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European	Journal	of	Chemistry	6	(1)	(2015)	21‐30	

	

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.6.1.21‐30.1137	

	
	

	

	

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

	 	 	

Novel	antimicrobial	and	anti‐acetylcholinesterase	dihydroisoxazoles		
from	(R)‐limonene	

Enis	Ben	Bnina	1,	Anis	Romdhane	1,	Majda	Daami‐Remadi	2	and	Hichem	Ben	Jannet	1,*	

1	Laboratoire	de	Chimie	Hétérocyclique,	Produits	Naturels	et	Réactivité,	Equipe:	Chimie	Médicinale	et	Produits	Naturels,	Faculté	des	Sciences	de	Monastir,	Université	
de	Monastir,	Avenue	de	l’Environnement,	5019	Monastir,	Tunisie	
2	UR13AGR09,	Production	Horticole	Intégrée	au	Centre	Est	Tunisien,	Centre	Régional	des	Recherches	en	Horticulture	et	Agriculture	Biologique	de	Chott‐Mariem,	
Université	de	Sousse,	4042,	Chott‐Mariem,	Tunisie	

*	Corresponding	author	at:	Laboratoire	de	Chimie	Hétérocyclique,	Produits	Naturels	et	Réactivité,	Equipe:	Chimie	Médicinale	et	Produits	Naturels,	Faculté	des	
Sciences	de	Monastir,	Université	de	Monastir,	Avenue	de	l’Environnement,	5019	Monastir,	Tunisie.		
Tel.:	+21.67.3500279.	Fax:	+21.67.3500278.	E‐mail	address:	hich.benjannet@yahoo.fr	(H.B.	Jannet).	

	
	

	 	

	 	 	

ARTICLE	INFORMATION	 	 ABSTRACT	

	
DOI:	10.5155/eurjchem.6.1.21‐30.1137	

Received:	22	August	2014	
Received	in	revised	form:	19	September	2014		
Accepted:	19	September	2014	
Published	online:	31	March	2015	
Printed:	31	March	2015	

	 We	 report	 herein	 the	 convenient	 procedures	 for	 the	 efficient	 and	 easy	 synthesis,	 and	 the
antimicrobial	and	the	anti‐acetylcholinesterase	evaluation	of	two	new	series	of	(R)‐limonene
derivatives.	A	substantial	modification	aimed	at	targeting	to	discover	novel	structures	with	a
better	antimicrobial	and	anti‐acetylcholinesterase	(anti‐AChE)	activities.	The	condensation	of
(R)‐limonene	(1)	with	various	arylnitrile	oxides	led,	via	the	1,3‐dipolar	cycloaddition	reaction,
conducted	with	 complete	 region‐specificity,	 to	 a	 series	 of	 new	 limonene‐dihydroisoxazoles,
2a‐h.	On	the	other	hand,	N‐alkylation	of	the	previously	prepared	limonene‐lactam	derivative
(3)	 yielded	 the	 corresponding	 dipolarophile	 (4),	 which	 affords	 by	 condensation	 with
arylnitrile	 oxides	 the	 expected	 new	 dihydroisoxazoles,	 5a‐h.	 The	 target	 compounds	 were
completely	 characterized	 by	 1H	 NMR,	 13C	 NMR	 and	 MS.	 All	 the	 synthesized	 heterocyclic
compounds	were	 tested	 for	 their	 antimicrobial	 and	 anti‐acetylcholinesterase	 activities.	 The
dihydroisoxazoles	2a	(IZ	=	13.25	mm,	cc	=	1	mg/mL)	and	5b	(IZ	=	13.75	mm,	cc	=	1	mg/mL)
exhibited	 the	 highest	 antifungal	 activity.	 The	 greatest	 anti‐acetylcolinesterase	 activity	 was
exhibited	by	2f	(IC50	=	82±3	µg/mL)	and	by	5a	(IC50	=	99±1	µg/mL).	

KEYWORDS	
Lactam	
(R)‐Limonene	
Antimicrobial	
Cycloaddition	
Dihydroisoxazoles	
Anti‐acetylcholinesterase	 Cite	this:	Eur.	J.	Chem.	2015,	6(1),	21‐30	

	
1.	Introduction	
	

Limonene	 is	 the	most	 abundant	monoterpene,	which	 has	
both	 an	 endocyclic	 and	 an	 exocyclic	 double	 bond	 [1].	 It	 is	
found	in	several	aromatic	plants	and	particularly	in	peels	and	
flowers	of	citrus	species	[2].	Several	studies	carried	out	proved	
that	 limonene	 presents	 various	 biological	 activities	 such	 as	
antifungal,	 anti‐aflatoxigenic,	 antioxidant	 [3]	 and	 anti‐
acaricide	 effects	 [4]	 it	 prevents	 also	 tumor	 development	
induced	 in	 the	mammary	glands	 [5]	 the	 skin	 [6]	 the	 liver	 [7]	
the	lungs,	the	forestomach	[8]	and	the	pancreas	[9].	Moreover,	
it	 has	 been	 reported	 that	 limonene	 has	 an	 antiproliferative	
effect	in	a	variety	of	cell	types,	such	as	melanoma,	gastric	and	
prostate	 cancer	 cells	 [10].	 Thus	 far,	 most	 efforts	 have	 been	
directed	 in	 characterizing	 its	 ability	 to	 prevent	 carcinogen‐
induced	cancer	[11].		

This	 monocyclic	 monoterpene	 drew	 the	 attention	 of	 the	
researchers	 to	 be	 able	 to	 develop	 it	 as	 being	 a	 precursor	 in	
several	 synthesis	 reactions	 leading	 important	molecules	 that	
find	 application	 in	 several	 fields	 [12]	 such	 as	 chiral	 vicinal	
diamines,	 amino	 alcohols	 and	 aminophosphines	 [13].	 The	
alkoxylation	 of	 limonene	 furnished	 1‐methyl‐4‐(α‐alkoxy‐

isopropyl)‐cyclohexenes	 [14]	 which	 are	 used	 as	 flavors	 and	
fragrances	for	perfume	and	cosmetic	products,	as	additives	for	
pharmaceuticals	 and	 agricultural	 chemicals,	 as	well	 as	 in	 the	
food	industry	[15].		

In	 the	 other	 hand,	 heterocyclic	 compounds	 have	 so	 far	
been	synthesized	mainly	due	to	their	wide	range	of	biological	
activities.	 In	 fact,	 much	 attention	 has	 been	 paid	 to	 the	
synthesis	 of	 heterocyclic	 compounds	 bearing	 nitrogen	 and	
oxygen	containing	ring	system	dihydroisoxazoles	(also	named	
isoxazolines)	 are	 of	 great	 interest	 because	 they	 have	 proven	
potential	 pharmaceutical	 leads	 [16,17]	 and	 versatile	
intermediates	 for	 the	 synthesis	 of	 different	 classes	 of	
functionalized	molecules	and	a	variety	of	bioactive	compounds	
[18].	 They	 are	 associated	 with	 diverse	 pharmacological	
activities	 such	 as	 human	 influenza	 A	 virus	 [19],	 anti‐
tuberculosis	 [20],	 antimicrobial	 [21,22]	 antidepressant	 [23],	
antimuscarinic	 [24],	 anti‐inflammatory	 [25]	 and	 herbicidal	
effects	[26].		

Several	methods	 for	 the	preparation	of	dihydroisoxazoles	
have	 been	 reported	 [27,28].	 Particularly,	 1,3‐dipolar	 cyclo‐
addition	reaction	represents	a	useful	synthetic	method	for	the	
preparation	of	five	membered‐ring	heterocyclic	compounds.		



22	 Bnina	et	al.	/	European	Journal	of	Chemistry	6	(1)	(2015)	21‐30	
	

	

 

Scheme	1	
	
	
In	recent	work,	the	fully	isoxazolines	were	prepared	by	the	

reaction	of	arylnitrile	oxides	with	alkenes	[29,30].		
In	continuation	of	our	research	directed	towards	the	study	

of	the	reactivity	of	some	natural	products,	in	the	present	work,	
(R)‐limonene	 was	 our	 starting	 material.	 To	 enrich	 the	
previously	 series	 of	 dihydroisoxazoles	 prepared	 from	
limonene	 [31],	 we	 have	 treated	 it	 with	 another	 series	 of	
arylnitrile	 oxides	 leading	 to	 new	 region‐specific	 limonene‐
dihydroisoxazoles,	 2a‐h.	 In	 the	 other	 hand,	 in	 order	 to	
continue	 the	 exploration	 of	 the	 exocyclic	 double	 bond	 in	
limonene,	 a	 lactamation	 reaction	 was	 carried	 out	
regiospecifically	 affording	 an	 original	 limonene‐lactam	
derivative	 (3)	 which	 was	 the	 subject	 to	 an	 N‐allylation	
reaction	yielding	a	novel	N‐allyl	limonene‐lactame	(4)	used	as	
a	 second	 precursor	 to	 access	 to	 a	 new	 series	 of	
dihydroisoxazoles,	5a‐h.	 Considering	 the	potential	 interest	 of	
antibacterial	 [32‐34]	 and	 antifungal	 [35,36]	 effects	 of	
dihydroisoxazoles,	 we	 focused	 our	 biological	 valorization	 on	
the	study	of	these	activities	for	all	the	synthesized	compounds.	
The	anti‐acetylcholinesterase	activity	of	compounds	2a‐h	and	
5a‐h	was	also	evaluated	and	discussed	in	this	work.		
	
2.	Experimental	
	
2.1.	Chemistry	
	

Mass	 spectra	 were	 obtained	 with	 ESI‐TOF	 (LCT	 Premier	
XE,	 Waters)	 using	 the	 reflectron	 mode	 in	 the	 positive	 ion	
mode.	Leucine‐enkephaline	peptide	was	employed	as	the	Lock	
Spray	lockmass.	1H	(300	MHz)	and	13C	(75	MHz)	NMR	spectra	
were	recorded	on	a	Bruker	AM‐300	spectrometer,	using	CDCl3	
as	 solvent	 and	 non	 deuterated	 residual	 solvent	 was	 used	 as	
internal	 standard.	 Chemicals	 shifts	 (δ)	 are	 given	 in	parts	 per	
million	(ppm)	and	coupling	constants	(J)	in	Hertz.		
	
2.1.1.	General	method	for	the	preparation	of	compounds	2a‐
h	[30]	
	

In	a	typical	procedure,	 to	a	stirred	solution	of	chlorinated	
oximes	(1.1	mmol)	and	limonene	1	(Aldrich	Chemical	Co.,	90%	
purity,	98%,	enantiomeric	excess,	ee)	(1	mmol	 in	dry	toluene	
30	mL),	was	added	dropwise	a	 solution	of	 triethylamine	 (1.1	
mmol)	in	5	mL	of	toluene.	The	mixture	wad	refluxed	for	2‐4	h	
under	nitrogen.	The	reaction	evolution	was	monitored	by	TLC.	
When	 all	 the	 starting	materials	were	 consumed,	 the	mixture	
was	cooled	to	room	temperature,	 the	solvent	was	evaporated	
off	and	the	crude	was	purified	by	chromatography	on	a	silica	
gel	column	using	petroleum	ether:	chloroform	(6:4,	v:v)	to	give	
the	desired	isoxazolines,	2a‐h	(Scheme	1).		

(R)‐5‐methyl‐5‐((R)‐4‐methylcyclohex‐3‐enyl)‐3‐phenyl‐4,5‐
dihydroisoxazole	(2a):	Color:	White	solid.	Yield:	67%.	1H	NMR	
(300	MHz,	CDCl3,	δ,	ppm):	1.40	(s,	3H,	H‐6),	1.65	(s,	3H,	H‐7'),	
1.84‐2.17	(m,	7H,	H‐1',	H‐2',	H‐5',	H‐6'),	2.90	(d,	1H,	J	=	16.5	Hz,	
H‐4a),	3.22	(d,	1H,	 J	=	16.5	Hz,	H‐4b),	5.39	(s,	1H,	H‐3'),	7.36‐

7.71	 (m,	 5H,	 Ar‐H).	 13C	 NMR	 (75	MHz,	 CDCl3,	 δ,	 ppm):	 23.3‐
30.5,	42.4,	43.6,	89.9,	120.0,	126.0‐134.3,	134.1,	155.8.	HRMS	
(ESI+):	 calcd.	 for	 (C17H21NO)+	 [M+Na]+	 278.1623,	 found:	
278.1638.	

(R)‐5‐methyl‐5‐((R)‐4‐methylcyclohex‐3‐enyl)‐3‐p‐tolyl‐4,5‐
dihydroisoxazole	(2b):	Color:	White	solid.	Yield:	73%.	1H	NMR	
(300	MHz,	CDCl3,	δ,	ppm):	1.29	(s,	3H,	H‐6),	1.55	(s,	3H,	H‐7'),	
1.73‐2.06	(m,	7H,	H‐1',	H‐2',	H‐5',	H‐6'),	2.41	(s,	3H,	H‐7''),	2.79	
(d,	1H,	J	=	16.5	Hz,	H‐4a),	3.11	(d,	1H,	J	=	16.5	Hz,	H‐4b),	5.30	
(s,	1H,	H‐3'),	7.09	(d,	2H,	J	=	8.1	Hz,	Ar‐H),	7.45	(d,	2H,	J	=	8.1	
Hz,	Ar‐H).	 13C	NMR	 (75	MHz,	CDCl3,	 δ,	 ppm):	23.6‐30.9,	 24.3,	
42.9,	43.0,	89.9,	120.4,	126.3‐140.2,	134.4,	156.0.	HRMS	(ESI+):	
calcd.	for	(C18H23NO)+	[M+Na]+	292.1780,	found:	292.1792.	

(R)‐3‐(4‐ethylphenyl)‐5‐methyl‐5‐((R)‐4‐methylcyclohex‐3‐
enyl)‐4,5‐dihydroisoxazole	(2c):	Color:	White	solid.	Yield:	76%.	
1H	NMR	(300	MHz,	CDCl3,	δ,	ppm):	1.24	(t,	3H,	J	=	7.5	Hz,	H‐8''),	
1.39	(s,	3H,	H‐6),	1.65	(s,	3H,	H‐7'),	1.83‐2.12	(m,	7H,	H‐1',	H‐2',	
H‐5',	H‐6'),	2.66	(q,	2H,	J	=	7.5	Hz,	H‐7''),	2.90	(d,	1H,	,	J	=	16.5	
Hz,	 H‐4a),	 3.21	 (d,	 1H,	 J	 =	 16.5	 Hz,	 H‐4b),	 5.39	 (s,	 1H,	 H‐3'),	
7.21	(d,	2H,	J	=	8.1	Hz,	Ar‐H),	7.58	(d,	2H,	J	=	8.1	Hz,	Ar‐H).	13C	
NMR	(75	MHz,	CDCl3,	δ,	ppm):	15.5,	23.3‐30.6,	42.6,	43.7,	89.6,	
120.1,	 126.5‐146.2,	 134.1,	 155.7.	 HRMS	 (ESI)+:	 calcd.	 for	
(C19H25NO)+	[M+Na]+	306.1936,	found:	306.1938.	

(R)‐3‐(4‐methoxyphenyl)‐5‐methyl‐5‐((R)‐4‐methylcyclo	
hex‐3‐enyl)‐4,5‐dihydroisoxazole	(2d):	Color:	White	solid.	Yield:	
80%.	1H	NMR	(300	MHz,	CDCl3,	δ,	ppm):	1.43	(s,	3H,	H‐6),	1.63	
(s,	3H,	H‐7'),	1.87‐2.16	(m,	7H,	H‐1',	H‐2',	H‐5',	H‐6'),	2.94	(d,	
1H,	J	=	16.5	Hz,	H‐4a),	3.25	(d,	1H,	J	=	16.5	Hz,	H‐4b),	3.83	(s,	
3H,	 H‐7''),	 5.39	 (s,	 1H,	 H‐3'),	 6.89	 (d,	 2H,	 J	 =	 8.7	 Hz,	 Ar‐H),	
7.61(d,	 2H,	 J	 =	 8.7	 Hz,	 Ar‐H).	 13C	 NMR	 (75	 MHz,	 CDCl3,	 δ,	
ppm):	23.2‐30.5,	 42.4,	 43.8,	 55.4,	 114.1‐128.0,	 120.0,	 134.3,	
160.8.	HRMS	(ESI+):	calcd.	for	(C18H23NO2)+	[M+Na]+	308.1619,	
found:	308.1630.	

(R)‐5‐methyl‐5‐((R)‐4‐methylcyclohex‐3‐enyl)‐3‐(4‐nitro	
phenyl)‐4,5‐dihydroisoxazole	 (2e):	 Color:	 White	 solid.	 Yield:	
82%.	1H	NMR	(300	MHz,	CDCl3,	δ,	ppm):	1.38	(s,	3H,	H‐6),	1.64	
(s,	 3H,	H‐7'),	 1.73‐2.17	 (m,	 7H,	H‐1',	H‐2',	H‐5',	H‐6'),	 2.91(d,	
1H,	J	=	16.8	Hz,	H‐4a),	3.25	(d,	1H,	J	=	16.8	Hz,	H‐4b),	5.39	(s,	
1H,	H‐3'),	7.79	(d,	2H,	J	=	9	Hz,	Ar‐H),	8.24	(d,	2H,	J	=	9	Hz,	Ar‐
H).	 13C	 NMR	 (75	 MHz,	 CDCl3,	 δ,	 ppm):	 23.4‐30.4,	 42.0,	 42.7,	
91.6,	119.7,	123.4‐148.3,	134.3,	154.4.	HRMS	(ESI+):	calcd.	for	
(C17H20N2O3)+	[M+Na]+	323.1474,	found:	323.1486.	

(R)‐3‐(4‐chlorophenyl)‐5‐methyl‐5‐((R)‐4‐methylcyclohex‐3‐
enyl)‐4,5‐dihydroisoxazole	(2f):	Color:	White	solid.	Yield:	87%.	
1H	NMR	 (300	MHz,	CDCl3,	 δ,	 ppm):	1.39	 (s,	 3H,	H‐6),	 1.65	 (s,	
3H,	H‐7'),	1.93‐2.11	(m,	7H,	H‐1',	H‐2',	H‐5',	H‐6'),	2.89	(d,	1H,	J	
=	16.5	Hz,	H‐4a),	3.19	(d,	1H,	J	=	16.5	Hz,	H‐4),	5.38	(s,	1H,	H‐
3'),	7.35	(d,	2H,	J	=	9	Hz,	Ar‐H),	7.58	(d,	2H,	J	=	9	Hz,	Ar‐H).	13C	
NMR	 (75	 MHz,	 CDCl3,	 δ,	 ppm):	 23.0‐30.8,	 42.8,	 43.7,	 90.6,	
120.2,	 128.0‐136.0,	 134.6,	 155.2.	 HRMS	 (ESI+):	 calcd.	 for	
(C17H20ClNO)+	[M+Na]+	312.1233,	found:	312.1242.	

	(R)‐3‐(furan‐2‐yl)‐5‐methyl‐5‐((R)‐4‐methylcyclohex‐3‐en	
yl)‐4,5‐dihydroisoxazole	(2g):	Color:	Gray	solid.	Yield:	70%.		

	



Bnina	et	al.	/	European	Journal	of	Chemistry	6	(1)	(2015)	21‐30	 23	
 

	

 
	

Scheme	2	
	
	
1H	NMR	(300	MHz,	CDCl3,	δ,	ppm):	1.38	(s,	3H,	H‐6),	1.65	

(s,	3H,	H‐7'),	1.82‐2.16	 (m,	7H,	H‐1',	H‐2',	H‐5',	H‐6'),	2.86	(d,	
1H,	J	=	16.8	Hz,	H‐4a),	3.18	(d,	1H,	J	=	16.8	Hz,	H‐4b),	5.39	(s,	
1H,	H‐3'),	6.47	(dd,	1H,	J1	=	3.3	Hz,	J2	=	1.8	Hz,	Ar‐H),	6.65	(d,	
1H,	J	=	3.3	Hz,	Ar‐H),	7.49	(d,	1H,	J	=	1.8	Hz,	Ar‐H).	13C	NMR	(75	
MHz,	CDCl3,	δ,	ppm):	23.1‐30.8,	42.7,	43.7,	δ	90.2	(C‐5),	111.3‐
112.0,	120.3,	134.5,	144.3‐146.0,	153.7.	HRMS	(ESI+):	calcd.	for	
(C15H19NO2)+	[M+Na]+	268.1416,	found:	268.1427.	

(R)‐5‐methyl‐5‐((R)‐4‐methylcyclohex‐3‐enyl)‐3‐(thiophen‐
2‐yl)‐4,5‐dihydroisoxazole	 (2h):	Color:	 Gray	 solid.	 Yield:	 68%.	
1H	NMR	 (300	MHz,	CDCl3,	 δ,	 ppm):	 1.39	 (s,	 3H,	H‐6),	 1.64	 (s,	
3H,	H‐7'),	1.87‐2.17	(m,	7H,	H‐1',	H‐2',	H‐5',	H‐6'),	2.90	(d,	1H,			
J	=	16.8	Hz,	H‐4a),	3.22	(d,	1H,	J	=	16.8	Hz,	H‐4b),	5.38	(s,	1H,	H‐
3'),	7.03	(dd,	1H,	J1	=	5.7	Hz,	J2	=	4.5	Hz,	Ar‐H),	7.14	(d,	1H,	J	=	
4.5	Hz,	Ar‐H),	7.34	(d,	1H,	J	=	5.7	Hz,	Ar‐H).	13C	NMR	(75	MHz,	
CDCl3,	δ,	ppm):	24.5‐31.4,	42.6,	43.7,	90.7,	116.1‐117.6,	120.3,	
133.9,	 151.9‐152.1,	 154.0.	 HRMS	 (ESI+):	 calcd.	 for	
(C15H19NOS)+	[M+Na]+	284.1187,	found:	284.1199.	
	
2.1.2.	General	method	for	the	preparation	of	(R)‐4‐methyl‐
4‐((R)‐4‐methylcyclohex‐3‐enyl)azetidin‐2‐one	(3)	[37]	
	

A	mixture	of	2.72	g	 (20.0	mmol)	of	 (R)‐limonene	 (1)	and	
2.84	g	(20.0	mmol)	of	CSI	was	stirred	in	dry	diethyl	ether	(50	
mL)	at	room	temperature	for	9	h.	Na2SO3	(3.78	g)	in	water	(50	
mL)	was	then	cautiously	added	dropwise	to	the	solution.	The	
pH	was	held	at	7‐8	by	the	addition	of	20%	aqueous	KOH.	After	
separation	of	the	organic	layer,	the	aqueous	one	was	extracted	
with	 diethyl	 ether	 (2×50	 mL).	 The	 combined	 organic	 layers	
were	dried	(Na2SO4),	evaporated	and	the	resulting	residue	was	
purified	 by	 chromatography	 on	 silica	 gel	 using	 chloroform:	
ethyl	acetate	(6:4,	v:v)	to	yield	compound	3	(Scheme	2).		

(R)‐4‐methyl‐4‐((R)‐4‐methylcyclohex‐3‐enyl)azetidin‐2‐one	
(3):	Color:	White	solid.	Yield:	78%.	1H	NMR	(300	MHz,	CDCl3,	δ,	
ppm):	1.52	(s,	3H,	s,	H‐5),	1.56‐1.94	(m,	7H,	H‐1',	H‐2',	H‐5',	H‐
6'),	1.71	(s,	3H,	H‐7'),	2.42	(d,	1H,	 J	=	14.4	Hz,	H‐3a),	2.66	(d,	
1H,	 J	=	14.4	Hz,	H‐3a),	5.37	(s,	1H,	H‐3'),	8.08	(s,	1H,	NH).	 13C	
NMR	 (75	 MHz,	 CDCl3,	 δ,	 ppm):	 21.3‐31.0,	 36.3,	 39.9,	 54.0,	
119.2,	 133.6,	 167.7.	 HRMS	 (ESI+):	 calcd.	 for	 (C11H17NO)+	
[M+H]+	180.1368,	found:	180.1380.	
	
2.1.3.	General	method	for	the	preparation	of	(R)‐1‐allyl‐4‐
methyl‐4‐((R)‐4‐methylcyclohex‐3‐enyl)azetidin‐2‐one	(4)	
[38]		
	

A	mixture	 of	 lactam	3	 (0.70	mmol),	 anhydrous	 DMF	 (12	
mL)	 and	 sodium	 hydride	 (1.3	 mmol)	 was	 stirred	 at	 room	
temperature	 in	 the	 argon	 atmosphere	 until	 evolution	 of	
hydrogen	had	ceased.	Then	the	allyl	bromide	(3.45	mmol)	was	
dropped	into	the	mixture	and	the	stirring	was	continued	for	1	
h.	 The	 mixture	 was	 poured	 into	 water	 and	 extracted	 with	
diethyl	 ether.	 The	 organic	 layer	was	 dried	 over	MgSO4.	 Then	
the	 solvent	 was	 removed	 and	 the	 residue	 was	 purified	 by	

chromatography	 on	 silica	 gel	 using	 chloroform‐ethyl	 acetate	
(9:1,	v:v)	to	produce	4	(Scheme	2).		

(R)‐1‐allyl‐4‐methyl‐4‐((R)‐4‐methylcyclohex‐3‐enyl)azeti	
din‐2‐one	 (4):	 Color:	 White	 solid.	 Yield:	 82%.	 1H	 NMR	 (300	
MHz,	CDCl3,	δ,	ppm):	1.35	(s,	3H,	H‐5),	1.62‐1.90	(m,	7H,	H‐1',	
H‐2',	H‐5',	H‐6'),	1.71	(s,	3H,	H‐7'),	2.42	(d,	1H,	 J	=	14.4Hz,	H‐
3a),	2.66	(d,	1H,	J	=	14.4Hz,	H‐3b),	3.68‐3.72	(m,	2H,	H‐6),	5.12	
(dd,	1H,	Jcis	=	10.2	Hz,	J2	=	1.2	Hz,	H‐8),	5.22	(dd,	1H,	Jtrans	=	17.1	
Hz,	 J2	=	1.8	Hz,	H‐8),	5.37	(s,	1H,	H‐3'),	5.77	(m,	1H,	H‐7).	 13C	
NMR	 (75	 MHz,	 CDCl3,	 δ,	 ppm):	 21.4‐30.2,	 38.6,	 46.1,	 116.2,	
120.3,	133.4,	134.2,	167.2.	HMRS	(ESI+):	calcd.	for	(C14H21NO)+	
[M+H]+	220.1682,	found:	220.1693.	
	
2.1.4.	General	method	for	the	preparation	of	compounds	5a‐
h	[30]	
	

In	 a	 typical	 procedure,	 to	 a	 stirred	 solution	 of	 arylnitrile	
oxide	 (1.2	mmol)	 and	 compound	4	 (1	mmol)	 in	 dry	 toluene	
(30	mL),	was	added	dropwise	a	solution	of	triethylamine	(1.2	
mmol)	 in	 5	mL	 of	 toluene.	 The	mixture	was	 refluxed	 for	 2‐4	
hours	under	nitrogen.	The	reaction	evolution	was	cheeked	by	
TLC.	 When	 all	 the	 starting	 materials	 were	 consumed,	 the	
mixture	 was	 cooled	 to	 room	 temperature,	 the	 solvent	 was	
evaporated	off	and	the	crude	was	purified	by	chromatography	
on	a	silica	gel	column	using	chloroform‐ethyl	acetate	(9:1,	v:v)	
to	yield	the	desired	cycloadducts,	5a‐h	(Scheme	3).	

(R)‐4‐methyl‐4‐((R)‐4‐methylcyclohex‐3‐enyl)‐1‐((3‐phenyl‐
4,5‐dihydroisoxazol‐5‐yl)methyl)azetidin‐2‐one	 (5a):	 Color:	
White	 solid.	 Yield:	 70%.	 1H	 NMR	 (300	 MHz,	 CDCl3,	 δ,	 ppm):	
1.40	(s,	3H,	H‐5'),	1.70	(s,	3H,	H‐7''),	1.79‐1.99	(m,	7H,	H‐1'',	H‐
2'',	H‐5'',	H‐6''),	2.38	(d,	1H,	J	=	14.7	Hz,	H‐3'a),	2.73‐3.07	(m,	
2H,	H‐4),	2.75	(d,	1H,	J	=	14.7	Hz,	H‐3'b),	3.22‐3.52	(m,	2H,	H‐
6),	5.30	(s,	1H,	H‐3''),	5.71‐5.84	(m,	1H,	H‐5),	7.33‐7.61	(m,	5H,	
Ar‐H).	13C	NMR	(75	MHz,	CDCl3,	δ,	ppm):	21.4‐30.2,	23.0,	23.8,	
39.1,	 39.5,	 54.5,	 55.6,	 61.8,	 120.5,	 127.8‐132.3,	 134.2,	 159.8,	
166.5.	HRMS	(ESI+):	calcd.	for	(C21H26N2O2)+	[M+H]+	339.1994,	
found:	339.1999.	

(R)‐4‐methyl‐4‐((R)‐4‐methylcyclohex‐3‐enyl)‐1‐((3‐p‐tolyl‐
4,5‐dihydroisoxazol‐5‐yl)methyl)azetidin‐2‐one	 (5b):	 Color:	
White	 solid.	 Yield:	 77%.	 1H	 NMR	 (300	 MHz,	 CDCl3,	 δ,	 ppm):	
1.41	(s,	3H,	H‐5'),	1.71	(s,	3H,	H‐7''),	1.79‐1.91	(m,	7H,	H‐1'',	H‐
2'',	H‐5'',	H‐6''),	2.39	(s,	3H,	H‐7''')	2.42	(d,	1H,	J	=	14.4	Hz,	H‐
3'a),	 2.76	 (d,	 1H,	 J	 =	 14.4	Hz,	 H‐3'b),	 2.77‐3.10	 (m,	 2H,	H‐4),	
3.18‐3.60	(m,	2H,	H‐6),	5.30	(s,	1H,	H‐3''),	5.70‐5.84	(m,	1H,	H‐
5),	7.14	(d,	2H,	J	=	8.1	Hz,	Ar‐H),	7.48	(d,	2H	J	=	8.1	Hz,	Ar‐H).	
13C	NMR	(75	MHz,	CDCl3,	δ,	ppm):	21.6‐30.3,	22.9,	23.9,	24.2,	
39.1,	 39.6,	 54.4,	 55.9,	 62.4,	 120.1,	 128.6‐130.8,	 134.3,	 139.8,	
160.6,	 166.3.	 HRMS	 (ESI+):	 calcd.	 for	 (C22H28N2O2)+	 [M+H]+	
353.2151,	found:	353.2162.	

(R)‐1‐((3‐(4‐ethylphenyl)‐4,5‐dihydroisoxazol‐5‐yl)methyl)‐
4‐methyl‐4‐((R)‐4‐methylcyclohex‐3‐enyl)azetidin‐2‐one	 (5c):	
Color:	White	solid.	Yield:	75%.		
	



24	 Bnina	et	al.	/	European	Journal	of	Chemistry	6	(1)	(2015)	21‐30	
	
	

N

O

1

2

3

4
5

6

7 8

6'

1'

2'

3'

4'

5'

7'

4

Ar N

Cl

O

H

N

O
1

2

3

4
5

6

6''

1'

2'

3'

4'
5'

7''

5a-h

O

N
Ar

1''

3''

4''

5''

5a: Ar= Ph

5b: Ar= p-PhCH3
5c: Ar= p-PhC2H5
5d: Ar= p-PhOCH3
5e: Ar= p-PhCl

5f: Ar= Pyrrole

5g: Ar= FuranToluene,

Et3N

5h: Ar= Thiophene

 
	

Scheme	3
	
	
1H	NMR	(300	MHz,	CDCl3,	 δ,	 ppm):	1.24	 (t,	3H,	 J	 =	7.2	Hz,	H‐
8'''),	1.40	(s,	3H,	H‐5'),	1.57‐1.89	(m,	7H,	H‐1'',	H‐2'',	H‐5'',	H‐
6''),	1.71	(s,	3H,	H‐7''),	2.44	(d,	1H,	J	=	15.3	Hz,	H‐3'a),	2.65	(q,	
2H,	J	=	7.2	Hz,	H‐7'''),	2.73	(d,	1H,	J	=	15.3	Hz,	H‐3'b),	2.75‐3.11	
(m,	2H,	H‐4),	3.81‐3.57	(m,	2H,	H‐6),	5.28	(s,	1H,	H‐3''),	5.68‐
5.82	(m,	1H,	H‐5),	6.96	(d,	2H,	J	=	8.1	Hz,	Ar‐H),	7.48	(d,	2H,	J	=	
8.1	 Hz,	 Ar‐H).	 13C	 NMR	 (75	MHz,	 CDCl3,	 δ,	 ppm):	 14.2,	 21.4‐
30.6,	23.1,	24.0,	31.8,	39.2,	39.6,	54.8,	55.8,	62.0,	119.8,	127.3‐
130.9,	 134.2,	 160.7,	 167.3.	 HRMS	 (ESI+)	 calcd.	 for	
(C23H30N2O2)+	[M+H]+	367.2307,	found:	367.2321.	

(R)‐1‐((3‐(4‐methoxyphenyl)‐4,5‐dihydroisoxazol‐5‐yl)	
methyl)‐4‐methyl‐4‐((R)‐4‐methylcyclohex‐3‐enyl)azetidin‐
2‐one	(5d):	Color:	White	solid.	Yield:	87%.	1H	NMR	(300	MHz,	
CDCl3,	δ,	ppm):	1.41	(s,	3H,	H‐5'),1.63‐1.98	(m,	7H,	H‐1'',	H‐2'',	
H‐5'',	H‐6''),	1.75	(s,	3H,	H‐7''),	2.52	(d,	1H,	J	=	14.7Hz,	H‐3'a),	
2.84	(d,	1H,	J	=	14.7	Hz,	CH2‐3'b),	2.85‐3.38	(m,	2H,	H‐4),	3.66‐
3.70	(m,	2H,	H‐6),	3.83	(s,	3H,	H‐7'''),	5.36	(s,	1H,	H‐3''),	5.72‐
5.83	(m,	1H,	H‐5),	6.90	(d,	2H,	J	=	8.7	Hz,	Ar‐H),	7.60	(d,	2H,	J	=	
8.7	 Hz,	 Ar‐H).	 13C	 NMR	 (75	 MHz,	 CDCl3,	 δ,	 ppm):	 21.9‐30.9,	
22.8,	23.5,	39.2,	39.5,	55.4,	56.3,	58.2,	61.8,	114.1,	121.7,	127.3‐
129.7,	 134.3,	 160.4,	 161.1,	 168.7.	 HRMS	 (ESI+):	 calcd.	 for	
(C22H28N2O3)+	[M+H]+	369.2100,	found:	369.2108.	

(R)‐1‐((3‐(4‐chlorophenyl)‐4,5‐dihydroisoxazol‐5‐yl)	
methyl)‐4‐methyl‐4‐((R)‐4‐methylcyclohex‐3‐enyl)azetidin‐
2‐one	(5e):	Color:	Yellow	solid.	Yield:	85%.	1H	NMR	(300	MHz,	
CDCl3,	δ,	ppm):	1.41	(s,	3H,	H‐5'),	1.61	(s,	3H,	H‐7''),	1.77‐1.99	
(m,	7H,	H‐1'',	H‐2'',	H‐5'',	H‐6''),	2.50	(d,	1H,	J	=	14.1	Hz,	H‐3'a),	
2.75	(d,	1H,	J	=	14.1	Hz,	CH2‐3'b),	2.84‐3.38	(m,	2H,	H‐4),	3.64‐
3.78	 (m,	2H,	H‐6),	 5.34	 (s,	 1H,	H‐3''),	 5.72‐5.83	 (m,	 1H,	H‐5),	
7.54	(d,	2H,	J	=	8.4	Hz,	Ar‐H),	7.67	(d,	2H,	J	=	8.4	Hz,	Ar‐H).	13C	
NMR	(75	MHz,	CDCl3,	δ,	ppm):	21.5‐30.4,	23.2,	23.8,	39.3,	39.7,	
55.7,	 59.6,	 62.4,	 121.3,	 δ	 127.8‐131.7,	 134.2,	 135.3,	 161.7,	
167.8.	 HRMS	 (ESI+):	 calcd.	 for	 (C21H25ClN2O2)+	 [M+H]+	
373.1605,	found:	373.1613.		

(R)‐1‐((3‐(1H‐pyrrol‐2‐yl)‐4,5‐dihydroisoxazol‐5‐yl)methyl)‐
4‐methyl‐4‐((R)‐4‐methylcyclohex‐3‐enyl)azetidin‐2‐one	 (5f):	
Color:	Crimson	solid.	Yield:	72%.	1H	NMR	(300	MHz,	CDCl3,	δ,	
ppm):	1.40	(s,	3H,	H‐5'),	1.62‐2.01	(m,	7H,	H‐1'',	H‐2'',	H‐5'',	H‐
6''),	 1.73	 (s,	 3H,	H‐7''),	 2.49	 (d,	 1H,	 J	 =	 11.4	Hz,	H‐3'a),	 2.77‐
3.08	(m,	2H,	H‐4),	2.84	(d,	1H,	J	=	11.4	Hz,	H‐3'b),	3.16‐3.52	(m,	
2H,	H‐6),	5.35	(s,	1H,	H‐3''),	5.77‐5.86	 (m,	1H,	H‐5),	7.04	 (dd,	
1H,	J1	=	3.6	Hz,	J2	=	2.4	Hz	,	Ar‐H),	7.18	(d,	1H,	J	=	3.6	Hz,	Ar‐H),	
7.36	(d,	1H,	J	=	2.4	Hz,	Ar‐H),	11.18	(s,	1H,	NH).	 13C	NMR	(75	
MHz,	 CDCl3,	 δ,	 ppm):	 21.8‐30.3,	 23.3,	 24.1,	 39.3,	 40.0,	 61.4,	
61.6,	 79.4,	 117.8‐119.7,	 119.8,	 134.2,	 167.0,	 168.7.	 HRMS	
(ESI+):	 calcd.	 for	 (C19H25N3O2)+	 [M+H]+	 328.1947,	 found:	
328.1959.	

(R)‐1‐((3‐(furan‐2‐yl)‐4,5‐dihydroisoxazol‐5‐yl)methyl)‐4‐
methyl‐4‐((R)‐4‐methylcyclohex‐3‐enyl)azetidin‐2‐one	 (5g):	
Color:	 White	 solid.	 Yield:	 68%.	 1H	 NMR	 (300	 MHz,	 CDCl3,	 δ,	
ppm):	1.39	(s,	3H,	H‐5'),1.57‐1.92	(m,	7H,	H‐1'',	H‐2'',	H‐5'',	H‐
6''),	1.69	(s,	3H,	H‐7''),	2.41(d,	1H,	J	=	11.1	Hz,	H‐3'a),	2.73‐3.07	

(m,	2H,	H‐4),	2.76	(d,	1H,	J	=	11.1	Hz,	H‐3'b),	3.22‐3.52	(m,	2H,	
H‐6),	5.31	(s,	1H,	H‐3''),	5.71‐5.84	(m,	1H,	H‐5),	7.18	(dd,	1H,	J	
=	3.6	Hz,	J	=	1.5	Hz,	Ar‐H),	7.18	(d,	1H,	J	=	3.6	Hz,	Ar‐H),	7.36	(d,	
1H,	J	=	1.5,	Ar‐H).	13C‐NMR	(75	MHz,	CDCl3,	δ,	ppm):	21.8‐30.3,	
23.1,	 24.0,	 39.3,	 39.9,	 61.5,	 61.7,	 79.2,	 108.6‐109.7,	 122.1,	
134.4,	 139.2‐143.9,	 165.7,	 167.3.	 HRMS	 (ESI+):	 calcd.	 for	
(C19H24N2O3)+	[M+H]+	329.1787,	found:	329.1799.	

(R)‐4‐methyl‐4‐((R)‐4‐methylcyclohex‐3‐enyl)‐1‐((3‐(thio	
phen‐2‐yl)‐4,5‐dihydroisoxazol‐5‐yl)methyl)azetidin‐2‐one	(5h):	
Color:	 Gray	 solid.	 Yield:	 74%.	 1H	 NMR	 (300	 MHz,	 CDCl3,	 δ,	
ppm):	1.41	(s,	3H,	H‐5'),	1.63‐1.97	(m,	7H,	H‐1'',	H‐2'',	H‐5'',	H‐
6''),	 1.69	 (s,	 3H,	H‐7''),	 2.51	 (d,	 1H,	 J	 =	 13.5	Hz,	H‐3'a),	 2.75‐
2.86	(m,	2H,	H‐4),	2.80	(d,	1H,	J	=	13.5	Hz,	H‐3'b),	3.38‐3.73	(m,	
2H,	H‐6),	5.35	(s,	1H,	H‐3''),	5.83	(m,	1H,	H‐5),	7.05	(dd,	1H,	J	=	
6	Hz,	 J	=	4.8	Hz,	Ar‐H),	7.19	(d,	1H,	 J	=	4.8	Hz,	Ar‐H),	7.39	(d,	
1H,	 J	 =	 6	Hz,	 Ar‐H).	 13C	NMR	 (75	MHz,	 CDCl3,	 δ,	 ppm):	 21.8‐
30.2,	23.2,	23.9,	39.3,	39.9,	61.4,	61.6,	79.4,	119.8,	127.4‐128.7,	
134.0,	 167.0,	 168.1.	 HRMS	 (ESI+):	 calcd.	 for	 (C19H24N2O2S)+	
[M+H]+	345.1635,	found:	345.1649.	
	
2.2.	Biological	activities	
	
2.2.1.	Antimicrobial	evaluation	
		

For	 the	 assessment	 of	 the	 antibacterial	 activity	 of	 (R)‐
Limonene	 1	 and	 the	 synthesized	 compounds,	 Burkholderia	
glathei	 153	 and	 Bacillus	 pumilus	 420	 Pseudomonas	
aureofaciens	499	were	used	as	bacterial	organisms.	They	were	
cultured	 at	 25	 °C	 on	 Nutrient	 Agar	 (NA)	 medium	 for	 48	 h	
before	use.	

For	 the	 antifungal	 test,	 five	 fungal	 species	 were	 used	
namely	 Aspergillus	 niger,	 A.	 flavus	 Penicillium	 digitatum,	
Trichoderma	 harzianum	 and	 Fusarium	 solani.	 They	 were	
cultured	at	25	°C	during	7	days	on	Potato	Dextrose	Agar	(PDA)	
medium	before	use.		

These	microorganisms	were	obtained	from	the	Laboratory	
of	 Phytopathology	 of	 the	 Regional	 Center	 of	 Research	 on	
Horticulture	and	Organic	Agriculture	of	Chott‐Mariem,	Tunisia.	
	
2.2.2.	Antibacterial	activity	
		

(R)‐Limonene	 1	 and	 the	 synthesized	 products	 were	
screened	 for	 their	 antibacterial	 activity	 using	 the	 agar	 disc	
diffusion	method	 [39].	Nutrient	Agar	 (NA)	medium	cooled	 at	
45	 °C	 was	 supplemented	 with	 a	 bacterial	 suspension	 (106	
CFU/mL)	 and	 poured	 into	 Petri	 plates.	 After	 solidification,	
sterile	Whatman	paper	discs	(diameter	6	mm)	were	placed	at	
the	 surface	 of	 the	 culture	medium	 and	 20	 μL	 of	 the	 product	
dissolved	 in	DMSO	 at	 different	 concentrations	 (250,	 500	 and	
1000	 μg/mL)	 were	 dropped	 onto	 each	 disc.	 The	 negative	
control	 plates	 had	 no	 product	 added	 to	 the	 filter	 paper	
whereas	in	the	positive	control	plates,	discs	were	impregnated	



Bnina	et	al.	/	European	Journal	of	Chemistry	6	(1)	(2015)	21‐30	 25	
 

with	 the	 same	volume	of	Ampicillin	 solution	 (5	mg/mL).	The	
treated	 Petri	 dishes	 were	 incubated	 at	 25	 °C	 for	 48	 h.	 The	
antibacterial	activity	was	evaluated	by	measuring	the	diameter	
of	 the	 inhibitory	 zones	 formed	 around	 the	 discs.	 The	
experiment	was	performed	in	triplicate.	
		
2.2.3.	Antifungal	activity	
	

Aspergillus	 niger,	 A.	 flavus	 Penicillium	 digitatum,	
Trichoderma	harzianum	and	Fusarium	solani	were	used	for	the	
screening	of	antifungal	activity	of	the	products	tested	by	using	
the	 disc	 diffusion	method	 [40].	 A	 conidial	 suspension	 of	 the	
tested	 fungi	 was	 prepared	 (104‐105	 CFU/mL)	 and	 added	 to	
PDA	medium	cooled	at	45	°C	and	poured	uniformly	into	Petri	
plates	 (diameter	 90	 mm).	 Sterilized	 paper	 discs	 (6	 mm,	
Whatman	No.	1	filter	paper)	were	 impregnated	with	20	µL	of	
the	 product	 dissolved	 in	 DMSO	 at	 different	 concentrations	
(250,	500	and	1000	μg/mL)	and	placed	on	 the	culture	plates	
whereas	 the	negative	control	plates	had	no	product	added	 to	
the	 filter	 paper.	 In	 the	 positive	 control	 plates,	 discs	 were	
imbibed	with	 the	 same	volume	of	 a	Carbendazim	suspension	
(0.5	 mg/mL).	 The	 diameter	 of	 the	 inhibition	 zone	 (mm)	
around	the	disc	was	measured	after	 incubation	at	25	°C	for	4	
days.	The	test	was	performed	in	triplicate.	
	
2.2.4.	Statistical	analyses	
		

Data	 of	 the	 antifungal	 and	 the	 antibacterial	 tests	 were	
analyzed	separately.	Data	were	subjected	to	one‐way	analysis	
of	 variance	 (ANOVA)	 according	 to	 a	 factorial	 design	 where	
fungal	 (or	 bacterial	 agents),	 compounds	 and	 the	
concentrations	used	were	the	three	fixed	factors.	Means	were	
separated	 using	 Student‐Newman‐Keul's	 (SNK)	 test	 at	 p	 ≤	
0.05.	
	
2.2.5.	Acetylcolinesterase	inhibition		
		

The	 acetylcholinesterase	 (AChE)	 inhibition	 of	 the	 two	
series	of	 the	separated	dihydroisoxazoles	2a‐h	 and	5a‐h	was	
determined	 using	 an	 adaptation	 of	 the	 method	 described	 in	
the	literature	[41].	90	μL	of	50	mM	Tris‐HCl	buffer,	pH	=	8,	30	
μL	 of	 the	 sample	 dissolved	 in	 MeOH	 and	 7.5	 μL	 of	
acetylcholinesterase	 solution	 containing	 0.26	 U/mL	 were	
mixed	 in	 a	 microwell	 plate	 and	 left	 to	 incubate	 for	 15	 min.	
Subsequently,	 22.5	 μL	 of	 a	 solution	 of	 acetylcholine	 iodide	
(AChI)	(0.023	mg/mL)	and	142	μL	of	3	mM	DTNB	were	added.	
The	absorbance	was	read	at	405	nm	 in	 the	presence	(Asample)	
and	 in	 the	 absence	 (Acontrol)	 of	 the	 tested	products	 and	when	
the	 reaction	 reached	 equilibrium.	 Eserine	 was	 used	 as	 a	
positive	control	and	water	served	as	a	negative	control	and	it	
was	considered	100%	activity.	The	 inhibition	percentage	(IP)	
is	given	as	follow:	
	
IP	=	100	‐	(Asample	/	Acontrol)	×	100	 	 	 (1)	
	
where	 Acontrol	 is	 the	 absorbance	 of	 the	 control	 reaction	
containing	all	reagents	except	the	tested	sample,	and	Asample	is	
the	 absorbance	 of	 the	 tested	 compounds.	 Tests	were	 carried	
out	in	triplicate.	
	
3.	Results	and	discussion		
	
3.1.	Chemistry	
		

We	report	here,	a	novel	one‐step	synthesis	of	isoxazolines	
by	 1,3‐dipolar	 cycloaddition	 reaction	 by	 condensing	 (R)‐
limonene	1	with	different	arylnitrile	oxides	in	the	presence	of	
triethylamine	in	refluxing	anhydrous	toluene	for	four	hours	to	
give	 a	 number	of	new	structural	 analogues	 of	5‐methyl‐5‐(4‐
methylcyclohex‐3‐enyl)‐3‐phenyl‐4,5‐dihydroisoxazoles	 2a‐h	
(Scheme	1	and	Table	1).	Under	these	experimental	conditions,	

the	reaction	revealed	in	all	cases	the	formation	of	a	mixture	of	
two	 diastereoisomers	 (R,R)	 and	 (R,S)	 not	 easily	 separable	
which	 were	 assigned	 as	 isoxazolines	 2a‐h	 based	 on	 their	
spectral	 data.	 The	 structure	 of	 compounds	 2a‐h	 has	 been	
assigned	from	their	analytical	data.	

In	 fact,	 ES‐HRMS	 of	 compound	 2d,	 given	 as	 an	 example,	
gave	 a	 pseudo‐molecular	 ion	 peak	 [M+Na]+	 at	m/z	 308.1630	
which	 is	 consistent	 with	 the	 molecular	 formula	 C18H23NO2.	
Furthermore,	 the	 1H	 NMR	 spectrum	 of	 this	 compound	 was	
compatible	 with	 the	 proposed	 structure.	 In	 addition	 to	 the	
signals	corresponding	to	 the	protons	 introduced	by	the	cyclic	
part	 of	 (R)‐limonene	 1,	 we	 observed	 the	 presence	 of	 new	
signals	consequent	 to	 the	methoxy	group	(δH	3.83	ppm,	s),	 to	
the	methylene	protons	H‐4a,b	(δH	2.94	ppm,	d,	J	=	16.5	Hz,	H‐
4a	 and	 δH	 3.25	 ppm,	 d,	 J	 =	 16.5	 Hz,	 H‐4b).	 A	 characteristic	
AA’BB’	pattern	for	aromatic	protons	was	observed	in	the	same	
spectrum.	 Examination	 at	 300	 MHz	 offered	 excellent	
resolution	with	two	doublets	at	δH	6.89	ppm	(2H,	d,	J	=	8.7	Hz;	
H‐3'',	H‐5'')	and	at	δH	7.61	ppm	(2H,	d,	J	=	8.7	Hz;	H‐2'',	H‐6'')	
all	 relative	 to	 the	 isoxazoline	moiety.	 The	 13C	NMR	 spectrum	
confirmed	 the	 above	 spectral	 data	 by	 the	 observation	 of	
signals	 at	 δC	 55.40	 ppm,	 δC	 160.82	 ppm	 and	 at	 δC	 114.08‐
127.95	ppm	attributable	to	the	methoxy,	 to	the	iminic	carbon	
(C‐3)	in	the	dihydroisoxazole	ring	and	to	the	aromatic	carbons,	
respectively.	 The	 duplication	 of	 the	 most	 indicated	 signals	
confirms	 the	 formation	 of	 the	 two	 diasatereoisomers	 (R,	 R)	
and	(R,	S).	The	reaction	was	diastereoselective.	

The	 β‐lactam	 3	 was	 prepared	 in	 good	 yield	 (78%)	 by	
treating	 (R)‐limonene	1	with	 chlorosulfonyl	 isocyanate	 (CSI)	
in	 anhydrous	 ether	 at	 room	 temperature	 for	 nine	 hours	
(Scheme	2).	The	structure	of	compound	3	was	established	on	
the	 basis	 of	 its	 spectroscopic	 data.	 Its	 positive	 ES‐HRMS	
showed	 a	 pseudo‐molecular	 ion	 peak	 at	 [M+H]+	 at	 m/z	
180.1380	 which	 is	 consistent	 with	 the	 molecular	 formula	
C11H17NO.	The	1H	NMR	spectrum	of	this	compound	showed	the	
appearance	of	 two	new	signals	at	δH	2.42	ppm	(1H,	d,	 J=14.4	
Hz)	 and	 δH	 2.66	 ppm	 (1H,	 d,	 J	 =14.4	 Hz)	 attributable	 to	 the	
non‐equivalent	 methylenic	 protons	 H‐3a	 and	 H‐3b	 of	 the	
lactam	 system,	 respectively,	 in	 addition	 to	 the	 characteristic	
signals	 of	 the	 protons	 introduced	 by	 the	 cyclic	 fragment	 of	
limonene	 1.	 Its	 13C	 NMR	 spectrum	 reinforced	 the	 proposed	
structure	by	the	appearance	of	the	signals	of	C‐2	(CO)	and	C‐3	
(CH2)	 at	 δC	 167.69	 ppm	 and	 δC	 39.92	 ppm,	 respectively,	 in	
addition	 to	 the	 carbon	 signals	 introduced	 by	 the	 limonene	
skeleton.	 The	 diastereo	 selectivity	 of	 the	 reaction	 was	
ascertained	 by	 the	 duplication	 of	 the	 most	 signals	 indicated	
above.	

The	required	dipolarophile	4	was	prepared	by	N‐allylation	
(Scheme	 2).	 Indeed,	 in	 our	 investigation,	 anhydrous	
dimethylformamide	was	 found	 to	 be	 an	 excellent	 solvent	 for	
the	 reaction	of	allylbromide	with	 lactam	3	 in	 the	presence	of	
NaH	used	as	a	base.	The	N‐allyllactam	4	was	obtained	in	88%	
yield	 and	 the	 reaction	 is	 completed	 after	 one	 hour	 at	 room	
temperature.	

The	structure	of	compound	4	was	established	on	the	basis	
of	 its	 spectroscopic	 data.	 Its	 ES‐HRMS	 spectrum	 gave	 a	
pseudo‐molecular	 ion	 peak	 [M+H]+	 at	 m/z	 220.1693	
compatible	with	the	molecular	formula	C14H21NO.	The	1H	NMR	
spectrum	 of	 compound	 4	 indicates	 the	 presence	 of	
characteristic	signals	of	the	precursor	3	skeleton	which	can	be,	
according	 to	 their	 chemical	 shifts	 and	 multiplicities,	 readily	
assigned	to	H‐3'	(δH	5.37	ppm,	1H),	H‐7'	(δH	1.71	ppm,	3H),	H‐5	
(δH	1.35	ppm,	3H).	In	addition	of	the	signals	corresponding	to	
the	 protons	 introduced	 by	 the	 lactam	 3,	 we	 revealed	 the	
appearance	of	signals	at	δH	2.42	ppm	(d,	1H,	J=	14.4	Hz)	and	at	
δH	2.66	ppm	(d,	1H,	J=	14.4	Hz)	relative	to	the	non‐equivalent	
protons	H‐3a	and	H‐3b,	of	the	lactam	system,	respectively.	The	
two	doublet	of	doublets	 at	δH	5.12	ppm	 (dd,	1H,	 Jcis=10.2	Hz,	
J2=1.2	Hz)	and	at	δH	5.22	ppm	(dd,	1H,	Jtrans=	17.1	Hz,	J2=1.8	Hz)	
was	attribuated	to	 the	 terminal	methylenic	protons	H‐8a	and	
H‐8b.		



26	 Bnina	et	al.	/	European	Journal	of	Chemistry	6	(1)	(2015)	21‐30	
	

	
Table	1.	Synthesis	of	compounds	2a‐h.	
Entry	 Compound	 Entry	 Compound	 Entry	 Compound	
1	

	

2a	67%	

2	

	
2b	73%	

3

	
2c	76%	

4	

	
2d	80%	

5	

	

2e	82%	

6

	
2f	87%	

7	

	

2g	68%	

8	

	

2h	70%	

	
	

The	multiplet	at	δH	5.77	ppm	(m,	1H)	was	assigned	to	the	
ethylenic	proton	H‐7.	On	the	other	hand,	C‐2	(167.19	ppm),	C‐
3	 (38.58	 ppm),	 C‐6	 (46.10	 ppm),	 C‐7	 (133.42	 ppm)	 and	 C‐8	
(116.23	 ppm)	 were	 readily	 assigned	 from	 the	 13C	 NMR	
spectrum.	

The	 dipolarophile	 4	 was	 then	 treated	 with	 various	
arylnitrile	 oxides	 generated	 in	 situ	 from	 aromatic	 oxime	
precursors	 under	 conventional	 conditions	 furnished	 the	
desired	isoxazolines	5a‐h	 in	good	yields	(68‐87%)	(Scheme	3	
and	 Table	 2).	 The	 reaction	 was	 regiospecific	 and	 disatereo‐
selective.	

The	 structures	 of	 these	 compounds	 were	 confirmed	
according	 to	 their	 spectral	 data.	 The	 1H	 NMR	 spectra	 of	
compounds	5a‐h	shows	duplication	of	most	signals	indicating	
the	 formation	 of	 a	 mixture	 of	 diastereoisomers	 due	 to	 the	
apparition	of	two	additional	stereogenic	centers,	(C‐4	and	C‐5).	
Attempts	 to	 separate	 these	 diastereoisomers	 by	 chromato‐
graphy	were	not	successful.	The	ES‐HRMS	of	compound	5h	as	
an	example	gave	a	pseudo‐molecular	 ion	peak	[M+H]+	at	m/z	
345.1649	 which	 is	 consistent	 with	 the	 molecular	 formula	
C19H24N2O2S.	 In	 the	 1H	 NMR	 spectrum	 of	 compound	 5h	we	
observed	a	multiplet	centered	at	5.83	ppm	attributable	to	the	
stereogenic	center	protons	(H‐5)	system	and	another	multiplet	
at	 δH	 2.75‐2.86	 (m,	2H)	 assigned	 to	 from	 the	 non‐equivalent	
methylenic	protons	H‐4	both	from	the	isoxazoline	moiety.	The	
N–CH2	 protons	 (H‐6)	 appeared	 as	 a	 multiplet	 at	 3.38‐3.73	
ppm.	Signals	at	δH	2.51	ppm	(d,	1H,	J=	13.5	Hz)	and	at	δH	2.80	
ppm	(d,	1H,	J=	13.5	Hz)	were	attributed	to	the	non‐equivalent	
protons	H‐3'a	and	H‐3'b	of	the	lactam	moiety.	We	revealed	the	
appearance	of	signals	at	δH	7.39	ppm	(d,	1H,	J=	3.9	Hz,	H‐4'''),	
at	δH	7.19	ppm	(d,	1H,	J=3.9	Hz,	H‐2''')	and	at	δH	7.05	ppm	(t,	
1H,	J=3.9	Hz,	H‐3''')	relative	to	the	protons	H‐4"',	H‐2"'	and	H‐

3"'	of	the	thiophene	system,	respectively.	13C	NMR	spectrum	of	
compound	5h	 exhibited	a	signal	at	61.36	ppm	corresponding	
to	 the	 N–CH2	 carbon	 (C‐6),	 two	 signals	 at	 39.25	 ppm	 and	
168.12	 attribuable	 to	 the	 lactam	 carbons	 C‐3'	 and	 C‐2',	
respectively,	 and	 two	 signals	 at	 39.9	 ppm	 and	 79.43	 ppm	
relative	 to	 the	 isoxazoline	 carbons	 C‐4	 and	 C‐5,	 respectively.	
The	 C=N	 carbon	 C‐3	 resonated	 at	 167.02	 ppm.	 The	 signals	
observed	between	127.37‐128.66	ppm	are	attributable	 to	 the	
thiophene	carbons.	The	duplication	of	some	signals	confirmed	
the	diastereo	selectivity	of	the	reaction.	
	
3.2.	Biological	activities	
	
3.2.1.	Antibacterial	activity	
		

Data	presented	 in	Table	3	 show	 that	 the	 inhibition	 zones	
induced	by	(R)‐Limonene	1,	lactam	3,	N‐alkyllactam	4	and	the	
different	 isoxazolines	 (2a‐h	 and	 5a‐h)	 tested	 against	 three	
batteries	Burkholderia	Glathei	153,	Bascillus	pumilus	 420	and	
Pseudomonas	aureofaciens	499	vary	significantly	(at	p	≤	0.01)	
depending	 on	 compounds,	 concentrations	 and	 the	 bacterial	
agents	 used.	A	 significant	 interaction	was	noted	between	 the	
three	fixed	factors.	

Table	3	indicates	also	that	the	antibacterial	activity	of	the	
tested	 compounds	 varied	 upon	 the	 concentrations	 used.	 In	
fact,	 visible	 inhibition	 zones	 were	 more	 evident	 since	 the	
concentration	of	250	μg/mL	and	 the	highest	 inhibitory	effect	
was	 recorded	 at	 the	 concentration	 of	 1000	 μg/mL	 with	 all	
compounds	tested.	

Burkholderia	Glathei	153	 and	Bascillus	pumilus	 420	were	
very	sensitive	 to	(R)‐limonene	1,	which	gave	inhibition	zones	
varying	from	32.5	to	33.75	mm.		



Bnina	et	al.	/	European	Journal	of	Chemistry	6	(1)	(2015)	21‐30	 27	
 

	
Table	2.	Synthesis	of	compounds	5a‐h.	
Entry	 Compound	 Entry	 Compound	
1	

N

O 1

2

3

4
5

6

6''

1'
2'

3'

4'
5'

7''

O
N

1''
2''

3''

4''

5''

5'''
6'''

2'''

1'''

3'''

4'''

	
5a	70%	

5

	
5e	85%	

2	

 
5b	77%	

6	

	
5f	75%	

3	

	
5c	75%	

7	

	
5g	68%	

4	

	
5d	87%	

8	

	
5h 74%

	
	
On	the	other	hand,	Pseudomonas	aureofaciens	499	shows	a	

resistance	to	(R)‐limonene	1	with	an	inhibition	zone	of	15.25	
mm.	

4,5‐Dihydroisoxazoles	 2a,	 2d,	 2e,	 2g,	 5a,	 5d,	 5e	 and	 5g	
showed	 significant	 inhibition	 zones	 (12.5‐14	 mm,	 concent‐
ration	of	1000	μg/mL)	against	Burkholderia	Glathei	153.	This	
activity	 may	 be	 related	 to	 the	 nature	 of	 the	 aromatic	 ring	
(phenyl,	 4‐methoxyphenyl,	 4‐chlorophenyl	 and	 furanyl,	
respectively)	 fixed	 at	 C‐3	 of	 the	 dihydroisoxazole	 moiety	 in	
these	compounds.	

The	growth	of	bacterial	Bascillus	pumilus	420	 is	 inhibited	
mainly	 by	 compounds	 2b,	 2d,	 2g,	 5b,	 5d	 and	 5g	 showing	
inhibition	zones	varying	between	12.5	and	15	mm.	The	nature	
of	 the	 aromatic	 ring	 (methylphenyl,	 4‐methoxyphenyl	 and	
furanyl,	respectively)	attached	to	C‐3	of	the	isoxazoline	system	
could	be	 the	 origin	 of	 the	noted	 activity	 in	 these	 compounds	
compared	to	the	others	cycloadducts.	

Pseudomonas	 aureofaciens	 499	 was	 found	 to	 be	
remarkably	 sensitive	 towards	 compounds	 2a,	2c,	2d,	2f,	2g,	
2h,	 5a,	 5c,	 5d,	 5f,	 5g	 and	 5h	 with	 inhibition	 zones	 ranging	
between	 12	 and	 17.25	 mm	 at	 the	 concentration	 of	 1000	
μg/mL.	 Compounds	2h	 (IZ	 =	 17.25	mm)	 and	5h	 (IZ	 =	 17.25	

mm)	 both	 bearing	 a	 thiophene	 ring	 at	 C‐3	 of	 the	
dihydroisoxazole	moiety,	showed	higher	antibacterial	activity	
against	 Pseudomonas	 aureofaciens	 499	 (IZ	 =	 15.25	 mm)	
compared	 to	 the	 other	 cycloadducts	 and	 to	 (R)‐limonene	 1.	
The	presence	of	 the	 thiophenyl	 ring	 in	both	2h	 and	5h	could	
be	the	origin	of	this	activity.	

Our	 results	were	 in	 good	 concordance	with	 that	 cited	 in	
the	 literature	 showing	 the	 important	 antibacterial	 activity	 of	
some	 dihydroisoxazoles	 [21,22].	 Table	 3	 also	 revealed	 that	
compounds	 2d,	 5d,	 2g	 and	 5g	 exhibited	 antibacterial	 effect	
towards	all	the	used	bacterial	agents.	
	
3.2.2.	Antifungal	activity	
		

Data	 shown	 in	Table	4	 revealed	 that,	 (R)‐limonene	1	 and	
all	 synthesized	 compounds	 were	 tested	 against	 five	 fungal:	
Aspergillus	 niger,	 Aspergillus	 flavus,	 Penicillium	 digitatum,	
Trichoderma	 harzanum	 and	 Fusarium	 solani.	 As	 above	
mentioned	for	the	antibacterial	activity,	the	inhibitory	effect	of	
the	tested	compounds	became	visible	since	the	concentration	
of	 250	 μg/mL	 and	 the	 highest	 inhibition	 of	 the	 tested	 fungi	
was	recorded	at	1000	μg/mL.	



28	 Bnina	et	al.	/	European	Journal	of	Chemistry	6	(1)	(2015)	21‐30	
	

	
Table	3.	Antibacterial	activity	of	compounds	1,	3,	4,	2a‐h	and	5a‐h	depending	on	the	concentrations	used	a.	
Compounds	 Concentration		

(µg/mL)	
Burkholderia	
glathei

Bacillus	
pumilus

Pseudomonas	
aureofaciens	

1	 250	 27.75	 24.25 10.25	
500	 29.75	 25.75	 11.75	
1000	 33.75	 32.5	 15.25	

3	 250	 7	 ‐	 7.5	
500	 9.75	 6.5	 9.75	
1000	 10.75 7.75 15.75	

4	 250	 ‐ ‐ 9.5	
500	 7.75 6.5 9.75	
1000	 13 10.25 16.25	

2a	 250	 7.5 6.25 7.25	
500	 10.5 7 8.5	
1000	 12.5 9 13.5	

2b	 250	 ‐	 8	 ‐	
500	 ‐	 10	 ‐	
1000	 7 12.5 8.25	

2c	 250	 6.75	 6.75	 10	
500	 8 6.75 11.5	
1000	 10 8.75 14	

2d	 250	 7.75 7.25 6.5	
500	 10 10 9.75	
1000	 12.5 15 12.5	

2e	 250	 8.5 6.75 7.25	
500	 10.75	 7.5	 9	
1000	 13.25 10.75 10.5	

2f	 250	 ‐	 6,5	 6.25	
500	 7.75	 9.25	 8.5	
1000	 8.75	 11.75	 13.5	

2g	 250	 9 9.25 9.75	
500	 12.75 12.75 10.25	
1000	 14 14.25 12	

2h	 250	 ‐ 6.5 8.75	
500	 ‐ 7.5 10.5	
1000	 8 9 17.25	

5a	 250	 7.5	 6.25	 7.25	
500	 10.5	 7	 8.5	
1000	 12.5	 9	 13.5	

5b	 250	 ‐ 8 ‐	
500	 ‐	 10	 ‐	
1000	 7 8.25	

5c	 250	 6.75 6.75 10	
500	 8 6.75 11.5	
1000	 10 8.75 14	

5d	 250	 7.75 7.25 6.5	
500	 10 10 9.75	
1000	 12.5	 15	 12.5	

5e	 250	 8.5 6.75 7.25	
500	 10.75	 7.5	 9	
1000	 13.25	 10.75	 10.5	

5f	 250	 ‐	 6,5	 6.25	
500	 7.75 9.25 8.5	
1000	 8.75 11.75 13.5	

5g	 250	 9 9.25 9.75	
500	 12.75 12.75 10.25	
1000	 14 14.25 12	

5h	
250	 ‐ 6.5 8.75	
500	 ‐	 7.5	 10.5	
1000	 8	 9	 17.25	

Ampicillin	b	 5000	 32.5	 30.5 31.5	
DMSO	 	 ‐ ‐ ‐	
a	Diameter	of	inhibition	zone	(IZ)	expressed	in	mm,	“‐”:	Not	active.	
b	Positive	control.	
	
	

Limonene	1	was	unable	to	reduce	the	growth	of	Aspergillus	
flavus	and	Trichoderma	harzianum	but	it	exhibited	a	significant	
effect	towards	Aspergillus	niger	(IZ	=	24.25	mm)	and	Fusarium	
solani	(IZ	=	25	mm).	All	synthesized	compounds	demonstrated	
variable	 inhibition	zones,	which	dominated	by	the	activity	2a	
and	2b	towards	Trichoderma	harzianum	(IZ	=	13.25	and	12.75	
mm,	respectively),	2b,	2d	and	2g	against	Penicillium	digitatum	
(IZ	 =	 13,	 12.25	 and	 13	 mm,	 respectively)	 and	 5b	 towards	
Aspergillus	 flavus	 (IZ	 =	 13.75	mm).	 The	 recorded	 results	 did	
not	 show	 any	 clear	 structure‐antifungal	 activity	 relationship	
and	 could	 be	 explained	 by	 the	 relative	 susceptibility	 of	 the	
microorganism	to	each	compound	tested.	
	
	
	

3.2.3.	Anti‐acetylcholinesterase	activity	
		
Inhibition	of	acetylcholinesterase	(AChE),	the	key	enzyme	

in	 the	 breakdom	 of	 acetylcholine,	 is	 considered	 one	 of	 the	
treatment	 strategies	 against	 several	 neurological	 disorders	
such	 as	 Alzheimer's	 disease,	 senile	 dementia,	 ataxia	 and	
myasthenia	 gravis	 [42,43].	 The	 acetylcholinesterase	 (AChE)	
inhibition	was	determined	using	an	adaptation	of	the	method	
described	 in	 the	 literature	 [41].	 Only	 dihydroisoxazoles	2a‐h	
and	5a‐h	were	 assayed	 for	 inhibition	 of	 acetylcholinesterase	
and	 the	 obtained	 results	 are	 shown	 in	 Table	 5.	 The	 IC50	 of	
compounds	 2a‐h	 ranged	 between	 82	 and	 280	 μg/mL,	 and	
those	of	compounds	5a‐h	varied	between	99	and	500	μg/mL.		

	
	



Bnina	et	al.	/	European	Journal	of	Chemistry	6	(1)	(2015)	21‐30	 29	
 

	
Table	4.	Antifungal	activity	of	compounds	1,	3,	4,	2a‐h	and	5a‐h	depending	on	the	concentrations	a.	
Compounds	 Concentration		

(µg/mL)	
Aspergillus		
niger	

Penicillium	
digitatum	

Trichoderma		
harzianum	

Fusarium	
solani	

Aspergillus	
flavus	

1	 250	 15		 7.5	 ‐	 13.75	 ‐	
500	 21	 9	 ‐	 23.25	 ‐	
1000	 24.25	 10.25	 ‐	 25	 ‐	

3	 250	 8.5	 ‐	 6.5	 6	 7.5	
500	 9.5	 ‐ 7.75 8.25	 8.75	
1000	 11.5	 ‐ 8.5 9 11.25	

4	 250	 8.25	 ‐ 6 ‐ 6.5	
500	 8.75	 ‐ 7 ‐ 7.75	
1000	 10.25	 8.25 8.75 7.75	 8.75	

2a	 250	 8.5	 ‐ 9.5 ‐ 7	
500	 11	 6.25 10.5 9 8.25	
1000	 12	 8	 13.25	 11.5	 10.25	

2b	 250	 8.5	 ‐ 8.75 ‐ 7.5	
500	 9.5	 7.75	 11.25	 ‐	 8.25	
1000	 11.75	 13	 12.75	 8.75	 9.5	

2c	 250	 7	 ‐ ‐ 7.75	 ‐	
500	 9	 ‐ 7.5 9.5 ‐	
1000	 10.25	 ‐ 8.5 10.5	 8.5	

2d	 250	 9.5	 ‐ 7 7.75	 8.75	
500	 11	 9 9.5 10.25	 9.5	
1000	 12.25	 12.25 11.5 11.75	 11	

2e	 250	 8	 ‐ ‐ ‐ ‐	
500	 9.25	 7	 ‐	 ‐	 7	
1000	 10.25	 8	 8	 8.5	 9	

2f	 250	 8	 ‐	 ‐	 ‐	 ‐	
500	 9.25	 ‐	 ‐	 8.25	 ‐	
1000	 11	 8.75 ‐ 10.75	 8.25	

2g	 250	 8.75	 7.25 8.25 7.25	 7.5	
500	 11.5	 10 11 9.5 10	
1000	 11.5	 13 13 11 10	

2h	 250	 8.75	 ‐ 7.25 7.75	 ‐	
500	 10.5	 ‐ 8.5 8.25	 8	
1000	 10.25	 8	 12	 10.75	 10.5	

5a	 250	 6.5	 ‐	 ‐	 8.25	 6.5	
500	 7.25	 ‐	 ‐	 9.75	 7.5	
1000	 8.25	 ‐ ‐ 12.5	 9.5	

5b	 250	 7	 ‐	 ‐	 ‐	 8	
500	 7.75	 7.5 7 ‐ 10	
1000	 9.25	 10 8.75 8.75	 13.75	

5c	 250	 8.5	 ‐ ‐ ‐ ‐	
500	 9	 6.25 9 ‐ 7.25	
1000	 10.5	 7.75 11.5 8 9	

5d	 250	 7.5	 ‐ ‐ ‐ 6.5	
500	 8.25	 ‐	 ‐	 ‐	 7	
1000	 10	 7.5 ‐ 9.25	 9.25	

5e	 250	 7.25	 ‐	 ‐	 ‐	 ‐	
500	 9.5	 ‐	 ‐	 ‐	 8.25	
1000	 10.5	 7.5	 ‐	 9.25	 9.25	

5f	 250	 7.25	 6 7.25 ‐ ‐	
500	 8	 6 9.25 7.75	 ‐	
1000	 9.5	 7.75 11.5 10.25	 8	

5g	 250	 7.5	 6 ‐ ‐ ‐	
500	 8.5	 6 9 8.25	 7	
1000	 9.75	 7.75 10.25 10.75	 8.75	

5h	
250	 7.75	 6	 ‐	 ‐	 6.75	
500	 9.5	 6	 7.25	 7.5	 8.75	
1000	 11.5	 6	 9.25	 9.25	 10.25	

Carbendazim	b	 500	 35	 ‐ 34 ‐ 35a	
DMSO	c	 	 ‐	 ‐ ‐ - -	
a	Diameter	of	inhibition	zone	(IZ)	expressed	in	mm,	“‐”:	Not	active.	
b	Positive	control.		
c	Solvent	(negative	control).	
	
Table	5.	Acetylcholinesterase	inhibition	capacity,	represented	by	IC50	(mg/mL)	of	2a‐h	and	5a‐h	a.	
Compounds	 IC50	(mg/mL)	 Compounds IC50	(mg/mL)	
2a	 175±2		 5a	 99±1	
2b	 172±3	 5b	 190±1	
2c	 170±1	 5c 110±2	
2d	 230±2	 5d	 175±4	
2e	 173±1	 5e	 140±1	
2f	 82±3	 5f 450±1	
2g	 250±2	 5g 500±3	
2h	 280±1	 5h 310±1	
Eserine	b	 0.018±0.002	
	a	Average±SD	were	obtained	from	three	different	experiments.	
	b	Positive	control.	
	
	

The	most	significant	inhibition	of	acetylcholinesterase	was	
induced	 by	 compounds	2f	 (IC50	 =	 82±3	µg/mL)	 in	which	 the	
aryl	 group	 fixed	 at	 C‐3	 of	 the	 dihydroisoxazole	 is	 the	 4‐

chlorophenyl,	and	5a	(IC50	=	99±1	µg/mL)	with	a	p‐tolyl	group	
at	 the	 same	 position.	 The	 compounds	2g,	2h,	5d,	5e	 and	5g	
have	a	close	inhibition.	



30	 Bnina	et	al.	/	European	Journal	of	Chemistry	6	(1)	(2015)	21‐30	
	

IC50	 values	 represent	 the	 concentration	 of	 inhibitor	
required	 to	 decrease	 enzyme	 activity	 by	 50%	 and	 are	 the	
mean	 of	 two	 independent	measurements,	 each	 performed	 in	
triplicate.	
	
4.	Conclusion	
	

In	this	paper,	we	described	the	synthesis	of	a	new	series	of	
dihydroisoxazoles	 from	 the	 (R)‐limonene	 via	 the	 1,3‐dipolar	
cycloaddition	 reactions	 exploring	 its	 disubstituted	 terminal	
double	 bond.	 On	 the	 other	 hand,	 the	 limonene‐lactam,	
previously	prepared	in	this	work,	was	N‐allylated	to	be	used	in	
a	1,3‐dipolar	cycloaddition	reaction	affording	an	original	new	
series	 of	 regiospecific	 dihydroisoxazoles.	 All	 synthesized	
compounds	 were	 evaluated	 for	 their	 antimicrobial	 and	 anti‐
acetylcholinesterase	 activities.	 The	 fusion	 of	 two	 biologically	
relevant	systems	as	they	are	the	limonene	skeleton	along	with	
the	 dihydroisoxazole	 ring	 may	 explain	 some	 significant	
activities	noted.	
	
Acknowledgement	
 

The	 authors	 acknowledge	 the	 Ministry	 of	 Higher	
Education,	 Scientific	 Research	 and	 Technology	 in	 Tunisia	 for	
the	financial	support.	
	
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