	 δ,	 ppm):	
166.3,	161.8,	153.3,	149.6,	141.3,	139.2,	135.6(2C),	134.1	(2C),	
133.2,	131.5,	128.6,	127.2,	126.2,	124.6,	123.4,	21.9.	MS	(EI,	m/z	
(%)):	 397	 [M+H]+.	 Anal.	 calcd.	 for	 C18H12N4O3S2:	 C,	 54.53;	 H,	
3.05;	N,	14.13.	Found:	C,	54.61;	H,	3.09;	N,	14.21%.	

5‐(4‐(Dimethylamino)benzylidene)‐2‐imino‐3‐(6‐nitrobenzo	
[d]thiazol‐2‐yl)thiazolidin‐4‐one	(6f):	Color:	Yellow.	Yield:	72%.	
1H	NMR	 (400	MHz,	CDCl3,	 δ,	 ppm):	 12.40	 (s,	 1H,	NH),	8.34	 (s,	
1H,	Ar‐H),	8.19	(d,	J	=	7.2	Hz,	1H,	Ar‐H),	8.01	(d,	J	=	7.2	Hz,	1H,	
Ar‐H),	7.72	(s,	1H,	CH),	7.69‐7.63	(m,	2H,	Ar‐H),	7.54	(d,	J	=	8.0	
Hz,	2H,	Ar‐H),	3.12	(s,	6H,	2CH3).	13C	NMR	(100	MHz,	DMSO‐d6,	
δ,	ppm):	168.7,	163.2,	156.4,	149.4,	144.5,	143.4,	138.6,	136.2,	
134.0	 (2C),	 133.4,	 130.4,	 127.9,	126.8	 (2C),	 125.1,	 123.4,	 44.1	
(2C).	 MS	 (EI,	 m/z	 (%)):	 426	 [M+H]+.	 Anal.	 calcd.	 for	
C19H15N5O3S2:	 C,	 53.63;	 H,	 3.55;	 N,	 16.46.	 Found:	 C,	 53.69;	 H,	
3.62;	N,	16.51%.	

	
	



554	 Samala	et	al.	/	European	Journal	of	Chemistry	5	(3)	(2014)	550‐556	
	
	

 
	
	

Figure	2.	General	synthetic	procedure.
	
	
2‐Imino‐3‐(6‐nitrobenzo[d]thiazol‐2‐yl)‐5‐(3,4,5‐trimethoxy	

benzylidene)thiazolidin‐4‐one	 (6g):	 Color:	 Light	 Yellow.	 Yield:	
83%.	1H	NMR	(400	MHz,	DMSO‐d6,	δ,	ppm):	13.03	(s,	1H,	NH),	
8.55	(s,	1H,	Ar‐H),	8.44	(d,	J	=	7.2	Hz,	1H,	Ar‐H),	8.26	(d,	J	=	7.6	
Hz,	1H,	Ar‐H),	7.72	(s,	1H,	CH),	7.54	(s,	2H,	Ar‐H),	3.96	 (s,	9H,	
3CH3).	 13C	 NMR	 (100	 MHz,	 DMSO‐d6,	 δ,	 ppm):	 168.4,	 162.3,	
154.8,	 154.2	 (2C),	 146.2,	 145.6,	 138.7,	 136.9,	 135.6,	 134.5,	
133.2,	132.6,	131.3,	130.6,	126.2	 (2C),	61.6,	61.2	 (2C).	MS	(EI,	
m/z	(%)):	473	[M+H]+.	 	Anal.	calcd.	for	C20H16N4O6S2:	C,	50.84;	
H,	3.41;	N,	11.86.	Found:	C,	50.92;	H,	3.45;	N,	11.92%.	

5‐(2‐Fluorobenzylidene)‐2‐imino‐3‐(6‐nitrobenzo[d]thiazol‐
2‐yl)thiazolidin‐4‐one	 (6h):	Color:	Brown.	Yield:	79%.	 1H	NMR	
(400	MHz,	DMSO‐d6,	δ,	ppm):	10.43	(s,	1H,	NH),	8.44	(s,	1H,	Ar‐
H),	8.31	(d,	J	=	7.2	Hz,	1H,	Ar‐H),	8.22	(d,	J	=	6.8	Hz,	1H,	Ar‐H),	
7.89	(t,	J	=	7.2	Hz,	1H,	Ar‐H),	7.81‐7.69	(m,	3H,	Ar‐H),	7.65	(d,	J	=	
7.6	Hz,	1H,	Ar‐H).	13C	NMR	(100	MHz,	DMSO‐d6,	δ,	ppm):	166.5,	
160.6,	 153.3,	 147.4,	 137.4,	 136.4,	 134.9,	 132.5,	 131.3,	 129.6,	
127.4,	 126.7,	 124.6,	 123.3,	 122.4,	 121.3,	 120.6.	 MS	 (EI,	 m/z	
(%)):	 401	 [M+H]+.	 Anal	 calcd.	 for	 C17H9FN4O3S2:	 C,	 50.99;	 H,	
2.27;	N,	13.99.	Found:	C,	51.10;	H,	2.29;	N,	14.06%.	

5‐(4‐Chlorobenzylidene)‐2‐imino‐3‐(6‐nitrobenzo[d]thiazol‐
2‐yl)thiazolidin‐4‐one	 (6i):	 Color:	 Light	 Yellow.	 Yield:	 72%.	 1H	
NMR	(400	MHz,	DMSO‐d6):	12.79	(s,	1H,	NH),	8.24	(d,	J	=	7.2Hz,	
1H,	Ar‐H),	8.12	(d,	J	=	6.8Hz,	1H,	Ar‐H),	7.99	(s,	1H,	Ar‐H),	7.81	
(d,	J	=	7.6Hz,	2H,	Ar‐H),	7.79	(s,	1H,	CH),	7.77	(d,	J	=	8.0Hz,	2H,	
Ar‐H).	 13C	 NMR	 (100	 MHz,	 DMSO‐d6,	 δ,	 ppm):	 165.3,	 158.8,	
154.4,	146.2,	139.3,	136.9,	135.1,	133.4,	132.8	(2C),	129.1	(2C),	
127.4,	125.1,	123.7,	121.4,	120.0.	MS	(EI,	m/z	(%)):	417	[M+H]+.	
Anal.	calcd.	for	C17H9ClN4O3S2:	C,	48.98;	H,	2.18;	N,	13.44	Found	
C,	49.03;	H,	2.23;	N,	13.53%.	

2‐Imino‐3‐(6‐nitrobenzo[d]thiazol‐2‐yl)‐5‐(3‐nitrobenzylide	
ne)thiazolidin‐4‐one	 (6j):	 Color:	 Yellow.	 Yield:	 84%.	 1H	 NMR	
(400	MHz,	DMSO‐d6,	δ,	ppm):	12.75	(s,	1H,	NH),	8.31	(s,	1H,	Ar‐
H),	8.16	(d,	J	=	7.2	Hz,	1H,	Ar‐H),	8.11	(s,	1H,	Ar‐H),	7.92	(d,	J	=	
7.2	Hz,	1H,	Ar‐H),	7.82	(s,	1H,	CH),	7.72‐7.62	(m,	3H,	Ar‐H).	13C	
NMR	(100	MHz,	DMSO‐d6,	 δ,	ppm):	167.1,	161.4,	156.2,	147.2,	
142.4,	 139.4,	 138.1,	 136.2,	 134.5,	 133.0,	 132.4,	 128.0,	 126.0,	
124.1,	123.6,	121.6,	120.4.	MS	(EI,	m/z	(%)):	428	[M+H]+.	Anal.	
calcd.	 for	 C17H9N5O5S2:	 C,	 47.77;	 H,	 2.12;	 N,	 16.39.	 Found:	 C,	
47.81;	H,	2.16;	N,	16.44%.	

2‐Imino‐3‐(6‐nitrobenzo[d]thiazol‐2‐yl)‐5‐((5‐nitrofuran‐2‐
yl)methylene)thiazolidin‐4‐one	 (6k):	Color:	Brown.	Yield:	70%.	
1H	NMR	(400	MHz,	CDCl3,	δ,	ppm):	12.33	(s,	1H,	NH),	8.31	(d,	J	=	
7.2	Hz,	1H,	Ar‐H),	8.21	(d,	J	=	7.6	Hz,	1H,	Ar‐H),	8.08	(d,	J	=	7.2	
Hz,	1H,	Ar‐H),	 7.99	 (s,	 1H,	 CH),	7.91	 (d,	 J	=	7.6	Hz,	1H,	Ar‐H),	
7.80	 (s,	 1H,	 Ar‐H).	 13C	NMR	 (100	MHz,	 CDCl3,	 δ,	 ppm):	 170.2,	
161.1,	 156.2,	 152.4,	 144.6,	 140.4,	 138.3,	 136.5,	 134.8,	 133.3,	
129.4,	126.4,	124.6,	123.6,	121.5.	MS	(EI,	m/z	(%)):	418	[M+H]+.	
Anal.	calcd.	for	C15H7N5O6S2:	C,	43.17;	H,	1.69;	N,	16.78.	Found:	
C,	43.22;	H,	1.72;	N,	16.82%.	

2‐Imino‐3‐(6‐nitrobenzo[d]thiazol‐2‐yl)‐5‐((5‐nitrothiophen‐
2‐yl)methylene)thiazolidin‐4‐one	(6l):	Color:	Yellow.	Yield:	72%.	
1H	NMR	(400	MHz,	CDCl3,	δ,	ppm):	12.44	(s,	1H,	NH),	8.29	(d,	J	=	
7.2	Hz,	1H,	Ar‐H),	8.17	(d,	J	=	7.6	Hz,	1H,	Ar‐H),	8.01	(d,	J	=	7.2	
Hz,	1H,	Ar‐H)	7.99	(s,	1H,	CH),	7.89	(d,	J	=	7.6	Hz,	1H,	Ar‐H),	7.72	
(s,	1H,	Ar‐H).	13C	NMR	(100	MHz,	CDCl3,	δ,	ppm):	171.3,	160.4,	
156.6,	 153.0,	 145.3,	 142.0,	 139.1,	 137.2,	 134.7,	 133.3,	 128.9,	
127.1,	125.2,	124.2,	121.9.	MS	(EI,	m/z	(%)):	434	[M+H]+.	Anal	
calcd.	 for	 C15H7N5O5S3:	 C,	 41.57;	 H,	 1.63;	 N,	 16.16.	 Found:	 C,	
41.62;	H,	1.69;	N,	16.24%.	
	
2.5.	In	vitro	MTB	screening	
	

Two‐fold	serial	dilutions	of	each	test	compound/drug	were	
prepared	 and	 incorporated	 into	 Middle‐Brook	 7H11	 agar	
medium	 with	 oleic	 acid,	 albumin,	 dextrose,	 and	 catalase	
(OADC)	 growth	 supplement	 to	 get	 final	 concentrations	 of	 50,	
25,	 12.5,	 6.25,	 3.13,	 1.56,	 and	 0.78	 μg/mL.	 Inoculum	 of	 M.	
tuberculosis	 H37Rv	 ATCC	 27294	 was	 prepared	 from	 fresh	
Middlebrook	 7H11	 agar	 slants	 with	 OADC	 (Difco)	 growth	
supplement	 adjusted	 to	 1	 mg/mL	 (wet	 weight)	 in	 Tween	 80	
(0.05%)	saline	diluted	 to	10−2	 to	give	a	concentration	of	∼107	
cfu/mL.	 Five	 microliters	 of	 this	 bacterial	 suspension	 was	
spotted	 onto	 7H11	 agar	 tubes	 containing	 different	 concent‐
rations	 of	 the	 drug	 as	 discussed	 above	 [10].	 The	 tubes	 were	
incubated	at	37	°C,	and	final	readings	(as	MIC	in	μg/mL)	were	
determined	after	28	days.	The	MIC	is	defined	as	the	minimum	
concentration	 of	 compound	 required	 to	 give	 complete	
inhibition	 of	 bacterial	 growth.	 This	 method	 is	 similar	 to	 that	
recommended	 by	 the	 National	 Committee	 for	 Clinical	
Laboratory	Standards	for	the	determination	of	MIC	in	triplicate.	

Susceptibility	testing	in	the	presence	of	the	efflux	inhibitors	
verapamil	was	carried	out	by	adding	the	respective	inhibitor	at	
sub	inhibitory	concentrations	(0.25	×	MIC)	to	the	MTB	cultures	
in	the	assay.	The	MICs	of	verapamil	>	200	μg	(taken	as	200	μg).	
	
2.6.	In	vitro	cytotoxicity	screening	
	

Some	 compounds	were	 further	 examined	 for	 toxicity	 in	 a	
RAW	264.7	cell	line	at	the	concentration	of	50	µM.	After	72	h	of	
exposure,	 viability	 was	 assessed	 on	 the	 basis	 of	 cellular	
conversion	of	MTT	into	a	formazan	product	using	the	Promega	
Cell	Titer	96	non‐radioactive	cell	proliferation	assay.	
	
3.	Results	and	discussion	
	
3.1.	Chemistry	
	

The	 target	 molecules	 were	 synthesised	 by	 following	 the	
three	step	synthetic	protocol	(Figure	2).	



Samala	et	al.	/	European	Journal	of	Chemistry	5	(3)	(2014)	550‐556	 555	
 

	
Table	1.	Biological	activities	of	synthesized	compounds.	

	
Compound	 R2	 Yield		

(%)	
M.p.		
(°C)	

C log P MTB	MIC	
(µM)	

MTB	MIC	(µM)	
(in	presence	of	verapamil)	

Cytotoxicity	at	100	µM	
(RAW	264.7	cells)	%	Inhibition	

4a	 2‐Hydroxyphenyl	 90	 208‐209	 4.64	 65.24		 ND	*	 ND	
4b	 4‐Hydroxyphenyl	 88	 221‐222	 4.64	 16.31	 ND	 22.60	
4c	 4‐Methoxyphenyl	 79	 189‐190 5.22 4.12 2.06 17.82	
4d	 4‐Benzyloxyphenyl	 93	 218‐220 6.99 6.88 6.88 19.04	
4e	 4‐Methylphenyl	 81	 205‐207 5.80 138.12 ND ND	
4f	 4‐(Dimethylamino)phenyl	 84	 234‐235 5.47 110.13 ND ND	
4g	 3,4,5‐Trimethoxyphenyl	 89	 208‐209 4.61 7.11 1.77 21.64	
4h	 2‐Fluorophenyl	 80	 201‐203 5.45 4.25 1.06 22.80	
4i	 4‐Chlorophenyl	 79	 209‐211 6.02 130.54 ND ND	
4j	 3‐Nitrophenyl	 76	 180‐181	 5.05	 63.45	 ND	 ND	
4k	 5‐Nitrofuran‐2‐yl	 92	 231‐232	 4.22	 4.07	 4.07	 26.72	
4l	 5‐Nitrothiophen‐2‐yl	 72	 241‐243	 4.78	 12.53	 ND	 24.62	
5a	 2‐Hydroxyphenyl	 88	 218‐220 1.99 117.3	 ND 12.86	
5b	 4‐Hydroxyphenyl	 79	 213‐214 1.99 39.1 ND 18.98	
5c	 4‐Methoxyphenyl	 84	 204‐205 2.58 3.78 1.89 14.56	
5d	 4‐Benzyloxyphenyl	 93	 241‐243 4.35 6.40 1.60 16.40	
5e	 4‐Methylphenyl	 89	 260‐262 3.16 63.12 ND ND	
5f	 4‐(Dimethylamino)phenyl	 76	 290‐291 2.82 118.48 ND ND	
5g	 3,4,5‐Trimethoxyphenyl	 88	 207‐208 1.96 3.31 0.82 12.40	
5h	 2‐Fluorophenyl	 78	 236‐238	 2.80	 3.90	 3.90	 16.86	
5i	 4‐Chlorphenyl	 81	 222‐223	 3.37	 59.95	 ND	 ND	
5j	 3‐Nitrophenyl	 88	 196‐198 2.41 14.63 ND 20.62	
5k	 5‐Nitrofuran‐2‐yl	 69	 232‐233	 1.58	 7.49	 3.74	 24.62	
5l	 5‐Nitrothiophen‐2‐yl	 76	 241‐242 2.14 7.21 3.60 18.72	
6a	 2‐Hydroxyphenyl	 83	 218‐220 3.54 44.85 ND 28.72	
6b	 4‐Hydroxyphenyl	 81	 227‐228 3.54 22.42 ND 16.32	
6c	 4‐Methoxyphenyl	 84	 232‐233 4.13 17.22 ND 22.30	
6d	 4‐Benzyloxyphenyl	 91	 208‐210 5.89 15.26 ND 20.63	
6e	 4‐Methylphenyl	 88	 249‐250 4.71 54.18 ND ND	
6f	 4‐(Dimethylamino)phenyl	 72	 281‐283	 4.37	 132.62	 ND	 ND	
6g	 3,4,5‐Trimethoxyphenyl	 83	 210‐213 3.51 7.40 3.7 14.80	
6h	 2‐Fluorophenyl	 79	 223‐225	 4.35	 4.26	 2.13	 20.62	
6i	 4‐Chlorphenyl	 72	 243‐244	 4.92	 85.30	 ND	 ND	
6j	 3‐Nitrophenyl	 84	 204‐206	 3.95	 66.31	 ND	 ND	
6k	 5‐Nitrofuran‐2‐yl	 70	 254‐255 3.13 6.81 1.70 18.92	
6l	 5‐Nitrothiophen‐2‐yl	 72	 241‐243 3.69 6.52 1.63 23.26	
Isoniazid	 0.72 0.72 ND	
Rifampicin	 0.24	 0.24	 ND	
Ethambutol	 7.64	 3.82	 ND	
Pyrazinamide	 50.77 ND ND	
Ciprofloxacin	 4.71 4.71 ND	
*	ND	indicates	not	determined.	
	
	

In	 the	 first	 step	 we	 treated	 the	 amines	 (1)	 like	 3‐
trifluromethyaniline,	 2‐amino‐5‐nitrothiazole	 and	 2‐amino‐6‐
nitrobenzothiazole	 with	 chloro	 acetylchloride	 without	 using	
any	 base	 under	 thermal	 conditions	 to	 get	 corresponding	
chloroacetamide	derivatives	(2).	

Refluxing	 of	 chloroacetamide	 derivatives	 (2)	 and	 potas‐
sium	thiocyanate	 in	acetone	 for	3	hours	yield	 the	correspond‐
ding	 cyclised	 3‐substituted‐2‐iminothiazolidine‐4‐ones	 (3).	 In	
final	 step	 we	 used	 Knoevenagal	 condensation	 [11]	 of	 the	
compound	3	with	various	substituted	aldehydes	using	NaOAc/	
acetic	acid	at	100	°C,	to	produce	title	compounds	(4a‐l,	5a‐l	and	
6a‐l).	These	reactions	were	also	successfully	carried	out	using	
piperidine/ethanol	 at	 90	 °C,	 but	 former	 reaction	 conditions	
favoured	 the	easy	purification,	due	 to	 the	 lower	solubility’s	of	
formed	 products	 in	 acetic	 acid.	 Direct	 filtration	 of	 reaction	
mixture	 and	 washing	 of	 residue	 with	 excess	 water,	 cold	
ethanol,	 ethers	 and	 hexanes	 produced	 the	 final	 compounds	
with	good	purity	and	in	excellent	yields	(Table	1).		
	
3.2.	Anti‐TB	activity	and	structure‐activity	relationship	
	

The	 compounds	 were	 screened	 for	 their	 in	 vitro	
antimycobacterial	 activity	 against	 M.	 tuberculosis	 H37Rv	 by	

microplate	 alamar	 blue	 assay	 method	 [12]	 for	 the	 determi‐
nation	 of	 MIC	 in	 duplicate.	 The	 minimum	 inhibitory	
concentration	 is	 defined	 as	 the	 minimum	 concentration	 of	
compound	 required	 togive	 complete	 inhibition	 of	 bacterial	
growth.	 MICs	 of	 the	 synthesized	 compounds	 along	 with	 the	
standard	 drugs	 for	 comparison	 are	 reported	 (Table	 1).	 The	
compounds	 showed	MIC’s	 ranging	 from	 3.31‐138.12	 µM;	 and	
fifteen	compounds	showed	promising	activity	with	MIC	of	 less	
than	 10	 µM.	When	 compared	 to	 original	 lead	 compound	 SID:	
24823007;	 all	 the	 compounds	 were	 found	 to	 be	 less	 active.	
When	 compared	 to	 standard	 first	 line	 antitubercular	 drug	
ethambutol	(MIC	of	7.64	µM),	fifteen	compounds	were	found	to	
be	more	 active	 and	when	 compared	 to	 pyrazinamide	 (MIC	 of	
50.77	 µM),	 twenty‐five	 compounds	 were	more	 active.	 All	 the	
molecules	were	found	is	less	active	than	isoniazid	(MIC	of	0.72	
µM)	 and	 rifampicin	 (MIC	 of	 0.24	 µM)	 but	 seven	 compounds	
were	more	active	than	DNA	gyrase	inhibitor	ciprofloxacin	(MIC	
of	4.71	µM).	Among	the	compounds,	2‐imino‐3‐(5‐nitrothiazol‐
2‐yl)‐5‐(3,	4,	5‐trimethoxybenzylidene)thiazolidin‐4‐one	(5g)	
was	found	to	be	the	most	active	compound	in	vitro	with	MICs	of	
3.31	µM.	 To	 study	 the	 SAR	we	prepared	 the	 compounds	with	
variations	 in	 N‐3	 and	 C‐5	 positions.	 In	 N‐3	 position	 we	 have	
tried	 with	 3‐trifluoromethyphenyl	 (4a‐l),	 5‐nitrothiazol‐2‐yl	



556	 Samala	et	al.	/	European	Journal	of	Chemistry	5	(3)	(2014)	550‐556	
	
(5a‐l)	and	6‐nitrobenzothiazol‐2‐yl	(6a‐l)	groups.	The	order	of	
activity	with	respect	to	N‐3	position	were;	6‐nitrobenzothiazol‐
2‐yl	 >	 3‐trifluoromethyphenyl	 >	 5‐nitrothiazol‐2‐yl	 group.	 In	
the	C‐5	position	we	have	prepared	molecules	with	phenyl	ring	
with	both	electron	donating	and	electron	with	drawing	groups	
and	 also	 with	 few	 heterocycles.	 In	 the	 case	 of	 phenyl	 ring	 at	
position	 C‐5;	 4‐hydroxyl	 substituent	 (4b,	 5b,	 and	 6b)	 were	
found	 to	 be	 two	 to	 four	 times	 more	 potent	 than	 2‐hydroxyl	
substituent	(4a,	5a,	and	6a).	Replacement	of	4‐hydroxyl	group	
with	 4‐methoxyl	 (4c,	5c,	 and	6c)	 and	4‐benzyloxy	 group	 (4d,	
5d,	 and	6d)	 increases	 the	activity;	whereas	methyl	group	(4e,	
5e	and	6e)	and	dimethylamino	group	(4f,	5f,	and	6f)	decreases	
the	 activity	 drastically.	 Tri	 substitution	with	3,4,5‐trimethoxyl	
group	 (4g,	5g,	 and	6g)	 showed	 good	 potency	with	MIC	 of	 <8	
µM.	 In	 the	 case	 of	 electron	 withdrawing	 groups;	 substitution	
with	2‐fluoro	group	(4h,	5h,	and	6h)	showed	good	activity	with	
MIC	of	<5	µM;	whereas	substituents	like	4‐chloro	group	(4i,	5i	
and	6i)	and	3‐nitro	groups	(4j,	5j	and	6j)	were	detrimental	for	
activity.	With	respect	to	heterocyclic	substituents;	we	prepared	
with	 5‐nitrofuran‐2‐yl	 and	 5‐nitrothiophen‐2‐yl	 groups	 and	
both	 of	 them	 provides	 good	 activity	 [13].	 We	 have	 also	
calculated	ClogP	with	Chemdraw	8.0	 for	all	 the	compounds	 to	
see	 the	 relationship	 between	 lipophilicity	 and	 antitubercular	
activity;	but	it	was	not	correlating	(Table	1).	

When	compared	 to	 lead	compound	SID:	24823007,	all	 the	
synthesized	compounds	showed	less	activity.	It	could	be	due	to	
involvement	 of	wide	 array	 of	 efflux	mechanisms	mediated	 by	
several	 ABC	 (ATP‐binding	 cassette)	 transporters	 and	 major	
facilitator	 superfamily	 (MFS)	proteins,	 or	 antibiotic‐modifying	
and	 ‐degrading	 enzymes,	 to	 name	 a	 few	 possibilities	 [14].	
Multiple	 drugs	 like	 verapamil,	 reserpine,	 phenothiazines	 such	
as	 thioridazine,	 and	 piperine	 have	 been	 shown	 to	 inhibit	
bacterial	efflux	pumps	in	vitro	[15].	In	general,	the	mechanisms	
by	 which	 these	 agents	 act	 are	 poorly	 understood.	 Several	
models	 have	 been	 proposed	 [16],	 such	 as:	 (1)	 direct	 binding	
and	inhibition	of	pump	assembly	or	function;	(2)	disruption	of	
the	 transmembrane	 gradients	 utilized	 by	 secondary	 trans‐
porters;	 (3)	 inhibitor	 binding	 to	 the	 antimicrobial	 compound;	
(4)	 competition	 for	 efflux.	 We	 have	 tested	 some	 selected	
compounds	 [MIC	 of	 <	 10	 µM],	 in	 presence	 of	 reported	 efflux	
pump	inhibitor	verapamil;	and	most	of	the	cases	MIC	decreased	
2	 to	 4	 fold	 when	 compared	 to	 absence	 of	 efflux	 pump.	 Most	
active	compound	5g	showed	MIC	of	0.82	µM	
	
3.3.	Cytotoxicity	study	
	

Compounds	 which	 showed	 MIC	 of	 less	 than	 50	 µM	 were	
further	 examined	 for	 cytotoxicity	 in	 a	 RAW	 264.7	 cell	 line	
(mouse	 leukaemic	 monocyte	 macrophage)	 at	 single	 concent‐
ration	of	100	µM.	We	have	selected	this	macrophage	cell	line	to	
test	 the	 toxicity	 because	 generally	MTB	 used	 to	 reside	 inside	
the	 macrophages	 and	 the	 new	 molecules	 should	 not	 possess	
any	 toxicity	 against	 macrophages.	 After	 72	 h	 of	 exposure,	
viability	 was	 assessed	 on	 the	 basis	 of	 cellular	 conversion	 of	
MTT	into	a	 formazan	product	using	the	Promega	Cell	Titer	96	
non‐radioactive	 cell	 proliferation	 assay.	 Most	 of	 the	 tested	
compounds	 were	 not	 cytotoxic	 to	 RAW	 264.7	 cells	 and	 their	
percentage	 growth	 inhibitions	were	 reported	 in	 Table	 1.	 The	
most	active	anti‐TB	compound	5g	has	shown	toxicity	of	12.4%	
at	100	µM	with	selectivity	index	of	>	30	for	MTB.		
	
4.	Conclusion	
	

In	this	study	we	have	designed,	synthesized	and	study	the	
SAR	of	various	inhibitors	of	MTB	based	on	the	lead	compound	
SID:	24823007	reported	by	TAACF.	Among	the	compounds,	2‐
imino‐3‐(5‐nitrothiazol‐2‐yl)‐5‐(3,4,5‐trimethoxybenzylidene)	
thiazolidin‐4‐one	 (19)	 was	 found	 to	 be	 the	 most	 active	
compound	 in	 vitro	 with	 MICs	 of	 3.31	 µM	 against	 log‐phase	
culture	of	MTB	and	also	non‐toxic	up	to	100	µM,	but	compound	
5g	 is	 less	 potent	 than	 lead	 compound	 SID:	 24823007.	

Compound	 5g	 showed	 MIC	 of	 0.82	 µM	 in	 presence	 of	 efflux	
pump	inhibitor.	Further	structural	optimization	required	to	get	
the	 compounds	with	 better	 potency	 than	 the	 lead	 compound	
and	 also	 to	 explore	 the	 possible	mechanism	 of	 action	 against	
various	MTB	essential	enzymes.	In	addition,	efflux	could	be	the	
rate‐limiting	step	in	the	discovery	of	novel	anti‐TB	compounds,	
as	 has	 already	 been	 recognized	 in	 the	 discovery	 of	 drugs	 for	
Gram‐negative	 bacterial	 infections.	 The	 discovery	 of	 new	
pumps	with	multiple	specificities	in	MTB	and	the	impact	these	
pumps	have	on	antitubercular	therapy	by	conferring	resistance	
to	many	of	the	new	molecules	discovered	necessitate	the	study	
of	efflux	mechanisms	as	an	important	therapeutic	target.		
	
Acknowledgements	
	

The	 authors	 are	 thankful	 to	 Department	 of	 Science	 &	
Technology	 (SR/S1/OC‐70/2010),	 New	 Delhi,	 India	 for	 their	
financial	 assistances.	 Ganesh	 Samala	 is	 thankful	 to	 Council	 of	
Scientific	&	Industrial	Research	for	providing	fellowship.	
	
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