untitled


European	Journal	of	Chemistry	6	(2)	(2015)	163‐168	

	

European	Journal	of	Chemistry	
ISSN	2153‐2249	(Print)	/	ISSN	2153‐2257	(Online)		2015	Atlanta	Publishing	House	LLC	‐	All	rights	reserved	‐	Printed	in	the	USA	

http://dx.doi.org/10.5155/eurjchem.6.2.163‐168.1224	

	
	

	

	

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

	 	 	

Synthesis	of	novel	cyanoacetamides	derivatives	and	their	urease	inhibition	
studies	

Abdul	Rauf	1,	Khalil	Ahmed	Nazish	1,	Faiz‐ul	Hassan	Nasim	1,		
Asma	Yaqoob	1	and	Ashfaq	Mahmood	Qureshi	2,*	

1	Department	of	Chemistry,	The	Islamia	University	of	Bahawalpur,	Bahawalpur,	63100,	Pakistan	
2	Institute	of	Chemical	Sciences,	Bahauddin	Zakariya	University,	Multan,	60800,	Pakistan	

*	Corresponding	author	at:	Institute	of	Chemical	Sciences,	Bahauddin	Zakariya	University,	Multan,	60800,	Pakistan.		
Tel.:	+92.61.3338863632.	Fax:	+92.61.9210069.	E‐mail	address:	shamashfaq@yahoo.com	(A.M.	Qureshi).	

	
	

	 	

	 	 	

ARTICLE	INFORMATION	 	 ABSTRACT	

	
DOI:	10.5155/eurjchem.6.2.163‐168.1224	

Received:	19	November	2014	
Received	in	revised	form:	11	January	2015	
Accepted:	18	January	2015	
Published	online:	30	June	2015	
Printed:	30	June	2015	

	 The	present	study	reports	a	convenient	approach	for	the	synthesis	of	cyanoacetamide	based
derivatives	 (7‐27)	 via	 two‐step	 process	 involving	 Knoevenagel	 reaction,	 followed	 by	 three
component	 reaction	 to	 avail	 desired	 compounds.	 All	 the	 synthesized	 compounds	 were
obtained	in	good	to	excellent	yield	and	extensively	characterized	employing	1H	NMR,	13C	NMR,
mass	 spectrometry	 and	 physical	 parameters.	 Further,	 these	 compounds	 were	 screened	 for
urease	inhibition.	All	of	the	synthesized	compounds	exhibited	good	to	excellent	urease	activity
notably	 compound	 15	 and	 19	 showed	 excellent	 urease	 inhibition	 activity	 with	 IC50	 value
~17.34	 μg/mL	 and	 36.75	 μg/mL	 in	 comparison	 to	 thiourea	 (used	 as	 standard)	 having	 IC50
value	~27.5	μg/mL.	

KEYWORDS	
Furan	
Thiophene	
Cyanoactamides	
Urease	inhibition	activity	
Knoevenagel	condensation	
N,N‐Dimethyl	barbituric	acid	

Cite	this:	Eur.	J.	Chem.	2015,	6(2),	163‐168	

	
1.	Introduction	
	

Cyanoacetamides	 are	 highly	 reactive	 compounds	 and	 are	
utilized	 mainly	 as	 reactants,	 reaction	 intermediates	 or	
synthons	 to	 attain	 variety	 of	 organic	 compounds.	 Cyano	
acetamide	 based	 moieties	 such	 as	 phenyl‐4,6‐dimethyl‐3‐
cyano‐2‐pyridones	 (1)	 and	 phenyl‐4,5,6‐trimethyl‐3‐cyano‐2‐
pyridones	 (2)	 (Figure	 1)	 and	 many	 others	 has	 interesting	
biological	 [1]	and	pharmaceutical	applications	such	as	potent	
anti‐microbial	[2,3]	and	were	found	useful	as	herbicidal,	plant	
growth	 regulators	and	also	active	against	neoplastic	disorder	
and	 used	 as	 kynurenine‐3‐hydroxykase	 inhibitors	 [4,5],	 anti‐
inflammatory,	antiviral,	antibacterial	[6],	antitumor,	neoplasm	
inhibitory	 [7],	 tyrosine	 kinase	 inhibitory,	 and	 analgesic	
properties,	and	for	the	treatment	of	disorders	related	to	vascu‐
logenesis	 [1,2].	 The	 synthetic	 utility	 of	N‐aryl	 and/or	 heteryl	
cyanoacetamides	in	various	organic	heterocycles	reveals	their	
potential	in	evolving	better	chemotherapeutic	agents	[8,9].	

Previously,	 we	 have	 reported	 various	 analogues	 based	
upon	barbituric	acid/thiobarbituric	acid	and	proposed	phara‐
macophores	 for	biological	 activities	 such	as	urease	 inhibition	
studies	 [10,11],	 which	 provoked	 further	 to	 look	 for	 other	
valuable	 functionalities	 which	 could	 be	 incorporated	 on	 to	

potent	 nucleus.	 Therefore,	 2‐cyano‐3‐phenylprop‐2‐enamide	
derivatives	prepared	by	Knoevenagel	condensation	of	various	
aromatic	and	heterocyclic	aldehydes	with	cyanoacetamide	are	
further	 condensed	 with	 barbituric	 acid,	 N,N‐dimethyl	 barbi‐
turic	 acid,	 thiobarbituric	 acid,	 malononitrile	 and	 4‐hydroxy	
coumarin	moieties.	
	

	

Figure	1.		Cyanoacetoamide	based	pyridones.	
	
2.	Experimental	
	
2.1.	Instrumentation	

	
All	 the	 synthesized	 compounds	 were	 purified	 and	 their	

physical	parameters	were	recorded.		
	



164	 Rauf	et	al.	/	European	Journal	of	Chemistry	6	(2)	(2015)	163‐168	
	

	 	

	
	

Scheme	1
	
	
Melting	 points	 were	 determined	 with	 an	 Electrothermal	

9100	apparatus	and	were	uncorrected.	Elemental	analysis	was	
performed	 using	 a	 Costech	 ECS	 4010	 CHNS‐O	 analyzer	 at	
analytical	 laboratory	 of	 HEJ	 research	 institute	 university	 of	
Karachi.	 Mass	 spectra	 were	 recorded	 on	 a	 FINNIGAN‐MAT	
8430	mass	 spectrometer	 operating	 at	 an	 ionization	 potential	
of	 70	 eV.	 IR	 spectra	 were	 recorded	 on	 a	 Shimadzu	 IR‐470	
spectrometer.	 1H	 and	 13C	 NMR	 spectra	 were	 recorded	 on	
BRUKER	DRX‐500	AVANCE	spectro‐meter	at	500.1	and	125.8	
MHz,	 respectively.	 1H	 and	 13CNMR	 spectra	were	 obtained	 on	
solution	 in	 DMSO‐d6	 using	 TMS	 as	 internal	 standard.	 Thin	
layer	chromatography	was	performed	with	Merck	silica	gel	60,	
230‐400	mesh.		
	
2.2.	Synthesis	of	novel	derivatives	of	cyanoacetamides	(7‐
27)	

	
General	 procedure:	 Freshly	 prepared	 2‐cyano‐3‐

phenylprop‐2‐enamide	(5)	derivatives	(0.3	moles)	were	added	
to	 a	 stirring	 mixture	 of	 triethyl	 orthoformate	 (TEF)	 (0.3	
moles),	active	methylene	component	(0.3	moles)	in	2‐butanol	
and	 the	 reaction	mixture	 was	 refluxed	 till	 the	 completion	 of	
the	 reaction	 monitored	 by	 TLC	 (Scheme	 1,	 Figure	 2).	 The	
reaction	was	stopped	and	the	hot	precipitate	were	filtered	off	
and	 further	 purified	 by	 extensive	 washing	 with	 boiling	
ethanol.	 The	 desired	 compounds	 were	 carefully	 dried	 and	
weighed.	

2‐Cyano‐3‐(3,	 4‐dimethoxyphenyl)‐N‐[(2,	 4,	 6‐trioxotetra	
hydropyrimidin‐5(2H)‐ylidene)methyl]prop‐2‐enamide	 (7):	
Yield:	89%.	Color:	Light	yellow.	M.p.:	307‐310	°C.	1H	NMR	(300	
MHz,	 DMSO‐d6,	 δ,	 ppm):	 12.44	 (d,	 1H,	 J	 =	 11.7	 Hz,	 =CNH‐),	
11.47	(s,	1H,	NH,	Barbituric	acid),	11.30	(s,	1H,	NH	Barbituric	
acid),	 8.48	 (d,	 1H,	 J	 =	 11.7	 Hz,	 =CHNH‐),	 8.46	 (s,	 1H,	 Ar‐
CH=C(CN)CO‐),	8.27	(s,	1H,	Ar‐H	),	7.82	(d,	1H,	 J	=	7.5	Hz,	Ar‐
H),	7.22	(d,	1H,	J	=	8.4	Hz,	Ar‐H),	3.89	(s,	3H,	OCH3),	3.82	(s,	3H,	
OCH3).	 13C	 NMR	 (300	 MHz,	 DMSO‐d6,	 δ,	 ppm):	 165.9	 (CO),	
163.5	 (CO),	 162.6	 (CO),	 160.2	 (CO),	 155.8	 (CH),	 154.3	 (C),	
150.5	(C),	148.8	(C),	146.2	(CH),	127.6	(CH),	116.4	 (C);	113.2	
(CH),	112.1	(CH),	100.3	(C),	98.7	(C),	56.0	(C),	55.4	(C).	MS	(EI,	
m/z	 (%):	 370	 (M+,	 89),	 232	 (17),	 216	 (100),	 188	 (31),	 173	
(13),	158	(11).	Anal.	calcd.	for	C17H14N4O6:	C,	55.14;	H,	3.81;	N,	
15.13.	Found:	C,	54.77;	H,	3.48;	N,	16.17%.	

2‐Cyano‐3‐(3,	 4‐dimethoxyphenyl)‐N‐[(4,	 6‐dioxo‐2‐thioxo	
tetrahydropyrimidin‐5(2H)‐ylidene)methyl]prop‐2‐enamide	(8):	
Yield:	90%.	Color:	Light	yellow.	M.p.:	236‐238	°C.	1H	NMR	(300	
MHz,	 DMSO‐d6,	 δ,	 ppm):	 12.56	 (s,	 1H,	 NH	 Barbituric	 acid),	
12.44	(d,	1H,	J	=	9.9	Hz,	=CHNH‐),	11.39	(s,	1H,	NH	Barbituric	
acid),	 8.50	 (d,	 1H,	 J	 =	 9.9	 Hz,	 =CHNH‐),	 8.18	 (s,	 1H,	 Ar‐
CH=C(CN)CO‐),	8.09	(s,	1H,	Ar‐H),	7.83	(d,	1H,	J	=	8.1	Hz,	Ar‐H),	
7.23	(d,	1H,	J	=	8.4	Hz,	Ar‐H),	3.89	(s,	3H,	‐OCH3),	3.82	(s,	3H,	‐

OCH3).	 13C	 NMR	 (300	 MHz,	 DMSO‐d6,	 δ,	 ppm):	 178.6	 (CS),	
176.4	 (CO),	 163.7	 (CO),	 160.8	 (CO),	 156.0	 (CH),	 154.4	 (C),	
150.5	(CH),	148.8	(C),	127.7	(CH),	125.4	(C),	116.4	(CH);	113.4	
(C),	112.2	(CH),	102.9	(C),	98.6	(C),	56.0	(C),	55.5	(C).	MS	(EI,	
m/z	(%)):	386	(M+,90),	216	(100),	188	(19),	173(8.3),	156	(7),	
130(5),	116.0	(4.1),	102.0	(3.0).	Anal.	calcd.	for	C17H14N4O5S:	C,	
52.84;	 H,	 3.65;	 N,	 14.50;	 S,	 8.30.	 Found:	 C,	 52.3;	 H,	 3.48;	 N,	
13.97;	S,	8.19%.	

2‐Cyano‐3‐(3,	 4‐dimethoxyphenyl)‐N‐[(2,	 4‐dioxo‐2H‐chro	
men‐3(4H)‐ylidene)methyl]	 prop‐2‐enamide	 (9):	 Yield:	 85%.	
Color:	Light	green.	M.p.:	266‐268	°C.	1H	NMR	(300	MHz,	DMSO‐
d6,	δ,	ppm):	13.37	(d,	1H,	J	=	11.4	Hz,	=CHNH‐),	8.79	(d,	1H,	J	=	
10.8	Hz,	=CHNH‐),	8.42	(s,	1H,	Ar‐CH=C(CN)CO‐),	8.02	(d,	1H,	J	
=	7.2	Hz,	ArH),	7.83	(s,	1H,	ArH),	7.77	(d,	2H,	J	=	7.2	Hz,	ArH),	
7.40	(d,	2H,	J	=	7.5	Hz,	ArH),	7.02	(d,	1H,	J	=	8.4	Hz,	ArH),	2.48	
(s,	 6H,	 OCH3).	 13C	 NMR	 (300	 MHz,	 DMSO‐d6,	 δ,	 ppm):	 161.6	
(CO),	 160.9	 (CO),	 156.6	 (CH),	 154.8	 (CO),	 154.1	 (2C),	 151.6	
(CH),	 149.8	 (2C),	 148.0	 (CH),	 136.7	 (CH),	 128.2	 (CH),	 126.3	
(CH),	 124.9	 (CH),	 123.1	 (C),	 117.5	 (CH),	 116.7	 (CH),	 114.9	
(CH),	104.0	(C),	96.9	(C),	55.7	(C),	54.6	(C).	MS	(EI,	m/z	(%)):	
404	(M+,	52),	216	(100),	202	(31.0)	188(16),	169.9	(17).	Anal.	
calcd.	 for	 C22H16N2O6:	 C,	 65.34;	 H,	 3.99;	 N,	 6.93.	 Found:	 C,	
65.89;	H,	3.54;	N,	6.37%.	

2‐cyano‐3‐(3,	 4‐dimethoxyphenyl)‐N‐((1,3‐dimethyl‐2,	 4,	 6‐
trioxotetrahydropyrimidin‐5(2H)‐ylidene)methyl)	 acrylamide	
(10):	Yield:	96%.	Color:	Pink.	M.p.:	294‐296	°C.	 1H	NMR	(300	
MHz,	DMSO‐d6,	δ,	ppm):	12.44	(d,	1H,	J	=	9.9	Hz,	=CHNH‐),	8.50	
(d,	1H,	J	=	9.9	Hz,	=CHNH‐),	8.41	(s,	1H,	Ar‐CH=C(CN)CO‐),	8.29	
(s,	1H,	ArH),	7.57	(d,	1H,	J	=	8.4	Hz,	ArH),	7.09	(d,	1H,	J	=	8.4	Hz,	
ArH),	 3.89	 (s,	 6H,	 OCH3),	 3.82	 (s,	 6H,	 NCH3).	 13C	 NMR	 (300	
MHz,	 DMSO‐d6,	 δ,	 ppm):	 173.7	 (CO),	 171.9	 (CO),	 165.6	 (CO),	
162.3	(CO),	159.5	(CH),	156.4	(C),	153.6	(CH),149.1	(C),	129.1	
(CH),	127.4	 (C),	 119.0	 (CH),	 116.4	 (C),	 112.1	 (CH),	 107.9	 (C),	
99.9	(C),	56.0	(C),	55.5	(C),	46.0	(C),	41.5	(C).	MS	(EI,	m/z	(%)):	
398	 (M+,	 52),	 230	 (9.1),	 216	 (100),	 202	 (31.0),	 188(16),	 173	
(13),	169.9	(17),	158	(11).	

2‐Cyano‐3‐(4‐hydroxy‐3‐methoxyphenyl)‐N‐[(2,	 4,	 6‐trioxo	
tetrahydropyrimidin‐5(2H)‐ylidene)	 methyl]	 prop‐2‐enamide	
(11):	Yield:	73%.	Color:	Yellow.	M.p.:	259‐260	°C.	1H	NMR	(300	
MHz,	 DMSO‐d6,	 δ,	 ppm):	 12.43	 (d,	 1H,	 J	=	 11.7	 Hz,	 =CHNH‐),	
11.46	(s,	1H,	NH	Barbituric	acid),	11.29	(s,	1H,	NH	Barbituric	
acid),	10.47	(s,	1H,	Phenolic),8.48	(d,	1H,	J	=	11.7	Hz,	=CHNH‐),	
8.41	(s,	1H,	Ar‐CH=C(CN)CO‐),	8.33	(s,	1H,	ArH),	7.72	(d,	1H,	J	=	
7.8	 Hz,	 ArH),	 7.00	 (s,	 1H,	 ArH),	 3.83	 (s,	 3H,	 OCH3).	 13C	 NMR	
(300	MHz,	 DMSO‐d6,	 δ,	 ppm):	 166.0	 (CO),	 163.8	 (CO),	 162.7	
(CO),	160.4	(CO),	156.0	(CH),	153.8	(C),	150.5	(C),	148.6	(CH),	
146.3	(C),	128.0	(CH),	123.0	(C),	116.3	(CH),	114.5	(CH),	100.0	
(C),	 97.6(C),	 55.6	 (C).	MS	 (EI,	m/z	 (%)):	 356.0	 (M+,	 42),	 216	
(17),	202	(78),	170	(100),	140.0	 (42),	114	(42),	103	(19),	76	
(24).	



Rauf	et	al.	/	European	Journal	of	Chemistry	6	(2)	(2015)	163‐168	 165	
 

	

	
7	

	
14	

	
	
21	

	
8	

	
15	

	
22	

	
9	

	
16	

	
	
23	

	
10	

	
	
17	

	
24	

	
11	

	
18	

	
25	

	
12	

	
19	

	
26	

N
H

O

O

O

O

CN

OCH3

HO

	
13	

	
	
20	

	
27	

Figure	2.	Compounds	7‐27	synthesised	via	one	pot	multicomponent	reaction.	
	
	
2‐Cyano‐N‐[(4,6‐dioxo‐2‐thioxotetrahydropyrimidin‐5(2H)‐

ylidene)methyl]‐3‐(4‐hydroxy‐3‐methoxyphenyl)prop‐2‐enamide	
(12):	Yield:	91%.	Color:	Yellow.	M.p.:	264‐265	°C.	1H	NMR	(300	
MHz,	 DMSO‐d6,	 δ,	 ppm):	 12.43	 (d,	 1H,	 J	=	 11.7	 Hz,	 =CHNH‐),	
11.80	(s,	1H,	NH	Barbituric	acid),	11.74	(s,	1H,	NH	Barbituric	
acid	),	10.80	(s,	1H,	Phenolic),	9.44	(s,	1H,	ArH),	8.50	(d,	1H,	J	=	
12.0	Hz,	=CHNH‐),	8.41	(s,	1H,	Ar‐CH=C(CN)CO‐),	7.73	(dd,	1H,	
J	=	8.4	Hz,	ArH),	6.99	(d,	1H,	J	=	9.1	Hz,	ArH),	3.83	(s,	3H,	OCH3).	
13C	NMR	(300	MHz,	DMSO‐d6,	δ,	ppm):	178.6	(CO),	177.8	(CO),	
163.8	(CO),	162.2	(C),	160.8	(C),	158.8	(CH),	156.2	(CH),	153.9	
(C),	 147.3	 (CH),	128.1	 (CH),	 123.0	 (C),	 116.3	 (CH),	 114.6	 (C),	
100.8	(C),	96.9	(C),	55.6	(CH).	MS	(EI,	m/z	(%)):	372	(M+,	12),	
202	 (23),	 171	 (100),	 142	 (12),	 113	 (15).	 Anal.	 calcd.	 for	
C16H12N4O5S:	 C,	 51.61;	 H,	 3.25;	 N,	 15.05.	 Found:	 C,	 50.97;	 H,	
3.21;	N,	15.35%.	

2‐Cyano‐N‐[(2,4‐dioxo‐2H‐chromen‐3(4H)‐ylidene)methyl]‐
3‐(4‐hydroxy‐3‐methoxyphenyl)prop‐2‐enamide	 (13):	 Yield:	
92%.	Color:	Light	orange.	M.p.:	260‐262	°C.	1H	NMR	(300	MHz,	
DMSO‐d6,	δ,	ppm):	13.37	(d,	1H,	J	=	11.4	Hz,	=CHNH‐),	10.86	(s,	
1H,	Phenolic),	 8.78	 (d,	1H,	 J	=	10.8	Hz,	=CHNH‐),	 8.47	 (s,	 1H,	
Ar‐CH=C(CN)CO‐),	 8.02	 (d,	 1H,	 J	 =	 7.2	 Hz,	 ArH),	 7.83	 (s,	 1H,	
ArH),	7.77	(d,	2H,	J	=	7.2	Hz,	ArH),	7.40	(d,	2H,	J	=	7.5	Hz,	ArH),	
7.02	(d,	1H,	 J	=	8.4	Hz,	1H,	ArH),	3.84	(s,	3H,	OCH3).	 13C	NMR	

(300	MHz,	 DMSO‐d6,	 δ,	 ppm):	 161.6	 (CO),	 160.9	 (CO),	 156.6	
(CH),	 154.8	 (CO),	 154.1	 (2C),	 151.6	 (CH),	 149.8	 (2C),	 148.0	
(CH),	 136.7	 (CH),	 128.2	 (CH),	 126.3	 (CH),	 124.9	 (CH),	 123.1	
(C),	 117.5	 (CH),	 116.7	 (CH),	 114.9	 (CH),	 104.0	 (C),	 96.9	 (C),	
55.7	 (C).	MS	 (EI,	m/z	 (%)):	390	 (M+,	26),	216	 (27),	202	 (65),	
170.0	(100),	142	(34),	114	(40),	92	(30),	76	(22).	Anal.	calcd.	
for	C21H14N2O6:	C,	 64.62;	H,	3.62;	N,	 7.18.	 Found:	C,	 64.24;	H,	
3.79;	N,	7.23%.	

2‐Cyano‐N‐[(1,3‐dimethyl‐2,4,6‐trioxotetrahydropyrimidin‐
5(2H)‐ylidene)	 methyl]‐3‐(4‐hydroxy‐3‐methoxyphenyl)prop‐2‐
enamide	 (14):	Yield:	75%.	Color:	Yellow.	M.p.:	235‐237	°C.	 1H	
NMR	(300	MHz,	DMSO‐d6,	 δ,	ppm):	12.42	 (d,	1H,	 J	=	11.4	Hz,	
=CHNH‐),	 10.81	 (s,	 1H,	 Phenolic),	 8.59	 (d,	 1H,	 J	 =	 12.0	 Hz,	
=CHNH‐),	8.44	(s,1H,	Ar‐CH=C(CN)CO‐),	7.80	(s,	1H,	ArH),	7.75	
(d,	1H,	J	=	8.4	Hz,	ArH),	7.50	(d,	1H,	J	=	8.4	Hz,	ArH),	3.83	(s,	3H,	
OCH3),	 3.32	 (s,	 6H,	 NCH3).	 13C	 NMR	 (300	 MHz,	 DMSO‐d6,	 δ,	
ppm):	 178.3	 (CO),	 176.1	 (CO),	 163.5	 (CO),	 161.9	 (CO),	 159.5	
(CH),	 157.6	 (C),	 156.1	 (CH),152.3	 (C),	 131.2	 (CH),	 127.4	 (C),	
122.0	(CH),	119.2	(C),	117.3	(CH),	110.7	(C),	99.9	(C),	53.9	(C),	
51.5	(C),	42.9	(C).	MS	(EI,	m/z	(%)):	384.0	(M+,	56),	216	(36),	
202	(89.9),	170	(100),	142	(44),	129	(15),	114	(56),	103	(25),	
77	(14).	Anal.	calcd.	for	C18H16N4O6:	C,	56.25;	H,	4.20;	N,	14.58.	
Found:	C,	55.87;	H,	4.08;	N,	14.17%.	



166	 Rauf	et	al.	/	European	Journal	of	Chemistry	6	(2)	(2015)	163‐168	
	

2‐Cyano‐N‐[(4,6‐dioxo‐2‐thioxotetrahydropyrimidin‐5(2H)‐
ylidene)	 methyl]‐3‐(furan‐2‐yl)prop‐2‐enamide	 (15):	 Yield:	
83%.	 Color:	 Yellow.	 M.p.:	 270‐271	 °C.	 1H	 NMR	 (300	 MHz,	
DMSO‐d6,	δ,	ppm):	12.57	(s,	1H,	NH	Barbituric	acid),	12.45	(d,	
1H,	 J	 =	 11.4	 Hz,	 =CHNH‐),	 11.41	 (s,	 1H,	 NHBarbituric	 acid),	
8.47	(d,	1H,	J	=	11.7	Hz,	=CHNH‐),	8.32	(d,	2H,	J	=	4.8	Hz,	ArH),	
8.18	(s,	1H,	Ar‐CH=C(CN)CO),	7.54	(d,	1H,	J	=	3.3	Hz,	ArH).	13C	
NMR	 (300	 MHz,	 DMSO‐d6,	 δ,	 ppm):	 178.6	 (CS),	 176.4	 (CO),	
163.7	 (CO),	 160.9	 (CO),	 152.3	 (CH),	 148.6	 (C),	 147.1	 (CH),	
139.6	(CH),	137.4	(C),	127.6	(CH),	114.9	(CH),	102.9	(C),	97.0	
(C).	MS	 (EI,	m/z	 (%)):	316.0	 (M+,	8),	146	 (100),	118	 (18),	90	
(60),	63.0	(12).	Anal.	calcd.	for	C13H8N4O4S:	C,	49.37;	H,	2.55;	N,	
17.71.	Found:	C,	50.04;	H,	2.44;	N,	17.29%.	

2‐Cyano‐N‐[(1,3‐dimethyl‐2,4,6‐trioxotetrahydropyrimidin‐
5(2H)‐ylidene)	 methyl]‐3‐(furan‐2‐yl)prop‐2‐enamide	 (16):	
Yield:	83%.	Color:	Brown.	M.p.:	263‐265	°C.	1H	NMR	(300	MHz,	
DMSO‐d6,	δ,	ppm):	12.42	(d,	1H,	J	=	12.0	Hz,	=CHNH),	8.56	(d,	
1H,	 J	=	12.0	Hz,	 =CHNH‐),	 8.42	 (s,	 1H,	Ar‐CH=C(CN)CO),	 8.33	
(d,	2H,	J	=	3.6	Hz,	ArH),	7.65	(d,	1H,	J	=	3.6	Hz,	ArH),	3.32	(s,	6H,	
NCH3).	 13C	 NMR	 (300	 MHz,	 DMSO‐d6,	 δ,	 ppm):	 172.9	 (CO),	
173.7	 (CO),	 166.17	 (CO),	 162.9	 (CO),	 157.0	 (CH),	 152.7	 (C),	
149.3	 (CH),	 143.6	 (CH),	 139.4	 (C),	 137.1	 (CH),	 124.4	 (CH),	
112.7	(C),	100.7	(C),	42.7	(C),	40.0	(C).	MS	(EI,	m/z	(%)):	328.0	
(M+,	 15),	 146	 (100),	 118	 (15),	 90	 (25).	 Anal.	 calcd.	 for	
C15H12N4O5:	 C,	 54.88;	 H,	 3.68;	 N,	 17.07.	 Found:	 C,	 54.77;	 H,	
3.48;	N,	17.17%.	

2‐Cyano‐N‐[(2,4‐dioxo‐2H‐chromen‐3(4H)‐ylidene)methyl]‐
3‐(furan‐2‐yl)	 prop‐2‐enamide	 (17):	 Yield:	 78%.	 Color:	 Green	
yellow.	M.p.:	240‐243	°C.	1H	NMR	(300	MHz,	DMSO‐d6,	δ,	ppm):	
13.36	 (d,	 1H,	 J	=	 11.7	Hz,	 =CHNH‐),	 8.76	 (d,	 1H,	 J	=	12.0	Hz,	
=CHNH‐),	8.39	(s,	1H,	Ar‐CH=C(CN)CO),	8.35	(d,	1H,	J	=	7.2	Hz,	
ArH),	8.03	(d,	1H,	J	=	7.8	Hz,	ArH),	7.79	(d,	1H,	J	=	7.2	Hz,	ArH),	
7.69	(d,	1H,	J	=	3.3	Hz,	ArH),	7.39	(t,	2H,	J	=	7.9	Hz,	ArH),	6.95	
(d,	1H,	J	=	1.8	Hz,	ArH).	13C	NMR	(300	MHz,	DMSO‐d6,	δ,	ppm):	
151.6	 (2CO),	 151.4	 (CO),	 149.6	 (C),	 148.7	 (C),	 140.0	 (2CH),	
136.7	 (CH),	 136.4	 (CH),	 127.5	 (CH),	 126.3	 (2C),	 124.9	 (CH),	
119.6	(CH),	117.6	(C),	117.5	 (C),	115.4	(C),	115.0	(C).	MS	(EI,	
m/z	 (%)):	 334.0	 (M+,	 26),	 216	 (3),	 188.1	 (14),	 146.0	 (100),	
121.0	 (24),	 90	 (28),	 63.0	 (34).	 Anal.	 calcd.	 for	 C18H10N2O5:	 C,	
64.67;	H,	3.02;	N,	8.38.	Found:	C,	64.54;	H,	3.09;	N,	8.23%.	

2‐Cyano‐3‐(furan‐2‐yl)‐N‐[(2,	 4	 ,6‐trioxotetrahydropyrimi	
din‐5(2H)‐ylidene)methyl]	 prop‐2‐enamide	 (18):	 Yield:	 91%.	
Color:	Gray.	M.p.:	251‐253	°C.	1H	NMR	(300	MHz,	DMSO‐d6,	δ,	
ppm):	 12.43	 (d,	 1H,	 J	 =	 11.4	 Hz,	 =CHNH),	 11.48	 (s,	 1H,	 NH	
barbituric	acid),	11.31	(s,	1H,	NH	barbituric	acid),	8.45	(d,	1H,	J	
=	11.7	Hz,	=CHNH‐	),	8.38	(s,	1H,	Ar‐CH=C(CN)CO),	8.30	(d,	1H,	
J	 =	 3.9	 Hz,	 ArH	 ),	 7.62	 (d,	 1H,	 J	=	 3.6	Hz,	 ArH),	 6.92	 (m,	 1H,	
ArH).	13C	NMR	(300	MHz,	DMSO‐d6,	δ,	ppm):	166.0	(CO),	164.0	
(CO),	162.6	(CO),	160.1	(CO),	151.2	(CH),	148.6	(CH),	146.1	(C),	
139.6	(CH),	135.9	(CH),	126.9	(CH),	121.4	(C),	115.0	(C),	100.4	
(C).	MS	(EI,	m/z	(%)):	300.0(M+	6),	146	(87),	90	(71),	63	(100).	
Anal.	calcd.	for	C13H8N4O5:	C,	52.01;	H,	2.69;	N,	18.66;	O,	26.65;	
Found:	C,	51.91;	H,	2.79;	N,	18.76.	

2‐Cyano‐N‐[(4,6‐dioxo‐2‐thioxotetrahydropyrimidin‐5(2H)‐
ylidene)methyl]‐3‐(thiophen‐3‐yl)prop‐2‐enamide	 (19):	 Yield:	
82%.	Color:	Gray.	M.p.:	246‐250	°C.	1H	NMR	(300	MHz,	DMSO‐
d6,	δ,	ppm):	12.57	(s,	1H,	NH	Barbituric	acid),	12.43	(d,	1H,	J	=	
11.8	Hz,	 =CHNH‐),	 11.42	 (s,	 1H,	NH	Barbituric	 acid),	 8.47	 (d,	
1H,	 J	=	11.7	Hz,	 =CHNH‐),	 8.32	 (s,	 1H,	Ar‐CH=C(CN)CO),	 8.16	
(d,	2H,	J	=	4.8	Hz,	ArH),	7.48	(s,	1H,	ArH).	13C	NMR	(300	MHz,	
DMSO‐d6,	 δ,	 ppm):	 178.6	 (CS),	 176.4	 (CO),	 163.7	 (CO),	 160.8	
(CO),	148.6	(CH),	147.2(CH),	143.5(C),	141.8	(CH),	138.8	(CH),	
135.9	 (C),	129.3	(CH),	115.8	 (C),	100.9	(C).	MS	(EI,	m/z	(%)):	
332.0	(M+,	15),	318.0	(64),	306.0	(26),	284.0	(13),	265.0	(41),	
162.0	(100),	134.0	(50).	Anal.	calcd.	for	C13H8N4O3S2:	C,	46.98;	
H,	2.43;	N,	16.86.	Found:	C,	46.63;	H,	2.31;	N,	16.53%.	

2‐Cyano‐3‐(thiophen‐3‐yl)‐N‐[(2,4,6‐trioxotetrahydropyrimi	
din‐5(2H)‐ylidene)	 methyl]	 prop‐2‐enamide	 (20):	 Yield:	 84%.	
Color:	Yellowish.	M.p.:	261‐263	°C.	1H	NMR	(300	MHz,	DMSO‐
d6,	 δ,	ppm):	12.44	 (d,	1H,	 J	=	11.7	Hz,	=CHNH‐),	11.48	 (s,	1H,	
NH	Barbituric	acid),	11.44	(s,	1H,	NHB	arbituric	acid),	8.46	(s,	

1H,	Ar‐CH=C(CN)CO),	8.33	(s,	1H,	=CHNH‐	),	8.16(d,	2H,	J	=	4.8	
Hz,	 ArH),	 7.43	 (d,	 1H,	 J	 =	 3.3	 Hz,	 ArH).	 13C	 NMR	 (300	 MHz,	
DMSO‐d6,	 δ,	 ppm):	 166.0	 (CO),	 164.6	 (CO),	 162.6	 (CO),	 160.0	
(CO),	 148.8(CH),	 143.5(CH),	 138.86	 (CH),	 134.9	 (CH),	 128.6	
(C),	116.7	(C),	102.0(C),	100.3	(C),	98.25	(C).	MS	(EI,	m/z	(%)):	
316.1	 (M+,	 10),	 176.0	 (15),	 162.0	 (100),	 134.0	 (51),	 90	 (38).	
Anal.	calcd.	for	C13H8N4O4S:	C,	49.37;	H,	2.55;	N,	17.71.	Found:	
C,	49.23;	H,	2.11;	N,	17.83%.	

2‐Cyano‐N‐[(2,4‐dioxo‐2H‐chromen‐3(4H)‐ylidene)methyl]‐
3‐(thiophen‐3‐yl)prop‐2‐enamide	(21):	Yield:	75%.	Color:	Light	
yellow.	M.p.:	261‐264	°C.	1H	NMR	(300	MHz,	DMSO‐d6,	δ,	ppm):	
13.35	(d,	1H,	 J	=	11.7	Hz,	=CHNH‐),	8.87	(s,	1H,	Ar‐CH=C(CN)	
CO),	8.39	(d,	1H,	J	=	7.8	Hz,	=CHNH‐),	8.20	(s,	1H,	ArH	),	8.02	(d,	
1H,	J	=	7.2	Hz,	ArH),	7.77	(m,	1H,	ArH),	7.60	(d,	1H,	J	=	7.5	Hz,	
ArH),	7.50	(m,	2H,	ArH),	7.36	(m,	1H,	ArH).	13C	NMR	(300	MHz,	
DMSO‐d6,	 δ,	 ppm):	 159.7	 (CO),	 154.7	 (CO),	 152.3	 (CO),	 151.4	
(C),	149.0	(CH),	142.0	(C),	139.1	(CH),	136.7	(CH),	133.0	(CH),	
132.3	 (CH),	 129.4	 (C),	 126.3	 (C),	 124.9	 (CH),	 124.0	 (CH),	
123.11	 (CH),	 116.6	 (CH),	 113.0	 (C),	 102.4	 (C).	 MS	 (EI,	m/z	
(%)):	 350.0	 (M+,	 5),	 176	 (24),	 162.0	 (100),	 134.0	 (60),	 121	
(24),	90	(28),	63	(34).	Anal.	calcd.	for	C18H10N2O4S:	C,	61.71;	H,	
2.88;	N,	8.00.	Found:	C,	61.19;	H,	2.35;	N,	7.69%.	

2‐Cyano‐N‐[(1,3‐dimethyl‐2,4,6‐trioxotetrahydropyrimidin‐
5(2H)‐ylidene)methyl]‐3‐(thiophen‐3‐yl)prop‐2‐enamide	 (22):	
Yield:	89%.	Color:	Yellow.	M.p.:	290‐292	°C.	1H	NMR	(300	MHz,	
DMSO‐d6,	δ,	ppm):	12.39	(d,	1H,	J	=	11.7	Hz,	=CHNH‐),	8.81	(s,	
1H,	Ar‐CH=C(CN)CO),	8.57	(d,	1H,	J	=	11.7	Hz,	=CHNH),	8.30	(d,	
1H,	J	=	4.8	Hz,	ArH),	8.18	(s,	1H,	ArH),	7.41	(d,	1H,	J	=	4.5	Hz,	
ArH),	 3.21	 (s,	 6H,	 NCH3).	 13C	 NMR	 (300	 MHz,	 DMSO‐d6,	 δ,	
ppm):	 166.0	 (CO),	 164.6	 (CO),	 162.6	 (CO),	 160.0	 (CO),	
150.0(C),	148.5(CH),	143.5(CH),	141.6	(CH),	138.9	(CH),	135.9	
(CH),	 134.9	 (C),	 129.3	 (C),	 116.7	 (C),	 100.1	 (C),	 97.3	 (C).	MS	
(EI,	m/z	(%)):	344.0	(M+,	6),	210.1	(9),	176.0	(24),	162.0	(100),	
134	 (65),	 90.0	 (31),	 58	 (35).	 Anal.	 calcd.	 for	 C15H12N4O4S:	 C,	
52.32;	H,	3.51;	N,	16.27.	Found:	C,	51.92;	H,	3.31;	N,	16.07%.		

2‐Cyano‐N‐(2,	 2‐dicyanoethenyl)‐3‐(thiophen‐3‐yl)prop‐2‐
enamide	(23):	Yield:	93%.	Color:	Yellow.	M.p.:	269‐271	°C.	 1H	
NMR	(300	MHz,	DMSO‐d6,	 δ,	ppm):	12.39	 (d,	1H,	 J	=	11.7	Hz,	
=CHNH‐),	 8.71	 (s,	 1H,	 Ar‐CH=C(CN)CO),	 8.28	 (d,	 1H,	 J	=	 11.7	
Hz,	=CHNH‐),	7.63	(s,	1H,	ArH),	7.37	(m,	1H,	ArH),	7.29	(m,	1H,	
ArH).	13C	NMR	(300	MHz,	DMSO‐d6,	δ,	ppm):	164.0	(CO),	162.5	
(C),	153.4	(CH),	143.5	(CH),	140.4	(CH),	138.6	(CH),	137.6	(C),	
135.3	(C),	134.8	(C),	129.2	 (CH),	116.6	(C),	114.3	(C).	MS	(EI,	
m/z	 (%)):	 254.0	 (M+,	 1),	 178.0	 (27),	 162.0	 (15),	 133.0	 (66),	
108.9	 (79),	90	 (17),	58.0	 (100).	Anal.	 calcd.	 for	C12H6N4OS:	C,	
56.68;	H,	2.38;	N,	22.03.	Found:	C,	55.98;	H,	2.39;	N,	22.73%.	

2‐Cyano‐3‐(3,	4,	5‐trimethoxyphenyl)‐N‐[(2,	4,	6‐trioxotetra	
hydropyrimidin‐5(2H)‐ylidene)	 methyl]	 prop‐2‐enamide	 (24):	
Yield:	80%.	Color:	Light	pink.	M.p.:	300‐302	°C.	1H	NMR	(300	
MHz,	 DMSO‐d6,	 δ,	 ppm):	 12.44	 (d,	 1H,	 J	=	 11.5	 Hz,	 =CHNH‐),	
11.49(s,	 1H,	NH	Barbituric	 acid),	 11.31	 (s,	 1H,	NH	Barbituric	
acid),	 8.47	 (s,	1H,	Ar‐CH=C(CN)CO‐),	 8.45	 (d,	1H,	 J	=	11.5	Hz,	
=CH‐NH),	 7.54	 (s,	 2H,	 ArH),	 3.83	 (s,	 6H,	 OCH3),	 3.79	 (s,	 3H,	
OCH3).	 13C	 NMR	 (300	 MHz,	 DMSO‐d6,	 δ,	 ppm):	 165.9	 (CO),	
164.4	 (CO),	 162.5	 (CO),	 159.9	 (CO),	 155.8	 (CH),	 152.9	 (C),	
150.2	 (C),	148.8	 (CH),	146.0	 (CH),	126.4	 (C),	116.0	 (C),	109.3	
(CH),	107.9	 (C),	101.0	(C),	100.5	 (C),	99.9	 (C),	67.0	 (C),	60.40	
(C),	 56.0	 (C).	 MS	 (EI,	m/z	 (%)):	 400.1(M+,	 4),	 262	 (15),	 215	
(11),	 181	 (35),	 155	 (35),	 144	 (13),	 128	 (13),	 117(16),	 105	
(15),	90(24),	85	(63).	Anal.	calcd.	 for	C18H16N4O7:	C,	54.00;	H,	
4.03;	N,	13.99.	Found:	C,	53.34;	H,	3.89;	N,	13.78%.	

2‐Cyano‐N‐[(4,6‐dioxo‐2‐thioxotetrahydropyrimidin‐5(2H)‐
ylidene)methyl]‐3‐(3,	4,	5‐trimethoxyphenyl)prop‐2‐enamide	
(25):	 Yield:	 95%.	 Color:	 Light	 orange.	 M.p.:	 228‐232	 °C.	 1H	
NMR	(300	MHz,	DMSO‐d6,	δ,	ppm):	12.57	(s,	1H,	NH	Barbituric	
acid),	 12.44	 (d,	 1H,	 J	 =	 10.2	 Hz,	 =CHNH‐),	 11.41	 (s,	 1H,	 NH	
Barbituric	acid),	8.49	(s,	1H,	Ar‐CH=C(CN)CO),	8.45	(d,	1H,	J	=	
6.6	Hz,	=CH‐NH),	7.54	(s,	2H,	ArH),	3.80	(s,	9H,	OCH3).	13C	NMR	
(300MHz,	 DMSO‐d6,	 δ,	 ppm):	 178.6	 (CS),	 176.4	 (CO),	 163.7	
(CO),	 160.7	 (CO),	 159.8	 (C),	 156	 (CH),	 152.8	 (C),	 148.5	 (CH),	
147.0	(CH),	142.9	(C),	126.4	(C),	116.0	(C),	109.3	(H),	101.1		



Rauf	et	al.	/	European	Journal	of	Chemistry	6	(2)	(2015)	163‐168	 167	
 

	
Table	1.	In‐vitro	urease	inhibition	of	compound	7‐27	*.	
Compound	 Inhibition	(%)	at0.5	mM	±	S.E.M.	 IC50,	µM	 Compound	 Inhibition	(%)	at0.5	mM	±	S.E.M.	 IC50,	µM	
7	 31.38±0.24	 ‐ 18 27.91±0.72 ‐	
8	 41.16±0.32	 ‐ 19 81.71±0.61 36.75±0.05
9	 18.15±0.81	 ‐ 20 83.14±0.53 195.14±0.11
10	 8.12±0.41	 21 34.44±0.21 ‐	
11	 23.10±0.89	 ‐	 22	 1.65±0.24	 ‐	
12	 ‐	 ‐	 23	 53.11±0.35	 ‐	
13	 24.88±0.61	 ‐ 24 25.12±0.87 ‐	
14	 5.74±0.78	 ‐	 25	 37.63±0.13	 ‐	
15	 88.61±0.24	 179.81±0.08	 26	 13.11±0.75	 	
16	 91.11±0.59	 17.34±0.01 27 7.51±0.87 ‐	
17	 34.57±0.63	 ‐ 	
*	Thiourea	used	as	standard	(IC50	value	of	27.5	μg/mL).	
	
	
(C),	100.8	(C),	60.40	(C),60.43	(C),	56.0	(2C).	MS	(EI,	m/z	(%)):	
416	 (M+	 4.17%),	 161	 (26.9),	 196	 (30),	 224	 (52),	 371.2(100).	
Anal.	calcd.	for	C18H16N4O6S:	C,	51.92;	H,	3.87;	N,	13.45.	Found:	
C,	51.04;	H,	3.26;	N,	13.88%.	

2‐Cyano‐N‐[(1,3‐dimethyl‐2,4,6‐trioxotetrahydropyrimidin‐
5(2H)‐ylidene)	 methyl]‐3‐(3,	 4,	 5‐trimethoxyphenyl)	 prop‐2‐
enamide	 (26):	Yield:	83%.	Color:	 Light	orange.	M.p.:	 277‐280	
°C.	1H	NMR	(300	MHz,	DMSO‐d6,	δ,	ppm):	12.44	(d,	1H,	J	=	12.0	
Hz,	=CHNH‐),	8.58	(d,	1H,	J	=	11.5	Hz,	=CH‐NH),	8.52	(s,	1H,	Ar‐
CH=C(CN)CO‐),	 7.58	 (s,	 2H,	 ArH),	 3.84	 (s,	 6H,	OCH3),	 3.81	 (s,	
3H,	OCH3),	3.21	(s,	6H,	CH3).	 13C	NMR	(300	MHz,	DMSO‐d6,	 δ,	
ppm):	 165.9	 (CO),	 164.4	 (CO),	 162.5	 (CO),	 159.9	 (CO),	 155.8	
(CH),	152.9	 (C),	 150.2	 (C),	 148.8	 (CH),	 146.0	 (CH),	126.4	 (C),	
116.0	 (C),	 109.3	 (C),	 107.9	 (C),	 101.0	 (C),	 100.5	 (C),	 99.9	 (C)	
60.40	(C),	56.0	(C).	MS	(EI,	m/z	(%)):	428	(M+,	22),	246	(100),	
215	 (50),	 188	 (28),	 161	 (26).	 Anal.	 calcd.	 for	 C20H20N4O7:	 C,	
56.07;	H,	4.71;	N,	13.08.	Found:	C,	55.37;	H,	4.26;	N,	13.09%.	

2‐Cyano‐N‐[(2,4‐dioxo‐2H‐chromen‐3(4H)‐ylidene)methyl]‐
3‐(3,	 4,	 5‐trimethoxyphenyl)prop‐2‐enamide	 (27):	 Yield:	 92%.	
Color:	Yellow.	M.p.:	260‐263	°C.	1H	NMR	(300	MHz,	DMSO‐d6,	
δ,	 ppm):	 12.57	 (d,	 1H,	 J	=	10.2	Hz,	 =CHNH‐),	 8.49	 (d,	 1H,	 J	=	
11.7	Hz,	=CH‐NH),	8.41	(s,	1H,	Ar‐CH=C(CN)CO‐),	7.98	(d,	1H,	J	
=	7.2	Hz,	ArH),	7.79	(s,	1H,	ArH),	7.75	(d,	2H,	J	=	7.2	Hz,	ArH),	
7.46	(d,	1H,	J	=	7.5	Hz,	ArH),	7.12	(d,	1H,	J	=	8.4	Hz,	ArH),	3.84	
(s,	6H,	OCH3),	3.83	(s,	3H,	OCH3).	13C	NMR	(300	MHz,	DMSO‐d6,	
δ,	ppm):	182.5	(CO),	164.1	(CO),	156.3	(CH),	154.7	(CO),	151.6	
(CH),	 136.6	 (CH),	 134.9	 (CH),	 133.0	 (CH),	 126.3	 (CH),	 125.0	
(C),	 124.8	 (CH),	123.9	 (C),	 123.0	 (CH),	 122.7	 (C),	 117.5	 (CH),	
116.7	 (CH),116.5	 (C),116.1	 (C),104.1	 (C),96.9	 (C),	 55.3	 (CH3),	
44.2	(CH3),	43.1	(CH3).	Anal.	calcd.	for	C23H18N2O7:	C,	63.59;	H,	
4.18;	N,	6.45.	Found:	C,	62.99;	H,	4.36;	N,	5.88%.	
	
2.3.	Urease	inhibition	assay	
	

Urease	 inhibition	 of	 newly	 synthesized	 compounds	 was	
determined	by	the	modified	phenol‐hypochlorite	method	[10‐
15]	(Table	1).	Urease	inhibition	curve	of	thiourea	was	used	as	
reference	at	varying	concentrations	i.e.,	100,	10,	1,	0.1	and	0.01	
µM.	The	reaction	mixtures	comprising	sample	solution	(2	μL),	
phosphate	 buffer	 (17	 μL)	 and	 sodium	 nitroprusside	 (20	 μL)	
were	 incubated	 at	 37	 °C	 for	 2	 h	 in	 well	 plates.	 Then	 Griess	
reagent	(20	μL,	0.3%	sulphanilic	acid	in	glacial	acetic	acid	and	
0.1%	Naphthyl	ethylene	diamine	(NED):	equal	volumes	of	both	
solutions	mixed	just	before	use)	was	added	to	each	well	plate	
and	 the	 mixture	 kept	 at	 room	 temperature	 for	 20	 min	 to	
develop	the	color.	The	absorbance	was	measured	at	528	nm	by	
using	an	ELISA	reader.	The	results	were	processed	using	Gen	5	
software.	 All	 the	 reactions	 were	 performed	 in	 triplicate.	
Positive	 control	 (containing	 known	 antiurease)	 and	 control	
(containing	all	samples)	were	run	in	parallel.	Negative	control	
was	 used	 to	 calibrate	 the	 instrument.	 The	%	 age	 antiurease	
activity	was	determined	by	using	the	Equation	(1).	
	
%	Antiurease	activity .		 	 	 	 .		 	 	

	 	
	x	100		(1)	

	

3.	Results	and	discussion		
	
3.1.	Synthesis	
	

Synthesized	cyanoacetamide	based	compounds	7‐27	were	
obtained	 in	 good	 to	 excellent	 yield	 (59‐87%)	 as	 colored	
precipitates	 from	 hot	 stirred	 reaction	 mixture	 via	 one	 pot	
multi‐component	 reaction.	 All	 the	 compounds	 were	
recrystallized	 in	 ethanol	 and	 were	 extensively	 characterized	
by	 their	 physical,	 spectral	 (IR,	 1H	 NMR,	 13C	 NMR,	 Mass)	 and	
elemental	 analyses	 data	 to	 establish	 their	 structures.	 The	
biological	 activities	 such	 as	 antibacterial,	 antiurease	 and	
antioxidant	 were	 also	 carried	 out	 and	 structural	 activity	
relationship	was	explored.	

A	 representative	 compound	 7’s	 structure	 elucidation	 is	
discussed.	The	1H	NMR	spectrum	of	compound	7	 justified	the	
proposed	 structure,	 which	 displayed	 the	 amide	 proton	
(=CHNH‐)	downfield	as	doublet	(J	=	11.7	Hz)	at	δ	12.44	ppm.	
The	pyrimidine	nonequivalent	amide	NH	protons	appeared	as	
separate	 singlet	 at	 δ	 11.30	 ppm.	 The	 second	 NH	 appeared	
slightly	 downfield	 at	 δ	 11.47	 ppm	due	 to	 the	 involvement	 of	
intermolecular	hydrogen	bonding	between	carbonyl	oxygen	of	
pyrimidine	 ring	 and	 methylidene	 amino	 function	 (N‐H).	 The	
methylidene	proton	 (=CHNH‐)	 appeared	 as	doublet	 at	 δ	 8.48	
ppm	 (J	 =11.7	 Hz)	 and	 the	 olefinic	 proton	 (Ar‐CH=C(CN)CO‐)	
appeared	 as	 a	 singlet	 at	 δ	 8.46	 ppm.	 In	 the	 spectrum	 of	
compound	7	aromatic	proton	appeared	downfield	as	a	singlet	
at	 δ	 8.27	 ppm	whereas	 other	 aromatic	 protons	 appeared	 as	
two	 separate	 doublets	 at	 δ	 7.22	 and	7.82	ppm.	The	methoxy	
protons	 appeared	 as	 two	 singlet	 at	 δ	 3.89	 and	 3.82	 ppm,	
respectively.	

The	 13C	 NMR	 spectrum	 of	 compound	7	 accounted	 17	 Cs,	
displayed	 two	carbonyl	 carbons	 (=CCO)	downfield	at	 δ	165.9	
and	163.5	ppm	whereas	(CNCCONH)	and	(NHCONH)	at	δ	162.6	
and	 160.18	 ppm.	 The	 carbon	 (CONHCH=)	 display	 signals	
downfield	at	δ	155.8	ppm.	The	aromatic	ring	carbons	(C‐OCH3)	
displayed	signals	at	δ	154.3	and	150.6	ppm,	respectively.	The	
barbituric	 acid	 ring	 carbon	 (COCCO)	 exhibited	 signals	 at	 δ	
148.8	ppm.	The	carbons	Ar‐CH=C(CN)	CO)	appeared	at	δ	146.2	
ppm.	 The	 aromatic	 ring	 carbons	 were	 observed	 at	 δ	 127.6,	
113.2	 and	 112.2	 ppm,	 respectively.	 The	 carbons	 Ar‐
CH=C(CN)CO	 and	 the	 ipso	 carbon	 next	 the	 group	 recorded	
signals	 at	 δ	 116.4	 and	 100.3	 and	 98.7	 ppm,	 whereas	 the	
methoxy	carbons	appeared	at	δ	56.0	and	55.4	ppm.	

The	mass	 spectrum	 of	 compounds	7	 recorded	molecular	
ion	 peak	 at	 370	 amu	 and	 rest	 of	 the	 fragmentation	 was	 in	
accordance	 with	 the	 proposed	 structure.	 Furthermore,	
elemental	 analysis	 was	 in	 agreement	 with	 the	 proposed	
structure	with	anal.	calcd.	for	C17H14N4O6:	C,	55.14;	H,	3.81;	N,	
15.13.	Found:	C,	54.77;	H,	3.48;	N,	17.17%.	
	
3.2.	Anti‐urease	activity	
	

The	chemical	nature	of	cyanoacetamide	based	compounds	
encouraged	us	 to	study	 the	antiurease	activities	of	our	newly	
synthesized	 compounds.	 Antiurease	 activity	 was	 carried	 out	



168	 Rauf	et	al.	/	European	Journal	of	Chemistry	6	(2)	(2015)	163‐168	
	
by	 using	 thiourea,	 with	 an	 IC50	 value	 of	 27.5	 μg/mL,	 as	 the	
standard.	 The	 synthesized	 compounds	 7‐27	were	 examined	
for	 their	 urease	 inhibition	 activity	 at	 0.5mM	 and	 IC50µM	
concentration	 (Table	 1,	 Figure	 3).	 It	 was	 considered	 that	 all	
these	 compounds	 could	 have	 the	 ability	 to	 bind	 with	 the	
enzyme’s	 active	 site	 and	 the	 synthesized	compounds	showed	
moderate	 to	 excellent	 urease	 inhibition.	 The	 compounds	 16	
and	 19	 proved	 to	 be	 the	 most	 potent	 urease	 inhibitor,	 with	
IC50	 values	 of	 17.34	 μg/mL	 and	 36.75	 μg/mL,	 respectively.	
These	values	were	comparable	to	the	27.5	μg/mL	value	of	the	
thiourea	 (standard)	 whereas	molecules	15	 and	20	 exhibited	
comparatively	moderate	antiurease	activities.	
	

	
	
Figure	3.	Most	active	urease	inhibition	shown	by	compound	15,	16,	19	and	
20.	
	
4.	Conclusion	
	

Various	cyano	acetamides	based	compounds	were	availed	
via	 three	component‐single	pot	reactions	 in	good	 to	excellent	
yield	and	extensively	characterized.	Among	the	21	synthesized	
compounds,	 five	 of	 them	 exhibited	 moderate	 to	 excellent	
urease	inhibition.	All	these	active	compounds	compromises	of	
barbituric	 acid,	 N,N‐dimethyl	 barbituric	 acid	 and	 thiobarbi‐
turic	 acid	 moiety	 and	 these	 are	 already	 known	 for	 their	
potency	for	urease	inhibition	[12‐15].	Compound	16	exhibited	
~91%	 inhibition	 and	 IC50	 value	 of	 17	 μg/mL	 which	 in	
comparison	 to	 standard	 thiourea	 is	 more	 potent,	 similarly,	
compound	 19	 exhibited	 82%	 inhibition	 and	 IC50	 value	 of	
36μg/mL.	 In	 comparison	 to	 the	 rest	 of	 the	 synthesized	
compounds,	compounds	16	and	19	have	furan	and	thiophene	
ring,	whereas	in	all	other	compounds	substituted	phenyl	rings	
are	present.	These	two	five	member	rings	containing	O	and	S	
generating	 an	 extra	 pocket	 to	 interact	 with	 Ni	 which	 is	 so	
critical	 for	 the	 urease	 enzyme	 activity	 [17].	 Therefore	 it	 is	
speculated	that	 furan‐CH=C(CN)CO‐	and	thiophene‐CH=C(CN)	
CO‐	 are	 two	 new	pharmacophore	which	 can	 be	 employed	 to	
enhance	urease	inhibition	activity.	
	
References	
	
[1]. Fadda,	A.	A.;	Bondock,	S.;	Rabie,	R.;	Etman,	H.	A.	Turk.	J.	Chem.	2008,	

32,	259‐286.		
[2]. Shishoo,	 C.	 J.;	 Devani,	 M.	 B.;	 Ananthan,	 S.;	 Jain,	 K.	 S.;	 Bhadti,	 V.	 S.;	

Mohan;	S.;	Patel,	L.	J.	Indian	J.	Chem.	B	1989,	28,	1039‐1047.		
[3]. Hazra,	K.;	Saravanan,	J.;	Mohan,	S.Asian	J.	Chem.	2007,	19,	3541‐3544.		
[4]. Zhuravel,	 I.	 O.;	 Kovalenko,	 S.	M.;	 Ivachtchenko,	A.	 V.;	 Balakin,	K.	 V.;	

Kazmirchuk,	V.	Bioorg.	Med.	Chem.	Lett.	2005,	15(24),	5483‐5487.		
[5]. Geissler,	A.	E.;	Huppatz,	 J.	L.;	Phillips,	 J.	N.	Pestic.	Sci.	1980,	11,	432‐

438.		
[6]. Shams	H.	Z.	,	Elkholy,	Y.	M.;	Azzam,	R.	A.;	Mohareb,	R.	M.	Phosphorus	

Sulfur	1999,	155,	215‐233.		
[7]. Ismail,	M.	M.	F.;	Ammar,	Y.	A.;	El‐Zahaby,	H.	A.	S.;	Eisa,	S.	I.;	Barakat,	S.	

E.	Arch.	Pharm.	Life	Sci.	2007,	340,	476‐482.		
[8]. Litvinov,	V.	P.	Russ.	Chem.	Rev.	1999,	68,	737‐763.		
[9]. Ghani,	U.	N.;	Atta‐ur‐Rahman;	Choudhary,	M.	I.;	Ullah,	N.;	James,	M.	N.	

J.	Mol.	Biol.	2001,	314(3),	519‐525.		
[10]. Qureshi,	A.	M.;	Qadir,	M.;	Rauf,	A.;	 Idrees,	M.;	Mumtaz,	S.;	Najam‐ul‐

Haq,	M.;	Aziz‐ur‐rehman;	 Ismail,	M.;	Athar,	M.;	Khushal,	 R.;	 Riaz,	 S.;	
Bokhari,	H.	Lett.	Drug	Des.	Discov.	2011,	8(10),	980‐987.		

[11]. Halliwell,	B.;	Gutteridge,	J.	M.	Meth.	Enzymol.	1990,	186,	1‐85.		
[12]. Rauf,	 A.;	 Liaqat,	 S.;	 Qureshi,	 A.	 M.;	 Yaqub,	 M.;	 Aziz‐ur‐Rehman,	

Hassan,	M.	U.;	Chohan,	Z.	H.;	Nasim,	F.	U.	H.;	Hadda,	T.	B.	J.	Med.	Chem.	
Res.	2012,	21,	60‐74.	

[13]. Shaukat,	A.;	Rasool,	N.;	Ullah,	A.;	Nasim,	F.	H.;	Yaqoob,	A.;	Zubair,	M.;	
Rashid,	U.;	Riaz,	M.	Molecules,	2013,	18,	14711‐14725.		

[14]. Garrat,	D.	 C.	Quantitative	 analysis	 of	 drugs.	 3rd	 edition,	 Chapman	&	
Hall	Ltd,	1964.	

[15]. Qureshi,	A.	M.;	Saira,	M.;	Rauf,	A.;	Ashraf,	M.;	Nasar,	R.;	Zahid,	H.	C.	J.	
Enzym.	Inhib.	Med.	Chem.	2015,	30,	119‐125.	

[16]. Rauf,	A.;	Ahmed.	F.;	Qureshi,	A.	M.;	Aziz‐ur‐Rehman;	Khan,	A.;	Qadir,	
M.	I.;	Choudhary,	M.	I.;	Chohan,	Z.	H.;	Youssoufi,	M.	H.;	Hadda	T.	B.	J.	
Chin.	Chem.	Soc.	2011,	58,	528‐537.		

[17]. Upadhyay,	L.	S.	B.	Ind.	J.	BioTech.	2012,	11,	381‐388.