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European	Journal	of	Chemistry	4	(4)	(2013)	454‐458	

European	Journal	of	Chemistry	
ISSN	2153‐2249	(Print)	/	ISSN	2153‐2257	(Online)		2013	EURJCHEM	

DOI:10.5155/eurjchem.4.4.454‐458.784	

	
	

	

	

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

	 	 	

Green	synthesis	of	novel	pyrazole	containing	Schiff	base	derivatives	as	
antibacterial	agents	on	the	bases	of	in‐vitro	and	DFT	

Salman	Ahmad	Khan	a,*,	Abdullah	Mohamed	Asiri	a,b,		
Abdulrhim	Alabbas	Basheike	a	and	Kamlesh	Sharma	c	

a	Chemistry	Department,	Faculty	of	Science,	King	Abdulaziz	University,	Jeddah	21589,	Saudi	Arabia	
b	Center	of	Excellence	for	Advanced	Materials	Research	(CEAMR),	King	Abdulaziz	University,	Jeddah	21589,	Saudi	Arabia	
c	Molecular	Science	Research	Institute,	School	of	Chemistry,	University	of	the	Witwatersrand,	Johannesburg,	South	Africa	

*Corresponding	author	at:	Chemistry	Department,	Faculty	of	Science,	King	Abdulaziz	University,	Jeddah	21589,	Saudi	Arabia.		
Tel.:	+966.56.8966770;	fax:	+966.2.6952292.	E‐mail	address:	sahmad_phd@yahoo.co.in	(S.A.	Khan).	

	
	

	 	

	 	 	
ARTICLE	INFORMATION	 	 ABSTRACT
Received:	25	March	2013	
Received	in	revised	form:	23	April	2013	
Accepted:	07	May	2013	
Online:	31	December	2013	

KEYWORDS	

	 A	series	of	pyrazole	containing	Schiff	bases	were	synthesized,	by	the	reaction	of	3,5‐dimethyl‐
1‐phenylpyrazole‐4‐carboxaldehyde	 and	 the	 corresponding	 active	 amines	 under	microwave
irradiation.	The	structures	of	 the	synthesized	compounds	were	established	by	spectroscopic
data	(FT‐IR,	1H	NMR,	13C	NMR	and	ESI‐MS)	and	elemental	analyses.	The	anti‐bacterial	activity
of	these	compounds	were	tested	in	vitro	by	the	disc	diffusion	assay	against	two	Gram‐positive
and	 two	 Gram‐negative	 bacteria,	 and	 then	 the	 minimum	 inhibitory	 concentration	 using
chloramphenicol	as	reference	drug.	All	 the	molecules	were	modeled	and	optimized	by	using
density	functional	theory,	DFT/B3LYP	method.	Calculated	descriptors,	 the	lower	unoccupied
molecular	 orbital	 and	 the	 density	 were	 used	 to	 interpret	 the	 antibacterial	 activity	 of	 the
compounds.	 The	 results	 showed	 that	 compound	 3	 is	 better	 inhibitor	 of	 both	 types	 of	 test
bacteria	as	compared	to	chloramphenicol.	

Pyrazole	
Schiff	bases	
Chloramphenicol	
Antibacterial	activity	
Density	functional	theory	
Minimum	inhibitory	concentration	

	
1.	Introduction	
	

Development	 of	 antibiotics	 for	 gram‐positive	 and	 gram‐
negative	bacteria	such	as	S.	aureus,	S.	pyogenes,	S.	typhimurium	
and	 E.	 coli	 are	 among	 the	 most	 powerful	 and	 successful	
achievements	 of	 modern	 science	 for	 the	 control	 of	 infectious	
diseases.	These	bacteria's	have	and	are	causing	serious	health	
problem	such	as	 food	poisoning,	 rheumatic,	salmonellosis	and	
diarrhea	 [1,2].	 More	 than	 50	 million	 people	 worldwide	 are	
infected	 and	 up	 to	 150,000	 die	 yearly	 due	 to	 these	 bacterial	
infections	 [3].	 Amoxicillin,	 norfloxacin,	 chloramphenicol,	
ciprofloxacin	 are	 the	 most	 common	 drugs	 used	 for	 these	
bacterial	 infections	but	are	associated	with	severe	side	effects	
[4].	 However,	 critical	 lateral	 effects	 have	 been	 described,	 i.e.	
neurological	 alterations	 produced	 by	 interaction	 of	 the	 drug	
with	the	central	nervous	system	[5].	Therefore,	 it	 is	necessary	
to	 search	 for	new	antibiotic	 agents.	Five	member	heterocyclic	
compounds	 such	 as	 pyrazole	 are	 important	 are	 important	
nitrogen	 containing	 compounds	 and	 numerous	 pyrazole	
derivatives	have	been	found	to	possess	considerable	biological	
activities	 [6],	such	as	antimicrobial	 [7‐9],	antiamoebic	[10,11],	
antinociceptive	 [12],	 anticancer	 [13],	 antidepressant	 [14]	 and	
anti‐inflammatory	 [15].	 On	 the	 other	 hand,	 pyrazoline	
derivatives	are	also	widely	used	in	materials	science	fields	such	
as	 non‐linear	 optics	 (NLO)	 [16],	 optical	 limiting	 [17],	 electro‐
chemical	 sensing	 [18],	 Langmuir	 films	 and	 photoinitiated	
polymerization	 [19].	On	 the	other	hand	a	pyrazole	ring	 linked	
to	five	or	six	member	heterocyclic	systems	with	an	azomethine	
group	also	showed	a	variety	of	biological	activity	such	as	anti‐
bacterial	 [20],	 anti‐fungal	 [21],	 anti‐inflammatory	 [22],	
antihypertensive	 [23],	 anti‐HIV	 [24],	 antitumor	 [25],	 and	
anticonvulsant	activities.	Thus,	the	pyrazole	containing	bi‐cyclic	

heterocyclic	Schiff	base	seems	to	be	a	possible	pharmacophore	
in	various	pharmacologically	active	agents.		

Aforementioned	 facts	 prompted	 us	 to	 synthesize	 new	
pyrazole	 containing	 heterocyclic	 Schiff	 bases	 from	 reaction	 of	
3,5‐dimethyl‐1‐phenylpyrazole‐4‐carboxaldehyde	 with	 the	
corresponding	 heterocyclic	 active	 amines	 under	 microwave	
irradiation	as	antibacterial	agent.		
	
2.	Experimental	
	

3,5‐Dimethyl‐1‐phenylpyrazole‐4‐carboxaldehyde,	 amino‐
phenazone,	 5‐amino‐3,4‐dimethylisoxazole,	 2‐amino	 pyridine,	
3‐amino	1,2,4	triazole	were	purchased	from	Acros	Organic	and	
2‐amino‐4,5,6,7‐tetrahydro‐1‐benzothiophene‐3‐carbonitrile,	
5,6‐diphenyl‐1,2,4‐triazin‐3‐amine	 were	 synthesized	 by	
published	 methods	 [26,27].	 The	 other	 reagents	 and	 solvents	
(A.R.)	 were	 obtained	 commercially	 and	 used	 without	 further	
purification	 except	 dimethylformamide	 (DMF),	 ethanol	 and	
methanol.	Melting	points	were	 recorded	on	a	Thomas	Hoover	
capillary	melting	apparatus	without	correction.	IR	spectra	were	
recorded	on	a	Nicolet	Magna	520	FTIR	spectrometer.	1H	and	13C	
NMR	 were	 recorded	 in	 CDCl3	 on	 a	 Bruker	 DPX	 600	 MHz	
spectrometer	using	TMS	as	internal	standard.	The	mass	spectra	
have	been	scanned	on	the	Waters	Micromass	Q‐T	of	Micro	(ESI)	
spectrometer.	 Elemental	 analyses	 were	 performed	 using	 a	
Carlo	 Erba	 EA1180	 microanalyser.	 We	 have	 been	 used	 the	
Microwave	Dual	Hertz	220	V,	50Hz	and	60Hz	for	the	reaction.		
	
2.1.	General	procedure	for	the	synthesis	of	Schiff	bases		
	

The	 Schiff	 base	derivatives	were	 synthesized	 by	using	 the	
literature	procedure.		



Khan	et	al.	/	European	Journal	of	Chemistry	4	(4)	(2013)	454‐458	 455	
 

	 	
Table	1.	Physicochemical	data	of	the	synthesized	compounds	(1‐7).		

	

	
	

Compound	 R	 Molecular	formula
	

M.p.	(oC)/
Crystallization	solvent	

Yield	(%) Reaction	Time	(min)

1	

	
	

C23H23N5O
	
	

206/CHCl3
	
	
	

88.5 3.0	

2	

	
	

C17H18N4O 105/CH3OH
	

86.4 2.5	

3	

	
	

C19H16N4S 145/CH2Cl2 82.4 2.0	

4	

	
	

C14H14N6	 136/CHCl3	
	
	

90.2	 3.0	

5	

	
	

C21H20N4S	 138/CH3OH	
	

89.5	 3.5	

6	

	
	

C17H16N4 132/CHCl3 86.2 5.0	

7	

	

C27H22N6	

	
	
	

175/CHCl3	 82.2	 4.0	

	
	
	

A	 mixture	 of	 3,5‐dimethyl‐1‐phenyl	 pyrazole‐4‐
carboxaldehyde	 (5.8	 mmol)	 and	 heterocyclic	 amines	 (5.8	
mmol)	 in	anhydrous	methanol	(15	mL),	 in	a	beaker	(100	mL),	
in	 the	 presence	 of	 few	 drop	 of	 acetic	 acid,	 and	 the	 reaction	
mixture	was	heated	 inside	 a	microwave	oven	 for	 2‐5	min.	 (at	
210	 Watts,	 i.e.	 30%	 microwave	 power).	 Progress	 of	 reaction	
was	monitored	 by	 TLC.	 After	 completion	 of	 the	 reaction	 and	
cooling,	 the	product	was	obtained	and	recrystallized	 from	 the	
proper	 solvent.	 The	 melting	 points,	 recrystallization	 solvent	
and	reaction	time	of	the	compounds	are	recorded	in	Table	1.	

4‐{[(E)‐(3,5‐Dimethyl‐1‐phenyl‐1H‐pyrazol‐4‐yl)methyli‐
dene]amino}‐1,5‐dimethyl‐2‐phenyl‐1H‐pyrazol‐3(2H)‐one	 (1):	
Color:	 Brown.	 FT‐IR	 (KBr,	 v,	 cm‐1):	 2924	 (C‐H),	 1641	 (C=C),	
1589	 (C=N),	 1130	 (C‐N).	 1H	 NMR	 (600	 MHz,	 CDCl3,	 δ,	 ppm):	
9.82	(s,	1H,	CHolefinic),	7.49‐7.29	(m,	10H,	Ar‐H),	3.13	(s,	3H,	N‐
CH3),	2.55	(s,	3H,	CH3),	2.53	(s,	3H,	CH3),	2.44	(s,	3H,	CH3).	13C	
NMR	(150	MHz,	CDCl3,	δ,	ppm):	161.20,	151.81,	151.15,	149.83,	
140.53,	139.18,	134.89,	129.15,	129.09,	127.82,	126.72,	125.19,	
124.18,	 119.67,	 117.59,	 36.07,	 13.55,	 11.69,	 10.13.	 MS	 (ESI,	
m/z,	 (%)):	387	(M+1,	62).	Anal.	calcd.	 for	C23H23N5O:	C,	71.67;	
H,	6.01;	N,	18.17.	Found:	C,	71.65;	H,	5.97;	N,	18.12%.	

N‐[(E)‐(3,5‐dimethyl‐1‐phenyl‐1H‐pyrazol‐4‐yl)methylidene]‐
3,4‐dimethylisoxazol‐5‐amine	 (2):	 Color:	 Brown.	 FT‐IR	 (KBr,	 v,	
cm‐1):	3063(C‐H),	1665	(C=C),	1597	(C=N),	1142	(C‐N).	1H	NMR	
(600	MHz,	CDCl3,	δ,	ppm):	8.84	(s,	1H,	CH	olefinic),	7.62‐7.48	(m,	

5H,	Ar‐H),	2.24	(s,	3H,	CH3),	2.11	(s,	3H,	CH3),	2.03	(s,	3H,	CH3),	
1.75	 (s,	 3H,	 CH3).	 13C	NMR	 (150	MHz,	 CDCl3,	 δ,	 ppm):	 185.26,	
153.76,	151.81,	144.77,	138.17,	129.35,	129.27,	128.77,	128.41,	
125.38,	 125.38,	 114.82,	 13.34,	 12.60,	 11.94,	 10.48,	 6.63.	 MS	
(ESI,	m/z,	 (%)):	 296	 (M+1,	 54).	 Anal.	 calcd.	 for	 C17H18N4O:	 C,	
69.37;	H,	6.16;	N,	19.03.	Found:	C,	69.32;	H,	6.12;	N,	18.98%.		

N‐[(E)‐(3,5‐dimethyl‐1‐phenyl‐1H‐pyrazol‐4‐yl)methylidene]‐
1,3‐benzothiazol‐2‐amine	(3):	Color:	Light	orange.	FT‐IR	(KBr,	v,	
cm‐1):	 3062	 (C‐H),	 1667	 (C=C),	 1598	 (HC=N),	 1118	 (C‐N).	 1H	
NMR	(600	MHz,	CDCl3,	δ,	ppm):	10.20	((s,	1H,	CH	olefinic),	7.52‐
7.40	(m,	9H,	Ar‐H),	2.55	(s,	3H,	CH3),	2.52	(s,	3H,	CH3).	13C	NMR	
(150	 MHz,	 CDCl3,	 δ,	 ppm):	 185.26,	 151.82,	 144.71,	 138.17,	
129.35,	129.20,	128.77,	125.39,	118.96,	12.59,	11.41.	MS	 (ESI,	
m/z,	(%)):	334	(M+1,	58).	Anal.	calcd.	for	C19H16N4S:	C,	68.65,	H;	
4.85;	N,	16.85.	Found:	C,	68.61;	H,	4.82;	N,	16.82%.	

N‐[(E)‐(3,5‐Dimethyl‐1‐phenyl‐1H‐pyrazol‐4‐yl)methylide‐
ne]‐4H‐1,2,4‐	 triazol‐3‐amine	 (4):	 Color:	 Light	 brown.	 FT‐IR	
(KBr,	v,	cm‐1):	3065	(C‐H),	2928	(C‐H),	1667	(C=C),	1544	(C=N),	
1177	 (C‐N).	 1H	 NMR	 (600	MHz,	 CDCl3,	 δ,	 ppm):	 10.16	 (s,	 1H,	
CHolefinic),	7.52‐7.46	(m,	5H,	Ar‐H),	7.50	(s,	1H,	CH),	2.55	(s,	1H,	
N‐H),	2.53	 (s,	 3H,	 CH3),	 2.51	 (s,	 3H,	 CH3).	 13C	NMR	 (150	MHz,	
CDCl3,	δ,	ppm):	185.26,	151.82,	144.77,	138.17,	129.35,	128.77,	
125.39,	 115.96,	 12.59,	 11.41.	 MS	 (ESI,	m/z,	 (%)):	 268	 (M+1,	
72).	Anal.	calcd.	for	C14H14N6:	C,	63.14;	H,	5.30;	N,	31.56.	Found:	
C,	63.12;	H,	5.28;	N,	31.52%.	



456	 Khan	et	al.	/	European	Journal	of	Chemistry	4	(4)	(2013)	454‐458	
	

	
Table	2.	Antibacterial	activity	of	pyrazole	Schiff	base	derivative	(1‐7),	positive	control	chloramphenicol	and	negative	control	(DMSO)	measured	by	the	Halo	Zone	
Test	(Unit,	mm).	
Compounds	 Corresponding	effect	on	microorganisms

S.	aureus	 S.	Pyogenes S.	typhimurium E.	coli	
1	 11.2±0.2	 10.5±0.2 9.6±0.3 9.6±0.4	
2	 11.4±0.2	 12.4±0.4 10.5±0.3 14.6±0.2
3	 18.2±0.3	 18.6±0.4 17.8±0.4 21.4±0.4
4	 11.5±0.3	 15.2±0.5 10.5±0.4 11.5±0.1
5	 17.2±0.2	 17.8±0.3 17.5±0.4 20.2±0.4
6	 12.5±0.4	 14.2±0.4 11.6±0.5 10.2±0.5
7	 12.4±0.3	 12.6±0.5	 12.8±0.2	 11.6±0.5	
Chloramphenicol	 17.0±0.5	 18.2±0.4 17.2±0.8 20.0±0.2
DMSO	 ‐	 ‐ ‐ ‐	
	
	
Table	3.	Minimum	inhibition	concentration	(MIC)	of	pyrazole	Schiff	base	derivative	(1‐7)	products,	positive	control:	chloramphenicol.	
Bacterial	Strain	 MIC	(μg/mL)	compound	 Positive	control	

	1	 2	 3 4 5 6 7
S.	aureus		 	512	 256	 16	 256		 32	 256	 128	 32	
S.	pyogenes		 	256	 64	 16	 512	 54	 512	 512	 32	
S.	typhimurium		 	256	 128	 32 256 32 256 128	 32	
E.	coli		 	512	 256	 16 128 32 64 256	 32	
	

	
	
(E)‐2‐(((3,5‐dimethyl‐1‐phenyl‐1H‐pyrazol‐4‐yl)methylene)	

amino)‐4,5,6,7‐tetrahydrobenzo[b]thiophene‐3‐carbonitrile	 (5):	
Color:	Dark	Yellow.	FT‐IR	 (KBr,	v,	 cm‐1):	3032	 (C‐H),	2937	 (C‐
H),	 1635	 (C=C),	 1595	 (C=N),	 1140	 (C‐N).	 1H	 NMR	 (600	MHz,	
CDCl3,	 δ,	 ppm):	 8.84	 (s,	 1H,	 CHolefinic),	 7.57‐7.48	 (m,	 5H,	Ar‐H),	
2.70‐1.82	(m,	8H,	‐CH2),	2.62	(s,	3H,	CH3),	2.54	(s,	3H,	CH3).	13C	
NMR	(150	MHz,	CDCl3,	δ,	ppm):	161.90,	152.21,	151.27,	142.87,	
138.60,	134.60,	130.51,	129.28,	128.45,	125.26,	115.86,	114.88,	
104.93,	25.18,	24.35,	23.17,	22.08,	13.0.	MS	(ESI,	m/z,	(%)):	362	
(M+1,	76).	Anal.	calcd.	for	C21H20N4S:	C,	69.97;	H,	5.59;	N,	15.54.	
Found:	C,	69.95;	H,	5.54;	N,	15.52%.		

N‐[(E)‐(3,5‐Dimethyl‐1‐phenyl‐1H‐pyrazol‐4‐yl)methylidene]	
pyridine‐2‐amine	 (6):	Color:	 Light	Brown.	FT‐IR	 (KBr,	v,	 cm‐1):	
3064	 (C‐H),	2929	 (C‐H),	1666	 (C=C),	1554	 (C=N),	1176	 (C‐N).	
1H	NMR	(600	MHz,	CDCl3,	δ,	ppm):	10.08	(s,	1H,	CHolefinic),	7.54‐
7.39	(m,	9H,	Ar‐H),	2.55	(s,	3H,	CH3),	2.53	(s,	3H,	CH3).	13C	NMR	
(150	 MHz,	 CDCl3,	 δ,	 ppm):	 185.26,	 151.81,	 144.77,	 138.17,	
129.35,	 128.76,	 125.38,	 117.96,	 12.60,	 11.41.	 MS	 (ESI,	 m/z,	
(%)):	278	(M+1,	62).	Anal.	calcd.	for	C17H16N4:	C,	73.89;	H,	5.84;	
N,	20.27.	Found:	C,	73.84;	H,	5.72;	N,	20.22%.		

(E)‐N‐((3,5‐dimethyl‐1‐phenyl‐1H‐pyrazol‐4‐yl)methylene)‐
5,6‐diphenyl‐1,2,4‐triazin‐3‐amine	 (7):	 Color:	 Orange.	 FT‐IR	
(KBr,	v,	cm‐1):	2937	(C‐H),	2988	(C‐H),	1659	(C=C),	1554	(C=N),	
1140	 (C‐N).	 1H	 NMR	 (600	 MHz,	 CDCl3,	 δ,	 ppm):	 9.92	 (s,	 1H,	
CHolefinic),	 7.51‐7.30	 (m,	 15H,	 Ar‐H),	 2.55	 (s,	 3H,	 CH3),	 2.53	 (s,	
3H,	 CH3).	 13C	NMR	 (150	MHz,	 CDCl3,	 δ,	 ppm):	 185.22,	 161.16,	
157.26,	150.75,	136.01,	135.37,	129.50,	129.35,	129.35,	128.77,	
128.53,	 128.37,	 125.38,	 116.62,	 12.59,	 11.41.	 MS	 (ESI,	 m/z,	
(%)):	430	(M+1,	72).	Anal.	calcd.	for	C27H22N6:	C,	75.33;	H,	5.15;	
N,	19.52.	Found:	C,	75.28;	H,	5.06;	N,	19.48%.		
	
2.2.	Organism	culture	and	in	vitro	screening	
	

The	antibacterial	activity	was	assessed	by	the	disc‐diffusion	
method	 with	 minor	 modifications.	 S.	 aureus,	 S.	 pyogenes,	 S.	
typhimurium	and	E.	coli	were	sub‐cultured	in	BHI	medium	and	
incubated	 for	 18	h	 at	 37	 oC,	 and	 then	 the	bacterial	 cells	were	
suspended,	 according	 to	 the	 McFarland	 protocol	 in	 saline	
solution	to	produce	a	suspension	of	about	10‐5	CFU/mL:	10	μL	
of	 this	 suspension	was	mixed	with	10	mL	 of	 sterile	 antibiotic	
agar	at	40	oC	and	poured	onto	an	agar	plate	 in	a	 laminar	flow	
cabinet.	 Five	 paper	 disks	 (6.0	mm	 diameter)	were	 fixed	 onto	
nutrient	agar	plate.	1	mg	of	each	test	compound	was	dissolved	
in	100	μL	DMSO	to	prepare	stock	solution	from	stock	solution	
different	 concentration	10,	 20,	 25,	 50,	 and	100	μg/μL	of	 each	
test	 compound	were	 prepared.	 These	 compounds	 of	 different	
concentration	 were	 poured	 over	 disk	 plate	 on	 to	 it.	
Chloramphenicol	 (30	 μg/disk)	 was	 used	 as	 standard	 drug	

(positive	control)	and	DMSO	poured	disc	was	used	as	negative	
control.	The	susceptibility	of	the	bacteria	to	the	test	compounds	
was	determined	by	the	formation	of	an	inhibitory	zone	after	18	
h	 of	 incubation	 at	 36	 oC.	 Table	 2	 reports	 the	 inhibition	 zones	
(mm)	 of	 each	 compound	 and	 the	 controls.	 The	 minimum	
inhibitory	 concentration	 (MIC)	 was	 evaluated	 by	 the	 macro	
dilution	 test	 using	 standard	 inoculums	 of	 10‐5	 CFL/mL.	 Serial	
dilutions	 of	 the	 test	 compounds,	 previously	 dissolved	 in	
dimethyl	 sulfoxide	 (DMSO)	 were	 prepared	 to	 final	 concent‐
rations	of	512,	256,	128,	64,	32,	16,	8,	4,	2	and	1	μg/mL	to	each	
tube	 was	 added	 100	 μL	 of	 a	 24	 h	 old	 inoculum.	 The	 MIC,	
defined	 as	 the	 lowest	 concentration	 of	 the	 test	 compound,	
which	 inhibits	 the	 visible	 growth	 after	 18	 h,	 was	 determined	
visually	 after	 incubation	 for	18h,	 at	37	 oC,	 and	 the	 results	 are	
presented	 in	 Table	 3.	 DMSO	 and	 chloramphenicol	 used	 as	
negative	and	positive	controls,	respectively.	
	
2.3.	Calculation	method	
	

The	 theoretical	 structures	 1	 to	 7	 were	 generated	 using	
Spartan’04	 (Version	 1.0.3)	 Windows	 with	 the	 aim	 to	 predict	
their	 antibacterial	 activities.	 The	 optimized	 geometries	 of	 all	
the	 molecules	 were	 calculated	 by	 using	 quantum	 chemical	
calculations	 including	 Semi‐empirical	 and	 Ab	 initio	 method.	
These	methods	have	been	previously	used	successfully	for	the	
small	 molecule	 calculations	 [28,29].	 All	 the	 molecules	 were	
modelled	 and	 sent	 to	 energy	minimization,	 and	 then	 all	 their	
possible	 conformers	were	obtained	using	Semi‐empirical	PM3	
calculations.	However,	only	the	most	lowest	energy	conformer	
of	 each	 molecule	 was	 submitted	 for	 the	 higher	 level	 of	
calculations	 that	 is	 Hartree‐Fock	 (HF)	 with	 3‐21G*	 basis	 set,	
and	 then	 to	HF/6‐31G*	basis	set.	The	resulting	wave	 function,	
Hessian	matrix,	and	geometry	of	 the	molecules	obtained	were	
used	 and	 submitted	 for	 final	 calculation	 that	 is	 Density	
Functional	 Theory	 (DFT)	 with	 6‐31G*	 (Figure	 1).	 Analytical	
vibrational	 frequency	 calculations	were	 performed	 for	 all	 the	
molecules.	 Exact	 zero	 imaginary	 vibrations	 characterize	
stationary	 points.	 The	 asterisk	 means	 that	 “d”	 polarized	
functions	were	added	for	C,	O,	N	and	S	atoms.	
	
3.	Result	and	discussion		
	
3.1.	Chemistry		
	

Schiff	base	derivatives	were	synthesized	by	the	reaction	of	
3,5‐dimethyl‐1‐phenylpyrazole‐4‐carboxaldehyde	 and	 the	
chosen	active	amines,	by	using	microwave	 irradiation	 in	 good	
yield	[30].		



Khan	et	al.	/	European	Journal	of	Chemistry	4	(4)	(2013)	454‐458	 457	
 

	

	
	

Figure	1.	Density	and	potential	map	of	all	Schiff	bases	1 to	7 calculated	with	DFT/	RB3LYP	method	is	shown.	
	
	
Conventional	 methods	 present	 several	 hurdles,	 such	 as	

toxic	reagents,	waste	disposal	problem,	strong	acidic	conditions	
and	 low	 selectivity	 makes	 these	 methods	 environmentally	
hazardous.	 In	 this	 respect	 solvent	 phase	 synthesis	 under	
microwave	 irradiations	 is	 considered	 as	 eco‐friendly	
alternative.	 The	 obtained	 compounds	 are	 stable	 in	 the	 solid	
state	 as	well	 as	 in	 the	 solution	 state.	 The	 structure	 of	 all	 the	
compounds	was	established	by	spectral	data	 (IR,	 1H	NMR,	 13C	
NMR	and	GC‐MS).	Assignments	of	selects	characteristic	IR	band	
positions	provide	significant	indication	for	the	formation	of	the	
Schiff	 base	 derivative.	 The	 FT‐IR	 spectra	 of	 pyrazole	 schiff	
bases	 1‐7	 showed	 absorption	 bands	 at	 2912‐2974	 cm‐1	 for	
aromatic	 C‐H	 and	 at	 1544‐1598	 cm‐1	 for	 azomethine	 group	 (‐
CH=N‐).	 The	 absence	 of	 absorption	 band	 at	 1700‐1750	 cm‐1	
also	 confirms	 the	 conversion	of	 ‐CHO	group	 to	 ‐CH=N‐	 group.	
Further	evidence	for	the	formation	of	Schiff	base	derivative	was	
obtained	 from	 the	 1H	 NMR	 spectra,	 which	 provide	 diagnostic	
tools	 for	 the	 positional	 elucidation	 of	 the	 protons.	 The	
structural	 assignments	 of	 the	 NMR	 spectra	 are	 given	 in	 the	
experimental	 section.	Assignments	of	 the	signals	are	based	on	
the	chemical	shifts	and	intensity	patterns.	The	1H	NMR	spectra	
of	Schiff	bases	showed	peaks	of	aromatic,	methyl,	and	olefinic	(‐
N=CH‐)	proton.	These	were	all	singlets	and	each	one	indicating	
intensity	 of	 one	 proton	 in	 600	 MHz	 1H	 NMR.	 The	 1H	 NMR	
spectrum	of	 Schiff	 bases	 (1‐7)	 showed	 sharp	 singlets	 at	 8.84‐
10.20	 ppm	 indicating	 the	 presence	 of	 azomethine	 (‐CH=N‐)	
proton.	The	two	sharp	singlets	at	2.03‐2.62	ppm	indicated	the	‐
CH3	group	attached	to	the	carbon.	The	appearance	of	multiplets	
at	7.29‐7.62	ppm	was	due	 to	 aromatic	 protons.	Moreover,	 13C	
NMR	 spectra	 showed	 signals	 in	 the	 range	 of	 114.82‐118.96	

ppm	 and	 at	 125.39‐130.15	 ppm	 due	 to	 aryl	 carbon	 and	
azomethine	 carbon,	 respectively.	 In	 the	 mass	 spectrum,	
compound	 1	 showed	 a	 peak	 at	m/z	 387,	 which	 matches	 its	
molecular	formula	C23H23N5O.	A	peak	at	m/z	296	was	observed	
for	 compound	 2	 which	 is	 in	 conformity	 with	 the	 molecular	
formula	C17H18N4O.	Mass	spectra	of	other	compound	are	given	
in	experimental	section.		
	
3.2.	Antimicrobial	activity	
	

The	 compounds	 (1‐7)	were	 tested	 for	 their	 antibacterial	
activities	 by	 disc‐diffusion	 method	 using	 nutrient	 broth	
medium	[contained	 (g/L):	beef	extract	3	g;	peptone	5	g;	pH	=	
7.0]	 [31].	 The	 Gram‐positive	 bacteria	 and	 Gram‐negative	
bacteria	utilized	in	this	study	consisted	of	S.	aureus,	S.	pyogenes,	
S.	typhimurium	and	E.	coli.	In	the	disc‐diffusion	method,	sterile	
paper	discs	(0.5	mm)	impregnated	with	compound	dissolved	in	
dimethylsulfoxide	 (DMSO)	 at	 concentration	 100	 μg/mL	 were	
used.	 Then,	 the	 paper	 discs	 impregnated	with	 the	 solution	 of	
the	compound	tested	were	placed	on	the	surface	of	the	media	
inoculated	with	the	microorganism.	The	plates	were	incubated	
at	35	οC	for	24	h.	After	incubation,	the	growth	inhibition	zones	
are	 shown	 in	 Table	 2.	 The	 Schiff	 base	 derivative	 was	 further	
checked	by	MIC	method.	The	results	are	presented	in	Table	3.	
	
3.3.	Computation	
	

Some	 selected	descriptors	 such	as	 total	 energy,	molecular	
weight,	LUMO,	volume	and	density,	calculated	with	DFT/B3LYP	
method	are	presented	in	Table	4	[32].		



458	 Khan	et	al.	/	European	Journal	of	Chemistry	4	(4)	(2013)	454‐458	
	

	
Table	4.	Table	of	all	selected	descriptors	used	in	this	study,	calculated	by	DFT/RB3LYP	method.	
Compound	 Total	energy	(au)	 Molecular	weight	 LUMO	(eV)	 Volume	(ų)	 Density	
1	 ‐1239.19998 385.471 ‐0.84 392.93 0.981
2	 ‐952.78993	 294.358 ‐1.25 313.48 0.939
3	 ‐1350.82148	 332.431	 ‐1.67	 360.55	 0.922	
4	 ‐870.36216	 266.308	 ‐1.47	 280.62	 0.949	
5	 ‐1429.42854	 360.485	 ‐1.66	 387.62	 0.930	
6	 ‐876.40910	 276.343	 ‐0.93	 283.14	 0.976	
7	 ‐1370.55273 430.515 ‐1.55 443.37 0.971

	
	
The	 antibacterial	 activity	 of	 the	 pyrazole	 Schiff	 bases	 is	 a	

function	 of	 LUMO	 (Lowest	 Unoccupied	 Molecular	 Orbital)	
energy	 and	 the	 density.	 Descriptor	 LUMO	 is	 an	 electronic	
parameter,	 which	 measures	 the	 electrophilicity	 of	 the	
molecules.	 When	 a	 molecule	 acts	 as	 a	 Lewis	 acid,	 incoming	
electrons	 are	 received	 in	 its	 LUMO.	 Molecules	 with	 low‐lying	
LUMO	are	more	able	 to	accept	electrons	 than	 those	with	high	
energy	LUMO,	and	thus	will	show	higher	activity.	Density	is	a	3‐
D‐spatial	 descriptor	 that	 is	 defined	 as	 the	 ratio	 of	 molecular	
weight	to	molecular	volume.	Density	reflects	the	types	of	atoms	
and	 how	 tightly	 they	 are	 packed	 in	 a	 molecule.	 Density	 is	
negatively	correlated	with	activity.	This	means	that	if	molecule	
is	compact,	 it	will	reduce	the	density	and	thereby	increase	the	
activity.	

Molecule	 3	 has	 the	 lowest	 LUMO	 energy	 and	 the	 lowest	
density	as	compared	 to	all	 the	other	molecules	studied	 in	 this	
study	 (see	 Table	 4).	 Thus	 one	 can	 conclude	 the	 highest	
antibacterial	 activity	 of	 compound	3.	 In	 addition,	 molecule	 5	
has	also	lower	LUMO	energy	and	the	lower	density	as	compare	
to	molecule	2,	4,	6	and	7.	Thus,	compound	5	will	show	higher	
antibacterial	 activity	 than	2,	4,	6	and	7.	However,	Molecule	1	
has	the	higher	LUMO	energy	and	the	higher	density.	Thus,	one	
can	conclude	its	lowest	antibacterial	activity.		
	
4.	Conclusions	
	

Heterocyclic	 Schiff	 bases	were	 synthesis	 and	 screened	 for	
antibacterial	activity	based	on	 in‐vitro	and	DFT/B3LYP	model.	
These	pyrazole	containing	Schiff	bases	were	synthesized	by	the	
reaction	 of	 3,5‐dimethyl‐1‐phenylpyrazole‐4‐carboxaldehyde	
with	 the	 corresponding	 active	 heterocyclic	 amines	 under	
microwave	 irradiation.	 The	 antibacterial	 activity	 of	 these	
compounds	 was	 examined	 using	 cultures	 of	 bacteria	 and	 the	
results	showed	that	the	sulphur	containing	pyrazole	Schiff	base	
increased	 the	 antibacterial	 activity.	 Among	 the	 seven	
compounds,	 thiazole	 containing	 pyrazole	 Schiff	 base	 (3)	
showed	 better	 antibacterial	 activity	 for	 both	 types	 of	 the	
bacterias	 (Gram‐positive	 and	 Gram‐negative)	 as	 compared	 to	
reference	 drug	 chloramphenicol.	 Our	 experimental	 results	
were	 found	 in	 good	 collaboration	with	 the	 theoretical	 results.	
Structure	 activity	 relationship	 studies	 revealed	 that	 pyrazole	
substituted	 derivatives	 play	 important	 role	 in	 antimicrobial	
activity.	 A	 little	 structure	 variation	 can	 cause	 immense	
difference	 in	 the	 activity	 of	 the	drug.	This	 approach	 can	open	
new	 vistas	 in	 the	 chemotherapy	 of	 the	 infective	 disease.	 The	
field	is	further	open	for	pharmacokinetics	and	clinic	studies	to	
establish	these	molecules	as	drugs	in	the	market.		
	
Acknowledgements	
	

The	 authors	 are	 grateful	 to	 the	 Department	 of	 Chemistry	
and	 Center	 of	 Excellence	 for	 Advance	 Materials	 at	 King	
Abdulaziz	University	for	providing	research	facilities.		
	
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