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European	Journal	of	Chemistry	2	(3)	(2011)	347‐355	

	
European	Journal	of	Chemistry	

ISSN	2153‐2249	(Print)	/	ISSN	2153‐2257	(Online)		2011	EURJCHEM	
DOI:10.5155/eurjchem.2.3.347‐355.168	

	
	

	

	

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

	 	 	

Synthesis	of	novel	pyrimidine	and	fused	pyrimidine	derivatives	

Mahmoud	Refaee	Mahmoud,	Ahmed	Kamel	El‐Ziaty,		
Mahmoud	Fawzy	Ismail*	and	Sayed	Ahmed	Shiba	

Chemistry	Department,	Faculty	of	Science,	Ain	Shams	University,	Abbassia,	Cairo,	11566,	Egypt	

*Corresponding	author	at:	Chemistry	Department,	Faculty	of	Science,	Ain	Shams	University,	Abbassia,	Cairo,	11566,	Egypt.	Tel.:	+0224831836;	fax:	+202.24831836.		
E‐mail	address:	fawzy2010@sci.asu.edu.eg	(M.F.	Ismail).	

	
	

	 	

	 	 	
ARTICLE	INFORMATION	 	 ABSTRACT	
Received:	15	June	2010	
Received	in	revised	form:	26	October	2010	
Accepted:	08	November	2010	
Online:	30	September	2011	

KEYWORDS	

	 4‐Amino‐2‐(benzylthio)‐6‐(4‐methoxyphenyl)pyrimidine‐5‐carbonitrile	 (1)	was	 prepared	 by
treatment	 of	 s‐benzylthiuronium	 chloride	 with	 2‐(4‐methoxybenzylidene)malononitrile	 in
ethanolic	 sodium	 hydroxide	 with	 hydrazine	 hydrate	 to	 afford	 the	 hydrazino	 derivative	 2,
which	 was	 allowed	 to	 react	 with	 different	 electrophilic	 reagents	 to	 give	 the	 pyrimidine
derivatives	 3‐12.	 The	 proclivity	 of	 (E)‐2‐cyano‐3‐(4‐nitrophenyl)acrylamide	 (13)	 towards
carbon	and	nitrogen	nucleophiles	was	also	investigated.	IR,	1H	NMR	and	mass	spectra	for	all
the	synthesized	compounds	were	discussed.	All	derived	compounds	were	investigated	for	anti
avian	 influenza	 (H5N1)	virus	 activity	 and	 compared	with	 zanamivir	 as	 control	 drug.	All	 the
synthesized	compounds	didn’t	possess	any	antiviral	activity.	

s‐Benzylthiuronium	chloride	
Activated	nitriles	
Pyrimidines	
Triazolopyrimidine	derivatives	
Antiviral	activity	
Cytotoxicity	

	
1.	Introduction	
	

The	 recent	wide	 applications	 of	 pyrimidine	 derivatives	 as	
anti‐tumor	[1],	anti‐HIV‐1	[2],	analgesic	[3],	anti‐depressive	[4],	
anti‐convulsant	 [5],	 anti‐microbial	 [6],	 herbicides	 [7,8],	 anti‐
inflammatory	 and	 antioxidant	 [3,9],	 beside	 their	 uses	 as	 a	
precursors	 in	 the	 synthesis	 of	 fused	 ring	 compounds	 like,	
triazolopyrimidines	as	antibacterial	agents	[10],	fungicidal	[11],	
and	 anti‐tumor	 agents	 [12],	 imidazopyrimidines	 as	
antimycobacterial	 [13]	and	pyridopyrimidines	as	antibacterial	
agents	[14]	make	them	worthy	to	be	synthesized	and	evaluated	
as	 drugs.	 We	 report	 here	 synthesis	 of	 pyrimidine	 and	 fused	
pyrimidines	 by	 developed,	 simple,	 convenient	 and	 efficient	
procedure.	 Also,	 this	 modified	 method	 is	 fast,	 cheap	 and	
unequivocal	preparation	with	improved	yields.	
	
2.	Experimental	
	
2.1.	Instrumentation	
	

Melting	points	 are	 uncorrected	 and	were	measured	 by	 an	
electric	 melting	 point	 apparatus	 (G‐K).	 The	 IR	 spectra	 were	
recorded	 on	 a	 Pye‐Unicam	 SP1200	 spectrophotometer	 using	
KBr	Wafer	technique.	The	1H	NMR	spectra	were	determined	on	
a	Varian	GEMINI	300	MHz	NMR	spectrophotometer	using	CDCl3	
or	 DMSO‐d6	 as	 solvent	 and	 TMS	 as	 an	 internal	 standard.	 All	
chemical	shifts	are	in	ppm	downfield	from	TMS.	The	elemental	
analysis	 was	 carried	 out	 in	 faculty	 of	 Science,	 Ain	 Shams	
University.	MS	were	 recorded	on	 Shimadzu	GC‐MS	QP1000EX	
instrument	 in	 micro	 analytical	 lab,	 Cairo	 University.	 The	
monitoring	of	the	progress	of	all	reactions	and	homogeneity	of	
the	synthesized	compound	was	carried	out	by	TLC.	
	
	

2.2.	Synthesis	
	
2.2.1.	4‐amino‐2‐(benzylthio)‐6‐(4‐methoxyphenyl)	
pyrimidine‐5‐carbonitrile	(1)	
	

To	a	 solution	of	 s‐benzylthiuronium	chloride	 (2.02	g,	 0.01	
mol)	 in	 water	 (10	 mL),	 sodium	 hydroxide	 (1	 N,	 10	mL)	 was	
added	 dropwise	 with	 shaking,	 the	 pale	 green	 precipitate	 so	
formed,	 was	 dissolved	 in	 warmed	 ethanol	 (10	 mL),	 then	 a	
solution	 of	 2‐(4‐methoxybenzylidene)malononitrile	 (1.84	 g,	
0.01	mol)	in	ethanol	(20	mL)	was	added	and	the	whole	mixture	
was	heated	under	reflux	for	5h.	The	solid	formed	after	cooling	
was	 collected	 by	 filtration	 and	 recrystallized	 from	 toluene	 to	
give	1	as	buff	crystals	(Scheme	1).	Yield:	82%.	M.p.:	171‐173	°C.	
FT‐IR	 (KBr,	 cm‐1):	 3461,	 3298	 υ(NH2),	 2204	 υ(C≡N),	 1626	
υ(C=N).	1H	NMR	(300	MHz,	CDCl3):	8.03	(d,	J=8.4	Hz,	2H,	Ar‐H),	
7.40‐7.27	(m,	5H,	Ar‐H),	7.00	(d,	J=8.7	Hz,	2H,	Ar‐H),	5.67	(s,	2H,	
CH2),	4.45	(br.s,	2H,	NH2,	 exchangeable	with	D2O),	3.89	(s,	3H,	
OCH3).	MS	 (EI,	m/z):	 348	 (M+).	Anal.	 Calcd.	 for	C19H16N4OS:	 C,	
65.50;	 H,	 4.63;	 N,	 16.08;	 S,	 9.20.	 Found:	 C,	 65.52;	 H,	 4.59;	 N,	
16.02;	S,	9.12%.	
	
2.2.2.	4‐amino‐2‐hydrazinyl‐6‐(4‐methoxyphenyl)	
pyrimidine‐5‐carbonitrile	(2)	
	

A	mixture	 of	 1	 (3.48	 g,	 0.01	mol)	 and	 hydrazine	 hydrate	
80%	(0.015	mol)	 in	ethanol	 (50	mL)	was	heated	under	 reflux	
for	3h.	The	colourless	solid	separated	on	hot	was	collected	by	
filtration	 and	 then	 recrystallized	 from	 dioxane	 to	 give	 2	 as	
colourless	crystals	(Scheme	2).	Yield	35%.	M.p.:	300‐302	°C.	FT‐
IR	 (KBr,	 cm‐1):	 3454,	 3302	 υ(NH2,	 NH),	 2194	 υ(C≡N),	 1634	
υ(C=N).	MS	 (EI,	m/z):	 256	 (M+).	Anal.	 Calcd.	 for	C12H12N6O:	 C,	
56.24;	H,	4.72;	N,	32.79.	Found:	C,	56.13;	H,	4.62;	N,	32.81%.	
	



348	 Mahmoud	et	al.	/	European	Journal	of	Chemistry	2	(3)	(2011)	347‐351	
	

	

	
	

Scheme	1
	
	
2.2.3.	4‐amino‐2‐(3,5‐dimethyl‐1H‐pyrazol‐1‐yl)‐6‐(4‐
methoxyphenyl)pyrimidine‐5‐carbonitrile	(3)	
	

A	mixture	 of	 compound	2	 (1.0	 g,	 3.9	mmol)	 and	pentane‐
2,4‐dione	 (0.4	 ml,	 3.9	mmol)	 in	 ethanol	 (25	mL)	 was	 heated	
under	reflux	for	6h.	The	solid	deposited	while	hot	was	collected	
by	 filtration	 and	 crystallized	 from	 dioxane	 to	 give	 3	 as	
colourless	crystals	(Scheme	2).	Yield	86%.	M.p.:	280‐282	°C.	FT‐
IR	(KBr,	cm‐1):	3477,	3273	υ(NH2),	2206	υ(C≡N),	1648	υ(C=N).	
1H	NMR	(300	MHz,	DMSO‐d6):	7.96	 (d,	 J=9	Hz,	2H,	Ar‐H),	7.13	
(d,	 J=8.8	 Hz,	 2H,	 Ar‐H),	 6.1	 (s,	 1H,	 =C4‐H),	 4.2	 (br.s,	 2H,	 NH2,	
exchangeable	with	D2O),	3.86	(s,	3H,	OCH3),	3.3	(s,	3H,	C3‐CH3),	
2.1	 (s,	 3H,	 C5‐CH3).	 MS	 (EI,	 m/z):	 320	 (M+).	 Anal.	 Calcd.	 for	
C17H16N6O:	C,	63.74;	H,	5.03;	N,	26.23.	Found:	C,	63.75;	H,	4.47;	
N,	26.25%.	
	
2.2.4.	N‐(5‐Cyano‐2‐(2,2‐diacetylhydrazinyl)‐6‐(4‐methoxy	
phenyl)pyrimidin‐4‐yl)	acetamide	(4)	
	

A	solution	of	2	 (1.0	g,	3.9	mmol)	 in	 freshly	distilled	acetic	
anhydride	 (10	 mL)	 was	 heated	 under	 reflux	 for	 6h.	 After	
cooling,	the	reaction	mixture	was	poured	on	ice	cold	water.	The	
crude	deposited	was	collected	and	crystallized	from	ethanol	to	
give	4	as	colourless	crystals	(Scheme	2).	Yield	76%.	M.p.:	182‐
184	 °C.	 FT‐IR	 (KBr,	 cm‐1):	 3368	 υ(NH),	 2216	 υ(C≡N),	 1716	
υ(C=O).	 1H	 NMR	 (300	 MHz,	 CDCl3):	 8.17	 (s,	 1H,	 NH,	
exchangeable	with	 D2O),	 8.03	 (d,	 J=8.8	 Hz,	 2H,	 Ar‐H),	 7.27	 (s,	
1H,	NH,	 exchangeable	with	D2O),	 7.03	 (d,	 J=8.8	Hz,	 2H,	 Ar‐H),	
3.91	 (s,	3H,	OCH3),	2.85	 (s,	3H,	CH3),	2.44	(s,	6H,	2COCH3).	MS	
(EI,	m/z(%)):	382	(M+).	Anal.	Calcd.	for	C18H18N6O4:	C,	56.54;	H,	
4.74;	N,	21.98.	Found:	C,	56.37;	H,	4.62;	N,	21.81%.	
	
2.2.5.	N'‐(4‐amino‐5‐cyano‐6‐(4‐methoxyphenyl)pyrimidin‐
2‐yl)furan‐2‐carbohydrazide	(5)	
	

A	mixture	of	2	 (1.0	g,	3.9	mmol)	and	 furoyl	chloride	(0.51	
mL,	3.9	mmol)	in	pyridine	(20	mL)	was	heated	under	reflux	for	
3h.	 After	 cooling	 the	 reaction	 mixture	 acidified	 on	 ice	 cold	
acetic	acid.	The	deposited	was	filtered	off,	washed	several	times	

with	 cold	 water	 and	 recrystallized	 from	 toluene	 to	 give	 5	 as	
pale	 yellow	 crystals	 (Scheme	2).	 Yield	52%.	M.p.:	 110‐112	 °C.	
FT‐IR	(KBr,	cm‐1):	3455,	3318,	3219	υ(NH2,	NH),	2206	υ(C≡N),	
1680	υ(C=O)	(hydrazide).	MS	(EI,	m/z):	350	(M+).	Anal.	Calcd.	
for	C17H14N6O3:	C,	58.28;	H,	4.03;	N,	23.99.	Found:	C,	58.35;	H,	
4.12;	N,	24.14%.	
	
2.2.6.	4‐amino‐2‐(1,3‐dioxoisoindolin‐2‐ylamino)‐6‐(4‐
methoxyphenyl)pyrimidine‐5‐carbonitrile	(6)	
	

A	mixture	 of	 2	 (1.0	 g,	 3.9	mmol)	 and	 phthalic	 anhydride	
(0.58	g,	3.9	mmol)	in	glacial	acetic	acid	was	heated	under	reflux	
for	one	hour.	The	white	solid	deposited	while	hot	was	collected	
by	 filtration	and	 then	recrystallized	 from	dioxane	 to	give	6	 as	
colourless	 crystals	 (Scheme	 2).	 Yield	 75%.	 M.p.:	 over	 300	 °C.	
FT‐IR	 (KBr,	 cm‐1):	 3414,	 3374,	 3333,	 3168	 υ(NH2,	 NH),	 2205	
υ(C≡N),	 1791,	 1731	 υ(C=O)	 (coupling	 bands).	 MS	 (EI,	 m/z):	
386	(M+).	Anal.	Calcd.	for	C20H14N6O3:	C,	62.17;	H,	3.65;	N,	21.75.	
Found:	C,	62.18;	H,	3.63;	N,	21.76%.	
	
2.2.7.	Ethyl	2‐(4‐amino‐5‐cyano‐6‐(4‐methoxyphenyl)	
pyrimidin‐2‐yl)hydrazinecarboxylate	(7)	
	

A	mixture	 of	2	 (1.0	 g,	 3.9	mmol)	 and	ethyl	 chloroformate	
(0.4	 mL,	 3.9	 mmol)	 in	 pyridine	 (20	 mL)	 was	 heated	 under	
reflux	 for	 6h.	 The	 reaction	 mixture	 was	 concentrated,	 and	
acidified	with	 cold	 dilute	 acetic	 acid.	 The	 solid	 separated	 out	
was	 filtered	 off,	 washed	 several	 times	 with	 cold	 water	 and	
recrystallized	 from	 ethanol	 to	 give	 7	 as	 colourless	 crystals	
(Scheme	 2).	 Yield	 84%.	 M.p.:	 201‐203	 °C.	 FT‐IR	 (KBr,	 cm‐1):	
3468,	 3345,	 3312,	 3173	 υ(NH2),	 2202	 υ(C≡N),	 1706	 υ(C=O)	
(ester).	 1H	 NMR	 (300	 MHz,	 DMSO‐d6):	 9.2‐9.1	 (d,	 2H,	 2NH,	
exchangeable	with	D2O),	 7.82	 (d,	 J=8.6	Hz,	 2H,	 Ar‐H),	 7.08	 (d,	
J=8.6	Hz,	2H,	Ar‐H),	4.1	 (q,	2H,	COOCH2CH3),4.0	 (br.s,	2H,	NH2,	
exchangeable	 with	 D2O),	 3.8	 (s,	 3H,	 OCH3),	 1.2‐1.05	 (t,	 3H,	
COOCH2CH3).	 MS	 (EI,	 m/z):	 328	 (M+).	 Anal.	 Calcd.	 for	
C15H16N6O3:	C,	54.87;	H,	4.91;	N,	25.60.	Found:	C,	54.82;	H,	4.86;	
N,	25.61%.	
	



Mahmoud	et	al.	/	European	Journal	of	Chemistry	2	(3)	(2011)	347‐351	 349	
 

 
 

	

N

N

Ar
NC

H2N S Ph

1

N

N

Ar
NC

H2N N
H
NH2

O O

Cl

O

O

py.N

N

Ar
NC

H2N NH N
H

O

O

N

N

Ar
NC

H2N N
H

H
N

O
O
Cl

O
O

N

N

Ar
NC

N
H

N
H
NH3COC

py.

O

O

O

6

5

7

4

3

2

EtOH/

EtOH/
N2H4.H2O

N

N

Ar
NC

H2N N
H
N

O

O

N

N

Ar
NC

H2N N N

MeMe

Ac
2 O

Ac
O
H

CH3

CH3
O

O

	
	

Scheme	2
	
	

	
	

Scheme	3
	
	
2.2.8.	7‐amino‐5‐(4‐methoxyphenyl)‐3‐thioxo‐2,3‐dihydro‐
[1,2,4]triazolo[4,3‐a]pyrimidine‐6‐carbonitrile	(8)	
	

A	mixture	of	2	 (1.0	g,	3.9	mmol)	and	carbon	disulphide	(5	
mL)	in	ethanolic	KOH	10%	(1.0	g	KOH	dissolve	in	1.0	mL	water	
and	9	mL	ethanol)	was	heated	under	reflux	on	water	bath	 for	
16h.	After	cooling	the	reaction	mixture	was	acidified	with	cold	
dilute	 acetic	 acid.	 The	 canary	 yellow	 solid	 that	 separated	 out	
was	 filtered	 off,	 washed	 several	 times	 with	 cold	 water	 and	
recrystallized	from	ethanol	 to	give	8	as	canary	yellow	crystals	
(Scheme	 3).	 Yield	 48%.	 M.p.:	 278‐280	 °C.	 FT‐IR	 (KBr,	 cm‐1):	
3292,	3213,	3198	υ(NH2,	NH),	2210	υ(C≡N),	1632	υ(C=N),	1259	
υ(C=S).	MS	(EI,	m/z):	298	(M+).	Anal.	Calcd.	 for	C13H10N6OS:	C,	
52.34;	H,	 3.38;	N,	 28.17;	 S,	 10.75.	 Found:	 C,	 52.35;	H,	 3.36;	N,	
28.19;	S,	10.74%.	
	

2.2.9.	7‐amino‐3‐(3,4‐dimethoxyphenyl)‐5‐(4‐methoxy	
phenyl)‐[1,2,4]triazolo[4,3‐a]	pyramidine‐6‐carbonitrile	(9)	
	

A	 mixture	 of	 2	 (1.0	 g,	 3.9	 mmol)	 and	 2‐(3,4‐
dimethoxybenzylidene)	 malononitrile	 (0.83	 g,	 3.9	 mmol)	 in	
ethanol	 (25	 mL)	 and	 drops	 of	 glacial	 acetic	 acid	 was	 heated	
under	 reflux	 for	6h.	After	 evaporation	of	 the	solvent	 in	vacuo,	
the	solid	obtained	was	collected	by	filtration	and	recrystallized	
from	toluene	to	give	9	as	pale	yellow	crystals	(Scheme	4).	Yield	
74%.	M.p.:	217‐220	 °C.	 FT‐IR	 (KBr,	 cm‐1):	 3460,	 3292	υ(NH2),	
2200	υ(C≡N),	1643	υ(C=N).	1H	NMR	(300	MHz,	DMSO‐d6):	8.12	
(s,	1H,	Ar‐H),	 7.86	 (d,	 J=8.4	Hz,	2H,	Ar‐H),	 7.30‐7.10	 (br.s,	2H,	
NH2,	exchangeable	with	D2O),	7.18‐6.98	(m,	4H,	Ar‐H),	3.83	(s,	
6H,	2OCH3),	3.79	(s,	3H,	OCH3).	MS	(EI,	m/z):	403	(M+1).	Anal.	
Calcd.	 for	 C21H18N6O3:	 C,	 62.68;	 H,	 4.51;	 N,	 20.88.	 Found:	 C,	
62.59;	H,	4.48;	N,	20.76%.	



350	 Mahmoud	et	al.	/	European	Journal	of	Chemistry	2	(3)	(2011)	347‐351	
	
	

	
	

Scheme	4	
	
	
2.2.10.	(E)‐2‐(2‐(4‐nitrobenzylidene)hydrazinyl)‐4‐amino‐6‐
(4‐methoxyphenyl)pyrimidine‐5‐carbonitrile	(10)	
	

A	mixture	of	2	 (1.0	g,	3.9	mmol)	and	p‐nitrobenzaldehyde	
(0.59	g,	3.9	mmol)	in	ethanol	(25	mL)	was	stirred	under	reflux	
for	10	min.	The	solid	separated	was	collected	by	filtration	and	
then	recrystallized	from	ethanol/dioxane	mixture	to	give	10	as	
yellow	crystals	(Scheme	5).	Yield	98%.	M.p.:	over	300	°C.	FT‐IR	
(KBr,	 cm‐1):	 3478,	 3294,	 3149	 υ(NH2),	 2201	 υ(C≡N),	 1647	
υ(C=N).	 1H	 NMR	 (300	 MHz,	 DMSO‐d6):	 11.7	 (s,	 1H,	 NH,	
exchangeable	with	 D2O),	 8.29	 (m,	 3H,	 (2H,	 Ar‐H)	 and	 N=CH),	
7.92	 (d,	 J=9	Hz,	 2H,	 Ar‐H),	 7.84	 (d,	 J=9	 Hz,	 2H,	 Ar‐H),	 7.6‐7.5	
(br.s,	2H,	NH2,	exchangeable	with	D2O),	7.10	(d,	J=9	Hz,	2H,	Ar‐
H),	3.84	(s,	3H,	OCH3).	MS	(EI,	m/z):	389	(M+).	Anal.	Calcd.	 for	
C19H15N7O3:	C,	58.61;	H,	3.88;	N,	25.18.	Found:	C,	58.52;	H,	3.75;	
N,	25.09%.	
	
2.2.11.	7‐amino‐5‐(4‐methoxyphenyl)‐3‐(phenylamino)‐
[1,2,4]triazolo[4,3‐a]pyrimidine‐6‐carbonitrile	(11)	
	

A	mixture	of	2	(1.0	g,	3.9	mmol)	and	phenyl	isothiocyanate	
(0.53	 mL,	 3.9	 mmol)	 in	 pyridine	 (25	 mL)	 was	 heated	 under	
reflux	for	6h.	After	cooling	the	reaction	mixture	was	poured	on	
ice	 cold	 acetic	 acid.	 The	 buff	 solid	 that	 separated	 out	 was	
filtered	 off,	 washed	 several	 times	 with	 cold	 water	 and	
recrystallized	from	benzene	to	give	11	(Scheme	5).	Yield	62%.	
M.p.:	 152‐315	 °C.	 FT‐IR	 (KBr,	 cm‐1):	 3204	 υ(NH2,	 NH),	 2204	
υ(C≡N).	 1H	 NMR	 (300	 MHz,	 DMSO‐d6):	 9.75	 (s,	 1H,	 NH,	
exchangeable	with	D2O),	7.49‐7.30	(m,	5H,	Ar‐H),	6.99‐6.96	(d,	
J=8.4	Hz,	2H,	Ar‐H),	6.56‐6.55	(d,	J=8.7	Hz,	2H,	Ar‐H),	4.9	(br.s,	
2H,	 NH2,	 exchangeable	 with	 D2O),	 3.34	 (s,	 3H,	 OCH3).	 MS	 (EI,	
m/z):	228	(M‐ph,	‐2CN).	Anal.	Calcd.	for	C19H15N7O:	C,	63.86;	H,	
4.23;	N,	27.44.	Found:	C,	63.87;	H,	4.20;	N,	27.45%.	
	
2.2.12.	N'‐(4‐amino‐5‐cyano‐6‐(4‐methoxyphenyl)pyrimidin‐
2‐yl)acetohydrazide	(12)	
	

A	mixture	of	2	 (1.0	g,	3.9	mmol)	and	diethyl	oxalate	(0.53	
mL,	3.9	mmol)	 in	n‐butanol	 (20	mL)	 in	 the	presence	of	 acetic	

acid	 (5	mL),	was	heated	under	 reflux	 for	10h.	After	 cooling,	 a	
colourless	 solid	 was	 separated	 out,	 filtered	 off,	 dryed	 and	
recrystallized	 from	 ethanol/dioxane	 mixture	 to	 give	 12	 as	
colourless	crystals	(Scheme	5).	Yield	83%.	M.p.:	268‐270	°C.	FT‐
IR	 (KBr,	 cm‐1):	 3448,	 3361,	 3299	 υ(NH2,	 NH),	 2204	 υ(C≡N),	
1668	 υ(C=O)	 (amide),	 1641	 υ(C=N).	 MS	 (EI,	 m/z):	 298	 (M+).	
Anal.	Calcd.	for	C14H14N6O2:	C,	56.37;	H,	4.73;	N,	28.17.	Found:	C,	
56.27;	H,	4.69;	N,	28.19%.	
	
2.2.13.	(Z)‐5‐(4‐nitrobenzylidene)‐6‐amino‐2‐thioxo‐2,3‐
dihydropyrimidin‐4(5H)‐one	(14)	and	7‐amino‐4‐hydroxy‐2‐
mercapto‐5‐(4‐nitrophenyl)‐5,8‐dihydropyrido[2,3‐d]	
pyramidine‐6‐carbonitrile	(15)	
	

A	mixture	of	13	 (2.17	g,	0.01	mol)	with	6‐amino‐2‐thioxo‐
2,3‐dihydropyrimidin‐4(1H)‐one	(1.43	g,	0.01	mol)	in	absolute	
ethanol	 (50	 mL)	 in	 the	 presence	 of	 piperidine	 (1.0	 mL)	 was	
heated	with	stirring	for	2h.	The	solid	separated	while	hot	was	
collected	 by	 filtration	 and	 recrystallized	 from	 dioxane	 to	 give	
14	as	yellow	crystals,	yield	(13%).	Acidification	of	the	alkaline	
filtrate	after	 evaporation	of	 the	solvent	with	cold	dilute	acetic	
acid	left	a	yellow	solid	which	filtered	off,	washed	several	times	
with	cold	water,	dried	and	recrystallized	from	DMF	to	give	15	
as	buff	crystals	(Schemes	6,7).	

(Z)‐5‐(4‐nitrobenzylidene)‐6‐amino‐2‐thioxo‐2,3‐dihydro	
pyrimidin‐4(5H)‐one	 (14):	 Yellow	 crystals.	 Yield	 13%.	 M.p.:	
236‐238	 °C.	 FT‐IR	 (KBr,	 cm‐1):	 3443,	 3410,	 3155	 υ(NH2,	 NH),	
1672	 υ(C=O),	 1632	 υ(C=N),	 1346	 υ(C=S).	 MS	 (EI,	 m/z):	 276	
(M+).	Anal.	Calcd.	for	C11H8N4O3S:	C,	47.82;	H,	2.92;	N,	20.28;	S,	
11.61.	Found:	C,	47.73;	H,	2.89;	N,	20.29;	S,	11.53%.	

7‐amino‐4‐hydroxy‐2‐mercapto‐5‐(4‐nitrophenyl)‐5,8‐
dihydropyrido[2,3‐d]	 pyramidine‐6‐carbonitrile	 (15):	 Buff	
crystals.	 Yield	 56.6%.	 M.p.:	 over	 300	 °C.	 FT‐IR	 (KBr,	 cm‐1):	
3431,	3325	υ(NH2),	2225	υ(C≡N),	1639	υ(C=N).	MS	(EI,	m/z):	
342	 (M+).	 Anal.	 Calcd.	 for	 C14H10N6O3S:	 C,	 49.12;	 H,	 2.94;	 N,	
24.55;	S,	9.37.	Found:	C,	48.89;	H,	2.87;	N,	24.36;	S,	9.26%.	
	
	
	



Mahmoud	et	al.	/	European	Journal	of	Chemistry	2	(3)	(2011)	347‐351	 351	
 

 
 

	 	

--

EtOH/

-

	
	

Scheme	5
	
	
2.2.14.	(E)‐2‐(5‐(4‐nitrobenzylidene)‐4‐oxo‐4,5‐dihydro	
thiazol‐2‐yl)acetonitrile	(17)	
	

A	 mixture	 of	 13	 (2.17	 g,	 0.01	 mol)	 with	 2‐(4‐oxo‐4,5‐
dihydrothiazol‐2‐yl)acetonitrile	 (1.4	 g,	 0.01	 mol)	 in	 absolute	
ethanol	 (50	 mL)	 in	 the	 presence	 of	 piperidine	 (1.0	 mL)	 was	
heated	 with	 stirring	 under	 reflux	 for	 8h.	 After	 cooling	 the	
reaction	mixture	 was	 poured	 on	 ice	 and	 acidified	with	 dilute	
acetic	acid.	The	separated	solid	was	filtered	off,	washed	several	
times	with	 cold	water	 and	 recrystallized	 from	ethanol	 to	 give	
17	 as	 red	 crystals	 (Scheme	7).	Yield	62.8%.	M.p.:	 194‐196	 °C.	
FT‐IR	 (KBr,	 cm‐1):	 2208	 υ(C≡N),	 1675	 υ(C=O).	 MS	 (EI,	 m/z):	
273	 (M+).	 Anal.	 Calcd.	 for	 C12H7N3O3S:	 C,	 52.74;	 H,	 2.58;	 N,	
15.38;	S,	11.73.	Found:	C,	52.71;	H,	2.48;	N,	15.41;	S,	11.73%.	
	
2.2.15.	1,2‐diamino‐5‐cyano‐4‐(4‐nitrophenyl)‐6‐oxo‐1,6‐
dihydropyridine‐3‐carboxamide	(18)	and	(3E,5Z)‐3‐amino‐
6‐cyano‐5‐(4‐nitrophenyl)‐7‐oxo‐2,7‐dihydro‐1H‐1,2‐
diazepine‐4‐carboxamide	(19)	
	

A	 mixture	 of	 13	 (2.17	 g,	 0.01	 mol)	 with	 2‐
cyanoacetohydrazide	(0.99	g,	0.01	mol)	in	absolute	ethanol	(50	
mL)	 in	 the	 presence	 of	 piperidine	 (1.0	 mL)	 was	 heated	 with	
stirring	 under	 reflux	 for	 3h.	 The	 yellow	 solid	deposited	while	
hot	was	collected	by	 filtration	and	recrystallized	 from	ethanol	
to	 give	 18.	 Acidification	 of	 the	 alkaline	 filtrate	 with	 ice	 cold	
dilute	acetic	acid	 left	a	yellow	solid	product	which	filtered	off,	
washed	several	times	with	cold	water,	dried	and	recrystallized	
from	toluene	to	give	19	(Scheme	7).	

1,2‐diamino‐5‐cyano‐4‐(4‐nitrophenyl)‐6‐oxo‐1,6‐dihydro	
pyridine‐3‐carboxamide	 (18):	 Yield	 46.5%.	 M.p.:	 201‐203	 °C.	
FT‐IR	(KBr,	cm‐1):	3494,	3403,	3358,	3289,	3186	υ(NH2),	2207	
υ(C≡N),	 1698,	 1656	 υ(C=O).	 MS	 (EI,	 m/z):	 314	 (M+).	 Anal.	
Calcd.	 for	 C13H10N6O4:	 C,	 49.69;	 H,	 3.21;	 N,	 26.74.	 Found:	 C,	
49.62;	H,	3.18;	N,	26.72%.	

(3E,5Z)‐3‐amino‐6‐cyano‐5‐(4‐nitrophenyl)‐7‐oxo‐2,7‐
dihydro‐1H‐1,2‐diazepine‐4‐carboxamide	 (19):	 Yield	 15.7%.	
M.p.:	 229‐230	 °C.	 FT‐IR	 (KBr,	 cm‐1):	 3329,	 3197	 υ(NH2,	 NH),	
2210	υ(C≡N),	1674	υ(C=O).	1H	NMR	(300	MHz,	DMSO‐d6):	8.26	
(d,	J=8.4	Hz,	2H,	Ar‐H),	7.55	(d,	J=8.7	Hz,	2H,	Ar‐H),	7.0	(br.s,	1H,	
NH,	exchangeable	with	D2O),	5.58	(br.s,	2H,	NH2,	exchangeable	
with	D2O).	MS	(EI,	m/z):	314	(M+).	Anal.	calcd.	for	C13H10N6O4:	C,	
49.69;	H,	3.21;	N,	26.74.	Found:	C,	49.67;	H,	3.16;	N,	26.77%.	
	
2.2.16.	6‐cyano‐7‐(4‐nitrophenyl)‐5‐oxo‐2‐thioxo‐1,2,3,5‐
tetrahydro‐[1,2,4]triazolo[1,5‐a]	pyridine‐8‐carboxamide	
(20)	
	

A	mixture	of	18	(1.0	g,	3.2	mmol)	and	carbon	disulphide	(5	
mL)	in	ethanolic	KOH	10%	(10	mL)	was	heated	under	reflux	on	
water	 bath	 for	 16h.	 After	 cooling	 the	 reaction	 mixture	 was	
poured	 on	 ice	 and	 acidified	 with	 dilute	 acetic	 acid.	 The	
precipitated	 solid	was	 filtered	 off,	 washed	 several	 times	with	
cold	water	and	recrystallized	from	methanol	to	give	20	as	light	
brown	crystals	(Scheme	7).	Yield	32%.	M.p.:	over	300	°C.	FT‐IR	
(KBr,	 cm‐1):	 3421	 υ(NH2),	 2213	 υ(C≡N),	 1690	 υ(C=O),	 1621	
υ(C=N),	 1342	 υ(C=S).	MS	 (EI,	m/z):	 322	 (M‐H2S).	 Anal.	 Calcd.	
for	 C14H8N6O4S:	 C,	 47.19;	 H,	 2.26;	 N,	 23.59;	 S,	 9.00.	 Found:	 C,	
47.15;	H,	2.24;	N,	23.54;	S,	8.98%.		



352	 Mahmoud	et	al.	/	European	Journal	of	Chemistry	2	(3)	(2011)	347‐351	
	
	

	

Scheme	6	
	
	
2.3.	Antiviral	activity	
	
2.3.1.	Methodology:	MTT	assay	(Cytotoxicity	assay)	
	

Samples	 were	 diluted	 with	 Dulbecco's	 Modified	 Eagle's	
Medium	(DMEM)	 to	desired	concentrations.	Stock	solutions	of	
the	 test	 compounds	were	 prepared	 in	 10	%	DMSO	 in	 ddH2O.	
The	cytotoxic	activity	of	the	extracts	was	tested	in	Madin	Darby	
Canine	 kidney	 (MDCK)	 cells	 by	 using	 the	 3‐(4,5‐
dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium	 bromide	 (MTT)	
method	 [15]	 with	 minor	 modification.	 Briefly,	 the	 cells	 were	
seeded	 in	 96	 well‐plates	 (100	 µL/well	 at	 a	 density	 of	 3×105	
cells/mL)	and	incubated	for	24	hrs	at	37	oC	in	5%	CO2.	After	24	
hrs,	cells	were	treated	with	various	concentrations	of	the	tested	
compounds	in	triplicates.	After	further	24	hrs,	the	supernatant	
was	 discarded	 and	 cell	monolayers	were	washed	with	 sterile	
phosphate	buffer	saline(PBS)	3	times	and	MTT	solution	(20	µL	
of	 5	 mg/mL	 stock	 solution)	 was	 added	 to	 each	 well	 and	
incubated	at	37	°C	for	4	hrs	followed	by	medium	aspiration.	In	
each	well,	 the	 formed	 formazan	 crystals	 were	 dissolved	with	
200	 µL	 of	 acidified	 isopropanol	 (0.04	 M	 HCl	 in	 absolute	
isopropanol).	 Absorbance	 of	 formazan	 solutions	 were	
measured	 at	 λmax	 540	 nm	 with	 620	 nm	 as	 a	 reference	
wavelength	using	a	multi‐well	plate	reader.	The	percentage	of	
cytotoxicity	 compared	 to	 the	 untreated	 cells	 was	 determined	
with	the	equation	(1).	
		
	%	Cytotoxicity	=	Abs.	of	cells	without	treatment‐Abs.	of	cells	with		 				(1)	
																																		Abs.	of	cells	without	treatment	
	

The	plot	of	%	cytotoxicity	versus	sample	concentration	was	
used	 to	 calculate	 the	 concentration,	 which	 exhibited	 50%	
cytotoxicity	(LD50).	
	
2.3.2.	Antiviral	assay	
	

The	 antiviral	 activity	 of	 the	 compounds	 was	 determined	
using	 cytopathogenicity	 (CPE)	 assay	 against	 avian	 influenza	
virus	 (H5N1).	 Stock	 solutions	 of	 the	 test	 compounds	 were	

prepared	in	DMSO	at	a	concentration	of	10	mg/mL.	Cells	grown	
to	 confluency	 in	 96‐well	 plates,	were	 infected	with	 100	 µL	 of	
stock	virus.	After	an	adsorption	period	of	2	hrs	at	37	 oC,	virus	
was	 removed	 and	 serial	 dilutions	 of	 the	 compounds	 were	
added.	 Maintenance	 DMEM	 with	 2%	 FBS	 was	 added	 (100	
µL/well).The	 cultures	 were	 further	 incubated	 at	 37	 oC	 for	 3	
days,	 until	 complete	 CPE	 was	 observed	 in	 the	 infected	 and	
untreated	virus	control.	The	determination	of	the	anti‐influenza	
virus	 activity	 of	 the	 compounds	 was	 based	 on	 virus‐induced	
cytopathogenicity	 of	 H5N1‐infected	 MDCK	 cells,	 measured	 at	
day	4	post	virus	infection	by	the	MTT	colorimetric	method	[16].	
An	absorbance	of	formazan	was	detected	by	a	multi‐well	plate	
reader	at	540	nm	with	620	nm	reference	wavelength.	
	
3.	Results	and	discussion	
	
3.1.	Synthesis	
	

The	utility	of	activated	nitriles	in	synthesis	of	a	wide	variety	
of	 heterocyclic	 systems	 [17‐26]	 encouraged	 us	 to	 synthesize	
pyrimidine	 derivatives	 from	 relatively	 simple	 starting	
materials.	 The	 title	 compound	1	 was	 prepared	 by	 reaction	 of				
2‐(4‐methoxybenzylidene)malononitrile	 with	 s‐benzylthiuro‐
nium	 chloride	 in	 refluxing	 ethanolic	 sodium	 hydroxide	
according	the	simple	mechanism	in	(Scheme	1).	

Hydrazinolysis	 of	 1	 afforded	 the	 sulfur	 free	 compound	
which	 identified	 as	 4‐amino‐2‐hydrazinyl‐6‐(4‐methoxy	
phenyl)pyrimidine‐5‐carbonitrile	(2)	[6]	(Scheme	2).	

Recently,	 it	 has	 been	 reported	 [27]	 that	 the	 hydrazino	
pyrimidines	can	be	considered	as	key	starting	materials	for	the	
synthesis	 of	 diverse	 nitrogen	 bridgehead	 compounds.	 This	
prompted	us	to	reinvestigate	the	proclivity	of	compound	2	with	
electrophilic	 reagents	 such	 as,	 pentane‐2,4‐dione,	 acetic	
anhydride,	 furoyl	 chloride,	 phthalic	 anhydride,	 ethyl	 chloro‐
formate,	 carbon	 disulphide,	 2‐(3,4‐dimethoxybenzylidene)	
malononitrile,	p‐nitrobenzaldehyde,	phenyl	isothiocyanate	and	
diethyl	 oxalate	 with	 the	 aim	 of	 preparing	 new	 pyrimidine	
derivatives	which	might	have	chemotherapeutic	and	biological	
evaluation.		



Mahmoud	et	al.	/	European	Journal	of	Chemistry	2	(3)	(2011)	347‐351	 353	
 

 
 

	

CN

NH2O

N
H

NH
O

H2N S

N

NH

O

H2N S

H

Ar

N

N

N
H

Ar H

H2N

NC

NH

O CN

NO

NC
Ar

NH2
NH2

NH2

O

18

(46.6%)

N N

NC
Ar

O NH2

O

NH2

19
(15.7%)

EtOH
pip.

14 15

13

Ar=C6H4-NO2:4

NO

NC
Ar

NH
HN

NH2

O

S

CS2
KOH/EtOH

20

N
S O

NC

N
S

O

NC

EtOH/pip.

ArH
17

Et
O
H/
pi
p.

H2
N

H H

16

N
S

O

NC

EtoH/

O2N
O

H

OH

SH

Ar

16

 
	

Scheme	7
	
	
Thus,	 treatment	 of	 the	 2‐hydrazino	 derivative	 2	 with	

pentane‐2,4‐dione	 in	 refluxing	 ethanol	 afforded	 4‐amino‐2‐
(3,5‐dimethyl‐1H‐pyrazol‐1‐yl)‐6‐(4‐methoxyphenyl)pyrimi‐
dine‐5‐carbonitrile	(3)	[6].	

Acetylation	 of	 compound	 2	 using	 freshly	 distilled	 acetic	
anhydride	 yielded	 the	 acetylated	 product	 (4).	 The	 mass	
spectrum	 of	 this	 product	 indicates	 the	 incorporation	 of	 three	
acetyl	groups	which	also	confirmed	from	its	1H	NMR	spectrum.	
On	the	other	hand,	treatment	of	2	with	furoyl	chloride	afforded	
the	monoacylated	product	(5).	However,	the	reaction	of	2	with	
phthalic	anhydride	in	refluxing	acetic	acid	gave	the	phthalimide	
derivative	 (6).	 Furthermore,	 compound	 2	 when	 treated	 with	
ethyl	 chloroformate	 yielded	 ethyl	 N‐pyrimidin‐2‐yl	 carbazate	
derivative	(7)	(Scheme	2).	

The	 structures	 4‐7	 were	 substantiated	 from	 the	 correct	
analytical	 and	 spectroscopic	 data	 (c.f.	 Exp.).	 The	 formation	 of	
compounds	4‐7	could	be	explained	on	the	basis	of	nucleophilic	
substitution	at	trigonal	carbon	(tetrahedral	mechanism).	

It	has	been	reported	that	heterocyclic	o‐aminocarbonitriles	
including	 furans,	 pyrimidines	 and	 quinazolines	 [28]	 reacted	
with	 carbon	 disulphide	 under	 different	 conditions	 to	 afford	
biologically	 interest	 fused	 thiazines	 and	 pyrimidinedithione.	
However,	 compound	2	was	 treated	with	 carbon	disulphide	 in	
ethanolic	 potassium	 hydroxide	 to	 yield	 7‐amino‐5‐(4‐
methoxyphenyl)‐3‐thioxo‐2,3‐dihydro‐[1,2,4]‐triazolo[4,3‐a]	
pyrimidine‐6‐carbonitrile	 (8)	 (Scheme	 5).	 This	 reaction	 could	
be	visualized	as	shown	in	(Scheme	3).	

7‐Amino‐3‐(3,4‐dimethoxyphenyl)‐5‐(4‐methoxyphenyl)‐
[1,2,4]triazolo[4,3‐a]pyrimidine‐6‐carbonitrile	 (9)	 was	
obtained	 in	 fairly	 good	 yield	 upon	 treatment	 of	 compound	2	
with	 2‐(3,4‐dimethoxybenzylidene)malononitrile	 in	 boiling	
ethanol	in	the	presence	of	few	drops	of	acetic	acid	(Scheme	5).	
The	structure	of	9	was	deduced	from	the	correct	analytical	and	

spectroscopic	data	 (c.f.	Exp.).	Formation	of	compound	9	 could	
be	postulated	as	shown	in	(Scheme	4).	

Refluxing	 the	 hydrazino	 derivative	 2	 with	 p‐nitro	
benzaldehyde	 in	 ethanol	 for	 10	 min	 afforded	 a	 crude	 yellow	
solid	 with	 molecular	 formula	 C19H15N7O3	 [M.+=	 389	 (6.5%)]	
which	detected	as	 (E)‐2‐(2‐(4‐nitrobenzylidene)hydrazinyl)‐4‐
amino‐6‐(4‐methoxyphenyl)pyrimidine‐5‐carbonitrile	 (10)	
(Scheme	5).	

In	 the	 context,	 it	 was	 claimed	 that	 the	 reaction	 of	 the	
hydrazino	 derivative	 with	 phenyl	 isothiocyanate	 in	 refluxing	
pyridine	 resulted	 in	 the	 formation	 of	 1,2,4‐triazolo‐	
pyrimidinthione	 or	 thiourea	 derivatives	 [27].	 Herein,	 when	
compound	 2	 was	 refluxed	 with	 the	 same	 reagent	 in	 boiling	
pyridine	 afforded	 the	 sulfur	 free	 product	 with	 molecular	
formula	C19H15N7O	[M.+=	357]	which	detected	as	7‐amino‐5‐(4‐
methoxyphenyl)‐3‐(phenylamino)‐[1,2,4]triazolo[4,3‐a]pyrimi‐
dine‐6‐carbonitrile	(11)	(Scheme	5).	

Treatment	of	2	with	diethyl	oxalate	 in	 refluxing	n‐butanol	
in	 the	 presence	 of	 acetic	 acid	 afforded	 the	 mono	 acetylated	
product	 (12)	 which	 indicates	 that	 no	 combination	 occurred	
between	compound	2	and	the	oxalate	in	the	presence	of	acetic	
acid.	This	confirmed	by	refluxing	2	with	acetic	acid	in	n‐butanol	
which	yielded	the	same	product	12	(Scheme	5).	

As	a	continuation	[29‐31]	of	our	study	on	the	reactivity	of	
activated	nitriles,	the	(E)‐2‐cyano‐3‐(4‐nitrophenyl)acrylamide	
(13)	 was	 prepared	 and	 allowed	 to	 react	 with	 carbon	 and	
nitrogen	nucleophiles.	Thus,	the	reaction	of	13	with	6‐amino‐2‐
thioxo‐2,3‐dihydropyrimidin‐4(1H)‐one	 in	 boiling	 ethanol	 in	
the	 presence	 of	 piperidine	 yielded	 a	 yellow	 solid	 product	
separated	on	hot	and	identified	as	the	pyrimidine	derivative	14	
(13%).	Acidification	of	 the	alkaline	 filtrate	 left	a	solid	product	
which	 detected	 as	 the	 pyrido[2,3‐d]pyrimidine	 derivative	 15	
(56.6%)	(Scheme	7).	



354	 Mahmoud	et	al.	/	European	Journal	of	Chemistry	2	(3)	(2011)	347‐351	
	

	

CN

NH2ONH

NC

+

Ar Ar
NC

NH2

O NH

NH2

a

b

NO

NC

Ar

NH2

NH2

NH

NO

NC

Ar

NH2
NH2

NH2

NO

NC

Ar

NH2
NH2

NH2

O

Michael adduct

N

(a)

1,6
-ex
o-d
ig

Cy
cli
zat
ion

1,7-exo-dig

Cyclization

(b)
O

O

13

-H213

18

(46.6%)

-H2

19

(15.7%)

O

O

N N

NC

Ar

O NH

O

NH2

H H

N N

NC

Ar

O NH2

O

NH2

H H

NH2

B

	
	

Scheme	8
	
	
The	reaction	was	assumed	to	proceed	via	Michael	addition	

of	 the	C‐5	of	pyrimidine	nucleus	to	 the	α,β‐unsaturated	nitrile	
13	 and	 subsequent	 1,6‐exo‐trig	 cyclization	 through	
nucleophilic	 addition	 of	 the	 amino	 group	 to	 the	 carboxamido	
carbonyl	group	(Scheme	6).	

Furthermore,	 treatment	 of	 13	 with	 2‐(4‐oxo‐4,5‐
dihydrothiazol‐2‐yl)	 acetonitrile	 (16)	 in	boiling	ethanol	 in	 the	
presence	of	catalytic	amount	of	piperidine	afforded	(E)‐2‐(5‐(4‐
nitrobenzylidene)‐4‐oxo‐4,5‐dihydrothiazol‐2‐yl)acetonitrile	
(17)	as	the	sole	product	(62.8%)	(Scheme	7).	The	structure	17	
was	chemically	supported	by	identity	with	an	authentic	sample	
resulted	from	the	condensation	of	16	with	p‐nitrobenzaldehyde	
(Scheme	7).	

N‐Amino‐2‐pyridones	 have	 proved	 to	 be	 useful	 synthetic	
intermediates	 [32‐35].	 However,	 there	 are	 few	 synthetic	
procedures	for	the	preparation	of	N‐amino‐2‐pyridones.	These	
compounds	 are	 usually	 obtained	 [36‐39]	 in	 low	 yields	 by	
reaction	 of	 hydrazine	 with	 2‐pyrones,	 which	 are	 in	 turn	
prepared	 in	 low	 yields	 from	 open	 chain	 compounds	 [35‐44].	
We	 report	 in	 this	 article	 one	 step	 synthesis	 of	 N‐amino‐2‐
pyridone	(18)	from	compound	13	with	2‐cyanoacetohydrazide.	
The	reaction	easily	performed	in	ethanol	at	room	temperature	
in	 the	 presence	 of	 piperidine	 by	 stirring	 for	 two	 hours.	 The	
separated	 solid	 was	 identified	 using	 analytical	 and	 spectral	
data	 as	 1,2‐diamino‐5‐cyano‐4‐(4‐nitrophenyl)‐6‐oxo‐1,6‐
dihydropyridine‐3‐carboxamide	 (18).	 Acidification	 of	 the	
mother	 liquor	 left	 another	 yellow	 solid	 which	 have	 the	 same	
molecular	formula	of	compound	18.	This	product	was	expected	
to	have	the	diazepinone	structure	19	(Scheme	7).	

The	 reaction	 may	 be	 assumed	 to	 proceed	 via	 Michael	
addition	 of	 2‐cyanoacetohydrazide	 to	 13	 and	 the	 resulting	
adduct	 undergoes	 in	 situ	 cyclization	 by	 nucleophilic	 attack	 at	
the	 cyano	 group	 to	 give	 the	 six	 membered	 ring	 which	 on	
aromatization	gives	rise	to	the	N‐amino‐2‐pyridone	(18)	(route	
a).	 A	 cyclization	 to	 seven‐membered	 diazepine	 ring	 must	 be	
favored	 by	 the	 higher	 nucleophilic	 character	 of	 the	 amino	

group	with	respect	to	the	CONH	group,	but	the	poor	yield	of	19,	
probably	 due	 to	 the	 lack	 of	 aromatic	 character	 of	 the	
diazepinone	ring	19	(Scheme	8).	

Beside	the	analytical	and	spectral	data	the	structure	18	was	
got	 a	 further	 chemical	 support	 by	 the	 reaction	 with	 carbon	
disulphide.	 However,	 when	 compound	 18	 was	 treated	 with	
carbon	 disulphide	 in	 ethanolic	 potassium	 hydroxide	 at	
refluxing	 temperature	 yielded	 1,2,4‐triazolo[1,5‐a]pyridine	
derivative	20	(Scheme7).	
	
3.2.	Biological	activity	test	results	
	

The	 results	 were	 expressed	 as	 the	 50%	 effective	
concentration	(EC50).	The	50%	effective	antiviral	concentration	
(EC50)	was	defined	as	the	compound	concentration	required	for	
protecting	 50%	 of	 the	 virus‐infected	 cells	 against	 viral	
cytopathogenicity.	 The	 therapeutic	 index	 was	 calculated	 by	
dividing	LD50	on	EC50	(Figure	1).	

Therapeutic	 index	 was	 calculated	 to	 all	 examined	
compounds	 and	 compared	 with	 Zanamivir	 as	 control	 anti‐
influenza	drug	as	shown	in	Table	1.	
	
Table	 1.	 Therapeutic	 index	 of	 the	 tested	 compounds	 comparing	 with	
zanamivir.	
Compound	 Therapeutic	index	
1 0.896257387	
2 0.449688858	
3 0.444114591	
4 0.437320824	
5 0.358697928	
6	 0.521603236	
7	 0.677685950	
8	 0.465886618	
9 0.533377287	
10 0.749826429	
11 0.598077678	
12 0.440400844	
Zanamivir 15.4	
	



Mahmoud	et	al.	/	European	Journal	of	Chemistry	2	(3)	(2011)	347‐351	 355	
 

 
 

	
Figure	 1.	 The	 50%	 effective	 concentration	 (EC50)	 and	 the	 concentration,	
which	 exhibited	 50%	 cytotoxicity	 (LD50)	 of	 the	 tested	 compounds.	 The	
results	were	compared	with	the	anti‐influenza	drug,	zanamivir.	Compounds	
1	and	10	showed	the	highest	therapeutic	index	than	the	other	compounds.	
	
4.	Conclusions	
	

The	 potentially	 synthetic	 value	 of	 the	 newly	 reported	
compounds,	 as	 excellent	 starting	 materials,	 stimulated	 our	
interest	to	utilize	them	for	the	synthesis	of	the	title	compounds,	
in	 anticipation	 that	 new	 compounds	 would	 exhibit	
pharmacological	activity.	All	 the	synthesized	compounds	were	
examined	 and	 compared	 with	 Zanamivir	 as	 control	 anti‐
influenza	 drug	 and	 only	 compounds	 1	 and	 10	 showed	 the	
highest	therapeutic	index	than	the	others.	
	
Acknowledgements	
	

The	 biological	 activity	was	 supported	 by	Mohamed	 A.	 Ali	
and	Ahmed	M.	Mostafa,	Division	of	Environmental	Researches,	
National	Research Centre,	Dokki,	Giza,	Egypt.	
	
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