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European	Journal	of	Chemistry	8	(1)	(2017)	82‐84	

	

European	Journal	of	Chemistry	
ISSN	2153‐2249	(Print)	/	ISSN	2153‐2257	(Online)		2017	Atlanta	Publishing	House	LLC	‐	All	rights	reserved	‐	Printed	in	the	USA	

http://dx.doi.org/10.5155/eurjchem.8.1.82-84.1537 

	
	

	

	

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

	 	 	

Synthesis	and	antimicrobial	activity	of		
1,3‐bis‐butyl‐5‐(4‐iodophenyl)‐1,3,5‐triazacyclohexane	

Leila	Lefrada	1,2,*,	Randolf	Köhn	3,	Souhila	Malki	1,2,	Wissam	Mazouz	1,4,		
Ahcene	Bouchemma	1,2	and	Meriem	Hadjem	1,2	

1	Laboratory	of	Applied	Chemistry	and	Materials	Technology	University	Larbi	Ben	M’hidi	of	Oum	El	Bouaghi,	04000,	Algeria	
2	Faculty	of	Exact	Sciences,	Department	of	Material	Science,	University	Larbi	Ben	M’hidi	of	Oum	El	Bouaghi,	Rue	de	Constantine,	04000,	Algeria	
3	Department	of	Chemistry	University	of	Bath,	Bath,	BA2	7AY,	United	Kingdom	
4	Department	of	Biology,	Faculty	of	Sciences,	University	Badji	Mokhtar,	2300	Annaba,	Algeria	

*	Corresponding	author	at:	Laboratory	of	Applied	Chemistry	and	Materials	Technology	University	Larbi	Ben	M’hidi	of	Oum	El	Bouaghi,	04000,	Algeria.		
Tel.:	+213.32421036.	Fax:	+213.32424213.	E‐mail	address:	aliel_2011@yahoo.fr	(L.	Lefrada).	

	
	

	 	

	 	 	

ARTICLE	INFORMATION	 	 ABSTRACT	

	
DOI: 10.5155/eurjchem.8.1.82-84.1537	

Received:	30	December	2016	
Received	in	revised	form:	21	January	2017	
Accepted:	22	January	2017	
Published	online:	31	March	2017	
Printed:	31	March	2017

 

	 This	work	describes	the	synthesis,	structural	characterization	and	antibacterial	activities	of
1,3,5‐triazacyclohexane	 type	 of	 new	 compound	 1,3‐bis‐butyl‐5‐(4‐iodophenyl)‐1,3,5‐
triazacyclohexane.	 The	 new	 triazacyclohexanes	 (R3TAC)	 with	 mixed	 aryl	 and	 alkyl	 N‐
substituents	 are	 synthesized	 by	 the	 reaction	 of	 a	 1:2	 mixture	 of	 4‐iodoaniline	 and	 n‐
butylamine	 with	 formalin.	 The	 synthesized	 compounds	 were	 characterized	 by	 spectral
analysis	IR,	1H	NMR	and	13C	NMR.	The	compound	was	screened	for	it’s	antibacterial	activity
against	Gram‐positive	and	Gram‐negative	bacteria	by	the	diffusion	method	on	agar	medium.	

KEYWORDS	
Formalin	
Synthesis	
n‐Butylamine	
4‐Iodoaniline	
Triazacyclohexane	
Antibacterial	activity	

Cite	this: Eur.	J.	Chem.	2017,	8(1),	82‐84

	
1.	Introduction	
	

The	 formation	 of	 1,3,5‐triazacyclohexane	 from	 primary	
amines	and	formaldehyde	has	been	known	for	more	than	one	
hundred	years	[1].	The	different	triazines	were	synthesized	in	
the	 laboratory	 according	 to	 the	 procedure	 described	
elsewhere	 [2].	 1,3,5‐Triazacyclohexanes	 can	 be	 employed	 as	
ligands	 for	 complexes	 used	 as	 catalyst	 in	 the	 polymerization	
and	 trimerization	 of	 olefines	 [3].	 Further,	 the	 interest	 in	
triazacyclohexanes	as	ligand	seems	to	growing	rapidly	[4‐8].	

Antibiotic	 resistance	 is	 a	 major	 problem	 in	 hospitals	 as	
well	 as	 in	 community	 settings	 [9].	 Considering	 the	 ever	
growing	 antibiotic	 resistance	 developed	 by	 many	 bacteria,	
there	is	an	immense	need	for	new	compounds	with	new	mode	
of	actions,	for	treatment	of	bacterial	infections	[10].	The	need	
for	 new	 antibiotics	 continues	 to	 be	 a	 still	 standing	 challenge	
[11].	1,3,5‐Triazacyclohexanes	containing	halides	exhibit	high	
biological	 activity	 since	 they	 contain	 CN	 group	 and	 halogen	
atom	 as	 pharmacophore.	 1,3,5‐Triazacyclohexane	 showed	 an	
activity	against	the	strains	of	microorganisms	used	[12].	
	

2.	Experimental	
	
2.1.	Instrumentation	

	
Purity	 of	 the	 compounds	 was	 checked	 by	 thin	 layer	

chromatography	 (TLC)	 using	 CH2Cl2:	 petroleum	 ether	 (4:1,	
v:v).	 IR	 spectra	 were	 prepared	 on	 the	Mattson	 Galaxy	 series	
FT‐IR	5000	spectrophotometer	using	KBr	discs.	NMR	spectra	
were	 recorded	 on	 Bruker	 spectrophotometer	 ARX	 500	 (500	
MHz	 for	 proton	 and	 100.62	 MHz	 for	 carbon).	 The	 chemical	
shifts	 (δ)	 are	 expressed	 in	 parts	 per	 million	 (ppm).	
Tetramethylsilane	 (TMS)	 is	 used	 as	 internal	 reference.	 The	
spectra	are	recorded	in	deuterated	chloroform	CDCl3	is	used	as	
solvent	(CHCl3:	δ	7.26	ppm,	CDCl3:	δ	77.0	ppm).	

	
2.2.	Synthesis	
	

Formaline	(37%,	3	mmol,	10	mL)	was	added	dropwise	to	a	
stirred	 solution	 of	 4‐iodophenylamine	 (10	 mmol)	 and	 n‐
butylamine	(20	mmol)	with	in	3	mL	of	ethanol.		

	



Lefrada	et	al.	/	European	Journal	of	Chemistry	8	(1)	(2017)	82‐84	 83	
 

	
Table	1.	Antibacterial	activity	of	gentamicin	expressed	as	the	diameter	of	the	inhibition	zone	in	mm	in	the	disk	sensitivity	assay.	
Bacterial	strains	 Gentamicin	
E.	coli		 31	mm
Resistent	S.	aureus		 28	mm
S.	aureus		 30	mm
	
Table	2.	Antibacterial	 activity	of	1,3‐bis‐butyl‐5‐(4‐iodophenyl)‐1,3,5‐triazacyclohexane	expressed	as	 the	diameter	of	 the	 inhibition	zone	 in	mm	 in	 the	disk	
sensitivity	assay.	
The	microbial	strains	 Concentrations	(mg/L)	

500 1000 2000	
E.	coli		 24	mm 22mm 20	mm	
Resistent	S.	aureus		 24 mm 22mm 23	mm	
S.	aureus		 10 mm 08mm 09	mm	

	

	
	

Scheme	1	
	
	
The	mixture	was	stirred	for	7	hours	at	20	°C.	The	resulting	

solution	 was	 evaporated	 on	 a	 rotary	 evaporator	 to	 dryness.	
Yield:	 89%.	 Boiling	 point:	 172‐174	 °C.	 Rf	 (Dichloromethane:	
petroleum	 ether,	 3:1,	 v:v):	 0.84.	 IR	 (KBr,	 ν,	 cm‐1):	 3085‐3060	
(Ar‐H),	2956‐2859	(CH3,	CH2),	1230‐1030	(C‐N),	817	(C‐I),	750	
(Ar‐H).	 1H	NMR	 (500	MHz,	 CDCl3,	 δ,	 ppm):	 0.90	 (t,	 6H,	 CH3),	
1.30	(m,	4H,	CH2),	1.42	(m,	4H,	CH2),	2.40	(t,	4H,	CH2),	3.39	(s,	
2H,	 C4H9‐N‐CH2‐N‐C4H9),	 4.05	 (s,	 4H,	 C4H9‐N‐CH2‐N‐Ar),	 6.71	
(d,	 2H,	 Ar),	 7.46	 (d,	 2H,	 Ar).	 13C	NMR	 (125.76	MHz,	 CDCl3,	 δ,	
ppm):	 13.88	 (CH3‐CH2),	 20.26	 (CH3‐CH2),	 29.57	 (C2H5‐CH2),	
51.83	 (C3H7‐CH2‐N),	 70.52	 (C4H9‐N‐CH2‐N‐C4H9),	 74.51	
(C4H9‐N‐CH2‐N‐Ar),	 82.62	 (C‐I),	 118.79	 (CH=C‐),	 137.62	
(CH=C‐),	149.67	(N‐C=).	
	
2.3.	Antibacterial	assays	

	
2.3.1.	Bacterial	strains	tested:	Germs	tested	to	detect	
antimicrobial	activity	of	compounds	
	

Escherichia	coli,	(also	known	as	E.	coli)	is	a	Gram‐negative,	
facultative	 anaerobic,	 rod‐shaped	 bacterium	 of	 the	 genus	
Escherichia	 that	 is	 commonly	 found	 in	 the	 lower	 intestine	 of	
warm‐blooded	 organisms	 (endotherms);	 Staphylococcus	
aureus	is	a	Gram‐positive	coccal	bacterium	that	is	a	member	of	
the	Firmicutes,	and	is	frequently	found	in	the	nose,	respiratory	
tract,	 and	 on	 the	 skin.	 It	 is	 often	 positive	 for	 catalase	 and	
nitrate	reduction.	Although	S.	aureus	is	not	always	pathogenic,	
it	 is	 a	 common	 cause	 of	 skin	 infections	 such	 as	 abscesses,	
respiratory	 infections	 such	 as	 sinusitis,	 and	 food	 poisoning.	
Pathogenic	 strains	 often	 promote	 infections	 by	 producing	
potent	protein	toxins,	and	expressing	cell‐surface	proteins	that	
bind	 and	 inactivate	 antibodies.	 The	 emergence	 of	 antibiotic‐
resistant	 strains	 of	 S.	 aureus	 such	 as	 Methicillin‐resistant	 S.	
aureus	(MRSA)	is	a	worldwide	problem	in	clinical	medicine.	

	
2.3.2.	The	culture	media	

	
The	 nutrient	 agar	 for	 the	 isolation	 and	 maintenance	 of	

bacterial	strains	and	the	Mueller	Hinton	agar	for	the	study	of	
the	susceptibility	of	bacteria	used	for	antimicrobial	tests.	
	
2.3.3.	Preparation	of	pre‐cultures	
	

Bacterial	 strains	 tested	 were	 grown	 in	 petri	 dishes	
containing	 nutrient	 agar.	 After	 18	 h	 incubation	 at	 37	 °C,	

bacterial	 suspensions	with	 an	optical	 density	 of	 1	McFarland	
were	 prepared	 for	 each	 microorganism	 in	 10	 mL	 of	 sterile	
physiological	saline.	

	
2.3.4.	Sensitivity	test	(Diffusion	on	agar	medium	method)		

	
Based	on	the	method	described	by	NCCLS	(1997),	different	

concentrations	of	compound	are	obtained	in	DMSO	(500,	1000	
and	 2000	 mg/L).	 The	 appropriate	 agar	 is	 poured	 into	 Petri	
dishes	 of	 90	 mm	 diameter	 and	 inoculated	 with	 a	 freshly	
prepared	pure	bacterial	suspension.	A	sterile	Whatman	paper	
disc	is	soaked	with	20	μL	of	each	dilution	and	gentamicin	disk	
(30	μg)	‐	antibiotic	aminoglycoside	active	against	a	variety	of	
bacteria‐used	as	a	positive	control.	All	the	discs	are	deposited	
on	 the	 surface	 of	 seeded	 agar,	 the	whole	 is	 incubated	 for	 24	
hours	at	37	°C.	Upon	application	of	the	discs,	the	extracts	and	
the	 antibiotic	 diffuse	 uniformly	 and	 after	 24	 hours	 of	
incubation,	 the	 presence	 of	 a	 circular	 zone	 of	 inhibition	 is	
sought.	
	
3.	Results	and	discussion		

	
3.1.	Synthesis	
	

The	 unsymmetrically	 substituted	 triazcyclohexanes	 1,3‐
bis‐butyl‐5‐(4‐iodophenyl)‐1,3,5‐triazacyclohexane	 were	 pre‐
pared	 from	 the	 condensation	 reaction	 of	 4‐iodophenylamine	
and	 n‐butylamine	 with	 formaldehyde	 in	 high	 yield	 (89%)	
(Scheme	 1).	 This	 compound	 is	 stable	 at	 room	 temperature.	
Unsymmetrically	 substituted	 products	 were	 obtained	
predominantly	 over	 the	 symmetrical	 triazacyclohexanes	 ‐	
much	 more	 than	 expected	 for	 statistical	 formation	 of	
triazacyclohexanes.	 This	 indicates	 higher	 stability	 for	 mixed	
substituents	[13‐15].		
	
3.2.	Biological	activity	

	
For	 the	 assessment	 of	 the	 antibacterial	 potential	 of	 the	

extracts	 studied,	 we	 choose	 to	 test	 them	 against	 many	
bacterial	 species,	 because	 each	 one	 has	 a	 particular	 cellular	
structures	 and	 metabolism	 [16].	 The	 sensitivity	 to	 different	
strains	 has	 been	 classified	 by	 the	 diameter	 of	 the	 inhibition	
zone	 is	 as	 follows:	 Diameter	 less	 than	 8	 mm:	 not	 sensitive;	
Diameter	 of	 9‐14	 mm:	 sensitive;	 Diameter	 15‐19	 mm:	 very	
sensitive;	Diameter	greater	than	20	mm:	extremely	sensitive.	

Results	 presented	 in	 the	 Table	 1	 and	 2	 showed	 that	
Staphylococcus	aureus	is	sensitive	against	1,3‐bis‐butyl‐5‐(4‐	



84	 Lefrada	et	al.	/	European	Journal	of	Chemistry	8	(1)	(2017)	82‐84	
	
	 	

	 	 	
	

Resistent	S.	aureus	 S.	aureus E.	coli.	
	

Figure	1.	Antibacterial	activity	of	1,3‐bis‐butyl‐5‐(4‐iodophenyl)‐1,3,5‐triazacyclohexane	in	the	disk	sensitivity	assay.	
	

	
iodophenyl)‐1,3,5‐triazacyclohexane,	while	resistant	Staphylo‐
coccus	 aureus	 and	 Escherichia	 coli	 are	 extremely	 sensitive	
against	our	compound.	Our	compound	showed	similar	activity	
to	that	of	the	positive	control,	which	has	a	broad	spectrum	of	
inhibition	against	a	variety	of	bacteria	(Figure	1).	

In	 summary,	 the	 new	 unsymmetrically	 substituted	
triazacyclohexanes	 1,3‐bis‐butyl‐5‐(4‐iodophenyl)‐1,3,5‐triaza	
cyclohexane	 was	 prepared	 from	 two	 amines	 and	 formal‐
dehyde.	 FT‐IR	 and	 NMR	 analyses	 of	 the	 compounds	 was	
reported	 and	 the	 solid‐state	 structures	 of	 some	 compound.	
The	 compound	 was	 targeted	 for	 their	 antibacterial	 activity	
against	 Gram‐positive	 and	 Gram‐negative	 bacteria	 by	 the	
diffusion	method	on	agar	medium	text	paragraph.	
	
Acknowledgements	
	

We	 thank	 the	 Ahcene	 Bouchemma	 director	 of	 the	
Laboratory	of	Applied	Chemistry	and	Materials	Technology	of	
the	 University	 Larbi	 Ben	 M’hidi	 of	 Oum	 El	 Bouaghi,	 Algeria.	
Research	Fund	(Project	No:	E03020130036).	
	
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